SECTION 8 Infectious diseases

8.1 Pathogenic microorganisms and the host 651

8.1 Pathogenic microorganisms and the host 651

8.1.1 Biology of pathogenic microorganisms 651

8.1.1 Biology of pathogenic microorganisms 651

8.1 Pathogenic microorganisms and the host CONTENTS 8.1.1 Biology of pathogenic microorganisms  651 Duncan J. Maskell and James L.N. Wood 8.1.2 Clinical features and general management of patients with severe infections  656 Peter Watkinson and Duncan Young 8.1.1  Biology of pathogenic microorganisms Duncan J. Maskell and James L.N. Wood ESSENTIALS Microorganisms are present at most imaginable sites on the planet, and have evolved to occupy these ecological niches successfully. A host animal is simply another ecological niche to be occupied. The ability to cause disease may in some cases be an accidental bystander event, or it may be the result of evolutionary processes that have led to specific mechanisms allowing the pathogen to ex- ploit the rich source of nutrients present in the host, and then be transmitted to another fresh host. Pathogenicity often relies on a series of steps, with specific and often distinct mechanisms operating at each of them. Some types of pathogen must adapt to the host environment by altering gene expression, and most must retain the ability to be transmitted readily between hosts. Specific mechanisms have evolved in microorgan- isms for the exploitation of the host and for evasion or avoidance of the innate and acquired immune systems. The advent and application of hyper-​rapid and ultra-​high throughput whole genome scale sequencing technologies is pro- viding a mass of information, which has started to change fundamen- tally our way of looking at infectious diseases and our understanding of how pathogens work. This should enable the development of new intervention strategies, especially vaccines and antimicrobials, but the complexity of some of the biological mechanisms involved may make this a difficult exercise. Furthermore, pathogens can vary and evolve rapidly, and thus are likely to remain one step ahead of these strategies. We live in a rapidly changing world. New pathogens will emerge to exploit new circumstances presented by changes in society, and ancient scourges will remain and re-​emerge to plague us. Many of the new infectious disease challenges will arise from animals, and will be zoonoses, at least in the early stages of their emergence. It is therefore probably more important in this field than in any other to develop the vision of ‘One Medicine’, with medical and veterinary clinicians and basic scientists working together, if we are to give our- selves the best chance of success in warding off threats from global infectious diseases. Introduction Microorganisms occupy almost all imaginable ecological niches. Microbes have been isolated from deep-​sea sites, where they sur- vive very high pressures, from extremely cold and extremely hot regions, where hyperthermophiles grow optimally at temperatures well in excess of 100°C, and even from rocks, where they can exploit chemical substrates for energy generation. It is no wonder, then, that microorganisms should also exploit other living organisms as po- tential habitats, from viruses that use other microorganisms as hosts (e.g. bacteriophage) through to microorganisms that occupy various ecological niches within and upon the mammalian body, sometimes to the benefit and sometimes to the severe detriment of the host. Microorganisms have been supremely successful in evolutionary terms and they contain enormous untapped reserves of biodiversity, much of which is to be found in those that we can neither isolate nor grow and which make up most microbes on the planet. Since micro- organisms reproduce much more rapidly than their mammalian hosts and have several specialized mechanisms for horizontal gene transfer, it is not surprising that they are often able to evolve quickly to stay one step ahead of any mechanisms that exist, or are invented by humans, to control them. The control of infectious diseases over the last century or so has been a major achievement, relying mainly on improved public health systems and social conditions, as well as on technological advances such as antimicrobial drugs and vaccines. Optimism about win- ning the battle with infectious diseases was bolstered by the eradi- cation of smallpox in 1977, achieved by a monumental worldwide public health and vaccination programme. But, as Aldous Huxley

652 SECTION 8  Infectious diseases wrote, ‘Hubris against the . . . order of Nature would be followed by its appropriate Nemesis’, and so it was that very soon afterwards we had to learn to cope with the global catastrophe that is AIDS, along with the resurgence of ancient killers such as tuberculosis, and the emergence of apparently new or re-​emergent threats such as bovine spongiform encephalopathy (BSE) and West Nile fever. In 2009, the world found itself dealing with the long-​predicted global pandemic of influenza which severely stretched and tested the in- genuity and organizational abilities of international human society in its attempts to control the impact of the virus. That this pandemic had a relatively minor impact, despite rapid and widespread dis- semination and transmission, was largely due to the generally mild nature of disease caused by this virus. Lessons for public health re- garding control are still being learned from this incident. Far more devastating, at least locally, has been the Ebola epidemic in three countries in West Africa between 2014 and 2016, a catastrophe that has had far reaching consequences on health service infrastruc- tures in those countries and on international responses to epidemic diseases. New disease threats will continue to emerge, leading to consequences that are difficult to predict, such as the emergence of Zika virus as a recent example. Pathogenicity in stages It is important to break down pathogenesis into different steps and stages. Most viral and bacterial pathogens enter the host via the mucosa of the respiratory, gastrointestinal, or genitourinary tracts, although some important pathogens are introduced by in- jection from insect vectors or through abraded or wounded skin. Most pathogens then must stick to a surface and have evolved struc- tures that enable this; these are usually constructed from proteins and many of them are complex and specialized. In bacteria, these protein molecules are known as adhesins and are often but not al- ways delivered at the end of long proteinaceous organelles called pili or fimbriae. The precise amino acid sequence of the adhesin, and hence its structure, can dictate which host and even which tissues within the host the bacterium sticks to and can, therefore, play a major role in dictating host range and tissue tropism. For example, enterotoxigenic Escherichia coli expressing K88 fimbriae will stick to piglet intestine and cause disease, those expressing K99 will stick to calf and lamb intestine, and those expressing colonization factor antigen (CFA) I and CFAII will stick to human intestine. Similarly, E. coli expressing P fimbriae (otherwise known as PAP pili) will stick efficiently to the human urinary tract and cause infection at that site. After initial loose adherence, enteropathogenic E. coli will stick more firmly to the intestinal surface via the non​fimbrial adhesin, intimin. The receptor for intimin on the host cell surface is a protein called Tir, which is itself an E. coli protein that has been translocated into the host cell via a specialized needle-​like structure, a type 3 secretory system (T3SS), which is itself closely related to bacterial flagella. This complex, coordinated series of events gives an insight into the extra- ordinary sequences that have evolved to enable bacteria to exploit their hosts as ecological niches. Viruses also rely on surface structures for host specificity, an ex- ample being influenza virus. Among several other mechanisms, the host range and tissue tropism of influenza virus is dependent on the structure of its haemagglutinin molecule. On the respiratory epithelium, haemagglutinin binds to sialic acid which is linked to galactose on the host cell surface via either an α2,3 or an α2,6 linkage. Human influenza viruses bind preferentially to the α2,6-​linked mol- ecule, which is abundant on human tracheal epithelium, whereas avian influenza viruses bind preferentially to the α2,3-​linked ver- sion, which is abundant on duck intestinal epithelium. The different binding capacities of the haemagglutinin molecules are also im- portant in the transmissibility of the virus, which is clearly a major element in determining whether or not an epidemic will occur. Interestingly, pig trachea expresses plenty of both types of molecule, which may explain in part why pigs are susceptible to both avian and human influenza viruses, although their distribution is different at different levels of the respiratory tract. An important nuance is that different viruses must bind to and infect the same cell in order for reassortment to result. While this raises significant questions for dogma that the pig may be a mixing vessel, it is interesting that the pig is the host of many viruses that have reassorted within swine, including the 2009 human pandemic virus. The precise cell tropism in the human respiratory tract for viruses of different host origin might also correlate with the type of disease caused, and possibly the amount of virus shed, leading both to different disease severities and potentially different transmission dynamics. Once established at a surface, pathogens have a wide array of possible strategies. They can stay at that surface and cause very little damage, and indeed be carried without causing any clinical signs. Bacteria such as Haemophilus influenzae and Neisseria meningitidis are good examples of this. Only as a result of some unknown and rare set of circumstances will these bacteria move into the blood- stream to cause septicaemia and sometimes meningitis. To survive and spread in the blood, bacteria have evolved a range of molecular strategies to inhibit the activation and activity of complement, and to avoid or resist phagocytosis. Alternatively, the bacteria can stay at the mucosal surface and cause considerable damage—​by direct invasion and destruction of the tissue, by inducing a damaging in- flammatory response, or by elaborating a toxin. The precise path- ology caused, and consequently the clinical signs that ensue, depends on the precise nature of the toxin and the site at which it has its effects. Thus, enterotoxigenic Escherichia coli makes labile toxins and stable toxins, which will usually result in watery diar- rhoea, whereas enterohaemorrhagic E. coli can make Shiga toxin, which is spread systemically and acts at a distance from the gut with severe consequences such as thrombocytopenia and kidney damage, leading to haemolytic uraemic syndrome. Other bacteria invade and spread systemically, finally lodging in particular tissues and causing direct pathology or inducing inflammatory responses that result in immunopathology (e.g. the lesions associated with systemic salmonella infections such as typhoid fever). These path- ologies often result from the binding of host receptors (pattern recognition receptors, PRRs) to relatively invariant structures on the invading organisms (pathogen-​associated molecular patterns, PAMPs), such as endotoxin, peptidoglycan, flagella, or in viruses’ double-​stranded RNA, leading to expression of a range of cytokines that mediate the inflammatory response. If this process gets out of control, or happens at the wrong time and in the wrong place, severe pathology can result. An example of this is the systemic inflamma- tion that leads to sepsis, with attendant tissue damage, circulatory collapse, and often death. Similarly, viruses bind to specific host cell receptors and the distribution of these receptors can determine the

8.1.1  Biology of pathogenic microorganisms 653 range of pathologies associated with the infection. For example, the henipaviruses bind to Ephrin B2, a specific N-​methyl-​D-​aspartate (NMDA) receptor that has a particular distribution, including in the central nervous system, which underlies the preponderance of encephalitis as a clinical manifestation of henipavirus infection in humans. The receptor binding of influenza viruses is considered earlier. The broad range of cellular tropism of filoviruses, such as Ebola virus, which is mediated through viral glycoprotein spikes, is also explained by the wide cellular distribution of the heparan sul- phate proteoglycans and T-​cell immunoglobulin and mucin domain 1 (TIM-​1) glycoproteins to which this virus can bind. Each of the different stages of bacterial infection relies on the pathogen being able to adapt physiologically and metabolically to the different niches in which it finds itself, and having the appro- priate structures to survive the onslaught of innate and adaptive immune responses. It is becoming increasingly apparent that many bacteria can adapt gene expression profiles rapidly, and have sophis- ticated molecular mechanisms for rapid switching of many of the structures that are required for virulence or are recognized by the immune system. Virulence factors versus fitness factors Almost any gene product that has been identified as being required for infection has been called a ‘virulence factor’ in the literature. However, this is imprecise and can be misleading. For bacteria, many of the genes required for host exploitation might be better considered as ‘fitness factors’, but are no less important in the consid- eration of infectious diseases and how to combat them. Bacteria can often grow outside their hosts and so not all their genes are neces- sarily required for fitness inside the host. Those that are include well recognized virulence factors such as adhesins and toxins, and also various metabolic pathways that enable the bacterium to survive for long enough and grow in the host to cause damage. Viruses, on the other hand, are obligate host parasites. They tend (with notable exceptions such as herpesviruses and poxviruses with genomes of 100–​200 kb) to have rather small genomes with few genes. Therefore, in most viruses, each gene is required for exploit- ation of the host in some way and is highly likely to be a fitness factor in the sense of evolutionary fitness. It may well be appropriate to consider them as virulence factors in pathogenesis. In considering virulence vs. fitness in evolution, we might ask, ‘Why do pathogens cause damage rather than simply existing in harmony with their host?’ This question might be framed better as, ‘What evolutionary pathway has resulted in pathogens that cause damage to their hosts?’ There are many possible answers. The pathogen might have evolved to exploit a particular ecological niche rather than a particular host, but has found itself by accident in a host, which it then damages almost as a bystander event. Another answer might be that by inducing a certain pathology the pathogen liberates more nutrients for itself and/​or facilitates its transmission to another host (preferably in most cases before it kills its original host). Whatever the truth is behind these evolutionary pathways, it is essential that people working with infectious diseases should recognize that there is more to the evolution of a pathogen than the acquisition of a toxin or two. Indeed, in pathogens that have evolved to infect a single host, such as Mycobacterium tuberculosis, Salmonella Typhi, or Bordetella pertussis, among others, comparison at the whole genome level with close relatives that have retained the capacity to infect multiple hosts suggests that gene loss relative to a recent common ancestor is a common feature of host adaptation. Adaptation to the environment A major shift in the minds of infectious disease researchers in re- cent years has been the realization that pathogens are far from the relatively static entities they were once thought to be. It is now clear, from many different examples, that bacterial pathogens sense the environment in which they live, and alter gene expression profiles accordingly to enable exploitation of and survival in that environ- ment. For example, a food-​poisoning bacterium such as Salmonella enterica might be living on a nutrient-​rich piece of meat, but at a cold temperature. The meat might then be cooked, providing the bac- terium with heat stress. If the meat is undercooked the salmonellae will survive the heat stress by expression of different heat-​shock op- erons, which incidentally might also lead to the expression of genes required to survive subsequent assaults in the host. On entry into the mouth, defences such as lysozyme and IgA must be overcome, and on entry into the stomach, a very low pH is encountered. Gene ex- pression will again change in the salmonellae such that genes for acid tolerance and acid resistance are now to the fore. Once the bacteria exit the stomach, the pH will change again, and they will be assaulted by bile salts and many other defence mechanisms until they arrive at their point of attachment to the small intestine. Although there are very few experimental data about these phenomena in actual host animals, experiments in vitro and a few experiments in cells or in animal models are beginning to reveal the complex changes that must take place in gene expression for a bacterium to establish itself in a host animal. Many of these changes are orchestrated through well-​understood environmental sensory and signal transduction systems. One of the most common is the two-​component sensory system. One compo- nent is a membrane protein that senses external environmental cues and the second is an intracellular protein that binds to DNA and either activates or represses the transcription and expression of sets of genes, usually called regulons. A signal is transmitted from the sensor to the activator/​repressor when the environment changes. Signal transduction is achieved via histidine protein kinases. These two-​component systems are very common in bacteria. Those bac- teria that can live in numerous environments tend to have many more of them (e.g. c.90 in Pseudomonas), whereas those bacteria that have become adapted to a lifestyle in a particular host have very few (e.g. 2 in Chlamydia, 1 in Mycoplasma) and there is often a concomi- tant loss of genomic size. A better understanding of how bacteria behave and of the genes that are actually expressed inside the host will very likely lead to breakthroughs in the design of new antimicrobials and vaccines. This is becoming an ever more important imperative, with major con- cern being expressed by medical and veterinary professionals, sci- entists, and politicians alike about the rapidly increasing prevalence of bacteria that have evolved to become resistant to the current set of antimicrobial drugs. There is a suggestion that the world might be on the brink of returning to a ‘pre-​antimicrobial’ era, with different bacterial infections becoming untreatable, and there are indeed

654 SECTION 8  Infectious diseases examples of bacteria that are resistant to all known antimicrobial drugs. A further consequence of resistance to antimicrobials is that many of our current therapies for a range of diseases, such as treat- ment for cancer, and major joint surgery, require extensive coverage by antimicrobial drugs so that secondary bacterial infections do not occur. If resistance continues to increase in prevalence, these ther- apies will also be severely compromised. Interaction with the immune system: 
Antigenic variation The survival of pathogens in hosts is made particularly challen- ging by the existence of the immune system. Pathogens have re- sponded to this challenge by evolving many specific mechanisms for the avoidance, evasion, or subversion of both innate and ac- quired host resistance mechanisms. For example, some viruses are inherently genetically unstable. The natures of the polymerases that replicate the RNA genomes of influenza virus and lentiviruses such as HIV result in variants being produced at a high rate. Many variants will be incompatible with the continued existence of the virus as an entity capable of reproduction. Consequently, many de- fective viruses can be detected, although they are usually missed by conventional consensus-​based sequencing approaches. Genetic changes may, however, not effectively alter the functionality of the viral protein other than to alter antigenicity and this may confer fitness benefits in the face of population-​level immunity, leading to selection of the variant viruses. In this way, over time, progressively varying viruses evade the immunity that develops in response to infection. Some viruses, such as influenza, have evolved segmented gen- omes. If two viruses happen to be occupying the same host cell, dif- ferent segments can rearrange, and a new virus can be assembled. This type of large-​scale reassortment leads to the so-​called ‘antigenic shifts’ that allow invasion of populations entirely immunologically susceptible to new variants, as occurred with the 2009 swine influ- enza H1N1 pandemic. At an entirely different level, within-​host antigenic variation is thought to be one mechanism that allows HIV to continue to be car- ried chronically in the face of what might appear to be a strong im- mune response. Many other pathogens may also escape the immune response by antigenic variation within the host. Bacteria such as Haemophilus influenzae, Neisseria meningitidis, and Campylobacter jejuni have evolved tracts of repetitive DNA in single base pair repeats or repeats of four or more base pairs. The number of these repeats can change, apparently randomly. This is a powerful mechanism that allows the existence of a population of bacteria with several dif- ferent antigens that may be ‘randomly’ expressed or not expressed in individuals within the population. This might be a kind of altru- istic evasion strategy whereby some members of the population are lacking a particular structure which is itself a target for the immune system, such that they will survive and thus continue the existence of that bacterial population. Other mechanisms involving recom- bination and gene conversion exist in other pathogens, leading to the expression of alternative antigenic versions of the same protein and underlying the cycling of different forms of, for example, variant surface glycoprotein in trypanosomes and the opportunistic fungal pathogen Pneumocystis jirovecii or pili in neisseriae. Pathogens have also evolved mechanisms to subvert the immune system by mimicking elements of the innate response. Good ex- amples are herpes and poxviruses that encode chemokine homo- logues and/​or chemokine receptor homologues or analogues. Genomes Many bacterial, viral, fungal, and protozoal genome sequences are now complete. Genome sequencing technologies are being used extensively to understand pathogens in detail and in completely new ways. The availability of genome-​scale sequence data for these pathogens has revolutionized our understanding of their biology and has opened up completely new methods of study and ways of thinking about how they interact with their hosts. Immediate bene- fits of having complete genome sequences include the obvious knowledge of the complete gene set. This means that we now ‘know’ every conceivable target for the immune system and every conceiv- able target for novel antimicrobial development. The real challenge for researchers and infectious disease physicians is to sift and un- ravel the whole mass of information and to select from it that which is genuinely useful. It might be better to invent strategies to let the host biology and the genome itself indicate which genes and antigens are likely to be useful as vaccine targets. Some of these methods are now being pub- lished and a good example is ‘reverse vaccinology’. Here, genes for outer membrane proteins from a pathogen of interest are selected using computer algorithms, cloned using the polymerase chain reac- tion or synthesized de novo, and the encoded protein expressed and purified. These proteins can then be used to interrogate sera from animals or humans that have been infected and are convalescent, to identify which of them is expressed as an antigen during infection, although this step is not essential. The proteins can subsequently be used to immunize animals with the intention of testing the resultant immune response for its ability to protect against virulent challenge in different infection models. The choice of read-​out and model is of course crucial if an effective vaccine for humans is to be designed. Despite many possible pitfalls, reverse vaccinology is an exciting technology platform with great promise for the exploitation of gen- omes to generate completely new candidate vaccines against bacteria. Indeed, a vaccine against group B meningococcus has been devel- oped and licensed for use in children based on this approach. Another fascinating story, emerging from the availability of many genome sequences derived from field isolates of bacteria has been the recognition of diversity within bacterial species and the evolu- tionary relationships between bacteria that this implies. It is clear that many bacterial species share their DNA promiscuously and that this can lead to rapid evolution of drug resistance and altered patho- genicity. Many tried and trusted schemes for classifying and typing bacteria need to be reassessed in the light of genomic information. Highly used typing schemes, such as the Kaufman–​White scheme for salmonellae, based on recognition of antigens on the bacteria by standardized antibodies, are being replaced by DNA-​based methods such as multilocus sequence typing, analysis of single nucleotide polymorphisms, and very high throughput sequencing based ap- proaches; the last of these will become the method of choice for many diagnostic applications once even newer sequencing technologies become established. DNA sequencing technology is improving at an

8.1.1  Biology of pathogenic microorganisms 655 astonishing rate, and ultra-​high-​throughput sequencing machines are now available that make the determination of a draft genome for a bacterium less expensive than a routine microbiological workup. Real-​time whole genome sequencing is already at a stage where it could be used to inform infection control measures. A study of Staphylococcus aureus in which 63 isolates of methicillin-​resistant Staphylococcus aureus (MRSA) were sequenced gave clear-​cut in- formation about the geographic origin of the isolates and definitive information about person-​to-​person spread in the hospital environ- ment. A similar study on Streptococcus pneumoniae, in which 240 genome sequences were determined, has been able to follow how the bacteria have adapted to clinical interventions, in the form of vaccines and antimicrobials, over very short time scales. This kind of approach is becoming routine in hospitals and is revolutionizing how we think about infections and their control. Genome sequencing approaches are also able to address questions of global importance, tracing the epidemiology of infectious disease outbreaks in time and space. For example, sequencing of isolates of Vibrio cholerae from around the globe has shown that the seventh pandemic clone of this organism originated in the Bay of Bengal and spread round the world, in at least three distinct waves. The outbreak of cholera in Haiti, beginning in 2010, became a politically charged event; and whole genome sequencing was able to show unequivocally that ra- ther than this outbreak being local and triggered by the earthquake that led to the breakdown of public health measures, the pathogen involved was actually derived from an organism prevalent in Nepal. This study also gave a clear glimpse into the role that international travel and glo- balization can play in altering local microbiological events. Genome sequencing will be a major tool in understanding and facing the challenge provided by the increasing rates of antimicrobial resist- ance, mentioned earlier. Whole genome sequencing of clinical isolates is being used to identify the presence of genes encoding resistance to antimicrobials, and the arrangements of those genes on different mo- bile genetic elements. This opens up new epidemiological studies, and new understanding of how bacteria, and indeed their genes, move be- tween humans, other animals, and the environment outside the host. Future challenges Most infectious diseases are unlikely to be eradicated in the near future. Even if they were, new infectious agents would inevitably evolve to exploit the rich environments presented by host animals. Infectious disease biology and medicine are currently undergoing a renaissance, driven by the revolution in genome science, allied to the real and present dangers still presented by many pathogens, and their evolution to become resistant to the drugs used to treat the dis- eases caused by them. They have the capacity to deliver sudden, se- vere global pandemics resulting in high global mortality. Emerging infections presenting acute public health problems are likely to be viral in nature, to have originated in an animal population and, therefore, at least initially, to be zoonoses, and to be spread quickly via air travel. Changing social conditions (e.g. increasing urbaniza- tion in certain parts of Africa), bring together animal and human populations that have rarely if ever been closely associated on a large scale. This brings with it an increased chance for microorganisms to be shared between these species, with a resulting increased chance of new pathogens emerging. The devastating effects of Ebola in West Africa from 2013 to 2016 exemplify this perfectly. Even relatively minor changes in societal behaviour can lead to major disease prob- lems. For example, changes to methods for preparing cattle feed led to the emergence of the BSE prion as a human disease problem in recent years in the United Kingdom and elsewhere. This analysis does not take into account the added problem of possible bioterrorism attacks, although the impact of these has not to date met the level of concern over them. To deal with these disease threats, the regulatory framework underlying the development and legal deployment of antimicrobials and vaccines will have to be adapted and evolved in step with the evolution of the diseases themselves. The pathogens are likely to win this race too! Artificial distinctions between ‘human’ and ‘vet- erinary’ medicine need to be removed. Most pathogens infect more than one species of host animal and certainly do not respect the anthropocentric division of research effort. The concept of ‘One Medicine’, introduced by Calvin Schwabe, is nowhere more per- tinent than in consideration of infectious diseases and the biology of pathogens, and is likely to be particularly important in dealing with the emerging antimicrobial resistance problem. A concerted effort is needed to deal with pathogens in all parts of the world and not just in the developed world. Inexpensive but ef- fective intervention strategies must be developed to defeat acute and chronic infectious diseases worldwide. The lives affected by patho- genic microorganisms in developing countries are just as valuable as those in more affluent areas, but are far more numerous. A focus on primary medical care and early detection of outbreaks would be far more beneficial than high-tech approaches that some claim will en- able us to predict which specific pathogens will cause future human pandemics. It remains our task, whether from a medical, veterinary, or basic science background, to try to understand how pathogenic microorganisms work, and to harness that knowledge to defeat, wher- ever possible and by whatever means, these ever-​adaptable scourges. FURTHER READING Bentley SD, Parkhill J (2015). Genomic perspectives on the evolution and spread of bacterial pathogens. Proc Biol Sci, 282, 20150488. Croucher NJ, et al. (2011). Rapid pneumococcal evolution in response to clinical interventions. Science, 331, 430–​4. Dean P, Maresca M, Kenny B (2005). EPEC’s weapons of mass subver- sion. Curr Opin Microbiol, 8, 28–​34. Galan JE, Wolf-​Watz H (2006). Protein delivery into eukaryotic cells by type III secretion machines. Nature, 444, 567–​73. Harris SR, et al. (2010). Evolution of MRSA during hospital transmis- sion and intercontinental spread. Science, 327, 469–​74. Janeway CA, Medzhitov R (2002). Innate immune recognition. Annu Rev Immunol, 20, 197–​216. Kuiken T, et al. (2006). Host species barriers to influenza virus infec- tions. Science, 312, 394–​7. Mora M, et  al. (2006). Microbial genomes and vaccine design:  re- finements to the classical reverse vaccinology approach. Curr Opin Microbiol, 9, 532–​6. Moxon R, Bayliss C, Hood D (2006). Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. Annu Rev Genet, 40, 307–​33. Murphy PM (2001). Viral exploitation and subversion of the immune system through chemokine mimicry. Nat Immunol, 2, 116–​22.

8.1.2 Clinical features and general management of

8.1.2 Clinical features and general management of patients with severe infections 656

656 SECTION 8  Infectious diseases Neumann G, Kawaoka Y (2006). Host range restriction and patho- genicity in the context of influenza pandemic. Emerg Infect Dis, 12, 881–​6. Roumagnac P, et al. (2006). Evolutionary history of Salmonella typhi. Science, 314, 1301–​4. van Riel D, et al. (2007). Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals. Am J Pathol, 171, 1215–​23. 8.1.2  Clinical features and
general management of patients with severe infections Peter Watkinson and Duncan Young ESSENTIALS Pathophysiological mechanisms The host response to an infection involves an intricate link between the inflammatory and coagulation systems, and mechanisms de- signed to limit damage to normal tissues. Key elements are: (1) the inflammatory cascade—​antigens from infecting organisms stimulate macrophages and monocytes (and other cells) via Toll-​like recep- tors to release tumour necrosis factor α. This results in a cascade of proinflammatory cytokine release which is a vital component of the host’s attempt to control and eradicate infection, but unfortunately can also result in damage to both infected and uninfected host tis- sues; (2) the anti-​inflammatory cascade—​a compensatory response involving anti-​inflammatory cytokines, soluble receptors, and re- ceptor antagonists directed against proinflammatory cytokines that is intended to localize and control the systemic proinflammatory response to the infection; (3) the coagulation cascade—​activated in an attempt to contain infection locally and prevent spread to other parts of the body; platelets are activated, procoagulant pathways are initiated, and anticoagulant mediators are downregulated; (4)  the anticoagulation cascade—​the coagulation response to sepsis is regu- lated via antithrombin, tissue factor pathway inhibitor, activated pro- tein C, and fibrinolysis. Clinical features Definitions—​(1) Systemic inflammatory response syndrome—​which can occur as a result of an infectious or non​infectious insult—​ requires the presence of at least two of the following: (a) hyper-​ or hypothermia, (b)  tachycardia, (c)  tachypnoea or hyperventilation, (d) leucocytosis, leucopaenia, or left shift. (2) Sepsis—​a suspected or confirmed infection plus criteria for systemic inflammatory response. (3) Severe sepsis—​sepsis resulting in the acute dysfunction of at least one organ system. (4) Septic shock—​infection resulting in hypoten- sion despite adequate fluid resuscitation. Management—​key elements are (1)  antibiotics—​often initiated empirically before culture results are available; (2)  control of the source of infection—​searching for the site of infection so that it can be eradicated should begin as soon as haemodynamic and respira- tory status are stabilized, antibiotics without source control often fail; (3) early resuscitation—​requiring (a) crystalloid infusions to maintain circulating volume, (b) vasopressors if arterial pressure remains low, and (4) other treatments—​organ support if needed, refining antibiotic treatment based on culture results, and measures to minimize further nosocomial infections. Introduction Although originally describing both localized and disseminated in- fections, the term sepsis is now more commonly used to describe the systemic response to a severe infection. The symptoms and signs of sepsis include fever and rigors, flushing and vasodilation, an ele- vated heart and respiratory rate, confusion, hypotension, and oli- guria. Occasionally a reduced core temperature and pallor occur with late presentations, especially in children. To these are added symptoms and signs relating to the specific infection site and patho- genic organism, such as a cough with russet sputum, pleuritic chest pain, cyanosis, and signs of pulmonary consolidation in a patient with pneumococcal pneumonia. The need for a reasonably precise definition of sepsis arose when drugs designed to modify the host response to severe infections (e.g. antibodies against endotoxin or tumour necrosis factor-​α, TNFα) were tested in clinical trials. These drugs were given as soon after presentation as possible, so a simple, clear definition of sepsis that did not require positive culture results was required to determine which patients entered the studies. Three concepts were developed; the systemic inflammatory response syndrome (SIRS), sepsis, and severe sepsis. SIRS, which can occur as a result of an infectious or non​infectious illness, is an indication of an activated inflammatory response. It requires any two or more of: (1) hyperthermia or hypo- thermia (core temperature ≥38°C or ≤36°C), (2) tachycardia (heart rate >90 beats/​min), (3) tachypnoea or hyperventilation (>30 respir- ations/​min or PaCO2 <4.2 kPa (32 mm Hg)), and (4) leucocytosis, leucopaenia, or left shift (≥12 or ≤4 × 109 white blood cells/​litre or

10% immature neutrophils). Sepsis is defined as a suspected or confirmed infection causing at least two of the SIRS criteria. Severe sepsis is used to describe sepsis resulting in the acute dysfunction of at least one organ system. When the infection results in hypotension despite adequate fluid resuscitation, the patient has septic shock. Patients with septic shock are a subset of those with severe sepsis. Terms such as septicaemia, ‘catheter shock’, blood poisoning, and toxaemia are less well defined. Although this set of definitions is still in use in clinical research, it has very limited applicability in clinical medicine. The SIRS component is very sensitive, but is not specific. Most acutely hospitalized patients demonstrate two or more SIRS criteria at some point, as do nearly all patients in critical care units. In addition, these clinically based diagnostic rules are of very limited value in patients who do not mount the conventional inflammatory response to sepsis, including immunocompromised patients and those at the extremes of age. As a result, recognition of sepsis still relies on a physician suspecting or confirming infection based on history, clinical signs, laboratory findings, and imaging. Variants of these definitions have been created for clinical trial entry criteria, management guidelines, and retrospective analyses of

8.1.2  Clinical features and general management of patients with severe infections 657 clinical and administrative databases. The difference in definitions in part explains the wide range of estimates of the incidence of sepsis and severe sepsis in developed healthcare systems. The incidence in the United States might lie between 300 and 1000 cases per 100 000 population per year. This incidence might be increasing over time as the population ages and more aggressive treatments are offered to older people. In part because of the limitations of this set of definitions, a model for risk stratification of patients with sepsis, based conceptually on the tumour, node, and metastasis (TMN) system used in oncology, has been proposed. The PIRO model (Predisposition, Infection, Response, and Organ dysfunction) can be used to generate a nu- meric score indicating the risk of mortality from sepsis at the point of presentation. It can also be used in clinical practice as a framework for assessing and describing patients with sepsis. Predisposing risk factors for infection include extremes of age, immunocompromising conditions or drugs, and anything that alters the risk of sepsis. Infection describes the likely source or pathogen and the site of in- fection; Response describes the physiological effects of the infection; and Organ dysfunction (renal, respiratory, and so on) describes organ system damage or failure caused by the infection. As yet, this classification/​descriptive system is not widely used. Very recently a revised set of diagnostic criteria for sepsis (‘Sepsis-​ 3’) was published. The new definition hinges on the SOFA score (Sequential Organ Failure Assessment), a severity scoring system used in critical care practice. A serum lactate above 2 mmol/​litre (18 mg/​dl) despite adequate fluid resuscitation is used as part of the definition of septic shock. The Sepsis-​3 definitions are still based on laboratory and physiological variables. They are as yet unproven for either clinical or research practice. Pathophysiology of infection Inflammatory cytokines mediate the physiological changes seen in patients with sepsis. A  consumptive coagulopathy, with microthrombi in the small vessels of various organs, is a common feature of severe sepsis. In infected patients, changes in the inflam- matory and coagulation systems are linked. Early on in an infection, cell wall antigens from infectious agents stimulate macrophages and monocytes to release TNFα. The cell wall components initially bind to transmembrane Toll-​like recep- tors (TLR2 and TLR4) found on the surface of macrophages, neu- trophils, fibroblasts, and some epithelial and endothelial cells. The cells then release TNFα into the circulation stimulating the release of other proinflammatory cytokines from macrophages and neutro- phils, especially interleukins 1 and 6 (IL-​1, IL-​6). This is frequently referred to as an inflammatory cascade. Clinical trials with anti-​Toll-​like receptor drugs showed no benefit, presumably because the cascade had already been activated at the time of administration. The infection-​triggered increase in circulating TNF​α is so short-​lived that most patients with sepsis have undetectable levels at the time of presentation. As a result, clin- ical trials using antibodies to TNF​α also showed no benefit. High levels of circulating proinflammatory cytokines cause the fever, tachycardia, and tachypnoea seen with severe infections. This inflammatory response is a component of the immediate host de- fence to severe infection, but directly and indirectly it can result in damage to both infected and uninfected host tissues. By about three days after presentation, the levels of IL-​6 and other proinflammatory cytokines are decreasing. However, the inflammatory state is main- tained by other mediators such as high-​mobility group box protein 1 (HMGB1), which further stimulates the release of proinflammatory cytokines but, in addition, regulates coagulation by inducing the se- cretion of plasminogen activator inhibitor type 1 (PAI-​1, procoagu- lant) and tissue plasminogen activator (fibrinolytic). There are checks on the inflammatory cascade. A compensatory anti-​inflammatory response exists to maintain homeostasis. TNFα and IL-​1 also stimulate leucocytes to release anti-​inflammatory mediators such as IL-​10, IL-​13, and transforming growth factor β (TGFβ) which exert a direct anti-​inflammatory effect on macro- phages and endothelial cells. They also inhibit monocyte presenta- tion of antigens to other immune cells. The inflammatory response is further controlled by the release of soluble receptors and receptor antagonists directed against proinflammatory cytokines. This com- pensatory anti-​inflammatory response, in the later stages of sepsis, can lead to a degree of immune paresis. The activation of the coagulation response is intricately linked to the inflammatory response. TNFα and IL-​1 both stimulate the release of tissue factor (also called factor III, thromboplastin) and activate endothelial tissue factor. Tissue factor (TF) combines with circulating factor VII to form a TF:VIIa complex. This in turn ac- tivates factor X, stimulating the formation of fibrin clots in the microcirculation via thrombin. Thrombin, TF:VIIa complex, and activated factor X also function as potent inflammatory mediators. Anticoagulant mechanisms such as protein C, antithrombin, and tissue factor pathway inhibitor are impaired. Both antithrombin and activated protein C have been tried as treatments for severe sepsis, but neither showed any efficacy. Diagnosis The key element of a diagnosis of sepsis remains a clinical suspi- cion of an infective process raised by findings in history and exam- ination. Laboratory studies can assist with the diagnosis. Most patients with sepsis have an increased white cell count, predomin- antly neutrophils. More rarely, in severe cases, a neutropaenia is seen. Immature neutrophils (band forms or a left shift) might be present as a result of increased white cell production. A modest thrombo- cytopaenia, associated with clotting abnormalities such as an in- creased activated partial thromboplastin time or increased ‘R’ time on a thromboelastograph are found in patients with an associated consumptive coagulopathy. Other non​specific abnormalities are often found. Hypotension, and dehydration, especially in late-​presenting patients, can cause a prerenal acute kidney injury so both plasma creatinine and urea might be elevated. In common with all critically ill patients, blood glucose is often elevated by increased circulating catechol- amines. An elevated arterial plasma lactate concentration is com- monly found, as the infection causes actual and functional tissue hypoperfusion. The increased capillary permeability that occurs in the lungs causes non​cardiogenic pulmonary oedema, a condition usually termed acute respiratory distress syndrome (ARDS). Body fluids sampled from sites of infection might show markers of infection that can be measured long before the results of cultures

658 SECTION 8  Infectious diseases of body fluids are available. For example, pleural fluid in patients with a bacterial empyema will have a reduced pH and contain in- flammatory cells. Cerebrospinal fluid from patients with bacterial or fungal meningitis might have raised protein and reduced glu- cose concentrations, often with increased white cells. Infected urine may show blood, nitrites, and leucocytes. Microscopy of body fluid samples treated with Gram’s stain might reveal Gram-​positive or Gram-​negative organisms. Patients with Legionella pneumophilia or Streptococcus pneumoniae pneumonia can have a specific bacterial antigen present in their urine. A major clinical challenge is distinguishing patients with infec- tions from patients with another reason for systemic inflammation caused by a non​infectious process, such as pancreatitis. The com- bination of the time taken to culture pathogenic organisms and the high false-​negative rate for culture-​based tests has led to a search for markers that identify patients with an infection quickly. Ideally the marker could be determined at the point of care or rapidly in a laboratory, and would give information about the pathogen species and antibiotic resistance. C-​reactive protein (so-​called because it reacts with the C-​ polysaccharide of pneumococci) plasma concentration increases within hours in response to acute infections. It has a short half-​life (19 hours) so concentrations largely reflect production rates, and so can be used to track the effects of treatment. However, C-​reactive protein is produced by hepatocytes in response to increases in plasma IL-​6 concentrations, and these also occur with tissue damage, inflamma- tion, and malignancy. Plasma IL-​6 levels themselves have been used as an entry criterion for clinical trials of antisepsis agents, but are not in clinical use. Procalcitonin, a polypeptide prohormone rises to very high values in response to bacteraemia or fungaemia. While procalcitonin ex- hibits a degree of specificity for bacterial infection, it can be raised as a result of other proinflammatory stimuli such as tissue damage or severe viral infections. Presepsin (a soluble C14 receptor subtype) is released into the circulation when monocytes are activated by lipopolysaccharide from Gram-​negative organisms, limiting its use as a rule-​in test to this class of pathogens. The soluble triggering receptor expressed on myeloid cells-​1 (sTREM-​1) can also increase early in sepsis. Presepsin and sTREM-​1 might, in future, be useful as markers of bacterial infection but remain research tools at present. The hallmark of sepsis is a positive culture from a normally sterile body fluid or site, such as blood, urine, or cerebrospinal fluid. However, culture-​based diagnosis is of little value in an acute setting. Blood cultures require careful techniques to ensure an ad- equate inoculum of blood and to avoid contamination. Culturing of micro-​organisms to the point where infection can be confirmed can then take 12–​72 hours, often with more time to confirm spe- cies and antibiotic sensitivity. Gram’s stain and other techniques to identify micro-​organisms in body fluids immediately after sampling are generally ‘rule-​in’ tests. If positive they make the diagnosis, but false negatives are common, especially in patients who have received antibiotics prior to collection of blood or body fluids for culturing. Culture results are rarely available to make early treatment deci- sions, and are more commonly used to fine tune the initial empiric antibiotic treatment. Molecular tests for pathogens, particularly bacteria, show par- ticular promise. These techniques allow the rapid detection of bacteria in blood and other clinical specimens, overcoming the de- lays and lack of sensitivity of conventional culture techniques. There are several techniques that are available commercially. Some decrease the time to identify pathogens but still requires the automated blood culture system to identify cultures with multiplying pathogens. For example, fluorescent in situ hybridization is a staining method for the detection of pathogens in positive blood cultures. Slides of posi- tive blood cultures are prepared, hybridized with fluorochrome-​ labelled oligonucleotide probes targeted at ribosomal RNA (rRNA), and visualized under a microscope. Broad-​range DNA amplification (PCR), detecting conserved sequences of bacterial/​fungal chromo- somal genes encoding ribosomal DNA, can detect bacteria in a few hours in blood cultures but secondary steps are needed to determine the species. Panels of primers can be used, with or without con- served sequences, to look for specific species or resistance patterns to make this process more efficient (multiplex techniques). Molecular techniques applied directly on whole blood samples for rapid identification of a circulating microorganism would be ideal for acute care. They are quicker and more sensitive than culture-​ based techniques. Available amplification techniques include both broad-​range and multiplex PCR. These approaches will give a re- sult in 3–​8 hours and have been shown to be sensitive and specific, though their impact on care pathways and outcomes is still under in- vestigation. The cost per test is high, and sample handling and DNA extraction remain a problem. The ideal solution, a simple point-​of-​ care or rapid-​turnaround laboratory device, with a wide range of detectable pathogens and antibiotic resistance markers, has yet to reach routine clinical medicine. Treatment of sepsis The mainstays of treatment for severe sepsis are resuscitation, early treatment with antibiotics, and source control. Resuscitation At first presentation a rapid ‘Airway, Breathing, Circulation’ assess- ment is needed. Not all patients with sepsis will require immediate resuscitation but a significant proportion will have an increased alveolar to arterial oxygen gradient. This might result from pul- monary infection, ARDS, or ventilation/​perfusion mismatching due to poor cardiac output. Supplemental oxygen may be required. Severe cases might require endotracheal intubation and artificial ventilation. The inflammatory cytokines released in sepsis cause vaso-​ and venodilatation. Increased insensible fluid losses from fever, sweating, vomiting and diarrhoea, and worsening microvascular permeability all contribute to decreased intravascular volume, manifesting as hypotension. Intravenous fluid remains the mainstay of cardiovas- cular resuscitation, and initial treatment of sepsis-​induced hypo- tension in an adult usually requires at least a litre of intravenous crystalloid solution given rapidly over half an hour. An average of about 4 litres is required in the first 6 hours after presentation. If hypotension does not resolve after 2 litres of fluid it might be ne- cessary to administer a pressor agent as a continuous infusion, usu- ally via a central venous catheter. The most commonly used pressor agent is noradrenaline (norepineprine) though some centres will augment this with vasopressin. Treatment with potent vasopressors

8.1.2  Clinical features and general management of patients with severe infections 659 needs suitably trained staff and physiological monitoring and so is usually confined to critical care or equivalent high-​care areas. The endpoint for discontinuing ‘aggressive’ fluid resuscitation re- mains ill-​defined. The increased capillary permeability associated with sepsis causes fluid to accumulate as peripheral oedema very quickly, and non​cardiogenic pulmonary oedema (ARDS) will be worsened by excess fluid. A bedside test of adequate fluid resusci- tation is to simply raise both legs to 45 degrees when the patient is supine. If the patient has received inadequate fluid resuscitation raising both legs will cause an immediate increase in blood pressure as venous blood returns to the thorax. An alternative is to assess the response to a rapid 250 ml fluid bolus. Patients who are adequately resuscitated will often begin to need less vasopressors and have an improving arterial lactate concentration, increasing urine output and resolving confusion. There have been attempts to codify the fluid and vasopressor treatment of patients with septic shock. Probably the best-​known regimen is early goal-​directed resuscitation, which was shown to considerably decrease mortality in one single-​centre study of pa- tients with severe sepsis and lactic acidosis or septic shock. The ‘goals’ were target values for central venous pressure, central venous oxygen saturation, and blood pressure. Three large, multicentre studies subsequently failed to show any benefit of early goal-​directed resuscitation over usual care. In the 1980s a reversible reduction in cardiac performance in some patients with severe sepsis was first recognized. Aided by the rise in echocardiography use on critical care units, this condition has been studied in more detail. It is a global, short-​lived (a few days) signifi- cant reduction in left ventricular inotropy, probably with modestly increased lusitropy (ventricular relaxation). Right ventricular dila- tation is often a feature. This condition is not caused by a cardiac oxygen supply/​demand mismatch or coronary artery disease, but is likely caused by high levels of circulating cytokines such as TNF​α
or IL-​1β. After diagnosis using transthoracic echocardiography, some centres will use β-​agonists (usually dobutamine infusions) to increase inotropy in these cases. Antibiotics Early administration of appropriate intravenous antibiotics reduces morbidity and mortality in patients with sepsis. In clinical trials, an appropriate antibiotic regimen is begun in a timely fashion in 85–​95% of occasions, but failure to do so is associated with a 25% higher mortality. Almost invariably the choice of initial antibiotics cannot be based on culture results. Most hospitals will have local prescribing advice based on the suspected site/​type of infection, local resistance patterns, where the patient developed the infection (community or nosocomial), whether the patient has any antibiotic allergies, the patient’s immune status, and the cost and local avail- ability of the drugs. Sometimes this advice needs modifying because of recent antibiotic use, laboratory findings, and previous micro- biology results. In general, initial antibiotic therapy should cover a broader spectrum of pathogens when the patient is critically ill or the certainty about the likely causative organism is low; for example, in neutropaenic sepsis the recommended initial empirical treatment is intravenous piperacillin with tazobactam. Once microbiological data are available, therapy should be changed promptly to the narrowest spectrum, least toxic, and least costly agent. When the risk of developing complications of sepsis is low patients should be switched early from intravenous to oral antibiotics. Selective oral decontamination and selective digestive decon- tamination are techniques that use antibiotics to reduce the burden of nosocomial infections in patients treated in critical care units. The benefits of this approach seem to depend on the healthcare settings in which it is used, and concerns remain about the risk of generating resistant strains of bacteria. As a result, this technique is not widely used. Source control Source control is a term used to describe removal of infected ma- terial. Antibiotics have little penetration into areas of devitalized tissue, or avascular collections of infected fluid. Common examples of infected fluid collections include infected urine retained behind a renal calculus, a gall bladder empyema, a thoracic empyema, or a leak of enteric content into the abdominal cavity. As soon as a patient’s haemodynamic and respiratory function are sufficiently stable any required imaging should be undertaken. Usually this in- volves ultrasound investigation or a computed tomography (CT) scan. In some patients, for example patients who have had prior sur- gery, it is sometimes difficult to distinguish between collections of sterile and infected fluid. Enhancement of the rim of the fluid collec- tion, gas formation, tissue oedema around the fluid collection, and large-​sized collections all suggest infected rather than sterile collec- tions on CT images. Significant collections of infected fluid require either surgical or radiological drainage. Patients who develop nosocomial sepsis, especially those who are immunocompromised, might have bacteraemia caused by infected indwelling devices such as intravascular catheters (catheter related bloodstream infections). If no other cause for the nosocomial infec- tion is found, these devices should be removed and replaced with new devices if they are still required. Many centres take blood cul- tures from central venous catheters before removing them, though a positive result could indicate colonization of the catheter rather than showing the catheter as the source of the bloodstream infec- tion, or could simply reveal a bacteraemia unrelated to the device. Culture of the distal portion of the catheter (tip culture) suffers from the same limitations. Simultaneous sampling of blood for cul- ture from the device and using venepuncture might show the same pathogen in both samples, and if the sample from the device flags positive earlier than the venepuncture sample the device is likely the source of infection. However, in practice if there is a suspicion of catheter-​related bloodstream infection, the device is usually just removed. Corticosteroid treatment The use of corticosteroids in patients with septic shock dates back to the 1970s, when high doses (30  mg/​kg methylprednisolone or equivalent) were used to reduce the inflammatory response to sepsis. The use of high-​dose corticosteroids declined as clinical trials failed to show a survival benefit. However, in the 1990s the use of lower doses to counter relative adrenal insufficiency (identified using a short adrenocorticotropic hormone (ACTH) stimulation test) gained popularity, though again trials failed to show benefit. The use of steroids is now mostly confined to patients who have se- vere septic shock which is not responding to fluid resuscitation or pressors. Moderate doses of steroids (200 mg hydrocortisone/​day or

660 SECTION 8  Infectious diseases equivalent) might allow less pressor use, but meta–​analysis of the 35 trials of steroids in sepsis to date suggests there is no survival benefit. Care bundles Activated protein C (drotecogin alfa) is a recombinant form of human activated protein C that has antithrombotic, anti-​inflammatory, and profibrinolytic properties. It was marketed for 10 years until it was withdrawn in 2011 after trials failed to confirm earlier studies that had suggested it improved survival in patients with severe sepsis. Part of the marketing strategy used by the manufacturers included funding the Surviving Sepsis campaign, designed to promote bun- dles of basic care for patients with sepsis. This campaign has now been taken on by the critical care community who update the core ‘bundle’ of interventions regularly. The core recommendations are listed in Boxes 8.1.2.1 and 8.1.2.2. Mortality from sepsis has reduced during the time in which sepsis bundles have been popularized. Sepsis bundles are complex pack- ages of treatments only appropriate for critical care or high-​care areas. Some centres have implemented a simpler set of six steps spe- cifically designed to facilitate early intervention in busy hospital and prehospital settings. This ‘Sepsis Six’ bundle has three diagnostic and monitoring steps and three therapeutic interventions: • Deliver high-​flow oxygen • Take blood cultures prior to antibiotics but do not delay treatment • Administer empirical intravenous antibiotics • Measure serum lactate • Start intravenous fluid resuscitation with crystalloid solutions • Commence urine output monitoring via either a catheter or chart Box 8.1.2.1  Initial resuscitation and treatment of patients with severe sepsis Initial resuscitation 1 Resuscitate patients with sepsis-​induced tissue hypoperfusion (hypo- tension persisting after initial intravenous fluid challenge or blood lac- tate concentration ≥4 mmol/​litre). Goals during the first 6 hours of resuscitation can include: a) Mean arterial blood pressure (MAP) ≥65 mm Hg b) Urine output ≥0.5 ml/​kg/​h c) Central venous pressure 8–​12 mm Hg. 2 In patients with elevated blood lactate levels use lactate levels to assess the effects of resuscitation. Diagnosis 1 Obtain cultures as clinically appropriate before antimicrobial therapy (if this will not delay starting antimicrobials by more than 45 minutes). 2 Obtain at least two sets of blood cultures (both aerobic and anaerobic bottles) before antimicrobial therapy with at least one sample drawn percutaneously and one sample drawn through each vascular access device, unless the device was recently (<48 hours) inserted. 3 Use rapid pathogen antigen tests (e.g. 1,3 β-​D-​glucan assay or mannan for invasive candidiasis, legionella or clostridium difficile antigens) where available. 4 Obtain prompt imaging to confirm a potential source of infection. Antimicrobial therapy 1 Administer empiric intravenous antimicrobials within the first hour after recognition of severe sepsis using one or more drugs that have activity against all likely pathogens. Use antimicrobials that pene- trate into tissues presumed to be the source of sepsis in adequate concentrations. 2 Reassess the antimicrobial regimen daily. Empiric combination therapy should not be administered for more than 3–​5 days. De-​escalate to the most appropriate single therapy as soon as the antimicrobial sus- ceptibility profile is known. 3 Duration of therapy is typically 7–​10 days; longer courses may be ap- propriate in patients who have a slow clinical response, undrainable foci of infection, bacteraemia with S. aureus; some fungal and viral infections or immunologic deficiencies, including neutropaenia. 4 Inflammatory markers may assist the clinician in the discontinuation of empiric antibiotics in patients who initially appeared septic, but have no subsequent evidence of infection. Source control 1 Seek a specific anatomical site of infection. Achieve source control within 12 hours if possible. 2 When source control is required, use the intervention associated with the least physiologic insult (e.g. percutaneous rather than surgical drainage of an abscess). 3 Remove vascular access devices if they are a possible source of sepsis. Nosocomial infection prevention 1 Use routine screening and monitoring of potentially infected seriously ill patients for severe sepsis to allow earlier implementation of therapy. 3 Instigate hospital–​based performance improvement efforts in severe sepsis. After Dellinger R et al. (2013) Crit Care Med 41(2): 580–​637. Box 8.1.2.2  Fluid, vasopressor, inotropes, and steroids in the treatment of patients with severe sepsis Fluid therapy of severe sepsis 1 Crystalloids are the initial fluid of choice in the resuscitation of severe sepsis. Do not use hydroxyethyl starch solutions. 2 Use an initial fluid challenge in patients with sepsis-​induced tissue hypoperfusion of 30 ml/​kg of crystalloids. Vasopressors 1 Use norepinephrine (noradrenaline) as the first-​choice vasopressor to maintain a mean arterial pressure of 65 mm Hg. 2 Consider using vasopressin 0.03 units/​minute as an intravenous infu- sion added to norepinephrine to either raise mean arterial pressure, or decrease norepinephrine infusion rates. 3 Phenylephrine is not recommended in the treatment of septic shock except in circumstances where norepinephrine is associated with ser- ious arrhythmias. 4 All patients requiring vasopressors should have an arterial catheter placed as soon as practical. Inotropic therapy 1 A trial of dobutamine infusion up to 20 micrograms/​kg/​min can be administered or added to vasopressor (if in use) in the presence myo- cardial dysfunction. Corticosteroids 1 Do not use intravenous hydrocortisone to treat adult septic shock pa- tients if adequate fluid resuscitation and vasopressor therapy restore hemodynamic stability. 2 In cases where this is not achievable, use intravenous hydrocortisone in divided doses. 3 Do not use the ACTH stimulation test to identify adults with septic shock who should receive hydrocortisone. 4 Taper off hydrocortisone treatment when vasopressors are no longer required. After Dellinger R et al. (2013) Crit Care Med 41(2): 580–​637.

8.1.2  Clinical features and general management of patients with severe infections 661 There is a clear emphasis on early treatment in both the campaign and the Sepsis Six bundle. Conclusion Although there have been many studies of therapeutic agents acting on the inflammatory cascade triggered by infection, to date none are in routine use. The mainstay of care still remains fluid resuscitation, timely and well-​chosen empiric antibiotics, and control of the source of infection. It is likely that PCR-​based rapid diagnostic tests will continue to advance so the time to identifica- tion of the pathogen species and its resistance pattern will continue to shorten. This will allow a reduction in the total use of broad-​ spectrum antibiotics. FURTHER READING Dellinger RP, et al. (2013). Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med, 41, 580–​637. Maurin M (2012). Real-​time PCR as a diagnostic tool for bacterial dis- eases. Expert Rev Mol Diagn, 12, 731–​54. Singer M, et al. (2016). The third international consensus definitions for sepsis and septic shock (Sepsis-​3). JAMA, 315, 801–​10.

8.10 Cestodes (tapeworms) 1520

8.10 Cestodes (tapeworms) 1520

8.10.1 Cestodes (tapeworms) 1520

8.10.1 Cestodes (tapeworms) 1520

8.10 Cestodes (tapeworms) CONTENTS 8.10.1 Cestodes (tapeworms)  1520 Richard Knight 8.10.2 Cystic hydatid disease (Echinococcus granulosus)  1529 Pedro L. Moro, Hector H. Garcia, and Armando E. Gonzalez 8.10.3 Cysticercosis  1533 Hector H. Garcia and Robert H. Gilman 8.10.1  Cestodes (tapeworms) Richard Knight ESSENTIALS Adult tapeworms maintain anchorage to the small-​gut mucosa of their vertebrate definitive host, and their eggs enter the faecal stream. Their life cycle then includes larval stages in two or more intermediate hosts. Serious disease can result when larval stages occur accidentally in humans, whereas infections by the adult stages often cause little harm. Two groups of cestode infect humans: the Cyclophyllidea and the Pseudophyllidea. The former have a terres- trial life cycle and cystic larvae; the latter have an aquatic cycle and worm-​like larvae. Cyclophyllidean tapeworms Taenia saginata The beef tapeworm is common in Africa, the Middle East, Asia, and South America. Transmission occurs where cattle have ac- cess to human faeces and where humans eat undercooked beef containing cysts. Symptoms include nausea, abdominal pain, and pruritis ani, but many people who are infected are asymptomatic. Diagnosis is by finding typical eggs in perianal swabs. Treatment is with niclosamide or praziquantel. Prevention is by health educa- tion concerning production and cooking of meat, also by proper sewage treatment and disposal. Mass treatment of selected or whole adult populations is the most effective short-​term measure when endemicity is high. Taenia solium The pork tapeworm is common in Africa, parts of Asia, and Central and South America. Infections occur when cysts in under- cooked pig meat are eaten. Symptoms, diagnosis, and treatment are similar to those of T. saginata. Cysticercosis (­chapter 8.10.3) occurs when eggs from the faeces of persons harbouring adult worms are ingested, producing cysts in striated muscle, subcuta- neous tissue, nervous system, and the eye. Other tapeworms These include (1)  Echinococcus granulosus—​the dog tapeworm, cause of hydatid disease (­chapter  8.10.2); (2)  Echinococcus multilocularis—​the fox tapeworm, cause of alveolar hydatid dis- ease; (3) genus Multiceps—​common tapeworms of dogs and foxes, cause of coenurosis; (4)  other gut tapeworms including Taenia asiatica and Hymenolepis nana. Pseudophyllidean tapeworms Diphyllobothriasis (‘fish or broad tapeworm infections’) Infection is now most common east of the Urals and follows in- gestion of infected undercooked fish. Infections are often asymp- tomatic, apart from passage of worm segments in stools, but there can be bowel upset or urticaria. Heavy or prolonged infections can lead to vitamin B12 deficiency and sometimes pernicious anaemia. Diagnosis is by finding eggs or proglottids in faeces. Treatment is with praziquantel or niclosamide and, if necessary, with vitamin B12. Sparganosis Spirometra tapeworms are parasites of canids and felids. Human infections are most common in East Asia. The plerocercoid larva grows as it migrates slowly in the tissues as a form of ‘larva migrans’, most frequently subcutaneously but also in brain, orbit, and else- where. Treatment is primarily surgical: anthelminthics give poor results and may aggravate neurological lesions. Introduction Adult tapeworms maintain anchorage to the host small-​gut mucosa by means the scolex, a holdfast structure. The rest of the body forms the strobila and consists of a chain of flattened proglottids, which proliferate just behind the scolex. The worms

8.10.1  Cestodes (tapeworms) 1521 change their site of attachment regularly, and are surprisingly motile. Gravid proglottids are lost from the end of the worm and are replaced by others that have grown and matured as they pass down the strobila. Each proglottid possesses a complete set of hermaphroditic sex organs and genital openings. Eggs accumulate in the uterus of gravid proglottids and only enter the faecal stream if the proglottids are disrupted. In many species the eggs enter the environment within intact proglottids. The life cycle of tape- worms includes two or more host species; the adult worm being in the definitive host and the larval stages being in the intermediate hosts. When larval stages occur in humans, serious disease can result. Two groups of cestode infect man: the Cyclophyllidea and the Pseudophyllidea. Cyclophyllidean tapeworms These include the beef and pork tapeworms, echinococcosis (hydatid) and several other species. The larval stages occur in vertebrates, adult worms have a scolex bearing a circlet of four suckers and usually a central evertible rostellum with one or more circlets of minute hooks (Figs. 8.10.1.1a, b, c). In many species the eggs enter the environment within intact proglot- tids. In either case the eggs are embryonated and contain a six-​ hooked hexacanth embryo. The egg shells have two membranes, but in Taenia the outer is lost early and the inner forms the thick embryophore. After ingestion by the intermediate host, eggs hatch and the released hexacanth embryo bores its way into the mucosa. The larval stages of the parasite (‘metacestode’) are generally cystic with an invaginated embryonic scolex—​the protoscolex. The cycle is completed when the larval stage, within the intermediate host or its tissues, is eaten by the definitive host; the protoscolex evaginates and attaches to the gut mucosa. Humans can be infected by adult parasites in the gut. More serious disease occurs when larval stages in the tis- sues:  1. Cysticercosis caused by Taenia solium (see next and Chapter 8.10.3). 2. Hydatid disease caused by the dog tapeworm Echinococcus granulosus (see Chapter 8.10.2). 3. Alveolar hydatid disease caused by the fox tapeworm Echinococcus alveolaris. 4.  Coenurosis caused by tapeworms of the genus Multiceps in canids and rabbits. Cyclophyllidean gut tapeworms In four gut species, humans are an obligatory part of the life­ cycle (Table 8.10.1.1), in the rest they are an accidental host (Table 8.10.1.2). The three Taenia species are anthropozoonoses because the cycle is maintained by an obligatory alternation be- tween human and non​human hosts. Phylogenetic studies suggest that Taenia tapeworms were acquired, before humans domesti- cated cattle and pigs, by hominid hunters intruding into predator–​ prey cycles involving perhaps lions and hyenas. Patients with Taenia infections pass proglottids in their faeces or experience their active migration per annum. The clinical importance the pork tapeworm relates mainly to cysticercosis, the occurrence of larval forms in human tissue (see Chapter 8.10.3). The dwarf tape- worm Hymenolepis nana infects an estimated 9 million people; there is normally no intermediate host. With any gut cestode, symptoms also result from local hypersen- sitivity reactions to the worm and its scolex, altered gut motility, and poorly defined systemic symptoms with an immunological basis. A blood eosinophilia up to 10% can occur. (a) (b) (c) Fig. 8.10.1.1  (a) Taenia saginata showing scolex with four suckers and no hooks. (b) Taenia solium showing scolex with four suckers and a double row of hooks. (c) Taenia solium detail of hooks. Courtesy of Professor Viqar Zaman.

section 8  Infectious diseases 1522 Taenia tapeworms Taenia saginata Geographical distribution The beef tapeworm T.  saginata is prevalent where cattle have access to human faeces and where humans eat undercooked beef. The highest prevalence is in Africa, particularly in eastern and north-​eastern parts; it is also common in many coun- tries in the Middle East, South America, and Southeast Asia. Prevalence is now very low in the United States of America, Canada, and Australia. It persists endemically in Europe; but prevalence increases progressively eastwards and into the former Soviet Union. Table 8.10.1.1  Major gut cestodes that infect humans Taenia saginata Beef tapeworm Taenia asiatica Asian tapeworm Taenia solium Pork tapeworm Hymenolepis nana Dwarf tapeworm Larval tapeworm Intermediate hosts Cattle, water buffalo, other bovids, reindeer Pig, wild boar Pig, wild boar; also in humans (cysticercosis) None but human and murine subspecies perhaps cross-​infect Type and size Cysticercus 7–​10 × 4–​6 mm Cysticercus 2 × 2 mm Cysticercus 5–​8 × 3–​6 mm Minute tailless cysticercoid 50 µm Location Muscle, viscera, brain (reindeer only) Viscera, mainly liver Muscle, brain, subcutaneous, eye, tongue Villi of small intestine Adult tapeworm Length 4–​12 m 4–​12 m 3–​5 m 25–​40 mm Number of proglottids 2000 (mean) 2000 (mean) 700–​1000 200 (mean) Gravid proglottid Longer than wide; 20–​30 × 5–​7 mm Longer than wide; 20–​30 × 5–​7 mm Longer than wide; 18–​25 × 5–​7 mm Wider than long; 0.8 × 0.2 mm Scolex No rostellum, no hooks Rostellum; no hooks Rostellum with double circlet of hooks Rostellum with single circlet of minute hooks Gravid uterus 15–​20 lateral branches 15–​20 lateral branches 7–​12 lateral branches Bilobed Egg (contains hexacanth embryo) Embryophore shell is radially striated and 31–​40 µm in diameter Embryophore shell is radially striated and 31–​40 µm in diameter Embryophore shell is radially striated and 31–​40 µm in diameter Oval, 30–​47 µm long; two shell membranes; 4–​8 filaments arise from each pole of inner membrane Table 8.10.1.2  Uncommon gut cestodes that infect humans Species Geographic distribution Definitive hosts Intermediate hosts Length and width
of tapeworm Shape of gravid proglottid Other features Bertiella mucronata South America, Cuba Primates Oribatid mites 15–​45 cm × 5–​10 mm Wider than long Inner eggshell bears bicornuate knob B. studeri South and Southeast Asia, Africa, Cuba Primates Oribatid mites 27–​30 cm × 6–​10 mm Much wider than long As above Dipylidium caninum Worldwide Dog, cat Fleas and dog louse 10–​70 cm × 2.5–​3 mm Elongate, wider in middle Double set of sex organs. Egg capsules with 8–​15 eggs Hymenolepis diminuta (rat tapeworm) Worldwide Rat Fleas, beetles, cockroaches 20–​60 cm × 3–​4 mm Much wider than long Egg like H. nana but yellow outer membrane and no filaments; 60–​85 µm Inermicapsifer madagascariensis Madagascar, Africa, Central America, Cuba Rats Arthropod 26–​42 cm × 2.6 mm Slightly elongate, white, and opaque Egg capsules with 6–​11 eggs Mathevotaenia symmetrica Thailand Rodents Beetles 13 cm × 1–​2 mm Elongate, wider in middle Capsule surrounds individual eggs Mesocestoides lineatus China, Japan, Korea Carnivores Mites (1st host); amphibia, reptiles, birds, rodents (2nd hosts) 40 cm × 1.5–​2 mm Longer than broad Single medioventral genital opening Mesocestoides variabilis Greenland, USA Carnivores Mites (1st host); amphibia, reptiles, birds, rodents (2nd hosts) 40 cm × 1.5–​2 mm Longer than broad Single medioventral genital opening Raillietina celebensis East Asia, Polynesia, Australia Rats Ant 16–​60 cm × 3 mm As above Egg capsules with 1–​4 eggs R. demerariensis Guyana, Cuba, Ecuador Rats Cockroach 16–​60 cm × 2–​3 mm As above Egg capsule with 8–​10 eggs

8.10.1  Cestodes (tapeworms) 1523 Epidemiology Most worms are solitary. Multiple worms are smaller and typically occur in high-​transmission areas, probably by simultaneous infec- tion. Viable eggs from human faeces persist on pasture for many months and can survive most forms of sewage treatment. Cattle have access to human faeces on farms, at camp sites and recreation areas, and on railway lines. Infected herdsmen can initiate epizootics. Eggs may be dispersed by flies and dung beetles, and seabirds can ingest proglottids in refuse or estuarine waters and deposit them in their faeces on inland pastures. In cattle the whitish, ovoid cysticerci become infective within 12 weeks and remain viable in the living host for 2 years; they are viable in stored, chilled meat for several weeks but are killed at –​20°C within 1 week. The prepatent period in man is 3 months and worms may live 30 years. Cattle develop protective immunity to new infection. Clinical features The whitish mature proglottids, approximately 2–​3 cm long, are actively motile, elongating and contracting (Fig. 8.10.1.2). Most patients experience active exit of single proglottids through the anus, others pass proglottids at defecation, often in short chains; free eggs also occur in faeces. Many have no other symptoms, but others complain of nausea and upper abdominal pains, often re- lieved by food. In children, impaired appetite can have nutritional consequences. Some patients have symptoms suggestive of hypo- glycaemia, namely dizziness and sweating. Pruritus ani is common. The worm can sometimes be visible on small-​bowel barium studies. Proglottids have been found in a variety of surgical specimens, including resected appendices, but a pathogenic role is usually diffi- cult to establish. They occasionally obstruct the small intestine, pan- creatic duct, or bile duct. Proglottids are recorded in the gallbladder, and eggs have been found in gallstones. Diagnosis The typical eggs (Fig. 8.10.1.3) can be found in faeces, but this is an insensitive method; perianal swabs are more useful. Eggs are indistinguishable from those of T. solium and T asiatica; patients should be asked to bring worm specimens. Unless the proglottid is fully gravid the number of uterine branches is an unreliable diag- nostic character. A better morphological distinction is the presence of a vaginal sphincter; this is absent in T. solium. In human surveys in endemic areas a 24-​h faecal collection after an anthelmintic will give the most reliable prevalence. Treatment Niclosamide, 2 g, is given to adults and older children as a single morning dose on an empty stomach; the tablets should be chewed. Children aged 2 to 6 years should receive 1 g, and those below 2 years 500 mg. The alternative is praziquantel, 5–​10 mg/​kg as a single dose after a light breakfast. After either drug the proximal part of the worm disintegrates in the gut and the scolex cannot be found. Failure of proglottids to reappear within 3 to 4 months indicates cure. Control This includes health education concerning raw beef, meat inspec- tion, sanitation and hygiene on cattle farms, and proper sewage treatment and disposal. Mass treatments of herd contacts, or whole adult populations, are the most effective short-​term measures when endemicity is high. T. saginata causes great economic loss to the beef industry in some developing countries. Vaccines may soon become available for use in cattle. Taenia asiatica This was first described in 1973 as a subspecies T. saginata from rural Taiwan, where raw pig or wild boar liver, but no beef, was eaten. It is now recognized as a separate species and known also to occur in Korea, China, northern Sumatra, Indonesia, Philipines, Thailand, India and Nepal. The cysticerci in pig viscera are very small. In immunodeficient mice T. asiatica eggs, but not those of T. saginata, produce cysticerci with hooked protoscolices. Eating uncooked pork with viscera from home-​killed pigs is a recognized risk factor. Symptoms and treatment are the same as for T. saginata. Fig. 8.10.1.2  Actively mobile, contracting proglottid of Taenia saginata found by a patient in the stool. Copyright D. A. Warrell. Fig. 8.10.1.3  Egg of Taenia. Courtesy of Professor Viqar Zaman.

section 8  Infectious diseases 1524 It appears not to cause cysticercosis in humans; possibly the species can hybridize with T. saginata. Taenia solium (see also Chapter 8.10.3) Generally less common than the beef tapeworm, the pork tape- worm T. solium is now very rare in North America and Western Europe, but it remains common in much of sub-​Saharan Africa, and in China, India, and other parts of Asia. It is highly prevalent in Mexico and other Latin American countries. Two genotypes are now recognized: the European type that has been introduced into the Americas and Africa since the 1500s, and the Asian type. Both types can produce neurocysticercosis, but only the latter causes subcutaneous cysticercosis. Epidemiology In pigs, muscle cysticerci produce ‘measly pork’ (Fig. 8.10.1.4). The cysts are most numerous in the tongue, masseter, heart, and diaphragm, but also occur in the brain. When eaten by humans in undercooked pork, the worms mature in 5 to 12 weeks. The eggs have the same resistant qualities as those of T. saginata. Human cysticercosis arises when eggs from the faeces of people infected with adult worms are ingested and hatch in the upper gut; humans thus become an accidental intermediate host. Conditions favouring cysticercosis include poor personal hy- giene, which facilitates external autoinfection, and contam- inated fingers among food handlers. Faecal pollution of the peridomestic environment, irrigation water, or cultivated veget- ables is also important. In parts of Africa, tapeworm proglottids are used in traditional medicine. In the absence of these factors, cases of cysticercosis may be very sporadic even when T. solium is common. Cysticercosis is a major health problem in Mexico, some South American countries, and to a lesser extent in Africa and Asia. In 1969, T. solium was introduced from Bali into the high- lands of Indonesian New Guinea, where the disease is now of great importance. Pathology of human cysticercosis Cysts occur especially in striated muscle, subcutaneous tissue (Asian genotype), the nervous system, and the eye. Many re- main clinically silent until the parasite dies after 3 to 5 years, when vigorous inflammatory and hypersensitivity reactions can occur; later, lesions may calcify. In the brain, particularly in the subarachnoid and the ventricular system, atypical racemose cysts may occur. They appear as irregular or grape-​like clusters of cysts that have no protoscolex; they can be mistaken pathologically for non​parasitic cysts. Clinical features Symptoms, if any, due to the adult worms are similar to those of T. saginata but are often milder and not associated with pruritus ani. The proglottids do not migrate actively per annum. Diagnosis Adult worm infection is detected as for T. saginata. Methods for detecting faecal antigen are available and have great potential use in epidemiological studies. Proglottid fragments can be identified using DNA probes. Treatment and control Adult worms are treated as for T. saginata. Because of the poten- tial risk of internal autoinfection vomiting must be avoided and an antiemetic is often recommended before treatment, together with a laxative 2 h after the medication. It should be remembered that the faeces will be potentially highly infective for several days, for both the patient and the attendants. Control measures in- clude meat inspection, health education, and population-​based chemotherapy. Local risk factors for human cysticercosis must receive special attention. Pigs can be treated with a single dose of oxfendazole and perhaps in the future given recombinant hexacanth vaccines. Hymenolepis nana The dwarf tapeworm, sometimes now placed in the genus Rodentolepis, is the most common cestode in humans; it is also the smallest. When worm loads are high, it causes more gut pathology than any other species. It is common in most developing and trop- ical countries. The life cycle normally involves only humans. Fully embryonated infective eggs are passed in the faeces; gravid proglot- tids normally disintegrate completely in the gut. Infection is com- monly direct, but also by the other faeco-​oral routes. Eggs hatch in the jejunum and the hexacanth embryo bores into a villus where it transforms into a cysticercoid larva. After 4 to 6 days it re-​enters the gut, everts the scolex, and attaches to the mucosa; eggs appear in the faeces within 12 days. The lifespan is 3 months. The eggs are delicate and survive less than 10 days in the environment. Prevalence is usually much higher in children than adults; outbreaks can occur in families and institutions. External autoinfection is common in high-​risk groups and enables high worm loads to build up. In addition, internal autoinfection occurs when there is gut stasis or retroperistalsis. Because of the importance of direct transmission, this infection may be common even in arid environments such as Western Australia. A similar parasite, recognized as a subspecies H. nana fraterna, occurs in the mouse but this normally has the flour beetle Tribolium as intermediate host, although direct mouse-​to-​mouse transmis- sion can occur. Both human and murine subspecies will infect these Fig. 8.10.1.4  ‘Measly pork’ showing numerous cysts in the pig’s muscle. Copyright Sornchai Looareesuwan.

8.10.1  Cestodes (tapeworms) 1525 beetles. The zoonotic potential of the murine subspecies is uncer- tain, as at least Australian human strains will not infect mice. Clinical features Heavily infected people, especially children, may harbour up to 1000 or more worms. Mucosal damage caused by both larval and adult worms leads to protein loss and sometimes malabsorption. Abdominal pains and anorexia are common. Immunosuppressant or steroid therapy, particularly in lymphoma patients, can lead to the development of bizarre cystic larval forms in the gut wall, mesenteric nodes, liver, and lungs. A similar condition can be produced in immunosuppressed mice. Diagnosis and treatment Eggs can be detected in faeces using concentration methods. Proglottids are rarely found in faeces, except after treatment. Praziquantel in a single dose of 25 mg/​kg is the most effective drug. If niclosamide is used, a 7-​day course is needed to ensure that larval stages are killed when they re-​enter the gut lumen. The dose on the first day is as for T. saginata; on the remaining days one-​half of this dose is given. Relapses often result from persistence of eggs in the patient’s environment. Uncommon gut cestodes Several species have been recorded in humans (Table 8.10.1.2). All have arthropods as intermediate hosts, the larval cysticercoid stage being in the haemocele; the full life cycles of some species are still uncertain. The normal definitive host becomes infected by eating the arthropod, intentionally or accidentally. The means by which humans become infected is sometimes not clear, but fleas, small beetles, and mites are easily overlooked in food. Dipylidium caninum infection occurs in children who have groomed their pets. Infections with Bertiella are mostly in owners of pet monkeys, but oribatid mites are common in fallen fruit, especially mangoes. Children may eat insects deliberately, and this appears to be the mode of infection by Raillietina in Bangkok. Beetles are used for medicinal purposes in parts of Thailand and Malaysia, and this is the most likely route by which Mathevotaenia is acquired. In many of these species the eggs are in capsules that are re- leased when the proglottid disintegrates in the gut, or more com- monly, in the faecal mass. Mesocestoides is unique among these parasites in that two intermediate hosts are required, and the genital opening is medioventral. Human Mesocestoides infections follow ingestion of raw viscera or blood from game, including birds, or from chickens. Many patients will present because they have passed proglot- tids. D. caninum actively migrates out of the anus, like T. saginata. Faecal examinations of people with abdominal complaints may reveal unusual eggs or egg capsules. Poorly defined systemic and allergic complaints are common. Treatment is as for T. saginata. Recognition of these parasites is of epidemiological interest and may indicate potential transmission of other zoonotic pathogens. It is certain that all these parasites are underreported. Unusual pro- glottids or eggs should be preserved in formol saline and sent to a parasitologist. Tissue cyclophyllidean tapeworms (For cysticercosis caused by Taenia solium, see earlier and Chapter 8.10.3. For hydatid disease caused by the dog tapeworm Echinococcus granulosus, see Chapter 8.10.2.) Alveolar hydatid disease caused by the fox tapeworm Echinococcus multilocularis Epidemiology Because of its high mortality and difficult clinical management this parasitic disease is important. Adult E. multilocularis tapeworms, which are smaller than those of E. granulosus, are widespread gut parasites of foxes and artic foxes in the Northern hemisphere, faecal eggs are ingested by rodents, especially voles and lemmings, which develop hepatic multilocular cysts; these intermediate hosts are in turn eaten by foxes to complete the cycle. Humans become acci- dental intermediate hosts when eggs contaminate food or water. Dogs and more rarely cats can also be infected by the adult parasite. The global annual incidence rate is now estimated to be 18 000, with 91% in China, and 1600 elsewhere, particularly Russia, Turkey, and Europe (Fig. 8.10.1.5), Prevalence of the parasite in European foxes is quite widespread, but human disease quite rare with about 60 cases annually in Germany and Switzerland and smaller num- bers in France, Poland, and the Baltic states and elsewhere. The principal rodent host in Europe is the common vole, but also musk- rats, nutria, and the beaver. There is concern that European beavers brought to the United Kingdom in reintroduction schemes might harbour this parasite. The raccoon dog (Nyctereutes) has been intro- duced to Europe and is now a potentially dangerous new host for the adult worm. Human cases are predominantly rural and focal, and can usually be attributed to ingestion of apples, berries, and salad vegetables or water sources contaminated by fox faeces; fur trappers may be in- fected during skinning of wild canids. Increases in populations of both rural and urban European foxes raise concerns, and in France where 407 human cases were diagnosed between 1982 and 2007, in- fected foxes are reported from Paris and on the Channel coast, but infected dogs are rare in France. Pathology Primary lesions are in the liver and consist of an amorphous mass of minute irregular spaces, containing little fluid, in an avascular adventitia but no surrounding fibrous capsule; unlike the lesions in the vole brood capsules and protoscolices are scarce. The lesion en- larges progressively with exogenous budding over a period of years, and may extend from the liver through the diaphragm or abdominal wall, sometimes metastasizing to the brain or elsewhere. Untreated, most cases are fatal but some resolve and may calcify. Clinical features and management Five or more years after presumed infection patients present with hepatomegaly, cholestatic jaundice, or less suggestive symptoms. Later there might be signs of liver failure or spread outside the liver. Liver ultrasound shows an irregular tumour with juxtaposed hypo-​ and hyperechoic areas, scattered calcification, and central necrosis. MRI can show the multivesicular nature of the lesion. Biopsy material

section 8  Infectious diseases 1526 can be stained with Periodic acid-​Schiff to show the germinal layer and parasite antigen detected by PCR. Immunodiagnosis gives good sensitivity and specificity. Treatment must aim at complete surgical excision with a 2 cm tissue margin whenever this is possible and be followed by albendazole for two years if resection is complete, and lifelong otherwise. The usual dose is 10–​20 mg/​kg daily in two divided doses. This drug does not kill the parasite directly but is parasitostatic. Patients are monitored by regular ultrasound, liver function tests, drug levels, blood counts, and perhaps serology. When complete removal is not possible then palliative care should be multidis- ciplinary, preferably in centres experienced in this condition. Liver transplantation has been successful, but the necessary immunosuppression facilitates parasite growth and albendazole cover must be given. Praziquantel is not used in humans with this infection but is used in baits for foxes as an effective control measure. Coenurosis caused by canid tapeworms of the genus Multiceps Epidemiology Multiceps multiceps is a common tapeworm in dogs, foxes, coy- otes, and other canids worldwide, M. serialis occurs mainly North American dogs and foxes, while M. brauni and M. glomerata infect African canids. The worms measure a metre or more in length. Gravid proglottids and eggs, passed in faeces are ingested by sheep and less commonly cattle, horses, goats, rabbits, or rodents and in these intermediate hosts the cystic larval stage or coenurus develop in the brain and other tissues. These cysts show characteristic mul- tiple invaginated scoleces. Sheep can devlop a fatal ataxia known as ‘gid’. Human infections, while uncommon, occur especially in Africa but also in sheep-​raising areas worldwide. They follow accidental ingestion of eggs from canid faeces and are commoner in children. Clinical features and management Coenurus cysts are usually unilocular and can measure 3  cm or more in diameter; they occur most commonly in the brain, spinal cord, eye, muscle, and subcutaneous tissue. The cysts of M. serialis are most frequent in subcutaneous tissue, and M. brauni in the eye. The clinical features are of a mass or space occupying lesion with the associated secondary effects. The cystic lesions must be differentiated from cysticerci and hydatid, but diagnosis will usually only be confirmed after surgical removal when parasitological methods including recent molecular techniques are used. Besides excision the role of praziquantel ap- pears promising. Taenia crassipes cysticercosis This is a common tapeworm of dogs, foxes, and felids; the inter- mediate hosts are rabbits and rodents in which the small cysticerci are in muscle and subcutaneous tissue, but sheep may also be in- fected and develop a fatal neurological disease. An important char- acteristic of this species is exogenous budding producing many daughter cysts. So far 10 human cases are reported; of whom 5 had Fig. 8.10.1.5  Global distribution of alveolar echinococcosis. Reprinted from Torgerson PR, et al. (2010). The Global Burden of Alveolar Echinococcosis. PLoS Negl Trop Dis 4(6): e722, © 2010 Torgerson et al.

8.10.1  Cestodes (tapeworms) 1527 AIDS and one lymphoma with cysts in muscle and subcutaneous tissue. In immunocompetent patients, cysts were found in the eye, but cysts in the human nervous system have not been reported so far. Treatment is by excision, but this is difficult because of the bud- ding. Cestocidal drugs may be ineffective. Pseudophyllidean tapeworms These include the fish tapeworms (Diphyllobothrium spp.) and the Spirometra species that cause sparganosis. When eggs of these parasites reach fresh water, they hatch into a coracidium, a spher- ical ciliated larva which when engulfed by small crustacean cope- pods becomes an elongated procercoid larva. If infected copepods are ingested by a fish, they become a motile plerocercoid or sparganum; fish-​eating mammals complete the life cycle when plerocercoids attach to the small-​gut musosa and become adult worms. The life cycle may also include non​fish vertebrates that ingest infected copepods. In addition, amphibia and snakes that ingest infected fish, become paratenic hosts in which the worm remains a plerocercoid; this is also the situation in humans who develop sparganosis. Adult pseudophyllidean worms have two sucking grooves or bothria on opposite sides of the scolex, the proglottids are usually much wider than broad, and the separate genital and uterine open- ings are on the mid-​ventral surface. Diphyllobothriasis (‘fish or broad tapeworm infections’) Epidemiology In 1970 there were five million cases of D. latum infection in cen- tral and eastern Europe, Finland, and the Baltic states maintained by ingestion of undercooked fish, particularly pike, perch, and burbot containing the plerocercoid larvae. Prevalence has since greatly de- clined but a recrudescence is now occurring in Switzerland, northern Italy, and eastern France as undercooked fish delicacies become popular; up to 5% of perch in some Alpine lakes are now infected. The larvae measure up to 20 mm and appear glistening opaque white and unsegmented; they occur throughout the fish tissues but mi- grate to muscle in dead fish. The adult worm, 2–​15 m in length and 5–​10 mm wide, is folded repeatedly within the small intestine. The scolex attaches to the ileum and the worm can grow 22 mm/​day and live 20 years. Prevalence has also declined in North American great lakes where the main fish host is pike, perch or walleye; the parasite was originally introduced by Scandinavian migrants. In Asia it is prevalent in all river basins east of the Urals. It has been introduced to Brazil and other South American countries where there have been recent outbreaks. Humans are infected by 13 species of Diphyllobothrium in addition to D. latum. The three most important, with their intermediate and non​human final hosts are: (1). D. nihonaiense in the North Pacific especially Japan, Pacific salmon, brown bears; (2). D. dendriticum is circumpolar, salmonelid, and coregonid fish, piscivorus birds espe- cially gulls and bears: this species is now regarded as an emerging infection. (3). D.  pacificum on Pacific Coast South America and Japan, marine fish, sea-​lions, and seals: human coprolites containing eggs of this species dating from 2000 BC have been found in Chile. In Japan 200 cases of infection by another diphyllobothrid worm, Diplogonoporus grandis, normally a parasite of whales, is reported following ingestion of raw anchovy or sardine. Clinical features, treatment, and prevention Many patients are unaware of their infection apart from noticing pro- glottids or long parts of the worm passed per annum, 20% complain of abdominal pain or discomfort, diarrhoea, or allergic symptoms such as urticaria. Rarely infections cause obstruction of the gut or bile duct. The worms cause dissociation of the vitamin B12-​intrinsic factor complex and 80% of the oral intake of the vitamin is taken up by the worm. Up to 40% of patients have a low serum B12 and a few with heavy or prolonged infections develop pernicious anaemia. Diagnosis is by parasitological examination of proglottids or faecal eggs (Fig. 8.10.1.6). Treatment is usually straightforward using a single dose of praziquantel 25 mg/​kg or niclosamide 2 g for adults and 1 g for children over 6 years; vitamin B12 may be necessary. Fish to be eaten raw or semi raw should be kept at –​20°C for 7 days or –​35°C for 15 hours to kill larvae; cooking to 55°C for 5 minutes will also kill them. Smoking fish will not kill larvae but brine storage will. The worldwide nature of fish trade, fish transport on ice, global warming, and El Nino may all facilitate outbreaks and introduction of these parasites to new areas. Sparganosis Epidemiology Spirometra tapeworms are parasites of canids and felids, S. erina­ ceieuropaei (syns. mansoni, erinacei) occurs throughout Asia and western Pacific, and S. mansonoides in the Americas. The second intermediate hosts are fish, snakes, and frogs or tadpoles; when these are eaten, or when snake tissue or frogs are applied to wounds, the eye, or vagina for ritualistic or medicinal reasons, the parasite may be transferred. Humans are also at risk by swallowing water containing infected copepods. By all these routes humans become infected with the plerocercoid stage of the parasite which con- tinues to grow and may reach 10 cm in length and 2 mm in width (Fig. 8.10.1.7); more than 1000 cases have been reported from China alone and is under reported elsewhere. Fig. 8.10.1.6  Egg of Diphyllobothrium latum. Courtesy of A. R. Butcher.

section 8  Infectious diseases 1528 Clinical features and management The worm migrates slowly in the tissues as a form of ‘larva migrans’; most frequently subcutaneously but also in viscera, the orbit, and the brain. More than 160 cases of central nervous system involve- ment have been reported and are being increasingly recognized as neuroimaging becomes widespread. Incubation periods may be long and subcutaneous migration may continue for years. Eye lesions may present as pain, proptosis, and blindness; brain lesions as seizures, headache, parasthesiae, or weakness. A rare form of sparganosis, in which the plerocercoid multiplies within the host, is now thought to be due to a separate species S. proliferum; prog- nosis is poor. In endemic areas the diagnosis may be suspected by clinical features, serological tests are not yet very specific. Rarely serial neuroimaging may demonstrate a tunnel through which the worm migrates (Fig. 8.10.1.8). More often diagnosis will be made on histology from an excision specimen or biopsy; unlike the cysts of cyclophyllidean tapeworms the worm is solid with no suckers or rostellar hooks, and no ‘bladder wall’. Treatment is primarily surgical, prognosis after excision of brain lesions can be good but the location of the lesion is critical. Praziquantel and albendazole or mebendazole generally give poor results and can aggravate neurological lesions. FURTHER READING Cyclophyllidean tapeworms Anita A, et al. (2014). Epidemiology and genetic diversity of Taenia asiatica: a systematic review. Parasit Vectors, 7, 45. Bagrade G, et al. (2016). Echinococcus multilocularis in foxes and raccoon dogs: an increasing concern for Baltic countries. Parasit Vectors, 9, 615. Brunetti E, Kern P, Vuitton AV (2010). Expert consensus for the diag- nosis and treatment of cystic and alveolar echinococcosis in hu- mans. Acta Tropica, 114, 1–​16. Campbell-​Palmer R, et al. (2015). Echinococcus multilocularis: detec- tion in live eurasian beavers (Castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions. PLoS One, 10, e0130842. Conraths FJ, et al. (2017). Potential risk factors associated with human alveolar echinococcosis: systematic review and meta-analysis. PloS Negl Trop Dis, 11, e0005801. Galán-​Puchades MT, Fuentes MV (2013). Lights and shadows of the Taenia asiatica life cycle and pathogenicity. Trop Parasitol, 3, 114–​9. Gawor J (2016). Alveolar echinococcosis in Europe and Poland. Threats to humans. Przegl Epidemiol, 70, 281–8. Hemphill A, et al. (2014). Treatment of echinococcosis: albendazole and mebendazole—​what else? Parasite, 21, 70. Hoberg EP (2006). Phylogeny of Taenia: species definitions and origins of human parasites. Parasitol Int, 55 Suppl, 23–​30. Hoberg EP, et al. (2001). Out of Africa: origins of the Taenia tape- worms in humans. Proc Roy Soc London B, 268, 781–​7. Muehlenbachs A, et al. (2015). Malignant transformation of Hymenolepis nana in a human host. N Engl J Med, 373, 1845–52. Pawlowski Z, Schultz MG (1972). Taeniasis and cysticercosis (Taenia saginata). Adv Parasitol, 10, 269–​343. Subianto DB, Tumada LR, Morgono SS (1978). Burns and epileptic fits associated with cysticercosis in mountain people of Irian Jaya. Trop Geogr Med, 30, 275–​8. Torgerson PR, et al. (2010). The global burden of alveolar echinococcosis. PLoS Negl Trop Dis, 4, e722. Umhang G, et al. (2013). Nutrias and muskrats as bioindicators for the presence of Echinococcus multilocularis in new endemic areas. Vet Parasitol, 197, 283–​7. Pseudophyllidean tapeworms Anantaphruti MT, et al. (2011). Human sparganosis in Thailand: an overview. Acta Trop, 118, 171–​6. Bennett HM, et al. (2014). The genome of the sparganosis tapeworm Spirometra erinaceieuropaei isolated from the biopsy of a migrating brain lesion. Genome Biolog, 15, 1–​17. Kuchta R, et  al. (2013). Tapeworm Diphyllobothrium dendriticum (Cestoda)—​neglected or emerging human parasite? PLoS Negl Trop Dis, 7, e2535. Liu Q, et al. (2015). Human sparganosis, a neglected food borne zoo- nosis. Lancet Inf Dis, 15, 1226–35. Scholz T, et al. (2009). Update on the human broad tapeworm (genus Diphyllobothrium), including clinical relevance. Clin Microbiol Rev, 22, 146–​60. Both groups of tapeworms Lescano AG, Zunt J. (2013). Other cestodes: sparganosis, coenurosis and Taenia crassiceps cysticercosis. Handb Clin Neurol, 114, 335–​45. Fig. 8.10.1.7  A sparganum surgically removed from a subcutaneous mass. Fig. 8.10.1.8  MRI scan of cerebral sparganosis. Coronal contrast-​ enhanced T1-​weighted image shows a tortuous curvilinear enhancing lesion (arrows) with surrounding low density of oedema and degeneration in the right frontal lobe.

8.10.2 Cystic hydatid disease (Echinococcus granul

8.10.2 Cystic hydatid disease (Echinococcus granulosus) 1529

8.10.2  Cystic hydatid disease (Echinococcus granulosus) 1529 8.10.2  Cystic hydatid disease (Echinococcus granulosus) Pedro L. Moro, Hector H. Garcia, and
Armando E. Gonzalez ESSENTIALS Cystic hydatid disease, caused by Echinococcus granulosus, is a zoo- notic disease principally transmitted between dogs and domestic livestock, particularly sheep. Humans are infected when they ingest tapeworm eggs, with disease occurring in most parts of the world where sheep are raised and dogs are used to herd livestock. Clinical features, diagnosis, and treatment—​the most common clin- ical manifestations are cysts in the liver (typically presenting with hepatomegaly) and/​or lung (presenting with cough, haemoptysis, and dyspnoea). Diagnosis is usually made on the basis of imaging techniques, supported by serological tests. Treatment options include
surgery, chemotherapy with anthelminthic agents, or—​for liver cysts—​ PAIR (puncture–​aspiration–​injection–​reaspiration). Prevention—​echinococcosis is a major public health problem in several countries. Control programmes have been aimed at educating dog owners to prevent their animals from having access to infected offal, along with praziquantel treatment of dogs. Vaccines against sheep hydatidosis and the dog tapeworm stage are prom- ising alternatives. Introduction Cystic hydatid disease is a zoonotic disease caused by infection with the larval stage (hydatid cyst) of the tapeworm Echinococcus granulosus. Hydatid cysts in liver and lung are frequent causes of human morbidity in endemic zones. Aetiology The lifecycle of E. granulosus requires two hosts. The adult tape- worm is found in the small intestine of the definitive host, usually dogs or other canids. It consists of only three to five proglottids, and measures between 3 and 7 mm long when fully mature. E. granu­ losus has remarkable biological potential; there may be as many as 40 000 worms in a heavily infected dog, each one of which sheds about 1000 eggs every 2 weeks. Dogs infected with echinococcus tapeworms pass eggs in their faeces that contaminate the soil and vegetation and remain viable for long periods in cold humid places. Intermediate hosts (sheep, cattle, horses, pigs, and other mammals, including humans) acquire hydatid disease by ingesting viable eggs of E. granulosus. Eggs hatch in the intestine, freeing oncospheres which penetrate the intestinal mucosa and are transported by the blood and lymphatic systems to the liver, lungs, and other organs, where they develop into cysts. Molecular studies using mitochondrial DNA sequences have iden- tified 10 distinct genetic types within E. granulosus. These include two sheep strains (G1, G2), two bovid strains (G3, G5), a horse strain (G4), the camelid strain (G6), a pig strain (G7) and the cervid strain (G8). A ninth genotype (G9) has been described in swine in Poland, and a tenth genotype (G10) in cervids. The sheep strain (G1) is the most cosmopolitan form that is most commonly associated with human infections. The other strains appear to be genetically distinct. The presence of distinct strains of E. granulosus may affect clinical aspects and control strategy. The risk of human infection differs as does its localization in the body, clinical expression, and geograph- ical distribution. Shorter maturation time of a given strain in dogs might reduce the duration of infection by the adult intestinal form so that shorter intervals might be required between rounds of ad- ministration of antiparasite drugs for control. Epidemiology Hydatid disease is an important cause of human morbidity, requiring costly surgical treatment. The infection is widely distributed in most parts of the world where sheep are raised and dogs are used to herd livestock. In the Americas, most cases have been reported from Argentina, Chile, Uruguay, Peru, and southern Brazil. Studies in Peru have revealed a prevalence of hydatid disease ranging from 5.7 to 8.9% in highland villagers, and as high as 32 and 89% in dogs and sheep, respectively. High prevalences of liver hydatid disease, with rates of up to 5.6%, have also been reported in north-​western Turkana in Kenya. Echinococcus is widespread in the Old World, particularly in Greece, Cyprus, Bulgaria, Lebanon, and Turkey. In the United States of America, most infections are seen in immigrants from endemic countries; however, sporadic autochthonous transmission is currently recognized in Alaska, California, Utah, Arizona, and New Mexico. Communities at higher risk of infection include those where sheep are raised extensively and where dogs are used to care for large flocks of livestock. Known risk factors for infection include feeding dogs with raw offal and access of dogs to sheep that die in the field (Fig. 8.10.2.1). The risk of infection is also linked to poor hygiene and intimate contact with dogs. In north-​western Turkana, dogs are allowed to stay within the house, and are used to clean up women’s menses and lick vomit from faces and diarrhoea from the Fig. 8.10.2.1  Epidemiological conditions for completion of the life cycle of echinoccocus: stray dogs waiting for sheep offal outside a slaughterhouse in Peru.

section 8  Infectious diseases 1530 anal regions of their children. Global losses due to hydatid disease have been estimated to be US $763 980 979. Pathogenesis The incubation period of human hydatid infections is highly vari- able and often prolonged for several years. Cysts have been reported to grow continuously. However, recent studies suggest that cyst growth is highly variable. Some cysts grow as much as 1 cm per year while other viable cysts showed no growth during 3 to 12 years of follow-​up. Most human infections remain asymptomatic; hydatid cysts are found incidentally at autopsy much more frequently than the re- ported local morbidity rates. The locality of the cysts, their size, and their condition determine the particular manifestations. Clinical features Hydatid cysts are most frequently seen in the liver (60–​70%) fol- lowed by the lungs (30–​40%). Signs of hepatic hydatid disease in- clude hepatomegaly with or without the presence of a mass in the upper right quadrant. Obstructive jaundice, mild epigastric pain, indigestion, and nausea might occur occasionally. Hydatid cysts can become secondarily infected with bacteria presenting as a hep- atic abscess. Features of lung involvement (Fig. 8.10.2.2) are cough, haemoptysis, dyspnoea, and fever. The ratio of liver to lung cysts varies from one geographical region to another: a liver to lung ratio of 1.4:1 has been observed in Peru, in contrast to the 3:1 to 13:1 ratio reported in Argentina and Uruguay. Brain cysts produce intracra- nial hypertension and epilepsy. Vertebral cysts compress the spinal cord causing paraplegia; bone cysts produce spontaneous fractures (Figs. 8.10.2.3 and 8.10.2.4) and deformity. Sudden rupture of cysts in the peritoneal cavity might result in peritonitis (Fig. 8.10.2.4), and rupture in the lungs can cause pneumothorax and empyema. Rupture can also cause allergic manifestations such as pruritus, oe- dema, dyspnoea, anaphylactic shock, and even death. Diagnosis Clinical findings, such as a space-​occupying lesion, and residence in an endemic region are suggestive of hydatid disease. Abdominal ultrasonography is the most widely used imaging technique for echinococcosis because of its widespread availability and useful- ness for defining number, location, dimensions, and vitality of cysts. Ultrasonography is the basis for the World Health Organization Informal Working Group on Echinococcosis (WHO-​IWGE) clas- sification of hydatid cysts and is the basis for treatment decisions involving hepatic cysts. Portable ultrasonography machines are used with good results in field surveys. Chest radiography is useful for diagnosis of lung cysts. CT scanning is very helpful, especially for diagnosis of atypical lesions (Fig. 8.10.2.4b). Serology Several serological tests have been developed for diagnosis of hydatid disease, including an enzyme immunoassay, which iden- tifies antibodies against antigen B or components of this antigen. Fig. 8.10.2.2  Plain chest radiograph showing a lung hydatid cyst displacing the heart. (a) (b) Fig. 8.10.2.3  (a) Pathological fracture of the femur caused by hydatid infection. (b) Hydatid cyst in muscle excised from around the femoral head (same case as shown in (a)). Copyright D. A. Warrell.

8.10.2  Cystic hydatid disease (Echinococcus granulosus) 1531 A western blot assay based on the identification of three specific antigens of 8, 16, and 21 kDa is currently used. Major drawbacks in serological diagnosis are low sensitivity for detection of lung hydatid cysts and cross-​reactivity with sera of patients with Taenia solium infection. Cyst rupture or secondary infection are strongly associated with a positive result in hydatid serology. In field sur- veys, serological tests should be used in combination with imaging techniques in order to detect most cases of hydatid disease. Parasitological diagnosis When serological assays are negative, identification of protoscolices, hooklets, or hydatid membranes can be done through percutaneous aspiration of liver cyst contents. It might also be possible to identify these structures from sputum samples of patients whose lung cysts have recently ruptured. Treatment Treatment of hydatid cysts should be stage-​specific and should take into account cyst size, stage, number, localization, presence of complications, as well as characteristics of the patient (e.g. com- pliance with long-​term follow-​up). The WHO-​IWGE reached a consensus on treatment of cystic echinococcosis which is an image-​based, stage-​specific approach and follows these options:

  1. surgery, 2) chemotherapy, 3) percutaneous treatment, 4) watch and wait. An imaging-​based classification system is recommended when using this approach (Box 8.10.2.1). The benefits and limi- tations of current treatment options have been reviewed by the WHO-​IWGE and others. Determining the radiologic stage of the cyst, using the WHO classification of the cyst stage, and the size of the cyst are the first steps in determining the treatment options. Surgery Surgical removal of hydatid cysts remains the treatment of choice in many countries. Surgery is the preferred treatment for (1) large cysts with multiple daughter vesicles (e.g. CE2, CE3b cysts of the WHO-​ IWGE classification); (2) single superficial hepatic cysts which may rupture spontaneously or through trauma; (3) infected cysts or cysts located in certain organs (i.e. lung, brain, kidney) or (4) cysts that communicate with the biliary tree. Surgery is contraindicated in pregnant women, patients with pre-​existing medical conditions that put them at operative risk, or those who have multiple cysts that are difficult to access. The usual surgical approach involves aspiration of cyst fluid and injection of a protoscolicidal agent into the cyst, usually 20% hypertonic saline solution or 90% alcohol, followed by evacuation of the fluid, prior to surgical excision. Major risks of sur- gical treatment include accidental spillage of fluid and scolices into the peritoneal cavity, which may lead to anaphylaxis or secondary peritoneal hydatidosis. Recurrence rates following surgery can be as (b) (a) Fig. 8.10.2.4  (a) Numerous subcutaneous, peritoneal, and renal hydatid cysts in an Argentine patient. (b) Contrast CT scan of the same patient. Courtesy of Professor Olindo Adriano Martino, Buenos Aires. Box 8.10.2.1  The World Health Organization 2001 classification of hepatic hydatid cysts is a useful for assessing stage of a hepatic hydatid cyst on ultrasound and to decide on appropriate management for it depending on the stage of cyst. Classification • CL —​  unilocular anechoic cystic lesion without any internal echoes and septations • CE 1 —​  uniformly anechoic cyst with fine echoes settled in it representing hydatid sand • CE 2 —​  cyst with multiple septations giving it multivesicular appearance or rossette appearance or honey comb appearance with unilocular mother cyst —​  this stage is the active stage of the cyst • CE 3 —​  unilocular cyst with daughter cysts with detached laminated mem- branes appearing as ‘water lily sign’ —​  this is the transitional stage of the cyst • CE 4 —​  mixed hypo and hyperechoic contents with absent daughter cysts, these contents give an appearance of ball of wool sign indicating the degenerative nature of the cyst • CE 5 —​  arch-​like thick partially or completely calcified wall —​  this stage of cyst is inactive and infertile

section 8  Infectious diseases 1532 high as 30%. Intraoperative mortality can vary from 0.5% to 4% and can be even higher with repeated interventions. Antihistamines are given as prophylaxis and suction cones have been used to prevent spillage. The efficacy of these methods is uncertain. Some experts recommend prophylactic chemotherapy prior to surgery Chemotherapy Benzimidazole compounds have been shown to be effective against some forms of hydatid disease. They can be used alone in patients with CE1 and CE3a cysts that are less than 5 cm in diameter. They are used in combination with percutaneous interventions CE2 and CE3b as well as larger CE1 and CE3a cysts. It is not indicated for CE4 or CE5 cysts. They can also be used in patients who are not candidates for, or refuse, surgery and those with multiple cysts. Albendazole should be administered in a dose of 10–​15 mg/​kg per body weight per day without interruption. This regime cures approximately one-​third of cases of liver hydatid disease and causes partial regression of cysts in another one-​third of patients. Because of its high scolicidal activity, albendazole is recommended as a prophylactic agent 1 to 3 months before surgical intervention. Albendazole is indicated when surgery is contraindicated. Mebendazole can also be used, although it is less effective than albendazole. Albendazole, mebendazole, and other benzimidazole compounds should not be used during early preg- nancy because of their potentially teratogenic effects at the doses and regimes used for hydatid disease. Since benzimidazoles are potentially hepatotoxic, liver enzymes should be monitored before and during treatment, every 2 weeks during the first 3 months and then monthly for one year. The optimal duration of treatment with albendazole is uncertain; one to three months might be appropriate, depending on clinical factors; up to six months can sometimes be re- quired. Fat fat-​rich meals facilitate absorption and bioavailability of albendazole. Adverse reactions (neutropenia, liver toxicity, alopecia, and others), have been observed in a few patients but are reversible with cessation of treatment. In addition to early pregnancy, contra- indications to chemotherapy include chronic hepatic diseases, and bone marrow depression. The combination of praziquantel and albendazole seems to show better efficacy than albendazole alone in a few human case series and experimental animal studies but further studies are needed before this approach can be recommended. Recent experimental studies in animals have shown that another benzimidazole compound, oxfendazole, has strong parasiticidal activity. Intermittent weekly therapy with oxfendazole was ef- fective in sheep hydatid disease, suggesting the possibility that daily therapy as currently used with albendazole might not be needed. Current studies are evaluating the safety and pharmacokinetics of oxfendazole in the treatment of humans. Percutaneous treatment There are two categories of percutaneous treatment. There is Percutaneous aspiration, injection, reaspiration (PAIR) and there is non-​PAIR percutaneous treatment. PAIR consists of percutaneous puncture using sonographic guid- ance, aspiration of substantial amounts of the cyst fluid, and injec- tion of a protoscolicidal agent, usually hypertonic saline for at least 15 min, followed by reaspiration of cyst contents. PAIR is indicated for patients with CE1 and C3a cysts that are larger than 5 cm in diameter. It can also be considered for those who cannot undergo surgery and for patients who refuse surgery, or those who have single or multiple cysts in the liver, abdominal cavity, spleen, kidney, and bones. PAIR is contraindicated for inaccessible or superficially located liver cysts and for inactive or calcified cystic lesions. It is also contraindicated for cysts that communicate with the biliary tree, CE2, CE3b, CE4, CE5, and lung cysts. Complications of PAIR in- clude secondary infection of the cavity, acute allergic reactions, and recurrence; however, these have been rare. Albendazole should be administered before PAIR treatment and one month after treatment. During PAIR antihistamines should be given to reduce the risk of allergic reactions if there is spillage of fluid. Good results have been reported with this procedure with no major complications. A meta-​ analysis comparing the use of PAIR and surgical treatment for liver hydatid cysts found fewer complications and a shorter hospital stay in the PAIR-​treated group. CE2 and CE3b cysts that are not surgically resected require non-​ PAIR percutaneous treatment in combination with chemotherapy. These two types of cyst respond poorly to PAIR. The goal of the non-​ PAIR percutaneous treatment is the removal of the entire endocyst and all daughter cysts. This requires a large bore catheter or a cutting device with an aspiration apparatus. The long-​term outcomes are unclear but studies of medium term outcomes suggest that treat- ment is successful. As with PAIR, this treatment is usually accom- panied by chemotherapy. Watch and wait Uncomplicated asymptomatic inactive cysts of the liver can be left untreated and monitored regularly using imaging techniques. The rationale for this approach is based on the observation that up to 20% of cysts become spontaneously inactive and remain so over time. CE4 and CE5 stage cysts are managed with this approach. Prevention and control Control programmes have been aimed at educating dog owners to prevent their animals from having access to infected offal. This ap- proach includes periodic treatment (every 45 days) of sheepdogs with 5 mg/​k of praziquantel, reduction in the dog population, close veterinary inspection of slaughterhouse facilities for the presence of dogs, and cremation of infected offal. Control programmes are in force in Argentina, Chile, and Uruguay. Partial success has been achieved. Control programmes in Cyprus, the Falkland Islands, New Zealand, and Tasmania have reduced the number of infected animals and the incidence of human infection. Serological tests such as the western blot for diagnosis of sheep hydatidosis and the coproantigen enzyme-​linked immunosorbent assay (ELISA) for canine echinococcosis are potentially useful for measuring the burden of disease and monitoring control pro- grammes in endemic regions. A  recent major advance has been the development of a recombinant vaccine (EG95) which seems to confer 96–​98% protection against challenge infection with oncospheres. Recent trials in Australia and Argentina using this vac- cine have reported that 86% of immunized sheep were completely free of viable hydatid cysts when examined 1 year later. The number of viable cysts was reduced by 99.3%. Similarly, a vaccine against the dog tapeworm stage has been developed and conferred 97 to 100% protection against worm growth and egg production in immunized dogs. A recent field trial of the EG95 vaccine in Argentina found that

8.10.3 Cysticercosis 1533

8.10.3 Cysticercosis 1533

8.10.3  Cysticercosis 1533 the prevalence of hydatidosis in sheep decreased from 26% before the vaccine was introduced to 8% 3 years after the vaccine was intro- duced. Although the results of these initial trials seem promising, further research is needed to assess the cost benefit of using these vaccines. FURTHER READING Allan JC, et al. (1992). Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology, 104, 347–​55. Brunetti E, Junghanss T (2009). Update on cystic hydatid disease. Curr Opin Infect Dis, 22, 497–​502. Brunetti E, et al. (2010). Expert consensus for the diagnosis and treat- ment of cystic and alveolar echinococcosis in humans. Acta Trop, 114, 1–​16. Brunetti E, et al. (2011). Cystic echinococcosis: chronic, complex, and still neglected. PLoS Negl Trop Dis, 5, e1146. Budke CM, Deplazes P, Torgerson PR (2006). Global socioeconomic impact of cystic echinococcosis. Emerg Infect Dis, 12, 296–​303. Craig PS, et al. (2007). Prevention and control of cystic echinococcosis. Lancet Infect Dis, 7, 385–​94. Frider B, Larrieu E, Odriozola M (1999). Long-​term outcome of asymptomatic liver hydatidosis. J Hepatol, 30, 228–​31. Gavidia CM, et al. (2008). Diagnosis of cystic echinococcosis, central Peruvian Highlands. Emerg Infect Dis, 14, 260–​6. Larrieu E, et al. (2013). Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: early impact and preliminary data. Acta Trop, 127, 143–​51. McManus DP, Thompson RCA (2003). Molecular epidemiology of cystic echinococcosis. Parasitology, 127, S37–​51. Macpherson CNL, et al. (1987). Portable ultrasound scanner versus serology in screening for hydatid cysts in a nomadic population. Lancet, ii, 259–​91. Moro PL, et  al. (1997). Epidemiology of Echinococcus granulosus
infection in the Central Andes of Peru. Bull World Health Org, 75, 553–​61. Morris DL, Taylor DH (1988). Optimal timing of post-​operative albendazole prophylaxis in E. granulosus. Ann Trop Med Parasitol, 82, 65–​66. Schantz PM, Williams JF, Posse CR (1973). Epidemiology of hydatid disease in southern Argentina. Comparison of morbidity indices, evaluation of immunodiagnostic tests, and factors affecting trans- mission in southern Rio Negro Province. Am J Trop Med Hyg, 22, 629–​41. Smego RA, et  al. (2003). Percutaneous aspiration-​injection-​ reaspiration-​drainage plus albendazole or mebendazole for hepatic cystic echinococcosis: a meta-​analysis. Clin Infect Dis, 27, 1073–​83. Thompson RCA, McManus DP (2002). Towards a taxonomic revision of the genus Echinococcus. Trends Parasitol, 18, 452–​7. Verastegui M, et  al. (1992). Enzyme-​linked immunoelectrotransfer blot test for the diagnosis of human hydatid disease. J Clin Microbiol, 30, 1557–​61. WHO-​Informal Working Group on Echinococcosis (2003). PAIR: puncture, aspiration, injection, re-​aspiration. An option for the treatment of cystic echinococcosis. World Health Organization, Geneva. Zhang W, et al. (2006). Vaccination of dogs against Echinococcus gran­ ulosus, the cause of cystic hydatid disease in humans. J Infect Dis, 194, 966–​74. 8.10.3  Cysticercosis Hector H. Garcia and Robert H. Gilman ESSENTIALS Cysticercosis, infection by larvae of the pork tapeworm Taenia solium (see Chapter 8.10.1), is the most common helminthic in- fection of the human central nervous system. It accounts for up to 30% of all seizures and epilepsy in endemic countries, and travel and immigration now lead to its more frequent presentation in in- dustrialized countries. Ingestion of raw or undercooked pork can lead to infection with the T. solium cysticercus, formerly known as ‘Cysticercus cellulosae’, which is an encysted immature tape- worm. Once attached to the person’s small intestine, the head, or scolex, evaginates from the cysticercus, anchors in the intes- tinal mucosa and develops segments (proglottids) to become an adult tapeworm. Proglottids discharged in the faeces contain tens of thousands of ova that can autoinfect the human host or pigs and, rarely, other susceptible mammals. Ingestion of T. solium ova by the faecal-​oral route in those infected with adult tapeworms or their close contacts can result in development of cysticerci in various tissues, but not an adult tapeworm. The ingested ova re- lease embryos that penetrate the intestinal mucosa and migrate in the blood stream to the brain (causing neurocysticercosis), muscles, and subcutaneous tissues. Only by ingesting T. solium ova can humans develop cysticercosis. Clinical features and diagnosis—​manifestations of neurocysticercosis depend on the number, location, size, and stage of the parasite cysts in the brain, as well as on the immunological response of the host. The most common syndromes are late-​onset epilepsy and intra- cranial hypertension. Diagnosis is based on brain imaging studies (CT or MRI) and supported by highly specific serology. Treatment and prognosis—​treatment is (1) symptomatic (e.g. anti- convulsants); shunts for intracranial hypertension in patients with hydrocephalus; and (2) antiparasitic—​albendazole or praziquantel, which are generally given with steroids to control cerebral oedema; but there is no role for these drugs in inactive neurocysticercosis (i.e. calcifications with or without enhancement on CT scan). Prognosis depends mainly on whether the cysts are intraparenchymal (better prognosis) or extraparenchymal (subarachnoid or intraventricular, poorer prognosis). Introduction Known since the Hippocratic era, cysticercosis is the most common helminthic infection of the human central nervous system. It is probable that the suspicion of its origins led some religions expressly to forbid the consumption of pork. Socioeconomic improvements eradicated the infection in Europe and North America. However, endemic Taenia solium taeniasis/​cysticercosis persists in most developing countries, where human cysticercosis is an important cause of epilepsy and other neurological morbidity, and porcine in- fections cause considerable economic losses to peasant farmers.

section 8  Infectious diseases 1534 Aetiology Cysticercosis is infection with the larval stage (cysticercus) of T. solium, the pork tapeworm (Chapter 8.10.1). In the life cycle of this two-​host zoonotic cestode (Fig. 8.10.3.1), humans are the only definitive host and harbour the adult tapeworm by ingesting cysts in infected pork, whereas pigs are intermediate hosts by ingesting eggs in human stools. The hermaphroditic adult T. solium inhabits the human small intestine. Its head, or scolex, bears four suckers and a double crown of hooks, connected by a narrow neck to a large body (strobila) between 2 and 4 m long, composed of several hundred proglottids (Chapter 8.10.1, Fig. 8.10.1.1b, c). Gravid proglottids, each containing 50 000–​60 000 fertile eggs, detach from the distal end of the worm and are excreted in the faeces. The cycle is com- pleted when pigs ingest stools contaminated with T. solium eggs. Once ingested by the pig, the invasive oncospheres in the eggs are liberated by the action of gastric acid and intestinal fluids and ac- tively penetrate the bowel wall, enter the bloodstream, and are car- ried to the muscles and other tissues where they develop into larval cysts (Chapter 8.10.1, and see Fig. 8.10.3.1). When humans ingest undercooked pork containing cysticerci, the larva evaginates in the small intestine, its scolex attaches to the intestinal mucosa, and it begins forming proglottids. By accidentally ingesting taenia eggs, humans can also act as intermediate hosts for T. solium and develop cysticercosis. Epidemiology The availability of neuroimaging studies and the subsequent devel- opment of specific serodiagnostic tests have resulted in the identi- fication of neurocysticercosis as a frequent neurological disorder in Latin America, Africa, and Asia, where the prevalence of active epilepsy is almost twice that in Western countries. Cysticercosis was introduced from Bali to the highlands of Papua, Indonesia nearly 40 years ago. Its seroprevalence is more than 20% in many communities. Neurocysticercosis is also an emerging problem in industrialized countries, seen mainly in immigrants from endemic areas, some of whom may spread the infection as tapeworm carriers. This applies to California and other southern areas of United States of America bordering Mexico. The main sources of human cysticercosis are faecal-​oral contam- ination in those carrying the tapeworm, or their contacts, and in- gestion of food contaminated with T. solium eggs. Epidemiological studies suggest that almost every newly diagnosed patient with cysticercosis has been infected by someone in their close environ- ment who is harbouring a T. solium and the tendency is to dismiss the role of environment or water in transmission. Airborne trans- mission of T. solium eggs and internal autoinfection by regurgitation of proglottids into the stomach have been suggested but not proved. Pathogenesis Any organ can be infected, but parasites survive more frequently in the nervous system, possibly because the reduced immune response. Signs and symptoms are caused by perilesional inflammation and oe- dema, mass effect, or obstruction of cerebrospinal fluid circulation. Although complete development of cysts takes 2 to 3 months, symp- toms usually develop years after the initial infection. This clinically silent period, and finding inflammation around cysts in symptomatic cases, suggests that in many cases symptoms are due to inflammatory processes associated with the recognition of the parasite by the im- mune system of the host (presumably progressing towards the death of the parasite) rather than to the presence of the parasite itself. Subarachnoid cysticerci elicit an intense inflammatory reaction causing thickening of basal leptomeninges. The optic chiasma and other cranial nerves are usually entrapped within this dense exudate, resulting in visual field defects and other cranial nerve abnormal- ities. The foramina of Luschka and Magendie can be occluded by the thickened leptomeninges, leading to hydrocephalus. Blood vessels can also be affected by the inflammatory reaction. The walls of small penetrating arteries are invaded by inflammatory cells, leading to a proliferative endarteritis with occlusion of the lumen, and this can result in cerebral infarction. Clinical features Neurocysticercosis is a pleomorphic disease, whose manifestations vary with the number, size, and topography of the lesions and the intensity of the host’s immune response to the parasites. Patients can be classified by the number, stage, and location of the cysticerci, and the presence or absence of associated inflammation or calcifications. Epilepsy, the most common presentation of neurocysticercosis, is usually the primary or sole manifestation of the disease. Seizures occur in 50–​80% of patients with parenchymal brain cysts or calci- fications but are less common in other forms of the disease. Other focal signs are less frequent and include pyramidal tract signs, sen- sory deficits, signs of brainstem dysfunction, and involuntary move- ments. These manifestations usually follow a subacute or chronic course, making neurocysticercosis difficult to differentiate clinically from neoplasms or other infections of the central nervous system. Focal signs can occur abruptly in patients who develop a cere- bral infarct as a complication of subarachnoid neurocysticercosis. Human (definitive host) Pig (intermediate host) Ingestion of infected pork, poorly cooked: taeniasis Ingestion of T. solium eggs by faecal contamination: human cysticercosis Ingestion of T. solium eggs or proglottids: porcine cysticercosis Fig. 8.10.3.1  Life cycle of T. solium.

8.10.3  Cysticercosis 1535 Subarachnoid cysticerci can reach 10 cm or more in diameter (‘giant’ cysticercosis, Fig. 8.10.3.2), and exert a mass effect. Neurocysticercosis may present with increased intracranial pressure, usually from hydrocephalus secondary to basal sub- arachnoid cysticercosis or intraventricular cysts, cysticercotic arachnoiditis, or granular ependymitis. In these cases, intracra- nial hypertension develops subacutely and progresses slowly. An encephalitic picture can result from overwhelming inflammation around many parasitic cysts, a syndrome that occurs more fre- quently in younger people, especially women. In contrast, some patients tolerate hundreds of intraparenchymal cysticerci with only minor symptoms. Ocular cysticercosis can involve the posterior segment, retina, vit- reous, subconjunctiva, orbit or eyelid (Fig. 8.10.3.3). Muscular pseudohypertrophy, a rare presentation, is caused by heavy cysticercal infection of skeletal muscles (Fig. 8.10.3.4) giving a ‘Herculean’ appearance. The few cases reported are come most frequently from India. Other apparent differences in clinical manifestations between Asia and Latin America include a high frequency of subcutaneous cysts and single degenerating brain lesions in Asia. Pathology The cysticerci are liquid-​filled vesicles consisting of vesicular wall and scolex (Fig. 8.10.3.5). The vesicular wall is composed of an outer, or cuticular, layer, a middle, or cellular, layer with pseudoepithelial structure, and an inner, or reticular, layer. The invaginated scolex has a head, or rostellum, armed with suckers and hooks, and a rudimentary body, or strobila, that includes the spiral canal. The macroscopic appearance of cysticerci varies in different lo- cations within the central nervous system. Cysticerci within the brain parenchyma are usually small and tend to lodge in the cere- bral cortex or basal ganglia (Fig. 8.10.3.6). Subarachnoid cysts might be small if located in the depths of cortical sulci, or grow to 5 cm or more in the basal cisterns or Sylvian fissures. Ventricular cysticerci are usually single, might or might not have a visible scolex, and might be attached to the choroid plexus or float freely in the ventricle. Spinal cysticerci are usually located in the sub- arachnoid space (rarely intramedullary). Here they can develop areas of arachnoiditis. Basal subarachnoid cysticerci can undergo a disproportionate growth of their membrane, with extension processes, resembling Fig. 8.10.3.2  Giant cysticercotic cyst (brain CT). Fig. 8.10.3.3  Intraocular cysticercosis: cysticercus in the anterior chamber of a Thai patient. Courtesy of the late Professor Sornchai Looareesuwan. Fig. 8.10.3.4  Heavy cysticercal infection of skeletal muscles. Courtesy of the late Professor Sornchai Looareesuwan.

section 8  Infectious diseases 1536 5 mm (a) (c) (e) (b) (d) Gd-enh Fig. 8.10.3.5  (a) Histopathology of a complete cysticercus removed by brain biopsy in a patient with recent onset of focal epilepsy (×4). (b) Structure of the cyst wall (×40). (c) Cerebral imaging CT enhanced. (d) MRI T2-​weighted. (e) MRI T1-​weighted with and without gadolinium enhancement. Copyright D. A. Warrell.

8.10.3  Cysticercosis 1537 a brunch of grapes (racemose cysticercosis, Fig. 8.10.3.7). In these cases, the scolex is frequently unidentifiable even by microscopy. Viable vesicular cysticerci elicit little inflammatory change in sur- rounding tissues because of active immune evasion mechanisms. The appearance of symptoms is interpreted as the result of immuno- logical attack from the host, in a process of degeneration that ends with the death of the parasite. Inflammatory changes in the parasite membrane and increased density of cyst fluid mark the transition between four defined stages: viable, colloidal, granular nodular, and calcified cyst. Viable cysts may coexist with degenerating cysts or calcifications. Laboratory/​imaging diagnosis The pleomorphism of neurocysticercosis makes it impossible to diagnose on clinical grounds alone. In endemic regions, late-​onset seizures in otherwise healthy individuals are highly suggestive of neurocysticercosis. Most of these patients are normal on neuro- logical examination. Routine neuroimaging and serological studies are, therefore, mandatory. Finding cysticerci outside the central ner- vous system (eye, subcutaneous tissue, muscle) assists the diagnosis of neurocysticercosis. Muscular and subcutaneous cysticerci are far less common in American than in African or Asian patients with neurocysticercosis. Neuroimaging CT and MRI have markedly improved diagnostic accuracy by providing objective evidence about the topography of the lesions and the degree of the host inflammatory response to the parasite. Imaging findings in parenchymal neurocysticercosis depend on the stage of involution of cysticerci. Viable cysticerci appear as rounded cystic lesions on CT (Fig. 8.10.3.2), hypointense on T1 and FLAIR sequences on MRI (Fig. 8.10.3.6), without associated enhance- ment, whereas degenerating parasites are seen as focal enhancing lesions surrounded by oedema (Fig. 8.10.3.4c–​e), and calcifications as hyperdense dots or nodules (Fig. 8.10.3.8). Disappearance of cyst fluid signals the degenerative phase and calcified nodules the re- sidual phase. Single or multiple ring-​like or nodular enhancing le- sions are non​specific and present a diagnostic challenge. Pyogenic brain abscesses, fungal abscesses, tuberculomas, toxoplasma ab- scesses, and primary or metastatic brain tumours may produce similar findings on CT or MRI. CT and MRI findings in subarachnoid neurocysticercosis are less specific. They include hydrocephalus, abnormal meningeal enhancement, and subarachnoid cysts. Cerebral angiography can show segmental narrowing or occlusion of major intracranial Fig. 8.10.3.6  Uncontrasted T1 MR image showing two intraparenchymal cysticerci with visible scolices. Fig. 8.10.3.7  Basal ‘racemose’ cysticercosis. Fig. 8.10.3.8  Calcified neurocysticercosis.

section 8  Infectious diseases 1538 arteries in patients with cerebral infarcts secondary to parasitic vasculitis. In neurocysticercosis there is rarely fever or signs of meningeal irritation; glucose levels in cerebrospinal fluid are usu- ally normal. MRI is generally better than CT for the diagnosis of neurocysticercosis, particularly in patients with basal lesions, brain- stem or intraventricular cysts, and spinal lesions. MRI is, however, less sensitive than CT for the detection of calcifications. Immunological tests Immunoblot (Western blot) using lentil-​lectin purified parasite glycoprotein antigens is the best available serological test for T. solium antibodies. It performs well with serum samples and is 98% sensitive in cases with more than one active lesion, and 100% specific. Its sensitivity may drop in patients with a single cyst. Other assays using unfractionated antigens (e.g. enzyme immunoassay, ELISA) suffer from poor specificity but are more reliable when performed with cerebrospinal fluid than serum. Antigen-​detection tests can provide a tool for serological moni- toring of antiparasitic therapy. Although results of serology and imaging studies might be similar, they evaluate different aspects of the disease and can be discordant in some patients. Intestinal tapeworm carriers, naturally cured patients, or non​neurological infections can have normal brain images but be positive serologic- ally. Those with only inactive lesions or a single cerebral lesion might be seronegative. Parasitological diagnosis A proportion (c.10–​15%) of patients with neurocysticercosis are tapeworm carriers at the time of diagnosis, and in another 10% or so a carrier can be detected in the household. Parasitological diagnosis is difficult: eggs and proglottids are shed only intermittently in stool and are frequently missed by routine stool examination. Stool as- says to detect parasite antigens are more sensitive than microscopy, but are not widely available. A recently described serological test for tapeworm carriers might improve detection. Diagnostic criteria A set of diagnostic criteria based on neuroimaging studies, sero- logical tests, clinical presentation, and exposure history has been proposed by Del Brutto and colleagues. Besides absolute demon- stration of the presence of the parasite, ‘major’ criteria (including typical findings on neuroimaging, demonstration of specific anticysticercal antibodies, or the presence of typical cigar-​shaped calcifications in muscle) are combined with ‘minor’ criteria and epidemiological data to suggest a probable or possible diagnosis. Application of these criteria should improve the consistency of diagnosis. Treatment Because of the clinical and pathological pleomorphism of neurocysticercosis, precise assessment of the viability and size of cysts, the location of parasites, and the severity of the host’s immune response is important before planning treatment. Symptomatic treatment is very important. Seizures secondary to parenchymal neurocysticercosis can usually be controlled with anticonvulsants. However, the optimal duration of anticonvulsant therapy in patients with neurocysticercosis has not been deter- mined, and it is difficult to withdraw this treatment. Prognostic factors associated with recurrence of seizures include the develop- ment of parenchymal brain calcifications, and occurrence of recur- rent seizures or multiple brain cysts before starting antiparasitic therapy. Antiparasitic agents destroy viable cysts and are associated with fewer seizures in the long-​term follow-​up. Antiparasitic treat- ment in patients with a single enhancing lesion seem to improve radiological resolution and decrease the chance of seizure relapses, albeit the magnitude of this effect is small. Albendazole is the drug of choice for antiparasitic treatment of cerebral cysticercosis (15 mg/​kg per day for 7 to 15 days, with steroids), although a re- cently described single-​day praziquantel regimen (75–​100 mg/​ kg, in three doses at 2-​h intervals, followed by steroids 6 h later) demonstrated similar cestocidal activity in patients with few cysts. The combination of albendazole with praziquantel is more ef- fective in patients with multiple parenchymal cysts. Longer courses may be required in patients with many lesions or subarachnoid cysticercosis. Transient worsening of neurological symptoms can be expected during antiparasitc therapy, secondary to the perilesional inflammatory reaction. There is no role for antiparasitic drugs in inactive neurocysticercosis (i.e. calcifications with or without en- hancement on CT scan) since the parasites are dead. Between the second and fifth day of antiparasitic therapy there is usually an exacerbation of neurological symptoms, attributed to local inflammation caused by the death of the larvae. For this reason, albendazole or praziquantel are generally given simultan- eously with steroids in order to control the oedema and intracranial hypertension. Serum levels of praziquantel decrease when steroids are administered simultaneously, an effect that does not occur with albendazole. However, there is no evidence that cysticidal efficacy is decreased. Serum levels of praziquantel or albendazole might be lowered by simultaneous antiepileptic drug (phenytoin or carba- mazepine) administration. Some forms of neurocysticercosis should not be treated with antiparasitic agents. In patients with severe cysticercotic encephal- itis, these drugs may result in worsening cerebral oedema and fatal herniation. In this case, the mainstay of therapy is high doses of corticosteroids or mannitol to decrease the inflammatory response. In patients with both hydrocephalus and parenchymal brain cysts, antiparasitic drugs should be started only after placement of a ven- tricular shunt in case the intracranial pressure increases as a result of drug therapy. Antiparasitic drugs must be used with caution in patients with giant subarachnoid cysticerci. In such patients, con- comitant steroid administration is mandatory to avoid cerebral in- farction. Albendazole can successfully destroy ventricular cysts, but the surrounding inflammatory reaction can cause acute hydroceph- alus if the cysts are located within the fourth ventricle or near the foramina of Monro and Luschka. Surgery is limited to ventriculoperitoneal shunts to relieve ob- structive hydrocephalus, and excision of single cysts (in the fourth ventricle or giant intraparenchymal cysts). However, shunts fre- quently malfunction. The protracted course in these patients and their high mortality rates (up to 50% in 2 years) is directly related to the number of surgical interventions required to change the shunts. Recently, neuroventriculoscopy has been employed as a less invasive option for resection of ventricular cysticerci.

8.10.3  Cysticercosis 1539 Prognosis Parenchymal cysticercosis has a good prognosis. Appropriately managed, seizures usually subside in time without sequelae. In contrast, extraparenchymal cysticercosis, and especially ra- cemose cysticercosis, has a poor prognosis, responding poorly to antiparasitic therapy, and leading to progressively deteriorating disease and death. Multiple courses of antiparasitic treatment and careful, prolonged follow-​up are crucial in this type of patients. Prevention and control Cysticercosis would not exist if pigs had no access to human faeces. However, this approach is hampered in endemic zones by the lack of sanitary facilities and veterinary inspection, and more importantly, because farmers tend to raise pigs under free-​range conditions in order to reduce the cost of feeding them. Intervention programmes have concentrated on mass chemotherapy to eliminate human taeniasis, but their results have not been sustained. New tools for control are oxfendazole, an effective and cheap single-​dose therapy for porcine cysticercosis, and the candidate porcine vaccines under trial by several groups. TSOL18, an oncosphere-​based vaccine de- veloped in Australia, may provide over 99% protection. A recent wide-​scale elimination program in Peru has provided initial evi- dence on the feasibility of focal elimination. Monitoring the effect of an intervention requires suitable indica- tors. Human seroprevalence does not reflect changes in infection patterns because antibodies persist for years, even after successful treatment. Similarly, symptoms can appear years after infection. Since the prevalences of human and porcine infection are strongly correlated, pigs are likely a better indicator for recent transmission. Possible future developments Although most cysts disappear after antiparasitic treatment, the antiparasitic efficacy of currently available regimes is incomplete. Data are missing on whether new drugs, combination therapy, or different schemes of albendazole or praziquantel can improve this efficacy. Schemes and doses of antiparasitic and steroid therapy need to be assessed in controlled trials targeted to specific types of neurocysticercosis. Systematic long-​term evaluation is needed to determine the impact of parasite destruction in seizure relapses in the short and long term, particularly considering the association between neurocysticercosis and mesial temporal sclerosis. The ef- ficacy and costs of comprehensive human–​porcine eradication pro- grammes must be assessed. FURTHER READING Del Brutto OH, et  al. (2001). Proposed diagnostic criteria for neurocysticercosis. Neurology, 57, 177–​83. Del Brutto OH, et al. (2006). Albendazole and praziquantel therapy for neurocysticercosis: a meta-​analysis of randomized trials. Ann Intern Med, 145, 43–​51. Evans C, et al. (1997). Controversies in the management of cysticercosis. Emerg Infect Dis, 3, 403–​5. Garcia HH, et  al. (2003). Taenia solium cysticercosis. Lancet, 362, 547–​56. Garcia HH, et al. (2004). A trial of anti-​parasitic treatment to reduce the rate of seizures due to cerebral cysticercosis. N Engl J Med, 350, 249–​58. Garcia HH, et al. (2014). Clinical symptoms, diagnosis, and treatment of neurocysticercosis. Lancet Neurol, 13, 1202–​15. Garcia HH, et al. (2014). Efficacy of combined antiparasitic therapy with praziquantel and albendazole for neurocysticercosis: a double-​blind, randomised controlled trial. Lancet Infect Dis, 14, 687–​95. Gonzalez AE, et al. (1997). Treatment of porcine cysticercosis with oxfendazole: a dose–​response trial. Vet Record, 141, 420–​2. Gonzalez AE, et  al. (2005). Vaccination of pigs to control human neurocysticercosis. Am J Trop Med Hyg, 72, 837–​9. Montano SM, et al. (2005). Neurocysticercosis: association between seizures, serology and brain CT in rural Peru. Neurology, 65, 229–​33. Nash TE, et al. (2006). Treatment of neurocysticercosis—​current status and future research needs. Neurology, 67, 1120–​7. Otte WM, et al. (2013). Drug therapy for solitary cysticercus granu- loma:  a systematic review and meta-​analysis. Neurology, 80,
152–​62. Salim L, et  al. (2009). Seroepidemiologic survey of cysticercosis-​ taeniasis in four central highland districts of Papua, Indonesia. Am J Trop Med Hyg, 80, 384–​8. Wender JD, et  al. (2011). Intraocular cysticercosis:  case series and comprehensive review of the literature. Ocul Immunol Inflamm, 19, 240–​5.

8.11 Trematodes (flukes) 1540

8.11 Trematodes (flukes) 1540

8.11.1 Schistosomiasis 1540

8.11.1 Schistosomiasis 1540

8.11 Trematodes (flukes) CONTENTS 8.11.1 Schistosomiasis  1540  David Dunne and Birgitte Vennervald 8.11.2 Liver fluke infections  1551  Ross H. Andrews, Narong Khuntikeo, Paiboon Sithithaworn,
and Trevor N. Petney 8.11.3 Lung flukes (paragonimiasis)  1558  Udomsak Silachamroon and Sirivan Vanijanonta 8.11.4 Intestinal trematode infections  1562  Alastair McGregor 8.11.1  Schistosomiasis David Dunne and Birgitte Vennervald ESSENTIALS Schistosomiasis is caused by trematode worms Schistosoma spp., whose life cycle requires a definitive vertebrate host and an inter- mediate freshwater snail host. Transmission to humans occurs through exposure to fresh water containing infectious larvae, which can penetrate intact skin before developing into blood-​dwelling adult worms. The disease is patchily distributed in parts of South America, Africa, the Middle East, China, and Southeast Asia, with about 200 million people infected and 20 million suffering severe consequences of infection. Clinical features Most infected people living in endemic areas have few (if any) overt symptoms, but clinical manifestations (when present) depend on the stage of infection. Larval invasion causes a transient immediate hypersensitivity reaction with intense itching (‘swimmer’s itch’) and rash (cercarial dermatitis). Early primary infection can cause a severe systemic reaction (acute schistosomiasis or Katayama fever) with fever, con- stitutional symptoms and (almost invariably) eosinophilia. Established infection can cause (1)  Urinary schistosomiasis (Schistosoma haematobium)—​active disease most commonly presents with painless, terminal haematuria; chronic disease is as- sociated with calcification, ulceration, and the development of papillomas in the bladder, and with ureteric fibrosis. (2) Intestinal schistosomiasis (S.  mansoni and S.  japonicum)—​clinical features include diarrhoea, hepatomegaly, and splenomegaly. (3)  Other manifestations—​these include (a)  nervous system—​myelopathy and radiculopathy; (b) lungs—​pulmonary hypertension and/​or cor pulmonale; (c) renal—​glomerulonephritis. Diagnosis Should be suspected with a history of exposure to potentially con- taminated water. Definitive diagnosis depends on direct microscopic detection of eggs in urine or stool samples, or in tissue biopsies. Antigen detection and polymerase chain reaction-​based tests can be useful in some circumstances. Treatment and prognosis Praziquantel is the drug of choice, with corticosteroids added in cases of Katayama fever. Acute schistosomiasis responds well, but chronic disease less so, but rapid re-​exposure and reinfection are common (particularly in young children) unless control measures are implemented at the community level. Prevention In areas of high transmission, population-​based chemotherapy or treatment of schoolchildren (who contribute most to ongoing trans- mission). Health education should be aimed at improving practices of water use and preventing indiscriminate urination and defecation. Introduction Schistosomiasis, also known as bilharzia, is caused by infection with parasitic trematode worms (flukes) of the genus Schistosoma. Disease is usually associated with acute or chronic infections con- tracted by exposure to fresh water containing infective cercarial larvae that penetrate intact skin and develop into blood-​dwelling worms. Most human infections are caused by one of three species, S. mansoni, S. haematobium, or S. japonicum. Two species, S. inter- calatum and S.  mekongi, are less significant. Schistosomiasis is patchily distributed in parts of South America, Africa, the Middle East, China, and South-​East Asia (Fig. 8.11.1.1). An estimated 800  million people are at risk of schistosomiasis worldwide, of whom at least 200 million are infected.

8.11.1  Schistosomiasis 1541 Diagnosis and treatment are often not available to exposed rural populations, and drug-​based control programmes are ham- pered by the continued susceptibility to reinfection of those who have been treated, particularly children. Human schisto- somiasis is most often an insidious and chronic disease with a range of pathological manifestations involving the intestine and liver, or the urogenital tract. Mortality estimates are difficult, but 20 000–​200 000 deaths might be directly associated with schisto- somiasis each year. Parasite life cycle The schistosome life cycle requires two host species: a definitive ver- tebrate host, in which adult male and female worms develop and sexual reproduction occurs, and an intermediate freshwater snail host, in which the parasite multiplies asexually. Transmission be- tween these hosts is achieved by two different free-​swimming larval stages. For species that infect humans, miracidia hatch from eggs excreted in the faeces or urine, and then seek out and infect snails. Cercariae are released from the snail and are able actively to pene- trate intact human skin. Snails Different schistosome species have their own, often very restricted, range of snail hosts. Schistosomiasis is thus closely associated with particular freshwater habitats, and its geographical distribution is restricted by the availability of particular snail species. S. man- soni and S.  haematobium are confined to aquatic snails (genera Biomphalaria and Bulinus, respectively) that inhabit ponds, lakes, irrigation canals, slow-​flowing streams, and rivers. S.  japonicum
is transmitted by amphibious snails of the genus Oncomelania that, in addition to a variety of freshwater habitats, are also present in damp soil and vegetation, such as paddy fields. Cercariae Once shed from freshwater snails, cercariae (Fig. 8.11.1.2) live for about 24 h, but their effective period of infectivity is probably shorter under field conditions. Cercarial behaviour and the timing of their release enhance their chance of contacting their vertebrate host of choice. Increasing temperature and light trigger the release of S. mansoni and S. haematobium cercariae during the day, and they use their tails actively to maintain their position near the water sur- face. S. japonicum cercariae are shed late in the day and are closely associated with the meniscus, perhaps reflecting their wider host range, as species specific for rodents are shed at night. Skin penetration, transformation,
and migration Contact with skin triggers adherence mechanisms, and proteolytic enzymes and muscular movements allow penetration of the skin in minutes. Penetration initiates transformation into a schistosomular Fig. 8.11.1.1  Global distribution of the schistosomes that affect humans. Fig. 8.11.1.2  The infective larva (cerceria) of Schistosoma mansoni, length approximately 200 µm. The head region has characteristic suckers; the muscular forked tail propels the free-​swimming larva, but is discarded during skin penetration. This larva will develop into an adult worm in a human host.

section 8  Infectious diseases 1542 larva, with loss of the tail and of the protective outer glycocalyx layer, and the addition of an extra lipid bilayer to the surface membrane of the parasite’s syncytial outer tegument. This tegument now forms the main parasite–​host interface and so has physiological and im- munological functions vital to long-​term survival in the mamma- lian bloodstream. These include uptake of nutrients, response to injury, and surface adsorption of host antigens to provide an im- munological disguise. Newly transformed schistosomula remain in the epidermis for several days before migrating, via the blood- stream, lungs, and systemic circulation, to the hepatic portal system. Here the schistosomula mature and differentiate into adult worms, pair, and migrate against the portal blood flow to the small venules draining the genitourinary tract (S. haematobium) or the large and, to a lesser extent, small intestine (S. mansoni, S. japonicum, S. inter- calatum, S. mekongi). Adult schistosomes Male and female worms are 1 to 2 cm long and morphologically dis- tinct. Paired worms remain permanently coupled, with the shorter, flatter, more muscular male gripping the female in its gynaecophoric canal (Fig. 8.11.1.3). Worms ingest blood cells into their blind-​ ending bifurcated gut, producing a haematin-​like pigment that is regurgitated into the blood. Adult worms have average lifespans in humans of 3 years (S. haematobium) to 7 years (S. mansoni), al- though active infections are reported in individuals who have left endemic areas more than 20 years previously. Schistosme eggs and miracidiae Female worms start to produce eggs between 5 and 12 weeks after infection, at rates of 300 (S. mansoni) to 3000 (S. japonicum) per day. A few days after an egg is laid, a single miracidium develops within the rigid eggshell, the shape and size of which is character- istic for each species. S. mansoni (Fig. 8.11.1.4) and S. haemato- bium eggs are ellipsoid, 65 × 150 µm, the former having a lateral spine and the latter a terminal spine. S. japonicum eggs are more spherical, 70 × 90 µm, with a small lateral knob that is not always apparent microscopically. Embryonated eggs pass from the venules into the gut or bladder lumen. This is facilitated by host immune responses to secreted egg antigens, as egg excretion is inhibited in immunosuppressed experimental hosts and HIV infected in- dividuals. The passage of the eggs causes tissue damage, as does the granulomatous reactions to eggs that fail to escape from the bloodstream and get swept into the liver by the portal blood flow. Eggs deposited in fresh water rapidly hatch in response to osmotic changes, releasing the miracidium. This ciliated and actively swim- ming larva lives for about 6 h, and can chemically detect the prox- imity of snails, modifying its swimming behaviour as it approaches a potential host. The parasite actively penetrates the snail’s tissues and transforms into a primary sporocyst. Asexual replication gives rise to daughter sporocysts that migrate to the snail’s hepatopan- creas where cercariae are asexually generated within each sporo- cyst. Thus, snails infected with a single miracidium release cercariae that are all of the same sex. Cercariae are first released from snails 3–​6 weeks after infection, depending on parasite species and am- bient temperature. Infected snails can shed hundreds of cercariae daily over several months. Epidemiology Distribution Schistosomiasis is associated with poor living conditions and inad- equate sanitation and water supply. Its distribution has changed over the last 50 years. In some areas sustained control strategies have been successful. However, environmental and climatic changes, develop- ment of water resources, population increases, and migration, have led to its spread into previously non​endemic areas or areas with a low rate of infection. S. japonicum and S. haematobium have decreased, whereas S. mansoni has increased to become the most prevalent and widespread species. S. japonicum has been controlled effectively in many areas and is now endemic only in China, where it is much re- duced, Indonesia, the Philippines, and Thailand. S. mekongi is found in Cambodia and Laos, and S. intercalatum is found in 10 countries within the rainforest belt of central Africa. S. mansoni is present in most countries of sub-​Saharan Africa, and in Madagascar, the Nile Delta and valley, as well as Saudi Arabia, Yemen, Oman, Libya, nor- thern and eastern Brazil, Suriname, Venezuela, and some Caribbean islands. S. haematobium is widespread in sub-​Saharan Africa and Fig. 8.11.1.3  Adult worms of S. mansoni. The shorter male encloses the female in its gynaecophoric canal, the characteristic haematin-​like pigment can be seen in the female worm’s gut. Fig. 8.11.1.4  Egg of S. mansoni containing a fully developed miracidium and showing the characteristic lateral spine of this species.

8.11.1  Schistosomiasis 1543 Madagascar, and is more prevalent than S. mansoni in North Africa and the Middle East. Transmission and epidemiology Each successful cercarial penetration of human skin has the po- tential to give rise to a single male or female adult worm, but it is probable that many cercariae die naturally in the epidermis. People tend to accumulate worms with continued exposure to infection. However, human populations in endemic areas do not just continue to accumulate worms with age. Intensities of infec- tion increase in children during their younger years (as estimated by numbers of excreted eggs), peaking around the age of 12 years, before falling to lower levels in adulthood (Fig. 8.11.1.5a). This is probably due to the death of older worms, which are not re- placed at a similar rate in older people. This age–​infection in- tensity profile is more pronounced if study populations are given chemotherapy to remove existing infections and then monitored for levels of reinfection over several subsequent years. In these circumstances, it is clear that young children are much more susceptible to reinfection than older children or adults, and that a striking change in susceptibility to reinfection occurs after 12 years of age. Age-​dependent resistance to infection The slower acquisition of worms in adulthood could be due to re- duced exposure to infection or to age-​dependent changes in innate resistance or acquired immunity. In many endemic areas children have more contact with water than adults, but careful observation of water-​associated behaviour has shown that age profiles of water contact are variable between communities, whereas profiles of re- infection intensities are remarkably consistent (Fig.  8.11.1.5b). This suggests that host-​related factors other than exposure influ- ence susceptibility to reinfection. This has been most convincingly shown in fishing communities in areas with high S. mansoni trans- mission on Lake Albert, Uganda. Here occupational water contact results in adults having greater exposure to infection than their children, yet, within 12 months of treatment, it is the children under 12 years of age that suffer much higher reinfection inten- sities. Current research is focused on assessing the relative roles of innate and acquired immunity in this age-​dependent resistance and whether the onset of puberty or the length of time spent living in endemic areas might be important. For example, it is not known if this age-​dependent resistance to infection holds true for travel- lers exposed to infection for the first time. Immune responses to schistosomes also differ between children and adults. Specific IgE and other characteristically Th2-​type responses against the para- site are associated with resistance to reinfection. Whatever mech- anisms underlie the contrasting susceptibilities of children and adults, continued exposure can be expected to result in reinfection, especially among younger children. Pathogenesis Schistosome eggs can be trapped in the tissues, often the walls of the intestines or, depending on species, the urinary bladder, ureters, and genital organs where they may be seen in cone biop- sies of the uterine cervix. The eggs of S. mansoni and S. japonicum are swept into the liver via the portal system, where they embolize into the portal radicles and give rise to vascular and granuloma- tous changes (Fig. 8.11.1.6). Granulomatous pyelophlebitis and peripyelophlebitis is responsible for development of portal hyper- tension, while granulomata with subsequent fibrosis may be re- sponsible for the periportal fibrosis. The characteristic lesion in the liver is a presinusoidal periportal fibrosis (Symmers’ fibrosis, Fig. 8.11.1.7). There is typically no bridging between the fibrous tracts, no nodule formation, and no hepatic cell damage. Increased portal pressure can result in the development of portosystemic 350 (a) 300 250 200 150 100 50 0 Geometric mean eggs per gram faeces (b) 12 10 6 4 2 Scaled geometric mean eggs per gram faeces 8 0 0 10 20 30 40 50 60 Mean age (years) 0 10 20 30 40 50 60 Mean age (years) Fig. 8.11.1.5  (a) Age–​intensity profiles of S. mansoni infection
from six communities in Kenya. (b) Age–​reinfection intensity
profiles of S. mansoni after chemotherapy in the same six communities in Kenya, assessed between 12 and 36 months after treatment. (a) From Fulford, AJ, et al. (1992). On the use of age-​intensity data to detect immunity to parasitic infections, with special reference to Schistosoma mansoni
in Kenya. Parasitology 105: 219–​227, reproduced with permission.

section 8  Infectious diseases 1544 collaterals and eggs can then pass directly from the portal vein to the pulmonary circulation (Fig. 8.11.1.8). Here the combin- ation of vascular and granulomatous changes is responsible for pulmonary hypertension. Clinical features Stage of invasion: Cercarial dermatitis
or ‘swimmer’s itch’ When cercariae penetrate the skin, they can cause a skin reaction, called cercarial dermatitis or ‘swimmer’s itch’. This is frequently seen after exposure to avian schistosomes, and is associated with the death of cercariae in the skin. It is seen both in areas endemic for human schistosomiasis and in non​endemic areas. In people exposed for the first time, the invasion causes a transient immediate hyper- sensitivity reaction with intense itching. Within 12 to 24 h, it is fol- lowed by a delayed reaction characterized by a small, red, pruritic, macular rash progressing to papules after 24 h. The rash can per- sist for up to 15 days and residual pigmentation might persist for months. Following repeated exposure, the signs and symptoms in- crease dramatically and start earlier. A similar reaction can be seen after re-​exposure to human cercariae, predominantly S.  mansoni and S. japonicum. Treatment, if needed, is symptomatic. Stage of maturation: Acute schistosomiasis
or Katayama fever The early stages of a primary infection can be associated with a severe systemic reaction that resembles serum sickness. This acute illness, called acute toxaemic schistosomiasis or Katayama fever, can occur following initial infection with any schistosome infecting humans, although it is more common in S. japonicum and S. mansoni infections. Acute schistosomiasis is most marked in primary infections in non​immune adults, but acute S. japoni- cum infection can occur in re-​exposed individuals. Symptoms appear 2 to 6 weeks after exposure. The clinical picture resembles an acute pyrexial illness with fever as a prime characteristic. The patient feels ill, and may have rigors, sweating, headache, mal- aise, muscular aches, profound weakness, weight loss, and a non​ productive irritating cough. Anorexia, nausea, abdominal pain, and diarrhoea can occur. Physical examination may reveal a generalized lymphadenopathy, an enlarged tender liver, and, sometimes, a slightly enlarged spleen Fig. 8.11.1.6  Schistosomal granuloma in the appendix. (a) (b) Fig. 8.11.1.7  The liver in S. mansoni infection in South Africa. Symmers’ clay pipe stem fibrosis: (a) macroscopic views; (b) microscopic view. Copyright Gareth Turner. Fig. 8.11.1.8  Schistosomal granuloma in the lung. Copyright Gareth Turner.

8.11.1  Schistosomiasis 1545 and an urticarial rash (Fig. 8.11.1.9). Eosinophilia is almost always present. Patients might become confused or stuporose or present with visual impairment or papilloedema. Severe cerebral or spinal cord manifestations may occur, and this is an indication for urgent investigative measures. Even light infections may cause severe illness and the syndrome can, in rare cases, be fatal. Differential diagnoses include infections such as typhoid (leuco- penia, no eosinophilia), brucellosis, malaria, infectious mononucle- osis, miliary tuberculosis, leptospirosis, and other conditions with fever of unknown origin. Fever and eosinophilia occur in trichin- osis, tropical eosinophilia, invasive ankylostomiasis, strongyloid- iasis, visceral larva migrans, and infections with Opisthorchis and Clonorchis species. Established infections Urogenital schistosomiasis (Schistosoma haematobium) The signs and symptoms of S. haematobium infection relate to the worms’ predilection for the veins of the urogenital tract, and result from deposition of eggs in the bladder, ureters, and to some extent the genital organs. In the phase of established infection two stages can be recognized: • an active stage mainly in children, adolescents, and younger adults with egg deposition in the urinary tract, egg excretion in the urine with proteinuria and macroscopic or microscopic haematuria • a chronic stage in older individuals with sparse or absent urinary egg excretion but the presence of urogenital tract pathology In the active stage many patients will have minimal symptoms. The most frequently encountered complaint is a painless, characteristic- ally terminal, haematuria, the prevalence and severity of which is related to the intensity of infection. In communities where S. hae- matobium is highly endemic, macroscopic haematuria among boys may be considered a natural sign of puberty. Dysuria, frequency, and suprapubic discomfort or pain is associated with schistosomal cystitis and may continue throughout the course of active infection. Initially the eggs may give rise to an intense inflammatory response in the mucosa. This may cause ureteric obstruction leading to hydroureter and hydronephrosis. Cytoscopy reveals friable masses or polyps ex- tending into the bladder, petechiae, and granulomas. These early in- flammatory lesions, including the obstructive uropathy, are usually reversible after treatment with antischistosomal drugs. The bladder lesions and obstructive uropathy can be visualized by ultrasonog- raphy (Fig. 8.11.1.10). As the infection progresses, the inflammatory component de- creases, possibly due to modulation by the host immune response, and fibrosis increases. Various changes occur in the bladder, including calcification, ulceration, and the development of papil- lomas. Cytoscopy reveals ‘sandy patches’ composed of large numbers of calcified eggs surrounded by fibrous tissue and an atrophic mu- cosal surface. The bladder lesions may lead to nocturia, precipitancy, retention of urine, dribbling, and incontinence. The ureters are less commonly involved, but ureteric fibrosis can cause irreversible ob- structive uropathy which can progress to uraemia. Bilateral ureteric involvement can occur. Despite damage to the ureters, symptoms are often absent or minimal. (a) (b) Fig. 8.11.1.9  Katayama fever (S. mansoni infection): (a) giant urticarial rash; (b) rash in a traveller. Courtesy of Dr Tom Doherty, London Hospital for Tropical Diseases. Fig. 8.11.1.10  Bladder pseudopolyps as seen by ultrasound in S. haematobium infection. Courtesy of Ms Hilda Kadzo, Kenyatta National Hospital, Nairobi, Kenya.

section 8  Infectious diseases 1546 Egg deposition can also cause granulomas and lesions to de- velop in the genital organs, most commonly in the cervix and vagina in women and the seminal vessels in men leading to syn- dromes termed female and male genital schistomasis, respectively. This may result in dyspareunia, abnormal vaginal discharge, con- tact bleeding, and lower back pain in women, and perineal pain, painful ejaculation, and haematospermia in men. Genital symp- toms like bloody discharge and genital itch are associated with S. haematobium infection in school-​age girls. Symptoms such as haematospermia and perineal discomfort have been described in travellers returning from Malawi. In some of these patients, eggs have been demonstrated in seminal fluid but not in urine. An asso- ciation between female genital schistosomiasis and HIV infection has been demonstrated but the impact of genital lesions caused by S. haematobium infection on the spread of HIV needs to be eluci- dated. Although small numbers of S. haematobium eggs are fre- quently detected in faeces and rectal biopsies, intestinal symptoms are uncommon. S. haematobium infection is associated with squamous cell car- cinoma of the urinary bladder and significant positive associations between the occurrence of urinary bladder cancer and infection with S.  haematobium has been reported in several case studies. S.  haematobium has been classified as definitely carcinogenic to humans (group 1 carcinogens). The aetiological significance of the parasite in the causation of this cancer is also supported by the finding that the prevalence of squamous cell carcinoma of the bladder is correlated with intensity of S. haematobium infection. Chronic inflammation occurring with continuous infection and reinfection plays a central role in the initiation of S. haematobium related bladder cancer. In the established stage, S.  haematobium should be distin- guished from renal tuberculosis with haematuria, haemoglobin- uria, and cancer of the urogenital tract. Differential diagnosis for genital schistosomiasis includes sexually transmitted infections and sexual abuse. Intestinal schistosomiasis In most early S. mansoni and S. japonicum infections, symptoms are mild or absent. Clinical features are generally encountered in those with high-​intensity infections. They include diarrhoea, sometimes with blood or mucus in the surface of the stool, abdominal discom- fort, and hypogastric pain or colicky cramps. Severe dysentery is rare, but can occur. The liver, especially the left lobe, may be en- larged; the spleen may also be enlarged, but is usually soft. At this stage, the condition is entirely reversible by antischistosomal treat- ment, but the relative lack of symptoms may cause it to pass un- noticed until irreversible complications set in. Later stages present as intestinal or hepatosplenic disease. Intestinal schistosomiasis is associated with granuloma formation (Fig. 8.11.1.6), inflammation, and fibrosis, primarily in the large intestine. Focal dense deposits of S. mansoni or S. japonicum eggs as well as eggs migrating through the intestinal wall provoke mucosal granulomatous inflamma- tion, pseudopolyposis, microulcerations, and superficial bleeding. The major clinical manifestation is intermittent diarrhoea with or without passage of blood or mucus, occasionally associated with protein-​losing enteropathy and anaemia. Intestinal schistosomiasis in S. japonicum infection can also involve the stomach, with gastric bleeding and pyloric obstruction. Differential diagnosis includes irritable bowel syndrome, amoeb- iasis, giardiasis, intestinal helminth infection, ulcerative colitis, Crohn’s disease, and tuberculosis. Hepatosplenic schistosomiasis is a manifestation of S. mansoni and S. japonicum infection caused by schistosome eggs trapped in liver tissue. The term covers two distinct clinical entities: early in- flammatory and late hepatosplenic disease with periportal fibrosis. Early inflammatory hepatosplenic schistosomiasis is the main cause of hepatosplenic schistosomiasis in children and adolescents. The liver is enlarged, especially the left lobe, and is smooth and firm. The spleen is enlarged, often extending below the umbilicus and firm or hard. Generally, no hepatic fibrosis can be demonstrated by ultrasonography. Early inflammatory hepatosplenic schistosom- iasis may be found in up to 80% of infected children and the se- verity is related to intensity of infection (Fig. 8.11.1.11). This type of hepatosplenomegaly may also be associated with concomitant chronic exposure to malaria. Presinusoidal periportal fibrosis (clay pipe stem or Symmers’ fi- brosis) (Figs. 8.11.1.12 and 8.11.1.7) develops later in life, gener- ally in young and middle-​aged adults with long-​standing intense exposure to infection. Patients with periportal fibrosis may excrete very few or no eggs in faeces. During the early stages the liver is enlarged, especially the left lobe; it is smooth, firm, and sometimes tender. Later, in many cases, it becomes small firm and nodular. The spleen is enlarged, often massively, due to passive congestion and reticuloendothelial hyperplasia (Fig. 8.11.1.12). The patient can be asymptomatic or might complain of a left hypochondrial mass with discomfort and anorexia. Anaemia might be present. There may be reduced growth, infantilism, and amenorrhoea, especially in S. japonicum infection. Severe hepatosplenic schistosomiasis can lead to portal hypertension, but hepatic function usually remains normal. Ascites, attributable both to the portal hypertension and to Fig. 8.11.1.11  Kenyan child with severe hepatosplenic schistosomiasis mansoni.

8.11.1  Schistosomiasis 1547 hypoalbuminaemia, may be seen, especially in S. japonicum infec- tion. Patients with severe hepatosplenic disease and portal hyper- tension can develop oesophageal varices detectable by endoscopy or ultrasound (Fig. 8.11.1.13). These patients might experience re- peated bouts of haematemesis, melaena, or both. This is the most severe, potentially fatal, complication of hepatosplenic schistosom- iasis, and death may result from massive loss of blood. Differential diagnoses of hepatosplenic schistosomiasis include kala-​azar (visceral leishmaniasis), tropical splenomegaly syndrome associated with malaria, leukaemia, lymphoma, alcoholic, or viral cirrhosis, and some of the haemoglobinopathies. Some regression of periportal fibrosis may occur after specific antischistosomal therapy, as judged by ultrasonography examination of the liver, but in most individuals with periportal fibrosis and clinical manifestations of se- vere hepatosplenic disease, regression does not occur. In comparison with S. japonicum and S. mansoni infections, clin- ical symptoms of disease in S. intercalatum infection are commonly mild or absent, and it is not regarded as a serious public health problem. Active infection is seen in children and adolescents and pathology is detected only in those with egg excretion exceeding 400 eggs/​g faeces. The usual clinical presentation is one of diarrhoea, often with blood in the stool and lower abdominal pain or discom- fort. S. mekongi infections are usually asymptomatic but may pro- duce a clinical picture similar to that of S. japonicum, although the infections are usually milder. Hepatosplenomegaly can occur. Other manifestations Nervous system manifestations Nervous system involvement in S.  mansoni and S.  haematobium infections most frequently affect the spinal cord following acute infection. This manifestation is not related to the intensity of infec- tion. Myelopathy and radiculopathy result from the inflammatory reaction, caused by the deposition of eggs around the spinal cord, and presents as an ascending flaccid paralysis with sensory level and sphincter involvement. The lesion is usually in the region of the cauda equina. Although paraparesis is seen most commonly during acute schistosomiasis, it might also be a late-​stage complication of S. mansoni infection in endemic areas with high rates of transmis- sion. Myelography, CT, and magnetic resonance imaging (MRI) are of diagnostic value. In acute cases lesions are seen on MRI scans as a diffuse swelling of the lumbar cord with central softening or cyst formation. The brain is the major site of central nervous system involvement in S. japonicum infections. About 2% of acutely infected patients experi- ence symptoms that mimic acute encephalitis or a focal neurological process. CT shows multiple enhancing lesions. In chronic infections, patients may present with focal brain lesions that can resemble tu- mours and present as focal epilepsy. These lesions contain masses of eggs and granulomas. Antischistosomal drugs, corticosteroids, and surgery are the types of treatment available for neuroschistosomiasis, and uncontrolled studies suggest that treatment with a combination of praziquantel and corticosteroids is effective. However, a con- sensus regarding the best treatment of the different presentations of neuroschistosomiasis has not been reached. Pulmonary manifestations Eggs may be deposited in the lungs. Granulomatous reactions and fibrosis develop in the pulmonary vasculature leading to pulmonary hypertension and/​or cor pulmonale (Fig. 8.11.1.8). This is normally seen secondary to hepatosplenic schistosomiasis in patients with portal fibrosis and portal hypertension, but pulmonary hyperten- sion can also result from accumulation of S. haematobium eggs in the lungs. A syndrome of cough with multiple small radiographic lesions and eosinophilia has been described. Symptoms include fa- tigue, palpitations, dyspnoea, cough, and sometimes haemoptysis. Patients may progress to decompensation with congestive cardiac failure. In endemic areas schistosomiasis must always be considered as a possible cause of pulmonary hypertension and cor pulmonale. Renal manifestations Glomerulonephritis can be associated with chronic S.  mansoni infection, especially hepatosplenic disease. Immunoglobulins, Fig. 8.11.1.12  Hepatic periportal fibrosis as seen by ultrasound in S. mansoni infection. Fig. 8.11.1.13  Oesophageal varices as seen by ultrasound in S. mansoni infection.

section 8  Infectious diseases 1548 complement components, and schistosome antigens are depos- ited in the mesangial area. The condition is manifested clinic- ally as proteinuria and/​or nephrotic syndrome, sometimes with hypertension. Miscellaneous manifestations Patients infected with any of the three major schistosome species and subsequently infected with salmonella may develop a prolonged intermittent febrile illness. Prolonged excretion of salmonella in the urine and intermittent bacteraemia has been demonstrated in S. hae- matobium infection. Treatment for the salmonella infection alone is often not effective without treatment of the underlying schistosome infection. Ectopic schistosomiasis lesions can sometimes be found in the skin, the peritoneum, or other organs. Diagnosis and clinical investigations Information about geographical area and history of exposure by wading, bathing, washing, or showering in potentially contamin- ated fresh water is important for diagnosis of schistosomiasis, espe- cially in travellers and immigrants. This can indicate the likelihood of infection and point to the schistosome species involved. A de- finitive diagnosis is made by the direct demonstration of schisto- some eggs by microscopy of urine or stool samples, biopsies or, on rare occasions, secretions such as seminal fluid. In epidemiological studies it is usually important to obtain quantitative estimates of egg output to provide information about intensity of infection within a population. Direct parasitological methods In S. haematobium infection, eggs can be detected in urine after fil- tration, sedimentation, or centrifugation followed by microscopy. Ideally, urine should be passed around midday and the terminal part of the stream examined. The most commonly used method in epidemiological studies in endemic areas is filtration of 10 to 20 ml of urine using a syringe and a polycarbonate (e.g. Nucleopore), poly- amide (e.g. Nytrel), or paper filter. Infection intensity is expressed as eggs/​10 ml of urine. This may not be sufficiently sensitive for detec- tion of low-​intensity infections in travellers. In such cases, diagnosis is often based on filtration of 24-​h urine samples. For S. mansoni, S. japonicum, S. mekongi, and S. intercalatum eggs in the faeces, sedimentation of the eggs followed by micros­ copy is a useful and simple technique. However, the Kato thick smear technique is the most widely used method in epidemio- logical studies. This is based on microscopic examination of a smear of a small but fixed amount of faecal sample (usually 20–​50 mg). Coarse particles and fibrous material are first removed from the sample by passing it through a sieve. A  fixed sample volume is obtained by the use of a template. This is placed on a microscope slide and squashed with either a piece of cellophane soaked in glycerol or a glass coverslip. After leaving the slide for 6 to 24 h to allow the preparation to clear, the eggs are counted and the level of infection expressed as eggs/​g faeces. Unfortunately, watery or diarrhoeal stools cannot be processed this way, and low-​ intensity infections may not be detected, since only small faecal samples are examined and eggs may be clumped unevenly in the stool. Increased sensitivity is obtained by increasing the number of samples examined. For diagnosis of light infections in previ- ously unexposed travellers, microscopic examination of a rectal tissue snip crushed between glass slides is often the most sensitive direct parasitological method. This method can also be used for biopsies. The crushed tissue sample is far better than a sectioned biopsy for the detection and identification of eggs. Other direct methods Detection of circulating schistosome antigens Sensitive enzyme immune assays (ELISA) have been developed to detect circulating schistosome antigens in serum or urine. These antigens, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), are derived from the gut of the adult schistosomes. The assays have almost 100% specificity and high sensitivity, and are excellent epidemiological tools as they provide a direct estimate of worm burden and can be used to monitor the efficacy of chemo- therapy since presence of CAA or CCA is an indication of an active infection. They are less well suited for diagnosis of light infections in travellers. A rapid point-​of-​care assay (Rapid Medical Diagnostics, Pretoria, South Africa) for CCA in urine is now commercially avail- able. It has been evaluated in several endemic locations and seems to be more sensitive than the Kato thick smear method in mapping S.  mansoni endemic areas and is now widely used for screening of infected communities in relation to mass drug administration programmes. Polymerase chain reaction-​based methods Polymerase chain reaction-​based detection of parasite DNA in stool or urine is more sensitive than parasitological methods and is now increasingly being employed for diagnosis and as a useful tool in epi- demiological studies. Multiplex polymerase chain reaction analysis which includes detection of several intestinal parasites in a single stool sample can be an advantage when diagnosing infections in travellers. Schistosome DNA can also be detected in vaginal lavage and cerebrospinal fluid samples for diagnosis of genital schistosom- iasis or neuroschistosomiasis. Indirect diagnostic techniques In S. haematobium infections, chemical reagent strips for detection of microhaematuria are widely used in endemic areas as a diag- nostic measure. The method can be used in areas of both high and low transmission and there is a consistent significant correlation between microhaematuria and intensity of infection. In intestinal schistosomiasis, blood may be found in the stools, but it is not as useful an indicator of infection. In urinary schistosomiasis, eosinophiluria, with high numbers of eosinophil granulocytes in the urine, is a characteristic finding. Detection of the eosinophil granule protein ECP (eosinophil cat- ionic protein) in urine has been used for the qualitative assessment of eosinophil infiltration of the bladder mucosa, and hence local in- flammation and can be used to follow post-treatment resolution of urinary tract morbidity in endemic areas. Eosinophilia, sometimes exceeding 50%, is often found in acutely infected travellers. In cases where eggs are difficult to find, eosino- philia plus a history of exposure might suggest the need for further examination for schistosomiasis including serodiagnosis.

8.11.1  Schistosomiasis 1549 Immunodiagnosis In cases of suspected schistosomiasis in which eggs have not been detected, serology can be used to demonstrate specific antibodies. An indirect immunofluorescence test using sections of adult worms for detection of specific immunoglobulins (IgM and IgG) is widely used. For travellers, a positive antibody result combined with a history of exposure should lead to treatment. Serodiagnosis is not useful in endemic areas because of the high levels of specific anti- bodies found in naturally exposed populations. Ultrasonography Ultrasonography is non​invasive, portable, has no biological hazards for the patient, and can be used to either complement or replace many invasive diagnostic techniques. It is the technique of choice for grading schistosomal periportal fibrosis, portal hypertension, hydronephrosis, and urinary bladder lesions. A protocol for stand- ardized investigations and methods of reporting has been produced by the World Health Organization (WHO) (http://​www.who.int/​ schistosomiasis/​resources/​tdr_​str_​sch_​00.1/​en/​). Ultrasonography is especially useful for monitoring decreases in morbidity after chemotherapy programmes. Lesions in the female genital tract, especially cervical lesions, can be visualized directly by colposcopy and a pocket atlas with pictures showing the lesions has been developed for use in health facilities in endemic areas (http://​www.who.int/​schistosomiasis/​resources/​ 9789241509299/​en/​). Endoscopy and cystoscopy can be used in demonstrating oe- sophageal varices in hepatosplenic schistosomiasis and urinary bladder lesions, respectively. CT and especially MRI are methods of choice when diagnosing neuroschistosomiasis Treatment The drug of choice is praziquantel, available as 600 mg tablets. It is administered orally and is effective against all schistosome spe- cies infecting humans. It is also effective for most other trematode infections and against adult cestodes. The drug is safe and well tolerated. The standard effective clinical regimen for praziquantel has been shown, in randomized control trials, to be 40 mg/​kg in divided oral doses over one day (2 × 20 mg/​kg does 4-​hourly) for S haematobium, S intercalatum and S mansoni, and 60 mg/​kg in divided oral doses over one day (2 × 30 mg/​kg either 4-​ or 6-​hourly or 3 × 20 mg/​kg doses 4-​hourly) for S mekongi and S japonicum. In patients who are not cured by the initial treatment, the same dose can be repeated at weekly intervals for 2 weeks. A repeat dose 6–​12 weeks later can be administered to cure prepatent infections, especially if eosinophilia or symptoms persist despite treatment. In schistosomiasis control programmes based on mass drug administration, a single dose of praziquantel (40  mg/​kg) is re- commended by WHO. Praziquantel has not been shown to be teratogenic in animals and based on extensive experience with the drug and review of the veterinary and human evidence WHO now recommends that pregnant and lactating women are treated during control campaigns. Similarly, WHO reports that there is growing evidence that infected children as young as 1 year old can be effectively treated with praziquantel without serious side effects; however, currently there is no paediatric formulation of praziquantel available. Any side effects are generally mild, resolving spontaneously over a few hours and rarely requiring medication. Gastrointestinal side effects include abdominal pain or discomfort and sometimes vomiting. They occur more frequently in individuals with high in- fection intensities. Urticarial skin reactions and periorbital oedema may occur in about 2% of treated individuals. General side effects including headache, dizziness, fever, and fatigue can also occur, but less frequently. As a general principle, all patients with acute schistosomiasis should be treated with praziquantel. Corticosteroids can be added in case of Katayama fever to suppress the hypersensitivity reac- tion. Since immature schistosomes are not susceptible to prazi- quantel, treatment should be repeated 4–​6 weeks later. Use of praziquantel for cerebral S. japonicum infections is effective, re- sulting in rapid dissipation of cerebral oedema and resolution of cerebral masses. However, corticosteroids and anticonvul- sants are sometimes needed in addition to praziquantel in cases with neuroschistosomiasis. Praziquantel should be administered with great caution in the case of concurrent neurocysticercosis. Chemotherapy is only part of the management of schistosomiasis-​ associated portal hypertension, since the main complications are due to obstructive pathology. Management of portal hypertension and prevention of bleeding from oesophageal varices is beyond the scope of this chapter. Praziquantel has largely replaced other drugs for treatment of schistosomiasis. Artemisinin derivatives are effective against immature stages (schistosomulae) of S. japonicum, S. mansoni, and S. haematobium and clinical trials in China has shown that repeated oral doses of artesunate or artemether prevented patent S. japonicum infections. In order to reduce the risk of inducing drug-​resistant malaria para- sites, artemisinin-​based combination therapies are used in treat- ment of malaria. So far two large-​scale trials have examined the effect of these therapies on S. haematobium in Mali and S. mansoni in Kenya, respectively. Taken together, the evidence suggests that the efficacy of artemisinin-​based combination therapies against the two major schistosome species is only moderate and inferior to a single dose of praziquantel, so these treatments should be reserved for the management of malaria. Prognosis Most infected people have few, if any, overt symptoms. Acute schis- tosomiasis can be fatal or can lead to severe residual damage to the nervous system if not treated, but responds well to antischistosomal therapy if started early. Early infections respond extremely well to treatment and the pathological lesions regress leaving little residual damage. However, in endemic areas individuals, particularly young children, are rapidly re-​exposed and reinfected, unless control meas- ures are taken at the community level. Chronic infections with se- vere periportal fibrosis respond less well to specific antischistosomal treatment, although some regression of hepatosplenic disease with periportal fibrosis has been seen after treatment. The lifetime prog- nosis is worst in patients with severe hepatosplenic schistosomiasis

8.11.2 Liver fluke infections 1551

8.11.2 Liver fluke infections 1551

8.11.2  Liver fluke infections 1551 Richter J (2003). The impact of chemotherapy on morbidity due to schistosomiasis. Acta Tropica, 86, 161–​83. Silva LC, et al. (2004). Treatment of schistosomal myeloradiculopathy with praziquantel and corticosteroids and evaluation by magnetic resonance imaging: a longitudinal study. Clin Infect Dis, 39, 1618–​24. Weerakoon KG, et al. (2015). Advances in the Diagnosis of Human Schistosomiasis. Clin Microbiol Rev, 28, 939–​67. World Health Organization (WHO) (2011). IARC monograph on the evaluation of carcinogenic risks to humans. Volume 100. A review of human carcinogens. Part B: biological agents. Schistosoma haemato- bium. International Agency for Research on Cancer, Lyon, France, pp. 377–​90. World Health Organization (WHO) (2015). Female genital schisto- somiasis: a pocket atlas for clinical health-​care professionals. WHO/​ HTM/​NTD/​2015.4. 8.11.2  Liver fluke infections Ross H. Andrews, Narong Khuntikeo,
Paiboon Sithithaworn, and Trevor N. Petney ESSENTIALS Liver flukes, otherwise known as trematodes, are leaf-​like hermaph- roditic flatworms. In certain parts of the world, the hepatobiliary system of humans is commonly infected by flukes of the genera Clonorchis and Opisthorchis and occasionally by other species
(Table 8.11.2.1). People acquire liver fluke infection by the ingestion of viable metacercariae that are found in second intermediate hosts through raw or partially cooked food, predominately freshwater cyp- rinid fish. These infections are usually diagnosed by finding eggs in the faeces. Unfortunately, eggs of many of these species cannot be differentiated from each other, nor can they be distinguished reli- ably from the eggs of certain intestinal trematodes. In such cases, definitive diagnosis can only be made if adult worms are recovered from the stools after anthelmintic treatment, at surgery, or at autopsy; parasitological texts should be sought for diagnostic details. The dis- eases caused by liver flukes range from asymptomatic, mild disease to fatal bile duct cancer. Currently the drugs of choice for treatment of liver fluke infection are praziquantel and triclabendazole. The medically important liver flukes are the food-​borne digenean trem- atodes of which, in recent times, two species have been acknow- ledged and listed as Group 1 carcinogens that cause bile duct/​liver cancer, cholangiocarcinoma; namely Opisthorchis vivverini sensu lato (sl; a species complex) and Clonorchis sinensis, which are distributed throughout Southeast Asia, China, and North Korea, and Opisthorchis felineus, which is distributed in Eastern Europe and Russia and has recently been implicated as causing cholangiocarcinoma. There are two larger trematodes of medical importance, Fasciola hepatica and Fasciola gigantica, which have worldwide distributions and have not been associated with cancer. These are more likely to present with abdominal pain or biliary symptoms and are usually acquired by in- gestion of aquatic plants, such as watercress, to which the infective metacercariae cling. Introduction The major human liver fluke diseases, which still remain neglected tropical diseases, are opisthorchiasis, clonorchiasis, and fascioliasis. They are transmitted by the fish/​food-​borne trematodes O. viverrini sl and C. sinensis found in continental Southeast Asia, China, and North Korea, and O. felinius, which is found in Eastern Europe and Russia. Currently 45 million people are estimated to be infected with liver flukes in Asia and Europe; 35 million are infected with C. sinen- sis, 10 million with O. viverrini sl and 1.2 million with O. felineus. It has been estimated that 67.3 million people are at risk of infection in Southeast Asia. It is important to note that the northeast of Thailand is the highest incidence world wide of cholangiocarcinoma (CCA) which is induced by O.  viverrini sl, with an estimate of between 10 000 and 20 000 deaths caused by CCA each year, predominately among the poor in regions that are resource limited. Individuals with premalignant pathology, i.e. periductal fibrosis, are considered as a high-​risk group for CCA. Alarmingly, it has been estimated that 700 million people world- wide are at risk of infection when the three species are considered. O. viverrini sl and C. sinensis have been classified as Group 1 car- cinogens. Moreover, O. felineus is also a significant human pathogen and recent evidence suggests that it may be carcinogenic in ani- mals and humans. The remaining liver flukes, Metorchis species and Dicrosidium, appear to be of medical importance only spor- adically. Importantly, the liver flukes and the diseases they cause, opisthorchiasis and clonorchiasis, as well as CCA, have received limited attention in a clinical/​diagnostic context, perhaps due to Table 8.11.2.1  Liver flukes infecting humans Species Geographical distribution Source of infection Clonorchis sinensis Eastern Asia Freshwater fish Dicrocoelium dendriticum Widespread Ants accidentally ingested with food Eurytrema pancreaticum Eastern Asia Grasshoppers Fasciola gigantica Asia, Africa Vegetation, e.g. watercress Fasciola hepatica Widespread Vegetation, e.g. watercress Metorchis conjunctus Canada Freshwater fish Opisthorchis viverrini sl Southeast Asia Freshwater fish Opisthorchis felineus Europe, Asia Freshwater fish Opisthorchis guayaquilaris Ecuador Freshwater fish Opisthorchis noverca India Freshwater fish Opisthorchis lobatus Lao PDR Freshwater fish Acknowledgement: We wish to thank David I. Grove and other past authors of this chapter for the significant contributions to the Oxford Textbook of Medicine.

section 8  Infectious diseases 1552 their extremely complex life cycles and because they are considered to be a ‘tropical’ local problem. It has been estimated that 17 million people are infected with the two larger human liver flukes, Fasciola hepatica and Fasciola gigan- tica. In a worldwide context including Europe, Africa, the Americas, and Oceania, 91 million people are estimated to be at risk of infec- tion. Fascioliasis, the disease caused by these liver flukes results in serious acute and chronic morbidity. F hepatica and F.  gigantica commonly infect domestic ruminants and wildlife. They also infect humans who represent accidental definitive hosts and usually are infected by eating aquatic fresh water plants from areas in which the water is contaminated with the faeces from animals harbouring Fasciola. Liver flukes belong to the subclass Digenea. These have sexual re- production in definitive human and animal hosts and asexual repro- duction in their first intermediate snail hosts. They have complex life cycles involving one or more intermediate hosts and many morpho- logical stages (see next). To date, control of liver flukes and treatment have been dependent on chemotherapy, with the drug of choice being praziquantel and the most successful drug for Fasciola being triclabendazole. During the past few years efforts have been made to improve sanitation systems and behaviour, and health promotion cam- paigns have been introduced to encourage the cooking of fish involved in the transmission of the opisthorchid liver flukes as important components of control to prevent reinfection. It is im- portant to appreciate that eating habits have cultural, social, and religious significance. As such, individual, village and community health perceptions of the detrimental effect and consequences of ‘raw’ or ‘partially’ cooked eating behaviour(s) are difficult to change. However, liver fluke control is a prerequisite for the reduc- tion of CCA. Opisthorchiasis and clonorchiasis Epidemiology Opisthorchis viverrini sl, O. felineus
(opisthorchiasis) and Clonorchis sinensis
(clonorchiasis) Opisthorchis viverrini sl, Clonorchis sinensis, and O.  felineus, are still important public health problems in many endemic areas. For instance, of the estimated 35 million people infected with C. sin- ensis globally, an estimated 15 million people are in China alone. Prevalence and intensity of infection usually increase with age, with initial infections occurring in the early teens. There is a higher prevalence and intensity of infection in males than females. Even though praziquantel is widely available and massive control ef- forts have been made, these liver flukes are still common and are a significant public health problem in many endemic areas. In add- ition to causing hepatobiliary disease, O. viverrini sl and C. sinen- sis are major aetiological agents of bile duct cancer, CCA, which, as stated earlier, is a leading cause of death in northeast Thailand. These three liver fluke species have similar egg morphologies, life cycles and pathogenesis, and distinction between them is usually based on the adult worm morphology. With respect to O. felineus, it is prevalent in animals throughout Europe and it has also been re- ported in people from Italy, eastern Germany, Poland, Kazakhstan, Russia, and the Ukraine, with high prevalences and intensities being reported in Siberia. During the past few years, control efforts in northeast Thailand have resulted in a decreased prevalence of O. viverrini sl infection from 35% in 1981, to 24 to 30% in 1992 and 18.6% in 1994 and 15.7% in 2009. However, some studies examining adult worms post- treatment and viable metacercariae in fish suggested that estimates of prevalence of infection may be influenced by misdiagnosis of minute intestinal fluke infections. O. viverrini sl, and minute intes- tinal fluke infection are common in Lao PDR, having an extensive distribution and a particularly high prevalence of infection in people in the southern region. During the past few years there has been mounting evidence from independent genetic, molecular, biological, and morphological studies that show that O. viverrini is not a single species but that it is species complex containing cryptic and/​or morphologically distinct species and defined genetic groups in specific wetlands in Thailand and Loa PDR. Whether the O. viverrini sl species/​genetic groups correlate with levels of CCA incidence, infectivity levels in fish and snails, varying levels of pathogenicity, and so on remains to be determined. Dishes of freshwater cyprinid fish that are eaten raw or dishes that contain partially cooked or fermented fish are a well-​established dietary tradition of people from Thailand, Lao PDR, Myanmar, Cambodia, and Vietnam. Raw cyprinid fish dishes may contain large numbers of metacercariae and are eaten occasionally, usually with the local alcoholic drink. Partially cooked fermented fish, which is eaten daily in many dishes, may also contain viable metacercariae and serve as a source of infection. Clonorchis sinensis remains prevalent and common in parts of Taiwan, Hong Kong, Vietnam, Macao, and China, even though it has largely been eliminated from Japan and significantly reduced in Korea. Human infection occurs in 24 provinces in China, predom- inately in the south (especially Guangdong and Guangsxi provinces) and the northeast (Henjian). Some Chinese people enjoy eating raw fish dipped in hot rice porridge, which is a popular dish, and children catch and eat them during play, the latter resulting in an unusual age-​related pattern of infection compared to the other two liver fluke species. Life cycle Adult liver flukes are found in the smaller intrahepatic bile ducts of definitive hosts, namely, people, cats, dogs, and other wild and domestic freshwater fish-​eating mammals (Fig. 8.11.2.1). Eggs pass down the bile duct of the definitive hosts into faeces and are fully embryonated upon excretion. Those that end up in fresh water can be located and eaten by snails were they hatch into miracidia. Normally, less than 1–​2% of snails are infected. Snails are the critical amplifying stage where the miracidia transform into sporocysts and rediae which multiply before becoming free-​swimming cercariae. Numerous cercariae are released by snails which swim about until they contact, attach, penetrate, and encyst in susceptible species of second intermediate host, in this case fresh water cyprinid fish, where they form metacercariae, the infective stage. Studies have found a wide variation in metacercarial prevalence (up to 100%) and intensity between seasons and different wetlands, and between different species of fish. Metacercariae are infective to

8.11.2  Liver fluke infections 1553 humans and other mammals if consumed with raw or insufficiently cooked fish. Once eaten, the metacercariae excyst, migrate up the duodenum through the ampulla of Vater and the extrahepatic bil- iary system to the intrahepatic bile ducts, where they mature to adult flat worms (Fig. 8.11.2.2). This occurs in about 1 month and the adult worms can live for many years. For instance, adult worms of C. sinensis might live for up to 40 years. Pathology and pathogenesis In heavily infected cases, liver enlargement and dilated subcapsular bile ducts with thick fibrotic walls can be seen grossly, and microscop- ically. Bile duct pathology is characterized by desquamation of the epi- thelial cells of secondary and tertiary ducts and chronic inflammation with infiltration by lymphocytes, monocytes, eosinophils, and plasma cells. Occasionally, along the bile ducts granulomatous inflammation can be observed around the eggs. At an early stage of infection, epithe- lial hyperplasia may be observed. In severe cases, adenomatous hyper- plasia, and goblet cell metaplasia may occur. The most prominent and significant histological feature of chronic infection is periductal fibrosis, which corresponds to periportal echoes detected by ultrason- ography. There have also been reports of inflammation, necrosis, and atrophy of hepatic cells. Fluke-​associated cholecystitis pathology in- volves fibrosis, infiltration of mast cells and eosinophils and mucosal hyperplasia of the gallbladder wall. Perforation of the gallbladder wall is not a common phenomenon in liver fluke infection. Adult worms Adult worms in humans, dog, and cat Cyprinid fish Second intermediate host In water and intermediate hosts Cercaria Redia Sporocyst Miracidium First intermediate host Adult in bile ducts Egg Fig. 8.11.2.1  Life cycle of O.viverrini sl. Fig. 8.11.2.2  Adult O. viverrini sl worms.

section 8  Infectious diseases 1554 and eggs have been found in the nidus of the gallbladder together with intrahepatic stones. (Figs. 8.11.2.3 and 8.11.2.4). Liver fluke mediated tissue damage may be caused by direct mechanical or chemical irritation and/​or an immune-​mediated re- sponse. The activities of the flukes contribute to biliary ulceration, which can be caused by injury due to the suckers of adult worms during feeding and migrating activities. Chemical irritation can occur via adult worm secretion and excretion of metabolic prod- ucts and wastes from the tegument and excretory openings into the bile, some of which are highly mitogenic to fibroblast, kidney, or biliary cell lines. The long-​observed hyperplasia of biliary epi- thelial cells in opisthorchiasis might be caused by these products. Additionally, the fluke excretory-​secretory products are known to be highly immunogenic. Marked inflammatory infiltration is as- sociated with excretory and secretory antigens in the intrahepatic and extrahepatic bile ducts in animals experimentally infected with liver flukes. Direct cytotoxic and mutagenic effects and in- creased cell proliferation may be caused by nitric oxide and other reactive oxygen intermediates produced by inflammatory cells during infection. An increase of 8-​nitroguanine (8-​NO2-​G) and 8-​oxo-​7,8-​dihydro-​2ʹ-​deoxyguanosine (8-​oxodG) is associated with liver fluke infection. This is further enhanced with repeated infection and/​or by praziquantel treatment, and is considered to be mutagenic. Highly mutagenic conditions for the chronically pro- liferating bile duct epithelium and enhanced hepatic activation of carcinogens in fibrosis areas may be caused by the increased en- dogenous production of N-​nitroso compounds. Independently, or in combination, these conditions provide an ideal environment for cancer development. Clinical features Most infections are asymptomatic and are diagnosed inciden- tally by examination for eggs in the faeces. Chronically infected people usually have few specific signs or symptoms, except for an increased frequency of a palpable liver. Biochemical and haem- atological features are unremarkable, even in people with heavy infections. On the other hand, ultrasonography reveals a high fre- quency of gallbladder enlargement, sludge, gallstones, and poor function in asymptomatic individuals. These are reversed within 10 months of praziquantel treatment if reinfection does not occur. In symptomatic cases of Opisthorchis and Clonorchis, infected in- dividuals generally experience pain in the right upper quadrant and have diarrhoea, loss of appetite, indigestion, and fullness. Severe cases may have general weakness, lassitude, weight loss, ascites, and oedema, while complications can include cholangitis, obstructive jaundice, intra-​abdominal mass, cholecystitis and gallbladder or intrahepatic stones (the latter are particularly fre- quent in clonorchiasis). Enhanced susceptibility to CCA is the most important clinical manifestation of liver fluke infection (Fig. 8.11.2.5). In Thailand, case-​control studies have shown a fivefold increased risk during infection at any intensity, while heavily infected individuals face a 15-​fold risk, which is also reflected geographically. For instance, the population-​adjusted frequency of CCA has been found to be six-​ and tenfold higher in females and males, respectively, in an en- demic province compared to a non​endemic area in Thailand. Even though such a large geographical association has not been reported for C. sinensis infection, accumulating evidence has shown that in- fection by C. sinensis is also strongly associated with CCA. An im- portant and not often reported point for O. felineus is that infection is acute and opisthorchiasis is associated with severe histopatho- logical changes. This is characterized by hepatosplenomegaly, ten- derness, up to 40% eosinophilia, chills, and fever, which occur early in infection and might be due to primary exposure to a large dose of metacercariae. Diagnosis Opisthorchis The traditional method used to diagnose liver fluke infection has been egg counts in faeces, usually using the Kato thick smear method, Stoll’s dilution, or the quantitative formalin ethyl acetate concentration techniques. Moderate to heavy infections are effect- ively detected by the three methods. Comparative studies in low-​ intensity areas, however, have shown that 70% of infections are detected with a single reading of the concentration and dilution techniques, while the sensitivity of 45% by the Kato technique was markedly lower. Results using Stoll’s dilution technique have shown Fig. 8.11.2.3  Egg of O. viverrini sl. Courtesy of AR Butcher. Fig. 8.11.2.4  Histological section of a gallstone showing masses of degenerate Clonorchis/Opisthorchis eggs.

8.11.2  Liver fluke infections 1555 a close correlation between worm burden and egg count with an es- timated egg output of 53 per gram of faeces per worm. Egg counts using the Stoll’s technique are generally higher than those of the con- centration technique. Although molecular detection of egg DNA is possible with high specificity, the presence of polymerase chain re- action (PCR) inhibitors may interfere with the PCR method. The eggs of C. sinensis and the Opisthorchis species cannot be dif- ferentiated. They are all yellow-​brown, 25–​35 µm long by 12–​19 µm wide and have a seated operculum with a small knob at the other end. Furthermore, they are extremely difficult to differentiate from eggs of flukes in the family Heterophyidae (see intestinal trema- tode infections, Chapter 8.11.4), although the latter tend to have a smoother egg shell, a less prominent shoulder at the operculum and the knob may be absent. The diagnosis can only be confirmed by examination of adult flukes. To date, egg detection is commonly used in surveys and treatment programmes. Recently, however, several immunodiagnostic tests have been developed for Opisthorchis and Clonorchis infections. Since parasite-​specific antibodies persist for a long after treatment and have potential cross-​reactivity to other parasites, serological tests are not routinely used for diagnosis. Recent reports on parasite antigen detection in faeces, as well as urine, provide more sensitive diagnoses for opisthorchiasis and clonorchiasis. Particularly, urine antigen detection provides a considerable practical advantage over conventional faecal examination. Early disease detection: CASCAP—​cholangiocarcinoma screening and care programme Recently there have been exciting developments in the early detec- tion of CCA of people at risk of O. viverrini infection, allowing for potentially curative surgery in early stage cancer and thus increasing the life span and quality of life of CCA patients. This has been achieved since the inception in 2014 of the Cholangiocarcinoma Screening and Care Program, CASCAP, at Khon Kaen University, northeast Thailand. CASCAP was developed and instigated because each year up to 20 000 cases of CCA are diagnosed in northeast Thailand. As most patients are first seen during the late stage of the disease, they have a five-​year survival of less than 10%. For more than three decades control and prevention programmes have been aimed at primary prevention. These programmes have focused on health education aimed at reducing the consumption of raw, fermented, or partially cooked freshwater cyprinid fish that are likely to be infected with O. viverrini sl. Studies at CASCAP have shown that early detection can significantly increase 5-​year survival. Before the inception of CASCAP 4 years ago, there were no strategies in place to increase the screening of the risk group or for early diagnosis of CCA. CASCAP represents a conceptual framework and extensive data base for health policy and strategies so that the opisthorchiasis and CCA problem can be administered and managed in a systematic and effective way. Management and treatment A single dose of praziquantel at 40 mg/​kg body weight is an effective treatment against opisthorchiasis and clonorchiasis. It is the regimen used most commonly in large-​scale treatment programmes. In China, however, higher doses of 120 mg/​kg over 2 days have been reported to be necessary to cure heavy Clonorchis infections. Side effects, including dizziness, vomiting, and abdominal pain, occur frequently, but are transient and rarely severe. Most stop after elim- ination of the adult worms which also usually results in the reduc- tion of most abnormalities of the gallbladder. Results from animal models have shown a relatively high efficacy of artesunate and artemether treatment against C. sinensis compared to O. viverrini sl infection. Recently, high efficacy of tribendimidine has been re- ported where a single dose of 200 mg (age below 14 years) or 400 mg (age above 14 years) resulted in a 99% egg reduction rate, which was equivalent to praziquantel in individuals with opisthorchiasis. Mebendazole (30 mg/​kg daily) or albendazole (400 mg twice daily) may be effective if given for several weeks. For O. felineus infections, hexachloroparaxylol (Chloxyle) has also been used extensively, but it may be less effective than praziquantel. For cholangiocarcinoma, surgery or biliary extraction at enteric retrograde cholangiopancreatography (ERCP) may be required in some patients with obstructive jaundice. To improve long-​term survival outcome, focus should centre on radical surgical techniques and perioperative care to improve the R0 resection rate and to min- imize postoperative morbidity and mortality along with screening tools to detect early lymph node negative cases. Prevention Prevention of human liver fluke infection can be facilitated by treatment (to reduce the excretion of eggs), early diagnosis (the de- velopment and introduction of new diagnostic techniques, in ultra- sonography, biomarkers, and so on), sanitation (to prevent eggs from reaching water sources) and health education (to modify people’s perception of the health problems associated with the eating of raw (a) (b) (c) Fig. 8.11.2.5  Hilar cholangiocarcinoma: (a) Magnetic resonance cholangiopancreatography (MRCP) image of tumour (b) enteric retrograde cholangiopancreatography (ERCP) image of tumour (c) histological section showing cholangiocarcinoma. Courtesy of M. Silva.

section 8  Infectious diseases 1556 or partially cooked fish). Reduction of metacercarial contamination of fish in the freshwater aquaculture industry can be achieved by the application of Hazard Analysis Critical Control Point principles and procedures. Freezing, irradiation and chemical treatment have also been suggested as treatment of raw fish. Because snails have a wide- spread geographical distribution and are resistant to adverse condi- tions, it is not considered feasible to use molluscicides for control. To have a significant longstanding effect, health education should be designed and delivered in a culturally sensitive manner aimed at modifying behaviour, as well as providing understandable scientific- ally informed information. To date, the large-​scale treatment efforts in endemic areas by public health ministries have had an impact on the intensity of all three infections. Fascioliasis Epidemiology: Fasciola hepatica and Fasciola gigantica (Fascioliasis) Eggs of Fasciola are excreted 3–​4 months after eating water plants which contain infective metacercariae. The life cycle is completed within 4–​6 months and moderate temperates, high humidity, and rainfall favour transmission. The most common infection of people with Fasciola is in villages and larger towns within rural areas, es- pecially sheep-​ and cattle-​buffalo grazing areas. Infection levels are dependent on the frequency of people eating plants (mainly water- cress in Europe, morning glory in Asia) from water bodies contam- inated with animal faeces. In most endemic areas, human infection is relatively rare, even where prevalence among domestic/​produc- tion animals is high. Where there have been outbreaks of F. hepatica in households and communities, they are often found to be asso- ciated with native, rather than cultivated, watercress. Of the high proportion of exposed people who become infected, some do not have symptoms, and infection may be the result of contaminated drinking water or cooking utensils. Infection by Fasciola infection is underestimated since eggs are often not detected by faecal examination. Results from community-​ based studies using improved diagnostic methods have detected areas with very high prevalence and intensity of infection in Bolivia, Peru, and Egypt. The frequent consumption of kjosco (raw water-​plant salad) by children tending their grazing animals in the high altitudes of the Bolivian Altiplano region has resulted in heavy infections. Studies have also indicated that infection can occur by drinking water contaminated with floating metacercariae. Infection can also occur by ingesting metacercariae that are attached to the food or kitchen uten- sils which were washed with water contaminated with metacercariae. Life cycle Fascioliasis is due to infection with the sheep liver fluke Fasciola he- patica or with Fasciola gigantica. Adult F. hepatica flukes 20–​30 mm by 8–​13 mm in size live in the large bile ducts and produce eggs which are passed in the stools. The eggs require a period of 9–​15 days for the miracidia to develop and hatch in water at 22–​25°C. They can remain viable for up to 9 months if kept moist and cool. The miracidia penetrate the tissues of various species of amphibious snails of the family Lymnaeidae and develop over the following 4 to 5 weeks from sporocyst, rediae, and daughter rediae to cer- cariae. The cercariae emerge from the snails and encyst on a variety of aquatic vegetation to become infective metacercariae. Many mammal species are susceptible to infection; however, sheep and cattle are the most important. Usually people are infected by eating watercress or by drinking water contaminated with metacercariae. Once eaten, the metacercariae excyst in the duodenum, penetrate the intestinal wall, and subsequently pass into the peritoneal cavity. They then invade the liver capsule and migrate through the hep- atic parenchyma to the bile ducts where they mature in about 3 to 4 months. These liver flukes have a life span of several years. F. gigan- tica is large, attaining a size of 25–​75 mm × 12 mm wide. The eggs are difficult to distinguish from those of F. hepatica and the life cycles of the two parasites are similar. Pathology and pathogenesis Fascioliasis is a serious medical condition because of the large size of the liver flukes, which reflect their origin as a flat worm of livestock. They cause considerable mortality in sheep and cattle. They also cause human morbidity which is dependent on the number of liver flukes and the stage of infection. The acute phase of infection oc- curs during migration of the immature liver flukes through the liver. Liver fluke ingestion and destruction of parenchymal tissue, sub- sequent haemorrhage, parasite death, and inflammatory responses which are largely mediated by eosinophils cause severe pathology. Repair mechanisms can result in extensive fibrosis, increased pres- sure atrophy of the liver, and periportal fibrosis. The chronic phase occurs when liver flukes are present in the bile ducts and tends to be less severe. Progressive inflammation (including bile duct prolif- eration, dilatation and fibrosis) is predominantly caused by mech- anical obstruction of the ducts, inflammatory responses, and the activity of proline. The liver flukes excrete proline in large quan- tities which may facilitate movement through the narrow ducts. Anaemia may result from blood loss through bile duct lesions and death caused by haemorrhaging in the bile duct is not common but occurs more frequently in children. Some liver flukes can migrate out of the intestine. If they do not locate in the liver they can form ectopic lesions in many tissues, causing nodules, granulomas, or mi- gration tracts which are often misdiagnosed as malignant tumours or gastric ulcers. During the chronic infection phase of proliferation, dilatation, fibrosis and calcification of the bile ducts and sequelae of partial obstruction may occur. Granulomas and abscesses can form around eggs trapped in the parenchymal tissue. Dead flukes have been detected inside calcified areas of tissue. Eosinophils can infiltrate the gallbladder wall, which may be thickened and oedema- tous with perimuscular fibrosis. Clinical features Fascioliasis can be symptomatic or asymptomatic, with more than 50% of cases subclinical (asymptomatic). Human infection can be classified as acute or chronic based upon clinical and labora- tory findings. Clinical symptoms can include upper abdominal or right costal margin pain and fever. Constitutional symptoms in- clude, urticaria, itching, respiratory symptoms, headache, malaise, weight loss, and night sweats and can occur about 2 months after ingestion of metacercariae and 1–​2 months prior to egg excretion. Hepatomegaly, splenomegaly, anaemia, and weakness are signs of the acute infection phase. Studies have revealed eosinophilia (>500/​mm3) and leukocytosis (>10 000/​mm3) in up to 80% of egg positive cases. For chronic infection beyond the latent phase, clinical symptoms are those of the complications of fascioliasis, for example,

8.11.2  Liver fluke infections 1557 ascending cholangitis, cholelithiasis, cholecystitis, pancreatitis, bil- iary cirrhosis, and hepatic fibrosis. Unlike the Opisthorchis species discussed in the previous sections, Fasciola species (fascioliasis) have not been associated with cancer. Typical fascioliasis is due to the mi- gration of immature worms through Glisson’s capsule and the liver parenchyma on their way to the bile ducts, where they mature and remain as adults (Fig. 8.11.2.6). In some circumstances immature worms reach ectopic destinations which lead to cutaneous or vis- ceral larva migrans, similar to strongyloidiasis and gnathostomiasis. Diagnosis Diagnosis of fascioliasis is usually by detection of eggs during faecal examination, by parasite-​specific antibody detection in a variety of immunodiagnostic assays, by radiological methods and by lapar- otomy. To enable effective differential diagnosis and to investigate sources of outbreaks, the dietary history is very useful. However, examination of faeces for eggs is limited as eggs are not excreted during the invasive stage of infection. This is when many people with severe symptoms present to medical facilities, and frequently eggs are undetectable during the chronic phase. It is uncertain whether the diagnostic techniques are not sensitive for very low egg out- puts in light infections (<100 eggs per gram) or if eggs are not being produced. It is very difficult to differentiate eggs from F. hepatica, F. gigantica, echinostomes, and Fasciolopsis. Additionally, to compli- cate matters, eggs may be passed after eating liver from infected ani- mals, which does not indicate infection. In cases such as these, any positive cases should be re-​analysed if liver has been eaten recently. Many immunodiagnostic tests using every available technique have been used for diagnosis, from skin tests to antibody and antigen de- tection assays targeting the somatic and excretory/​secretory antigens of adult worms. Most of these methods claim more than 90% sensi- tivity. Problems resulting from cross-​reactivity with other trematode infections are avoided through the use of purified specific antigens, cystatin-​treated plates or specific antibody subclasses. A  Fasciola-​ specific serologic response develops within 2 to 4 weeks of infection, whereby confirmation of infection can be done 5 to 7 weeks before eggs appear in the faeces. In the chronic phase, positive serology can also be used to detect infection when egg release might be irregular or absent. A significant advantage of immunodiagnostic methods over para- sitological techniques is that they can detect early, prepatent infec- tions. They can also detect infection at chronic stages where there is irregular or no egg output. Unlike other parasitic infections, antibody levels decline rapidly after successful treatment. As a consequence, the assays are likely to detect only active infection and as such serology is likely to revert to negative within a year after successful treatment. Additionally, biochemical and blood cell microscopy techniques can also support diagnosis. Eosinophilia, leukocytosis, and elevated inflammatory markers are frequent in acute infections, whereas an- aemia and/​or elevated serum hepatic transaminases, bilirubin, and al- kaline phosphatase are infrequently present during chronic infection. As presentations are not markedly different from hepatobiliary disease of other origin(s), clinical diagnosis can be difficult and fascioliasis might not be considered by clinicians in regions where human infections are uncommon. Studies have shown that lapar- otomy and radiological imaging by ultrasonography, endoscopic retrograde cholangiopancreatography and percutaneous cholangi- ography can be useful for diagnosis as they allow detection of the lesions of acute and chronic fascioliasis and at times eggs (by lapar- otomy) or worms in the hepatobiliary system. Management and treatment The regimen of choice against fascioliasis is a single dose of triclabendazole, 10 mg/​kg body weight, as it is active against both immature and adult worms. It has a high cure rate and there are only temporary and mild adverse reactions following treatment. Even at relatively high doses praziquantel has been found to be ineffective against fascioliasis. Efficacy is frequently variable and difficult to as- sess, which arises from different sensitivities of adult and migrating worms, the size and thick tegument of Fasciola, damaged hepatic function and variation in clinical presentation. In the past, bithionol was most often used against fascioliasis with dosages varying form 30–​50 mg/​kg body weight per day ad- ministered in three divided doses on alternate days for 10–​15 days dehydroementine at a dose of 1 mg/​kg daily for 10 days, adminis- tered intramuscularly or subcutaneously, has been reported to be successful against acute infection, but moderate to severe side effects have been observed. Additionally, both drugs and multiple courses are often required for successful outcome. Some clinical trials have shown that nitazoxanide is effective; for instance, one study found that 97% of people were free of Fasciola eggs in faeces after 30 days of treatment. However, further studies are required to confirm these results. Depending on when people present, other drugs given be- fore the fasciolicide could be useful in recovery, for instance, pred- nisolone (5–​10  mg/​day) may alleviate toxaemia. Antibiotics are frequently administered to treat acute cholangitis due to secondary bacterial infection. Previously, chloroquine was administered be- cause it rapidly relieved symptoms of acute disease but, unfortu- nately, it does not eradicate Fasciola species. Prevention The eventual control of Fasciola should aim at introducing a stra- tegic treatment or immunization programme for livestock and other Fig. 8.11.2.6  Fasciola hepatica fluke retrieved by ERCP from bile duct. Courtesy of A. Bailey.

8.11.3 Lung flukes (paragonimiasis) 1558

8.11.3 Lung flukes (paragonimiasis) 1558

section 8  Infectious diseases 1558 herbivorous animals that maintain the zoonotic life cycle of Fasciola species. This is encouraging given the advances in veterinary vaccine development over recent years. Currently, comprehensive immun- ization of livestock is under serious consideration by some countries, thereby reducing human infection and economic losses to the para- site. Control of the snail vectors using molluscicides is not considered practical in most situations. In areas where fascioliasis is prevalent, introduction of a health education programme to discourage human consumption of raw, wild watercress and other edible water plants may prove successful. Some Western countries have instigated strict controls on the commercial production of water plants. This would assist in preventing the expansion of endemic areas in developing countries. Increased data from community-​based studies assessing seroprevalence, and an increased informed understanding of fasciol- iasis and its diagnostic difficulties by clinicians, would markedly re- duce the magnitude to which fascioliasis affects human health. FURTHER READING Andrews RH, et al. (2008). Opisthorchis viverrini: an underestimated parasite in world health. Trends Parasitol, 24, 497–501. Hughes T, et  al. (2017). Opisthorchiasis and cholangiocarcinoma in Southeast Asia: an unresolved problem. Intern J Gen Med, 10, 227–37. Johansen MV, Lier T, Sithithaworn P (2015). Towards improved diag- nosis of neglected zoonotic trematodes using a one health approach. Acta Trop, 141 (Pt B), 161–9. Khuntikeo N, et al. (2015). Cohort profile: cholangiocarcinoma screen­ ing and care program (CASCAP). BMC Cancer, 15, 1475–87. Khuntikeo N, et al. (2018). Current Perspectives on Opisthorchiasis Control and Cholangiocarcinoma Detection in Southeast Asia. Front Med, 5, 117. doi:10.3389/fmed.2018.00117. Mekky MA, et al. (2015). Human fascioliasis: a re- emerging disease is upper Egypt. Am J Trop Med Hyg, 93, 76–9. Petney TN, et al. (2016). Foodborne trematodes: a diverse and challenging group of neglected parasites. Trans R Soc Trop Med and Hyg, 110(1), 1–3. Sithtithaworn, P, et al. (2008). Food borne Trematodes. In Cook G and Zumla A. eds. Manson’s Tropical Diseases, p1461–76, Saunders Ltd, Elsevier, Canada. Sayasone S, et al. (2016). Efficacy and safety of tribendimidine against Opisthorcis viverrini: two randomised, parallel group, single-blind, dose ranging, phase 2 trials. Lancet Infect Dis, 16, 1145–53. Shin SH, et al. (2016). Development of two FhSAP2 recombinant based assays for immunodiagnosis of human chronic fascioliasis. Am J Trop Med Hyg, 95, 852–5. 8.11.3  Lung flukes (paragonimiasis) Udomsak Silachamroon and Sirivan Vanijanonta ESSENTIALS Paragonimiasis is an infection by flukes of the genus Paragonimus, with foci of disease in Asia, Africa, and Central and South America. Humans acquire infection by eating metacercariae in improperly cooked freshwater crabs or crayfish. Acute inflammatory and allergic symptoms are rarely serious and usually resolve spontaneously. Chronic manifestations may be (1)  pulmonary—​most remark- ably with a chronic, productive cough with jam-​like, brownish-​red sputum; and (2)  extrapulmonary—​most importantly in the cen- tral nervous system, often presenting with seizures. Diagnosis is by demonstrating ova in sputum, stool, or pleural fluid. Serology can be used to support the diagnosis, especially in extrapulmonary paragonimiasis. Treatment with praziquantel is almost always ef- fective. Prevention is by health education and the mass treatment of infected people in an endemic area. Introduction Lung fluke infection is caused by Paragonimus spp. of which there are more than 40 that cause disease in mammals and about 16 species causing human disease. Paragonimus westermani is the most common and widespread. Other species prevalent in some region are P.  heterotremus in Southeast Asia, P.  africanus and P. uterobilateralis in West Africa, P. skrjabini in China, P. kellicotti in North America, and P. mexicanus and P. caliensis in Central and South America. Aetiology and life cycle Adult flukes are reddish-​brown and pea-​shaped. They are 0.8–​1.6 cm in length, 0.4–​0.8 cm in width, and 0.3–​0.5 cm thick (Fig. 8.11.3.1). Typically, they are encapsulated in cysts adjacent to the bronchi. Ova (Fig. 8.11.3.2) are expelled through the bronchi and expectorated with sputum or swallowed and passed in the faeces. They hatch in fresh water after a few weeks. The resulting miracidia then infect various species of freshwater snail in which they form sporocysts, rediae, and daughter rediae. Metacercariae develop in susceptible freshwater crabs and crayfish (Fig. 8.11.3.3). Human infection re- sults from ingestion of viable metacercariae in raw or insufficiently cooked crabs and crayfish. Metacercariae excyst in the intestine, Fig. 8.11.3.1  Adult fluke of Paragonimus westermani, approximately 1 cm long. Courtesy of Dr Prayong Radamyos, Faculty of Tropical Medicine, Mahidol University, Bangkok.

8.11.3  Lung flukes (paragonimiasis) 1559 then pass through the peritoneal cavity, diaphragm, and pleural cavity, before finally encysting in the lung. Tunnels may be formed during their migration. Encysted flukes mature over a period of 6–​8 weeks and eggs are produced in 10–​12 weeks. The circuitous routes of migration allow young flukes to lodge and mature in ectopic locations. The reservoir hosts are wild and domestic mammals. Pigs, wild boars and deer are paratenic hosts in which the flukes remain immature and reside in the muscles. When human consume these meats raw, the young flukes mature into adult worms. Epidemiology The three major foci of this disease are Asia, Africa, and Central and South America. Human paragonimiasis is not uncommon in North America. Both imported and endemic cases have been reported in recent years. Paragonimus kellicotti infection was associated with ingestion of raw crayfish from fresh water river system within the Mississippi River Drainage Basin with the largest number of cases from Missouri. Pathogenesis and pathology While they migrate, larvae cause irritation, acute inflammatory reactions, traumatic tracts, pressure effects, haemorrhage, and ne- crosis in affected tissues. Acute, diffuse, fibrinoexudative peritonitis may also occur. Abscess cavities containing young flukes are then formed and become enclosed in a fibrous capsule. Mature cysts ad- jacent to the bronchi may rupture and their contents then drain into the bronchial system. Single or multiple cysts may occur, usually in the lower lobes of the lungs. Extrapulmonary pathological changes may be caused by aberrant migratory flukes. Cysts, abscesses, and granulomas may be found in the abdominal viscera, subcutaneous tissue, muscles, genital organs, and brain. Clinical features The clinical manifestations are divided into acute and chronic phases. The acute phase occurs after consumption of metacercariae. The in- cubation period varies from a few days to weeks. The severity of symptoms usually correlates with the worm load. Invasion and mi- gration by young flukes cause inflammatory and allergic responses such as fever, rashes, urticaria, migratory swelling, abdominal pain, cough, and chest pain. Acute symptoms are rarely serious and usually resolve spontaneously. A large proportion of cases are asymptomatic. Chronic manifestations may be pulmonary and extrapulmonary. Pulmonary paragonimiasis The most remarkable clinical feature is a chronic, productive cough with jam-​like, brownish-​red sputum (Fig. 8.11.3.4). Other symp- toms include breathlessness and chest pain. Pleural effusion, em- pyema, or hydropneumothorax can occur. Occasionally, patients Fig. 8.11.3.2  Ovum of Paragonimus westermani, approximately 100 µm long. Courtesy of Dr Prayong Radamyos, Faculty of Tropical Medicine, Mahidol University, Bangkok. Fig. 8.11.3.3  Metacercaria of Paragonimus westermani in a freshwater crab. Courtesy of Dr Prayong Radamyos, Faculty of Tropical Medicine, Mahidol University, Bangkok. Fig. 8.11.3.4  Typical appearance of sputum coughed up by a patient with pulmonary paragonimiasis. Courtesy of the late Professor Sornchai Looareesuwan.

section 8  Infectious diseases 1560 experience haemoptysis following heavy work or exertion. Physical examination usually shows few abnormalities. Extrapulmonary paragonimiasis Aberrant migration of young flukes to other organs causes extrapulmonary paragonimiasis. The most common and im- portant site is the central nervous system. Presentation of cere- bral paragonimiasis depends on the site of the lesion. Seizures are common. Increased intracranial pressure induces persistent in- tense headache, nausea, vomiting, papilloedema, diplopia, and loss of visual acuity. Mental disturbances of the schizoid and paranoid type may develop. Involvement of the basal meninges results in in- creased intracranial pressure, hydrocephalus, arterial thrombosis, and stroke. Involvement of intrabdominal organs such as spleen, liver, and small and large intestine causes non​specific symptoms and signs. Migratory subcutaneous nodules might occur. Spinal involvement presents with back pain, paralysis, and sensory impairment of the lower extremities. Differential diagnosis Pulmonary paragonimiasis should be differentiated from other conditions presenting with chronic cough productive of bloody or rusty sputum, notably pulmonary tuberculosis, bronchiectasis, lung abscess, and tumour. Paragonimiasis is frequently misdiag- nosed as tuberculosis but does not respond to antituberculosis treatment. Patients usually look relatively healthy. A careful his- tory of residence or travel to endemic areas and eating habits aids diagnosis. Cerebral paragonimiasis should be differentiated from cere- bral cysticercosis, hydatid disease, meningoencephalitis, brain ab- scesses, and tumours. Subcutaneous paragonimiasis may resemble gnathostomiasis, sparganosis, loiasis, or onchocerciasis. Clinical investigation Blood counts typically show leucocytosis with eosinophilia. Sputum is thick, gelatinous, rust-​coloured, or bloody. Microscopic examination shows necrotic tissue, blood, leucocytes, Charcot–​ Leyden crystals, and ova. In pleuropulmonary paragonimiasis, examination of the pleural fluid may be pathognomonic. This shows an exudate with eosinophils in variable proportions (12–​75%). Typically, it has an elevated protein (6–​7 g/​dl), low glucose level (<10 mg/​dl), low pH (<7.10), and elevated lactic dehydrogenase (>1 000 U/​litre). Parasite ova may be found in the pleural fluid. A minority of symptomatic patients have normal chest radio- graphs. Abnormal findings include linear infiltrations, exudative pneumonia, localized pleural effusions, and nodular or cystic lesions. These lesions are found predominantly in the basilar and peripheral regions of both lower lung fields. Cysts may be single or multiple. The most characteristic radiographic feature is a ring shadow with a crescent-​shaped opacity along one side of the heart border (Fig. 8.11.3.5). Multiple cysts may aggregate, producing a soap-​bubble appearance. Other findings are pleural thickening and calcification. Fibroatelectasis resembling tubercu- losis may occur. CT is more sensitive for detecting these abnormalities. Cystic nodules are clearly seen even when they are invisible in plain radiographs (Fig. 8.11.3.6). Focal bronchiectasis is commonly (b) (a) Fig. 8.11.3.5  (a) Posteroanterior radiograph of a patient with pulmonary paragonimiasis showing thick-​walled cystic lesions in the right perihilar area and left upper lobe. Patchy infiltration and pleural thickening are also seen in the right lower lobe. (b) Enlargement of the left upper lobe cystic lesion. Copyright Dr Udomsak Silachamroon. Fig. 8.11.3.6  CT scan of a patient with paragonimiasis, showing multiple cystic lesions in the subpleural region. Copyright Professor Sirivan Vanijanonta.

8.11.3  Lung flukes (paragonimiasis) 1561 found. Worm migration tracts may be identified. Paragonimiasis is one of the benign lesions that may give an increased uptake in a fluorodeoxyglucose positron emission tomography (FDG-​PET) scan and can mimic malignant lung tumour. Characteristic CT findings in cerebral paragonimiasis are conglomerate, multiple ring-​shaped enhancing lesions with sur- rounding oedema (Fig. 8.11.3.7). Demonstration of Paragonimus infection Detection of characteristic eggs in sputum, stool, or pleural fluid con- firms the diagnosis. Paragonimus eggs are golden brown in colour and ovoid in shape with an operculum at one end (size 80–​120 × 50–​60 µm) (see Fig. 8.11.3.2). Egg detection rate is low (12–​62%) but repeating the examination results in a higher yield. Paragonimus eggs can be detected by Ziehl–​Neelsen staining of sputum sample and this appears superior to the standard wet smear technique. Expectoration of the intact fluke is rare. Various serological tests have been developed to aid the diag- nosis. The complement fixation test is sensitive and can be used for evaluation of treatment response, but it is now rarely avail- able. Currently enzyme-​linked immunosorbent assay (ELISA) and immunoblot are commonly applied for various kinds of parasitic disease. These tests are highly sensitive (90–​100%) and specific (>90%). Species differentiation could be achieved by these tests. They are essential for diagnosis of extrapulmonary paragonimiasis. Positive results persist for some time after suc- cessful treatment (4–​24 months) so the response to treatment cannot be evaluated. Definitive diagnosis can be made by demonstrating ova in sputum, stool, pleural fluid, or tissue biopsy. In cases where eggs are not detected or paragonimiasis is extrapulmonary, compatible clin- ical findings with positive serology are accepted as diagnostic. Treatment The drug of choice is praziquantel in a dose of 75 mg/​kg per day in three divided doses for 2 to 3 days with a cure rate of nearly 100%. Symptoms improve within a few days. Eggs disappear from the sputum in a few weeks. Radiological improvement takes months, depending on the extent and chronicity of the disease. However, some patients with long duration of respiratory symptoms, high ELISA titre, and multiple pulmonary lesions might relapse after treatment. The cure rate can be improved by giving a second course of praziquantel. Urticaria or a transient increase in eosinophilia is occasionally seen, indicating a reaction to dead parasites. Allergy to praziquantel is rare but when it occurs without an available al- ternative, desensitization to praziquantel should be considered. Convulsions, coma, and behavioural changes may develop during treatment of cerebral paragonimiasis as a result of brain oedema and increased intracranial pressure. Dexamethasone is suggested for this reaction. Repeated thoracentesis in combination with chemotherapy is required for patients with large pleural effusions. Chronic pleural effusion or empyema may resist chemotherapy because penetration of the drug is limited by pleural thickening. Surgical decortication may be indicated in such cases. Triclabendazole (dose of 10 mg/​kg twice a day for 1–​2 days), a drug for treatment of fascioliasis, was reported to be as effective as praziquantel for pulmonary paragonimiasis and better tolerated by the patients. When available, it might be considered as an alternative to praziquantel. Bithionol and niclofalan are also effective. Prognosis Pulmonary paragonimiasis is rarely fatal. The lesions may calcify or resolve completely in a few years. Cerebral paragonimiasis may cause chronic morbidity such as epilepsy, mental changes, and neurological sequelae. Prevention and control Effective control measures are directed towards interruption of the life cycle. However, control and eradication of intermediate hosts is impracticable; health education, changes in social and dietary cus- toms, and the mass treatment of infected people in an endemic area are therefore more effective for prevention and control. FURTHER READING Chai JY (2013). Praziquantel treatment in trematode and cestode infec- tions: an update. Infect Chemother, 45, 32–​43. Diaz JH (2013). Paragonimiasis acquired in the United States: native and nonnative species. Clin Micobiol Rev, 26, 493–​504. Fig. 8.11.3.7  Cerebral CT scan of a patient with paragonimiasis, showing multiple ring-​shaped enhancing lesions with surrounding oedema. Courtesy of Professor Seung-​Yull Cho, Suwon, Korea.

8.11.4 Intestinal trematode infections 1562

8.11.4 Intestinal trematode infections 1562

section 8  Infectious diseases 1562 Im JG, Chang K, Reeder M (1997). Current diagnostic imaging of pul- monary and cerebral paragonimiasis, with pathological correlation. Semin Roentgenol, 32, 301–​24. Gong Z, et al. (2017). Paragonimiasis in children in southwest China. A retrospective case reports review from 2005 to 2016. Medicine, 96, 25(e7265). Keiser J, et al. (2005). Triclabendazole for the treatment of fascioliasis and paragonimiasis. Expert Opin Invest Drugs, 14, 1513–​26. Kim TS, et al. (2005). Pleuropulmonary paragonimiasis: CT findings in 31 patients. Am J Roentgenol, 185, 616–​21. Kyung SY, et al. (2011). A paragonimiasis patient with allergic reac- tion to praziquantel and resistance to triclabendazole:  successful treatment after desensitization to praziquantel. Korean J Parasitol, 49, 73–​7. Nagayasu E, et al. (2015). Paragonimiasis in Japan: a twelve-​year retro- spective case review (2001–​2012). Intern Med, 54, 179–​86. Oh IJ, et al. (2011). Can pleuropulmonary paragonimiasis be cured by only the 1st set of chemotherapy? Treatment outcome and clinical features of recently developed pleuropulmonary paragonimiasis. Intern Med, 50, 1365–​70. Slesak G, et al. (2011). Ziehl–​Neelsen staining technique can diagnose paragonimiasis. PLoS Negl Trop Dis, 5, e1048. Watanabe S, et al. (2003). Pulmonary paragonimiasis mimicking lung cancer on FDG-​PET imaging. Anticancer Res, 23, 3437–​40. 8.11.4  Intestinal trematode
infections Alastair McGregor ESSENTIALS It is notoriously difficult to estimate the prevalence of intestinal trematode infection. The most widely accepted figures suggest that 40–​50 million people worldwide are infected with at least one of these organisms. The great majority of infections are found in trop- ical South and East Asia, mostly as a result of local culinary prac- tice. The incidence of trematode infections is probably changing as a result of migration, increased transportation, altered dietary habits, and other factors. The most important intestinal flukes are Fasciolopsis buski and members of the families Echinostomatidae and Heterophyidae. As with all the flukes, these parasites have com- plicated lifecycles with a definitive host (which can be human) and two intermediate hosts. The first intermediate host is generally a snail. Infection is acquired through the ingestion of the second intermediate host—​undercooked freshwater fish, molluscs, frogs, or vegetation contaminated with live metacercariae. Fasciolopsis buski, one of the largest (at 20–​77 mm) and most important flukes, is acquired by ingestion of contaminated water plants. Heavy in- fections may cause abdominal discomfort and diarrhoea, but most infections are entirely asymptomatic. Diagnosis of intestinal trematodiasis is with microscopy of faecal concentrates for ova, but it is extremely difficult to distinguish the eggs of organisms within the same family. Praziquantel is the drug of choice for all of these infections, which can be prevented by thoroughly cooking poten- tially infected foodstuffs. Introduction Parasitic trematodes are divided according to their target organ into blood, liver, lung, and intestinal flukes. With the exception of blood flukes (Schistosoma spp.), infections with these para- sites are together classed as the food-​borne trematodiases and are recognized as a neglected tropical disease by the World Health Organization. Approximately 76 species of intestinal fluke belonging to 14 fam- ilies have been recorded as parasites of humans, making intestinal flukes the largest and most diverse group within the food-​borne trematodes. In clinical terms, however, they are probably the least important members given the often benign nature of infection and the association of other flukes (Clonorchis, Opisthorcis, and Schistosoma spp.) with cancers. Intestinal trematode infections are widespread but are most common in Asia. The lifecycles of these organisms are complex and fascinating. The definitive host acquires infection through the in- gestion of food containing metacercariae, a stage in which the larva is dormant and encysted. The vector is most commonly a fresh- water fish, mollusc, or amphibian, although some species encyst on aquatic plants. Infections occur when these metacercariae are not killed during food preparation, which may be because the food is eaten raw or undercooked, or because processing (smoking, drying, pickling) does not kill the encysted parasites. Metacerceriae mature into adult flukes in the definitive host. Ova from these flukes ma- ture into miracidia if they find themselves in water once excreted in the faeces of the definitive host. The parasite passes through the first intermediate host (a snail) and then encysts in the second, awaiting ingestion by the definitive host. In general, the severity of symptoms relates to the parasite burden, which is entirely the product of the numbers of ingested metacercariae. Symptoms are non​specific and include abdominal discomfort and diarrhoea. Most infected individuals are asymptom- atic and might only be identified when screened. Diagnosis The diagnosis of intestinal fluke infections is usually based on recovery of eggs from stools. Unfortunately, ova from species within a given family often look very similar and it may only be possible when using routine laboratory methods to identify an in- fection to family level such as a heterophyid or echinostomatid egg. Definitive identification requires recovery of adult worms expelled after anthelmintic treatment. Identifying characteristics are provided in parasitology texts although taxonomy is confusing and many of these trematodes have been named on more than one occasion.

8.11.4  Intestinal trematode infections 1563 Treatment Praziquantel has been shown to be effective with several of these in- fections and is the drug of first choice. It is given in a dose of 20 mg/​ kg orally after a meal, perhaps repeated once or twice. Flukes are usu- ally expelled the following day. The role of triclabendazole, which is the drug of choice for Fasciola spp. is not yet clear. Other alternatives which are less likely to be effective include niclosamide 150 mg/​kg orally for 1 or 2 days and albendazole 200 mg orally for 2 days. Prevention These fluke infections can be prevented by thoroughly cooking po- tentially infected foodstuffs. Echinostomiasis This term covers infections with flukes of the family Echinostomatidae. There are more than 30 genera in this family and nearly 20 species have been reported to infect humans (Table 8.11.4.1). These spe- cies vary in size from 1 to 20 mm in length. Echinostomes live in the intestines of various birds and mammals. Eggs are passed in the stools and the miracidium develops, hatches and enters a snail (the first intermediate host) when these eggs reach water. Within the snail, the parasite then develops through the stages of sporo- cyst, mother rediae, and daughter rediae, and eventually cercariae. The cercariae leave the snail and in turn infect second intermediate hosts to become encysted metacercariae. Suitable hosts vary with the species of fluke but include various species of gastropod snails, bivalves, frogs and fish, or they encyst on vegetation. Humans are infected after ingestion of inadequately cooked food containing these metacercariae. In humans, mature worms live in the small bowel, particularly the jejunum, where they may cause a variable amount of mucosal damage. Heavy worm loads may cause abdominal discomfort, flatu- lence, and diarrhoea. Eggs (80–​150 × 50–​75 µm in size) are passed in the stools (Fig. 8.11.4.1). They are yellow-​brown, ellipsoidal, thin-​shelled, and operculate and contain an immature embryo; they cannot be reliably differentiated from each other or from those of the intestinal fluke Fasciolopsis buski or the liver flukes Fasciola hepatica and F. gigantica. Table 8.11.4.1  Intestinal trematodes belonging to the family Echinostomatidae that infect humans Species Geographical distribution Definitive hosts other than humans Source of infection Size of adults (mm) Size of eggs (µm) Acanthoparyphium tyosenense Korea Birds Freshwater molluscs 2–​4 × 0.5–​0.7 84–​110 × 60–​69 Artyfechinostomum (Paryphostomum) malayanum India, South East Asia Rats, pigs Freshwater snails 4.8–​8.4 × -​ 96 × 64 Echinochasmus fujianensis ( = liliputanus) East Asia Dogs, cats, foxes, pigs Water, raw freshwater fish 1.5–​2.1 × 0.47–​0.56 Echinochasmus japonicus East Asia Cats, dogs, rodents, chickens Freshwater fish 0.6–​0.9 × 0.16–​0.18 77–​90 × 51–​57 Echinochasmus liliputanus China, Middle East Dogs, cats Freshwater fish 1.5–​2 × 0.5 66–​80 × 43–​46 Echinochasmus perfoliatus Asia, Egypt Cats, dogs, foxes,
rats, pigs Freshwater fish 4.0–​5.5 0.85–​1.1 99–​125 × 58–​74 Echinochasmus (Echinoparyphium) recurvatum Egypt, East Asia Birds, mammals Amphibians, freshwater molluscs 1.9–​7.3 × 0.4–​0.9 88–​111 × 54–​75 Echinostoma cinetorchis East Asia Rats Amphibians, freshwater snails 5.6–​21.2 × 1.3–​3.7 96–​100 × 61–​70 Echinostoma echinatum Indonesia, Brazil Rats, birds Freshwater molluscs 13–​22 × 2.5–​3.0 92–​124 × 65–​76 Echinostoma hortense East Asia Dogs, rats Freshwater fish, amphibians 8.2–​14 × 0.9–​1.6 110–​126 × 61–​70 Echinostoma ilocanum Southeast Asia, China Dogs, rats, mice Freshwater snails 4–​8 × 0.55–​1.0 86–​116 × 52–​72 Echinostoma macrorchis Japan Rats Freshwater snails 3.3–​4.2 × 0.68–​0.86 81–​89 × 54–​58 Echinostoma malayanum Southeast Asia, China Rats Freshwater snails, tadpoles, fish 5–​10 × 2.5 137 × 75.5 Echinostoma revolutum Asia Ducks, geese, chickens, rats Amphibians, freshwater molluscs 21–​26 × 2.0–​3.5 104–​112 × 64–​72 Episthmium caninum Thailand Dogs Fish 1.0–​1.5 × 0.40–​0.75 84 × 50–​60 Himasthla muehlensi USA Birds Molluscs 11–​18 × 0.41–​0.67 114–​149 × 62–​85 Hypoderaeum conoideum Thailand Ducks, fowl Amphibians, freshwater molluscs 6–​12 × 1.3–​2.0 95–​108 × 61–​68 Isthmiophora melis Romania, China, USA Rodents and carnivores Tadpoles, fish 5.5–​7.5 × 1.20 132–​154 × 75–​85

section 8  Infectious diseases 1564 Fasciolopsiasis This infection, caused by Fasciolopsis buski, is endemic in Asia. The adult fluke (20–​70 × 8–​20 mm in size; Fig. 8.11.4.2) is found in the small intestine of humans and pigs. When eggs are passed in the stools and reach water, the miracidium develops, hatches, and enters the first intermediate host, a freshwater snail of the species Segmentina, Hippeutis, and Gyraulis, among others. In the snail, the miracidium then develops through the stages of sporocyst and rediae and, after 8 weeks or so, cercariae escape from the snail. The cercariae encyst on water plants and develop into metacercariae over 4 weeks. Infection is acquired by ingestion of infected uncooked edible plants such as water caltrop (Trapa species), water chestnut Eliocharis tuberosa, water bamboo Zizania aquatica, and watercress Neptunia oleracea. Fifty years ago, it was estimated that 10 million people were in- fected with this parasite. The current prevalence is entirely unknown. Fasciolopsiasis occurs most commonly in areas where people keep pigs and raise and eat freshwater plants. The adult worms attach themselves to the mucosa of the upper small bowel where they may cause inflammation and erosion and provoke a mucous intestinal discharge. Light infections are gener- ally asymptomatic but heavy worm burdens can be associated with anorexia, nausea, abdominal discomfort and diarrhoea, or even in- testinal obstruction. Rarely, heavy infections may cause small bower perforation. Stools may be foul-​smelling and contain undigested food. In severe cases, a protein-​losing enteropathy is associated with ascites, generalized oedema, and prostration. Eggs (130–​140 × 80–​85 µm in size) are passed in the stools (Fig. 8.11.4.3). These are yellow-​brown, ellipsoid, thin-​shelled, and operculate and contain an immature embryo; they cannot be reli- ably differentiated from those of the intestinal echinostomes or of the liver flukes F. hepatica and F. gigantica. Heterophyiasis This term may be conveniently used to include all infections with flukes of the family Heterophyidae, although some infections are more precisely known by the generic name of the infecting or- ganism (e.g. metagonimiasis). These are small flukes, generally less than 1 to 2 mm in length. Almost 30 species in this family have been reported to infect humans (Table 8.11.4.2). These infec- tions are found in many places but are most common in Asia and Fig. 8.11.4.1  Egg of Echinostoma ilocanum. All echinostome eggs look similar, as do those of Fasciolopsis and Fasciola species. Courtesy of P Radomyos. Fig. 8.11.4.2  Adult Fasciolopsis buski, 6.5 cm in length. Courtesy of P Radomyos. Fig. 8.11.4.3  Egg of Fasciolopsis buski. Note its similarity to ova of Fasciola species and echinostomes. Courtesy of AR Butcher.

8.11.4  Intestinal trematode infections 1565 Egypt. Metagonimus yokogawai is believed to be the most common heterophyid infection. Heterophyids live in the intestines of various mammals and birds. When eggs are passed in the stools, they contain a ciliated mira- cidium which hatches when ingested by a freshwater or brackish-​ water snail, the first intermediate host. Snails susceptible to Heterophyes include Pirenella conica, Cerithidea cingulata, and Tympanotonus micropterus. Semisulcospira libertina and Thiara granifera are host to Metagonimus spp. In the snail, the miracidium then develops through the stages of sporocyst and one or two gen- erations of rediae until leaving the snail as cercariae. The cercariae in turn invade tissues of the second intermediate host, various spe- cies of freshwater or coastal salmonoid and cyprinoid fish. These include mullet (e.g. Mugil cephalus) and minnow (Gambusia spe- cies) for Heterophyes species, and carp (e.g. Carassius carrasius) and sweet fish Plecoglossus altivelis in the case of Metagonimus species. Humans are infected after ingestion of inadequately cooked fish containing metacercariae, which mature in the flesh or scales of the fish. The adult worms attach to or invade the mucosa of the upper small bowel where they may cause granulomatous inflammation and erosion. Light infections are generally asymptomatic but heavy worm burdens may be associated with anorexia, nausea, abdominal discomfort, and mucous diarrhoea. Occasionally ova deposited in the bowel wall enter blood vessels and embolize to other tissues. Eggs have been found in the heart and central ner- vous system and rarely in the blood. In cases of heterophyiasis described in the Philippines, cardiac failure was associated with subepicardial haemorrhages, myocardial damage caused by oc- clusion of vessels by ova, and eggs stuck to a thickened, calcified mitral valve. Neurological features include focal cerebral disturb- ances and transverse myelitis. Table 8.11.4.2  Intestinal trematodes belonging to the family Heterophyidae that infect humans Species Geographical distribution Definitive hosts other than humans Source of infection Size of adults (mm) Size of eggs (µm) Apophallus donicus USA Dogs, cats, rats, foxes, rabbits Fish 1.1–​1.3 × 0.58–​0.72 35 × 25 Acanthotrema felis Korea Cats Fish 0.43–​0.46 × 0.27–​0.29 13–​15 × 25–​28 Acanthotrema (Stictodora) tridactyla Arabia Cats Fish Centrocestus armatus East Asia Cats, dogs, rodents, herons Fish 0.35–​0.63 × 0.18–​0.29 28–​32 × 16–​17 Centrocestus caninus Taiwan Dogs, cats, rats Fish 0.4–​0.45 × 0.21–​0.25 32–​35 × 17–​20 Centrocestus cuspidatus Egypt, Taiwan Chickens, rats Fish 0.5–​0.8 × 0.25–​0.35 30–​35 × 15–​20 Centrocestus formosanus East Asia Rats, cats, dogs, chickens, ducks Fish, frogs 0.42–​0.47 × 0.21–​0.25 0.24–​0.42 × 0.21–​0.25 Centrocestus kurokawai Japan Dogs, rodents (experimental) Fish 0.35–​0.51 × 0.18–​0.23 33–​40 × 17–​21 Centrocestus longus Taiwan Dogs, cats (experimental) Fish 0.6 × 0.15 41 × 22 Cryptocotyle lingua Greenland Cats, dogs, rats Fish 1.2–​2.0 × 0.4–​0.9 42–​48 × 20–​22 Haplorchis pleurolophocerca Egypt Cats Fish 0.32–​0.42 × 0.14–​0.17 29–​32 × 15–​18 Haplorchis pumilio Southeast Asia, Egypt Dogs, cats, birds Fish 0.45–​0.89 × 0.2–​0.4 24–​28 × 12–​15 Haplorchis taichui Asia Dogs, cats Fish 0.47–​0.64 × 0.18–​0.22 20–​33 × 11–​17 Haplorchis vanissimus Philippines Fish 0.38–​0.51 × 0.25–​0.31 25–​30 × 18–​21 Haplorchis yokogawai Asia Dogs, cats Fish 0.47–​0.64 × 0.18–​0.22 20–​33 × 10–​17 Heterophyes heterophyes Egypt, Asia Cats, dogs, rats, foxes, weasels, birds Fish 1.0–​1.7 × 0.3–​0.4 28–​30 × 15–​17 Heterophyes nocens East Asia Dogs, cats, rats Fish 0.9–​1.1 × 0.4–​0.5 28 × 15.5 Heterophyopsis continua East Asia Dogs Fish 2.0–​2.1 × 0.24–​0.28 25–​26 × 14–​16 Metagonimus minutus Taiwan Cats, mice Fish 0.43–​0.50 × 0.25–​0.40 21–​24 × 12–​15 Metagonimus miyatai Korea Fish 0.9–​1.3 ×.04–​0.6 28–​32 × 16–​19 Metagonimus takahashii Korea Dogs, cats, rats, birds Fish 0.84–​1.48 × 0.42–​0.72 28–​34 × 17–​21 Metagonimus yokogawai Asia, Europe Dogs, cats, pigs, pelicans Fish 1.0–​2.5 × 0.40–​0.75 26–​28 × 15–​17 Phagicola sp. Brazil Dogs Fish Procerovum calderoni Philippines Cats, dogs Fish 0.47–​0.55 × 0.25–​0.26 21–​25 × 11–​15 Procerovum varium Japan Cats, birds Fish 0.26–​0.38 × 0.13–​0.16 25–​29 × 12–​18 Pygidiopsis summa Korea Birds, cats, dogs, rats Fish 0.49–​0.76 × 0.25–​0.44 21–​23 × 11–​14 Stellantchasmus falcatus Asia, Hawaii Dogs, cats Fish 0.59 × 0.23 21–​23 × 12–​13 Stellantchasmus formosanus Taiwan Cats, rats Fish 0.32–​0.56 × 0.13–​0.21 18–​24 × 20–​22 Stellantchasmus pseudocirratus Hawaii, Philippines Dogs, cats Fish 0.3–​0.6 × 0.2–​0.3 18–​21 × 9–​12 Stictodora fuscata East Asia Cats, birds Fish 0.59 × 0.23 36–​38 × 22–​23 Stictodora lari Korea Seagulls Fish 0.70–​0.86 × 0.27–​0.36 28–​37 × 17–​20

section 8  Infectious diseases 1566 Eggs (20–​40 × 10–​20 µm in size) are passed in the stools (Fig. 8.11.4.4). They are yellow-​brown, elongated, opercu- late, and contain a miracidium. Eggs of members of the family Heterophyidae cannot be reliably differentiated from each other. Furthermore, they are extremely difficult to differentiate from eggs of Clonorchis sinensis and Opisthorchis species although heterophyids tend to have a smoother egg shell and a less prom- inent shoulder at the operculum, and the abopercular knob may be absent. Other intestinal fluke infections There are another dozen or so species of intestinal flukes be- longing to various families that have been reported to infect hu- mans (Table 8.11.4.3). All appear to be very uncommon and little is known about their epidemiology, although some probably exist in pockets of hyperinfestation, exploiting peculiarities in local behavioural, culinary, and animal husbandry practices. As with other fluke infections, definitive identification depends upon recovery of adult worms, as excreted ova lack discriminatory features (Fig. 8.11.4.5). This is most commonly achieved by treat- ment with praziquantel. Fig. 8.11.4.4  Egg of Metagonimus yokogawai. All heterophyid eggs look similar, as do those of Clonorchis sinensis and Opisthorchis viverrini. Courtesy of P Radomyos. Table 8.11.4.3  Families of intestinal trematodes containing species that are uncommon human pathogens Species Geographical distribution Definitive hosts other than humans Source of infection Size of adults (mm) Size of eggs (µm) Brachylaimidae Brachylaima cribbi South Australia Mice, birds Land snails 6–​12 × 0.3–​0.5 28–​30 × 16–​17 Gastrodiscidae Gastrodiscoides hominis Asia, Nigeria Pigs, rats, monkeys, deer Water plants 4–​8 × 3–​4 150 × 72 Gastrothylacidae Fischoederius elongatus China Ruminants Aquatic plants 9–​20 × 3–​6 110–​140 × 60–​80 Gymnophallidae Gymnophalloides seoi Korea Birds Oysters 0.4–​0.5 × 0.2–​0.3 20–​25 × 11–​15 Lecithodendriidae Phaneropsulus bonnei Thailand, Indonesia Bats, monkeys Dragonflies 0.48–​0.78 × 0.22–​0.35 27–​29 × 10–​12 Phaneropsulus spinicirrus Thailand 0.55–​0.76 × 0.43–​0.63 27–​33 × 13–​16 Prosthodendrium molenkampi Thailand, Indonesia Bats, monkeys, rats Dragonflies, damselflies 30 × 15 Microphallidae Gynaecotya squataroloe Korea Birds Crabs 560–​690 × 285–​361 21 × 17 Spelotrema ( = Carneophallus) brevicaeca Philippines Birds Crabs 0.5–​0.7 × 0.3–​0.4 15–​16 × 9–​10 Nanophyetidae ( = Troglotrematidae) Nanophyetus salmincola Russia, North America Dogs, foxes, birds Fish 1–​2 × 0.3–​0.5 80 × 40 Neodiplostomidae Neodiplostomum seoulens Korea Freshwater snails Frogs, snakes 0.8–​1.2 × 0.4–​0.5 86–​99 × 55–​63 Paramphistomatidae Watsonius watsoni Southern Africa Monkeys Water plants? 8–​10 × 4–​5 120–​130 × 75–​80 Plagiorchidae Plagiorchis harinasutai Thailand Insect larvae 1.75–​1.87 × 0.60–​0.65 32–​34 × 16–​18 (continued)

8.11.4  Intestinal trematode infections 1567 FURTHER READING Chai JY (2007). Intestinal flukes. In:  Murrell KD, Fried B (eds). Food-​borne parasitic zoonoses, p. 429. Springer, New York, NY. Chai JY, et al. (2009). Foodborne intestinal flukes in Southeast Asia. Korean J Parasitol, 47, Suppl, S69–​S102. Keiser J, Utzinger J (2009). Food-​borne trematodiases. Clin Microbial Rev, 22, 466–​83. Sripa B, et  al. (2010). Foodborne trematodiases in Southeast Asia: epidemiology, pathology, clinical manifestation and control. Adv Parasitol, 72, 305–​50. Toledo R, Fried B (eds) (2014). Digenetic trematodes: advances in experi- mental medicine and biology, Vol. 766. Springer, New York, NY. Photographs of various stages of these parasites
and diagrams of life cycles may be found at several excellent websites: Centers for Disease Control and Prevention. http://​www.dpd.cdc.gov/​ DPDx/​HTML/​Image_​Library.htm Korean Society for Parasitology. http://​www.atlas.or.kr Fig. 8.11.4.5  Egg of Brachylaima cribbi. Courtesy of AR Butcher. Species Geographical distribution Definitive hosts other than humans Source of infection Size of adults (mm) Size of eggs (µm) Plagiorchis javensis Indonesia Birds, bats Insect larvae 1.8 × 0.7 36 × 22–​24 Plagiorchis muris Japan Birds, dogs, rats Snails, aquatic insects 0.8–​2.0 × 0.24–​0.84 36 × 21 Plagiorchis philippinensis Philippines Birds, rats Insect larvae 1.5–​2.0 × 0.39–​0.44 28–​30 × 19–​21 Strigeidae Cotylurus japonicus China Birds Snails Table 8.11.4.3  Continued

8.12 Nonvenomous arthropods 1568

8.12 Nonvenomous arthropods 1568

ESSENTIALS Most medically important arthropods are insects (including mos- quitoes, midges, other flies, bedbugs, and other true bugs, lice, fleas, and cockroaches) or arachnids (spiders, ticks, mites, scorpions). Clinical features Arthropod-​related problems include the following: (1) injuries from direct contact (bites, stings, and other penetrating or crushing in- juries from spines, bristles, or pincers) and the consequences of such contact (envenoming, allergic reactions, secondary infection of wounds, and transmission of infectious agents); (2)  infestation of the patient’s body, skin, hair, clothes, or immediate environment (myiasis, canthariasis, tungosis, pediculosis, and so on); (3) inhalant allergy; (4) hygiene and aesthetic issues; and (5) the psychological phenomena of delusion and phobia. Treatment and prevention—​general aspects Broad principles of management include: (1) Identification of the problem and the kind of arthropod involved. (2)  The immediate treatment—​if necessary—​of allergic reactions or secondary infection. (3)  Appreciation of consequences of exposure, such as transmis- sion of infectious agents; many species of dipterans (flies)—​including mosquitoes, blackflies, sand flies, tsetse flies, and horse flies—​bite humans, and in some regions some of these are important vectors. (4) Use of antimalarials or vaccines and the development of strat- egies to avoid further contact, including eradication of infestations, changes in behaviour, use of repellents and clothing that covers the skin, and bed nets. Travellers and their clinicians should be aware of the risks posed by arthropod-​borne infections and ways to prevent them in particular geographical areas. Particular conditions True bugs (Hemiptera)—​bedbugs infest dwellings and bite at night: pa- tients may complain of mysterious skin lesions and sleeplessness, and a special search may be necessary to find the bugs. In South America, triatomine bugs bite at night and are vectors of trypanosomiasis. Ticks (Ixodoidea)—​these attach themselves while feeding and are noticed by the patient. They are important vectors of many infec- tions, which are often specific to particular genera or species of tick and confined to particular geographical areas. In Europe, tick-​related infections include Lyme borreliosis and tick-​borne encephalitis. Infestations—​clinically important infestations include the fol- lowing. (1) Scabies (infestation of the skin by scabies mites) and pediculosis (infestation of the hair or clothing by head or body lice)—​these are cosmopolitan in distribution and usually managed by use of topical acaricides or insecticides, although resistance is a growing problem. (2) Fleas—​the human flea is now rare in the developed world, but infestation of dwellings with cat fleas is com- monly reported. Tungosis is a condition of tropical areas where jigger fleas (not to be confused with similarly named trombiculid mites) burrow into the feet or under the toenails of those who walk about barefoot. (3) Fly and beetle larvae—​myiasis, which is the infestation of the body by the larvae (maggots) of dipteran flies, is classified as (a) benign when self-​limiting or malign when there is destructive tissue invasion, (b) according to anatomical site (dermal, wound, orbital, ophthalmic, urogenital, intestinal), and (c) according to the species involved. Canthariasis—​infestation of the body by beetles or beetle larvae—​is clinically similar to myiasis and is rarely reported. Other aspects—​some synanthropic insects, especially certain spe- cies of fly, cockroach, and pharaoh’s ants, have been implicated in the passive transmission of infections (e.g. shigellosis and hepatitis A). It is generally considered to be in the interests of good hygiene to control these insects in healthcare settings or where food is prepared. Introduction Most arthropods are harmless, but there is a select group of medic- ally significant species. Invertebrates with jointed limbs belong to the phylum Arthropoda. Most of the medically important arthropods are in the classes Insecta (insects) or Arachnida (spiders, ticks, mites, scorpions). Some members of the class Chilopoda (centipedes) may bite humans, and some of the larger members of the Crustacea (crabs, lobsters) may cause injury with pincers or spines. Although they are classified as a separate group, phylum Pentastomida, there is some evidence that the parasitic tongue worms may actually be highly specialized crustaceans (Chapter 8.13). Categories of med- ical significance include: envenoming by bites or stings (Chapter 10.1.2); allergic reactions to bites, stings, hairs, or inhaled allergens; transmission of infectious agents; infestation; the pain and trauma from bites or penetrating spines; phobia and delusory parasitosis. Arthropods may cause nuisance by their presence or the noises they may make, or by being perceived as unhygienic. To allow a logical approach to the management of arthropod-​related issues it is helpful to identify the species involved, although as this may not always be 8.12 Non​venomous arthropods John Paul

8.12  Nonvenomous arthropods 1569 possible, generic approaches may be developed to the management of problems. Bites Arthropod bites are common and often trivial, but bites may be important when associated with envenoming (Chapter 9.2), sensi- tization (leading to pruritus, excoriation, and secondary infection), anaphylaxis, or the transmission of infectious agents. Biting insects may simply be a nuisance (e.g. it may be difficult to tolerate swarms of biting flies, making it difficult to work outdoors and dangerous to operate machinery). Immune response varies with age, past ex- posure, and other factors. Management may be directed towards treatment of the bite, if necessary (topical corticosteroids, sys- temic antihistamines), considering the risk of transmitted infec- tion and prevention of further bites (eradication of ectoparasites, change in behaviour to avoid exposure, repellents, special clothing, insecticide-​impregnated bed nets). It is often possible to associate bites with infesting ectoparasites, such as arthropods which remain attached (ticks) or predatory bloodsuckers that are highly visible (mosquitoes, midges, and blackflies, when swarming) and which cause immediately painful bites (tsetse flies, some mosquitoes, tabanid flies). It is harder to ascribe a cause to bites from arthropods which bite at night or when the patient is asleep (some mosquitoes, sand flies, bedbugs, triatomine bugs) or from arthropods that are inconspicuous and do not cause immediately painful bites (harvest mites, some fleas, some biting flies). Bites of larger arthropods typic- ally have a central punctum and a surrounding area of inflammation and are pruritic. In cases of uncertainty it may be necessary to obtain a dermatological opinion to exclude other diagnoses, including or- ganic disorders, artefact, and delusion. Bloodsucking flies (Diptera) Many flies are haematophagous (Table 8.12.1). Bloodsucking flies include members of the suborder Nematocera (mosquitoes, sand flies, balck flies, biting midges) and the suborder Brachycera (horse flies, clegs, snipe flies, stable flies, and keds). All bloodsucking flies are at least a nuisance: the bites are often painful and associated with sensitization. More importantly, biting flies may transmit infection. Mosquitoes (Culicidae) are vectors of filariasis and numerous viral diseases, including yellow fever and dengue fever. Mosquitoes of the genus Anopheles transmit malaria. Depending on species and loca- tion, mosquitoes bite at various times of the day. Mosquitoes may be controlled by reducing their access to stagnant water needed for development of their larval stages and by application of insecticides Table 8.12.1  Bloodsucking flies Family Representative genera (and species) Associated agent or condition Suborder Nematocera Culicidae (mosquitoes) Subfamily Anophelinae Anopheles Malaria, brugian and bancroftian filariasis Subfamily Culicinae Culiseta Western equine encephalitis Culex Bancroftian filariasis Mansonia Brugian filariasis Aedes Eastern equine encephalitis, dengue fever, yellow fever, bancroftian filariasis Haemagogus Yellow fever Sabethes Yellow fever Phlebotomidae (sand flies) Phlebotomus Leishmania spp. Lutzomyia Leishmania spp., Bartonella bacilliformis Simuliidae (blackflies) Simulium Onchocerca volvulus, Mansonella ozzardi, haemorrhagic syndrome of Altimira Ceratopogonidae (biting midges) Culicoides Dipetalonema perstans, Mansonella ozzardi Suborder Brachycera Tabanidae (horse flies, clegs) Haematopota Tabanus Pangonia Chrysops Loa loa Rhagionidae (snipe flies) Symphoromyia Atherix Spaniopsis Austroleptis Glossinidae (tsetse flies) Glossina African trypanosomiaisis Calliphoridae (Congo floor maggot) Auchmeromyia luteola Muscidae Stomoxys calcitrans (stable fly) Hippoboscidae Melophagus ovinus (sheep ked) Lipoptena cervi (deer ked)

section 8  Infectious diseases 1570 Fig. 8.12.1  Reaction to blackfly (Simulium sp.) bites, 48 h after exposure. Algonquin, Ontario, Canada. to dwellings. Use of permethrin-​impregnated bed nets has been shown to reduce malaria transmission. Sand flies (Phlebotominae) are mainly tropical and subtropical in distribution and transmit leishmaniasis. In South America, sand flies of the genus Lutzomyia transmit Bartonella bacilliformis. Blackflies (Simuliidae) occur worldwide and are vectors of Onchocerca volvulus and Mansonella ozzardi. Blackfly larvae require well-​oxygenated water. Female blackflies pierce the skin and suck blood from the edge of the punc- ture. Substances in blackfly saliva inhibit platelet aggregation, im- pair the final common pathway of the coagulation cascade, and encourage vasodilatation. The bites, oozing blood, have a charac- teristic appearance and may be associated with severe reaction, sometimes referred to as simuliosis or simuliotoxicosis. Puncture sites often become surrounded by a wide zone of haemorrhagic ery- thema and oedema (Fig. 8.12.1). Rarely, haemorrhagic shock may occur. In Brazil, the haemorrhagic syndrome of Altimira has been epidemiologically associated with exposure to blackflies. Blackfly saliva appears to contain immunomodulating substances. In Brazil, the autoimmune condition ‘fogo selvagem’ (a form of pemphigus foliaceus) occurs in simuliid-​infested areas (Fig. 8.12.2a). In Britain, blackflies are rarely troublesome to humans except in certain lo- calities. In southern England, the Blandford fly, Simulium postica- tum occurs on the river Stour, Dorset, on tributaries of the river Thames in Oxfordshire, and on other rivers. In 1993, 16% (22% female, 9% male) of Blandford’s inhabitants reported bites. Use of the biological larvicide Bacillus thuringiensis as a control measure was associated with a marked drop in the number of people com- plaining of bites. In Scotland, S. reptans, S. argyreatum, and other species may bite humans. Biting midges (Ceratopgonidae) (Fig. 8.12.3) are vectors of the filarial worms Mansonella (Dipetalonema) perstans and M. ozzardi. In Africa, tabanid flies transmit Loa loa. Tsetse flies are vectors of African trypanosomiasis (see Chapter 8.8.11). Deer keds Lipoptena cervi are highly evolved louse-​like flies with biting mouth parts that feed on deer. Occasionally, deer keds bite people, such as forest workers, hunters, or entomologists (Fig. 8.12.2b). Deer ked derma- titis is a condition where itchy papules exist for weeks to months at the site of bites. Eventually, papules resolve without specific treat- ment. It has been suggested that Bartonella schoenbuchensis may have a role in the aetiology of the conditions as this agent has been detected in deer keds but not as yet in humans. Prevention When visiting locations where biting flies are troublesome, bites may be avoided to some extent by wearing clothing that covers the skin and by use of repellents. True bugs (Hemiptera) Bedbugs The common bedbug Cimex lectularius (Fig. 8.12.4) is cosmopol- itan. In recent years, reports of infestations in developed coun- tries such as the United Kingdom and United States of America have increased. The resurgence has been linked to the emergence of resistance to pyrethroid insecticides used for bedbug control. Infestation may be unrelated to lack of general hygiene but as- sociated with translocation of personal effects or furniture. The (a) (b) Fig. 8.12.2  (a) Endemic Pemphigus foliaceus (‘fogo selvagem’ meaning ‘wild fire’) in a man from a rural area of São Paulo State, Brazil infested with Simulium flies. (b) Deer ked or deer fly without wings (Lipoptena cervi Diptera, Hippoboscidae). (a) Copyright DA Warrell. (b) Copyright J Paul.

8.12  Nonvenomous arthropods 1571 tropical bedbug C.  hemipterus occurs in tropical and subtrop- ical countries. Epidemiological studies have failed to produce clear evidence of bedbugs as vectors of infections, such as hepa- titis B. They are nocturnal, hiding during the day and feeding at night. Although in some cases bites may go unnoticed and there may be no allergic reaction, bedbugs may cause sleeplessness, and the bites may cause pain and swelling and, exceptionally, dissem- inated bullous eruptions (Fig. 8.12.5). Rooms that are heavily in- fested may acquire an unpleasant odour. Bugs may be found by making special searches at night or by seeking their hiding places during the day. They resemble lentils superficially, being round and flat. Adults reach a length of about 5 mm. Nymphs pass through five instars to reach adulthood after about 4 months. Bedbugs can live for 6 months without feeding, becoming paper-​thin. Related bugs which occasionally bite humans derive from pigeons, bats, and martins (C. columbarius, C. pipistrelli, and Oeciacus hirundinis respectively). Infestation may be managed by restricting access of host species to dwellings, but in the United Kingdom, for example, bats are protected under the Wildlife and Countryside Act. Prevention and control  Pyrethroid insecticides are widely used to control bedbugs but emergence of resistance means that they are becoming unreliable. As a consequence, alternatives including carbamates, arylpyrroles, neonicatinoids, and organophophates are being used increasingly although availability varies from country to country. Cone-​nose bugs Most of the 129 species of cone-​nose bugs (family Reduviidae, sub- family Triatominae) occur in the Americas. Seven species occur in Asia and one species, Triatoma rubrofasciata, is cosmotropical. Many triatomines are obligate feeders on the blood of vertebrates. Triatomines transmit South American trypanosomiasis. Important vector species are Rhodnius prolixus, T.  infestans, T.  brasiliensis, T. dimidiata, and Panstrongylus megistus. The bugs infest dwellings, hiding in crevices during the day and biting at night. Dwellings may be heavily infested: in Columbia, 11 403 specimens of R. prolixus were reported from a house occupied by nine people, all of whom were seropositive for trypanosomiasis. As well as transmitting tryp- anosomiasis, triatomines may cause significant blood loss to occu- pants of infested buildings. Prevention  Dwellings are deinfested with insecticides and con- structed to offer few hiding places for the bugs (Chapter 8.8.11). Ticks (Ixodoidea) Hard ticks (Ixodidae) and soft ticks (Argasidae) occur worldwide. Stages of the life cycle are egg, larva (six-​legged), and nymph and adult (both eight-​legged). Ticks attach and feed with a barbed hypostome and detach when engorged. Smaller stages and ticks in inconspicuous sites, such as the perineum may feed unobserved. Bites are usually painless but may result in local sensitization, sec- ondary infection, and transmission of infectious agents, including numerous viruses, rickettsias, and Lyme disease (Table 8.12.2). Local reaction to bites may be confused with erythema migrans of Lyme disease, (which expands as a ring with a central punctum—​ see Chapter 8.6.33). Ticks may be removed by gripping with forceps (or, in the field, with finger and thumbnail), between the skin and Fig. 8.12.5  Erythematous macules of bedbugs. Courtesy of D Hill, Adelaide, South Australia. Fig. 8.12.3  Reaction to midge (Ceratopogonidae) bites, 24 h after exposure. Sligachan, Isle of Skye, United Kingdom. Fig. 8.12.4  Bedbugs Cimex lectularus. Copyright J Paul.

section 8  Infectious diseases 1572 the tick’s head and pulling gently. Special tools for removing ticks have been made widely available by the pet industry and such de- vices should work just as well with humans. Toothed devices that work in the manner of combs or forceps that are curved in pro- file (tick tweezers) have the advantage of allowing removal while avoiding squeezing the tick. Careless removal may detach the head or hypostome, leaving a potential source of inflammation and sec- ondary infection. In the United Kingdom, the ticks most often found on humans are the sheep tick Ixodes ricinus (a vector of Lyme dis- ease) and the hedgehog tick I. hexagonus (Fig. 8.12.6). Prevention When visiting tick-​infested places, bites may be avoided by tucking trousers into boots and wearing light-​coloured clothing which makes ticks highly visible. After visiting tick-​infested habitats, searches of the body allows prompt removal of ticks which reduces the chance of disease transmission. Harvest mites (Trombiculidae) In the United Kingdom, larvae of the harvest mite Neotrombicula autumnalis are a common cause of bites in late summer, especially in chalk downland. They are tiny and seldom noticed, crawling rap- idly on to the body, attaching (often under tight-​fitting clothes), injecting proteolytic enzymes, feeding on tissue fluid, and then detaching, leaving pruritic, sometimes bullous lesions hours later. For many victims, the cause of irritation remains a mystery. Red bugs or chiggers (confusingly, a term also applied to the flea Tunga penetrans) are names given to trombiculids in the Americas. Bites to the penis, associated with swelling and dysuria, have been described in the paediatric literature as ‘summer penile syndrome’. In Asia, trombilucids are vectors of scrub typhus. Prevention Where trombiculids are troublesome, tucking trousers into boots and applying diethyltoluamide or other repellents may be partially effective. Notorious ‘mite islands’ densely infested with trombiculids in cleared areas of jungle should be avoided. Accidental bites Arthropods which do not normally bite humans but can inflict painful but usually trivial bites when provoked by handling (e.g. by children and entomologists), include predatory true bugs such as the water Table 8.12.2  Ticks and tick-​borne diseases Genus and species Geographical distribution Associated infections Argasidae (soft ticks) Ornithodoros spp. Widely distributed Endemic relapsing fever Ixodidae (hard ticks) Amblyomma hebraeum Africa Tick typhus Amblyomma cajennense Americas Rocky mountain spotted fever Dermacentor andersoni North America Colorado tick fever, Rocky Mountain spotted fever Dermacentor marginatus Palaearctic Tick typhus, Omsk haemorrhagic fever Dermacentor silvarum Eastern Palaearctic Tick typhus, tick-​borne encephalitis Dermacentor variabilis North America Rocky Mountain spotted fever Haemaphysalis concinna Palaearctic Tick typhus Haemaphysalis spinigera India Kyasanur Forest disease Haemaphysalis turturis India Kyasanur Forest disease Hyalomma spp. Old World Crimean-​Congo haemorrhagic fever Ixodes scapularis Eastern North America Lyme disease Ixodes pacificus Western North America Lyme disease Ixodes ricinus Western Palaearctic Lyme disease, tick-​borne encephalitis, louping ill Ixodes persulcatus Eastern Palaearctic Tick-​borne encephalitis, Omsk haemorrhagic fever Rhipicephalus sanguineus Cosmopolitan Tick typhus Fig. 8.12.6  Upper side of hedgehog tick Ixodes hexagonus, to show sucking mouthparts (hypostome). Copyright J Paul.

8.12  Nonvenomous arthropods 1573 boatman Notonecta glauca and the assassin bug Reduvius personatus in the United Kingdom and wheel bugs Arilus spp. in the Americas; larger beetles (Coleoptera); dragonflies (Odonata); and bush-​crickets (Orthoptera) such as the wartbiter Decticus verrucivorus. Spines used in defence by the great silver diving beetle Hydrous piceus and larger tropical grasshoppers of the subfamily Cyrtacanthridinae can cause penetrating injury when handled. Pincers of larger crabs and lobsters (Crustacea) can cause crushing injuries of digits and their spines may cause penetrating injury. Infestation Sites of infestation include the hair, body surface, and immediate environment (ectoparasites:  lice, fleas); the skin and subdermis (scabies, tungosis, dermal myiasis); wounds, tissues, and orifices (myiasis); and the gastrointestinal tract (myiasis, canthariasis). With ectoparasites, the main problems are related to their bites: diag- nosis and management may be based on the identification of the ectoparasite. Delusory parasitosis is a condition in which the patient becomes convinced of infestation by parasites despite reassurance by the doctor and absence of clinical or laboratory evidence. Scabies The agent of human scabies, a chronic infestation, is the human sca- bies mite Sarcoptes scabiei var. hominis. Scabies mites adapted to other hosts, such as Sarcoptes scabiei var. canis, cause a self-​limiting pruritus in humans. Clinical manifestations of scabies are caused by the adult female mite that burrows through the epidermis. The adult female is oval and about 0.33 mm long (Fig. 8.12.7). The fe- male lives for about 1 month, burrowing and ovipositing daily. The burrow may extend to 1 cm in length. Six-​legged larvae hatch after a few days and moult to become eight-​legged nymphs and later eight-​legged adults. Adult males are smaller than females, do not burrow, and die after mating on the epidermis. Scabies is cosmo- politan in distribution. Prevalence rates vary but may be higher in conditions of overcrowding and following social disruption in war- time. Outbreaks may occur in nursing homes and hospitals. Most cases must be acquired by close contact, as the mites do not survive long away from the body. The main presenting symptom is pruritus which occurs with sensitization about 1 month after the onset of in- festation. Symptoms may be worse at night and after a hot bath or shower. Burrows commonly occur in web spaces between the fingers and on the wrists but may be very widespread. There is often evi- dence of excoriation but the appearance of the skin is variable and may show secondary infection, eczematization, lichenification, and papulovesicles (Figs. 8.12.8 and 8.12.9). Careful examination may Fig. 8.12.7  Adult specimen of Sarcoptes scabei. Courtesy of RV Southcott, Adelaide, South Australia. Fig. 8.12.8  Secondarily infected scabies in mother and child. Fig. 8.12.9  Papulovesicular lesions of scabies.

section 8  Infectious diseases 1574 reveal burrows and mites. Diagnosis may be confirmed by micros- copy of scrapings from affected areas, especially interdigital spaces, but many cases are atypical and a dermatological opinion may be required to exclude other causes. Immunosuppressed patients, including transplant recipients and patients with AIDS, are prone to crusting or so-​called Norwegian scabies in which crusting lesions of scales and mites accumulate over the hands, feet, and other sites such as eyebrows, but the patient suffers relatively little discomfort. Such cases, and presumably their fomites, are highly contagious. Occasionally the mites Dermanyssus gallinae and Ornithonyssus spp., whose normal hosts are birds, bite humans, causing lesions that resemble scabies. Treatment Treatment of scabies is by topical application of acaricides. Aqueous lotions of 0.5% malathion or 5% permethrin are currently recom- mended in the United Kingdom, given as two treatments a week apart. γ-​Benzene hexachloride is also effective. The lotion is ap- plied to the whole body surface of all affected people and left on for 24 h before being washing off. Itching may persist for several weeks and requires a topical counterirritant and corticosteroid (e.g. crotamiton and hydrocortisone) and a sedating antihistamine (chlorphenamine at night). Ivermectin (200 µg/​kg single dose) is used for Norwegian scabies and in patients whose severe excori- ations make topical treatment intolerably irritating and painful. During outbreaks, it may be necessary to treat whole cohorts of pa- tients or healthcare teams. Louse infestation Lice are obligate parasites of animals. They bite using piercing mouthparts to feed on blood or tissue fluids. Three species, of cosmopolitan distribution, are associated with humans: the pubic louse Pthirus pubis, the body louse (or clothing louse) Pediculus humanus (Fig. 8.12.10), and the head louse P. capitis. Body and head lice are morphologically similar and are treated by some au- thors as subspecies or forms of P. humanus. Lice complete their life cycle on their host. Adult females deposit eggs (nits) on hair shafts (pubic and head lice) or on clothing (body louse). Larvae hatch after about 1 week, begin to feed and over the course of about 2 weeks, undergo several moults before reaching adulthood. Adult females live for about 1 month and may lay about 100 eggs. Egg cases re- main where attached and may persist after successful treatment of infestation. Most infestations are probably acquired through close contact with an infested case, but some cases may result from con- tact with clothing, bedclothes, or hairbrushes containing living lice or their eggs, which may be attached to shed hairs. In addition to the aesthetic and social drawbacks of louse infestation, medical problems common to all three taxa relate to sensitization of the host to louse antigens from bites and the resulting pruritus which may lead to excoriation and secondary infection. Louse bites have a central punctum and surrounding small red macule. Body lice may transmit several agents, including those of endemic typhus (Rickettsia prowazekii), trench fever (Bartonella quintana), and relapsing fever (Borrelia recurrentis). Pubic lice (crab lice) The lice (Pthirus pubis) attach themselves to pubic hairs. Rarely, lice may be found on eyebrows, eyelashes (phthirosis palpebrarum), axillary, head, or chest hair. Eggs are deposited on hair shafts. Most infestations are probably acquired through sexual contact with an infested case. Children may acquire phthirosis at atypical sites through close contact with adults. Lice seldom stray from the body. Transmission is possible but unlikely without close contact with an infested case. The main symptom is pruritus, sometimes with excoriation and secondary infection. Grey patches (maculae caeruleae) may occur on the skin. Diagnosis is by observation of the lice, which may be difficult to find, or of eggs or egg cases at- tached to hair shafts. Adults are 1 to 2 mm long. The anterior legs are smaller than the other two pairs. The body is squat and crablike (body length, excluding head, c.1.2 times body width) (Fig. 8.12.11). The original description contained a printing error (pthirus) for phthirus (Greek: louse). Fig. 8.12.10  Louse Pediculus humanus. Head lice and body lice are morphologically similar. Copyright J Paul. Fig. 8.12.11  Adult specimen of Pthirus pubis. Courtesy of D Hill, Adelaide, South Australia.

8.12  Nonvenomous arthropods 1575 Treatment Aqueous carbaryl, permethrin, phenothrin, or malathion is applied to the whole body and left on for 1–​2 days. This is repeated a week later to kill newly hatched larvae. Sexual contacts must be treated. Head lice Head lice infest the scalp and rarely other body sites. They lay their eggs at the base of hair shafts. Infestation is more common in chil- dren than in adults and more common in females than in males. Prevalence rates vary but may be very high in certain communities or institutions, such as schools. Prevalence rates may be high despite good standards of hygiene. Most cases probably occur as a result of close contact. The main symptom is pruritus which may be associ- ated with excoriation, secondary infection, and lymphadenopathy. Diagnosis is by observation of lice, which generally remain close to the scalp, or of eggs or egg cases, attached to hairs (Fig. 8.12.12). A fine comb (nit comb) may be used to collect material to make the diagnosis. Adults are 3–​4 mm long. Treatment Insecticide lotion (malathion, permethrin, phenothrin, dimeticone, or carbaryl) is applied to the scalp overnight. This is repeated a week later to destroy newly hatched larvae. Permethrin failure has been reported from many parts of the world. Compared with labora- tory reference strains, lice collected from infestations failing to re- spond to permethrin have shown relative resistance to the agent. In Israel, there is evidence that permethrin resistance may be due to monooxygenase plus nerve insensitivity resistance mechan- isms. Malathion resistance has been reported and may be due to a malathion-​specific esterase. Pediculocides should be used with caution in children and asthmatics. Regular and fastidious use of a nit comb may be used (on its own or in combination with a pediculocide) to treat infestation. There is much anecdotal evidence, that combing can be effective, and it avoids concerns of pediculocide toxicity and resistance, but a study in Wales showed combing to be less effective that chemical treatment. In institutions, coordinated treatment campaigns may be required to prevent reinfestation. Body lice Body lice infest clothing and body hair. They lay their eggs on clothing, often along seams. Body lice are morphologically like head lice but slightly larger. Body louse infestation is associated with poor hygiene and social deprivation, as may occur in wartime. Transmission occurs as a result of close contact or through contact with infested clothing. Bites occur on the body, resulting in pruritus which may be associated with excoriation, eczematization, and secondary infection. Diagnosis is confirmed by finding lice, usually on clothing. Treatment Infestation may be treated by topical application of carbaryl or malathion to the whole body, repeated a week later to kill newly hatched larvae. Hot washing of clothing will destroy adults and early stages. Fleas (Siphonaptera) Fleas are bloodsucking ectoparasites. There are thousands of species, adapted to various host animals. Adults are a few millimetres long, brown, laterally compressed, and typically very active. Adults move through the fur or under clothing but can survive in the environment for long periods without feeding. Eggs are dropped to the ground, where the larvae develop, feeding on organic matter. The pupa may remain in the environment for long periods before the adult emerges. Increasing standards of hygiene in developed countries have made the human flea Pulex irritans a rarity. Most flea bites in Britain are now due to cat and dog fleas, Ctenocephalides felis
(Fig. 8.12.13) and C. canis, either through direct exposure to an in- fested animal or to an environment exposed to an infested animal, possibly months previously. Flea bites result in intense pruritus at the bite site. There is a central punctum and there may be bulla for- mation (Fig. 8.12.14). Flea bites often occur in groups. Although patients may not witness fleas, clues that bites have been caused by fleas include intense pruritus, the appearance of bites in small linear groups, and a history of exposure to a flea-​ridden animal or its do- main. Troublesome bites may be treated with topical corticosteroids and systemic antihistamines. There is circumstantial evidence that Fig. 8.12.12  Nits attached to hair. Photograph from a patient with pediculosis showing several hair fibres with numerous egg cases attached. Courtesy of D Hill, Adelaide, South Australia. Fig. 8.12.13  Cat flea Ctenocephalides felis, a common cause of flea bites in humans. Copyright J Paul.

section 8  Infectious diseases 1576 cat fleas may act as vectors of Bartonella henselae, the agent of Cat-​ scratch disease. In the Netherlands cat fleas were shown to harbour Bartonella clarridgeiae and Rickettsia felis, both of which have been reported, albeit rarely, as agents of human disease. Prevention Good domestic hygiene is important. Infested animals and envir- onments should be treated with insecticides. Certain species of flea are vectors of several infectious diseases including plague and murine typhus. Tungosis Tungosis is infestation by a flea Tunga penetrans, known as the jigger, chigger, chigoe (popular names shared with trombiculid mites) or sand flea. Tungosis is a zoonosis that affects a range of domestic ani- mals as well as humans. The gravid female, about 1 mm long, bur- rows into exposed skin (usually the foot), or under a toenail, and swells to about 1 cm in diameter, causing local inflammation and discomfort. The wearing of footwear prevents infestation. In the developed world tungosis is regarded as an easily treatable condi- tion that is occasionally seen in returning travellers. Lesions may be enucleated surgically, and the diagnosis confirmed by histology. Local remedies in endemic areas (tropical Africa and the Americas) of shelling out fleas may leave cavities prone to secondary infection and lethal tetanus. In endemic areas of the tropics, such as in parts of Brazil and Uganda, high levels of infestation may cause significant disability and morbidity. Myiasis Myiasis is the infestation of living animals by the larvae of flies (Diptera). Useful schemes of classification of myiasis include those based on the anatomical site (dermal, subdermal, wound, naso- pharyngeal, orbital, ophthalmic, aural, urogenital, pulmonary, in- testinal) and on the species of fly involved. Myiasis caused by flies whose larvae are obligate parasites of living tissues may be termed specific or primary myiasis. Myiasis associated with larvae which feed on decaying organic matter may be termed opportunistic or secondary myiasis. Myiasis due to larvae which find their way into the body (especially the gastrointestinal tract) by chance may be called accidental myiasis. Of the many species listed as possible agents (Table 8.12.3), most are opportunists whose saprophagous larvae feed on decaying organic matter, which might include nec- rotic wound tissue. Opportunists usually confine themselves to dead tissue and may even benefit the healing process. There is no dip- terous obligate intestinal parasite of man. Intestinal myiasis may be caused by coprophagous larvae which invade the rectum or by resilient maggots, such as those of the false stable fly Muscina stabulans and the cheese skipper Piophila casei which survive when swallowed in food and may cause intestinal dis- turbance and scarring. Intestinal myiasis may be spurious, following diagnosis based on observation of rapidly hatching larvae on freshly passed faeces. Rat-​tailed maggots, larvae of drone flies Eristalis spp., are sometimes referred for identification to laboratories and numerous case reports link these maggots to intestinal myiasis. As the maggots naturally live in aqueous environments that are rich in organic matter, the finding of maggots in latrines may represent spurious association in some cases. Flies from several genera, not- ably Fannia, may cause urogenital myiasis. Scuttle flies (Phoridae) have been reported to cause pulmonary myiasis, possibly following inhalation of the gravid female fly. A small number of flies are obli- gate parasites of living tissues and a few species are closely associated with, but not specific to, humans. Many cases of myiasis are benign, self-​limiting, and relatively harmless, but aural, nasopharyngeal, and malign wound myiasis are potentially lethal entities that may require removal of the larvae and possibly reconstructive surgery. Myiasis is diagnosed by observing dipteran larvae in a lesion. Identification of larvae may require entomological expertise but management of the patient, which depending on the type of lesion, may involve the re- moval of larvae, surgical exploration, debridement, or treatment of secondary infection, should be based on clinical assessment. Dermal myiasis The human bot fly Dermatobia hominis is a common cause of dermal myiasis in the American tropics. The female fly lays her eggs on biting arthropods, such as mosquitoes. The eggs hatch when in con- tact with skin into which the larva burrows. The larval stage lasts about 10 weeks, a boil with a small aperture forming as the larva grows. Such boils are not infrequently seen in Europeans returning from the neotropics. The larva may grow to more than 1 cm in length (Fig. 8.12.15). An early symptom is sporadic pain caused by the spiny larva. Unless in an unusual anatomical site, such as close to the eye, infestation is generally harmless. Secondary infection of the Fig. 8.12.14  Flea bites; erythematous macropapule with central bite point visible. Courtesy of D Hill, Adelaide, South Australia.

8.12  Nonvenomous arthropods 1577 wound is the most common complication. Larvae may be removed through a simple incision. Alternatively, a commercially available snake venom extractor (not recommended for snake bite) has been shown to serve as a useful tool for removal of the larvae. Remedies which include application of raw meat or glue to the lesion may not be successful. Squeezing may rupture the larva to evoke a local granulomatous reaction. The tumbu fly Cordylobia anthropophaga is widespread in the Afrotropical region. There have also been rare reports of apparent acquisition in Spain and Portugal. The female oviposits on sand and also on drying clothes. Ironing destroys eggs. Contact with viable ova on clothing leads to infestation. The larvae pierce the skin and grow rapidly. An uncomfortable boil forms which oozes serosanguinous fluid (Fig. 8.12.16). Fever and lymphadenopathy may occur. Larvae reach maturity in about 10 days. Larvae may be removed through a simple incision, but with care it may be possible to express larvae following application of petroleum jelly (Fig. 8.12.17). The larvae of warble flies Hypoderma spp. occasionally cause dermal myiasis in humans. Larvae of horse bot flies Gasterophilus spp. cannot complete their life cycle in humans but they can pierce human skin, where they wander for a week or so, causing intense itching (creeping eruption). Wound myiasis Many dipterous species are known to cause wound myiasis, but most of them are facultative feeders on necrotic tissue and are rarely Table 8.12.3  Flies associated with myiasis in humans Genus and species Common name Distribution Type of myiasis Psychodidae Telmatoscopus albipunctatus Moth fly Widely distributed Intestinal, nasal Phoridae Megasalia Scuttle flies Cosmopolitan Wound, intestinal, urogenital, pulmonary Syrphidae Eristalis tenax Common drone fly Widely distributed Rectal Piophilidae Piophila casei Cheese skipper Widely distributed Intestinal Muscidae Fannia canicularis Lesser house fly Cosmopolitan Urogenital Musca domestica House fly Cosmopolitan Wound, intestinal Muscina stabulans False stable fly Cosmopolitan Intestinal Stomoxys calcitrans Stable fly Cosmopolitan Intestinal Calliphoridae Auchmeromyia luteola Congo floor maggot Africa Sanguinivorous Calliphora spp. Bluebottles Widely distributed Wound, intestinal Cochliomyia hominivorax New World screw worm Americas Primary Cochliomyia macelleria Secondary screw worm Americas Wound Cordylobia anthropophaga Tumbu fly Africa Subdermal Cordylobia rodhaini Lund’s fly Africa Subdermal Chrysomya bezziana Old World screw worm Africa, Asia Wound, auricular Lucilia spp. Green bottles Widely distributed Wound Sarcophagidae Wohlfahrtia magnifica Wohlfahrt’s myiasis fly Southern Palaearctic Primary Wohlfahrtia vigil Grey flesh fly North America Dermal Wohlfahrtia nuba Southern Palaearctic Wound Sarcophaga spp. Flesh flies Widely distributed Intestinal, wound Gasterophilidae Gasterophilus spp. Horse bot fly Widely distributed Dermal (creeping), tracheopulmonary Cuterebridae Cuterebra spp. Rabbit bot fly Americas Subdermal, nasal, tracheopulmonary Dermatobia hominis South American bot fly Neotropics Subdermal Oestridae Oestrus ovis Sheep nasal bot fly Widely distributed Ocular myiasis Hypoderma spp. Warble flies Holarctic Dermal (creeping), ophthalmic, oral

section 8  Infectious diseases 1578 destructive to the host although the presence of maggots in a wound may cause distress. Debridement of necrotic tissue will control such infestation. In contrast, under controlled conditions, clinicians may introduce maggots to promote healing. Causes of malign myiasis include the New World screw worm Cochliomyia hominivorax in the Americas, the Old World screw worm Chrysomya bezziana, and Wohlfahrt’s wound myiasis fly Wohlfahrtia magnifica in the Old World. Their larvae are obligate parasites of living tissue. Eggs are laid on wounds, in ears, and on mucous membranes. The larvae (Fig. 8.12.18) burrow in groups into healthy tissue, causing widespread destruction which may be mutilating or fatal (Fig. 8.12.19). Secondary bacterial infection or secondary wound myiasis may ensue. All species may cause naso- pharyngeal, aural, orbital, genital, and malign wound myiasis. Infestation is best avoided by cleaning and dressing wounds as they occur. Treatment involves surgical removal of the larvae, de- bridement of affected tissue, and treatment of secondary infection. Reconstructive surgery may be required. Ophthalmomyiasis (ocular myiasis) Fewer than 5% of cases of human myiasis affect the eye. Usually only external structures such as the lids and conjunctivae are in- fested but some fly larvae can penetrate the conjunctiva or sclera, causing corneal ulceration and damage to anterior and posterior internal structures that may result in blindness. The usual cause of ophthalmomyiasis externa is Oestrus ovis, the cosmopolitan sheep and goat nasal botfly, whose natural host is herbivorous mammals. Although most common in tropical developing countries (especially North Africa, the Middle East, and the Caribbean) it still occurs rarely in Western cities. Female flies eject their larvae into the nostrils of the host, where they mature. Human victims may give a history of having been buzzed in the face or struck on the eye by an insect and later of developing irritation and redness of the eye, foreign body sensation, pain, lacrimation, palpebral oedema, and signs of puru- lent conjunctivitis or a stye (hordeolum). O. ovis larvae rarely develop Fig. 8.12.15  Two third larval instars of the human bot fly Dermatobia hominis (c.13 mm long) extracted from a facial ‘boil’ in a European who had been visiting Guyana. Fig. 8.12.16  Skin lesion caused by larva of the Tumbu fly Cordylobia anthropophaga in a Peruvian man who had been working in Zambia. Copyright DA Warrell. Fig. 8.12.17  Larvae of African tumbu fly Cordylobia anthropophaga, a common agent of dermal myiasis. Fig. 8.12.18  Larvae of the New World screw worm Cochliomyia hominivorax (c.8 mm long) extracted from the wound illustrated in Fig. 8.12.19. These were sent to the Natural History Museum in London where they were identified. Larvae of the second myiasis species (C. macellaria) were also found in the sample and were probably collected from the edges of the wound. Courtesy of Dr Martin JR Hall, Medical and Veterinary Division, Natural History Museum, London.

8.12  Nonvenomous arthropods 1579 beyond the first instar in humans and so symptoms are self-​limiting, but they may be more rapidly relieved by slit lamp examination and removal of larvae which cling to the conjunctivae and may cause fol- licular conjunctival reaction and pseudomembrane formation. Other causes of human ophthalmomyiasis include Rhinoestrus purpureum, Dermatobia hominis, Hypoderma spp., (Oestridae); and Cochlyomyia hominis, Lucilia spp., Phormia spp. (Calliphoridae). Larvae of Hypoderma, Cochlyomyia, Dermatobia, and Oedemagena tarandi are more dangerous as they may burrow into the eye, resulting in pain, nausea, and destruction. They must be surgically removed. Nosocomial myiasis Hospitalized patients with exposed wounds, ulcers, or medical de- vices that breach the skin are susceptible to wound myiasis. Patients with impaired mobility or decreased levels of consciousness are also susceptible to infestation of the airways and urogenital tract. Cases of nosocomial myiasis have been reported from developed countries as well as from hospitals in resource-​poor settings. Many reported cases involve infestation with opportunistic species including mem- bers of the genera Lucilia, Sarcophaga, and Musca. Cases of infest- ation with the New World screw worm Cochliomyia hominivorax in hospitalized patients have been reported in the Americas. Flies are attracted to necrotic tissues and readily gain access through open windows but may even reach patients through open doorways in air-​conditioned rooms. Canthariasis Infestation of the body by beetles (Coleoptera) or their larvae is called canthariasis. Clinically, it may resemble myiasis but is much rarer. Larvae swallowed with food may dwell temporarily in the intes- tines, causing discomfort and may be detected in excreta. Beetles occasionally invade orifices. In Sri Lanka, scarabid dung beetles have been reported to invade the rectum. A specimen of the Asian carabid ground beetle Scarites sulcatus was recovered from the vagina of a women complaining of vaginal discharge who had visited Pakistan (Fig. 8.12.20). In Israel, the dung beetle Maladera matrida has been reported to invade the external auditory canal. In Oman, two cases of invasion of the external auditory canal by the ground beetle Crasydactylus punctatus were reported. In one case, the beetle reached the middle ear causing sensorineural hearing loss. (See also venomous coleoptera, Chapter 10.1.2.) Allergy A wide range of immunological responses to arthropod bites has been described, from local pruritus to anaphylaxis. The dead re- mains, cast skins (exuviae), and faeces of many arthropods include sensitizing agents. They may act as contact or inhalant allergens, following domestic or occupational exposure resulting in derma- titis, conjunctivitis, rhinitis, and asthma. Allergic patients may show specific IgE antibody to a wide range of domestic pests including house flies, clothes moths, cockroaches, carpet beetles Anthrenus sp., silverfish Ctenolepisma longicaudata, and house dust mites Dermatophagoides spp. Dermatophagoides spp. are a common cause of allergy in the United Kingdom and exposure to cockroach al- lergens in household dust has been associated with asthma in the United States of America. Following mass emergence, non​biting midges (Chironimidae) and the exuviae of mayflies (Ephemeroptera) and caddis flies (Trichoptera) may act as inhalant allergens. Chironimid midges occur worldwide and are especially troublesome in the Sudan, where Cladotanytarsus lewisi (green nimitti midge) breeds in dammed stretches of the Nile, causing seasonal epidemic allergy. Chironimid haemoglobin has been shown to be allergenic. The rearing of chironimid larvae as food for fish has been associated with occupa- tional allergy. Entomologists who collect insects by sucking them into pooters may develop inhalant allergy to their subject of study. Occupational Fig. 8.12.20  An Asian carabid beetle Scarites sulcatus, from a patient complaining of vaginal discharge; a rare example of genital canthariasis. Fig. 8.12.19  Fatal myiasis (New World screw worm). Historical illustration of a 50-​year-​old Honduran woman who complained of a small chronic ulcer on the right cheek; on admission to hospital she was found to have a huge ulcer exposing the bones of the face and forehead and destroying the tissues of the cheek and face, right eye, and orbit. More than 300 larvae were removed (see Fig. 8.12.18). From Harrison JHH (1908). A case of myiasis. J Trop Med Hyg, XI, 20.

section 8  Infectious diseases 1580 exposure to deer keds Lipoptena cervi has been associated with al- lergic rhinoconjunctivitis in forest workers in Finland. Larvae of the beetles Tenebrio molitor (mealworm) and Alphitobius diaperinus (lesser mealworm), which are reared for fish bait and animal food, have been associated with rhinoconjunctivitis, contact urticaria, and asthma. Beetles which infest stored grain, including Tenebrio moli- tor, Tribolium confusum (confused flour beetle), Sitophilus sp. (grain weevil), and Alphitobius diaperinus have been associated with occu- pational allergy in grain workers or bakers. Allergy has been associ- ated with other beetles, including Dermestes peruvianus (hide beetle), Gibbium psylloides (mite beetle), and Harmonia axyridis (Asian lady- bird). Insect allergy can be investigated by skin prick tests, measure- ment of allergen-​specific serum IgE, and monitoring of respiratory function following allergen exposure. Insects and hygiene Synanthropic insects which feed or wander over faeces, wounds, and food may serve as passive vectors of bacterial and viral dis- eases. Such insects include pharaoh’s ants Monomorium pharao- nis, flies, and cockroaches (Dictyoptera). Despite many reports of the isolation of pathogenic bacteria and viruses from these in- sects, there have been few epidemiological studies to define their importance as passive vectors, although is generally accepted that the presence of these insects in hospitals should be monitored and controlled. Flies Many species of fly (especially of the suborder Cyclorrhapha), fre- quent human and animal food, wounds, eyes, and faeces. Such flies vomit and defecate where they feed. Numerous pathogenic bacteria and viruses have been isolated from flies, suggesting that they may act as passive vectors of bacterial and viral diseases. A controlled study in the Gambia, where fly control was associated with fewer new cases of trachoma, suggested that flies may act as vectors of the trachoma agent Chlamydia trachomatis. In the Gambia, Musca sor- bens is the most common eye-​visiting fly. In Pakistan, a controlled study showed fly control to be significantly associated with a re- duction in incidence of childhood diarrhoeal illness. In Israel, fly control was associated with a reduction in cases of shigellosis. Flies may be controlled by using insecticides or fly traps in dwellings and latrines. Ants Pharaoh’s ants Monomorium pharaonis L.  commonly infest hos- pitals, where they invade sterile packs and wound dressings. They are potential passive vectors:  bacteria including salmonella and staphylococcus have been isolated from these ants, which should therefore be controlled with insecticides. In Iran, ants of the genus Pheidole have been associated with sudden localized hair loss from the scalp. Patients in different parts of the country reported awakening to find collections of hair on their pillows and ants on their beds or scalps. Cockroaches Cockroaches are omnivorous scavengers. A  few of the 3500 described species have become cosmopolitan synanthropes. The main pest species are the common cockroach Blatta orientalis, the American cockroach Periplaneta americana, the German cockroach Blattella germanica, and the banded cockroach Supella longipalpa. Other species may be locally important (e.g. Ectobius lapponi- cus), described by Linnaeus as infesting dried fish in Lapland. The common pest species are mostly of tropical origin and require tem- peratures of 25–​33°C, but B. orientalis will tolerate 20°C. In cooler climates they are restricted to permanently heated areas and can occur in large numbers in hospitals and in sewers. Many patho- genic viruses, including poliomyelitis virus and coxsackie A virus, and bacteria, including Shigella spp., have been isolated from cock- roaches. There is evidence that cockroaches acted as vectors of hepatitis A during an outbreak in California and of Salmonella typh- imurium on a paediatric ward in Belgium. Cockroaches are potential allergens, 7.5% of healthy individuals being skin-​test positive in one study. Cockroaches wander over sleepers and are attracted to nasal and oral secretions. Herpes blattae is a dermatitis described from Réunion and attributed to cockroach allergy. Cockroaches some- times wander into ears and nostrils, where they become trapped or reluctant to leave. Lignocaine (lidocaine) spray is reported to hasten the exit of such visitors. Eye-​frequenting moths and beetles Like the oriental eye fly (Siphunculina funicola, Diptera, Chloropidae), some nocturnal moths of the families Pyralidae, Noctuidae, and Geometridae in Africa and Southeast Asia habitually feed on the lachrymal secretions of animals. They may visit human eyes, causing a certain amount of discomfort, and may transmit eye infections, including trachoma and viral conjunctivitis. They may also cause mechanical damage to the cornea. The moths stimulate the flow of secretions by vibrating and probing with their probosces. Implicated species include Lobocraspis griseifulva, Arcyophora spp., and Filodes fulvidorsalis. Calyptra eustrigata is a skin-​piercing, bloodsucking noctuid moth from Malaya. Such Lepidoptera may be avoided by sleeping under a net. In Australia, a beetle, Orthoperus sp. has been associated with corneal erosion. FURTHER READING Aguilera A, et al. (1999). Intestinal myiasis caused by Eristalis tenax. J Clin Microbiol, 37, 3082. Auerbach PS (ed) (2012). Wilderness medicine, 6th edition. Elsevier Mosby, Philadelphia, PA. Boggild A, et al. (2002). Faruncular myiasis: a simple and rapid method for extraction of intact Dermatobia hominis larvae. Clin Infect Dis, 35, 336–​8. Doggett SL, et al. (2012). Bed bugs: clinical relevance and control options. Clin Microbiol Rev, 25, 164–​92. Feldmeier H, et al. (2003). Severe tungosis in underprivileged commu- nities: case series from Brazil. Emerg Infect Dis, 9, 949–​55. Liebold K (2003). Alopecia induced by ants. Trans Roy Soc Trop Med Hyg, 93, 427. Roberts DT (ed) (2000). Lice and scabies: a health professional’s guide to epidemiology and treatment. Public Health Laboratory Service, London. Rosenstreich DL, et al. (1997). The role of cockroach allergy and ex- posure to cockroach allergen in causing morbidity among inner-​city children with asthma. N Engl J Med, 336, 1356–​63.

8.12  Nonvenomous arthropods 1581 Roth LM, Willis ER (1957). The medical and veterinary importance of cockroaches. Smithsonian Miscellaneous Collection, 134, 1–​147. Smith KGV (ed) (1973). Insects and arthropods of medical importance. Natural History Museum Publications, London. Thomas, J. et al. (2015). Scabies: an ancient global disease with a need for new therapies. BMC Infect Dis, 15, 250. Tijsse-​Klasen E, et  al. (2011). Absence of zoonotic Bartonella
species in questing ticks:  first detection of Bartonella clarridgeiae
and Rickettsia felis in cat fleas in the Netherlands. Parasit Vectors, 4, 61. Zumpt F (1965). Myiasis in man and animals in the Old World. Butterworth, London.

8.13 Pentastomiasis (porocephalosis, linguatulosis

8.13 Pentastomiasis (porocephalosis, linguatulosis/ linguatuliasis, or tongue worm infection) 1582

ESSENTIALS Pentastomiases or porocephaloses are emerging zoonotic infec- tions caused by branchiuran maxillopod crustacean parasites. Linguatula serrata (‘tongueworm’)—​ cosmopolitan, infecting upper respiratory tracts of the definitive hosts, canids. Nymphs discharged in nasal secretions are taken up by herbivorous animals, the inter- mediate hosts, which pass on the infection when they are eaten. Humans may be infected by eating raw liver and other offal of sheep, goats, and other animals, soon after which acute allergic obstructive naso-​laryngo-​pharyngitis (halzoun or marrara syndrome) may de- velop. Larvae can be found in sputum and vomitus. Armillifer spp.—​these are confined to Africa and Southeast Asia, where they infect the respiratory tracts of snakes. Humans are in- fected by drinking snake-​polluted water, eating raw snake, or handling snakes, a common practice in some communities. Most infections are asymptomatic, but massive infections may produce symptoms of an acute abdomen and are rarely fatal by causing in- testinal obstruction or enterocolitis. Nymphs are detected at lapar- otomy or autopsy and (calcified) on abdominal radiographs. Treatment and prevention—​aside from standard measures for hypersensitivity phenomena, there is no specific treatment, although praziquantel, mebendazole, and albendazole have been tried. Surgical intervention may be necessary in cases of obstruction and with intraocular infections. Prevention is by thoroughly cooking all meat of whatever origin, by boiling drinking water and by avoiding intimate contact with snakes. Introduction Pentastomida are dioecious, obligate parasites that are cur- rently grouped in subclass Branchiura (fish lice and cycloids) of class  Maxillopoda, subphylum Crustacea, phylum Arthropoda. Common names are ‘pentastomes’ (referring to two pairs of hooks above the mouth that give the impression of five stomata; Fig. 8.13.1) or ‘tongue worms’ (alluding to the tongue-​like appearance of some, such as adult Linguatula). Adult pentastomes inhabit the upper re- spiratory tracts of their end hosts (vertebrates such as reptiles, fish, birds and mammals) where they feed on blood and other tissues. Their larvae infect internal organs of vertebrate or arthropod inter- mediate hosts. Pentastomes appear to have coevolved with other maxillopodan/​branchiuran parasites and their vertebrate hosts. There are about 100 living species in the orders Cephalobaenida (e.g. genus Raillietiella) and Porocephalida (e.g. genera Linguatula, Armillifer, Porocephalus, Leiperia, and Sebekia). Humans are ac- cidental dead-​end hosts to some nine species of these emerging zoonotic parasites, causing infections termed pentastomiasis, porocephalosis, linguatulosis, or linguatuliasis. Ninety percent of human visceral pentastomiasis is attributable to Linguatula serrata 8.13 Pentastomiasis (porocephalosis, linguatulosis/​linguatuliasis,
or tongue worm infection) David A. Warrell Fig. 8.13.1  Adult pentastomid showing mouth (arrowed) and lateral hooks giving the appearance of five stomata. Scanning electron micrograph, ×400. Courtesy of Professor Viqar Zaman.

8.13  Pentastomiasis 1583 or Armillifer armillatus. Nasopharyngeal pentastomiasis (‘Halzoun’ or ‘Marrara syndrome’) is caused by L. serrata. Pentastomiasis used to be regarded as a rare condition, but it is emerging as an increasingly important zoonosis in Africa (e.g. in Nigeria and DR Congo) and China, while imported cases are being reported in Europe and North America in immigrants from Africa. Aetiology Linguatula species Linguatula serrata occurs in Europe, the Middle East, Africa, and North, Central, and South America. The names ‘linguatula’ and ‘tongueworm’ reflect the 72–​92 annular grooves and flattened shape, particularly of the adult female. Dogs, foxes, and wolves, the defini- tive (final) hosts, harbour adults and nymphs in their upper respira- tory tract and shed them in their nasal secretions, saliva, and faeces. Rodents and other small herbivorous mammals ingest the ova with vegetation. They hatch in the lumen of the gut, releasing larvae that moult, burrow into the tissues and encyst. When these intermediate hosts are eaten by definitive carnivorous hosts, nymphs hatch from the cysts and migrate to the lungs and nasopharynx where they mature. Clinical features Visceral pentastomiasis When humans ingest ova of Linguatula, such as by eating uncooked meat, larvae hatch in the gut, burrow through its wall, migrate through the tissues, and encyst especially in the liver. Second-​ or third-​stage larvae cause symptoms through obstruction or com- pression (e.g. in biliary, gastrointestinal, or respiratory tracts, men- inges, eye, or brain). Visceral pentastomiasis is often asymptomatic but fever, abdominal pain and distension, ascites, and anorexia have been reported with hepatomegaly, peripheral neutrophil and eosino- phil leucocytosis, raised serum IgE, and anaemia. At laparotomy, autopsy, or by imaging, multiple small sharp-​bordered nodules are found in lungs, liver, and abdominal lymph nodes. Nasopharyngeal pentastomiasis (Lebanon ‘halzoun’,
Sudan ‘marrara syndrome’) Ingestion of cysts containing third-​stage larvae in raw liver or lymph nodes from sheep, goats, cattle, camels, and lagomorphs can cause nasopharyngeal pentostomiasis, known as ‘halzoun’ (halzun means ‘snail’ in Arabic) in Lebanon and ‘marrara syndrome’ in the Sudan. This has also been reported from Greece, Turkey, North Africa, Egypt, Jordan, Iran, and elsewhere. In the human stomach, larvae escape from the cysts and migrate up the oesophagus to the naso- pharynx mucosa. Within minutes to a few hours of eating the in- fected viscera, there is intense irritation of the upper respiratory and gastrointestinal tracts causing coughing, sneezing, rhinorrhoea, retching, vomiting, lacrimation, haemoptysis, epistaxis, cervical lymphadenopathy, transient deafness, difficulty in speaking, dys- phagia, wheezing, dyspnoea, and oedema of the face and oro- pharynx. The larvae, which are 5–​10 mm long, can be found in sputum and vomitus. Patients usually recover in 1 or 2 weeks, but fatal acute upper airway obstruction is reported. Clinical features suggest a hypersensitivity reaction. Flukes (Fasciola hepatica and Dicrocoelium dendriticum) and nematodes (Mammomonogamus laryngeus) ingested in raw sheep and goat liver, and aquatic leeches (Limnatis nilotica and Dinobdella ferox) (see Chapter 10.4.2) have been implicated in halzoun but cannot explain the classic syndrome. Very rarely, larvae may mature to adulthood in the human nasal cavity, causing bleeding and obstruction. Armillifer and Porocephalus species These are annulated parasites (Fig. 8.13.2a). Adult males and the much larger females (up to 20 cm long) inhabit the respiratory and digestive tracts of snakes (Fig. 8.13.3), especially those of the genera Python, Lamprophis/​Boaedon (African house snakes), Naja (cobras) (Fig. 8.13.4), Bitis (African vipers) (Fig. 8.13.2b), Bothrops (Latin American lanceheads) (Fig. 8.13.5), Crotalus (North American Fig. 8.13.3  Whip snake Demansia atra (Papua New Guinea) bringing up a pentastome. Copyright Mark O’Shea. (a) (b) Fig. 8.13.2  Armillifer armillatus. (a) Left: two adults found in the lungs of a rhinoceros viper; Right: calcified nymph from the mesentery of a Ghanaian patient. (b) Rhinoceros viper (Bitis rhinoceros). Copyright DA Warrell.

section 8  Infectious diseases 1584 rattlesnakes) and other vertebrates. Ova are shed in the snake’s nasal secretions and faeces and are picked up by rodents, monkeys, other small herbivorous mammals, and geckoes. Larvae encyst in the tis- sues of these intermediate hosts and will develop to the nymph stage if ingested by another animal, but develop to adults only in snakes. Human infections Humans are infected by drinking water contaminated with ova or by ingesting living encysted larvae in raw or undercooked snake meat, blood, or gallbladders or by handling snakes either in tropical snake farms or in villages in parts of West Africa where they kept as sa- cred totems. Raw snake meat is eaten habitually or as part of ju ju rituals in Africa (Nigeria, Côte d’Ivoire, Benin, Cameroon, and DR Congo) and in South-​East Asia, especially by the Temuan tribe of Malaysian aborigines. Ingested ova hatch in the gut, releasing larvae which moult, burrow into the tissues where they encyst as nymphs and usually degenerate after a few years. Epidemiology The prevalence of infection can be judged by discovering calcified nymphs (Fig. 8.13.2) on radiographs of the abdomen and chest (Fig. 8.13.6). These appear as discrete, crescent-​shaped, soft tissue calcifications, 4 to 8 mm in size. In West Africa they are seen par- ticularly in the right upper quadrant and are situated beneath the peritoneum covering the liver. In Ibadan, Nigeria, they were seen in 1.4% of randomly selected straight abdominal films (7% in men aged 50–​59 years) and in DR Congo in 1%. However, the preva- lence of encysted nymphs or larvae at autopsy was 12–​22.5% in DR Congo, 33% in Nigerian patients dying of malignancy, 7.8–​12.6% in Cameroon, and 45.5% among Malaysian Orang Asli. Cysts are (a) (b) Fig. 8.13.4  (a) Pentastomes from the lungs of (b) an Egyptian cobra Naja senegalensis. Fig. 8.13.5  Pentastomes found in the respiratory tract of lancehead vipers Bothrops spp., Manaus, Brazil. Copyright DA Warrell. Fig. 8.13.6  Typical radiographic appearance of calcified nymphs of Armillifer armillatus in the abdominal cavity of a Ghanaian patient. Courtesy of Dr GM Ardran.

8.13  Pentastomiasis 1585 found most commonly in liver (Fig. 8.13.7), mesentery, gut wall, peritoneum, spleen, kidneys, omentum, and lungs. In Ibadan, pentastomiasis was the third most common cause of hepatic cirrhosis. In Côte d’Ivoire, seroprevalence was 4.2%. Recently, an epidemic of A. grandis infections was described in Sankuru district of DR Congo. Human infections with the larvae or nymphs of the following species of Armillifer have been reported: • A. agkistrodontis—​(length 10 mm, 7–​9 annular rings) China (in the snake Deinagkistrodon acutus) • A.  armillatus  —​(length 9–​23  mm; 18–​22 annular rings) West and Central Africa (Senegal, the Gambia, Ghana, Benin, Nigeria, Cameroon, DR Congo, Zimbabwe), Egypt, and the Arabian Peninsula • A. grandis—​(length 9–​15 mm, >25 annular rings) Central Africa (DR Congo) • A.  moniliformis—​(length 12–​20  mm, 30 annular rings) Asia (Malaysia, Borneo, Philippines, Indonesia, Tibet, Australia). Intermediate hosts include monkeys, otters, rats, house geckoes, and cockroaches • A. najae—​India Human infections with the following species of Porocephalus have been reported: • P. crotali—​(38–​40 body segments) • P. taiwani—​(length 4–​5 mm, 10–​11 spiral rings) Clinical features Most infections are entirely asymptomatic. However, migration of large numbers of larvae from the gut into the tissue and their deaths, releasing parasite antigens and provoking hypersensitivity, may produce a variety of symptoms including fever, night sweats, persistent cough, abdominal pain and tenderness, vomiting, diar- rhoea, and obstructive jaundice. Some patients have mild blood eosinophilia. Massive infection, perhaps following ingestion of a gravid female, can cause acute abdominal symptoms prompting laparotomy at which hundreds of wriggling nymphs may be dis- covered beneath the visceral peritoneum covering the liver and spleen, in liver parenchyma, mesentery, intestinal wall, abdom- inal lymph nodes, lungs, or pleura. Serious inflammatory and obstructive effects have been described in the gut, peritoneum, mesentery, liver and biliary tract, lungs, pleura, pericardium, myocardium, central nervous system, and eye (see next). These may be due partly to hypersensitivity. The few reported fatal cases resulted from massive liver infections, mechanical intestinal ob- struction, or haemorrhagic enterocolitis complicated by secondary Gram-​negative septicaemia. A suggested association between Armillifer infection and colonic or other malignancies has not been substantiated. Ocular pentastomiasis About 20 cases have been reported from the United States, Ecuador, Europe, Israel, India, Brazil and DR Congo (L. serrata, A. armilla- tus, A.  grandis). Adnexal infections (e.g. subconjunctival, associ- ated with peri-​orbital oedema) were easily removed but intraocular infections usually caused blindness through iritis and uveitis with goniosynechiae and secondary glaucoma, iridodonesis and lens subluxation, and vitreous, subretinal, or retinal detachments. Early surgical extraction of nymphs is recommended. Other pentastomid infections Human infections with Leiperia cincinnalis have been described in Africa. Subcutaneous infections by Railliettiella gehyrae and R. hemi- dactyli occur in Vietnam and by Sebekia species in Costa Rica. In Vietnam, infection with Railliettiella spp. results from swallowing small live lizards for medicinal purposes. Diagnosis The radiographical appearances of calcified pentastomid nymphs are distinctive (Fig. 8.13.6). They are not found in muscle, distinguishing pentastomiasis from cysticercosis. Pentastomes may be discovered at laparoscopy, surgery, or autopsy and dis- tinguished from helminths macroscopically or in tissue sections. In the liver (Fig. 8.13.7), intestinal wall, mesentery, mesenteric lymph nodes, peritoneum, or lung, viable encysted larvae, or granulomas containing necrotic pentastomes or their moulted cuticles may be found. Initially, encysted larvae excite little or no tissue reaction, but the granulomas are surrounded by hyalinized or calcified fibrous tissue. Serological tests are being developed, while a specific polymerase chain reaction that targets the 18S small subunit rRNA gene might be useful even in formalin-​fixed paraffin-​embedded tissue. Fig. 8.13.7  Encysted nymph/​larva of Armillifer armillatus in human liver. The outer layer of the parasite (arrowed) lines the cyst wall. Acidophilic glands (ag), intestine (in), ×21. Armed Forces Institute of Pathology photograph, negative number 75–​2703.

section 8  Infectious diseases 1586 Treatment There is no specific treatment, although ivermectin, praziquantel, mebendazole, and albendazole have been used with apparent suc- cess. Obstruction and compression should be relieved surgically. Hypersensitivity phenomena should be treated with adrenaline (epi- nephrine), antihistamines, and corticosteroids. Prevention Pentostomiasis can be prevented by thoroughly cooking all meat of any origin and boiling or filtering drinking water. Eating sheep’s lymph nodes is proscribed by the Shi’ite Muslims of Lebanon. Other zoonoses transmitted from reptiles to humans The most important of these is salmonellosis transmitted to humans by the faecal–​oral route or by scratches and bites, from chelonians (tortoises, turtles, terrapins) and from snakes and lizards, espe- cially iguanas. In the United Kingdom, 38% of imported tortoises (Testudo spp.) contain salmonella. In the United States of America, where 8 million reptiles are kept as pets, contact with reptiles and amphibians accounts for an estimated 74 000 (6%) of the approxi- mately 1.2 million sporadic human salmonella infections that occur there annually. The banning by the United States Food and Drug Administration of commercial distribution of small turtles has prevented an estimated 100 000 cases of salmonellosis among chil- dren each year. Although salmonellosis usually causes self-​limiting gastroenteritis, septicaemia or meningitis can occur especially in infants and immunocompromised people. Species associated with reptile salmonellosis include S.  enterica serotype Typhimurium, S. enterica serotype Pomona, and S. enterica subspecies diarizonae. Other infections transmissible from reptiles to humans in- clude Arizona hinshawii (in snake powder, Pulvo de Vibora, made from rattlesnakes), Plesiomonas shigelloides, Edwardsiella tarda, leptospirosis, Q fever, sparganosis, capillariasis, strongyl- oidiasis, mesocestoidiasis, sarcocystiasis, and infestation with the mite Ophionyssus natricis. Potential zoonoses include mycobac- teria, pseudomonas, other aeromonas species, proteus, and some togaviruses (such as Western equine encephalitis in garter snakes in western North America) and herpesviruses. FURTHER READING Chen SH, et  al. (2009). Multi-​host model-​based identification of Armillifer agkistrodontis (Pentastomida), a new zoonotic parasite from China. PLoS Negl Trop Dis, 4, e647. Lai C, et al. (2010). Imaging features of pediatric pentastomiasis infec- tion: a case report. Korean J Radiol, 11, 480–​4. Lavrov DV, et al. (2004). Phylogenetic position of the pentastomida and (pan)crustacean relationships. Proc Biol Sci, 271, 537–​44. Magnino S, et al. (2009). Biological risks associated with consumption of reptile products. Int J Food Microbiol, 134, 163–​75. Palmer PES, Reeder MM (eds) (2001). Pentastomida. In: The imaging of tropical diseases with epidemiological, pathological and clinical cor- relation, Vol. 2, pp. 389–​95. Springer, Berlin. Pantchev N, Tappe D (2011). Pentastomiasis and other parasitic zoonoses from reptiles and amphibians. Berl Munch Tierarztl Wochenschr, 124, 528–​35. Riley J (1986). The biology of pentastomids. Adv Parasitol, 25, 45–​128. Schacher JF, Khalil GM, Salman S (1965). A field study of Halzoun (parasitic pharyngitis) in Lebanon. J Trop Med Hyg, 68, 226–​30. Sulyok M et  al. (2014). Ocular pentastomiasis in the Democratic Republic of the Congo. PLoS Negl Trop Dis, 8, e3041. Tappe D, Büttner DW (2009). Diagnosis of human visceral pentastomiasis. PLoS Negl Trop Dis, 5, e320. Tappe D, et al. (2011). Diagnosis of human visceral pentastomiasis. Emerg Infect Dis, 17, 251–​4. Tappe D, et al. (2011). Transmission of Armillifer armillatus ova at snake farm, The Gambia, West Africa. Emerg Infect Dis, 17, 251–​4. Tappe D, et al. (2014). Imported Armillifer pentastomiasis: report of a symptomatic infection in the Netherlands and mini-​review. Travel Med Infect Dis, 12, 129–​33. Tappe D, et al. (2015). Molecular diagnosis of abdominal Armillifer grandis Pentastomiasis in the Democratic Republic of Congo. J Clin Microbiol, 53, 2362–​4. Vanhecke C, Le-Gall P, Le Breton M, Malvy D (2016). Human pentastomiasis in Sub-Saharan Africa. Med Mal Infect, 46, 269–75. Yagi H, et al. (1996). The Marrara syndrome: a hypersensitivity reac- tion of the upper respiratory tract and buccopharyngeal mucosa to nymphs of Linguatula serrata. Acta Trop, 16, 127–​34. Yao MH, Wu F, Tang LF (2008). Human pentastomiasis in China: case report and literature review. J Parasitol, 94, 1295–​8.

SECTION 9 Sexually transmitted diseases Section editor: Jackie Sherrard 9.1 Epidemiology of sexually transmitted
infections   1589 David Mabey and Anita Vas-​Falcao 9.2 Sexual behaviour   1597 Catherine H. Mercer and Anne M. Johnson 9.3 Sexual history and examination   1600 Gary Brook, Jackie Sherrard, and Graz A. Luzzi 9.4 Vaginal discharge   1603 Paul Nyirjesy 9.5 Urethritis   1606 Patrick Horner 9.6 Genital ulceration   1610 Patrick French and Raj Patel 9.7 Anogenital lumps and bumps   1613 Henry J.C. de Vries and Charles J.N. Lacey 9.8 Pelvic inflammatory disease   1622 Jonathan D.C. Ross 9.9 Principles of contraception   1626 Zara Haider

8.2 The patient with suspected infection 662

8.2 The patient with suspected infection 662

8.2.1 Clinical approach 662

8.2.1 Clinical approach 662

8.2 The patient with suspected infection CONTENTS 8.2.1 Clinical approach  662 Christopher J. Ellis 8.2.2 Fever of unknown origin  664 Steven Vanderschueren 8.2.3 Nosocomial infections  669 Ian C.J.W. Bowler and Matthew Scarborough 8.2.4 Infection in the immunocompromised host  673 Jon Cohen and Elham Khatamzas 8.2.5 Antimicrobial chemotherapy  684 Maha Albur, Alasdair MacGowan, and Roger G. Finch 8.2.1  Clinical approach Christopher J. Ellis ESSENTIALS Infection is most often suspected when patients present with pyrexia and is certainly the most common cause of this presentation, whether in hospitalized patients or those in the community. The other principal causes of fever are primary inflammatory conditions and malignancy, but infections are likely to be most rapidly progressive and acutely life-​ threatening, and hence must be the physician’s first concern. The clinical approach to patients with likely infection begins with a focused history, leading on to a clinical examination which assesses the extent of the physiological derangement and looks for a focus of infection. Standard physiological measures define likely sepsis (see Chapter 8.1.2), which is the commonest reason for their sudden de- rangement in hospitalized patients. Investigations should be phased and must not delay the start of potentially life-​saving treatment, the response to which must be carefully followed, especially when treatment has to be started before a complete or certain diagnosis is possible, and compared with the likely speed of response for the putative condition being treated. There is increasing evidence that delays in initiating appropriate therapy, especially antimicrobial medication and circulatory support, increase mortality. Introduction No diagnostic challenge better illustrates the power of traditional clinical methods than the patient with possible infection; clin- icians rarely find themselves in a situation where there is poten- tially so much urgency in establishing a working hypothesis and management plan. It is vital to keep in mind that previously healthy people with life-​threatening infections may have few symptoms other than malaise, and little in the way of abnormality on examination other than an altered body temperature and tachycardia. However, at this point only decisive intervention will prevent a rapid decline into circulatory collapse, coagulopathy, and multiple organ failure with a high risk of death. What suggests that the patient’s life is in danger? Standard observations (vital signs) are usually valuable pointers to life-​threatening situations and have been combined to define sepsis syndromes (Box 8.2.1.1). Observations made routinely on hospitalized patients are now co- dified to produce early warning track and trigger systems, such as the modified early warning score, which highlight developing sepsis. Box 8.2.1.1  Sepsis syndromes Sepsis (systemic inflammatory response syndrome) Defined by two or more of: • Temperature more than 38°C or under 36°C • Pulse rate more than 90/​min • Respiratory rate more than 20/​min • Leucocyte count more than 12 or under 4 × 109/​litre Severe sepsis Sepsis with one or more of: • Hypotension • Confusion • Oliguria • Hypoxia • Acidosis • Disseminated intravascular coagulation Septic shock • Severe sepsis with hypotension despite fluid resuscitation

8.2.1  Clinical approach 663 In general hospitals, the development of sepsis is currently the most common single reason for patients’ scores reaching the trigger point. Although these alarm calls are important, they will not identify all patients in whom urgent treatment is vital. Table 8.2.1.1 lists some conditions that typically present in a bland and non​specific manner although the patient may be only a day or two from death if not treated appropriately. Infection may be mimicked by life-​threatening non​infectious con- ditions. Primary vasculitic conditions commonly present with fever and skin infarcts identical to those seen in patients with endocarditis, while cerebral systemic lupus erythematosus is clinically indistin- guishable from an infective encephalitis. Conditions such as DRESS syndrome (drug reaction eosinophilia and systemic symptoms) may be precipitated by antibiotics, and also mimic severe sepsis. Management before diagnosis Acute medicine has been described as ‘the art of making suffi- cient conclusions on insufficient information’. When confronted with a patient who fulfils the criteria for severe sepsis, attention must be paid to oxygenation, circulatory support, and intravenous antimicrobials, even if the clinician is still some way from a de- finitive diagnosis. In these fraught circumstances, organizational confusion can lead to vital actions being omitted or delayed. In particular, the prescriber should ensure the prompt administra- tion of intravenous antimicrobials and deliver a fluid challenge to patients with hypovolaemia, bearing in mind that up to 40% of effective circulating volume can be lost, usually through vasodila- tation, before vital signs register more than tachycardia. One litre of saline given over 30 min, and repeated if the pulse rate does not fall, is an appropriate prescription for an adult in this situation. The history of the illness Once immediate life-​saving measures are in hand, if they are indi- cated, a thorough, focused history must include several questions that are not routinely asked (Table 8.2.1.2). Clinical examination and chest radiograph Examination of a patient with suspected infection can be both rapid and comprehensive. Having noted the vital signs, the clinician can proceed from head to toe. Temporal arteries should be examined in patients over the age of 50 years with fever and headaches; the mouth should be examined for poor dentition and oral candidiasis (a pointer to possible HIV infection), and the heart for murmur(s). Chest examination might reveal signs of consolidation or an effusion. Examination of the abdomen should pay particular attention to liver enlargement and/​or tenderness and include several firm blows over Table 8.2.1.1  Conditions presenting in a non​specific manner Condition Key clue Malaria Travel history Early meningococcaemia Early purpura Bacteraemia Rigor (i.e. visible shivering for at least 10 s) Fasciitis Tenderness to pressure beyond apparent bounds of cellulitis Toxic shock syndrome The patient has fainted on standing (because of incipient shock); erythematous rash Table 8.2.1.2  Questions that must be asked concerning the history of the illness Open question Possible significance Have you travelled? All cases of falciparum malaria imported into non​endemic areas are in people who have visited malarial areas in the preceding 3 months; most are within 1 month. In the UK, 90% of these infections will have been acquired in sub-​Saharan Africa Approximately one-​half of the cases of legionnaires’ disease in the UK are in patients who have returned from Europe or Turkey in the preceding 2 weeks Have you been sexually active? Unprotected sex with a new partner (or a promiscuous regular partner) in the previous 2 months increases the probability of primary HIV and secondary syphilis Have you been exposed to crowds of new social contacts (e.g. university freshers week, new military recruits, or large military deployments)? Increased probability of meningococcal or pneumococcal infection Have you been hospitalized or have you received medical attention recently? Fever following the start of medication raises the possibility of drug fever (typically when a course of penicillin is extended beyond 1 week) Recent administration of antibacterial drugs predisposes to Clostridium difficile colitis Acquisition of a resistant strain of bacteria (e.g. extended-​spectrum β-​lactamase-​producing or carbapenemase-​producing bacteria) Dental work predisposes to endocarditis Previous splenectomy predisposes to fulminating pneumococcal septicaemia Infection of surgical wounds, retained surgical material, or prostheses Partial treatment of an abscess, most commonly intra-​abdominal or retroperitoneal, including psoas abscess Is the illness remittent? Characteristically remittent conditions, including vivax malaria, systemic Still’s disease, lymphoma Temporary improvement with antibacterial drugs suggests a possible ‘collection’, a concealed abscess

8.2.2 Fever of unknown origin 664

8.2.2 Fever of unknown origin 664

664 SECTION 8  Infectious diseases the ribs overlying the posterior surface of the liver which may elicit tenderness in patients with posterior liver abscesses. Tenderness in the right iliac fossa might indicate bowel-​related sepsis, such as an appendix mass, while bimanual examination may reveal an enlarged or tender kidney. The patient’s posture should be noted; flexion of the hip points to a possible psoas abscess. Examination of the peri- neum is mandatory in febrile neutropenic patients; it may reveal septic necrosis spreading from the rectum. Enlarged lymph nodes should be carefully sought in the neck and in the axilla, where they are easily overlooked. The entire skin should be inspected for rash or for areas of inflammation. The spine should be palpated and percussed looking for angulation or tenderness. The nervous system should be examined if there is evidence of menin- gitis, encephalitis, or focal neurological symptoms. Investigations These should be phased in the interests of both time and money and to avoid misleading false-​positives. Initial blood tests must include a specific test for malaria if the patient has travelled to an endemic area. Blood samples for culture should be taken before antibiotics are started. Patients who have been started on antibiotics before in- vestigation should have them stopped, provided it is judged safe to do so, and blood taken for culture 24 h later. The chest radiograph should be inspected on admission. A chest radiograph may reveal areas of consolidation in patients without any respiratory symptoms and hilar lymphadenopathy in patients without palpably enlarged nodes elsewhere. Conversely, a normal chest radiograph does not exclude early pneumonia. All patients should have a properly taken midstream or clean-​catch urine sample sent for analysis. Liaison with the microbiology laboratory is essential, and prompt delivery of specimens is a priority. Investigations involving cell counts (i.e. cerebrospinal fluid and urine microscopy), must be car- ried out on the day they were obtained. Initial investigations are therefore: • Background—​full blood count, urea and electrolytes, liver func- tion tests, C-​reactive protein • Specific—​blood culture, malaria test if indicated, urine analysis, chest radiograph In assessing the results of these tests, the clinician must be aware of the significance of collateral effects, such as thrombocytopenia in dissem- inated intravascular coagulation and malaria, and moderate elevations of transaminases in bacteraemia from any focus. The nature of bacteria isolated from blood culture often indicates the need for attention to a likely source (e.g. Streptococcus viridans to endocarditis, Streptococcus milleri to endocarditis or liver abscess, Streptococcus bovis to both endo- carditis and neoplasm of the colon), while a mixture of Gram-​negative rods and anaerobes points to liver abscess or gut-​related sepsis. Second phase investigations If the initial investigations do not point to a particular focus, imaging of the abdomen should be performed. Ultrasound examination is good for detecting fully liquefied liver abscesses and hydronephrosis and may point to focal sepsis or enlarged nodes. If ultrasound is negative, a CT scan should be considered. Increasingly PET/CT scanning is used in the investigation of unexplained fever. Therapeutic trials A therapeutic trial of an antibacterial may be indicated when, for example, the patient reports temporary improvement following a previous course and the investigations outlined above have proved unhelpful. The spectrum covered by the previous antibiotic should be taken into consideration when selecting the trial agent. For example, a response to flucloxacillin might suggest the need for a more protracted course of antistaphylococcal therapy. It is essential to compare the re- sponse to treatment with the response expected in the condition that has been provisionally diagnosed. In most bacterial infections pyrexia will settle within 48 h of starting appropriate antibacterial therapy, but there are notable exceptions, including typhoid fever, any abscess with a volume of more than about 10 ml, and conditions in which there is a significant host response to the infection, such as the development of pleural effusion in patients with pneumococcal pneumonia. A trial of antituberculosis chemotherapy is routine in patients in whom this infection is likely on clinical grounds, while awaiting cul- ture results. It should also be considered when a tissue biopsy reveals granulomata. Finally, when the history suggests the possibility of systemic Still’s disease with criteria either fulfilled or approximated, or when a pa- tient over the age of 50 has intermittent fever and symptoms con- sistent with giant cell arteritis, a trial of corticosteroids should be considered. This should not be delayed, but if the patient does not show clear improvement within 5  days of starting prednisolone 60 mg daily, the trial should be stopped. Such a course carries only a small risk of significant adverse effects, and the likelihood of infec- tion ‘lighting up’ is, in practice, very small. 8.2.2  Fever of unknown origin Steven Vanderschueren ESSENTIALS Fever of unknown origin refers to a prolonged febrile illness that per- sists without diagnosis after careful initial assessment. Although over 200 causes have been described, including rare diseases, most cases are due to familiar entities presenting in an atypical fashion. Causes of fever of unknown origin—​the ‘big three’ are (1) infections—​ including tuberculosis, endocarditis, abdominal and hepatobiliary infections and abscesses, complicated genitourinary tract infections, pleuropulmonary infections, bone and joint infections, salmonellosis, cytomegalovirus, Epstein–​Barr virus, and HIV; (2) tumours—​including lymphoma; and (3) multisystem inflammatory conditions—​including connective tissue diseases, vasculitic syndromes, and granulomatous disorders. A miscellaneous category including factitious fever, ha- bitual hyperthermia, and drug fever deserves consideration early in

8.2.2  Fever of unknown origin 665 a patient’s workup, since timely recognition may avert invasive and expensive procedures. Clinical approach to the patient with fever of unknown origin—​ the clinician must rely on a very careful and thorough clinical his- tory and examination that does not neglect any part of the body, followed by appropriately targeted investigations directed by
knowledge of the broad spectrum of diseases and local epidemi- ology. As advocated by Sutton’s law—​‘go where the money is’—​ the approach should follow any possible diagnostic clues, which
may sometimes be subtle. If clues are absent or prove misleading, then screening imaging techniques can focus further investigation, but a rigid algorithm and a blind pursuit of increasingly complex tests are ill-​advised. Likewise, therapeutic trials without firm founda- tion are rarely diagnostically rewarding. If the diagnosis in a stable patient remains elusive despite vigorous effort, a watchful waiting approach is warranted as most patients with fever of persistently unknown origin do well. Definition Original definition Most fevers are readily explained or resolve rapidly. Fever with un- clear cause or source at first sight should not be labelled fever (or pyrexia) of unknown (or undetermined) origin (FUO). Defined properly, true FUO is uncommon and is encountered once or twice a month at most teaching hospitals. A strict definition, which should not be changed too rapidly, is necessary for comparison of litera- ture data and to guide clinicians faced with this rather rare clinical problem. The three criteria initially proposed by Petersdorf and Beeson in 1961 are: (1) an illness of at least 3 weeks’ duration, (2) a fever (temperature more than 38.3°C on at least three occasions), and (3) no established diagnosis after 1 week of hospital investiga- tion. The first criterion eliminates acute, self-​limiting, frequently viral diseases and the second eliminates habitual hyperthermia, an entity commonly diagnosed at that time. Update of the initial definition In 1991, Durack and Street suggested modification of the third criterion to an uncertain diagnosis after at least three outpatient visits or at least 3 days in hospital. This revision reflected trends in medical practice, including a shift towards outpatient manage- ment, advances in diagnostic techniques, and an accelerated pace of investigation. They also divided FUO into four groups: classic FUO, nosocomial FUO, neutropenic FUO, and HIV-​associated FUO. In the last three groups the case mixture differs from that of classic FUO, and the predominance of nosocomial and op- portunistic infections in these often frail patients frequently justifies early empirical antimicrobial therapy. The present chapter focuses on classic, community-​acquired FUO in immunocompe- tent adults. Contemporary definition of classic fever of unknown origin Recently, it has been suggested that the third criterion should be changed from a quantitative to a qualitative one, specifying which particular examinations are necessary before an unsolved prolonged febrile illness classifies as FUO, rather than an arbitrary number of hospital days or outpatient visits. These minimum re- quirements (Box 8.2.2.1) should be adapted to regional, mainly infectious, epidemiological factors. Finally, a protracted unex- plained febrile illness with fever below 38.3°C but with persist- ently raised inflammatory markers should probably be approached similarly. These proposed changes culminated in a modern defin- ition of classic FUO (Box 8.2.2.2), which can be used for the next few decades. Causes Diagnostic spectrum The list of differential diagnoses is among the longest and most challenging in internal medicine, encompassing more than 200 entities. Common and uncommon causes of FUO in adults are listed in Boxes 8.2.2.3 and 8.2.2.4. These causes are conveniently classified into five categories:  (1) infections, (2)  malignancies, Box 8.2.2.1  Minimum diagnostic evaluation to qualify as fever of unknown origin • Comprehensive history (including accompanying symptoms, travel history, sexual risk behaviour, profession, hobbies, contact with ani- mals (pets, birds, insects) and ill persons, family history, use of medi- cations and illicit drugs, past medical and surgical history, transfusion, presence of foreign material) • Meticulous physical examination (eyes, mucosal surfaces, temporal arteries, skin, hands and nails, lymph nodes, thyroid, heart, lungs, ab- domen, genitalia, rectal examination, musculoskeletal system, neuro- logical examination, vascular examination) • Erythrocyte sedimentation rate, C-​reactive protein, serum protein electrophoresis • Complete blood count, including differential and platelet count • Routine blood chemistry, including creatinine, sodium, potassium, lac- tate dehydrogenase, bilirubin, liver enzymes, creatine kinase • Antinuclear and antineutrophil cytoplasmic antibodies • Urinalysis, including microscopic examination • Routine blood and urine cultures taken while not receiving antibiotics, cultures of other normally sterile fluids (e.g. from joints, pleura, or cere- brospinal space) whenever appropriate • Tuberculin skin test or interferon-γ release assay (IGRA) • Chest radiograph • Abdominal ultrasonography (including pelvis) • Further evaluation of any abnormalities detected by above tests (e.g. HIV serology, hepatitis serology, echocardiography in case of cardiac murmur, blood smear for malaria in the traveller, Epstein–​Barr virus, and cytomegalovirus serology in case of reactive lymphocytosis) Box 8.2.2.2  Modern definition of classic fever of unknown origin • Illness of more than 3 weeks duration • Temperature of at least 38.3°C, or lower temperature with laboratory signs of inflammation, on at least three occasions • No diagnosis or reasonable (eventually confirmed) diagnostic hypoth- esis after an initial diagnostic investigationa • Exclusion of nosocomial fevers and severe immunocompromise aSee Box 8.2.2.1.

666 SECTION 8  Infectious diseases (3)  non​infectious inflammatory diseases, (4)  miscellaneous causes,  and (5)  undiagnosed cases. Infections predominated in earlier case series, in paediatric series, and in series from developing countries and from secondary care hospitals. In recent series from western European and Japanese referral centres, non-​ infectious inflammatory disease (comprising connective tissue disorders, vasculitides, and granulomatous disorders) surpassed infections as the most prevalent category. Despite innovative rapid microbiological techniques, old and emerging infectious dis- eases will remain an important source of FUO, due to increasing global travel, migration, implantation of devices, and resistance of microorganisms. Somewhat counterintuitively, the proportion of undiagnosed cases is highest in referral centres and has risen over recent decades, amounting to 25–​50% of cases. This apparent loss of diagnostic yield is partially attributable to the improved diagnostic armamentarium that reveals the aetiology or source well before a febrile illness turns into FUO. Yet the cause of some prolonged fevers remains unknown despite vigorous clinical Box 8.2.2.3  Common causes of classic fever of unknown origin in adults Infections • Tuberculosis • Endocarditis • Abdominal and hepatobiliary infections and abscesses • Complicated genitourinary tract infections • Pleuropulmonary infections • Bone and joint infections • Salmonellosis (including typhoid fever) • Cytomegalovirus, Epstein–​Barr virus, HIV Neoplasms • Haematological -​ Non-​Hodgkin’s lymphoma -​ Hodgkin’s disease -​ Leukaemia • Solid -​ Adenocarcinoma (e.g. colon, kidney) Metastatic Non​infectious inflammatory diseases • Connective tissue diseases -​ Adult-​onset Still’s disease -​ Polymyalgia rheumatica -​ Rheumatoid arthritis -​ Sjögren’s syndrome -​ Systemic lupus erythematosus • Vasculitis syndromes -​ Giant cell arteritis -​ Polyarteritis nodosa -​ Granulomatosis with polyangiitis • Granulomatous disorders -​ Inflammatory bowel disease -​ Sarcoidosis Miscellaneous • Drug fever • Habitual hyperthermia • Factitious fever • Subacute thyroiditis • Venous thromboembolism • Haematoma Box 8.2.2.4  Rare causes of fever of unknown origin in adults Infections • Bartonellosis (including Bartonella henselae, B. quintana), brucellosis, campylobacteriosis, gonococcaemia, melioidosis, meningococcemia, listeriosis, tularaemia, yersiniosis • Chlamydial infections (including psittacosis), ehrlichioses, rickettsioses, Coxiella burnetii (Q fever) • Non​tuberculous mycobacteria, leprosy • Febris recurrens, leptospirosis, Lyme disease, rat-​bite fever, syphilis • Actinomycosis, nocardiosis, Whipple’s disease • Human herpesvirus type 8, parvovirus B19 • Aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococ­ cosis, histoplasmosis, mucormycosis, pneumocystosis, sporotrichosis • Amoebiasis, babesiosis, echinococcosis, fascioliasis, malaria, leish- maniasis, schistosomiasis, toxocariasis, toxoplasmosis, trichinosis, trypanosomiasis • Malakoplakia, xanthogranulomatous pyelonephritis • Central nervous system infection, dental infection, upper respiratory tract infection, wound infection • Intravenous catheter infection, infected vascular graft, mycotic aneurysm Neoplasms and related conditions • Haematological malignancies -​ Angioimmunoblastic T-​cell lymphoma -​ Intravascular lymphoma -​ Amyloidosis -​ Hypereosinophilic syndrome -​ Multiple myeloma -​ Myelodysplastic syndromes -​ Myelofibrosis • Solid tumours -​ Atrial myxoma -​ Hepatoma -​ Renal cell carcinoma -​ Other (more than 30 reported), with or without necrosis, with or without metastases Non​infectious inflammatory diseases • Connective tissue diseases -​ Acute rheumatic fever -​ Crystal-​induced arthropathy -​ Eosinophilic fasciitis -​ Felty’s syndrome -​ Mixed connective tissue disease -​ Inflammatory myositis -​ Relapsing polychondritis -​ Seronegative spondylarthropathy • Vasculitis syndromes -​ Behçet’s disease -​ Henoch–​Schönlein purpura -​ Mixed cryoglobulinaemia -​ Takayasu’s arteritis -​ Urticarial vasculitis Miscellaneous • Addison’s disease, hyperparathyroidism, hyperthyroidism, hypothal- amic hypopituitarism, phaeochromocytoma • Erythema multiforme, erythema nodosum, linear IgA dermatosis, Sweet’s disease • Castleman’s disease, inflammatory pseudotumour of lymph nodes, Kikuchi’s disease • Vogt–​Koyanagi–​Harada syndrome (continued)

8.2.2  Fever of unknown origin 667 efforts. In larger series, even autopsy failed to unravel the cause of the FUO in a substantial minority. Subpopulations The cause of FUO differs among subpopulations. The importance of geographical origin and the immune status of the host have already been alluded to, and age matters as well. In older people, giant cell arteritis, tuberculosis, malignancies, and drug fever are important considerations, while in younger adults, viral infections, particularly cytomegalovirus infection, adult-​onset Still’s disease, habitual hyper- thermia, factitious fever, and undiagnosed cases are more prevalent. In recurrent or episodic FUO, defined as at least two episodes of fever with fever-​free intervals of at least 2 weeks and seeming remis- sion of the underlying illness, traditional causes such as infections and malignancies are less frequently implicated. Recurrent FUO is especially challenging, as a final diagnosis is established in no more than one-​half of the patients. As the duration of the fever increases, the likelihood of an infectious cause decreases. Common diseases prevail Although the possible aetiologies of FUO are myriad, a limited list of disorders (Box 8.2.2.3) accounted for the great majority of diag- noses in published series. Most patients do not have esoteric dis- eases, unfamiliar to the clinician, but rather are exhibiting atypical manifestations of common illnesses. A few examples may illustrate this point. The forms of tuberculosis that give rise to FUO are often disseminated disease, yet without the characteristic miliary pattern on chest radiograph, or extrapulmonary disease without clear lo- calizing features; tuberculin skin tests and sputum smears are often negative. The forms of endocarditis that enter the FUO spectrum are frequently culture-​negative or are caused by fastidious organ- isms; a new regurgitant murmur or signs of peripheral emboli are frequently absent. Leukaemia presents as an FUO characteristically in the aleukaemic phase. Giant cell arteritis may manifest with con- stitutional symptoms only (anorexia, weight loss, fever), without polymyalgia or arteritic signs and symptoms, and without a strik- ingly elevated erythrocyte sedimentation rate. Likewise, in sub- acute thyroiditis, localizing symptoms and signs may be subtle or nonexistent. Approach to the adult with classic fever of unknown origin Ruling out the ‘little three’ For didactic and practical purposes, it is convenient to split the aeti- ologies into the ‘big three’ and the ‘little three’. The ‘big three’ are infections, neoplasms, and non​infectious inflammatory diseases, which together represent the bulk of diagnoses. The ‘little three’ comprise factitious fever, habitual hyperthermia, and drug fever. While these three causes are numerically less important, consid- ering them from the start may prevent painstaking and invasive investigations. For this reason, at an early stage, fever should be verified, temperature charts recorded, and an effort made to stop all nonessential medications and switch essential ones to unrelated alternatives. Factitious fever Due to either manipulation of the thermometer or self-​induced disease (e.g. by self-​injection of contaminated materials), this characteristically occurs in young women, often health profes- sionals. Discrepancy between symptoms and clinical and labora- tory findings raises the suspicion of fraudulent fever. Unexplained polymicrobial bacteraemia, serial episodes of bacteraemia by dif- ferent pathogens, or recurrent soft tissue infections suggest self-​ induced infection. Habitual hyperthermia This is also seen mainly in young women who complain of ‘flu-​like’ and functional symptoms. In this syndrome, which overlaps with chronic fatigue syndrome and fibromyalgia, the diurnal variation in body temperature is maintained. Evening temperatures are on average 0.5°C higher than morning temperatures, body temperature rises especially following physical and intellectual activity, the re- sponse to antipyretics is poor, and temperatures only occasionally exceed 38.3°C. Laboratory evaluation, including acute-​phase react- ants, is entirely unremarkable. Drug fever Virtually any drug can cause fever, with the possible exceptions of digitalis and aminoglycosides. The mechanisms are multiple and often poorly understood, with hypersensitivity being most common. Examples of drugs causing FUO include anticonvul- sants, antimicrobials (such as minocycline, β-​lactams, vanco- mycin, sulphonamides, and nitrofurantoin), antihistamines, non​steroidal anti-​inflammatory drugs (including salicylates), antihypertensives (hydralazine, methyldopa), antiarrhythmics (quinidine, procainamide), and allopurinol. Patients may have been on the offending drug for prolonged periods. Fever is rarely the sole Box 8.2.2.4  Continued • Giant haemangioma • Dissecting aneurysm • IgG4-​related disorders • Thrombophlebitis • Cholesterol embolism, polytetrafluoroethylene (Teflon) embolism, silicone embolism • Antiphospholipid syndrome • Cyclic neutropenia, haemolytic anaemia, haemoglobinopathies, macrophage activation (haemophagocytic) syndrome, vitamin B12 deficiency • Schnitzler’s syndrome • Dressler’s syndrome (postmyocardial infarction syndrome) • Cerebrovascular accident, epilepsy • Alcoholic hepatitis, autoimmune hepatitis, cirrhosis (with active ne- crosis), primary sclerosing cholangitis • Extrinsic allergic alveolitis, hypersensitivity pneumonitis, interstitial pneumonia • Hereditary periodic fever syndromes (familial Mediterranean fever, tumour necrosis factor receptor-​1-​associated periodic syndrome, hyper-​IgD syndrome, Muckle–​Wells syndrome, familial cold autoin­ flammatory syndrome) • Gaucher’s disease, Fabry’s disease • Hypertriglyceridaemia • Erdheim–​Chester disease

668 SECTION 8  Infectious diseases manifestation but may be accompanied by rash, urticaria, mucosal ulceration, eosinophilia, and other haematological abnormalities, hepatic or renal dysfunction, or pulmonary involvement. Phenytoin and carbamazepine are notorious for inducing a pseudolymphoma syndrome. Some patients with drug fever look severely ill and toxic, while others look and feel surprisingly well. Withdrawal of the offending drug usually results in defervescence within 72–​96 hours. Rechallenge is generally safe unless organ damage (e.g. hepatitis or interstitial nephritis) has occurred, but is rarely performed in clin- ical practice. Formal allergy testing is sometimes used to confirm the diagnosis of drug allergy, particularly if the patient appears to have multiple drug allergies or is likely to require treatment with a particular drug or related drugs in the future. Fever characteristics While recording and monitoring of body temperature are imperative, fever height and pattern do not contribute much to diagnosis. The few entities that have a distinctive fever pattern (e.g. non-​falciparum malaria or cyclic neutropenia) are rare, as are fever patterns thought to be characteristic of other diseases, such as Pel–​Ebstein fever (a relapsing fever that disappears and reappears at intervals of several days) in Hodgkin’s disease. Other features that lack diagnostic dis- crimination among the numerous sources of FUO are the presence of night sweats, weight loss, chills, and relative bradycardia (a heart rate lower than expected for the degree of fever). The naproxen test was proposed on the assumption of a selective antipyretic activity against neoplastic fever, but in clinical practice the accuracy of this test too is too low to be discriminatory. Go where the money is The diagnostician confronted with FUO should keep in mind Sutton’s law:  ‘go where the money is’. Possible diagnostic clues elicited from the history, physical examination, and the prelim- inary diagnostic evaluation (Box 8.2.2.1) should, of course, guide further investigation, but many cases become a FUO because these clues are misleading. Whenever possible, the clinician should strive to achieve microbiological or pathological confirmation. Any sus- pected focal abnormality that is accessible should be aspirated or biopsied. Close communication with the microbiologist and the pathologist will increase the diagnostic yield. Molecular methods, such as polymerase chain reaction studies, are increasingly an asset in selected cases. When diagnostic clues are either absent or misleading, an indi- vidualized approach is preferable. Indeed, there are no useful or evidence-​based rigid algorithms. Screening imaging techniques Imaging is used primarily to localize abnormalities for further evalu- ation. Due to the higher spatial resolution compared with chest radio- graphs and ultrasound of the abdomen, CT scanning of thorax or abdomen is useful when looking for focal disease, mainly infectious or neoplastic. In the near future, the role of MRI in the work-​up of FUO is anticipated to grow as its benefits relative to CT are demar- cated. Nuclear imaging studies are potential tools for FUO workup. The choice between a whole variety of radiopharmaceuticals depends on local availability, cost, and skill. We do not advocate tracers that are more specific for infections, such as labelled leucocytes, because a var- iety of inflammatory and neoplastic conditions enter the differential diagnosis, not just infections. In particular, 18F-​fluorodeoxyglucose positron emission tomography (FDG-​PET) is an established inflam- mation tracer technique in FUO, yielding the diagnosis in 25–​40% of patients and performing at least as well as gallium scintigraphy. However, unlike gallium, fluorodeoxyglucose is taken up in vasculitic lesions in large blood vessels (giant cell arteritis and Takayasu’s arter- itis), which are classic causes of FUO. The combined and integrated use of FDG-PET and CT improves diagnostic accuracy. Selective testing The imaging studies may unmask hidden infectious, neoplastic, and inflammatory foci, but endoscopic techniques (e.g. gastrointes- tinal endoscopy, bronchoscopy), selective radiographs (e.g. of teeth, sinuses, sacroiliac joints), or contrast studies (e.g. gastrointestinal series, arteriography) should be ordered only when there is a well-​ founded and specific clinical suspicion. They should not be used as routine tests for FUO. This is even more the case for invasive pro- cedures such as mediastinoscopy, thoracoscopy, or laparoscopy, techniques that are being replaced increasingly by less invasive ultra- sound echoendoscopy, or CT-​guided biopsy. Nowadays, explora- tory laparoscopy is restricted to exceptional situations (e.g. when peritoneal carcinomatosis or tuberculosis are suspected and other tests have failed). Likewise, biopsies of lymph nodes, bone marrow, or liver, and lumbar puncture can be diagnostic, but should not be performed blindly, in the absence of firm suspicion of pathological involvement. The only biopsy that may be routinely performed is temporal artery biopsy in a patient over the age of 50 with a pro- longed unexplained fever and vigorous acute-​phase response, even in the absence of arteritic symptoms. Giant cell arteritis is one of the most frequent diagnoses in this age group and carries a serious risk of visual loss and other ischaemic complications. Watchful waiting An undirected pursuit of often increasingly costly and invasive tests is discouraged. Instead, when the diagnosis remains in doubt, all data (including those from other hospitals) should be critically re- viewed, and history taking, physical examination, and some basic tests (e.g. white blood cell count with differential, creatine kinase, urinalysis, chest radiograph) repeated in an effort to find clues that were previously overlooked or inapparent. There is no substitute for observing, talking to, and thinking about the patient. If the diagnosis cannot be established after intelligent thorough investigation, an ex- pectant approach is justified if the patient’s condition is stable. In published series, most patients with FUO who left hospital without a diagnosis did remarkably well. Therapeutic trials Therapeutic trials are seldom diagnostically rewarding and tend to obscure, rather than illuminate. In contrast to the approach to fever in immunocompromised patients (Chapter  8.2.4), the gen- eral goal when dealing with classic FUO is to ascertain the diag- nosis before starting therapy. Antipyretics, mainly non​steroidal anti-​inflammatory drugs, may be symptomatically useful but rarely aid diagnosis. Blind administration of corticosteroids is discour- aged. Infections such as tuberculosis may seemingly respond ini- tially, only to deteriorate thereafter. Most patients have already had a failed trial of antibiotics before referral to secondary or tertiary care. Defervescence following administration of an antimicrobial

8.2.3 Nosocomial infections 669

8.2.3 Nosocomial infections 669

8.2.3  Nosocomial infections 669 agent is rarely diagnostic as the spectrum generally involves more than a single microorganism. Moreover, fevers caused by infections, such as disseminated tuberculosis or culture-​negative endocar- ditis, may wane only several days after starting appropriate therapy. Spontaneous resolution of fever may coincide with a therapeutic trial, which is another argument against its routine use. The excep- tion to the rule of withholding empirical therapy in classic FUO is the severely deteriorating patient. In such situations, antituberculosis chemotherapy is warranted, since tuberculosis is probably the most common cause of avoidable death in adults with classic FUO. Corticosteroid treatment is the next step in case of further deterior- ation of the clinical condition. Prognosis Not surprisingly, the outcome of classic FUO is highly variable and depends on the underlying disease. In the series of Larson et al. from the 1980s, for instance, only 9% of patients with malignan- cies were long-​term survivors, while 78% of patients with infec- tions and 88% of patients with FUO in other categories were alive after 1 year. Older age carries a worse prognosis. In a series from the 1990s, haematological malignancies (especially non-​Hodgkin’s lymphoma), constituted 12% of diagnoses but accounted for almost 60% of deaths. Treatable causes of death have included abdominal abscesses, endocarditis, vasculitis, pulmonary embolism, and espe- cially tuberculosis. Most patients who cannot be diagnosed do well and over two-​ thirds have no recurrence of symptoms. Among the rest, a subgroup has clinical features suggesting protracted non​infectious inflam­ matory conditions, without meeting accepted diagnostic criteria for any particular disease. Most of these fevers respond to corticosteroid therapy. FURTHER READING Arnow PM, Flaherty JP (1997). Fever of unknown origin. Lancet, 350, 575–​80. Cunha BA (2007). Fever of unknown origin: clinical overview of clas- sical and current concepts. Infect Dis Clin N Am, 21, 867–​915. Durack DT, Street AC (1991). Fever of unknown origin: reexamined and redefined. Curr Clin Top Infect Dis, 11, 35–​51. Hayakawa K, Ramasamy B, Chandrasekar PH (2012). Fever of un- known origin: an evidence-​based review. Am J Med Sci, 344, 307–​16. Hirschmann JV (1997). Fever of unknown origin in adults. Clin Infect Dis, 24, 291–​302. Knockaert DC, Vanderschueren S, Blockmans D (2003). Fever of un- known origin in adults: 40 years on. J Intern Med, 253, 263–​75. Knockaert DC, et al. (1992). Fever of unknown origin in the 1980s: an update of the diagnostic spectrum. Arch Intern Med, 152, 51–​5. Larson EB, Featherstone HJ, Petersdorf RG (1982). Fever of undeter- mined origin:  diagnosis and follow-​up of 105 cases, 1970–​1980. Medicine, 61, 269–​92. Petersdorf RB, Beeson PB (1961). Fever of unexplained origin: report on 100 cases. Medicine, 40, 1–​30. Vanderschueren S, et al. (2003). From prolonged febrile illness to fever of unknown origin: the challenge continues. Arch Intern Med, 163, 1033–​41. 8.2.3  Nosocomial infections Ian C.J.W. Bowler and Matthew Scarborough ESSENTIALS Hospital-​acquired or nosocomial infections—​defined for epidemio- logical purposes as infections manifesting more than 48 hours after hospital admission—​are common. They affect 1.4  million people worldwide, involve between 5 and 25% of hospitalized patients
at any one time and are associated with considerable morbidity, mortality, and cost. The most common sites of nosocomial infection are the urinary tract, surgical wounds, and the lower respiratory tract. Most are bacterial in origin, the most common species being Escherichia coli, Staphylococcus aureus (including methicillin-​resistant Staphylococcus aureus), enterococci, Pseudomonas aeruginosa, and coagulase-​ negative staphylococci. The principal risk factors are extremes of age, the severity of underlying acute disease (e.g. neutropenia, organ system failure), and chronic medical conditions (especially diabetes, renal failure, and alcohol abuse). Between 15 and 30% of nosocomial infections are preventable, and hospital practitioners have a duty of care to minimize the risk of infection for their patients. Systematic surveillance to assess the incidence and prevalence of such infections, together with a regu- larly audited organized programme to minimize their impact, should be an important part of every hospital’s quality assurance system. All staff should receive regular education to ensure that they recognize that infection control is ‘everyone’s business’ and hospital managers must ensure appropriate staffing and resources to provide: • access to advice from appropriately trained experts in infection control • surveillance of infection with regular feedback of the data to staff • isolation of patients with infections, with appropriate arrange- ments for their nursing and medical management • appropriate arrangements for carrying out procedures likely to in- crease the risk of infection (e.g. insertion of central venous lines) • policies for outbreak management Definitions Nosocomial infections, as distinct from community-​acquired in- fections, are defined for epidemiological purposes as infections manifesting more than 48 hours after admission to hospital. More rarely, nosocomial infections can affect hospital staff; in such in- stances they are defined as infections acquired through exposure at work. Some nosocomial infections may not be so easily identified as hospital acquired; for example, hospital-​acquired hepatitis B in- fection may not become clinically apparent until months after the patient has been discharged because of the prolonged incubation period. Healthcare-​associated infections are those that present in non-​ hospitalized patients who have had extensive or recent healthcare contact. They include infections in nursing home or long-​term care

670 SECTION 8  Infectious diseases facility residents, and infections arising within 90 days of discharge from hospital or 30 days from hospital attendance. Iatrogenic infections are acquired as the direct consequence of a therapeutic intervention (e.g. insertion of a urinary catheter). Opportunistic infections are caused by organisms that do not ordinarily harm healthy people; they occur in people with impaired immune defences. Endogenous (autogenous) infections are produced by the patient’s normal flora. Exogenous infections result from transmission of organisms to the patient from elsewhere. Although in practice it may not always be possible to distinguish endogenous from exogenous infections, this differentiation must be attempted because of important implications for infection control. Scale and costs of nosocomial infections Rates of nosocomial infections between 4 and 6.4 per 100 admissions have been reported. The urinary tract, surgical wounds, and the lower respiratory tract are the most common sites (Table 8.2.3.1). In the United States of America, an estimated 75 000 deaths per year are dir- ectly attributable to nosocomial infection. In 2013 the cost associated with nosocomial infection in the United States was estimated at $9.8 billion, most of which was attributed to delayed discharge from hos- pital. Rapid changes in healthcare provision mean that the frequency and nature of nosocomial infection are changing. The increasing trend towards early discharge, particularly for surgical patients, can lead to an underassessment of the disease burden. New interventions provide new opportunities for infection. For instance, flexible endo- scopes, which have revolutionized the investigation and manage- ment of a wide variety of diseases, can transmit hepatitis B between patients if the endoscopes are not appropriately decontaminated between procedures. In 2015 heater/cooler equipment used during cardio-pulmonary bypass was implicated in the transmission of Mycobacterium chimaera resulting in mediastinitis and endocarditis. Host and environmental factors The principal risk factors are extremes of age and the severity of the underlying disease (e.g. neutropenia, organ system failure). The ageing population in more developed countries has had a major im- pact on the prevalence of hospital-​acquired infection. In multivariate analysis, certain medical diagnoses, including diabetes mellitus, renal failure, and alcohol abuse, are strongly associated with risk. Treatment itself lower host defences (e.g. surgical incisions, bladder catheterization, mechanical ventilation, and neutropenia following cancer chemotherapy). The increasing use of prosthetic devices (e.g. intravascular catheters, cardiac valves and pacemakers, vascular grafts, and joint replacements) which facilitate formation of biofilm by certain bacteria, can also subvert normal defence mechanisms. Patients with similar clinical problems, who are likely to share similar risk factors for infection, tend to be nursed together for convenience, so the introduction of a microorganism into such a group can rapidly infect several patients. A good example is the rapid spread of norovirus gastroenteritis in geriatric wards. A poorly maintained hospital envir- onment is a threat to vulnerable patients; for instance, in units caring for patients with solid organ transplants, outbreaks of legionellosis can result from defective air conditioning and hot water systems. Microorganisms and use of antibiotics Bacteria are the most frequently implicated pathogens in nosocomial infections. These include Escherichia coli, Staphylococcus aureus, enterococci, Pseudomonas aeruginosa, Klebsiella spp, coagulase-​ negative staphylococci, and Clostidium difficile, in decreasing order of frequency. Viruses, fungi, and protozoa play a minor part. Whether endogenous or exogenous, the organisms causing noso- comial infection are usually part of a patient’s normal colonizing flora and it is often difficult to distinguish infection from coloniza- tion using bacteriological tests alone. The organisms are frequently multidrug resistant, since the widespread use of antibiotics in hos- pitals gives these strains a selective advantage. Empirical antibiotic therapy should accommodate the shift towards more resistant col- onizing flora in hospitals, particularly in burns units and intensive care units. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-​resistant S. aureus (MRSA), and enterococci are often resistant to multiple antimicrobials, making them difficult and expensive to treat. Increasing international travel means that organisms which were previously geographically restricted (e.g. NDM E.  coli and Klebsiella pneumoniae in India) are becoming increasingly common globally. These organisms may be resistant to nearly all antibiotics, including carbapenems, and can spread when patients are transferred between countries and between centres. It is im- portant that such organisms are detected by screening cultures at the time of admission, so that appropriate precautions to prevent their spread can be implemented. Antibiotic resistance in bacteria causing nosocomial infection is a topic of worldwide concern. There is increasing emphasis on im- plementation of ‘stewardship’ programmes to ensure antibiotics are targeted appropriately and indiscriminate use avoided. Table 8.2.3.1  Rates and sites of nosocomial infection in three regions European Union, Norway, Iceland,
and Croatia (2011) UK (2011) USA (2011) Rates: cases/​100 admission sites (% of all infections) 5.7 6.4   4 Lower respiratory tract infection 24 23 22 Surgical wound infection 20 16 22 Urinary tract infection 19 17 13 Other 37 44 47

8.2.3  Nosocomial infections 671 Principles of hospital infection control The principal aim of the hospital infection control programme is to prevent nosocomial infection. The identification and typing of iso- lates causing nosocomial infection allows recognition of organisms that are epidemiologically linked. Invasive multidrug-​resistant or- ganisms, such as MRSA, often require infection control measures to prevent their spread and so minimize the use of expensive, some- times toxic, antibiotics required for their prophylaxis and treatment. Epidemic infections account for less than 10% of the nosocomial disease burden but attract professional and media interest because they are unusual. They are amenable to measures that interrupt the spread of infection, such as the use of gowns and gloves, and meticulous hand hygiene. Care of colonized or infected patients in single rooms or an isolation ward is a physical means of preventing spread. Alternatively, patients infected with the same organism can be grouped together (cohorted) and attended to by a group of nurses not involved with un- infected patients. Identification of additional carriers and elimination of colonization may be necessary to control some epidemic outbreaks. There have been no randomized trials demonstrating the efficacy of such measures, but many observational studies support their use. Endemic nosocomial infections are more difficult to control. The size of the problem may not be apparent either because attack rates in individual units may be low or because infection is seen as a normal consequence of certain interventions. It is important that information about endemic infections is collected systematically in a comprehensive surveillance programme, analysed, disseminated, and discussed so that preventive strategies can be improved. Control measures are applied to selected patients according to risk (e.g. cor- rectly timed antimicrobial prophylaxis and meticulous sterile tech- nique in prosthetic joint replacement surgery). Site of nosocomial infections Urinary tract A bacterial count of at least 105 organisms/​ml in a freshly voided urine sample indicates infection, although counts as low as 102 or- ganisms/​ml are included by some classifications. The presence of any organisms in a sample taken from a urinary catheter at the time of insertion or from a suprapubic aspirate may indicate infection. Indwelling urinary catheters account for 80% of nosocomial urinary tract infections, and 80% of patients catheterized for longer than 7 days develop bacteriuria (bacteria in the urine). Most patients with catheter-​related urinary tract infection remain asymptomatic, but 20–​30% develop the symptoms of urinary tract infection and about 1 in 100 of these develops bacteraemia. Instrumentation of the urinary tract is also a risk factor for urinary tract infection. The main source of organisms is the periurethral flora, with E. coli reported as the dominant pathogen in all studies. Treatment is with broad-​ spectrum antimicrobials administered empirically after obtaining appropriate cultures and later adjusted according to the results of bacteriological studies. Asymptomatic patients need not be treated. Since the most important risk factor is the duration of catheteriza- tion, avoiding catheterization (or ensuring their early removal) is the most effective means of preventing nosocomial urinary tract infec- tion. In addition, catheters should be inserted aseptically, attached to a closed sterile drainage system, and placed on uninterrupted gravity drainage. Suprapubic or intermittent urethral catheterization are also sometimes employed to reduce the risk of nosocomial infection. Some practitioners advocate a single prophylactic dose of antibiotic at the time of urinary catheter insertion or exchange in men to prevent bacteraemia. In other settings prophylactic antibiotics have not been shown to prevent infection for more than a few days. Catheters coated with antimicrobials such as silver have been shown to reduce infection rates in some patient groups, but their cost-​effectiveness is disputed. Surgical wound infection The diagnosis of a surgical wound infection usually requires the pres- ence of spreading erythema or purulent discharge from a wound, but rates vary according to the definition used. Internationally agreed diagnostic criteria are used for high-​quality clinical and epidemio- logical studies Most wound infections result from direct inoculation of organ- isms into the wound at the time of surgery. The main risk factor is the degree of wound contamination at operation. Operations may be ‘clean’ (e.g. herniorrhaphy), ‘clean–​contaminated’ (e.g. appendicec- tomy which requires incision of bowel), or ‘contaminated’ (e.g. gross spillage from the gastrointestinal tract during surgery). S. aureus is the most common pathogen complicating clean surgery, for which rates below 2% are expected. ‘Contaminated’ surgery is often associ- ated with polymicrobial infections, especially with E. coli and mixed anaerobes originating from the patient’s gastrointestinal tract; rates of infection following contaminated surgery are reported to be be- tween 5–​25%. Other risk factors include age, obesity, the duration of the operation, and the presence of a remote infection. Wound infections usually present with local symptoms and signs (pain, erythema, pus, dehiscence) and with general features of infec- tion, such as fever. Appropriate cultures, including blood cultures, are taken, pus is drained, and broad-​spectrum antimicrobials are given empirically, directed at the likely flora but later adjusted ac- cording to bacteriological results. Prevention is by meticulous aseptic surgical techniques. Prophylactic antimicrobials, given no more than 2 hours before the surgical incision, have been shown to reduce wound infection rates by between two-​ and fivefold for clean–​contaminated and contam- inated procedures, and in clean surgery when a prosthesis is inserted (e.g. joint replacement, vascular graft insertion). Nosocomial pneumonia Pneumonia is defined clinically by the production of purulent sputum, signs of respiratory consolidation, a fall in arterial Po2, and the appearance of new infiltrates on the chest radiograph. Between 0.55 and 1.5% of patients admitted to hospital develop lower respira- tory tract infections. Crude case fatality rates of between 20 and 30% are quoted, but death occurs most commonly as a result of underlying disease. Patients who are intubated and ventilated have a high risk of developing pneumonia as a result of aspiration of bacteria colon- izing the upper respiratory and gastrointestinal tracts. The organisms causing ventilator-​associated pneumonia are usually acquired after admission to hospital and the bacteria are often more antibiotic-​ resistant than community-​acquired organisms. Examples of organ- isms causing nosocomial pneumonia are listed in Table 8.2.3.2. Culture of expectorated sputum or tracheal aspirates is poorly predictive of the bacterial cause of nosocomial pneumonia, which

672 SECTION 8  Infectious diseases is best determined by quantitative culture of specimens obtained by sampling the terminal airways (e.g. by bronchoalveolar lavage). Initially, broad-​spectrum antimicrobials should be given empiric- ally. Once the susceptibility of the causative pathogen has been de- termined, specific antimicrobial treatment can be instituted. The risks of nosocomial pneumonia can be reduced by a var- iety of strategies, including avoidance of intubation and the use of non​invasive ventilation techniques. For those who are intubated, continuous aspiration of subglottic secretions and nursing in the semi-​recumbent position have been shown to be effective. Selective decontamination of the digestive tract by the administration of non​absorbable antibiotics has shown modest mortality benefit in ventilated patients in countries where resistance rates are low, but very limited advantage in areas where there is a high prevalence of multidrug-​resistant organisms. Short courses of antibiotics at the time of intubation have been shown to be effective in certain pa- tient groups. Epidemic nosocomial pneumonia usually results from bacterial contamination of respiratory equipment, such as nebu- lizers, ventilators, or bronchoscopes. It is best prevented by ensuring single-​use respiratory devices, by cleaning and disinfecting equip- ment, and by hand hygiene before and after every patient contact. Intravascular device-​associated infections Bacteraemia is the most important intravascular device-​associated infection; it varies in prevalence from about 0.04% for subcuta- neous central venous lines to about 0.2% for peripheral intravenous cannulae, and approximately 10% for temporary non​tunnelled cen- tral venous haemodialysis catheters. The duration of intravascular access is the most significant risk factor. Bacteria usually gain entry by direct spread from the skin surface in- cision along the subcutaneous catheter tunnel to its tip in the blood vessel. Less commonly, line infection results from contamination of connecting devices; this is particularly important in catheters with sub- cutaneous cuffs, such as Hickman catheters, where the peri-​luminal route of infection is less likely. The organisms that most frequently cause intravenous device-​related bacteraemia are coagulase-​negative staphylococci, S. aureus, Pseudomonas spp., and Candida spp. Line-​related infection most commonly presents with features of bacteraemia. In a minority of cases, there are clear signs of local in- flammation or thrombophlebitis at the insertion site. Management most commonly involves taking blood cultures, removal of the catheter (with culture of the tip), and empirical antimicrobials. Sometimes, long-​term intravenous catheters, such as Hickman lines, can be ‘sterilized’ by administering parenteral antibiotics into the line as ‘antibiotic lock’ therapy. Superficial infections which are restricted to the insertion site can sometimes be treated with antibiotics and line retention. Tunnel infections usually require removal of the line. Prevention of line-​associated infections is best achieved by using aseptic techniques during insertion, maintaining high standards of line care, and removing catheters as soon as possible. Before insertion, the skin should be prepared with a reliable disinfectant such as an al- coholic solution of chlorhexidine. For insertion of long lines, the op- erators should wash their hands, use a large sterile drape to isolate the insertion site, and wear sterile gloves, gown, face mask, and hat. Central venous catheters are usually removed only if blocked or suspected as a source of sepsis. The skin at the exit site of peripheral intravascular devices should be checked daily and the device removed if infection is suspected. Subcutaneous tunnelling, use of a cuffed device (Hickman line), use of subcutaneous access (e.g. portacaths), and use of antimicro- bial coated lines can all reduce the infection rate significantly. Replacing the entire intravenous delivery set every 72 hours is sufficient to reduce sepsis secondary to intraluminal contamination of ‘giving’ sets. Prosthetic device-​related infection Infections of prosthetic devices such as heart valves, vascular grafts, cerebrospinal fluid shunts, artificial lenses, and joint replacements are usually caused by the normal skin flora. The devices become coated with a layer of host-​derived macromolecules such as fibronectin and fibrin which have specific adhesion receptors for bacteria, particularly staphylococci. Once attached, these organisms multiply on the sur- face of the coated prosthesis forming a biofilm. Microbes embedded in biofilm are relatively inactive metabolically and far less susceptible to antibiotics as compared to planktonic or free-​living bacteria. The formation of biofilm, therefore, confers significant phenotypic resist- ance to medical therapy even if the organisms appear susceptible to antibiotics in vitro. Apart from those involving an intraocular lens, such infections are rarely cured with antimicrobial therapy alone and frequently require removal of the prosthetic device. Bacteria gain access to prosthetic devices by direct inoculation, usu- ally at the time of surgery, or less commonly by settling on the pros- thesis after haematogenous spread. Direct inoculation at surgery can be responsible for prosthetic device infections presenting more than 1 year after insertion since the organisms involved are usually skin commensals of low virulence (e.g. coagulase-​negative staphylococci). Prevention is by avoiding contamination of the wound at sur- gery and by using strict aseptic surgical techniques. In ortho- paedic implant surgery, a large randomized controlled trial showed that an ultraclean air supply to the operating theatre is of benefit. Prophylactic antimicrobials given at the time of surgery have also been shown to reduce the risk of prosthetic joint infections. Antibiotic-​associated diarrhoea Up to 30% of patients treated with antibiotics will develop diarrhoea as a result of the disturbance of the complex gut flora. In a few, loss of ‘colonization resistance’ predisposes to acquisition of Clostridium difficile. Colonization by this organism is usually harmless, but in about 3% of patients, particularly older people, the organism may overgrow and produce a cytotoxin resulting in colitis. The clinical picture varies from mild diarrhoea with fever to fulminating colitis with dilatation of the colon (toxic megacolon) requiring colectomy. More severe disease and a greater likelihood of relapse are associated with a quinolone-​resistant clone of C. difficile, Table 8.2.3.2  Causative organisms identified in samples
obtained at bronchoscopy or tracheal aspiration (percentage
of all pneumonias) USA (2007) Staphylococcus aureus including MRSA 27 Pseudomonas aeruginosa 18 Escherichia coli and other Enterobacteriaceae 15 Acinetobacter spp.   8 Stenotrophomonas maltophilia   7 Streptococci   3 Other species 22

8.2.4 Infection in the immunocompromised host 673

8.2.4 Infection in the immunocompromised host 673

8.2.4  Infection in the immunocompromised host 673 which produces large amounts of toxin due to the deletion of a regu- lator gene tcdC. C.  difficile-​associated diarrhoea delays discharge from hospital by about one week. Since attack rates in older patients are around 5% and relapse can occur in up to 25%, the disease can have a major impact on hospital resources (see Chapter 8.6.24). Diagnosis is by two-​stage testing; a stool sample is first tested for the presence of the organism by polymerase chain reaction or enzyme-​linked immunosorbent assay, and, if positive, is then tested for the presence of cytotoxin. This strategy has sensitivity of 95% but limited specificity for C. difficile disease: toxin may be found in the stool of asymptomatic patients, and for many weeks after full recovery in those with symptoms. Patient management includes ad- equate rehydration, avoiding drugs which inhibit gut motility and stopping the provoking antibiotics. Treatment is with oral vancomycin, fidaxomicin, or metronida- zole. Surgical review is required for severe cases. Relapse may occur in up to 25% of cases but is less common (15%) if fidaxomicin is used as the initial therapy. The use of intravenous immunoglobulin and faecal transplant are controversial but there is growing evidence in favour of their use in severe or recurrent disease. Prevention is by restricting the use of antibiotics according to agreed and audited protocols. The importance of antibiotic restric- tion has been emphasized by recent studies using bacterial whole genome sequencing, which show that the C.  difficile strains cul- tured from symptomatic patients in hospital are extremely diverse; nearly half of all patients were infected with their own unique strain rather than one acquired from other patients in the hospital. Hand washing after patient contact, isolation of patients with diarrhoea, and cleaning the ward environment are employed on microbiological grounds, despite a lack of prospective studies showing their efficacy. Nosocomial bacteraemia Bacteraemia may occur secondarily to the infections mentioned earlier. The incidence is approximately 3 per 1000 hospital admis- sions. The case fatality is about 40%, but varies with the severity of the underlying disease and comorbidities, being as low as about 2% in obstetric patients. Most cases are related to a urinary catheter, intra- vascular catheter, or postsurgical infection. Management should include the identification and, if possible, removal of the infective focus, as well as appropriate antimicrobial therapy after obtaining blood and other relevant samples for microbiological culture. Future developments The increasing cost of healthcare will drive governments to impose mandatory surveillance and targets for reduction of selected nosoco- mial infections as these measures are highly cost-​effective. In United Kingdom this has been manifest by legislation (‘The Health Act 2006: A code of practice for the prevention and control of healthcare-​associated infection’) which mandates hospitals to have in place processes for the continuous improvement of infection rates. The UK government has published ‘care bundles’ of ‘high impact interventions’ outlining evidence-​based practice for how this can be achieved. Coincident with the implementation of these measures since 2006, MRSA bacter- aemia and C. difficile infection rates have declined significantly in the United Kingdom. Rapidly developing techniques of molecular biology are likely to reveal more clearly the relationship between hospital patients and the organisms which infect them, pointing the way to new risk-​reducing strategies. Whole genome sequencing will improve our understanding of transmission pathways, virulence, and patho- genicity of the organisms involved, and will enable targeted interven- tions. Recent sequencing studies have shown that the global increase in Candida auris infections is due to the co-evolution of multiple separate clades, each with different polymorphisms for antifungal resistance. Human genetic studies may also identify polymorphisms which pre- dispose certain individuals or groups of individuals to infection. FURTHER READING Chand M, et al. (2017). Insidious risk of severe Mycobacterium chimaera infection in cardiac surgery patients. Clin Infect Dis, 64, 335–42. Edgeworth JD, et al. (2007). An outbreak in an intensive care unit of a strain of methicillin-​resistant Staphylococcus aureus sequence type 239 associated with an increased rate of vascular access device-​ related bacteraemia. Clin Infect Dis, 44, 493–​501. Eyre DW, et al. (2013). Diverse sources of C. difficile infection iden- tified on whole-​genome sequencing. N Engl J Med, 369, 1195–​205. Flores C, et  al. (2006). A CXCL2 tandem repeat promoter poly- morphism is associated with susceptibility to severe sepsis in the Spanish population. Genes Immun, 7, 141–​9. Harris SR, et al. (2010). Evolution of MRSA during hospital transmis- sion and intercontinental spread. Science, 327, 469–​74. Lockhart SR, et al. (2017). Simultaneous Emergence of Multidrug- Resistant Candida auris on 3 Continents Confirmed by Whole- Genome Sequencing and Epidemiological Analyses. Clin Infect Dis, 64(2), 134–40. Magill SS, et al. (2014). Multistate point-​prevalence survey of health care-​associated infections. N Engl J Med, 370, 1198–​208. World Health Organization (2005). Global patient safety challenge: 2005–​ 6/​world alliance for patient safety. World Health Organization, Geneva. Jarvis WR (2007). Bennett and Brachman's hospital infections, 5th edition. Lippincott Williams & Wilkins, Philadelphia, PA. Websites Department of Health England. High Impact Interventions. http://​ webarchive.nationalarchives.gov.uk/​+/​http://​www.dh.gov.uk/​en/
​Publicationsandstatistics/​Publications/​PublicationsPolicyAnd Guidance/​DH_​078134 Department of Health (2006). UK Health Act 2006. http://​www.legislation. gov.uk/​ukpga/​2006/​28/​contents Information on New Delhi metallo-​beta-​lactamase 1. http://​en.wikipedia. org/​wiki/​New_​Delhi_​metallo-​beta-​lactamase_​1. 8.2.4  Infection in the immunocompromised host Jon Cohen and Elham Khatamzas ESSENTIALS The term ‘immunocompromised host’ embraces a group of overlapping conditions in which the ability to respond normally to

674 SECTION 8  Infectious diseases an infective challenge is in some way impaired. This includes patients with underlying conditions such as protein–​calorie malnutrition and diabetes, as well as organ transplant recipients, those with haem- atological malignancies and others receiving therapeutic immuno- suppression, and patients with HIV infection. Many patients have multiple risk factors that increase the risk of opportunistic infection. General clinical approach A high level of awareness is essential for the management of patients who are immunocompromised; infections can progress very quickly, the early physical signs are often muted, and the microbiology can be confusing. Aside from a full history and detailed physical exam- ination, assessment should take account of risk factors such as the depth and duration of neutropenia, and the dose and duration of immunosuppressive therapies as well as history or exposure to antimicrobial agents or colonisation with antimicrobial resistant or- ganisms. It is particularly helpful to try to form a judgement of how quickly the condition is progressing. Patients must be reviewed fre- quently and will often require empirical antimicrobial therapy, but when possible it is better to try to establish the cause of the infection before starting treatment. This is partly because the differential diag- nosis is wide and choosing the right treatment depends on knowing the causative organism, and partly because it is not uncommon for multiple organisms of different types to be involved. Particular clinical syndromes Fever of unknown origin—​this is common in patients with neutro- penia, with the risk of bacteraemia being most acute when the neutrophil count falls to less than 0.1 × 109/​litre; in 50% of cases an organism is never identified. Empirical antibiotic therapy is vital and needs to be directed against both Gram-​negative and Gram-​ positive organisms. The risk of invasive fungal infection rises if fever persists, in which case empirical antifungal therapy is justified. Fever and new pulmonary infiltrates—​this is a challenging problem with a wide range of potential causes depending on the clinical setting, including conventional respiratory pathogens, nosocomial pathogens, ‘atypical’ organisms, mycobacteria and re- lated organisms, viruses, fungi, parasites, and also non​infectious causes such as pulmonary oedema, pulmonary haemorrhage,
pulmonary emboli/​infarction and drug toxicity. The clinical and radiological features are very rarely pathognomonic, hence there should be a low threshold for performing a diagnostic procedure such as bronchoalveolar lavage. Acute neurological syndromes—​these include both (1)  meningoencephalitis—​associated with conventional bac- terial infections, listeriosis, and tuberculosis, as well as fungi such as cryptococcus and candida; and (2)  space-​occupying lesions—​ caused by, for example, toxoplasma, aspergillus, and nocardia. Gastrointestinal syndromes—​these are frequent and include (1)  stomatitis—​the three most common causes (candida, herpes simplex virus and chemotherapy-​induced mucositis) are clinically indistinguishable and can coexist; (2) diarrhoea—​graft-​versus-​host disease is very difficult to distinguish from infective causes in haem- atopoietic stem cell transplant (HSCT) recipients; (3) abnormalities of liver function tests—​mild derangements are a common accom- paniment to many systemic infections, but hepatitis is a particular feature of both toxoplasmosis and cytomegalovirus infection. Prevention This is an integral part of the management of patients who are im- munosuppressed and, depending on context, comprises interven- tions such as nursing them in single rooms and chemoprophylaxis (e.g. co-​trimoxazole to prevent pneumocystis and valganciclovir to prevent cytomegalovirus), but perhaps the single most important factor is being aware of the different and often subtle presentations of infection in this vulnerable group of patients. Classification The term ‘immunocompromised host’ has no formal definition but it embraces a group of overlapping conditions in which the ability to respond normally to an infective challenge is in some way impaired. By convention, this does not include otherwise healthy individuals whose only risk factor is a genetic polymorphism which may confer a slightly enhanced risk, for instance, to malaria or tuberculosis. It is helpful to think of immunocompromised patients as falling into one of several distinct groups (Fig. 8.2.4.1). Primary immunodeficiency syndromes These are patients with congenital defects in immunity that render them more susceptible to infection. At the most extreme, children with severe combined immunodeficiency have virtually no func- tioning cellular or humoral immunity and, if unprotected, they will die from infection within a few months of birth. In contrast, some patients with chronic granulomatous disease, an inherited defect in neutrophil function, remain undiagnosed until early adult life. A complete description of the diagnosis and management of this group of disorders is given in Chapter 5.2. AIDS HIV causes AIDS which is a model for an acquired defect of cellular immunity leading to an increased risk of infection. Although there are inevitably parallels with other groups of immunocomprom- ised patients, there are particular issues both in the diagnosis and management of infection in AIDS that warrant separate discussion (Chapter 8.5.23). Infection related to the underlying condition The notion of opportunistic infection in the immunocomprom- ised host is most familiar with haematological malignancy or organ Immunocompromised host AIDS Primary immunodeficiency syndromes Related to the underlying disease Related to therapeutic immunosuppression Secondary immunodeficiency syndromes Fig. 8.2.4.1  A classification of the immunocompromised host.

8.2.4  Infection in the immunocompromised host 675 transplantation, discussed in detail next. Less obvious, but prob- ably more numerous, are the many physiological conditions and other diseases associated with an increased incidence of infection (Box 8.2.4.1). These immune defects are usually mixed and fre- quently poorly characterized. The susceptibility to infection varies considerably both in the pattern and severity of infection that oc- curs, but the clinical problem is real enough. For example, in mal- nutrition infection due to mycobacteria and salmonella is more common, and pneumocystis pneumonia was first described in children with protein–​calorie malnutrition. There is an extensive literature documenting multiple defects of host defence in asso- ciation with alcohol abuse; clinically, this is reflected in an excess of lower respiratory tract infections with Streptococcus pneumo- niae, Mycobacterium tuberculosis, and Klebsiella pneumoniae. In Cushing’s disease, the excess endogenous steroid production can result in a pattern of opportunistic infections that mirrors that seen in patients receiving corticosteroid therapy (see next). Diabetes mellitus is a good example of a disease that is frequently compli- cated by infection, typically with staphylococcal skin abscesses. In myeloma and chronic lymphocytic leukaemia, the primary defect is hypogammaglobulinaemia. This is manifested clinically by an excess of bacterial infections, typically those caused by en- capsulated organisms such as S. pneumoniae and H. influenzae. In contrast, patients with rheumatoid arthritis, systemic lupus erythematosus, or polyarteritis nodosa predominantly have im- paired cellular immunity, although because they also commonly receive treatment with immunosuppressive drugs it can be very difficult to attribute cause and effect. Finally, patients who have had their spleen removed or who have functional (or more rarely congenital) asplenia are at increased risk of certain infections caused by particular organisms, notably S. pneumoniae and H. in- fluenza. The degree of risk is related to the underlying cause; overall, approximately 5% of patients will have a serious infec- tion, but this varies from 1.5% following traumatic splenectomy to as high as 25% in patients with thalassaemia. Serious infections are most common during the first 5 years following splenectomy and particularly during the first year, but overwhelming post-​ splenectomy infection can occur decades after the surgery. Infection complicating therapeutic immunosuppression In addition to the well-​recognized risk groups, such as those with haematological malignancy or allograft recipients, infective com- plications of immunosuppression are now being recognized in a much broader range of patients. Conditions as diverse as severe skin disease, asthma, inflammatory bowel disease, and rheumatoid arthritis are routinely treated with immunosuppressive drugs such as prednisolone, azathioprine, ciclosporin, cyclophosphamide, and biological agents such as antitumour necrosis factor (anti-​TNF) drugs. These patients are not so profoundly immunosuppressed as HSCT recipients, but they are certainly at risk of opportunistic infections. Immunosuppressed patients have multiple risk factors; a bone marrow transplant recipient may have been neutropenic, receiving corticosteroids and ciclosporin for management of graft-​versus-​ host disease, and have an indwelling right atrial catheter for feeding purposes. Clearly each of these factors represents a substantial and very different type of risk factor for infection and it is important to remember that, in such patients, multiple pathogens can cause disease simultaneously. Further complexity comes from the recog- nition that some opportunistic infections can themselves be im- munosuppressive; for instance, cytomegalovirus reactivated in the context of critical illness can itself reduce cell mediated immunity. Factors such as the precise nature and intensity of the immunosup- pressive regimen, anatomical and/​or surgical considerations, and the premorbid status of the patient will all have some influence on the pattern of opportunistic infections that occur. For instance, BK virus is a human polyoma virus that can cause renal allograft re- jection but virtually never causes clinical problems in other organ recipients; liver transplantation is notable for the high incidence of invasive candida infections, and toxoplasmosis is recognized to be a particular problem following cardiac transplantation. The increasingly widespread use of biological agents led to the rec- ognition that these too have particular risk profiles for causing opportunistic infection (Table 8.2.4.1) although in many cases ex- perience is still very limited. Finally, there is an extensive, although still somewhat confused literature indicating that a variety of single nucleotide polymorphisms can either increase, or reduce suscepti- bility to opportunistic infection, for example, invasive aspergillosis in stem cell transplant recipients. The following sections describe the management of some of the common clinical syndromes that present as infection in immunosuppressed patients. Common clinical syndromes A general approach to management Infections in immunosuppressed patients can progress with frightening rapidity; the early physical signs are often muted and the microbiology can be confusing. Patients need to be reviewed fre- quently and will often need empirical therapy, but this need not be totally ‘blind’; a structured and informed assessment will generally allow a logical response to what are the most likely pathogens. Most hospitals will have antimicrobial policies to guide empirical therapy in immunocompromised patients. Box 8.2.4.1  Examples of conditions associated with impaired immune responses and an increased risk/​severity of infection • Alcohol abuse and severe liver disease • Severe burns • Cushing’s disease • Cystic fibrosis • Primary infections with respiratory viruses such as influenza • Diabetes mellitus • Down’s syndrome • Extremes of age • Haemodialysis • Intravenous drug abuse • Malnutrition • Obesity • Pregnancy • Psychological stress • Sarcoidosis • Spinal cord injury • Splenectomy • Trauma/​surgery/​critical care • Uraemia

676 SECTION 8  Infectious diseases History This might reveal exposure to community-​acquired infections such as varicella zoster or tuberculosis, which can be particularly severe in the immunocompromised patient. Note should be made of any past history of infection; bronchiectasis, for instance, can be very troublesome in transplant recipients. A detailed travel history is im- portant; patients who have visited certain parts of the United States of America might have been exposed to the systemic mycoses such as histoplasmosis or coccidioidomycosis, which are unfamiliar to many clinicians. Visitors to Central America or the Far East, even many years ago, might have acquired an asymptomatic infection with the helminth Strongyloides stercoralis; immunosuppression can lead to overt disease (the hyperinfection syndrome) with a high mortality (see next). Physical examination This might be unhelpful as immunosuppressed patients often do not mount a good inflammatory response. Thus, there might be only a low-​grade fever, a thin serous exudate may suffice for pus, and mild abdominal tenderness can be the only sign of peri- tonitis. Nevertheless, careful, and if necessary repeated clinical examination is worthwhile, as signs of inflammation might be- come apparent only when immune function returns. The pres- ence of mucositis is strongly associated with risk of bacteraemia. Particular attention should be paid to new skin lesions. In neu- tropenic patients, bacteraemias can be accompanied by striking embolic lesions (Fig. 8.2.4.2); pseudomonas infections (and less commonly klebsiella and aeromonas) can cause a focal necrotic cellulitis called ecthyma gangrenosum. Fungal infections pre- sent as indolent locally invasive lesions; aspergillus infections often have a black eschar. The perianal area and the insertion sites of indwelling catheters repay careful examination. Aspiration and/​or biopsy of any new skin lesion in immunosuppressed pa- tients are well worthwhile, since they might quickly point to an otherwise inapparent diagnosis. Lymphadenopathy is always im- portant and will usually require aspiration or biopsy. It can be a manifestation of a lymphoproliferative condition, post-​transplant lymphoproliferative disease (PTLD), arising as a consequence of the intense immunosuppressive regimens now in widespread use. Epstein–​Barr virus infection or reactivation play a major role in the pathogenesis of PTLD. Underlying disease This can provide valuable clues. Neutropenia is a major risk factor for infection and renders the patient susceptible to bacteraemia, particularly with Gram-​negative organisms such as Escherichia coli and Pseudomonas aeruginosa, often due to mucosal transloca- tion. A patient with an obstructing bronchial neoplasm might de- velop a lung abscess due to inadequate drainage. Corticosteroids are used widely; when given in doses exceeding 15–​20 mg daily for Fig. 8.2.4.2  Disseminated Gram-​negative sepsis in a neutropenic patient. Table 8.2.4.1  Biological agents and the association with opportunistic infection. Some risks are well recognized: anti-​TNF agents and tuberculosis for instance. In many other cases the evidence suggests that there is a very small but nevertheless clearly increased risk Name of agent Target molecule Clinical indication Risk of infection Infliximab; etanercept; adalimumab; certolizumab; golimumab TNF RA, IBD, AID Mycobacteria; Listeria; Nocardia, Salmonella, fungi; PCP; hepatitis B/​C; herpesviruses Anakinra IL-​1 receptor RA; AID Mycobacteria; fungi; herpesviruses Abatacept T-​cells RA; AID Mycobacteria; fungi; herpesviruses Tocilizumab IL-​6 RA Mycobacteria; fungi; herpesviruses Rituximab CD20 RA; AID; B-​cell lymphoproliferative disorders Hepatitis B reactivation; PML; occasionally other OIs Alemtuzumab CD52 CLL; NHL; multiple sclerosis HSV; VZV; PCP; CMV; PML Bortezomib NF-​kB Myeloma; NHL VZV Natalizumab α 4 integrin Multiple sclerosis PML Vedolizumab α 4 integrin IBD Nasopharyngeal infections; occasionally other OIs Etrolizumab α 4 integrin IBD Nasopharyngeal infections; occasionally other OIs Secukinumab; ixekizumab; brodalumab IL-​17 Psoriasis Nasopharyngeal infections Canakinumab IL-​1 Juvenile RA Minor infections? (insufficient data) TNF, tumour necrosis factor; NF-​kB, nuclear factor kappa B; IL, interleukin; RA, rheumatoid arthritis; IBD, inflammatory bowel disease; AID, autoimmune diseases; CLL, chronic lymphatic leukaemia; NHL, non-​Hodgkins’s lymphoma; PCP, Pneumocystis jirovecii pneumonia; PML, progressive multifocal leukoencephalopathy (JC virus); OI, opportunistic infection; HSV, herpes simplex virus; VZV, varicella zoster virus; CMV, cytomegalovirus.

8.2.4  Infection in the immunocompromised host 677 long periods they increase susceptibility to infections with viruses, fungi, parasites, and bacteria such as Mycobacterium tuberculosis and Pneumocystis jirovecii, all organisms normally associated with impaired cellular immune defences. Duration of immunosuppression This often has a profound effect on the type of infection that oc- curs, and is well illustrated by comparing the ‘timetables’ of infec- tions in renal transplant recipients with patients receiving an HSCT (Fig. 8.2.4.3). In the first 6 weeks after renal transplantation bac- terial infections predominate, typically surgical complications of the procedure or urinary infections. Between 6 weeks and 6 months post-​transplantation the patient is most at risk from the ‘classic’ op- portunistic infections; as time continues and the intensity of im- munosuppression declines, typical community-​acquired infections become more common. In HSCT, the initial period of neutropenia is characterized by bacterial infections; later, when many patients re- ceive high-​dose steroids for graft-​versus-​host disease, cytomegalo- virus, and fungal infections (candida and aspergillus) develop. Speed of progression An assessment of this is helpful in both differential diagnosis and in deciding on empirical therapy. In neutropenic patients, the onset of fever is usually an indication for immediate empirical antibiotic therapy (see next). In contrast, the response to a fever and new pul- monary infiltrates in a patient who is 8 months postrenal transplant- ation will depend on the pace of the illness. Rapid deterioration over the space of a few hours will suggest a bacterial infection or a non​infectious cause, and will need urgent therapy; a more indolent presentation would point to a fungal or mycobacterial aetiology, and treatment can be delayed for a short period to try and establish the diagnosis. Investigations It is important that the diagnostic laboratories be made aware of the clinical problem since handling of specimens from immunosup- pressed patients—​and interpretation of the results—​will often differ substantially from routine procedures. Fever of unknown origin In neutropenic patients, fever is often the first and only sign of bac- teraemia, and prompt action is necessary. In this setting, a fever of unknown origin is defined as a single measurement of 38.3°C or greater, or a temperature of 38°C or greater sustained for 1 h and not obviously due to an identifiable cause such as concomitant blood transfusion. The risk of bacteraemia is directly related to the depth of the neutropenia; the incidence of infection rises when the neutrophil count falls to below 0.5 × 109/​litre, and is particularly severe when the count falls to less than 0.1 × 109/​litre (Fig. 8.2.4.4). Some years ago, the most common bloodstream isolates were Gram-​negative bacteria such as E. coli and klebsiella, generally derived from the patient’s gut flora, and P.  aeruginosa, a common environmental pathogen. Gram-​negative bacteraemia in neutropenic patients carried a very high mortality and led to the introduction of several preventative strategies such as the use of prophylactic antibiotics and colony-​stimulating factors. Although these approaches have not been entirely successful and might have contributed to the increasing incidence of multiresistant enterobacteriaceae, the in- cidence of Gram-​negative bacteraemias has declined substantially, and in most units Gram-​positive organisms, notably coagulase-​ negative staphylococci (Staphylococcus epidermidis) are now the most common isolates. Importantly though, tissue-​based infec- tions such as pneumonia continue to be caused predominantly by Gram-​negative bacteria. Renal transplantation Bone marrow transplantation BACTERIA OPPORTUNISTIC INFECTIONS VIRUSES FUNGI FUNGI 0 1 2 3 4 5 6 Months HSV CMV VZV Deep candidiasis Aspergillosis, trichosporon etc. Wound, UTI, pneumonia Pneumonia*, UTI, M. tuberculosis GNR bacteraemia GPC Oral candida S. pneumoniae N. meningitidis Cytomegalovirus Mycobacteria Pneumocystis Listeria Nocardia Candida, Aspergillus Toxoplasma (Interstitial pneumonitis) UTI = urinary tract infection

678 SECTION 8  Infectious diseases Clinical features are frequently unhelpful. Sometimes a focus will be suggested by erythema around the point of entry of an indwelling catheter, a finding often associated with staphylococcal infec- tion. Septic shock is infrequent, although it can be associated with viridans streptococci; interestingly, endocarditis is rare. Blood cultures should be drawn before treatment is begun. Ideally two sets should be obtained, at least one of which should be from a peripheral vein (rather than an indwelling catheter), although this is not always possible. Culturing larger volumes of blood (e.g. 30 ml compared to the more conventional 10 ml) will increase the yield. Appropriate samples must also be taken from other potential foci of infection. Nevertheless, it has been one of the enduring frustra- tions of this subject that even the most rigorous of microbiological investigations in the febrile neutropenic patient will yield only 40–​50% of positive cultures. The explanation for this is unknown; some studies have suggested that it is due to endotoxaemia in the absence of bacteraemia, but the data are inconclusive. What is clear, however, is that treatment must begin before the results of the cul- tures are available. The choice of the initial empirical antibiotic regimen for the febrile neutropenic patient has been the subject of intense in- vestigation. The ideal regimen will be safe and have good bac- tericidal activity against all the common pathogens. No single regimen is perfect; much will depend on the availability (and cost) of antibiotics in a given institution, and on local patterns of antibiotic susceptibility. Well-​validated hospital-​based regimens include the combination of an antipseudomonal penicillin plus an aminoglycoside or the use of single agents such as a third-​ or fourth-​generation cephalosporin (e.g. ceftazidime or cefepime), a carbapenem such as meropenem, or a β-​lactam/​β-​lactamase in- hibitor combination (e.g. piperacillin–​tazobactam). The Infectious Diseases Society of America has published helpful guidelines on the management of these patients (see Further reading). All these regimens are very active against the common Gram-​ negative organisms, but are relatively ineffective at treating antimicro- bial resistant Gram-​positive bacteria, such as coagulase-​negative staphylococci, meticillin (methicillin)-​resistant Staphylococcus au- reus (MRSA) and vancomycin-​resistant enterococci, that are now- adays common problems in many units. Unfortunately, there are only a very limited number of drugs that are reliably active against these organisms. Some clinicians have advocated adding an anti-​Gram-​ positive agent such as vancomycin to the initial empirical regimen; disadvantages of this approach include the toxicity of vanco- mycin, which may not be justified, particularly because coagulase-​ negative staphylococci rarely cause death, and the increasing rate of glycopeptide-​resistant organisms. Based on available evidence, guidelines recommend that unless there are strong grounds for con- sidering MRSA infection, vancomycin can usually be withheld until the results of blood cultures are known. An important development in practice has been the risk assess- ment of febrile neutropenic patients. The goal is to distinguish those high-​risk patients that need hospital admission and parenteral anti- biotics, from a low-​risk group (<5% risk of complications) who can be managed as outpatients with oral therapy. Patients who are as- sessed as being in a low-​risk group can be managed either with a brief period of inpatient parenteral therapy followed by rapid conversion to oral agents, or by oral therapy from the outset. A suitable regimen is the combination of a fluoroquinolone plus amoxicillin-​clavulanate. In patients who respond to the initial regimen, the treatment should be continued for at least 7 days, and ideally until the neutrophil count has returned to over 0.5 × 109/​litre. Sometimes this is not possible; the patient may have a persistent or unresponsive neutropenia (e.g. aplastic anaemia, or following HSCT). In these patients, treatment is usually cautiously stopped after an arbitrary period such as 14 days. However, there is undoubtedly a tension between the need to main- tain adequate antimicrobial cover during high risk periods and the wish to minimise exposure in line with the principles of good anti- biotic stewardship. In some parts of the world, multi-resistant Gram negative bacteria are re-emerging as threats to neutropenic patients. A common problem is the patient who continues to have high swinging fevers after 48–​72 h of broad-​spectrum antibacterial anti- biotics. The patient must be carefully re-​evaluated: Has some new clin- ical sign appeared? Could there be a resistant organism or an occult source of the sepsis? Simply changing the antibiotic regimen or adding vancomycin in the absence of any evidence to support these moves is not supported by clinical trial data. In this situation, invasive fungal infection becomes more likely and empirical addition of an antifungal agent with activity against moulds should be considered. Most centres will have their own antifungal policies and the choice of agent should take into account local epidemiology, antifungal prophylaxis, and screening strategies for pre-​emptive treatment. Recommended drugs include triazoles such as voriconazole, isavuconazole, amphotericin B formulation, or echinocandins but the emergence of newly recog- nized multi-resistant species such as Candida auris means that pre- cise diagnosis is mandatory in order to choose the most appropriate treatment. The Infectious Diseases Society of America and European Conference on Infections in Leukaemia have published comprehen- sive guidelines (see Further reading). Fever of unknown origin in the non​neutropenic immunosup- pressed patient presents as a completely different problem. Fever in this setting is rarely immediately life-​threatening, and the wide Percentage of patient days with infection Granulocyte level Relapse Total Remission <100 100– 500 500– 1000 1000– 1500

1500 60 50 40 30 20 10 Fig. 8.2.4.4  Relationship between neutrophil count and the risk of invasive Gram-​negative infection. From Bodey GP, et al. (1966). Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia. Ann Intern Med, 64,
328–​40, with permission.

8.2.4  Infection in the immunocompromised host 679 differential diagnosis means that it is generally better to pursue the cause rather than embark on empirical therapy. Fever and new pulmonary infiltrates The development of fever and new pulmonary infiltrates is one of the most challenging clinical problems in this group of patients. Pneumonia is a very common infective cause of death in immuno- compromised patients. In the presence of diffuse airspace disease, the mortality approaches 50% irrespective of the underlying defect in host defence, although the epidemiology varies both between dif- ferent patient groups and at different times reflecting the intensity of the immunosuppression (Table 8.2.4.2). The condition can progress extremely quickly, and conventional diagnostic procedures can be unhelpful. The list of possible causes is so daunting (Box 8.2.4.2) that clinicians might be tempted to use multiple empirical anti- microbial agents, sometimes to the patient’s detriment. It is often not possible to ‘guess’ with any certainty the precise cause of the problem (indeed, it can be dangerous to do so, since it is not uncommon for multiple causes to be present simultaneously), but by considering the available information one can construct a ‘short list’, which will guide further investigation and treatment. The initial evaluation should follow the approach outlined earlier, in particular making an assessment of the intensity of the immuno- suppression and the speed of progression of the pulmonary disease as well as the use of prophylactic agents, such as those advocated for prevention of invasive fungal infections. The main purpose of this is to determine the need for empirical therapy, either because the clinical picture is suggestive of a ‘simple’ bacterial pneumonia or because of a potentially more serious progressive cause of uncertain aetiology. Factors that would favour a bacterial aetiology include the presence of neutropenia, a rapidly developing clinical evolution (e.g. deterioration over a period of 12 h), progressive hypoxia, or a chest radiographic appearance that has worsened significantly over a short period. High fever is not necessarily a part of this syndrome; indeed, it is important to emphasize that this rapidly evolving clinical picture is not inevitably due to infection. Non​infective causes  such as acute lung haemorrhage or pulmonary oedema can present in an identical fashion, and the most appropriate therapy might be diuretics rather than antimicrobials. However, antimicrobials will often need to be given as well because of what has been termed ‘infection-​provoked relapse’. In immunologic- ally mediated diseases such as systemic lupus erythematosus or antiglomerular basement membrane (GBM) disease (Goodpasture’s syndrome) infection can precipitate a relapse of the underlying dis- ease. Thus, the development of fever and new pulmonary shadows in a patient with anti-​GBM disease might be primarily due to lung haemorrhage associated with a rise in anti-​GBM antibodies, but this in turn can be precipitated by an infection that need not ne- cessarily be in the lung. Treatment must be directed both towards improving oxygenation and the underlying infection. Blood cultures should always be obtained, and sputum obtained if it is available. A chest radiograph and arterial blood gas analysis are essential. The initial treatment will be dictated by the clinical circumstances, but the temptation to use a complex regimen to provide very broad-​spectrum cover is best avoided. Rapid clinical Table 8.2.4.2  Aetiology of the ‘febrile pneumonitis’ syndrome in different patient groups Renal transplantation Bone marrow transplantation Less than 1 month Aspiration Nosocomial LRTI Aspiration Nosocomial LRTI Aspergillus 1–​3 monthsa Cytomegalovirus Pneumocystis Aspergillus Nocardia Mycobacteria Mucor Cytomegalovirus Pneumocystis Aspergillus Respiratory syncytial virus Mycobacteria Mucor Non​infective causesb More than 3 monthsa Influenza Legionella Common respiratory bacteria Varicella zoster GVHD Common respiratory bacteria and viruses GVHD, graft-​versus-​host disease; LRTI, lower respiratory tract infection. a Six months in renal transplant recipients. b Includes idiopathic interstitial pneumonitis in bone marrow transplant recipients. Modified from Wilson WR, Cockerill FR 3rd, Rosenow EC 3rd (1985). Pulmonary disease in the immunocompromised host (2). Mayo Clin Proc, 60, 610–​31. Box 8.2.4.2  Causes of fever and new pulmonary infiltrates in the immunocompromised host Infections Bacterial • Conventional respiratory pathogens -​ S. pneumoniae, H. influenzae, klebsiella • Nosocomial pathogens -​ E. coli, pseudomonas, legionella • ‘Atypical’ organisms -​ Chlamydia psittaci, C. pneumoniae, mycoplasma • Mycobacteria and related organisms -​ M. tuberculosis, non​tuberculous mycobacteria, nocardia Viral • Herpes viruses -​ Cytomegalovirus, herpes simplex virus, varicella zoster virus • Respiratory viruses -​ Respiratory syncytial virus, influenza and parainfluenza viruses, adenovirus, measles Fungi • Systemic mycoses -​ Blastomycosis, histoplasmosis, coccidioidomycosis • Opportunistic mycoses -​ Pneumocystis jirovecii, candida, aspergillus, mucor, cryptococcus • Other rare fungi -​ Trichosporon, Pseudallescheria boydii/Scedosporiosis Parasites • Strongyloides stercoralis, Toxoplasma gondii Non​infective causes Pulmonary pathology • Pulmonary oedema, pulmonary infarction/​emboli, pulmonary haemorrhage • Primary or secondary malignancy Other causes • Drugs (e.g. sulphonamides, methotrexate, bleomycin, procarbazine, cyclophosphamide, sirolimus) • Activity of the underlying disease (e.g. systemic lupus erythematosus) • Radiation pneumonitis

680 SECTION 8  Infectious diseases deterioration is usually caused by bacterial infections; a combin- ation of piperacillin–​tazobactam plus a macrolide will usually be appropriate. The addition of vancomycin might be necessary if there are clinical or epidemiological grounds to be concerned about MRSA infection. Unusual (‘opportunistic’) organisms such as mycobacteria, nocardia, or cytomegalovirus rarely cause such a rapid clinical deterioration and it is extremely difficult to distin- guish them on clinical grounds alone. For these reasons, the add- ition of further empirical agents is usually not warranted. In patients in whom immediate empirical therapy is not neces- sary, additional diagnostic procedures can be done. These should in- clude serological tests for atypical organisms (including histoplasma and coccidioides in patients who have been in endemic areas), swabs of the upper respiratory tract for viruses and examination of blood for cytomegalovirus DNA. The radiographic appearances are rarely sufficiently specific as to suggest a precise diagnosis, although they can provide helpful pointers. Thus, a bilateral interstitial midzone infiltrate associated with marked hypoxia is typical of pneumonia due to Pneumocystis jirovecii (previously called P.  carinii), and a pleura-​based infarct is suggestive of aspergillus. However, there are pitfalls in relying on the radiographic appearance alone in guiding the choice of therapy. First, no radiographic appearance is path- ognomonic of any single pathological process; for example, cyto- megalovirus or pulmonary oedema can mimic pneumocystis, and legionella pneumonia cannot be distinguished from aspergillus. Second, multiple agents can be present simultaneously, and each may require separate treatment. Other imaging techniques such as high-​resolution CT can often provide useful additional informa- tion on the extent of the process, and might sometimes point to the cause (e.g. the ‘halo sign’ associated with invasive aspergillosis, or the ‘ground glass’ appearances of pneumocystis; see Fig. 8.2.4.5). Radiological abnormalities and the presence of a fever not re- sponding to appropriate antibiotics should prompt further inves- tigations to try to make a specific diagnosis by obtaining material directly from the bronchial tree. In most cases the method of choice is bronchoscopy with bronchoalveolar lavage. This should be insti- gated early, before clinical deterioration of the patient with progres- sive hypoxia precludes invasive procedures. It will provide adequate material without incurring a serious risk of bleeding (many such patients are thrombocytopenic). In most series, bronchial brush or transbronchial biopsy specimens produce only a marginal increase in the diagnostic yield, and are usually not done unless the clinical picture is suggestive of a non​infective process such as an infiltrating tumour. Close liaison with the microbiology laboratory is very im- portant because additional diagnostic procedures will need to be performed. These should include conventional cultures including fungal cultures, cytology, as well as antigen-​based assays detecting fungal cell wall products (galactomannan, β-​glucan) and molecular polymerase chain reaction-​based techniques detecting viruses and fungi. Acute neurological syndromes Many conventional and opportunistic pathogens can lead to neuro- logical infection in immunocompromised patients. Although there is some degree of overlap, the underlying defect in host defence is often a good indicator of the likely cause (Table 8.2.4.3). The clinical features might help suggest the diagnosis. Meningitic syndromes are more likely to be associated with conventional bacterial infections, listeriosis, and tuberculosis, as well as fungi such as cryptococcus and candida. In contrast, infections with toxoplasma, aspergillus, or nocardia more com- monly present as space-​occupying lesions. Pure encephalitic syn- dromes are less common, but can occur with herpes simplex virus or rarely human herpesvirus 6.  Rhinocerebral mucormycosis is a progressive, destructive infection caused by mucor and re- lated moulds that usually begins in the paranasal sinuses and spreads caudally to involve the orbits or the frontal lobes of the brain (Fig. 8.2.4.6). It is seen particularly in patients with un- controlled diabetes mellitus or as a complication of neutropenia. Progressive multifocal leukoencephalopathy is a subacute neuro- logical disease caused by the JC polyomavirus. It presents with the insidious onset of impairment of speech, ­vision, and higher functions without evidence of raised intracranial pressure. The condition progresses inexorably, usually leading to death in about 6 months. Bacterial infections generally proceed rapidly, while fungi and parasites pursue a more indolent course. However, exceptions to this are common and there is no substitute for obtaining a precise diag- nosis. Examination of the skin (see next) and fundoscopy may be valuable. Retinitis is not usually a feature of systemic infection with toxoplasma; in contrast, candida endophthalmitis can be the only manifestation of deep-​seated infection (Fig. 8.2.4.7). Examination of the cerebrospinal fluid is mandatory. A  high index of suspicion is necessary, since the clinical features of menin- gitis are often muted in these patients. An unexplained low-​grade fever and mild headache might be the only clues; frank meningism, photophobia, or focal neurological signs occur late. Examination of the cerebrospinal fluid should include direct microscopy and cul- ture for bacteria, mycobacteria, and fungi, a cryptococcal antigen Fig. 8.2.4.5  Pneumocyctis jirovecii pneumonia. Chest radiograph showing bilateral reticular infiltration in characteristic central distribution in a patient with autoimmune disease on corticosteroid therapy.

8.2.4  Infection in the immunocompromised host 681 test, antigen tests for S. pneumoniae, and the demonstration of spe- cific antibody production or DNA sequences by the polymerase chain reaction (e.g. for herpes simplex, polyomaviruses, and toxoplasma). Certain organisms are notable for their absence on direct micros- copy: mycobacteria are seen in less than 10% of cases, and nocardia and aspergillus only very rarely. A predominance of lymphocytes suggests partially treated bacterial infection, tuberculosis, or a viral aetiology. A low cerebrospinal fluid glucose points to bacterial men- ingitis or tuberculosis but is not specific. Sometimes the only ab- normality is a modest elevation of the cerebrospinal fluid protein; this should never be ignored, even in the seeming absence of other features of neurological infection. Where appropriate, cytological examination of the cerebrospinal fluid should be done to exclude carcinomatous or leukaemic meningitis, which can mimic an acute infective presentation. Certain neurological infections are often associated with pul- monary disease; these include legionella, tuberculosis, aspergillus, mucor, and nocardia. A contrast-​enhanced CT brain scan should be performed. Focal, usually contrast-​enhancing lesions are par- ticularly associated with pyogenic abscesses and toxoplasmosis. Tuberculomas can appear as single lesions. MRI is superior to CT scanning, particularly for abnormalities of the brain stem (e.g. the basal meningitis associated with cryptococcal infection), and fre- quently reveals lesions in toxoplasmosis that are not seen on CT scans. MRI can be particularly helpful in avoiding a brain biopsy when a diagnosis of progressive multifocal leukoencephalopathy is considered. Any new skin lesions should be biopsied, and a nasal biopsy might reveal mucor. An electroencephalogram is rarely helpful. Brain bi- opsy is done very rarely; it should not be considered unless empirical therapy has failed and there is a real prospect of therapeutic benefit to the patient. If the cerebrospinal fluid is non​diagnostic but bacterial infec- tion cannot be excluded, empirical antibiotics should be given im- mediately. Ceftriaxone (together with amoxicillin to provide cover for listeria) is used first-​line but a carbapenem such as meropenem should be considered in patients who have recently received broad-​ spectrum antibacterials or in whom nocardia is a possibility. Serological tests for toxoplasmosis are not specific in this setting, and if the infection is suspected it is better to start empirical therapy with pyrimethamine and sulfadiazine. Cerebral aspergillosis and Table 8.2.4.3  Organisms causing neurological infections in different patient groups Bacteria Fungi Parasites Viruses Neutropenia Gram-​negative Enterobacteriaceae Candida Aspergillus Mucor T cell/​monocyte defect Listeria Cryptococcus Toxoplasma Varicella zoster Legionella Aspergillus Strongyloides Herpes simplex Nocardia Mucor Polyomavirus Mycobacteria Coccidioides Human herpesvirus-​6 Splenectomy S. pneumoniae H. influenzae Neisseria (b) (a) Fig. 8.2.4.6  Invasive mucormycosis. (a) Clinical appearances. (b) CT scan showing extensive sinus involvement.

682 SECTION 8  Infectious diseases mucormycosis have a very poor prognosis; treatment should be begun with high-​dose amphotericin B, and surgical debridement considered if possible. Herpes simplex virus should be treated with aciclovir; the effectiveness of foscarnet for HHV-​6 is not es- tablished. There is no effective treatment for progressive multifocal leukoencephalopathy. Acute gastrointestinal syndromes The organisms associated with specific gastrointestinal syndromes in immunocompromised patients are shown in Table 8.2.4.4. Severe stomatitis is a common complaint in immunosuppressed patients. The three most common causes, candida, herpes sim- plex virus, and chemotherapy-​induced mucositis, are clinically indistinguishable and can indeed coexist. For these reasons, the diagnosis should always be confirmed by microscopy and culture. Herpetic stomatitis in particular can be atypical in these patients; the classic appearance of groups of small vesicles is unusual, and a more common presentation is ulceration, which can be extensive (Fig. 8.2.4.8). In profoundly immunosuppressed patients such as HSCT  recipients, oral candidiasis is very common, and in ­patients who are seropositive before transplantation, reactivation of herpes simplex virus is almost universal. For these reasons, anti- viral prophylaxis is usually given. Both herpes simplex virus and candida can cause oesophagitis, generally (but not exclusively) as an extension of oral disease. If necessary, oesophagoscopy with brush cytology and/​or biopsy is the investigation of choice. Proven oesophageal candidiasis should be regarded as ‘invasive’ disease and treated with systemic antifungals (fluconazole or an echinocandin). Many organisms can cause acute diarrhoeal syndromes; in add- ition, non​infective conditions such as radiation enteritis, drugs, and graft-​versus-​host disease must be included in the differential diagnosis. There are no distinguishing clinical features of note, and diagnosis depends on microbiological examination of the faeces. The diarrhoea due to Clostridium difficile is usually caused by a cytotoxin resulting in pseudomembranous colitis. However, patients with leukaemia or aplastic anaemia might develop neutropenic en- terocolitis (previously called typhlitis), a fulminating invasive col- itis characterized by diffuse dilatation and oedema of the bowel walls, haemorrhage, ulceration, and a high mortality. Classically this has been associated with clostridial bacteraemia, in particular Clostridium septicum, but other clostridia, including C. difficile, and even Gram-​negative bacteria can also be found. Strongyloides stercoralis is a nematode that can be carried asymptomatically for many years after exposure. Strongyloidiasis has been recognized as a complication of human T-​lymphotropic virus 1 (HTLV-​1) infection, and also occurs secondary to immuno- suppression (typically with high-​dose corticosteroids and in solid organ transplant recipients). A rise in the worm burden results in the hyperinfection syndrome, which can present as pneumonitis or intermittent intestinal obstruction. The movement of the worms through the gut wall can carry with them enteric bacteria, resulting in polymicrobial bacteraemia and Gram-​negative meningitis when the worms penetrate the blood–​brain barrier. Giardiasis is particularly associated with hypogammaglobulinaemia, and curiously is rarely seen in other groups. Cryptosporidia, micro­ sporidia, and isospora are now well-​recognized causes of severe and Fig. 8.2.4.7  Candida endophthalmitis (arrows indicate fungal microcolonies). Table 8.2.4.4  Gastrointestinal syndromes in the immunocompromised host Bacteria Fungi Parasites Viruses Oral infection Candida Herpes simplex virus Diarrhoeal syndromes Neutropenic enterocolitis Candida Giardia lamblia Enterovirus C. difficile Isospora belli Adenovirus Salmonella Shigella Campylobacter Shiga toxin producing E. coli Cryptosporidium Cytomegalovirus Non​tuberculous mycobacteria Microsporidium Rotavirus Hepatic syndromes Candida Toxoplasma gondii Cytomegalovirus Hepatitis B, C, and E viruses Herpes simplex virus Varicella zoster virus Epstein–​Barr virus

8.2.4  Infection in the immunocompromised host 683 sometimes chronic diarrhoea in AIDS patients, but can also occur in other less severely immunocompromised patients. Among the viruses the most problematic is cytomegalovirus. Cytomegalovirus can cause a severe colitis, and in these cases ganciclovir is beneficial. Ideally the diagnosis should be confirmed by biopsy, but ultimately might depend on the result of a therapeutic trial since demonstra- tion of the organism does not necessarily indicate that it is causing disease. Mild abnormalities of liver function tests are a common accom- paniment to many systemic infections, but hepatitis is a particular feature of both toxoplasmosis and cytomegalovirus infection. An increased prevalence of hepatitis B has been found in patients on chronic haemodialysis (10%) and those with Hodgkin’s disease (8%) and lepromatous leprosy (20%). The acute hepatitic episode is mild, often anicteric, and might pass unnoticed. However, persistent viral replication and the development of complications associated with chronic infection are more likely. Cirrhosis secondary to hepatitis C is currently the third most common indication for liver transplant- ation, however the prognosis is improving with the advent of new combinations of different classes of directly acting antiviral agents. Unfortunately, recurrence of infection post-​transplantation is al- most inevitable and requires specific approaches to prevention and treatment. Chronic hepatitis E virus infection has also been reported in solid organ transplant recipients. Epstein–​Barr virus replication can be detected in 20–​30% of solid organ transplant recipients and up to 80% of those who receive antithymocyte globulin and high doses of immunosuppressants. Clinical manifestations range from a benign mononucleosis syndrome to hepatitis to post-​transplant lymphoproliferative disorder. Acute urinary tract syndromes In renal transplant recipients the most common site of infection is the urinary tract, giving rise to complications such as bacteraemia, graft pyelonephritis, and cystitis. Recurrent infections should prompt investigations for anatomical abnormalities. Haemorrhagic cystitis can occur following HSCT. In the early post-​transplantation period it is almost exclusively related to drug toxicity. However, its occurrence in the late post-​transplantation period can be a mani- festation of graft-​versus-​host disease or viral infections. Polyoma BK and adenovirus are implicated most commonly. Prevention of infection Approaches designed to prevent infection in immunosuppressed pa- tients have assumed increasing importance. In general, meticulous adherence to infection prevention and control practices are essential to prevent hospital-​acquired infections. For profoundly neutropenic Fig. 8.2.4.8  Severe herpetic stomatitis in a patient with lymphoma. Table 8.2.4.5  Infection prevention strategies in organ transplant recipients and patients with neutropenia Strategy Comment Bacterial infections Bacterial sepsis in neutropenia Oral quinolones Re-​emerging following earlier concerns with efficacy and risk of resistance High-​efficiency particulate air (HEPA)-​filtered rooms Very expensive and no clear advantage in survival Adherence to intravenous catheter care bundles Overwhelming postsplenectomy sepsis Immunization (pneumococcal, Hib, and meningococcal) and oral penicillin Tuberculosis Isoniazid In exposed or high-​risk patients, especially if receiving prolonged high-​dose corticosteroids Viral infections Herpes simplex, cytomegalovirus Aciclovir, ganciclovir, valganciclovir Dose and drug varies depending on specific indication Hepatitis B reactivation Lamivudine, entecavir, tenofovir In risk groups (B-​cell depleting agents, anthracycline derivates, monoclonal antibodies) Influenza Immunization Not routine except in high-​risk groups Fungal infections Candida, aspergillus Fluconazole, itraconazole, voriconazole, posaconazole, and liposomal amphotericin B Choice and duration of drug depends on type of transplant or haematological risk group Pneumocystis jirovecii Co-​trimoxazole Used for both bone marrow transplantations and in some solid organ transplantations

8.2.5 Antimicrobial chemotherapy 684

8.2.5 Antimicrobial chemotherapy 684

684 SECTION 8  Infectious diseases patients, measures including nursing them in single rooms and taking great care to avoid nosocomial acquisition of infection from staff, visitors, or other patients is simple but effective. Chemoprophylaxis for a wide range of bacterial, viral, and fungal pathogens has had a major impact (Table 8.2.4.5). Guidelines regarding immunization in immunocompromised hosts have been drawn up by the Infectious Diseases Society of America (see Further reading). In addition, rou- tine screening of transplant recipients and donors should include serological tests for cytomegalovirus, hepatitis B, and HIV. FURTHER READING Baker TM, et al. (2016). The Growing Threat of Multidrug-Resistant Gram-Negative Infections in Patients with Hematologic Malignancies. Leuk & Lymph, 57, 2245–58. Davies JM, Barnes R, Milligan D (2002). Update of guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen. Clin Med, 2, 440–​3. Heinz WJ, et al. (2017). Diagnosis and empirical treatment of fever of unknown origin (FUO) in adult neutropenic patients: guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO). Ann Hematol, 96, 1775–92. Jaksic B, et  al. (2006). β lactam monotherapy versus β lactam-​ aminoglycoside combination therapy for fever with neutropenia: systematic review and meta-​analysis. BMJ, 326, 1111–​19. Kalil AC, et al. (2018). Severe infections in critically ill solid organ transplant recipients. Clin Microbiol Infect, https://doi.org/10.1016/j. cmi.2018.04.022 Mikulska M, et al. (2018). Fluoroquinolone prophylaxis in haemato- logical cancer patients with neutropenia: ECIL critical appraisal of previous guidelines. J Infect, 76, 20–37. Pappas PG, et al. (2016). Clinical practice guideline for the manage- ment of Candidiasis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis, 62, e1–e50. Patterson TF, et al. (2016). Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis, 63, e1–e60. Rubin LG, et al. (2014). 2013 IDSA clinical practice guideline for vaccin- ation of the immunocompromised host. Clin Infect Dis, 58, 309–​18. 8.2.5  Antimicrobial chemotherapy Maha Albur, Alasdair MacGowan, and
Roger G. Finch ESSENTIALS The practice of medicine changed dramatically with the avail- ability of effective antimicrobial agents. Often fatal diseases, such as infective endocarditis, became treatable; much minor com- munity infectious morbidity became readily controlled; for ex- ample, urinary tract infection; many surgical procedures became much safer, and developments in solid organ and bone marrow transplantation became possible. However, the very success of antimicrobial chemotherapy has led to anti-​infective overuse and misuse. In some countries, antibiotics are freely available to the public for purchase ‘over the counter’, with few controls or guid- ance to ensure their safe and effective use. In many others there are poorly developed antimicrobial stewardship programmes. The emergence and spread of antimicrobial resistance worldwide and the decline in development and licensing of new antimicrobials over the last 30 years has threatened the future successful treat- ment of bacterial infections. Antimicrobial drugs Pharmacological characteristics and antimicrobial spectrum—​antibacterial drugs can be divided according to their mode of action into those that (1) inhibit cell wall synthesis (e.g. β-​lactams such as penicillins, cephalosporins, carbapenems, and monobactams); (2) interfere with protein synthesis (e.g. tetracyclines, aminoglycosides); (3) inhibit bac- terial nucleic acid synthesis (e.g. fluoroquinolones); and (4) act on metabolic pathways (e.g. sulphonamides and trimethoprim). The antimicrobial spectrum of a drug is determined by the mode of ac- tion and ability to reach the relevant bacterial target site. Antibiotics active against a few particular bacteria species are considered narrow spectrum (e.g. benzylpenicillin), while others are active against many species and are labelled broad spectrum (e.g. meropenem). Some antimicrobials are only active against anaerobically dividing bacteria (e.g. metronidazole). Clinical effectiveness—​to be effective clinically, sufficient drug must reach the infection site and show antibacterial activity. The pharmacokinetic characteristics of absorption, distribution, metab- olism, and excretion are critical to defining dose, efficacy and, often, safety. Poorly absorbed agents are often administered parenterally, some topically. Hydrophobicity and hydrophilicity are important in defining tissue and extracellular fluid concentrations, as are factors such as molecular size and pH. Highly protein-​bound drugs such as flucloxacillin achieve lower tissue concentrations in selected body sites. The pharmacodynamic properties with drug pharmacokinetics determine microbiological efficacy. Pharmacodynamics describes the antibacterial effect of a drug at various concentrations and is as- sessed by measurement such as minimum inhibitory concentration, persistent antibiotic effects, whether a drug kills or inhibits bacteria, as well as the risk of emergence of resistance. Excretion, metabolism, and drug monitoring—​many drugs are metabolically degraded in the liver and/​or excreted by the kidney via glomerular filtration or tubular secretion. It should therefore be anticipated that dose modification might be necessary to avoid ad- verse events and preserve microbiological efficacy in patients with compromised hepatic or renal function. Therapeutic drug moni- toring is important in ensuring therapeutic and non​toxic concentra- tions of some drugs (e.g. aminoglycosides and glycopeptides) but is not restricted to these drugs or patients with poor excretory organ function. Antiviral, antifungal, and antiparasitic drugs—​the availability of drugs to treat herpesvirus infections (herpes simplex, varicella–​zoster and cytomegalovirus), and the development of new drugs active against influenza, hepatitis viruses, influenza viruses, and HIV have revolutionized the management of viral infections. Advances in the

8.2.5  Antimicrobial chemotherapy 685 management of invasive fungal disease have seen the reliance on polyenes (e.g. amphotericin), eclipsed with the availability of several azoles and triazoles and echinocandins. In the case of many para- sitic diseases, advances have been extremely slow, but the import- ance of malaria has led to new compounds being developed (e.g. the artemisin derivatives), also new ways of using established drugs in combination. Resistance to antimicrobial drugs Resistance mechanisms—​loss of efficacy through acquisition of re- sistance mechanisms is unique to antimicrobial drugs. There are four main types: (1) drug inactivation or destruction, (2) target site alteration, (3) reduced cell wall permeability (porin mutation) or in- creased removal from the cell (efflux resistance); and (4) inhibition as a result of metabolic bypass. Individual drugs can be subject to one or more mechanisms of resistance, which may vary by infecting microorganism. Spread of resistance—​genetic mutations that confer resistance do not just affect the target pathogen in the treated individual. They can disseminate both horizontally and vertically as a result of person-​to-​person or indirect spread of the pathogen. Spread through genetic mechanisms via plasmids, transposons, integrons, and phages between bacteria of the same and different species are common, as is spread between genera. Likewise, resistance mech- anisms can spread to organisms making up the normal flora of the gut and skin. Clinical impact—​antibiotic resistance is of considerable medical and public concern, and affects all aspects of medicine. Infections become unresponsive to initial therapy, sometimes with fatal con- sequences in the seriously ill. In others, reassessment and alternative therapy with agents that are often more toxic and more expensive are required, leading to increased morbidity and increased costs through prolonged hospitalization. The spread of resistant pathogens within hospitals, nursing homes, and the community is a very significant concern. Increasing rates of multidrug-​resistant E. coli and Klebsiella species infections are present in many countries, including those in North America, Europe, and Asia. Public concern has led to major government initiatives in the United Kingdom, European Union, United States of America, and many other countries in efforts to con- tain resistant pathogens. Prescribing of antimicrobial drugs A set of principles has emerged to support safe and effective pre- scribing, covering issues of choice of drug, dose and route of ad- ministration, duration of therapy, strategies to minimize adverse reactions, and what factors need to be considered should initial treatment fail. The complexity of modern therapeutics has led to the development of formularies and practice guidelines, the latter increasingly being evidence based, with the goals of minimizing the risks of emergence of antibiotic resistance, ensuring effective and safe therapy, and supporting cost-​effective treatment. These prin- ciples are often supported by active steps in both hospital and the community to manage antibiotic use via routine monitoring, specific audits, clinical feedback, drug restriction, and educational initiatives. These activities make up antibiotic stewardship programmes, which are now increasingly common worldwide. Introduction The discovery and clinical application of antimicrobial chemo­ therapeutic agents is one of the major achievements in medicine. Life-​threatening infections such as infective endocarditis and ty- phoid fever are now treatable, whereas before they were generally fatal. Likewise, the morbidity associated with many infectious dis- eases of a less life-​threatening nature, such as urinary tract infec- tions, skin and soft tissue infections, and bone and joint sepsis, has been substantially reduced. Major advances in medicine, such as solid organ and especially bone marrow transplantation, as well as the use of cancer chemotherapy, have become safer because of the availability of effective antimicrobial agents. In the field of surgery, perioperative prophylactic use of antibiotics has reduced the risk of infections complicating procedures such as large bowel and gall bladder surgery, vaginal hysterectomy, caesarean section, and im- plant surgery such as the insertion of prosthetic heart valves, joints, and neurosurgical shunting devices. Antimicrobial chemotherapy is the use of antibiotics and chemotherapeutic substances to control infectious disease. The term ‘antibiotic’ was coined by Waksman to describe a substance derived from naturally occurring microorganisms and possessing anti- microbial activity in high dilution. The latter characteristic is essen- tial in defining its selective toxicity to other microorganisms. True antibiotics include penicillin, derived from the mould Penicillium notatum, streptomycin from Streptomyces griseus, and the cephalo- sporins from Cephalosporium spp. Many chemotherapeutic sub- stances with antimicrobial activity have been artificially synthesized, such as the sulphonamides, quinolones, oxazolidinones, and iso- niazid. However, the term ‘antibiotic’ is loosely applied to both the true antibiotics and other antimicrobial agents. Anti-​infectives are among the most widely prescribed drugs, ac- counting for a projected international expenditure of $190 billion by 2025. In the United Kingdom, around 80% of all prescribing is in the community where the emphasis is largely on oral agents; the re- mainder are used in hospitals where there is a greater emphasis on injectable drugs. More than 125 different antibiotics are available, but a relatively small number are necessary to deal with most prescribing needs. It is important that clinicians who prescribe these drugs are familiar with the principles of antimicrobial chemotherapy and that they adopt a continuous learning approach throughout their pro- fessional lives to ensure safe and effective prescribing. Table 8.2.5.1 summarizes the agents available for the treatment of bacterial, myco- bacterial, fungal, viral, protozoal, and helminthic infections. More agents have been developed for the treatment of viral infections, but globally viral, fungal, and parasitic infections predominate. In recent years, there have been major advances in the availability of antiviral drugs, particularly for the treatment of the influenza, herpes viruses, and HIV. Likewise, safe and effective systemic antifungal agents have resulted from the discovery of azoles, triazoles, and echinocandins. The very success of antimicrobial chemotherapy has led to wide- spread and often excessive use, particularly in community prac- tice where prescribing is largely empirical and clinical distinction between viral and bacterial infections is difficult. Antibiotics are used extensively in animal husbandry both for the treatment and prevention of infectious disease. This has raised concerns about the

686 SECTION 8  Infectious diseases emergence and spread of antibiotic resistance, which affects many classes of antibiotic, may be intrinsic to a particular pathogen, or may result from genetic mutation. Resistance can be caused by en- zymatic inactivation (β-​lactamase), failure of drug penetration into the bacterial cell (porin mutation), alteration of the target binding site (e.g. penicillin-​binding protein alteration in penicillin-​resistant Streptococcus pneumoniae), or from efflux resistance whereby the drug is extruded from the bacterial cell (e.g. chloroquine-​resistant Plasmodium falciparum). Organisms can also develop alternative metabolic pathways which bypass drug inactivation. Resistance may be transferable between the same species or genera but may also spread between genera. Coding for multiple antibiotic resistance has been increasingly observed and results from several mechanisms, in particular plasmid transfer. Despite the advances in antimicrobial chemotherapy, fresh chal- lenges remain. These include the treatment of viral causes of en- teric infection, hepatitis A and E, and viral meningitis, all of which are still without effective chemotherapy. Tuberculosis and malaria are among the world’s major infectious disease killers and here problems of antibiotic resistance have escalated. In the case of tu- berculosis, the continuing reliance on lengthy and complex regi- mens continue to frustrate disease management as a result of cost, toxicity, and patient compliance with these regimens. Recent ad- vances include the development and licensing of new agents with novel mechanisms of action: for tuberculosis, bedaquiline, which blocks adenosine S-​triphosphate synthesis; and delamanid, which blocks manufacture of mycolic acids and destabilize the bacterial cell wall. Among the more worrying trends in antibiotic resistance is the emergence of methicillin-​resistant Staphylococcus aureus (MRSA), vancomycin-​resistant enterococci, and multidrug-​resistant Gram-​ negative species such as extended-​spectrum β-​lactamases or Delhi metallo-β-​lactamase (NDM)-​producing E.  coli, KPC produ- cing Klebsiella sp., and IMP-​ or VIM-​producing P.  aeruginosa. Streptococcus pneumoniae is another community pathogen which has rapidly become less sensitive to penicillins, macrolides, and fluoroquinolones causing clinical failures when causing menin- gitis or otitis media. Internationally, multidrug-​resistant (MDR) and extensively drug-​resistant (XDR) tuberculosis, and multidrug-​ resistant salmonellae, including Salmonella typhi, are also of major concern. Resistance is not confined to bacteria. Fungal resistance is increasing (e.g. Candida albicans and C.  krusei to fluconazole). Resistance of the HIV to the nucleoside, non​nucleoside, and pro- tease inhibitors is rapidly emerging with many treatment-​naive pa- tients acquiring virus resistance to one or more agents. Antiviral resistance resulting in virological treatment failure is now a major factor responsible for progression of HIV disease. Table 8.2.5.1  Antimicrobial agents available by class or indication effective against bacterial, fungal, viral, protozoal, and helminthic infection (indicative number of agents availablea) Antibacterial (78) Antifungal (10) Antiviral (52) Antiprotozoal Anthelminthics (15) Penicillins Polyenes Hepatitis B & C agents Antimalarials Anticutaneous larva migrans Cephalosporins Triazoles Herpes virus agents Amoebicides Antihydatid agents Carbapenems Echinocandins HIV nucleoside analogues Trichomonacides Antistrongyloidiasis Monobactams Tetracyclines Triazoles HIV non​nucleoside agents Antigiardials Antithreadworm/​hookworm Aminoglycosides Flucytosine HIV protease inhibitors Leishmaniacides Ascaricides Macrolides HIV fusion entry inhibitor Trypanocides Filaricides Ketolides HIV integrase inhibitors Lincosamides Triazoles Ribavirin Antipneumocystis agents Schistosomicides Chloramphenicol Terbinafine Amantadine/​rimantadine Taeniacides Sodium fusidate Foscarnet Glycopeptides cidofovir Daptomycin Linezolid Neuraminidase inhibitors Quinupristin/​dalfopristin Colistin Sulphonamides Trimethoprim Antituberculous Antileprotic Nitroimidazoles Quinolones Urinary antiseptics a Based on agents listed in the British National Formulary (https://​www.bnf.org).

8.2.5  Antimicrobial chemotherapy 687 Pharmacology Mode of action Knowledge of the pharmacological mode of action of an anti- microbial agent permits an understanding of the diverse mech- anisms of microbial inhibition and the opportunities for drug resistance. This is best established for antibacterial and antiviral agents. In the case of antifungal and, especially, antiparasitic agents the modes of action are less well defined. This reflects the process of drug discovery whereby an understanding of the biochemical and molecular action of agents derived from natural or chemical sources has not always been a priority in establishing efficacy and safety, especially with regard to older agents. Antibacterial drugs Antibacterial agents may affect cell wall or protein synthesis, nu- cleic acid formation, or may act on critical metabolic pathways (Table 8.2.5.2). The β-​lactams (penicillins, cephalosporins, carbapenems, and monobactams) and the glycopeptides (vancomycin, teicoplanin, telavancin, oritavancin, dalbavancin), inhibit cell wall synthesis. The β-​lactams, which share the common β-​lactam ring, act on cell wall transpeptidases to inhibit cross-​linking of peptidoglycan. The glycopeptide antibiotics act at an earlier stage of cell wall syn- thesis by binding to acyl-​d-​alanyl-​d-​alanine. Despite both being cell wall active, the glycopeptides, such as vancomycin, are less bac- tericidal agents than the β-​lactams, such as flucloxacillin, against Staphylococcus aureus. Inhibitors of protein synthesis Antibacterial agents that inhibit protein synthesis act on the 30S ribosomal subunit responsible for binding mRNA, or the 50S subunit which binds aminoacyl tRNA. The aminoglycosides, tetracyclines, and macrolide antibiotics are the most widely used inhibitors of protein synthesis. Chloramphenicol, clindamycin, and the recently introduced oxazolidinones (linezolid, tedizolid) also act at this site. Inhibitors of nucleic acid Nucleic acid synthesis is targeted by quinolones, metronidazole, and rifampicin. The bacterial DNA gyrase is essential for the super- coiling of bacterial DNA. This, together with the enzyme topoisom- erase IV, are the major targets for the quinolones. These enzymes are absent in humans, explaining the selective activity of these drugs. Rifampicin and other rifamycins interfere with DNA-​dependent RNA polymerase, preventing chain initiation. Table 8.2.5.2  Microbial site of action and targets for selected antibacterial drugs Site of action Drugs Target Cell wall peptidoglycan Penicillins Transpeptidase Cephalosporins Transpeptidase Vancomycin Acyl-​d-​alanyl-​d-​alanine Teicoplanin Acyl-​d-​alanyl-​d-​alanine Telavancin Binds late stage peptidoglycan precursors and disrupts bacterial membrane Oritavancin Inhibits transglycosylation and transpeptidation and disrupts bacterial membranes Dalbavancin D-​alanyl-​D-​alanine Fosfomycin UDP-​N-​acetylglucosamine 3 enolpyruvyl transferase Daptomycin Binds to bacterial membranes Colistin Binds to bacterial membranes Ribosome Chloramphenicol Peptidyl transferase of 50S subunit Pleuromutilins Peptidyl transferase of 50S subunit Clindamycin 50S ribosomal subunit transpeptidation Linezolid Blocks initiation phase Tedizolid Blocks initiation phase Macrolides 50S ribosomal subunit Tetracyclines Ribosomal A site Aminoglycosides Initiation complex and translation Mupirocin Isoleucyl-​transfer RNA synthetase Fusidic acid Elongation factor G Nucleic acid Fluoroquinolones DNA gyrase Metronidazole DNA strands Rifampicin RNA polymerase Folic acid synthesis Sulphonamides Pteroic acid synthetase Trimethoprim Dihydrofolate reductase

688 SECTION 8  Infectious diseases Metabolic inhibitors The best known metabolic inhibitors are the sulphonamides and tri- methoprim which interfere with folic acid synthesis by sequentially inhibiting the enzymes dihydropteroic acid synthetase (EC 2.5.1.15) and dihydrofolate reductase (EC 1.5.1.3). The two drugs act sequen- tially on the metabolic pathway, resulting in a combined antibiotic effect. The selective activity of these compounds is dependent on the fact that humans are unable to synthesize folic acid and require pre- formed folic acid in their diet. Antiviral agents Viruses live and replicate within the host cell. Antiviral chemo- therapy therefore presents a particular challenge if it is to be selectively toxic. The cycle of viral replication provides several op- portunities for therapeutic intervention. Most available antiviral agents are nucleoside analogues, largely used in the treatment of HIV or herpesvirus infections (Table 8.2.5.3). The growth in num- bers of antiviral agents has greatly benefited from HIV-​related re- search through the identification of new drug targets (Fig. 8.2.5.1). Table 8.2.5.3  Mode of action of selected antiviral drugs Drug Target virus Antiviral activity Aciclovir HSV, VZV Nucleoside analogue Cidofovir HSV and CMV Nucleoside analogue Famciclovir VZV Nucleoside analogue Foscarnet CMV Inhibits DNA polymerase Ganciclovir CMV Nucleoside analogue Valaciclovir HSV, VZV Valyl ester of aciclovir Valganciclovir CMV Valyl ester of ganciclovir Interferon HBV, HCV Induce interferon stimulated genes and block viral protein synthesis Adefovir HBV Nucleotide reverse transcriptase inhibitor Entecavir HBV Nucleoside analogue Telbivudine HBV Nucleoside analogue Ribavirin HCV, RSV Inhibits replication of DNA and RNA viruses, inhibits initiation and elongation of RNA fragments Boceprevir HCV Binds to NS3 serine protease of HCV Telaprevir HCV Binds to NS3 serine protease of HCV Oseltamivir Influenza A and B Inhibits viral neuraminidase Zanamivir Influenza A and B Inhibits viral neuraminidase Amantadine Influenza A Uncoating and assembly Rimantadine Influenza A Uncoating and assembly Abacavir HIV Nucleoside reverse transcriptase inhibitor Didanosine HIV Nucleoside reverse transcriptase inhibitor Emtricitabine HIV Nucleoside reverse transcriptase inhibitor Lamivudine HIV, HBV Nucleoside reverse transcriptase inhibitor Stavudine HIV Nucleoside reverse transcriptase inhibitor Tenofovir HIV Nucleoside reverse transcriptase inhibitor Zalcitabine HIV Nucleoside reverse transcriptase inhibitor Zidovudine HIV Nucleoside reverse transcriptase inhibitor Delavirdine HIV Non​nucleoside reverse transcriptase inhibitor Efavirenz HIV Non​nucleoside reverse transcriptase inhibitor Etravirine HIV Non​nucleoside reverse transcriptase inhibitor Nevirapine HIV Non​nucleoside reverse transcriptase inhibitor Rilpivirine HIV Non​nucleoside reverse transcriptase inhibitor Amprenavir HIV Protease inhibitor Atazanavir HIV Protease inhibitor Darunavir HIV Protease inhibitor Fosamprenavir HIV Protease inhibitor Indinavir HIV Protease inhibitor (continued)

8.2.5  Antimicrobial chemotherapy 689 Interference with cell surface attachment through ligand blockade of surface receptors provides a theoretical, but so far unfulfilled, target. Penetration into the host cell may be through a process of trans- location or direct fusion between the outer lipid membrane of the virus and the cell membrane, before uncoating and release of viral nucleic acid. Replication differs among viruses, thereby providing several therapeutic options. Viral mRNA becomes translated into multiple copies of viral proteins encoded by the viral genome either as a result of virus-​specific enzymes or by co-​opting host-​derived protein. For example, HIV employs its own reverse transcriptase to convert RNA to DNA before integration into the host cell chromo- some. Transcription and translation follow. Before the virus can be released, new viral particles must be assembled for which host cell proteins and mechanisms of phosphorylation and glycosylation may be recruited. The protease inhibitors act at this stage and have been particularly successful. Virus release is the result of either transpor- tation and budding or host cell lysis. Antifungal agents The polyene antifungals (amphotericin B and nystatin) act on er- gosterol within the fungal cell membrane. Ergosterol is largely absent from bacteria and humans, explaining the selective tox- icity of these agents. The azole antifungals include the imidazoles (e.g. clotrimazole, miconazole, and ketoconazole) and the tri- azoles (fluconazole, itraconazole, voriconazole and posoconazole) which bind preferentially to fungal cytochrome P450 to inhibit Drug Target virus Antiviral activity Lopinavir HIV Protease inhibitor Fosamprenavir HIV Protease inhibitor Nelfinavir HIV Protease inhibitor Ritonavir HIV Protease inhibitor Saquinavir HIV Protease inhibitor Tipranavir HIV Protease inhibitor Enfurvitide HIV Fusion entry inhibitor Raltegravir HIV Integrase inhibitor Dolutegravir HIV Integrase inhibitor Elvitegravir HIV Integrase inhibitor Maraviroc HIV CCR5 antagonist CMV, cytomegalovirus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; RSV, respiratory syncytial virus; VZV, varicella zoster virus Table 8.2.5.3  Continued Fusion inhibitor: enfuvirtide Penetration Uncoating Reverse transcription Integration Transcription Translation Assembly and release Complete HIV peptide Glycoprotein Glycosylation and cleavage Host chromosome Proviral DNA Viral mRNA Unintegrated dsDNA Genomic RNA cDNA HIV virion Receptor/ Coreceptor Building particle Nucleosides: abacavir, didanosine, emtricitabine lamivudine, stavudine, tenofovir, zalcitabine, zidovudine Nonnucleosides: efavirenz, nevirapine Protease inhibitors: amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, tipranavir Fig. 8.2.5.1  Sites of inhibition of HIV replication by current antiretroviral drugs.

690 SECTION 8  Infectious diseases 14-​α-​methylsterol demethylation to ergosterol. The echinocandins (e.g. caspofungin, micafungin, and anidulafungin) act on fungal cell wall β (1–​3) d-​glycan to inhibit growth. Antiparasitic agents The mechanism of action of many antiparasitic drugs is only par- tially known. Among the antimalarials, chloroquine interferes with the metabolism and utilization of haemoglobin by malaria parasites. It also concentrates within parasite acid vesicles and raises internal pH, inhibiting parasite growth. Amodiaquine is similar in structure to chloroquine and there is cross-​resistance between the two drugs. Quinine acts by depressing oxygen uptake and carbohydrate metabolism and by intercalating into DNA, disrupting para- site replication and transcription. Mefloquine is a quinolone methanol compound structurally similar to quinine. Primaquine disrupts mitochondria, disrupts DNA, and eliminates the tissue exoerythrocytic forms of Plasmodium falciparum. The exact mech- anism of action of lumefantrine is unknown but it may inhibit the formation of β-​haematin by complexing with haemin. Sulfadoxine–​pyrimethamine inhibits tetrahydrofolate synthesis. Atovaquone inhibits parasite electron transport in mitochon- dria, resulting in inhibition of adenosine triphosphate (ATP) and nucleic acid synthesis. Proguanil inhibits dihyrofolate reductase. Together, atovaquone and proguanil affect the erythrocytic and exoerythrocytic stages of parasite development. Artemisin derivatives include artemether, arteether, dihydroartemisinin, and artesunate. They appear to act by binding iron, breaking down peroxide bridges leading to the gen- eration of free radicals that damage parasite proteins. They kill all blood stages of Plasmodium spp. and have the fastest parasite clearance times of any antimalarial. Metronidazole is active against several protozoa such as Entamoeba histolytica and Giardia lamblia as well as anaerobic bacteria. It acts as an electron sink, by reduction of its 5-​nitro group activated by nitroreductase within the target pathogen, thus interrupting DNA synthesis. Among the anthelmintic drugs, piperazine and praziquantel act by selectively inducing muscle paralysis in the target helminth. Others, such as thiabendazole, inhibit parasitic ATP synthesis and energy production. Antimicrobial spectrum of activity The antimicrobial spectrum of an agent is dependent on target site susceptibility among pathogenic organisms at clinically achievable drug concentrations. Some microorganisms are intrinsically re- sistant to certain antibiotics. For example, the aminoglycosides are inactive against anaerobic bacteria because cell entry is an energy-​ dependent process relying on respiratory quinones, which are ab- sent in anaerobic bacteria. The antimicrobial spectrum of a drug in part dictates its clinical indications. While information on this spectrum is more easily de- termined in vitro, in vivo efficacy can only be confirmed through clinical use, which can be supported by preclinical model data during drug development. For example, in vitro, Salmonella typhi is susceptible to gentamicin, but the drug is not effective clinically. Narrow-​spectrum and broad-​spectrum agents There are few truly narrow-​spectrum agents. Fusidic acid, the glycopeptides (vancomycin and teicoplanin), daptomycin, and linezolid target specific Gram-​positive pathogens and are mainly used to treat microbiologically confirmed infections. Broad-​spectrum agents, such as the fluoroquinolone anti- biotics and the parenteral cephalosporins such as cefotaxime or ceftriaxone, are active against many Gram-​positive and Gram-​ negative species of pathogens. Metronidazole has activity against many anaerobic bacteria and, because of this restricted activity, is considered to have a narrow spectrum. The aminoglycosides, al- though active against staphylococci and aerobic Gram-​negative bacilli, are inactive against streptococci and anaerobes and are, therefore, frequently prescribed in combination. The carbapenems (imipenem, meropenem, doripenem, and ertapenem) possess the broadest spectrum of activity which includes most aerobic and anaerobic bacterial pathogens. Ertapenem differs from the other carbapenems in its lack of activity against Pseudomonas aeruginosa. Broad-​spectrum agents are often used empirically in the initial management of severe infection. However, they frequently affect the normal flora so that superinfection with Clostridium difficile and yeasts are more likely to arise. Narrow-​spectrum drugs are used as definitive therapy, that is, after a microbiological diagnosis is reached but are also used in combination as empiric therapy. Susceptibility testing Antibiotic susceptibility testing of clinical isolates is important for appropriate prescribing and for gathering epidemiological data on the burden of antimicrobial resistance. It is determined in vitro by using either broth-​based or agar-​based methods. Pathogens are ex- posed to known concentrations of an antibiotic and their degree of inhibition compared to a standard control. Disc susceptibility testing is a widely used method. Zones of inhibition around the antibiotic-​containing disc are measured, compared to a standard, and the pathogen designated sensitive, resistant, or of intermediate susceptibility to the drug. Currently, such methods require the iso- late to be tested in pure culture. It is, therefore, difficult to obtain information on the susceptibility of a pathogen in less than 36–​48 h from sample collection. The minimum inhibitory concentration (MIC) in milligrams per litre (L) provides more precise in vitro information on the activity of a drug against a bacterial pathogen. It is more time consuming and costly to determine, although automated systems and commercial strip tests are available (Fig. 8.2.5.2). Defining susceptibility by MIC determination permits greater predictive benefit in the treatment of certain infections such as gonorrhoea, bacterial endocarditis, and pneumococcal meningitis. Knowledge of the in vitro suscepti- bility of common pathogens to antimicrobial agents (Fig. 8.2.5.3) is helpful in selecting drug therapy but is only relevant to the achiev- able drug concentrations, which is important in predicting perform- ance as discussed next. Combined drug therapy In hospital practice, it is common to combine agents when dealing with mixed infections or where initial broad-​spectrum empir- ical therapy is required. Another important reason for combining

8.2.5  Antimicrobial chemotherapy 691 drugs is to prevent the emergence of antibiotic resistance, such as in the treatment of tuberculosis, HIV, and malaria. Antituberculosis regimens have been developed to ensure that naturally occurring minority populations of Mycobacterium tuberculosis resistant to iso- niazid or rifampicin do not emerge during therapy. By combining isoniazid and rifampicin with pyrazinamide and ethambutol for the initial phase of therapy (2 months), resistance is usually avoided. Therapy can be restricted to isoniazid and rifampicin for the con- tinuation phase (4  months). The regimen is extended in those patients unable to tolerate pyrazinamide and in the treatment of tu- berculous meningitis (Box 8.2.5.1). HIV infection is treated with multidrug regimens. The success of highly active antiretroviral therapy, in which nucleoside analogues and protease inhibitors are combined in a three-​drug regimen, is not only based on greater efficacy of the combined regimen but also on its ability to slow the emergence of drug-​resistant mutants. The non​nucleoside reverse transcriptase inhibitors, such as efavirenz, appear to be equally effective in combination with nucleoside Fig. 8.2.5.2  Staph. aureus resistant to penicillin (MIC 8 mg/​litre) on the left and sensitive to vancomycin (MIC 1.0 mg/​litre) on the right, as demonstrated by a commercial antibiotic gradient strip test. Penicillin Ampicillin/amoxicillin Amoxicillin/clavulanate Flucloxacillin Cefuroxime Cefotaxime Ceftazidime Erythromycin Clindamycin Tetracyclines Vancomycin/teicoplanin Linezolid Gentamicin/tobramycin/ netilmicin/amikacin Co-trimoxazole Trimethoprim Ciprofloxacin Staph. aureus (penicillin- resistant) Staph. aureus (meticillin- resistant) Strep. pneumoniae Strep. pyogenes Enterococcus faecalis Neisseria gonorrhoeae N. meningitidis Haemophilus influenzae E. coli Klebsiella spp. Proteus mirabilis Serratia spp. P. aeruginosa Bacteroides fragilis Sensitive Resistant Sensitive but not appropriate therapy Some strains resistant Fig. 8.2.5.3  Sensitivity of selected pathogenic bacteria to some common antibacterial agents.

692 SECTION 8  Infectious diseases analogues but have a lower barrier to resistance. The options for treating HIV infection are summarized in Box 8.2.5.2 (see also Chapter 8.5.23). Occasionally, drugs are combined for the purpose of achieving a synergistic effect based on evidence that the in vitro activity of the combination is shown to be greater than the sum of the activity of the individual agents. Most drugs in combination will simply be additive in effect. One of the more frequently prescribed syner- gistic combinations is that of penicillin (or ampicillin) and strepto- mycin (or gentamicin) in the treatment of endocarditis caused by Enterococcus spp. The aminoglycoside alone is generally inactive against enterococci but in combination with ampicillin achieves synergistic killing (Fig. 8.2.5.4). A similar effect is employed in the treatment of viridans streptococcal endocarditis with this combination. Another widely used example of synergistic inhibition is the combined effects of an antipseudomonal β-​lactam, such as ceftazidime or piperacillin, and an aminoglycoside, such as genta- micin, tobramycin, or amikacin. This combination can be used to treat documented or suspected P. aeruginosa infections occurring in neutropenic states complicating bone marrow transplantation, cytotoxic chemotherapy, and burn wound infections. Antibiotic resistance General considerations Antibiotic resistance has been recognized since the introduction of effective antibiotics. For example, penicillin-​resistant strains of S. aureus became widespread shortly after the introduction of this agent; penicillin-​sensitive strains are now uncommon. Resistant strains of Gram-​negative bacteria, such as E. coli, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas aeruginosa are commonly found in high-​dependency and other hospital units where they may cause outbreaks. Conventional approaches to controlling these infections may be unsuccessful, leading to contagion through­ out the healthcare system. The emergence of extended-​spectrum β-​lactamase-​producing E. coli and Klebsiella in the last 15 years in hospital and community practice has led to an increase in the use of carbapenem antibiotics which may, in turn, be a driver for the emer- gence and spread of carbapenemase (NDM, KPC, and OXA-​48) producing Klebsiella and E. coli. Other problems include the emer- gence of penicillin-​resistant pneumococci, β-​lactamase-​producing Haemophilus influenza, and multidrug-​resistant gonococci. At present, there is great international concern among profes- sionals, politicians and, increasingly, the public about antibiotic re- sistance. In the United Kingdom there is a 5-​year strategy to address antimicrobial resistance. This led to several initiatives including: (1) reducing the use of antibiotics, particularly in the treatment of minor upper respiratory tract infections in the community; Box 8.2.5.1  Tuberculosis treatment regimens for pulmonary and extrapulmonarya tuberculous infection caused by Mycobacterium tuberculosis Initial phase (2 months) • Isoniazid (with pyridoxine) • Rifampicin • Pyrazinamide • Ethambutol Continuation phase (4 months) • Isoniazid • Rifampicin a Central nervous system infection should be treated with isoniazid (with pyridoxine), rifampicin, pyrazinamide, and ethambutol for 2 months then isoniazid and rifampicin for 10 months. Tuberculosis, NICE Guideline—​13 Jan 2016 (nice.org.UK/​guidance/​fig33). Box 8.2.5.2  HIV infection: Initial treatment regimens for antiretroviral-​naive patientsa Two nucleoside reverse transcriptase inhibitorsb Plus Boosted protease inhibitorc Or Non​nucleoside reverse transcriptase inhibitord Or Integrase inhibitore a See Table 8.2.5.3 for agents available. b The recommended NRTI backbone is tenofovir + emtricitabine. Alternative NRTI backbone is abacavir + lamivudine. c The recommended boosted PI combinations are atazanavir + ritonavir or darunavir + ritonavir. d The recommended NNRTI is rilpivirine (provided the viral load <100 000). Alternative agent is efavirenz. e Any one of the licensed integrase inhibitor (dolutegravir, elvitegravir, or raltegravir). 0 0 Log10 number colony - forming units/ml 1010 108 106 104 102 4 8 Gentamicin 2 mg/litre Control Ampicillin 0.5 mg/litre Ampicillin 0.5 mg/litre + gentamicin 2 mg/litre 12 Time (h) 16 20 24 Fig. 8.2.5.4  Effects of ampicillin (0.5 mg/​litre) and gentamicin
(2 mg/​litre) alone and in combination on a strain of Enterococcus faecalis from a patient with infective endocarditis. A synergistic effect is observed with the combined agents.

8.2.5  Antimicrobial chemotherapy 693 (2) education strategies for prescribers and the public; (3) better enforcement of infection control policies; (4) improved antibiotic stewardship for hospitals; (5) monitoring and feedback of drug use in community practice; (6) introduction of infection control and antibiotic stewardship targets for state funded healthcare providers with linked financial incentives or penalties. Within the European Union, similar measures have been proposed. However, antibiotic resistance is a global problem. An increasing number of multidrug-​ resistant infections caused by Salmonella and Mycobacterium tu- berculosis are being imported from developing countries where the availability and prescribing of antibiotics is less controlled. The recent emergence of extensively drug-​resistant tuberculosis (XDR-​ TB) and multidrug-​resistant gonococci is a major cause for concern. Antibiotic resistance drives changes in patterns of prescribing and is a major impetus to the pharmaceutical industry in its search for new therapies. Microorganisms differ in their ability to develop resistance, which may affect a particular drug, a class, or multiple classes of antibiotics. Genetic mutations select for antibiotic re- sistance, which frequently occurs under the influence of antibiotic pressure. The major mechanisms of resistance are summarized in Table 8.2.5.4. Resistance to single or multiple antibiotics can be ei- ther chromosomally or plasmid mediated, or both. In turn, genes might code for resistance to a single or to multiple antibiotics. In addition to plasmid-​mediated resistance, other transposable genetic elements (transposons) and insertion sequences (integrons) incap- able of self-​replication might exist within a chromosome, plasmid, or rarely bacteriophage. Resistance genes are most frequently transferred between or- ganisms by conjugation. This occurs between the same or different species of bacteria and also between different genera. Other mech- anisms of transferring resistance are transformation in which naked DNA released during cell lysis is taken up by other bacteria or very rarely transduction via a bacteriophage. Transposon-​mediated resistance reflects transfer of discreet sequences of DNA between chromosomes or plasmids whereby in- dividual or groups of genes can be inserted into the host bacterial cell. Integrons may contain one or more gene cassettes which carry determinants of combinations of resistance genes within the bac- terial chromosome, plasmid, or transposons. The antibiotic resist- ance genes are bound on each side by conserved segments of DNA. These individual resistance genes can be inserted or removed be- tween the conserved structures and act as expression vectors for antibiotic resistance genes. The molecular mechanisms of antibiotic resistance are legion and the ability of drug-​resistant microorganisms to survive, dissem- inate, and cause disease varies widely. In many instances, antibiotic resistance might give a survival advantage only in the presence of continued antibiotic exposure to such agents. This is reflected in the occurrence of epidemic infections in high-​dependency units such as intensive care facilities where antibiotic usage is often high. However, it is also clear that once the genetic mechanism for evading antimicrobial activity has been acquired, it is rarely lost and adds to the continuously expanding genetic memory that has steadily eroded the efficacy of many antimicrobial drugs. Enzymatic inactivation Aminoglycoside-​modifying enzymes include adenylating, acetyl- ating, and phosphorylating enzymes. Gentamicin is the most susceptible and amikacin the least susceptible to such inactiva- tion. However, the largest group of inactivating enzymes are the β-​lactamases (EC 3.5.2.6) which hydrolyse the β-​lactam ring common to all penicillins, cephalosporins, carbapenems, and monobactams. Penicillinase was the first β-​lactamase to be iden- tified and is the reason why most strains of S. aureus are resistant to this drug. Another important β-​lactamase is TEM-​1, which is responsible for resistance to ampicillin by Haemophilus influen- zae or E. coli. The major impetus to the development of the broad-​ spectrum penicillins and cephalosporins was to extend their activity by resisting inactivation by β-​lactamases present in many aerobic Gram-​negative bacilli. However, new inactivating enzymes con- tinue to emerge, including the extended-​spectrum β-​lactamases, which are now limiting the clinical utility of third-​generation cephalosporins. A further example is the carbapenemase group of β-​lactamases which hydrolyse imipenem, meropenem, doripenem, and ertapenem, as well as other β-​lactams. Impermeability resistance Drug uptake of antibiotics such as the β-​lactams, tetracyclines, and fluoroquinolone antibiotics by bacteria is through protein channels (porins) which cross the outer membrane. Alterations in the per- meability of the outer membrane of Gram-​negative bacteria is an increasingly important mechanism of drug resistance. Mutations in porin structure are responsible for resistance among pathogens such as P. aeruginosa and Enterobacteriaceae. Alterations in target site Another important mechanism of resistance is mutational modifi- cation of drug binding sites. This affects susceptibility to β-​lactams, erythromycin, chloramphenicol, fluoroquinolones, and rifampicin. Erythromycin and chloramphenicol bind to the bacterial 50S ribo- somal subunit which is subject to genetic mutation. In contrast, the quinolones target DNA gyrase which is subject to subunit struc- ture alteration resulting in one variety of resistance to drugs such as ciprofloxacin. The increasing resistance to penicillin among Strep. pneumoniae is the result of reduced binding of penicillin to sev- eral binding proteins (PBP2a and PBP2x). Staph. aureus resistance Table 8.2.5.4  Examples of resistance mechanisms for selected antibiotics Enzymatic/​inactivation Altered target site Altered permeability Efflux Metabolic bypass Aminoglycosides Erythromycin β-​Lactams Tetracycline Sulphonamides β-​Lactams Chloramphenicol Quinolones Quinolones Trimethoprim Chloramphenicol Fusidic acid β-​Lactams Colistin β. lactams

694 SECTION 8  Infectious diseases to methicillin is due to the presence of penicillin-​binding protein (PBP2a) which has reduced affinity for methicillin and other β-​ lactams and is encoded by the mecA gene. The problem of vancomycin-​resistant enterococci, which largely affects Enterococcus faecium, is the result of the production of en- zymes (ligases) which permit continued cell wall synthesis despite the presence of vancomycin. To date, five different genes have been found responsible for this phenomenon (vanA to vanE) which result in different phenotypic patterns of resistance to the glycopeptides vancomycin and teicoplanin. The transfer of the vanA gene from E. faecium to S. aureus has resulted in the emergence of vancomycin-​ resistant S. aureus (VRSA), but very few clinical cases have been re- ported in the last decade. Metabolic bypass resistance Bacteria must synthesize folic acid from the precursor p-​aminobenzoic acid. The sulphonamide antibiotics competitively inhibit the enzyme dihydropteroate synthetase. Trimethoprim acts on the same metabolic pathway by inhibiting dihydrofolate reductase. The sequential inhibi- tory effects of trimethoprim and sulfamethoxazole (co-​trimoxazole) result in synergistic bactericidal activity against many pathogens. Resistant organisms are able to synthesize their own enzymes thereby evading such competitive inhibition. Surveillance of antibiotic resistance Information on the susceptibility of pathogenic microorganisms is important. Such data can provide information on the relative fre- quency of pathogens and the pattern of susceptibility to prescribed agents. Surveillance, therefore, has a role in guiding prescribing, in developing prescribing policies, and in identifying and moni- toring organisms that are subject to infection control measures. On a broader front, surveillance is also of value in alerting industry, public-​private partnerships, researchers, and healthcare planners to the need for new drug and vaccine strategies for disease control. To be of maximum benefit, surveillance needs to be linked to a de- fined geographical base, which might simply reflect the catchment area of specimens submitted to a particular laboratory, providing information on the trends in community and hospital isolates. Within hospitals, more specific information can be provided about susceptibility patterns in high-​dependency units, where antibiotic consumption is often greater, and more resistant pathogens such as Klebsiella, Serratia, Enterobacter, and Acinetobacter spp. and P. aeruginosa are found. Among Gram-​positive pathogens, entero- cocci, and Staph. aureus present the main challenges to prescribing and infection control practice. National networks of surveillance often vary in their focus and include data on Gram-​negative pathogens such as Escherichia coli and P. aeruginosa, Staph. aureus, penicillin resistance among pneumococci, and, more recently, vancomycin-​resistant entero- cocci. There are important international networks which col- lect information on such pathogens as Legionella pneumophila and Mycobacterium tuberculosis. Drug-​resistant tuberculosis is increasingly prevalent in the United Kingdom and elsewhere. Antimicrobial surveillance, based on specimens referred to la- boratories for clinical diagnostic purposes, can be limited if, for example, very few specimens are sent (i.e. otitis media), labora- tory networks are poorly developed (i.e. in some middle-​income countries) or data extraction is difficult. In these cases, special arrangements may be necessary to collect isolates and perform testing just for surveillance purposes. Surveillance of resistance to antiviral agents is largely confined to HIV in a few countries. Patient-​specific data are increasingly sought in those with HIV infection to assess drug failure, guide change in management, and direct primary therapy in selected cases of person-​ to-​person and mother-​to-​infant transmission. Determination of phenotypic resistance is still costly and time consuming, and most data relate to genotypic patterns of resistance to antiretroviral drugs among HIV isolates. Pharmacokinetics To be effective, antimicrobial agents must achieve therapeutic concentrations at the site of the target infection. This might be lo- calized to a single anatomical site, such as the bladder or the cere- brospinal fluid, or involve major organs, such as the lung. Infections can also be generalized and affect many body sites. Drug selection must also take into consideration the fact that pathogens such as Mycobacterium tuberculosis, Legionella pneumophila, Listeria monocytogenes, and Salmonella typhi replicate intracellularly. Antimicrobial drugs can be administered parenterally, orally, or topically to the skin, oral and genital mucosae, external auditory meatus, conjunctiva, and by intraocular or intravesical applica- tion. In the case of systemically active agents, the effective drug concentrations are determined by the standard pharmacokinetic parameters of absorption, distribution, metabolism, and elim- ination. Since selective toxicity is crucial to safe prescribing, the dose regimen for each agent aims to avoid concentrations toxic to the host but inhibitory to the microorganism. This ‘therapeutic window’ varies by drug. Bioavailability The rate and degree of absorption from the gastrointestinal tract is not only important for plasma concentrations reflected in the pharmacokinetic parameters of Cmax and Tmax of a drug, but also for potential adverse effects on the bowel (Table 8.2.5.5). For example, ampicillin, the first of the aminopenicillins, commonly caused gastrointestinal side effects, most notably diarrhoea. These effects have been reduced by increasing the bioavailability of the active drug through the introduction of hydroxyampicillin (amoxicillin) and various esters and prodrugs of ampicillin. Some agents such as cephalexin, doxycycline, linezolid, and quin- olone antibiotics are extremely well absorbed, achieving 80–​100% bioavailability. In the case of some quinolones, the excellent bio- availability has raised the possibility of treating with oral antibiotics some severely ill patients who might normally require parenteral therapy. In contrast, drugs which are poorly bioavailable, such as cefixime and cefuroxime axetil, not only have a higher incidence of gastrointestinal side effects, but also are more likely (although not uniquely) to select for C. difficile-​associated disease. Distribution Most drugs are distributed in the blood via the plasma before gaining access to the extracellular fluid. Tissue concentrations of a particular agent are affected by pH, drug ionizability, lipid solubility, and the presence of an inflammatory reaction whereby the capillary

8.2.5  Antimicrobial chemotherapy 695 fenestrations are increased in size. In the case of agents administered intravenously by infusion or by bolus injection, the distribution phase is rapid in comparison with orally, rectally, or intramuscularly administered drugs. Drugs which are poorly lipophilic, such as the β-​lactams and aminoglycosides, achieve low concentrations in tis- sues such as the brain. However, the β-​lactams achieve therapeutic concentrations in the cerebrospinal fluid as a result of the inflamma- tory reaction which accompanies meningitis. Drugs can also be taken up intracellularly, as in the case of macrolides and quinolones, resulting in a large volume of distribu- tion compared to drugs confined to the extracellular space, such as the β-​lactams and aminoglycosides. This is important in relation to the treatment of intracellular pathogens such as Mycoplasma pneu- moniae, Legionella pneumophila, and Mycobacterium tuberculosis, which can only be effectively treated by drugs that are concentrated and remain biologically active within the cell. The plasma half-​life (T½), which is the time required for the con- centration of a drug in the plasma to fall by one-​half, is affected initially by drug distribution and its rate of elimination as a result of metabolism and excretion. This in turn affects the time taken to reach steady state. In the treatment of life-​threatening infections, it is important that steady state kinetics are achieved rapidly and the administration of a loading dose may be required. This ap- plies to the use of agents such as intravenous quinine in the case of life-​threatening malaria and colistin for the treatment of serious Gram-​negative infections where the pharmacokinetic behaviour can be altered by the severity of the disease in comparison with healthy subjects (Fig. 8.2.5.5). Drugs are commonly distributed in the blood and tissues bound to plasma proteins, mostly albumin, and they vary in their degree of protein binding. With agents such as flucloxacillin and ceftriaxone it exceeds 95%. The importance of protein binding lies in the fact that the active moiety is the unbound drug. Dissociation from the bound to the unbound state is usually rapid, but this equilibrium may af- fect drug performance at certain sites such as the joints. The rela- tionship between protein binding and drug performance has been emphasized from studies of the pharmacodynamics of drug activity (see next). Metabolism Antibiotics, like other drugs, are degraded at various sites in the body but predominantly within the liver. Degradation involves con- jugation, hydrolysis, oxidation, glucuronidation, or dealkylation, according to the particular drug. Members of the hepatic cyto- chrome P450 group of enzymes play a dominant role in this process. Drug metabolites are usually, but not always, biologically inactive. For example, cefotaxime is degraded to desacetylcefotaxime and clarithromycin to hydroxyclarithromycin, both of which are Table 8.2.5.5  Bioavailability and intestinal elimination of some commonly prescribed antibacterial drugs after oral administration Drug Bioavailability (%) Intestinal elimination Penicillins Amoxicillin 80–​90 Concentrated up to 10-​fold in bile Ampicillin 50 Concentrated up to 10-​fold in bile Flucloxacillin 80–​90 Negligible Cephalosporins Cefalexin 80–​100 Concentrated up to 3-​fold in bile Cefixime 40–​50 Concentrated up to 50-​fold in bile Cefuroxime axetil 30–​40 Bile concentrations of up to 80% of serum Quinolones Ciprofloxacin 70–​85 Concentrated up to 10-​fold in bile Levofloxacin 99–​100 <5% dose found in bile Moxifloxacin 90 60% dose, concentrated 2–​6-​fold in bile Nalidixic acid 90–​100 Biliary concentrations similar to serum Other antibacterials Linezolid 100 1–​5 concentrations in bile Erythromycin 18–​45 Concentrated up to 300-​fold in bile Clarithromycin 50–​70 Metronidazole 80–​95 Concentrations in bile similar to serum Rifampicin 90–​100 Concentrated up to 1000-​fold in bile Sulfamethoxazole 70–​90 Concentrations in bile 40–​70% of serum Tetracycline 75 Concentrated up to 10–​30-​fold in bile Doxycycline 95 Concentration in bile 10–​30-​fold serum Trimethoprim 80–​90 Concentrated up to 2-​fold in bile Note that drugs which are well absorbed may still achieve high concentrations in the faeces because of secretion into bile or other enteral secretions.

696 SECTION 8  Infectious diseases biologically active and contribute to the overall antibacterial activity of these agents. Excretion Most drugs are excreted in the urine by glomerular filtration, tubular secretion, or a combination of these mechanisms. Thus, high con- centrations of drug will often be present in the urine; this has thera- peutic importance in the treatment of urinary tract infections. Urinary pH affects the biological activity of many drugs (e.g. the ac- tivity of ciprofloxacin is markedly reduced at pH 5.5). Tubular excre- tion can be blocked by probenecid. This was formerly used to ensure higher plasma concentrations of penicillin and is still recommended in alternative treatment regimens for gonorrhoea when single doses of amoxicillin are prescribed. It is also important to note that any re- duction in glomerular filtration rate will affect not only urinary con- centrations of drug but also the plasma half-​life and, in turn, serum concentrations of drugs which are primarily excreted by this route. In the case of antibiotics such as the aminoglycosides and vanco- mycin, the dose must be reduced in renal failure. Biliary excretion is another important route for drug elimination either as the active compound or as a microbiologically active or inactive metabolite. Reabsorption from the gastrointestinal tract can result in enterohepatic recirculation, which in turn may affect plasma half-​life. Drugs which achieve high concentrations in the bile are effective in the treatment of infections at this site such as cholecystitis. However, biliary obstruction or hepatic impairment can reduce therapeutic efficacy and require dose reduction to avoid toxic effects. Examples include clindamycin, efavirenz, mefloquine, and tetracyclines. Therapeutic drug monitoring of some antibiotics is essential in order to ensure therapeutic yet non​toxic concentrations. This ap- plies particularly to aminoglycosides which have a relatively narrow therapeutic index. Trough concentrations of gentamicin in excess of 1 mg/​litre, indicate reduced rates of renal elimination with the subse- quent increased risk of accumulation in kidney and inner ear which are associated with nephrotoxicity and ototoxicity. Vancomycin is also frequently monitored, particularly in patients with impaired renal function. Therapeutic drug monitoring is also increasingly used with other drug classes to optimize efficacy especially as it has been increasingly recognized patients with severe sepsis may well have highly variable pharmacokinetics. Pharmacodynamics Pharmacodynamics describes the effect of changes in drug concen- tration on antimicrobial effect or toxicological effect. However, the interrelationship between drug, microorganism, and the infected host creates the microbiological outcome dynamic. Antibiotics are unique in therapeutics in that they are targeted at an invading microorganism which may be present at a particular site or be more widely distributed in the body. The host’s response to infection might modify the pharmacokinetic handling of a drug. Many anti- biotics have a measurable effect on a variety of bacterial and host cell functions, even at subinhibitory concentrations. It is difficult to establish the exact role that these factors play clinically, but they are likely to contribute to the overall effect of an antibiotic. Macrolides, such as erythromycin, illustrate this point since they affect a variety of virulence characteristics (Table 8.2.5.6) as well as affecting the host’s response to infection. Exposure of microorganisms to sublethal concentrations of an antibiotic may temporarily inhibit growth which recommences fol- lowing removal of the drug. The time to recovery is known as the persistent antibiotic effect. This varies with the drug and the micro- organism; for example, the quinolones have a longer postantibiotic effect than β-​lactams (Table 8.2.5.7). The relevance of this obser- vation to the in vivo situation, where plasma drug concentrations 0 0 5 10 15 Severe malaria IV IM Uncomplicated malaria Healthy subjects Plasma quinine (mg/litre) t 2 4 6 Time (h) 8 10 12 β18 h tβ16 h tβ11 h Fig. 8.2.5.5  Average plasma quinine concentrations following administration of a loading dose of 20 mg (salt)/​kg to patients with severe and uncomplicated malaria, compared with those predicted to occur in normal subjects. From White NJ (1992). Antimalarial pharmacokinetics and treatment regimens.
Br J Clin Pharmacol, 34, 1–​10, with permission. © 1992 The British Pharmacological Society. Table 8.2.5.6  Effect of macrolides on bacterial virulence at subinhibitory concentrations Factor Effect Factor Effect Adhesins (pili, fimbriae) ↓ Exoenzyme production: Fibronectin binding ↓ Elastase ↓ Alginate production ↓ Protease ↓ Exotoxin A production ↓ DNAse ↓ β-​Haemolysin activity ↓ Coagulase ↓ Serum susceptibility ↑ Leukocidin ↓ Flagellar function ↓ From Shyrock TR, Mortensen JE, Baumholtz M (1998). The effects of macrolides on the expression of bacterial virulence mechanisms. J Antimicrob Chemother, 41, 505–​12.

8.2.5  Antimicrobial chemotherapy 697 are often well above the inhibitory concentration and are sustained through repeat doses, remains uncertain. It may have greater rele- vance to tissue concentrations, which tend to be lower than plasma concentrations. The persistent antibiotic effect certainly contrib- utes to the effects of agents that are administered once daily, such as gentamicin. The relationship between the pharmacokinetic characteristics of a drug and bacterial inhibition is critical to therapeutic outcome (Table 8.2.5.8). In the case of agents such as penicillins and ceph- alosporins, the time that drug concentrations are maintained above the MIC predicts the response. This contrasts with agents such as the quinolones and aminoglycosides, where it is more important to achieve high Cmax to MIC ratios or area under the concentration curve (AUC) to MIC ratios. Modelling the MIC of a particular or- ganism against the dose response curve for a drug (Fig. 8.2.5.6) has established several important pharmacodynamic parameters, which have been supported by studies in preclinical models and man. For example, dosage regimens of quinolones such as ciprofloxacin, levofloxacin, and moxifloxacin have been based on pharmaco- dynamic data. The ratio of AUC to MIC was defined in preclinical studies and was used to predict effect in man against S. pneumoniae or aerobic Gram-​negative rods. This was also predictive of outcome in human studies. The importance of protein binding for drug per- formance has also emerged as an important modifying factor in this modelling. The AUC to MIC ratio of the free drug is the most useful predictor of response. The manner in which these ratios differ for selected quinolones is shown in Table 8.2.5.9. Principles of use In comparison with many other classes of drugs, antimicrobial agents are usually prescribed in short courses ranging from a single dose to a few days. Prolonged therapy is required for certain infec- tions such as tuberculosis and bone and joint infections, and for HIV infection treatment is lifelong. Most antibiotic prescribing, especially within community prac- tice, is empirical. Even among patients in hospital, where there are greater opportunities for diagnostic precision based on laboratory investigations, the exact nature of the infection is established in only a minority of cases. Most therapeutic prescribing requires a pre- sumptive clinical diagnosis that, in turn, is linked to a presumptive microbiological diagnosis based on knowledge of the usual micro- bial causes of such infections. Among the most widely treated in- fections are those affecting the upper and lower respiratory tracts, the urinary tract, and skin and soft tissues for which the likely mi- crobial aetiology is restricted. For example, urinary tract infections arising in the community are usually caused by E. coli and other Gram-​negative enteric pathogens and, less commonly, by entero- cocci or Staphylococcus saprophyticus. Local knowledge of the sus- ceptibility of these pathogens to commonly used agents such as trimethoprim, ampicillin, and a quinolone such as ciprofloxacin is Table 8.2.5.7  Postantibiotic effects (h) of selected drugs against Staph. aureus, E. coli, and P. aeruginosa Drug Staph. aureus E. coli P. aeruginosa Ampicillin 1.7 0.1 NT Cefotaxime 1.4 0.2 0.3 Ciprofloxacin 2.0 2.1 2.4 Erythromycin 3.1 NT NT Gentamicin 2.0 1.8 2.2 Imipenem 2.6 0.5 1.5 Rifampicin 2.8 4.2 NT Vancomycin 2.2 NT NT NT, not tested. Table 8.2.5.8  Summary of major pharmacodynamic differences between aminoglycosides and β-​lactams Pharmacodynamic measurement Aminoglycosides β-​Lactam Rate of bacterial killing Rapid and dose related Slower with little or no increase at higher doses Number of bacteria killed per dose administered Concentration-​dependent over a wide concentration range Little increase in degree or rate of killing at concentrations above 4–​5 × MIC Postantibiotic effect Concentration-​dependent over a wide concentration range
for Gram-​positive and Gram-​negative pathogens Unpredictable in Gram-​negative bacteria, always short with little or no increase related to concentration Experimental models Large, infrequent doses more effective than smaller, more frequent doses which supports once-​daily dosing Frequent (hourly) injection or constant infusion most effective Ambrose et al., 2007 Clinical trials Cmax/​MIC ratios linked to clinical outcomes T>MIC linked to clinical outcomes Clinical trials with amikacin, gentamicin, and netilmicin have shown single daily dosing to be effective as multidosing Increased use of prolonged or continuous infusion dosing regimen in critical care settings Ambrose et al., 2007. Peak (c max) Area under the curve (AUC) exceeding MIC Minimum inhibitory concentration (MIC) Time Time above MIC Drug concentration Fig. 8.2.5.6  Relationship between the minimum inhibitory concentration (MIC) of a drug and its pharmacokinetic profile. Mouton et al., 2005.

698 SECTION 8  Infectious diseases helpful in recommending initial empirical antibiotic management. (Table 8.2.7.10). In more severe infections, such as community-​acquired pneu- monia, prompt empirical therapy is essential. Although the range of possible pathogens is more extensive (Table 8.2.5.11), Strep. pneu- moniae predominates and must always be targeted. Assessment of severity, based on validated criteria, assists in defining the initial em- pirical antibiotic regimen. This is illustrated by the British Thoracic Society’s recommendations for the initial empirical antibiotic man- agement of community-​acquired pneumonia (Table 8.2.5.12). The use of empirical therapy depends on the ease with which a clinical diagnosis can be made, as well as disease severity and drug toxicity. In the case of herpesvirus infections, the empirical use of aciclovir for the treatment of mucocutaneous herpes simplex in- fections or of shingles in older people is now common. However, it would be inappropriate to start treatment for HIV or cytomegalo- virus infections without laboratory support for these diagnoses in view of the toxicity and cost of the antiviral agents used to treat these infections. Antibiotic prophylaxis Antibiotics are used widely in the prevention of infection, in associ- ation with surgery, and in a range of medical conditions (see earlier). Antibiotic prophylaxis is used for selected surgical procedures where the risk of infection, although relatively low, is of serious import should it occur. Examples include prosthetic joint implantation and cardiac surgery in which prosthetic valves and intracardiac patches are inserted. The principles of antibiotic prophylaxis are based on the selection of an agent active against the known potential target pathogen(s). The drug should be present in high concentrations at the site and time of surgery and be relatively free from adverse reactions. One dose is generally effective but further doses may be needed depending on the length of the procedure. No regimen can be effective against all potential pathogens, hence the importance of postoperative follow-​up. Medical reasons for prophylaxis are also important; for example, prophylaxis is the use of low-​dose suppressive therapy to prevent Pneumocystis jirovecii pneumonia in those with advanced HIV in- fection. Co-​trimoxazole is the preferred agent; dapsone, atovaquone, or inhaled pentamidine are also used. Anatomical or functional asplenia is associated with a 12.6-​fold increased incidence of severe sepsis compared with the general population. This risk is related to the patient’s age and, in those after splenectomy, the reason for surgery and the period of time that has elapsed. Young children are particularly at risk, but this de- clines substantially after the age of 16 years. Hence the recommen- dation that immunization be supplemented with prophylactic oral penicillin (erythromycin for the intolerant) to prevent fulminant pneumococcal sepsis which predominates. Other recommended vaccines include Haemophilus influenzae type b (Hib) and menin- gococcal vaccination. Apart from good evidence for the benefit of prophylaxis in children with sickle cell disease, there is poor support Table 8.2.5.9  Pharmacokinetic and pharmacodynamic parameters of some recent quinolone antibacterial drugs Drug (dose mg) Protein binding (%) MIC90 Strep. pneumoniae AUC total
(mg/​h per L) AUC free
(mg/​h per litre) AUC/​MIC
(total drug) AUC/​MIC (free drug) Gatifloxacin (400) 20 0.5 51.3 41.0 102.6 82 Levofloxacin (500) 25 2.0 72.5 54.4   36.2 27.2 Moxifloxacin (400) 48 0.25 26.9 14.0 107.6 56.0 AUC, area under the concentration curve; AUIC, AUC to MIC ratio or area under the inhibitory concentration of total and free (unbound) drugs; MIC90, minimum inhibitory concentration active against 90% of isolates tested. Table 8.2.5.10  Prevalence of antibiotic resistance in 193 E. coli isolate from urine samples referred by general practice in North Bristol MIC (mg/​litre) %Sensitive Range MIC50 MIC90 Amoxicillin 0.5–​>128 8

128 52.9 (45.9–​59.9) Co-​amoxiclav 0.5–​>128 4 64 65.3 (58.6–​72.0) Ciprofloxacin 0.008–​>128 0.03 32 82.3 (76.9–​87.7) Nitrofurantoin 1–​>128 8 16 98.6 (96.9–​100) Cefradine 4–​>128 8 16 91.7 (87.8–​95.6) Trimethoprim 0.12–​>128 0.5 128 58.0 (51.0–​65.0) Co-​trimoxazole 0.015–​>32 0.12 32 63.7 (56.9–​70.5) Fosfomycin 0.25–​32 0.5 1 100 Cefixime 0.008–​64 0.25 1 93.7 (90.3–​97.1) Doxycycline 0.5–​>128 2 32 —​ Mecillinam 0.12–​>128 0.05 16 85.7 (80.8–​90.6) From Chin TL, et al. (2015). Prevalence of antibiotic resistance in Escherichia coli isolated from urine samples routinely referred by general practitioners in a large urban centre in South-​West England. J Antimicrob Chemother, 70(7), 2167–​2169, by permission of Oxford University Press.

8.2.5  Antimicrobial chemotherapy 699 Table 8.2.5.11  Microbiological aetiology (%) of adult community-​acquired pneumonia in the United Kingdom Pathogens Community (n = 236) Hospital (n = 1137) ICU (n = 185) Strep. pneumoniae 36.0 39.0 21.6 Haemophilus influenzae 10.2 5.2 3.8 Legionella spp. 0.4 3.6 17.8 Staph. aureus 0.8 1.9 8.7 Moraxella catarrhalis ? 1.9 ? Enterobacteriaceae 1.3 1.0 1.6 Mycoplasma pneumoniae 1.3 10.8 2.7 Chlamydophila pneumoniae ? 13.1 ? Chlamydophila psittaci 1.3 2.6 2.2 Coxiella burnetii 0 1.2 0 Viruses 13.1 12.8 9.7 Influenza A and B 8.1 10.7 5.4 Mixed 11.0 14.2 6.0 Other 1.7 2.0 4.9 None 45.3 30.8 4.0 ICU, intensive care unit. Reproduced from Lim WS, et al. (2009). The British Thoracic Society guidelines for the management of community-​acquired pneumonia in adults. Thorax, 64 Suppl III, 1–​61, and Lim WS, et al., 2015. Annotated BTS Guideline for the management of CAP in adults (2009). Summary of recommendations (www.brit-​thoracic.org.uk). Copyright © 2009, BMJ Publishing Group Ltd and the British Thoracic Society, with permission from BMJ Publishing Group Ltd. Table 8.2.5.12  Preferred and alternative initial empirical treatment regimens for community-​acquired pneumonia as recommended by the British Thoracic Society (2009 and 2015) Pneumonia severity (based on clinical judgement supported by CURB65 severity score) Treatment site Preferred treatment Alternative treatment Low severity (e.g. CURB65 = 0–​1 or CRB65 score = 0, <3% mortality) Home Amoxicillin 500 mg tds orally Doxycycline 200 mg loading dose then 100 mg orally or clarithromycin 500 mg bd orally Low severity (e.g. CURB65 = 0–​1, <3% mortality) but admission indicated for reasons other than pneumonia severity (e.g. social reasons/​unstable comorbid illness) Hospital Amoxicillin 500 mg tds orally If oral administration not possible: amoxicillin 500 mg tds IV Doxycycline 200 mg loading dose then 100 mg od orally or clarithromycin 500 mg bd orally Moderate severity (e.g. CURB65 = 2, 9% mortality) Hospital Amoxicillin 500 mg–​1.0 g tds orally plus clarithromycin 500 mg bd orally If oral administration not possible: amoxicillin 500 mg tds IV or benzylpenicillin 1.2 g qds IV plus clarithromycin 500 mg bd IV Doxycycline 200 mg loading dose then 100 mg orally or levofloxacin 500 mg od orally or moxifloxacin 400 mg od orallya High severity (e.g. CURB65 = 3–​5, 15–​40% mortality) Hospital (consider critical care review) Antibiotics given as soon as possible Co-​amoxiclav 1.2 g tds IV plus clarithromycin 500 mg bd IV (If Legionella is strongly suspected, consider adding levofloxacinb) Benzylpenicillin 1.2 g qds IV plus either levofloxacin 500 mg bd IV or ciprofloxacin 400 mg bd IV OR Cefuroxime 1.5 g tds IV or cefotaxime 1 g tds IV or ceftriaxone 2 g od IV, plus clarithromycin 500 mg bd IV (If Legionella is strongly suspected, consider adding levofloxacinb) bd, twice daily; IV, intravenous; od, once daily; qds, four times daily; tds, three times daily. a Following reports of an increased risk of adverse hepatic reactions associated with oral moxifloxacin, in October 2008 the European Medicines Agency (EMEA) recommended that moxifloxacin ‘should be used only when it is considered inappropriate to use antibacterial agents that are commonly recommended for the initial treatment of this infection’. b Caution –​ risk of QT prolongation with macrolide-​quinolone combination. Reproduced from Lim WS, et al. (2009). The British Thoracic Society guidelines for the management of community-​acquired pneumonia in adults. Thorax, 64 Suppl III, 1–​61, and
Lim WS et al., 2015. Annotated BTS Guideline for the management of CAP in adults (2009). Summary of recommendations (www.brit-​thoracic.org.uk). Copyright © 2009, BMJ Publishing Group Ltd and the British Thoracic Society, with permission from BMJ Publishing Group Ltd.

700 SECTION 8  Infectious diseases for efficacy in other populations of asplenic patients. There remain, therefore, differences of opinion about the recommendation for the continued use of chemoprophylaxis in adults, although some rec- ommend that a period of two years is appropriate. Issues of cost, compliance, and drug-​resistant pathogens add further fuel to the de- bate. What is clear is that the patient or legal guardian(s) should be educated concerning this risk. Dose selection Few antibacterial drugs are specific to a single pathogen, or site of infection, hence the dosage regimen must capture a range of sus- ceptibilities of the various target microorganisms as well as likely concentration in different body sites to ensure a successful response. The dosage regimen for new antibiotics is determined initially by pharmacokinetic studies in healthy volunteers combined with pre- clinical pharmacodynamic studies to determine the dominant pharmacodynamic index (Cmax/​MIC, AUC/​MIC, T>MIC) and the size of this index for antibacterial effect. This is supplemented by in- formation from standardized animal models that simulate infections such as lung infection, peritonitis, endocarditis, meningitis, otitis media, and sepsis-​complicating neutropenia. In man, information on drug penetration into the lung extracellular lining fluid, urine, cerebrospinal fluid, and other tissue spaces are needed. Dosing re- gimens ultimately need to be validated in clinical trials to measure safety and efficacy and confirm the preclinical pharmacokinetic-​ dynamic findings. Bactericidal versus bacteriostatic agents In the treatment of many common community infections which are usually of mild or moderate severity, the choice of either a bacterio- static or a bactericidal antibiotic is of limited importance. However, in patients with severe infection, particularly when complicating an immunocompromised state, a bactericidal agent must be used. This applies particularly to those with severe neutropenia which is a common accompaniment of cytotoxic chemotherapy, especially in the treatment of haematological malignancies and following haem- atopoietic stem cell transplantation. Another important indication for selecting a bactericidal regimen is in the treatment of infective endocarditis; although the infected vegetations are in the blood- stream, they are relatively protected from host phagocytic control. Effective penetration into the fibrin–​platelet mass requires high con- centrations of a bactericidal drug to sterilize the infected vegetations. Duration of treatment The duration of therapy for many common infections has not been rigorously determined. The treatment of many common conditions is based on custom and practice and often varies internationally. The trend over the last 25–​30 years has been to shorten duration of therapy, where possible, and this is likely to continue in the future. The duration of treatment has been more thoroughly determined in the following cases: • Gonococcal urethritis responds promptly to single-​dose treat- ment with agents such as ceftriaxone, azithromycin, or a quin- olone antibiotic such as ciprofloxacin or ofloxacin. • Uncomplicated urinary tract infection, particularly when affecting women of childbearing years, responds promptly to selected agents such as nitrofurantoin and fluoroquinolones. Although bacteriuria can be eliminated with a single dose, the symptoms of dysuria and frequency take longer to subside, hence a 3-​day course is preferred. • Pharyngitis caused by Streptococcus pyogenes improves symp- tomatically within a few days of antibiotics such as penicillin, but eradication of the infecting organism from the throat often takes up to 10 days. It is acknowledged that this presents major difficul- ties with regard to drug compliance. • For pulmonary tuberculosis the current recommendation of an initial 2-​month treatment with rifampicin, isoniazid, pyrazinamide, and ethambutol (reducing to isoniazid and rifam- picin for a further 4 months provided the isolate is confirmed to be susceptible) is based on extensive clinical trials (Box 8.2.5.1). • In cases of bacterial endocarditis, knowledge of the in vitro sus- ceptibility of the infecting organism is crucial in determining dose, duration, and outcome of therapy. Highly penicillin-​ sensitive strains (MIC ≤0.1 mg/​litre) of viridans streptococci are treated effectively with a 2-​week regimen of parenteral penicillin and gentamicin or 4–​6 weeks of parenteral penicillin alone. Less sensitive strains should be treated with parenteral penicillin for a total of 4–​6 weeks, plus 2 weeks IV gentamicin. If the infecting organism is an enterococcus, a minimum of 6 weeks’ treatment with parenteral penicillin (or ampicillin) and aminoglycoside is essential. Infections caused by Staph. aureus are a particular challenge since the severity is highly variable and yet the potential for metastatic infection and chronicity, as in the case of osteomyelitis, must be kept in mind. The isoxazolyl penicillins such as flucloxacillin are pre- ferred, with the use of combination therapy still largely unproven. Clindamycin is a useful alternative agent. Many Staph. aureus in- fections of the skin and soft tissues respond promptly to 7 day oral therapy and no antibiotics are needed if surgery is used to drain cutaneous abscesses. Where there is a severe systemic response to infection, parenteral therapy is appropriate initially. Where there is evidence of blood stream infection and dissemination, treatment should be extended for periods of up to 4–​6 weeks. In the case of septic arthritis, antibiotics should be given promptly and joint aspiration carried out, sometimes repeatedly, to avoid damage to the articular cartilage. The duration of therapy has not been rigorously determined. Most infections will resolve in 2–​3 weeks. One of the most challenging infections is staphylococcal osteomyelitis. To avoid chronicity, it is customary to treat for 4–​6 weeks. Treatment is generally administered parenterally, at least ini- tially; outpatient intravenous antibiotic therapy is also widely used in the treatment of bone and joint infection employing agents such as ceftriaxone, teicoplanin which can be administered once daily. However, the role of outpatient intravenous antibiotic therapy com- pared to oral step-​down therapy remains controversial and subject to ongoing clinical trials. For most infections, the duration of therapy remains uncertain. However, many mild to moderate uncomplicated infections will defervesce within a 3-​ to 5-​day period suggesting that 5–​7 days of treatment is usually adequate. There is little evidence to suggest that treatment periods of 10–​14 days, or longer, are any more effective. They are also likely to be associated with an increased risk of side effects, superinfection, and the selection of antibiotic-​resistant or- ganisms, as well as being more costly.

8.2.5  Antimicrobial chemotherapy 701 The parenteral administration of antibiotics is appropriate in the management of severe life-​threatening infections and when oral therapy is contraindicated, such as in the postoperative period, if the patient is vomiting, or where gastrointestinal absorption cannot be relied on. However, the need for continued parenteral therapy should be reviewed regularly. In the treatment of many common in- fections, the acute features of infection such as temperature, tachy- cardia, and an elevated circulating neutrophil count usually improve within a period of 48 to 72 h. Provided there is no contraindication to oral therapy, this should be considered early in the course of pa- tient management. The advantages are not just in the reduced cost of medication; the risk of intravenous line associated complications, such as infection, is also eliminated, discharge from hospital may be hastened and the need to deliver outpatient intravenous therapy removed. Adverse drug reactions Overall, antimicrobial agents have an outstanding record of safety. Nonetheless, no drug is without the potential for side effects. The risk varies by agent and sometimes by dose, while host genetic fac- tors and pathophysiological status can also be important. Oral antibiotics are largely used in the community where they are generally well tolerated and used in the treatment of minor infec- tions in large populations. Injectable agents selected for short-​course perioperative prophylaxis have a well-​established safety record. However, agents such as the antiviral drugs and amphotericin B carry a higher risk of more serious adverse drug reactions, which must be balanced against the life-​threatening nature of their target infections. While drug safety is assessed during drug development, the full repertoire of adverse reactions becomes apparent only during widespread clinical use, hence, the importance of adverse drug re- action reporting systems. In the United Kingdom, the ‘yellow card’ system has been very successful and relies on voluntary reporting of possible adverse drug events to the Medicines & Healthcare Products Regulatory Agency (http://​www.mhra.gov.uk) by doctors, dentists, coroners, pharmacists, nurses (including midwives and health visitors), radiographers, optometrists, and, most recently, pa- tients. It is important to distinguish between adverse event reporting and adverse drug reaction reporting. The latter is more difficult to establish with certainty and may require rechallenge, which raises medical and ethical concerns. It is essential to enquire about previous drug reactions as well as other forms of drug toxicities before prescribing. The relation- ship to a previously prescribed drug requires careful assessment. Hypersensitivity is among the more common of drug reactions and, in the case of β-​lactam drugs, appears to be more a function of the five-​membered thiazolidine ring (Fig. 8.2.5.7) of the penicillin molecule, since hypersensitivity reactions are less common with the cephalosporins which have a six-​membered dihydrothiazine ring. The monobactam aztreonam has no ring structure and hypersensi- tivity reactions appear to be rare. However, it is important to note that accelerated systemic hypersensitivity reactions (anaphylaxis) can be life-​threatening, such that any previous association with a β-​lactam drug is an absolute contraindication to the use of all β-​lactams. Some drug toxicities are genetically determined. For example, people who are genetically slow acetylators of isoniazid are more at risk of side effects such as peripheral neuropathy. Those genetically deficient in the enzyme glucose-​6-​phosphate dehydrogenase (EC 1.1.1.49) are at risk of drug-​induced haemolysis. This risk is more common in those of African, Mediterranean, or Far Eastern descent. Hence, it is important to screen for this red cell enzyme deficiency before the administration of oxidant drugs such as primaquine. Adverse drug reactions may not always be acute in their pres- entation but reveal themselves after prolonged drug exposure. Oral flucloxacillin and co-​amoxiclav when administered for sev- eral weeks, particularly in older patients, are more likely to induce drug-​associated hepatotoxicity. Likewise, parenteral formulations of selected drugs may be more toxic than their oral formulation, as is the case with fusidic acid where prolonged parenteral administra- tion frequently gives rise to hepatotoxicity. Concentration-​dependent adverse reactions (Table 8.2.5.13) are more likely to occur in the presence of organ system failure. Penicillins O COOH O -Lactam ring Thiazolidine ring C R NH S Cephalosporins O O R’ COOH -Lactam ring Dihydrothiazine ring C R NH S β β carbapenems R O N COOH R lactam β Monobactams O O N SO3H -Lactam ring C R NH β Fig. 8.2.5.7  Chemical structure of the β-​lactam antibiotics (penicillins, cephalosporins, and monobactams) identifying the common β-​lactam ring component which is subject to hydrolysis by β-​lactamases.

702 SECTION 8  Infectious diseases Aminoglycoside toxicity is more common in older people, in those with pre-​existing renal failure, and after repeated aminoglycoside doses or other nephrotoxic drugs. Concentration-​dependent bone marrow suppression characterizes the use of chloramphenicol whereby pancytopenia arises when plasma concentrations are in ex- cess of 25 mg/​litre. This is to be distinguished from the idiopathic aplastic anaemia that is a rare accompaniment of chloramphenicol use, but unfortunately is rarely reversible. Much has been learned about the structure–​activity determinants of drug toxicity. For example, the quinolone antibiotics as a class have the potential to induce phototoxicity, arthrotoxicity, central nervous system toxicity, cardiotoxicity, and interact with agents such as caffeine, theophylline, and non​steroidal anti-​inflammatory drugs (Fig. 8.2.5.8). Knowledge of such predictors has led to the se- lection of agents with safer structural profiles. Despite this, adverse drug reactions have led to the withdrawal or modification of the licensed indications for several quinolones, notably temafloxacin, trovafloxacin, and sparfloxacin, emphasizing the importance of clin- ical recognition and reporting of adverse events. Few infectious conditions require lifelong therapy. The man- agement of HIV infection has challenged this paradigm. To date, drugs directed at the causative viruses or complicating opportun- istic infections are suppressive rather than achieving eradication. Table 8.2.5.13  Dose-​related adverse effects of selected antimicrobials Drug Adverse effect Comment Antibacterial drugs General Superinfection by yeasts or C. difficile; selection of drug-​resistant bacteria from the normal flora These are universal adverse effects of antibacterial drugs and are generally related to the duration of exposure β-​Lactams Myelosuppression Neutropenia may occur after 1–​2 weeks of high-​dose IV therapy Drug fever Occurs during prolonged (>1 week), high-​dose IV therapy (e.g. endocarditis) Central nervous stimulation/​convulsions Can occur with overdose in renal failure Aminoglycosides Nephrotoxicity; ototoxicity Monitoring of serum concentrations minimizes but does not avoid toxicity; risk of toxicity is related to the duration of the dose and concomitant therapy Vancomycin Nephrotoxicity; ototoxicity May potentiate aminoglycoside nephrotoxicity Macrolides (e.g. erythromycin) Gastrointestinal stimulation This is a prokinetic effect of erythromycin which does not occur with all macrolides Ototoxicity; cardiac arrhythmias Only with high-​dose IV therapy Drug interactions Increased serum concentrations of theophylline and cyclosporin Quinolones (e.g. ciprofloxacin) Central nervous stimulation Quinolones are weak GABA antagonists; this effect is potentiated by coadministration with NSAIDs, especially fenbufen Drug interactions May inhibit metabolism of theophylline Oxazolidinone (e.g. linezolid) Anaemia, neutropenia, thrombocytopenia; neuropathy; lactic acidosis Limit treatment to 28 days to reduce risk of haematological toxicity Antifungal/​antiprotozoal/​antiviral drugs Deoxycholate Amphotericin B Nephrotoxicity Decreased creatinine clearance and renal potassium wasting are universal at clinically effective doses Rigors/​hyperthermia/​hypotension Related to the rate of infusion Ketoconazole Inhibition of steroid synthesis Occurs with prolonged (>1 week) high-​dose therapy Aciclovir Central nervous adverse effects; crystalluria Rare except with high-​dose IV therapy Quinine Hypoglycaemia GABA, γ-​aminobutyric acid; NSAID, non​steroidal anti-​inflammatory drug. Effect of F on side-effect profile has not been reported Influence phototoxicity and genetic toxicity Metal binding and chelating site controls interactions with antacids, milk, iron supplements No side effects associated with this position Controls theophylline interaction and genetic toxicity Controls phototoxicity (major) and genetic toxicity. Minor effect in NSAID interactions Controls GABA binding (major), NSAID interaction (major), Theophylline interaction (major), genetic toxicity F R5 R1 R2 X8 R7 N O O C OH Fig. 8.2.5.8  Structure–​activity side-​effect relationships of the fluoroquinolone antibacterial drugs. GABA, γ-​aminobutyric acid; NSAID, non​steroidal anti-​inflammatory drug. Redrawn from Domagala JM (1994). Structure–​activity and structure–​side-​effect relationships for the quinolone antibacterials. J Antimicrob Chemother, 33, 685–​706, by permission of Oxford University Press.

8.2.5  Antimicrobial chemotherapy 703 It is also important to note that the drugs used in the treatment of HIV are often licensed with limited information concerning their long-​term safety. The potential for adverse reactions and especially interactions is considerable and requires careful atten- tion to their detection and management. This has become an in- creasingly important challenge as life expectancy for those with HIV infection improves. It is important to balance drug safety while encouraging compliance and the maintenance of a reason- able state of health. Failure of antibiotic therapy Antimicrobial therapy may fail for several reasons. The agent selected may be inappropriate for the particular infection and fail to inhibit the target organism, or fail to reach the site of infection in sufficient concentration. For example, drugs such as nitrofurantoin and pivmecillinam, while achieving high urinary concentrations, fail to deal adequately with parenchymatous infection of the kidney or bacteraemia which may complicate acute pyelonephritis. The prostate also presents a chemotherapeutic challenge owing to the relatively low pH (c. 6.4) in chronic bacterial prostatitis. Drugs which are weak bases, such as trimethoprim either alone or in combination with sulfamethoxazole (co-​trimoxazole), are pre- ferred, especially since they are also lipid soluble. Ciprofloxacin has similar characteristics and has also produced favourable re- sults. However, treatment of acute bacterial prostatitis sometimes needs to be prolonged (4 weeks and occasionally longer), espe- cially if there is a history of chronic relapsing infection. The drug may be appropriate, but the dose selected may be in- adequate. This may apply to such conditions as unsuspected bac- terial endocarditis where high-​dose parenteral antibiotic is required. Likewise, the concentration of penicillin required to deal with pneumococcal meningitis greatly exceeds that effective in the treat- ment of pneumococcal pneumonia; occasionally the two diseases coexist. Infections caused by Legionella pneumophila and Chlamydia spp. require drugs that achieve high intracellular concentrations such as the fluoroquinolones, macrolides, or tetracyclines. Resistance emerging during treatment is an uncommon cause of clinical failure but should be considered. Drug-​resistant Mycobacterium tuberculosis can develop on therapy as a result of the emergence of minority populations of organisms resistant to such first-​line drugs as rifampicin and isoniazid. The current multidrug regimens are, in part, designed to avoid this occurrence. Likewise, in those with HIV infection, drug-​resistant virus is an increasingly important cause of treatment failure and requires good compliance with multidrug regimens to slow its rate of emergence. Failure of the patient to take the drug is an important consideration, particularly in these two infections; non​adherence can lead to the development of drug resistance. Mixed infections are commonly associated with intra-​abdominal sepsis and occasionally with infections of the lung. They may fail to respond to treatment unless the regimen covers the full range of bacterial pathogens. In the case of intra-​abdominal sepsis, the regimen should be active against anaerobic as well as aerobic bac- terial pathogens. Another important cause of antibiotic failure is the continued presence of a focus of infection. This may be an abscess that requires surgical drainage or the removal of an implanted medical device such as an intravascular catheter. Much more serious is infection of a prosthetic heart valve, hip joint, or central nervous system shunt where revision surgery carries significant risks. Many antibiotics fail to achieve therapeutic concentrations within abscess cavities, or are pH sensitive. Implant-​associated infections present a similar chal- lenge since bacteria often replicate slowly within a biofilm that is protective against normal host defences. Finally, it should be remembered that a persistently elevated tem- perature in the presence of what appears to be adequate antibiotic treatment can reflect drug fever or indeed fever complicating a non-​ infectious diagnosis. This emphasizes the importance of monitoring the response to treatment and repeated patient assessment. Practice guidelines and formularies The plethora of therapeutic agents currently available presents a considerable challenge to the prescriber. Guidance on the choice of agent and the management of disease is becoming increasingly im- portant. This is not only to ensure that the selection of treatment is appropriate for the target infection and consistent with current patterns of antimicrobial susceptibility but also that it reflects an ac- ceptable safety profile as well as being sensitive to the appropriate use of healthcare resources. Such guidance is increasingly provided designed for local use, within either a hospital or a community practice. It is often combined with a formulary which lists available drugs, and the institution’s antibiotic policy which describes the ob- jectives of its stewardship programme, how antibiotic use and resist- ance is monitored, ongoing audits and education, and how the whole process is managed. The guidelines frequently offer information on preferred and alternative regimens for particular infections. Within hospital practice, it is common for such formularies to identify drugs which may be prescribed freely according to specific indications and those for which expert advice an infection disease specialist should be sought. The latter applies particularly to drugs that require specific skill and experience in their use, need drug levels to be monitored, or are expensive. For example, the treat- ment of deep-​seated fungal infections with amphotericin B requires careful clinical assessment and guidance on dosage and moni- toring. Likewise, the treatment of HIV infection is increasingly a specialist area. Antibiotics which are expensive to prescribe such as parenteral or drugs, or last resort, carbapenems may be restricted. The guidelines will also have recommendations for the timing of transfer from parenteral to oral therapy in order to minimize the use of injectable agents. Formularies and guidelines have an educational role and allow the prescriber to become familiar with indications and safety of the most commonly used agents. Their use should be supported by educational activities both at undergraduate and postgraduate level. Ideally, the selection of agents for inclusion in the formulary should be based on sound evidence of efficacy, safety, and economic benefit. However, such evidence-​based medicine is often lacking or incomplete for commonly treated infections, since clinical trials of antibiotics, although increasingly robust in their design, are largely conducted to support licensing requirements rather than to ad- dress clinical use. They generally demonstrate the non​inferiority of a new agent in comparison with existing therapies. As a result, the

704 SECTION 8  Infectious diseases recommendations of formularies and practice guidelines are based on a matrix of information derived from knowledge of the in vitro profile of an agent, its pharmacokinetic-​dynamic parameters, its clinical and microbiological efficacy, and its safety profile. This, in turn, is modified by custom and practice which explains why there is local and, sometimes, national and international variation in re- commendations for some common indications such as community-​ acquired pneumonia. In low and middle-​income countries, where medical resources are much more limited, greater reliance is placed on low-​cost agents. The World Health Organization regularly updates its list of recommended essential drugs which includes anti-​infective agents (Table 8.2.5.14). Despite the emphasis on low-​cost agents, the drugs offered cover most infections and prescribing needs of these countries. The agents available in individual countries often vary according to local interpretation of the needs for these ‘essential’ drugs. Recent developments in economically advanced countries have included an assessment of healthcare technologies for current management, national need, and the resources available. In the United Kingdom, the National Institute of Clinical Excellence (NICE, https://​www.nice.org.uk) to assesses a variety of healthcare technologies including procedures as well as new therapies. Such assessments place greater emphasis on ensuring that new tech- nologies are evaluated in a manner that more closely resembles clinical practice as well as demonstrating economic benefit, in contrast to drug licensing which addresses the quality, safety, and efficacy of new therapies. This emphasis is likely to require a greater partnership between healthcare systems and pharma- ceutical companies to ensure that the place of new technologies is rapidly assessed and that their use is consistent with healthcare strategies. FURTHER READING Albur MS, et al. (2012). Factors influencing the clinical outcome of methicillin-​resistant Staphylococcus aureus bacteraemia. Eur J Clin Microbiol Infect Dis, 31, 295–​301. Ambrose PG, et al. (2007). Pharmacokinetics-​pharmacodynamics of antimicrobial therapy: it’s not just for mice anymore. Clin Infect Dis, 44, 79–​86. Bennett WM, et al. (1994). Drug prescribing in renal failure: dosing guidelines for adults, 3rd edition. American College of Physicians, Philadelphia, PA. Chin TL, et al. (2015). Escherichia coli isolated from urine samples routinely referred by general practitioners in a large urban centre in South-​West England. J Antimicrob Chemother, 70, 2167–​9. Davies JM, et al. (2011). Review of guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen: prepared on behalf of the British Committee for Standards Table 8.2.5.14  The World Health Organization (2015) model list of essential drugs (anti-​infectives) Anthelmintics/​antifilarials/​ antischistosomal and others Antibacterials Antitubuculosis medicines Antifungals Antivirals Antiprotozoal Albendazole Amoxicillin±clavulanic acid Ethambutol Amphotericin B Aciclovir Diloxanide Levamisole Ampicillin Isoniazid Co-​trimoxazole Abacavir Metronidazole Mebendazole Benzathine benzylpenicillin Pyrazinamide Fluconazole Lamivudine Miltefosine Niclosamide Benzylpenicillin Rifampicin Flucytosine Stavudine Stibogluconate Praziquantel Cephalexin Rifabutin Griseofulvin Tenofovir Amodiaquine Pyrantel Cefazolin Rifapentine Nystatin Zidovudine Artemether ± lumefantrine Diethylcarbamazine Cefixime Streptomycin Efavirenz Artesunate ± amodiaquine±mefloquine Ivermectin Ceftriaxone Nevirapine Chloroquine Triclabendazole Cloxacillin Atazanavir Primaquine Phenoxymethyl/​penicillin Darunavir Quinine Procaine penicillin Ritonavir Proguanil Azithromycin Saquinavir Pyrimethamine Chloramphenicol Oseltamivir Sulfadiazine Ciprofloxacin Ribavirin Pentamidine Clarithromycin Valganciclovir Suramin Doxycycline Entecavir Eflornithine Erythromycin Sofosbuvir Melarsoprol Gentamicin Daclatasvir Nifurtimox Metronidazole Dasabuvir Benznidazole Nitrofurantoin Ribavirin Spectinomycin Co-​trimoxazole Trimethoprim

8.2.5  Antimicrobial chemotherapy 705 in Haematology by a working party of the Haemato-​Oncology Task Force. B J Haem, 155, 308–​17. Domagala JM (1994). Structure–​activity and structure–​side-​effect re- lationships for the quinolone antibacterials. J Antimicrob Chemother, 33, 685–​706. Finch RG, Williams RJ (1999). Baillière’s clinical infectious diseases: an- tibiotic resistance. Baillière Tindall, London. Finch RG, et  al. (2010). Antibiotic and chemotherapy, 9th edition. Churchill Livingstone, Edinburgh. Freifeld AG, et al. (2011). Clinical practice guideline for the use of anti- microbial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis, 52, 427–​31. Gould FK, et al. (2011). Guidelines for the diagnosis and antibiotic treatment of endocarditis in adults: a report of the Working Party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother, 67, 269–​89. Grayson ML, et al. (2010). Kucer’s The use of antibiotics, 6th edition. CRC Press, Boca Raton, FL. Kerr KG (1999). The prophylaxis of bacterial infections in neutropenic patients. J Antimicrob Chemother, 44, 587–​91. Lim WS, et  al. (2015). Annotated BTS guideline for the manage- ment of CAP in adults 2015. https://​www.brit-​thoracic.org.uk/​ standards-​of-​care/​guidelines/​bts-​guidelines-​for-​the-​management-​ of-​community-​acquired-​pneumonia-​in-​adults-​update-​2009/​ annotated-​bts-​guideline-​for-​the-​management-​of-​cap-​in-​adults-​ 2015/​ Mouton JW, et al. (2005). Standardisation of pharmacokinetic/​pharma- codynamic (PK/​PD) terminology for anti-​infective drugs: an up- date. J Antimicrob Chemother, 55, 601–​7. Nahid P, et al. (2016). Official American Thoracic Society/​Centers for Disease Control and Prevention clinical practice guidelines: treatment of drug-​susceptible tuberculosis. Clin Infect Dis, 63, e147–​95. National Institute for Health and Care Excellence (NICE) (2006). Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control. Clinical guideline [CG33]. https://​www.nice.org.uk/​CG033 National Institute for Health and Care Excellence (NICE) Short Clinical Guidelines Technical Team (2008). Prophylaxis against infective endocarditis:  antimicrobial prophylaxis against infec- tive endocarditis in adults and children undergoing interventional procedures. National Institute for Health and Clinical Excellence, London. Russell AD, Chopra I (1996). Understanding antibacterial action and resistance, 2nd edition. Ellis Horwood, London. Shyrock TR, Mortensen JE, Baumholtz M (1998). The effects of macrolides on the expression of bacterial virulence mechanisms. J Antimicrob Chemother, 41, 505–​12. Standing Medical Advisory Committee Subgroup on Antimicrobial Resistance (1998). The path of least resistance. Department of Health, London. White NJ (1992). Antimalarial pharmacokinetics and treatment regi- mens. Br J Clin Pharmacol, 34, 1–​10. Wise R, Honeybourne D (1999). Pharmacokinetics and pharmaco- dynamics of fluoroquinolones in the respiratory tract. Eur Respir J, 14, 221–​9. World Health Organization (2007). Model list of essential medi- cines, 15th edition. http://​www.who.int/​medicines/​publications/​ essentialmedicines/​eu

8.3 Immunization 706

8.3 Immunization 706

ESSENTIALS Immunization is one of the most successful medical interventions ever developed: it prevents infectious diseases worldwide. Mechanism of effect—​the basis for the success of immunization is that the human immune system is able to respond to vaccines by pro- ducing pathogen-​specific antibody and memory cells (both B and T cells) which protect the body should the pathogen be encountered. Clinical practicalities—​most currently licensed vaccines contain live or killed bacterial or viral constituents, bacterial polysaccharides, or bacterial toxoids, while new types of vaccines are being developed that contain DNA. Most vaccines are delivered directly into skin or muscle via needles, or they are administered orally. New edible vac- cines and vaccines delivered via the skin without the use of needles are being developed. Who should be immunized?—​vaccines can be used in a targeted way (i.e. only for those at high risk), or they can be recommended for mass immunization of whole populations. The latter approach may eventually lead to complete eradication of an infectious disease, as was the case with smallpox: polio eradication is the next global chal- lenge. Vaccines that can interrupt the transmission of a pathogen between individuals are able to provide indirect protection, with the benefit of vaccination extending beyond the vaccinated population (e.g. infant immunization with pneumococcal vaccine has reduced the burden of disease in adults). Global perspective—​the Expanded Program on Immunization, set up by the World Health Organization to define which vaccines should be delivered in resource poor countries, has done much to increase coverage of vaccination among infants most at risk of infec- tious diseases. The evaluation of immunization programmes includes measurement of vaccine coverage, continuing surveillance for vaccine-​ preventable infections, seroprevalence studies to assess population immunity, and systems for monitoring and reporting adverse events. Introduction Infectious diseases remain a major cause of mortality and mor- bidity worldwide. The prevention of certain infectious diseases by effective immunization programmes is one of the major triumphs of 20th-​century medicine. Most of this was achieved in the final third of that century, during which rapid strides in the understanding of the biology and pathogenicity of infectious agents or their compo- nents, and improved techniques for their purification, led to the de- velopment of safe and effective vaccines. The greatest triumph in the field of immunization was the eradication of smallpox. In 1959 the World Health Organization (WHO) declared its intention to eradi- cate smallpox, and in 1966 began to allocate sufficient resources to accomplish this ambitious goal. Thirteen years later, in 1979, the global eradication of smallpox was officially declared. Effective vac- cines can eliminate infectious diseases, but to do this they must be implemented and used appropriately. Of the more than 12 mil- lion children under the age of 5 years who die annually, 2.4 million die of diseases that could be prevented by vaccines already avail- able through the WHO’s Expanded Programme on Immunization. While rapid advances in vaccine science have introduced new tech- niques such as DNA vaccines, delivering vaccines to those most at risk must remain a priority. Immunology of active immunization Both non​specific (innate) and specific (adaptive or acquired) im- mune systems are responsible for protecting humans against infec- tious diseases. The ability of the adaptive immune system to refine its antigen-​recognition domains and establish immunological memory is the basis of successful active immunization. The antigen-​specific component of the immune system contains both cellular and hu- moral elements (secreted antibody), whose relative importance dif- fers depending on the nature of the infecting organism. Cellular responses are induced when antigen-​presenting cells, such as dendritic cells, present antigens to T cells. T cells do not respond to soluble, unmodified antigens, and only recognize pep- tide antigens in association with major histocompatability com- plex (MHC) molecules. Two major forms of MHC molecules exist. Most nucleated cells produce MHC class I molecules, which stimulate a subset of T cells that produce the CD8 differentiation antigen. These T cells recognize and lyse infected target cells, hence their designation as cytotoxic T lymphocytes. By contrast, MHC class II molecules are produced by cells that participate in the immune response, and are recognized via a subset of T cells 8.3 Immunization David Goldblatt and Mary Ramsay

8.3  Immunization 707 producing the CD4 differentiation antigen. A major role of such T cells is to augment the immune response, and so they are known as T helper cells. Several subsets of T helper cells have been de- scribed: T helper 1 cells are involved in cytotoxic and delayed-​type hypersensitivity responses, T helper 2 cells support antibody pro- duction, follicular helper T cells provide help to B cells enabling them to develop into antibody-​secreting plasma cells, while Th17 cells are important for protection against bacteria and fungi at mucosal surfaces. Immunoglobulin receptors on the surface of B cells are able to recognize soluble antigens, and so initiate the process of B-​cell activation and differentiation. During differentiation, naive B cells become antibody-​secreting plasma cells. In addition, B cells endocytose antigen bound to their surface immunoglobulins, and re-​express it in the form of small peptides on the surface of the B cell in the context of MHC class II molecules. Thus, B cells act as antigen-​presenting cells and recruit T-​cell help. The signals and sol- uble factors that result from such T-​cell help drive the B-​cell process of affinity maturation and memory formation. This takes place in the germinal centres of lymph nodes, where there is intimate contact between B cells, T cells, and dendritic cells. It is here that memory B cells are formed and then migrate to the bone marrow, spleen, and the submucosa of the respiratory tract and gut. On re-​encountering the antigen, memory B cells undergo rapid activation and differenti- ation into plasma cells, and secrete large amounts of switched, high-​ affinity antibody. Thus, the ideal vaccine antigen will lead to the activation, replica- tion, and differentiation of T and B lymphocytes. Ideally the antigen will persist in lymphoid tissue, conformationally intact, to allow the continuing production of cells that secrete high-​affinity antibody, and the generation of memory cells. Vaccine antigens The ideal vaccine antigen is safe, with minimal side effects, pro- motes effective resistance to the disease (although it does not neces- sarily prevent infection), and promotes lifelong immunity. It needs to be stable and remain potent during storage and shipping, and also has to be affordable to allow widespread use. Most currently li- censed vaccines contain live or killed bacterial or viral constituents, bacterial polysaccharides, or bacterial toxoids (Table 8.3.1). Live vaccines are ideal for certain diseases, as replication in the body mimics natural infection, thereby inducing appropriate and site-​specific immunity. Live vaccines must be attenuated to remove the danger of clinical disease, but retain the beneficial effects of inducing immunity. Some live vaccines may be spread from person to person, and thus enhance herd immunity, although such spread may endanger immunocompromised individuals, in whom live vac- cines should be avoided. Live vaccines are inherently less stable than killed vaccines, and the possibility of reversion of vaccine virus to the wild type exists (as in polio). Killed vaccines do not carry the risk Table 8.3.1  Currently licensed vaccines for use in humans Vaccine type Live vaccines Killed/​subunit vaccines Viral Rubella Poliomyelitis (Salk) Measles Influenza Poliomyelitis (Sabin) Rabies (human diploid cell) Yellow fever Hepatitis A Mumps Hepatitis B Varicella zoster (chicken pox) Japanese encephalitis Rotavirus Human papillomavirus Herpes zoster (shingles) Tick-​borne encephalitis Dengue yellow fever vaccine Hepatitis E Bacterial Bacillus Calmette–​Guérin Cholera Typhoid Typhoid Neisseria meningitidis group B Cholera Pertussis Borrelia burgdorferi Anthrax Plague Bacterial polysaccharides Haemophilus influenzae type b Neisseria meningitidis group A, C, Y, W135 Streptococcus pneumoniae Rickettsial Typhus Bacterial toxoid Diphtheria Tetanus Parasitic Malaria

708 SECTION 8  Infectious diseases associated with person-​to-​person spread, and are inherently more stable, but often require two or three doses to induce optimal im- munity, especially when used in the first year of life. New developments in vaccine antigens Developments in molecular biology have begun to revolutionize the field of vaccine science, and provide a glimpse of the future, when the traditional reliance on live attenuated viral vaccines or purified bac- terial or viral products as vaccine antigens may be reduced. The first licensed vaccine to contain recombinant genetic material was the hepatitis B vaccine. Despite the licensing of highly effective plasma-​ derived hepatitis B vaccines in the early 1980s, fears about safety, and their high cost, led to the search for other hepatitis B vaccines. Several vaccine manufacturers used recombinant DNA technology to express hepatitis B surface antigen in other organisms, which led to the development of new vaccines. DNA itself has also attracted interest as a vaccine antigen. The po- tential of this approach was discovered by chance in 1989 during a gene therapy experiment, when it was shown that a gene inserted directly into a mammalian cell could induce the cell to manufacture the protein encoded by that gene. In early experiments, DNA was in- jected directly into muscle, and the resulting immune response was measured. DNA vaccines can induce protective immunity to a variety of pathogens in animals, but data in humans are limited. As DNA has the theoretical potential to be incorporated into the host’s genetic makeup and subvert the genetic working of cells, safety concerns have delayed studies in humans. Phase I studies, however, have as- sessed DNA vaccines designed to protect against hepatitis B, herpes simplex type 1 and 2, HIV, influenza, and malaria. So far clinical trials have proved disappointing, either because the level of the re- sponse was inadequate or because excessive doses of DNA were re- quired to achieve an adequate response. This poor immunogenicity of DNA vaccines remains a major hurdle. Prime-​boost strategies where the immune system is primed with a vector coding for one antigen and then subsequently boosted with a different vector or the antigen itself has been the one area of promise in this field and has been applied to malaria, HIV, and new tuberculosis vaccines. The abundant information now available about the genomic makeup of pathogens has ushered in a new era that has been termed ‘reverse vaccinology’. Using information from the pathogen genome, sequences coding for likely protective antigens have been cloned into expression systems, expressed and screened as vaccine antigens using animal models. A related but alternative technique involves highly representative small-​fragment genomic libraries that are expressed to display frame-​selected epitope-​size peptides on a bacterial cell surface. These are then screened with disease-​relevant high-​titre sera and the candidate antigens recognized are assessed further for their potential as vaccine antigens. This approach has been described for several bacteria including Neisseria meningitidis, Staphylococcus aureus, and epidermidis; Streptococcus pyogenes, aga- lactiae, and pneumoniae; Enterococcus faecalis; Helicobacter pylori; Chlamydia pneumoniae; the enterotoxigenic Escherichia coli; and Campylobacter jejuni. The first vaccine to be developed using this approach (4C-​MenB, Bexsero® GSK Biologicals) was licensed in the European union in 2013. The vaccine is designed to offer broad coverage against inva- sive strains of serogroup B meningococcal infection. This vaccine contains three recombinant proteins (Neisserial adhesin A (NadA), factor H binding protein (FHbp), and Neisserial heparin binding antigen), identified using reverse vaccinology; the fourth compo- nent is from an outer membrane vesicle vaccine that was used suc- cessfully in New Zealand. The vaccine has been used on a wide scale in response to an outbreak in Quebec, and in a university outbreak in the United States. In September 2015, the United Kingdom be- came the first country to incorporate this vaccine into its routine infant programme. A second vaccine against serogroup B menin- gococcal infection (MenB-​FHbp Trumemba, Pfizer) was approved for use by the US Food and Drug Administration (FDA) in October 2014. MenB-​FHbp consists of two recombinant FHbp antigens, one from each subfamily (A and B), and provides another option for protecting older children (aged 10 years or above) from this serious disease. Despite these new technologies, vaccines for some pathogens are proving difficult to develop. Vaccines for HIV remain a major health priority, but phase III clinical trials to date have proved disappointing. The RV144 HIV vaccine trial is the only phase III vaccine trial that has shown a modest protection (31%) against HIV infection. It was conducted in Thailand where more than 16 000 participants re- ceived four priming injections of a recombinant canarypox vector vaccine plus two booster injections of a recombinant glycoprotein 120 subunit vaccine. Despite the relatively modest effect of the vac- cine, the large study size will permit the evaluation of correlates of protection which are vital for the ongoing effort to find a safe and effective vaccine. Improved vaccines for tuberculosis are a priority as the Bacillus Calmette–​Guérin (BCG) vaccine provides imperfect protection. Several recombinant BCG constructs have entered clinical trials and several new subunit vaccines, formulated as adjuvanted or viral vectored vaccines and designed to be used with BCG in a ‘prime-​ boost’ approach are currently in clinical trials. The recent unpre- cedented epidemic of Ebola virus disease epidemic in West Africa in 2014–​2015 acted as a stimulus for the first large scale use of a viral vectored vaccine. A live attenuated recombinant vesicular sto- matitis virus (VSV), where the G-​protein associated with viru- lence was replaced by the Ebola glycoprotein from the Zaire strain (rVSV–​EBOV) was used in phase III clinical trials in both Guinea and Sierra Leone. Ring vaccination of contacts with a single dose of rVSV–​EBOV was shown to provide 100% efficacy when given im- mediately in a cluster, when compared to those clusters when vac- cination was delayed. Phase III trials of a second vaccine (cAd3-​EBO Z), using a replication-​deficient adenovirus chimpanzee serotype 3 (cAd3) vector expressing Ebola glycoprotein from the Zaire strain, were also planned in Liberia, but progress has been slow due to small numbers of cases of Ebola virus disease. To provide the potential for longer-​term protection, the prime-​boost strategy, using two doses of vaccine each delivered on a different viral vector, are now underway. New developments in vaccine delivery Research into different routes of vaccine delivery has been driven by the limitations of the parenteral route. These include the difficulty associated with the use of live viral vaccines in the first 6–​9 months

8.3  Immunization 709 of life (because of the neutralizing effect of passively transferred ma- ternal antibody) and the difficulty and expense of delivering mass immunization by injection. Mucosal delivery of vaccine via the intranasal route has been studied for several antigens, including measles, influenza, rubella, varicella, and Streptococcus pneumoniae. The induction of local immunity for pathogens that either enter the body via the nasopharynx (measles, influenza) or are commonly carried in the nasopharynx (S. pneumoniae) is attractive. Edible vaccines are attracting increasing attention, providing as they do both a means of antigen production and delivery. Studies in animals, and phase I studies in humans, have demonstrated their ­potential. Mice fed with potatoes expressing a non​toxic fragment of the cholera toxin developed mucosal antibodies to the toxin, which reduced diarrhoea on challenge with whole cholera toxin. Humans fed raw potatoes expressing the B subunit of enterotoxi- genic Escherichia coli also showed mucosal immune responses and an increase in neutralizing antibody levels. There are some problems with stability, but edible vaccines are a potentially simple and con- venient method of vaccine delivery on a wide scale. The requirement for increased immunogenicity of existing vac- cines has driven the search for better adjuvants. Until recently the only adjuvants in widespread use have been aluminium salts. New, safe adjuvants with acceptable safety profiles are finally appearing in vaccines and include oil in water emulsions such as ASO3 used in influenza vaccines, and a combination of aluminium hydroxide and monophosphoryl lipid A (ASO4), used for human papilloma virus vaccine and hepatitis B vaccine. Experience with H1N1 influenza vaccines, containing another oil in water adjuvant (MF-​59), have demonstrated good immune response to a novel strain of influenza with only a single dose, thus enabling vaccine to provide protection earlier than expected in the recent pandemic. The aim of immunization programmes Once a vaccine has been developed and shown to be effective it can be used in different ways. Many vaccines are used selectively in groups of the population who are at increased risk of infection (e.g. because of occupation or travel) or of severe consequences of the disease (e.g. because of an underlying medical condition). Other vaccines are employed for mass immunization targeting the whole population. Mass immunization can eradicate, eliminate, or control an infectious disease. Eradication, the state where a disease and its causal agent have been removed from the natural environment, has been achieved only for smallpox. Once eradication has been certi- fied, mass immunization programmes can cease, and resources can be transferred to other programmes. The next target for the WHO is the global eradication of polio- myelitis. Characteristics that favour eradication are the absence of an animal host, the absence of a carrier state, and lifelong protec- tion given by vaccination. The polio eradication campaign has in- volved the use of National Immunization Days (NIDs), on which live attenuated polio vaccine is delivered to a high proportion of the childhood population on a single day. Millions of children have been immunized with trivalent oral polio vaccine (against types 1, 2, and 3) during NIDs. This had led to the successful interrup- tion of poliovirus transmission in many previously endemic coun- tries and no cases of type 2 poliovirus (WPV2) have been detected since 1999. In 2015, WPV2 was declared eradicated, but, as eradica- tion of wild virus nears, concern about the persistent circulation of vaccine-​derived polioviruses (cVDPVs) have become greater. July 2015 marked an important step on the road to a world without polio. No cases of wild poliovirus infection had occurred in Nigeria for a whole year, and infection remains endemic in only two countries—​ Afghanistan and Pakistan. During 2015, however, more countries were affected by cVDPVs than by the wild polio viruses. Therefore, as part of the global endgame, a switch from the use of trivalent oral polio vaccine to the bivalent version, without the type 2 component, is planned from April 2016. For some infections, eradication by immunization is not possible. A good example is tetanus, where the agent is distributed widely in the environment. For these programmes the aim is to control in- fection to the point where it no longer constitutes a public health burden. To maintain control, immunization must be continued indefinitely. For diseases that are transmitted from person to person, a good immunization programme provides protection by conferring both individual and herd immunity. For many vaccines, herd immunity can be achieved by vaccinating a high proportion of the childhood population; older individuals are generally immune as a result of previous natural infection. If such a situation can be sustained, transmission of the infection may be interrupted, and elimination or eradication becomes possible. If vaccine coverage or efficacy is sub- optimal, however, then, in the absence of natural transmission, the number of susceptible people will gradually increase. Eventually, the proportion of susceptible people (those who did not receive vaccine or who failed to respond to it) may reach a level sufficient to support an epidemic. Although the size of these epidemics may be small by prevaccine standards, the average age of those infected will be higher than in the prevaccine era. For infections that have more severe con- sequences in older individuals the morbidity associated with such outbreaks can be substantial. A tragic example of this was observed in Greece, where mass vaccination against rubella in childhood was recommended from 1975. Implementation was poor, however, and during the 1980s coverage was below 50%. The low level of coverage was sufficient to interrupt transmission for several years, but by the time rubella infection recurred in 1993, a high proportion of preg- nant women were susceptible to rubella and an epidemic of con- genital rubella syndrome occurred. Other potential negative consequences of achieving high vaccine coverage, and therefore high herd immunity, have been described. One negative impact may be that pressure is created on an organism to mutate, or that other strains may expand to fill an ecological niche left, for example, by eradicating nasopharyngeal carriage. The introduction of a 7-​valent pneumococcal conjugate vaccine into the routine infant immunization programme of the United States of America, and the associated surveillance for invasive pneumo- coccal disease, has revealed not only the direct impact of the vaccine in reducing disease in vaccinated children, but also a huge indirect effect, which has resulted in the reduction of invasive pneumococcal disease in unvaccinated adults. A similar experience, however, has been followed by an increase in serotypes not covered by the vaccine in several countries. Vaccines with higher valencies (covering 10 or 13 serotypes) are now being employed; long-​term surveillance to monitor whether these too lead to replacement is essential. Another negative impact of the introduction of vaccination may be disruption

710 SECTION 8  Infectious diseases of asymptomatic transmission and reduction in natural boosting of immunity. This can result in protection from vaccine waning earlier than expected, and therefore in resurgences in disease in older indi- viduals. This may partially explain some of the recent outbreaks of mumps described in several countries with high vaccine coverage. Although it is clear that morbidity associated with mumps in vac- cinated individuals is substantially reduced, waning immunity may prevent the long-​term elimination of mumps infection. Delivery of immunization programmes In 1974 the WHO launched the Expanded Programme on Immuni­ zation, in recognition of the major contribution of vaccines to public health. At the start of the programme fewer than 5% of the world’s infants were immunized against the six target diseases: diphtheria, tetanus, whooping cough, polio, measles, and tuberculosis. Between 1990 and 1997, around 80% of the 130 million children born each year were immunized by their first birthday, preventing around 3 million deaths each year. Each year, more than 500 million immun- ization contacts occur with children, and these have provided an op- portunity for the delivery of other primary healthcare interventions. During the 1990s the Expanded Programme on Immunization added immunization against yellow fever and hepatitis B to its target diseases. As a major barrier to using new vaccines is access to sus- tainable funding, the introduction of the new vaccines has been slower, particularly in the poorest countries in greatest need. In 2001, an aid consortium known as the Global Alliance for Vaccines and Immunization has been supporting vaccination programmes in around 75 of the poorest countries. This model has helped to reduce the cost and the risk of producing vaccines for developing coun- tries, by supporting long-​term contracts at high volumes and low unit costs. In 2012, the World Health Assembly endorsed the Global Vaccine Action Plan which aims to prevent millions of deaths by 2020 through more equitable access to existing vaccines for people in all communities. The plan involves four main goals: to strengthen routine immunization to meet vaccination coverage targets; to ac- celerate control of vaccine-​preventable diseases with polio eradica- tion as the first milestone; to introduce new and improved vaccines; and to spur research and development for the next generation of vaccines and technologies. Six strategic objectives of the Global Vaccine Action
Plan 2011–​2020 • All countries commit to immunization as a priority. • Individuals and communities understand the value of vaccines and demand immunization as both their right and responsibility. • The benefits of immunization are equitably extended to all people. • Strong immunization systems are an integral part of a well-​ functioning health system. • Immunization programmes have sustainable access to predictable funding, quality supply, and innovative technologies. • Country, regional, and global research and development innov- ations maximize the benefits of immunization. The plan recognizes that, for mass immunization to achieve its aims, high and uniform coverage of immunization must be reached and sustained. The level of coverage of immunization is associated with a variety of factors, including the sociodemographic characteristics of the population, the organization of health services, knowledge among health professionals, and parental attitudes. Sociodemographic factors that may influence vaccine coverage include deprivation, maternal education, and family size. Centrally coordinated health services with few barriers to access, and standard record systems with facilities for call and recall are likely to achieve higher vaccine coverage. Health professionals with accurate know- ledge of the indications and true contraindications to immunization are important. Excessive lists of contraindications for diphtheria–​ tetanus–​pertussis immunization in the newly independent states of the former Soviet Union contributed to a massive resurgence of diphtheria in the early 1990s. The number of cases rose from 2000 in 1990 to over 47 000 in 1994; 2500 deaths from diphtheria occurred between 1990 and 1995. Whether or not parents decide to have their children vaccinated depends on their perceptions of the severity of the disease and of the safety and effectiveness of the vaccine. Knowledge of parental per- ceptions can be used successfully to target health promotion cam- paigns. When coverage is high, the incidence of vaccine-​preventable disease declines, and parental perception of the severity of that dis- ease may decrease. In this situation, concerns about the safety of the vaccine become paramount and can lead to a decline in vaccine coverage. Such a situation arose in the United Kingdom in the early 1970s, when concern about the safety of pertussis vaccine led to a fall in coverage. This resulted in resurgence of the disease, with con- sequent mortality and morbidity (Fig. 8.3.1). Over the next decade vaccine coverage improved again, and the incidence of the disease fell to the lowest levels ever. In 2003–​2004, concern about the safety of the polio vaccine led to the suspension of the programme in northern Nigeria. This led to an outbreak of polio in west and central Africa and the re- introduction of poliovirus into 22 previously polio-​free countries. By 2005, after massive efforts from the international community, successful campaigns were launched to stop these outbreaks and transmission was contained in all but six of these countries. In 2008, a further polio outbreak occurred in Nigeria, leading to persistent importations into neighbouring countries and re-​ established transmission in Angola, Chad, the eastern part of 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 Notifications 0 80 40 0 – 40 Coverage 1940 1943 1946 1949 1952 1955 1958 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 Notifications Vaccine coverage (%) *Provisional Year Fig. 8.3.1  Whooping cough cases and vaccine coverage in England and Wales between 1940 and 1998.

8.3  Immunization 711 Democratic Republic of Congo, and southern Sudan. This experi- ence illustrates the major global implications of failure to sustain confidence in vaccination. Evaluation of immunization programmes Evaluation of an immunization programme may include the meas- urement of vaccine coverage, surveillance of disease incidence, as- sessment of prevalence of immunity, and the monitoring of adverse events. Vaccine coverage Timely measurement is important for monitoring trends in vaccine coverage and identifying pockets of low coverage. Low coverage may be apparent before any increase in disease incidence is observed. Since the late 1970s, three outbreaks of poliomyelitis have been ob- served among groups in the Netherlands with religious objections to immunization. Despite national coverage of 96% for the measles, mumps, and rubella (MMR) vaccine, the same group has been the focus of a large epidemic of measles. Between April and December 1999, 1750 cases of measles occurred in the Netherlands, compared with only 9 in the whole of 1998. Disease surveillance Once an immunization programme has been implemented, dis- ease incidence data can be used to monitor the effectiveness of the strategy. For example, the dramatic decline in the incidence of invasive Haemophilus influenzae infection described in both the Netherlands and the United Kingdom can be used to demonstrate the impact of conjugate vaccination. The age distribution of infec- tion may change, as children above or below the target age form an increasing proportion of those infected. Various epidemiological methods, including case–​control studies, cohort studies, and the screening method can be used to estimate the efficacy of the vac- cine in the field. The impact of vaccines should be monitored in age groups other than those targeted by vaccination, to determine the effect of herd immunity. Neisseria meningitidis group C (Men C) polysaccharide-​conjugate vaccine was introduced into the United Kingdom in 1999 and by 2002 all under the age of 25 years in the population had been offered the vaccine. The effect of invasive Men C disease reduction was, however, seen in all ages, both those dir- ectly immunized and those protected by reduced transmission of the bacterium from the nasopharynx in vaccinated individuals to the rest of the population (Fig. 8.3.2). Seroprevalence studies Seroprevalence studies are used to assess population immunity to infection. Such immunity results either from immunization or from natural infection. This can detect groups that include a high proportion of susceptible individuals, who may be the focus of future outbreaks. In 1991, seroprevalence studies in the United Kingdom identified that a large proportion of school-​age children was susceptible to measles, and therefore that an epidemic of mea- sles was likely. A large campaign was mounted in November 1994 to immunize children from 5 to 16 years of age. The number of cases of measles fell rapidly and remained at low levels over the next 5 years. Adverse events The monitoring of adverse events is important for maintaining public confidence in an immunization programme and for detecting rare events that could not be identified before licensing the vaccine. The detection of such events may lead to the withdrawal of certain vaccines. In August 1998, a quadrivalent vaccine using reassortant rhesus rotavirus strains was licensed for use in the United States of America and recommended for the mass immunization of infants. During prelicensing studies, five cases of intussusception had been reported in around 10 000 recipients, compared with only 1 in al- most 5000 controls; this difference was not statistically significant. During postlicensure surveillance, however, 15 cases were reported to the Vaccine Adverse Event Reporting System. On 22 October 1999, a review of scientific data concluded that there was an in- creased frequency of intussusception in the 1–​2 weeks after vac- cination, which led to withdrawal of the first licensed vaccine. New rotavirus vaccines that appear to be less likely to produce intussus- ception are now licensed. Adverse events may be linked to the ac- tive antigen or to other contents of the vaccine. Concerns in Finland about an observed excess of cases of narcolepsy occurring after the adjuvanted pandemic influenza vaccine suggest that the novel adju- vant MF-​59 may be a trigger. A live attenuated tetravalent chimeric vaccine designed to prevent Dengue infection (Dengvaxia®) was li- censed in 2016 and has been used for immunization of children in the Phillipines. Its use has been associated with more severe disease in vaccine recipients who have never been exposed to Dengue. The immunization campaign has been suspended while this association and safety data are evaluated further. FURTHER READING Bernier RH, Hinman AR (1988). Assessing vaccine efficacy in the field: further observations. Epidemiol Rev, 10, 212–​41. Centers for Disease Control and Prevention (2005). Direct and indirect effects of routine vaccination of children with 7-​valent pneumococcal 0 100 200 300 400 500 600 700 800 97/98 98/99 99/00 00/01 '01/02 '02/03 '03/04 '04/05 '05/06 '06/07 '07/08 '08/09 09/10 10/11 11/12 12/13 13/14 14/15 No of cases Year 20 + yrs Under 20 Fig. 8.3.2  Cases of invasive disease due to Neisseria meningitidis capsular group C infections England 1998/​1999–​2014/​15 epidemiological years (July to June). Data from Public Health England: https://​www.gov.uk/​government/​uploads/​ system/​uploads/​attachment_​data/​file/​470612/​Table_​8_​Invasive_​meningococcal_​C_​ infections_​lab_​reports_​_​England_​by_​age_​group_​_​_​epi_​year.pdf

712 SECTION 8  Infectious diseases conjugate vaccine on incidence of invasive pneumococcal disease—​ United States, 1998–​2003. MMWR Morb Mortal Wkly Rep, 54, 893–​7. Chen RT (1999). Vaccine risks: real, perceived and unknown. Vaccine, 17, S41–​6. Czerkinsky C, et al. (1999). Mucosal immunity and tolerance: rele- vance to vaccine development. Immunol Rev, 170, 197–​222. Henao-​Restrepo AM, et  al. (2015). Efficacy and effectiveness of an rVSV-​vectored vaccine expressing Ebola surface glycoprotein: in- terim results from the Guinea ring vaccination cluster-​randomised trial. Lancet, 386, 857–​66. Ladhani SN, et al. (2015). The introduction of the meningococcal B (MenB) vaccine (Bexsero(®)) into the national infant immunisation programme—​new challenges for public health. J Infect, 71, 611–​4. Lehtinen M, et  al. (2012). Overall efficacy of HPV-​16/​18 AS04-​ adjuvanted vaccine against grade 3 or greater cervical intraepithelial neoplasia: 4-​year end-​of-​study analysis of the randomised, double-​ blind PATRICIA trial. Lancet Oncol, 13, 89–​99. Leitner WW, Ying H, Restifo NP (1999). DNA and RNA-​based vac- cines: principles, progress and prospects. Vaccine, 18, 765–​77. Pilishvili T, et al. (2010). Active bacterial core surveillance/​emerging infections program network. sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J Infect Dis, 201, 32–​41. Rappuoli R, Black S, Lambert PH (2011). Vaccine discovery and trans- lation of new vaccine technology. Lancet, 378, 360–​8. Rerks-​Ngarm S, et  al. (2009). Vaccination with ALVAC and AIDSVAX to prevent HIV-​1 infection in Thailand. N Engl J Med, 361, 2209–​20. Tacket CO, et al. (1998). Immunogenicity in humans of a recom- binant bacterial antigen delivered in a transgenic potato. Nat Med, 4, 607–​9. Wichmann O, et al. (2017). Live-attenuated tetravalent dengue vaccines: The needs and challenges of post-licensure evaluation of vaccine safety and effectiveness. Vaccine, 35(42), 5535–42.

8.4 Travel and expedition medicine 713

8.4 Travel and expedition medicine 713

ESSENTIALS Tourists, business people, pilgrims, and visitors to friends and relatives are making increasing numbers of trips to tropical and developing parts of the world, where the risk and range of infectious and envir- onmental diseases and injuries may be much higher than in Western countries. The aim of travel and expedition medicine is to reduce risk through education, appropriate immunizations, and other medical advice, hence enhancing the enjoyment and achievements of trav- elling abroad. Explorers, expeditioners, and wilderness travellers face the greatest health challenges, but risk can be minimized by technical competence, careful planning, training in practical medical skills, and rehearsing emergency evacuation. Pretravel advice—​this requires precise information about the mode of travel, geographical itinerary, and the purpose of the visit, and must take into account the age, background health, and immunocom- petence of the traveller. Important provisions are (1) a first-​aid kit, (2) sun-​block, (3) insect repellent, (4) treatments for motion sickness, jet lag and high altitude sickness, (5) supplies of regular medications for chronic medical conditions, and (6) generous, comprehensive travel insurance. Pretravel immunization—​this involves (1) boosting childhood vac- cinations (e.g. tetanus, poliomyelitis, and diphtheria); (2) adding pro- tection against hepatitis A (and B in those at risk of parenteral or sexual exposure) and infections endemic in the areas to be visited (e.g. yellow fever in equatorial Africa and South America, Japanese encephalitis in Southeast Asia, tick-​borne encephalitis in northern Europe and Asia, Neisseria meningitidis in the meningitis belt of Africa, typhoid in South Asia, and rabies in most parts of the world). Pregnancy and immunodeficiency present particular problems of vulnerability to infections and restrict the use of live vaccines. Reducing the risk of infections—​food and water hygiene are crucial for prevention of travellers’ diarrhoea, the most common medical problem likely to be encountered. Avoidance of bites by disease vectors such as mosquitoes and ticks and use of appropriate prophylactic drugs reduces the risk of malaria and many other trop- ical infections. The risks of emerging infectious diseases such as avian influ- enza, Middle East respiratory syndrome coronavirus (MERS-​CoV), and Ebola need to be understood and managed. Recent years have seen the emergence of medical tourism, where people travel to an- other country to undergo surgical or medical treatment, and this has been associated with the acquisition of multidrug resistant bacteria. Underestimated hazards of travel include sexually transmitted infec- tions, psychiatric illness, drowning, and road traffic accidents. Introduction International tourism has grown prodigiously over the last few years. In 2014 international tourist arrivals exceeded 1.1 bil- lion, and this number is predicted to be 1.8 billion by 2030. Approximately 30% were to tropical or subtropical developing countries. United Kingdom citizens make 60 million visits abroad each year, 8% of these to developing countries which carry a higher risk of illness (600-​fold increased risk in Mexico, 1835-​fold in the Indian subcontinent) than travel to European countries such as France. It has been estimated that 50–​75% of short-​term trav- ellers to tropical or subtropical countries become unwell, usually because of an infection. Those travelling outside Europe need to be provided with adequate medical advice to minimize the risks of their journeys, while back at home, admitting physicians should consider a broader range of differential diagnoses, diagnostic tests, and specific treatments. Among the more common infectious dis- ease health risks faced by travellers to developing countries are traveller’s diarrhoea, malaria, dengue fever, acute lower respiratory tract infection, hepatitis A, gonorrhoea, and animal bites with ra- bies risk (Table 8.4.1). Pretravel advice This can be obtained from a variety of sources, but ideally should be sought from medical practitioners and clinics with a special interest in travel medicine. Other sources include the embassies of the countries to be visited, travel agencies, and, increasingly, the internet (see ‘Further reading’). People travel for a variety of reasons:  business travel, pilgrimage, gap-​year and educational travel, and tourism are all increasing. Many members of the immi- grant communities of Western countries travel to visit their friends 8.4 Travel and expedition medicine Susanna Dunachie and Christopher P. Conlon

714 SECTION 8  Infectious diseases and relatives abroad; these travellers are less likely to seek pretravel advice, but may be more vulnerable to endemic diseases in the tropics because of the living conditions at their destination. At the pretravel clinic, the clinician elicits details about the pro- posed journey and the individual traveller’s previous health and requirements. Such discussions allow a proper risk assessment to be made, so that advice and immunizations can be appropriately tailored. Issues that should be considered include general health ad- vice, an assessment of the problems posed by different climates or environments, and the route, type, and duration of travel. Specific advice will include details of the necessary immunizations, and pro- tection against malaria and other relevant diseases. It is important to discuss what might be done if the traveller were to fall ill while abroad or become unwell after their return. Travellers should be encouraged to take out generous and specific travel and health in- surance, including cost of repatriation in case of serious illness or accident. General advice about health First-​aid kit Travellers should carry a basic first-​aid kit that should include anti- septic solution/​wipes; bandages; plasters; proprietary drugs for pain relief, diarrhoea, constipation, dyspepsia, allergy, and itch; sun- screen preparations; water purification tablets; and insect repellents. Motion sickness Antiemetic drugs such as cyclizine or hyoscine are effective, but they may cause sedation and a dry mouth. Long-​acting transdermal skin patches containing scopolamine or antiemetics that can be absorbed through the buccal mucosa are preferable. Air travel and jet lag Long-​haul air flights lead to jet lag: sleep disturbance, fatigue, a feeling of light-​headedness and unreality, and poor concentration. Table 8.4.1  Immunizations Vaccine Type Route Primary course Booster Routine Combined tetanus, polio, diphtheria, pertussis, and Haemophilus influenzae b DTaP/​IPV/​Hib IM/​SC Three doses at monthly intervals 10 years (maximum 5 total doses) Influenza Killed virus IM Single dose Yearly Pneumococcal 23-​valent polysaccharide IM/​SC Single dose Repeat in those at high risk Pneumococcal 13-​valent conjugate polysaccharide IM/​SC Three doses at 2, 4, and 13 months (not licensed for adults) Repeat in those at high risk Measles, Mumps and Rubella (MMR) Live attenuated virus IM Two doses Meningococcal disease Conjugate ACWY IM Single Every 3–​5 years Those previously immunized with polysaccharide vaccines should be boosted with conjugate vaccine Travel Combined diphtheria, tetanus, and polio DTP IM/​SC 10 years Hepatitis Aa Killed virus IM Two doses, 6–​12 months apart Probably not required Hepatitis B Adsorbed IMb Three doses at 0, 1, and 6 months Single booster at 5 years (may not be required) Japanese B encephalitis Killed virus Vero cell vaccine IM/​SC Two doses on days 0, and 28 1–​2 years, later boosting uncertain Rabies Killed virus IMb/​IDb, c 0.1 ml Three doses on days 0, 7, and 28 Rarely indicated in travellers; consider boost at 10 years Tick-​borne encephalitis Killed virus IM Two doses 4 weeks apart, then at 9–​12 months Every 3 years Tuberculosis: BCGd Attenuated ID Single dose None Cholera Inactivated O1 strain plus recombinant B toxin subunit PO Two doses 1 week apart 6 months Typhoid Live Ty21a strain (attenuated) PO Three doses on alternate days Every 3 years Typhoida Capsular Vi polysaccharide IM Single dose Every 3 years Yellow fever Live virus (attenuated) SC Single dose Not needed ID, intradermal; IM, intramuscular; PO, oral; SC, subcutaneous. a Combined hepatitis A and typhoid vaccines are available. b Should not be given into the buttock; deltoid or anterior thigh preferred. Double the dose for immunocompromised patients, or those on dialysis. c Efficacy reduced if given with chloroquine antimalarial prophylaxis. d Evidence of efficacy only in children under 5 years of age.

8.4  Travel and expedition medicine 715 These symptoms may be attributable to a hangover if exces- sive alcohol has been drunk on the flight. A short-​acting seda- tive, such as zopiclone or zolpadem, taken for the first couple of nights after flying, helps to re-​establish a regular sleeping pat- tern. Some ­travellers have found that melatonin is helpful, but obtaining ­products with the active ingredient can be a problem. The ­appropriate timing of exposure to daylight and meals can speed up the adjustment of circadian rhythms. People with dia- betes might need advice on adjusting their insulin regimen or diet for changes in time zones, as might patients taking other regular medications. Modern aircraft ventilation systems have high-​ quality filtration systems to remove microorganisms and deliver much lower rates of recirculated air than air-​conditioned build- ings. However, transmission of air-​borne pathogens including the common cold, influenza, and tuberculosis from immediate neighbours can occur. The risk of deep vein thrombosis from pro- longed immobility and dehydration can be reduced by wearing tailored elastic stockings, moving about as much as possible, and frequently drinking water. Regular medications Patients with chronic illnesses such as diabetes, asthma, or HIV should take plenty of their current medications, as these may not be available abroad, and pack vital supplies in hand luggage. Patients should carry a letter from their physician outlining the condition, itemizing the medications to be carried and providing contact details. Food and water hygiene Strict food and water hygiene are important for travellers to coun- tries with relatively poor sanitation. ‘Boil it, peel it, or forget it’ is a useful adage for the traveller, but is sometimes difficult to imple- ment without causing offence when receiving hospitality. Foods to be avoided are raw or rare meat, fish, and other seafood (but see further on in this chapter), food that has been stored unrefrigerated since it was cooked, ‘street food’ unless freshly boiled or fried, un- pasteurized dairy products, cold sauces and dressings, raw salads and vegetables, and unpeeled fruits or even peeled tomatoes. Water purification tablets and many types of portable water filters are avail- able. Beverages made with boiled water are generally safe, whereas bottled water and, especially, ice cubes are unreliable. Treated water should always be used, even for tooth cleaning and washing fruit. Unfortunately, marine toxins are not destroyed by heat and so high-​ risk seafoods, such as puffer fish, large reef fish, and shellfish gath- ered while there is a ‘red tide’, should be avoided. Viral gastroenteritis (e.g. norovirus) is transmitted by contamin- ated food and water, person-​to person contact, aerosolization, and environmental contamination. Outbreaks frequently occur in con- fined places such as all-​inclusive resorts and on cruise ships. Good hand hygiene and avoiding contaminated food, water, and environ- ments reduce the risk of transmission. Climatic and environmental extremes Sun and heat Travellers should be reminded of the risks of sun exposure, and en- couraged to dress and behave appropriately, to use sunscreen, and to wear a hat or other head protection in the sun. They must keep adequately hydrated and be aware of the risk of heatstroke. Several days of relative inactivity are needed to acclimatize safely to hot climates. Swimming Apart from the risk of drowning (Chapter 10.4.4), or being bitten, stung, or attacked by aquatic animals (Chapter 10.1.2), swimmers and bathers can be exposed to waterborne diseases such as schis- tosomiasis and leptospirosis in fresh water, together with the pos- sibility of ingesting water and contracting gastrointestinal illnesses, even in swimming pools. Generally, swimming in chlorinated water is to be preferred. Schistosomiasis (bilharzia) occurs in Africa (including Madagascar), the Middle East, eastern South America, China, and Southeast Asia. Infection is acquired by both bathing and washing with fresh water in lakes and sluggish rivers. Vector-​borne diseases Travellers should be warned about the risk of diseases transmitted by the bites of mosquitoes (e.g. malaria, dengue fever, chikungunya, Zika virus, Japanese encephalitis) and ticks (e.g. tick-​borne enceph- alitis, Lyme disease, rickettsioses) and advised how to avoid bites. Dengue and chikungunya are transmitted predominantly by Aedes mosquitoes that breed well in urban areas and frequently bite during the day. Both diseases have increased markedly in the past decade and international travel has enhanced their spread. The global distribution of chikungunya has expanded rapidly in recent years, with outbreaks in the Americas and Caribbean since 2013 and in the Pacific since 2014. Smaller outbreaks in South Europe and the southern US states have occurred. A large Zika virus epidemic in South America has been associated with an increased risk of micro- cephaly when women are infected in pregnancy. High altitude At high altitudes, snow blindness and severe sunburn can occur under clear skies, even at very low ambient temperatures. Those going to high altitudes should acclimatize slowly and build up their level of physical activity gradually (see Chapter 10.4.6). They should be aware of the symptoms and signs of altitude sickness. Acetazolamide in an adult dose of 250 mg twice a day, starting 12 h before starting the ascent, is effective prophylaxis for mild mountain sickness, especially if the traveller has to ascend rapidly (e.g. flying from sea level to more than 3000 m). But gradual ascent allowing acclimatization is preferable, and if severe symptoms develop there is no substitute for rapid descent. In the tropics, heat, dehydration, and salt depletion may cause additional problems. Wilderness, game parks, and safaris Careful planning should include provision of navigational aids to avoid getting lost; appropriate clothing, equipment, and vehicle; and adequate food and drink. Wilderness travellers may be exposed to a variety of environmental dangers including unpredictable meteoro- logical extremes, including flash floods and lightning strikes (see Section 10), unfriendly local inhabitants, and attacks by large wild animals. Personal safety and security Travellers should be aware of risks of crime, political instability, and travel safety at their destination. They should keep up to date

716 SECTION 8  Infectious diseases with travel advice from their host country (e.g. https://​www.gov.uk/​ foreign-​travel-​advice) and be aware that their travel insurance may be invalid if they travel to regions against the Foreign Office’s advice. If travelling to regions at increased risk of terrorism or civil unrest, keeping in touch with events at their destination through news out- lets and social media is advisable. Road traffic accidents are a leading cause of death and severe in- jury in travellers abroad, and travellers should be advised to assess the safety of a mode of transport before embarking on a journey. Alcohol and drug use while travelling raise the risk of being a victim of theft or assault and the risk of acquiring sexually transmitted in- fections. Safety standards for activities such as diving and hazardous sports may vary from international standards. Blood-​borne and parenteral infections In many developing countries, blood-​borne pathogens such as hepatitis B and C viruses, HIV, and, in some areas, malaria, trypano- somiasis, and other infections are prevalent. Screening of donated blood may not be rigorous, and needles may be reused, sometimes without adequate sterilization. As a result, travellers going far off the beaten track should consider taking sterile disposable needles, can- nulas, and syringes. A covering letter from a doctor may allay the suspicion of customs officials that they are to be used for drug abuse. Sexually transmitted infections Surveys indicate that 4 to 20% of travellers have casual sex while abroad, and that these acts are unprotected on 50% of occasions. One result is about seven new cases of HIV per 100 000 travellers per year. For United Kingdom residents, the risk of acquiring HIV is 300 times greater while travelling abroad than at home. Between 14 and 25% of cases of gonorrhoea and syphilis diagnosed in Europe are imported. Travellers are more likely than usual to engage in un- protected sexual activity, especially when disinhibited by alcohol or other recreational drugs. Since sexually transmitted infections, including HIV, are highly prevalent in many holiday resorts (not only in prostitutes), good-​quality condoms, often not available when travelling, should be carried and used. Pretravel advice should in- clude a discussion of the risks of unsafe sex. Immunizations Childhood vaccinations The traveller’s record of childhood immunizations should be re- viewed (Chapter 8.3). Many adults will require booster doses for tetanus, polio, and diphtheria and may not have been adequately immunized against measles or mumps. Over the past few years, outbreaks of mumps and measles have occurred in many coun- tries. Pertussis occurs globally and resurgences of disease in popu- lations have been reported in countries with good vaccine coverage, demonstrating lack of lifelong protection from a primary course. Previous travel immunizations should be noted, so that they are neither repeated unnecessarily, nor allowed to lapse. Hepatitis A The incidence of hepatitis A in developing countries ranges from 6 to 30 cases per 100 000 unprotected travellers per month of stay. Active immunization is safe, effective, and durable. Those who have received a full course of immunization will probably not need any further boosting doses. Hepatitis B This is a risk to medical or laboratory staff whose work involves contact with human blood and to those staying for prolonged periods, such that there is a possibility of receiving unsafe injec- tions or unscreened blood transfusions. Hepatitis B is also a risk of unprotected sexual activity. Vaccination in these circumstances is sensible. Yellow fever This is the only vaccination for which an internationally valid cer- tificate is statutorily required for entry into countries where the dis- ease is endemic, and for travellers from those places. Yellow fever remains endemic in tropical Africa and South America, but not in Asia (see Fig. 8.4.1). There have been reports of adverse events asso- ciated with yellow fever vaccine, particularly in older people. Such reactions may be more common in those with thymic dysfunction or with other types of immune defect. There have also been inter- national shortages of the vaccine. Studies support immunity of at least 35 years being conferred by a single dose of yellow fever vaccine, and in 2014 the World Health Organization (WHO) adopted the recommendation to remove the requirement for a 10-​year booster from the International Health Regulations, but up to date certificate requirements for individual countries should be checked from a reliable source such as Public Health England or the Centers for Disease Control and Prevention. Cholera Vaccination is no longer required by international regulations. Earlier vaccines were of little use, and although there is now a li- censed oral vaccine, it is really only necessary for those, like aid workers in refugee camps, who have a high risk of exposure. Typhoid This potentially serious infection remains prevalent in Pakistan, India, Bangladesh, Indonesia, and Nepal, where the incidence of infection is approximately 1 in 3000 per month of stay. Those staying for long periods in rural areas, and especially those visiting friends and relatives abroad, are at greatest need of vaccination. Meningococcal disease In the meningitis belt of sub-​Saharan Africa, from Senegal to Sudan, and in some other areas, cool, dry-​season meningococcal meningitis outbreaks are so predictable that immunization is re- commended for travellers living or working closely with local people. The quadrivalent meningococcal vaccine (covering sero- groups A, C, W135, and Y) is recommended. The ACWY conju- gate meningococcal vaccine has proved safe and immunogenic in all age groups, including infants. The quantity and quality of the antibody response is superior to that produced by polysaccharide vaccines and their use reduces meningococcal carriage, reducing transmission. Following outbreaks associated with the Hajj over the past few years, pilgrims to Mecca are required to be immunized and provide proof.

8.4  Travel and expedition medicine 717 Rabies Pre-​exposure rabies vaccination is being used increasingly (see Chapter 8.5.10). Although the risk of transmission is fairly low, the lack of effective treatment for rabies encephalitis, and the fear en- gendered by bites by dogs, and in many parts of the world by bat bites, justifies considering immunization. Prophylactic vaccination does not remove the need for postexposure treatment if a poten- tially rabid bite is received, but it simplifies the treatment and usually avoids needing postexposure rabies immune globulin which is un- available in many endemic tourist destinations. Fig. 8.4.1  Yellow fever vaccination recommendations for (a) Africa and (b) South America from http://​www.nathnac. org/​pro/​factsheets/​yellow.htm and WHO (2011) http://​www.who.int/​ith/​en/​ WHO (2011) http://www.who.int/ith/en/

718 SECTION 8  Infectious diseases Other encephalitides Vaccination against Japanese encephalitis and tick-​borne encephal- itis may be considered after reviewing the travel itinerary and risk of exposure (Table 8.4.1). A new Vero cell-​derived vaccine against Japanese encephalitis (IXIARO) was licensed in 2009. Prevention of malaria Both travellers and non​specialist physicians must be educated about the prevention and recognition of malaria (see Chapter 8.8.2). It is important to be aware of the need to prevent mosquito bites by all possible means: wearing appropriate clothing, application of insect repellents to exposed skin and clothing, and the use of insecticide-​ impregnated bed nets and insecticide sprays or vaporizers in the sleeping quarters. UK residents who were born in malaria-​endemic regions and return to visit friends and family at particular risk be- cause they may not appreciate that any naturally acquired immunity has waned. Guidelines for antimalarial chemoprophylaxis are regularly up- dated (see ‘Further reading’, Websites) and for travel to areas where the risk of malaria, although finite, is low, standby antimalarial treat- ment rather than prophylaxis is increasingly recommended. In areas of low incidence (fewer than 10 cases of malaria per 1000 of the local population per year) such as Central America and Southeast Asia, the risk of taking antimalarial drugs outweighs the risk of infection and so reliance is placed on antimosquito measures and carrying a course of standby emergency treatment if the traveller develops symptoms suggestive of malaria while out of reach of medical care. Travellers should be educated about the risks of fake and ineffective drugs for malaria for sale in some endemic regions. Prevention and management of travellers’ diarrhoea Diarrhoea is the most common health problem of travellers. Symptoms are usually mild, lasting only about 3–​5  days, but holiday and business plans may be disrupted (Box 8.4.1). The most common cause is enterotoxigenic Escherichia coli (ETEC). Salmonella spp., Campylobacter spp., Shigella spp., and other patho- genic E. coli are also common. Protozoan pathogens, such as Giardia intestinalis, Entamoeba histolytica, Cryptosporidium parvum, and Cyclospora cayetanensis are less common causes. Viruses, particu- larly norovirus and rotavirus, are increasingly recognized causes of travellers’ diarrhoea in adults. Fish and shellfish poisoning cause similar symptoms to each other, starting within minutes or hours of exposure. Strict food and water hygiene reduce the risk of gastroenteritis. Heating water to 100 °C will kill most pathogens, as will chemical treatment with chlorine or iodine (iodine is contraindicated in pregnant women and some patients with thyroid disease). Water filters are also effective. Antimicrobials such as fluoroquinolones, azithromycin (for fluoroquinolone-​resistant campylobacter in South and South-​East Asia) or rifaximin, a poorly absorbed rifamycin de- rivative provide some protection, but are not cheap, may cause side effects, cannot be taken for prolonged periods, and may encourage antimicrobial resistance. Colloidal bismuth salts are cheaper, safer, and reasonably effective, but the large volumes are inconvenient. An experimental transcutaneous heat-​labile enterotoxin vaccine reduced the frequency and severity of travellers’ diarrhoea but oral killed Vibrio cholerae vaccine had little effect despite inducing crossimmunity to ETEC. Treatment involves maintaining an adequate fluid intake and using sachets of oral rehydration salts that can be made up with boiled water. Eating solid food may stimulate bowel action by the gastrocolic reflex. Antidiarrhoeal agents such as codeine phosphate and loperamide often relieve symptoms sufficiently to allow normal activities to continue. Short courses of empirical antimicrobials (e.g. ciprofloxacin, 500 mg for 3 days, adults only), azithromycin, or rifaximin can be useful, particularly for patients with underlying diseases. Combination of an antimicrobial, such as rifaximin, with loperamide has proved more effective symptomatically. Localized abdominal pain, high fever, and bloody diarrhoea are indications for seeking medical help immediately. Special groups of travellers Immunocompromised travellers Except for asplenic patients, immunocompromised travellers—​ including those who have recently received chemotherapy or radiotherapy—​should not be given live vaccines such as yellow fever, oral polio, and oral typhoid. Killed or synthetic vaccines are safe. Those patients with mild to moderate immune suppression will probably make a reasonable response to immunization; those with more severe immunosuppression may still make a useful, though less durable, response. Influenza, pneumococcal, and Haemophilus influenzae b conjugate vaccines are recommended, as these patients’ Box 8.4.1  Some causes of travellers’ diarrhoea Bacteria Enterotoxigenic and enteroaggregative Escherichia coli (c.15–​80%) Aeromonas spp., Plesiomonas spp. Campylobacter jejuni Salmonella typhi Other Salmonella spp. Shigella spp. Vibrio parahaemolyticus Clostridium difficile Protozoa Cryptosporidium parvum Cyclospora cayetanensis Entamoeba histolytica Giardia intestinalis Plasmodium falciparum Other Rotavirus/​norovirus Schistosoma mansoni Strongyloides stercoralis Irritable and inflammatory bowel disease Tropical sprue Food allergy Drug side effects, Fish/​shellfish toxins

8.4  Travel and expedition medicine 719 risk of respiratory infection and bacteraemia is increased. Studies show that immunosuppressed patients can make a response to hepa- titis A immunizations, although the durability of this response is again uncertain. People with HIV will often make a good response if they are on antiretroviral medication and have made a good CD4 count. Asplenic individuals should be on prophylactic antibiotics, such as amoxicillin, particularly if travelling, and should be dis- suaded from travelling to areas with high rates of malaria transmis- sion, as they are more likely to get severe disease if infected. Immunocompromised patients should carry antimicrobials with them for treating respiratory or gastrointestinal infections, should seek medical help when abroad, and should carry a letter from their physician outlining their condition and medication. Pregnant travellers Commercial airlines will not normally convey a woman who is 36 weeks or more pregnant, without a covering letter from her midwife or physician. Insurance to cover the cost of delivery abroad should be considered. If possible, pregnant women should avoid travelling to areas where diseases are prevalent that pose a special risk in their condition, such as malaria and hepatitis E. The risk–​benefit assessment of immunizations and chemo- prophylaxis is of particular importance for the pregnant woman and the fetus. Live vaccines should be avoided, but if there is a genuine risk of yellow fever the vaccine should be given, as there is no rec- ognized associated teratogenicity. Inactivated polio vaccine may be given parenterally, and tetanus immunization is safe. The old heat-​ killed whole cell vaccine should be avoided but the modern polysac- charide capsular Vi vaccine is safe. Pneumococcal, meningococcal, and hepatitis B vaccines are safe in pregnancy, as is γ-globulin. Malaria is especially dangerous in pregnant women. Chloroquine and proguanil are safe prophylactic drugs, and quinine in normal therapeutic doses is safe for treatment. Artemisinin derivatives are not known to be safe in the first trimester. Atovaquone-​proguanil and doxycycline are not recommended in pregnancy. A study of mefloquine in pregnancy (predominantly first trimester) showed no difference in birth defects compared to controls, but many would still advise caution in the first trimester of pregnancy. Pregnant women should take special care with food and drink when abroad, as dehydration may threaten the fetus. There are concerns about congenital goitre when pregnant women use iodine to purify water; the maximum recommended daily intake is 175 μg. Loperamide as an antidiarrhoeal agent is safe, but antimicrobials such as tetracyc- lines and quinolones should be avoided. Extremes of age Young children should have completed their routine immuniza- tions before travelling if possible. Malaria chemoprophylaxis is re- commended for all ages. Yellow fever vaccine should be given only to children older than 9 months, as a few cases of vaccine-​associated encephalitis have occurred in younger children. Most other vaccines, including rabies, are safe. Hepatitis A is rarely symptomatic in children under 5 years old. Families planning to live in developing countries should consider Bacille Calmette–​Guérin (BCG) vaccination for chil- dren under 5 years of age to reduce the risk of tuberculous meningitis. Older people should have the same immunizations as younger adults, and should take antimalarial drugs. They are more prone to respiratory infections, and should therefore be given influenza, pneumococcal, and Haemophilus influenzae vaccines. Jet lag and changes in time zones may be very disturbing. Older people are more likely to have an underlying medical condition requiring medication. It is important that sufficient supplies of medicines are taken abroad and that the patient has a detailed list of these medi- cines and their dosages in case the tablets are lost or stolen. They should carry the name and contact address of their home physician, in case of emergency. Hajj pilgrimage Every year more than two million Muslim people from almost 200 countries undertake pilgrimage to Mecca, Saudi Arabia, making it the world’s largest mass gathering. Some travellers will be eld- erly and/​or immunocompromised. Travellers must show a valid International Certificate of Vaccination with the quadrivalent meningococcal vaccine. Children under 15 years of age must also submit proof of polio vaccination. Up-​to-​date requirements are outlined on the webpage of the Saudi Arabian embassy:  http://​ wwwnc.cdc.gov/​travel/​yellowbook/​2016/​select-​destinations/​ saudi-​arabia-​hajj-​pilgrimage. The most common cause of illness in Hajj pilgrims is pneu- monia, and vaccination against influenza is advised alongside pneumococcal vaccine for at-​risk groups. Middle East respiratory syndrome coronavirus (MERS-​CoV) was first identified in Saudi Arabia in 2012, with a possible role of camels in disease transmis- sion. Travellers should be advised to avoid contact with camel prod- ucts and to practise good hand and respiratory hygiene to reduce the risk of respiratory illnesses. Other risks for Hajj pilgrims include heat exhaustion and the risk of crush injury in stampedes due to overcrowding. Explorers and expeditions Because of their adventurous aims, expeditions are likely to involve exposure to greater environmental extremes and hazards than or- dinary travel. Expeditions usually take place in areas remote from even rural health centres, and so a greater responsibility for dealing with medical problems will devolve to the expedition members. The explorer’s greatest fear may be to fall victim to a lethal tropical dis- ease or an attack by a wild animal, but the reality is much more mun- dane:  road traffic accidents, mountaineering disasters, drowning, and attacks by humans claim the most lives. The prevention and treatment of medical problems must be planned well in advance. Detailed advice and information can be obtained from several organizations, such as the Expedition Advisory Centre (Geography Outdoors) of the Royal Geographical Society in London, from clubs specializing in mountaineering, cave exploring, diving, and other activities, and from books, journals, and websites. All expeditions should have a designated medical officer, and all their members should receive first-​aid training aimed at the par- ticular needs of the expedition. The basics are clearing the airway, controlling bleeding, treating shock, relieving pain, and moving the injured person without causing further damage. Expedition medical kits should be more comprehensive than those carried by ordinary tourists and travellers. Lists of essential drugs are given in Johnson et al. (2008) and an adequate water supply must be assured or taken

720 SECTION 8  Infectious diseases if the expedition is into desert areas. The need for further equipment to facilitate rescue such as a collapsible stretcher, and communica- tion systems such as satellite phones must be evaluated. A covering letter on official notepaper, signed by a doctor, may be helpful in allowing drugs, even apparently innocuous ones such as codeine, through customs and explaining the need for needles and syringes. The medical facilities nearest to the site of the exped- ition must be identified and contacted in advance. An emergency plan must be drawn up for the first-​aid treatment and evacuation of severely ill or injured expedition members. In some areas, flying doctor and air evacuation services (such as the African Medical and Research Foundation (AMREF) in East Africa) are available. Medical insurance must be generous and comprehensive, and in- clude repatriation of the injured. Before leaving their home country, expedition members should have a thorough dental check and treat- ment for any outstanding medical or surgical problems. Control of chronic medical problems such as diabetes mellitus, hypertension, and asthma should be stabilized. In selecting members for an exped- ition, the most important attributes are experience, possession of the necessary technical skills (e.g. diving and mountaineering), physical fitness, and proven psychological stability under stress. It is advis- able always to appoint a reliable local agent in the country where the expedition will take place, and a home agent in their country of origin to facilitate communication if problems arise. Illness in returning travellers Details are needed about the countries visited, the activities under- taken while travelling, immunizations, and antimalarials taken. Common problems are fever, rash, diarrhoea, and eosinophilia (Tables 8.4.2 and 8.4.3, and Box 8.4.2). The most important diagnosis to exclude in a traveller from the tropics with a fever is malaria. In travellers with acute diarrhoea, a dietary history, assessment of hydration state, stool microscopy and culture, abdominal films, and sigmoidoscopy may be needed. There are many possible causes (see Box 8.4.1). Patients with chronic diar- rhoea may be infected with Giardia spp., Cryptosporidium spp., Entamoeba histolytica, shigellae, or salmonellae. Investigations should include a search for Clostridium difficile, especially if the Table 8.4.2  Causes of fever in returned travellers Tropical infections Other infections Non​infective causes Short incubation; <3 weeks Endocarditis Connective tissue disease African trypanosomiasis Pneumonia Drug reaction Brucellosis Prostatitis Factitious Chikungunya fever Dengue fever Sexually transmitted infection Inflammatory bowel disease Haemorrhagic fevers (e.g. Ebola, Lassa) Sinusitis Malignancy Hepatitis A Urinary tract infection Malaria Influenza Relapsing fevers EBV /​ CMV Tick/​scrub typhus Typhoid Leptospirosis Malaria Middle East respiratory syndrome
coronavirus (MERS-​CoV) Melioidosis Long incubation; >3 weeks Amoebic abscess Brucellosis Coccidioidomycosis Filariasis Hepatitis A, B, or C HIV Leishmaniasis Malaria Melioidosis Schistosomiasis (Katayama fever) Tuberculosis Typhoid

8.4  Travel and expedition medicine 721 patient took antimicrobials while abroad. A  minority of patients may develop postinfective enteropathy, the most common problem being secondary lactose intolerance. Rarely, bacterial overgrowth or tropical sprue develops. The most common causes of eosinophilia are allergy, drug reac- tions, and helminths (Box 8.4.2). Emerging infectious diseases in returning travellers The 21st century has seen the emergence of new and underrecognized diseases including severe acute respiratory syndrome (SARS), MERS-​CoV, Zika virus, and avian influenza. Melioidosis, a Gram-​ negative sepsis with high case fatality caused by Burkholderia pseudomallei, has presented in travellers returning from a wide dis- tribution of tropical countries alongside hyperendemic regions in Southeast Asia and Northern Australia. The outbreak of Ebola virus in West Africa since 2014 has under- scored the need for physicians to consider viral haemorrhagic fever specifically in travellers returning with a fever from affected areas. Being able to reassure the public and healthcare workers where no significant risk of viral haemorrhagic fever is present is important. Up-​to-​date information on risk assessment and maps can be found on the Public Health England website:  https://​www.gov.uk/​gov- ernment/​collections/​viral-​haemorrhagic-​fevers-​epidemiology-​ characteristics-​diagnosis-​and-​management. FURTHER READING Auerbach PS (ed) (2011). Wilderness medicine, 6th edition. Mosby Elsevier, Philadelphia, PA. Barwick R (2004). History of thymoma and yellow fever vaccination. Lancet, 364, 936. Chen LH, et al. (2011). Vaccination of travelers: how far have we come and where are we going? Expert Rev Vaccines, 10, 1609–​20. Chiodini PL, et al. (2015). Guidelines for malaria prevention in travel- lers from the United Kingdom. Public Health England, London. Conlon CP (2001). The immunocompromised traveler. In: DuPont HL, Steffen R (eds) Textbook of travel medicine and health, 2nd edition. BC Becker, London. Dawood R (2012). Travellers’ health: how to stay healthy abroad, 5th edition. Oxford University Press, Oxford. Freedman DO, Chen LH, Kozarsky PE (2016). Medical considerations before international travel. N Engl J Med, 375, 247–​60. Hill DR, Ford L, Lalloo DG (2006). Oral cholera vaccines: use in clin- ical practice. Lancet Infect Dis, 6, 361–​72. Imray CHE, et al. (2015). Extreme expedition and wilderness medi- cine. Lancet, 386, 2520–​5. Johnson C, et al. (eds) (2008). Oxford handbook of expedition and wil- derness medicine. Oxford University Press, Oxford. Johnston V (2009). Fever in returned travellers presenting in the United Kingdom:  recommendations for investigation and initial management. J Infect. 59, 1–​18. Khatami A, Pollard AJ (2010). The epidemiology of meningo- coccal disease and the impact of vaccines. Expert Rev Vaccines, 9, 285–​98. Paredes-​Paredes M, et al. (2011). Advances in the treatment of trav- elers’ diarrhea. Curr Gastroenterol Rep, 13, 402–​7. Ross AGP, et al. (2013). Enteropathogens and chronic illness in re- turning travelers. N Engl J Med, 368, 1817–​25. Sacks R (2010). Jet lag. N Engl J Med, 362, 440–​2. West JB, et al. (2012). High altitude medicine and physiology, 5th edi- tion. Arnold, London. Wilderness & Environmental Medicine (formerly Journal of Wilderness Medicine) (1990–​). Published for the Wilderness Medical Society by Elsevier, London. Wills K (2013). Outdoor first aid: a practical manual: essential know- ledge for outdoor enthusiasts. Pesda Press, Bangor, UK World Health Organization (WHO) (2013). Vaccines and vaccina- tion against yellow fever. WHO position paper—​June 2013. Weekly Epidemiological Record, 88, 269–​84. Wu D, Guo C-​Y (2013). Epidemiology and prevention of hepatitis A in travelers. Journal of Travel Medicine, 20, 394–​99. Websites General travel advice Centers for Disease Control and Prevention. Travelers’ Health. http://​ wwwn.cdc.gov/​travel/​ National Travel Health Network and Centre (NaTHNaC). Protecting the Health of British Travellers. http://​www.nathnac.org National Travel Health Network and Centre. The Yellow Book. http://​ www.nathnac.org/​yellow_​book/​01.htm Public Health England. https://​www.gov.uk/​topic/​health-​protection/​ infectious-​diseases Royal Geographical Society. Expedition Advisory Centre. https://​www. rgs.org/​in-​the-​field/​advice-​training/​ The International Society of Travel Medicine. http://​www.istm.org Table 8.4.3  Causes of rash in returning travellers Infective Non​infective Cutaneous larva migrans; myiasis Contact allergy Cutaneous leishmaniasis Drug reaction Dengue fever Erythema multiforme Dermatophytes Insect bites Primary HIV infection Sunburn Lyme disease Meningococcal disease Mycobacteria Scabies/​lice Sexually transmitted infections Tick/​scrub typhus Tinea versicolor Typhoid/​paratyphoid Box 8.4.2  Infective causes of eosinophilia in travellers Angiostrongylus (Parastrongylis) spp. Ascaris spp. Echinococcus spp. Filariasis (onchocerciasis) Gnathostoma spp. Hookworm and other gut nematodes Pulmonary eosinophilia Schistosomiasis Strongyloides spp. Trichinosis Trichuris spp. Visceral larva migrans

722 SECTION 8  Infectious diseases World Health Organization. International Travel and Health. http://​ www.who.int/​ith/​ Malaria Centers for Disease Control and Prevention. Malaria. http://​www.cdc. gov/​malaria/​ Public Health England. Malaria prevention guidelines for travellers from the UK. https://​www.gov.uk/​government/​publications/​malaria-​prevention-​
guidelines-​for-​travellers-​from-​the-​uk https://​www.gov.uk/​government/​collections/​malaria-​guidance-​
data-​and-​analysis World Health Organization (2015). Guidelines for the treatment of malaria, 3rd edition. http://​www.who.int/​malaria/​publications/​ atoz/​9789241549127/​en/

8.5 Viruses 723

8.5 Viruses 723

8.5.1 Respiratory tract viruses 723

8.5.1 Respiratory tract viruses 723

8.5 Viruses CONTENTS 8.5.1 Respiratory tract viruses  723 Malik Peiris 8.5.2 Herpesviruses (excluding Epstein–​Barr virus)  734 J.G.P. Sissons 8.5.3 Epstein–​Barr virus  754 Alan B. Rickinson and M.A. Epstein 8.5.4 Poxviruses  764 Geoffrey L. Smith 8.5.5 Mumps: Epidemic parotitis  769 B.K. Rima 8.5.6 Measles  772 Hilton C. Whittle and Peter Aaby 8.5.7 Nipah and Hendra virus encephalitides  784 C.T. Tan 8.5.8 Enterovirus infections  787 Philip Minor and Ulrich Desselberger 8.5.9 Virus infections causing diarrhoea and vomiting  797 Philip R. Dormitzer and Ulrich Desselberger 8.5.10 Rhabdoviruses: Rabies and rabies-​related
lyssaviruses  805 Mary J. Warrell and David A. Warrell 8.5.11 Colorado tick fever and other arthropod-​borne reoviruses  819 Mary J. Warrell and David A. Warrell 8.5.12 Alphaviruses  821 Ann M. Powers, E.E. Ooi, L.R. Petersen, and D.J. Gubler 8.5.13 Rubella  827 Pat Tookey and J.M. Best 8.5.14 Flaviviruses excluding dengue  830 Shannan Lee Rossi and Nikos Vasilakis 8.5.15 Dengue  845 Bridget Wills and Yee-Sin Leo 8.5.16 Bunyaviridae  852 James W. Le Duc and D.A. Bente 8.5.17 Arenaviruses  862 Jan H. ter Meulen 8.5.18 Filoviruses  870 Jan H. ter Meulen 8.5.19 Papillomaviruses and polyomaviruses  877 Raphael P. Viscidi, Chen Sabrina Tan, and Carole Fakhry 8.5.20 Parvovirus B19  886 Kevin E. Brown 8.5.21 Hepatitis viruses (excluding hepatitis C virus)  889 Matthew Cramp, Ashwin Dhanda, and Nikolai V. Naoumov 8.5.22 Hepatitis C virus  896 Paul Klenerman, Katie J.M. Jeffery, Ellie J. Barnes, and Jane Collier 8.5.23 HIV/​AIDS  901 Sarah Fidler, Timothy E.A. Peto, Philip Goulder, and
Christopher P. Conlon 8.5.24 HIV in low-​ and middle-​income countries  933 Alison D. Grant and Kevin M. De Cock 8.5.25 HTLV-​1, HTLV-​2, and associated diseases  941 Kristien Verdonck and Eduardo Gotuzzo 8.5.26 Viruses and cancer  945 Robin A. Weiss 8.5.27 Orf and Milker’s nodule  947 Emma Aarons and David A. Warrell 8.5.28 Molluscum contagiosum  949 David A. Warrell and Christopher P. Conlon 8.5.29 Newly discovered viruses  951 Susannah J.A. Froude and Harriet C. Hughes 8.5.1  Respiratory tract viruses Malik Peiris ESSENTIALS Viral respiratory infections, including rhinovirus, coronavirus, adeno- virus, respiratory syncytial virus, human metapneumovirus, para- influenza viruses, and influenza viruses, are a substantial cause of morbidity worldwide. Transmission occurs through direct contact, contaminated fomites, and large airborne droplets, with long-​range

724 section 8  Infectious diseases transmission by small particle aerosols reported in at least some in- stances of influenza and severe acute respiratory syndrome. Clinical syndromes affect the upper and/​or lower respiratory tract, including coryza, pharyngitis, croup, bronchiolitis, and pneumonia. Each syndrome can potentially be caused by several viruses, and each respiratory virus can be associated with different clinical syn- dromes. Measles is a major cause of lower respiratory tract infections and fatality in tropical countries. Diagnosis—​nasopharyngeal aspirates, washes, and swabs are su- perior to throat and nose swabs for diagnosis, with virus detected by culture or detection of antigen or nucleic acid (e.g. polymerase chain reaction-​based methods). Sputum is a useful specimen for viruses predominantly affecting the lower respiratory tract (e.g. Middle East respiratory syndrome). New respiratory viruses con- tinue to be discovered, but some acute respiratory infections have no identifiable aetiology, and some patients have multiple respira- tory viruses detectable in the respiratory tract in association with their disease—​whether these have a synergistic role in pathogenesis remains unclear. Particular respiratory tract viruses Influenza—​types A and B are clinically important causes of human disease; the viral envelope contains two glycoproteins, haemagglu- tinin (H) and neuraminidase (N), which are critical in host immunity and used to designate viral subtype (e.g. H1N1). Potential to cause pandemics makes influenza type A an unique challenge for global public health. Typically causes an illness associated with fever, chills, headache, sore throat, coryza, non​productive cough, myalgia, and sometimes prostration. It can cause pneumonia directly or by sec- ondary bacterial infections. Oseltamivir and zanamivir result in a re- duction of 1–​2 days in the time to alleviation of symptoms when administered within the first 48 h of illness, but even later com- mencement of therapy might still confer clinical benefit in severe influenza illness. Disease can be prevented by influenza vaccine, which contains antigens from the two subtypes of human influenza A (H3N2 and H1N1) and the Victoria and Yamagata lineages of influ- enza B viruses, but the composition of the vaccine must be updated on an annual basis to keep abreast of change in the surface antigens of the virus, and annual reimmunization is required. Synergic inter- action with Streptococcus pneumoniae enhances pathogenesis, and pneumococcal conjugate vaccine reduces hospitalization associated with respiratory viruses. Respiratory syncytial virus—​a major cause of bronchiolitis and pneumonia in infants. Infection in adults is often mild, but during the respiratory syncytial virus season (winter months in temperate regions) it is an important cause of lower respiratory tract disease in older people. It may be lethal (as can other respiratory viruses) in patients immunocompromised following organ or blood and marrow transplants (but is not a significant problem in patients with AIDS). Severe acute respiratory syndrome—​this novel coronavirus of ani- mals adapted to efficient human transmission and spread world- wide, causing a global outbreak in 2003 of an illness characterized by lower respiratory tract manifestations, severe respiratory failure, and death in about 10% of cases. Public health interventions interrupted viral transmission and it is no longer transmitting within humans, but the precursor virus remains in the animal reservoir (bats, Rhinolophus spp.) and could readapt to cause human disease in the future. Middle East respiratory syndrome coronavirus is endemic in drom- edary camels and causes zoonotic disease in the Arabian peninsula and the Middle East, sometimes leading to outbreaks associated with transmission between humans, especially within healthcare facilities. Travel-​associated cases have been reported in other countries. While the virus can infect all ages, severe clinical disease predominantly manifests in older people or in those with comorbidities. Introduction Viral respiratory infections are amongst the most common afflic- tions of humankind. They are the most frequent reasons for med- ical consultations, are believed to account for 30% of work absences and school absenteeism, and are a major reason for antibiotic pre- scriptions. Longitudinal family studies suggest that a person has, on average, 2.4 respiratory viral infections per year, a quarter of them leading to a medical consultation. The synergistic interactions between viruses and bacteria in pathogenesis are being increas- ingly recognized, for example that between influenza virus and Streptococcus pneumoniae or Staphylococcus aureus. With the excep- tion of influenza in elderly people, these viral infections are not a major cause of mortality in otherwise healthy people in the devel- oped world, but it is estimated that they contribute to over 1 million deaths annually in the developing world. The term ‘respiratory virus’ for the purpose of this discussion will include those that have the respiratory tract as their primary site of clinically relevant pathology. Taxonomically, they belong to six virus families (Table 8.5.1.1) and are global in distribution. Other viruses cause systemic disease with respiratory tract involvement as part of an overall disseminated disease process in patients who are im- munocompetent (e.g. measles, Hantavirus pulmonary syndrome) or immunocompromised (e.g. cytomegalovirus). These are dealt with elsewhere. A respiratory virus may cause a range of clinical syndromes. Conversely, a respiratory syndrome may be caused by more than one virus. The major viral respiratory syndromes and their common aetiological agents are shown in Table 8.5.1.2. Although season- ality may differ, the patterns of disease seen in tropical countries are similar; but a notable difference is the role of measles as a major cause of lower respiratory tract infections and fatality in the tropics. The anatomical demarcation between upper and lower respira- tory tract infections is the larynx. Influenza, respiratory syncytial virus, parainfluenza virus and adenoviruses are well-​recognized causes of lower respiratory tract infection in adults as well as in children, although many other respiratory viruses might do so occasionally. Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and zoonotic avian influenza (e.g. H5N1, H5N6, H7N9) are unusual in that lower respiratory manifestations predominate over the involvement of the upper respiratory tract. With newer molecular-​based approaches to pathogen discovery, new respiratory viruses continue to be recognized. Some recently recognized viruses have been long endemic in humans (e.g. human metapneumovirus, coronaviruses NL-​63 and HKU1, bocavirus) while others are novel pathogens, newly emergent as causes of human infections such as SARS, MERS, and avian influenza.

8.5.1  Respiratory tract viruses 725 Transmission The routes of respiratory virus transmission are through direct contact, contaminated fomites, and large airborne droplets (mean diameter >5 µm, range of transmission <1 m). There remains con- troversy over the potential for the spread of viruses such as influ- enza over longer distances by small particle aerosol (mean diameter <5 µm), but even here, large droplets, direct contact, and fomites are Table 8.5.1.1  Respiratory tract viruses: summary of classification, incubation period, duration of infectivity, and diagnostic options Virus Classification (virus family) and composition of virus Subgroups, serotypes, and subtypes Incubation period (days) Duration of virus shedding in immunocompetent patients (days) Options for laboratory diagnosisa Rhinovirus Picornaviridae Non​enveloped RNA viruses

150 serotypes phylo-​genetically divided into three groups: A, B, and C 1–​2 days 5–​6 days by culture; 50% remain positive by RT-​PCR for 2 weeks or longer RT-​PCR ( viral culture is less sensitive and only possible with some rhinoviruses) Enterovirus Picornaviridae Non​enveloped RNA viruses 112 serotypes distributed in groups A–D Few days Up to 2 weeks from respiratory tract, much longer in faeces RT-​PCR. Viral culture less sensitive and not possible for some types unless animal inoculation is used. Coronavirus Coronaviridae. Enveloped RNA viruses 6 types (OC43, 229E, NL-​63, HKU-​1, SARS-​CoV, MERS-​CoV) 4–​5 days 5–​8 days. SARS-​CoV and MERS-​CoV can be detectable by RT-​PCR for many weeks. RT-​PCR Respiratory syncytial virus (RSV) Paramyxoviridae Enveloped RNA virus Subgroup A and B 5 days 6–​7 days Culture Rapid antigen detection,a RT-​PCR Serology: useful in adults but less so in infants Human metapneumovirus Paramyxoviridae Enveloped RNA virus Serotypes A and B ND ND RT-​PCR Viral antigen detection (not readily available) Parainfluenza Paramyxoviridae Enveloped RNA virus Type 1, 2, 3, 4a, 4b 3–​6 days 7 days Culture Rapid antigen detection,a RT-​PCR Serology: useful in adults but less so in infants Influenza Orthomyxoviridae Enveloped RNA virus Types A, B, C Human influenza A subtypes currently in circulation are H1N1 and H3N2 Average 2–​3 (range 1–​7) c. 5 days in adults c. 7 days in children Culture Rapid antigen detectiona, RT-​PCR Serology Adenovirus Adenoviridae Non​enveloped DNA virus Subgroups A–​G Types 1–​54 Average 10 (range 2–​15) Days–​weeks (from respiratory tract), weeks–​months (in faeces) Culture Rapid antigen detection,a RT-​PCR, Serology Bocavirus Parvoviridae Non​enveloped DNA virus Four species, bocavirus (BCoV) types 1–​4 ND Prolonged virus shedding for weeks or months RT-​PCR ND, not defined. a Best sensitivity from nasopharyngeal aspirates or nasopharyngeal swabs (in that order). Throat swabs give lower sensitivity. Table 8.5.1.2  Viral aetiology of common respiratory syndromes Virus Coryza Pharyngitis Croup Bronchiolitis Pneumonia Rhinovirus +++a ++

Rare Coronavirus ++ +

++ +++ +++ (children);

++ ++ (children) Parainfluenza 1 + ++ +++ + Parainfluenza 2 + ++ ++ + Parainfluenza 3 + ++ ++ ++ ++ (children) Influenza A/​B + ++ ++ + ++ (more severre in children and elderly) a Frequency of cases caused by the virus: +++ the major cause (>25%); ++ a common cause (5–​25%); + an occasional cause; blank, rare cause or not reported. Data adapted from Treanor 2009.

726 section 8  Infectious diseases probably more important. Occasionally, SARS-​CoV and MERS-​ CoV appear to have spread by small particle aerosols, although droplets and fomites probably contributed to the major part of the transmission of these diseases. Adenoviruses are transmitted by the faeco-​oral route as well as by direct contact and large droplets. Factors increasing transmission of respiratory viruses include the time of exposure, close contact (e.g. spouse, mother), crowding, family size, and lack of pre-​existing immunity (including lack of breastfeeding). School-​age children often introduce an infection into the family and the beginning of school term might affect transmis- sion patterns in the community. Infected children shed higher titres of viruses than adults. The duration of virus excretion is shown in Table 8.5.1.1. Infectivity usually precedes the onset of clinical symp- toms. Immunocompromised patients shed virus for a longer time. Seasonality Some respiratory viruses have a predictable seasonality, which varies regionally. For example, influenza A is a typically winter disease in temperate regions, a spring/​summer disease in the subtropics (e.g. Hong Kong) and occurs all year round (e.g. Singapore) or predom- inantly in the rainy season (e.g. Thailand) in the tropics. The basis for such seasonality is unclear, but climatic factors such as high hu- midity and temperature may help virus survival in small particle aerosols or droplets, and on contaminated surfaces. Factors affecting population congregation such as commencement of school term and seasonal effects on social behaviour might also play a role. Laboratory diagnosis A well-​collected specimen is the first and often most important deter- minant in successful laboratory diagnosis. Nasopharyngeal aspirates (secretions aspirated from the back of the nose into a mucus trap), nasopharyngeal washes, and nasopharyngeal swabs are superior to throat and nose swabs for the diagnosis of many respiratory viruses. They offer the advantage that rapid (‘same day’) diagnosis for sev- eral viruses is possible provided the appropriate methods are avail- able. Swabs for viral culture are placed in viral transport medium immediately upon collection and kept cool (around 4°C) until pro- cessed. Sputum appears to be a useful specimen for viruses that pre- dominantly cause lower respiratory disease (e.g. MERS-​CoV, avian influenza H7N9). More invasive specimens such as endotracheal aspirates, bronchoalveolar lavage, or lung biopsy, when available, usually provide better information. For example, in patients with influenza pneumonia during the 2009 pandemic, endotracheal as- pirate specimens sometimes provided positive results even when the upper respiratory tract specimens were negative. However, the likely site of pathology must be kept in mind—​the more invasive specimen is not always better. Laboratory methods used for detecting a virus in clinical spe- cimen/​s are viral culture, antigen detection, and nucleic acid de- tection (e.g. polymerase chain reaction (PCR)-​based methods). The widespread use of molecular methods for viral detection has led to recognition that some viruses that are difficult to culture (e.g. coronaviruses and some rhinoviruses and enteroviruses) are found more often in patients with acute respiratory disease than previously recognized. Similarly, these methods have allowed the discovery of novel viruses associated with respiratory disease (e.g. coronaviruses NL-​63, HKU1, bocavirus). They have also revealed that infection with multiple viruses is relatively common. These findings neces- sitate a reassessment of the clinical relevance of positive PCR re- sults. Relevant questions include how commonly these viruses are detectable by these methods in age-​matched healthy controls and how long viruses remain detectable after infection. It is important to understand the relevance of detection of multiple pathogens in a respiratory specimen. Are these viruses synergistic in pathogenesis or is one more important than another? Many of these questions re- main to be resolved. Demonstration of rising antibody titres in paired sera is used to diagnose some respiratory virus diseases, but serology is imprac- ticable for others such as rhinoviruses where the large number of antigenically distinct serotypes have no common immunodominant antigen(s). However, adenoviruses and influenza viruses, though having many antigenic types or variants, have common antigen(s) and a single antigen can detect serological responses to many of them. IgM assays are not routinely available for diagnosis of respira- tory viral diseases. Serology is also helpful in assessing the clinical relevance of a virus detected in a respiratory specimen (see earlier) by helping differentiating recent infection from more remote events. ‘Near patient testing’ is becoming a reality for some viruses (e.g. influenza, respiratory syncytial virus or RSV) with availability of tests that can be performed in a general practice setting. These be- come more relevant with the greater availability of antiviral drugs. Rhinoviruses Rhinoviruses belong to the Picornavirus family and are adapted to replicate at temperatures of 33–​35°C, as found in the external air- ways. Until recently, more than 150 serotypes of rhinoviruses were recognized phylogenetically clustered into groups A, B, and C; group C viruses are non​cultivable and recently discovered by mo- lecular methods. Several rhinovirus types will circulate in a region at any given time. Epidemiology Rhinoviruses remain one of the most common infections of hu- mans: 0.5 infections per person per year is a conservative estimate. Secondary attack rates in families can be around 50% overall and 70% in those who are antibody negative. They were thought to cause mainly mild community infections, but are being recognized increas- ingly as the commonest viral agent detected by RT-​PCR in children hospitalized with acute respiratory illness. Many of these represent coinfections with other potential respiratory pathogens. As rhino- viruses are often detectable by RT-​PCR for weeks after initial infec- tion (50% remain positive at 2 weeks), the aetiological significance of this finding is unresolved and more studies with relevant control populations are needed. Rhinoviruses are important triggers of ex- acerbation of asthma and chronic obstructive pulmonary disease. Immunity In experimental challenges, immunity is serotype specific. Homologous type specific protection lasts for at least 1 year and correlates with serum IgA, IgG, and secretory IgA antibody levels.

8.5.1  Respiratory tract viruses 727 Pathogenesis Viral replication occurs predominantly in the ciliated epithelial cells of the nasopharynx. The structure of the epithelium is preserved. Mucosal secretions associated with coryza appear to be due to the release of inflammatory mediators and neurogenic reflexes. It was thought that the preference of the virus for a lower tem- perature for replication restricted it to the upper respiratory tract. However, this is not strictly true. The virus has been isolated from the lower respiratory tract (including bronchial brushings) and viral RNA has been demonstrated by in situ hybridization in bronchial epithelial cells. Rarely, the virus has been isolated post-​mortem from lungs of immunocompromised patients. Clinical manifestations Rhinorrhoea, nasal obstruction, pharyngitis, and a cough are common features of rhinovirus infections. Fever and systemic symptoms are rare, but more common in older people in whom disease can be more severe. Rhinoviruses are a major cause of exacerbations of asthma and chronic obstructive respiratory disease in adults. Lower respira- tory tract symptoms are uncommon in healthy young adults, but may occur in children (bronchiolitis), the immunocompromised, and older people. Rhinovirus infections associated with wheezing in the first 3 years of life is predictive of asthma in later childhood. Treatment and prevention There are no established antiviral drugs for treatment and manage- ment is symptomatic. Topical interferon-​α prevents symptoms if given before onset of disease, but cannot be used for prophylaxis over prolonged periods because of side effects. Inhaled interferon-β is currently in clinical trials in adult asthmatics on inhaled cortico- steroids with a history of clinical deterioration following upper re- spiratory infections. Pleconaril is a viral capsid-​binding agent that blocks viral attachment and uncoating and has had modest benefit in clinical trials, but concerns over drug interactions have prevented its licensing. Vapendavir is a drug with similar action that is cur- rently in clinical trials for rhinovirus infections. Antibiotics are ineffective in preventing bacterial complications of the common cold. Mucopurulent discharges are part of the natural course of the common cold and are not an indication for antimicrobial treatment, unless it persists (e.g. >10 days). Given the considerable number of rhinovirus serotypes, vaccination is not an option. Enteroviruses Enteroviruses and rhinoviruses (see earlier) are genera within the family Picornaviridae. Enteroviruses have long been known as causes of central nervous system infections, myocarditis, or exan- thema rather than as a respiratory pathogen, the latter role being assigned to rhinoviruses. As many enteroviruses fail to replicate in cell culture, the wider use of molecular diagnosis has revealed an increased role of enteroviruses in acute respiratory infections. Clinically, patients present with rhinitis, cough, fever, sore throat, or otitis media. There remains a need for studies of age-​matched controls to better establish the clinical relevance of these molecular tests. In comparative studies done on the duration of shedding of enteroviruses and rhinoviruses, fewer enterovirus infected children continue to shed virus for longer than 2 weeks while 50% of rhino- virus infections do. This suggests that a positive enterovirus RT-​PCR result in the respiratory tract is probably more likely to be clinically relevant than one for rhinovirus. Recently there have been outbreaks of acute respiratory illness, predominantly in children, caused by en- terovirus 68, manifesting as cough, wheezing, and hypoxemia some patients requiring hospitalization and intensive care. This virus may also cause flaccid paralysis. Coronaviruses Six human coronaviruses are currently known, four of them being new viruses discovered since the SARS outbreak in 2003. Coronaviruses are taxonomically subdivided into four groups; the human coronaviruses 229E and NL-​63 are alphacoronaviruses while OC43, HKU1, SARS-​CoV, and MERS-​CoV are β-coronaviruses. There are no known human γ- or deltacoronaviruses yet recognized. Human coronaviruses OC43 and 229E have long been recognized as important causes of the common cold but coronaviruses cause a range of respiratory illnesses. SARS-​CoV and MERS-​CoV area newly emerged pathogens of zoonotic origin. Human coronaviruses are difficult to culture from clinical specimens and laboratory diag- nosis largely relies on molecular methods. Epidemiology Infection with OC43 and 229E occur in early childhood and 85 to 100% of adults have antibody to both virus types. NL-​63 has a similar epidemiology but less is presently known of HKU1. SARS-​ CoV emerged from an animal reservoir, adapted to human trans- mission, and caused a global outbreak in 2003 that affected 29 countries across five continents. Determined public health inter- ventions interrupted transmission of this virus and it is no longer transmitting within humans. However, the precursor virus remains in the animal reservoir (bats, Rhinolophus spp.) and these could, at some future date, readapt to cause human disease. MERS-​CoV was first recognized in a patient with fatal pneumonia in Saudi Arabia in 2012 but the virus has been endemic in dromedary camels for many decades. Zoonotic human infection has so far been confined to the Arabian Peninsula or the Middle East and might sometimes be mild and not recognized. Severe disease occurs mainly in older people on in those with underlying comorbidities. Clusters of human trans- mission have occurred within healthcare facilities. Patients who acquired infection in the Middle East and travelled elsewhere have sometimes caused outbreaks in other parts of the world. Immunity Volunteer reinfection studies with 229E show that 1 year after ini- tial infection, protection from reinfection and illness following a challenge from the homologous virus is incomplete. Comparable data are not available for the newly recognized NL-​63, HKU1, or SARS-​CoV. Pathogenesis In common with rhinoviruses, coronaviruses 229E induce little or no damage to the respiratory mucosa. The mucosal discharge is caused by the release of mediators from affected host cells. SARS-​CoV and MERS-​CoV have a predilection to infect alveolar pneumocytes in

728 section 8  Infectious diseases the lower respiratory tract and consequently caused a severe viral pneumonia. Disease severity of SARS was markedly age related. Children had mild disease whereas those over 50 years had a poor prognosis. The basis for this age-​related pathogenesis is unknown. The virus receptor for 229E is CD13, both SARS-​CoV and NL-​63 utilize the human ACE-​2 molecule for virus entry while MERS-​CoV binds to human DPP4 (CD26). Clinical findings Coronaviruses 229E and OC43 typically cause upper respiratory tract infection and the common cold but also cause a range of other respiratory manifestations and are significant pathogens in elderly people. NL-​63 and HKU1 cause both upper and lower respiratory disease. NL-​63 appears to be an important cause of croup, bronchio- litis, and pneumonia. HKU1 appears to be an important pathogen particularly in those with underlying respiratory complications. SARS typically presented with lower respiratory tract manifest- ations and radiological changes with minimum involvement of the upper respiratory tract. Many patients had diarrhoea resulting from viral replication in the gastrointestinal tract. Overall case fatality was 9.6%. Terminal events were severe respiratory failure associated with acute respiratory distress syndrome and multiple organ failure. Age, comorbidities, and viral load in the nasopharynx and serum during the first 5 days of illness correlated with an adverse prognosis. Clinical features of MERS are broadly similar to those of SARS; fever, chills, or rigors, cough (dry or productive), and shortness of breath being the common presenting symptoms. Diarrhoea or vomiting were reported by around one-​third of patients. Upper respiratory symptoms are uncommon. Chest radiographic abnor- malities can include unilateral or bilateral hilar infiltrates, patchy in- filtrates, segmented or lobar opacities or ground glass opacities, with the lower lobes being generally more affected than the upper lobes, early in the illness. Lymphopenia, thrombocytopenia and high lac- tate dehydrogenase levels are seen in around one-​third of patients. Reported case fatality ranges from 30 to 40%, but this is probably because milder cases are not being recognized. Treatment and prevention There are presently no clinically validated antiviral treatments for human coronaviruses disease, although several drugs have been documented to have in vitro activity against SARS-​CoV and MERS-​ CoV and some (ribavirin, interferon, HIV protease inhibitors) have been used in uncontrolled settings with inconclusive results. Passive immunotherapy is currently being explored for treatment of MERS. Several experimental vaccines were developed for SARS, but with its disappearance from the human population, the incentive to take these forward to human clinical trials and licensing has waned. Vaccines for MERS-​CoV for humans and for camels are in preclin- ical trials. Adenoviruses Currently there are 54 adenovirus types classified in six groups (A–F​). Adenoviruses in subgroups A to D cause respiratory, ocular, hepatic, genitourinary, or gastrointestinal system disease in immunocompe- tent or immunocompromised individuals. Only respiratory diseases are considered here. Productive replication and excretion of infectious virus can occur for a prolonged period (see next). In addition, adenoviruses can es- tablish chronic persistence or ‘latency’, the virological basis and clin- ical significance of which is poorly understood. Epidemiology Adenovirus infections are common during childhood (usually sero- types 1, 2, 5 in early childhood, 3 and 7 during school years or later), but continue to occur throughout life. Reinfection with the same serotype occurs but is usually asymptomatic. Serotypes 1, 2, 5, and 6 are typically endemic, types 4 and 7 more typically associated with outbreaks, and type 3 can occur in either situation. Recently, adeno- virus 14p1 (previously designated 14a) has been spreading in the United States of America and elsewhere and is associated with more severe disease especially within military facilities. Clinical features Adenovirus respiratory illness often leads to upper respiratory tract infection with coryza and sore throat. Fever may last up to 2 weeks. The sore throat may be exudative and clinically difficult to differ- entiate from streptococcal infection. Adenoviral infection may pre- sent as pharyngoconjunctival fever. Otitis media is a complication in children. Unlike other respiratory viral infections, adenoviruses may be associated with elevated white blood cell counts (exceeding 15 × 109/​litre), C-​reactive protein, or erythrocyte sedimentation rate, and thus more easily confused with bacterial diseases. Though uncommon, pneumonia can occur sporadically or in epi- demics (e.g. caused by serotypes 4 and 7), particularly in closed com- munities such as the military where stress and physical exertion may predispose to lower respiratory tract involvement. Community out- breaks of adenoviral pneumonia have been reported. Radiological appearance varies from diffuse to patchy interstitial infiltrates and pleural effusion may be present. Adenovirus type 7 pneumonia can lead to permanent lung damage, including bronchiectasis, bron- chiolitis obliterans, and unilateral hyperlucent lung syndrome. Adenoviral infection may disseminate and present as ‘septic shock’ in neonates. Manifestations in immunocompromised pa- tients include hepatitis (especially in liver transplant recipients), colitis and haemorrhagic cystitis (in stem cell and organ transplant recipients) in addition to pneumonia. The serotypes associated with disease in these patients may differ from those typically found in the immunocompetent patient, and include the subgroup B2 serotypes 11, 34, and 35. With improving control of other common viral dis- eases of immunocompromised patients (e.g. cytomegalovirus), the role of adenovirus infections is being increasingly appreciated. Isolation or PCR detection of an adenovirus from a clinical spe- cimen presents a challenge in interpretation. Adenoviruses are ex- creted for a prolonged period after initial infection, especially, but not exclusively, from faeces. In children, one-​third of patients shed viruses for longer than 1 month and 14% longer than 1 year. The clinical significance of a positive result depends on the specimen, the method, and the serotype. Isolation of viruses from the respiratory tract carries greater significance than that from faeces. Patients who have symptomatic adenoviral diseases have higher viral loads than those with asymptomatic carriage. Thus, a rapidly growing virus, a positive antigen detection test from a respiratory specimen (both re- flecting higher virus load), or a detectable serological response all point to greater clinical significance.

8.5.1  Respiratory tract viruses 729 Immunocompromised patients might be infected with unusual serotypes. The detection of the virus in the peripheral blood or in multiple body sites suggests greater clinical significance and is an indication that therapeutic intervention needs to be considered. Treatment and prevention Most adenoviral infections in immunocompetent patients are self-​ limited and require no specific therapy; however, some infections, especially but not exclusively in immunocompromised patients, are severe and life-​sthreatening. Intravenous cidofovir has been used for treatment of adenoviral infections in the immunocompromised, but nephrotoxicity and neutropenia limit its use. Orally administered brincidofovir (lipid ester derivative of cidofovir) appears to provide better clinical outcome with fewer side effects, but clinical trials are ongoing. Live attenuated oral vaccines containing serotypes 4 and 7 (associ- ated with outbreaks in military conscripts) are now used for military personnel in the United States and are safe and effective, but not li- censed for general use. Respiratory syncytial virus Respiratory syncytial virus (RSV) infects human and non​human primates and was first isolated from a chimpanzee with a ‘cold’. The virus has two surface glycoproteins on its envelope (G and F) and the immune responses to them correlate with protection. Two sub- groups (A and B) are recognized on the basis of antigenic differences of the G glycoprotein. Epidemiology Over two-​thirds of infants acquire RSV infection during the first year of life. Of patients hospitalized with RSV disease, 75% are younger than 5 months. The peak of morbidity occurs around 2–​4 months of age, a time when passive maternal antibodies protect against most other viral infections. It is associated with significant morbidity worldwide and to between 66 000 and 200 000 deaths annually, pre- dominantly in low-​ and middle-​income countries. Primary infec- tion does not lead to solid immunity and reinfection is common. The first reinfection can still be associated with lower respiratory tract involvement. Subsequent reinfection occurs throughout life leading to asymptomatic or upper respiratory tract infection. However, sig- nificant diseases may result in the immunocompromised or elderly. Immunity Both antibody and cell mediated immunity are important in pro- tection. Antibody to the G protein prevents attachment of viruses to the cellular receptor, but immunity to the F protein is required to prevent cell to cell spread via fusion of virally infected cells. Cell mediated immunity is important in eliminating established viral infection. Pathogenesis The virus leads to a ballooning degeneration of the ciliated epithe- lial cells, lymphocytic infiltration, and necrosis of the epithelium. There is oedema and increased secretion from the mucous cells and the formation of plugs of mucous and cellular debris in the bronchi- oles. This results in obstruction and air trapping leading to collapse or overdistension of the distal alveoli. Cells throughout the respira- tory tract are affected but the alveoli are spared unless there is RSV pneumonia. The pathogenesis of RSV bronchiolitis still remains controversial. Severe RSV bronchiolitis is strongly associated with subsequent childhood asthma. RSV appears to promote type 1 hypersensitivity responses following subsequent exposure to unrelated antigens. Clinical features RSV infections of infants may lead to bronchiolitis and pneumonia. Bronchiolitis in infants is associated with expiratory wheeze, sub- costal recession, hyperinflation of the chest, nasal flaring, and hyp- oxia with or without cyanosis. Fever is not prominent in one-​half of the patients. Complete obstruction of a small airway leads to subsegmental atelectasis. Apnoea can occur (particularly in prema- ture infants or in those <3 months of age) and might precede the de- velopment of bronchiolitis. Interstitial pneumonitis is uncommon but carries a bad prognosis. Otitis media is a common complication of RSV infection in children. Infants at highest risk from severe RSV disease are those aged under 6 months, those with pre-​existing con- genital heart disease, chronic lung diseases (e.g. bronchopulmonary dysplasia), and those born premature. Infection in adults is often asymptomatic or leads to upper re- spiratory tract infection. However, during the RSV season, it is an important cause of lower respiratory tract infection in adults and elderly people and it is estimated to cause 2 to 9% of the hospitaliza- tions and deaths associated with pneumonia in elderly individuals. Much of this morbidity is clinically indistinguishable from influenza. RSV (as well as parainfluenza and influenza) infections in the immunocompromised patient can be life threatening. They usu- ally occur during community outbreaks, but a significant propor- tion are nosocomially acquired. The disease typically commences as an upper respiratory tract infection but can progress to involve the lower respiratory tract with more serious consequences. Factors that increase risk of disease progression appear to include stem cell and organ transplant recipients who acquire the infection in the period prior to engraftment and oncology patients with neutrophil counts less than 0.5 × 109/​litre. Those immunocompromised by HIV appear to tolerate community acquired respiratory viruses better than on- cology patients and transplant recipients. Treatment and prevention Ribavirin has activity against RSV in vitro. Administration of small particle aerosols via a mist tent, mask, oxygen hood, or ventilator has been recommended because it results in much higher concen- trations in the respiratory tract than can be achieved by intravenous administration. However, there seems little therapeutic benefit of ribavirin therapy in RSV disease in immunocompetent children or adults. In patients at high risk for severe RSV disease such as stem cell transplant recipients, inhaled ribavirin together with intra- venous immune globulin (selected batches with high neutralizing antibody titre) appeared to be beneficial when compared to histor- ical controls. Intermittent delivery (2 hours’ therapy every 8 hours) appeared to be more effective in preventing progression to lower re- spiratory tract disease than continuous administration. Ribavirin is a potential teratogen and there are concerns over healthcare worker exposure. Oral ribavirin is currently being explored as an alternative to inhaled ribavirin in this setting.

730 section 8  Infectious diseases Monthly intravenous administration of a polyclonal immune globulin enriched in neutralizing antibodies to RSV (RespiGam) or a humanized monoclonal antibody to RSV (palivizumab) during the RSV season protects against disease of the lower respiratory tract and otitis media in children with pre-​existing risk factors. Palivizumab appears to be more effective than RespiGam and there is less of a problem with fluid overload in children with chronic heart disease. High-​titre RSV intravenous immunoglobulin by itself is ineffective in treatment of established RSV disease. Newer modifications de- rived from Palivizumab with higher virus neutralizing competence (Motavizumab; MEDI-​524) and longer half-​life in the circulation (MEDI-​557) have been developed but are not licensed for clinical use, in part because of increased cutaneous side effects observed with Motavizumab. Candidate vaccines for RSV are undergoing clinical trials at pre- sent but none is yet available for routine use. Experience of early trials with inactivated RSV vaccines that led to enhanced RSV dis- ease, rather than protection continues to haunt the field. Parainfluenza virus Parainfluenza viruses, despite their name, are not related to influ- enza viruses, and are more akin to respiratory syncytial virus with which they are classified (Table 8.5.1.1). They carry two envelope glycoproteins: HN containing both haemagglutinin and neuramin- idase activity, and F carrying fusion activity. Epidemiology The total impact on hospitalization of children by all four types of parainfluenza viruses taken together is similar to that of RSV but, in contrast to RSV, their impact is in later infancy and childhood. In temperate countries, parainfluenza virus type 3 occurs annu- ally and infects two-​thirds of all infants in their first year of life. Parainfluenza types 1 and 2 tend to occur in alternate years and infection is acquired more slowly over childhood. Reinfection with parainfluenza viruses occurs, but rarely leads to lower respiratory tract infection. Pathogenesis The virus is confined to the respiratory epithelial cells, macrophages, and dendritic cells within the respiratory tract. Dissemination is rarely documented even in immunocompromised patients. Immunity Reinfection with parainfluenza viruses continues throughout life. Presence of virus-​specific IgE in nasopharyngeal secretions has been implicated in the development of parainfluenza croup or bronchiolitis. Clinical features Parainfluenza type 1 predominantly causes croup, while types 2 and 3 also cause bronchiolitis and pneumonia. Croup (or laryngotracheobronchitis) in children is associated with fever, hoarseness, and a barking cough, and may progress to inspiratory stridor due to narrowing of the subglottic area of the trachea. The differential diagnosis is epiglottitis due to Haemophilus influenzae type b. Parainfluenza type 4 infection is less common, but causes bronchiolitis and pneumonia in children, often in those with underlying disease. Reinfection in adults, when symptomatic, is a coryzal illness with hoarseness being prominent. Parainfluenza viruses (type 3 in par- ticular) are significant causes of lower respiratory tract infection in adults when the virus is active in the community. As with RSV, parainfluenza viruses cause problems in immuno- compromised patients. Lower respiratory tract involvement is associated with wheezing, rales, dyspnoea, and diffuse intersti- tial infiltrates, and a fatal outcome in one-​third of patients with stem cell transplants. When pneumonia occurs, the histological appearance of the lung is that of a giant cell or an interstitial pneumonia. Treatment and prevention The need for specific antiviral therapy arises, particularly in the im- munocompromised. Ribavirin is effective in vitro and was associ- ated with a reduction of viral replication in vivo in anecdotal cases but there are no controlled trials documenting its clinical efficacy. DAS181 is a sialidase that removes the sialic acid receptors neces- sary for parainfluenza virus attachment to the cell membrane. It is currently in clinical trials for treatment of parainfluenza disease in immunocompromised patients. There are no options for prevention at present, either using vac- cines or passive immunization. A  live attenuated bovine-​derived vaccine strain is currently undergoing clinical trials. Human metapneumovirus Human metapneumovirus (HMPV) belongs to the genus Metapneumovirus within the virus family Paramyxoviridae, sub- family Pneumovirinae. It closest known relative is the avian pneumovirus, an upper respiratory tract disease of turkeys and among human viruses is RSV which also belongs to the subfamily Pneumovirinae. It was first recognized in 2001 but is a virus that has circulated unrecognized in humans for many decades. There are at least two serotypes A and B which are antigenically distinct and ap- pear to provide partial cross-​protection. Epidemiology The virus is ubiquitous and most children have been infected with one or both serotypes by the age of 5 years. HMPV is a common cause of hospitalization of children under 5 years of age and ac- counted for 12% of all lower respiratory tract infection hospitaliza- tion in one long-​term study. However, the incidence in any given year may vary widely. The peak age for HMPV morbidity is between 6 and 12 months, which is later than that for RSV (2–​4 months). Symptomatic reinfection is common through life. Infection is com- monest in the winter months in temperate regions and in late spring or summer in subtropical areas. Clinical manifestations Clinical features of HMPV are similar to that of RSV and range from upper respiratory tract infection to bronchiolitis and pneumonia. In common with rhinovirus and RSV, HMPV appears to trigger ex- acerbations of asthma. Diarrhoea, vomiting, rash, febrile seizures, conjunctivitis, and otitis media have been reported. HMPV has on

8.5.1  Respiratory tract viruses 731 one occasion been isolated as the sole pathogen from the brain in a patient with encephalitis. Risk factors for severe HMPV disease in children are: aged less than 2  years, gestational age less than 37 weeks, and underlying comorbidities. HMPV can cause respiratory disease in elderly or immunocom- promised individuals, and those with underlying conditions at any age. Since HMPV is difficult to grow in vitro, laboratory diagnosis is reliant on the detection of viral RNA in clinical specimens by mo- lecular methods. Treatment and prevention There are currently no available vaccines. As with RSV, the F and G proteins are the main targets of the neutralizing antibody response and while the former is antigenically conserved, the latter is more variable. Thus the F protein has been the focus of vaccine devel- opment. Ribavirin has comparable in vitro activity against HMPV as against RSV but there is no clinical trial data that demonstrates therapeutic efficacy. Influenza viruses Influenza viruses contain a segmented RNA genome. Types A, B, and C are antigenically distinct; of these, types A and B are clin- ically important causes of human disease. The viral envelope con- tains two glycoproteins, the haemagglutinin (H) and neuraminidase (N) which are critical in host immunity. While protective antibody to H and N are largely subtype specific, antibody to conserved re- gions of the H stalk region appear to provide cross-​subtype protec- tion and are currently being targeted for passive immunotherapy and vaccine development. The M2 transmembrane protein is also found on the virion surface and can provide broadly cross-​reactive immunity following experimental immunization but does not ap- pear to elicit a significantly protective host response following nat- ural infection. Human influenza viruses are designated by the virus type, place of isolation, strain designation, year of isolation, and the H and N antigen subtype, for example, A/​Sydney/​5/​1995 (H3N2). There are two lineages of influenza B, the Yamagata and Victoria lineages, which are antigenically distinct with only partial cross-​protection. Epidemiology The H and N genes of influenza types A and B undergo mutational change resulting in the emergence of antigenic variants (‘antigenic drift’). Every few years, a variant successful in evading the prior im- munity of the human population emerges, to cause a global epidemic. Influenza viruses have a marked winter seasonality in temperate re- gions, making the disease burden of the virus more obvious. The more diffuse seasonality in tropical and subtropical regions leads to an obscuring of the clinical impact of the virus, leading to the il- lusion in some quarters that influenza is less significant in warmer climates. However, careful epidemiological studies demonstrate that the burden of mortality and morbidity in temperate and tropical re- gions are very similar. In those 65 years or older, influenza is as- sociated with approximately one excess death per 1000 population annually in both the temperate and tropical regions. In aquatic birds, the natural reservoir of the virus, 16 H and 9 N subtypes of influenza A are found. Recently, influenza viruses have been found in bats that carry H17, H18 and N10, N11 subtypes. From 1918 to 1957, human influenza A viruses carried H1N1 surface antigens. In 1957, this virus acquired the novel H, N, and additional polymerase gene (PB1) from an avian influenza virus through gen- etic reassortment of its segmented genome giving rise to the H2N2 subtype virus (‘antigenic shift’). As the human population lacked im- munity to these novel viral antigens, this led to the ‘Asian flu’ pan- demic. A similar reassortment event gave rise to the H3N2 virus and the ‘Hong Kong influenza’ pandemic of 1968. Although all three influ- enza pandemics of the 20th century resulted in significant morbidity and mortality, the toll exacted by the ‘Spanish flu’ of 1918 was particu- larly horrendous—​over 40 million deaths, greater than that of both World Wars combined. Since influenza B (and C) have no significant zoonotic reservoirs, antigenic shift and pandemics do not occur. In early 2009, a novel H1N1 virus of swine origin gave rise to the first pandemic of the 21st century. The pandemic arose in Mexico and rapidly spread worldwide along routes of air-​travel. Unlike the two previous pandemics (1957, 1968) that arose through gen- etic reassortment of an avian virus with the prevailing human sea- sonal influenza virus, the pandemic virus of 2009 arose through reassortment between swine viruses previously documented in North America (so called ‘triple reassortant’ swine viruses that con- tained virus gene segments of swine, avian and human origin) and ‘Eurasian-​swine’ viruses. Although the H1 haemagglutinin of both seasonal human and swine influenza viruses was originally derived from the 1918 ‘Spanish flu’ H1N1 virus, they had antigenically di- verged during their subsequent evolution in these two hosts so that the contemporary seasonal human H1N1 virus offered little cross-​protection against the pandemic H1N1 virus of swine origin. However, people born prior to the 1950s had substantial cross-​ protection against the novel pandemic virus, presumably derived by infection with H1N1 viruses circulating in the first half of the 20th century. Thus, the pandemic was associated with explosive out- breaks in children and young adults while there was less infection in older adults. When infection did occur, severity of disease in older adults was much more severe than that in children. Overall, the 2009 pandemic was less severe than previous pandemics. However, com- plications, severe illness, and fatalities did occur, especially in those who were pregnant or with underlying comorbidities including asthma and other lung disease, cardiovascular diseases, diabetes, neurological disorders, autoimmune disorders, and morbid obesity. While some of those with severe disease had secondary bacterial infections, others developed a primary viral pneumonia leading to acute respiratory distress syndrome. Avian viruses (e.g. subtype H5N1, H9N2, H7N7, H7N9, H5N6) can zoonotically infect humans occasionally without undergoing prior reassortment with existing human strains. Currently, an H5N1 virus that is highly pathogenic for chickens has become en- trenched in poultry flocks in several Asian and African countries and continues to zoonotically transmit to humans, often causing se- vere disease. Similarly, a descendent of this virus, H5N6 is now be- coming dominant in poultry and causing zoonotic human disease. Such transmission has so far not led to sustained human-​to-​human transmission, which is the prerequisite for the generation of a new pandemic. However, recent studies with experimentally mutated H5N1 viruses have shown that these viruses can acquire droplet

732 section 8  Infectious diseases transmission capacity in ferrets, the best available surrogate for vir- uses with human transmission potential. H7N9 viruses emerged in 2013 and have caused zoonotic disease, many of those with severe disease are elderly or have underlying comorbidities (unlike H5N1 disease). Pathogenesis Viral replication occurs in the columnar epithelial cells leading to its desquamation down to the basal cell layer. The pathology typically involves the upper respiratory tract and the tracheobronchial tree. Infection results in decreased ciliary clearance, impaired phago- cyte function, and increased adherence of bacteria to viral infected cells, all of which promote the occurrence of secondary bacterial infection. While there may be differences in viral virulence, pre-​existing cross-​reactive immunity is a major determinant in reducing dis- ease severity. Virus dissemination outside the respiratory tract is uncommon with human influenza viruses. However, zoonotic in- fections with the avian H5N1 virus may disseminate, and virus has been often detected in the gastrointestinal tract and occasionally in the central nervous system. Immunity Infection by an influenza virus results in long-​lived immunity to homologous reinfection. However, the continued antigenic change in the virus allows it to keep ahead of the host immune response. Cross-​immunity to ‘drifted’ strains within the same H or N subtype may provide partial protection, but there is believed to be little cross-​ protection between different subtypes. Local and systemic antibody responses and cytotoxic T cells contribute to host protection. Clinical features The severity of influenza ranges from asymptomatic infection, through the typical influenza syndrome, to the complications of influenza. Although it cannot always be distinguished from other viral infections on clinical grounds, the typical influenza syndrome is relatively characteristic in the adult. It is associated with fever, chills, headache, sore throat, coryza, non​productive cough, my- algia, and sometimes prostration. The onset of illness is abrupt and the fever lasts 1–​5 days. The pharynx is hyperaemic but has no ex- udate. Cervical lymphadenopathy is often present and crackles or wheezing are heard in around 10% of patients. While the acute illness usually resolves in 4–​5 days, cough and fatigue can persist for weeks afterwards. Common (>10% of symptomatic patients) complications of influ- enza include otitis media (in children) and exacerbation of asthma, chronic airways obstruction, and cystic fibrosis. Less common complications are acute bronchitis, primary (viral) and secondary (bacterial) pneumonia, myocarditis, febrile convulsions, encephal- opathy, encephalitis, and myositis (especially in patients with influ- enza B infection). Age, prior immunity, virus strain, the presence of underlying diseases, pregnancy, and smoking all influence mor- bidity and severity. Treatment and prevention Antiviral therapy Antiviral drugs with proven clinical efficacy for treatment of influ- enza A are the ion channel (M2) blockers that interfere with viral uncoating (amantadine, rimantadine) and the neuraminidase in- hibitors (e.g. zanamivir, oseltamivir) which block virus release from infected cells. The neuraminidase inhibitors are also active against influenza B, while amantadine and rimantadine are only active against influenza A. However, seasonal H3N2 and H1N1 viruses increasingly acquired resistance to amantadine and rimantadine and the current 2009 pan- demic H1N1 which is has now replaced the previous seasonal H1N1 virus in humans is resistant to amantadine and rimantadine. Thus, these are no longer drugs of choice in the treatment or prophylaxis of human influenza. Oseltamivir resistance to seasonal H1N1 vir- uses emerged in early 2008 and spread worldwide but this strain has largely been replaced by the pandemic H1N1 that emerged in 2009. Thus the influenza A (pandemic H1N1 virus and seasonal H3N2 viruses) and influenza B viruses now remain sensitive to oseltamivir. Although resistant pandemic H1N1 viruses have been occasionally reported, these have not so far become dominant within the human population. Zanamivir remains uniformly effective against seasonal and pandemic influenza viruses. Zanamivir is administered by inhalation and oseltamivir orally. Inhaled zanamivir can occasionally cause bronchospasm in those with underlying airways disease or asthma. In patients infected with viruses sensitive to these drugs, zanamivir or oseltamivir treatment commenced within the first 48 h of disease onset leads to a 1 to 2 days reduction in the time to alleviation of clinical symptoms and also reduces incidence of influenza associated complications. Some studies have indicated benefit in reducing the complications of in- fluenza even for patients in whom treatment commenced after the second day of clinical illness. However, the sooner the drugs are used, the better the chance of clinical benefit. With a virus such as the highly pathogenic H5N1 virus which can disseminate beyond the respiratory tract, a sys- temically administered drug (oseltamivir) is likely to be superior to one administered by inhalation (zanamivir). Oseltamivir provides clinical benefit in H5N1 disease, earlier commencement being as- sociated with improved outcome. Parenteral therapy (intravenous peramivir or zanamivir) may be preferable for treatment of severe influenza. Intravenous peramivir is presently licensed for clinical use in Japan, South Korea, and United States. Baloxavir marboxil is a virus endonuclease inhibitor (with mech- anisms of action that is different to other licensed influenza drugs) and is approved for use in Japan. Aspirin should be avoided in children with influenza because of the increased risk of Reye’s syndrome. Vaccines Influenza vaccine is a trivalent or quadrivalent vaccine containing antigens from the two currently circulating subtypes of human influ- enza A (H3N2 and H1N1) and B viruses. Trivalent vaccines would have the lineage of influenza B (Victoria or Yamagata) considered most likely to be dominant in the forthcoming influenza season while the quadrivalent vaccines include both Victoria and Yamagata lin- eage viruses in the vaccine. To keep abreast of change in the surface antigens of the virus, vaccine composition must be modified on an annual basis and annual reimmunization is required. This updating of the vaccine is achieved through a collaborative effort of the global in- fluenza virus surveillance network coordinated by the World Health Organization (WHO). As a result of this surveillance, the WHO

8.5.1  Respiratory tract viruses 733 makes recommendations of candidate vaccine viruses twice annually for vaccine production for the northern and southern hemispheres. Vaccines currently in use are based on antigen derived from vir- uses grown in embryonated eggs or (less commonly) cell cultures and contain detergent-​treated virus (split virus vaccines) or puri- fied surface antigens (subunit of surface antigen vaccines). These vaccines have fewer side effects than killed vaccines containing the whole virus which were used in the past and are licensed for use in anyone 6 months of age or older. Previously unvaccinated children require two doses at least 1 month apart, whereas a single dose ap- pears adequate for adults. These vaccines are generally safe, the most common side effect being soreness at the injection site lasting a few days. Vaccine efficacy is best when there is a good antigenic match between the vaccine and outbreak virus. Adjuvented (e.g. MF59) vaccines are used to enhance immunogenicity in older people or for dose-​sparing in pandemic contexts. An intranasally administered, cold-​adapted, live attenuated vac- cine is now also licensed for use in those aged 2 to 49 years and offers the advantages of broader cross-​protection across antigenic drifted viruses as well as easier administration and greater patient acceptability. Immunogenicity and clinical protection are better in healthy young adults compared to patients with chronic renal failure and immunocompromised or elderly patients (all groups most at need of the vaccine). However, the vaccine is still effective in reducing in- fluenza and pneumonia-​related hospitalization and mortality in eld- erly people and is cost-​saving. An additional option for protecting such high-​risk individuals is the immunization of children and care- givers in contact with these individuals. In young adults, vaccination is associated with decreased absenteeism from work. The duration of protection is limited and therefore vaccine administration should be timed to precede the expected peak of influenza activity. Influenza vaccine recommendations vary from country to country. Most countries recommended annual seasonal influenza vaccine for those groups at highest risk of influenza related complications including (1) those aged 6 months to 5 years of age; (2) those aged 65 years or older; (3) pregnant women who will be in the second or third trimester during the influenza season; and (4) those with chronic medical conditions including persons with chronic disorders of pulmonary or cardiovascular systems (except hypertension), those with renal dysfunction, haemoglobinopathies, metabolic disorders, or immunodeficiency, and those aged 6 months to 18 years who are on long-​term aspirin therapy. Furthermore, vaccine is also recom- mended for healthcare workers and for persons living or caring for those at high risk, who may transmit influenza to such high-​risk in- dividuals. Some countries (e.g. United States) recommend influenza vaccine to all ages over 6 months of age, irrespective of risk factors as long as there are no contraindications to vaccination. Bocavirus and polyomavirus KI and WU Human bocavirus is a member within a newly discovered genus Bocavirus within the family Parvoviridae. As with other parvoviruses, they are relatively resistant to inactivation by acid or alkaline pH or moderate heat (e.g. 56°C). Molecular detection by PCR in respiratory clinical specimens is the main option for diagnosis. The virus can also be sometimes detected in serum. There are four species of human bocaviruses (HBoV). There is evidence demonstrating an association between HBoV1 and respiratory disease in children, while HBoV2 is associated with gastroenteritis. However, because HBoV1 is shed in the human respiratory tract for prolonged periods, HBoV1 infec- tions are often (>80%) coinfections with other respiratory pathogens. High viral loads and viremia are often associated with symptoms and viremia may be associated with systemic manifestations such as en- cephalopathy. The peak age of detection is in children aged 6 months to 2 years and occasionally in adults. KI and WU are two novel polyomaviruses recently discovered in the respiratory tract of patients with acute respiratory infections. There are found in a proportion of children and adults with acute re- spiratory infection but often found as coinfections with other known respiratory pathogens. Their contribution to disease causation is still unclear. Nosocomial infection Respiratory viruses are efficient nosocomial pathogens. Though paedi- atric units face the brunt of the problem, adult wards are not exempt. Transmission can occur from patient to patient, patient to staff, and staff to patient, with visitors making their own contribution. Although influenza and RSV are the most notorious among the endemic respira- tory viruses, even rhinoviruses cause problems when transmitted to immunocompromised patients. Once infected, immunocompromised patients have a prolonged period of viral shedding and pose a signifi- cant risk of transmission to other high-​risk patients. Transmission of many respiratory virus infections occurs by large respiratory droplets gaining access to the mucosa of a susceptible in- dividual. Large respiratory droplets have a relatively short dispersal range (<1 m). On the other hand, direct hand contact is an important means of transmission within healthcare settings and adherence to strict hand-​washing is the most critical preventive measure. Gloves will only be effective if they are changed between patients. Cohorting infected patients, either by symptoms (during the outbreak season) or by rapid viral diagnostic results, is useful. Influenza A vaccination of healthcare workers, especially those caring for high-​risk children, is to be recommended. Staff education is vital, including awareness of the fact that some of these viruses manifest themselves as a mild ‘cold’ in adults, and that infected staff members can transmit to pa- tients under their care. The most dramatic example of the impact of nosocomial trans- mission with a respiratory virus occurred with SARS and MERS where healthcare facilities served as a major hub of virus transmis- sion. Much of this transmission was preventable by implementation of good infection control and prevention practices including droplet and contact precautions, although protection from small particle aerosols was important when carrying out aerosol-​generating pro- cedures such as intubation. FURTHER READING Abed Y, Boivin G (2006). 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8.5.10 Rhabdoviruses Rabies and rabies- related ly

8.5.10 Rhabdoviruses: Rabies and rabies- related lyssaviruses 805

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Kingdom-​agnostic metagenomics and the importance of complete characterization of enteric microbial communities. Gastroenterology, 146, 1459–​69. Offit PA (1994). Rotaviruses: immunological determinants of protec- tion against infection and disease. Adv Virus Res, 44, 161–​202. Patel MM, et al. (2011). Intussusception risk and health benefits of rota- virus vaccination in Mexico and Brazil. N Engl J Med, 364, 2283–​92. Payne DC, et  al. (2013). Norovirus and medically attended gastro- enteritis in U.S. children. N Engl J Med, 368, 1121–​30. Ramani S, et al. (2015). Mucosal and cellular immune responses to Norwalk virus. J Infect Dis, 212, 397–​405. Richardson V, et al. (2010). Effect of rotavirus vaccination on death from childhood diarrhea in Mexico. N Engl J Med, 362, 299–​305. Robinson CM, Pfeiffer JK (2014). Viruses and the microbiota. Annu Rev Virol, 1, 55–​69. Ruvoën-​Clouet N, Belliot G, Le Pendu J (2013). 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European Society for Paediatric Infectious Diseases consensus recommendations for rotavirus vaccination in Europe. Pediatr Infect Dis J, 34, 635–​43. Virgin HW (2014). The virome in mammalian physiology and disease. Cell, 157, 142–​50. Wold WSM, Ison MG (2013). Adenoviruses. In: Knipe DM, et al. (eds) Fields virology, 6th edition, pp. 1732–​67. Lippincott Williams & Wilkins, Philadelphia, PA. Yunus MA, et al. (2010). Development of an optimized RNA-​based murine norovirus reverse genetics system. J Virol Methods, 169, 112–​18. Zaman K, et al. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-​blind, placebo-​controlled trial. Lancet, 376, 615–​23. 8.5.10  Rhabdoviruses: Rabies and rabies-​related lyssaviruses Mary J. Warrell and David A. Warrell ESSENTIALS The Rhabdoviridae are a large family of RNA viruses, two genera of which infect animals: the genus Lyssavirus contains rabies and rabies-​related viruses that cause at least 55 000 deaths annually in Asia and Africa. Transmission and epidemiology The risks and problems posed by rabies and other lyssaviruses vary across the world. Viruses can penetrate broken skin and intact mucosae. Humans are usually infected when virus-​laden saliva is in- oculated through the skin by the bite of a rabid animal, usually a dog.

806 section 8  Infectious diseases Although the greatest threat to man is the persistent cycle of infection in stray dogs, several other terrestrial mammal species are reservoirs of infection. In the Americas, bat viruses are also classic species 1 rabies and insectivorous bats have become the principal vectors of infection to humans in the United States of America. Elsewhere in the world, there is increasing evidence of widespread rabies-​related lyssavirus infection of bats. Unrecognized infection of organ donors has proved fatal to transplant recipients. Clinical features After a highly variable incubation period (usually 20–​90 days), pro- dromal symptoms include itching at the site of the healed bite wound. These are followed by symptoms of either furious or para- lytic rabies, reflecting whether infection of the brain or spinal cord predominates. Furious rabies—​the diagnostic symptom is hydrophobia, a com- bination of jerky inspiratory muscle spasms, associated with terror, initially provoked by attempts to drink water. Patients may suffer gen- eralized arousal, during which they become wild, hallucinated, fugi- tive, and on rare occasions aggressive. Paralytic rabies—​flaccid ascending paralysis develops, starting in the bitten limb. Diagnosis The diagnosis can be made during life using rapid laboratory methods such as immunofluorescence of punch biopsy specimens of skin taken from a hairy area. Polymerase chain reaction tests are used increasingly to detect rabies in saliva and skin biopsy material. However, lack of facilities hampers the confirmation of disease in developing countries where the diagnosis usually relies on recog- nition of hydrophobic spasms and other clinical features of furious rabies. Paralytic disease is rarely identified. Rabies has been misdiag- nosed as cerebral malaria, or even drug abuse. Management and prognosis The few human survivors of rabies encephalomyelitis had received vaccine and, with two exceptions, were left with severe neurological sequelae. Only one unvaccinated patient bitten by a bat in North America has made a good recovery. However, dog rabies virus in- fection remains fatal in man. Patients with furious rabies rarely live more than one week without intensive care but survival can be up to one month with paralytic disease. Infected neurons remain vi- able but dysfunctional, and no treatment has proved effective experimentally. Management—​intensive care treatment may be appropriate for patients infected by a bat in the Americas if they present early or are already seropositive. Other patients with rabies should be sedated heavily and given adequate analgesia to relieve their pain and terror. Prevention Highly effective methods for control and prevention of rabies are available. Control of rabies in domestic dogs—​99% of human rabies deaths could be prevented by controlling the transmission of dog rabies, but education and resources are lacking. Pre-​exposure prophylaxis—​a two or three-​dose course of rabies vaccine is recommended for travellers and indigenous people in dog rabies endemic areas, but the cost is often prohibitive. Postexposure prophylaxis—​at the time of a bite, correct cleaning of the wound and optimum postexposure immunization virtually elim- inate the risk of rabies. Effective prophylaxis demands urgent wound cleaning with copious amounts of soap and water, followed by vac- cine and rabies immunoglobulin. A new 2 visit, four-​site intradermal postexposure vaccine regimen could increase the availability of af- fordable treatment. Epidemiology Rabies is a zoonosis of mammals that remains endemic in most parts of the world (Fig. 8.5.10.1). A cycle of infection is maintained in several reservoir species, of which the domestic dog is by far the most important. Many wild mammals including bats are also in- dependent rabies reservoirs (sylvatic infection) with identifiable strains of virus. Any mammalian species is potentially susceptible to rabies and may be a vector (e.g. a cat infected by a dog may then bite and infect a person). However, there is no persistent virus transmis- sion between cats. The animal source of human disease depends on the likelihood of contact with an infected species. Hence domestic dog rabies viruses are the source of 99% of human cases worldwide, mainly in Africa and Asia, but also in parts of South America. Rabies control programmes can reduce the risk of rabies in domestic ani- mals to such an extent that wild animals (e.g. insectivorous bats in the United States of America), become the principal vectors of infec- tion to humans. Rabies in wild mammals is usually spread by bites or by ingestion of infected prey. Rabies and rabies-​related viruses The Lyssavirus genus currently includes the classic rabies virus, spe- cies 1, and 15 rabies-​related species that are continent-​specific in Europe, Australasia, and Africa, and are, with one exception, zoo- noses of bats (see ‘Rabies-​related viruses known to infect humans’). New unclassified lyssaviruses are now emerging. No rabies-​related viruses have been found in the Americas and only there do bats have the classic rabies species. All terrestrial rabies reservoir mammal species (dogs and wildlife) carry classic rabies, except for the rare Mokola virus in Africa. Countries currently reported as rabies-​free include Iceland, Cyprus, and most other Mediterranean islands, Singapore, Sabah, Sarawak, Antarctica, Oceania (including New Guinea and New Zealand), Hong Kong islands (but not the New Territories), Japan, South Korea, and Caribbean islands with the notable exceptions of Cuba, the Dominican Republic, Grenada, Haiti, and Trinidad and Tobago. The British Isles, together with other Western European countries, Scandinavia, and Australia have no rabies in terrestrial species, but do harbour rabies-​related lyssaviruses in bats (Fig. 8.5.10.1). Lyssavirus-​seropositive bats have been found in every country where surveillance has been carried out, so unusual con- tact with bats should be considered as a rabies risk anywhere. Inadvertent, usually illegal importation of infected mammals is a global risk. Cyclical epizootics of rabies may result from an uncontrolled in- crease in the population of the key reservoir species, such as the fox epizootic in Europe in the late 20th century. This started in Poland and spread across France, but it has now been eliminated from

8.5.10  Rhabdoviruses 807 Western Europe. Outbreaks in dogs have followed social unrest and the movement of refugees. Although the fox is one of the species most susceptible to rabies, about 3% of animals survive the infection and become immune. Seropositive bats are not uncommon, and rabies antibody has been found in several other species, exceptionally even in dogs. There is no evidence that animals can become chronically infected or be in- fectious carriers, although an apparently healthy animal may be in- fectious during the prodromal stage of infection. Wild mammal reservoir species Wild mammal reservoir species vary in different areas. North America Reservoir species in the central United States of America and California are striped skunks Mephitis mephitis and, to a lesser ex- tent, spotted skunks Spilogale putorius; in Arizona and Texas grey foxes Urocyon cinereoargenteus and red foxes Vulpes vulpes; and in Alaska arctic foxes Alopex lagopus. However, in the east, rabies is most commonly found in raccoons Procyon lotor that transmit it to skunks and foxes. In North America many insectivorous bats are reservoirs of classic rabies virus, including big brown bats Eptesicus fuscus, Mexican free-​tailed bats Tadarida brasiliensis mexicana, little brown bats Myotis lucifugus, and silver-​haired bats Lasionycteris noctivagans whose virus is the main cause of human rabies infec- tions in the United States of America (see next) where bat infection has been found in every state. Latin America and the Caribbean Dog rabies persists in some urban areas of South America des- pite control programmes. The three species of true vampire bats Desmodus rotundus, Diaemus youngi, and Diphylla ecaudata (Desmodontinae) occur from sea level to over 3500 m but usu- ally under 1500 m only in Mexico, Central and South America, and some Caribbean islands (Fig. 8.5.10.2). The common vam- pire bat D. rotundus (Fig. 8.5.10.3) is the main reservoir of vampire bat rabies in Trinidad, Mexico, and Central and South America, where humans are occasionally bitten (Fig. 8.5.10.4). Carnivorous bats of the family Megadermatidae, such as the Indian ‘vampire’ Megaderma lyra, have given rise to the myth that vampires occur elsewhere. In Latin America, thousands of cattle are lost each year from vampire bat-​transmitted paralytic rabies (derriengue) with locally serious economic consequences. Mongooses Herpestes auropunctatus are reservoirs of sylvatic ra- bies in Central America, Grenada, Puerto Rico, Cuba, Haiti, and the Dominican Republic. Fig. 8.5.10.1  Global distribution of rabies and rabies-​related lyssaviruses which infect humans. Red: Rabies in terrestrial mammal species (Lyssavirus classic rabies) and bat infections by other lyssavirus species. Yellow: Terrestrial and bat rabies are all the classical species. Green: Bat lyssaviruses, Australian bat lyssavirus European bat lyssaviruses and Irkut, only. White: No lyssaviruses reported. Isla de Margarita Trinidad Fig. 8.5.10.2  Distribution of the three species of true vampire bats (Desmodontinae).

808 section 8  Infectious diseases Africa and Asia Dog rabies predominates but there is sylvatic rabies in Africa in foxes, wolves, jackals, and small carnivores of the families Mustelidae and Viverridae (e.g. the yellow mongoose Cynictis penicillata in South Africa), and in Asia in wolves, jackals, ferret-​badgers Melogale moschata in China and Taiwan, and palm civets Paradoxurus her- maphroditus in Indonesia. Europe Foxes, wolves, raccoon dogs Nyctereutes procyonoides, and insect- ivorous bats are infected (see also ‘Rabies-​related viruses known to infect humans’). Rodents There are reports of rabies virus being isolated from wild rodents in many countries, especially in China. They are unlikely to be a reservoir species. There are very rare Chinese reports of rodent-​ transmitted human rabies diagnosed clinically. Monkeys Monkey bites are very common in tourists, especially to Asia. Rabies has been reported in non​human primates but they are not reser- voirs of infection except for marmosets Siamiri sciuerus in Brazil, which have caused human cases. A variety of other species have been reported to be infected in South America, Africa, and Asia, but transmission to humans has very rarely been clinically diagnosed only in India and Sri Lanka. However, the risk of rabies should al- ways be considered, especially if an animal is behaving abnormally or the bite is severe. Incidence of human rabies The true incidence of human rabies throughout the world is not re- flected in official figures; 59 000 deaths annually have been estimated to occur in Asia and Africa, including about 20 000 in India alone. High mortalities also occur in Bangladesh and Pakistan, and the in- cidence has been rising in China. Surveillance is minimal, especially in Africa. The World Health Organization (WHO) no longer issues country-​specific data. In Latin America, the risk of canine rabies persists in Brazil, Bolivia, Dominican Republic, Peru, Guatemala, and Haiti. Vampire bat rabies has been reported recently in Peru, Ecuador, Mexico, and Brazil. In the United States of America there are on average two human deaths annually. Among 24 indigenous infections occurring in 10 years, 21 (88%) were caused by insect- ivorous bats. Europe reported 68 deaths in the last 15 years, mainly from the Russian Federation, Ukraine, and Georgia. Rabies was ap- parently eliminated from the United Kingdom by 1903, but since 2000 there have been five imported cases and one indigenous human European bat lyssavirus infection. Virology The Rhabdoviridae are a family of more than 100 bullet-​shaped RNA viruses found in vertebrates, insects, and plants (Fig. 8.5.10.5). Two genera infect animals, Vesiculovirus and Lyssavirus. Vesicular stomatitis virus is a vesiculovirus of cattle and horses, which oc- casionally causes an influenza-​like illness in farmers or laboratory workers. The genus Lyssavirus contains rabies and rabies-​related viruses. The rabies virion is approximately 180 × 75 nm. Its core is a single spiral strand of negative non​segmented RNA associated with a nu- cleoprotein, a phosphoprotein, and an RNA polymerase to form a helical ribonucleoprotein complex. This is enveloped in a matrix protein, host cell-​derived lipid, and a coat of protruding glycopro- tein (G) molecules bearing spikes or knobs 10 nm long. The compos- ition of the glycoprotein determines viral virulence. The virus is readily inactivated by ultraviolet light, drying, boiling, most organic lipid solvents including at least 45% ethanol, soap solu- tion, detergents, hypochlorite, and glutaraldehyde solutions. Fig. 8.5.10.3  Desmodus rotundus common vampire bat (Peru). Courtesy of Dr Vargas Meneses, Lima, Peru. Fig. 8.5.10.4  A typical puncture wound inflicted by vampire bat, Madre de Dios, Peru. Courtesy of Dr Vargas Meneses, Lima, Peru.

8.5.10  Rhabdoviruses 809 Genetic sequencing techniques allow the identification of diverse strains of rabies and rabies-​related viruses from different geograph- ical areas and vector species. Transmission Virus can penetrate broken skin and intact mucosae. Humans are usually infected when virus-​laden saliva is inoculated through the skin by the bite of a rabid dog or other mammal (Fig. 8.5.10.6a, b). Saliva from a rabid animal can infect if the skin is already broken (e.g. by the animal’s claws). In North America, contact with bats leading to rabies has passed unnoticed; only 39% of patients reported a bat bite and 34% had no history of exposure to bats. Animals can be infected through the gastrointestinal tract, but there is no evi- dence that this happens in humans. Inhalation of aerosolized virus created by infected nasal secretions of bats may be a method of transmission among cave-​dwelling bats. In Texas, two men died of rabies after visiting caves inhabited by millions of Mexican free-​tailed bats Tadarida brasiliensis mexicana, some of which were rabid; however, fleeting bat contact is more likely to have caused the infection. Two laboratory workers in the United States of America developed rabies after inhaling aerosol- ized fixed strains of rabies virus during the preparation of vaccines. The accidental use of vaccine in which the virus was not inactivated has led to fixed virus rabies (rage de laboratoire), for example, in Fortaleza, Brazil in 1960. Transmission of rabies between people has been proved in 16 cases of tissue transplantation from donors who had died of un- diagnosed neurological diseases. Six recipients of infected corneal grafts developed retro-​orbital headache on the side of the graft 22–​ 39 days after transplantation and died soon afterwards (other infec- tions spread by corneal grafts include Creutzfeldt–​Jakob disease and cryptococcosis). In the United States and Germany, eight recipients of kidney, liver, lung, pancreas, or even just a segment of iliac artery, developed rabies encephalitis. Rabies was not suspected in the three donors despite high risk histories of rough travel in India, a bat bite, or raccoon hunting. Recreational drug abuse was detected in two. Rabies developed 18 months after receiving the raccoon rabies in- fected kidney graft, but 3 other recipients of the donor’s organs were unaffected. Transplant related deaths also occurred in China and the Middle East. Postexposure prophylaxis following corneal trans- plants from infected donors has been successful. Considering that the saliva, respiratory secretions, and tears of ra- bies patients contain virus, it is surprising that there is no documented case of the disease being spread to intimate relatives and nurses. Transplacental infection has been observed in animals but has only been reported once in humans. Several women with rabies en- cephalitis have given birth to healthy babies. The transmission of rabies from mother to suckling infant via the breast milk has been suspected in at least one human case and is well known in animals. Pathogenesis The mechanism by which the highly neurotropic rabies virus en- ters the nervous system and travels into the brain and out again to Fig. 8.5.10.5  Rhabdoviruses. Virion of rabies virus. (Note the surface projections composed of glycoprotein (G). The marker line is 100 nanometres long) (a) (b) Fig. 8.5.10.6  Bites inflicted by rabid dogs in (a) Nigeria, and (b) Thailand. These wounds carry a high risk of rabies with a short incubation period. Copyright D. A. Warrell.

810 section 8  Infectious diseases many organs is intriguing. The virus may replicate locally in muscle cells or attach directly to nerve endings. It can bind to many types of receptors including the nicotinic acetylcholine receptors at motor endplates, which may concentrate the virus at this postsynaptic site before entry into the presynaptic axon terminal, possibly via the neural cell adhesion molecule. Several other neuronal binding mechanisms may be involved. Once inside peripheral nerves, virus travels in a strictly retrograde direction up the axon. The dynein mo- lecular motor transports the virus within vesicles towards the cell soma, where replication begins. How the virus components move along the dendrite to emerge at the next synapse as complete virions is unknown. Rabies virus is experimentally inaccessible to serum antibody while concealed in the peripheral nerves. On reaching the central nervous system, the virus replicates mas- sively within neurons and continues to be transmitted cell to cell trans-​synaptically. Dramatic symptoms can appear before histo- pathological changes are apparent. Viral virulence is inversely re- lated to neuronal apoptosis. Rabies alters host cell gene expression, but the mechanisms of gross neuronal dysfunction are speculative. Centrifugal spread of virus from the central nervous system, appar- ently in somatic and autonomic efferent nerves, deposits virus in many tissues including skeletal and cardiac muscle, adrenal medulla where infection may be clinically significant, and also in kidney, retina, cornea, pancreas, taste buds, respiratory tract, and the skin in nerve twiglets around hair follicles (see ‘Laboratory diagnosis’). At this stage, productive viral replication occurs, with budding from outer cell membranes in the salivary glands, and then rabies may be transmitted by bites to other mammals. Viraemia has been detected very rarely, only in animals, and is not thought to be involved in pathogenesis or spread. Immunology Immunological response to rabies infection in humans Some patients die without any detectable immune response, because rabies virus evades and suppresses the immune system. Antibody might become detectable in serum seven days or more after the onset of illness and in cerebrospinal fluid a little later. It may rise to high levels in patients whose lives are prolonged by intensive care. A small amount of rabies-​specific IgM is sometimes detectable, but is not useful as a means of diagnosis. There is little evidence of a lymphocyte-​mediated immune re- sponse to rabies encephalitis. A pleocytosis appears in only 60% of patients, with a mean leucocyte count of 75 × 103/​mm. Peripheral blood lymphocyte transformation is minimal if any. Experimentally, in fatal rabies there is inhibition of innate immunity, particularly interferon activity, and the few immune lymphocytes entering the brain undergo apoptosis, whereas survival is associated with in- creased permeability of the blood–​brain barrier, neutralizing anti- body in the brain, expression of rabies glycoprotein, and apoptosis of infected neurons. Also in animals, latent infections can be reacti- vated by corticosteroids and stress. This provides a possible explan- ation for occasional reports of long incubation periods Immunological response to rabies vaccination The viral glycoprotein induces neutralizing antibody, which is de- tectable from 2 weeks after the start of primary immunization. In animal studies, the neutralizing antibody titre is the best available measure of protection against death. A titre of 0.5 IU/​ml indicates specific seroconversion and is the WHO minimum acceptable level after vaccination. A relatively low response occurs in about 3% of the population, in immunosuppressed patients, and in older people. The nucleoprotein antigens also stimulate antibody that is cross-​reactive between lyssaviruses, whereas glycoprotein antibody is more strain-​ specific. Transient low levels of interferon may be induced after the first dose of rabies vaccine. Although neutralizing antibody is un- doubtedly protective in the early stages after inoculation of virus, it may be deleterious once central nervous system infection is estab- lished. In animals, acceleration of the terminal phase of the enceph- alitis (‘early death phenomenon’) is associated with the presence of low titres of rabies antibody in serum. Rabies in animals All warm-​blooded animals can be infected with rabies, but their sus- ceptibility varies. However, only mammals are infected naturally. In dogs, the incubation period ranges from 5 days to 14 months, but is usually between 3 and 12 weeks. The first symptom, as in many humans, is intense irritation at the site of the infection. Despite the popular idea of the ‘mad’ rabid dog, probably only a minority de- velop furious rabies. There is an early and striking change in the dog’s behaviour with dysphagia, ptosis, altered bark, paralysis of the jaw, neck, and hind limbs (Fig. 8.5.10.7), hypersalivation, congested conjunctivae, pruritus, shivering, trembling, snapping at imaginary objects, pica, and extreme restlessness causing the animal to wander miles from home. Dogs with furious rabies attack inanimate objects, often seriously injuring their mouths in the process. The virus has Fig. 8.5.10.7  Dog with paralytic rabies showing paralysis of the limbs and hypersalivation. Copyright D. A. Warrell.

8.5.10  Rhabdoviruses 811 been found in the saliva 3 days before symptoms appear, and the animal usually dies within the next 7 days. This is the basis for the traditional 10-​day observation period for dogs that have bitten humans. Very rare old reports from India, Ethiopia, and Nigeria of persistent or intermittent excretion of virus in the saliva of apparently healthy dogs have not been confirmed by subsequent thorough searches. ‘Oulou fato’, a clinical variant of canine rabies with reduced virulence, was seen in West Africa 50 years ago. Rabid foxes lose their fear of humans and the majority develop paralytic rabies. An extreme degree of furious rabies is seen in 75% of infected cats. Cattle usually develop paralytic symptoms with dys- phagia, hypersalivation, groaning, trembling, colic, diarrhoea, ten- esmus, and rectal prolapse. Most other domestic ungulates develop paralytic symptoms. Horses often show furious features with sexual excitement. Most wild animals, like foxes, lose their fear of humans and may appear tame. Rabid skunks, raccoons, badgers, martens, and mongooses may become very aggressive. Dysphagia and in- ability to drink is common in rabid animals, but they do not exhibit hydrophobia. Clinical features in humans The incubation period ranges from 4 days to many years, but it is be- tween 20 and 90 days in three-​quarters of cases. It tends to be shorter after bites on the face (average 35 days) than after those on the limbs (average 52 days). Prodromal symptoms Often, the first symptom is itching, pain, or paraesthesia at the site of the healed bite wound (Fig. 8.5.10.8). Non​specific prodromal symptoms include fever, chills, malaise, weakness, tiredness, head- ache, photophobia, myalgia, anxiety, depression, irritability, and symptoms of upper respiratory tract and gastrointestinal infections. Subsequently, symptoms of either furious or paralytic rabies will de- velop, depending on whether the spinal cord or brain are predom- inantly infected. Furious rabies Furious rabies is the more common presentation. Most patients have the diagnostic symptom of hydrophobia, which is a com- bination of inspiratory muscle spasm, associated with terror (Fig. 8.5.10.9a–​f). Initially provoked by attempts to drink water, this reflex can be excited by a variety of stimuli including a draught of air (‘aerophobia’), water splashed on the skin, irritation of the re- spiratory tract or, ultimately, by the sight, sound, or even mention of water. The inspiratory spasm is violent and jerky. The neck and back are extended, the arms thrown up, and the episode may end with the patient in a position of extreme extension—​opisthotonos, having a generalized convulsion complicated by cardiac or a re- spiratory arrest. Patients experience hyperaesthesia and, at times, generalized arousal during which they become wild, hallucinated, fugitive, and sometimes aggressive (Fig. 8.5.10.10). This behaviour alternates with periods of mental lucidity during which patients may become distressingly aware of their predicament. Despite these dramatic symptoms, attributable to brainstem encephalitis, conventional neurological examination may prove to be completely normal, leading to the false assumption of conversion disorder. Reported abnormalities include meningism, cranial nerve lesions (especially III, VI, VII, IX–​XII), upper motor neuron lesions, fasciculation, and involuntary movements. Disturbances of the hypothalamus or autonomic nervous system are reflected by hypersalivation (Fig. 8.5.10.11a, b), sweating, lacrimation, hypertension or hypoten- sion, hyperthermia or hypothermia, diabetes insipidus or inappro- priate secretion of antidiuretic hormone, and rarely, priapism with spontaneous orgasms, satyriasis, or nymphomania. Hypersexuality suggests similar aetiology to the Klüver–​Bucy syndrome created in rhesus monkeys by bilateral ablation of the hippocampus. Without supportive treatment, about one-​third of the patients will die during a hydrophobic spasm during the first few days. The rest lapse into coma and generalized flaccid paralysis, and rarely survive for more than a week without intensive care. Paralytic or dumb rabies This is the clinical pattern recognized in less than one-​fifth of human cases except in the case of bat-​transmitted rabies, especially vampire bat infection, which is usually paralytic. Patients may become liter- ally dumb (‘rage muette’) because their laryngeal muscles are para- lysed, but symptoms are quieter (‘rage tranquille’) than in furious rabies. The largest reported outbreak was in Trinidad between 1929 and 1936 when there were 53 human cases, initially misattributed to poliomyelitis or botulism; others have been described from Mexico, Guyana, Brazil, Peru, Ecuador, Bolivia, and Argentina. The paralytic form of rabies was also seen in patients with postvaccinal rabies, in the two patients who inhaled fixed virus, and is said to be more likely to develop in patients who have received antirabies vaccine. After the same prodromal symptoms, especially fever, headache, and local par- aesthesias, flaccid paralysis develops, usually in the bitten limb, and ascends symmetrically or asymmetrically with pain and fasciculation in the affected muscles and mild sensory disturbances. Paraplegia and sphincter involvement then develop, and finally fatal paralysis of deglutitive and respiratory muscles (Fig. 8.5.10.12). Hydrophobia is unusual, but may be represented by a few pharyngeal spasms in the terminal phase of the illness. Even without intensive care, patients with paralytic rabies have survived for up to 30 days. Other manifestations and complications Respiratory system Asphyxiation and respiratory arrest can complicate the hydrophobic spasms or generalized convulsions of furious Fig. 8.5.10.8  This man developed intense itching in the right leg, provoking scratching and excoriation, 8 weeks after being bitten in that limb by a rabid dog. He died with furious rabies a few days later. Copyright David A. Warrell.

812 section 8  Infectious diseases rabies and the bulbar and respiratory paralysis of dumb rabies. Bronchopneumonia is a predictable complication if life is pro- longed by intensive care, but a primary rabies pneumonitis may occur. Various abnormal patterns of respiration have been de- scribed, including cluster and apneustic breathing. There are some similarities to palatal myoclonus. Pneumothorax may complicate inspiratory spasms. Cardiovascular system A variety of dangerous cardiac arrhythmias have been reported, including supraventricular tachycardias, sinus bradycardia, atrioventricular block, and sinus arrest, together with T wave and ST segment changes (Fig. 8.5.10.13). Hypotension, pulmonary oedema, and congestive cardiac failure are attributable to myocarditis. Nervous system Raised intracranial pressure resulting from cerebral oedema or in- ternal hydrocephalus has been reported in a few cases, but spinal fluid opening pressure is usually normal and papilloedema is rarely seen. There is clinical and electrophysiological evidence of diffuse axonal neuropathy, consistent with histological appearances of de- generation of peripheral nerve ganglia and axons. (b) (a) (c) (d) (e) (f) Fig. 8.5.10.9  Hydrophobic spasms (a–​e) in a 14-​year-​old Nigerian boy with furious rabies. Note the violent contraction of inspiratory muscles (sternomastoids and diaphragm) depressing the xiphisternum. (f) In a Thai man who had just asked to drink water. Copyright D. A. Warrell.

8.5.10  Rhabdoviruses 813 Gastrointestinal system ‘Stress’ ulcers and the Mallory–​Weiss syndrome are possible explan- ations for the haematemesis often reported in rabies. Clinical and differential diagnosis Rabies should be suspected in any patient who develops neuro- logical symptoms after being bitten by a mammal in a rabies en- demic area. However, some patients fail to remember that they have been bitten and others may be infected while they are asleep possibly by contact with lip mucosae (North American insectiv- orous bats) or near-​painless bites by vampire bats in parts of Latin America. Furious rabies Pathognomonic inspiratory spasms with associated emotional re- sponse are provoked by asking the patient to swallow accumulated saliva or by directing a draught of air on to the face. • Psychiatric conditions:  Rabies encephalitis has been misdiag- nosed as a variety of psychiatric conditions, including conversion disorder and behavioural disturbances attributed to recreational drugs. Conversely, patients with a morbid fear of rabies (rabies phobia, lyssaphobia, pseudohydrophobia) may simulate the more melodramatic features of the disease but hydrophobia is unlikely to be mimicked accurately, the incubation period after the bite (hours or a few days) is usually much too short for rabies enceph- alitis, and the prognosis is, of course, excellent. • Otolaryngological conditions:  Pharyngeal and upper airway symptoms of hydrophobia may be misinterpreted as pharyngitis or laryngitis so that the patient is referred to an otolaryngologist. • Tetanus: This can also follow an animal bite and is similar to ra- bies in some respects, especially the pharyngeal form of cephalic tetanus (‘hydrophobic tetanus’). It is distinguished by its shorter incubation period (usually less than 15 days in severe tetanus), Fig. 8.5.10.10  Episode of intense arousal in a Nigerian patient with furious rabies. Copyright D. A. Warrell. (a) (b) Fig. 8.5.10.11  Autonomic overactivity in rabies encephalomyelitis. (a) Sweating and hypersalivation. (b) Salivation and lacrimation. Copyright D. A. Warrell. Fig. 8.5.10.12  Paralytic rabies. Copyright D. A. Warrell.

814 section 8  Infectious diseases the presence of trismus, the persistence of muscle rigidity between spasms, the absence of meningoencephalitis (cerebrospinal fluid is universally normal), and the better prognosis. • Other encephalopathies/​encephalitides: The typical encephalitic progression from severe headache to continuous coma is un- usual in furious rabies. Hydrophobia with intermittent excita- tion and lucid intervals of full consciousness does not occur in other encephalitides. Among children with suspected cerebral malaria in Malawi, some were proved by biopsy to have died of rabies. Toxic encephalopathies: Delirium tremens, some drugs (pheno- thiazines, amphetamines, modafinil, cocaine, and other recre- ational drugs), and plant poisonings (e.g. thorn apple, Datura stramonium) can cause excitable and aggressive behaviour that might be confused with rabies. Paralytic rabies Other causes of ascending (Landry-​type) paralysis may enter the differential diagnosis. • Postvaccinal encephalomyelitis (see next): This usually develops within 2 weeks of the first dose of the now rarely used nervous tissue rabies vaccines. • Poliomyelitis:  Objective sensory disturbances are absent, and fever rarely persists after paralysis has developed. • Acute inflammatory polyneuropathy (Guillain–​Barré syn- drome): Cerebrospinal fluid examination will help to distinguish this condition. • Cercopithecine herpesvirus (B virus) encephalomyelitis:  Bites and other types of contact with Asian macaque monkeys (genus Macaca), especially rhesus (M. mulatta) and cynomolgus (M. fas- cicularis) transmit this dangerous infection. The incubation period (3–​4 days) is usually shorter than in rabies and symptoms develop within 1 month of contact. Vesicles may be found in the monkey’s mouth and at the site of the bite, and the diagnosis can be confirmed virologically. Pathology The brain, spinal cord, and peripheral nerves show ganglion cell de- generation, perineural, and perivascular mononuclear cell infiltra- tion, neuronophagia, and glial nodules. Inflammatory changes are most marked in the midbrain and medulla in furious rabies and in the spinal cord in paralytic rabies. Negri bodies (Fig. 8.5.10.14), eosinophilic intracytoplasmic in- clusions, function as viral factories containing rabies RNAs and translated proteins. They can be demonstrated by haematoxylin and eosin stains in histological sections of grey matter in up to 75% of human cases, especially in hippocampal pyramidal cells and cere- bellar Purkinje cells. In view of the appalling prognosis of rabies encephalitis, neuro­ nolysis is often surprisingly mild and patchy, and death can occur without any inflammatory response. Vascular lesions such as throm- bosis and haemorrhage have also been described. The brainstem, limbic system, and hypothalamus appear to be most severely affected and, in paralytic disease, the spinal cord and medulla. Outside the nervous system, there is focal degeneration of salivary and lacrimal glands, pancreas, adrenal medulla, and lymph nodes. An interstitial myocarditis with round cell infiltration is found in about 25% of cases. Laboratory diagnosis If a mammal suspected of being rabid has bitten, scratched, or other- wise may have infected a person, it should be killed, and its brain examined without delay. The best way to detect rabies antigen in Fig. 8.5.10.13  Electrocardiogram in a Nigerian patient with furious rabies
showing sinus tachycardia, atrial and ventricular premature beats, and a wandering atrial pacemaker. Copyright D. A. Warrell. Fig. 8.5.10.14  Street virus in human cerebellar Purkinje cells as
seen with the light microscope. Several Negri bodies can be seen
(one is arrowed). Magnification × 615. Courtesy of Armed Forces Institute of Pathology 73–​12 330.

8.5.10  Rhabdoviruses 815 acetone-​fixed brain impression smears is by the direct immuno- fluorescent antibody (IFA) test. Alternatively, if no fluorescent microscope is available, rapid enzyme immunodiagnosis can be used. Virus isolation takes about 4 days in cell culture. In humans, rabies can be confirmed early in the illness by dem- onstration of viral antigen by the direct IFA test in frozen sections of full-​thickness skin biopsies taken from a hairy area, usually the nape of the neck. Specific diagnostic staining is seen in nerve twiglets around the base of hair follicles (Fig. 8.5.10.15). This rapid method is positive in 60–​100% of cases, and no false-​positive results have been reported. Antigen can also be found in brain biopsies, but tests on corneal impression smears are very unreliable. Reverse transcription polymerase chain reaction (RT-​PCR) is now used to detect rabies in saliva, skin biopsy material, and occasionally cerebrospinal fluid. During the first week of illness, virus may be detected in saliva, brain, cerebrospinal fluid, and very rarely urine. Rabies antibodies are not usually detectable in serum or cerebrospinal fluid before the eighth day of illness in unvaccinated patients. The IFT antibody test can cross-​react with other viruses, so low levels alone are not diagnostic. Serum antibody may leak into the cerebrospinal fluid in patients with postvaccinal encephalomyelitis, but a very high titre suggests a diag- nosis of rabies. A specific IgM test has not proved useful diagnostically. Prognosis There is no specific antirabies therapeutic agent. Rabies was formerly regarded as a universally fatal disease, but there are reports of 10 cases of recovery or prolonged survival following intensive care. The diagnoses were made serologically except in three cases where virus was identified by PCR. Nine patients who had received some vaccine before the onset of symptoms, survived months or years with inten- sive care. However, seven had profound neurological impairment but two recovered. A boy in Ohio USA infected by an insectivorous bat in 1970, had delayed treatment with duck embryo vaccine and completely recovered after intensive care therapy. Also, a Turkish man who had one dose of vaccine 4 days after a dog bite, was said to have recovered completely from rabies encephalitis, according to a brief report. Neutralizing antibody was present on the second day in hospital and he recovered spontaneously without intensive care. The first unvaccinated patient to recover from rabies has re- turned to near normal life following intensive care and antiviral therapy. She was bitten by a bat in Wisconsin in 2004, had no ra- bies prophylaxis, and developed typical encephalitis without hydro- phobia. Rabies neutralizing antibody was detected on the sixth day of illness. Treatment comprised coma induction and antiviral drugs. She made a slow recovery over 5 years and has returned to normal life, although with minor neurological deficits. The antiviral treatments have not proved effective against rabies experimentally; however, she developed antibody at an early stage of the disease. Her treatment possibly maintained her vital functions until her immune response eliminated the virus, probably with loss or mal- function of infected neurons. In animal experiments, American bat rabies virus infection differs from that of canine virus in that it is slower to evolve and progress, virus replication is not restricted to neurons, and histopathological changes are milder. This suggests that the virus may be less pathogenic and may also explain the re- covery of the boy in Ohio bitten by a bat. It is likely that he too had rabies antibody present at an early stage of illness. Treatment No treatment has yet proved effective in animal models. Antiserum, antiviral agents, interferon-​α, corticosteroid, and other immuno- suppressants have proved useless. Human rabies of canine origin remains 100% fatal in unvaccinated patients. The ‘Milwaukee’ treatment protocol used in Wisconsin has since been used unsuc- cessfully in 30 other patients with rabies encephalitis who were infected by bats or dogs. There is no evidence that it is superior to supportive intensive care. Until a new treatment is proved effective experimentally, palliation of the patient’s symptoms and immunization of contacts is recommended. Patients must be sedated heavily and given adequate analgesia to relieve their pain and terror. Intensive treatment may be appropriate for patients infected by an American bat, who present early, and are already seroposi- tive. Intensive care is inappropriate for canine virus infection, especially in developing countries, and the cost is prohibitive. If intensive care is undertaken, the aim is to prevent complications such as cardiac arrhythmias, cardiac and respiratory failure, raised intracranial pressure, convulsions, fluid and electrolyte disturb- ances, including diabetes insipidus and inappropriate secretion of antidiuretic hormone, and hyperpyrexia. A future treatment could be intrathecal live attenuated rabies virus to induce intracerebral neutralizing antibody to eliminate infection. Fig. 8.5.10.15  Diagnosis of human rabies during life. Vertical section through a hair follicle and shaft showing fluorescence of nerve cells around the follicle indicating the presence of rabies antigen. Magnification × 250. Copyright M. J. Warrell.

816 section 8  Infectious diseases Control of rabies in animals The elimination of dog rabies would reduce the human mortality by over 99% and drastically reduce the need for human vaccin- ation. Rabies control has been achieved most effectively where the principal reservoir is the domestic dog, as in 19th-​century United Kingdom, Malaysia, and Japan, and since then in other areas including Western Europe, Taiwan, North America, and parts of urban Latin America. In countries where rabies is enzootic The control strategy depends on the local pattern of rabies oc- currence in wild and domestic animals. Education and publicity about rabies is always needed. Domestic animals can be protected by regular vaccination. Owned dogs can be muzzled or kept off the streets. People should be discouraged from keeping wild car- nivores such as skunks, raccoons, coatis, and mongooses as pets. Unnecessary contact with mammals should be avoided (e.g. stroking stray dogs or apparently friendly wild animals, exploring bat-​infested caves). Culling reservoir species has proved an un- popular and ineffective method of long-​term control. Impressive reduction of urban rabies in stray dogs has proved possible in India by vaccination, population control, and reducing available food and shelter by removing refuse. Effective oral vaccination of dogs is not yet practicable. Control of sylvatic rabies has been achieved by vaccination of key wild animal reservoir populations with live oral vaccines dis- tributed in bait. Repeated campaigns distributing attenuated rabies vaccine have eliminated fox rabies in Western Europe, and vaccinia-​ recombinant vaccine expressing rabies glycoprotein has been used in North American coyotes, foxes, and raccoons. New vaccines are being developed for other species. Vaccination of bats is unlikely to be feasible. Vampire bat rabies is controlled by destroying roosts and poisoning the bats with anticoagulants. In countries where rabies is not endemic The inadvertent importation of a mammal incubating rabies is a universal risk. The movement of potential vectors, especially do- mestic dogs and cats, wild carnivores, and bats, should be strictly controlled. Serological evidence of successful vaccination should be provided for imported mammals, or they should be vaccinated on arrival and quarantined. Prevention of human infection Pre-​exposure prophylaxis Pre-​exposure vaccination is the most effective form of rabies pre- vention. No rabies deaths have been reported in anyone who had had pre-​exposure vaccine and then postexposure booster doses. It is recommended for people who handle imported animals, workers in zoos and rabies laboratories, and those who are resi- dent in or intend to travel to dog rabies-​endemic areas, especially children. Others particularly at risk in certain areas include veter- inarians, dog catchers, farm workers, cave explorers, naturalists, and animal collectors. In dog rabies-​endemic areas, pre-​exposure prophylaxis is advisable but is rarely used. Travellers should be educated to seek immediate local medical help if they are bitten, scratched, or licked by mammals. However, recommendations vary in different areas and local advice may be unreliable. Tissue culture vaccine and especially rabies immune globulin may not be readily available. Primary pre-​exposure vaccine course A course of three doses of tissue culture rabies vaccine (see next) is given intramuscularly (IM) into the deltoid, or the anterolateral thigh in children, on days 0, 7, and 21–28. The 2018 WHO recommenda- tions include a 2 dose IM regimen, on days 0 and 7. An effective economical alternative is intradermal (ID) injections of 0.1 ml, 3 doses at the same intervals, but the latest recommendations give a 2-site, one week ID alternative regimen on days 0 and 7. If the ID injection is too deep to produce a papule, withdraw the needle and repeat the procedure. Rabies vaccines do not contain preservatives. Strict aseptic precautions are mandatory to avoid contamination. One vaccine ampoule can be shared but must be used within a day or discarded. If chloroquine is being taken for malaria prophylaxis (unlikely today), or in other cases of suspected immunosuppression, the intramuscular route must be used. Many travellers cannot afford three doses of an expensive vaccine, so the economical intradermal route is ideal for family, student, or other groups who can be vaccin- ated on the same day. Pre-​exposure booster doses A booster dose 1–​2 years after the primary course enhances and prolongs the presence of antibody. Although the titre falls more rapidly after intradermal than intramuscular inoculation, the re- sponse to a booster dose is still prompt. Confirmation of serocon- version is recommended only if immunosuppression is suspected. Further booster doses for those at higher risk may be given ID or IM at intervals of 2–​10 years. Boosters are not necessary if the ra- bies neutralizing antibody level is at least 0.5 IU/​ml. Laboratory staff at high risk should have more frequent serology tests. Travellers who will have rapid access to vaccine if exposed need not have further immunization, but if medical resources will be unreliable, a booster vaccination should be given before departure if 5 years have elapsed since the previous dose. A personal record of immunization must be kept, and urgent treatment is essential after possible exposure. Lyophilized rabies vaccine is relatively stable even at tropical ambient temperatures. It is sensible to take a dose on expeditions to remote rabies endemic areas. An extra emergency injection can then be given immediately after a risky encounter with an animal. If more than one person is exposed, the ampoule can be shared by giving multiple ID doses to each, using the whole dose (see Postexposure prophylaxis’, next). This does not replace the normal postexposure treatment, which must still be given as soon as possible. Postexposure prophylaxis Despite intensive care, rabies encephalomyelitis of canine origin re- mains 100% fatal in unvaccinated patients. At the time of the bite, however, correct cleaning of the wound (see next) and optimum postexposure immunization reduce the risk of rabies to nearly zero compared to about 35–​57% for untreated bites by proven rabid ani- mals. The risk varies with the biting species and the site and severity of the bites. It is highest following bites to the head by proven rabid

8.5.10  Rhabdoviruses 817 wolves, which carries a case fatality exceeding 80% in unvaccinated people. The decision to give postexposure treatment depends on an assessment of the risk of infection. Intact skin is a barrier against the virus. Ask about the precise geographical location of the exposure; its severity, whether it was a bite or lick on broken skin; the site of the lesion; and the nature, appearance, behaviour, and fate of the biting animal, and whether it had been recently vaccinated against rabies (Box 8.5.10.1). The animal’s brain must be tested for rabies if possible. If there is any doubt, the patient should be given full postexposure prophylaxis, even if the bite is several months old. If the exposure was more than a year previously no rabies immuno- globulin is needed. The aim of prophylaxis is to neutralize inoculated virus before it can enter the nervous system. Wound cleaning and active and passive immunization must be implemented as soon as possible. Wound cleaning This is effective in killing virus in superficial wounds, but is often neglected. First aid includes vigorous cleaning of the wound with soap or detergent and water under a running tap for at least 5 min. Foreign material should be removed and a viricidal agent such as povidone iodine, or 40 to 70% alcohol, should be applied liber- ally. Quaternary ammonium compounds such as benzalkonium chloride are inactivated by soap and so are not recommended. Hospital treatment of wounds involves thorough exploration, debridement, and irrigation of deep lesions, if necessary under local or general anaesthetic. Suturing should be avoided or de- layed, and the wound left without occlusive dressings. Attention should be given to tetanus prophylaxis and the large range of viral, bacterial, and fungal pathogens particularly associated with mammal bites. These include Cercopithecine herpesvirus (B virus) from Asian macaques (Chapter 8.5.2); Pasteurella multo- cida (Chapter  8.6.18), Francisella tularensis (Chapter  8.6.19), Streptobacillus moniliformis, and Spirillum minus (Chapter 8.6.13) from rodents; and Pasteurella multocida, Capnocytophaga cani- morsus, and Bartonella henselae (Chapter 8.6.42) from dogs and/​ or cats. Most of the bacteria are sensitive to amoxicillin/​clavulanic acid, cefoxitin, or tetracycline. Active immunization Rabies vaccines Three highly immunogenic tissue culture vaccines that meet the WHO recommended standards are purified chick embryo cell (PCEC) vaccine, purified Vero cell rabies vaccine (PVRV), and human diploid cell vaccine. Several tissue culture vaccines are produced, mainly for national use, in China, India, Japan, and Russia. Obsolete nervous tissue rabies vaccines are not sanctioned by the WHO, but suckling mouse brain (Fuenzalida) vaccine is still used in a few countries in South America and in Algeria. Daily subcuta- neous doses for 7–​14 days, followed by booster doses, are usually given over the abdominal wall. Neurological reactions including postvaccinal encephalomyelitis could still occur. Postexposure tissue culture vaccine regimens The IM Essen regimen has become 4 × 1-​ml (PVRV 0.5 ml) doses injected into the deltoid (or anterolateral thigh in children) on days 0, 3, 7 and 14–28. There is no change to the alternative 2-​1-​1 IM regimen, of two full doses injected into the deltoids on day 0, and one dose on days 7 and 21. The intramuscular regimens are unaffordable in many countries. However, economical multisite ID methods are available, requiring less vaccine than the IM regimens. Each of the intradermal injection sites drains to a different group of lymph nodes, intended to stimu- late more lymphoid tissue to produce antibody. Aseptic precautions are required as for pre-​exposure ID treatment. The simplified four-​site ID regimen replaces the eight-​site ID regimen. It consists of a whole ampoule of vaccine divided be- tween four intradermal injections over the deltoid and the thigh or suprascapular areas. The volume per site is about 0.1 ml for PVRV and the equivalent dose for vaccines containing 1 ml per ampoule is 0.2 ml. On day 7, two intradermal injections of 0.1/​0.2 ml in the deltoid areas are followed by a single intradermal dose on day 28. Since giving half the dose, 0.1 ml of PCECV (1 ml/​ampoule) was found to be immunogenic in trial conditions, there is a wide safety margin in case of inexperience with ID injection technique or for immunosupressed patients. Without sharing ampoules, a maximum of three doses are needed. The two-​site ID regimen was designed for use with PVRV. A dose of 0.1 ml for PVRV, or 0.2 ml for vaccines formulated in vials con- taining 1 ml, is given ID at two sites in the deltoid area on days 0, 3, 7 and 28. An ID dose of 0.1 ml per site is usually used with PCEC 1 ml vaccine but higher-​potency vaccines are demanded by most coun- tries using this lower dose. Omitting the day 28 dose is now permitted by the WHO. The 2-site ID regimen becomes 3 visits in one week, and vaccine vials can be shared on each occasion. The 4-site ID regimen becomes 2 visits: 4-site ID using a whole vial on day 0 and 2-site using half a vial on day 7. For possibly im- munosuppressed patients, a day 28 single site ID booster dose is still advisable. For all other vaccines, the manufacturer’s instructions should be followed. Box 8.5.10.1  Specific postexposure prophylaxis for use in a rabies endemic areaa following contact with a domestic or wild rabies vector species, whether or not the animal is available for observation or diagnostic tests Minor exposure (including minor scratches, or abrasions without bleeding) • Start vaccine immediately • Stop treatment if animal remains healthy for 10 days • Stop treatment if animal’s brain proves negative for rabies by appro- priate laboratory tests Major exposure (including licks of broken skin or mucosa, minor bites on arms, trunk or legs, or major severe bites, i.e. multiple or on face, head, fingers, or neck) • Immediate rabies immune globulin and vaccine • Stop treatment if domestic cat or dog remains healthy for 10 days • Stop treatment if animal’s brain proves negative for rabies by appro- priate laboratory tests a This scheme is a simplification of the recommendations of the World Health Organization Expert Consultation on Rabies (2018).

818 section 8  Infectious diseases Postexposure vaccine boosting regimen for people who have already received vaccination If a complete pre-​exposure or postexposure course of a potent tissue culture vaccine has been given in the past, or if the neu- tralizing antibody level has been over 0.5 IU/​ml, rabies immune globulin is not required and only two doses of tissue culture vac- cine are given IM on days 0 and 3. Alternatively, a the one day booster regimen is four 0.1 ml ID injections in deltoid and thigh areas. Vaccine should not be wasted, so if immediate sharing is not possible, a whole vial of vaccine is divided between four intra- dermal sites. Side effects of tissue culture vaccines Mild and transient local redness, itching (especially after ID injec- tion), or pain at the site of injection are not uncommon. Influenza-​like symptoms and rashes are infrequent. Type I immediate hypersensi- tivity occurs rarely during primary courses. No fatal reactions have been reported. Very rarely neurological symptoms including poly- neuritis or Guillain–​Barré syndrome, have been reported in patients receiving tissue culture vaccines but no more frequently than for other commonly used virus vaccines. Passive immunization: Rabies immune globulin Rabies immune globulin (RIG) has proved valuable in providing protection before neutralizing antibody has been actively gener- ated, presumably by neutralizing rabies virus during the first week after initial vaccination. It is recommended as part of primary postexposure treatment, but it is vital following severe bites (on the head, neck, hands, and multiple or deep bites) (see Box 8.5.10.1). The dose of human RIG is 20 IU/​kg body weight and for equine RIG is 40 IU/​kg. Reactions to equine and human RIG have been observed in 1.8% and 0.09% of recipients, respectively, and serum sickness in 0.72% and 0.007%, respectively. These are not predicted by previous ID hypersensitivity testing and so this must not be used. Adrenaline (epinephrine) should always be available in case of reactions. All the RIG is infiltrated into and around the bite wound if ana- tomically possible, but any remaining is injected intramuscularly preferably into the thigh, not the buttock, at a site distant from the vaccine. RIG should be given at the start of vaccination. If it is given hours or days before the first dose, the active immune response will be impaired but it can be given up to 7 days after the first vaccine dose. RIG is prohibitively expensive and is not avail- able or affordable for 99% of people in developing countries for whom postexposure treatment is indicated. If supplies of RIG are limited or if it is unaffordable, wound infiltration alone may be given. Failures of postexposure prophylaxis Deaths from rabies have occurred despite prophylaxis. Failures are attributable to delay in starting vaccination, incomplete vaccine course, use of a substandard vaccine or omission of RIG. Failure to wash the wound or infiltrate RIG around it, injection of vaccine into the buttock, or impaired immune responsiveness of the pa- tient may also contribute. Vaccine protection against rabies-​related lyssaviruses may be less efficient than against the classic rabies spe- cies (see next), but no case of vaccine failure has been attributed to this phenomenon. Rabies-​related virus infections of humans The genus Lyssavirus contains 12 species: type 1, classic rabies and 15 rabies-​related species, but only 6 of these in three phylogroups are known to have infected man (Table 8.5.10.1). Rabies-​related viruses occur in Africa, Europe, Asia and Australia. They are not found in the Americas. With the exception of Mokola, all are viruses of bats. Phylogroup I contains classic rabies and five rabies-​related bat species which are known to cause rabies-​like encephalitis in humans. In phylogroup II, Mokola virus of shrews causes a milder disease in man. European bat lyssaviruses have occasionally been detected in terrestrial species, but diagnostic tests are available only in specialized laboratories, infection is rarely suspected, and the routine tests for classic type 1 rabies virus may be weakly positive or negative. The true prevalence of lyssaviruses is unknown. Only 13 human cases of rabies-​related virus infections have been reported, and disease is likely to remain unrecognized and misdiagnosed. African lyssaviruses • Mokola virus (species 3 phylogroup II) has been isolated from shrews (Crocidura spp.) and rodents, as well as cats and dogs Table 8.5.10.1  Lyssaviruses known to infect humans Species phylogroup I Virus Distribution Reservoir mammal species Human deaths 1 Rabies Almost worldwide Terrestrial mammal species and bats in
the Americas Unknown 60 000? 4 Duvenhage virus Africa Insectivorous bat 3 5 European bat lyssavirus type 1 Northern and Eastern Europe Insectivorous bat 2 6 European bat lyssavirus type 2 Western Europe and Scandinavia Insectivorous bat 2 7 Australian bat lyssavirus Australia Fruit bats and insectivorous bats 3 10 Irkut virus Eurasia Insectivorous bat 1 Phylogroup II 3 Mokola virus Africa Shrew, rodent 2?

8.5.11 Colorado tick fever and other arthropod- bo

8.5.11 Colorado tick fever and other arthropod- borne reoviruses 819

8.5.11  Colorado tick fever and other arthropod-borne reoviruses 819 which are presumably vectors. It was isolated from a child with meningitis who recovered, and from another with fatal encephal- itis. Mokola virus also caused mild disease in a rabies-​vaccinated laboratory worker. • Duvenhage virus (species 4) has been identified in three people, all of whom had had skin lesions inflicted by bats and had developed a fatal illness with clinical features identical to rabies encephalitis. European bat lyssaviruses Infected insectivorous bats have been found in Europe since 1954. The European bat lyssavirus (EBLV) group comprises species 5 (also known as EBLV 1) and species 6 (EBLV 2), which have subgroups a and b. EBLV type 1a is found across Northern and Eastern Europe from the Netherlands to Russia; EBLV type 1b in the Netherlands, France, and Spain; EBLV type 2a in the Netherlands and the United Kingdom; EBLV type 2b rarely in Switzerland and Scandinavia. Five unvaccinated people with bat bites died of encephalitis indis- tinguishable from rabies: two infected in Russia, one each in the Ukraine, Scotland, and Finland. Australian bat lyssavirus Australian bat lyssavirus (species 7) has been found in fruit bats (genus Pteropus) (Fig. 8.5.10.16) and insectivorous bats in Eastern Australia since 1996. It caused a fatal rabies-​like encephalitis in three people who had handled bats. FURTHER READING Banyard AC, et al. (2011). Bats and lyssaviruses. Adv Virus Res, 79, 239–​89. Gautret P, et al. (2014). Rabies in nonhuman primates and potential for transmission to humans: a literature review and examination of selected French national data. PLoS Negl Trop Dis, 8, e2863. Gnanadurai CW, et al. (2015). Novel approaches to the prevention and treatment of rabies. Int J Virol Stud Res, 3, 8–​16. Helmick CG, Tauxe RV, Vernon AA (1987). Is there a risk to contacts of patients with rabies? Rev Infect Dis, 9, 511–​18. Kaplan C, Turner GS, Warrell DA (eds) (1986). Rabies:  the facts,
revised edition. Oxford University Press, Oxford. Manning SE, et al. (2008). Human rabies prevention—​United States, 2008: recommendations of the Advisory Committee on Immunization Practices. MMWR Recomm Rep, 57(RR-​3), 1–​28. Nel LH, Markotter W (2007). Lyssaviruses. Crit Rev Microbiol, 33, 301–​24. Schnell MJ, et al. (2010). The cell biology of rabies virus: using stealth to reach the brain. Nat Rev Microbiol, 8, 51–​61. Warrell DA, et al. (1976). Pathophysiologic studies in human rabies. Am J Med, 60, 180–​90. Warrell MJ (2012). Current rabies vaccines and prophylaxis sched- ules: preventing rabies before and after exposure. Travel Med Infect Dis, 10, 1–​15. Warrell MJ, Warrell DA, Tarantola A (2017). The imperative of palli- ation in the management of rabies encephalomyelitis. Trop Med Infect Dis, 2, 52. World Health Organization (2018). Expert consultation on rabies. Third report. World Health Organ Tech Rep Ser, 1012. Zeiler FA, Jackson AC (2016). Critical appraisal of the milwaukee protocol for rabies: This failed approach should be abandoned. Can J Neurol Sci, 43, 44–51. 8.5.11  Colorado tick fever and other arthropod-​borne reoviruses Mary J. Warrell and David A. Warrell ESSENTIALS Human pathogens are found in six genera of Reoviridae:
Reovirus, Rotavirus, Orthoreovirus, and three arthropod-​borne genera—​Coltivirus (Colorado tick fever, Salmon River virus, and Eyach viruses), Orbivirus (Kemerovo group, Changuinola, Orungo, and Lebombo) and Seadornavirus (Banna virus). Colorado tick fever—​common in parts of north-​western North America; acquired from hard tick (ixodid) bites, most often by hikers and campers, presenting 3–​6 days later with sudden fever, rigors, generalized aches, myalgia, headache and backache, rashes (12%), and gastrointestinal symptoms (20%). Diagnosis is con- firmed by detection of viral antigen in erythrocytes or serum, or Fig. 8.5.10.16  Pteropid fruit bats (flying foxes) (Pteropis) roosting, the natural reservoir of Nipah, Hendra, and Menangle paramyxoviruses, and of Australian bat lyssavirus. Copyright David A. Warrell.

820 section 8  Infectious diseases by serodiagnosis. Management is symptomatic. Illness usually resolves in 10–​14  days, but convalescence may be prolonged. Prevention is by avoiding, repelling, or rapidly removing ticks; no vaccines are available. Coltiviruses Colorado tick fever The virus responsible for Colorado tick fever or ‘mountain fever’ is an 80-​nm double-​shelled particle covered with capsomeres. The icosahedral core contains 12 segments of double-​stranded negative-​sense RNA. The virus can infect human erythro- cytes and this may also occur with the other coltiviruses and orbiviruses. Colorado tick fever is a zoonosis involving hard (ixodid) ticks (principally Dermacentor andersoni, but also D. occidentalis, D. parumapertus, D. albipictus, and others) and wild mammals, including porcupines, deer, coyotes, squirrels, chipmunks, deer mice, and other rodents. Ticks pass Colorado tick fever virus transstadially and transovarially. Epidemiology Colorado tick fever is acquired from tick bites in western and north-​western parts of the United States of America (including California) and Canada (British Columbia and Alberta). Very rarely, it has been caused by an infected blood transfusion. In the United States of America over 10 years up to 2012, 75 cases were reported mostly in Wyoming and other western states. Although underreporting is suspected, the incidence seems to have been declining. Hikers and campers are at special risk in rodent-​ and tick-​infested terrain. The prevalence of antibody to Colorado tick fever among shepherds was 32%. The highest incidence is from May to July when ticks are most active. Infection usually confers lasting immunity. Clinical features In adults, the infection is nearly always mild, but in children it is occasionally severe but rarely fatal. Three to six days after the tick bite (extreme range 1–​19 days) there is a sudden fever for about 3 days, with rigors, generalized aches, myalgia, headache, and back- ache. In one-​half of the patients there is a biphasic fever. Rashes then appear in up to 12% of patients, usually a transient peripheral maculopapular rash or petechiae on flexor surfaces of arms but can be widespread and it may be hyperaesthetic. Gastrointestinal symp- toms occur in 20% of patients. Laboratory findings include leuko- penia with relative lymphocytosis, occasional thrombocytopenia, and mild lymphocyte pleocytosis. The illness usually resolves in about 10–​14  days, but conva- lescence may be prolonged. Severe manifestations include en- cephalitis, meningitis, or drowsiness, sometimes associated with gastrointestinal symptoms, spontaneous bleeding, thrombocyto- penia, and disseminated intravascular coagulation. Fatalities are rarely reported in children and one immunosuppressed adult. Late, possibly immunological effects include myocarditis, pericarditis, pleurisy, arthritis, and epididymitis. Colorado tick fever infection may precipitate abortion or transplacental infec- tion, but the teratogenic effects reported in mice have not been observed in humans. Diagnosis Viral antigen may be detected in erythrocytes by immunofluor- escence 1–​120 days after the start of symptoms. Erythrocyte pre- cursors are infected in the marrow, but their survival is apparently not affected. Virus can be isolated from the blood and, if there is cen- tral nervous system involvement, the cerebrospinal fluid. Colorado tick fever virus produces a cytopathic effect on several cell lines, but intracerebral injection of ground blood clot or preferably washed erythrocytes into suckling mice is more sensitive for diagnostic iso- lation. Antigen can be detected in serum during acute infections, especially the first 2 weeks after onset, by polymerase chain reaction (RT-​PCR), but enzyme-​linked immunosorbent assay techniques have been less sensitive. Neutralizing antibody and specific IgM en- zyme immunoassays become positive after 14–​21 days and the IgM disappears after 45 days. Differential diagnosis Many other tick-​borne acute febrile illnesses, some with rashes and nervous system involvement, can be acquired in the area en- demic for Colorado tick fever. These include Rocky Mountain spotted fever, tularaemia, Lyme disease, and relapsing fever. Tick paralysis caused by D. andersoni and other ixodid ticks presents as a poliomyelitis-​like, ascending, flaccid paralysis that is unlikely to be mistaken for the meningitic or encephalitic syndromes of Colorado tick fever. Treatment The symptomatic treatment of fever and pain should exclude sali- cylates in case of thrombocytopenia. Tribavirin (ribavirin) inhibits the replication of Colorado tick fever virus experimentally, but its use in humans has not been reported. Immunity is long-​lasting. Convalescent patients should not donate blood for 6 months after their illness. Salmon River virus This virus is closely related to Colorado tick fever virus. It was iso- lated from a patient with similar symptoms in Idaho. Eyach This European coltivirus has been found in ticks in Germany and France and is likely to be an adapted variant Colorado tick fever virus introduced from America. There is serological evidence of human infection in Czechoslovakia causing meningoencephalitis or neuropathies. Orbiviruses Although the respective antibodies to the tick-​borne Great Island virus and insect-​borne Corripata orbiviruses have been found in humans, there is no evidence of the diseases’ pathogenicity.

8.5.12 Alphaviruses 821

8.5.12 Alphaviruses 821

8.5.12  Alphaviruses 821 Kemerovo group Kemerovo group viruses have been isolated from ixodid and hyalomma ticks in Russia and Central Europe. They cause benign fe- brile illnesses and, occasionally, meningitis or encephalitis in spring and early summer when ticks are active. Rodents and birds are in- volved in the zoonotic cycle. The closely related Tribeč and Lipovník virus distributions range from Siberia to central Europe. Some healthy humans are sero- positive and so they may cause occasional fever or meningitis. The clinical features and epidemiology are similar to the flavivirus tick‐ borne encephalitis infection. Oklahoma tick fever is another Kemerovo virus rarely causing fe- brile illness in the United States of America. Changuinola There is a single report of human febrile illness with the orbivirus Changuinola in Panama. The virus has been isolated from phlebotomine flies and mammals in that area. Orungo Orungo virus is found mainly in West Africa but also in Uganda and the Central African Republic. Up to 75% of some human popu- lations are seropositive. The clinical effects are unknown, but fever, headache, myalgia, nausea, and diarrhoea occur in some people. There is no rash or jaundice. It is transmitted by Anopheles, Aedes, and other mosquitoes. Monkeys, sheep, and cattle may be infected. Lebombo This orbivirus was isolated from one febrile child in Nigeria. Lebombo is also found in mosquitoes and rodents. Seadornaviruses These viruses from Southeast Asia and Indonesia include Banna virus from China, which has been isolated from patients with en- cephalitis. In China, 20 new cases of Banna virus were identified in areas where Japanese encephalitis virus is endemic. These two en- cephalitis viruses share a common vector, Culex tritaeniorhynchus, and they may be clinically confused. Banna virus cases may be un- detected during a Japanese encephalitis virus outbreak. Prevention Tick-​borne infections are prevented by avoiding, repelling with diethyltoluamide, and rapidly removing ticks. No vaccines are avail- able. Long-​sleeved, tight-​fitting clothing should be worn in the high-​risk areas and the body should be checked for ticks at frequent intervals. The nucleoside analogue, 3′-​fluoro-​3′-​deoxyadanosine, inhibits replication in vitro. FURTHER READING Attoui H, et  al. (2005). Coltiviruses and seadornaviruses in North America, Europe, and Asia. Emerg Infect Dis, 11, 1673–​9. Hubálek Z, Rudolf I (2012). Tick-​borne viruses in Europe. Parasitol Res, 111, 9–​36. Libikova H, et al. (1978). Orbiviruses of the Kemerovo complex and neurological diseases. Med Microbiol Immunol, 166, 255–​63. Liu H, et al. (2010). Banna virus, China, 1987–​2007. Emerg Infect Dis, 16, 514–​7. McGinley-​Smith DE, Tsao SS (2003). Dermatoses from ticks. J Am Acad Dermatol, 49, 363–​92. Romero JR, Simonsen KA (2008). Powassan encephalitis and Colorado tick fever. Infect Dis Clin North Am, 22, 545–​59. Silva SP, et  al. (2014). Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil. J Gen Virol, 95, 2251–​9. Yendell SJ, Fischer M, Staples JE (2015). Colorado tick fever in the United States, 2002–​2012. Vector Borne Zoonotic Dis, 5, 311–​6. 8.5.12  Alphaviruses Ann M. Powers, E.E. Ooi, L.R. Petersen, and D.J. Gubler ESSENTIALS There are 31 registered alphaviruses belonging to the family Togaviridae, 16 of which are known to cause human infection. They are RNA viruses with global geographical distribution and complex transmission cycles, usually between wild or domestic animals and one or more mosquito species; humans are infected by mosquito bites and are often inci- dental hosts that do not contribute to the maintenance of the virus. They cause a spectrum of clinical manifestations ranging from non-​ specific febrile illness to chronic arthralgia to acute encephalitis and death. Diagnosis of infection is made by several methods including (1) serologically by detection of IgM and/​or IgG antibodies, (2) virus isolation, (3) molecularly using reverse transcription–​polymerase chain reaction, or (4) by immunohistochemistry on tissue samples. Old World alphaviruses, including chikungunya, Ross River, Sindbis, Barmah Forest, Mayaro, and o’nyong-​nyong, generally have mammals as their natural vertebrate host and cause acute febrile illness charac- terized by rash and arthritis. Clinical management is symptomatic; pre- vention and control is by reducing vector mosquito populations and by avoiding mosquito bites. Several efforts to develop vaccines for chikun- gunya and Ross River viruses are in progress and are at different stages of development. The New World alphaviruses, eastern and western equine enceph- alitis viruses, generally have birds as their natural vertebrate hosts, while the Venezuelan equine encephalitis complex viruses have rodents as their natural hosts. About 2% of adults infected with eastern equine enceph- alitis virus (less for other viruses) develop encephalitis which can be fatal, with permanent neurological sequelae in many survivors. As with the Old World alphaviruses, management is symptomatic; prevention and control is by reducing vector mosquito populations and by avoiding mosquito bites. Various vaccines have been used in laboratory workers and others at high risk of exposure. New generation vaccines are in development.

822 section 8  Infectious diseases Introduction The genus Alphavirus of the family Togaviridae is comprised of 31 registered viruses, 16 of which are known to cause human in- fection (Table 8.5.12.1). Alphaviruses are lipid-​enveloped virions with a diameter of 60–​70 nm whose genome is a molecule of single-​ stranded, positive-​sense RNA approximately 12 000 nucleotides in length. Most alphaviruses are maintained in nature in complex trans- mission cycles between wild or domestic animals and one or more mosquito species. Humans are infected when the infected mosquito bites them and transmits the virus via their saliva. Patients develop high viraemias with some alphaviruses and this may contribute to the transmission cycle by infecting mosquitoes. The epidemiology and geographical distribution of the alphaviruses depend on several Table 8.5.12.1  Known disease associations of alphaviruses Virus Geographical distribution Disease in humans Outbreaks Other features Aura South America No Barmah Forest Australia SFI, arthropathy Yes Clinically similar to Ross River virus infection Bebaru Malaysia No Laboratory infection only Cabassou French Guiana No Chikungunya Africa, Asia, South America, Central America, Caribbean, South Pacific SFI, arthropathy Yes Large outbreaks in urban settings Eastern equine encephalitis North America on Atlantic and Gulf Coasts, Caribbean SFI, encephalitis Yes Isolated cases or small outbreaks occur mainly in North America Eilat Israel Insect only alphavirus Everglades Florida SFI, encephalitis No Member of the Venezuelan equine encephalitis antigenic complex Fort Morgan Colorado, California, Nebraska, Oklahoma No Getah Asia SFI No Highlands J North America SFI No Madariaga South America, Central America SFI Formerly South American variants of eastern equine encephalitis virus Mayaro South America, Caribbean SFI, arthropathy Yes Middelburg South, West, and Central Africa No Mosso das Pedras Brazil, Argentina No Member of the Venezuelan equine encephalitis antigenic complex Mucambo Trinidad, South America SFI No Member of the Venezuelan equine encephalitis antigenic complex Ndumu Africa No Onyong-​nyong East and West Africa, Zimbabwe SFI, arthropathy Yes Igbo Ora virus is a variant of onyong-​nyong Pixuna Brazil, Argentina SFI No Rio Negro Argentina No Ross River Australia, South Pacific SFI, arthropathy Yes Periodic epidemics in South Pacific Salmon Pancreas disease North Atlantic No Semliki Forest Sub-​Saharan Africa SFI, encephalitis No Sindbis Africa, East Mediterranean, South and Southeast Asia, Australia, Europe SFI, arthropathy Yes Subtypes includes Babanki, Kyzylagach, Ockelbo Southern elephant seal Antarctica No Tonate French Guiana SFI, encephalitis No Member of the Venezuelan equine encephalitis antigenic complex Trocara South America No Una South America, Trinidad No Venezuelan equine encephalitis Northern South America, Central America, Mexico SFI, encephalitis Yes Epidemics are caused by epizootic virus strains (Subtypes IAB and IC) Western equine encephalitis North and South America SFI, encephalitis Yes Human disease rare outside of North America and Brazil; Whataroa New Zealand, Australia No SFI, systemic febrile illness. Adapted from Griffin D (2007). Alphaviruses. In: Knipe DM, Howley PM (eds) Fields virology, 5th edition, vol. 1, pp. 1023–​67. Lippincott Williams & Wilkins, Philadelphia.

8.5.12  Alphaviruses 823 factors including the presence of suitable amplifying hosts, the pres- ence and feeding behaviour of suitable arthropod vectors, and the frequency of exposure of non​immune reservoir hosts and humans to infected vectors. Alphavirus infections are not directly commu- nicable between humans. Many alphavirus infections in humans are asymptomatic, but alphaviruses can cause a spectrum of clinical illness ran- ging from non​specific febrile illness, often with rash, myalgia, or arthralgia, to frank encephalitis, haemorrhage, and death. They cause two main clinical syndromes:  Old World alphaviruses generally cause illness characterized by rash and arthritis while New World alphaviruses are generally associated with neuroinvasive disease. No specific therapy is available. Vaccines for some alphaviruses are used in animals, although none have been licensed for humans. Laboratory diagnosis Alphavirus infections are diagnosed serologically by detection of IgM and/​or IgG antibodies. All alphaviruses have some common antigenic determinants that may result in cross-​reactions in immunodiagnostic tests. Neutralization tests are typically confirma- tory for serological diagnosis in areas where multiple alphaviruses are endemic/​enzootic. Isolation of virus from acute-​phase serum is possible with some alphaviruses, but they are seldom recovered from the central nervous system, including cerebrospinal fluid, ex- cept from fatal cases. Virological diagnosis can also be made using polymerase chain reaction and immunohistochemistry on tissue samples. Alphaviruses associated with arthritis and rash Chikungunya Aetiology and epidemiology Chikungunya virus has a nearly global distribution and is trans- mitted primarily by day-​biting Aedes sp. mosquitoes. The primary vertebrate reservoir hosts remain to be conclusively determined, ­although non​human primates such as monkeys and baboons are likely candidates in sylvatic environments in Africa. In urban surroundings, the virus is transmitted between humans by Aedes aegypti and Ae albopictus mosquitoes. Explosive urban epidemics occur during the rainy season in long endemic areas and year-​ round in previously chikungunya-​free areas. Since 2004, chi- kungunya virus spread extensively, beginning with outbreaks on the East Coast of Africa then moving to the islands of the Indian Ocean, India, and Southeast Asia. This epidemic was exacerbated by a new variant of the virus containing a single amino acid mu- tation in the envelope protein. This mutation increased infectivity for Ae albopictus, a mosquito that has spread throughout the tropics and subtropics and has a wider distribution in urban, semiurban, and rural habitats than Ae aegypti, which favours urban environ- ments. In 2007, it reached a subtropical country (Italy) for the first time. The activity in Italy involved local transmission by Ae albopic- tus mosquitoes resulting in 205 cases and one death. The outbreaks continued to spread, finally reaching the Americas (Caribbean) in 2013. However, the outbreaks in the Americas were not an ex- tension of the Indian Ocean outbreaks but rather an independent introduction of the Asian genotype into the Western Hemisphere. Within 1 year, chikungunya virus had spread to 44 countries in the Americas and caused an estimated 1.1 million cases. Serological surveys following outbreaks have shown antibody prevalences gen- erally ranging from 30% to 70%. Clinical characteristics ‘Chikungunya’ means ‘that which bends up’ in Makonde, an East African language, and refers to the crippling arthralgia that char- acterizes the disease. After an incubation period of 2–​3 days (range 1–​12  days), there is sudden and high (>39°C) fever and severe arthralgia. Arthralgias are polyarticular, with the knees, ankles, elbows, and small joints of the hands and feet most commonly affected. A useful sign is pain on squeezing the wrists (tenosyno- vitis). Headache, injected pharynx, gastrointestinal symptoms, and myalgias can be frequent during the acute illness. Rashes, typically on the trunk and limbs, occur in about one-​half of the patients, usually during the second to fifth day of illness. They are variable in appearance:  papular or maculopapular erythemas (blanching as in dengue), vesicular, bullous, dyshidrotic, keratolytic, pur- puric and hyperpigmented associated with facial oedema, ery- thema nodosum, and aphthous ulcers. Arthralgia might last several months and is associated with effusions and bursitis; a few patients have symptoms 5  years after infection. Haemorrhage, meningo- encephalitis, Guillain–​Barré polyradiculopathy, myocarditis, and hepatic and renal complications are uncommon but may be fatal. Rheumatological manifestations are less frequent in children. Conjunctival suffusion and cervical or generalized lymphadenop- athy can occur. Serological surveys suggest that asymptomatic in- fections can occur but typically in less than 20% of those infected. Neonatal infection has occurred from mothers ill shortly before or at the time of delivery resulting in more severe manifestations in the newborns. Diagnosis Leukopenia and elevation of liver and muscle enzymes are common early in infection. Detection of viral RNA by reverse transcription–​ polymerase chain reaction (RT-​PCR) is particularly useful for diagnosis given the high titres and long duration of viremia. Haemagglutinin inhibition and IgM antibodies will be present in nearly all patients by the seventh day of illness. IgM antibodies detectable in serum by IgM antibody capture enzyme-​linked im- munosorbent assay (MAC-​ELISA) may persist for 6 months after infection. Virus isolation and RT-​PCR both provide confirmed diagnosis. Prevention, control, and treatment Prevention and control can only be achieved by reducing vector mosquito populations in the large urban centres of the tropics and by avoiding mosquito bites. The American military devel- oped an investigational vaccine, but it is not licensed for general use. Several new vaccines using a variety of approaches including subunit vaccines, chimeric vaccine, virus-​like particle vaccines, and DNA vaccines are all in late stage development. There is no specific treatment. Anti-​inflammatory drugs might relieve arthralgia and a range of potential therapeutic agents are being evaluated.

824 section 8  Infectious diseases Ross River virus Aetiology and epidemiology This virus causes ‘epidemic polyarthritis’ in Australia, south-​western Pacific islands, and Fiji. Aedes vigilax and Culex annulirostris are im- portant vectors in Australia and Ae scutellaris complex mosquitoes in some south Pacific islands, although the virus has been isolated from more than 30 mosquito species. An epidemic in various Pacific islands in 1979 to 1980 affected more than 50 000 people with up to 60% of the population affected on some islands. An average of 5000 cases is reported annually from Australia. Explosive outbreaks and viraemias in humans implicate virus transmission from human to human by certain mosquitoes. Outbreaks tend to be associated with periods of increased rainfall. Camping is a significant risk factor in tropical Australia; however, recent outbreaks have reached the borders of major coastal urban areas where human expansion has brought populations closer to vector habitats. Clinical characteristics The incubation period ranges from 2 to 21 days (7–​9 days on average). The illness begins suddenly with fever and arthralgias predomin- antly in the ankles, wrists, knees, fingers, and feet. A maculopapular rash occurs in about one-​half of patients within 2 days of onset and is most prominent on the trunk and limbs, but can cover the entire body; the rash may progress to small vesicles. Myalgias, headache, anorexia, nausea, and tenosynovitis are common, but the tempera- ture is only slightly elevated. Arthralgia generally resolves within 3 to 6 months. Symptomatic infection is rare in children. Diagnosis Isolation of virus from serum is possible for the first few days of illness. IgM antibodies will be detected by MAC-​ELISA within 5–​10 days of onset. Complement fixation, haemagglutinin inhib- ition, and neutralization tests may be useful, particularly when paired serum samples are available. Virus isolation and PCR are confirmatory. Prevention, control, and treatment Avoidance of mosquito bites and peridomestic mosquito control can effectively reduce the risk of infection. No specific treatment is available. Non​steroidal anti-​inflammatory drugs might relieve symp- toms. One study suggested that corticosteroids might hasten recovery. Vaccines against this virus are in preclinical stages of development. Sindbis Aetiology and epidemiology Sindbis virus is widely distributed in Africa, India, tropical Asia, Australia, and Europe. However, clinical disease is reported only in geographically restricted areas where specific variants are de- scribed. In Europe, the main vectors to humans are late summer, ornithophilic mosquitoes of the genera Culex and Culiseta. High antibody prevalences in regions of Africa suggest that human ex- posure is common. Several outbreaks have been noted since the ori- ginal identification of the virus in 1952. Clinical characteristics In northern Europe, symptomatic disease is recognized from Sweden (Ockelbo disease), through Finland (Pogosta disease), to the former Karelian Autonomous Soviet Socialist Republic (Karelian fever). The clinical features include mild fever, rash, arthralgia, my- algia, malaise, headache, and pruritus. The maculopapular rash progresses from trunk to extremities and vesicles can occur on the palms and soles. Ankle, finger, wrist, and knee joints are most com- monly affected. While disease symptoms are typically mild, prom- inent rheumatic symptoms, sometimes persisting for several years, have been noted in Europe and South Africa. Diagnosis Haemagglutinin inhibition and IgM antibodies will be present in nearly all patients by the eighth day of illness. IgM antibodies de- tectable in serum by MAC-​ELISA may persist for 6 months after infection. Virus can be infrequently detected by culture or RT-​PCR from blood or skin lesions. Prevention, control, and treatment Avoidance of mosquito bites can reduce the risk of infection. No specific treatment is available. Barmah Forest virus Since its first recognition as a cause of human disease in 1988, the geographical distribution of Barmah Forest virus has expanded re- cently in Australia. It causes sporadic disease and epidemics, with up to 300 serologically confirmed cases. The disease resembles that of Ross River virus infection, although the rash tends to be more florid and true arthritis is less common. The illness is prolonged in some patients. Little is known about the ecology of Barmah Forest virus, although outbreaks have coincided with Ross River virus outbreaks and the virus has been identified in the same mosquito species. Mayaro virus Mayaro virus has been isolated from humans, wild vertebrate reser- voir species, and Haemogogus sp. mosquitoes, the principal vectors, in Trinidad, Colombia, Brazil, Suriname, Guyana, French Guiana, Peru, Bolivia, Venezuela, and most recently in Haiti. Seroprevalence is high in human populations in many forested areas of South America. The clinical presentation resembles chikungunya, onyong-​ nyong, Ross River, Barmah Forest, and Sindbis virus infections. In an outbreak in Pará, Brazil, after an incubation period of about a week, fever, chills, headache, arthralgia, myalgia, and lymphadenopathy developed and persisted for 2–​5 days. Arthralgia was almost uni- versal and could last for months. Small joints in the extremities were principally involved. It was accompanied by joint oedema in 20% of cases, causing severe temporary disability. Maculo-​ or micropapular rashes appeared on the fifth day and lasted for 3–​4 days in two-​thirds of the cases, more in children (Fig. 8.5.12.1). All patients had leuco- penia and a minority had mild thrombocytopenia and albuminuria. Viraemias as high as 5.0 log/​ml suggested that humans might be amplifying hosts for this virus. In other outbreaks, eye pain, diar- rhoea, and vomiting were additional features. Onyong-​nyong virus From 1959 to 1962, this virus caused an epidemic in Uganda, Kenya, Tanzania, and Malawi involving approximately 2  million people. The virus was also isolated in 1978 from Anopheles funestus mosqui- toes in Kenya after a long period of no apparent onyong-​nyong virus activity. In 1996–​1997, an outbreak occurred in Uganda. However,

8.5.12  Alphaviruses 825 in West Africa, variants known as Igbo Ora have been found but have not been associated with large outbreaks. In 2003, a small out- break occurred among refugees in the Côte d’Ivoire and a human infection was confirmed in Chad in 2004. Onyong-​nyong is closely related to chikungunya and produces a similar illness, although fever is less pronounced and cervical lymphadenopathy is very common. An funestus and An gambiae transmit the virus; onyong-​nyong virus is the only alphavirus to utilize anopheline vectors. Alphaviruses associated with neuroinvasive disease Eastern equine encephalitis Aetiology and epidemiology The virus is widely distributed throughout eastern North America and the Gulf Coast. In North America, it is maintained in a bird–​ mosquito cycle in hardwood swamps in coastal areas from the Great Lakes and southeastern Canada to the Gulf Coast. Recent studies have suggested that snakes may serve as overwintering reservoirs in southern states. In the United States of America human infections are usually sporadic, and small outbreaks occur each summer mostly along the Atlantic and Gulf Coasts; outbreaks of equine disease are common in Florida. In recent years, 1–​21 cases have been reported annually. In North America, wild birds and Culiseta melanura mosquitoes maintain the virus in hardwood swamps, but a variety of mosquito species act as bridge vectors to humans and domestic animals. The newly named Madariaga virus was formerly known as South American eastern equine encephalitis virus. Madariaga virus is less associated with human or equine disease and is likely main- tained in a transmission cycle distinct from eastern equine enceph- alitis virus in North America. Clinical characteristics Most infections are inapparent. The incubation period exceeds 1 week with a prodromic period that lasts up to 11 days before high fever and neurologic symptoms appear. About 2% of infected adults and 6% of children develop encephalitis. Eastern equine encephalitis is the most severe of the arboviral encephalitides, with a mortality of 30–​ 70% in those that develop encephalitis. Symptoms and signs include dizziness, decreasing level of consciousness, tremors, seizures, and focal neurological signs. Death can occur within 3–​5 days of onset of neurological symptoms. Lifelong neurological sequelae are common in non​fatal encephalitis and include convulsions, paralysis, and cog- nitive impairment. Illness due to eastern equine encephalitis in South America (Madariaga virus infection) appears to be less severe. Diagnosis Cerebrospinal fluid pressure can be raised, protein levels are increased, sugar is normal, and pleocytosis exists (up to 2000 cells/​mm3). IgM antibodies are readily detected in serum or cerebrospinal fluid by ELISA. Paired serum samples can be tested by haemagglutinin inhib- ition, ELISA, or neutralization tests. Horse or pheasant deaths and the proximity to swamps provide clues to the diagnosis. Prevention, control, and treatment Prevention depends on the avoidance of mosquito bites and mos- quito control in suburban areas. Inactivated vaccines have been used successfully in horses, and an investigational inactivated vaccine has been used experimentally in laboratory workers and others at high risk of exposure. No specific treatment is available. Venezuelan equine encephalitis antigenic complex Aetiology and epidemiology Six subtypes (I–​VI) within the Venezuelan equine encephalitis anti- genic complex have been identified. Five antigenic variants exist within subtype I (IAB, IC, ID, IE, IF). These subtypes and variants are classified as epizootic or enzootic, based on their apparent viru- lence and epidemiology. Epizootic variants of subtype I (IAB and IC) cause equine epizootics and are associated with more severe human disease. Only subtypes IAB, IC, ID, and IE are classified as Venezuelan equine encephalitis virus; all other subtypes are distinct viral species. Enzootic strains (ID, IE, IF (Mosso das Pedras virus), II (Everglades virus), III (Mucambo virus [A, B, D], Tonate virus [B]‌), IV (Pixuna virus), V (Cabassou virus), VI (Rio Negro virus)) do not cause epizootics in horses, but can produce sporadic disease in humans. Large epizootics (IAB and IC) have occurred in equines in northern countries of South America and Central America, sometimes reaching the United States of America. In 1969–​1972, a massive epizootic extending from Ecuador to Texas killed more than 200 000 horses and caused several thousand human infections. In 1995, a large epizootic, which began in Venezuela and spread to (a) (b) Fig. 8.5.12.1  Mayaro virus infection acquired in the Peruvian Amazon, showing maculopapular rash that first appeared on the palms of the hands on the fifth day spreading first to arms, knees, and then to entire body and lasting 3 days, accompanied by arthralgia and swelling of the fingers and feet, later affecting the knees. Rash is similar in appearance to that seen in chikungunya, onyong-​nyong, Sindbis, and Ross River cases. Courtesy of Dr Celie Manuel.

826 section 8  Infectious diseases Colombia, affected thousands of horses, and caused approximately 90 000 human infections. Epizootic strains are carried by a wide var- iety of mosquitoes including Aedes, Mansonia, and Psorophora spp. Horses are the principal amplifying hosts during epizootics but are not amplifying hosts for enzootic transmission. Enzootic strains are maintained in a cycle involving Culex (Melanoconion) mosquitoes and rodents. Subtype IE Venezuelan equine encephalitis virus has caused some small equine outbreaks in Mexico, but it is still con- sidered an enzootic subtype. Clinical characteristics (epizootic virus infections) After an incubation period of 1–​6 days, there is a brief febrile illness of sudden onset characterized by malaise, nausea, or vomiting, head- ache, and myalgia. Acute symptoms last 2–​5 days, and generalized asthenia up to 3 weeks. Clinically, Venezuelan equine encephalitis can be indistinguishable from dengue or other arboviral diseases. Among those with clinical illness, less than 0.5% of adults and less than 4% of children develop encephalitis. Nausea and vomiting, nu- chal rigidity, ataxia, convulsions, paralysis, and death may occur. Long-​term sequelae following encephalitis are uncommon. Diagnosis (epizootic virus infections) A marked leukopenia is universal, often accompanied by neutropenia and thrombocytopenia, with moderate lymphocytosis in the cerebro- spinal fluid. Virus can be detected by isolation or by RT-​PCR from serum or throat swab within the first few days of illness. Paired sera can be tested by haemagglutinin inhibition and neutralizing tests. Specific IgM can be detected by MAC-​ELISA in the second week of illness. Prevention, control, and treatment Equine immunization has been effective in controlling epizootic disease. Venezuelan equine encephalitis is highly infectious by the aerosol route and many laboratory infections have occurred. Investigational live attenuated and inactivated vaccines have been used in laboratory workers. People in affected areas should avoid mosquito bites. No specific treatment is available. Western equine encephalitis Aetiology and epidemiology Western equine encephalitis virus is found in North and South America, but human disease is rare outside North America and Brazil. Previously, summer outbreaks tended to occur with flooding, which increases breeding of Culex mosquitoes (particularly Culex tarsalis in the western United States of America). Large outbreaks of western equine encephalitis in humans and horses occurred in the western United States of America in the 1950s and 1960s; however, a declining horse population, equine vaccination, viral mutations, and improved vector control have reduced the reported number of human cases to zero in recent years. Clinical characteristics The ratio of apparent to inapparent infection in adults is less than 1 in 1000; however, this ratio increases to 1:1 in infants under 1 year of age. Following an incubation period of about 7 days, headache, vomiting, stiff neck, and backache are typical; restlessness and irrit- ability are seen in children. Weakness and hyporeflexia are common. Convulsions occur in 90% of affected infants and 40% of affected children between 1 and 4 years, but are rare in adults. Recovery in 5–​10 days is common, but convalescence may be protracted. Although rare in adults and older children (<1%), sequelae are common in newborns, with one-​half of those with encephalitis being left with convulsions and/​or severe motor or intellectual deficits. Congenital infection during the third trimester resulting in encephalitis in the infant has been described. The overall case fatality rate is 3–​7%. Diagnosis Clinical laboratory findings in western equine encephalitis are often unremarkable. IgM antibodies are readily detected in serum by ELISA. Paired sera can be tested by haemagglutinin inhibition, IgG ELISA, or neutralization tests for a rise in titre. Virus can occasion- ally be isolated from serum or cerebrospinal fluid but odds of isola- tion decrease with the onset of neurologic symptoms. Isolation from the brain post-​mortem is common. Prevention, control, and treatment Prevention of western equine encephalitis relies on mosquito con- trol and the avoidance of mosquito bites. A licensed vaccine is avail- able for horses. An investigational inactivated vaccine has been used for laboratory staff and others at high risk of exposure. No specific treatment is available. FURTHER READING Aichinger G, et al. (2011). Safety and immunogenicity of an inacti- vated whole virus Vero cell-​derived Ross River vaccine: a random- ized trial. Vaccine, 29, 9376–​84. Centers for Disease Control and Prevention (2006). Eastern equine en- cephalitis: New Hampshire and Massachusetts, August–​September 2005. MMWR Morb Mortal Wkly Rep, 55, 697–​700. Griffin D (2007). Alphaviruses. In: Knipe DM, Howley PM (eds) Fields virology, 5th edition, Vol. 1, pp. 1023–​67. Lippincott Williams & Wilkins, Philadelphia, PA. Halsey ES, et al. (2013). Mayaro virus infection, Amazon Basin region, Peru, 2010–​2013. Emerg Infect Dis, 19, 1839–​42. Kiwanuka N, et  al. (1999). O’nyong-​nyong fever in South-​Central Uganda, 1996–​1997: clinical features and validation of a clinical case definition for surveillance purposes. Clin Infect Dis, 29, 1243–​50. Laine M, et al. (2004). Sindbis virus and other alphaviruses as cause of human arthritic disease. J Int Med, 256, 457–​71. Petersen LP, Powers AM (2016). Chikungunya:  epidemiology. F1000Research, 5(F1000 Faculty Rev), 82. Pialoux G, et al. (2007). Chikungunya, an epidemic arbovirosis. Lancet Infect Dis, 7, 319–​27. Powers AM (2011). Genomic evolution and phenotypic distinctions of Chikungunya viruses causing the Indian Ocean outbreak. Exp Biol Med (Maywood), 236, 909–​14. Powers AM (2018). Vaccine and Therapeutic Options to Control Chikungunya Virus. Clin Microbiol Rev, 13, e00104–16. Schwartz O, Albert ML (2010). Biology and pathogenesis of chikun- gunya virus. Nat Rev Microbiol, 8, 491–​500. Suhrbier A, Jaffar-​Bandjee MC, Gasque P (2012). Arthritogenic alphaviruses—​an overview. Nat Rev Rheumatol, 8, 420–​9. Tesh RB, et al. (1999). Mayaro virus disease: an emerging mosquito-​ borne zoonosis in tropical South America. Clin Infect Dis, 28, 67–​73. Weaver SC, et al. (2004). Venezuelan equine encephalitis. Annu Rev Entomol, 49, 141–​74. Zacks MA, Paessler S. (2010). Encephalitic alphaviruses. Vet Microbiol, 140, 281–​6.

8.5.13 Rubella 827

8.5.13 Rubella 827

8.5.13  Rubella 827 8.5.13  Rubella Pat Tookey and J.M. Best ESSENTIALS Rubella is caused by an enveloped RNA virus, for which humans are the only known host. Transmission is by airborne droplet spread, with infection seen predominantly in spring and early summer in temperate zones. Postnatally acquired infection—​presents after incubation of
14–​21  days with rash (maculopapular, usually beginning on
the face before spreading to the trunk and extremities), lymph- adenopathy (suboccipital and posterior cervical), and mild fever. Sore throat, coryza, cough, conjunctivitis, and arthralgia may be seen. The illness is usually mild. Management is symptomatic. Rubella in pregnancy—​in the first 10 weeks of gestation
this is associated with a 90% risk of congenital fetal abnormal- ities, most typically comprising sensorineural hearing loss, alone or combined with cataracts and/​or cardiac anomalies. Clinical diagnosis is unreliable, hence rapid investigation is essential when a woman develops a rubella-​like illness in the first 16 weeks of pregnancy, comprising (1)  testing of maternal serum for rubella IgG and IgM antibodies; and sometimes (2) amniotic fluid and/​or fetal blood testing; and (3) ultrasonography to de- tect fetal defects. If a fetus is infected, termination of pregnancy is considered. Prevention—​live attenuated rubella vaccines provide protec- tion to about 95% vaccinees and are usually given in combin- ation with measles or measles and mumps vaccines. Vaccination of greater than 80% of children is required to prevent circulation of rubella virus. Healthcare workers and women of childbearing
age whose rubella status is unknown (including recent immi- grants) should also be targeted for measles, mumps, and rubella vaccination. Immunization of pregnant women is contraindi- cated, but women found to be susceptible at antenatal testing should be offered measles, mumps, and rubella vaccination after delivery. Introduction Rubella is a mild exanthematous disease of little clinical signifi- cance. However, infection in early pregnancy can result in multiple congenital abnormalities, often referred to as ‘congenital rubella syndrome’. As a result of the widespread use of rubella vaccine, con- genital rubella syndrome is now rare in many countries. Aetiology Rubella is caused by rubella virus, an enveloped RNA virus, which is classified in its own genus Rubivirus within the family Togaviridae. There are no major antigenic differences among ru- bella virus isolates, although at least seven genotypes have been described. Epidemiology Humans are the only known host for rubella virus. In temperate zones the infection is seen predominantly in spring and early summer. Before the introduction of rubella vaccine, rubella was endemic in virtually all countries. Epidemics were superimposed on the endemic infection every 4 to 9 years and pandemics every 10–​30 years. In most populations, in the absence of a mass im- munization programme, 10–​20% of women are still susceptible to rubella infection when they reach childbearing age. A review by the World Health Organization in 2008 estimated that more than 110 000 infants were born with congenital rubella syndrome each year with the highest burden being in the WHO African and Southeast Asian regions. Postnatally acquired infection The rash usually begins on the face and spreads to the trunk and then the extremities; the pink maculopapular lesions are initially discrete but later tend to coalesce. The suboccipital and posterior cervical lymph nodes are characteristically enlarged. Mild fever, sore throat, coryza, cough, and conjunctivitis may be present; symptoms are usually mild and last 3–​7 days. There may be a pro- drome with malaise and fever, especially in adults. There is no spe- cific treatment. Transient arthralgia with or without arthritis occurs in up to 70% of postpubertal women, but is less common in men and chil- dren. Less common complications include thrombocytopenia with or without purpura, postinfectious encephalitis, transverse myelitis, and rarely the Guillain–​Barré syndrome. When rubella is acquired in early pregnancy congenital infection might occur (see next). Rubella is clinically indistinguishable from several other infec- tions and 20–​50% of infections are subclinical. Therefore, a his- tory of clinically diagnosed rubella infection is unreliable. The incubation period is 14–​21 days. The exact mode of trans- mission is uncertain but airborne spread by the respiratory route is likely and close contact is usually necessary for transmission. Individuals are most infectious just before the onset of symptoms, and the infectious period lasts from about a week before to a week after the rash appears. Infection usually produces lifelong immunity; however, when rubella is circulating reinfection may occur and is usually asymptomatic. Congenital infection Risk to the fetus The possible consequences of rubella in pregnancy are the birth of an infant with congenital rubella infection with or without congenital defects, the birth of a normal infant, or spontaneous abortion. Infection before conception is not a risk to the fetus. Spontaneous abortion may occur when rubella is acquired early in pregnancy. When maternal infection occurs during the first 10 weeks of pregnancy the rate of fetal infection is about 90%; it then declines until the last few weeks of pregnancy when the rate rises

828 section 8  Infectious diseases again. Virtually all of those infected during the first 10 weeks of pregnancy are likely to have congenital defects, but the risk de- clines over the next 6 weeks. After 16 weeks’ gestation, even sen- sorineural hearing loss and growth retardation are rare, and no abnormalities have been demonstrated following serologically confirmed maternal infection after 18 weeks’ gestation. Most pro- spective studies of the risk to the fetus have been carried out on women with symptoms, but asymptomatic primary infection is thought to carry a similar risk. Following maternal reinfection in pregnancy the risk of transmis- sion to the fetus is probably less than 10% and the risk of damage less than 5%, although it may be higher following symptomatic reinfection. Clinical features Congenital rubella is typically associated with cataracts, cardiac anomalies, and sensorineural hearing loss, and the term congenital rubella syndrome refers to this classic triad of defects. The terato- genic effects may result in a variety of defects (Box 8.5.13.1), but sensorineural hearing loss alone or combined with other abnor- malities is most common. Severe multiple problems are more likely when infection occurs early in pregnancy. Some defects, particularly sensorineural hearing loss, may not develop or become apparent until late infancy or childhood. Other reported late-​onset problems include diabetes mellitus, thyroid dysfunction, autism, and other behavioural and psychi- atric disorders. A  rare progressive rubella panencephalitis has also been reported. Laboratory diagnosis The diagnosis of congenital rubella infection is relatively easy if suspected early, but more difficult to confirm after 3 months of age. The presence of rubella IgM antibody in early infancy is vir- tually diagnostic of congenital infection because acquired infec- tion is rare at this age. Using sensitive assays, rubella IgM may be detected in 85% of infected infants at 3–​6 months and about 30% at 6–​12 months of age. The presence of IgG antibody alone is not diagnostic since it is likely to indicate passively transferred ma- ternal antibody, but persistence of IgG between 6 and 12 months is strongly suggestive of congenital infection. When abnormalities present late, a presumptive diagnosis can be made based on a com- patible clinical picture and the presence or persistence of rubella IgG antibodies in a young child who has not yet been vaccinated. Congenital infection can also be diagnosed by detection of virus during the first months of life when it can be isolated or detected by polymerase chain reaction from a variety of specimens including nasopharyngeal swabs, urine, oral fluid, and conjunctival fluid. Congenitally infected infants shed large amounts of virus from the oropharynx and may be a source of infection for many months; viral shedding occasionally persists for more than a year. Management of rubella-​like illness during pregnancy Appropriate management of a rash illness in pregnancy will de- pend on the local epidemiology of rubella. Routine antenatal rubella antibody screening is not designed to identify rubella in- fection in pregnancy, and specific diagnostic investigations are needed. Pregnant women with a rubella-​like rash should be in- vestigated simultaneously for rubella and parvovirus B19, since they are clinically indistinguishable and even women previously reported to be immune should be investigated in case of laboratory error. Blood should be tested for rubella IgG and IgM antibodies. Rising IgG or detectable IgM antibody indicates recent infection; a positive IgM result alone should be confirmed with a second serum sample. Pregnant women who are susceptible or of unknown ru- bella antibody status and are in contact with a rubella-​like illness should also be investigated as rapidly as possible. The detection of rubella IgM in a woman without a rash or history of contact should be interpreted with caution as rubella IgM may persist for some months or even years after infection or vaccination, or the IgM may be due to cross-​reaction with autoantibodies or other viral IgM antibodies. Investigations must be done in consultation with a virologist who should be aware of the date and type of con- tact, stage of pregnancy, and history of previous immunization and testing. Prenatal diagnosis of congenital infection using amniotic fluid and/​or fetal blood may sometimes be indicated. Ultrasound examination may detect such defects as microcephaly, dystrophic calcification, cataracts, microphthalmos, hepatosplenomegaly, and intrauterine growth restriction. Box 8.5.13.1  Most common defects associated with congenital rubella Classic triad • Deafness -​ Sensorineural -​ Central auditory • Abnormalities of the cardiovascular system -​ Patent ductus arteriosus -​ Pulmonary stenosis -​ Pulmonary arterial hypoplasia • Abnormalities of the eye -​ Retinopathy -​ Cataracts -​ Microphthalmos -​ Iris hypoplasia Other defects • Growth retardation • Microcephaly • Mental retardation • Speech defects Other signs in the neonatal period and infancy • Low birthweight • Hepatosplenomegaly • Jaundice • Meningoencephalitis • Rash • Thrombocytopenia with or without purpura • Adenopathies • Bony radiolucencies • Hypogammaglobulinaemia • Pneumonitis

8.5.13  Rubella 829 Prevention Rubella can be prevented by live attenuated rubella vaccines. The RA27/​3 strain is commonly used and this produces antibodies in about 95% of recipients; protection is probably lifelong in most vaccinees. Rubella vaccine is usually combined with measles or mea- sles and mumps (MMR) vaccines. In children, rubella vaccine causes few side effects. Low-​grade fever and rash are occasionally reported, and transient arthralgia has been seen in about 3% of vaccinees; there have also been rare re- ports of myositis and vasculitis. Joint symptoms are more common in adult women, affecting up to 60% of vaccinees, but are transient and less severe than following naturally acquired rubella. When rubella vaccines were first licensed in the late 1960s, universal childhood vaccination was implemented in the United States of America with the aim of eliminating rubella. A different strategy was pursued elsewhere, and the selective programmes established in Australia and some European countries targeted prepubertal girls and women of childbearing age. This provided individual protection while allowing the continued circulation of wild virus and the acquisition of natural immunity by most individ- uals. When the combined MMR vaccine became available, many countries with high vaccine uptake moved to a universal offer of MMR vaccine for children in the second year of life, usually with a second dose offered preschool or later. The MMR vaccine was introduced into the United Kingdom schedule in 1988, and uptake by the age of 24 months reached 92% between 1992 and 1996. The schoolgirl programme was discon- tinued in 1996 and replaced by the offer of a second MMR for four-​ year-​olds. Uptake of MMR subsequently declined to a low point of 80% in 2003, because of unfounded concerns about safety; however, by 2005 there were signs of recovery and uptake had increased to over 92% by 2011/​12. Although the circulation of rubella virus has dropped to very low levels since the introduction of MMR, prolonged periods of low vaccine uptake may lead to outbreaks of rubella in the future, putting susceptible pregnant women in the United Kingdom at risk. Nevertheless, there have been dramatic declines in the num- bers of susceptible pregnant women, rubella-​associated termin- ations, and children born with congenital rubella syndrome. Fewer than five congenitally infected infants were reported on average each year between 1990 and 1999, compared with about 50 per year in the 1970s. Between 2000 and 2014 about 20 cases were identified, and in over half of these the infant’s mother acquired infection abroad. Termination of pregnancy associated with rubella disease or contact is also now a rare occurrence. In 2012 the World Health Organization published a strategic plan for the global elimination of measles, rubella, and congenital rubella, with the goal of achieving both measles and rubella elimination in at least five of the six WHO Regions by 2020, and laying out specific strategies focusing on vaccination coverage, evaluation, and surveil- lance, outbreak management, public engagement, and research and development. This built on previous initiatives, particularly the ex- perience gained in the WHO Region of the Americas, where, with no evidence of endemic transmission of rubella or congenital rubella in five consecutive years, elimination of rubella and congenital ru- bella was confirmed in 2015. The WHO European Region renewed its commitment to the elimination goal in 2014 with the adoption of a European Vaccine Action Plan 2015–​2020, and reported that 23 of the Region’s 53 member states had interrupted endemic transmis- sion of rubella, based on 2013 reporting. Vaccination in pregnancy There have been persistent concerns that the vaccine virus might be teratogenic if given during pregnancy. Although vaccinees cannot infect other susceptible individuals, the virus can cross the placenta. Data from studies of children born to several hundred women inadvertently vaccinated up to 3 months before conception or during pregnancy show less than 3% with serological evidence of congenital infection, and no reported case of abnormalities attrib- utable to congenital rubella. At least 80 of these infants were born to women vaccinated in the month of conception, probably the period of greatest vulnerability. These data suggest that the likely maximum theoretical risk of rubella-​associated abnormalities is less than 5%. Likely developments • Elimination of rubella by further countries • Introduction of rubella vaccine in additional countries worldwide • Use of mathematical models to guide rubella vaccination strat- egies in different countries • Use of genotyping to track the source of rubella outbreaks as coun- tries approach elimination of rubella virus • Development of techniques for the diagnosis of congenital rubella syndrome after the age of 3 months FURTHER READING Banatvala JE, Brown DWG (2004). Rubella. Lancet, 363, 1127–​37. Banatvala JE, Peckham C (eds) (2007). Rubella viruses: perspectives in medical virology, vol. 15. Elsevier, London. Best JM, Icenogle JP, Brown DWG (2009). Rubella. In: Zuckerman AJ, et al. (eds) Principles and practice of clinical virology, 6th edition, pp. 561–​92. John Wiley & Sons, Chichester. Cooper LZ, Alford CA (2006). Rubella. In:  Remington JS, et  al.
(eds) Infectious diseases of the fetus and newborn infant, 6th edition, pp. 894–​926. Elsevier, Saunders, Philadelphia, PA. Department of Health (2013). Rubella. In: Immunisation against in- fectious disease—​‘the green book’. https://​www.gov.uk/​government/​ collections/​immunisation-​against-​infectious-​disease-​the-​green-
​book Goodson JL, et  al. (2011). Rubella epidemiology in Africa in the prevaccine era, 2002–​2009. J Infect Dis, 204(Suppl 1), S215–​25. HPA Rash Guidance Working Group (2011). Guidance on viral rash in pregnancy: investigation, diagnosis and management of viral rash illness, or exposure to viral rash illness, in pregnancy. http://​www.hpa. org.uk/​web/​HPAwebFile/​HPAweb_​C/​1294740918985 Robertson SE, et al. (2003). Rubella and congenital rubella syndrome: global update. Pan Am J Public Health, 14, 306–​15. World Health Organization (2012). Global measles and rubella: strategic plan: 2012–​2020. WHO, Geneva. http://​www.measlesrubellainitiative. org/​wp-​content/​uploads/​2013/​06/​Measles-​Rubella-​Strategic-​ Plan.pdf

8.5.14 Flaviviruses excluding dengue 830

8.5.14 Flaviviruses excluding dengue 830

830 section 8  Infectious diseases 8.5.14  Flaviviruses excluding dengue Shannan Lee Rossi and Nikos Vasilakis ESSENTIALS Dengue and dengue haemorrhagic fever (see Chapter 8.5.15) are the most important and widespread human diseases caused by an arbovirus, causing a broad spectrum of illness ranging from asymptomatic to severe and fatal haemorrhagic disease. It is pri- marily an urban disease transmitted among humans by the highly domesticated Aedes aegypti mosquito. Japanese encephalitis virus—​has a widespread distribution throughout Asia; is the most important cause of arboviral en- cephalitis; is maintained in a cycle involving Culex mosquitoes and water birds; only about 1% of infections are symptomatic, with manifestations ranging from a febrile illness with headache, through aseptic meningitis, to encephalitis, and death. Many sur- vivors have residual neurological abnormalities. There is no spe- cific treatment. Vaccination should generally be offered to people spending a month or more in endemic areas, especially if travel includes rural areas. Yellow fever virus—​found in tropical America and Africa; forest/​ jungle transmission cycle involves canopy-​dwelling mosquitoes and monkeys, urban cycle involves humans as the vertebrate host and Aedes aegypti as the principal vector; 5% of infections present clinic- ally with a viraemic illness, which may be followed after a transient period of remission by relapse with shock, neurological deterior- ation, jaundice, haemorrhagic manifestations, and renal failure. Treatment is symptomatic. A live, attenuated, single-​dose vaccine is highly effective. Zika virus was relatively rare until the last decade but now has a large distribution. In 2015–​2016, it was responsible for large out- breaks in South America. Like dengue virus, it is largely spread by the A. aegypti mosquito and, in most people is either asymptomatic or causes a febrile illness with a rash and arthralgia. However, it is now known to be associated with congenital defects, particularly microcephaly, and with Guillain–​Barré syndrome. There is no spe- cific treatment and no vaccine available. West Nile virus—​found in Africa, the Middle East, Asia, Australia (Kunjin is a subtype of West Nile virus), parts of Europe and the Americas; maintained in a cycle involving Culex mosquitoes and water birds; most infections are asymptomatic, but 20% develop a febrile illness, and 1% neuroinvasive disease including meningitis, encephalitis, and acute flaccid paralysis. There is no specific treat- ment. Several equine vaccines are available, and human vaccines are in clinical trials. Other important mosquito-​borne flaviruses include Murray Valley, St Louis encephalitis, and Rocio virus. Tick-​borne flaviviruses Tick-​borne encephalitis, louping ill, Powassan encephalitis—​geo- graphical distribution determined by that of relevant hard tick vectors; rodents are the principal vertebrate hosts, with occupa- tional and vocational pursuits favouring tick exposure as risk fac- tors for human disease; most infections are subclinical, but a non​ specific influenza-​like illness may be followed, after a few days of apparent recovery, by aseptic meningitis or meningoencephalitis that may lead to permanent paralysis in some cases. Treatment is supportive. Effective inactivated vaccines are available for tick-​ borne encephalitis. Tick-​borne haemorrhagic fevers—​these include Kyasanur Forest disease and Alkhumra (strictly Al Khumra) and Omsk haemorrhagic fevers. Introduction The family Flaviviridae currently consists of four recognized genera: Flavivirus, Pestivirus, Hepacivirus, and Pegivirus. Although members of the family have a large host range that includes both vertebrates and invertebrates, only members of the genus Flavivirus are known as arboviruses, vectored either by mosqui- toes or ticks. The remaining genera in the family are exclusively found in mammals, and their diversity has greatly expanded with recent virus discoveries. The genus Flavivirus comprises 92 virus species, of which over 40 can cause human infection (Table 8.5.14.1). Many of these include important human patho- gens such as Zika (ZIKV), dengue (DENV), yellow fever, West Nile (WNV), and Japanese encephalitis virus. Flaviviruses are small spherical particles of approximately 40–​50 nm in diameter, whose genome is a positive sense single stranded RNA of c.11 kb that encodes three structural proteins and seven nonstructural proteins. Based on epidemiological and phylogenetic analyses (Fig. 8.5.14.1), the flaviviruses are classified into four groups: (1) those that are mosquito-​borne, (2) those that are tick-​borne, (3) those for which no arthropod vector has been demonstrated, and (4)  those with a restricted host range transmission among arthropods (insect-​specific) without the involvement of verte- brates. All flaviviruses of human importance belong to the first two groups; the last two groups contain viruses found only in other vertebrates or in arthropods, respectively. Most flaviviruses are maintained in nature within two ecologic- ally and evolutionarily distinct transmission cycles between ver- tebrates (wild or domestic animals or humans) and one or more hematophagous arthropod vectors. The transmission cycles in- clude: (i) an enzootic, sylvatic cycle, where the virus circulates be- tween arboreal mosquitoes and non​human primates; and (ii) and a human or urban cycle, between humans and peridomestic/​ domestic mosquitoes. Representative transmission cycles for Zika and yellow fever viruses are depicted in Fig. 8.5.14.2. Humans mostly become infected when infected arthropod vectors feed on them and, for most of the flaviviruses, humans do not develop high enough viremias and are not thought to contribute to the transmission cycle. However, some flaviviruses such as dengue, yellow fever, and Zika viruses do produce high viremias in hu- mans, which allow maintenance through a mosquito–​human–​ mosquito transmission cycle. Transmission of some flaviviruses directly from one person to another through blood transfusion or organ transplantation as well as in utero or via sexual contact has also been documented. The epidemiology and geographical distribution of members of the genus flavivirus depends on several factors including: (i) the

8.5.14  Flaviviruses excluding dengue 831 Table 8.5.14.1  Taxonomy of flaviviruses Group Species name Strain name, synonyms, and tentative species names Abbreviation Mosquito-​borne viruses Aroa virus group Aroa virus Aroa virus AROAV Bussuquara virus BSQV Iguape virus IGUV Narajal virus NJLV Dengue virus group Dengue viruses Dengue virus 1 DENV-​1 Dengue virus 2 DENV-​2 Dengue virus 3 DENV-​3 Dengue virus 4 DENV-​4 Japanese encephalitis virus group Cacipacore virus Cacipacore virus CPCV Japanese encephalitis virus Japanese encephalitis virus JEV Koutango virus Koutango virus KOUV Murray Valley encephalitis virus Alfuy virus ALFV Murray valley encephalitis virus MVEV St Louis Encephalitis virus St Louis Encephalitis virus SLEV Usutu virus Usutu virus USUV West Nile virus Kunjin virus KUNV West Nile virus WNV Yaounde virus Yaounde virus YAOV Kokobera virus group Kokobera virus Kokobera virus KOKV Stratford virus STRV Ntaya virus group Bagaza virus Bagaza virus BAGV Ilheus virus Ilheus virus ILHV Rocio virus ROCV Israel Turkey meningoencephalitis virus Israel Turkey meningoencephalitis virus ITV Ntaya virus Ntaya virus NTAV Tembusu virus Tembusu virus TMUV Zika virus Zika virus ZIKV Yellow fever virus group Sepik virus Sepik virus SEPV Wesselsbron virus Wesselsbron virus WSLV Yellow fever virus Yellow fever virus YFV Probably mosquito-​borne Kedougou virus group Kedougou virus Kedougou virus KEDV Edge Hill virus group Banzi virus Banzi virus BANV Bouboui virus Bouboui virus BOUV Edge Hill virus Edge Hill virus EHV Jugra virus Jugra virus JUGV Saboya virus Potiskum virus POTV Saboya virus SABV Uganda S virus Uganda S virus UGSV Tick-​borne viruses Mammalian tick-​borne virus group Gadgets Gully virus Gadgets Gully virus GGYV Kyasanur Forest disease virus Kyasanur Forest disease virus KFDV Alkhumra haemorrhagic fever virus AHFV Langat virus Langat virus LGTV (continued)

832 section 8  Infectious diseases Group Species name Strain name, synonyms, and tentative species names Abbreviation Louping ill virus Louping ill virus LIV British subtype LIV-​Brit Irish subtype LIV-​IR Spanish subtype LIV-​Spain Turkish sheep encephalitis virus subtype TSEV Greek goat encephalitis virus subtype GGEV Omsk haemorrhagic fever virus Omsk haemorrhagic fever virus OHFV Powassan virus Powassan virus POWV Royal Farm virus Royal Farm virus RFV Tick-​borne encephalitis virus Tick-​borne encephalitis virus TBEV European subtype TBEV-​Eu Far Eastern subtype TBEV-​FE Siberian subtype TBEV-​Sib Seabird tick-​borne virus group Meaban virus Meaban virus MEAV Saumarez Reef virus Saumarez Reef virus SREV Tyuleniy virus Tyuleniy virus TYUV Probably tick-​borne Kadam virus group Kadam virus Kadam virus KADV Viruses with no known arthropod vector Entebbe bat virus group Entebbe bat virus Entebbe bat virus ENTV Sokoluk virus SOKV Yokose virus Yokose virus YOKV Modoc virus group Apoi virus Apoi virus APOIV Cowbone Ridge virus Cowbone Ridge virus CRV Jutiapa virus Jutiapa virus JUTV Modoc virus Modoc virus MODV Sal Vieja virus Sal Vieja virus SVV San Perlita virus San Perlita virus SPV Rio Bravo virus group Bukalasa bat virus Bukalasa bat virus BBV Carey Island virus Carey Island virus CIV Dakar bat virus Dakar bat virus DBV Montana myotis leukoencephalitis virus Montana myotis leukoencephalitis virus MMLV Phnom Penh bat virus Batu Cave virus BCV Phnom Penh bat virus PPBV Rio Bravo virus Rio Bravo virus RBV Viruses tentatively placed in the Flavivirus genus Mammalian tick-​borne Karshi virus KSIV Mosquito-​borne Spondweni virus SPOV Insect/​Mosquito Specific Flaviviruses Aedes flavivirus AEFV Cell fusing agent virus CFAV Culex flavivirus CXFV Kamiti River virus KRV Table 8.5.14.1  Continued (continued)

8.5.14  Flaviviruses excluding dengue 833 presence of suitable amplifying hosts, (ii) the presence, density and feeding behaviour of suitable arthropod vectors, and (iii) the frequency of exposure of immunologically naive vertebrate reser- voir hosts susceptible to infection. Following World War II, glo- balization of trade and travel, uncontrolled human population growth and urbanization, changes in land and water use, changes in agricultural practices, new irrigation systems and deforest- ation and unsustainable vector control programmes have pro- duced fertile conditions for the explosive increase in incidence and geographical expansion of the flaviviruses (Fig. 8.5.14.3). Two dramatic examples in the last 20 years are the introduction and subsequent spread of the West Nile and Zika viruses in the western hemisphere. Flavivirus infections in humans can result in a wide spectrum of manifestations ranging from asymptomatic infection or clin- ical illness ranging from non​specific febrile illness, fever with rash or arthralgia or both, haemorrhagic fever, hepatitis, encephal- itis, and death. For most flaviviral infections no specific therapy is available, however, prompt supportive treatment and proper management may substantially reduce mortality from some flavivirus infections. Laboratory diagnosis All flaviviruses have common group epitopes on the pre/​mem- brane, envelope, and non​structural 1 (NS1) proteins that result in extensive cross-​reactions in serological tests. The specificity of antibodies ought to be confirmed by the gold standard test of specificity, the plaque reduction neutralization test (PRNT). However, in areas where multiple flaviviruses are hyperendemic/​ enzootic, antibody-​based assays, including the PRNT assay, are non​informative. The most common diagnostic assay based on serology for acute flavivirus infections is the IgM antibody capture enzyme-​linked immunosorbent assay (MAC-​ELISA), which is cost-​effective and does not require specialized laboratory settings. IgM-​specific anti- bodies are usually detectable 5 to 8 days after onset of symptoms. Group Species name Strain name, synonyms, and tentative species names Abbreviation Nakiwogo virus NAKV Quang Binh virus QBV Mercadeo virus MECDV Hanko virus HANKV Nienokoue virus NIEV Palm Creek virus PCV Ilomantsi virus ILOV Marisma mosquito virus MMV Donggang virus DONV LaTina virus LTNV Long Pine Key virus LPKV Kampung Karu virus KKV Nhumirim virus NHUV Barkedji virus BJV Culiseta flavivirus CsFV Parramatta River virus PaRV Yamadai flavivirus YDFV Yunnan Culex flavivirus YNCxFV Culex theileri flavivirus CtFV Ochlerotatus caspius flavivirus OCFV Xishuangbanna flavivirus XFV Viruses with no known arthropod vector Chaoyang virus CHAOV Lammi virus LAMV Ngoye virus NGOV Nounané virus NOUV Tamana bat virus TABV Table 8.5.14.1  Continued

834 section 8  Infectious diseases -Insect-specific viruses -No known vector -Tick-borne -Vectored by ‘Old World’ Stegomyia Spp. -Vertebrate host unknown -Vectored by Stegomyia Spp. -Vectored by Culex Spp. Fig. 8.5.14.1  Maximum-​likelihood phylogenetic tree of representative members of the genus flavivirus. Bootstrap values are shown for most clades. All horizontal branch lengths are drawn to scale; bar, 0.05 nucleotide substitutions per site. The tree is midpoint-​rooted for purposes of clarity only.

8.5.14  Flaviviruses excluding dengue 835 However, because IgM antibodies can persist for one or more months after infection with most flaviviruses, their presence does not necessarily confirm current infection. Therefore, people with detectable IgM antibodies are considered recent or presumptive cases. Confirmatory laboratory diagnosis of most flaviviruses re- quires isolation of the virus, detection of specific viral RNA by nu- cleic acid amplification or of specific antigen in autopsy tissues by immunohistochemistry. Mosquito-​borne flavivirus infections of human health importance Japanese encephalitis virus Aetiology and epidemiology Japanese encephalitis virus is the most important cause of arboviral encephalitis with several thousand cases annually. Japanese enceph- alitis has a widespread distribution throughout Asia, and its distri- bution has expanded in recent years with outbreaks in the Pacific, Australia, Nepal, and western India, putting at risk of infection close to 3 billion people (Fig. 8.5.14.4). Virus transmission occurs pri- marily in rural agricultural areas, often associated with rice pro- duction and flooding irrigation. Although transmission can occur year-​round in the tropics, peaking during the rainy season, in tem- perate regions, Japanese encephalitis virus transmission is seasonal with disease incidence peaking in the summer and fall. Japanese encephalitis virus is antigenically related to several other flaviviruses that may have similar geographic distribution. The virus is maintained in a transmission cycle involving Culex mosquitoes and wading birds, and is transmitted to humans by Culex mosquitoes, primarily species of the Culex tritaeniorhynchus complex which breed in rice fields. Humans are considered dead-​ end hosts, because they do not develop high enough viremia to in- fect feeding mosquitoes, whereas pigs are considered the primary amplifying host in the peridomestic environment. There are several genotypes of Japanese encephalitis viruses that circulate in distinct geographical areas. Clinical characteristics Most Japanese encephalitis virus infections are asymptomatic and only about 1% of all infections develop clinical illness, which ranges from febrile illness with headache, aseptic meningitis, encephalitis, and death. The incubation period lasts 6–​16 days, before onset of symptoms presented by high fever, change in mental status (leth- argy), nausea and vomiting, and headache (prodromal state) lasting several days. The onset of neurological signs, altered state of con- sciousness and delirium reflects damage to the thalamus, brain stem, and cerebral cortex. In most patients seizures are generalized, although at their onset can be more focal. In 30% of the patients, facial and cranial nerve palsies, and acute flaccid paralysis are ob- served. This poliomyelitis-​like illness may be the only neurological manifestation of the illness or may proceed or accompany enceph- alitis. Respiratory dysregulation, coma, abnormal plantar reflexes, ZIKV YFV Sylvatic Zone of emergence Urban TOT TOT ?? Ae. africanus (Africa) Ae. luteocephalus (Africa)* Ae. metallicus (Africa) Ae. opok (Africa) Ae. vittatus (Africa)* Ae. simpsoni complex (Africa) Homo sapiens Ae. aegypti aegypti (global) Alouatta spp (S. America) Colobus spp (Africa) Cercopithecus spp. (Africa) G. senegalensis (Africa) H. janthinomys (S. America) H. leucocelaenus (S. America) S. chloropterus (S. America) Ae. africanus (Africa)* Ae. bromeliae (Africa) Ae. taylori (Africa)* Ae. africanus (Africa)* Ae. dalzieli (Africa) Ae. furcifer (Africa)* Ae. luteochephalus (Africa)* Ae. vittatus (Africa) Rhesus spp (Africa) Chlorocebus sabaeus (Africa) Cercopithecus spp (Africa) Colobus guereza (Africa) Erythrocebus patas (Africa) Pongo borneo (SE Asia) ?? Ae. apicoargenteus (Africa) Ae. furcifer (Africa)* Ae. hirsitus (Africa) Ae. metallicus (Africa) Ae. opok (Africa) Ae. taylori (Africa)* Ae. unilineatus (Africa) Ma. uniformis (Africa) An. coustani (Africa) Cx. perfuscus (Africa) Ae. aegypti aegypti (global) Ae. albopictus?? Ae. polynensiensis (Polynesia) Ae. hensilii (Polynesia) Homo sapiens Ae. Furcifer (Africa)* Ae. Furcifer (Africa)* Fig. 8.5.14.2  Transmission cycles of Zika and yellow fever viruses, flaviviruses with significant human health impact. ToT, transovarial transmission; *, indicates major vectors; in red: vectors in either transmission cycle; in green: vectors implicated as bridge vectors.

836 section 8  Infectious diseases and prolonged convulsions are associated with a poor prognosis. Defervescense occurs in the second week of the illness and is char- acterized by gradual improvement of neurologic manifestations and recovery, although long-​term sequelae may remain. Overall, up to 70% of survivors have residual neurological abnormalities including behavioural changes, and psychological deficits. Curiously, in some patients clinical relapse has been observed several months following recovery from acute illness, suggesting persistent infection, likely of peripheral mononuclear cells. Laboratory examination during the first week of illness shows modest levels of peripheral leukocytosis and hyponatraemia due to dysregulated antidiuretic hormone (ADH) secretion. Cerebrospinal fluid is clear and elevated pleocytosis is observed. Around 10–​40% of cases are fatal usually within the first week of illness. Children less than 10 years of age are more likely to die, and if they survive, are more likely to have residual neurological defects. Congenital infection during the first and second trimester of pregnancy has led to fetal death and spontaneous abortion, whereas infection in the third trimester has been associated with normal fetal outcomes. Common complications during Japanese encephalitis virus infections include concurrent bacterial and parasitic infections, or tuberculosis, which could complicate man- agement of the illness. Diagnosis The differential diagnosis in Japanese encephalitis virus infec- tions includes other viral encephalitides including arboviruses (e.g. dengue with encephalopathy, WNV, Murray Valley enceph- alitis), herpes, and enteroviral infections (mostly entrovirus 71), cerebral malaria, and bacterial infections. Travel history, season, location of residency may provide clues for diagnosis. Nucleic acid testing (NAT) is useful only in the early acute stage of illness. Serology offers accurate and specific diagnosis. MAC-​ELISA is nearly 100% sensitive in paired blood and cerebrospinal fluid samples obtained 1–​2 weeks after the onset of illness. However, as discussed earlier (‘Laboratory diagnosis’ section), due to cross-​ reaction with other flaviviruses, results may be difficult to inter- pret, especially in patients living in hyperendemic settings, where other related flaviviruses, such as SLEV, WNV, and dengue are cocirculating. Prevention and control There are safe and efficacious vaccines against Japanese enceph- alitis, ranging from inactivated to attenuated. The formulation offered will depend on the country providing the vaccine. For ex- ample, the live attenuated SA14-​14-​2 has been successfully used in endemic Asian countries. However, an inactivated vaccine derived Fig. 8.5.14.3  Global geographic distribution of medically important flaviviruses.

8.5.14  Flaviviruses excluding dengue 837 from cell culture is the only version offered in the United States and requires two doses. It is highly recommended by the Centers for Disease Control and Prevention (CDC) to receive this traveller’s vaccine if the stay will be longer than a month or will occur in high transmission areas and times. Typically, short-​term stays in highly urbanized areas during a low-​transmission time of the year does not warrant traveller’s vaccination. As with all vector-​born viral infections, preventing the vector’s bite is one of the best ways to prevent infection. Wearing mosquito repellent as well as long-​sleeved clothing will work well. Treatment There is no treatment specifically tailored to combat Japanese en- cephalitis disease. If a patient is in the hospital, supportive care is administered. Fluid and pain medication are typically offered to patients. Yellow fever virus Aetiology and epidemiology Although yellow fever was first described in the 17th century, it re- mains to this day an important human disease in vast areas of Africa and South America (Fig. 8.5.14.4), with 200 000 infections and 30 000 deaths every year; nearly 90% of these occurring in Africa. Today nearly a billion humans are at risk of infection every year. In 1900, the anthropophilic mosquito Aedes aegypti was proven to be involved in the transmission of yellow fever. The virus was isolated in 1927 and a vaccine developed in 1937, leading to the award of the Nobel Prize to its inventor (Max Theiler). The virus is present in tropical America and Africa, but has not been reported in Asia. The transmission cycles of yellow fever include: (i) an enzootic, sylvatic cycle, where the virus circulates between arboreal mosquitoes and non​human primates; and (ii) a human or urban cycle, between hu- mans and peridomestic/​domestic mosquitoes. Transovarial trans- mission of mosquitoes had been demonstrated in both transmission cycles and may provide a mechanism for the maintenance of the transmission cycles in interepidemic periods (Fig. 8.5.14.2). The American yellow fever originated from the Old World as a result of sailing ships. Between 1986 and 2016, a series of outbreaks in Nigeria caused an estimated 100 000 cases, with attack rates in affected areas of 30/​ 1000 and case fatality rates exceeding 20%. Similar epidemics occur in regular intervals in South America, in what are termed travel- ling epizootics. The disease affects several hundred people annually, principally young men working in forest areas exposed to arboreal mosquitoes. In the past 10 years, yellow fever was reported in the Democratic Republic of the Congo, Angola, Côte d’Ivoire, Central African Republic, Liberia, Cameroon, Guinea, Uganda, Peru, Brazil, Argentina, and Paraguay. At the time of writing there is another out- break in central and northern Brazil, resulting in several deaths to date. These events have led to severe vaccine shortages leading to frac- tional dose vaccine administration. Additionally, as ecotourism has increased in recent years, yellow fever in unvaccinated travellers from Areas with Risk of Japanese Encephalitis and Yellow Fever Virus Endemic Yellow Fever Virus Tranmission Risk Japanese Encephalitis Risk No Known Risk 0 Kilometers N Robinson Projection 2,000 4,000 Fig. 8.5.14.4  Global distribution of Japanese encephalitis virus. Source data from WHO Fact sheet No 386, ‘Japanese encephalitis’, December 2015. Copyright © WHO 2012.

838 section 8  Infectious diseases North America and Europe has become more common. Furthermore, the recent detection of YF-viremic Chinese workers returning to Asia from Angola raises serious concerns for the establishment of a YFV transmission cycle in Asia. The prospect of such an event will be cata- strophic as it will put a third of the world’s population at risk who are currently immune-naive. Clinical characteristics In some patients, yellow fever infection is asymptomatic or presents as a mild, undifferentiated febrile illness. The incubation period lasts 3–​6 days, and in its classic form, is characterized by an abrupt onset of chills, fever, headache, viremia, photophobia, lumbosacral pain, nausea, prostration, generalized myalgia, facial flushing, red tongue, and conjunctivitis. The moderately ill begin to recover after a period of 3–​4 days. However, in severe cases this recovery is transient, also known as period of remission, only to relapse with jaundice, albumin- uria, oliguria, bradycardia (Faget’s sign), delirium, stupor, metabolic acidosis, shock, and haemorrhage. The haemorrhagic manifest- ations are caused by decreased synthesis of clotting factors and may be complicated by disseminated intravascular coagulation, and can vary from petechial lesions to epistaxis, bleeding gums, and haema- temesis. This relapse is known as the period of intoxication and the prognosis in such cases is poor, as the case fatality rate is between 20 to 50%. Pathology includes midzonal hepatic necrosis and eosino- philic degeneration of Councilman bodies, and acute renal tubular necrosis, although renal failure has rarely been reported. Focal myo- carditis, brain swelling, and petechial haemorrhages contribute to the clinical picture. Diagnosis The differential diagnosis of yellow fever includes typhoid, lepto- spirosis, tick-​borne relapsing fever, typhus, Q fever, malaria, severe viral hepatitis, Rift valley fever, Crimean-​Congo haemor- rhagic fever, Lassa, Marburg, and Ebola fever. Yellow fever can be diagnosed through serology (haemagglutination inhibition, compliment fixation and the PRNT) by virus isolation or NAT. As discussed earlier (‘Laboratory diagnosis’ section), due to cross-​ reaction with other flaviviruses, results might be difficult to inter- pret, especially in patients living in hyperendemic settings. Virus isolation can be attempted from blood, which should be collected within the first 4 days of illness. A variety of techniques are avail- able for virus isolation, such as intracerebral inoculation of new- born Swiss mice or inoculation into susceptible vertebrate or arthropod cell lines. In fatal cases, post-​mortem histopathological examination of the liver may provide conclusive diagnosis, with or without immunocytochemical staining for viral antigen, al- though similar liver pathology has been observed in fatal dengue cases. Liver biopsy is contraindicated as it may lead to severe haemorrhage. Prevention and control Vaccination is recommended by the World Health Organization (WHO) for residents of yellow fever endemic areas; travellers to endemic areas should also be vaccinated. A live attenuated vac- cine, known as the 17D vaccine, has been available since 1937 and is delivered as a single 0.5-​ml subcutaneous dose with minimal side effects, although mild reactions, such as headache, myalgia and low-​grade fever occurring in 5–​10% of vaccines have been reported. Vaccination results in lifelong immunity. Until recently official WHO recommendations suggested a booster dose should be given every 10 years, but this changed in 2016. The contraindi- cations to the use of 17D vaccine are altered immune states (e.g. immunosuppressed individuals and pregnancy) and hypersensi- tivity to eggs. If the vaccine is inadvertently given during preg- nancy, recipients should be closely monitored. Vaccination is also contraindicated in children less than 6 months of age, due to in- creased risk of postvaccine encephalitis. Vaccine-​associated vis- cerotropic disease is more common in patients with a history of thymic tumour and thymectomy and is contraindicated in these groups. Fatal outcomes following vaccination have been rarely re- ported. However, neurological involvement, presenting as enceph- alitis and Guillain–​Barré syndrome and viscerotropic disease have been reported mainly among older vaccinees. Besides vaccination, reducing the densities of the anthropophilic mosquito vector Ae aegypti in tropical urban settings through fumigation and other sophisticated vector control approaches are effective methods in controlling epidemics. Treatment There is no specific antiviral therapy available and treatment is sup- portive. Intensive medical treatment is be required for severe cases presenting with acidosis, shock, and metabolic imbalance. Patients with renal failure might require dialysis. Intensive care is a challenge and difficult to provide as many epidemics occur in remote areas of Africa and the Americas. YF disease is regarded a disease of inter- national public health significance, requires quarantine of affected patients and notification of public health officials as soon as possible so that vector eradication and mass immunization can be carried out as promptly to prevent further epidemics. Zika virus Aetiology and epidemiology Zika virus (ZIKV) is a mosquito-​borne flavivirus first discovered in the Zika forest of Uganda in 1947 during an investigation of enzo- otic yellow fever. The transmission cycles of ZIKV include: (i) an enzootic, sylvatic cycle, where the virus circulates between arboreal mosquitoes and non​human primates; and (ii) a human or urban cycle, between humans and peridomestic/​domestic mosquitoes. Transovarial transmission of mosquitoes had been demonstrated in both transmission cycles and may provide a mechanism for the maintenance of the transmission cycles in interepidemic periods (Fig. 8.5.14.2). Importantly, the recent pandemic documented horizontal transmission through sexual contact, a novel mode of arbovirus transmission. Current studies are assessing the impact of sexual transmission on the epidemic potential of ZIKV, and its contribution to the severity of congenital Zika syndrome. Until 2007, only 14 sporadic human cases were reported, although sero- logical studies and virus isolation from mosquitoes suggested wide- spread ZIKV circulation in Africa and Asia. The first major ZIKV outbreak was detected in 2007 in Yap Island, followed by another outbreak in same year in Gabon. These outbreaks were followed by 2013–​2014 epidemic in French Polynesia which quickly spread to in New Caledonia, the Cook Islands, Easter Island, Vanuatu, and the Solomon Islands. In early 2015, the first ZIKV infections were

8.5.14  Flaviviruses excluding dengue 839 described in Brazil, which quickly became an explosive epidemic spreading throughout the hemisphere. As of January 2017, autochthonous ZIKV infections had been re- ported in all countries and territories in the Americas, with the ex- ception of mainland Chile and Canada. Many countries in North America and Europe have also reported hundreds of imported cases (Fig. 8.5.14.5). Clinical characteristics It has been reported that up to 80% of ZIKV infections can be asymp- tomatic. Symptomatic ZIKV infection is characterized by a self-​limited illness with mild clinical manifestations, including fever, lethargy, eye pain, conjunctivitis, rash, muscle aches, and arthralgia. In rare instances ocular and auditory abnormalities, brain ischaemia, myelitis, and men- ingoencephalitis have also been reported. Many of these symptoms are similar to those of DENV and other flavivirus infections, which share geographic distribution range and often have the potential to cocirculate with ZIKV. This has confounded differential clinical diag- nosis. Symptoms can last for several days to a week and often patients do not become ill enough to see a medical provider. Infection with Zika very rarely results in fatal outcomes unless there are underlying comorbitities. Infection with Zika leads to lifelong immunity. In some severe cases, ZIKV infection leads to Guillain–​Barré syndrome (GBS), an autoimmune polyradiculoneuropathy (see Chapter 8.5.3). Patients typically present with reduction or absence of deep tendon reflexes and can also develop cranial nerve disorders. A  cluster of GBS cases was identified retrospectively during the French Polynesia outbreak of 2013; however, an explosive increase in GBS incidence was documented in several American countries where ZIKV circulation has occurred. The underlying factors that influence the association of GBS and ZIKV infection are not fully understood. However, it has been suggested that sequential arbo- virus infections may exacerbate the immune response and trigger an immunopathogenic process attacking peripheral nerves, thus leading to the onset of GBS. Up to now, ZIKV-​induced GBS has been transient in duration and most patients fully recover following intra- venous immunoglobulin therapy. Of greater concern is the large increase of microcephaly cases first reported in Brazil, with about 20-​fold increase in incidence from 2014 to 2015. The linkage between ZIKV infection and micro- cephaly is supported by evidence from clinical, epidemiological, and experimental studies. Microcephaly refers to a head that is smaller than expected. The size of the head is typically defined by the occipito-​frontal head circumference, which can be measured Fig. 8.5.14.5  Global distribution of Zika virus.

840 section 8  Infectious diseases in the fetus by ultrasound or in the neonate using a tape. In ob- stetrical practice, ultrasound measurements for various fetal struc- tures between the 10% and 90% centiles are usually considered within the normal range. The Society for Maternal Fetal Medicine recommends that fetal microcephaly be defined as a fetal head cir- cumference three standard deviations or more below the mean for gestational age, and that the diagnosis be considered certain if the head circumference is five standard deviations or more below the mean. The development of microcephaly depends on an insult that affects brain growth, a dynamic process that may take several weeks to become apparent. An ultrasound close to the time of insult may not show any findings. Birth defects following ZIKV infection in utero include micro- cephaly, calcium deposits in the brain indicating possible brain damage, excess fluid in the brain cavities and surrounding the brain, absent or poorly formed brain structures, abnormal eye de- velopment, or other abnormalities resulting from damage to brain that affects nerves, muscles, and bones, such as arthrogryposis and hearing loss. In rare instances ZIKV infection in utero may lead to hydranencephaly, hydrops fetalis, and fetal demise. The constella- tion of these congenital abnormalities has now been termed con- genital Zika syndrome (CZS). Diagnosis The differential diagnosis includes other arboviruses, such as dengue, chikungunya, and mayaro virus infections. The US Centers for Disease Control and Prevention recommends the Zika MAC-​ ELISA to be used for the qualitative detection of Zika virus IgM antibodies in serum or cerebrospinal fluid. However, as discussed earlier (‘Laboratory diagnosis’ section), due to cross-​reaction with other flaviviruses, results might be difficult to interpret, especially in patients living in hyperendemic settings. Presumed positive, equivocal, or inconclusive tests must be confirmed by the PRNT assay, which might also be not interpretable in patients living in hyperendemic settings. The most sensitive method of ZIKV detection requires the detection of ZIKV genetic material (virus RNA) by NAT, including the reverse transcription polymerase chain reaction (RT-​PCR) or the Trioplex assay (described next). Viral RNA can be detected early in the course of illness on serum collected within a narrow window of 5–​6  days after symptom onset. NAT testing can also be conducted on urine samples collected, with a patient-​matched serum specimen, less than 14  days after symptom onset. Semen samples in infected males have been shown to be positive by NAT up to 6 months postinfection. A negative NAT result does not ex- clude ZIKV infection and serum should be analysed concurrently with serological tests. Virus isolation can be attempted from blood collected within the first 4 days of illness. A variety of techniques are available for virus isolation, such as intracerebral inoculation of newborn Swiss mice or inoculation into susceptible vertebrate or arthropod cell lines. For asymptomatic pregnant women, NAT testing is recom- mended on serum and urine within 2 weeks of the date of last possible exposure (e.g. travel to areas with active ZIKV transmis- sion). Pregnant women who present to their obstetric care provider two or more weeks after exposure, and have been found to be IgM positive, are strongly recommended to be tested by NAT. In areas with active ZIKV transmission, asymptomatic pregnant women should undergo serologic testing (MAC-​ELISA) as part of their rou- tine obstetric care in the first and second trimester. Given that the differential diagnosis includes dengue and chikun- gunya infections, major arboviroses that cocirculate with ZIKV, the Trioplex RT-​PCR, a laboratory test designed to detect Zika virus, dengue virus, and chikungunya virus RNA, is highly recommended. The Food and Drug Administration (FDA) has not cleared or ap- proved this test, but it is currently authorized for use under an emer- gency use authorization. Prevention and control Controlling the Zika pandemic is a major challenge, as the cornerstone of its success is based solely on interrupting its trans- mission cycle. Recent attempts to control dengue, which shares a similar or identical urban transmission cycle, by relying on con- trolling its arthropod vectors has largely failed. However, some sophisticated vector control approaches, such as release of gen- etically modified, or Wolbachia-​infected mosquitoes have shown promise to reduce mosquito populations, but these campaigns take months if not years to implement. The potential for pro- longed presence of the virus in semen indicates the potential existence of alternative routes of human-​human transmission. The risk of being infected with ZIKV can be reduced by using mosquito repellents, wearing long sleeves and trousers while spending time outdoors. Although there is no licensed vaccine currently available, sev- eral approaches that have successfully led to efficacious flavivirus vaccines are currently pursued, including but not limited to live attenuated, inactivated, and chimeric virus vaccines, as well as subunit vaccines representing ZIKV proteins, DNA vaccines ex- pressing viral proteins, and other viral vectors expressing viral antigens. It should be noted that each vaccine approach has its pros and cons, complementary approaches should be explored simul- taneously to advance effective vaccines for ZIKV. Additionally, no clinically approved antiviral drug therapy is currently avail- able for treatment of ZIKV. However, two recent studies suggested that several repurposed FDA-​approved drugs previously shown to have antiflaviviral activity (e.g. bortezomib, ivermectin, and mycophenolic acid), showed promise in inhibiting ZIKV infect- ivity. While there are several approaches being considered, ef- fective countermeasures (vaccines and antivirals) may take years for final approval. Treatment No specific therapy is available, but supportive treatment can reduce morbidity and mortality. St. Louis encephalitis (SLEV) Aetiology and epidemiology St. Louis encephalitis virus (SLEV) is a mosquito-​borne virus that is found through the Americas. In 2005, an outbreak in Argentina was the first confirmed case of St. Louis encephalitis disease (SLE) outside of North America. A year later, an outbreak was observed in Brazil. However, on an annual basis, most SLEV cases occur in the United States, mostly in the eastern and central states, where urban-​ centred outbreaks have recurred since the 1930s; in the western states

8.5.14  Flaviviruses excluding dengue 841 transmission is more of an endemic nature. While only a handful of cases are reported annually in the United States, the largest epidemic of SLEV ever recognized in the United States took place in 1975, with nearly 2000 cases reported. SLEV is maintained in nature within a mosquito-​avian cycle. The species of Culex mosquitoes that transmits SLEV depends upon the geographic location. Within the United States, Culex pipiens, Culex quinquefasciatus, Culex nigripalpus, and Culex tarsalis are the main vectors. These mosquitoes prefer feeding from avian spe- cies, but will non​discriminately feed from other mammals, rep- tiles, and amphibians. As a result, many non​reservoir or amplifying hosts can seroconvert to SLEV infection. However, both wild and peridomestic birds can develop viremia sufficient to maintain the transmission cycle. This allows the virus to travel along avian mi- gration patterns, which may help to explain the large geographic range of this virus. Although the geographic range of the virus ex- tends from Canada to Argentina, human cases have almost exclu- sively occurred in the United States. Clinical characteristics The incubation period for St. Louis encephalitis disease (SLE) is be- tween 5 to 15 days. Of those infected, less than 1% produce symp- tomatic illness. Patients develop rapid onset of symptoms including fever, headache, malaise, dizziness, and nausea. Approximately a week later, patients can recover completely or progress to a neuro- logic disease characterized by meningism, tremor, abnormal re- flexes, ataxia, cranial nerve palsies, convulsions (especially in children), stupor, and coma. The disease burden is highest for older people where c.90% of symptomatic patients develop encephalitis. The overall case fatality rate is between 5–​15% and increases with patient age. Underlying diseases such as hypertension, diabetes, and alcoholism affect the outcome. Diagnosis SLE can be confirmed during the viraemic phase by collecting serum and testing for the presence of viral RNA by NAT. Isolations from tissues aside from cerebrospinal fluid and brain are difficult to make, so direct virus isolation may not be useful. Serum and cerebrospinal fluid IgM tests are available through the CDC and for purchase from commercial vendors. SLE is a report- able disease in the United States. Other evidence, such as a history of mosquito bites or even the time of year, may be useful. Prevention and control There is no licensed vaccine to protect against SLE. The most ef- fective course of action is to prevent mosquito bites by wearing mos- quito repellents and avoiding peak mosquito biting times. Treatment There is no antiviral or treatment for SLE. Only supportive care can be offered to patients. West Nile virus (WNV) Aetiology and epidemiology West Nile virus (WNV) can be found worldwide and across every continent except Antarctica. It was first isolated in Africa and was responsible for small outbreaks there and across the Middle East and India. In 1999, the virus caused an epidemic in New York City and eventually became endemic in North America. Other outbreaks in the 1990s also expanded the virus’ range across Europe and into Asia. A cluster of infections in Argentina were also observed. A variant of WNV, Kunjin virus, is found in Australia. Like SLEV, WNV is maintained between Culex species mosqui- toes and birds. The virus is quite unique in that is vectored by many species of mosquitoes and has been found in mosquitoes from the Aedes, Anopholes, Coquillettidea, Culiseta, Deinocerites, Mansonia, Orthopodomyia, Psorophora, and Uranotaenia genera. Likewise, al- though the major disease and mortality burden is placed on cor- vids, WNV has been found in hundreds of bird species in the United States alone. This promiscuousness has allowed WNV to become endemic worldwide. The wide range of feeding preferences from infected Culex mos- quitoes has resulted in many other vertebrates becoming infected and seroconverting to WNV. Horses and humans can be infected and succumb to illness, but cannot produce a high enough vir- emia to contribute to the transmission cycle, and are considered ‘dead-​end’ hosts. Human infection can occur through a variety of mechanisms. The most common cause of infection is via an infected mosquito bite. Babies can be infected from the mother in utero, during birth or by breastfeeding. Furthermore, any transplantation of infected tissues, such as organ transplantation and blood transfusion can re- sult in infection. West Nile disease is also a reportable disease in the United States. Clinical characteristics The incubation period for WNV varies between 2 days to 2 weeks. Infection results in clinical symptoms in only 20% of individuals. Of those showing West Nile disease, symptoms include fever, headache, malaise, body aches, and vomiting. Some present with a rash. Most patients recover fully but the recovery time can vary greatly and even last months. Less than 1% of patients will have neurologic complications, including meningitis and/​or en- cephalitis, seizures, paralysis, and coma. Acute flaccid paralysis and Guillain–​Barré syndrome have also been associated with WNV infection. Disease severity is age dependent and increases substantially after the age of 60 yrs. Long-​term sequelae are not uncommon following resolution of symptoms, including memory loss, muscle weakness, depression, and other neurological defects. The case fatality rate among patients with neurological disease is c.10%. Interestingly, Kunjin disease is generally subclinical and progression to encephalitis is rare. Diagnosis Diagnostics tests to confirm WNV infection are similar to most other flaviviruses. During the viraemic phase, serum can be used to culture the virus and viral RNA genomes can be detected by NAT. In neurologic cases, cerebrospinal fluid may also be used in lieu of serum. The presence of IgM in the serum or cerebrospinal fluid can also be used to confirm recent infection, although this type of antibody may be long-​lived (>30 days) in some patients, so cau- tion must be used when trying to determine the date of infection. IgG testing by ELISA and PRNT to measure total antibody and

842 section 8  Infectious diseases neutralizing antibody titres, respectively, are also useful for con- firming previous WNV infection. It is best to compare the titres taken at two different times after infection to look for an increase in WNV-​specific titres since flaviviruses exhibit strong cross-​ reactivity with one another. Prevention and control There is no currently licensed vaccine for human use, despite over a dozen years’ worth of direct research to develop one. Several candidates are currently in clinical trials. A horse vac- cine has been useful in preventing equine disease. Infection via tissue transplantation has been dramatically reduced due to in- tensive screening. As most WNV infections occur via mosquito bites, vector con- trol has been a main priority of many individuals and governments. Using mosquito repellent and avoiding mosquito bites at peak biting times are the best ways to prevent infection. Mosquito spraying and destruction of breeding sites have been strategies employed by local communities. Treatment There is no effective and licensed countermeasure for West Nile disease. Supportive care is offered to hospitalized patients, par- ticularly those with neurologic complications, and may require re- spiratory and rehydration support. Other mosquito-​borne infections Ilheus virus (ILHV) Aetiology and epidemiology Ilheus virus (ILHV) was first isolated in 1944 from mosquitoes of the genera Ochlerotatus and Psorophora collected near the town of Ilheus, Bahia, Brazil. ILHV was also isolated from other mos- quito species, including the genera Culex, Sabethes, Haemagogus, and Trichoprosopon, and from a variety of birds in different countries in Latin America. ILHV is believed to be maintained in zoonotic cycles between birds and mosquitoes in Central and South America. Human infection with ILHV has been reported in Trinidad, Panama, Colombia, French Guyana, Brazil, Ecuador, and Bolivia. Clinical characteristics lheus virus causes mainly asymptomatic infections in human. In mild cases patients present with malaise, asthenia, conjunctival in- jection, vesicular rash, facial oedema, arthralgia, myalgias, bone pain, abdominal pain, headache, earache and gastrointestinal or re- spiratory symptoms lasting ≈1 week. In severe cases, either the cen- tral nervous or cardiac system can be affected. However, long-​term sequelae or deaths have not been described. Diagnosis Differential diagnosis includes dengue, St. Louis encephalitis, yellow fever, or influenza. Laboratory diagnosis of ILHV infections is through serological based assays, such as MAC and IgG ELISA and testing of serum to detect virus-​specific antibodies. Virus may be detected by NAT during the viraemic phase of illness. Prevention and control The risk of infection can be reduced with ILHV by using mosquito repellents, wearing long sleeves and trousers while spending time outdoors. Treatment There is no specific treatment for ILHV and patients are maintained by supportive care. Anti-​inflammatory drugs may be effective under certain conditions. Tick-​borne infections of the CNS Tick-​borne encephalitis virus Aetiology and epidemiology Tick-​borne encephalitis viruses (TBEV) are endemic across Europe and Asia, affecting dozens of countries. There are three antigenically similar viruses that comprise the TBEV subtypes: European (TBEV-​ Eu), Siberian (TBEV-​Sib), and Far Eastern (TBEV-​FE). The Far Eastern subtype is also known as Russian spring-​summer enceph- alitis virus. Each subtype may also have coevolved with a specific vector which has restricted its geographic range. TBEV-​Eu is trans- mitted primarily by the hard tick Ixodes ricinus whereas TBEV-​Sib and TBEV-​FE use Ixodes persulcatus ticks. Ticks in the Dermacentor and Haemaphysalis genera may also contribute to TBEV mainten- ance in nature. These ticks serve as both the reservoir and vector for TBEV as transtadial and transovarial transmission has been observed. These ticks feed from small rodents as well as livestock. Humans become infected following the bite from an infected tick or by consuming tainted milk and milk-​based products. Transmission from mother to fetus in humans has been observed. It is estimated that thousands of people are infected by TBEV each year. Clinical characteristics The incubation period of tick-​borne encephalitis (TBE) is typically 1–​2 weeks. Many of the patients infected with TBEV have mild and non​specific symptoms, which in some cases may be biphasic. The ini- tial viraemic phase is characterized by non​specific illness including fever, headache, muscle aches, malaise, and nausea with or without vomiting. Leukopenia and thrombocytopenia are also common. About 8 days later, approximately one-​third of patients will progress to the neurologic phase, accompanied by meningitis, encephalitis, or meningoencephalitis. Long-​term sequalae are not uncommon. In general, all subtypes of TBEV result in similar clinical disease (resulting in a case fatality rate c.1–​2%) but the TBEV-​FE subtype can result in more severe symptoms and a higher case fatality rate (5–​20%). Most of these deaths occur if severe neurologic disease is present. Diagnosis Virus may be detected by direct isolation or indirectly by RT-​ PCR during the viraemic phase of illness. IgM, indicative of a

8.5.14  Flaviviruses excluding dengue 843 recent infection, can also be used to confirm infection and is detected by ELISA. The cerebrospinal fluid may also contain virus and antibodies and should be tested in addition to serum. A patient history of a recent tick bite might also help, but is not always noticed. Prevention and control Inactivated vaccines are available in several parts of the world, but not the United States. Immunization is recommended for those who live in endemic areas. Multiple doses are often required to reach vaccine efficacy, and boosters may be required to main- tain immunity. Each country has its own guidelines for initial dose and booster times. Evading tick bites by wearing repellents, wearing appropriate clothing, and avoiding tick-​infested areas is recommended. It is also advised to avoid unpasteurized milk. Treatment There is no specific treatment for TBEV and patients are maintained by supportive care. Anti-​inflammatory drugs may be effective under certain conditions. Powassan virus Aetiology and epidemiology The virus was first isolated from the brain of a patient who died in Powassan, Ontario. The virus is transmitted between Ix. cookei (Ixricinus complex) ticks and rodents. Humans are infected when they come into contact with ticks during outdoor activities. Many people who become infected with Powassan virus remain asymp- tomatic. Approximately 60 cases of Powassan virus disease were reported in the United States in the past 10 years. Most cases have occurred in the Northeast and Great Lakes region. However, docu- mented Powassan virus cases have also occurred in Russia where the primary vector is Ix. persulcatus. Clinical characteristics The incubation period (time from tick bite to onset of illness) ranges from about 1 week to 1 month. Signs and symptoms of infection can include fever, headache, vomiting, weakness, confusion, loss of coordination, speech difficulties, seizures, and memory loss. Powassan virus can infect the central nervous system and cause en- cephalitis and meningitis with a 10% fatality rate. Approximately 50% of the infected patients have permanent neurological symp- toms, such as recurrent headaches, muscle wasting, and memory problems. Diagnosis Diagnosis is often based on the patient’s clinical presentation, travel history, activities, and epidemiologic history of the loca- tion where infection likely occurred. Laboratory diagnosis of Powassan virus infections is through serological based assays, such as MAC-​ELISA, MIA (microsphere-​based immunoassay), and IgG ELISA, testing of serum and/​or cerebrospinal fluid to de- tect virus-​specific antibodies. In fatal cases, NAT, histopathology with immunohistochemistry and virus culture of autopsy tissues can be conclusive. Prevention and control The risk of Powassan virus infection is greatly reduced by using tick repellents, wearing long sleeves and trousers, avoiding bushy and wooded areas, and doing thorough tick checks after spending time outdoors. Treatment There is no specific treatment or vaccine available at present. People with severe Powassan virus illness often require hospitalization to receive respiratory support, intravenous fluids, or medications to reduce swelling in the brain. Louping ill virus (LIV) Aetiology and epidemiology This virus, isolated in 1931, is primarily of veterinary importance, mainly affecting sheep, but sometimes other animals. It is a member of the tick-​borne encephalitis virus complex and is predominantly found in Ireland, western Scotland, northern England, and Norway. The usual vector is Ix. ricinus. Naturally occurring human infections are relatively rare but infection via laboratory exposure is not unknown. Most infec- tions relate to human contact with animals or with sheep blood. Clinical features Clinical disease in humans is relatively mild and might just present as an influenza-​like illness. Neurological disease, either presenting as a lymphocytic meningitis or encephalitis, with ataxia and stupor, is the most commonly reported syndrome. Cases resembling polio- myelitis have also been reported. Rare fatal infections have occurred. Diagnosis Detection of specific IgM or demonstration of a rise in titre of IgG over time can aid in the diagnosis but molecular tests are more sen- sitive and specific. Prevention and control Most control measures focus on vector control, reducing tick density. There is a vaccine for sheep as well. For humans, appropriate clothing and tick avoidance are important, along with care in handling dis- eased animals. Treatment There is no known treatment other than symptomatic relief. Tick-​borne infections with haemorrhagic manifestations Kyasanur forest disease virus (KFV) Aetiology and epidemiology Kyasanur Forest disease virus (KFDV) is endemic to the southern part of India as the name hails from the Kyasanur Forest from which it was originally isolated from in 1957. The virus is transmitted in a tick-​rodent cycle. The hard-​bodied forest tick, Haemaphyalis spinigera is the primary vector. Small animals like mice, rats, and shrews maintain the virus. Monkeys are extremely susceptible to

844 section 8  Infectious diseases KFDV infection, which is typically lethal and associated with large epidemics. Dead-​end livestock hosts like cattle, goats, and sheep may become infected but are not thought to influence human dis- ease. Human infections occur when they are bitten by an infected tick or come into contact with a KFDV-​infected animal. Clinical characteristics Kyasanur Forest disease (KFD) is similar to Omsk haemorrhagic fever in that the disease presents non​specically with fever, headache, myalgia, cough, hypotension, and dehydration. This lasts for a few days (3–​4) after the incubation period (3–​8 days). A biphasic illness is observed in some patients (c.10–​20%), which appears in the third week of illness and is characterized by fever and signs of enceph- alitis. The case fatality rate is c.3–​5%. Unlike Omsk haemorrhagic fever, no major sequalae are observed. Diagnosis Blood taken during the viraemic phase (between days 3–​12 of symp- toms) can be amplified by RT-​PCR to detect viral RNA. After this phase, antibodies against KFDV can be detected by ELISA or PRNT assays. Prevention and control A formalin-​inactiated vaccine for KFDV is available. This vaccine is provided in endemic areas and requires multiple doses; two doses will provide c.62% efficacy whereas three doses increases to 83% effi- cacy. Avoidance of tick bites by applying repellants and checking for ticks when in the forest, as well as avoiding sick animals, is advised. Treatment No specific treatment is available. Supportive care for patients including maintaining hydration and preventing excessive bleeding. Alkurma haemorrhagic fever Aetiology and epidemiology Relatively little is known about Alkurma haemorrhagic fever virus (ALKV). It was first observed in 1995 in Saudi Arabia. The full geo- graphic range of ALKV is not fully known but serology is limited to within the countries of the Persian Gulf. The transmission cycle is also unknown. A recent study has shown Ornithodoros savignyi ticks contained the ALKV viral RNA. The vertebrate host has not yet been determined. Clinical characteristics A clear clinical picture of this disease is limited based upon the small number of documented cases. Alkurma haemorrhagic fever presents with fever, headache, joint, muscle and retro-​orbital pain, and vomiting. Low platelets and white blood cells are observed and some patients progress to haemorrhagic fever or encephalitis. The low number of cases may obscure the actual case fatality rate, which currently is c.30%. Diagnosis Diagnosis is made by detecting the viral RNA in the blood by RT-​ PCR. The presence of anti-​ALKV antibodies can be made by ELISA or neutralization assays. Prevention and control There is no vaccine for ALKV. The best method of prevention is avoiding tick bites. As more is known about the life cycle of ALKV, this may change. Treatment Treatment is supportive with no specific countermeasure currently available. Omsk haemorrhagic fever virus Aetiology and epidemiology Omsk haemorrhagic fever virus is a member of the TBE serogroup and as such is transmitted by ticks. The virus is endemic to a few regions of the Russian Federation, including Kurgan, Tyumen, Omsk, and Novosibirsk. Transmission can occur with or without the tick vectors. Demacentor reticulatus, Demacentor marginatus, and Ixodes apronophrus ticks are the primary vectors of transmis- sion. Small rodents, like muskrats and voles, are the vertebrate hosts for Omsk haemorrhagic fever virus. Humans become exposed to the virus upon the feeding of an infected tick, or by the direct physical exposure to the bodily fluids of an infected rodent. The latter in- fection route is often associated with hunting. Interestingly, Omsk haemorrhagic fever virus can also be transmitted via the milk of in- fected goats and sheep. No human-​to-​human transmission has been described. Clinical characteristics Omsk haemorrhagic fever presents with a Kyasanur Forrest-​like disease, with non​specific signs and symptoms including fever, headache, chills, muscle pain, and bleeding. This can last up to 3–​4 days after the incubation period of approximately 3–​8 days. During this time, low blood pressure is observed along with low RBC, WBC, and platelet counts. After this initial phase, some pa- tients may continue onto the second phase of illness characterized by fever and/​or encephalitis. Some patients develop long-​term sequalae including hearing and hair loss. The case fatality rate is between 0.5 to 3%. Diagnosis The best diagnostics tests for confirming infection are like other flavivirus infections. Testing the blood early during the disease al- lows for the detection of live virus by plaque assay/​virus isolation and/​or PCR. After viraemia, detection of antibodies by ELISA or PRNT are the best assays to confirm infection. Prevention and control There is no vaccine specifically targeting Omsk haemorrhagic fever. However, the TBE vaccines may provide sufficient cross-​reactivity. Infection is best controlled by avoiding tick bites and hunting musk- rats, especially in the winter. Treatment The only treatment for Omsk haemorrhagic fever is supportive care, including the administration of fluids for hydration in the case of a haemorrhagic disease.

8.5.15 Dengue 845

8.5.15 Dengue 845

8.5.15  Dengue 845 FURTHER READING Aliota MT, et al. (2017). Zika in the Americas, year 2: What have we learned, What gaps remain? A report from the Global Virus Network. Antivir Res, 144, 223–46. Barrows N, et al. (2016). Repurposed drug candidates to treat ZIKV infection in pregnancy. Cell Host Microbe, 20, 259–​70. Brasil P, et al. (2016). Zika virus infection in pregnant women in Rio de Janeiro. N Engl J Med, 375, 2321–34. Beck AS, Barrett AD (2015). Current status and future prospects of yellow fever vaccines. Expert Rev Vaccines, 14, 1479–​92. Cao-​Lormeau et al. (2016). Guillain–​Barré syndrome outbreak asso- ciated with Zika virus infection in French Polynesia: a case-​control study. Lancet, 387, 1531–​9. Franca GVA et al. (2016). Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation. Lancet, 388, 891–​7. Griffiths MJ, Turtle L, Solomon T (2014). Japanese encephalitis virus infection. Handb Clin Neurol, 123, 561–​76. Halstead SB, Thomas SJ (2011). New Japanese encephalitis vaccines: alternatives to production in mouse brain. Expert Rev Vaccines, 10, 355–​64. Holbrook MR (2012). Kyasanur forest disease. Antiviral Res, 96, 353–​62. Kasabi G (2013). Coverage and effectiveness of Kyasanur forest disease (KFD) vaccine in Karnataka, south India, 2005–​10. PLoS Negl Trop Dis, 7, 1–​4. Madani TA (2005). Alkhumra (Alkhurma) virus infection, a new viral hemorrhagic fever in Saudi Arabia. J Infect, 51, 91–​7. Mead PS, et al. (2018). Zika virus shedding in semen of symptomatic infected men. N Engl J Med, 378, 1377–85. Patel H, et al. (2015). Long-​term sequelae of West Nile virus-​related illness: a systematic review. Lancet Infect Dis, 15, 951–​9. Piantadosi A, et al. (2016). Emerging cases of powassan virus enceph- alitis in New England: clinical presentation, imaging, and review of the literature. Clin Infect Dis, 62, 707–​13. Pierson TC, Diamond MS (2013). Flaviviruses. In: Knipe DM, Howley PM (eds) Fields virology, 6th edition. Wolters Kluwer/​Lippincott Williams & Wilkins, Philadelphia, PA. Vasilakis N, Weaver SC (2017). Flavivirus transmission cycles fo- cusing on Zika. Curr Opin Virol, 22, 30–​5. Weaver SC et al. (2015). Zika virus: history, emergence, and pro- spects for continued outbreaks in the Americas. Antiviral Res, 130, 69–​80. 8.5.15  Dengue Bridget Wills and Yee-Sin Leo ESSENTIALS Dengue is the most important mosquito-​borne viral infection to affect humans. It is an RNA virus in the Flavivirus genus, family Flaviviridae. There are four closely related but serologically distinct viral serotypes, all of which may cause disease. Following infec- tion with one serotype there is lifelong immunity to that serotype but the possibility of more severe disease during a subsequent in- fection with a different serotype. The primary mosquito vector is
Aedes aegypti. Recent estimates suggest around 100 million symptom- atic, and many more asymptomatic, infections occur annually world- wide. The disease is hyperendemic in many large Asian cities, and is also a significant problem in the Pacific region and in the Americas. Clinical features and diagnosis—​among symptomatic cases a wide variety of clinical manifestations are seen, ranging from mild febrile illness through to severe and potentially fatal disease. Of note, a systemic vascular leak syndrome may develop around the time of defervescence, with the resulting plasma leakage sometimes severe enough to cause life-​threatening dengue shock syndrome. Altered haemostasis and thrombocytopenia are typically present, some- times accompanied by clinically significant bleeding. Severe hepatic, neurological, and cardiac complications are also seen in some cases, but are less common. Diagnosis depends on viral isolation, detection of viral antigen or viral RNA, or serological testing. Management and prevention—​treatment is supportive, with particular emphasis on careful fluid management. In particular, prompt volume resuscitation is essential for patients with dengue shock syndrome, together with regular monitoring of the pulse rate, blood pressure, and haematocrit, to minimize the risk of fluid over- load. A vaccine with moderate efficacy has recently been licensed in a few countries, but current prevention efforts continue to rely on elimination of potential vector breeding sites, biological and chem- ical vector control strategies, and avoidance of mosquito bites. Introduction and aetiology Dengue is the most important mosquito-​borne viral infection of hu- mans. The name is originally a West Indian Spanish word, derived from Ki Swahili ‘ka dinga pepo’ (‘a kind of cramping plague’) that was brought from Africa to the Caribbean. In the British West Indies it was called ‘dandy fever’ because of the stiff posture of its victims. Later, in Cuba, dengue was termed ‘quebranta huesos’ or ‘break-​bone fever’ because of the severe myalgias and arthralgias that often occur. The dengue virus is a single stranded, positive-​sense RNA virus; four closely related but serologically distinct viral serotypes (DENV-​1, 2, 3, and 4) together constitute one subgroup of the genus Flavivirus, family Flaviviridae. The virus comprises three structural proteins, together with seven non​structural proteins that are ex- pressed in infected cells but do not form part of the mature virion. There is only transient cross-​protective immunity between the four serotypes, so people living in endemic areas can be infected several times during their lifetime. Epidemiology Humans are infected with dengue viruses by the bite of Aedes mos- quitoes, principally Ae. aegypti and Ae. albopictus. Other vertebrate

846 section 8  Infectious diseases species are generally not susceptible to infection. The most im- portant vector globally, Ae. aegypti, is a highly domesticated tropical mosquito that lays its eggs in artificial water containers commonly found in and around homes. The adult mosquitoes rest indoors and prefer to feed on humans during daylight hours, with peak biting ac- tivity in the early morning and late afternoon. A mosquito acquires DENV when it bites a viremic individual; the virus then replicates in the mid-​gut before disseminating to the saliva after 7–​12 days so that onward transmission can occur when the mosquito takes its next blood feed. Higher plasma viremia in the original infected in- dividual appears to be a significant factor in facilitating a productive infection in the mosquito. Aedes mosquitoes are nervous feeders, and if interrupted they will return to the same or different person(s) to continue feeding. Thus, during a single blood meal several indi- viduals may become infected, making Ae. aegypti a highly efficient epidemic vector. Epidemics of febrile illness attributed to dengue have been re- ported at intervals over the last 200 years across Asia, Africa, and North America, likely reflecting progressive expansion in the global distribution of the mosquito vectors. From the 1950s on- wards a new clinical syndrome, characterized by vascular leakage and bleeding and given the name dengue haemorrhagic fever (DHF), began to emerge in Southeast Asia. The first epidemic of DHF in the Americas appeared in 1981 in Cuba, associated with the arrival of a new Asian genotype of DENV-​2 different from the American strain. Since that time the geographic footprint of dengue, encompassing a range of clinical syndromes, has con- tinued to expand across the tropics and subtropics, a phenom- enon largely attributable to the effects of urbanization and climate change on mosquito ecology. Thus dengue is now hyperendemic in most Asian cities, with epidemics occurring every 3 to 5 years superimposed on background endemic transmission. It has also become established as a significant problem in the Pacific region and in the Americas, and outbreaks have been reported from Africa, the Arabian Peninsula, and the warmer parts of Europe. The spread of Ae. albopictus to new areas of the world including southern Europe, America, and Africa may explain the rise in dengue transmission in these areas. Today in excess of 2.5 billion people, or around 40% of the world’s population, live in tropical and subtropical areas of the world where they are at risk for infection, and approximately 100 million clinically apparent dengue infections are estimated to occur annually across more than 100 countries (Fig. 8.5.15.1). For more than 50 years dengue has been a leading cause of hospital- ization among children in many of these endemic countries, but in parallel with the changing epidemiology and transmission dy- namics described earlier, different clinical presentations are also emerging. A shift towards clinical disease in older age groups has been reported in Singapore, Indonesia, Bangladesh, and Thailand, and likewise many of the reported cases from the Americas are among adults rather than children. The nature and frequency of the complications now seen likely reflects age-​related differences in intrinsic physiology, as well as the greater likelihood for older patients to have underlying comorbidities. Thus, DSS remains more common in children and young adults, while clinically sig- nificant bleeding and/​or severe organ involvement typically occur in older adults. Although low mortality rates (below 1% for severe disease) are usual in experienced hands, higher rates are still re- ported from some regions, as well as among high-​risk groups such as infants and older people. Pathogenesis All four DENV serotypes can cause disease. Infection with one serotype elicits immunity to that serotype but does not provide long-​term cross-​protective immunity to the remaining serotypes. Severe disease occurs predominantly in patients experiencing a second or subsequent infection with a dengue serotype different from their first infection, or else in infants with transmitted ma- ternal antibody experiencing their first infection. The generally accepted antibody-​dependent enhancement hypothesis suggests that residual heterotypic non​neutralizing antibodies bind to the new virus, enhancing its infectivity by increasing the efficiency of binding and uptake of virus–​antibody complexes through Fc receptors on blood monocyte or tissue macrophage cells, thus amplifying viral replication. The resulting increase in viral load drives an immunopathogenic cascade that alters microvascular function in some way, resulting in the characteristic systemic vascular leak syndrome and coagulopathy. Rapid mobilization of serotype cross-​reactive memory T cells has been suggested as an alternative mechanism to trigger the inflammatory cascade. Other factors considered to influence disease severity include dif- ferences in viral virulence, molecular mimicry, and immune com- plex and/​or complement-​mediated dysregulation, as well as age, genetic predisposition, obesity, and pre-​existing or underlying comorbidities. However, the pathogenesis of the vascular leakage and coagulopathy associated with severe infections remains poorly understood and as yet no mechanism has been identified that links the established immunological derangements with a de- finitive effect on microvascular structure or function. The con- tribution of various viral proteins, in particular non​structural protein 1 (NS1), to pathogenesis is a major focus of current re- search efforts. Clinical manifestations Infection with dengue virus can cause a wide spectrum of symp- toms ranging from mild febrile illness to severe and fatal disease, although most infections are thought to be asymptomatic. In the past, symptomatic disease was conventionally separated into two major clinical syndromes, dengue fever and dengue haemorrhagic fever (DHF), with case definitions and management guidelines for these entities published by the World Health Organization (WHO). The pathognomonic feature of DHF is an increase in vascular permeability causing plasma leakage, which, in a small proportion of cases, is severe enough to result in hypovolaemic shock. In addition, to qualify for a diagnosis of DHF, a patient must have some evidence of bleeding and a platelet count below 100 × 109/​litre. Due to practical difficulties in using the old WHO scheme a revised classification system was adopted in the WHO

8.5.15  Dengue 847 dengue guidelines published in 2009. The new scheme classifies symptomatic disease into dengue and severe dengue, in line with several other complex diseases such as malaria and pneumonia. It is hoped that this simpler system will be useful for triage, aid clinical management, and improve the quality of surveillance and epidemiological data. Symptomatic dengue is primarily a disease of older children and adults. After an incubation period of around 4 to 7 days symptoms Fig. 8.5.15.1  Global evidence consensus, risk, and burden of dengue in 2010. Reprinted by permission from Macmillan Publishers Ltd: Nature (Bhatt S et al. (2013) The global distribution and burden of dengue. Nature 496, 504–​507), copyright © 2013.

848 section 8  Infectious diseases start suddenly and typically follow three phases—​an initial febrile phase, a critical phase around the time of defervescence, and a spon- taneous recovery phase. Febrile phase There is sudden onset of high fever often accompanied by facial flushing, headache, retro-​orbital pain, lumbosacral pain, severe malaise, myalgias, joint and bone pains, anorexia, altered sense of taste, mild sore throat, nausea, and vomiting. Young children may experience the high fever, but are generally less symptom- atic than older individuals. Some patients may have a transient rash or skin flush in early illness (Fig. 8.5.15.2a). Other find- ings associated with infection may include generalized lymph- adenopathy, mild haemorrhagic manifestations (e.g. petechiae or easy bruising, Fig. 8.5.15.3a, b), and palpable hepatomegaly but rarely splenomegaly. Jaundice is rare. Clinical laboratory find- ings during the first week include thrombocytopenia and leuko- penia, often with moderate elevation of hepatic transaminases. Creatine kinase may also be elevated, sometimes markedly so in patients with severe myalgia. Critical phase Most patients recover around the time of defervescence, usually between 3 and 7 days after fever onset. However, in a small propor- tion of cases a vasculopathy characterized by endothelial dysfunc- tion and a transient vascular leak syndrome becomes apparent at this time, marking the onset of the critical phase. The vascular leak syndrome manifests with increasing haemoconcentration, hypoproteinaemia, pleural effusions, and ascites; in severe cases the circulating plasma volume is so compromised that the patient develops hypovolaemic shock—​this is, DSS (Fig.  8.5.15.4a, b). When the pulse pressure narrows to less than 20 mm Hg with a rapid weak pulse and/​or impaired peripheral perfusion, or if the patient becomes hypotensive due to the reduced plasma volume, then he/​she is defined as having DSS. If fluid resuscitation is not instituted promptly the ongoing depletion of plasma becomes critical, the systolic pressure falls rapidly, and irreversible shock and death may follow. However, with judicious fluid manage- ment most patients make a full recovery. Warning signs that the patient may be developing severe disease include persistent vomiting, lethargy, worsening abdominal pain, and increasing hepatomegaly. Haemorrhagic manifestations are common during the crit- ical period but are often limited to the presence of skin petechiae or bruising, or a positive tourniquet test. Mucosal bleeding (e.g. epistaxis, gum bleeding, gastrointestinal bleeding, haematuria, menorrhagia) can occur, but is rarely clinically significant in chil- dren except in association with profound shock. However, adults tend to experience more severe bleeding problems than children (Fig. 8.5.15.5a, b), and gastrointestinal bleeding and/​or menor- rhagia may be significant even in patients with little evidence of vascular leakage. Moderate to severe thrombocytopenia is usual in all age groups, with nadirs below 20 × 109 /​litre not uncommon during the critical period followed by rapid improvement during the recovery phase. An increase in the activated partial thrombo- plastin time and a reduction in fibrinogen levels are also frequently noted. However, these findings are not indicative of classic dis- seminated intravascular coagulation and the true nature of the coagulopathy remains unclear. Other laboratory investigations show similar but usually more profound abnormalities to those seen in uncomplicated cases. Recovery phase The increase in vascular permeability is transient and reverts to normal after approximately 24–​48 hours. Fluid is reabsorbed quite rapidly, often with an obvious diuresis, and the patient generally improves. However, particular care is needed with parenteral fluid therapy at this time to minimize the risk for development of respira- tory compromise due to fluid overload. A second rash, varying in appearance from scarlatiniform to maculopapular, might be noted around day 6 to 7 of illness, typ- ically on the extremities although sometimes involving the trunk and face (Fig. 8.5.15.2b). The rash blanches on pressure and may be accompanied by intense pruritus. Florid rashes can take some weeks to resolve, often with marked skin desquamation on the extremities. Other complications, notably blurred vision and sometimes transient visual loss, are also reported infrequently during the recovery phase. These symptoms generally resolve with time. (a) (b) Fig. 8.5.15.2  (a) Blanching erythematous rash on the back of a young adult with dengue in the febrile phase. (b) Classic convalescent rash in two Singaporean adults with dengue: marked erythema highlighting islands of white skin, usually most obvious on the extremities. (a) Copyright Dinh The Trung. (b) Copyright IIDE-​TTSH.

8.5.15  Dengue 849 Other syndromes Moderate hepatitis is common across the spectrum of dengue dis- ease, but severe hepatitis and acute liver failure are also sometimes seen. Renal impairment in the absence of severe plasma leakage or shock develops occasionally, usually secondary to severe muscle involvement with rhabdomyolysis. Neurological involvement is usually attributed to an encephalopathy (e.g. hypoxic, hepatic), but true encephalitis does occur, albeit infrequently. Various cardiac manifestations have been described, ranging from rare fulminant myocarditis to relatively common functional myocardial impair- ment, arrhythmias, and subclinical myocarditis. Increasingly, with systematic application of appropriate diagnostic techniques, rec- ognition of these less common clinical manifestations of dengue is improving. Severe dengue Under the new WHO scheme, patients who recover without com- plications are classified as having dengue, while those who experi- ence any one of the following problems are classified as having severe dengue: plasma leakage resulting in shock, and/​or fluid accumula- tion sufficient to cause respiratory distress; severe bleeding; severe organ impairment (e.g. liver failure, fulminant myocarditis, and so on). In children most deaths from dengue occur due to profound shock, particularly if the situation is complicated by fluid overload. In such cases severe bleeding and organ involvement sometimes de- velop secondary to shock. However, major bleeding or severe organ impairment occurring in the absence of shock are seen almost exclu- sively in adults. Differential diagnosis The differential diagnosis during the acute phase of illness includes influenza, Epstein–​Barr virus, measles, rubella, typhoid, leptospir- osis, rickettsial infections, malaria, other viral haemorrhagic fevers, primary HIV infection, and meningococcaemia. Arboviral infec- tions such as Chikungunya and Zika must be considered, particu- larly since these viruses are also transmitted primarily by Aedes mosquito species. Laboratory diagnosis During the early febrile stage (up to about day 5 of illness) labora- tory confirmation of dengue relies either on viral isolation, or on detection of viral RNA or a viral antigen such as NS1 by RT-​PCR in blood. After this time IgM antibody capture enzyme-​linked (a) (b) Fig. 8.5.15.3  (a) Petechial rash on the leg of a Vietnamese child with dengue. (b) Conjunctival petechiae in a Vietnamese adult with dengue. (a) Copyright Dinh The Trung. (b) Copyright D. A. Warrell. (a) (b) Fig. 8.5.15.4  (a) Vietnamese child with severe DSS, pleural effusions, ascites, oedema, and bruising at venepuncture sites. He required crystalloid and colloid infusions, inotropic support, and nasal continuous positive airway pressure but made a good recovery. (b) Chest X-​ray of a Vietnamese teenager after resuscitation for DSS, showing a large right pleural effusion. (a) Copyright B. A. Wills. (b) Copyright Dinh The Trung.

850 section 8  Infectious diseases immunosorbent assay (MAC-​ELISA) is the most widely used serological test for dengue diagnosis; seroconversion or a rising titre of dengue-​specific IgM in paired samples indicates acute infection. Patients with a secondary infection (either dengue or another flavivirus infection) often develop high levels of IgG anti- bodies in the acute phase and the IgM response may be less in- tense. Serological diagnosis is also complicated by the existence of flavivirus cross-​reactivity, making it necessary to perform tests for other locally prevalent flaviviruses in parallel with dengue ser- ology. Because antidengue antibodies persist for several months, diagnosis based on a single positive MAC-​ELISA result should be considered provisional. Rapid serological tests are now available but, in common with conventional serological tests, might not be- come positive until towards the end of the first week of illness. By contrast ELISA tests that detect circulating NS1 can be used from the first few days after fever onset; these tests are generally very specific, although sensitivity is variable depending on the infecting viral serotype and the concurrent humoral immune response. In primary (first) infections the sensitivity of NS1 detection in the fe- brile phase typically exceeds 90%, but it may be considerably lower (60–​80%) in secondary infections when an anamnestic serologic response to the previous virus is present. Commercially available rapid diagnostic kits that detect both NS1 and IgM/​IgG exploit the differing evolution of these responses to extend the window of de- tection through the acute illness. They are easy to use with quick turnaround time and are often employed at the point-​of-​care; sensi- tivity is improved (>90%) compared to NS1 rapid testing alone, with only minor loss of specificity. Management Good supportive care, with a particular focus on careful fluid man- agement, is critical for a favourable outcome. For patients with mild disease, oral rehydration is usually sufficient. Fever should be con- trolled using conventional methods. If an antipyretic is indicated, paracetamol is the preferred agent, with both aspirin and non-​ steroidal anti-​inflammatory drugs being contraindicated. Given the likelihood of thrombocytopenia and deranged coagulation, intra- muscular injections should be avoided (Fig. 8.5.15.5c). In the absence of particular features of concern, most patients can be managed with daily review as outpatients. However, the threshold for admission for infants, older people, and individuals with chronic diseases should be low. Persistent vomiting, severe abdominal pain, mucosal bleeding, or severe bleeding into the skin or subcutaneous tissues, a rapidly rising haematocrit, or a marked drop in the platelet count, indicate the need for admission for close observation and frequent monitoring of vital signs and haemato- crit. Judicious parenteral fluid therapy is indicated for those with a rapidly rising haematocrit. For patients with established DSS, prompt restoration of circu- lating plasma volume is crucial, followed by carefully controlled maintenance fluid therapy to support the circulation at a level just sufficient to maintain critical organ perfusion until vascular per- meability reverts to normal. Unfortunately, fluid overload with re- spiratory compromise is a common complication and one of the major contributors to mortality. Thus, the volume of parenteral fluid must be kept to the minimum required to maintain cardio- vascular stability and adequate urine output during the phase of active leakage, and as soon as reabsorption begins, usually about 1 to 2 days later, intravenous fluids should be stopped. Isotonic crystalloid solutions should be used initially, with colloid solu- tions reserved for patients presenting with severe DSS and those (a) (b) (c) Fig. 8.5.15.5  (a) Major bleeding at the insertion site of a venous line in a Vietnamese teenager with severe DSS. (b) Extensive subconjunctival haemorrhages in a Vietnamese adult with dengue and markedly deranged coagulation. (c) Large haematoma developing in a Singaporean adult with dengue, at the site where an intramuscular injection of an antipyretic agent had been given. (a) and (b) Copyright Dinh The Trung. (c) Copyright IIDE-​TTSH.

8.5.15  Dengue 851 who fail to improve with crystalloid therapy. Correction of meta- bolic acidosis, electrolyte imbalances, and hypoglycaemia are also essential. As confirmed in a recent randomized trial, platelet con- centrates are not indicated, even for profound thrombocytopenia unless there is overt bleeding, as the thrombocytopenia usually improves rapidly during the recovery phase without intervention. However, in the event of significant bleeding, transfusion of fresh blood, platelets, and other blood products might be indicated, but should be undertaken with great care because of the risk of fluid overload. No specific pharmacologic agents are available as yet for the treatment of dengue. Current research is focused on two main therapeutic approaches: first, reduction in viraemia through use of antiviral drugs, and second, immune modulation to suppress the immunopathogenic cascade that is thought to be responsible for the severe manifestations. Unfortunately, the findings of sev- eral recent studies that investigated use of antiviral agents in adults with confirmed dengue have been disappointing—​separate ran- domized blinded trials of chloroquine, balapiravir, and celgosivir versus matched placebo, with treatment commenced during the febrile phase, failed to demonstrate either a clinical benefit, or to alter virological or host inflammatory kinetics. With respect to immune modulation, corticosteroid therapy showed no convincing benefit on mortality from shock in several small clinical trials during the 1980s. A more recent safety study of early prednisolone use during the viraemic phase indicated no effect of steroids on viral clearance mechanisms. In addition, although the study was not powered to assess efficacy, there was no reduction in the incidence of shock or other recognized complications of dengue, suggesting that any protective effect of early steroid use is small. In another recent randomized placebo-​controlled trial lovastatin, an agent thought to have pleiotropic immunomodulatory effects that could improve endothelial function, was investigated in adults with dengue. It proved to be safe and well tolerated, but again there was no evidence of a beneficial effect on clinical manifestations of the disease. Vaccines A vaccine that simultaneously induces sustained protective im- munity against all four serotypes is needed. The only vaccine currently licensed, Dengvaxia, is a tetravalent formulation of at- tenuated yellow fever 17D vaccine strains expressing dengue enve- lope proteins. Several large phase III trials of this vaccine showed modest protection, but with rather limited efficacy against disease caused by DENV-​2. Additional analyses, performed after deploy- ment of the vaccine in several countries, indicated that naïve vac- cine recipients had a significantly higher risk for hospitalization/ severe dengue upon exposure to a subsequent wild-type infec- tion, compared to the unvaccinated trial participants regardless of age. While acknowledging that the vaccine could still have sig- nificant population-level benefits in high seroprevalence settings, WHO’s current position is that Dengvaxia should only be adminis- tered to individuals with clear evidence of prior dengue infection. Alternative candidates in clinical development include two other live attenuated virus vaccines, noninfectious vaccines and recom- binant subunit vaccines. Outcome Most patients with dengue make a full recovery. Even those with DSS and/​or significant bleeding usually do well provided they re- ceive appropriate supportive care from experienced healthcare per- sonnel during the critical phase of the illness. For severe dengue mortality rates below 1% are usual in experienced centres. Adults may go on to experience several weeks of extreme tired- ness, weakness, skin desquamation, pruritus, and depression during convalescence after infection, but there are no permanent sequelae. In general, children recover more rapidly and do not experience such problems. Prevention Although major efforts are being directed towards development of safe and effective dengue vaccines, it seems unlikely that large-​scale deployment will occur in the near future. In the meantime, disease prevention efforts continue to rely on elimination of potential vector breeding sites together with biological and chemical vector control strategies. Community control of Ae aegypti by eradication of mos- quito larvae from standing water sources is recommended, but has proved difficult to achieve and sustain in contemporary tropical urban settings. Insecticide-​treated bed nets have limited use since Ae aegypti mosquitoes are primarily daytime feeders. An alternative approach involving release of biologically or genetically modified mosquitoes designed to invade and displace wild mosquito popu- lations is showing promise. When introduced into Aedes mosqui- toes the intracellular bacterium Wolbachia reduces dengue viral replication in the mosquito, thereby limiting its ability to transmit the virus. Large studies are currently underway in Australia and Vietnam to investigate the practicalities, effectiveness, and poten- tial limitations of introducing Wolbachia infected mosquitoes into wild local mosquito populations. Genetically engineered male Aedes mosquitoes are also entering field trials in the Cayman Islands, with promising results. A combination of host protective strategies and vector control initiatives are likely to be required to reduce dengue transmission. Avoidance of mosquito bites in areas infested with Ae aegypti by using repellents containing N,N-​diethyl-​3-​methylbenzamide or picaridin and protective clothing are the most effective preventive measures for the traveller. FURTHER READING Akey DL, et al. (2014). Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science, 343, 881–​5. Bhatt S, et al. (2013). The global distribution and burden of dengue. Nature, 496, 504–​7.

8.5.16 Bunyaviridae 852

8.5.16 Bunyaviridae 852

852 section 8  Infectious diseases Deen JL, et al. (2006). The WHO dengue classification and case defin- itions: time for a reassessment. Lancet, 368, 170–​3. Ferguson NM, et al. (2015). Modeling the impact on virus transmis- sion of Wolbachia-​mediated blocking of dengue virus infection of Aedes aegypti. Sci Transl Med, 7, 279ra37. Hadinegoro SR, et al. (2015). Efficacy and long-​term safety of a dengue vaccine in regions of endemic disease. N Engl J Med, 373, 1195–​206. Halstead SB (1965). Dengue and hemorrhagic fevers of Southeast Asia. Yale J Biol Med, 37, 434–​54. Halstead SB, Nimmannitya S, Cohen SN (1970). Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med, 42, 311–​28. Low JG, et al. (2014). Efficacy and safety of celgosivir in patients with dengue fever (CELADEN):  a phase 1b, randomised, double-​blind, placebo-​controlled, proof-​of-​concept trial. Lancet Infect Dis, 14, 706–​15. Lye DC, et al. (2017). Prophylactic platelet transfusion plus sup- portive care versus supportive care alone in adults with dengue and thrombocytopenia: a multi-centre, open-label, randomised super- iority trial. Lancet, 389, 1611–18. Mongkolsapaya J, et al. (2003). Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med, 9, 921–​7. Nguyet MN, et al. (2013). Host and viral features of human dengue cases shape the population of infected and infectious Aedes mosqui- toes. Proc Natl Acad Sci USA, 110, 9072–​7. Rouvinski A, et  al. (2015). Recognition determinants of broadly neutralizing human antibodies against dengue viruses. Nature, 520, 109–​13. Simmons CP, et al. (2012). Current concepts: dengue. N Engl J Med, 366, 1423–​32. Sridhar S, et al. (2018). Effect of dengue serostatus on dengue vaccine safety and efficacy. N Engl J Med, 379, 327–40. Whitehorn J, et al. (2016). Lovastatin for the treatment of adult patients with dengue: a randomized, double-​blind, placebo-​controlled trial. Clin Infect Dis, 62, 468–​76. WHO revised SAGE recommendation on use of dengue vaccine. WHO Geneva 2018. Wills BA, et al. (2005). Comparison of three fluid solutions for resusci- tation in dengue shock syndrome. N Engl J Med, 353, 877–​89. World Health Organization (1997). Dengue haemorrhagic fever: diag- nosis, treatment, prevention and control. World Health Organization, Geneva. World Health Organization (2009). Dengue: guidelines for diagnosis, treatment, prevention and control. World Health Organization, Geneva. Yauch LE, Shresta S. (2014). Dengue virus vaccine development. Adv Virus Res, 88, 315–​72. 8.5.16  Bunyaviridae James W. Le Duc and D.A. Bente ESSENTIALS Based on the ICTV 9th report (2011), viruses of the family Bunyaviridae are the largest family of RNA viruses with more than 350 named isolated. They contain a three-​segmented, single-​stranded, negative-​sense RNA genome. They are divided into five genera, of which four are known to include human pathogens—​ Orthobunyavirus, Phlebovirus, Hantavirus, and Nairovirus. These viruses are found throughout the world and are transmitted between verte- brate hosts and to humans through the bite of infected arthropod vectors (mosquitoes, ticks, others), or from infectious excreta of ro- dents and other small mammals, and rarely person to person. Many are transmitted from infected arthropod vector females to the next generation by transovarial transmission, thereby surviving adverse environmental conditions and leading to marked seasonal distribu- tion of disease. There are few vaccines or drugs available to protect against infection. Prevention is by avoidance of exposure to poten- tially infected arthropod and small mammal vectors. Clinical features Bunyaviridae cause a variety of clinical illnesses, ranging from self-​ limited febrile disease to severe, life-​threatening haemorrhagic fever, acute respiratory distress, or encephalitis. The most important human diseases include those caused by: La Crosse virus—​the most common cause of ‘California encephal- itis’, most cases of which are relatively mild and with good prognosis; treatment is supportive. Oropouche fever—​causes epidemics of febrile illness, sometimes with meningitis, throughout the Amazon Basin and elsewhere in tropical America; prognosis is good; treatment is supportive. Haemorrhagic fever with renal syndrome—​caused by four distinct viruses (Hantaan, Dobrava, Puumala, Seoul); Hantaan and Dobrava cause the most severe disease, characterized sequentially by (1) fe- brile phase with features including headache, myalgias, petechiae and conjunctival haemorrhage, (2) hypotensive phase with shock, (3)  oliguric phase, when one-​third of cases have severe haem- orrhage, (4) diuretic phase, (5) convalescent phase, which may be prolonged; ribavirin is effective if started early in disease. Inactivated vaccines against hantaviruses are available for use in Asia. Hantavirus pulmonary syndrome—​most commonly reported from the western United States, Canada, Central and South America; symptoms are primarily those of acute unexplained adult respiratory distress syndrome; treatment is supportive; mortality is 20–​40%. Other diseases caused by Bunyaviridae—​these include sandfly fever, Rift Valley fever, severe fever with thrombocytopenia syndrome, and Crimean–​Congo haemorrhagic fever. Some viruses of the family (e.g. Rift Valley fever virus and Nairobi sheep disease virus), are important pathogens of domestic animals. Viral taxonomy and vectors Based on the 9th ICTV report of 2011, the family Bunyaviridae currently contains around 350 viruses, and is divided into five genera (Table 8.5.16.1). The family name, and that of the genus Orthobunyavirus, is derived from the type species Bunyamwera virus, which was isolated in Uganda from Aedes mosquitoes. The other genera are Hantavirus named after Hantaan virus (the cause of Korean haemorrhagic fever), Nairovirus after Nairobi sheep dis- ease virus, Phlebovirus after phlebotomus or sandfly fever virus, and Tospovirus after tomato spotted wilt virus. All members of the family share structural, biochemical, and genetic properties,

8.5.16  Bunyaviridae 853 such as a spherical enveloped virion 80–​120 nm in diameter (Fig. 8.5.16.1) and a genome of single-​stranded negative-​sense RNA divided into three segments (L, M, S). Members of dif- ferent genera vary substantially in their biological and bio- chemical properties and in their mechanisms of replication. Orthobunyaviruses, nairoviruses, and phleboviruses, which to- gether make up most of the family, are all arthropod-​borne animal viruses (arboviruses). These circulate in a wide variety of dif- ferent vertebrate hosts and are transmitted between vertebrates, including humans, by the bites of blood-​sucking arthropods, principally mosquitoes for orthobunyaviruses, sandflies and ticks for phleboviruses, and ticks for nairoviruses. Hantaviruses are zoonotic agents infecting rodents and other small mammals. They may spread to humans who are in close contact with infected small mammal excreta. Tospoviruses are arthropod-​transmitted plant viruses of no known medical importance. Viruses within the larger genera are further subdivided into serogroups; orthobunyaviruses have at least 18 serogroups and Table 8.5.16.1  The family Bunyaviridae: its genera, serogroups, vectors, and viruses infecting humans Genus Serogroup Vector Viruses infecting humans Orthobunyavirus (over 150) Anopheles A (12) Mosquito Tacaiuma Anopheles B (2) Mosquito Bakau (5) Mosquito Bunyamwera (33) Mosquito Bunyamwera, Calovo, Germiston, Ilesha, Maguari, Ngari, Shokwe, Tensaw, Wyeomyia Bwamba (2) Mosquito Bwamba, Pongola C group (14) Mosquito Apeu, Caraparu, Itaqui, Madrid, Marituba, Murutucu, Nepuyo, Oriboca, Ossa, Restan California (14) Mosquito California encephalitis, Guaroa, Inkoo, Jamestown Canyon, Keystone, La Crosse, snowshoe hare, Tahyna, trivittatus Capim (10) Mosquito Gamboa (8) Mosquito Guama (12) Mosquito Catu, Guama Koongol (2) Mosquito Minatitlan (2) Mosquito Nyando (2) Mosquito Nyando Olifantsvlei (5) Mosquito Patois (7) Mosquito Simbu (24) Mosquito Oropouche, Shuni Tete (5) Mosquito Turlock (5) Mosquito Unassigned (3) Mosquito Hantavirus (39) Hantaan (39) None Amur, Andes, Araraquara, Bayou, Bermejo, Black Creek, Canal Choclo, Dobrava, Hantaan, Juquitiba, Laguna Negra, New York, Lechiguanas, Maciel, Monongahela, Oran, Prospect Hill, Puumala, Saaremaa, Seoul, Sin Nombre Nairovirus (32) Crimean–​Congo (3) Tick Crimean–​Congo haemorrhagic fever, Hazara Dera Ghazi Khan (6) Tick Hughes (10) Tick Soldado Nairobi sheep disease (3) Tick Dugbe, Ganjam, Nairobi sheep disease Qalyub (3) Tick Sakhalin (7) Tick Avalon Thiafora (2) Tick Phlebovirus (57) Phlebotomus (44) Sandflya Alenquer, Candiru, Chagres, Corfou, Punta Toro, Rift Valley fever, sandfly fever Naples, sandfly fever Sicilian, severe fever with thrombocytopenia syndromea, Heartland, Toscana Uukuniemi (13) Tick Uukuniemi, Zaliv-​Terpeniya Tospovirus (1) Thrips Unassigned (53) Mosquito Bangui, Kasokero, Tataguine Tick Bhanja, Issyk-​kul Keterah, Tamdy, Wanowrie Numbers in parentheses indicate the approximate number of viruses in the genus or serogroup. Bold type indicates the type species and viruses causing major disease in humans. a Mosquito vector for Rift Valley fever virus; tick suspected for severe fever with thrombocytopenia virus and Heartland virus.

854 section 8  Infectious diseases nairoviruses have 7 (Table 8.5.16.1). Of over 60 Bunyaviridae that are known to infect humans, the type species and those causing major human diseases are shown in bold type in Table 8.5.16.1 and are described in more detail. Table 8.5.16.2 lists the distribution of the remaining viruses that cause minor human infections with their principal arthropod vectors. The habitats of the different viruses and their vectors range from arctic to tropical. The enzootic cycles of arbo- viruses are poorly understood. Most viruses undergo alternate cycles of replication in vertebrate and invertebrate hosts, but transovarial and transstadial transmission within some mosquitoes, ticks, and phlebotomine flies, and venereal transmission from vertically in- fected male mosquitoes to uninfected females is also known to occur. Most arboviruses have a narrow host range, occur within a limited area, and are transmitted by specific vectors to a limited number of vertebrate hosts, but some viruses infect a wider host range, are transmitted by more than one type of vector, and may occur in more than a single continent. Tick transmission predominates in Asia, but is unknown in South or Central America, and although some Bunyaviridae have been isolated in Australia, none is known to in- fect humans in that continent. Viruses of this family are among the most common, apparently emerging, diseases. Following viral entry, whether through the skin after the bite of an infected arthropod or by another route, there is replication in draining lymph nodes, which can be enlarged, and then viraemia. Symptoms develop when virus is deposited and replicates in other sites. The viruses are killed by bleach, phenolic disinfectants and detergents, autoclaving, boiling, and γ-​irradiation. Enzymes such as nucleases also inactivate these viruses. Biosafety level 3 is recommended for handling most human pathogens with the ability to spread by aerosol (e.g. hantaviruses and Oropouche virus), but level 4 is required for Crimean–​Congo haem- orrhagic fever virus. Added precautions are necessary when handling hantavirus-​infected animals and virus concentrates. Genus Orthobunyavirus Viruses of the genus Orthobunyavirus are primarily transmitted by mosquitoes and can cause mild febrile disease or infections with cen- tral nervous system involvement. Two orthobunyaviruses, Akabane and Aino viruses in the Simbu serogroup, produce congenital de- formities in sheep, goats, and cattle in Japan, Australia, Africa, and the Middle East. However, there is no evidence that any member of the genus or family produces teratogenic effects in humans, but there is concern that Oropouche virus, a Simbu serogroup pathogen of Central and South America, may be a threat to pregnant women. Studies with Bunyamwera and similar viruses show reassortment within the three-​segmented genome when two closely related vir- uses infect the same cell, either in nature or in the laboratory. When Fig. 8.5.16.1  Electron micrograph of Crimean–​Congo haemorrhagic fever virus. Magnification × 400 000. Courtesy of Dr D. S. Ellis. Table 8.5.16.2  Bunyaviridae causing only mild or trivial infections in humans, arranged on a geographical basis Africa North America Central America South America Europe Asia Bangui (M) Avalon (T) Fort Sherman Alenquer (P) Bhanja (T) Batai (M) Bhanja (T) Keystone (M) Madrid (M) Apeu (M) Calovo (M) Bhanja (T) Dugbe (T) Prospect Hill Nepuyo Candiru (P) Corfou (P) Issyk-​Kul (T) Germiston (M) Tensaw (M) Ossa (M) Caraparu (M) Tamdy (T) Ganjam (T) Ilesha (M) Trivittatus (M) Restan (M) Catu (M) Uukuniemi (T) Hazara (T) Kasokero (M) Soldado Guama (M, P) Keterah (T) Nairobi sheep disease Trivittatus (M) Guaroa (M) Wanowrie (T) Nyando (M) Itaqui Zaliv-​Terpeniya (M, T) Pongola (M) Maguari (M) Shokwe (M) Marituba (M) Shuni (M) Murutucu (M) Tataguine (M) Oriboca Thiafora Restan (M) Wanowrie (T) Tacaiuma (M) Wyeomyia (M) M, virus transmitted by mosquitoes; P, virus transmitted by phlebotomine flies; T, virus transmitted by ticks.

8.5.16  Bunyaviridae 855 such events occur in nature they can impede the diagnosis and may lead to a change of virulence of the newly emerging virus. Laboratory studies of this phenomenon have been used to analyse the molecular basis of virulence for vertebrate and invertebrate hosts. Bunyamwera virus Symptoms A mild febrile illness, usually with headache, joint and back pains, sometimes with a rash, and occasionally with mild involvement of the central nervous system. Serological surveys indicate widespread human infection in sub-​Saharan Africa, but it is rarely recognized. Laboratory infections have been recorded. A reassortant virus de- rived from Bunyamwera virus is Ngari virus. Ngari virus, a close relative to Cache Valley virus endemic in North America, was first isolated from male Aedes simpsoni mosquitoes in Southeastern Senegal in 1979 and has been associated with large outbreaks of viral haemorrhagic fever with gastrointestinal bleeding in Kenya and Somalia. Treatment and prognosis No treatment is necessary and the prognosis is normally good. California encephalitis, Inkoo, Jamestown Canyon,
La Crosse, Tahyna, and snowshoe hare viruses The aforementioned viruses, and perhaps others currently unrecog- nized, are responsible for the clinical condition known as California encephalitis. The viruses are widely distributed through­out many parts of North America, Europe, and Eurasia. In the United States of America most reported human infections are due to La Crosse virus in North Carolina, Ohio, West Virginia, Tennessee, Wisconsin, and Minnesota, with nearly 700 neuroinvasive cases reported from 23 states from 2007 to 2016, or approximately 40–​120 cases reported annually (Fig. 8.5.16.2). Most occurred in children, usually in boys, although Jamestown Canyon virus is found more often in adults. There is nearly always a history of outdoor exposure during warmer months in areas where woodland mosquitoes are prevalent. The in- cubation period is 5–​15 days. Most cases of La Crosse encephalitis are relatively mild with headache, fever (2–​3 days usually), and vomiting, progressing to lethargy, behavioural changes, and occasional brief seizures, followed by improvement. Severe cases (10–​20%) are more frequent in children under 16 years old that develop sudden fever and headache, disorientation, and seizures during the first 24 h of illness, sometimes progressing to coma and requiring intensive supportive care. Overall, about 50% of symptomatic children have seizures with status epilepticus in 10–​15%. The case fatality rate approaches 1%. Residual seizures occur in 6–​13%, persistent hemiparesis in about 1%, and cognitive dysfunction in a few. In appropriate epidemio- logical settings, the disease should be considered in children pre- senting with aseptic meningitis or encephalitis. In Europe, Tahyna virus is widely distributed in Austria, former Czechoslovakia, France, Germany, Italy, Norway, Romania, former Yugoslavia, and the former Soviet Union. Seroprevalence exceeds Fig. 8.5.16.2  La Crosse virus neuroinvasive disease average annual incidence per 100 000 population by county, 2007-2016. Counties are shaded according to incidences ranging from less than 1.00, 1.00 to 2.49 and greater than 2.50 per 100 000 population. Source: ArboNET, Centers for Disease Control and Prevention, accessed 28 June 2018.

856 section 8  Infectious diseases 95% in parts of former Czechoslovakia, and is about 50% in the Rhone valley in France and the Danube basin near Vienna, but overt disease is seldom recognized. Inkoo virus is prevalent in Finland and also in neighbouring regions of Russia. Most adult Lapps have antibodies. Small children may have signs of central nervous system involvement during acute infection. Antibodies reactive with California serogroup viruses have also been found in human sera collected in Sri Lanka, China, and in the far northern latitudes of Eurasia where several California serogroup viruses have been iso- lated from mosquitoes, some related to Inkoo and Tahyna viruses, but others to snowshoe hare virus. In another Russian study of c.50 people, mainly 14–​30 years old, with infections caused by California serogroup viruses, about two-​thirds had influenza-​like illnesses without central nervous system involvement, while the remaining one-​third had aseptic meningitis. Control, treatment, and prognosis Measures to limit mosquito breeding are useful in endemic regions. No vaccines are available, and there is no specific treatment. Fluid and electrolyte balance must be maintained, and anticonvulsive drugs might be required to control seizures. Oropouche virus Symptoms Before 1961, Oropouche virus was known to have caused only a mild fever in a single forest worker in Trinidad, but that year it was re- sponsible for a substantial epidemic in the Belém area of northern Brazil, where c.7000 people were affected. Over the ensuing decades, massive epidemics of febrile illness have been recorded throughout the Amazon Basin and beyond, with many thousands infected. Symptoms include headache, generalized pain including back pain, prostration, and fever (40°C). Rash, meningitis, or meningism occa- sionally accompany infection. Illness lasts from 2 to 5 days, occasion- ally with protracted convalescence. No fatalities have been reported. Control, treatment, and prognosis No vaccine is available. Transmission is probably by the biting midge Culicoides paraensis and outbreaks appear to be a consequence of agricultural development where accumulated organic waste from cacao and banana production provides ideal breeding sites for Culicoides, leading to massive populations and subsequent epidemic Oropouche disease. Measures to reduce Culicoides breeding may be beneficial. Treatment is supportive and the prognosis is good, al- though convalescence can be protracted. Genus Hantavirus Haemorrhagic fever with renal syndrome Hantaan virus of the genus Hantavirus is the cause of Korean haem- orrhagic fever in Korea. The Hantaan River is near the demilitarized zone between North and South Korea where the virus was first re- covered in 1976 from its rodent host Apodemus agrarius. The clin- ical diseases caused by Hantaan and related viruses in the Eurasian continent have long been known by different synonyms: epidemic haemorrhagic fever, Korean haemorrhagic fever, or nephropathia epidemica, but haemorrhagic fever with renal syndrome is pre- ferred. Four distinct viruses are responsible for most recognized cases: Hantaan virus, found primarily in Asia; Dobrava virus in an enclave of disease in the Balkan region and sparsely elsewhere in Europe; Puumala virus in Scandinavia, western Russia, and much of Europe; and Seoul virus, probably found globally wherever un- controlled populations of Rattus norvegicus exist. A few cases of haemorrhagic fever with renal syndrome have been associated with Saaremaa virus in Europe and Amur virus in Asia. Hantaan and Dobrava viruses cause severe life-​threatening disease with mortality of about 5%, reaching up to 30% in select populations. Puumala virus infections are less severe, although patients still re- quire admission to hospital, but fewer than 1% of admitted patients die. Seoul virus is thought to be the least severe of the pathogenic strains of Old World hantaviruses, although it has been associated with human deaths. Each hantavirus is associated with a particular rodent host: Hantaan virus with the striped field mouse Apodemus agrarius; Dobrava virus with the yellow-​necked mouse Apodemus flavicol- lis; Puumala virus with the bank vole Myodes glareolus; and Seoul virus with the Norway rat Rattus norvegicus. Humans are infected by aerosols of rodent excreta, or rarely by rodent bites. It is seen among adult men in rural environments and may be an occupa- tional disease. Those at greatest risk include farmers, woodcutters, shepherds, and, especially, soldiers in the field. Most hantavirus disease is seasonal, with a peak incidence in late autumn and early winter, although the Balkan form is found most often during summer months in Greece and adjacent countries. Symptoms The incubation period for hantaviruses is variable; it is usually 12–​16 days but it can be up to 2 months. Severe disease, typically associated with Hantaan or Dobrava virus infections in Asia or the Balkans, is characterized by five phases: 1 Febrile: 3-​ to 7-​day duration 2 Hypotensive: lasting from a few hours to 3 days 3 Oliguric: from 3 to 7 days 4 Diuretic: from a few days to weeks 5 Convalescent: prolonged Signs and symptoms of the febrile phase include fever, mal- aise, headache, myalgia, back pain, abdominal pain, nausea and vomiting, facial flushing, petechiae, and conjunctival haemorrhage (Fig. 8.5.16.3). In the hypotensive phase, patients have nausea, vomiting, tachycardia, hypotension, blurred vision, haemorrhagic signs, and shock. About one-​third of fatalities occur during this phase. In the oliguric phase, nausea and vomiting may persist and blood pressure may rise. Renal failure develops with anuria, and about one-​third of cases have severe haemorrhage (epistaxis, gastrointestinal, cutaneous, or bleeding at other sites). Nearly half of the deaths occur during the oliguric phase. In the diuretic phase, urine output increases to several litres per day. Convalescence is protracted and it might be months before full strength and function are regained. Not all the phases are seen in the less severe forms of the disease. The milder forms of haemorrhagic fever with renal syndrome, such as nephropathia epidemica due to Puumala virus, follow a similar

8.5.16  Bunyaviridae 857 but less severe course, with abrupt onset of fever of 38–​40°C, head- ache, malaise, backache, and generalized abdominal pain. Back or loin pain is especially common. Signs of renal failure are usually not as pronounced, and the need for renal dialysis varies. Transient blurred vision occurs in about 10% of cases. Infection due to Seoul virus follows a similar course, but may present with more evidence of liver involvement. There is no evidence of person-​to-​person transmission. Treatment and prognosis Admission to hospital, avoidance of trauma and unnecessary movement, close observation, and careful supportive care are es- sential for patient survival. Treatment is phase specific, with special attention to fluid balance and volume, and control of hypotension and shock. Renal dialysis may be required. Antiviral therapy using ribavirin has been shown to be effective if started early in disease. Recovery is protracted but usually complete, with the exception of Seoul virus infection which might carry an increased risk of chronic renal disease, hypertension, or stroke. Hantavirus pulmonary syndrome Hantavirus pulmonary syndrome, first reported from the United States of America in 1993, also occurs in Canada, Central and South America. The initial cases had a mortality of more than 50%, but rates have declined to 20–​40% as clinical experience has increased. Most disease was reported from the western United States of America and Canada, and more recently from Argentina, Chile, Brazil, and other Central and South American countries. Sin Nombre virus was first associated with hantavirus pulmonary syndrome, but many add- itional hantaviruses have now been recognized as likely causes of this syndrome (Table 8.5.16.1). As Old World hantaviruses are generally associated with specific microtine rodents (subfamilies Arvicolinae and Microtinae: voles, lemmings, muskrats, rats, and their allies, dis- tributed worldwide), so each American hantavirus appears to be as- sociated with a specific sigmodontine host (Sigmodontinae: cotton rats and their allies found in the western hemisphere). Apparent human-​to-​human transmission of Andes virus occurred during an outbreak in southern Argentina, including transmission to medical staff. Protective precautions are recommended when treating sus- pected cases of hantavirus pulmonary syndrome. Symptoms Symptoms are primarily those of acute unexplained adult respira- tory distress syndrome and cardiogenic shock, rather than the expected renal disease. Non​specific prodromal features of fever, myalgia, and malaise may last 3–​5  days, with nausea, vomiting, and abdominal pain, often accompanied by dizziness. On admis- sion, physical examination of patients with confirmed infection reveals fever (more than 38°C), tachycardia (more than 100 beats/​ min), tachypnoea (more than 20 breaths/​min), and often hypo- tension (systolic pressure less than 100 mmHg), with audible rales in the chest. Laboratory findings include hypoxia, leukocytosis, haemoconcentration, thrombocytopenia, atypical lymphocytosis, elevated transaminases, and prolonged prothrombin time. Chest radiography shows progression from subtle interstitial findings to bilateral frank pulmonary oedema; pleural effusions are usually pre- sent (Fig. 8.5.16.4). Thrombocytopenia and haemoconcentration are independent statistical predictors of hantavirus pulmonary syn- drome, although not infallible. In a patient with rapidly progressive pulmonary oedema, a blood smear showing four of the following five characteristics is a highly sensitive and specific means of establishing the diagnosis of hantavirus pulmonary syndrome: (1) thrombocyto- penia, (2) haemoconcentration, (3) lack of toxic granulation in neu- trophils, (4) more than 10% immunoblasts, and (5) myelocytosis. Disease progresses rapidly once the lungs begin to fill, and death is commonly seen 24–​48 h after admission, or sooner if there is hyp- oxia or circulatory failure. The severity of disease correlates with the degree of pulmonary oedema on chest radiography. Hypotension and shock may occur independently in patients whose hypoxaemia is medically controlled. Treatment and prognosis Treatment is supportive, ideally in a modern intensive care unit, with careful management of hypoxia, fluid balance, and shock. About two-​thirds of patients require intubation and mechanical (a) (b) Fig. 8.5.16.3  Patient with acute Korean haemorrhagic fever, showing extensive conjunctival haemorrhages (a) and facial swelling (b). Courtesy of Professor H. W. Lee.

858 section 8  Infectious diseases ventilation. Fluid loss into the lungs leads to haemoconcentration, but infusion of fluids exacerbates pulmonary oedema; therefore, fluids should be administered cautiously with careful monitoring. Limited experience suggests that intravenous ribavirin has little ef- fect on the course of hantavirus pulmonary syndrome, perhaps be- cause of the speed with which the disease progresses. Control Prevention involves avoidance of infected rodents either through effi- cient rodent control programmes in cities, for Seoul virus, or mainten- ance of clean campsites so that waste food is not allowed to accumulate and attract rodents. Nationally approved inactivated vaccines, reported to be safe and effective against hantaviruses, are available for use in Asia. Genus Nairovirus The genus Nairovirus, named after Nairobi sheep disease, is an acute haemorrhagic gastroenteritis affecting sheep and goats in East Africa, with transmission by the sheep tick Rhipicephalus appendiculatus. It has caused laboratory infections, but the genus includes Crimean–​ Congo haemorrhagic fever virus and several other viruses known to infect humans; for example, Ganjam virus, almost indistinguish- able from Nairobi sheep disease virus but first isolated in India from Haemaphysalis intermedia ticks collected from healthy goats; Hazara virus, recovered from Ixodes redkorzevi ticks collected from the vole Alticola roylei in a subarctic habitat at an altitude of 3660 m in the Kaghan valley of Hazara district, Pakistan; Dugbe virus, isolated in Nigeria from Amblyomma variegatum ticks collected from healthy cattle; and Soldado virus, repeatedly isolated from a variety of bird ticks but recently linked to a mild illness in humans. Crimean–​Congo haemorrhagic fever virus The virus was first recognized as a cause of an acute febrile haemor- rhagic disease affecting humans in the Crimean region of the former Union of Soviet Socialist Republics, transmitted by ticks and carrying a mortality of 5–​30%. In Africa, Congo virus was first isolated in the then Belgian Congo (now Democratic Republic of the Congo) from the blood of a local 13-​year-​old boy, and it caused a moderately severe laboratory infection. Related viruses were isolated in Uganda where more laboratory infections occurred, one of which ended fatally after a severe haematemesis. In Asia, a virus indistinguishable from Congo virus was isolated from pools of ticks collected from a variety of wild and domestic animals in western Pakistan. Crimean haemorrhagic fever virus was later proved to be serologically indistinguishable from Congo virus, hence the use of the term Crimean–​Congo haemor- rhagic fever virus. Different strains of this virus have been associated with outbreaks of severe and sometimes fatal disease in the Crimea, Rostov, and Astrakhan regions of Russia, in Albania, Bulgaria, and the Balkans, in East, West, and South Africa, in Iran, Iraq, and western Pakistan, and in China. From 2002 to 2015, c.9700 cases were re- ported in Turkey, although it was virtually unknown there previously. A U.S. soldier serving in Afghanistan was infected in 2009, treated at a medical facility in Germany, but died. Two healthcare providers had nosocomial infections with mild or no symptoms. In 2016, two cases of Crimean–​Congo haemorrhagic fever, including one fatal infection, were diagnosed in Spain. The virus had been detected in Hyalomma lusitanicum ticks from Spain 4 years earlier. Most infec- tions are seen among farmers or abattoir workers and acquired by tick bites or exposure to viraemic animal blood, but infections have occurred in both hospitals and laboratories. Symptoms The incubation period is 3 to 7 days. Fever usually starts suddenly and is normally continuous, although occasionally it is remittent or biphasic. Other clinical features are headache, nausea, vomiting, joint pains, backache, photophobia, circulatory disorders, thrombocytopenia, and leukopenia. Haemorrhagic manifestations are common. Patients show cutaneous petechiae and extensive ecchymoses, and bleed from nasal, gastric, intestinal, uterine, and urinary tract mucosae (Fig. 8.5.16.5). Patients may present with acute abdominal pain, mimicking an acute surgical emergency, and operating room staff have become infected and died through exposure to infected blood or secretions at operation. The mortality is about 5–​30%, but may be up to 40% or higher in hos- pital or nosocomial outbreaks. Transient hair loss has been reported. Control, treatment, and prognosis No internationally licensed vaccine is available. Avoidance of tick bites may reduce the risk of infection. In hospital outbreaks, meticu- lous attention to the containment of infected secretions is essential and barrier nursing should be used. Overt disseminated intravas- cular coagulation usually indicates a poor prognosis, and haema- temesis, melaena, and somnolence are significantly more common in fatal cases. Supportive therapy is essential, with monitoring of Fig. 8.5.16.4  Chest radiograph of a patient with early hantavirus pulmonary syndrome (left), and the same patient 24 h later (right) showing development of bilateral perihilar alveolar oedema. Courtesy of Dr Loren Ketai.

8.5.16  Bunyaviridae 859 fluid and electrolyte balance. The antiviral ribavirin has been recom- mended, however, efficacy is circumstantial. Limiting injections and avoidance of aspirin or other drugs affecting coagulation may reduce bleeding. Patients who recover can have residual polyneur- itis persisting for months, but eventual recovery is to be expected. Laboratory investigations with live virus require biological safety level 4 containment. Genus Phlebovirus At least ten different phleboviruses are known to infect humans (see Table 8.5.16.1). Pappataci fever, sandfly fever, or phlebotomus fever was recognized as a clinical entity in the Mediterranean area during the 19th century, and the association with Phlebotomus papa- tasi sandflies was demonstrated by showing that filtrates of human blood reproduced the disease in human volunteers. It was thought that humans were the only vertebrate host, but antibody studies in- dicate that gerbils, cattle, and sheep may also be infected. Sandfly fever Naples virus was isolated from human serum collected during an outbreak of sandfly fever in Naples, and the sandfly fever Sicilian virus was isolated from American troops with a similar disease in Palermo, Sicily. The two viruses have many common properties, but are serologically quite distinct. Sandfly fever is widespread throughout the Mediterranean area, and also occurs in Iran, Turkey, Bangladesh, India, Pakistan, and the southern states of Russia. Toscana virus, serologically related to the Naples virus, is found in countries bordering the Mediterranean; it is notable for its ability to infect the central nervous system, especially in central Italy where it is thought to be responsible for at least 80% of acute summertime infections of the central nervous system in children. The viruses that cause sandfly fever do not occur in the New World, but in South and Central America a similar clinical condition follows infection with Alenquer, Candiru, Chagres, and Punta Toro viruses. Rift Valley fever has long been known as a disease of domestic animals, mainly sheep, in East Africa, which occasionally spreads to farm workers and others handling infected animals. The infec- tion is endemic, but seldom recognized, in many wild game ani- mals in Africa. Rift Valley fever virus differs from the sandfly fever viruses, Punta Toro virus, and most other members of the genus in being normally transmitted by mosquitoes rather than sandflies. Uukuniemi and Zaliv-​Terpeniya viruses are tick-​transmitted; the only evidence that Uukuniemi virus can infect humans is the finding of specific antibodies in some human sera collected in Estonia and in former Czechoslovakia. Severe fever with thrombocytopenia syndrome virus and Heartland virus are a newly discovered tick-​ borne phleboviruses found in China, South Korea and Japan, and the United States of America, respectively, and primarily affecting farmers living in rural areas. Zaliv-​Terpeniya virus was isolated from bird ticks collected on an island in the Sea of Okhotsk, Sakhalin region, and there is some evidence that it may be pathogenic to humans. Sandfly fever Naples virus and sandfly fever Sicilian virus Symptoms After an incubation period of 2–​6 days, fever starts abruptly with chills, nausea and vomiting, epigastric pain, and often severe gen- eralized headache leading to incapacitating prostration. Fever of 38–​40°C usually resolves after 2–​3 days, but may be biphasic and persist for a week. There is no rash, but small haemorrhages into the skin and mucous membranes may be seen. Photophobia and eye pain occur, lymphadenopathy is often seen, and the liver may be tender although jaundice is rare. The disease is self-​limiting, with complete recovery. No deaths have been attributed to either sandfly fever Naples virus, or sandfly fever Sicilian virus. Rift Valley fever virus Following its initial isolation in 1930 as the agent of enzootic hepa- titis of domestic animals in Kenya, Rift Valley fever virus was rec- ognized as the cause of sporadic human infections in East, Central, and West Africa, with a particular tendency to infect laboratory workers handling the virus. In East and Central Africa, the virus has been isolated from a variety of mosquito species and it is capable of persisting in mosquito eggs during the dry season, emerging when larvae hatch in the rainy season. From 1951 to 1956 there were severe epizootics in lambs in southern Africa, and many human cases oc- curred. Further human cases with several deaths were seen in South Africa in 1975, and a major outbreak occurred in East Africa fol- lowing El Niño flooding in 1997 to 1998, apparently seeding a ‘virgin (a) (b) Fig. 8.5.16.5  Turkish patients with Crimean–​Congo haemorrhagic fever showing petechiae (a) and extensive ecchymoses (a, b) on the arms and thorax. Courtesy of Professor D. I. H. Simpson.

860 section 8  Infectious diseases soil’ outbreak in Saudi Arabia and Yemen in 2000. In 1997–​1998 and 2006–​2007 there were epizootics in Kenya, Tanzania, Burundi, and Somalia. In the recent epidemic, 684 cases with 155 deaths were re- ported in Kenya (case fatality 23%), 264 cases with 109 deaths in Tanzania (case fatality 41%), and 114 cases with 51 deaths in Somalia (case fatality 45%). Heavy rains in East Africa during 2006–​2007 triggered another outbreak with many human and animal cases. Continuing the roughly 10 year cycle, an outbreak affecting humans and domestic animals was reported in north-eastern Kenya in 2018. In the Central African Republic in 1969, a virus isolated from Mansonia africana mosquitoes and named Zinga virus was associ- ated with several cases of haemorrhagic fever; Zinga virus was later shown to be a strain of Rift Valley fever virus. In West Africa, Rift Valley fever virus was isolated from mosquitoes in Nigeria and from bats in Guinea, but despite the presence of antibodies in human sera collected in Nigeria and Senegal, human disease was unrecognized until 1987 when a substantial epidemic occurred in Mauritania, with further epi- demics in following years. In 1977 the virus spread, apparently for the first time, into Egypt, producing a major epizootic in domestic ani- mals, principally sheep and goats but also cattle, and causing about 600 human deaths within 3 months. The virus has been detected intermit- tently since then in Egypt. The principal known vector is the mosquito Culex pipiens. Both the Egyptian and the Mauritanian epidemics ap- peared to be linked to major ecological changes following the construc- tion of the Aswan Dam on the Nile and dams on the Senegal River. Symptoms After an incubation period of 3–​6 days, fever starts abruptly with shivering, nausea and vomiting, epigastric pain, arthralgia, and often severe generalized headache. The fever may be biphasic, with temperatures between 38 and 40°C, and may remain elevated for at least a week. There is no rash, but small haemorrhages appear on mucous membranes. Photophobia and eye pains occur. There may be conjunctival inflammation, and a central serous retinitis leading to central scotoma and sometimes to retinal detachment can occur late in disease. The fundus may show macular exudates that are slow to disappear. There is often a lymphadenopathy and although the liver is frequently involved and may be tender, jaundice is rare. Convalescence may be protracted but is usually uncomplicated. A few patients develop severe disease with haemorrhage, enceph- alitis, or eye lesions. Haemorrhagic disease presents as mentioned, but progresses with cutaneous and mucous membrane petechiae, ecchymoses (Fig.  8.5.16.6a), gastrointestinal haemorrhage, and jaundice with severe liver and renal dysfunction often progressing to disseminated intravascular coagulation, hepatorenal syndrome, and death. Patients with encephalitis usually recover from acute febrile disease only to present within a few days to 2 weeks later with head- ache, meningism, confusion, and fever, often leading to residual defects or ending in death. Ocular complications are characterized by rapid onset of decreased visual acuity with scotomas due to ret- inal haemorrhage, exudates, and macular oedema (Fig. 8.5.16.6b). These are also seen after apparent recovery from the initial disease. About one-​half of these patients have some degree of permanent visual loss. Death from Rift Valley fever was rarely recognized be- fore the 1977 outbreak in Egypt, but the Mauritanian epidemics with mortality due to jaundice and haemorrhagic manifestations, and the recent East African and Arabian Peninsula outbreaks with several hundred suspect fatalities establish it as a life-​threatening infection. Control, treatment, and prognosis Veterinary vaccines have been used for some years, and formalin-​ inactivated vaccines have also had limited use for the prevention of disease in laboratory workers and others exposed to high risk of in- fection. Improved vaccines based on molecular techniques are under development. Treatment is supportive. Although there are no reports of nosocomial transmission, barrier nursing would be a sensible precaution. Severe fever with thrombocytopenia syndrome virus Severe fever with thrombocytopenia syndrome first came to the at- tention of Chinese health officials in 2009 when cases appeared in rural areas of Henan and Hubei provinces in central China. Recently cases were diagnosed in South Korea and Japan. Adult farmers appear to be at greatest risk. Severe fever with thrombocytopenia syndrome virus was isolated from ticks (Haemaphysalis longicor- nis), but not from mosquitoes, and molecular characterization of the virus suggests that it is distantly related both to viruses of the sandfly fever complex and to Uukuniemi virus. In cen- tral China between 2011–2016 over 5300 laboratory-confirmed cases were reported, most occurred from May to July, with about 75% of patients over 50 years of age. Transmission by tick bite (a) (b) Fig. 8.5.16.6  Severe Rift Valley fever. (a) Cutaneous petechiae and ecchymosis. (b) Severe central retinal lesion. Courtesy of Professor D. I. H. Simpson.

8.5.16  Bunyaviridae 861 is suspected; however, person-​to-​person transmission has been reported following contact with bloody secretions or vomited blood of patients. Symptoms Symptoms of Severe fever with thrombocytopenia syndrome in- clude fever, thrombocytopenia, and leucopenia, followed by multiorgan failure in severe cases. Patients may present with fever, fatigue, conjunctival congestion, diarrhoea, abdominal pain, pro- teinuria, and haematuria in addition to thrombocytopenia and leucopenia. Multiorgan failure may develop, rapidly leading to death in 12–​47% of hospitalized cases based on limited data. Characteristics of less severe disease among non​hospitalized cases have yet to be determined. A robust humoral immune response oc- curs and neutralizing antibodies persist in surviving patients for at least 1 year. Onset of disease occurred 6–​13 days after contact with blood of infected persons where person-​to-​person transmis- sion was suspected. Control, treatment, and prognosis Assuming transmission from infected ticks, control and prevention should focus on avoidance of tick bite. Barrier nursing and standard precautions against contact with blood and other potentially infec- tious bodily fluids are prudent precautions. There is no vaccine and no specific treatment and susceptibility to antiviral drugs like ribavirin has yet to be evaluated. Laboratory testing of clinical specimens and experimental manipulation of severe fever with thrombocytopenia syndrome virus should be done under appropriate containment. Heartland virus A novel phlebovirus was recently isolated from two patients seen independently from north-​western Missouri, United States, and each hospitalized with fever, fatigue, diarrhoea, moderate to se- vere thrombocytopenia and leukopenia. Both reported a history of recent tick bite prior to onset of illness. Both patients noted fa- tigue, short-​term memory difficulty, and anorexia following hos- pital discharge. Additional cases were diagnoses in 2012–​2013 and fatal infections have occurred. Virus was isolated from leukocytes collected from both patients on day 2 of hospitalization and found by phylogenetic analysis to be a novel phlebovirus closely related to severe fever with thrombocytopenia syndrome virus. Heartland virus was detected in nymphs of the tick, Amblyomma america- num. Epidemiological and clinical characteristics of human in- fection due to Heartland virus, as well as optimum treatment and prevention remain to be determined. Unassigned viruses and viruses causing only minor disease in humans The great majority of the viruses listed in Table 8.5.16.2 cause only a mild febrile illness, but the following show certain additional features. Bhanja virus (Phlebovirus) This virus was first isolated from Haemaphysalis intermedia ticks collected from healthy goats in India, but has since been isolated in Sri Lanka, Africa, and Europe. Infection of goats is widespread in Italy and the Balkans where there have been several reported human cases, including some with severe neurological disease. Laboratory infections have also occurred. Bhanja virus is related to Heartland and severe thrombocytopenia syndrome viruses. Bwamba virus (Orthobunyavirus) This was first isolated in Uganda in 1941 and is very widespread throughout sub-​Saharan Africa. More than 75% of adult human sera collected in Nigeria and over 95% of human sera collected in Uganda and Tanzania have antibodies against Bwamba virus. The original cases showed fever, headache, generalized pain, and con- junctivitis but no rash, although a rash has been described in the Central African Republic. No fatalities have been reported. Nyando virus (Orthobunyavirus) This virus was first isolated from mosquitoes in Kenya. It has since been isolated from humans in the Central African Republic where it caused fever, myalgia, and encephalitis. Tataguine virus (unassigned) This causes fever, rash, and joint pains in at least five African coun- tries (Cameroon, Central African Republic, Ethiopia, Nigeria, and Senegal). Wanowrie virus (unassigned) This virus was first isolated in India from Hyalomma marginatum ticks collected from sheep. It has also been isolated in Egypt and Iran, and in Sri Lanka where it was recovered from the brain of a 17-​year-​old girl who died following a 2-​day fever with abdominal pain and vomiting. FURTHER READING Bunyavirus overviews Freiberg AN, Bente DA, Le Duc JW (2014). Bunyaviruses: hantaviruses and others. In: Kaslow RA, et al. (eds) Viral infections of humans. Springer Science and Business Media, New York, NY. California encephalitis and Oropouche viruses Calisher CH, Thompson WH (eds) (1983). California serogroup viruses:  progress in clinical and biological research, vol. 123. Liss, New York, NY. Carpenter S, et al. (2013). Culicoides biting midges, arboviruses and public health in Europe. Antiviral Res, 100, 102–​13. Gaensbauer JT, et al. (2014). Neuroinvasive arboviral disease in the United States: 2003–​2012. Pediatrics, 134, e642–​50. LeDuc JW, Pinheiro FP (1989). Oropouche fever. In: Monath TP (ed) The arboviruses: epidemiology and ecology, Vol. 4, pp. 1–​14. CRC Press, Boca Raton, FL. Haemorrhagic fever with renal syndrome and Hantavirus pulmonary syndrome Jonsson CB, Figueiredo LTM, Vapalahti O (2010). A global perspec- tive on hantavirus ecology, epidemiology and disease. Clin Microbiol Rev, 23, 412–​41. Kruger DH, et al. (2015). Hantaviruses—​globally emerging pathogens. J Clin Virol, 64, 128–​36. Peters CJ (1998). Hantavirus pulmonary syndrome in the Americas. Emerg Infections, 2, 17–​64.

8.5.17 Arenaviruses 862

8.5.17 Arenaviruses 862

862 section 8  Infectious diseases Crimean–​Congo haemorrhagic fever Bente DA, et al. (2013). Crimean–​Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diver- sity. Antiviral Res, 100, 159–​89. Conger NG, et al. (2015). Health care response to CCHF in US soldier and nosocomial transmission to health care providers, Germany, 2009. Emerg Infect Dis, 21, 23–31. Swanepoel R (1995). Nairovirus infections. In:  Porterfield JS (ed) Exotic viral infections, pp. 285–​93. Chapman & Hall, London. Sandfly fever, Rift Valley fever, and severe thrombocytopenia syndrome Bartelloni PJ, Tesh RB (1976). Clinical and serologic responses of vo- lunteers infected with phlebotomus fever virus (Sicilian type). Am J Trop Med Hyg, 25, 456–​62. Bird BH, McElroy AK (2016). Rift Valley fever virus:  unanswered questions. Antiviral Res, 132, 274–​80. Madani TA, et  al. (2003). Rift Valley fever epidemic in Saudi Arabia: epidemiology, clinical and laboratory characteristics. Clin Infect Dis, 37, 1084–​92. Mansfield KL, et al. (2015). Rift Valley fever virus: a review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33, 5520–​31. McMullan LK et al. (2012). A new phlebovirus associated with severe febrile illness in Missouri. N Engl J Med, 367, 834–​41. Yu X-​J, et al. (2011). Fever with thrombocytopenia associated with a novel Bunyavirus in China. N Engl J Med, 364, 1523–​32. 8.5.17  Arenaviruses Jan H. ter Meulen ESSENTIALS Arenaviruses are zoonotic RNA viruses that are distributed worldwide and are adapted to various rodent genera. Some are highly patho- genic and cause haemorrhagic fevers that are endemic in restricted regions of a few countries. Humans are thought to become infected mainly through inhalation of aerosolized rodent urine or dust par- ticles to which infectious urine has dried, or by ingestion of contamin- ated foodstuff: prevention therefore depends on rodent control and avoidance of contact with rodents, their excreta, and nesting materials. Clinical approach—​because arenaviruses cause diseases that start insidiously and therapy is life-​saving, they should be considered in all patients with fever of unknown origin and a history of possible exposure in the well-​known endemic areas. Specific infections Lassa fever—​reservoir is a small rodent (Mastomys natalensis); occurs
regularly in rural areas of Nigeria, Liberia, Sierra Leone, and the Republic of Guinea, but may occur also in other West African countries. Clinical picture is highly variable and can be difficult to distinguish from other febrile infections, but may include chest pain, nausea/​vomiting/​ diarrhoea/​abdominal pain, facial swelling, pulmonary oedema, and bleeding. Case fatality is 15–​30%, but may be reduced by up to 90% through prompt administration of ribavirin. Irreversible sensorineural deafness is a frequent complication. Body fluids of patients are highly infectious and Lassa virus has been transmitted directly from person-​ to-​person, hence strict ‘barrier nursing’ measures are required and (if possible) patients with severe disease and bleeding should be man- aged in a negative-​pressure room by personnel wearing appropriate protective gear, including respiratory filters; postexposure prophylaxis with ribavirin should be considered. No vaccine is available. Lymphocytic choriomeningitis virus infection—​reservoir is the house mouse. Most commonly causes an influenza-​like illness, some- times with subsequent aseptic meningitis or encephalomyelitis. Intrauterine infection has resulted in non​obstructive hydrocephalus with periventricular calcifications, chorioretinitis, and psychomotor retardation. Use of ribavirin has not been systematically evaluated. South American haemorrhagic fevers—​the reservoir(s) for Argentinian haemorrhagic fever is the vesper mouse, for Bolivian haemorrhagic fever Calomys callosus, and for Venezuelan haem- orrhagic fever the cotton rat and the cane mouse. These cause an influenza-​like illness with marked skin erythema and (in almost half of cases) haemorrhagic manifestations; a late neurological cerebellar syndrome occurs in about 10%. Treatment with convalescent-​phase plasma is very effective in Argentinian haemorrhagic fever, and ribavirin may be effective. A live attenuated vaccine for Argentinian haemorrhagic fever is licensed in Argentina. Introduction Arenaviruses are pleomorphic enveloped negative-​stranded seg- mented RNA viruses with a characteristic internal granular structure, hence their family name Arenaviridae (Latin areno- sus = sandy). A newly suggested taxonomy divides the Arenaviridae family into the genera Mammarenavirus and Reptarenavirus, whose reservoirs are mainly rodents and reptiles (i.e. snakes), respectively. Mammalian arenaviruses are grouped into New World and Old World arenaviruses based on their geographical distributions as well as their serological and phylogenetic differences. While most of these viruses do not cause human disease, nine species are as- sociated with neurological and haemorrhagic diseases in humans. Lymphocytic choriomeningitis virus (LCMV) is distributed world- wide and occasionally causes acute central nervous system (CNS) disease and congenital malformations and has been transmitted through solid organ transplantation. Lassa virus in West Africa, and Junin, Machupo, Guanarito, Sabia, and Chapare viruses in South America cause viral haemorrhagic fevers. Certain rodent species are the principal hosts of arenaviruses and shed them lifelong in high titres in their urine. Humans are thought to become infected mainly through inhalation of aerosolized rodent urine or dust particles to which infectious urine has dried, or by ingestion of contaminated foodstuff. Human-​to-​human transmission occurs with some of the viruses. In geographically confined endemic rural areas, sporadic infections with these viruses occur regularly and are often linked to seasonal agricultural activities. Novel related viruses are emerging from time to time in previously unaffected areas. In 2000, three patients from California were fatally infected with a novel arena- virus related to Whitewater Arroyo virus, originally isolated from

8.5.17  Arenaviruses 863 rodents in New Mexico. In 2008, a novel, genetically distinct, and highly pathogenic arenavirus (named Lujo virus) was isolated from a Zambian patient hospitalized with a fatal Lassa fever-​like illness in South Africa, who transmitted the infection to several care givers. Aetiology, genetics, pathogenesis, and pathology Common to all arenavirus haemorrhagic fevers is disruption of vas- cular endothelial integrity, originating most likely from the release of endogenous mediators from infected macrophages or endothe- lial cells, and resulting in extravasations of fluid into extravascular spaces (‘capillary leakage syndrome’). Coagulation disorders are subtler than in filovirus infections and disseminated intravascular coagulopathy is not observed. Platelets are dysfunctional despite their adequate or only mildly depressed numbers, and evidence for a soluble protein inhibitor of platelet function, presumably of host origin, has been described in Lassa virus and Junin virus infections. Experimentally, Lassa virus-​infected non​human primates also showed a marked decrease in endothelial prostacyclin production. Arenaviruses initially infect macrophages and immature dendritic cells, compromising the ability of the latter to mature and stimu- late T-​cell responses. Infected dendritic cells seem to be eliminated by immunopathological mechanisms, correlating with a decline in the number of T lymphocytes and destruction of the architecture of lymphatic organs, which may explain the overall poor immune response throughout the course of Lassa virus infection. Studies of human survivors of Lassa virus infections indicate a general im- munosuppression characterized by low levels of type I IFN and pro-​ inflammatory cytokines such as tumour necrosis factor α (TNFα) or interleukin 1β (IL-​1β). Altered levels of IFNγ-​induced protein 10 (IP-​10), IL-​6 or IL-​8 are observed in fatal Lassa virus cases and the reduction of cytokines is paralleled by a reduced expression of costimulatory proteins in DCs, such as CD86, which results in the failure to activate CD4+ and CD8+ T lymphocytes. In LCMV and Lassa virus infections, neutralizing antibody re- sponses are absent or appear late, likely due to masking of neutral- izing epitopes by heavy glycosylation of the viral envelope protein. In contrast, infection with Junin virus induces neutralizing anti- bodies. Hence, immune plasma is used for passive immune therapy of this disease. Arenaviruses replicate in many epithelial cell types with only modest cytopathic effect and there is ominous absence of an inflam- matory response in infected organs. Autopsy of Lassa virus-​infected non​human primates shows pulmonary congestion, pleural effusion, and pericardial oedema, and effusion. Major microscopic lesions are necrotizing hepatitis and interstitial pneumonia. The degree of hepatic damage is not sufficient to implicate hepatic failure as the cause of death. In Junin virus infection, there are large areas of intra-​ alveolar or bronchial haemorrhage, petechiae on organ surfaces, and ulcerations of the digestive tract, although bleeding is not massive. Pneumonia with necrotizing bronchitis or pulmonary emboli is ob- served in half of the cases. Haemorrhage and a lymphocytic infiltrate have been observed in the pericardium, and splenic haemorrhage is common. Renal damage occurs in about half of the fatal cases and consists of severe structural damage in the distal tubular cells and collecting ducts with relative sparing of the glomeruli and proximal tubules. Neurological involvement during the acute phase of the disease is common in the South American haemorrhagic fevers, but there is no evidence of direct viral infection of the CNS. In Lassa fever, neurological complications, mainly sensorineural deafness, are very common during convalescence and are thought to be due to immunopathology. There is evidence that Lassa virus persists, at least for some time, because it has been isolated in human urine for up to 60 days. In one report of a fatal human LCMV infection there was peri- vascular macrophage infiltration in multiple areas of the brain and antigen was observed in the meninges and cortical cells. LCMV has been recovered from the CNS of newborn children with malformations. Epidemiology Lassa fever Clinical cases of Lassa fever are reported regularly in rural areas of Nigeria, Liberia, Sierra Leone, and the Republic of Guinea, but might also occur in other West African countries. Rare cases and evidence of exposure of humans have been documented in neighbouring countries (i.e. Benin, Burkina Faso, Côte d’Ivoire, Ghana, Mali, and Togo) and in some of them Lassa virus has been isolated from ro- dents. The reservoir of Lassa virus is a small rodent (Mastomys natal- ensis) that lives in and around human dwellings. In West Africa, 300 000 to 500 000 Lassa virus infections are estimated to occur annually, resulting in approximately 150 000 clinical cases, ranging in severity from flu-​like illness to haemorrhagic fever, and approxi- mately 5000 deaths. In endemic areas, 75% of all Lassa virus infec- tions are probably asymptomatic, with an overall mortality of 1–​5%. Lassa fever patients are not infectious during the incubation period and quite close contact with body fluids is required for person-​to-​ person spread of the virus. However, airborne transmission, prob- ably through direct contact with droplets produced during heavy coughing and presumably originating from Lassa pneumonitis, has been reported in a few instances. It has recently been estimated in an endemic area that up to 20% of hospitalized Lassa fever cases origin- ated from human-​to-​human transmission in the community. Most of these seem to have been caused by a few ‘super spreaders’, as only 5% of human cases resulted in an effective reproduction number greater than 1, with a maximum value of up to 12. Expatriates working in endemic areas have imported Lassa fever into Europe and North America. Lymphocytic choriomeningitis virus infection The distribution of LCMV is highly variable within populations of its natural host Mus musculus. From infected mouse colonies, LCMV spreads to humans in rural settings or when human habi- tats are substandard in urban areas. Infected laboratory and pet rodents have also been associated with disease in humans, and aerosol transmission might have occurred. Clinical cases of LCMV infection seem to be rare in the United States of America, even though 9.0% of house mice and 4.7% of residents had measurable antibodies in the Baltimore area in the 1990s. Person-​to-​person spread has not been demonstrated. Intrauterine LCMV infection has resulted in fetal or neonatal death, as well as hydrocephalus and chorioretinitis in infants, and the virus may be a more frequent cause

864 section 8  Infectious diseases of CNS disease in newborns than previously recognized. Two clus- ters of transplantation-​associated transmission of LCMV have been reported. Argentine haemorrhagic fever The endemic area of Argentine haemorrhagic fever (caused by Junin virus) comprises the provinces of Buenos Aires, Córdoba, Santa Fe, and La Pampa. The major rodent hosts of Junin virus are the agrarian rodents (vesper mice) Calomys musculinus and C. laucha. Most human cases are male agricultural workers. About 21 000 cases have been reported since the early 1960s, averaging about 360 a year with wide annual fluctuations. Peak incidence is during summer and early autumn. Overall human antibody preva- lence is about 12% and about 30% had no history of typical illness. Occasional hospital or family epidemics have occurred, but cases have not been observed outside of Argentina. Recent introduction of a live attenuated vaccine has reduced the incidence of the disease dramatically. Bolivian haemorrhagic fever Bolivian haemorrhagic fever (caused by Machupo virus) is limited to rural areas of Beni department in Bolivia. The only known res- ervoir is Calomys callosus. The largest known epidemic of Bolivian haemorrhagic fever, involving several hundred cases, followed a marked and unusual increase in the Calomys population in homes in the town of San Joaquin in 1963 and 1964. This seems to have been a unique event, and there were almost no further cases until 1994, when there was an outbreak in north-​eastern Bolivia. Since all ages and both sexes are affected, it can be assumed that most patients were infected in their homes. Person-​to-​person spread is rarely re- ported. A novel virus, tentatively designated Chapare virus, was iso- lated from a fatal haemorrhagic fever case near Cochabamba. The virus is genetically related to Sabia virus from Brazil; its rodent host and geographical distribution are currently unknown. Venezuelan haemorrhagic fever Venezuelan haemorrhagic fever (caused by Guanarito virus) is en- demic to the southern and south-​western parts of Portuguesa state and adjacent regions of Barinas state in Venezuela. From 1989 to 1995, a total of 105 confirmed or probable cases of Venezuelan haemorrhagic fever were reported, of which 34% were fatal. All ages and sexes were infected suggesting that transmission had occurred in and around houses. The incidence peaked each year between November and January, during the period of major agricultural activity. In addition, epidemic activity of the illness appears cyc- lically every 4 to 5 years. The cotton rat Sigmodon alstoni and the cane mouse Zygodontomys brevicauda are the rodent reservoirs. Seroprevalence in humans living in the state of Portuguesa is below 2%. Human-​to-​human transmission has not been reported. Other arenavirus infections Sabia virus was isolated in 1990 from a fatal case in São Paulo, Brazil. Its natural distribution and host are still unknown. One patient who acquired the infection in the laboratory treated himself immediately with ribavirin, and made a rapid and full recovery. Whitewater Arroyo virus was isolated in 1996 from white-​ throated wood rats or pack rats (Neotoma albigula and Neotoma spp.) collected in McKinley County, New Mexico. A related virus caused three fatal human infections in California in 1999 and 2000; they are believed to be rare events because the abundance and habits of wood rats suggest that potential contact with humans is limited. One patient reportedly cleaned rodent droppings in her home during the 2 weeks before illness onset; no history of rodent contact was solicited for the other two patients. Several other arenaviruses isolated from North American rodents have not yet been shown to cause human infections. Dandenong virus, a new arenavirus related to LCMV, has recently been isolated in Australia from patients who had received organ trans- plants from a deceased donor who had travelled in Eastern Europe. Prevention Rodent control In endemic areas, rodent control is essential and direct contact with rodents, their excreta, and their nesting materials should be avoided. Management of infected patients Safe and orderly care of the ill and adequate disinfection proced- ures should be instituted early (barrier nursing, guidelines from Centres for Disease Control and World Health Organization, see Box 8.5.17.1), with effective surveillance of high-​risk contacts and prompt isolation of further cases. Direct person-​to-​person trans- mission occurs in Lassa fever and, although rare, has been docu- mented for some New World viruses. Nosocomial transmission can occur through direct contact with an infected patient’s blood, urine, or pharyngeal secretions. If possible, patients with severe disease and bleeding should be placed in a negative-​pressure room and all personnel should wear protective gear with P3 filters for respiratory protection. High-​risk contacts are associated with percutaneous or mucosal contact with blood or body fluids. Medium-​risk contacts (unprotected contact with blood or body fluids) may safely be ob- served for development of persistent high fever for 3 weeks from the last date of contact by daily temperature measurement and tele- phone reporting. Ribavirin postexposure prophylaxis There are no evidence-​based data to support oral ribavirin as postexposure prophylaxis, but, anecdotally, a German physician seroconverted asymptomatically under ribavirin prophylaxis after examining a coughing Lassa fever patient without respiratory pro- tection and gloves (medium-​risk contact). Prophylaxis should be given to high-​risk contacts of Lassa fever and South American haemorrhagic fever patients, and offered to medium-​risk contacts of Lassa fever patients on an individual basis. One recommended dosage is 600 mg orally four times a day for 10 days. Temporary side effects of this regimen were rash, tachycardia, myalgia, diar- rhoea, and abdominal pain. In one case, there may have been an association between ribavirin and worsening of a pre-​existing tachyarrhythmia. Among 16 people there were reversible increases in plasma bilirubin concentrations in 11 and a decrease in haemo- globin concentration in 9. One person stopped prophylaxis after 4 days because of jaundice, and in another the serum lipase concen- tration increased.

8.5.17  Arenaviruses 865 Vaccines Experimental vaccines based on different viral vector systems have protected against lethal challenge with Lassa virus in animal models. The most impressive results to date have been obtained with a replication competent recombinant vaccine based on ves- icular stomatitis virus, which also forms the backbone of an Ebola vaccine that showed efficacy in a phase 2 trial. A single injection of this vaccine expressing the Lassa virus glycoprotein protected non​ human primates against lethal challenge and was cross-​protective against several Lassa virus strains. A live attenuated vaccine (can- didate No. 1) against Junin virus is licensed in Argentina and pro- duced by the Maiztegui Institute, Pergamino. It was tested in over 200 000 volunteers, showed an estimated effectiveness of 95.5%, and may be cross-​protective against Machupo virus only. The Salk Institute, Swiftwater, PA, also produced some quantities of the vaccine, which has an investigational new drug status from the United States Food and Drug Administration for high-​risk populations. Clinical features Lassa fever The clinical picture of Lassa fever is highly variable and may be very difficult to distinguish from other febrile infections. Following an incubation period of 7–​21 days, Lassa fever begins insidiously with fever, weakness, malaise, severe usually frontal headache, and a painful sore throat. One-​half of patients develop joint and lumbar pain and a non​productive cough. Severe retrosternal chest pain, nausea with vomiting or diarrhoea, and abdominal pain are also common. Respiration rate, pulse rate, and temperature are elevated, and blood pressure may be low. There is no characteristic rash; pe- techiae and ecchymoses are not seen. About one-​third of patients will have conjunctivitis. More than two-​thirds of patients have pharyn- gitis, one-​half with exudates; the posterior pharynx and tonsils are dif- fusely inflamed and swollen, but there are few ulcers or petechiae (Fig. 8.5.17.1). The abdomen is tender in half of the patients. Neurological signs in the early stages are limited to a fine tremor, most marked in the lips and tongue. Thirty per cent (30%) of patients progress to a prostrating illness 6–​8 days after onset of fever, usually with persistent vomiting and diarrhoea. Patients are often dehydrated with elevated haematocrit. Proteinuria occurs in two-​thirds of patients, with mod- erately elevated blood urea nitrogen. About one-​half of Lassa fever pa- tients have diffuse abdominal tenderness without localizing signs or loss of bowel sounds. The severe retrosternal or epigastric pain seen in many patients may be due to pleural or pericardial involvement. Facial and conjunctival swelling develop, and severe pulmonary oedema and adult respiratory distress syndrome are common in fatal cases, with gross head and neck oedema, stridor, and hypovolaemic shock (Fig. 8.5.17.2a, b). Renal and hepatic failure are not seen. Bleeding is seen in only 15–​20% of patients and is restricted to mucosal surfaces, conjunctiva, and gastrointestinal and/​or genital tracts. Over 70% of patients have abnormal electrocardiograms (non​specific ST-​segment and T-​wave abnormalities, ST-​segment elevation, generalized low voltage complexes, and changes reflecting electrolyte disturbance), but none correlate with disease severity or outcome. There is no Box 8.5.17.1  Principles of barrier nursing in resource-​poor settings (World Health Organization) Protective clothing • Double gloves • (Single-​use) gown • Plastic apron • Mask (P3 protection) • Goggles • Disinfect within isolation area or destroy (single-​use) material Handwashing • After each patient contact or contact with infected material • Rinse in disinfectant, then wash with soap and water • Disinfectant/​washing facilities must be located just outside isolation rooms Instruments • Individual thermometer for each patient, keep in receptacle with disinfectant • Disinfect stethoscope and sleeve of sphygmomanometer between each use • Place all reusable instruments in disinfecting fluid after use Bed covering and linen • Use of plastic sheet to cover and protect entire mattress is essential • Disinfect after discharge or death of the patient • Place bedding and linen in plastic bag for sterilization (soak in disin- fectant, boil, or autoclave) Food • Food should be supplied by hospital, not relatives • Each patient must have own eating utensils • Wash and disinfect in isolation area • Dispose of uneaten food Charts/​records • Keep outside isolation area Disinfection methods • Household bleach—​viruses causing viral haemorrhagic fevers are killed by exposure to a 1:10 solution for 1 min, or to a 1:100 solution for 10 min • Heat sterilization—​if autoclave not available, boil at 100°C for 20 min Fig. 8.5.17.1  Lassa fever: pharyngitis. Copyright D. A. Warrell.

866 section 8  Infectious diseases clinical evidence of myocarditis. Neurological signs are infrequent but carry a poor prognosis; they progress from confusion to severe en- cephalopathy with or without general seizures and without focal signs (Fig. 8.5.17.3). There has been a report of an imported fatal Lassa fever case presenting with only neurological symptoms. Cerebrospinal fluid is usually normal, apart from a few lymphocytes. Pneumonitis and pleural and pericardial rubs develop in early convalescence in about 20% of hospitalized patients, sometimes associated with congestive cardiac failure. Lassa virus is present in the breast milk of infected mothers, and neonates are therefore at risk of congenital, intrapartum, and puer- peral infection. Lassa fever may be difficult to diagnose in children. In very young babies marked oedema has been reported. Laboratory findings A normal mean white blood cell count on admission to hospital (6×109/​litre) may mask early lymphopenia with later relative or abso- lute neutrophilia as high as 30×109/​litre. Thrombocytopenia is mod- erate, even in severely ill patients, but platelet function is markedly depressed. The ratio of aspartate aminotransferase (AST, SGOT) to alanine aminotransferase (ALT, SGPT) is as high as 11:1. Prothrombin times, glucose, and bilirubin levels are nearly normal, excluding bio- chemical hepatic failure. Platelet and fibrinogen turnover are normal and there is no indication of disseminated intravascular coagulopathy. Complications and sequelae Nearly 30% of patients develop unilateral or bilateral deafness beginning during convalescence. About one-​half show a near or complete recovery after 3–​4  months, but the other one-​half re- main permanently deaf. Many patients also show transient cere- bellar signs during convalescence, particularly tremors and ataxia. Other complications include uveitis, pericarditis, orchitis, pleural effusion, ascites, and acute adrenal insufficiency. Prognosis The case fatality rate of hospitalized patients in West Africa is approximately 15%, but it exceeds 50% in patients with haemor- rhage. CNS manifestations carry a poor prognosis. Lassa fever is a common cause of maternal mortality in parts of West Africa. Mortality is 20% in the first trimester and 30% in the second tri- mester of pregnancy, with fetal loss occurring in 87%, apparently not varying with the trimester. Mortality was reduced fourfold in women who spontaneously or were therapeutically aborted. High viral titres in serum (exceeding 104 TCID50/​ml), AST (SGOT) raised above 150 U/​litre, and bleeding, each worsen the prognosis, with the combination of high viral titres and high AST (SGOT) carrying a risk of death of approximately 80%. High neu- trophil counts (more than 30 × 109/​litre) may be observed in these patients. In a recently published series of 284 Lassa fever patients treated in a dedicated facility in Nigeria, overall case-fatality rate was 24%, with a 1.4 times increase in mortality risk for each 10 years of age, reaching 39% for patients older than 50 years. 28% of patients had acute kidney injury, which was strongly associated with poor outcome, and 37% had CNS manifestations, respect- ively. Normalization of creatinine concentration was associated with recovery. Elevated serum creatinine (OR 1·3; p=0·046), as- partate aminotransferase (OR 1·5; p=0·075), and potassium (OR 3·6; p=0·0024) were independent predictors of death. Fig. 8.5.17.3  Lassa fever: generalized oedema and encephalopathy in a pregnant woman in Sierra Leone. Copyright D. A. Warrell. (a) (b) Fig. 8.5.17.2  (a) Lassa fever: facial and generalized oedema and hypovolaemic shock in a pregnant woman in Sierra Leone. (b) Lassa fever: facial oedema in a child. (a) Copyright D. A. Warrell. (b) Courtesy of Dr S. Mardel.

8.5.17  Arenaviruses 867 In most patients with imported Lassa fever treated in developed countries, diagnosis and ribavirin therapy have often been delayed, and the patients have died despite full supportive care. Lymphocytic choriomeningitis virus infection An influenza-​like illness is the most common clinical presentation of LCMV. Fever (up to 40°C) with rigors is always present. Frequently noted are malaise, retro-​orbital headache, photophobia, lumbar myalgias, anorexia, nausea, bradycardia, and pharyngeal injection without exudate. Mild non-​tender cervical or axillary lymphaden- opathy may occur. Up to 50% of patients have vomiting, sore throat, and dysaesthesias, and one​quarter of patients complain of chest pains and cough, associated with pneumonitis. Arthritis, parotitis, orchitis, myocarditis, rash, and alopecia have also been noted. In some pa- tients, the disease is biphasic with subsequent aseptic meningitis of about one week’s duration or encephalomyelitis in a smaller number of cases. Other neurological manifestations such as myelitis, Guillain–​ Barré syndrome, and sensorineural deafness have been reported. The onset of CNS disease can also occur without any prodrome. Intrauterine infection This has resulted in non​obstructive hydrocephalus with periven- tricular calcifications, chorioretinitis, and psychomotor retardation. No cardiac abnormalities were observed. Some mothers had a his- tory of febrile illness during pregnancy. Transplantation-​associated lymphocytic choriomeningitis virus infection In two clusters of cases, the solid organ transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within 3 weeks after transplantation. Diarrhoea, peri-​incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died 9–​76 days after transplantation. Prognosis Patients with aseptic meningitis almost always recover without sequelae, but 25–​30% of patients with encephalitis have neurological residua. South American haemorrhagic fevers In Argentine and Bolivian haemorrhagic fevers, after an incubation period of 7–​16 days, there is insidious development of malaise, chills, fever, severe myalgia, anorexia, lumbar pain, epigastric pain, abdom- inal tenderness, conjunctivitis, retro-​orbital pain often with photo- phobia, and constipation. Nausea and vomiting occur frequently after 2–​3  days of illness. There is no lymphadenopathy, spleno- megaly, sore throat, or cough, but there is high fever (up to 40°C), marked erythema of the face, neck, and thorax, and conjunctivitis (Fig. 8.5.17.4). Respiratory symptoms are uncommon. Petechiae ap- pear by the fourth or fifth day of the illness. There may be a pharyn- geal enanthema, but pharyngitis is uncommon. The infection either resolves after about 6  days or progresses to severe disease. South American haemorrhagic fevers are associated with haemorrhagic manifestations in nearly half of patients: gingival haemorrhages (Fig. 8.5.17.5), epistaxis, metrorrhagia, petechiae (Fig. 8.5.17.6), ecchymoses, purpura, melaena, and haematuria. Severe cases have nausea, vomiting, intense proteinuria, microscopic haematuria, Fig. 8.5.17.4  Argentine haemorrhagic fever: facial swelling and erythema. Courtesy of Professor D. I. H. Simpson. Fig. 8.5.17.5  Argentine haemorrhagic fever: petechial haemorrhages. Courtesy of Professor D. I. H. Simpson. Fig. 8.5.17.6  Argentine haemorrhagic fever: gingival bleeding. Courtesy of Professor D. I. H. Simpson.

868 section 8  Infectious diseases oliguria, and uraemia. Fatal cases develop hypotensive shock, hypo- thermia, and pulmonary oedema. Renal failure has been reported but glomerular filtration rates, renal plasma flow, and creatinine clear- ance are usually normal. There is some electrocardiographic evidence of myocarditis. Fifty per cent of patients have neurological symptoms during the second stage of illness, such as tremors of the hands and tongue, progressing in some patients to delirium, oculogyration, and strabismus. Meningeal signs and cerebrospinal fluid abnormalities are rare. This late neurologic syndrome can also follow treatment with immune plasma, the usual treatment in endemic areas. The clinical presentation of Venezuelan haemorrhagic fever is similar. Patients are toxic and usually dehydrated, with pharyngitis, conjunctivitis, cervical lymphadenopathy, facial oedema, or petechiae. Laboratory findings Thrombocytopenia (below 150 × 109/​litre) and neutropenia (range 0.8–​6.6 × 109/​litre) are almost invariable. Bleeding and clot retrac- tion times are concomitantly prolonged. Although reductions of levels of factors II, V, VII, VIII, and X, and of fibrinogen are observed, alterations in clotting functions are usually minor and full-​blown disseminated intravascular coagulopathy is not a feature. Complications and sequelae A late neurological syndrome in about 10% of cases, consisting mainly of cerebellar signs, is associated with treatment using high-​titre antiserum. Among survivors of South American haem- orrhagic fevers, convalescence typically takes 1–​3  months, with weight loss, fatigue, autonomic instability, and occasional hair loss. Mild permanent damage to acoustic centres has been detected in a small group of patients. Prognosis In endemic areas, the case fatality rate of Argentine haemorrhagic fever is 15–​30% for untreated hospitalized patients and 1% for pa- tients who received plasma therapy. CNS manifestations carry a poor prognosis. The case fatality rate of Bolivian haemorrhagic fever is higher. In one series of hospitalized patients with Venezuelan haemorrhagic fever, the case fatality rate was reported to be 33% despite vigorous supportive care. Argentine haemorrhagic fever is reported to be severe in pregnancy. Whitewater Arroyo-​like virus Illnesses were associated with non​specific febrile symptoms including fever, headache, and myalgias. Within the first week of hospitalization, lymphopenia was observed in all three patients, and thrombocytopenia (30–​40 ×109/​litre) was seen in two. All three pa- tients had acute respiratory distress syndrome and two developed liver failure and haemorrhagic manifestations. All patients died 1–​8 weeks after becoming unwell. Criteria for diagnosis and differential diagnosis Due to the variable clinical presentation of arenavirus infections, the diseases should be suspected in any patient presenting with a severe fe- brile illness and evidence of vascular involvement (low blood pressure, postural hypotension, petechiae, haemorrhagic diathesis, flushing of face and chest, non​dependent oedema). Sore throat, abdominal symptoms, and CNS symptoms are likewise important. For many re- gions in the world, the major differential diagnosis is malaria. Lassa fever Lassa fever should be suspected in a patient living in or coming within the incubation period (7–​21 days) from rural areas in Sierra Leone, Liberia, Nigeria, the Republic of Guinea, and adjacent terri- tories, and presenting with otherwise unexplained high fever (above 38.5°C), pharyngitis with dry cough and chest pain, or abdom- inal pain and diarrhoea, facial oedema, mucosal bleeding, or CNS symptoms. In West Africa, fever with pharyngitis, proteinuria, and retrosternal chest pain had a predictive value for Lassa fever of 81% and a specificity of 89%. Due to the variable clinical picture of Lassa fever, there are many differential diagnoses including severe mal- aria, typhoid fever, rickettsial diseases, relapsing fevers, shigellosis, leptospirosis, meningococcaemia, and gram-​negative sepsis. Viral haemorrhagic fevers such as yellow fever, Rift Valley fever, and Marburg and Ebola virus infections are much more likely to cause haemorrhage, disseminated intravascular coagulopathy, and severe liver dysfunction than Lassa fever. South American haemorrhagic fevers These should be considered in patients coming from endemic areas of Argentina (particularly male agricultural workers), Bolivia, Venezuela, and Brazil who present with unexplained fever and a bleeding diathesis. Differential diagnoses are similar to those for Lassa fever and, in addition, yellow fever and dengue fever must be considered. Appearance of the blanching maculopapular rash and a shorter duration of fever differentiate dengue from the early stages of arenavirus infections. The combination of a platelet count of less than 100 × 109/​litre and a white blood cell count of less than 2.5 × 109/​litre has a sensi- tivity of 87% and a specificity of 88% for Argentine haemorrhagic fever. These criteria are recommended when screening Argentine haemorrhagic fever patients for treatment with immune plasma or ribavirin in endemic areas. LCMV infection should be considered in patients presenting in autumn or winter with a biphasic disease characterized by fever and persistent meningeal signs, particularly if there is a history of rodent contact. Other rat bite fevers (Chapter 8.6.31) enter the differential diagnosis. Laboratory diagnosis Laboratory diagnosis of arenavirus infection is by isolation of virus from serum, demonstration of a fourfold rise in antibody titre, or high-​titre IgG antibody with virus-​specific IgM antibody in asso- ciation with compatible clinical disease. More recently, detection of viral sequences by reverse transcriptase–​polymerase chain reaction (RT-​PCR), or by detection of viral proteins using an enzyme-​linked immunosorbent assay system have been introduced. In LCMV in- fection, viremia can persist for approximately 15 days. Virus titres in the cerebrospinal fluid are lower and present for a shorter period of time. Indirect fluorescent or complement fixation antibodies appear 2–​3 weeks after the onset of the illness and reach their peak titres 5–​6 weeks after the onset of illness before becoming undetectable after a few months. Neutralizing antibodies appear 2–​6 weeks after onset of symptoms and persist for 6 months to 5 years. Acute and convalescent sera can be tested for increases in antibody titres, and

8.5.17  Arenaviruses 869 an increase in the specific IgM in blood and cerebrospinal fluid is diagnostic. For handling of clinical specimens from suspected cases, see Table 8.5.17.1. Treatment Lassa fever While the broad-​spectrum antiviral ribavirin has been evaluated in Lassa fever patients and is currently recommended for treat- ment, recent studies have also shown very good efficacy of the drug Favipiravir in animal models. Favipiravir (T-​705) is a broad-​ spectrum antiviral developed by Toyama Chemical Co Ltd. It has been approved in Japan and is now in phase III of clinical develop- ment in the United States for the treatment of complicated or re- sistant flu. Ribavirin is effective but must be given as early as possible while laboratory confirmation of the diagnosis is pending. It is ad- ministered by intravenous infusion as a 2-​g loading dose followed by 1 g every 6 h for 4 days, then 0.5 g every 8 h for 6 more days. Another recommended intravenous regimen is an initial dose of 30 mg/​kg fol- lowed by 15 mg/​kg every 6 h for 4 days, followed by 7.5 mg/​kg every 8 h for 6 days. Rigors can occur if the drug is infused too rapidly. Oral ribavirin doses are a 2-​g loading dose followed by 4 g/​day in four div- ided doses for 4 days followed by 2 g/​day for six doses. Oral ribavirin is believed to be only half as effective as intravenous therapy. A five-​ to tenfold decrease in the case fatality ratio was demonstrated in pa- tients treated with ribavirin compared to untreated patients when therapy was given within the first 6 days of illness. Patients with high AST (SGOT) and viraemia, who were treated within the first 6 days of illness, had a 5 to 9% case fatality, and a 26–​47% fatality when treated after 6 days, compared with 52–​78% when untreated. Ribavirin is contraindicated in early pregnancy because of potential teratogenicity, but the fetus rarely survives the infection. Fluid, elec- trolyte, respiratory, and osmotic imbalances should be corrected, and full intensive care support, including mechanical ventilation, offered as required. However, even vigorous support might be insuf- ficient to prevent fatal progression of advanced disease. Interferon-​α has shown efficacy against arenaviruses in animal models but only if given within a couple of days of challenge. A syn- ergistic effect with suboptimal doses of ribavirin was observed. Lymphocytic choriomeningitis virus infection Ribavirin treatment has not been evaluated in human CNS disease caused by LCMV. In a cluster of transplantation-​associated systemic LCMV infections, one recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. South American haemorrhagic fevers Convalescent-​phase plasma has been shown to be highly successful in Argentine haemorrhagic fever, reducing the mortality from 15–​ 30% to 1% in patients treated in the first 8 days of illness. Efficacy is directly related to the concentration of neutralizing antibodies, and delayed treatment is less successful. Availability of appropriately screened plasma may, however, be a problem. Ribavirin is effective against the causative Junin virus in experimentally infected pri- mates, but does not prevent CNS involvement. In one small double-​ blind trial with 18 patients, mortality was 12.5% in those treated compared to 40% in the placebo group. One human case of Bolivian haemorrhagic fever has been successfully treated with ribavirin and Venezuelan haemorrhagic fever is also likely to respond. Other issues Lassa fever is a truly neglected re-​emerging disease that has a consid- erable impact on West African healthcare systems and the economy of affected rural areas. The absence of local diagnostic capacity and the high price of intravenous ribavirin preparations are the main barriers to the introduction of specific therapy to endemic areas. Areas of uncertainty or controversy The pathogenic events leading to plasma leakage, bleeding, and shock are not well understood in arenavirus infections, compared to filoviruses The pathological basis of the sensorineural hearing Table 8.5.17.1  Inactivation of blood/​serum from viral haemorrhagic fever patients for laboratory analysis Material Examination Inactivation Blood Thick film Add formalin to a final concentration of 1% to solution used for lysis of erythrocytes Blood Thin film Methanol fixation Blood Leucocyte count 1:100 in 3% acetic acid, 15 min room temperature Serum/​plasma Serological tests Heat for 60 min at 60°Ca 0.25% β-​propiolactone (final concentration), 30 min 37°C Serum/​plasma Clinical chemistry Heat for 60 min at 60°Cb 0.25% β-​propiolactone (final concentration), 30 min 37°Cc 0.1% Triton X-​100 (final concentration), 60 min room temperatured a Loss of reactivity. Heating at 56°C for 1 h preserves antibody reactivity better but leaves sample with residual infectivity. Only recommended if sample can be safely handled in biological safety level 2 cabinet (laminar air flow). b No influence on sodium, potassium, magnesium, urea, creatinine, urate, bilirubin, glucose, C-​reactive protein. Reduced levels of bicarbonate, aspartate aminotransferase, calcium, phosphate, albumin, total protein. Measurement not possible for alkaline phosphatase, alanine aminotransferase, γ-​glutamyl transpeptidase, creatine kinase. c Liver enzyme values reduced by 20%. pH and bicarbonate not useful. d Influence on clinical chemistry not evaluated.

8.5.18 Filoviruses 870

8.5.18 Filoviruses 870

870 section 8  Infectious diseases loss, which occurs in up to 30% of Lassa fever patients, was recently shown in a mouse model to be associated with mild damage to the cochlear cells and significant degeneration of the spiral ganglion cells of the auditory nerve. The T-​cell response to Lassa virus is sus- pected to play a role in the pathology. Likely developments in the near future The novel pyrazine derivative Favipiravir (T-​705, Toyama Chemical Company Ltd), is likely to be tested in clinical trials for efficacy against Lassa fever. Given the success of the Ebola vaccine based on recombinant vesicular stomatitis virus (Ebola-​VSV), a Lassa-​VSV vaccine which has shown promise in non​human primates might ad- vance to the clinic as well. FURTHER READING Bonthius DJ, et al. (2007). Congenital lymphocytic choriomeningitis virus infection: spectrum of disease. Ann Neurol, 62, 347–​55. Fischer SA, et al. (2006). LCMV in transplant recipients: transmission of lymphocytic choriomeningitis virus by organ transplantation.
N Engl J Med, 354, 2235–​49. Kerber R, et al. (2015). Research efforts to control highly pathogenic arenaviruses:  a summary of the progress and gaps. J Clin Virol, 64, 120–​7. McCormick JB, et  al. (1986). Lassa fever:  effective therapy with ribavirin. N Engl J Med, 314, 20–​6. McCormick JB, et al. (1987). A case-​control study of the clinical diag- nosis and course of Lassa fever. J Infect Dis, 155, 445–​55. Safronetz D, et al. (2015). A recombinant vesicular stomatitis virus—​ lassa fever vaccine protects guinea pigs and macaques against chal- lenge with geographically and genetically distinct Lassa viruses. PLoS Negl Trop Dis, 17, 9, e0003736. Westover JB, et al. (2016). Low-​dose ribavirin potentiates the antiviral activity of favipiravir against hemorrhagic fever viruses. Antiviral Res, 126, 62–​8. Wilson MR, Peters CJ. (2014). Diseases of the central nervous system caused by lymphocytic choriomeningitis virus and other arena- viruses. Handb Clin Neurol, 123, 671–​81. 8.5.18  Filoviruses Jan H. ter Meulen ESSENTIALS Filoviruses are large RNA viruses, of which Ebola virus and Marburg virus cause the most severe forms of viral haemorrhagic fever and have been best-​studied because of fear of their misuse as bioter- rorism agents. These are zoonotic viruses with reservoirs, most likely fruit-​eating bats, in the rainforests of tropical Africa, where they cause sporadic infections and outbreaks among great apes and humans. Epidemiology—​The primary mode of transmission of Ebola virus to humans often involves contact of hunters with dead animals that serve as amplifying hosts, especially gorillas, chimpanzees, and forest antelopes, whose meat is consumed as ‘bush meat’.
Contact with bats has been implicated for both Marburg and Ebola virus. However, the viruses are highly infectious and are transmitted from the index case and subsequently from person to person by all body fluids, including sweat, respiratory droplets, and semen. The viruses have been found to persist in convalescent patients for many months. Clinical features and therapy—​Because filovirus infections cause a range of severe symptoms with overt haemorrhage occurring only in a subset of patients, the terms Ebola virus disease and Marburg virus disease are now being used instead of Ebola or Marburg haemorrhagic fever. Clinical presentation of Ebola virus disease and Marburg virus disease is similar, initially as an influenza-​like illness, often with gastrointestinal symptoms, followed by develop- ment of a maculopapular rash and haemorrhagic manifestations developing in approximately half of patients, including epistaxis, gum-​bleeding, haematemesis, melaena, petechiae, and ecchymoses. There is no licensed specific antiviral treatment. Broad-​spectrum
antivirals, monoclonal antibodies and inhibitory RNAs have recently been evaluated in small studies or on a compassionate use basis with varying results. Intensive supportive care and treatment of compli- cations are very important to improve survival. Survivors often suffer for prolonged periods of time from various sequelae and may experi- ence relapses of symptoms, which collectively are called post-Ebola syndrome. The viruses can persist in semen for months, requiring precautions in convalescence to prevent sexual transmission. Diagnosis and prevention—​The clinical diagnose of viral haem- orrhagic fever or Ebola virus disease/​Marburg virus disease re- quires the immediate instalment of the strictest barrier nursing procedures and notification of public health authorities. Care must be taken in both drawing and handling blood specimens, which must be inactivated before performing routine laboratory tests, and samples must be shipped immediately to a reference laboratory for diagnosis by detection of virus by cell culture, viral antigen by enzyme-​linked immunoabsorbent assay, and viral RNA by polymerase chain reaction. A prophylactic Ebola vaccine based on a recombinant, replication-​competent vesicular sto- matitis virus has shown high efficacy in ring vaccination during the last epidemic and is anticipated to be licensed by Merck & Co. Introduction Filoviruses are large, enveloped, negative-​stranded, non​segmented RNA viruses with a characteristic thread-​like morphology, hence the family name Filoviridae (Latin filum = thread). They comprise of five species of Ebola virus, named Zaire, Sudan, Tai Forest, Bundibugyo, and Reston, and one species of Marburg virus. They are now among the best-​studied agents of viral haemorrhagic fevers, mainly because of fear of their misuse as bioterrorism agents (Chapter 10.5.13). The first appearance of these viruses was in Marburg, Germany, in 1967, when laboratory, medical, and animal care personnel exposed to tissues

8.5.18  Filoviruses 871 and blood from African Green monkeys (Cercopithecus aethiops) were infected. In 1976 and 1979, epidemics of a haemorrhagic disease with very high mortality in the northern Democratic Republic of the Congo (then Zaire) and in southern Sudan were found to be due to two strains of a related filoviruses, named Ebola virus. Over the next 10 years, rare, sporadic cases of filovirus infections in Africa were the only continuing evidence of the existence of these viruses. Another species of the virus, Ebola virus Reston was imported on four oc- casions between 1989 and 1996 with wild-​caught monkeys (Macaca fascicularis) from Mindanao, Republic of the Philippines, to animal facilities in the United States of America and Italy. This virus, which is highly lethal for monkeys, has caused asymptomatic infections in pigs and animal keepers. Since 1990, both Ebola virus and Marburg virus disease have re-​emerged across tropical Africa between lati- tudes 5° north and 5° south, causing several devastating outbreaks, and in December 2013 the Zaire strain of Ebola virus emerged in the forests of the Republic of Guinea, West Africa, and triggered the lar- gest and longest human epidemic of Ebola virus infection recorded to date. Across several countries in West Africa, over 28 000 people were infected and more than 11 000 died. This fatality rate of less than 50% was lower than in most previous outbreaks. Taken together, Ebola virus outbreaks or sporadic human cases have been recorded in Côte d’Ivoire, DRC, Gabon, Guinea, Liberia, Sierra Leone, Sudan, and northern Uganda and Marburg virus disease cases in Uganda, Kenya, Angola, and DRC. The largest Marburg virus disease outbreak to date has occurred in Uige, Angola, with more than 250 people infected and a case-​fatality rate of close to 90%. Bats or non​human primates represent the most likely species in- volved in the occurrence of sporadic human outbreaks. Zoonotic transmission to the human host likely occurs through hunting and meat consumption (‘bush meat’), while human-​to-​human transmission efficiently propagates Ebola virus through mucosal contact with infected body fluids. The risk of transmission con- tinues following death; hence, corpses remain at high risk and must be handled in accordance with full infection control procedures. Aetiology, genetics, pathogenesis, and pathology Filovirus infections are characterized by massive, unchecked, and destructive replication of virus in several organs, profound im- munosuppression due to infection of immune cells and apop- tosis of infected and non​infected cells, and triggering of a cascade of immune-​mediated mechanisms resulting in a cytokine storm, endothelial damage, and coagulopathy culminating in shock and organ failure. The immunological and pathological events in end-​ stage filoviral disease resemble, in several aspects, those of bacterial sepsis: systemic inflammation (increased levels of proinflammatory cytokines, e.g. IL-​6 and IL-​8, and the anti-​inflammatory cytokine IL-​10), immune dysfunction (increased susceptibility to secondary bacterial infections, lymphocyte apoptosis), coagulopathy (in- creased D-​dimers, thrombomodulin, ferritin, disseminated intra- vascular coagulation, thrombocytopenia), endothelial dysfunction (vascular leak with hypovolemia) and organ dysfunction (renal insufficiency, hepatic dysfunction, respiratory failure, neurologic dysfunction). Through minute lesions in the skin and mucosa, the pantropic filo- viruses infect initially dendritic cells, monocytes, and macrophages. Lymphocytes are spared from the infection. Ebola virus and Marburg virus disease infected dendritic cells fail to mature to the antigen-​presenting stage and do not produce proinflammatory cyto- kines required for activation of natural killer cells and T cells. At the molecular level, the expression of viral proteins interferes with the production of interferon-​α (IFN-​α) and β, and with the ability of these and IFN-​γ to induce an antiviral state in cells. Dendritic cells show no increase in costimulatory molecules such as CD40, CD86, and interleukin 12 (IL-​12). The early immune response dysfunction originating in dendritic cells is aggravated by continued replication of filoviruses in monocytes and macrophages, accompanied by the secretion of non​inhibited proinflammatory cytokines and activa- tion of polymorphonuclear leucocytes. This accumulated release of proinflammatory mediators culminates in a ‘cytokine storm’, causing thrombocytopenia and endothelial injury, for example, through the action of tumour necrosis factor-​α (TNFα). Fatal human Ebola cases showed a marked elevation of serum levels of IFN-​γ, IL-​2, and IL-​ 10, whereas elevated IFN-​α, TNFα, and IL-​6 were associated with fatalities in some, but not all, studies. Increased blood levels of ni- tric oxide, which has been shown to contribute to hypotension, cardiodepression, and vascular hyporeactivity in sepsis, were also found to be associated with mortality. The likely reason for the vari- ations of cytokine and chemokine release observed in vivo, as well as in experimentally infected primary human cells, is currently un- known genetic differences of the host. One study reported that HLA-​ B07 and HLA-​B14 alleles were associated with survival, whereas HLA-​B67 and HLA-​B15 were associated with lethality in Ebola virus-​infected patients. Both humans and experimentally infected non​human primates show massive apoptotic death of non​infected CD4+, CD8+, and NK cells in the blood and peripheral lymph nodes, a phenomenon which has been termed ‘bystander apoptosis’. Lymphocyte apoptosis was thought to be responsible for an elimin- ation of adaptive immune responses; however, studies in transgenic mice have not confirmed it as a major factor in the pathogenesis of disease. In addition, there appears to be also massive apoptotic death of infected macrophages. The expression of tissue factor is upregulated in infected monocytes and triggers the extrinsic pathway of coagulation. The procoagulant state amplifies the production of proinflammatory cytokines and the development of vascular leakage, which further provokes activation of coagulopathy. The terminal stage of the disease is therefore charac- terized by plasma leakage, disseminated intravascular coagulopathy, and bleeding. It is thought that triggering the aforementioned cas- cade of events is more critical to the development of the observed pathology than direct organ damage due to cytopathic virus replica- tion. However, infection of the liver and adrenal glands impairs the synthesis of clotting factors and steroids, thus aggravating haemor- rhage and shock. Whether infection of endothelial cells contributes to the overall pathology remains controversial. At autopsy, both Marburg and Ebola-​infected humans and pri- mates show widespread haemorrhagic diathesis of skin, membranes, and soft tissue. Extensive necrosis with little infiltration is seen in parenchymal cells of many organs, including liver, spleen, kidneys, and gonads. The most characteristic histopathological features are seen in the liver. Large disseminated deposits of viral antigen can be found in different organs, including the sweat glands and the skin. Virus is also detectable in pneumocytes and as cell-​free virions in the alveoli.

872 section 8  Infectious diseases Spleen and lymph nodes show various degrees of lymphoid de- pletion with extensive vascular follicular necrosis. Fatal infection is marked by absence of specific IgG and presence of low levels of spe- cific IgM in only 30% of cases, whereas in human survivors early and increasing levels of Ebola-​specific IgM and IgG is followed by activation of cytotoxic T cells. During two outbreaks in Gabon, asymptomatic seroconversion with polymerase chain reaction (PCR)-​proven infection occurred in several people who mounted an early, strong but transient inflammatory response, with high levels of proinflammatory cytokines. This unexpected observation and data from animal models suggest that a tightly controlled, tran- sient early type I IFN and proinflammatory cytokine response can induce protective antiviral innate and adaptive immune responses. All of this points to great variability in individual host susceptibility to infection and reinfection based on innate immunity, as well as the viral load to which the individual is exposed during a challenge or rechallenge. The recent successful immunization against Ebola virus disease in animal models using different vaccine modalities revealed that humoural immunity plays the major role in protection against Ebola virus infection, whereas cell-​mediated immunity plays a supporting role, becoming more prominent when vaccine induced antibody levels are suboptimal. Epidemiology Central African non​human primates and monkeys are victims of Ebola virus, as are other animals such as bushpigs, porcupines, and antelopes living in the tropical rainforest. Data from wildlife sur- veillance show that epizootics occur more often than previously thought and that Ebola virus has caused massive declines of gor- illas and chimpanzees. Phylogenetic analysis of the viruses further suggests that the outbreaks are epidemiologically linked and that Ebola virus, strain Zaire, has spread south-​westward since 1976 in a wave-​like manner from Yambuku, its site of appearance in the DRC, to the Republic of the Congo and to Gabon at a speed of approxi- mately 50 km per year. This argues against the hypothesis that Ebola virus-​Z was resident, but undetected, in the central African forest block before the mid-​1970s. The exact source of the 2014 outbreak in West Africa has not been confirmed but likely involves exposure to a colony of Angolan free-​tailed bats (Mops condylurus) roosting in a tree. Evidence has also accumulated that fruit-​eating bats (Hypsignathus monstrosus, Epomops franqueti, Myonycteris torquata and others) are one, but possibly not the primary, natural reservoir of Ebola virus, and hunting of bats for human consumption has been linked to an Ebola virus outbreak in DRC in 2007. Recently, Ebola virus Reston was detected in domestic swine in the Philippines and a few asymptomatic human infections were reported. The pathogen- icity of the virus for these animals and their possible role in a trans- mission cycle are currently not known. The primary mode of transmission of Ebola virus to humans often involves contact of hunters with dead animals, especially chimpan- zees, whose meat is consumed as ‘bush meat’. In several outbreaks, however, the mode of infection of the index case could not be elu- cidated. The index cases usually transmit the virus to caring family members, often women, who come into contact with blood and body fluids. These are highly infectious, so that the average rate of secondary cases generated from the index case is around 10–​20%, but might be considerably higher. Occasionally, the virus has been spread through sexual contact. Nosocomial spread through im- properly sterilized reusable syringes or other medical equipment has caused explosive Ebola epidemics in Sudan and the Democratic Republic of the Congo. The mortality among surgical staff operating on Ebola virus disease patients misdiagnosed as having acute ab- dominal conditions was also extremely high. Nursing activities and preparing the corpse for burial carry a high risk of infection, as do burial practices which include touching of the corpse and collectively washing hands in a common bowel thereafter. There is no epidemio- logical evidence that Ebola or Marburg viruses are transmitted as true, small particle aerosols between humans. However, direct mu- cosal exposure to droplets generated by a patient during coughing poses a considerable risk of infection. A meta-​analysis of all publi- cations on the household secondary attack rate (SAR) during Ebola epidemics estimated the overall SAR at 12.5%, with the greatest risk factor being the provision of nursing care (SAR, 47.9%). According to this analysis, 27.1% of all Ebola infections are asymptomatic and these individuals are unlikely to transmit the virus. Ebola virus has been cultured from aqueous humour, saliva, breast milk, urine, and semen of infected patients; in addition, viral RNA has been found in stool, tears, and sweat, and in rectal, conjunc- tival, vaginal, and skin swabs. Because large amounts of virus can be found in skin, and sweat may contain the virus, touching an infected person might result in transmission. Infected persons can shed virus for prolonged periods of time after infection (several weeks to months). The virus has been cultured from semen up to 82 days after illness onset. Sexual transmission has, so far, only been documented in a single case, based upon which infectious virus may persist in semen for 179 days. Mother-​to-​child transmission by breastfeeding in survivors of Marburg virus has been reported, and the potential for transmission through breast milk has also been suggested for Ebola. Viable Zaire Ebola virus was detected in aqueous humour 14 weeks after the onset of Ebola virus disease and 9 weeks after the clearance of viremia. However, samples of conjunctivae and tears tested negative for Ebola virus, which supports previous studies sug- gesting that patients who recover from Ebola virus disease pose no risk of spreading the infection through casual contact. Marburg virus disease epidemiology is similar to that of Ebola virus. Evidence of infection has been detected in fruit-​eating bats (Rousettus aegyptiacus) from Uganda and Kenya, and in insectiv- orous bats in DRC (Miniopterus inflatus, Rhinolophus elocuens). However, epizootics have not been observed in mammals. Contact with bats during mining activities was reported for several index cases of Marburg haemorrhagic fever, in accordance with cave roosting of R. aegyptiacus, a habit that is not observed in the bat spe- cies implicated in Ebola virus transmission. Until 2000, the viral ori- gins of cases could be traced to eastern Africa. However, in 2005 the largest outbreak of Marburg haemorrhagic fever occurred in Uige, Angola, expanding the known range of the disease to the far western edge of the Congo basin. Continuing population movements in cen- tral Africa, destruction of the rainforest, and increased consumption of ‘bush meat’ increase the likelihood of future filovirus outbreaks. In 2008 a fatal and a non​fatal case of Marburg haemorrhagic fever occurred in the Netherlands and the United States of America, respectively, imported by tourists who had visited a bat-​roosting cave in Uganda (Python cave, Queen Elizabeth Park). Touching bat

8.5.18  Filoviruses 873 excrement or being hit by low-​flying bats were identified as possible risk factors for acquisition of the infection. Recently, a genetically distinct filovirus was discovered in Spain in dead insectivorous bats (Miniopterus schreibersii) and named Lloviu virus. There is currently no evidence of human infections with this virus. Prevention In endemic areas, avoidance of contact with bats and their excrements, with dead and diseased monkeys, and control of monkey sellers are currently the only feasible options for prevention. In case of outbreaks, interruption of person-​to-​person spread of the virus is essential for control. Early institution of safe and orderly care of the ill, using barrier nursing and disinfection procedures, should be set up with effective surveillance of high-​risk contacts and prompt isolation of further cases (e.g. barrier nursing, guidelines from the Centers for Diesase Control and Prevention (CDC) and World Health Organization (WHO); see Chapter 8.5.17 and Box 8.5.17.1). In fully equipped hospitals, patients must be placed in negative-​pressure rooms and all personnel must wear protective gear with FP3 filters for respiratory protection (Fig. 8.5.18.1). Cutaneous or mucosal contact with blood or body fluids from an Ebola patient poses a high risk. Contacts must be followed up for development of persistent high fever for 3 weeks from the last date of contact by daily temperature measurement. Development of vaccines against filoviruses has recently made astonishing progress, driven by the public health emergency of the West African Ebola virus outbreak in 2014/​15. Two recom- binant viral vectored vaccines, a replication defective adenovirus and a replication-​competent vesicular stomatitis virus (VSV) each expressing the glycoprotein of Ebola virus were tested during the outbreak. The latter was recently reported to have 100% efficacy in the preliminary analysis of a phase 2/​3 trial employing a ring-​ vaccination cluster-​randomized design during the Ebola virus epi- demic in Guinea and its licensure could be as early as 2018. The manufacturer Merck & Co. has made the vaccine available during 2016 for ring vaccinations following the occurrence of several iso- lated cases of Ebola virus disease in West Africa after the epidemic had been declared over, and again in 2018 in the most recent out- break of Ebola in the DRC. Its single-​dose regimen and proof of effectiveness from 10 days postimmunization make it an attractive candidate for use in an outbreak campaign. A drawback of this vac- cine is that is requires storage at –70 oC. Protection against Marburg virus disease infection in animal models has been much easier to achieve using a variety of vac- cines, including recombinant proteins, than against Ebola virus. This is probably due to the slightly slower replication of the virus in these models. Given the success with the VSV-​vectored Ebola virus vaccine (see earlier), this approach will likely be extended to a multifilovirus vaccine comprising of three species of Ebola virus and of Marburg virus disease. Clinical features Marburg virus disease and Ebola virus cause identical clinical dis- eases. After an incubation period of 5 to 12 days, the disease starts suddenly with fever, headache, myalgia, and extreme fatigue. Early signs also include conjunctivitis, bradycardia, and sore throat, often associated with severe swelling and dysphagia, but no exudative pharyngitis. Severe nausea, vomiting, abdominal pain, and profuse watery diarrhoea are common (Fig. 8.5.18.2). Around the fifth day, a perifollicular, non​itching, maculopapular rash frequently appears on the trunk, back, and shoulders, spreading to the face and limbs and becoming confluent (Fig. 8.5.18.3). It may be difficult to see and has a measles-​like appearance on dark skin. The rash fades in 3–​10 days and is followed by a desquamation in survivors. In about half of the patients, haemorrhagic manifestations occur between the fifth and seventh day, including epistaxis, gum-​bleeding, haematemesis (Fig. 8.5.18.4), melaena, petechiae, ecchymoses (Fig. 8.5.18.5), haem- orrhages from needlesticks and post-​mortem evidence of visceral Fig. 8.5.18.1  Personal protective equipment in use in Sierra Leone during Ebola epidemic in 2015. Courtesy of Dr Alastair Moore.

874 section 8  Infectious diseases haemorrhagic effusions. While clinically significant haemorrhage occurs in only a minority of patients, coagulopathy appears to be a typical feature of Ebola virus disease. Dehydration and prostration are frequent; patients show the ghost-​like facial expression typical of the disease. During the first week, the temperature remains high around 40°C, falling by lysis during the second week, to rise again between days 12 and 14. Other clinical signs during the second week include hepatosplenomegaly, oedema, orchitis, scrotal or la- bial reddening, myocarditis, and pancreatitis. Jaundice is not a fea- ture. A poor prognosis is marked by haemorrhagic signs, oliguria or anuria, chest pain, shock, tachypnoea, and neurological symp- toms (sudden hearing loss, blindness, painful paraesthesia, intract- able hiccups). Death in shock usually occurs 6–​9 days after onset of clinical disease. Infection in pregnancy results in high maternal mortality and virtually 100% fetal death. Central nervous system in- volvement has led to hemiplegia and disorientation, and sometimes frank psychosis. Causes of death remain poorly understood but are likely to be due (in combination or alone) to a combination of septic shock (leaky gut?) and multiorgan failure (direct cytopathic effect). The recovery of Marburg and Ebola disease is prolonged with arth- ralgia or persistent arthritis, ocular disease (ocular pain, photo- phobia, hyperlacrimation, loss of visual acuity, uveitis), hearing loss, and orchitis occurring as late manifestations. Neurological ab- normalities in survivors seem to be frequent; on neurological exam most common findings reported are abnormal smooth pursuits and saccades, tremor, abnormal reflexes, and sensory abnormal- ities. In a minority of survivors ongoing seizures, evidence of stroke (including hemiparesis, hemianopsia, and cranial nerve abnormal- ities), and parkinsonism have been described. Other symptoms of the so-called post-Ebola syndrome include abdominal pain, anorexia, Fig. 8.5.18.2  Severe vomiting and diarrhoea in a patient with Ebola virus disease in Sierra Leone. Courtesy of Dr Alastair Moore. Fig. 8.5.18.3  Rash of Ebola haemorrhagic fever acquired through a laboratory accident. Courtesy of Professor D. I. H. Simpson. Fig. 8.5.18.4  Haemorrhage and oedema of face and neck in Marburg haemorrhagic fever. Courtesy: Professor S. Stille. Fig. 8.5.18.5  Ecchymoses in a patient with Ebola virus disease. Courtesy of Professor D. I. H. Simpson.

8.5.18  Filoviruses 875 headache sleep disturbances, dizziness, itchiness/rashes, impotence, numbness, retroorbital pain, and muscular weakness. Serious but re- versible personality changes have been recorded in a few survivors, namely confusion, anxiety, depression, and aggressive behaviour. Blindness has been reported as a sequel. Preliminary data from a study tracking 1500 Ebola survivors for up to 5 years in Liberia (PREVAIL) show that 68% have neurological complications, 60% eye problems, and 55% musculoskeletal disorders. Both Ebola virus and Marburg virus disease have been iso- lated from the anterior chamber of the eye and from seminal fluid many weeks after the onset of clinical disease and there have been documented cases of sexual transmission. The shedding of Ebola virus RNA has been detectable in semen and vaginal fluid by PCR for many months, with approximately one-​quarter of the nine par- ticipants of one study having positive findings on quantitative RT-​ PCR at 7–​9 months after onset, but not by virus isolation. Patients should, therefore, refrain from sexual activities during early conva- lescence. Another lesson to emerge from the 2014 epidemic is that some survivors experience serious symptoms after their recovery from the main disease episode, suggesting that viral persistence in certain compartments of the body is more serious in some survivors than previously recognized. A British nurse who developed Ebola virus meningitis more than 9 months after surviving acute Ebola virus disease was readmitted 14 months past the initial infection to a high-​containment unit for treatment of late disease complications. Ebola virus can persist in the central nervous system and be trig- gered to reactivate or to escape immune surveillance, or both. It is not clear how, or whether, post-​Ebola virus disease immunity is af- fected by the stage of treatment or type of therapy given. Haematological studies reveal early leucopenia, thrombocyto- penia accompanied by abnormal platelet aggregation, subsequent relative neutrophilia, and the appearance of atypical lymphocytes. Liver enzymes are elevated (AST/​SGOT >ALT/​SGPT) consistent with histopathological evidence of hepatitis (Fig. 8.5.18.6), but al- kaline phosphatase and bilirubin levels are usually normal or only slightly elevated. Disseminated intravascular coagulation is a prom- inent manifestation of Ebola virus infection in primates (prolonged prothrombin (PT) and partial thromboplastin time (PTT), D-​dimers, fibrin split products), and elevated D-​dimers and thrombocytopenia are consistently observed in early stages of illness in humans. Fibrin deposition has been documented at autopsy. Detailed studies of the coagulation disorder have been performed in two patients treated in intensive care in the United Kingdom and both had evidence of a consumptive coagulopathy. As this resolved, thromboelastography demonstrated that both developed a marked hypercoaguable state, which was treated with low molecular weight heparin. Neither case developed any clinical evidence of venous thromboembolic disease or complications from anticoagulation. Currently the frequency of occurrence and clinical importance of the hypercoagulable state is unknown. In non​human primates, a rapid decline in plasma protein C levels was observed in Ebola virus infection, preceding clinical symptoms. Differential diagnosis and criteria for diagnosis Clinically, filovirus infections can be confused with non​viral in- fections such as severe malaria, typhoid fever, shigellosis (‘diarrhée rouge’ in francophone Africa), leptospirosis, rickettsial diseases, meningococcaemia, Gram-​negative sepsis, and other conditions re- sulting in disseminated intravascular coagulation. There is overlap of clinical presentation with other viral haemorrhagic fevers. Ebola virus disease or Marburg virus disease should be suspected in a pa- tient living in or coming from, within the incubation period, a known endemic area (currently Angola, Côte d’Ivoire, the Democratic Republic of the Congo, Gabon, Sudan, Kenya, and Uganda, Guinea, Sierra Leone, Liberia, Ivory Coast) and presenting with other- wise unexplained high fever (above 38.5°C) and vascular involve- ment (subnormal blood pressure, postural hypotension, petechiae, haemorrhagic diathesis, flushing of face and chest, non​dependent oedema). Reported contact with another viral haemorrhagic fever patient or a known viral haemorrhagic fever vector is obviously a very important risk factor. During outbreaks, more specific case def- initions are typically being developed (see Box 8.5.18.1). The case definition for suspected Ebola virus infection might change during the course of an outbreak and differ from that of pre- vious outbreaks. Because viral haemorrhagic fever is a purely clinical diagnosis which requires the immediate instalment of barrier nursing proced- ures and notification of public health authorities, rapid laboratory con- firmation is mandatory. Care must be taken in drawing and handling blood specimens since virus titres can be extremely high and the virus is stable for long periods, even at room temperature. During the first week of clinical illness, virus is easily detected by cell culture, viral antigen by enzyme-​linked immunoabsorbent assay, and viral RNA by PCR, and commercially available field tests to detect antigen in blood or cadaveric oral fluids (OraQuick Ebola Rapid Antigen test, Orasure Fig. 8.5.18.6  Hepatic histology in Ebola haemorrhagic fever. Courtesy of Professor D. I. H. Simpson. Box 8.5.18.1  Signs and symptoms for suspecting Ebola virus infection during the 2014 outbreak (developed by WHO, CDC USA, Médecins Sans Frontières) 1 Fever + contact with a known Ebola virus disease case, or 2 Fever + at least three of the following: Headaches, lethargy, dyspnoea, dysphagia, dyspepsia, loss of appetite, myalgia/​arthralgia, vomiting, diarrhoea, hiccups, or 3 Any person with unexplained bleeding, or 4 An unexplained death

876 section 8  Infectious diseases Technologies Inc, USA) or RNA in blood (RealStar Zaire EBOV PCR, Altona Diagnostics, Germany) have recently been used successfully to identify EBOV during the 2018 outbreak in DRC. Impressively, the circulating Ebola virus strain could be sequence-confirmed by a local laboratory within 10 days using nanopore-sequencing tech- nology (MinION device, Oxford Nanopore Technologies, UK). Blood samples must be handled and shipped to a reference labora- tory using special precautions (triple packaging: primary, secondary, and outer container with absorbent material in between) and must be inactivated for performing routine laboratory tests (Chapter 8.5.17, Table 8.5.17.1). In fatal human Ebola virus cases, antiviral IgM and IgG antibodies were detected in 46% and 30% of patients, respectively. However, in the age of rapid and accurate molecular diagnostics, ser- ology does not play a major role in diagnosis of the disease. Virus can also be visualized by immune-histochemistry in formalin-fixed skin biopsies taken from the axilla or nape of the neck. For handling of clinical specimens from suspected cases, see Chapter 8.5.17, Table 8.5.17.1. Treatment Conceptually, therapy of Ebola virus disease and Marburg virus dis- ease consists of specific antiviral approaches, modulation of the host immune response, and symptomatic treatment. Currently, no specific antiviral licensed therapy is available. However, several small mol- ecule and biological drugs were evaluated in small trials during the recent outbreak in West Africa, or were given on a compassionate use basis. Favipiravir (T-​705) is a broad-​spectrum antiviral developed by Toyama Chemical Co Ltd., which has been approved in Japan and is now in phase III of clinical development in the United States for the treatment of complicated or resistant influenza. In a non​comparative, proof-​of-​concept trial, in which all patients received favipiravir along with standardized care (10-​day treatment with a loading dose of 6000 mg on day 1 and a maintenance dose of 2400 mg/​day for adults), it had no effect on survival in patients with a viral load of greater than 7.7 log 10 copies/​ml. However, it had a possible effect on patients with lower viral loads (20% mortality in treatment group vs. 30% in pretrial controls). Toxicity was not reported in this trial. TKM-​Ebola, devel- oped by Arbutus biopharma (formerly known as Tekmira), belongs to a new therapeutic class based on RNA interference technology. This drug is composed of two small interfering RNAs (siRNAs), which si- lence the Ebola virus viral polymerase and VP35 genes by inhibiting mRNA translation and enhancing host cell-​mediated viral mRNA destruction. As siRNAs are very unstable, they are encapsulated and protected in lipid nanoparticles coated with polyethylene glycol mol- ecules. TKM-​Ebola has been used in the United States in two adult patients as compassionate treatment in combination with extensive supportive care and convalescent plasma. The two patients survived despite severe disease-​related clinical and biological alterations. A phase II, single-​arm clinical trial in Sierra Leone to evaluate the ef- ficacy of TKM-​Ebola in patients was discontinued because of a low probability of demonstrating an overall therapeutic benefit. ZMapp is a cocktail of three humanized monoclonal antibodies with strong neutralizing in vitro and in vivo activity against the Zaire strain of Ebola virus. In a randomized controlled trial in West Africa, mortality in the ZMapp-​treated participants who received a fixed dose of 50 mg/​ kg administered every 3 days was 40% lower (8 of 36; 22% mortality) than in participants receiving standard of care alone (13 of 35; 37%). However, due to a smaller-​than-​intended sample size because of en- rolment problems, this difference did not reach statistical significance. Serum from Ebola virus survivors contains varying levels of low titre neutralizing antibodies and Ebola virus-​infected non​human primates have been successfully treated up to 48 hours after a lethal Ebola virus challenge with multiple doses of concentrated, species-​ matched, polyclonal immunoglobulin G obtained from vaccinated rhesus macaques that had survived challenge with a lethal Ebola virus dose. In a non​randomized, comparative study in West Africa, 84 patients of various ages (including pregnant women) with con- firmed Ebola virus disease received two consecutive transfusions of 200–​250 ml of ABO-​compatible convalescent plasma, with each unit of plasma obtained from a separate convalescent donor. The transfusions were initiated on the day of diagnosis or up to 2 days later. No significant improvement in survival was observed in the treated group (risk of death 31% vs. 38% in control group), however, the level of neutralizing antibodies against Ebola virus in the plasma was unknown at the time of administration. A summary of all experimental Ebola virus drugs which were evaluated in clinical trials during the last epidemic has been re- cently published (Cardile et al., 2017). Among the post-exposure treatments monoclonal antibodies seem to demonstrate the highest level of efficacy, whereas for relapsed or convalescent patients who are shedding filoviruses faviparivir, siRNAs and or the adenosine analogue GS-5734 (Remdesivir, Gilead Sciences) may be more appropriate. Fluid, electrolyte, respiratory, and osmotic imbalances should be managed carefully. Patients may require full intensive care sup- port, including mechanical ventilation, along with blood, plasma, or platelet replacement. The maintenance of intravascular volume is a particular challenge, but every effort is justified since the crisis is short lived, and complete recovery can be expected in survivors. Treatment of all concurrent (tropical) infections is important. Recommendations on how to best treat Ebola patients requiring critical care delivered by experienced multidisciplinary teams (e.g. using Trexler isolator tents), have been published (Fig. 8.5.18.7). Meticulous adherence to infection prevention guidelines and thor- ough training of staff is key to prevent nosocomial infections. Fig. 8.5.18.7  An example of an isolation unit for transporting a patient with Ebola virus disease. Courtesy of Dr Alastair Moore.

8.5.19 Papillomaviruses and polyomaviruses 877

8.5.19 Papillomaviruses and polyomaviruses 877

8.5.19  Papillomaviruses and polyomaviruses 877 Prognosis The case fatality of filovirus infections is extremely high and possibly de- pendent on the infecting species, with up to 90% for Ebola virus Zaire and Marburg virus disease Angola. Among 27 patients with Ebola virus dis- ease who were cared for in the United States or Europe, close monitoring and aggressive supportive care that included intravenous fluid hydration, correction of electrolyte abnormalities, nutritional support, and critical care management for respiratory and renal failure were needed; 81.5% of these patients who received this care survived (Uyeki et al., 2016). Common denominators of survival in filovirus-​infected macaques are maintenance of D-​dimer levels, maintenance of protein C activity (>50%), maintenance of levels of proinflammatory/​procoagulant cyto- kines, and low viral load. Areas of uncertainty/​controversy None of the trials conducted with antiviral drugs during the 2014 Ebola virus epidemic produced results sufficient for licensure, so their future development and availability during the next filovirus outbreak is uncertain. Likely developments over the next 5–​10 years The licensing of a recombinant, vesicular stomatitis virus-​based Ebola virus vaccine is to be expected in the United States of America within 1–​2 years. FURTHER READING Cardile AP, et al. (2017). Will there be a cure for ebola? Annu Rev Pharmacol Toxicol, 57, 329–​48. Cross RW, et al. (2018). Post-exposure treatments for Ebola and Marburg virus infections. Nat Rev Drug Discov, 17, 413–31. Lamontagne F (2018). Evidence-based guidelines for supportive care of patinets with Ebola virus disease. Lancet, 391, 700–08. 8.5.19  Papillomaviruses and polyomaviruses Raphael P. Viscidi, Chen Sabrina Tan, and Carole Fakhry ESSENTIALS Papillomaviruses and polyomaviruses are small, non​enveloped, double-​stranded DNA viruses. Human papillomavirus There are nearly 200 human papillomavirus types that infect epi- thelia of skin and mucous membranes. They infect only humans, and cause conditions including the following: Skin warts and verrucas—​caused by types 1 and 2; infection initi- ated when, after minor skin abrasions, for example, the basal cells of the epithelium come in contact with infectious virus. Anogenital warts—​caused by types 6 and 11; transmitted by direct sexual contact, these are the most common sexually trans- mitted infection; present clinically as multiple exophytic le- sions or as subclinical flat lesions. Can be treated topically with podophyllin or imiquimod, or by ablative surgical methods. Recurrences are common. A highly efficacious prophylactic vac- cine is available. Cervical cancer—​the second most common tumour in women worldwide; most often caused by types 16 and 18, whose DNA can be recovered from nearly all cases of invasive disease and squa- mous intraepithelial lesions of the cervix, which precede invasive cancer. Prevention is by cervical screening and vaccination (two highly effective vaccines are available). Other cancers—​ human papillomaviruses can cause cancers at other lower anogenital tract sites and in the oropharynx. Human papillomavirus DNA is often detected in non​melanoma skin cancers, but it is not known whether this is pathogenic. Respiratory papillomatosis—​caused by types 6 and 11; usually in- volves the vocal cords, leading to presentation with hoarseness or voice change; may rarely cause life-​threatening airway obstruc- tion; mainstay of treatment is surgical removal of papillomas, which commonly recur. Human polyomaviruses Exposure to polyomaviruses is nearly universal: primary infection
is most likely asymptomatic and occurs in childhood and then
persist as latent infections, primarily in the kidney epithelial cells, producing disease in the context of immunosuppression. BK virus—​can cause (1) nephropathy and renal failure in renal transplant patients; management is by gradual reduction in im- munosuppression, but more than 50% of patients lose their allo- graft, and (2)  ureteral stenosis; (3)  haemorrhagic cystitis in bone marrow transplant patients. JC virus—​causes progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system that is usually relentlessly progressive and fatal. Progressive multifocal leukoence­ phalopathy is most often seen in patients with HIV/​AIDS, but re- cently reported as a rare complication of treatment with many immunoregulatory monoclonal antibodies, but most commonly with natalizumab given to patients with multiple sclerosis or Crohn’s disease. Merkel cell polyomavirus has recently been implicated as the aetiological agent of Merkel cell cancer, a rare aggressive skin tumour. Trichodysplasia spinulosa-​associated polyomavirus causes a skin disease, trichodysplais spinulosa, in paediatric heart transplant patients. Other polyomaviruses include Washington University polyomavirus, Karolinska Institute polyomavirus, Malawi polyomavirus, and St Louis polyomavirus, named for the geographic location where the viruses were isolated; and human polyomavirus 6, 7, 9, 12, and 13, named in order of their discovery. These have been isolated from both sterile sites such as blood (human polyomavirus 9)  and non​sterile sites, including nasopharynx (Washington University polyomavirus and Karolinksa Institute polyomavirus) and stool (Malawi and St. Louis polyomaviruses). It is currently unclear if these polyomaviruses cause specific human disease.

878 section 8  Infectious diseases Introduction Papillomaviruses and polyomaviruses are small, spherical, non-​ enveloped, doubled-​stranded DNA viruses that multiply in the cell nucleus. The two virus groups are unrelated. Papillomaviruses infect surface epithelia and produce disease at these sites. Polyomaviruses cause viraemia and spread, after initial multiplication at the site of entry, to affect internal organs such as the kidney, the brain, or skin. Viruses of both families produce experimental tumours in la- boratory animals and both viruses are responsible for some cancers in humans, although only papillomavirus-​associated cancers are common. Within each family the viruses are immunologically re- lated and share nucleotide similarity. Nearly 200 human papillomaviruses have been recognized, about 35 of which infect mucous membranes (genital and respiratory tracts, and the oral cavity) and the remainder infect skin. Human papillomaviruses cause skin warts, genital warts, respiratory papil- lomas, and papillomas at other mucosal sites (e.g. mouth, eye). In addition, infection with some genital tract human papillomaviruses causes cervical cancer, one of the most common female malignan- cies in the world, as well as a proportion of cancers at other genital tract sites and the oropharynx. JC virus is the aetiological agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease occurring in immunodeficient people. BK virus is associated with haemorrhagic cystitis in bone marrow transplant recipients, and with nephrop- athy and renal failure in renal transplant recipients. Merkel cell polyomavirus is implicated as the aetiological agent of Merkel cell cancer, a rare aggressive skin tumour. Trichodysplasia spinulosa-​ associated polyomavirus is found in the rare skin disease of the same name and may play a role in the development of the disease. It is not known if the other human polyomaviruses cause disease. Human papillomaviruses (HPVs) Human papillomaviruses cannot be propagated in tissue culture and require nucleic acid hybridization assays for their identification. Their double-​stranded circular genome contains about 8000 bp, ­divided into an early region, necessary for transformation, a late region, encoding for capsid proteins, and a regulatory region, con- taining control elements (Fig. 8.5.19.1). Open reading frames of the viral genome are located on one strand: E1 to E8 in the early region and L1 and L2 in the late region. The functions assigned to the dif- ferent open reading frames are listed in Table 8.5.19.1. Human papillomaviruses infect only humans. They show a marked degree of cellular tropism. Mucosal human papillomaviruses do not readily infect cutaneous epithelia and cutaneous human papillomaviruses are rarely present on mucous membranes. Infection is initiated when, after minor trauma (e.g. during sexual intercourse or after minor skin abrasions), the basal cells of the epi- thelium come in contact with infectious virus. The virus stimulates the proliferation of basal cells. The early region open reading frames are expressed in all layers of the infected epithelium, but expression of the late region open reading frames and synthesis of viral particles occur only in the upper differentiating and keratinizing layers. Important disease associations and characteristics of mucosal HPVs are listed in Table 8.5.19.2. The burden of human cancers attributable to HPVs is shown in Table 8.5.19.3. The genital tract is the reservoir for all but a few mucosal human papillomaviruses, and genital human papillomavirus infections constitute the most common viral sexually transmitted infections. Genital human papillomaviruses may sometimes infect non​anogenital mucosal sites (e.g. the respiratory tract, the mouth, and the conjunctiva). Transmission of genital tract HPV types 6 and 11 from an infected mother to the baby at birth results in juvenile-​onset recurrent re- spiratory papillomatosis. Infection with two types, HPV-​13 and HPV-​32, appears to be confined to the oral cavity. Table 8.5.19.4 lists disease associations of cutaneous HPVs, which are transmitted by direct contact with infected tissue or by contact with a contaminated object. Anogenital warts Anogenital warts (condylomas) are the most commonly recognized clinical manifestations of genital HPV infections. More than 90% of condylomas result from infections with HPV-​6 and HPV-​11. In the LCR E6 E7 E1 E2 E5 E4 L2 L1 HPV-16 P97 AE 1000 2000 3000 4000 5000 6000 7000 7904/1 AL Fig. 8.5.19.1  Genomic map of HPV-​16. On the inner circle, P97 represents the transcriptional promoter and AE and AL designate early and late polyadenylation sites. The location of the early region open reading frames (E1–​E8), the late region open reading frames (L1, L2), and of the long control or regulatory region (LCR) are shown. Reproduced from Shah KV, Howley PM (1996). Papillomaviruses. In: Fields BN, et al. (eds) Fields virology, vol. 2, pp. 2077–​109. Lippincott-​Raven, Philadelphia,
with permission. Table 8.5.19.1  Functions of human papillomavirus open reading frames Function ORF Major capsid protein and neutralizing epitopes L1 Minor capsid protein L2 Replication of viral DNA E1, E2 Regulation of transcription E2 Coding for late cytoplasmic protein E4 Cellular proliferation E5 Transformation E6, E7 Not known E3, E8 ORF, open reading frame. Modified from Shah KV, Howley PM (1996). Papillomaviruses. In: Fields BN, et al. (eds) Fields virology, vol. 2, pp. 2077–​109. Lippincott-​Raven, Philadelphia.

8.5.19  Papillomaviruses and polyomaviruses 879 United States of America, there are more than a million annual consultations for anogenital warts. Epidemiology Genital and anal warts are most common between the ages of 16 and 24 years. They are transmitted by direct sexual contact. Anogenital warts in children can also be due to close but non​sexual contact within a family but, in many cases, sexual abuse by an infected adult is responsible. Clinical features The incubation period is between 3 weeks and 8 months (mean  = 2.8 months). In men, condylomata acuminata (exophytic condyl- omas) most often appear on areas exposed to coital trauma, the glans penis, coronal sulcus, prepuce, and terminal urethra. The soft fleshy vascular tumours are usually multiple and may coalesce into large masses (Fig. 8.5.19.2). Sessile or papular warts are more likely to occur on dry areas such as the shaft of the penis (Fig. 8.5.19.3). The raised pink or grey lesions, 0.5–​3 mm in diameter, may occur alone or with exophytic condylomas. Subclinical HPV lesions (flat condylomas) are identified by examining the genitalia with magnification after the application of 5% aqueous acetic acid solution. The affected areas are slightly raised and shiny white (acetowhite), with a rough surface. Flat condylomas affect the same areas as exophytic condylomas. Perianal warts are usually exophytic and in moist conditions around the anus may reach a large size. In 50% of cases, condylomas also appear in the anal canal. Areas of acetowhite epithelium indi- cative of subclinical HPV infection may be associated with perianal warts or occur alone. In women, exophytic condylomas (Fig. 8.5.19.4) appear at the fourchette and adjacent areas, and may spread to the rest of the vulva, the perineum, anus, vagina, and cervix. Multiple sessile warts may affect the labia and perineum. Subclinical HPV infection presents as slightly raised acetowhite lesions: the fissuring of these may cause dyspareunia. About 15% of women with vulval warts have exophytic condylomas on the cervix. Subclinical infection is more common, and consists of acetowhite lesions with punctation due to capil- lary loops, which can be identified by colposcopy. Large, exophytic vulval condylomas may develop during pregnancy and can become so large that they compromise delivery. Most regress post-​partum. Even with therapy (see next), recurrence of genital warts occurs within 3 months in 25–​67% of cases. Recurrences are often at sites of previous genital warts and are attributed to persistent infection that then reactivates. Diagnosis and management Genital warts must be distinguished from Fordyce’s spots, fibro­ epithelial polyps, molluscum contagiosum, and the papillar lesions of secondary syphilis. Lesions that appear atypical or respond poorly to treatment must be biopsied early. Associated sexually transmitted infections must be excluded. Sexual partners should be examined. Intraepithelial neoplasia must also be excluded. Cervical cytological examination should always be done on women with vulval warts and on female partners of men with penile warts. Treatments for genital warts can be classified as topical, immu­ nomodulatory, or surgical. Podophyllin and podophyllotoxin, which are derived from the root of the mayapple plant, are antimitotic agents that disrupt viral activity by inducing local tissue necrosis. Patient-​applied topical podophyllotoxin, 0.5%, has a clinical cure rate of 56%; however, recurrence rates range from 23 to 65%. Disadvantages of podophyllin compounds include local Table 8.5.19.2  Mucosal human papillomaviruses: chief clinical associations Clinical association Viral type(s) Exophytic condyloma; respiratory papillomas; oral and conjunctival papillomas HPV–​6, –​11 Cervical cancer: High-​risk infections HPV–​16, –​18, –​31, –​45, –​33, –​35, –​39, –​51, –​52, –​56, –​58, –​59 Low-​risk infections HPV–​6, –​11, –​40, –​42, –​43, –​44, –​54, –​61, –​70, –​72, –​81 Vulval, vaginal, penile, anal, and oropharyngeal cancers HPV–​16 Focal epithelial hyperplasia of the oral cavity HPV-​13, -​32 Modified from Shah KV, Howley PM (1996). Papillomaviruses. In: Fields BN, et al. (eds) Fields virology, vol. 2, pp. 2077–​109. Lippincott-​Raven, Philadelphia. Includes material from The Oxford textbook of medicine, 3rd edition, pp. 3366–​9. Table 8.5.19.3  Cancers attributable to HPV infection in 2002 Site Attributable to HPV (%) Total cancers Attributable to HPV % of all cancers Cervix 100 492 800 492 800 4.54 Penis 40 26 300 10 500 0.10 Vulva, vagina 40 40 000 16 000 0.15 Anus 90 30 400 27 300 0.25 Oropharynx 50 52 100 26 500 0.25 All sites c.5 10 862 500 Modified from Parkin DM and Bray F (2006). The burden of HPV-​related cancers. Vaccine, 24, Suppl 3, S11–​S25.

section 8  Infectious diseases 880 adverse reactions, risk of systemic absorption, and teratogenicity. Imiquimod, a topical treatment for genital warts, induces macro- phages to secrete cytokines, principally interferon-​α, and is thought to work by stimulation of a cell-​mediated immune response against HPV. Imiquimod is as effective as podophyllin for initial clearance of genital warts and results in a lower recurrence rate. The side ef- fect profile of imiquimod is benign. Warts can be destroyed by cryo- therapy with liquid nitrogen, electrocautery, electrodessication, scissor excision, or carbon dioxide laser therapy. Although these ab- lative therapies are successful in initially removing genital warts, re- currences are common. In a comparative trial, imiquimod 5% cream alone or in combination with ablative treatments was superior to ablation alone in reducing the recurrence rate of successfully treated anogenital warts. A  prophylactic vaccine that prevents 100% of genital warts due to HPV-​6 and HPV-​11, if administered prior to exposure to HPV, is now available (see next). Respiratory papillomatosis This rare disease may have onset in childhood or in adult life. It is most common in children under the age of 5 years. It may become life-​threatening if it obstructs the airways. Papillomatosis usu- ally involves the vocal cords and the patient presents with hoarse- ness or voice change. Papillomas might recur despite surgical removal. HPV-​6 and HPV-​11, genital tract HPVs that are responsible for most of the exophytic genital warts, also cause respiratory papillomatosis. Patients with juvenile-​onset disease are infected at birth during passage through an infected birth canal. In adult-​onset disease, transmission may occur by sexual contact. Respiratory papillomas rarely progress to invasive cancer. Irradiation of papil- lomas with X-​rays (a practice now discontinued) increased the risk of malignancy. Caesarean delivery for mothers who are found to have genital warts or are infected with HPV-​6 or HPV-​11 would reduce the risk of juvenile-​onset respiratory papillomatosis, but it is not gen- erally recommended because of the small risk of disease following perinatal infection. The mainstay of treatment is surgical removal of papillomas; however, recurrence of lesions is common. Various adjunct therapies have been tried, including interferon-​α, indole-​3-​ carbinol, cidofovir, and photodynamic treatment. These therapies Table 8.5.19.4  Cutaneous human papillomaviruses: chief clinical associations Clinical association Viral type Deep plantar wart HPV–​1 Common wart HPV–​2, –​4 Mosaic wart (superficial spreading wart) HPV–​2 Flat warts HPV–​3, –​10, –​28, –​41 Macular plaques of epidermodysplasia verruciformis HPV–​5, –​8, –​9, –​12, –​14, –​15, –​17, –​19, –​20, –​21, –​22, –​23, –​24, –​25, –​36, –​47, –​50 Squamous cell carcinoma HPV–​5,–​8, –​20, –​36, –​38 Modified from Shah KV, Howley PM (1996). Papillomaviruses. In: Fields BN, et al. (eds) Fields virology, vol. 2, pp. 2077–​109. Lippincott-​Raven, Philadelphia. Fig. 8.5.19.2  Condylomata acuminata (exophytic condylomas) of the penis. Fig. 8.5.19.3  Sessile (papular) warts of the penis.

8.5.19  Papillomaviruses and polyomaviruses 881 have had only modest success in reducing the need for surgery or recurrence. It is anticipated that a child born to a mother who has received the HPV Gardasil vaccine, will have a markedly reduced risk of developing respiratory papillomatosis. Cervical cancer (See Chapter 5.1.) Human papillomavirus DNA is recovered from nearly 100% of cases of invasive cervical cancer and squamous intraepithelial lesions of the cervix, which precede invasive cancer. The viral genome is present in the tumour cells of primary as well as metastatic cervical cancer. The progression from low grade squamous intraepithelial lesions to invasive cancer may take more than 10 years; human papillomaviruses are found throughout this disease process. The vir- uses are recovered much less frequently from cytologically normal women of comparable age. In prospective studies of women with normal cervical cytology, the presence of HPV is a strong risk factor for the subsequent development of squamous intraepithelial lesions. Certain HPV types are preferentially associated with invasive cancers. From their distribution in normal individuals and in preinvasive and invasive cervical disease, genital tract HPVs have been categorized as high-​risk, or low-​risk types (Fig. 8.5.19.5; Table 8.5.19.2). HPV-​16 and HPV-​18 are the predominant viruses in invasive cancers and account for 40–​60% and 5–​20%, respect- ively, of HPV-​positive cancers in different studies. About a dozen additional types of HPV are found in small proportions of invasive cancers. The low-​risk HPVs are almost never detected in invasive cervical cancers. Comparisons of different HPV types for their ability to trans- form human keratinocytes in vitro show that HPV-​16 and HPV-​18, types most clearly associated with naturally occurring cervical can- cers, also have the greatest oncogenic potential in laboratory studies. The transforming functions of HPVs are localized to open reading frames E6 and E7; these are the viral genes consistently expressed in naturally occurring HPV-​positive cancers. The viral genome is in- tegrated into the cellular DNA in most cervical cancers. The break in the circular viral genome that is required for integration occurs most frequently in the E1/​E2 region and results in an enhanced expression of the transforming E6 and E7 open reading frames. The transforming HPV proteins E6 and E7 interact with cellular tumour suppressor proteins p53 and Rb, respectively. The oncogenic effect of HPVs is mediated largely by their ability to inactivate the tumour suppressor proteins which normally regulate the cell cycle. Epidemiology Human papillomavirus infections of the genital tract are extremely common in sexually active populations. A history of multiple sexual partners, and having a male sexual partner who has many sexual partners, are the man risk factors for a woman for the acquisition of HPVs. In young sexually active women, point prevalence (single sampling) of HPV infection as measured by the detection of HPV DNA in genital tract specimens by the sensitive polymerase chain reaction (PCR) may be as high as 40%, and the cumulative preva- lence (multiple sampling of women over time) may be as high as 80–​90%. The prevalence decreases with increasing age. Most of these infections are found in women with normal cervical cytology and undoubtedly resolve without leaving a trace. Only a small propor- tion of infections persists and progresses to squamous intraepithelial lesions and then to invasive cancer. The cofactors that might be as- sociated with progression to cancer include smoking, use of oral contraceptives, parity, and presence of other sexually transmitted infections. Human immunodeficiency virus (HIV) infection and associated immunosuppression, leads to a much higher prevalence, and longer persistence, of HPV infections, and to greater incidence of squamous intraepithelial lesions. Prevention and control Screening for cervical cytological abnormalities by cervical smear and treatment of preinvasive and invasive cancers identified by Fig. 8.5.19.4  Condylomata acuminata of the vulva. 16 0 10 20 30 40 50 60 70 Contribution to cervical cancer (%) 18 45 31 33 52 58 35 59 HPV Type 56 39 51 73 68 66 X Unk Fig. 8.5.19.5  Percentages of cervical cancer cases attributed to the most frequent HPV types in all world regions combined. X includes the rare types 40, 42, 53, 54, 55, 83, and 84. ‘Unk’ includes specimens that were positive for HPV DNA but could not be genotyped by current methods. Data from Munoz N, et al. (2004). Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer, 111, 278–​85.

882 section 8  Infectious diseases screening, have been credited with the decrease in incidence of cer- vical cancer and mortality due to the disease that has been observed in many developed countries over the last 40–​50 years. Cervical cancer screening by detection of DNAs of high-​risk HPV types has been shown to have greater sensitivity than Pap smear for the detec- tion of preinvasive lesions and invasive cancer. Primary screening with HPV testing or HPV cotesting with cytology is replacing cy- tology screening in many developed countries. Some screening algorithms include testing HPV-​positive women for HPV16/​18 genotypes with immediate referral of HPV 16/​18 positive women to colposcopy and a 1-​year follow-​up for other high-​risk HPV-​positive women. Because the infrastructure required for the screening pro- gramme is costly, the World Health Organization has recommended that screening and treatment be performed at a single visit (‘screen and treat approach’) using HPV tests where available and affordable or visual inspection with acetic acid, a low-​cost test that has shown sensitivity comparable to Pap testing. Prophylactic vaccines The discovery that the L1 coat protein of papillomaviruses could as- semble into a virus-​like particle, when expressed as a recombinant protein, and the demonstration that immunization of rabbits, cattle, and dogs with virus-​like particles of their respective papillomaviruses protected against papillomavirus-​induced disease, stimulated the development of vaccines for human papillomaviruses. L1 virus-​like particles appear to induce very limited cross-​neutralization against other genotypes necessitating a multicomponent vaccine to provide coverage against disease caused by more than one type. Two HPV L1 virus-​like particle vaccines have been developed commercially; Cervarix is a bivalent HPV-​16/​18 L1 virus-​like particle vaccine and Gardasil is a quadrivalent HPV16/​18/​6/​11 L1 virus-​like particle vaccine. Both vaccines are generally safe and well tolerated and are highly immunogenic. Both vaccines have demonstrated truly re- markable efficacy, preventing nearly 100% of incident infections and preinvasive cervical cancers due to the HPV types in the vaccines. However, protection from incident infection or disease from non-​ vaccine types is restricted and the vaccines have no effect on preva- lent infection and disease. Gardasil is also 100% effective in women and men in preventing genital warts associated with HPV 6/​11. No clinical trials have been conducted of HPV vaccines for prevention of oropharyngeal cancers. Since genital HPV infection is sexually transmitted, the vaccines ideally should target prepubertal girls and boys, aged 11–​12. Gardasil is approved for use in boys and girls, whereas Cervarix is only approved for use in girls. The vaccines are also recommended for young women 13–​26 years of age and young men up to age 21, because many of them may not yet have been ex- posed to the HPV types in the vaccines. To extend coverage to other high-​risk HPV types, a non​avalent vaccine (Gardasil 9) has been de- veloped, which contains VLPs for types 31, 33, 45, 52, and 58, in addition to the four types in Gardasil. The durability of the immune response engendered by HPV vaccines and thus the possible need for a booster in vaccinated individuals is unknown. Because protec- tion may wane over time and because vaccination does not protect against the HPV types not included in the vaccines, screening pro- grammes will need to be maintained, but the strategy may change with longer intervals between screening and a greater emphasis on HPV DNA testing as a screening method. Therapeutic vaccines Human papillomavirus-​associated cancers express HPV E6 and E7 proteins in their tumour cells. Candidate therapeutic vaccines targeted to these proteins are being developed for the treatment of high grade squamous intraepithelial lesions, invasive cervical cancer, and HPV-​associated oropharyngeal cancers. Cancers at other lower anogenital tract sites Human papillomavirus infections are very common on the vulva, vagina, penis, perineum, and anus. Synchronous neoplasia at mul- tiple sites in the female lower genital tract is almost always asso- ciated with HPVs, especially HPV-​16. Carcinoma of the vulva is aetiologically heterogeneous. Vulval cancers occurring in younger women are associated with HPVs, but the typical squamous cell carcinoma of the vulva in older women is not. Neoplasia of the anal canal, seen frequently in HIV-​seropositive homosexual men, is strongly associated with HPVs. Cancer of the oropharynx A subset of head and neck cancers is aetiologically linked to high-​risk HPVs, most often HPV-​16. HPV-​positive head and neck cancers are distinct from HPV-​negative head and neck cancers. HPV-​positive cancers tend to arise in the oropharynx, specifically the tonsil and base of tongue. Patients with HPV-​positive head and neck cancers tend to be younger and have less tobacco and alcohol exposure, the traditional risk factors of HPV-​negative head and neck cancers. Additionally, a greater proportion of HPV-​positive head and neck cancers tend to occur in men. As compared to HPV-​negative cancers, the HPV-​ positive cancers are characterized by basaloid, non-​keratinizing well differentiated pathology, less frequent p53 and Rb mutations, and better prognosis. Demonstration of HPV genome in tumour cells, presence of HPV transcripts, and immunostaining for cellular p16 characterize HPV-​caused cancers. These cancers are increasing in in- cidence in the United States, as well as in other developed countries around the world. It is estimated that the number of HPV-​caused oro- pharyngeal cancers in men and women will exceed the number of cer- vical cancers in the United States of America by 2020. Skin warts (See Chapter 23.10.) Skin warts and verrucas may occur anywhere on the skin and are morphologically diverse. They are most common in older chil- dren and young adults. Except in the rare condition known as epidermodysplasia verruciformis (see next), they almost never be- come malignant. Most regress within 2 years. Specific HPV types are strongly associated with specific types of warts (Table 8.5.19.4). Epidermodysplasia verruciformis This is a rare, lifelong disease in which a patient has extensive warty involvement of the skin that cannot be resolved. It generally begins in infancy or childhood with multiple, disseminated polymorphic wart-​like lesions on the face, trunk, and extremities that tend to be- come confluent. The warts are either flat or reddish-​brown macular plaques that resemble pityriasis versicolor. In about a third of the cases, foci of malignant transformation occur in macular plaques in areas of the skin exposed to sunlight. The tumours are slow growing and rarely metastasize.

8.5.19  Papillomaviruses and polyomaviruses 883 Epidermodysplasia verruciformis (EV) is often a familial dis- ease. Patients sometimes have a history of parental consanguinity. A  susceptibility locus has been mapped to chromosome 17q25.3 and truncating mutations in either of two novel adjacent genes, EVER1 and EVER2, are associated with the disease in different pedigrees. The function of the gene products of EVER1 and EVER2 and how they confer increased risk for EV are unknown. Recent studies suggest the EVER proteins may be involved in zinc homoeo- stasis. A second putative susceptibility locus is located on chromo- some 2p21-​p24. The flat warts yield the same HPV types as those of normal individuals, but a very large number of HPVs that are seldom encountered in normal individuals are recovered from the macular plaques (Table 8.5.19.3). It is unclear how patients with epidermodysplasia verruciformis become infected with these par- ticular papillomaviruses. The factors that contribute to the occur- rence of carcinoma in these patients therefore include a genetic defect, infection with specific HPVs (e.g. HPV-​5 and HPV-​8), and exposure of the affected area to sunlight. Non​melanoma skin cancers HPV DNA has been detected in 30–​50% of non​melanoma skin can- cers in immunocompetent populations and in up to 90% of non-​ melanoma skin cancers from immunocompromised populations, in particular organ transplant recipients. The HPV prevalence is gener- ally higher in squamous cell carcinoma than in basal cell carcinoma. The sequences represent cutaneous HPV types, EV-​associated HPVs, and many novel HPV sequences. No single HPV type pre- dominates and there is no evidence of high-​risk types analogous to those seen in cervical cancer. The amount of HPV DNA in skin tu- mours is very low, indicating that not every tumour cell harbours an HPV genome. Because HPV DNA is frequently detected in normal skin samples, it is not clear to what extent HPVs contribute to the development of non​melanoma skin cancers. Ultraviolet (UV) light is considered the most significant risk factor for non​melanoma skin cancers. Cutaneous HPVs, through the anti-apoptotic activity of their E6 gene, may act as cocarcinogens by preventing elimination of cells with UV-​induced DNA damage. Human polyomaviruses In 1971, BK virus was isolated from the urine of a renal transplant recipient with the initials ‘BK’, who developed ureteral fibrosis and obstruction, and JC virus was recovered from the brain of a patient (‘JC’) with progressive multifocal leukoencephalopathy. Human polyomaviruses, KI virus and WU virus, were detected in respira- tory tract secretions of children by using molecular techniques. The viruses were detected in upper respiratory tract specimens in the presence of other recognized respiratory tract pathogens and thus their role in disease is unclear. In 2008, another human polyomavirus, Merkel cell virus, was identified in tumour cells from patients with Merkel cell carcinoma, a rare aggressive skin cancer. Trichodysplasia spinulosa is a rare skin disease primarily af- fecting immunosuppressed patients and presenting as follicular-​ based papules and keratin spicules widespread on the face, along with variable degrees of alopecia and dysmorphism. In 2010, a new human polyomavirus, designated trichodysplasia spinulosa-​ associated polyomavirus, was identified in the trichodysplasia spinulosa lesions of a heart transplant recipient. Two new human polyomaviruses, designated type 6 and 7, were detected in the skin of healthy persons. Another new human polyomavirus, type 9, was identified in the blood and urine of a renal transplant patient. Subsequently the virus was independently identified in the skin of a Merkel cancer patient. Malawi polyomavirus was isolated from stool and wart samples, St Louis polyomavirus was isolated from stool, human polyomavirus 12 from liver tissue of a patient with colon cancer, and human polyomavirus 13 from muscle endothelial cells. No diseases have been associated with polyomavirus types 6, 7, 9, 12, 13, or the Malawi, and St Louis polyomaviruses. All the new polyomaviruses were detected by using a variety of molecular tech- niques that do not rely on prior knowledge of the DNA sequence of the virus. Polyomaviruses have a double-​stranded DNA genome of about 5000 bp, which is divided into an early region encoding viral large T proteins, a late region encoding viral capsid proteins VP1, 2, and 3, and a non​coding regulatory region (Fig. 8.5.19.6). The large T pro- teins regulate viral transcription, initiate viral DNA replication, and mediate inactivation of host cell tumour suppressor proteins, which contribute to the oncogenic potential of polyomaviruses. The viral regulatory region contains elements for viral DNA replication and promoters for transcription of early and late genes, as well as binding sites for cellular transcription factors, which determine the host and tissue tropism of polyomaviruses. The early and late regions are transcribed from different strands of the viral DNA. Although BK and JC viruses are homologous for 75% of their nucleotide sequence, the infections are readily distin- guishable by both serological tests and specific PCR detection with unique primers. Infection occurs in childhood and is largely subclinical. Most chil- dren acquire antibodies to BK virus by the age of 10; infection with JC virus occurs at a later age. Infection is believed to occur by the oral route. Both viruses establish latent, often lifelong, infection in the kidney, and are often shed in the urine of immunocompetent healthy individuals. Reactivation in immunodeficient individuals is RR Agno BKV Small t Large T 5141 bp VP1 VP2 VP3 Fig. 8.5.19.6  Genomic map of BK polyomavirus. The genome is approximately 5 kb in size. Bidirectional transcription generates proteins of the viral capsids (VP1, VP2, and VP3) in one direction, and proteins for transcriptional controls (large T and small t) in the opposite direction. The non​coding region (RR) contains binding sites for host transcription factors. The Agno region makes a small protein that may be involved in viral replication.

884 section 8  Infectious diseases responsible for most associated illnesses. The viruses are reactivated in pregnancy, but without any apparent harm to the mother or the newborn. Polyomavirus-​associated illnesses Nephropathy in renal transplant recipients This condition is associated most often with BK virus and rarely with JC virus. It occurs in 3 to 10% of renal transplant recipients and re- sults in a loss of the allograft in 50 to 80% of the affected patients. The recent increase in the incidence of this complication is related to the introduction of new and intensive immunosuppressive therapies. Pathologically, the disease is characterized by inclusion-​bearing en- larged nuclei in renal tubular and glomerular epithelial cells which are readily detected by microscopy (Fig. 8.5.19.7). Systematic moni- toring of the patients for BK virus post kidney transplant has pre- dictive value for the risk of nephropathy and can guide decisions to reduce immunosuppression to suppress further viral reactivation. Early curtailment of BK viral replication prevents progression to nephropathy. Haemorrhagic cystitis in bone marrow transplant recipients Haemorrhagic cystitis in bone marrow transplant recipients is asso- ciated with BK virus infection. Large amounts of BK virus are shed in urine during the haemorrhagic episodes. Haemorrhagic cystitis usually occurs after engraftment and is associated with allogeneic transplant, graft-​versus-​host disease, and myeloablative condi- tioning regimen. Patients often present with haematuria, dysuria, urgency, or suprapubic pain. Complications include severe bleeding, urinary tract obstruction, and renal failure. With use of increased immunosuppression and umbilical cord blood transplantation, BK virus is also now associated with additional renal diseases, such as nephropathy. BK virus reactivation associated diseases can affect up to 15.9% of haematopoietic stem cell transplant recipients. No antiviral therapy is available for BK virus. Patients are treated with symptom relief and decrease of immunosuppressants Progressive multifocal leucoencephalopathy (See Chapters 23.5, 24.10.2, 24.11.2, and 24.11.4.) JC virus causes progressive multifocal leukoencephalopathy, a subacute demyelinating disease of the central nervous system occurring in individuals with impaired cell-​mediated immunity. Until the advent of AIDS, it was a rare disease found mainly in older patients with lymphoproliferative disorders or chronic diseases. Because progressive multifocal leukoencephalopathy is a complica- tion in 1–​2% of AIDS cases, it is a more common disease and is seen much more frequently in younger patients. It has also been recog- nized in children who have inherited immunodeficiency diseases or have AIDS. Recently, progressive multifocal leukoencephalopathy has been recognized as a complication in patients treated with monoclonal antibodies, such as those with Crohn’s disease or mul- tiple sclerosis treated with natalizumab, which inhibits migration of cells across the blood–​brain barrier. Other monoclonal antibodies and immune modulatory drugs have also been associated with rare cases of progressive multifocal leukoencephalopathy. The key pathogenetic event in progressive multifocal leukoence­ phalopathy is the cytocidal JC virus infection of oligodendrocytes, which are responsible for the production and maintenance of myelin. This leads to foci of demyelination that tend to coalesce and (a) (b) Fig. 8.5.19.7  Histology of BK nephropathy. (a) Kidney tissue
stained with Hematoxyline and eosin shows widespread but patchy
interstitial oedema and a mixed inflammatory cell infiltrate with occasional neutrophils. Tubules show extensive viral cytopathic changes with accompanying acute tubular injury and necrosis. (b) Staining with antibody to SV40 large T antigen, which cross
reacts with BK large T antigen, shows numerous cells positive for
viral protein. (a) (b) Fig. 8.5.19.8  A lesion of progressive multifocal leukoencephalopathy showing oligodendrocytes with enlarged, deeply staining nuclei (arrow) and giant astrocytes (left), and a crystalloid array of JC virus particles in an infected oligodendrocyte nucleus (right). Reproduced, with permission, from Shah KV (1992). Polyomavirus, infection and immunity. In: Roitt IM (ed) Encyclopedia of immunology, pp. 1256–​8. Academic Press, New York.

8.5.19  Papillomaviruses and polyomaviruses 885 eventually involve large areas of the brain. Infected oligodendro- cytes, containing large inclusion-​bearing nuclei filled with abundant virus particles, surround the foci of demyelination (Fig. 8.5.19.8). Enlarged astrocytes often show bizarre nuclear changes but are mostly virus negative. They are found within the foci of demyelin- ation. JC virus is disseminated haematogenously to the central ner- vous system. Progressive multifocal leukoencephalopathy starts insidiously. Early signs and symptoms indicate the presence of multifocal asym- metrical lesions in the brain and involve impairment of vision and speech, and mental deterioration. The disease is usually relentlessly progressive and fatal within 3–​6 months, but rarely may become stabilized, with survival for many years. Diagnosis is made based on images (Fig. 8.5.19.9), clinical presentations. Detection of JC virus in cerebrospinal fluid or brain biopsy also aids in the diagnosis. There are no effective antivirals against JC virus. However, viral rep- lication can be stopped in patients who can immune reconstitute, such as HIV patients after treatment with antiretroviral therapy, and natalizumab-​treated patients after plasma exchange. Other diseases caused by JC virus  Although exceedingly rare, JC virus granule cell neuronopathy, where JC viruses infect and des- troy cerebellar granule cell neurons, has been reported worldwide. Patients present with cerebellar-​associated neurological deficits. In addition, cases of JC virus encephalopathy, meningitis, and neph- ropathy have all been reported. Role of polyomaviruses in human tumours The role of polyomaviruses in human tumours is the subject of de- bate. JC virus and BK virus are oncogenic for laboratory animals and they transform cultured cells. There are reports of finding JC virus DNA in brain and colon tumours and BK virus DNA in pros- tate, bladder, and brain tumours, as well as neuroblastomas and insulinomas. However, a reproducible and consistent aetiological association of either virus with any human tumour has not been demonstrated. The Merkel cell virus provides a more convincing example of a polyomavirus-​induced human tumour, since the viral genome was found to be integrated into tumour cell DNA, a key event in experimental polyomavirus-​induced animal tumours. Further supporting the carcinogenic potential of the virus is the observation that survival of virus-​positive Merkel cancer cell lines is dependent on expression of the viral oncoprotein. The virus has been detected in approximately 80% of Merkel cell cancers world- wide. Serological studies have revealed that exposure to Merkel cell polyomavirus is very high in human populations. Thus, the precise role of the virus and modifying cofactors in the aetiology of Merkel cell carcinoma remains to be established. FURTHER READING Berger JR, et al. (1998). Progressive multifocal leukoencephalopathy in patients with HIV infection. J Neurovirol, 4, 59–​68. Bosch FX, et  al. (2002). The causal relation between human papillomavirus and cervical cancer. J Clin Pathol, 55, 244–​65. Chatuevedi AK, et al. (2002). Human papillomavirus and rising oro- pharyngeal cancer incidence in the United States. J Clin Oncol, 29, 4294–​301. D’Souza G, et  al. (2007). Epidemiological evidence that human papillomavirus is a cause of oropharyngeal squamous cell carcinomas. N Engl J Med, 356, 1944–​56. Feng H, et al. (2008). Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science, 319, 1096–​100. Hirsch HH, Steiger J (2003). Polyosmavirus BK. Lancet Infect Dis, 3, 611–​23. Koutsky LA, et al. (2002). A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med, 347, 1645–​51. Munoz N, et al. (2004). Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer, 111, 278–​85. Randhawa P, Brennan DC. (2006). BK virus infection in transplant re- cipients: an overview and update. Am J Transplant, 6, 2000–​5. Yousry TA, et al. (2006). Evaluation of patients treated with natalizumab for progressive multifocal leukoencephalopathy. N Engl J Med, 354, 924–​33. (a) (b) Fig. 8.5.19.9  Brain fluid attenuation inversion recovery (FLAIR) MRIs in axial (a) and sagittal (b) planes of a 36-​year-​old man with AIDS and progressive multifocal leukoencephalopathy proven by detection of JC virus DNA in cerebrospinal fluid by PCR.

8.5.2 Herpesviruses (excluding Epstein– Barr virus

8.5.2 Herpesviruses (excluding Epstein– Barr virus) 734

734 section 8  Infectious diseases Dowell SF (ed) (1998). Principles of judicious use of antimicrobial agents for pediatric upper respiratory tract infections. Pediatrics, 101 Suppl, 163–​84. Falsey AR, Walsh EE (2006). Viral pneumonia in older adults. Clin Infect Dis, 42, 518–​24. Gern JE (2010). The ABCs of rhinoviruses, wheezing, and asthma.
J Virol, 84, 7418–​26. Grohskopf LA, et  al. (2015). Prevention and control of influenza with vaccines:  recommendations of the Advisory Committee on Immunization Practices, United States, 2015–​16 influenza season. MMWR Morb Mortal Wkly Rep, 64, 818–​25. Hayden FG (2013). Advances in antivirals for non-​influenza respiratory virus infections. Influenza Other Respir Viruses, 7 Suppl 3, 36–​43. Hayden FG, et al. (2018). Baloxavir marboxil for uncomplicated influ- enza in adults and adolescents. N Engl J Med, 379, 913–23. Hui DS, Zumla A (2015). Emerging respiratory tract viral infections. Curr Opin Pulm Med, 21, 284–​92. Ison MG, Lee N (2011). Influenza 2010–​11: lessons from the 2009 pan- demic. Cleveland Clinic J Med, 77, 812–​20. Kim YJ, Boeckh M, Englund JA (2007). Community respiratory virus in- fections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immuno- deficiency virus infection. Semin Respir Crit Care Med, 28, 222–​42. Madeley CR, Peiris JSM, McQuillin J (1996). Adenoviruses. In: Myint S, Taylor-​Robinson D (eds) Viral and other infections of the human respiratory tract, pp. 169–​90. Chapman & Hall, London. Mallia P, Johnston SL (2006). How viral infections cause exacerbation of airway diseases. Chest, 130, 1203–​10. Nicholson KG, Webster RG, Hay AJ (eds) (1998). Textbook of influ- enza. Blackwell Scientific, Oxford. Peiris JSM, et al. (2006). Severe acute respiratory syndrome (SARS). In: Scheld WM, Hooper DC, Hughes JM (eds) Emerging infections 7, pp. 23–​50. ASM Press, Washington DC. Principi N, Esposito S (2014). Paediatric human metapneumovirus in- fection: epidemiology, prevention and therapy. J Clin Virol, 59, 141–​7. Siddell S, Myint S (1996). Coronaviruses. In: Myint S, Taylor-​Robinson D
(eds) Viral and other infections of the human respiratory tract,
pp. 141–​67. Chapman & Hall, London. Treanor J (2009). Respiratory infections. In: Richmond DD, Whitley RJ, Hayden FG (eds) Clinical Virology, 3rd edition, pp. 7–​27. ASM Press, Washington, DC. Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010). Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection. N Engl J Med, 362, 1708–​19. 8.5.2  Herpesviruses (excluding Epstein–​Barr virus) J.G.P. Sissons† ESSENTIALS Eight human herpesviruses, all with a linear double-​stranded DNA genome and divided into α-​, β-​, and γ-​subfamilies on the basis of genomic and biological properties, share the capacity to produce latent infection. The diseases they cause may result from primary infection, or reactivation of the virus from latency, and tend to be more severe in immunosuppressed patients. Diagnosis of the various herpesvirus infections may be made on clinical grounds alone, by culture or demonstration of viral particles by electron microscopy of relevant samples, by serological testing, or now more routinely by polymerase chain reaction-​based tests. Herpes simplex viruses (HSV) These two α-​herpesviruses infect epithelial cells and become latent in the central nervous system. (1) HSV-​1—​transmitted by direct contact with infected secretions from a carrier; predomin- antly causes orofacial infections; becomes latent in the trigeminal ganglion; reactivation may give rise to recurrent orolabial mu- cosal ulcers (‘cold sores’) on the lips or skin around the mouth; is the commonest identified cause of acute sporadic encephalitis occurring in immunocompetent subjects in Western countries. (2)  HSV-​2—​usually acquired through sexual contact and is the predominant cause of genital HSV infection, which may also be recurrent. Treatment of both HSV-​1 and HSV-​2 is with aciclovir, which is preferentially phosphorylated in HSV-​infected cells, or other newer related drugs (famciclovir and valaciclovir). Oral treatment is used in immunocompetent patients, but intravenous therapy is indi- cated in severe infections, encephalitis, and in immunosuppressed patients. Varicella-​zoster virus (VZV) This α-​herpesvirus is presumed to spread by the respiratory route and, after an incubation period of 10–​20 days, causes varicella (chickenpox), predominantly an exanthematous disease of child- hood, but which may be complicated in adults by pneumonitis and encephalitis. The virus becomes latent in dorsal root ganglia after primary infection, whence it can reactivate to cause herpes zoster (shingles), with pain, erythema, and vesicular lesions occurring in a dermatomal distribution, particularly in elderly and immunosup- pressed individuals. Treatment of severe varicella or herpes zoster is with aciclovir, with higher doses being required than for HSV. A live attenuated VZV vaccine is available: this induces 90% pro- tection from natural varicella in children, and also diminishes the incidence of zoster and postherpetic neuralgia when given to older age groups. Human cytomegalovirus (HCMV) This β-​herpesvirus is the largest human herpesvirus. Infection is spread by close contact with body fluids of infected individ- uals: from 50 to 100% of adults are seropositive, depending on socioeconomic and sexual risk, with myeloid lineage cells being a major site of HCMV latency. Primary infection in children and adults is usually asymptomatic, but infectious mononucleosis clinically indistinguishable from that caused by primary Epstein–​Barr virus infection can be produced (see Chapter 8.5.3), and HCMV can pro- duce severe disease in two particular situations. (1) Fetal infection—​ congenital HCMV infection occurs in around 0.5 to 1% of live births in developed countries; most infected babies are asymptomatic, but classical ‘cytomegalic inclusion disease’ has a high mortality † It is with great regret that we report that J.G.P. Sissons died on 25 September, 2016.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 735 and surviving infants have mental, visual, and hearing impairment. (2) Infection in patients who are immunosuppressed patients—​the most serious forms are pneumonitis in bone marrow transplant re- cipients and retinitis in HIV/​AIDS patients. Specific treatment for HCMV is usually with ganciclovir, which requires intravenous ad- ministration and has limiting side effects including myelotoxicity; valganciclovir has higher oral bioavailability and is particularly used for prophylaxis. Human herpesvirus 6 and 7 (HHV-​6 and 7) These are β-​herpesviruses, genetically related to HCMV and most probably transmitted via maternal saliva. Unlike any other herpes- virus family members, HHV-​6 genome DNA can be found cova- lently integrated into the cell chromosomes in about 1% of the population. Primary infection with HHV-​6 in young children is associated with roseola infantum (exanthem subitum, sixth disease), and also with a febrile illness without rash. More than 90% of children are seroposi- tive for HHV-​6 by 2 years of age. HHV-​6 reactivation may occur in immunosuppressed solid organ and bone marrow transplant recipi- ents, but it is not clear that HHV-​6 causes disease in these patients. HHV-​6 sensitivity to antiviral drugs corresponds with that of HCMV, but no treatment is usually required. HHV-​7 has been associated with some cases of roseola, but there is no other evidence for its being pathogenic. Human herpesvirus-​8 (HHV-​8) This member of the rhadinovirus (γ 2-​herpesvirus) family is the most recently discovered human herpesviruses, having been iso- lated from Kaposi’s sarcoma tissue in 1994. The mechanism of transmission is probably by saliva and sexual contact; reported seroprevalence is around 50% or more in many African adult popu- lations, but 5% or less in blood donors in the United Kingdom and the United States of America, with intermediate rates in Italy and other Mediterranean countries. HHV-​8 (as other γ-​herpesviruses such as Epstein–​Barr) is potentially oncogenic:  it is clearly associated with (1)  Kaposi’s sarcoma—​HHV-​8 can be detected by PCR in the blood of nearly all cases. Manifests clinically as purplish-​brown macules, papules and
plaques, and is described in four clinical settings: (a) the classic form—​typically presents in elderly Mediterranean or Jewish men with lesions on the extremities and an indolent course; (b)  the endemic African form—​accounts for 10% of cancer in equatorial Africa and is similar clinically to the classic form of the disease; (c)  in patients with immunodeficiency states such as transplant recipients—​lesions are more widespread and rapidly progressive, but visceral involvement is unusual; and (d) the AIDS-​associated form—​with widespread cutaneous lesions, involvement of the oral mucosa, visceral lesions in the lungs or gastrointestinal tract, and sometimes rapid progression. Kaposi’s sarcoma lesions may regress with antiretroviral treatment, withdrawal of immuno- suppression, and the disease can also be treated with radiation therapy and (for widespread cutaneous or visceral disease) with chemotherapy. (2)  Primary effusion lymphoma—​HHV-​8 is pre- sent in the tumour cells of all cases of this rare and aggressive type of B-​cell lymphoma that presents in patients with AIDS. (3) Multicentric Castleman’s disease (angiofollicular lymph node hyperplasia)—​HHV-​8 is present in most cases of this condition, es- pecially those associated with HIV. Cercopithecine herpesvirus 1 This α-​herpesvirus (formerly named herpes B virus) is closely re- lated to HSV, and found in Old World monkeys, its natural host. Transmission to humans from monkey bites results in a high inci- dence of severe disease, with progressive encephalitis. Treatment is with aciclovir or ganciclovir, but morbidity and mortality are high. Human herpesviruses The Herpesviridae family is widely distributed in the animal kingdom. More than 100 have been isolated from humans, pri- mates, and other mammals, and from reptiles and fish. Comparative sequence analysis suggests they have been coevolving with their in- dividual hosts for millions of years. Eight human herpesviruses have been identified to date (Table 8.5.2.1). Shared genomic and bio- logical properties divide the herpesviruses into three subfamilies, the α-​, β-​, and γ-​herpesvirinae. All the herpesviruses have a linear double-​stranded DNA genome contained inside an icosahedral capsid that is surrounded by a protein tegument. The outer lipid envelope contains virus glycoprotein spikes. These large viruses have genomes consisting of unique segments of DNA flanked by inverted repeats, and en- code most of the proteins needed for replication. All herpesviruses share an important biological feature, their capacity to produce la- tent infection in their natural host, during which the viral genome persists in cells, usually as a closed circle (episome), expressing only a limited subset of virus genes. This property results in their ability to produce lifelong infection in different types of cell, depending on the individual virus, and thus to persist in the popu- lation. Herpesvirus disease may result from primary infection, or reactivation of the virus from latency, and tends to be more severe in immunosuppressed patients. The γ-herpesviruses can induce cell transformation, and are associated with specific tumours. Herpes simplex virus infections Historical background ‘Herpes’ derives from the Greek, meaning to creep or crawl, ap- parently used since antiquity to describe the evolution of the skin lesions caused by herpes simplex virus (HSV) and varicella-​zoster virus. HSV was the first of the herpesviruses to be isolated, in the 1930s, although the transmission of infection to animals had been demonstrated in 1919. The serological distinction between the two types, HSV-​1 and HSV-​2, and the association of HSV-​2 with genital herpes, was made in the 1960s. HSVs are now some of the most in- tensively studied human viruses. Aetiology HSV has a genome size of 150 kbp, and codes for about 80 pro- teins. The genomes of HSV-​1 and HSV-​2 are largely colinear, but

736 section 8  Infectious diseases have only about 50% genomic homology. Gene expression occurs in three temporally regulated phases: immediate-​early, early, and late. Immediate-​early proteins are largely regulatory proteins that prepare the cell to produce further virus. The early genes code particularly for enzymes involved in the replication of virus DNA, and the late genes for the structural proteins of the virion. Antigenic differences in the surface glycoprotein G are used to distinguish between HSV-​1 and HSV-​2. The release of progeny virus is normally accompanied by cell death (i.e. the infection is lytic). The virus infects a relatively wide range of cells in vitro, and can also infect experimental animals, allowing studies of its pathogenesis. Epidemiology HSV is a ubiquitous virus, widely distributed in populations throughout the world. Although animals can be infected experi- mentally, there are no natural animal hosts, and humans are the only reservoir. Transmission occurs when a susceptible person has direct contact with infected secretions from an HSV carrier, usually from oral, genital, or skin lesions, to mucous membranes or abraded skin of the recipient. HSV carriers can excrete virus asymptomatically, and 1–​15% of adult carriers excrete HSV at any one time. Conventionally, the prevalence of infection is assessed by demonstrating antibody to HSV-​1 or HSV-​2. The prevalence of HSV-​1 increases with age, although the time of acquisition of HSV-​1 antibody varies depending on socioeconomic factors. Seroprevalence in early life is higher among lower socioeconomic groups, 70–​90% of children having antibodies by the age of 10, whereas only about 30% of children in higher socioeconomic groups have antibodies by this time. By mid-​life, 80–​90% of people are HSV-​1 seropositive. HSV-​2 infection is usually acquired through sexual contact; consequently, seroconversion correlates with the onset of sexual activity, and a progressive increase in seroprevalence to HSV-​2 begins in adolescence. The number of sexual contacts is a major risk factor for the acquisition of HSV-​2. Cumulative seropreva- lence rates in adults vary from 10 to 80%, depending on the popu- lation and risk factors. HSV can be transmitted to neonates by infection (usually HSV-​2) from maternal genital secretions at the time of delivery. The mothers are most often asymptomatic excretors of the virus who have no his- tory of genital herpes. Pathogenesis HSV infects and replicates in epithelial cells at the site of inocula- tion onto mucous membranes or abraded skin, with an incubation period of 4–​6 days before clinical lesions appear. There is a marked local inflammatory response, but viraemia and dissemination may occur in the immunocompromised host. Following local epithelial replication, HSV enters the peripheral sensory nerves innervating the site of replication, and ascends the axons by retrograde trans- port to reach the dorsal root ganglia, or the trigeminal ganglion in the case of oral or conjunctival inoculation. The virus then becomes latent in the sensory ganglia, but despite extensive study, the mech- anism of virus latency remains uncertain. Latent HSV DNA is largely in an inactive state, with minimal gene expression. RNA species called latency-​associated transcripts, and their processed microRNAs (miRNAs), are the only detectable tran- scripts. These have no detectable protein product, and their dele- tion from the genome does not prevent the establishment of latency, although reactivation is impaired. These transcripts are believed to help globally suppress viral gene expression to maintain latency. Latent HSV is carried for the lifetime of the host, but may be re- activated in response to certain stimuli, including stress, menstru- ation, ultraviolet light, and immunosuppression. Upon reactivation, infectious virus is produced, travels down the peripheral nerves by anterograde axonal transport, and replicates in the epithelial cells at the nerve ending. The neuronal latency of HSV and varicella-​zoster virus is an ex- tremely effective method of virus persistence. Latent virus in neur- onal cells appears to be inaccessible to the immune response, and as it does not replicate is not susceptible to the action of antiviral drugs. In normal HSV carriers, reactivation at local sites is thought to be controlled by a specific effector T-​lymphocyte response. However, HSV DNA encodes proteins that interfere with antigen processing by the class I MHC pathway, and are presumed to help the virus evade the T-​cell immune response. There is no good evidence that the immune response to HSV of people who have symptomatic re- activation episodes differs from that of asymptomatic carriers. Clinical features Primary infection with HSV is often asymptomatic; among sexu- ally active subjects, only 60% of primary infections with HSV-​1, and 40% with HSV-​2, are symptomatic. HSV-​1 is the predominant cause Table 8.5.2.1  The human herpesviruses Common name Designation Subfamily Genome size (kbp) Site of latency and persistence Herpes simplex virus 1 Human herpesvirus 1 α 152 Neurons (sensory ganglia) Herpes simplex virus 2 Human herpesvirus 2 α 152 Neurons (sensory ganglia) Varicella zoster virus Human herpesvirus 3 α 125 Neurons (sensory ganglia) Epstein–​Barr virus Human herpesvirus 4 γ 172 B lymphocytes (oropharyngeal epithelium) Human cytomegalovirus Human herpesvirus 5 β 235 Blood monocytes (and possibly epithelial cells) HHV6 Human herpesvirus 6A and 6B β 170 Monocytes, T lymphocytes HHV7 Human herpesvirus 7 β 145 –​ Kaposi’s sarcoma-​associated herpesvirus Human herpesvirus 8 γ 230 Uncertain

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 737 of orofacial infections, whereas HSV-​2 is the usual cause of genital HSV infection, but the clinical manifestations overlap. Gingivostomatitis This is the most common clinical form of primary infection with HSV-​1. It is most often seen in children, following an incubation period of 2–​12 days. Primary infection may be associated with a con- siderable systemic reaction, involving fever, sore throat, pharyngeal oedema, and redness. Painful vesicles appear a few days later on the pharynx and oral mucosa, the lips, and the skin around the mouth (Fig. 8.5.2.1). There may be cervical lymphadenopathy. Affected pa- tients may have difficulty in eating, and the lesions last from 3 days to 2 weeks. The differential diagnosis includes other causes of pha- ryngitis, including bacterial pharyngitis and herpangina (from Coxsackie A virus infection). Anterior vesicles and ulceration af- fecting the lips and skin around the mouth are more suggestive of HSV infection. Stevens–​Johnson syndrome and severe aphthous ul- ceration may appear similar, and staphylococcal impetigo affects the skin around the mouth, but is not associated with oral ulceration. Reactivation of HSV may give rise to recurrent orolabial lesions, appearing as intraoral mucosal ulcers, but more frequently as the classical cold sore on the lips or skin around the mouth. A tingling sensation in the area of impending ulceration may precede the ap- pearance of vesicles by 1 to 2 days. The lesions usually recur at the same site in individual patients. Around 25% of HSV-​1 seroposi- tive people develop recurrent orolabial lesions. The majority have only one or two reactivation episodes per year, although a minority (<10%) have more than one attack per month. The episodes are not associated with systemic symptoms, and diagnosis is usually straightforward. Infection at other cutaneous sites Herpetic whitlow HSV infection of the finger, herpetic whitlow, may complicate pri- mary oral or genital herpes by autoinoculation of virus, or may occur through occupational exposure (e.g. in nursing, medical, and dental staff). There is oedema, erythema, and local tenderness of the infected finger. Lesions at the fingertip may be confused with pyo- genic bacterial paronychias and incised, which is contraindicated for herpetic whitlow, and may even spread infection. Herpes gladiatorum This is mucocutaneous HSV infection occurring by transmission of virus via skin trauma resulting from wrestling or other contact sports. Eczema herpeticum HSV infections of the skin are more severe in patients with pre-​ existing skin disease. In patients with eczema, burns, or other blis- tering skin diseases, HSV infection may become disseminated. Cutaneous HSV infection can be confused with herpes zoster, although the latter is usually easy to diagnose by its unilateral dermatomal distribution. Herpes simplex and erythema multiforme About 15% of all cases of erythema multiforme are preceded by a symptomatic attack of recurrent herpes simplex, and in susceptible people the characteristic rash can be induced by the intradermal in- oculation of inactivated herpes simplex virus antigen. The rash of erythema multiforme starts several days after the onset of the her- petic vesicles, and in severe cases can involve the mucous mem- branes (Stevens–​Johnson syndrome). The frequency of these attacks can be reduced by aciclovir prophylaxis. Keratitis HSV keratitis is characterized by the acute onset of pain, blurred vi- sion, conjunctival injection, and dendritic ulceration of the cornea (Fig. 8.5.2.2). It can cause corneal blindness, and treatment is ur- gent. Topical aciclovir is the drug of choice; topical steroids may make the infection worse. HSV can also cause an acute necrotizing retinitis, usually only seen in immunosuppressed people, including those with HIV infection. Genital herpes Primary genital HSV infection is sexually transmitted, and may be associated with systemic symptoms such as fever, headache, and myalgias. Symptoms tend to be more severe in women than men. There is local pain and itching, dysuria, vaginal discharge, and in- guinal lymphadenopathy, with vesicles and ulcers on the vulva, peri- neum, vagina, and cervix, and sometimes on the skin of the buttocks (Fig. 8.5.2.3). In males, primary HSV lesions are vesicles on the shaft or glans of the penis, and there may be associated urethritis. HSV-​2 causes most genital infections, with a variable smaller pro- portion resulting from HSV-​1. Only 40% of primary HSV-​2 genital infections are symptomatic. In patients who have had prior HSV-​1 infection, the symptoms of primary genital herpes tend to be less severe. HSV has been isolated from the urethra in 5% of women with urethral syndrome, in the absence of obvious genital lesions. (a) (b) Fig. 8.5.2.1  Herpes simplex gingivostomatitis: (a) and (b).

738 section 8  Infectious diseases Other manifestations of genital tract disease resulting from primary HSV infection are, rarely, endometritis and salpingitis in women, and prostatitis in men. HSV proctitis may follow rectal intercourse. There is anorectal pain and discharge, with ulcerative lesions visible on sigmoidos- copy. Perianal lesions are seen in immunosuppressed patients, and spreading perianal HSV infection and HSV proctitis occur in HIV-​ infected patients. Recurrent genital herpes is frequent in the first year after primary genital disease (90% for HSV-​2 and 55% for HSV-​1). Thereafter, the recurrence rate tends to decrease with time, to around three to four attacks per year for HSV-​2, but fewer for HSV-​1. Severe recurrent genital herpes is particularly troublesome to women. The complications of primary genital HSV infection include sa- cral radiculomyelitis, with urinary retention and hyperaesthesia of the perineal area, which usually resolves over several weeks. Aseptic meningitis requiring admission to hospital occurs in up to 7% of women and 2% of men, although suggestive symptoms are more common. Occasionally, and more seriously, transverse myelitis may occur. HSV encephalitis (See also Chapter 24.11.2.) Encephalitis is the most serious type of disease produced by HSV in the immunocompetent host, and has an estimated annual inci- dence of two to three cases per million. It is the most commonly iden- tified cause of acute sporadic encephalitis in Western countries. The great majority of cases are caused by HSV-​1. A biphasic age incidence is reported, with higher rates between the ages of 5 and 30 years, and in those older than 50 years. The clinical features are of focal enceph- alitis, with acute onset of fever, confusion, and unusual behaviour, impaired consciousness, and possibly focal neurological abnormal- ities. However, there are no specific features, and the diagnosis of HSV should be considered in any patient with possible encephalitis. The cerebrospinal fluid shows lymphocytic pleocytosis, although neutrophils and red cells may also be present, with a raised protein level. CT scans of the brain may show changes in the temporal lobe; MRI is a more sensitive method of detection. The electroenceph- alogram classically shows spike and slow-​wave activity localized in the temporal lobes. The definitive way of establishing the diagnosis is brain biopsy. In the original trial of aciclovir for the treatment of HSV encephalitis, brain biopsy was an entry criterion, but confirmed the diagnosis in only 50% of clinically suspected cases. Since the ad- vent of effective non​toxic chemotherapy for HSV, brain biopsy is very rarely used. There is good correlation between a positive poly- merase chain reaction (PCR) test for HSV DNA in the cerebrospinal fluid, and a diagnosis of HSV encephalitis by brain biopsy and virus isolation. Evidence of intrathecal production of specific HSV anti- body is also diagnostic, but as it is usually not detectable until 1 week after onset, PCR-​based diagnosis is more useful. Serum or cerebro- spinal fluid titres of antibodies to HSV do not usually increase in the first week of the illness. In practice, the diagnosis is established by a compatible clinical picture, evidence of characteristic temporal lobe involvement on CT or MRI, and electroencephalogram (EEG), and by PCR-​based detection of HSV DNA in the cerebrospinal fluid. The pathological features are of focal haemorrhagic necrotizing encephalitis affecting the temporal lobes. The pathogenesis of HSV encephalitis remains uncertain. Up to one-​half of patients have Fig. 8.5.2.3  Genital herpes in the natal cleft. Courtesy of the late Dr B. E. Juel-​Jensen. (a) (b) Fig. 8.5.2.2  Herpes simplex keratitis: (a) disciform, and (b) dendritic. Courtesy of the late Dr B. E. Juel-​Jensen.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 739 primary infection, and in the rest the disease is presumed to result from reactivation. However, where HSV has been isolated from the brain and mouth simultaneously in the same patient, the two isolates differ by restriction endo nuclease analysis in about 30% of cases, suggesting a new exogenous virus infection in an already seroposi- tive patient. HSV DNA can be detected at autopsy in the brains of normal virus carriers, and the factors precipitating HSV encephal- itis are not known. Immunosuppression is not usually associated with HSV encephalitis, which predominantly affects apparently immunocompetent adults, and very rarely patients with advanced HIV infection. However, rare mutations affecting the Toll-​like re- ceptor 3 signalling pathway causing autosomal recessive UNC-​93B and TLR3 deficiencies and autosomal dominant TLR3 and TRAF3 deficiencies have been associated with primary HSV encephalitis in children. Treatment with intravenous aciclovir should be started immedi- ately if HSV encephalitis is clinically suspected, without waiting for confirmation of the diagnosis (in doses as described next; see ‘CNS infections’). The untreated mortality from HSV encephalitis is more than 70%, and very few survivors make a full neurological recovery. Intravenous aciclovir was established to be more effective than the previous best therapy of vidarabine in a randomized trial reported in 1986. Mortality in the aciclovir-​treated group was 28%, although a lower Glasgow coma score on entry carried a higher risk of mor- tality. However, only 38% of those who received aciclovir had fully recovered at 6 months. There is still a high incidence of permanent neurological sequelae, particularly seizures, defects of memory, and personality changes, and the prognosis of HSV encephalitis remains poor. Meningitis HSV can cause aseptic meningitis, which is quite independent of, and not associated with progression to, HSV encephalitis. It is most commonly associated with primary genital HSV-​2 infection, in which the incidence of proven HSV meningitis is 7% in women and 2% in men. There is pleocytosis, usually lymphocytic, but neu- trophils may predominate in early meningitis. HSV may be isolated from the cerebrospinal fluid by culture, but is now more reliably de- tected by PCR for HSV DNA. In a high proportion of patients with Mollaret’s meningitis (recurrent aseptic meningitis of unknown aetiology; Chapter 24.11.2), HSV DNA is reported to be detectable in the cerebrospinal fluid by PCR. The role of HSV in this syndrome remains uncertain. Neonatal HSV infection and pregnancy The incidence of neonatal HSV infection is approximately 1 in 3500 deliveries per annum in the United States of America, but appears to be lower in the United Kingdom, at 1 in 6600 live births. About 70% of cases are caused by HSV-​2, and result from fetal acquisition of HSV-​2 from maternal genital secretions during delivery. Most infants with neonatal HSV are born to mothers without clinically evident HSV infection. The risk of transmission from women with symptomatic primary HSV or clinically evident recurrent HSV-​ 2 infection is about 50 and 20%, respectively. A small proportion (c.10%) of infections is acquired postnatally through contact with people with active lesions. Neonatal HSV infection may appear as lesions on the skin, eye, and mouth, or as encephalitis or disseminated visceral infection. Although initial superficial infection may progress to visceral infec- tion, visceral infection can present without cutaneous lesions, and the diagnosis should be considered in severely ill neonates. Untreated, visceral infection has a high mortality (around 60%). Primary infec- tion in early pregnancy can lead to congenital HSV infection, which is rare, but can produce serious congenital abnormalities. HSV in immunosuppressed patients HSV infections in immunosuppressed people are usually because of reactivation, rather than primary infection. They tend to be more severe, are more likely to progress, and take longer to heal than in the immunocompetent host. Clinical manifestations in patients with HIV infection include severe perineal, orofacial, and oesophageal infection. HSV pneumonitis, hepatitis, and colitis are also described in immunosuppressed patients. Pathology The histological appearance of HSV infection remains the same, whether it is primary or recurrent. There is ballooning of infected cells, with condensed chromatin in the cell nuclei; intranuclear inclusion bodies (Cowdry type A  bodies) may be seen; and multinucleated giant cells form. Varicella-​zoster virus produces a similar appearance. Laboratory diagnosis Definitive diagnosis is made by virus isolation. Swabs from ves- icular fluid or other body fluids in virus transport medium can be inoculated into tissue culture, producing typical cytopathic effects. Electron microscopy of negatively stained vesicle fluid is rapid, but will not differentiate HSV from varicella-​zoster virus. The use of PCR-​based techniques to detect viral DNA is becoming more wide- spread. It is particularly applicable to the detection of HSV DNA in cerebrospinal fluid. Serological tests for antibody to HSV are useful only for making a retrospective diagnosis. Seroconversion provides proof of primary infection, and the absence of antibody to HSV-​1 or HSV-​2 rules out a diagnosis of recurrent HSV infection. However, making a diag- nosis of reactivation by demonstrating rising antibody titres is of limited value. Treatment The introduction of aciclovir heralded a new era of specific anti- viral drugs, and superseded the drugs previously used for the treatment of HSV infections, such as vidarabine and idoxuridine. Aciclovir is an acyclic nucleoside that is preferentially phosphor- ylated to the monophosphate in HSV-​infected cells by the virus-​ encoded thymidine kinase. Cellular kinases then phosphorylate the monophosphate to the triphosphate, which is incorporated into nas- cent HSV DNA, where it acts as a chain terminator; aciclovir also directly inactivates the HSV DNA polymerase. Two newer, related drugs with the same mechanism of action are famciclovir, a prodrug of penciclovir, and valaciclovir, the valyl ester of aciclovir, which has greater bioavailability and less frequent dosage. All these drugs are relatively free of side effects, although intravenous aciclovir can crystallize in the renal parenchyma and produce renal impairment; it should be given by infusion over an hour, and patients should be adequately hydrated. The doses should be reduced in patients with renal impairment.

740 section 8  Infectious diseases Primary mucocutaneous infection In primary oral and genital infection, aciclovir 200 mg 5 times daily given orally for 10 to 14 days from the onset reduces the severity of infection, the duration of symptoms, and the duration of viral shed- ding. There is little evidence that the treatment of primary infec- tion reduces the incidence of subsequent symptomatic reactivation episodes. If swallowing is difficult, intravenous aciclovir (5 mg/​kg 8 hourly) may need to be given. Famciclovir 250 mg 3 times daily or valaciclovir 500 mg twice daily are alternatives. Symptomatic reactivation of mucocutaneous infection The treatment of recurrent infections in immunocompetent hosts is often unnecessary, as the symptoms are usually very mild. However, aciclovir can shorten the duration of symptoms if it is given very early in the course of the recurrence, prefer- ably during the prodrome before vesicles appear. Oral aciclovir is effective, and anecdotal reports suggest that topical aciclovir is effective symptomatically. The same dosage as desribed for pri- mary infection can be given for 5 days. Patient-​initiated courses of single-​day famciclovir (1 g twice daily) or 3-​day valaciclovir (500 mg twice daily) have been shown to be effective for recur- rent genital HSV. Long-​term suppressive therapy This can be considered in immunocompetent patients with genital herpes who have frequent reactivation episodes. Trials of aciclovir in recurrent genital herpes have shown that a dose of 400 mg twice daily significantly reduces the frequency of attacks. However, pa- tients may be able to find a lower effective dose, and in some, 200 mg daily prevents attacks. Because there is some evidence that resistant virus is a problem in this population, it is advisable to stop treat- ment for a month every 6 to 12 months. Valaciclovir 500 mg daily or famciclovir 250 mg twice daily are alternatives. CNS infections For HSV encephalitis, intravenous aciclovir (10 mg/​kg 8 hourly for 14–​21 days) should be given to any patient in whom the diagnosis is clinically suspected (see ‘HSV encephalitis’ earlier). For HSV men- ingitis, intravenous aciclovir 5 mg/​kg 8 hourly can be used, with conversion to oral valaciclovir 1 g twice daily when improvement occurs, for a total of 10 days. Systemic infection in the immunosuppressed Oral treatment, as for primary HSV, can be used for mild mucocutaneous infection, but for more severe and for visceral in- volvement, intravenous aciclovir 5 mg/​kg 8 hourly should be used. After resolution, continued prophylaxis is usually necessary until immunocompetence is restored, particularly in patients with HIV. Aciclovir resistance Resistance of HSV to aciclovir develops readily in vitro, but is clinically rare; it results from mutations in the HSV thymidine kinase or DNA polymerase genes. It is seen almost exclusively in immunocompromised patients who have received prolonged aciclovir prophylaxis, especially those with HIV infection, and is manifest as unresponsive or worsening HSV disease despite treat- ment with aciclovir. There is usually cross-​resistance to famciclovir and valaciclovir, and intravenous foscarnet is the most useful alternative drug in severe infection caused by resistant HSV, al- though it is more usually used for human cytomegalovirus (see ‘Human cytomegalovirus’, next). Prevention and control No vaccine is licensed for HSV, although a gD (glycoprotein D) based vaccine reduced new HSV2 infection in seronegative women, and other candidates are approaching phase III trials. There is par- ticular interest in the use of vaccines for postinfective immunization to reduce the frequency of recurrent genital HSV attacks. This has proved possible in guinea pigs. Special problems in pregnant women Prevention of neonatal HSV infection is best achieved by preventing genital HSV infection late in pregnancy. There is no reason to give aciclovir prophylactically to women with a history of recurrent genital herpes who are asymptomatic, as the incidence of neonatal HSV infection is low in their children. However, women with clin- ically apparent genital herpes in the last trimester (and probably at any other time in pregnancy) can be treated with aciclovir, although the drug is not licensed for treatment in pregnancy. Women with no clinical lesions may have a vaginal delivery, but the presence of active lesions at the time of labour is an indication for Caesarean section. Babies born to mothers with clinically apparent genital HSV infec- tion, or with a history of recurrent genital HSV infection, should be screened for HSV by cultures from the nasopharynx and eyes after birth. Proven neonatal HSV infection should be treated with high-​dose intravenous aciclovir (20 mg/​kg per day every 8 h for 21 days). Varicella-​zoster virus infection Historical background There are clinical descriptions of varicella (chickenpox) and herpes zoster (shingles) in very early medical literature, although the skin lesions of herpes simplex and herpes zoster were grouped together under the term herpes. The similarities between the exanthematous rashes associated with smallpox and varicella meant they were not distinguished until the late 19th century. The characteristic clin- ical appearance of shingles, in a dermatomal distribution, was rec- ognized as a discrete entity in the early Greek literature. The term zoster is derived from the Greek word for a girdle, and shingles from the Latin cingere meaning to encircle. In 1892 von Bocquet observed that children developed vari- cella after contact with adults with herpes zoster, and in 1925 it was shown that vesicular fluid from patients with zoster, inoculated into susceptible people, produced chickenpox. The idea that zoster re- sulted from the reactivation of latent virus remaining in the tissues following childhood varicella was put forward by Garland in 1943, and strengthened by the work of Hope-​Simpson, a British general practitioner. Varicella-​zoster virus (VZV) was isolated in 1958, and Weller and colleagues showed the similarity between viral isolates from varicella and zoster patients. Restriction endonuclease ana- lysis showed that the isolates from chickenpox and from later zoster in the same immunocompromised patient were identical. The long interval between the two illnesses has prevented such studies in im- munocompetent people.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 741 Aetiology VZV is structurally similar to other members of the herpesvirus family. The genome is a linear double-​stranded DNA of 125 kbp. VZV is an α-​herpesvirus, and encodes sets of genes that are largely colinear to those of HSV, and are also expressed in immediate-​early, early, and late phases. The virus is closely cell associated, and spreads from cell to cell in tissue culture. Epidemiology VZV infects only humans, which are thus the only reservoir. The virus is presumed to spread by the respiratory route. Varicella is pre- dominantly a disease of childhood, affecting both sexes; and 90% of cases occur in children under the age of 13 years. The incubation period is about 2 weeks (with a range of 10–​20 days); patients are infectious for about 48 h before the vesicles appear, and remain so for 4 to 5 days afterwards, until all the vesicles have crusted over. The secondary attack rate in susceptible contacts with an index case in the household is 70 to 90%. The prevalence of VZV varies in dif- ferent ethnic groups. In Europe, about 10% of the population over 15 years old is seronegative, and consequently susceptible to infec- tion, although in tropical countries only 50% of young adults may be seropositive. Varicella in adults is uncommon in Europe, and less than 2% of all cases occur in patients older than 20 years. Subclinical infection is unusual, and accounts for less than 5% of all infections, but the disease may be mild, and in some surveys only 10% of people with a negative history were in fact seronegative for VZV. One attack of chickenpox usually confers lifelong immunity. After primary infection, VZV becomes latent in dorsal root ganglia. Reactivation appears clinically as herpes zoster, which is a common disease affecting all age groups, but particularly older and immunosuppressed people; about 20% of the population will ex- perience an attack. There is no evidence that exposure to people with active VZV infection predisposes to herpes zoster in their contacts, but a seronegative person may catch varicella from contact with the vesicles of a patient with shingles. Nosocomial varicella infection is well recognized, and the isolation of patients with varicella, and im- munocompromised patients with herpes zoster, should be ensured in hospitals. Local unidermatomal zoster is less likely to cause infec- tion, and consequently to need isolation. Pathogenesis During primary infection, initial virus replication probably occurs in the epithelial cells of the upper respiratory tract mucosa, followed by a phase of viraemia during which VZV can be isolated from leucocytes, and the disseminated rash appears. In the skin, the virus infects capillary endothelial cells, and adjacent fibroblasts and epi- thelial cells. During the viraemic phase, virus may spread to visceral organs, including alveolar epithelial cells, and transient subclinical hepatitis is probably a normal feature of varicella. VZV encephal- itis may be a feature of primary infection, particularly affecting the cerebellum. Patients usually recover completely from encephalitis (unlike that associated with HSV), and it has been suggested its pathogenesis may be immune mediated. Following recovery from primary infection, the virus persists for life in a latent state in dorsal root ganglia. VZV reaches the ganglia by retrograde axonal trans- port from the skin lesions during primary infection, and all dorsal root ganglia and the trigeminal ganglion can potentially carry latent VZV in neurones and possibly in satellite cells. As with other herpesviruses, the host response is critical in con- taining the initial infection. Cellular immunity is important, since varicella may be progressive in patients with severely impaired T-​ cell immunity. Both CD4 and CD8 cytotoxic T lymphocytes spe- cific for VZV are present in normal people carrying latent VZV. The cellular immune response presumably plays a part in controlling reactivation, since impaired T-​cell immunity increases the risk of developing zoster, and of having vesicles in multiple dermatomes, and cutaneous dissemination of reactivated virus. The increasing incidence of herpes zoster with age may reflect waning cellular im- munity to VZV. Clinical features Primary infection and varicella The most striking feature of varicella is the rash, which is centripetal (mainly on the trunk). The lesions are initially present on the face and scalp, before progressing to the trunk and later to the limbs (Fig. 8.5.2.4). A macular erythematous rash, papules, and vesicles may all be present together. Individual lesions progress from being papules to vesicles to pustules, and then crust over. The scabs nor- mally separate after 10 days, without scarring. The systemic symp- toms associated with varicella vary considerably. In most children there is a mild illness with fever. Adults characteristically have a more severe illness, with myalgia, headache, arthralgia, malaise, and higher fever, with the complications listed next. Symptoms may precede the rash by 1 to 2 days. (a) (b) Fig. 8.5.2.4  Severe chickenpox: (a) and (b). Copyright D. A. Warrell.

742 section 8  Infectious diseases Complications of varicella The principal complications of varicella in immunocompetent pa- tients are pneumonitis and encephalitis. Pneumonitis  In a prospective study, 6% of young adults with chickenpox had respiratory symptoms, although 16% had changes on chest radiography, but the rate of admission to hospital for pneumonia in adults with varicella is only about 0.3%. Patients present with dyspnoea, cough, hypoxia, and bilateral infiltrates on the chest radiograph, occurring 1 to 6 days after the appearance of the rash. Hypoxia may be more severe than expected from the physical signs or the chest radiograph. The interstitial pneumon- itis can progress to respiratory failure requiring artificial ventila- tion and intensive care (Fig. 8.5.2.4a), but it is more commonly transient, resolving completely within 2 to 3 days. Varicella pneu- monia is said to be more common in smokers. Fatalities are rare, and VZV pneumonia is not associated with long-​term respiratory problems. Benign nodular calcification throughout the lung occa- sionally follows. Encephalitis  Central nervous system involvement during varicella most commonly presents as acute cerebellar ataxia within 1 week of onset of the rash, although it may appear up to 21 days after the rash. It resolves completely over 2 to 4 weeks. A frequency of 1 in 4000 children aged less than 15 years has been quoted. The cerebrospinal fluid of these patients shows lymphocytosis and elevated protein concentration. More serious encephalitis can occur in 0.1 to 0.2% of cases of varicella. This begins earlier in the course of infection than cere- bellar ataxia, with headache, vomiting, confusion, and impaired consciousness. There is evidence of diffuse cerebral oedema, but no defined pattern of CT or MRI abnormality. The encephalitis may be progressive, and the mortality is between 5 and 20%, with neuro- logical sequelae in up to 1% of survivors. Varicella meningitis can occur. Other rarely reported neuro- logical complications include optic neuritis, transverse myelitis, and Reye’s syndrome. Other complications  Primary VZV infection may be compli- cated by acute thrombocytopenia, with petechiae, purpura, haem- orrhage into vesicles, and other haemorrhagic manifestations. The platelet count can remain low for weeks after the illness has resolved. Secondary infection of the skin lesions with Staphylococcus aureus or Streptococcus pyogenes may occur. Purpura fulminans is a rare complication associated with arterial thrombosis and haemorrhagic gangrene (Fig. 8.5.2.5). Nephritis and arthritis have been reported as occasional complications, and myocarditis, pericarditis, pancrea- titis, and orchitis are even more rare. Special problems in  pregnant women  Varicella in pregnant women can be severe, with a maternal mortality of 1%. Varicella in the first trimester can cause varicella embryopathy. Affected infants may have a scarred, atrophic limb, microcephaly, cortical atrophy, and eye defects including chorioretinitis, microophthalmia, and cataracts. The autonomic nervous system may be damaged. Varicella embryopathy is rare; in recent reported series the risk was about 1 to 2% in mothers with varicella in the first 20 weeks of pregnancy. Varicella-​zoster immunoglobulin should be considered for pregnant women in contact with varicella, and varicella in pregnancy should be treated with aciclovir on a named-​patient basis. Neonatal vari- cella occurs in babies whose mothers contract varicella just before or after delivery, and is most severe when maternal disease appears from 2 to 7 days after delivery. Herpes zoster The clinical syndrome caused by the reactivation of VZV from sen- sory ganglia is herpes zoster. Typical prodromal localized pain or paraesthesia is followed by erythema and vesicular lesions occurring in a dermatomal distribution. The thoracic dermatomes, especially T4 to T12, are involved in about 50% of cases (Fig. 8.5.2.6); the lumbosacral dermatomes in about 16%; and the cranial nerves (mainly the Vth) in 14 to 20% of patients (Fig. 8.5.2.7a). The first symptoms are usually paraesthesia and shooting pains in the af- fected dermatome, which precede the eruption of vesicles by several days, occasionally 1 week or more. Erythematous maculopapular lesions then appear and quickly evolve into a vesicular rash, nearly always in a unilateral dermatome, with no vesicles beyond the Fig. 8.5.2.5  Varicella purpura fulminans. Courtesy of the late Dr B. E. Juel-​Jensen. Fig. 8.5.2.6  Herpes zoster (shingles) of the T4 dermatome. Copyright D. A. Warrell.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 743 midline. The vesicles usually form scabs after 3 to 7 days, and these separate after 2 weeks or so, but there is sometimes a more severe locally necrotic reaction (Fig. 8.5.2.8). There is a risk of secondary infection, particularly with Staphylococcus aureus. There may be malaise and low-​grade fever, but laboratory investigations usu- ally show no abnormalities, although up to 40% of patients with uncomplicated zoster may have lymphocytes and elevated protein in the cerebrospinal fluid. Involvement of the mandibular branch of the Vth cranial nerve can give intraoral lesions on the palate (Fig. 8.5.2.7b), floor of the mouth, and tongue. Involvement of the geniculate ganglion results in Ramsay Hunt syndrome, with pain and vesicles in the external auditory meatus, a loss of taste in the anterior two-​thirds of the tongue, and a lower motor neurone VIIth cranial nerve palsy. Complications of zoster Ophthalmic zoster  VZV reactivation from the trigeminal ganglion can affect the ophthalmic division of the trigeminal nerve, resulting in ophthalmic zoster (Fig. 8.5.2.7a). The features include conjunctivitis, anterior uveitis, keratitis, and sometimes iridocyclitis, with sec- ondary glaucoma and panophthalmitis. However, these latter sight-​ threatening complications of ophthalmic zoster are unusual. A rare association with ophthalmic zoster is granulomatous cerebral angiitis, which can be associated with arterial thrombosis; cerebral angiog- raphy shows segmental narrowing in the cerebral arteries on the side of the ophthalmic zoster occurring weeks after the rash. CT may show cerebral infarcts, particularly in the middle cerebral artery territory, and contralateral hemiparesis can occur. (a) (b) Fig. 8.5.2.8  Herpes zoster of the Vth cranial nerve, showing severe necrotic effects: (a) acutely, and (b) after recovery. Courtesy of the late Dr B. E. Juel-​Jensen. (a) (b) Fig. 8.5.2.7  Herpes zoster of the Vth cranial nerve: (a) ophthalmic division, and (b) lesions on the palate. Courtesy of the late Dr B. E. Juel-​Jensen.

744 section 8  Infectious diseases Motor zoster  Weakness or paralysis can sometimes be associated with zoster, and results from the involvement of the anterior horn cells in the same segment of the spinal cord as the involved dorsal root ganglion. Depending on the segment involved, this can lead to a monoparesis affecting the upper or lower limb, or to diaphrag- matic palsy (with the involvement of C5/​6). Paralysis usually re- covers completely, although the outlook for the recovery of facial nerve palsy is more variable. It is suggested VZV may be responsible for some cases of idiopathic VIIth nerve (Bell’s) palsy. Autonomic zoster  Lumbosacral herpes zoster can be associated with neurogenic bladder, and acute retention of urine. This may be accompanied by haemorrhagic cystitis resulting from vesicles on the bladder wall. Intestinal ileus and obstruction may occur. Zoster meningoencephalitis  Meningoencephalitis may accom- pany zoster at any site, and is heralded by impaired consciousness, headache, photophobia, and meningism. The interval from the onset of skin lesions to symptoms is around 9 days, but may be as long as 6 weeks. Symptomatic encephalitis usually lasts around 2 weeks, and is nearly always followed by full recovery without neurological sequelae. Transverse myelitis, although rare, can occur at any level of the spinal cord. Postherpetic neuralgia  The incidence of postherpetic neuralgia rises with the increasing age of the patient. It is uncommon in young people, but can occur in 50% of patients older than 50 years. It is characterized by pain in the affected dermatome persisting for 1 month or more after the acute attack of zoster has resolved. The pain may be steady and burning, or paroxysmal and stabbing in na- ture; it may occur spontaneously, or be triggered by stimuli such as temperature or touch. Zoster sine herpete  This term refers to radicular pain similar to that experienced in zoster, but without the antecedent skin lesions of zoster. It was originally applied to patients who did have ob- vious zoster, but had dermatomal pain in areas distinct from those where there was rash. However, it is more commonly applied to patients with radicular pain and no rash at all. There have been reports describing the use of PCR testing for the detection of VZV DNA in the cerebrospinal fluid of patients with presumed zoster sine herpete. The literature is anecdotal, and it is difficult to regard zoster sine herpete as a diagnostic entity unless there is good evidence for VZV involvement (e.g. by the detection of VZV DNA in cerebro- spinal fluid and/​or blood mononuclear cells). It should be included in the differential diagnosis of radicular pain of unknown cause. Any possible mechanism is speculative. VZV infection in immunosuppressed patients  In patients with immunosuppression, particularly of cellular immunity, vari- cella can be much more severe. The skin lesions are more diffuse (Fig. 8.5.2.9), and can take up to 3 times as long to heal. There may be visceral dissemination to the lungs, liver, and central nervous system. Patients with lymphoma undergoing chemotherapy are particularly susceptible. Herpes zoster in immunosuppressed patients is also more severe than in healthy subjects. Before effective antiviral therapy was avail- able, skin lesions were more extensive and could take several weeks longer to heal. Dissemination, presumably because of viraemic spread, with widespread skin lesions as in varicella, occurs in 10 to 40% of patients. Cutaneous dissemination is more likely to be asso- ciated with visceral dissemination to the same sites as those associ- ated with varicella. Patients with HIV infection or AIDS are prone to multidermatomal zoster, which can be one of the defining features of AIDS. VZV retinitis  This is a combination of pain and blurred vision in one eye, with progressive necrotizing retinitis seen on ophthalmos- copy. Adjacent cutaneous zoster indicates the diagnosis, but occa- sionally VZV retinitis occurs in immunocompetent patients as the sole manifestation of VZV reactivation. VZV retinitis may be diffi- cult to distinguish from cytomegalovirus (CMV) retinitis. A severe form of the disease, seen particularly in patients with HIV infection, and named progressive outer retinal necrosis, is associated with a high incidence of retinal detachment, and may require treatment with ganciclovir, as aciclovir is often ineffective. Differential diagnosis Varicella is usually recognized relatively easily. Other causes of a vesicular rash are generalized herpes simplex in the immunosup- pressed patient, and enteroviral disease, particularly hand, foot, and mouth disease caused by Coxsackie virus infection, but the rash on the hands and feet is unlike that of varicella, which has a centripetal distribution (Chapter 8.5.8). Human cases of infection with animal pox viruses (monkey pox and camel pox) have rarely been described (Chapter 8.5.4). Localized pain before the appearance of shingles or in zoster sine herpete may be severe enough to suggest myocardial ischaemia, or lung or intra-​abdominal pathology if it involves the thoracic dermatomes. Pathology The histological appearance of VZV infection is similar or indistin- guishable from that of HSV infection. Fig. 8.5.2.9  Herpes zoster varicelliformis. Courtesy of the late Dr B. E. Juel-​Jensen.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 745 Laboratory diagnosis The diagnosis of varicella and herpes zoster is usually made on clin- ical criteria alone. Virus can be seen in vesicular fluid by electron mi- croscopy, or isolated in culture. A serological diagnosis of varicella can be made by demonstrating seroconversion or VZV IgM anti- body. Urgent serology is needed to confirm the seronegative status of contacts at risk of severe VZV infection, to determine the need for VZV immunoglobulin (see ‘Prevention and control’, next). PCR-​ based tests for the detection of VZV DNA are available, and are of most use in testing cerebrospinal fluid in cases of suspected central nervous system disease. Treatment Pruritus may be alleviated by calamine lotion and antihistamines in patients with chickenpox. Fingernails should be closely cut to min- imize scratching. Skin care is important to prevent secondary bac- terial infection in patients with varicella and zoster. Aspirin should be avoided in children with chickenpox because of the risk of Reye’s syndrome. Strong analgesia may be needed in patients with zoster. VZV is sensitive to the nucleoside analogues aciclovir, famciclovir, and valaciclovir; as for HSV, VZV encodes a thymidine kinase that preferentially phosphorylates these drugs in infected cells. The median 50% inhibitory concentration of aciclovir against HSV is 0.1 µM, but is 2.6 µM against VZV, so 800 mg orally is necessary to achieve inhibitory concentrations. The treatment recommendations for varicella and herpes zoster are summarized in Box 8.5.2.1. Varicella Whether to treat normal children with varicella (who are the great majority of patients) has been much debated; the argument can be made that the disease is not always mild and it is not possible to predict which child may have a severe case. Therapy with aciclovir is safe, and although it has been suggested that widespread treat- ment with antivirals might result in viral resistance, or failure to develop normal immune responses, there is no evidence of this in controlled trials. Treatment with aciclovir begun within 24 h of the onset of the rash leads to a 25% decrease in the duration and se- verity of chickenpox. The argument for treating all adolescents and adults is easier, as chickenpox is more severe for them than it is for young children. Chickenpox in neonates, children with leukaemia, and transplant recipients should always be treated with aciclovir. Intravenous aciclovir limits the visceral spread of the virus if given immediately on diagnosis. Treatment in these immunosuppressed patients can be changed from intravenous to oral aciclovir once the fever has settled, if there is no evidence of visceral varicella. Herpes zoster The major justification for the antiviral treatment of herpes zoster in immunocompetent patients has been to limit postherpetic neur- algia. Although there are difficulties in accurately and objectively quantifying the pain of postherpetic neuralgia, trial data indicate that aciclovir, valaciclovir, and famciclovir can limit the duration of zoster-​associated pain, and that valaciclovir is slightly more ef- fective. All three drugs accelerate the healing of cutaneous lesions by 2 days over placebo; valaciclovir and famciclovir have the advantage of more convenient dosage, as well as probably being slightly more effective. Patients over the age of 50 years with zoster have the highest risk of postherpetic neuralgia, and so should be offered antiviral treat- ment. Younger patients may warrant treatment if they have marked pain. All patients with ophthalmic zoster should be treated urgently with antivirals, even if they present relatively late, as aciclovir re- duces the incidence of keratitis. Immunosuppressed patients with herpes zoster should receive intravenous aciclovir to prevent cuta- neous and visceral dissemination. Valaciclovir and famciclovir may be used if zoster presents in a localized form in less severely im- munosuppressed patients. Corticosteroids have been advocated in patients with herpes zoster, in order to reduce the severity of postherpetic neuralgia. However, the addition of oral prednisone to aciclovir slightly in- creases the rate of healing of skin lesions, but does not affect the incidence of postherpetic neuralgia; a role for corticosteroids thus remains unproven. Established postherpetic neuralgia can be man- aged with analgesics, tricyclic antidepressants, and other agents used for neuropathic pain, such as gabapentin and pregabalin, which were effective for the treatment of postherpetic neuralgia in large placebo-​controlled trials. Although the use of opioids for the treat- ment of neuropathic pain is controversial, several studies support their efficacy and safety; oxycodone and tramadol have been shown to be superior to placebo for the treatment of postherpetic neuralgia. Topical agents such as lidocaine 5% patches and topical capsaicin have been useful in ameliorating postherpetic neuralgia, but are un- satisfactory for use as sole agents (see also Chapter 24.12). Prevention and control Varicella-​zoster immune globulin, prepared from high-​titre immune human serum, has been shown to prevent or ameliorate varicella in seronegative people at high risk, such as immunocompromised Box 8.5.2.1  The use of aciclovir in varicella-​zoster infections Indications for intravenous aciclovir (10 mg/​kg, 8 hourly) Chickenpox: • Immunocompromised patients • Neonatal chickenpox • Chickenpox with systemic complications • Severe chickenpox in adults and in pregnancy (5 mg/​kg, 8 hourly) Shingles: • Severe shingles in immunocompromised patients • Multidermatomal shingles • Shingles complicated by ocular, motor, autonomic, or systemic involvement • VZV retinitis (severe forms in AIDS may require foscarnet or ganciclovir) Indications for oral aciclovir (800 mg, 5 times daily) • Uncomplicated chickenpox (except for mild chickenpox in children) • Uncomplicated shingles in patients over 45 years • Uncomplicated shingles in immunosuppressed patients • Shingles presenting with severe pain Infections not requiring active antiviral treatment • Uncomplicated mild chickenpox in children • Patients presenting more than 48 h after the appearance of the last le- sion, or when all lesions have crusted • Uncomplicated shingles in patients under 45 years • Postherpetic neuralgia

746 section 8  Infectious diseases people and pregnant women. It should be given to seronegative immunodeficient patients (including those on high-​dose cortico- steroid treatment), and pregnant women with definite contact with varicella. It should be administered within 10 days (preferably 2–​ 4 days) of exposure. Neonates whose mothers have had varicella less than 1 week before delivery, or within 28 days after delivery are also recommended to receive varicella-​zoster immune globulin. A VZV vaccine is available; a live attenuated vaccine containing the Oka strain of VZV. It confers 90% protection from natural varicella when administered to susceptible immunosuppressed people (such as patients with leukaemia and lymphoma receiving chemotherapy), but it produces rash in up to 40% of these recipients. In immunized healthy children the risk of subsequent varicella after community exposure is reduced to less than 5%, and the vaccine-​induced rash is much less common (about 5% of recipients). This vaccine is li- censed in Japan, some European countries, and the United States of America, where it is recommended for the routine immunization of children aged 12 to 18 months. However, in the United Kingdom it is recommended only for use in seronegative healthcare workers and children over 1 year in contact with individuals at high risk of severe varicella. Trials have shown that the postinfective immuniza- tion of subjects aged 60 years or over diminishes the incidence of zoster and postherpetic neuralgia, and the vaccine is now licensed in the United States of America for the prevention of herpes zoster in this age group. The nosocomial transmission of VZV by patients with varicella requiring admission to hospital is a significant risk, as 10% of adults are seronegative. Nursing and managing patients with varicella in hospital should be restricted to those staff known to be seropositive for VZV. Patients with varicella in hospital should ideally be isolated in negative-​pressure rooms to prevent airborne transmission. Human cytomegalovirus infection Historical background The syndrome of congenital cytomegalovirus infection, cytomegalic inclusion disease, was described in children with fatal infection in 1904, but the intranuclear inclusions were attributed to a protozoan parasite. In 1921, the pathologist Goodpasture suggested that the inclusions in the parotid glands of infants were caused by a virus, because a filterable agent produced similar histology in guinea pig salivary glands, and the lesions were attributed in 1926 to ‘salivary gland virus’. Human cytomegalovirus (HCMV) was finally isolated in 1956, and so named by Weller for the characteristic owl’s-​eye, or cytomegalic inclusions it produces in the nuclei of infected cells. HCMV produces little morbidity in immunocompetent people, but can produce severe disease in the fetus if infection is acquired in utero, and in immunosuppressed patients. Aetiology HCMV is the largest human herpesvirus, with a linear double-​ stranded DNA genome of 250 kb encoding more than 200 proteins. Mammalian cytomegaloviruses are species specific, and so HCMV cannot be studied in animal models. The most widely studied la- boratory strain, AD169, shows significant genomic variation from recent clinical isolates, which possess an additional 15 kb of DNA. HCMV replicates slowly compared with other herpesviruses, and gene expression occurs sequentially in immediate-​early, early, and late phases. Epidemiology Following primary infection, HCMV persists for life as a latent in- fection, with periodic asymptomatic excretion of virus in saliva, breast milk, urine, semen, and cervical secretions. Infection is spread by close contact with these body fluids. In developing coun- tries, HCMV is usually acquired in childhood, and nearly 100% of young adults are seropositive. In developed countries, serocon- version progresses with age, but seroprevalence is higher in lower socioeconomic groups. Overall, about 50% of adults are seroposi- tive. In childhood, HCMV is acquired from breast milk or con- tact with infected children excreting virus in their saliva or urine. Children in day nurseries transmit the virus to each other, and to susceptible adult carers. Later, sexual transmission becomes a major route of infection, and seroprevalence approaches 100% in homo- sexual men, and sex workers. Blood and blood products from normal seropositive donors can transmit HCMV. Transfusion recipients at risk of HCMV disease now usually receive screened seronegative blood; otherwise the risk of transfusion-​related HCMV infection is 2.5% per unit of blood. The virus is carried in leucocytes, and leukodepletion of blood greatly reduces the risk of HCMV transmission. The technique is also being widely adopted as a preventive measure against transmis- sible spongiform encephalopathies. Finally, solid organ and bone marrow transplants from seropositive donors can transmit HCMV, producing particularly severe disease in seronegative recipients. Pathogenesis Current evidence suggests myeloid lineage cells are a principal site of HCMV latency, and that virus may be reactivated from dendritic cells and monocytes as they differentiate. Endothelial cells, possibly epithelial and other cells, may also be sites of latency. The immune response is critical for controlling infection in the normal host. Normal immunocompetent individuals infected with HCMV mount a strong T-​cell response, with very high frequencies of cytotoxic (CD8+) T lymphocytes in the peripheral blood targeted particularly at the HCMV major tegument protein pp. 65, and the major immediate-​early protein IE1. Impairment of this response is associated with the risk of disseminated infection. HCMV possesses multiple immune-​evasion genes, whose products interfere with the class I MHC antigen-​processing pathway, and recognition by natural killer (NK) cells, which may help the virus reactivate by delaying T-​ and NK-​cell recognition of infected cells. Antibody probably limits blood-​borne dissemination of HCMV, as maternal IgG appears to be especially important in preventing viral transmission to the fetus. Subclinical reactivation occurs frequently in the normal host, but is controlled by the immune response. Immune deficiency, particu- larly of the T-​cell response, such as iatrogenic or disease-​induced immunosuppression, may allow uncontrolled replication and re- sult in HCMV disease. Pathology is presumably produced by the direct cytopathic effects of the virus, although indirect effects pro- duced by soluble virus-​encoded proteins or the host response are also possible. The presence of HCMV in a diseased organ does not necessarily implicate the virus as a cause; reactivation of the virus

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 747 can sometimes be non​pathogenic, and reflects its being a bystander, coexisting with another pathogenic process. Clinical features of HCMV disease Primary infection in immunocompetent subjects Primary infection in children and adults is asymptomatic in most cases, but HCMV can produce an illness clinically indistinguishable from infectious mononucleosis caused by primary Epstein–​Barr virus (EBV) infection, typically with fever, myalgia, cervical lymph- adenopathy, and mild hepatitis. Tonsillopharyngitis is much less common than in primary EBV infection, and lymphadenopathy and splenic enlargement are less prominent features. The fever lasts 2 to 3 weeks, but can persist for up to 5 weeks. In developed countries an increasing proportion of HCMV seroconversion illness is seen in older adults, and the diagnosis should still be considered in patients aged over 50 years. Myocarditis, pneumonitis, and aseptic menin- gitis are rare complications. A proportion (5–​10%) of patients with Guillain–​Barré syndrome show serological evidence of primary HCMV infection; they are more likely to have antibodies to the GM2 ganglioside than other patients with Guillain–​Barré syndrome, and a causal relationship has been postulated. Primary HCMV infection acquired from blood transfusion re- sults in a similar clinical picture occurring 3 to 6 weeks after transfu- sion, and is usually self-​limiting in the normal host. To distinguish between primary HCMV infection and other causes of mononucle- osis syndromes, such as EBV and toxoplasmosis, requires sero- logical testing (the Paul–​Bunnell and monospot tests are negative in HCMV mononucleosis). HCMV disease in immunosuppressed patients HCMV infection is most severe in immunosuppressed patients, particularly solid organ and bone marrow transplant recipients, and those with AIDS, all of whom have impaired T-​lymphocyte func- tion. This strongly supports the importance of T cells in controlling infection. Solid organ transplant recipients The risk of HCMV disease is 3 to 5 times greater in a seronegative recipient receiving a graft from a seropositive donor, and it causes much more severe infection than in a seropositive recipient who has a reinfection or reactivation of latent virus. Many centres pair seronegative donors with seronegative recipients, although this is often thwarted by organ shortage. Clinically, there may be specific organ involvement, which is not seen in normal patients. Interstitial pneumonitis caused by HCMV is rare, except in bone marrow transplant recipients, and carries a poor prognosis; gastrointestinal disease includes oesophagitis, gastritis and peptic ulceration, and colitis; and HCMV retinitis may occur in severely immunosup- pressed patients. HCMV has been reported to be associated with increased graft rejection and renal artery stenosis in renal trans- plant recipients; with accelerated coronary artery stenosis in heart transplant recipients; and with vanishing bile duct syndrome in liver transplant recipients. However, none of these associations is definitively established as causal. Bone marrow transplant recipients HCMV disease is a major problem in allogeneic bone marrow trans- plant recipients, with a 30 to 50% incidence of clinically significant infection. It is a lesser problem in autologous bone marrow trans- plant. If the donor and/​or recipient is seropositive there is a risk of HCMV disease, but if both donor and recipient are seronegative, in- fection can be prevented if solely HCMV-​seronegative blood prod- ucts are used to support the patient. Pneumonitis is the most serious manifestation of HCMV infection after bone marrow transplant, occurring in 10 to 15% of allogeneic bone marrow transplant recipi- ents, with a mortality of 80% without antiviral therapy. The clinical presentation is interstitial pneumonitis in the absence of any other identifiable pathogen, with increasing arterial hypoxaemia, and progression to respiratory failure. It is suggested that graft versus host disease may contribute to the lung injury in HCMV pneumon- itis in bone marrow transplant recipients. The relationship between HCMV and graft versus host disease is controversial, with sugges- tions that HCMV may predispose to graft versus host disease, and vice versa. Patients with AIDS HCMV disease is one of the most frequent opportunistic infections in patients with advanced HIV infection, of whom 40% develop sight-​ or life-​threatening HCMV disease. A CD4 count of less than 50/​µl carries a high risk of disease, although the widespread use of antiretroviral therapy in developed countries means that relatively few patients now have such low CD4 counts, and the incidence of HCMV disease in patients with AIDS has declined significantly. HCMV retinitis has been seen in up to 25% of patients with AIDS not receiving effective antiretroviral therapy. Haemorrhagic retinal necrosis spreads along retinal vessels, and threatens sight when disease encroaches on the macula (Fig. 8.5.2.10). The clin- ical effect is visual impairment, and the risk of retinal detachment and haemorrhage is increased, hence those with low CD4 counts should have regular fundoscopy to detect retinitis before it be- comes symptomatic. Diagnosis is made by the ophthalmological detection of typical retinal changes, preferably with accompanying evidence of HCMV viraemia. In the absence of treatment, HCMV retinitis almost invariably progresses to affect both eyes and des- troy vision. HCMV is reported to produce diffuse encephalitis in AIDS patients, but although the virus is sometimes seen in neuronal cells at autopsy, encephalitis attributable to HCMV is relatively rare in clinical practice by comparison with the other causes of encephalitis in AIDS. HCMV can also produce a progressive Fig. 8.5.2.10  CMV retinitis.

748 section 8  Infectious diseases radiculopathy, causing low-​back pain that radiates to the area supplied by the affected spinal nerve root, and the development of flaccid paraparesis. In the gastrointestinal tract, HCMV is associated with oesopha- gitis, gastritis, and enterocolitis, and virus can be seen in biopsies from these sites, usually in shallow ulcers. HCMV pneumonitis is rare in patients with AIDS, suggesting that there must be additional factors to account for its frequency in bone marrow transplant recipients. Congenital and neonatal HCMV infection HCMV infection of the neonate may be congenital from intrauterine infection, perinatal from transmission during birth, or postnatal from breast milk. The frequency of congenital HCMV infection in developed countries is around 0.5 to 1% of live births, resulting from either primary maternal infection in pregnancy, or from reactiva- tion of HCMV in a previously infected mother during pregnancy. The risk of primary maternal infection in pregnancy is about 1%, and it carries a 40% risk of congenital infection. Fetal infection is more likely to be severe following primary infection in early preg- nancy, whereas the risk of symptomatic congenital infection is much lower, although not absent, from reactivation of maternal HCMV. Pre-​existing maternal immunity limits spread to the fetus. Approximately 5 to 20% of congenitally infected babies are symp- tomatic at birth. In its most severe form, usually in babies of mothers with primary maternal infection, the clinical features of congenital HCMV are: microcephaly; chorioretinitis; nerve deafness; hepa- titis with jaundice and hepatosplenomegaly; and thrombocyto- penia with petechiae. This classical cytomegalic inclusion disease has a high mortality, and 80% of all infants symptomatic at birth who survive have serious sequelae, such as learning, visual, and hearing impairment. However, most congenitally infected babies are asymptomatic at birth, and only 5 to 15% of these subsequently develop sequelae on long-​term follow up, the most common being sensorineural deafness, which also occurs in isolation in otherwise normal babies. Perinatally or postnatally acquired HCMV infection is rarely symptomatic or associated with long-​term sequelae, if the mother is seropositive. Pathology On light microscopy, typical HCMV-​infected cells appear large, with a relative reduction in cytoplasm, and nuclei that contain prominent inclusions surrounded by a clear halo (described as owl’s-​eye in- clusions). These cells contain replicating virus, and are associated with active infection and disease; they are diagnostic when seen in biopsies of affected organs. In patients dying of severe disease, histological evidence of HCMV involvement can be found in most organs, whereas it infects a restricted range of cells in vitro. Malignancy Although associations between HCMV and malignancy have been postulated in the past, there is currently no good evidence to asso- ciate the virus with any human malignancy. Laboratory diagnosis Primary infection is usually diagnosed by the detection of IgM anti- body to HCMV in the absence of IgG antibody; there is a marked atypical lymphocytosis (mainly increased CD8+ T cells), but heterophile antibody (as detected in primary EBV infection by the monospot or Paul–​Bunnell tests) is absent. IgG antibody is a useful marker of HCMV carriage, but titres do not rise reliably in disease; IgM antibody, a marker of primary infection, is also sometimes found with reactivation in immunosuppressed patients, and serology is of limited use in confirming HCMV disease in these patients. Culture of virus from urine may only indicate asymptomatic reactivation, but culture from the blood buffy coat suggests HCMV disease. The virus can never be cultured from the blood of normal HCMV car- riers, and culture from an organ site (such as bronchoalveolar lavage fluid) may indicate locally active infection. Rapid culture methods such as DEAFF (detection of early antigen fluorescent foci), which uses a monoclonal antibody against an immediate-​early viral protein, or shell vial tests (centrifuging sam- ples onto cell cultures) are now used less often. PCR techniques are increasingly used to detect and quantify the HCMV load in blood or plasma, and this is now the standard assay for detecting HCMV in most laboratories. As virus can never be detected in plasma (as opposed to leucocytes) in normal carriers, the presence of HCMV DNA in plasma indicates active viral replication. Detection of virus in biopsy specimens by histological and immunohistological tech- niques implies active HCMV infection in the relevant tissue. In practice, HCMV disease is usually diagnosed by the combin- ation of an appropriate clinical syndrome, and detection of HCMV DNA by quantitative PCR above a threshold level in blood or plasma, or in biopsies from involved organs, in the absence of any other likely causal microbial pathogen. Treatment Several drugs are now available for the treatment of HCMV disease. Aciclovir has little in vitro activity against HCMV, which does not possess a thymidine kinase (see ‘Herpes simplex virus infections’, earlier), and has no place in therapy (although valaciclovir is used in prophylaxis; see ‘Antiviral prophylaxis’, next). Ganciclovir, another nucleoside analogue, is monophosphorylated in infected cells by the UL97 gene product of HCMV, and is active against HCMV; its most limiting side effect is myelotoxicity, with leukopenia and thrombocytopenia, but it has many other potential side effects, including azoospermia, and intravenous administration is necessary. Valganciclovir, a valyl ester prodrug of ganciclovir, has much higher oral bioavailability, and produces equivalent plasma concentrations to intravenous ganciclovir; it is thus useful for prophylaxis. Resistance to ganciclovir results from a mutation in the HCMV DNA polymerase, or in the UL97 gene, and is seen mainly in immunosuppressed patients in whom prolonged use is necessary. An alternative drug to ganciclovir is foscarnet, a competitive in- hibitor of the viral DNA polymerase, which shows no cross-​resistance with ganciclovir. This also must be given intravenously, and its side effects include renal impairment and hypocalcaemia. Cidofovir, a nucleotide analogue acting on the viral DNA polymerase, is highly nephrotoxic (probenecid must be given concurrently to prevent ir- reversible renal damage), and therefore relatively infrequently used. Ganciclovir resistance remains a significant problem, and hence other drugs with in vitro activity against HCMV are currently being studied, and have been used in initial small clinical trials, although none is yet licensed for HCMV infection: these include maribavir, brincidofovir, and letermovir. Interestingly, both leflunomide, and artesunate have been shown to have some anti-​CMV activity.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 749 Primary infection In the immunocompetent host this usually requires no specific anti- viral treatment, although occasionally, severe primary infection may lead to hospitalization and require treatment. HCMV disease in immunosuppressed patients Whether due to primary or secondary infection, or reactivation, this is usually treated with ganciclovir or foscarnet for 2–​3 weeks, with full-​dose induction intravenous therapy; for ganciclovir this is 5 mg/​kg every 12 h and for foscarnet 60 mg/​kg every 8 h. Oral valganciclovir 900 mg twice daily is an equivalent dose to intra- venous ganciclovir. Secondary prophylaxis may well be needed if immunosuppression persists (see ‘Prevention and control’, next). HCMV pneumonitis in bone marrow transplant recipients This responds poorly to ganciclovir or foscarnet alone, but the com- bination of full-​dose ganciclovir with intravenous immunoglobulin has been reported to reduce mortality. Specific anti-​CMV immuno- globulin was initially used, then other trials suggest normal pooled intravenous immunoglobulin was equally effective, and recent re- ports question whether IVIg confers any additional benefit. Many centres monitor bone marrow transplant recipients, especially of allogeneic grafts, for CMV viraemia, and commence preemptive therapy with ganciclovir if viraemia is detected before the develop- ment of symptomatic or obvious organ disease. HCMV retinitis in AIDS This is treated with an induction course of ganciclovir or foscarnet (both drugs have also been used in combination) or valganciclovir 900 mg twice daily for 21 days. Continued prophylaxis is needed to prevent relapse until significant recovery of the CD4 count can be induced with antiretroviral therapy; valganciclovir 900 mg daily is most convenient. Implantable intraocular devices providing sus- tained release of ganciclovir into the vitreous humour have also been used. The use of combination antiretroviral therapy in HIV-​infected patients is associated with much improved long-​term control of HCMV infection. However, the syndrome of immune-​recovery vitritis, characterized by posterior segment inflammation, can occur in patients with previously treated CMV retinitis when their CD4 count reconstitutes on antiretroviral therapy. Congenital HCMV infection Treating symptomatic congenital HCMV infection with ganciclovir (8 or 12 mg/​kg daily for 6 weeks) reduces the excretion of CMV in the urine, but viruria returns to near pretreatment levels after cessation of therapy. Hearing improvement may occur, but the role of antiviral therapy in congenital HCMV infection remains to be established. Prevention and control The problem posed by HCMV in immunosuppressed patients has led to several approaches to prophylaxis. Antiviral prophylaxis There is a definite case for primary prophylaxis in solid organ and bone marrow transplant recipients at high risk of disease (seronega- tive recipients of a seropositive graft, or seropositive recipients), and in AIDS patients with fewer than 100 CD4 cells/​µl. Ganciclovir has been widely used, and valganciclovir 900 mg daily is effective in many of these settings. Despite limited in vitro activity against HCMV, and lack of efficacy as therapy, oral valaciclovir has been shown to provide significant prophylaxis against HCMV disease in renal transplant recipients, and is licensed for this use. Passive immunization CMV hyperimmune globulin is reported to reduce the risk of HCMV disease in renal transplant recipients, but is expensive and little used in practice. There are reports that HCMV-​specific T-​cell immunity can be reconstituted in bone marrow transplant recipients by the adoptive transfer of virus-​specific T lymphocytes from the immune donor, but this is still the subject of investigational studies. Active immunization A live laboratory strain (Towne) of HCMV has been tested as an experimental candidate vaccine in renal transplant recipients, with some evidence of protective immunity, perhaps equivalent to having previous natural HCMV infection. A more recent phase II trial of recombinant glycoprotein B based HCMV vaccine in seronegative women showed it has the potential to decrease incident cases of ma- ternal and congenital CMV infection. However, there is currently no available licensed vaccine. Special problems in pregnant women Pregnant women who are seronegative should avoid contact with possibly infected children in day-​nursery settings, although this may be impractical. Ganciclovir must not be used in pregnancy. Human herpesvirus 6 and 7 Human herpesvirus 6 Human herpesvirus 6 (HHV-​6) was first isolated in 1986 from cul- tured human lymphocyte lines, and named human B lymphotropic virus, a misnomer since it is trophic principally for T cells, al- though replication also occurs in macrophages, glial cells, and EBV-​transformed B cells. HHV-​6 is widely distributed in humans. Primary infection causes roseola infantum (also known as exanthem subitum or sixth disease), an aetiological association first described in Japanese children in 1988. Unlike any other herpesvirus, HHV-​ 6 genome DNA can be found covalently integrated into the cell chromosomes in about 1% of the population. Aetiology HHV-​6 has typical herpesvirus morphology, and is genetically clas- sified in the β-herpesvirus subfamily. Two types of isolate, HHV-​ 6A and HHV-​6B, are now clearly distinguished by their genetic sequence, and some variation in biological properties. HHV-​6B is associated with roseola, whereas HHV-​6A has not been associated with disease. Epidemiology There is high seroprevalence of HHV-​6 in all populations. More than 90% of children are seropositive at 2 years of age. The virus (usually the HHV-​6B variant) can be detected in peripheral blood

750 section 8  Infectious diseases mononuclear cells by PCR-​based tests in nearly all healthy people. It is most probably transmitted via maternal saliva, although intra- uterine and perinatal transmission could occur. There is also evi- dence that chromosomally integrated maternal HHV6 can be transmitted in the germline, although the clinical significance is un- certain. The virus is not detectable in breast milk. Pathogenesis HHV-​6 probably replicates in regional lymphoid tissue in the oro- pharynx during primary infection, and can be found in circulating lymphocytes. The virus replicates in vitro in CD4 + T-​cell lines, but during persistent infection in the normal adult, virus can be de- tected by PCR in both CD4 + T cells and monocytes/​macrophages in peripheral blood, which are probably the principal site of car- riage during persistent infection. The mechanism of viral latency is uncertain. Although HHV-​6 cannot usually be isolated by culture from the peripheral blood of normal people, specific DNA is easily de- tected in blood during immunosuppression, indicating reactivation of HHV-​6. The mechanism by which HHV6 produces its clinical manifestations remains unclear. Clinical features Primary infection with HHV-​6 in young children is associated with roseola, and also with a febrile illness without rash. Roseola infantum (exanthem subitum, sixth disease) Roseola is an acute illness of infants and young children, typically 3 to 5 days of high fever with upper respiratory tract symptoms, and sometimes cervical lymphadenopathy. As the fever subsides, a rash appears and lasts for 1 to 3 days. The rash is diffuse, macular, or maculopapular, and appears similar to that of rubella. There is mild atypical lymphocytosis and there may be neutropenia. Infections may rarely be complicated by febrile convulsions, meningitis, encephal- itis, and hepatitis; the last is usually mild, but occasionally severe. Roseola has been estimated to occur in only 10 to 20% of children, and primary HHV-​6 infection is commonly subclinical. Febrile illness Fever without rash is a more usual manifestation of primary HHV-​6 infection than roseola. In a North American study, 10% of 1600 fe- brile children under the age of 3 years (including 20% of those aged 6–​12 months) presenting with acute febrile illness were diagnosed as primary HHV-​6 infection, but only 17% of them had clinical roseola. Febrile convulsions It is suggested HHV-​6 may have a particular association with febrile convulsions in young children. Primary HHV-​6 infection was re- ported to account for one-​third of all the febrile seizures in children up to the age of 2 years; however, there were no seizures in 81 chil- dren with primary infection in a prospective cohort. HHV-​6 DNA can be detected in the cerebrospinal fluid of children with primary infection, and any association may be because HHV-​6 specifically infects the nervous system, rather than solely because of high fever. HHV-​6 infection in immunosuppressed patients Several studies have shown increases in antibody titres to HHV-​ 6, and increased HHV-​6 DNA levels in the peripheral blood of immunosuppressed solid organ and bone marrow transplant re­cipients. In bone marrow transplant recipients, HHV-​6 has been associated with fever, rash, graft versus host disease, encephalitis, delayed engraftment, marrow suppression, and pneumonitis. It is not clear whether HHV-​6 plays a specific aetiological role in all these syndromes; the evidence is perhaps stronger for a causal role in encephalitis. There is also good evidence that HHV-​6 reactivates in patients with advanced HIV infection and AIDS, but again there is less firm evidence that this is associated with disease. Other disease associations Studies of chronic fatigue syndrome and multiple sclerosis have not provided convincing evidence of any significant aetiological associ- ation with HHV-​6. Differential diagnosis Primary HHV-​6 infection may be confused with many febrile child- hood illnesses accompanied by a rash. Roseola may also be misdiag- nosed as a sensitivity reaction to recent antibiotic treatment. Other virus infections (EBV, HCMV) may also be associated with atypical lymphocytes and a mononucleosis syndrome. Pathology HHV-​6 replicates in vitro in cells originating from the central ner- vous system, particularly glial cell lines. HHV-​6 DNA can be de- tected in the brains of apparently normal people, suggesting viral persistence in the central nervous system. No distinctive histopath- ology has yet been attributed to HHV-​6. Malignancy HHV-​6 DNA has been detected in the blood of patients with sev- eral lymphoproliferative disorders, but this probably reflects re- activation rather than any causal association with the tumour. HHV-​6 DNA has been reported in some tumour tissues, including the nodular sclerosis variant of Hodgkin’s disease, but without a convincing aetiological association between the virus and any tumour. Laboratory diagnosis Most assays for HHV-​6 antibody do not distinguish between anti- body to HHV-​6A and HHV-​6B, and may cross-​react with anti- bodies to HHV-​7. Seroconversion is evidence of primary infection. IgM assays for HHV-​6 antibody are not reliable indicators of pri- mary infection, as some HHV-​6 carriers may periodically have IgM antibody. Although HHV-​6 can be cultured from peripheral blood mono- nuclear cells during acute primary infection, few laboratories will undertake this. PCR-​based techniques for the detection of HHV-​6 DNA in plasma and cerebrospinal fluid are the method of choice for clinical diagnosis, and are becoming more widely available. Treatment HHV-​6 sensitivity to antiviral drugs corresponds with that of cytomegalovirus. Thus, HHV-​6 replication is inhibited in vitro by ganciclovir and foscarnet, but not aciclovir; however, there are no controlled clinical trials of these drugs. Their use may be considered for immunosuppressed patients with suspected HHV-​6-​associated pneumonitis.

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 751 Prevention and control There are no preventive measures for HHV-​6 transmission. It seems unlikely that there will be a case for the development of a vaccine because infants may be infected so early in life, while they still have maternal antibody. Special problems in pregnant women Nearly all pregnant women will be carriers of HHV-​6. There is no evidence that HHV-​6 infection harms the fetus or the neonate. Human herpesvirus 7 Human herpesvirus 7 (HHV-​7) was isolated in 1990, and is a β-herpesvirus similar to, but distinct from, HHV-​6. HHV-​7 pre- dominantly infects CD4+ T cells and can be reactivated from latency by T-​cell activation. Although there is serological crossreactivity between HHV-​6 and HHV-​7, data indicate that HHV-​7 infects nearly all children, but later than HHV-​6, with more than 90% being infected by the age of 5 years. The virus is excreted in saliva. HHV-​7 has been associated with some cases of roseola, which it was reported to cause in Japanese infants with a previous episode of roseola proven to be caused by HHV-​6. There is no further evidence of pathogenicity. The best method of diagnosis is PCR-​based testing of serum or cerebrospinal fluid. Laboratory tests for HHV-​6 often detect HHV-​ 7 by multiplex PCR. There is no reason to consider any treatment for HHV-​7. Human herpesvirus 8 Human herpesvirus 8 (HHV-​8) is the most recently isolated of the human herpesviruses; Chang and colleagues reported the detection of novel DNA sequences with homology to herpesviruses in Kaposi’s sarcoma tissue in 1994. Initially named Kaposi’s sarcoma-​associated herpesvirus, it was subsequently designated HHV-​8. It is genetic- ally most closely related to a well characterized simian herpesvirus (herpesvirus saimiri), and less so to EBV; it has consequently been assigned to the rhadinovirus (γ2-​herpesvirus) subfamily. Current culture techniques are unreliable, but the virus can be detected by PCR. Serological assays depend on the use of infected cell lines or synthetic antigens from predicted open reading frames. The seroepidemiology, biology, and disease associations of the virus are still being analysed, but HHV-​8 is clearly associated with Kaposi’s sarcoma, a tumour that has long been suspected of having a viral aetiology; with primary effusion lymphoma; and with multicentric Castleman’s disease. Reported associations with multiple myeloma and other cancers are unconfirmed. Aetiology HHV-​8 has the characteristic morphology of a herpesvirus. The viral genome is composed of a 141 kbp unique segment flanked by mul- tiple 801 bp direct repeats. Sequence analysis suggests that HHV-​8, like other herpesviruses, is an ancient human virus; comparative analysis of the variable genes ORF-​K1 and K15 indicates there are at least four virus subtypes, A to D, reflecting the migrationary di- vergence of modern human populations. HHV-​8 contains genes homologous to mammalian genes encoding cell-​cycle regulatory proteins (the cyclins), chemokines, and inhibitors of apoptosis. On the evidence to date, the normal cellular site of latency of HHV-​8 almost certainly includes the B cell. Epidemiology The emerging epidemiology of HHV-​8 suggests it is less ubiqui- tous than other human herpesviruses. Initial serological assays de- tected antibodies to a latent nuclear antigen; assays using lytic-​cycle antigens gave higher rates of seroprevalence, and newer assays using multiple HHV-​8 antigens are currently being applied. Current data suggest a seroprevalence of 90% or more in patients with Kaposi’s sarcoma, and 40% in HIV-​positive homosexual men without Kaposi’s sarcoma. Seroprevalence in healthy adults is reported as being more than 50% in African adults in West Africa, 20% in black South African blood donors, and 53% in HIV-​positive and -​negative adults in Uganda. Seroprevalence is 5% or less in blood donors in the United Kingdom and the United States of America, with inter- mediate rates in Italy and other Mediterranean countries. HHV-​8 can be detected by PCR in nearly all cases of Kaposi’s sarcoma, but is less easy to detect in the blood of normal carriers. The usual route of transmission is probably saliva and sexual con- tact, but intravenous drug use, blood transfusion, and organ trans- plantation also transmit the virus. A latent nuclear antigen-​based assay detected seroconversion to HHV-​8 in HIV-​infected homo- sexual men at a median of 33 months before they subsequently de- veloped Kaposi’s sarcoma. HHV-​8 infection in children correlates with seropositivity in their mothers, but whether this reflects ver- tical or horizontal transmission is uncertain. Pathogenesis There has been much uncertainty over the cell of origin of Kaposi’s sar- coma, but the spindle cells of which the tumour is largely composed are thought to be of lymphatic endothelial origin. In Kaposi’s sarcoma tumour tissue, HHV-​8 DNA and latent nuclear antigen are present in every spindle cell, suggesting an aetiological role for the virus. In HIV-​associated Castleman’s disease, the HHV-​8 latent nuclear antigen is present in immunoblasts in the mantle zone of the tu- mour. HHV-​8 is present in the tumour cells of all cases of primary effusion lymphoma so far studied (although so is EBV), and HHV-​8 latently infected cell lines derived from these tumours can be in- duced to release infectious virus. These clear associations of virus DNA with tumour cells suggest a definite oncogenic role for HHV-​ 8. HHV-​8 latent transcripts, including latency-​associated nuclear antigen, viral cyclin, viral FLIP, and virus-​encoded microRNAs, promote cell proliferation and prevent apoptosis, whereas lytic proteins, such as viral G protein-​coupled receptor, K1, and virus-​ encoded cytokines (viral interleukin-​6 and viral chemokines) con- tribute to the characteristic angioproliferative and inflammatory Kaposi’s sarcoma lesions through a mechanism known as paracrine neoplasia. It has been suggested that HHV-​8 may be involved in the patho- genesis of multiple myeloma, but this association is unproven, as is an association with primary pulmonary hypertension. The individual HHV-​8 subtypes are not associated with any distinct pathology. Clinical features Apart from these malignancies, the only reported clinical syndrome accompanying primary or reactivated HHV-​8 infection is fever

752 section 8  Infectious diseases and bone marrow graft failure in immunosuppressed transplant recipients. Kaposi’s sarcoma Kaposi’s sarcoma appears as purplish-​brown macules, papules, or plaques. It is described in four characteristic clinical settings:  the classical form in older Mediterranean or Jewish men, the endemic African form (accounting for 10% of cancer in equatorial Africa), in patients with immunodeficiency states, such as transplant recipients, and the AIDS-​associated form. In the classical and African forms there are lesions on the extremities; systemic and mucosal involve- ment is rare, and the disease is indolent. In immunosuppressed pa- tients (other than those with AIDS) the lesions are more widespread and more rapidly progressive, although visceral involvement is still unusual, and lesions may regress if immunosuppressive drugs are stopped. AIDS-​associated Kaposi’s sarcoma is seen predominantly in homosexual men in Western countries, but is commonly associated with heterosexually acquired HIV infection in African countries. The clinical signs are widespread cutaneous lesions, with involvement of the oral mucosa (see Chapter 8.5.23, Figs. 8.5.23.12 and 8.5.23.13), and visceral lesions may occur in the lungs or gastrointestinal tract. Progression can be much more rapid than the other forms. HHV-​8 has been isolated from all four types of Kaposi’s sarcoma. Primary effusion lymphomas Previously known as body-​cavity based lymphomas, these are a rare and aggressive type of B-​cell lymphoma in patients with AIDS. They present as lymphomatous effusions of the peritoneal, pleural, or pericardial spaces, usually without any identifiable tumour mass. HHV-​8 is present in the tumour cells of all cases so far studied, al- though so also is EBV. Castleman’s disease or angiofollicular lymph node hyperplasia This can be localized, and is amenable to curative excision. However, a multicentric form is seen particularly in HIV-​infected patients, and is more aggressive. HHV-​8 is found in a high proportion of these multicentric cases, especially those associated with HIV. Pathology No distinctive histopathology has been identified for HHV-​8 inde- pendent of the pathology of the tumours with which it is associated. Laboratory diagnosis HHV-​8 can best be detected by PCR-​based tests. The antibody assays described earlier may become commercially available in the near future. Treatment In vitro assays in HHV-​8-​infected lymphoma cell lines indicate that HHV-​8 replication is moderately sensitive to foscarnet, ganciclovir, and cidofovir. AIDS patients treated with foscarnet and ganciclovir may be less likely to develop Kaposi’s sarcoma. Antiviral drugs are not an established treatment for HHV-​8 tumours. Kaposi’s sarcoma confined to the skin can be treated with radio- therapy or intralesional α-​interferon. More widespread cutaneous or visceral disease can be treated with single-​agent or combination chemotherapy. The treatment of Kaposi’s sarcoma in AIDS pa- tients is discussed in Chapters 8.5.23 and 8.5.24. Kaposi’s sarcoma lesions may regress with antiretroviral treatment, possibly because of improved cellular immunity resulting from the reduction in HIV load. Prevention and control Given the uncertainty around the epidemiology and disease associ- ations of HHV-​8, prevention and control are not yet possible. No spe- cial problems of infection have been identified in pregnant women. Cercopithecine herpesvirus 1 (herpes B virus) Cercopithecine herpesvirus 1 is the formal name now given to herpes B virus (replacing the previous term, herpesvirus simiae), the natural hosts of which are members of the Macaca genus of Old World monkeys. It produces minimal disease in its natural hosts, but its transmission to humans results in a high incidence of severe dis- ease. Although more than 30 other herpesviruses have been isolated from non​human primates, none of these has been unequivocally ­associated with a disease in humans. The virus was first isolated in 1932 from the brain of Dr W B, who died of encephalitis after a bite from a macaque (hence the name herpes B virus). There have since been about 45 cases of human infection resulting from accidental transmission from captive monkeys. Aetiology Herpes B virus is an α-​herpesvirus closely related to HSV, and ­appears to behave in an analogous manner to HSV in its natural pri- mate host. Herpes B virus can also infect and produce disease in other non​human primates and small mammals. Epidemiology Herpes B virus is enzootic in Old World monkeys of the Macaca genus, principally rhesus (M. mulatta) and cynomolgus (M. fascicu- laris) macaques. The epidemiology in its primate host is similar to that of HSV in humans, with 80% or more of natural and captive adult monkeys being infected. Infected monkeys may develop ves- icular oral lesions, and can shed virus intermittently from oral, con- junctival, and genital secretions. Rhesus and cynomolgus macaques have been quite widely used in medical research, particularly for the development of polio vaccine in the mid-​1950s, and in the late 1980s following the AIDS epidemic, for studies of retroviruses. Nearly all the reported human cases re- sulted from occupational exposure through bites and scratches in workers handling monkeys, but transmission from needlestick in- juries and a splash in the eye have also been reported. One case of human-​to-​human transmission apparently occurred by inoculation onto inflamed skin. Two clusters of infection have been described in the United States of America (in 1987, involving the case of human-​to-​human transmission, and 1989). A seroprevalence study of more than 300 monkey handlers showed that none was seropositive, and asymp- tomatic infection documented by seroconversion appears to be ex- tremely uncommon. Clinical features The incubation period, from occupational exposure to the devel- opment of symptoms, has usually been 3 to 5 days, but can range

8.5.2  Herpesviruses (excluding Epstein–Barr virus) 753 from 3 to 30 days. Cutaneous vesicles may occur at or near the site of inoculation, accompanied by regional lymphadenitis. In the first 2 weeks, fever, malaise, headache, and abdominal pain are common, but the dominant and characteristic features are progressive multi- focal haemorrhagic myelitis, and encephalitis. Visceral spread of herpes B virus is recorded in fatal cases. The untreated mortality is 80%. The history of monkey bite may lead to a suspicion of rabies (Chapter 8.5.10). It is not clear whether herpes B virus in humans can become latent and then be reactivated. Viral shedding has recurred when antiviral treatment was stopped relatively early, so most patients have been maintained on antivirals for long periods. Laboratory diagnosis As herpes B virus is a category 4 pathogen, viral culture and isolation are only attempted in a few designated laboratories: in the United Kingdom at the Central Public Health Laboratory, Colindale, London; and in the United States of America at Georgia State University, Atlanta. Monkeys with suspected infection should have serum antibody tests. Serodiagnosis in humans is difficult because of antigenic crossreactivity between herpes B virus and HSV. The inoculation site should ideally be biopsied for culture and analysis. PCR-​based methods are available in specialized centres, and are the standard for definitive diagnosis. Treatment Although injuries from macaques carry the risk of herpes B virus in- fection, most captive macaque colonies are now maintained free of the virus. A suspected contaminated wound should be debrided and cleaned with chlorhexidine or iodine soap. Postexposure prophy- laxis may be initiated if the monkey is suspected to be positive for herpes B virus, and there is skin puncture or mucosal exposure. There may be a case for initiating immediate antiviral treatment if infection in the monkey is suspected, or for a deep wound. Aciclovir and ganciclovir both inhibit herpes B virus replication in vitro. For postexposure prophylaxis, valaciclovir 1 g 8 hourly is re- commended for at least 2 weeks. If symptomatic disease is suspected or proven, intravenous aciclovir is recommended if CNS symptoms are absent (15 mg/​kg 8 hourly), and ganciclovir if CNS symptoms are present (5 mg/​kg every 12 h). Treatment has been associated with the limitation of disease, and recovery, in some patients, but pro- longed oral therapy with aciclovir or valaciclovir is advised to limit the risk of reactivation. Prevention and control Those working with macaques should follow standard procedures to avoid infection. The screening of newly imported monkeys, and the creation of colonies of macaques free of herpes B virus, are now becoming standard practice. FURTHER READING Herpes simplex virus infections Casanova JL, et  al. (2011). Human TLRs and IL-​1Rs in host de- fense: natural insights from evolutionary, epidemiological, and clin- ical genetics. Annu Rev Immunol, 29, 447–​91. Corey L, Wald A (2009). Current concepts:  maternal and neonatal herpes simplex virus infections. N Engl J Med, 361, 1376–​85. Lakeman FD, Whitley RJ (1995). Diagnosis of herpes simplex en- cephalitis:  application of polymerase chain reaction to cerebro- spinal fluid from brain-​biopsied patients and correlation with disease. NIAID collaborative antiviral study group. J Infect Dis, 171, 857–​63. Langenberg AGM, et al. (1999). A prospective study of new infec- tions with herpes simplex virus type 1 and type 2. N Engl J Med, 341, 1432–​8. Pellett PE, Roizman B (2007). The Herpesviridae: a brief introduc- tion. In: Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2479–​99. Lippincott, Williams and Wilkins, Philadelphia, PA. Roizman B, et al. (2007). Herpes simplex viruses. In: Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2501–​601. Lippincott, Williams and Wilkins, Philadelphia, PA. Zhang SY, et al. (2007). Human toll-​like receptor-​dependent induction of interferons in protective immunity to viruses. Immunol Rev, 220, 225–​36. Varicella-​zoster virus infection Cohen JI, et al. (2007). Varicella-​zoster virus. In: Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2773–​818. Lippincott, Williams and Wilkins, Philadelphia, PA. Cunningham AL, et  al. (2016). Efficacy of the herpes zoster sub- unit vaccine in adults 70 years of age or older. N Engl J Med, 375, 1019–​32. Gilden DH, et  al. (2000). Medical progress:  neurologic complica- tions of the reactivation of varicella-​zoster virus. N Engl J Med, 342, 635–​46. Gilden DH, et al. (2002). The protean manifestations of varicella-​zoster virus vasculopathy. J Neurovirol, 8 Suppl 2, 75–​9. Kimberlin DW, Whitley RJ (2007). Varicella-​zoster vaccine for the pre- vention of herpes zoster. N Engl J Med, 356, 1338–​43. Tseng HF, et al. (2016). Declining effectiveness of herpes zoster vaccine in adults aged >60 years. J Infect Dis, 213, 1872–​75. Wood MJ, et al. (1994). A randomised trial of acyclovir for 7 days or 21 days with and without prednisolone for treatment of acute herpes zoster. N Engl J Med, 330, 901–​5. Human cytomegalovirus infection Boeckh M, Ljungman P (2009). How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood, 113, 5711–​9. Crumpacker CS, Wadhwa S (2005). Cytomegalovirus. In:  Mandell GL, Bennett JE, Dolin R, (eds). Principles and practice of infec- tious diseases, pp. 1786–​801. Elsevier Churchill Livingstone, Philadelphia, PA. Hodson EM, et al. (2008). Antiviral medications for preventing cyto- megalovirus disease in solid organ transplant recipients. Cochrane Database Syst Rev, 2, CD003774. Kotton CN, et al. (2013). Updated international consensus guidelines on the management of cytomegalovirus in solid-​organ transplant- ation. Transplantation, 96, 333–​60. Mocarski ES, et al. (2007). Cytomegaloviruses. In: Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2701–​72. Lippincott, Williams and Wilkins, Philadelphia, PA. Pass RF, et al. (2009). Vaccine prevention of maternal cytomegalovirus infection. N Engl J Med, 360, 1191–​9. Sinclair J, Sissons JGP (2006). Latency and reactivation of human cyto- megalovirus. J Gen Virol, 87, 1763–​79.

8.5.20 Parvovirus B19 886

8.5.20 Parvovirus B19 886

886 section 8  Infectious diseases 8.5.20  Parvovirus B19 Kevin E. Brown ESSENTIALS Parvovirus B19 (B19V) is a small DNA virus that replicates in eryth- roid progenitor cells, with virus-​induced cytotoxicity stopping red cell production. It only infects humans, is endemic in most places, and is transmitted predominantly by the respiratory route. In healthy people it causes the rash illness, erythema infectiosum, also known as ‘fifth disease’ or ‘slapped cheek disease’, associated with minimal drop in haemoglobin, but in patients with increased red cell turnover (e.g. haemolytic anaemia or haemoglobinopathy), it causes transient aplastic crisis; in immunocompromised patients it causes chronic an- aemia; and following maternal infection it leads to hydrops fetalis or fetal loss. Treatment is supportive in most instances, but reduction in iatrogenic immunosuppression and/​or intravenous immunoglobulin may be appropriate in some cases. No vaccine is available. Introduction Parvovirus B19 (B19V) is a member of the Parvoviridae, small (c.22 nm), non​enveloped, icosahedral-​shaped viruses (Fig. 8.5.20.1), with a linear single-​stranded DNA genome of about 5000 nucleo- tides. At least five types of parvovirus infect humans: B19V; adeno-​ associated viruses; human partetraviruses (Parv4/​5) and bocaviruses, and the recently described human bufavirus. To date, only B19V and human bocavirus 1 (HBoV1) have definitively been shown to be a human pathogen. HBoV1 is a respiratory pathogen, associated with respiratory infections and wheezing in young children. Aetiology, pathogenesis, and pathology Based on viral sequence, B19V can be divided into three distinct genotypes (1, 2, and 3). Genotypes 2 and 3 are infrequently detected in Europe or the United States of America. No differences in patho- genicity are observed between the different genotypes, and they are all a single B19V serotype. B19V replication occurs primarily in erythroid progenitors, with the specificity in part due to the limited tissue distribution of the B19V receptor, blood group P antigen (globoside). Infection leads to high titre viraemia (>1012 virus particles/​ml or IU/​ml) (Fig. 8.5.20.2), and the virus-​induced cytotoxicity stops red cell production. In immunocompetent people, viraemia and arrest of erythropoiesis is transient, and resolves as the antibody response is mounted. In those with normal erythropoiesis, the drop in haemoglobin is minimal, but in patients with increased red cell turnover, infection induces a transient crisis with severe anaemia (Fig. 8.5.20.2b). Similarly, in the fetus or anyone who does not mount a neutralizing antibody response which halts the lytic infec- tion, erythroid production is compromised and patients develop chronic anaemia (Fig. 8.5.20.2c). The immune-​mediated phase of illness begins 2–​3 weeks postinfection as the IgM response peaks, and the rash of fifth dis- ease, arthralgia, and/​or frank arthritis appear. The B19 receptor is found on other cell types, including megakaryocytes, endothelial cells, placenta, myocardium, and liver. B19 infection at these sites may be responsible for some of the more unusual presentations. Rare people who lack P antigen are naturally resistant to B19V. Epidemiology B19V exclusively infects humans, and the virus is endemic in virtually all parts of the world. Transmission is predomin- antly via the respiratory route, prior to the onset of the rash or arthralgia. About 50% of 15-​year-​old children have detectable IgG, increasing to more than 90% of older people. In pregnant women there is an estimated annual seroconversion rate of ap- proximately 1%. The secondary infection rate within households approaches 50%. Prevention High titre B19V is not unusual in blood, and transmission occurs via transfusion, particularly of pooled components. B19V is resistant to heat and solvent/​detergent inactivation. Plasma pools Fig. 8.5.20.1  Typical appearance of parvovirus B19, with characteristic 22 nm icosahedral particles. Courtesy of Dr Hazel Appleton, Virus Reference Department, Public Health England.

8.5.20  Parvovirus B19 887 are currently screened by nucleic acid testing and high titre pools are discarded. Clinical features The clinical manifestation of B19V infection varies widely, depending on the host (Table 8.5.20.1). Most of these infections are asymptomatic. In healthy, immunocompetent people, B19 in- fections causes erythema infectiosum, also known as ‘fifth disease’ or ‘slapped cheek disease’ due to the characteristic facial rash which appears several days after a minor febrile prodrome. The rash may spread and develop a lacy reticular appearance, but the intensity and distribution of the rash varies and is difficult to distinguish from other viral exanthems. Rarely the rash can present as papular-​ purpuric gloves and socks syndrome; see Fig. 8.5.20.3. (a) 15 13 11 9 7 5 3 1 B19 virus 100 Clinical manifestations Clinical manifestations Clinical manifestations 10 8 6 4 14 Haemoglobin (g%) Reticulocytes (g%) 10 8 6 4 14 Haemoglobin (g%) Reticulocytes (g%) 10 0.2 0 4 14 Haemoglobin (g%) Reticulocytes (g%) 10 10 10 Days Fever, chills headache myalgia Rash arthralgia Inoculation or infection 6 2 20 Normals 10 Days Infection 6 2 20 TAC 10 Days Symptoms of anaemia Symptoms of anaemia Infection 6 2 20 PRCA 10 IgM and IgG IgG IgG (b) 15 13 11 9 7 5 3 1 B19 virus (c) 15 13 11 9 7 5 3 1 B19 virus IgM IgM 0 10 50 B19 antibodies 0 10 50 100 B19 antibodies 0 10 50 100 B19 antibodies Fig. 8.5.20.2  Schematic of the time course of B19 infection in (a) erythema infectiosum (EI), (b) transient aplastic crisis (TAC), and (c) pure red cell aplasia (PRCA) or chronic anaemia. The B19 virus titres are given in log 10 IU/​ml. From Young NS, Brown KE (2004). Parvovirus B19. N Engl J Med, 350, 586–​97. Copyright © 2004 Massachusetts Medical Society. Reprinted with permission. Table 8.5.20.1  Diseases associated with parvovirus B19 infection and methods of diagnosis Disease Host(s) Pathogenesis IgM IgG Quantitative PCR Fifth disease Healthy children Immune-​mediated Positive Positive

104 IU/​ml Polyarthropathy syndrome Healthy adults
(especially women) Immune-​mediated Positive within 3 months of onset Positive 104 within 3 months of onset Transient aplastic
crisis (TAC) Patients with increased erythropoiesis Erythroid cytotoxicity Often >1012 IU/​ml, but rapidly decreases Persistent anaemia/​ pure red cell aplasia Immunocompromised patients Impaired neutralizing antibody Negative/​weak positive Negative/​weak positive Often >1012 IU/​ml, but should be >106 IU/​ml in the absence of treatment Hydrops fetalis Fetus Erythroid cytotoxicity and impaired neutralizing antibody Positive amniotic fluid or tissue

888 section 8  Infectious diseases In adults, the ‘slapped cheek’ may not be apparent. Although uncommon in children, a symmetrical polyarthropathy, affecting the small joints of the hands and occasionally the ankles, knees, and wrists occurs in c.50% of adults, more often in women than men. Resolution usually occurs within a few weeks, but recurring symptoms can continue for months. Patients with increased erythropoiesis (i.e. those with haemo- lytic anaemia or haemoglobinopathy) develop transient aplastic crisis, with symptoms of acute anaemia. Bone marrow examin- ation reveals an absence of erythroid precursors and the presence of characteristic giant pronormoblasts. In the immunocompromised (i.e. patients with HIV, leukaemia, and following transplantation), B19 infection may lead to chronic anaemia or pure red cell aplasia. Patients have persistent anaemia with reticulocytopenia, absent or low levels of B19 IgG, high levels of B19 DNA in serum, and often scattered giant pronormoblasts in the bone marrow. Transient neutropenia, lymphopenia, and thrombocytopenia may be seen, and B19V occasionally causes a haemophagocytic syndrome. Infection with B19V during pregnancy can lead to hydrops fetalis and fetal loss. The risk of transplacental fetal infection is about 30%, and the risk of fetal loss, predominantly in the second trimester, 9%. The risk of congenital infection is less than 1%. Although B19V does not appear to be teratogenic, there are anecdotal reports of eye damage and central nervous system abnormalities. Cases of con- genital anaemia have also been described. B19V probably causes 10–​20% of all cases of non​immune hydrops. B19V infection is rarely associated with hepatitis, vasculitis, myocarditis, glomerulosclerosis, and central nervous system disease. Diagnosis In immunocompetent people, B19V infection is usually diag- nosed by the detection of B19 IgM antibodies (Table 8.5.20.1). IgM can be found at the time of rash in erythema infectiosum and by the third day of transient aplastic crisis in patients with haem- atological disorders. IgM remains detectable for about 3 months. B19 IgG appears by the seventh day of illness and remains for life. Detection of B19 DNA should be used for the diagnosis of early transient aplastic crisis or chronic anaemia. Although levels fall rapidly with the development of the immune response, low levels of DNA (103 IU/​ml or less can be detectable by polymerase chain reaction (PCR) for months and even years after infection, even in healthy people), so a quantitative PCR should be used for diag- nosis. At the height of viraemia, more than 1012 B19 DNA IU/​ml of serum can be detected, but titres fall rapidly within 2 days. Patients with aplastic crisis or B19-​induced chronic anaemia generally have more than 105 IU/​ml B19 DNA. Treatment No antiviral drug is available, and treatment is often only symptomatic. B19-​induced transient aplastic crisis may require blood transfusions, and intrauterine blood transfusion can prevent fetal loss in some cases of fetal hydrops. In patients on chemotherapy, stopping treat- ment temporarily may result in an immune response and resolution, but if unsuccessful or inapplicable, intravenous human normal im- munoglobulin may cure or improve persistent B19 infection. These patients and those with transient aplastic crisis should be considered infectious. Administration of immunoglobulin is not beneficial for erythema infectiosum or B19-​associated polyarthropathy. Prevention in the future No vaccine is currently approved for parvovirus B19. A  vaccine based on viral-​like particles expressed in yeast cells is in develop- ment and may overcome some of the problems with the earlier insect-​cell based vaccine. FURTHER READING Brown KE, et al. (1994). Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen). N Engl J Med, 330, 1192–​96. Chandramouli S, et al. (2013). Generation of a parvovirus B19 vaccine candidate. Vaccine, 31, 3872–​78. Kurtzman GJ, et al. (1987). Chronic bone marrow failure due to per- sistent B19 parvovirus infection. N Engl J Med, 317, 287–​94. Maple PA, et al. (2014). Identification of past and recent parvovirus B19 infection in immunocompetent individuals by quantitative PCR and enzyme immunoassays:  a dual-​laboratory study. J Clin Microbiol, 52, 947–​56. Young NS, Brown KE (2004). Parvovirus B19. N Engl J Med, 350, 586–​97. Fig. 8.5.20.3  A very unusual presentation of parvovirus B19 infection
is papular-​purpuric gloves and socks syndrome. From Gutermuth J, et al. (2011). Papular-​purpuric gloves and socks syndrome. Lancet, 378, 198, Copyright © 2011, with permission from Elsevier.

8.5.21 Hepatitis viruses (excluding hepatitis C vi

8.5.21 Hepatitis viruses (excluding hepatitis C virus) 889

8.5.21  Hepatitis viruses (excluding hepatitis C virus) 889 8.5.21  Hepatitis viruses (excluding hepatitis C virus) Matthew Cramp, Ashwin Dhanda, and
Nikolai V. Naoumov ESSENTIALS The group of hepatitis viruses includes five unrelated human viruses (A to E), which differ in their genome organization, biology, and epidemiology, while being united by their hepatotropism. About 10–​15% of cases of viral hepatitis are considered as non-​A to E hepatitis, whose aetiology is still unknown, but the search for which has led to the identification of several new viruses (e.g. hepatitis G virus or GB virus-​C, TT, and SEN viruses) of uncertain pathogenic significance. Clinical aspects of viral hepatitis are discussed in Chapter 15.22.1. Hepatitis A virus This is a single-​stranded RNA virus with four genotypes in humans. Hepatitis A virus replicates primarily in hepatocytes and is excreted via the biliary system into the faeces, where it can be found in high concentrations prior to clinical symptoms. Hepatitis A virus causes acute hepatitis with significant morbidity and occasional mortality. Antihepatitis A virus IgG remains detectable after acute infection and provides protective immunity. Hepatitis B virus Hepatitis B virus is the smallest human DNA virus. Ten genotypes, designated A to J, have been determined, with variable geograph- ical distribution. The virus is non​cytopathic, with virus-​specific cel- lular immunity being the main determinant for the outcome of infection and in those cases who resolve. An effective immune re- sponse controls hepatitis B virus replication and prevents liver dis- ease developing. The natural evolution of chronic infection includes four consecutive phases:  (1) early ‘immunotolerant’ phase—​high levels of virus replication and minimal liver inflammation; (2) im- mune reactive phase—​significant hepatic inflammation and ele- vated serum aminotransferases without viral clearance; with some patients progressing to (3) ‘non​replicative’ phase—​seroconversion to anti-​HBe; undetectable or low level of viraemia (below 2000 IU/​ml by polymerase chain reaction-​based assays); resolution of hepatic inflammation; and (4) HBeAg-​negative chronic hepatitis B—​due to the emergence of viral mutations; characterized by fluctuating serum hepatitis B virus DNA and serum alanine aminotransferase levels, and progressive liver disease. Hepatitis C virus See Chapter 8.5.22. Hepatitis delta virus This is a defective virus with a single-​stranded circular RNA genome. Eight genotypes have been determined in humans; genotype 1 is most common in the Western world and genotype 2 is predom- inant in East Asia. Hepatitis delta virus infection is always associ- ated with hepatitis B virus infection, either arising concurrently with hepatitis B virus as coinfection or after hepatitis B virus as super- infection. Clinical manifestations vary from acute to fulminant hepatitis and from an asymptomatic carrier state to progressive chronic liver disease. Diagnosis is based on the detection of serum HDAg and serum hepatitis D virus RNA. The optimal treatment of hepatitis delta virus is uncertain. Prevention is by vaccination against hepatitis B virus. Hepatitis E virus This is a single-​stranded RNA virus. Hepatitis E virus is widely dis- tributed and transmitted by the faeco-​oral route and may be zoo- notic, with evidence of infection in pigs, cattle, and sheep in endemic regions. It causes acute viral hepatitis which can be fulminant in those who are pregnant, malnourished, or have existing liver dis- ease. Chronic infection has been reported in solid organ transplant recipients, patients with haematological malignancies and HIV. Immunity can be transient and might wane if acquired in childhood. An effective vaccine has been developed but is not yet commercially available. Introduction Viral hepatitis (Fig. 8.5.21.1) is an ancient disease which re- mains a major health problem worldwide. Five viruses have been identified as aetiological agents and named A, B, C, D, and E (Table 8.5.21.1). These unrelated human viruses differ in their genome organization, biology, and epidemiology, while being united by their hepatotropism. Approximately 10–​15% of cases with viral hepatitis are considered as non-​A to E hepatitis and the aetiology is still unknown. The search for additional hepatitis agents led to the identification of several new viruses, named hepa- titis G virus (HGV or GB virus-​C), TT, and SEN viruses. These viruses have been detected in high proportions of the general population and their pathogenic role, if any, remains uncertain. Thus, the search for new hepatitis agents responsible for the small proportion of cases with cryptogenic hepatitis continues. Clinical aspects of viral hepatitis are discussed in Chapter 15.22.1. Fig. 8.5.21.1  Acute viral hepatitis with jaundice and subconjunctival haemorrhages. Copyright D. A. Warrell.

890 section 8  Infectious diseases Hepatitis A virus (HAV) HAV particles were first discovered by immune electron mi- croscopy in 1973 in stool samples of patients with hepatitis A. The virus is classified in the genus Heparnavirus of the family Picornaviridae. The genome of HAV is a single-​stranded, linear RNA of approximately 7474 nucleotides (Table 8.5.21.1). This includes a 5′ untranslated region (5′ UTR) of 742 nucleotides, followed by a single, long, open reading frame (ORF, 6681 nu- cleotides) which encodes a polyprotein of 2227 amino acids, and a short 3′ non​coding region (63 nucleotides). After translation, HAV polyprotein undergoes multiple cleavages by a virally-​ encoded enzyme, 3C protease. The polyprotein contains three functionally separate domains. At the N-​terminal end is domain P1 which includes the major structural polypeptides of HAV in Table 8.5.21.1  Main characteristics of hepatitis viruses Virus Family Morphology Genome Proteins Antibodies Pathogenesis Specific features HAV Picornaviridae 27 nm non-​ enveloped, spherical particles Single-​stranded linear RNA, 7474 nt Four capsid proteins, viral polymerase, and proteases Anti-​HAV Non-​ cytopathic virus Immune-​mediated acute hepatitis No chronic infection Effective vaccines available HBV Hepadnaviridae 42 nm particle with nucleocapsid (core) and outer envelope (surface) Partially double-​ stranded, circular DNA, 3200 nt Envelope Major protein (HBsAg) Middle protein (PreS2 + S) Large protein (PreS1 + S2 + S) Nucleocapsid (HBcAg) HBeAg non-​ structural, soluble protein Anti-​HBs Anti-​HBc Anti-​HBe Non-​ cytopathic virus Immune-​mediated acute and chronic hepatitis Weak T-​cell reactivity—​a dominant cause for persistent viral replication In chronic infection spontaneous evolution from HBeAg(+) to anti-​HBe(+) phase Mutant strains (surface, precore, polymerase) evolve under selection pressure DNA integration into host genome Transactivation of cellular genes Effective vaccines available 22 nm spherical and filamentous subviral particles Envelope proteins only Anti-​HBs HCV Flaviviridae 50–​60 nm enveloped spherical particles Single-​stranded linear RNA, approx. 9500 nt Structural Envelope 1 (E1) Envelope 2 (E2) Nucleocapsid (core) Anti-​E1 Anti-​E2 Anti-​core Usually non-​ cytopathic virus Neutralizing antibodies (?) High degree of virus heterogeneity (genotypes and quasi species) High propensity to chronic infection No integration in host genome Six major genotypes Non​structural NS2 NS3 NS4 NS5 Anti-​NS3 Anti-​NS4 Anti-​NS5 T-​cell reactivity—​ major role for resolution of acute infection HDV Resembles viroids and plant viruses 35–​37 nm enveloped particles Single-​stranded circular RNA, 1700 nt HD-​Ag (nucleocapsid) HBsAg (envelope) Anti-​HD Anti-​HBs Direct cytopathic and/​or immune-​ mediated liver injury Defective RNA virus Requires help from HBV for providing the envelope HBsAg HEV Caliciviridae 32–​34 nm
non​enveloped spherical particles Single-​stranded linear RNA, 7500 nt ORF1—​
non​structural proteins ORF2—​structural proteins ORF3—​unknown function Anti-​HEV Probably immune mediated (?) Enterically transmitted hepatitis mainly in Asia, Middle East, and Central America Chronic infection can be seen in immunocompromised HGV/​GBV-​C Flaviviridae ? Single-​stranded linear RNA, 9400 nt Conserved E2 No core protein Anti-​E2 Primary site of replication unknown Does not cause hepatitis Can establish chronic infection No clear pathogenic role TTV Circinoviridae (?) ? Single-​stranded, circular DNA, approx. 3850 nt ? ? Does not cause hepatitis Can establish chronic infection High degree of virus heterogeneity No clear pathogenic role NS, non​structural; nt, nucleotides; ORF, open reading frame.

8.5.21  Hepatitis viruses (excluding hepatitis C virus) 891 the following sequence—​VP2, VP3, and VP1. A fourth very small polypeptide, VP4, which is presumed to be involved in HAV capsid formation, is located at the extreme N-​terminal end of the polyprotein. These four structural polypeptides assemble into a viral capsid containing 60 copies of each. It is not known how the viral RNA is incorporated into the virion, but both empty and RNA-​containing capsids have been observed in most virus pre- parations. The other P2 and P3 domains of the viral polyprotein include at least six separate proteins which are involved in viral replication. These include 2B and 2C helicase, 3A and 3B pro- teins, 3C (the viral protease), and 3D (an RNA-​dependent RNA polymerase). Hepatocytes are the predominant site of HAV replication in vivo. Recent data indicate that HAV might also replicate within the epithelial cells of the gastrointestinal tract. However, the mechanism by which HAV reaches the liver remains unknown. The maximal HAV replication in hepatocytes occurs before serum aminotransferases rise. The virus is excreted via the biliary system into the faeces where it can be found in high concentrations around 1–​2 weeks before the onset of clinical symptoms. Viraemia is pre- sent from the earliest phase of infection and is due to HAV repli- cation within hepatocytes. HAV differs from other picornaviruses because of its non​cytolytic replication. Liver injury is immune me- diated by natural killer cells, virus-​specific CD8+ cytotoxic T cells, and non​specific inflammatory cells recruited to the liver. At the onset of clinical symptoms there is a humoral immune response and antibodies to structural HAV proteins (anti-​HAV) are detect- able in patients’ serum. Initially, these are mainly IgM antibodies (IgM anti-​HAV) which usually persist for approximately 6 months. During convalescence, anti-​HAV IgG becomes predominant and remains detectable indefinitely, representing protective immunity to HAV. An effective vaccine generating lasting protective im- munity is widely available. Hepatitis B virus (HBV) Two discoveries related to HBV mark the beginning of the under- standing of hepatitis viruses. In 1965, Baruch Blumberg identified the hepatitis B surface antigen (HBsAg) of HBV, initially termed ‘Australia antigen’, and in 1970 the complete virion (a 42 nm par- ticle) was identified by Dane and colleagues using electron micros- copy. HBV belongs to a virus family named Hepadnaviridae, which includes similar hepatotropic DNA viruses specific for woodchucks, ground squirrels, and Pekin ducks. An estimated 240 million people are chronically infected with HBV globally, mainly in East Asia and sub-​Saharan Africa, where up to 10% of the adult population is infected. Genome organization The HBV genome contains only 3200 nucleotides and is the smallest DNA virus (Table 8.5.21.1). One of the DNA strands, known as the ‘minus’ strand, is almost a complete circle and contains four overlapping ORFs encoding enveloped (pre-​S/​S), core (precore/​ core), polymerase and X proteins (Fig. 8.5.21.2). The other (‘plus’) strand is shorter and varies in length. The envelope ORF contains three start codons which separate the pre-​S1, pre-​S2, and S regions, encoding the large (L), middle (M), and small (S) envelope proteins respectively. The surface gene encodes the major envelope protein (HBsAg), which has 226 amino acids. The translation product of the pre-​S2 and S gene is the middle en- velope protein and the product of pre-​S1, pre-​S2, and S gene is the large envelope protein. In addition to the complete virion, a much greater amount of non​infectious, 22 nm in diameter, spherical, and 1800 1600 1400 1200 1000 800 600 400 200 3182 3000 2800 2600 2400 2200 2000 1814 EcoRI 0 DR 1 Poly A S1P S2P – + Oligoribo- nucleotide 5′ Enh I XP DR 2 Enh II CP Pre-core/core gene Polymerase gene X-gene Surface gene Pre-S2 Pre-S1 Fig. 8.5.21.2  Schematic representation of hepatitis B virus genome. CP, core promoter; DR1, direct repeat 1; DR2, direct repeat 2; EcoRI, restriction site for EcoRI enzyme used as a starting point for numbering; EnhI, enhancer I; EnhII, enhancer II; S1P, pre-​S1 promoter; S2P, pre-​S2 promoter; XP, X gene promoter.

892 section 8  Infectious diseases filamentous subviral particles are produced in infected hepatocytes. HBsAg and the middle envelope protein are present in all viral and subviral particles, while the large protein is present in the virions and in some subviral filaments. The pre-​S1 domain of the large envelope protein is a key determinant for binding and recently the sodium taurocholate cotransporting polypeptide (NTCP), a multiple trans- membrane transporter predominantly expressed in the liver, has been shown to be the receptor on the plasma membrane of hepatocytes. The precore/​core ORF has two start codons which encode two closely related proteins. Translation from the preC start codon produces a precursor molecule, designated precore protein. In the endoplasmic reticulum this protein undergoes two proteolytic steps at the N-​ and at the C-​terminal ends, and the resultant polypeptide is secreted from hepatocytes as hepatitis B e-​antigen (HBeAg). This is a non​structural protein, which is not essential for viral replica- tion. Translation from the C start codon results in the nucleocapsid protein (HBcAg), which has 183 amino acids. In the cytoplasm of hepatocytes HBcAg assembles spontaneously into nucleocapsid particles. HBeAg and HBcAg share about 90% of the amino acids but differ substantially in their conformation. The polymerase ORF encodes the HBV polymerase protein with 832 amino acids. It has three functional domains—​terminal pro- tein, reverse transcriptase, and RNAse H activity. The X ORF en- codes a protein with 154 amino acids. Recent studies have shown that the X protein is essential for viral replication through degrad- ation of the Smc5/6 restriction factor. Ten genotypes of HBV (designated with the letters A to J) have been determined. The variations involve approximately 10% of the genome. Data on the geographical distribution indicate that geno- type A is predominant in central and northern Europe, genotypes B and C in Asia, genotype D in the Mediterranean basin, and genotype E in Africa. Viral replication Following HBV entry into hepatocytes, the nucleocapsid is trans- ported to the nucleus (Fig. 8.5.21.3). Cellular enzymes repair the open circular HBV DNA into covalently closed circular DNA (cccDNA), which serves as a template for the synthesis of pregenomic and mes- senger RNAs. Viral DNA does not integrate into the host genome as part of the normal replication cycle. The pregenomic RNA is trans- ported to the cytoplasm and serves as mRNA for translation of new core and polymerase proteins. When these three components (pregenomic RNA, core, and polymerase proteins) reach sufficient quantities, they assemble into nucleocapsid particles, with the polymerase protein being directly involved in the pregenomic RNA encapsidation. Inside the particles the pregenomic RNA is reverse transcribed into DNA ‘minus’ strand, while the RNA template is simultaneously degraded by RNAse H. Finally, the ‘plus’ strand is produced which completes a new, partially double-​stranded, HBV DNA. Some of the newly synthesized nucleocapsids with HBV DNA are transported back to the nucleus, which maintains a stable pool of cccDNA. Others are enveloped and leave the cell as new virions. cccDNA persists in the nucleus of infected hepatocytes, even after HBsAg and HBeAg loss, and is considered to be the main mechanism of HBV chronicity. The replication strategy used by hepadnaviruses differs from that of retro- viruses in two main aspects: 1) integration into the host genome is not obligatory during replication; 2) functional mRNAs are produced from several internal promoters of the circular DNA genome. Recent progress in the understanding of the mechanisms of viral entry and replication have identified a variety of new targets for drug therapy that are directed towards a functional cure (sustained loss of HBsAg with or without surface antibody seroconversion). These in- clude entry inhibitors, inhibitors of cccDNA formation and transcrip- tion and targeting the X protein and intracellular trafficking. See Lok et al reference in Further Reading for a contemporary detailed review. 3.5 kb RNA pregenome Subgenomic RNAs 2.4 kb 2.1 kb Envelope proteins NUCLEUS ccc DNA Core protein P protein Reverse transcriptase (–) Strand DNA DNA polymerase (+) Strand DNA Endoplasmic reticulum Subviral particles HBV HBV Fig. 8.5.21.3  Replicative cycle of hepatitis B virus.

8.5.21  Hepatitis viruses (excluding hepatitis C virus) 893 Host immune response and pathogenesis HBV is a non​cytopathic virus. The virus-​specific cellular immune response mainly determines the outcome of infection. Both human leukocyte antigen class  I  and class  II-​restricted T-​cell responses are strong and directed to multiple viral antigens in patients with acute self-​limited hepatitis B. Despite clearance of serum HBsAg, HBV DNA remains detectable by polymerase chain reaction (PCR) in most cases, and HBV-​specific CD4+ and CD8+ T-​cell reactivity has been demonstrated 10–​20 years after the time of acute infection. Cytokines released from these cells, especially interferon-​γ, have been shown to exert a non​cytolytic inhibition on HBV replication without causing cell death. Thus, eradication of HBV may be rare, but an effective immune response controls HBV DNA expression and there is no liver disease. Patients with chronic HBV infection (defined by detection of HBsAg in serum for longer than 6 months) show weak virus-​specific T-​cell reactivity, which is the dominant cause for HBV persistence. This ineffective response, together with antigen non​specific inflammatory cells recruited at the site of in- flammation, is responsible for the progression of liver damage. The humoral immune response involves antibodies directed at different HBV antigens (Table 8.5.21.1). The clinical significance is based on several aspects: (1) diagnosis—​the antibody profile in the serum, together with the result of HBsAg and HBeAg, is used to define different phases of HBV infection; (2) prophylaxis—​the development and the level of the protective antibody (anti-​HBs) is used to monitor the response to vaccination; (3) pathogenesis—​the humoral immune response contributes to viral elimination from the circulation by forming immune complexes. In some cases, the tissue deposition of antigen-​antibody complexes is responsible for extrahepatic pathology such as glomerulonephritis, polyarteritis nodosa, arthritis, and skin changes. Evolution of chronic HBV infection HBV-​host interactions change over time, typically in four consecu- tive phases, which are characterized by different levels of HBV rep- lication and associated liver disease. The early ‘immunotolerant’ phase with HBeAg positivity, and high levels of viral replication with HBV DNA levels as high as 107 or 108  IU/​ml, but normal liver enzymes, usually occurs in infants after vertical or peri-​natal transmission and lasts until adolescence. This phase is often short-​ lived or absent when HBV is contracted during adulthood. It is associated with minimal liver inflammation or fibrosis. However, recent studies have suggested this phase may not be as benign as previously thought; there have been reports of histologically ac- tive chronic hepatitis in ‘immunotolerant’ children and there is an absence of a tolerogenic T-​cell pattern. In early adulthood, there is enhanced immune reactivity to the virus, characterized by sig- nificant hepatic inflammation, elevated or fluctuating levels of serum aminotransferases, and rapid progression of hepatic fibrosis. HBeAg remains positive and HBV DNA level is usually lower but still greater than 2000 IU/​ml. This immune reactive HBeAg posi- tive phase can last for weeks to years. Currently, antiviral therapy is only recommended during this immune active stage of the in- fection. Some patients will progress spontaneously to the next ‘non​replicative’ or low-​replicative phase, manifested by serocon- version to anti-​HBe, undetectable or less than 2000  IU/​ml vir- aemia, and resolution of hepatic inflammation. In a proportion of patients, HBeAg loss may be due to the emergence of mutations in the core promoter and/​or in the precore region (usually the G1896A stop codon), which prevent the translation of HBeAg. These HBe-​ minus mutants are replication competent and when viraemia levels are high, they cause HBe-​negative chronic hepatitis B. The latter is characterized by fluctuating serum HBV DNA levels, mirrored by serum alanine aminotransferase (ALT) fluctuations, and progres- sive liver disease. Finally, the patient might achieve HBsAg loss to enter an ‘occult’ phase of chronic infection. During this phase anti-​ HBc antibodies with or without anti-​HBs antibodies are detected. There might be low levels of viral replication although HBV DNA is generally undetectable. Replication-​competent HBV genomes remain within hepatocytes and immune reactivation can occur in response to immunosuppression. Hepatitis C virus (HCV) (See Chapter 8.5.22.) Hepatitis D virus (HDV) HDV is a defective virus that causes acute and chronic liver disease only in association with hepatitis B virus. This unique pathogen was discovered in 1977 by Mario Rizzetto in liver biopsies from patients with hepatitis B. HDV particles contain the viral RNA nucleocapsid, which is hepatitis delta antigen (HDAg), and an outer envelope (HBsAg), which is provided by the helper virus HBV. The HDV genome is a single-​stranded, circular RNA (Table 8.5.21.1), and is the smallest known animal virus genome. Because of a high degree of internal complementarity, 70% of the nucleotides are base-​paired. This gives an unusual, rod-​like structure of the HDV genome. HDV RNA replicates via RNA-​directed RNA synthesis by transcription of genomic RNA to a complementary antigenomic delta RNA. The latter serves as a template for subsequent genomic RNA synthesis. HDV produces a single protein, hepatitis delta antigen (HDAg), which is encoded by the antigenomic RNA. RNA editing of the antigenomic RNA allows the virus to make two forms of HDAg—​ small (HDAg-​S, 195 amino acids) and large (HDAg-​L, 214 amino acids). Both forms are present in the virions and have different func- tions in the HDV replicative cycle. HDAg-​S facilitates HDV RNA replication, while HDAg-​L inhibits replication and is required for assembly of the virion. Although the formation of delta virions re- quires the helper function of HBV, the replication of HDV RNA within the cell can occur without HBV. Global coinfection prevalence is estimated at 5% but this might be an underestimate due to a lack of standardized testing for HDV in HBV-​infected patients. Prevalence in some parts of Europe has recently been reported to be increasing, probably due to patterns of immigration from endemic regions, while in other countries, such as Taiwan, it is decreasing due to an active hepatitis B immunization programme and systematic screening of blood products. Eight phylogenetically distinct HDV genotypes have been identi- fied. The most widespread is genotype 1, identified in Africa, Asia, Europe, and North America, which is associated with a broad spec- trum of chronic liver disease with chronic infection established in 70–​90% of patients. Genotype 2 is found only in East Asia and seems to cause mild hepatitis delta. Genotype 3 is found exclusively in nor- thern parts of South America and is associated with particularly severe hepatitis. At least five additional HDV genotypes have been

894 section 8  Infectious diseases described; their clinical features are less well characterized than genotypes 1 to 3. Clinical manifestations vary from acute to fulminant hepatitis and from an asymptomatic carrier state to progressive chronic liver dis- ease. Clinical outcome might be related to different HDV genotypes. Persistent HDV replication is associated with annual rates of devel- opment of cirrhosis and hepatocellular carcinoma of 4% and 2.8%, respectively. Diagnosis is based on the detection of serum HDAg (detectable in acute infection), serum HDV RNA, and anti-​HDV antibodies. In practice, HDV RNA is the most reliable and available test since standardized methods to analyse HDAg and anti-​HDV antibodies do not exist. The optimal treatment of HDV is uncer- tain. Interferon-α is the only licensed treatment, but responses have been poor. Pegylated interferon might be better, with randomized controlled trials demonstrating a sustained virological response in 17–​43% of patients after 6 months of treatment. Addition of nucleo- side analogues does not appear to be beneficial. Prevention of HDV is by vaccination against HBV. Host immune response and pathogenesis HDV can infect a person either simultaneously with HBV (coinfection) or as superinfection of a person with chronic HBV infection. Because HDV requires the helper function of HBV, the duration of delta infection is determined by the duration of HBsAg positivity. Analogous to the antibodies to HBV nucleocapsid (anti-​ HBc), antibodies to HDAg are not protective. Chronic HDV infec- tion is accompanied by high titres of IgG anti-​HD. A high serum level of IgM anti-​HD indicates acute delta infection or exacerbation of chronic hepatitis D. The relative role of cellular immune reac- tions to HDAg, HBV antigens, or both in the immunopathogenesis of hepatitis D is not fully understood. The lack of liver pathology in transgenic mice expressing HDV and data from experimental infections suggest that HDV is not cytopathic. This is supported by the experience with patients undergoing liver transplantation for HDV cirrhosis. Although HDV recurs universally in the graft, necroinflammation is absent unless HBV recurs as well. The pres- ence of microvesicular steatosis in severe hepatitis D indicates a possible direct cytopathic effect in some circumstances. Hepatitis E virus (HEV) HEV was first identified in 1983 by immune electron microscopy in the faeces of patients and classified in the genus Hepevirus, family Hepeviridiae. HEV is an icosahedral, non​enveloped single-​ stranded RNA virus that is 27–​34  nm in diameter. The HEV genome is approximately 7500 nucleotides and contains three ORFs (Table 8.5.21.1). ORF1 encodes non​structural proteins involved in virus replication—​helicase and RNA-​dependent RNA polymerase. ORF2, comprising approximately 2000 nucleotides, codes for the major structural proteins. ORF3 has 328 nucleotides and also ap- pears to code for a structural protein. The genomic organization of HEV is different from HAV and HCV because the structural and non​structural proteins are coded by discontinuous, partially overlapping ORFs. HEV is widely distributed with the highest incidence of infection in Asia, Africa, the Middle East, and Central America. In developed countries including the United Kingdom, France, and Japan, acute HEV is more common than HAV. The seroprevalence of HEV was thought to be low at 1–​2% but recent epidemiological data using improved assays suggest a wide geographic variation with sero- prevalence ranging from 4.8 to 16% in the United Kingdom, up to 40% in the southwest of France. In developing countries seropreva- lence rates are around 30% but there is variation depending on the population studied. It is spread by the faeco-​oral route in endemic areas. Person-​to-​person transmission is uncommon. HEV can be transmitted by blood transfusion, particularly in endemic areas. HEV RNA was found in 0.7% of pooled plasma from donors from England and is estimated to be present in 1 in 3200 to 7000 blood donors in Europe. Unlike HAV, HEV infection might be zoonotic. HEV RNA has been found in the faeces of wild pigs and serological evidence of infection was found in pigs, cattle, and sheep in endemic regions. Transmission has been reported in the context of consump- tion of undercooked meat. Four genotypes of HEV have been identified, which show 25% nucleotide variability. Geographically, genotype 1 has been isolated from tropical countries in Asia and Africa and is responsible for large outbreaks and epidemics. Genotype 2 was found in Mexico, whereas genotype 3 has worldwide distribution, including America, Asia, and Europe and is responsible for the non​travel associated sporadic infections seen in these endemic areas. Genotype 4, in con- trast, has been found only in Asia. Genotypes 3 and 4 infect humans as well as pigs and other mammals while genotypes 1 and 2 are found only in humans. Vaccine development has been helped as all HEV strains share at least one major, serologically cross-​reactive epitope. HEV usually causes an acute self-​limited infection, although ful- minant infection can occur. Fulminant infection is more common in pregnancy, malnutrition, and in those with pre-​existing liver dis- ease. In pregnancy, acute HEV genotype 1 and 2 infections are as- sociated with a high maternal mortality of 20–​25% due to obstetric complications or fulminant hepatic failure, as well as a high rate of stillbirth. The same obstetric risk is not apparent for HEV genotype 3 and 4 infections, the reasons for which are not clear. Chronic HEV infection, defined as positive HEV RNA for at least 6 months, is well described in solid organ transplant recipients, in immunosuppressed individuals in the context of haematological malignancy or HIV infection. Chronic HEV can result in significant chronic liver disease. Treatment with ribavirin for 3 months can be effective, with a recent retrospective case series reporting a sustained virological response in 78% of cases. The primary site of HEV replication is not fully understood. Following intravenous HEV inoculation in experimental models, serum aminotransferases levels rise after 24–​38 days. Expression of HEV antigens has been detected in the cytoplasm of hepato- cytes as early as 7 to 10 days after inoculation. Experimental data indicate that during an initial phase with high HEV replication the virus might be released from hepatocytes into bile, which occurs be- fore the elevation of liver enzymes and morphological changes in the liver. The virus shedding appears to end with the normalization of serum aminotransferases. HEV RNA is detectable in the stool 1 week before the onset of illness and persists for 2 weeks afterwards. HEV RNA is detectable in the serum by real-​time PCR of virtu- ally all patients within 2 weeks of the onset of hepatitis. Prolonged periods of viraemia, between 4 to 16 weeks, have also been reported. The detection of anti-​HEV by enzyme immunoassays, involving recombinant HEV antigens or synthetic peptides, is the most fre- quently used method for diagnostic purposes and for epidemio- logical studies. However, these tests are unreliable in the diagnosis

8.5.21  Hepatitis viruses (excluding hepatitis C virus) 895 of chronic HEV infection in immunocompromised individuals and HEV RNA testing is recommended. During acute infection, the humoral immune response gradually develops in parallel with the ALT rise. The serum level of anti-​HEV IgM reaches a maximal titre around the time of peak ALT levels and is detectable for 5–​6 months. Although the IgG anti-​HEV response persists for several years after the acute phase, the natural history of protective immunity to HEV is not fully established. In contrast to HAV, hepatitis E shows an unusually high attack rate among adults, suggesting that immunity to HEV, if acquired in childhood, may wane. Vaccines against HEV are being developed and two large ran- domized controlled trials have shown 96% preventive efficacy in en- demic settings. A vaccine has been licensed in China for use in high risk populations since 2011 but is not yet recommended in a 2015 World Health Organization position paper due to lack of safety and efficacy data in individuals less than 16 or over 65 years old, as well as no long-​term efficacy data. There is no evidence for the efficacy of pre-​ or postexposure prophylaxis with immune globulin for the prevention of HEV. New hepatitis-​associated viruses GB virus-​C (GBV-​C) or hepatitis G virus (HGV) The genome of GBV-​C was identified in 1995 by molecular hybrid- ization techniques in the serum of a patient with the initials GB. In parallel, another group of investigators identified the genome of a new RNA virus, named hepatitis G virus. The comparison of HGV and GBV-​C genomes revealed very high homology, both at nucleotide (86%) and amino acid level (100%). It is now accepted that they represent two isolates of the same virus. GBV-​C/​HGV is an RNA virus with a single ORF encoding a polyprotein of ap- proximately 3000 amino acids (Table 8.5.21.1). Together with another two RNA viruses, GBV-​A and GBV-​B, it belongs to the Flaviviridae and these three viruses show various similarities with HCV. Specific features of the GBV-​C/​HGV genome include ab- sence of core gene (nucleocapsid); long 5′ and 3′ NTR and lack of poly A tail. Unlike HCV, this virus has a very conserved E2 re- gion. GBV-​C has a global distribution with a high prevalence in the North American blood donor population. Longitudinal studies have shown that GBV-​C/​HGV can establish chronic infection with RNA persistence in serum for up to 15 years. A proportion of pa- tients clear the virus spontaneously and develop anti-​E2 reactivity, which is used as a marker of past infection. Anti-​E2 also seems to confer protective immunity. A large body of evidence suggests that GBV-​C/​HGV does not cause liver disease. Evidence suggests a protective effect of GBV-​C in patients coinfected with HIV. The protective effect might be related to maintenance of an intact T-​ helper 1 cytokine profile, induction of HIV-​1 inhibitory cytokines and interference with HIV-​1 replication; the therapeutic implica- tions of these findings remain unclear. TT virus (TTV) TTV was identified in 1997 by investigators in Japan. By applying the methodology used for the identification of GBV-​C, they de- tected the genome of a new DNA virus in the serum of a patient with cryptogenic posttransfusion hepatitis. The patient’s initials (TT) prompted the name of this new virus and a causative role for acute and chronic hepatitis was suggested. TTV and its smaller variant are now spelt as Torque teno virus (TTV) and Torque teno mini virus (TTMV), after the Latin for ‘thin necklace’. The TTV genome is circular, single-​stranded DNA of approxi- mately 3850 nucleotides (Table 8.5.21.1). Three partially open reading frames have been predicted, but TTV proteins have not been expressed so far. It is suggested that TTV belongs to the genus Anellovirus in the Circinoviridae family. TTV DNA has been de- tected in non​human primates and in farm animals. The primary site of TTV replication and the biological nature of TTV are still unknown. Unlike other DNA viruses, TTV shows remarkable genomic vari- ability. Phylogenetic analyses of TTV isolates have identified at least 20 genotypes, which differ between each other by more than 40% of the DNA sequences. As recombinant viral proteins are not available, the diagnosis of TTV infection is based on the detection of TTV DNA by PCR. TT virus population is very heterogeneous, and fre- quently a mixed infection with 3 to 5 TTV genotypes is present in one patient. TTV infection is ubiquitous in more than 90% of adults world- wide. The virus was initially thought to have mainly a parenteral route of transmission, although the high prevalence of TTV in- fection in the general population indicates the importance of non-​ parenteral routes as well. The prevalence of TTV infection was shown to increase with age in paediatric and adult groups. The pathogenic role of TTV, if any, is unknown. Analysis of liver histology in patients with TTV infection, longitudinal studies, as well as experimental TTV inoculation in chimpanzees all demon- strate that this virus does not cause hepatitis. Possible associations with other diseases, such as severe idiopathic inflammatory myop- athies, systemic lupus erythematosus, pancreatic cancer, diabetes mellitus, laryngeal cancer, and periodontal disease have been re- ported. TTV may replicate in the respiratory tract of children and has been implicated in acute respiratory diseases in infants and ex- acerbations of asthma and bronchiectasis. However, TTV remains an example of a human virus with no clear disease association. SEN virus (SEN-​V) SEN-​V is a recently discovered single-​stranded DNA virus, distantly related to TTV, with a worldwide distribution. Eight genotypes of SEN-​V, designated A to H, have been identified. SEN-​V is trans- mitted via transfusion of blood products and parenteral contact. Interest in SEN-​V was triggered by the initial reports that two SEN-​ V genotypes, SEN-​V-​D and H, were associated with posttransfusion non-​A, non-​E hepatitis. No causative agent and no evidence of hepa- titis due to SEN-​V infection have yet been established. FURTHER READING Cristina J, Costa-​Mattioli M (2007). Genetic variability and molecular evolution of hepatitis A virus. Virus Res, 127, 151–​7. Hino S, Miyata H (2007). Torque teno virus (TTV): current status. Rev Med Virol, 17, 45–​57. Kamar N, et al. (2014). Ribavirin for chronic hepatitis E virus infection in transplant recipients. N Engl J Med, 370, 2447–​8. Kamar N, et al. (2017). Hepatitis E virus infection. Nat Rev Dis Primers, 3, 17086.

8.5.22 Hepatitis C virus 896

8.5.22 Hepatitis C virus 896

896 section 8  Infectious diseases Lok AS, et al. (2017). Hepatitis B: from discovery to regulatory ap- proval. J Hepatol 67, 847–61. Rehermann B (2013). Pathogenesis of chronic viral hepatitis: differen- tial roles of T cells and NK cells. Nat Med, 19, 859–​68. Rehermann B, Nascimbeni M (2005). Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol, 5, 215–​29. Rizzetto M (2016). The adventure of delta. Liver Int, 36 Suppl 1, 135–​40. Taylor JM (2006). Structure and replication of hepatitis delta virus RNA. Curr Top Microbiol Immunol, 307, 1–​23. 8.5.22  Hepatitis C virus Paul Klenerman, Katie J.M. Jeffery, Ellie J. Barnes,
and Jane Collier ESSENTIALS Hepatitis C virus is a major cause of liver disease worldwide. It is estimated that globally 170 million people are affected. Infection is parenteral. In many countries most recent acquisition is in people who inject drugs, but transmission worldwide also occurs in in healthcare settings due to the reuse or inadequate sterilization of medical equipment and the transfusion of unscreened blood and blood products. Sexual transmission also occurs, particularly in men who have sex with men. The virus has a tendency to be- come persistent in most of those infected. However, a substantial minority (around 25%) do clear the virus as a result of effective in- nate and adaptive immune responses at the time of acute infec- tion. In those with persistent infection, the clinical course is quite variable. Most individuals will develop some degree of hepatic in- flammation and fibrotic liver disease, of whom a fraction will go on over time do develop cirrhosis, with an excess risk of hepatocellular carcinoma. Cofactors that predispose to increased progression in- clude coinfection with HIV and alcohol use. Historically therapy was a combination of Interferon-α, delivered weekly as a pegylated compound, and ribavirin. Cure rates were 50–​80% dependent on viral genotype, with significant side effects. Recently, oral therapies targeted against viral gene products such as protease, and NS5A and NS5B polymerases given for 2–​3 months are much better tol- erated and have increased cure rates to greater than 90% across a range of viral genotypes. Introduction Hepatitis C virus (HCV) is a major global pathogen. The only known natural host is man, although it has been possible to in- fect chimpanzees experimentally. The origin of the virus in human populations is not well established, but the huge genetic diversity and global distribution, together with analyses of the viral mo- lecular clock, suggest that it has coevolved with human populations for centuries. However, recent spread through changes in med- ical practice and intravenous drug use have created an emerging health problem, recognized since the 1990s. The capacity of the virus to persist in the face of host innate and adaptive immune responses has made it difficult to develop vaccines. Recently there have been major improvements in the efficacy of treatment regi- mens, but these are still expensive, and some are associated with major side effects. A key task is to identify those who are infected, and those most likely to benefit from the available therapies, taking into account the observed progression and the likely response to treatment. Historical perspective The presence of HCV as an infectious entity, previously known as non-​A, non-​B hepatitis, had been recognized for many years before its discovery by Kuo and Houghton in 1988. It was quickly appreciated that HCV was a major infectious agent and the de- velopment of antibody-​based assays allowed an assessment of its prevalence to be made, as well as allowing the development of screening tools for blood products. Molecular techniques for detection of viral RNA in blood identified most of those infected as chronic viral carriers, while sequencing and bioinformatics approaches led to the description of diverse viral genotypes. The inability to culture the virus proved a major obstacle to be over- come, but the development of a replicon system by Bartenschlager in 1999 proved a significant breakthrough, allowing a dissection of viral replication in vitro. However, no infectious virus system was available until 2005, when several groups took advantage of an unusual Japanese strain (JFH-​1), to develop cell culture infec- tious systems. Aetiology, genetics, pathogenesis, and pathology HCV is a positive-​sense, single stranded RNA virus. It is classed in- dividually as a Hepacivirus and genetically related to flaviviruses, such as dengue. Related hepaciviruses have been found in dogs, horses and mice, and a human virus that is closely related to both hepaciviruses and human pegiviruses (human hepegivirus 1) has re- cently been identified in blood transfusion recipients. The viral RNA genome is approximately 10 kb in length and comprises a long single open reading frame. The genome is typ- ically divided into structural and non​structural proteins. The structural proteins–​contained within virions—​comprise core and envelope (E1 and E2). The latter are glycosylated, form a heterodimer, and are important targets for antibodies. They are also highly variable and contain sites known as hypervariable re- gions (HVR 1 and 2), which evolve rapidly under antibody se- lection pressure. The non-​structural proteins contain enzymes with defined protease and helicase activity, as well as a viral polymerase. Viral replication is initiated using an internal ribosomal entry site in the 5’ untranslated region (5’UTR). The latter is a highly conserved area, which varies slightly between genotypes and thus has become an important target for molecular diagnostics. The

8.5.22  Hepatitis C virus 897 polymerase replicates the virus through a double stranded inter- mediate, which is a substantial trigger for host innate responses. However, the virus can disable triggering of one of these pathways (RIG-​I) through the action of the protease, which cleaves a cellular target (CARDIF). One further important feature of replication is that it is highly error-​prone, thus within any one individual the virus exists as a swarm of closely related variants, sometimes de- scribed as ‘quasispecies’. HCV replicates largely in hepatocytes. The existence of virus in other cell types, including lymphocytes and dendritic cells, and within the central nervous system, has been described. However, the contribution of such sites to disease pathogenesis has not been defined. Several cellular receptors for HCV have been described. These include CD81 (a member of the tetraspanin family with signalling properties on lymphocytes), Claudin 1, Occludin, the low-​density lipoprotein receptor, DC-​Sign, and a macrophage scav- enger receptor. After natural or experimental infection, there is a substantial period, lasting weeks to months, where virus may be detect- able, without any apparent clinical, biochemical, or immuno- logical disturbance. During this time, virus may replicate to high levels in blood and within the liver, indicating that the direct cytopathic effects of the virus, in the absence of host immune responses, are minimal. This silent phase is followed by the onset of acute hepatitis, which may or may not be clinically apparent. Detailed intrahepatic studies in animal models (not possible in man), reveal that the first responses at this stage of infection are mediated by innate immune mediators (interferons, NK cells). Polymorphisms in the region of interferon-​lambda 3 (IL28B) have a major impact on spontaneous resolution, suggesting a critical role for this cytokine in early viral control. This innate response is followed by an influx of T cells (both CD4 + and CD8 +). In studies of human acute hepatitis C, the emergence of highly activated virus-​specific CD8 + T cells correlates quantitatively and temporally with the peak of the alanine transaminase (ALT). This suggests that at this stage the tissue damage is largely a result of the host T-​cell response. The subsequent events vary substantially between different pa- tients, but three clinical patterns are observed—​clearance of virus below the level of detection in blood, persistence of virus without host control, or an intermediate state, where virus is transiently controlled, with relapse (Fig. 8.5.22.1). The immunological events that determine these different outcomes are not fully understood, but the association of specific HLA genes both Class II (such as HLA DR11/​DQ3) and Class I (such as HLA A3, B27 and B57) with spontaneous resolution point to the importance of host T-​cell re- sponses. It is generally considered that T-​cell responses that are broader and more sustained in number and function are most likely to be successful in viral control. B-​cell responses are also likely to be involved. However, the rapid emergence of viral escape mutants in the hypervariable envelope regions may limit the efficacy of neu- tralizing antibody responses in containing viral replication. Viral mutation within T-​cell epitopes is also a major cause of persistence in the face of T-​cell responses, although other phenomena such as T-​cell exhaustion and the emergence of regulatory T-​cell subsets also contribute to T-​cell failure. In those where virus is cleared below the level of detection long term, around 25% overall, antibody and T-​cell responses may be HCV Load HCV detection threshold Weeks Weeks Weeks ALT HCV Load ALT HCV Load ALT Upper limit of normal (ALT) Acute-resolving Acute-persistent Acute-transient Control-persistent Fig. 8.5.22.1  Distinct clinical outcomes following acute hepatitis C virus (HCV) infection. Some patients are able to eliminate virus following infection (upper panel) but the majority develop persistent infection (middle panel). A subgroup have a period of transient control but ultimately develop long-​term carriage (lower panel).

898 section 8  Infectious diseases detected for many years. In most individuals, virus persists after acute hepatitis, in the presence of antibody responses. T-​cell re- sponses in blood during chronic disease are weak but within the liver, infiltrates of T cells may be found. The pathology of HCV is highly variable between patients, and there is no diagnostic staining pattern, but it is typified by portal tract infiltrates of T and B cells, with the emergence in some cases of lymphoid follicles within liver tissue. Histologic scores (Ishak’s, Metavir) have been developed to quantify the degree of liver damage. These comprise a measure of the degree of hepatic inflammation (typically portal tract infiltration, ‘interface’ hepa- titis, lobular infiltration, and necrosis), together with the degree of hepatic fibrosis. The viral genotype is thought to have some influence on the pathogenesis, with genotype 3 associated with the development of hepatic steatosis, increased inflammation and fibrosis, and more rapid progression to cirrhosis. Epidemiology HCV is thought to infect around 170 million individuals world- wide. Spread is parenteral, and largely associated with needle use and exposure to infected blood products. Mother to child spread does occur but at relatively low rates (around 3–​5%) (2–​3-​fold higher in the context of HIV coinfection), and sexual spread is documented but rates are also low (see next). Thus, the risk groups are those exposed to infected blood products (recipients of un- screened blood/​plasma fractions, haemophiliacs) and contam- inated needles (people who inject drugs (PWID), participants in parenteral therapy programmes, nosocomial spread). Of these, in the west, PWID groups have particularly high rates of acquisi- tion and represent the main current focus of the infection. In some countries, most notably Egypt, medical programmes have been re- sponsible for spread of HCV in specific groups, and the prevalence of HCV in Egypt is the highest worldwide, up to 20–​30% in some communities. In the last decade, epidemic spread of HCV infec- tion among HIV + men who have sex with men has been observed in many countries globally. HCV has evolved into multiple genotypes (1–​6) and subtypes. Molecular typing techniques can trace the spread of individual strains within populations (including infections from a single source). Thus the Egyptian outbreak is genotype 4a, the older cir- culating western strains were typically genotype 1a and 1b, and the more recent strains acquired in Western PWID populations are 3a. Genotype 3 strains were originally linked to Southeast Asia, where genotype 6 is still largely found. Genotype 2 and Genotype 5 have remained localized strains, in West Africa and South Africa, respectively, but all strains are to some extent found globally. Prevention Primary prevention There are no licensed vaccines to prevent HCV infection although some are in development. Primary prevention of HCV worldwide depends on ensuring a safe blood supply and scrupulous atten- tion to the provision of sterile injection devices for all necessary healthcare interventions. The provision of sterile injection equip- ment has been shown to reduce the prevalence of HCV in people who inject drugs. HCV is not transmitted by typical household ex- posures but household contacts of HCV-​positive individuals are advised to avoid sharing razors and toothbrushes. Sexual trans- mission of HCV is inefficient, and studies show a low prevalence (average: 1.5%) of HCV infection in long-​term spouses of patients with chronic HCV infection who had no other risk factors for in- fection. No intervention has been clearly shown to decrease the risk of mother to child transmission of HCV, and breastfeeding is not discouraged. In healthcare settings, equipment, facilities, and policies should be in place to reduce the risk of percutaneous injury to staff. Postexposure prophylaxis Immune globulin is not effective in preventing HCV infection postexposure. Individuals exposed to the virus (e.g. via a percutan- eous injury), or perinatally, should be offered follow-​up testing for HCV. The average risk of infection following a percutaneous injury is 1.8% (range 0–​7%) and those who become infected should be offered early antiviral therapy. Clinical features Acute hepatitis C Acute HCV infection is clinically indistinguishable from other causes of acute viral hepatitis, and may therefore present with a pro- drome of fever, myalgia, and malaise. However, the acute phase is usually asymptomatic, and compared with hepatitis A or B it is un- common to develop the classical symptoms of jaundice, pruritus, pale stools, and dark urine. Serum levels of transaminases can be markedly elevated, although levels of up to 10 times the upper limit of normal would be more usual. Fulminant hepatic failure in acute HCV is rare. There is evidence that patients with symptomatic acute HCV in- fection have a higher rate of spontaneous viral clearance than those with asymptomatic infection. Overall approximately one in four patients will clear the virus spontaneously. Serum transaminases usually remain elevated at around twice the upper limit of normal in those who fail to clear the virus, although may normalize completely. Chronic infection Chronic infection with HCV is defined as the persistence of HCV RNA in blood for greater than six months. Most patients with chronic HCV infection will be unaware of their diagnosis, and many will have been tested following an incidental finding of abnormal liver function tests or have tested positive on rou- tine screening (e.g. for blood donation), or having given a his- tory of potential HCV exposure, such as intravenous drug use. Such patients will either be asymptomatic or have non​specific symptoms such as fatigue. Symptoms suggestive of liver dis- ease are unlikely to be present unless cirrhosis has developed. Hypoalbuminaeamia, thrombocytopaenia, and coagulopathy are

8.5.22  Hepatitis C virus 899 suggestive of cirrhosis, although this can only be diagnosed de- finitively with a liver biopsy. HCV is associated with several extrahepatic manifestations, the best documented of which are HCV related lymphoproliferative dis- orders, most commonly characterized by mixed cryoglobulinaemia. Although studies suggest a high prevalence of serum cryoglobulins in HCV-​positive patients, they are generally present at low levels with absent or only mild symptoms. Occasionally patients will pre- sent with neuropathies, arthralgias, and purpura and more severe cases may involve the kidney. B-​cell non-​Hodgkin’s lymphomas, porphyria cutanea tarda, Sjogren’s syndrome, and lichen planus have been found more commonly in association with HCV infec- tion than in HCV negative control groups in some studies, and other studies suggest autoimmune thyroiditis and type II diabetes mellitus are found with a higher frequency than expected in HCV-​positive individuals. Prognosis The rate of progression of HCV is highly variable. Risk factors for progression include older age at acquisition of infection, male gender, presence of immunosuppression including coinfection with HIV and concurrent heavy alcohol consumption. It is estimated that between 7 to 20% will develop cirrhosis within 20 years of infec- tion. Progression is higher in transfusion associated hepatitis than in community acquired HCV (i.e. people who inject drugs). However, some patient groups, such as a cohort of Irish women infected in 1977 through contaminated blood products, show rates very much lower than this with only 3% developing cirrhosis within 20 years of infection. Once cirrhosis develops, 80% will develop complications such as ascites and variceal bleeding within 10 years, and once complica- tions have developed, 50% will develop liver failure within a further 5 years. Hepatocellular carcinoma only occurs in the presence of cir- rhosis with an incidence of 1–​5% per year. HIV coinfected patients progress more rapidly to liver failure once complications of cirrhosis have occurred. Liver transplantation is indicated for decompensated HCV cirrhosis and in cirrhotics who develop small hepatocellular carcinomas despite good liver function. In the absence of therapy, infection universally recurs in the transplanted liver and pro- gression to cirrhosis occurs in about 10% of transplant recipients within 5 years. Diagnosis Serology Initial diagnosis of HCV infection is usually made by detecting HCV-​antibodies directed against recombinant HCV proteins in highly sensitive screening immunoassays. In low prevalence popu- lations, the probability of a false positive antibody test is high, and supplementary confirmatory tests should be performed. The de- velopment of an antibody response can take up to 2 months in im- munocompetent individuals, and can be delayed or not occur at all in immunocompromised individuals such as those with HIV infection or on haemodialysis. By six months, 97% of those infected will have developed an antibody response. It is good practice to confirm HCV-​antibody positivity with a second antibody test and a test for active infection, such as viral RNA or hepatitis C antigen, and also to test a second independent sample. Hepatitis C antigen detection is becoming a widely available immunoassay and correl- ates highly with the detection of HCV RNA, although it may lack some sensitivity at low HCV viral loads (<1000 IU/​ml). Hepatitis C antigen testing can be used to rapidly confirm active hepatitis C infection in the context of a newly detected HCV-​antibody. As it re- duces the ‘window period’ when compared with antibody only tests, the antigen test is particularly useful for screening blood donations in resource poor settings. Specialist laboratories may be able to pro- vide further immunoassays which examine responses to different antigens, or line/​strip immunoblots which have individual synthetic or recombinant antigens applied as separate lines to a solid phase; serologic responses to different HCV antigens can be distinguished. However, the diagnosis of HCV positivity can be established with a high degree of specificity with a combination of antibody and antigen/​RNA assays. HCV RNA testing Nucleic acid-​based tests are the gold standard for the diagnosis of acute and ongoing chronic HCV infection. HCV RNA can be detected by polymerase chain reaction (PCR) as early as 2 weeks postinfection before the development of antibody responses. Commercial assays now produce quantitative results with in- creasingly sensitive limits of detection. Methods used to detect HCV RNA include reverse transcription PCR (RT-​PCR), and transcription-​mediated amplification. Although quantitative re- sults may be important in predicting the response to interferon-​ based therapies, they are not useful for predicting disease severity or long-​term progression in contrast to HIV infection. Some coun- tries have successfully introduced nucleic acid-​based testing of pools of samples for blood donation screening. Pretreatment evaluation HCV virus genotyping is essential prior to treatment as it predicts duration of treatment and response (see next). HCV genotyping can be performed by line probe assays utilizing reverse hybridization and nucleic acid probes, or by viral sequencing which has the ad- vantage of identifying rare intergenotypic recombinant strains. In the era of directly acting antiviral agents (DAAs), next-​generation sequencing that can provide full viral sequences can be used to track specific mutations linked to drug resistance. Resistance testing pretreatment is not in common clinical use (except in the case of simeprevir) but the detection of specific resistance patterns will be under close review as the DAA drugs become more com- monly used. Non​invasive methods of assessing the degree of liver fibrosis using serum markers of fibrosis, assessment of liver stiffness using an ultrasound probe or newer magnetic resonance techniques (e.g. Fibroscan™, FibroTest™, and MRI) are good negative predictors of significant fibrosis. Liver biopsy is usually reserved for those indi- viduals where non​invasive markers suggest scarring to exclude cir- rhosis, where hepatocellular carcinoma surveillance may be needed, or those who may have dual liver pathology such as associated

900 section 8  Infectious diseases non-​alcoholic fatty liver disease. Polymorphisms in IL28B are as- sociated not only with spontaneous clearance but also clearance associated with interferon-​based treatment regimens. IL28B also impacts on highly effective directly acting antiviral agent regimens but its effect is less evident. Treatment The aim of treatment of HCV is to eradicate HCV RNA from serum. Although loss of viraemia is associated with improvement in liver histology, if cirrhosis is present, the risk of hepatocellular carcinoma remains after successful treatment with antiviral therapy, although the risk is reduced. Screening for hepatocellular carcinoma should, therefore, continue in patients with cirrhosis during and after treatment. HCV viraemia can re-​emerge within 3  months of stopping treatment (relapse) but individuals who remain HCV RNA nega- tive 3 months after completing therapy are considered to be cured (sustained response) and will remain HCV RNA negative, unless reinfected. Chronic hepatitis C Interferon-α and ribavirin have been the mainstay of treatment for over a decade with historical cure rates of 45–​70% but have now largely been replaced by better tolerated and more effective direct acting antiviral drugs (DAAs) for most genotypes. Interferon-​α induces the expression of multiple genes that have antiviral and antiproliferative action including those encoding RNAases, 2’-​5’ oligo-​adenylate synthase, and pro- tein kinase R. It is administered as pegylated interferon which has the advantage of being given once weekly. There are two pegylated interferons, 2a and 2b, which are modified interferon-​ α molecules with different side chains which lengthen their half-​life. Ribavirin is a guanosine analogue which, when used alone, does not reduce HCV RNA levels but, when combined with interferon-​α, improves the sustained virological response compared to interferon-​α monotherapy and might also improve cure rates in cirrhotic patients when used with the direct acting antiviral therapies. The oral directly acting antiviral agents are inhibitors of the hepatitis C viral gene proteins such as the NS3 protease, NS5A and NS5B polymerases (Fig. 8.5.22.2). They are typically given in 2 or 3 drug combinations for 8 to 12 weeks. In genotype 1 the overall sustained response rate is more than 90% even in the presence of cirrhosis. Cure rates are probably slightly lower, at 70% in decom- pensated more advanced cirrhosis, but historically this patient group could not be treated because interferon treatment is asso- ciated with worsening liver failure. Drug combinations in current clinical practice include e.g. ledipasvir plus sofosbuvir, sofosbuvir with simeprevir, ombitasvir with ritonavir boosted paritaprevir and dasabuvir. It is currently unclear whether shorter courses of 4–​6 weeks of therapy will be effective in some patient groups. In geno- type 2 treatment with one DAA, sofosbuvir, with ribavirin for 12 weeks is associated with an 87% cure rate. Genotype 4 has a cure rate of 90% with two oral directly acting antiviral agents—​ombitasvir and paritaprevir—​boosted with ritonavir for 12 weeks in non​cirrhotics, and 24 weeks in presence of cirrhosis. Genotype 3 has proven the more difficult virus to eradicate with the newer DAAs. A  shorter 3-​month course of pegylated α-interferon given with sofosbuvir and ribavirin has a cure rate of greater than 85% even in the presence of cirrhosis. Daclatasvir also has genotype 3 efficacy when given with sosfosbuvir and ribavirin. Newer NS5A inhibitors with pan-genotypic activity (such as Velpatasvir) show good clinical efficacy against genotype 3 in combination with other DAAs. Cure rates in the presence of HIV coinfection and in the presence of immunosuppression ap- pear similar to non​infected immunocompetent patients with the new DAAs. With interferon-​based regimens, side effects of treatment were common and quality of life was universally affected, although many individuals were able to continue working during therapy. Treatment with the α-interferons is associated with fatigue, depres- sion, and mood swings. Other side effects include rashes and thy- roid abnormalities. Bone marrow suppression was common with such regimens and led to the need for dose reductions. This was a particular problem in patients with cirrhosis who were already pancytopaenic before the start of treatment. Interferon-​α is also contraindicated in renal and cardiac transplant recipients because of the risk of inducing acute cellular rejection. Ribavirin causes haemolysis and frequently leads to a 2–​3 gram drop in haemoglobin during treatment and, because of its renal ex- cretion, it is contraindicated in renal failure. In contrast, the new directly acting antiviral agents have few side effects, mainly limited to headache and nausea. However, drug interactions are common and a careful history of prescribed and over the counter medication is needed pretreatment. Potential drug interactions can be checked at https://​www.hep-​druginteractions.org. Acute hepatitis C The results of treating acute HCV are much better than chronic infec- tion. Although as many as 50% of patients with acute symptomatic C E1 E2 NS2 NS5A NS3 NS4A NS5B NS3/4A Protease inhibitor Paritaprevir (ritonavir boosted) Grazoprevir Voxilaprevir Glecaprevir Simeprevir NS5A Polymerase inhibitor Ombitsavir Elbasvir Ledipasvir Velpatasvir Pibrentasvir Daclatasvir (Odalasivir)* NS5B Polymerase inhibitor Dasabuvir Sofosbuvir (AL-335)*

8.5.23 HIV AIDS 901

8.5.23 HIV/ AIDS 901

8.5.23  HIV/AIDS 901 HCV may clear the virus spontaneously within the first 3 months, treatment is advocated for acute HCV as it is very effective. It should be started between 12 and 24 weeks of infection. This allows suffi- cient time to assess whether spontaneous resolution has occurred, without losing the clinical benefit of early treatment. Genotype does not affect response and historically cure rates as high as 80–​95% can be achieved with 6 months of pegylated interferon monotherapy. It may be that short treatments of the oral directly acting antiviral agents for less than 8 weeks will be as effective in eradicating HCV in the acute setting. Area of uncertainty or controversy The degree of resolution of severe fibrosis and the risk of hepatocellular carcinoma after effective cure with directly acting antiviral agents will need further study, in particular to define at what point screening for primary liver cancer might no longer be necessary. Also, to what extent the immune system recovers from the immunomodulation created during chronic infection is not known. The best strategy to identify and treat all patients with costly directly acting antiviral agents has yet to be identified and repre- sents an important public health challenge. Although pre-​existing or emergent drug resistance to directly acting antiviral agents does not appear to be a common problem currently, the potential emergence of drug resistance means this will require close moni- toring over time. Likely developments over the next 5–​10 years The development of new DAA combinations and rescue regimens which are active against a range of genotypic variants and rare sub- types is important as drug resistance is likely to be a problem in a small proportion of patients. Access to more accurate HCV gene sequencing will also be im- portant when targeting drug resistance. Accessibility and affordability of the newer HCV treatments on a global scale is essential if cirrhosis and hepatocellular carcinoma are to be prevented in most of the world’s HCV infected popu- lation. Further development of current prophylactic vaccine ap- proaches will depend on the outcome of current clinical trials in at-​risk groups. FURTHER READING Afghal N, et al. (2014). Ledipsavir and sofosbuvir for untreated HCV genotype 1 infection. N Engl J Med, 370, 1889–​98. Benvegnu L, et al. (2004). Natural history of compensated viral cir- rhosis: a prospective study on the incidence and hierarchy of major complications. Gut, 53, 744–​9. Duggal P, et al. (2013). Genome wide association study of spontan- eous resolution of hepatitis C virus infection Ann Intern Med, 158, 235–​45. Feld JJ, Hoofnagle JH (2005). Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature, 436, 967–​72. Foster GR, et al. (2015). Efficiacy of sofosbuvir plus ribavirin with or without peginterferon-​alpha in patients with hepatitis C virus geno- type 3 infection and treatment-​experienced patients with cirrhosis and hepatitis C virus genotype 2 infection. Gastroenterology, 149, 1462–​70. Gee I, Alexander G (2005). Liver transplantation for hepatitis C virus related liver disease. Postgrad Med J, 81, 765–​71. Heim MH, Thimme R (2014). Innate and adaptive immune responses in HCV infections. J Hepatol, 61, S14–​25. Johnson RJ, et  al. (1993). Membranoproliferative glomeruloneph- ritis associated with hepatitis C virus infection. N Engl J Med, 328, 465–​70. Liang TJ, Ghany MG (2014). Therapy of hepatitis C back to the future. N Engl J Med, 370, 2043–​47. Micallef JM, Kaldor JM, Dore GJ (2006). Spontaneous viral clearance following acute hepatitis C infection: a systematic review of longitu- dinal studies. J Viral Hepatol, 13, 34–​41. Poordad F, et  al. (2014). ABT-​450r-​ombitasvir and dasabuvir with ribavirin for hepatitis C with cirrhosis. N EnglJ Med, 370, 1973–​82. Poynard T, Bedossa P, Opolon P (1997). Natural history of liver fibrosis progression in patients with chronic hepatitis C.  The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet, 349, 825–​32. Wakita T, et al. (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med, 11, 791–​6. Wiese M, et  al. (2014). Evaluation of liver disease progression in the german hepatitis C virus (1b) contaminated anti-​D cohort at 35 years after infection. Hepatology, 59, 49–​57. Zignego AL, et al. (2007). Italian Association of the Study of Liver Commission on Extrahepatic Manifestations of HCV infection: a general overview and guidelines for a clinical approach. Dig Liver Dis, 39, 2–​17. 8.5.23  HIV/​AIDS Sarah Fidler, Timothy E.A. Peto, Philip Goulder, and
Christopher P. Conlon ESSENTIALS Since its discovery in 1983, the human immunodeficiency virus (HIV) has been associated with a global pandemic that has affected more than 78 million people and caused more than 39 million deaths. Globally, 36.9 million (31.1 million–​43.9 million) people were living with HIV at the end of 2017. An estimated 0.8% of adults aged 15–​ 49 years worldwide are living with HIV, although the burden of the epidemic continues to vary considerably between countries and re- gions. Sub-​Saharan Africa remains most severely affected, with nearly 1 in every 20 adults living with HIV and accounting for nearly 71% of the people living with HIV worldwide. The impact of HIV in some African countries has been sufficient to reverse population growth

902 section 8  Infectious diseases and reduce life expectancy into the mid-​thirties, although HIV inci- dence has recently declined in some of these high-​prevalence coun- tries. However, there are large-​scale epidemics of HIV elsewhere (e.g. India, the Russian Federation, and Eastern Europe). By the end of 2017, globally, 21.7  million (19.1–22.6 million), 59% (44–73%) of all people living with HIV were accessing antiretroviral therapy, while 1.8 million people became newly HIV infected and 940 000 (670 000–1.3 million) died from HIV-​related illnesses; a fall of 49% from the peak in 2004. Transmission Worldwide, the principal mode of transmission is heterosexual inter- course. Other risk factors for acquisition of HIV include unprotected sex between men, injecting drug use, transfusion of contaminated untested blood products, and mother-​to-​child transmission. Cellular biology HIV-​1 (derived from a simian immunodeficiency virus in the chim- panzee) and HIV-​2 (animal reservoir the sooty mangabey monkey) belong to the lentivirus subfamily of retroviruses. The viral genes in infectious particles are carried as RNA, but upon infection of the host cell, reverse transcriptase catalyses the synthesis of a double-​ stranded DNA viral genome that is inserted into the chromosomal DNA of the infected cell by viral integrase. Genomic structure—​HIV has only nine genes: (1) gag—​encoding the core proteins p17, p24, and p15; (2) pol—​encoding the enzymes protease, reverse transcriptase, and integrase; (3) env—​encoding en- velope glycoproteins (gp120 and gp41); (4) two major regulatory genes—​tat and rev—​encoding proteins that are not assembled into the virus but are essential for replication in the cell; (5) four accessory genes, whose functions are not clearly understood. HIV receptors and cellular tropism—​CD4 is the cell-​surface re- ceptor for HIV, which binds to it via gp120; gp41 is then thought to effect membrane fusion. However, another cellular component Page: 621 or coreceptor is required, and different substrains of HIV (even those isolated from the same patient) exhibit specific tropisms for different cell types in culture, dependent on the ability of each particular substrain to bind to particular chemokine receptor family coreceptors. Sexual HIV transmission is usually with a CCR5-​using viral variant, and with advanced untreated disease this tends to tran- sition to CXCR4 using species. Naturally occurring deletions in the host genome encoding CCR5 (delta-​32 base pair deletion) confers natural resistance to HIV susceptibility. Knowledge of the cell biology of HIV has facilitated the develop- ment of pharmacological agents that have transformed the disease from a uniformly fatal illness to a chronic condition in those coun- tries able to provide antiretroviral treatment. Diagnostic tests and screening Reliable tests that detect HIV antibodies and antigen are used for diagnosis and screening. In all countries, around a quarter of the people living with HIV are unaware of their HIV status. For those unaware of their status, therefore not accessing antiretroviral therapy, there remains an increased risk of sexual and perinatal transmis- sion. Targeted screening programmes are needed to encourage HIV testing for this unreached group to achieve zero HIV-related deaths and zero new infections. Due to viral heterogeneity diagnostic tests for HIV focus on the detection of HIV-​specific antibodies rather than viral detection itself. Point-​of-​care technology has transformed up- take of HIV diagnosis and is now commonly used in many coun- tries and is increasingly available for self-​testing using finger prick blood drops or oral swab testing. HIV antibody testing misses the diagnosis of acute infection, prior to the development of detectable levels of antibodies. In such cases, combination antigen (p24) anti- body testing is performed on venous blood samples and detection of HIV RNA or DNA polymerase chain reaction may also be used. HIV antibody/​antigen combined testing is used for confirmation of point-​of-​care tests. Clinical features Primary HIV infection—​a few weeks after acquisition of HIV, many people develop a non​specific influenza-​like illness (seroconversion illness/​acute retroviral syndrome), often accompanied by a transient macular or maculopapular rash affecting the upper body. Rarely, there are neurological complications and severe immunodeficiency with secondary opportunistic infections. Most do not seek medical help, and diagnosis is often missed. Non​specific symptoms can be mistakenly attributed to influenza, upper respiratory tract infections, or glandular fever. Several such indicator diseases should trigger the offer of an HIV test, particularly among men-​having-​sex-​with-​men and those from high-​prevalence regions. The natural history of untreated HIV disease was referred to as a period of ‘Clinical latency’ typically lasting 8–​10 years before de- velopment of further illness. This period is often asymptomatic, but some individuals have persistent generalized lymphadenopathy, and
may develop minor opportunistic conditions affecting the skin
and mucous membranes (e.g. viral warts, oropharyngeal candid- iasis, and oral hairy leucoplakia). During this period, in the absence of antiretroviral therapy there is progressive immune destruction,
particularly depleting circulating CD4+ T cells from normal levels (>900 cells/​cc3 to <200 cells/​cc3. Once the CD4 count has fallen to less than 350 cells/​cc3 there is an associated significant risk of HIV-​ related opportunistic disease and AIDS. Progression to symptomatic HIV disease (AIDS)—​the value of making a distinction between AIDS and HIV infection in the era of antiretroviral therapy is no longer relevant: It is more useful to con- sider untreated progressive HIV disease as a continuous spectrum. Complications of late-​stage HIV disease include (1) opportunistic infections—​for example, pneumocystis pneumonia, oesophageal candidiasis, cerebral toxoplasmosis, and cytomegalovirus retinitis; (2) opportunistic tumours—​ for example, Kaposi’s sarcoma and non-​ Hodgkin’s lymphoma; and (3) direct HIV effects—​ for example, HIV-​ associated neurocognitive disorders. Clinical management and prognosis CD4+ T-​lymphocyte count (CD4 count) and plasma HIV-​1 viral load are the two laboratory markers with the best prognostic value: (1) CD4 count—​this is an indicator of HIV-​related immune impairment, with decline to below 200/​cc3 associated with the risk of life-​threatening opportunistic infection; based on the findings from recent trial data (START trial) antiretroviral treatment is currently recommended for all people living with HIV irrespective of CD4 count or clinical stage. (2)  Viral load—​quantitative estimation of HIV RNA in the blood plasma is the key tool in monitoring the effectiveness of therapy,

8.5.23  HIV/AIDS 903 which aims to maintain suppression of viral RNA at undetectable levels (<20–​40 copies/​ml, depending on the assay). The choice of initial antiretroviral regimen should take into account the results of baseline genotypic resistance testing, comorbidity, coinfections, and drug interactions as well as cost. Prognosis—​the outlook for people with HIV infection has been transformed since the late 1990s by the advent of highly active anti- retroviral therapy, but access to antiretroviral drugs continues to be difficult in less-​resourced countries (see Chapter 8.5.24). Antiretroviral therapy (ART) for effective viral suppression requires combinations of three drugs from more than 20 agents that are now available: a minimum of three, drawn from at least two drug classes, is required. Initial regimens usually include (1) a backbone of two nucleoside analogues (inhibitors of HIV reverse transcriptase), for example, tenofovir and emtricitabine or abacavir and lamivudine with either (2) a non​nucleoside reverse transcriptase inhibitor (e.g. efavirenz), (3) an integrase inhibitor (e.g. Raltegravir or Dolutegravir), (4)  or a boosted protease inhibitor (e.g. Darunavir and ritonavir). Factors considered when selecting the initial antiretroviral combin- ation include potential drug interactions with other medications, presence of renal or hepatic dysfunction, the presence of cardio- vascular risk factors and coinfections with other viruses, in particular hepatitis B and C, and avoidance of predictable drug toxicities. An important development has been the availability of simplified regi- mens involving small numbers of coformulated tablets taken once daily. Adherence to treatment and avoidance of suboptimal therapy (such as regimens involving fewer than three active agents, or use of agents in the presence of HIV mutations conferring resistance) are important in avoiding treatment failure. Other drugs for HIV—​(1) New agents in established drug classes—​ these have activity against HIV despite mutations conferring resist- ance to other drugs in the class, for example, etravirine and (integrase inhibitors) elvitegravir and dolutegravir. Also, tenofovir alafenamid, a better tolerated, less toxic compound with activity comparable to tenofovir DF, has become available and has been shown to have sig- nificantly less renal and bone toxicity. In addition, novel fusion inhibi- tors currently in phase I and II clinical trials and long-​acting injectable formulations are under trial. Adverse reactions to antiretroviral drugs—​are relatively common and include: (1) short-​term reactions—​gastrointestinal disturbances, rashes, and neuropsychiatric reactions that might require early ad- justments to the treatment regimen; (2)  longer-​term reactions—​ metabolic complications include (a)  mitochondrial toxicity; (b) disturbances of lipid and glucose metabolism associated with a risk of cardiovascular disease including myocardial infarction—​the absolute risk is small but requires consideration in patients with pre-​ existing cardiovascular risk factors; (c) renal impairment (e.g. with tenofovir); (d) metabolic bone disease. (3) Paradoxical reactions—​ called immune reconstitution syndromes—​these can occur in up to 20% of patients starting treatment, in particular those with severely impaired immune function prior to therapy, and include new or worsening inflammatory symptoms, especially in patients who have opportunistic infections such as tuberculosis. Earlier HIV diagnosis and initiation of antiretroviral therapy will reduce the cases of im- mune reconstitution syndromes. Recent clinical trials have identified a clinical benefit of initiation of antiretroviral therapy irrespective of CD4 T-​cell count at the time of HIV diagnosis and this has been in- corporated in the most recent World Health Organization antiretro- viral therapy guidelines of 2015. Coinfections involving HIV and tuberculosis, hepatitis B, or hepatitis C are common and require specialized treatment due to drug interactions and cotreatment requirements. Integrase inhibi- tors have limited drug interactions with tuberculosis treatments and are the preferred agents in the presence of tuberculosis. Hepatitis B treatment includes the use of tenofovir plus emtricitabine for most coinfected patients, enabling easier treatment of both HIV and hepatitis B. When to start antiretroviral therapy—​Historically there has been an important balance between the potential drug related toxicities and the individual clinical benefit of treatment. As antiretroviral therapy has become less toxic, easier to take (currently many ef- fective regimens are one coformulated pill once a day) clinical trials have explored the risks vs. benefits of starting antiretroviral therapy at higher CD4 count thresholds. The recently reported START trial identified in a large international randomized controlled study that immediate antiretroviral therapy for patients with CD4 T-​cell counts greater than 500 cells/​cc3 still conferred overall individual clinical benefit compared with deferral until a CD4 threshold of 350 cells/​ cc3. The results of this trial have now been incorporated into the World Health Organization and many national antiretroviral therapy guidelines and have resulted in the recommendation of immediate initiation of antiretroviral therapy for all people living with HIV, irre- spective of CD4 count. HIV prevention Strategies to raise awareness and provide education, and promote risk reduction, underpin HIV control programmes worldwide. HIV viral load is the most critical determinant of onward HIV transmis- sion. Reduction of HIV viral load using antiretroviral therapy for people living with HIV has been shown to be highly effective at preventing sexual transmission within both heterosexual and men who have sex with men. In addition, the use of antiretroviral therapy or components of it for HIV exposed, but uninfected individuals as postexposure prophylaxis or pre-​exposure prophylaxis are also highly effective at preventing establishment of infection among high-​ risk exposed individuals. Control of coexistent sexually transmitted genital ulcers and other genital infections reduces HIV transmission. Similarly, mother-​to-​child transmission can be reduced to below 1% if antiretroviral treatment is administered to the mother during preg- nancy, delivery is by planned caesarean section (vaginal delivery can be an option if HIV viral load is below the detection threshold), and breastfeeding is avoided. Despite decades of research, an effective HIV vaccine is not available. Introduction Acquired immunodeficiency syndrome was first recognized in 1981 in the United States of America, when outbreaks of pneumo- cystis pneumonia and Kaposi’s sarcoma were reported in men who have sex with men in New York and California. The variety of unusual infections and other conditions declared a new form

904 section 8  Infectious diseases of cellular immunodeficiency and was described as the acquired immune deficiency syndrome (AIDS). In 1983, the causative retro- virus was isolated and subsequently named human immunodefi- ciency virus (HIV). At the time of its discovery, HIV was already widespread, the earliest infections probably having occurred be- fore the 1950s. In 1986, a second retrovirus causing AIDS, HIV-​2, was identified in West Africa. It remains largely confined to this region, while HIV-​ 1 is the cause of the world pandemic of AIDS. HIV infection may be regarded as a zoonosis: HIV-​1 is derived from a simian immuno- deficiency virus in the chimpanzee (Pan troglodytes troglodytes), and the animal reservoir for HIV-​2 is the sooty mangabey monkey (Cercocebus atys). Epidemiology The global HIV-​1 pandemic has had the greatest impact in developing countries (see Chapter  8.5.24). The World Health Organization (WHO) estimated that in 2010, 33.3 million people were living with HIV worldwide, of whom 22.5 million were in sub-​Saharan Africa (Fig. 8.5.23.1). Worldwide, the WHO esti- mated there were 2.6 million new HIV infections in 2009, of which 1.8 million were in sub-​Saharan Africa. Globally, AIDS caused 1.8 million deaths of which 1.3 million were in sub-​Saharan Africa. New HIV infections have fallen by 35% since 2000. Worldwide, 2 million (1.9–​2.2 million) people became newly in- fected with HIV in 2014, down from 3.1 million (3.0–​3.3 million) in 2000. New HIV infections among children have declined by 58% since 2000. Worldwide, 220 000 (190 000–​260 000) children became newly infected with HIV in 2014, down from 520 000 (470 000–​580 000) in 2000. In North America, Western Europe, and Australasia the epi- demic began among men who have sex with men and injecting drug users. However, in these regions the proportion attributable to heterosexual transmission subsequently increased. In 2015, over 100 000 people were living with HIV in the United Kingdom, nearly a quarter of whom were unaware of their diagnosis. The proportion of newly diagnosed cases in the United Kingdom attributed to het- erosexual transmission rose steadily from 1999 onwards, largely due to increased numbers arriving from countries with high prevalence, and increased HIV detection through routine antenatal testing. However, injecting drug use, unprotected sex between men, and unprotected paid sex remain important modes of transmission in Europe, as well as in Asia and Latin America. Fig. 8.5.23.1  World distribution of HIV, 2013. Copyright © WHO 2014.

8.5.23  HIV/AIDS 905 In contrast, HIV transmission in regions with the highest preva- lence rates, such as sub-​Saharan Africa, is predominantly hetero- sexual and perinatal. The estimated overall adult prevalence there is 6%, rising to between 20 and 30% in some countries such as Botswana and Zimbabwe, where AIDS has curtailed population growth and life expectancy had fallen into the mid-​30s. Swaziland (population 1 million) is the country with the highest adult sero- prevalence, of 33%. The country with more infected people than any other is believed to be South Africa (6.4 million adults living with HIV, adult seroprevalence 20%), although in India, where preva- lence data are less easily accessible, the overall numbers might be similar (despite adult seroprevalence below 1%). The overall prevalence of HIV has risen worldwide in recent years because new transmissions exceed AIDS-​related deaths, and because successful antiretroviral treatment has reduced mortality. However, there have been notable declines in some high-​prevalence countries such as Kenya, Zimbabwe, and parts of India, and a level- ling off in other parts of sub-​Saharan Africa, attributable to preven- tion efforts. Uganda is seen as one of the best examples of a country where prevalence has declined significantly, believed to be, at least in part, due to the timely government campaign of public educa- tion, although more recently this reduction in incidence has not been sustained. The global distribution of HIV is currently characterized by very variable rates of prevalence and scattered areas of very high trans- mission in epidemics. Consequently, the risk of acquisition of HIV is also highly variable from region to region. In some countries, such as Russia and Ukraine, HIV has caused large-​scale epidemics and transmission rates remain high. The risk of onward transmis- sion is especially high soon after sexual acquisition of HIV, when plasma viral load is high, and the virus is present in genital secre- tions in sexually active people, often unaware of their changed HIV status. Therefore, it is particularly important to detect primary HIV infection in population screening programmes. HIV transmission continues at a high level in many countries because of poverty, low condom usage, high rates of other sexually transmitted infections, and higher risk behaviour such as unprotected paid sex and use of non​sterile injecting drug equipment. HIV-​2 is endemic in parts of West Africa and is also prevalent in Angola, Mozambique, France, and Portugal. In other parts of the world, the prevalence is very low, although it is present in India. The clinical features of HIV-​2 are similar to those of HIV-​1, but some pa- tients with HIV-​2 appear to progress much more slowly than those with HIV-​1 for unknown reasons. Most of the currently available antiretroviral therapies (ART) are not effective against HIV-​2. Variation of HIV-​1 RNA sequences has been identified, leading to a classification of 11 sequence subtypes (or clades), A to K, of the main group M, and N (new) and O (outlier) as two quite distinct groups in west central Africa. The subtypes have varying geograph- ical distributions. For instance, subtypes A and D are found in cen- tral Africa, B in North America and Europe, and E in Thailand. More people are infected with clade C virus than any other, being the pre- dominant clade of virus in southern Africa as well as India. A new group, P, was designated after the discovery of a novel HIV strain in a female from Cameroon in France in 2009, which was closely related to the simian immunodeficiency virus found in gorillas. Study of the genetic and geographical divergence of subtypes has shed light on the emergence and global spread of HIV. Cellular biology of HIV The viral replication cycle HIV-​1 (Fig. 8.5.23.2) and HIV-​2 belong to the lentivirus subfamily of retroviruses. Retrovirus implies a ‘backwards’ step in biological information during viral replication attributable to its enzyme, re- verse transcriptase. As with all retroviruses, the viral genes in infec- tious particles are carried as RNA, but upon infection of the host cell, reverse transcriptase catalyses the synthesis of a double-​stranded DNA viral genome (Fig. 8.5.23.3). Insertion of the DNA genome into the chromosomal DNA of the infected cell is effected by viral integrase. The integrated provirus may remain latent, particularly in resting lymphocytes. In actively infected cells, however, RNA tran- scripts and proteins are synthesized, leading to the formation of new virus particles. The core proteins derived from the gag and pol genes are made as large polypeptides that are then cleaved into smaller ­components  representing the enzymes and building blocks of the virus. This cleavage is achieved by the viral protease. The unique reverse transcriptase and protease are targets of antiretro- viral therapy (see next). Reverse transcriptase inhibitors such as abacavir and lamivudine affect an early step in HIV replication, whereas the protease inhibitors, such as darunavir, block a late stage of virus assembly (Fig. 8.5.23.3). Compounds that inhibit any stage of HIV replication, without being too toxic to the in- fected person, are potential antiviral drugs. Agents have been developed to block viral entry (e.g. fusion entry inhibitors and CCR5 receptor antagonists) and integration into the host cell DNA (integrase inhibitors). Although regarded as a complex retrovirus, HIV has only nine genes (Fig. 8.5.23.4). The three structural genes are gag, pol, and env, encoding the core proteins p17, p24, and p15, the enzymes (protease, reverse transcriptase, and integrase), and the envelope glycoproteins (gp120 and gp41), respectively. The major regulatory genes tat and rev encode proteins that are not assembled into the virus but are essential for replication in the cell. The Tat protein acts in positive feedback to enhance transcription of viral RNA from the DNA provirus, while the Rev protein helps the efficient transport of Fig. 8.5.23.2  Electron micrograph of HIV-​1. Reproduced by courtesy of H. Gelderblom.

906 section 8  Infectious diseases viral RNA from the nucleus to the cytoplasm. Either of these pro- teins could be a suitable target for antiviral therapy, particularly Tat, because the synthesis of all the other viral proteins depends on its activity. The functions of the four accessory genes of HIV are less well understood. Vif encodes a protein assembled in virus particles that appears necessary for the infectivity (‘viral infectivity factor’) at a stage soon after entry. Vif binds and hastens the degradation of the cellular protein APOBEC which, in the absence of Vif, hypermutates HIV, thereby disabling it. Nef also affects an early postentry function; it is not needed by laboratory-​adapted HIV strains or if virus enters via endosomal vesicles rather than fusing with the outer cell membrane. It also down-​regulates surface ex- pression of the primary cell-​surface receptor for HIV, the CD4 antigen, by drawing CD4 into clathrin-​coated pits. Vpu similarly interacts with CD4, promoting its degradation by directing it to the ubiquitin–​proteasome pathway. Vpr has dual functions; first, it directs the preintegration complex of the virus, containing the newly synthesized DNA, into the nucleus so that it can integrate into chromosomal DNA; second, it blocks cell proliferation in the G2 phase of the cell cycle, thereby enhancing the amount of viral progeny released per cell. Unlike HIV-​1, HIV-​2 and the simian immunodeficiency viruses (SIV) lack vpu, but have an alternative gene, vpx. HIV-​2 Vpr leads the viral genome into the cell nucleus, but does not arrest the cell cycle. These proteins presumably recognize cellular proteins and some of these interactions are species-​specific. Thus, the Vpr and Vif proteins in SIV of African green monkeys do not function in human cells, while the equivalent proteins of SIV from sooty man- gabey monkeys work well in human cells. This could explain why sooty mangabey SIV was able to infect humans and become HIV-​2, whereas the more widespread African green monkey SIV has not led to a zoonosis. Another difference is that HIV-​1 incorporates the cellular protein cyclophilin A (the target of the drug ciclosporin A) into virus particles, where it may cooperate with Vif and is required for steps early in the infection. In contrast, HIV-​2 does not contain cyclophylin A and replicates well without it. HIV receptors and cellular tropism CD4 is the cell-​surface receptor for HIV; it is expressed on T-​helper lymphocytes, the cells that become depleted in AIDS. CD4 is also expressed (to a lesser extent but sufficient to permit infection) on macrophages, Langerhans dendritic cells in mucous membranes, and brain microglial cells. These are the other target cells for HIV infection. CD4 is necessary to initiate HIV infection but is not suf- ficient to allow the virus to fuse with host cell membranes: another cellular component or coreceptor is required. Different substrains of HIV, even those isolated from the same patient, exhibit specific tropisms for different cell types in cul- ture. All isolates can infect primary CD4 lymphocytes, but only some infect macrophages while others can infect cell lines estab- lished from CD4+ leukaemic cells. Macrophage-​tropic strains pre- dominate early in the course of HIV infection, and may be more transmissible from person to person. They do not cause CD4 lymphocytes to fuse together in culture and hence are referred to as non-​syncytium-​inducing (NSI) strains. In contrast, many HIV isolates established from late-​stage infection rapidly adapt in culture to infect T-​cell lines and are syncytium-​inducing (SI). Approximately 50% of patients with AIDS develop SI strains in addition to NSI strains. The differences in cellular tropism and SI/​NSI phenotype occur in all HIV subtypes or clades, which ap- pear to reflect geographical variation of HIV rather than specific biological properties of the virus. The complex cellular tropism of HIV has been explained by the discovery that different members of the chemokine receptor family act as coreceptors to CD4 for HIV entry into cells. Chemokines are chemoattractant, locally acting hormones or cytokines that bind to one or more receptors which are structurally related to olfactory and neurotransmitter receptors. Following binding to the CD4 re- ceptor, primary NSI strains use CCR5, the chemokine receptor for macrophage-​inhibitory proteins (MIP-​1α, MIP-​1β) and RANTES. In contrast, the SI strains of HIV use the CXCR4 coreceptor, the re- ceptor for another chemokine, stromal-​derived factor-​1 (SDF-​1). Other receptors such as CCR3 (the receptor of eotaxin) can be used by some NSI strains. High levels of MIP-​1α or -​β in the blood correlate with relative resistance to HIV infection. Some exposed yet uninfected individ- uals are homozygous for an inherited defect of the CCR5 receptor involving a 32 bp deletion in the CCR5 gene. This mutation is present in approximately 20% of white people (approximately 1% of white Receptor RNA RT inhibitors DNA Chromosome Proteins Protease inhibitors HIV virus particle RNA Fig. 8.5.23.3  Replicative cycle of HIV. HIV-1 LTR GAG POL VIF VPR VPU TAT REV ENV NEF LTR HIV-2 LTR GAG POL VIF VPR VPX ENV NEF LTR TAT REV kbp 0 1 2 3 4 5 7 6 8 9 Fig. 8.5.23.4  HIV genome map.

8.5.23  HIV/AIDS 907 people are homozygous), but is not found in African and Asian populations. Individuals who are homozygous for the deletion are healthy, indicating that the CCR5 receptor is not essential for the development of immune competence, probably because MIP-​1 and RANTES can also bind to alternative receptors. However, homo- zygotes are genetically resistant to infection by NSI strains of HIV, and the few homozygotes with Δ32 deletions who are HIV-​positive appear to have been infected with SI strains that utilize CXCR4 in- stead. Other, more subtle, mutations in the promoter region of the CCR5 gene allowing only low levels of coreceptor expression may confer relative resistance to HIV infection and also, if infection oc- curs, slower the progression to AIDS. A blood group antigen on red blood cells, the Duffy antigen receptor for chemokines (DARC), was shown to be another non-​ human leukocyte antigen (HLA) genetic factor influencing HIV transmission and disease progression. In addition to forming a receptor for certain HIV-​suppressive and proinflammatory chemokines such as RANTES, DARC serves as the red cell receptor for Plasmodium vivax malaria and consequently nonexpression of DARC on red cells (Duffy negative phenotype) confers complete resistance to P. vivax. As a result of selection pressure from malaria, most West Africans and two-​thirds of African Americans do not express DARC on red cells (although expression is preserved on endothelial cells). The DARC-​negative red cell phenotype is associated with an increased risk of acqui- sition of HIV-​1; however, it is also associated with slower HIV-​ related disease progression. The outer envelope glycoprotein, gp120, is the molecule on HIV that binds to CD4 and subsequently to the coreceptor. Gp120 is an- chored to the viral envelope via gp41, the viral protein that is thought to effect membrane fusion. The gp120–​gp41 is present in the viral envelope as a trimeric complex. SI strains Page: 625 have a gp120–​ gp41 structure that is less stable than NSI strains, readily undergoing conformational change on binding to CD4. This property makes SI strains more sensitive to neutralization by gp120 antibodies and also to inactivation by soluble forms of recombinant CD4, which were once seen as promising therapeutic agents. NSI strains, however, are more resistant. Mutations in the V3 loop of gp120 can convert NSI strains to SI strains. These mutations arise naturally during progres- sion to AIDS and may allow HIV to switch to infect different cell types via new coreceptors. The natural chemokines act as competitive inhibitors of HIV entry; certain chemically modified chemokines and chemical ana- logues act as strong HIV inhibitors without triggering the down- stream signalling of the receptor. This has led to a new class of anti-​HIV drugs, called CCR5 receptor antagonists. Diagnosis of HIV infection Following seroconversion, antibody to envelope protein persists indefinitely in the serum and forms a highly specific test for HIV infection. Most laboratories use one or more sensitive enzyme im- munoassay tests that detect HIV-​1 and HIV-​2 antibodies and p24 antigen as the initial screening test. Positive screening tests are usu- ally referred to a specialist laboratory for additional tests to confirm the presence of HIV antibodies. Most seroconversions occur within 3 months of infection, and very rarely up to 6 months. Routine diagnostic tests, if negative, should be repeated 6 to 12 weeks after the last possible exposure. Additional tests using HIV RNA and DNA can also detect acute infection more accurately. If primary infection is suspected, or after high-​risk exposure, add- itional tests may be indicated (see ‘Primary HIV infection’, next). Point-​of-​care HIV testing is now a standard testing technology globally and involves a finger prick sample or oral swab. This has dramatically increased uptake of HIV testing especially in high-​ prevalence resource-​limited settings where task shifting away from clinically trained healthcare facility testing has enabled enhanced uptake of HIV testing. In many settings HIV self-testing using oral swab testing or in some settings finger prick sampling or testing is also available. Globally around 25% of people living with HIV are unaware of their HIV infection; in the United Kingdom the estimated number of people with undiagnosed HIV infection has reduced from 13 300 in 2015 to around 10 400 in 2016, with most of the decline seen in gay and bisexual men in London, and in black African heterosexual women. Detection of HIV is important for timely intervention with antiretroviral treatment, to reduce HIV- related morbidity and mortality and reduce the risk of perinatal transmission, and to promote behavioural change to protect sexual partners. Unfortunately, HIV remains a stigmatized condition and acceptance of testing is limited by potential distress and disruption of domestic, social, and professional lives. Psychological support and counselling may be needed, especially around the time of diag- nosis. However normalization of HIV testing and the availability of immediate treatment might serve to enhance acceptability of testing. Many industrialized countries are developing strategies to in- crease HIV detection. In the United Kingdom, it is standard prac- tice that all patients attending sexual health clinics for screening or treatment for sexual infections are offered HIV testing, and ‘opt out’ testing in genitourinary medicine (GUM) clinics and ante- natal settings has dramatically improved testing rates. It is re- commended that HIV testing should be considered in all general medical admissions to hospital, particularly in individuals from high-​risk groups and those presenting with ‘indicator’ conditions such as Epstein–​Barr virus, cytomegalovirus (CMV) upper re- spiratory tract infections, and pneumonia. Testing should also be considered for all adults registering with a general practice if the HIV prevalence in the local population exceeds 2 in 1000 (typ- ically larger cities). In the United States of America, where more than 25% of those infected are unaware of their HIV infection, the Centers for Disease Control and Prevention (CDC) has re- commended that HIV testing should be routinely offered in all healthcare settings. In patients with unexplained symptoms that could be caused by HIV, testing is essential for diagnosis so that appropriate treatment can be provided. If the patient is too ill to give consent, testing is justified on the grounds of being in the patient’s best interest. As there is still stigma associated with HIV, confidentiality must be maintained; disclosure of HIV-​positive status is acceptable only in the medical interests of the patient and in general with their knowledge and consent. Patients unwilling to inform their sexual partners should be advised of the possible legal implications of non​disclosure if transmission occurs. In the United Kingdom, there have been successful prosecutions of indi- viduals knowingly exposing their partners to HIV.

908 section 8  Infectious diseases Transmission of HIV Viral transmission across a mucosal membrane is relatively ineffi- cient when compared to other sexually transmitted infections (gon- orrhoea or syphilis) but once established, dissemination to the local, and then regional lymph nodes occurs rapidly, within a few days. Transmission occurs usually with a clonal infection which rapidly diversifies due to the lack of fidelity of the reverse transcriptase en- zyme into a new quasi-​species. The clonality of recently transmitted virus can be used in research studies to estimate how recently the infection occurred. Infected antigen presenting cells and CD4+ T cells rapidly traffic to regional and local lymph nodes carrying virus with them and the lymph node is the active site of viral transfer to uninfected CD4-​bearing cells. For this reason postexposure prophy- laxis (PEP) must be initiated as soon as possible after exposure to optimize efficacy although current guidelines still recommend pre- scription up to 72 hours after exposure. Mucosal delivery of ART agents through vaginal rings impregnated with antiretroviral agents is under trial as a prevention tool. Clinical presentation and features Primary HIV infection Between 2 and 6 weeks after exposure to HIV, 50 to 70% of those infected develop a transient, often mild, non​specific illness (some- times called seroconversion illness or acute retroviral syndrome) similar to infectious mononucleosis, with fever, malaise, myalgia, lymphadenopathy, and pharyngitis. However, unlike infectious mononucleosis, over 50% of people develop a rash, typically ery- thematous, maculopapular, and affecting the face and trunk. Other rashes and patterns of distribution, and oral and genital ulcers have also been reported. The illness begins abruptly and usually lasts for 1–​2 weeks, but may be more protracted. Most cases are so mild that patients do not seek medical help. In addition, abnormal liver function tests are common and neurological complications can occur and include acute encephalitis, lymphocytic menin- gitis, and peripheral neuropathy. Severe or long-​lasting illness and neurological involvement are associated with accelerated pro- gression to AIDS and a bad prognosis, which might be influenced by immediate antiretroviral therapy. A transient decrease in CD4 lymphocytes is usual during primary illness. Occasionally, this can be substantial and associated with opportunistic infections such as oral or oesophageal candidiasis, and rarely pneumocystis pneu- monia (PCP). There are few data on primary infection features associated with HIV-​2. Diagnosis requires a high index of suspicion. Primary HIV in- fection is a time of high viraemia (typically 105–​106 viral par- ticles/​ml) during which antibodies to HIV may initially be absent (Fig. 8.5.23.5). However, combined antigen-​antibody fourth-​generation tests can detect p24 antigen as well as HIV anti- bodies and can often detect p24 antigen in advance of antibody early in primary infection. Serum antibodies to the core and surface proteins of the virus usually appear within 2 to 6 weeks. However, if primary infection is suspected and the initial antibody test is nega- tive, additional tests may be required; rapid diagnosis can be pro- vided by detecting HIV viraemia using tests for HIV RNA or proviral cDNA (by polymerase chain reaction, PCR), which might confirm HIV infection before antibodies become detectable. Aggressive therapy of primary HIV infection with antiretroviral drugs does not eradicate the infection but, on theoretical grounds, might alter the natural history. After primary infection, in the ab- sence of intervention, the viral load becomes relatively stable after 6 to 9 months at an average level of 30 000 HIV RNA copies/​ml plasma (Fig. 8.5.23.5). The plasma HIV RNA level at this virological steady state or ‘set point’ is of prognostic importance. Starting ART in primary HIV infection confers a better chance of immune Eclipse I 0 106 105 Viral Load (virious/ml of plasma) 104 103 102 101 107 0 10 20 30 40 Days since Infection 50 100 1000 Viral RNA + Gag p24 antigen + ELISA + Western blot ± Western blot + p31— Western blot + p31+ Viral load Strain-specific neutralizing antibodies gp41 Antibodies Antibody—antigen complexes Inflammatory cytokines CD8 T-cell responses Acute-phase reactants Viral latency II III IV V VI Fig. 8.5.23.5  Natural history of HIV infection showing changes in viral and immune parameters. Reproduced from Cohen MS et al. (2011). Acute HIV-​1 Infection. NEJM, 364, 1943–​54, Copyright © 2011 Massachusetts Medical Society.

8.5.23  HIV/AIDS 909 function normalization in terms of CD4 count recovery as well as CD4:CD8 ratio. Early HIV infection Following the primary illness or subclinical seroconversion, there usually follows an asymptomatic period lasting an average of 10 years without antiretroviral therapy. Although a time of clinical latency, there is intense viral turnover: 109 to 1010 viral particles are replaced daily and the half-​life of circulating CD4 lymphocytes is substantially reduced. During the asymptomatic period, physical examination may be normal, but about one-​third of patients have persistent general- ized lymphadenopathy. The enlarged nodes, caused by a reactive follicular hyperplasia, are usually symmetrical, mobile, and non-​ tender. The cervical and axillary nodes are most commonly af- fected. Nodes that are markedly asymmetrical, painful, or rapidly enlarging should be biopsied to exclude tumours such as lymphoma and opportunistic infections such as tuberculosis. Symptoms of progressive HIV infection can be prevented by anti- retroviral treatment. In the absence of treatment, patients often de- velop minor opportunistic conditions affecting the skin and mucous membranes. These are also common throughout the later stages of HIV disease. They include a range of infections: fungal (e.g. tinea, pityrosporum), viral (e.g. warts, molluscum contagiosum, herpes simplex, herpes zoster), and bacterial (e.g. folliculitis, impetigo); and also eczema, seborrhoeic dermatitis, and psoriasis. Oral hairy leucoplakia usually appears as corrugated greyish-​ white lesions on the lateral borders of the tongue in homosexual men. The condition is symptomless and non​progressive, but can be a clue to HIV seropositivity. Epstein–​Barr virus DNA has been dem- onstrated in these lesions. One of the characteristic clinical presentations of HIV disease is a sore mouth and throat due to oropharyngeal candidiasis (oral thrush) (Fig. 8.5.23.6). This is a sign of worsening immunodefi- ciency and might be recurrent. Candida albicans is usually re- sponsible, but other species (e.g. Candida glabrata) are sometimes implicated. There is an increased incidence of peri-​dontal disease in those with untreated HIV, including inflammation of the gums (gingivitis) and the more serious and extensive periodontitis that can lead to loss of teeth. Two distinctive forms are associ- ated with HIV: a linear gingival erythema that causes a typical red band along the gum line, and in advanced immunosuppression, necrotizing ulcerative periodontitis which may require extensive debridement and antimicrobials. Recurrent oropharyngeal aph- thous ulceration is common and can be painful. Recurrent ulcers can occur in the oesophagus and other parts of the gastrointes- tinal tract. They usually respond to local or systemic cortico- steroid therapy. Resistant cases may respond to thalidomide. The availability of antiretroviral therapy has reduced the need for spe- cific treatment. Later on in the course of untreated HIV infection, intermittent or persistent non​specific constitutional symptoms might develop, which include lethargy, anorexia, diarrhoea, weight loss, fever, and night sweats. These symptoms can presage severe opportunistic in- fections or tumours. Generally, disease progression is slow in HIV-​2 infections and has a much longer period of clinical latency. The rate of CD4 count de- cline is much slower. Progression to symptomatic HIV disease (AIDS) Various staging systems for HIV infection and case definitions of AIDS were developed and modified as understanding of the pathogenesis and natural history increased (Fig. 8.5.23.7). The CDC in the United States of America listed a range of specific diseases and other criteria, such as a CD4 lymphocyte count of less than 200/​mm3 (0.2 × 109/​litre), as indicative of AIDS. AIDS-​ defining illnesses were essential for surveillance when HIV status was frequently unknown, the natural history of HIV infection was poorly understood (the proportion developing opportunistic complications was uncertain), and disease-​modifying drugs were not available. Antiretroviral therapy improves the clinical condition and survival, even when started after progression to AIDS. These fac- tors have undermined the epidemiological value and prognostic Fig. 8.5.23.6  Oral candidiasis. By courtesy of the late Dr B. E. Juel-​Jensen. 1.00 0.75 0.50 0.25 0 Cumulative survival probabilty Pre-ART period ART period 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time from seroconversion (years) Fig. 8.5.23.7  Natural history of HIV. Estimated proportions of individuals surviving from HIV-​1 seroconversion in the pre-​ART era. ART, antiretroviral therapy. Reprinted from CASCADE collaboration (2000). Survival after introduction of HAART in people with known duration of HIV-​1 infection. The CASCADE Collaboration. Concerted Action on SeroConversion to AIDS and Death in Europe. Lancet, 355, 1158–​59, Copyright © 2000, with permission from Elsevier.

910 section 8  Infectious diseases importance of a strict AIDS case definition. Therefore, the current value of making a distinction between AIDS and HIV infection at other stages is questionable, especially in industrialized countries. It is more useful to consider progressive HIV disease as a continuous spectrum. However, clinical criteria to identify symptomatic HIV disease and AIDS were needed in resource-​poor countries, if laboratory confirmation of HIV was not possible. The WHO therefore adopted clinical case definitions for AIDS surveillance in resource-​limited countries, based on clinical manifestations with or without labora- tory confirmation of HIV infection. This approach has been super- seded by the WHO clinical staging system, which assumes that an HIV test has been done. Rapid HIV tests can now be done even in field conditions. For surveillance, it is suggested that HIV case reporting should supersede AIDS case reporting, though that may not yet be possible everywhere. Once clinical progression occurs in HIV-​2 infection, the clinical features are similar to those seen in HIV-​1. Non​clinical progression The average CD4 decline without treatment is approximately 67 cells/​year. Although the average time between infection with HIV and CD4 count less than 200 cells/​cc3 or the development of AIDS is about 10 years, approximately 20% of patients progress rapidly to AIDS within 5 years and 10–​15% remain clinically well for 15–​20 years. Age is an independent risk factor for disease pro- gression; acquisition of HIV in later life is associated with a less favourable prognosis. Long-​term healthy survivors previously called ‘non​progressors’, and this subgroup generally represents the tail end of a normal distribution of progression rates. Ultimately for most individuals previously defined as ‘non​progressors’ clin- ical disease does become evident. Although several investigators have reported virological, genetic, and cellular and humoral im- munological factors that may be associated with non​progression, limitations in study design have made it difficult to identify what is responsible. In white cohorts of antiretroviral treatment-​naive, HIV-​ infected persons who show unusually successful control of HIV to levels of below 50 copies/​ml plasma, more than 50 to 90% express one or both of the HLA class I alleles HLA-​B5701 and HLA-​B2705. The protective value of these HLA class I al- leles is related to the importance of the CD8+ T-​cell response in successful immune control of HIV, and in particular where the CD8+ T-​cell response includes broad targeting of epitopes in the conserved internal Gag protein. Infected persons ex- pressing the HLA class  I  alleles HLA-​B3502 or B5802 tend not to make Gag-​specific CD8+ T-​cell responses, and progress to AIDS significantly more rapidly. In addition to defining the nature of the CD8+ T-​cell response, the particular HLA alleles expressed have important influences on the natural killer cell re- sponse against HIV, also affecting rates of progression to HIV disease. Non-​HLA genes that affect rates of progression include the macrophage chemokine receptor CCR5 gene mutation (see ‘Cellular biology of HIV’,) associated with non​progression in the heterozygous state. In the setting of universal ART, the distinc- tion of ‘non​progressors’ has now become clinically less relevant. However, a better understanding of natural viral control is still of great interest in terms of HIV cure. Late complications and their management Pneumocystis jiroveci pneumonia (See Chapter 8.7.5.) P. jirovecii (previously Pneumocystis carinii) pneumonia (PCP), one of the hallmarks of AIDS, is now less common because of pri- mary prophylaxis and antiretroviral therapy. Some 85% of cases occur in patients with CD4 counts below 200/​mm3, and mostly at counts below 100/​mm3. Symptoms typically include increasing shortness of breath, dry cough, and fever, usually developing subacutely over a few weeks. Malaise, fatigue, weight loss, and chest pains, or tightness may occur. Chest signs are usually minor (crackles) or absent. The characteristic chest radiograph shows bi- lateral mid-​zone interstitial shadowing (Fig. 8.5.23.8), but can be normal. Other appearances include localized infiltrates or con- solidation, upper lobe shadows resembling tuberculosis, nodular lesions, and pneumothorax; effusions are very rare. The arterial oxygen saturation is usually less than 95% at rest or falls after exercise. Infection with P. jirovecii is associated with an interstitial inflam- matory infiltrate and progressive impairment of lung function. The diagnosis can sometimes be confirmed by microscopy of sputum, which is induced by nebulized saline in properly ventilated isolation rooms (to read more about reducing the risk of tuberculosis trans- mission, see ‘Multidrug-​resistant tuberculosis’, next). P.  jirovecii cysts and trophozoites are visualized by the use of special stains. If the result is negative, fibreoptic bronchoscopy with bronchial lavage is indicated (Fig. 8.5.23.9); other causes of lung disease or coexistent infection might also be diagnosed by this technique, including tuberculosis, fungal infections, and Kaposi’s sarcoma. Immunofluorescence using monoclonal antibodies, or DNA ampli- fication by PCR, may improve diagnostic sensitivity when compared with conventional staining techniques. In a minority of patients with Fig. 8.5.23.8  Chest radiograph: Pneumocystis jirovecii pneumonia.

8.5.23  HIV/AIDS 911 P. jirovecii pneumonia the diagnosis is not confirmed but treatment is given empirically. High-​dose co-​trimoxazole (120 mg/​kg daily in divided doses) for 3 weeks is the first-​line treatment for pneumocystis pneumonia. Oral therapy is often adequate, but in moderate and severe cases the drug should be given intravenously. A switch to oral therapy can be made if fever, symptoms, and oxygenation have improved after 7–​10 days. Adverse reactions to co-​trimoxazole—​especially neutropenia, anaemia, rash, and fever—​occur in up to 40% of pa- tients, usually after 6–​14 days. Intravenous pentamidine (4 mg/​kg per day) is the second-​line choice for patients who do not tolerate co-​trimoxazole. Patients intolerant of co-​trimoxazole and pentamidine can be treated with clindamycin plus primaquine or dapsone plus trimetho- prim. These regimens have only been evaluated in patients with mild to moderate pneumocystis pneumonia, as has atovaquone, an antiprotozoal drug that is active against P. jirovecii. Although slightly less effective than co-​trimoxazole, atovaquone causes fewer adverse effects. In patients with moderate or severe pneumocystis pneumonia, ad- junctive high-​dose corticosteroids reduce morbidity and mortality. If the arterial partial pressure of oxygen (Pao2) is less than 9.3 kPa or the alveolar–​arterial oxygen gradient is greater than 4.7 kPa, oxygen, intravenous methylprednisolone or oral prednisolone should be given for 5 to 10 days. Patients who develop respiratory failure re- quire ventilatory support. After treatment for pneumocystis pneu- monia has been completed, secondary prophylaxis should be given to prevent recurrence. This can be discontinued if there is a good response to antiretroviral treatment, with a rise in the CD4 count sustained above 200/​mm3. Bacterial pneumonia The risk of bacterial pneumonia is increased in HIV, especially if the CD4 count is below 200/​mm3. The most common cause is Streptococcus pneumoniae, although Haemophilus influen- zae and Moraxella catarrhalis are also relatively common, and Staphylococcus aureus, Klebsiella spp., and other Gram-​negative rods are important causes in advanced HIV disease. Rare causes in- clude nocardia spp. and Rhodococcus equi. The presentation might be atypical, and radiological appearances frequently include diffuse infiltrates that resemble pneumocystis pneumonia, as well as more typical segmental or lobar patterns. Cavitation with abscess forma- tion, pleural effusion, and empyema can occur. HIV predisposes to recurrent invasive pneumococcal infections with bacteraemia; recurrent bacterial pneumonia in a 12-​month period is an AIDS-​ defining condition. Chronic lung damage with bronchiectasis and colonization by Pseudomonas aeruginosa have been reported. Other pulmonary complications Disseminated fungal infections, including Cryptococcus spp., can involve the lungs (see Chapter 8.7.2). In endemic areas, histoplas- mosis, coccidioidomycosis, and disseminated Taralomyces marnef- fei infection need to be considered (see next). Invasive Aspergillus fumigatus infections can occur in patients with advanced HIV dis- ease who have additional risk factors such as severe neutropenia. Patients usually have severe systemic illness. The radiographic appearances in all these fungal infections are usually non​specific. Bronchoalveolar lavage might be needed for diagnosis. HIV-​ associated lymphocytic interstitial pneumonitis (LIP) causes dif- fuse abnormalities, usually in children but occasionally in adults. Increasingly recognized are chronic lung abnormalities in adoles- cents who were infected vertically with HIV. Cryptogenic organ- izing pneumonia is a steroid-​responsive cause of lung infiltrates, probably a tissue response to various underlying conditions, which has also been reported in HIV and may be confused with pneumo- cystis pneumonia. Tuberculosis (See Chapter 8.6.26.) The interaction between HIV and tuberculosis (TB) was recognized early in the HIV epidemic. Studies in central Africa in the mid-​1980s showed that more than 60% of newly diagnosed tuberculosis patients were HIV positive at a time when the background seroprevalence of HIV in the population was much lower. Injecting drug users were shown to have an increased risk of developing active tuberculosis if they were HIV positive. After decades of progressive decline in the incidence of tuberculosis in the United States of America, notifica- tions increased during the mid-​1980s, soon after the emergence of the HIV epidemic. A similar trend was subsequently observed in Western Europe. Globally, tuberculosis remains the most frequent life-​threatening opportunistic infection in AIDS. Most cases of tuberculosis in HIV-​positive individuals represent reactivation of dormant bacilli. However, molecular typing of iso- lates of Mycobacterium tuberculosis by restriction fragment length polymorphism analysis suggests that up to 40% are new infections. The WHO estimates that one-​third of the world’s HIV-​positive population is coinfected with tuberculosis. In communities where M.  tuberculosis is endemic those who are immunosuppressed by HIV have an increased risk of relapsing or contracting new infec- tions. Where the background prevalence of tuberculosis is low, the disease is uncommon in HIV-​positive patients unless they become exposed (e.g. through travel). Testing for HIV should be done in all patients presenting with active tuberculosis, and tuberculosis should be considered as a cause of unexplained symptoms in pa- tients with HIV. Active tuberculosis can occur at any time during the course of HIV infection. In early-​stage HIV, it is more likely to present with the typical clinical features: subacute history of cough, fever, and Fig. 8.5.23.9  Pneumocystis jirovecii cysts in bronchoalveolar lavage aspirate.

912 section 8  Infectious diseases weight loss, upper lobe cavitary disease and/​or pleural disease on chest radiographs, and a positive skin test to tuberculin. In late-​stage HIV, infected patients are more likely to present atypically with un- usual chest findings, extrapulmonary involvement, and cutaneous anergy. The chest radiograph might be normal in up to 40% of cases and, when abnormal, upper lobe involvement is less common. Sputum smears should be examined for acid-​fast bacilli, but are less likely to be positive in HIV. Blood cultures can sometimes be posi- tive for M. tuberculosis. Patients with HIV and TB are more likely to relapse after com- pletion of therapy and to die prematurely if their HIV disease is not treated. Patients with advanced HIV infection are more likely to de- velop extrapulmonary tuberculosis involving lymph nodes, pericar- dium, liver, bone marrow, or meninges. Diagnosis can be difficult and frequently relies on invasive procedures to obtain appropriate specimens. The role of interferon-​γ release assays (IGRA) in the diagnosis of HIV-​associated TB is uncertain. The standard 6-​month regimen of four antituberculosis drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) is generally effective in patients with HIV, unless there is resistance to one or more of these first-​line drugs. The drug regimen might need to be adjusted once in vitro sensitivity results are known. For fully sensi- tive organisms, after 2 months on four drugs, isoniazid and rifam- picin should be continued for a further 4 months. It should be noted that rifampicin interacts with many of the antiretroviral drugs, particularly protease inhibitors, and these interactions need to be considered in deciding on drug regimens. Patients with pulmonary tuberculosis should, ideally, be isolated initially. Contact tracing is important; HIV-​positive contacts of a smear-​positive TB case are at particular risk and should be offered isoniazid preventive therapy (unless the index case has multidrug-​resistant TB). The tuberculin skin test might be negative, especially if the CD4 count is low, as might interferon-​γ release assays (IGRA). Up to 20% of patients with HIV experience adverse reactions to antituberculosis drugs. In HIV-​positive patients with tuberculosis in Africa, the sulpha-​based drug thiacetazone has been associated with serious skin reactions, including toxic epidermal necrolysis and fatal cases of Stevens–​Johnson syndrome. Whereas response rates for conventional short-​course tuberculosis treatment in industrialized countries are similar to those achieved in HIV-​negative patients, in resource-​limited countries and where compliance is less easily achieved, cure rates are lower and there is a risk that resistance will develop. Several countries have adopted a ‘directly observed therapy’ strategy to address this problem. Supervised drug administration is a component of this strategy but political commitment, secure drug supply, and good organization are needed for this to be effective. The optimal timing of the initiation of antiretroviral treatment in patients presenting with HIV-​associated TB has been controversial. Early initiation of ART increases the pill burden and risks immune reconstitution inflammatory syndromes (IRIS), whereas starting too late risks HIV disease progression. However, recent studies have clarified this area and have shown that in patients with TB and HIV whose initial CD4 count is less than 50/​mm3, ART and TB therapy should be started together. Although the incidence of IRIS reactions is increased, this does not increase mortality. Concomitant adminis- tration of prednisolone in this setting might reduce the risk of IRIS. For patients with CD4 counts greater than 200/​mm3, antiretroviral therapy can be deferred for 8 to 12 weeks. HIV therapy should not be delayed beyond 12 weeks, however. It should be noted that these studies were mainly carried out in patients with pulmonary TB and a study in TB meningitis showed no benefit with immediate ART and an increased frequency of severe adverse effects. Multidrug-​resistant tuberculosis Over 15 outbreaks of multidrug-​resistant tuberculosis (MDR-​TB) have been reported since the late 1980s. MDR-​TB isolates are re- sistant to at least two first-​line antituberculosis drugs, most com- monly isoniazid and rifampicin, and are often resistant to several agents. Most have occurred in HIV units in hospitals, but there have been outbreaks in prisons, drug treatment centres, and nursing homes. Most documented outbreaks have been in the United States of America. Elsewhere, over 200 people were involved in Buenos Aires, Argentina, and another outbreak affected over 100 people in Lisbon, Portugal. In MDR-​TB outbreaks, healthcare workers are at risk of becoming infected. Initially, the mortality among HIV-​ positive patients was very high (up to 93%), but the outcome has subsequently improved because of more rapid diagnosis and treat- ment with at least four drugs to which the M. tuberculosis isolate is sensitive in vitro. More recently, extensively drug-​resistant TB (XDR TB) in HIV-​infected patients in South Africa has caused outbreaks, with 100% mortality in those with HIV. To prevent outbreaks of MDR-​TB, special precautions are re- quired when HIV-​positive patients with possible tuberculosis are admitted to hospitals. Diagnosis must not be delayed, appropriate treatment must be started as soon as possible, and drug resistance identified, although this can be difficult in resource-​limited settings. Precautions include the isolation of patients in negative-​pressure rooms, use of respiratory protection for staff, and special care during certain procedures such as bronchoscopy or nebulized pentami- dine administration. With effective treatment, patients rapidly be- come non​infectious, but precautions need to be continued until the sputum is repeatedly culture-​negative. Mycobacterium avium complex In the absence of antiretroviral treatment, patients with advanced HIV infection and CD4 counts below 50/​mm3 are at high risk of dis- seminated Mycobacterium avium complex (MAC) infection, particu- larly in industrialized countries where historically it was reported to develop in up to 40% of patients with AIDS. M. avium is a ubiquitous environmental organism of low pathogenicity that can be isolated from domestic water supplies. Infection is likely to be through the gastrointestinal tract. MAC infection becomes widely disseminated in those with advanced HIV and causes fever, night sweats, weight loss, diarrhoea, abdominal pain, anaemia, disturbed liver function, and reduced overall survival. The organism can usually be cultured from blood or bone marrow, or might be recognized as acid-​fast ba- cilli in tissue biopsies (e.g. from lymph node, small bowel, or liver). It is unclear why the diagnosis is uncommon in low-​income countries; high mortality from other opportunistic infections at earlier stages of immunosuppression could be partly responsible. MAC infection is intrinsically resistant to most first-​line antituberculosis drugs. Comparative trials suggest that initial therapy should be with two or three drugs:  clarithromycin or azithromycin and ethambutol should be used, and additional rifabutin or a quinolone (e.g. ciprofloxacin) considered. In severely ill patients, intravenous amikacin might be useful as the third agent.

8.5.23  HIV/AIDS 913 In the absence of antiretroviral treatment, lifelong treatment is be required to prevent relapse; but if immunity is restored by highly active antiretroviral therapy, such maintenance therapy can be dis- continued. Early studies in the pre-​ART era showed some benefit with azithromycin prophylaxis in those with low CD4 counts but now it is more appropriate to use ART to improve the immune system. Other non​tuberculosis mycobacteria Other mycobacteria, notably Mycobacterium kansasii, Myco-​bacte- rium genavense, and Mycobacterium celatum, can cause opportun- istic infections in those with HIV. M. genavense, which colonizes pet birds, was discovered in European patients with HIV and causes fever, diarrhoea, and severe weight loss. HIV does not seem to affect the incidence or natural history of leprosy (Mycobacterium leprae). Gastrointestinal disease Oesophageal candidiasis Oesophagitis presents with retrosternal pain on swallowing, and in patients with HIV is most commonly caused by C.  albicans. Oesophageal candidiasis indicates advanced immunosuppres- sion and is an AIDS-​defining condition. The diagnosis should be suspected in a patient with oral candida and dysphagia, and may be supported by barium swallow or confirmed by endoscopy and biopsy. Treatment is with oral azole antifungal agents such as fluconazole. It might recur and in patients with severe immunosup- pression, and in the absence of antiretroviral treatment, candida can become resistant to prolonged azole treatment. Resistance tends to develop gradually and can be monitored by in vitro testing. Such pa- tients require treatment with high doses of fluconazole or one of the newer azoles. Azole-​resistant oro-​oesophageal candidiasis has be- come rare since the advent of antiretroviral therapy. Echinocandins or amphotericin can be used in such cases. The differential diagnosis of oesophageal candidiasis includes oesophagitis caused by cytomegalovirus (CMV) or herpes sim- plex virus (HSV), which require specific antiviral therapy, and aphthous ulceration, which might respond to oral prednisolone or thalidomide. Intestinal infections Some infections are much more common in HIV disease than in other settings. Cryptosporidium parvum can lead to cholera-​ like diarrhoea. An ascending cholangitis might occur with fever, pain, and jaundice and have the imaging appearance of scler- osing cholangitis. Other protozoan parasites, such as Cystisospora belli and Cyclospora cayetanensis, may also cause diarrhoea, as can microsporidia. Cytomegalovirus can cause an acute colitis with pain and bloody diarrhoea. Sigmoidoscopy shows ulceration and biop- sies show characteristic CMV inclusions. Tuberculosis can also pre- sent as intestinal disease or peritoneal infection. HIV enteropathy Many patients with HIV, especially in the tropics, present with diar- rhoea and malnutrition leading to wasting in the absence of detect- able gastrointestinal opportunist infections. Biopsies often show villous blunting and increased inflammatory cells in the lamina propria of the small bowel and functional tests suggest increased bowel permeability. The pathogenesis of this enteropathy is poorly understood, but might involve cytokine activation secondary to HIV infection. HIV and the nervous system (See Chapter 24.11.4) The nervous system is a major site of involvement for direct and in- direct complications of HIV at all stages of infection. All parts of the nervous system may be affected. In advanced HIV, opportunistic infections and tumours (lymphoma), and tissue damage caused by HIV replication in the brain and spinal cord, are important and rela- tively common during progressive HIV disease. Cerebral toxoplasmosis (See Chapter 8.8.4.) Cerebral infection with the intracellular protozoan Toxoplasma gon- dii is the most frequent infection of the central nervous system in AIDS, occurring when the CD4 count is below 200/​mm3. It usually results from reactivation of toxoplasma cysts in the brain, leading to the formation of focal lesions that are typically multiple but may be single. Symptoms develop subacutely and include focal neurological disturbance, headache, confusion, fever, and convulsions. On com- puted tomography (CT) scan the lesions appear as ring-​enhancing masses with surrounding oedema (Fig. 8.5.23.10). MRI is more sensitive and frequently detects lesions not visible on CT. Serum antibodies to toxoplasma spp. are usually detectable; their absence makes the diagnosis unlikely but does not exclude it. Detection of toxoplasma DNA in cerebrospinal fluid by PCR is being evaluated as a diagnostic test. The principal differential diagnosis is primary cere- bral lymphoma; other causes of focal brain lesions in AIDS include cryptococcoma, cerebral abscess (including infection with nocardia spp.), tuberculoma, progressive multifocal leukoencephalopathy, and neurosyphilis. Brain biopsy is required for a definitive diagnosis, but is rarely performed. As toxoplasmosis is by far the most common treatable cause of focal cerebral lesions in HIV, it is standard practice to treat for this and only consider biopsy if there is no clinical im- provement in 7–​10 days. Fig. 8.5.23.10  Cerebral toxoplasmosis: ring enhancement and surrounding cerebral oedema (CT with contrast).

914 section 8  Infectious diseases The condition responds well if treatment is started early; a combin- ation of sulfadiazine at 4 to 6 g/​day and pyrimethamine at 50–​75 mg/​ day is the treatment of choice. More than 40% of patients experi- ence adverse effects, especially rash and nephrotoxicity caused by sulfadiazine. The haematological toxicity of pyrimethamine might be reduced by adding folinic acid (10 mg/​day). If sulpha drugs are not tolerated, clindamycin with pyrimethamine is an effective al- ternative. Corticosteroids can be used to reduce cerebral oedema in patients with large lesions and serious mass effects, but this is controversial. Treatment is usually given for 3 to 6 weeks, and in the absence of effective antiretroviral treatment, relapse is common after stopping. In these circumstances, lifelong maintenance treatment is usually required using pyrimethamine (25–​50 mg/​day) with a sulpha drug or clindamycin. However, these can be discontinued if antiretroviral treatment leads to sustained immunological recovery. The use of pri- mary prophylaxis against PCP also reduces the risk of toxoplasmosis. Cryptococcal meningitis (See Chapter 8.7.2.) Although infection of the central nervous system with Cryptococcus neoformans can occur in the absence of immunodeficiency, it most commonly arises in association with HIV infection. Before the wide- spread use of azole antifungals for mucosal candidiasis, it accounted for 5 to 10% of opportunistic infections in patients with AIDS. The presentation is usually subacute and may be subtle and non​specific with headache, vomiting, and mild fever, and few neurological signs. Less frequently, psychiatric disturbance, convulsions, cranial nerve palsies, truncal ataxia, or focal intracerebral lesions can occur. Neck stiffness is unusual. The diagnosis is made by detecting cryptococci in the cerebrospinal fluid by India ink staining, detection of crypto- coccal antigen in the cerebrospinal fluid (uniformly positive), and culture. Cryptococcal antigen is also usually detectable in serum. C. neoformans in patients with AIDS causes minimal inflammation, so the white cell count of the cerebrospinal fluid is often only mildly raised and the protein and glucose levels of the cerebrospinal fluid may be normal. A randomized, controlled trial showed that the combination of amphotericin B and flucytosine was superior to amphotericin B alone or fluconazole alone for the treatment of cryptococcal men- ingitis. Amphotericin B and flucytosine together lead to more rapid sterilization of the cerebrospinal fluid, but are not as well tolerated as fluconazole. Resistance of cryptococci to fluconazole is very rare. Adverse reactions to amphotericin are frequent, especially fever, myalgia, renal impairment, and electrolyte disturbances. Close monitoring is required. Lipid formulations of amphotericin are in- creasingly used and have become the standard of care. Raised intra- cranial pressure is associated with clinical deterioration and the risk of blindness: repeated lumbar punctures and, sometimes, lumbar or ventricular shunting are needed in these circumstances. Steroids have been shown to be ineffective and possibly deleterious. Without secondary prophylaxis, cryptococcal meningitis relapses in 50 to 80% of patients with HIV in the absence of antiretroviral treatment. Oral fluconazole (200 mg/​day) is effective for mainten- ance, and can be discontinued when antiretroviral treatment leads to sustained immunological recovery. A recent trial has found that oral fluconazole is safe and effective as primary prophylaxis against cryptococcal disease in patients awaiting or starting antiretroviral therapy in Uganda. Progressive multifocal leukoencephalopathy Progressive multifocal leukoencephalopathy is a progressive demyelinating condition of advanced HIV disease caused by JC virus, a polyomavirus cytopathic for oligodendroglia. It presents with focal neurological deficits, personality changes, or ataxia; headache and mass effects are absent. Brain MRI, the investigation of choice, usually shows multiple white matter lesions. JC virus is detectable in cerebrospinal fluid by PCR, but this is not usually ne- cessary for diagnosis. There is no specific treatment. Survival of less than 6 months is usual, but progression might sometimes be halted or reversed by highly active antiretroviral therapy. The other human polyomavirus, BK virus, is a very rare cause of encephalitis and interstitial nephropathy in AIDS. Primary cerebral lymphoma These are B-​cell non-​Hodgkin lymphomas that are associated with Epstein–​Barr virus, which is usually detectable in the cerebrospinal fluid by PCR. Lymphoma of the central nervous system may present in a manner similar to toxoplasmosis, with focal signs or seizures. CT or MRI of the brain often reveals a single space-​occupying lesion but the disease is typically multifocal. Without treatment the prog- nosis is only a few weeks. Treatment with radiotherapy and steroids, with or without chemotherapy, and antiretroviral therapy, prolongs the median survival to several months, but longer-​term survival is exceptional. HIV-​associated neurological disorders (HAND) HIV can infect the nervous system directly, leading to a variety of clinical problems. The virus targets macrophages in the nervous system but can also infect astrocytes, but non​productively. Most patients dying of AIDS show histological evidence of brain in- volvement including neuronal loss. It is estimated that up to 50% of those with HIV have some form of neurological disorder. So-​ called HAND (HIV-​associated neurological disorder) is split into three types. The most common is asymptomatic neurocognitive impairment where individuals have abnormalities demonstrated by neuropsychological testing. Some of these abnormalities can be moderately severe and impact on daily living, including the ability to adhere to treatment regimens. A smaller subset, up to 12% of those with HIV in one study, have more marked problems, termed mild neurocognitive disorder. These individuals have worse scores on testing and usually have symptoms and functional impairment. At the extreme are those with HIV-​associated dementia (HAD), previ- ously called AIDS dementia complex or HIV encephalopathy. Risk factors for HAND include a lower nadir CD4 count and older age at time of infection. Other comorbidities also play a role, such as cardiovascular disease, but no clear genetic influences have been identified. Diagnosis is largely clinical, along with neuropsychological testing when available. Although abnormalities in the cerebro- spinal fluid have been reported, such as elevated protein levels, lymphocytosis, and detectable virus, none of these are consistent or diagnostic. Virus levels in the cerebrospinal fluid do not cor- relate with disease.

8.5.23  HIV/AIDS 915 There has been a lot written about ART and the effect on HAND. The CASCADE study showed that the incidence of HAD had dimin- ished with the advent of ART but had not disappeared completely. Nevertheless, it is not clear that asymptomatic neurocognitive im- pairment is affected much by treatment. Various ART drugs have been assessed to have better central nervous system penetration efficacy (CPE), such as tenofovir, emtricitabine, and dolutegravir but it is not really clear if choosing drug combinations with good CPE affects outcomes. There are examples of patients developing neurocognitive problems while on ART who have detectable virus in the cerebrospinal fluid that is resistant to the ART, but these are very rare. Other psychological/​psychiatric problems include anxiety, panic attacks, and depression. Psychotherapy might be helpful. Antidepressants are needed in severe cases. Acute psychosis is rare. Dystonic reactions to various drugs, such as metoclopramide, are more common in patients with HIV. In the late stages of HIV disease, the differential diagnosis of HIV-​ associated dementia includes cytomegalovirus (CMV) encephal- itis. This usually presents with rapidly progressive confusion and dementia, impaired consciousness, fever, cranial nerve lesions, and convulsions. MRI shows necrotizing periventriculitis; protein levels in cerebrospinal fluid might be elevated and CMV DNA is detect- able in the cerebrospinal fluid by PCR. Ganciclovir and other anti-​ CMV agents might reduce progression. Peripheral neuropathy and myelopathy Peripheral neuropathy can occur at any stage of HIV infection, even with primary infection, but is most common in advanced disease, when 10 to 15% of patients have a distal symmetrical sen- sorimotor neuropathy of axonal type causing pain and paraesthesia that can limit walking and, less often, distal weakness and atrophy. Mononeuritis multiplex and acute inflammatory demyelinating polyneuropathy resembling the Guillain–​Barré syndrome are also described, generally at an earlier stage. Drugs used in patients with HIV, including stavudine, didanosine, and vincristine, might cause or exacerbate peripheral neuropathy. HIV-​related autonomic neur- opathy can cause postural hypotension, diarrhoea, impotence, impaired sweating, and bladder symptoms. CMV infection in pa- tients with AIDS presents with a lumbosacral polyradiculopathy causing sacral paraesthesiae and numbness, lower limb weakness, and urinary retention that might progress to flaccid paraparesis if untreated. HIV can involve the spinal cord directly, causing a vacuolar myel- opathy. This usually presents with bilateral leg weakness and sen- sory symptoms, usually paraesthesias, and might progress to spastic paraparesis, ataxia, and incontinence. Rarely, a myopathy can occur. Ocular disease Cytomegalovirus retinitis Without antiretroviral therapy, up to 30% of patients with AIDS (and a CD4 lymphocyte count below 50/​mm3) develop reactivation of CMV in the form of a destructive and blinding retinitis. This is rare in other types of immunosuppression. It usually presents with blurring of vision, scotomas, floaters, or flashing lights. The charac- teristic retinal changes are patches of irregular retinal pallor, caused by oedema and necrosis, and haemorrhages in a perivascular distri- bution (Fig. 8.5.23.11). The retinitis usually starts peripherally and progresses rapidly to involve the macula and whole retina, leading to blindness. Complications include retinal detachment, branch retinal artery occlusion, persistent iritis, and cataract. CMV retinitis should not be confused with cotton wool spots (HIV retinopathy)—​small, pale retinal lesions without haemorrhages that commonly occur in patients with HIV. These are benign and often come and go. The diagnosis of CMV retinitis is clinical, based on the character- istic retinal appearance (see Chapter 25.1). CMV viraemia may be detectable by PCR, and high or rising CMV viral load is associated with an increased risk of developing retinitis and other CMV disease. Anti-​CMV drugs (ganciclovir, foscarnet, cidofovir) are virustatic. Before the availability of antiretroviral drug therapy, the aim of treatment was to stop progression rather than to cure disease. First-​ line treatment is with intravitreal ganciclovir injection or implant; oral valganciclovir is often given in conjunction with intravitreal ganciclovir as patients treated without systemic therapy may develop contralateral or extraocular CMV disease. Oral valganciclovir alone may be used as an alternative in patients without sight-​threatening disease. Other effective treatments include intravenous ganciclovir, foscarnet, or cidofovir. Intravenous ganciclovir and forscarnet require administration via a central vein, whereas foscarnet can be given via a peripheral vein, The main adverse effects of ganciclovir are bone marrow suppres- sion, in particular neutropenia, central nervous system symptoms, abnormal liver function tests, fever, and rash. Foscarnet is associ- ated with decreased renal function, electrolyte abnormalities, and infusion-​related nausea. With the advent of ART, CMV retinitis is much less common in developed countries. Sustained suppression of HIV viral load and improvement in immune status can allow discontinuation of main- tenance treatment. New manifestations of ocular CMV, such as vitritis, have been reported in patients starting ART (see ‘Immune reconstitution syndromes’, next). Other ocular syndromes Acute retinal necrosis is a rare condition originally reported in reactivation of varicella zoster virus in otherwise healthy adults. In patients with advanced HIV infection, it is usually preceded by dermatomal herpes zoster and typically presents with blurring of Fig. 8.5.23.11  CMV retinitis.

916 section 8  Infectious diseases vision and pain in the affected eye. Progressive necrotizing ret- initis leads to visual deterioration that may be associated with uveitis. An outer retinal necrosis syndrome with little ocular in- flammation also occurs in patients with AIDS. There is a high risk of visual loss and retinal detachment. Both eyes can be affected. Suspected acute retinal necrosis should be treated with intra- venous aciclovir. Acute toxoplasma choroidoretinitis can resemble CMV retinitis, but the retinal scarring that follows treatment is distinctive. The dis- ease is more common in countries such as Brazil and France where the background prevalence of toxoplasmosis is much higher than in the United Kingdom. Choroidoretinitis is also a rare complica- tion of histoplasmosis and cryptococcosis, and uveitis can occur in syphilis. HIV-​related tumours HIV is an oncogenic virus and even for individuals with high CD4 counts (>500 cells/​cc3) there appears to be an increased risk of developing cancer compared to uninfected individuals. In the re- cently reported START trial where individuals were randomly al- located to immediate or deferred ART initiation with CD4 counts greater than 500, those in the deferred arm had an increased risk of cancer. Kaposi’s sarcoma Kaposi’s sarcoma characteristically presents as multiple, purplish, nodular skin lesions (Fig. 8.5.23.12). Lesions start as small, pink, deep purple, or brown macules, and develop into nodules or plaques that may ulcerate. They also occur on mucosal surfaces, most com- monly on the hard palate. Local or regional oedema and lymph node enlargement may occur. Mucocutaneous lesions are cosmetically and psychologically important but are rarely of clinical importance (Fig. 8.5.23.13). However, visceral disease, which most commonly affects the lungs and gastrointestinal tract, is an important cause of morbidity and even mortality. Lung lesions cause dyspnoea, cough, or haemoptysis, and gut involvement may cause abdominal pain, bleeding, or a rare protein-​losing enteropathy. Extensive visceral in- volvement can cause constitutional symptoms such as fevers, night sweats, and weight loss. Kaposi’s sarcoma rarely affects the central nervous system. In industrialized countries, Kaposi’s sarcoma is over 2000 times more common in HIV-​infected individuals than in the general population. Classic Kaposi’s sarcoma in HIV-​negative individ- uals occurs in middle-​aged and older men of Eastern European or Mediterranean origin. Endemic Kaposi’s sarcoma in Africa has been known for decades. It is predominantly a disease of older men that has a fairly indolent course. HIV-​related Kaposi’s sar- coma, on the other hand, is a more aggressive disease and oc- curs mostly in those people who have acquired HIV via a sexual route, namely gay and bisexual men and in younger African men and women. The epidemic of Kaposi’s sarcoma in Central and East Africa exactly mirrors the HIV epidemic in these regions. Kaposi’s sarcoma is rare in intravenous drug users and very rare in recipients of blood products, including those with haemophilia. These epidemiological features suggested a sexually transmissible aetiological agent. (d) (c) (b) (a) (e) Fig. 8.5.23.12  Kaposi’s sarcoma. (a, b) cutaneous Kaposi’s sarcoma in a white man; (c, d), cutaneous Kaposi’s sarcoma in a Zimbabwean; (e) invasive Kaposi’s sarcoma in a Kenyan. Copyright D. A. Warrell.

8.5.23  HIV/AIDS 917 In 1994, a new herpesvirus, human herpesvirus 8 (HHV-​8), was found in HIV-​related Kaposi’s sarcoma and was soon detected in the lesions of all forms of Kaposi’s sarcoma. Seroepidemiological studies show that HHV-​8 is common only in certain geographical regions, corresponding to where Kaposi’s sarcoma was endemic before the era of HIV. HHV-​8 is detectable in saliva but less often in semen. This might explain why both sexual and other routes of transmis- sion occur. In Africa, where HHV-​8 infection is common, it is trans- mitted perinatally from mother to child. Kaposi’s sarcoma lesions are characterized by proliferating spindle cells of lymphatic and blood vascular endothelial origin, thin-​walled slit-​like vascular spaces, infiltration by lymphocytes and plasma cells, and extravasated red cells. Multiple lesions appear synchron- ously in widely dispersed areas. The clonality of Kaposi’s sarcoma lesions has not been fully resolved. Although some studies have sug- gested a monoclonal origin, others have shown a mixed picture and the lesions may be reactive proliferative rather than truly cancerous. HHV-​8 is detectable in spindle cells and flat endothelial cells lining the vascular spaces of Kaposi’s sarcoma lesions. It is likely that the virus triggers the release of cellular and virus-​encoded cytokines that promote the proliferation of spindle cells. Antiretroviral therapy has led to a dramatic reduction in the fre- quency and mortality of Kaposi’s sarcoma in developed countries. In early Kaposi’s sarcoma, the progression is often halted or reversed by starting antiretroviral treatment alone. Otherwise, cutaneous le- sions may be left untreated or treated with local radiotherapy, cryo- therapy, or intralesional vinblastine. Widespread skin or visceral disease is usually treated by systemic chemotherapy, usually with a liposomal anthracycline such as daunorubicin or doxorubicin, which are more effective than the previously used combination of vincristine and bleomycin. Paclitaxel, a taxane, is potentially more toxic than liposomal anthracyclines, but can be useful as a second-​line agent after treatment failure. Treatment of dissemin- ated Kaposi’s sarcoma has not been considered to be curative, but remissions might be induced by a combination of ART and sys- temic chemotherapy. Non-​Hodgkin’s lymphoma Non-​Hodgkin lymphoma develops in 3 to 10% of HIV-​positive patients, an incidence 60 to 100 times higher than in the general population. Most tumours are extranodal and, histologically, 60% are large cell B-​cell lymphomas; 30% are Burkitt’s type and the rest are of T-​cell or non-​B-​, non-​T-​cell origin. Some 50% are associated with Epstein–​Barr virus infection and are more aggressive with a shorter survival. A minority of HIV-​related lymphomas are associ- ated with HHV-​8. They present as body cavity lymphomas, causing pleural or peritoneal effusions (primary effusion lymphoma). Patients on highly active antiretroviral therapy have a reduced risk of developing non-​Hodgkin lymphoma, and consequently the in- cidence of HIV-​related lymphomas in developed countries has de- clined in recent years. HIV-​associated lymphoma outside the central nervous system may respond well to standard lymphoma chemotherapy regimens, in addition to antiretroviral therapy. Response is better in those who are less immunosuppressed (CD4 above 200/​mm3 and no pre- vious AIDS diagnosis). Opportunistic infections cause many deaths during chemotherapy. Lower dose or less toxic chemotherapy proto- cols are sometimes advocated for patients with more advanced HIV disease. Non-​AIDS-​defining cancers It is now apparent that HIV increases the frequency of several can- cers other than those that have been categorized as AIDS-​defining (i.e. Kaposi’s sarcoma, non-​Hodgkin’s lymphoma, and cervical car- cinoma), and non-​AIDS-​defining cancers cause a high proportion of deaths in HIV patients in industrialized countries (50% in a re- cent study from France). These include Hodgkin’s disease, particu- larly of the mixed cellularity type and associated with Epstein–​Barr virus. Disseminated disease with a poor prognosis seems to be more frequent than for HIV-​negative Hodgkin’s disease. Other cancers that arise with higher frequency than in HIV-​ negative individuals include hepatocellular carcinoma (caused by hepatitis C and hepatitis B); lung carcinoma; skin tumours (basal cell carcinoma, squamous cell carcinoma, and malignant melanoma); and cancers of the head and neck, probably associated with human papillomavirus (HPV), especially type 16. There is an increased in- cidence of squamous cell carcinoma of the conjunctiva in patients with HIV infection, especially in Africa. HPV is also associated with a higher incidence of cervical intraepithelial neoplasia (CIN) and predisposition to cervical carcinoma in HIV-​infected women, and the higher incidence of vulvar intraepithelial neoplasia in women; and also anal carcinoma and its precursor anal intraepithelial neo- plasia, which occurs at a greatly increased frequency in HIV-​positive gay men. Other cancers that might arise with greater frequency, but where this is less clear cut, include colorectal cancer, testicular cancer, myeloma, and acute myeloid leukaemia. Cancers that do not arise with greater frequency include prostate, bladder, and breast. It should be noted that even when ART is started at high CD4 counts, the incidence of malignancy seems not to change, suggesting that HIV itself is associated with an increased risk of cancer. Fig. 8.5.23.13  Palatal Kaposi’s sarcoma. Copyright D. A. Warrell.

918 section 8  Infectious diseases Miscellaneous conditions Castleman’s disease Castleman’s disease (angiofollicular lymph node hyperplasia) is a lymphoproliferative condition that can be HHV-​8 related and, in the multicentric form, is associated with HIV. It can sometimes be difficult to distinguish from Kaposi’s sarcoma. Bacillary angiomatosis Disseminated infection with Bartonella henselae, the principal agent of cat-​scratch disease, is the cause of bacillary angiomatosis, an HIV-​associated condition that typically causes multiple sub- cutaneous vascular lesions, fever, liver lesions (bacillary peliosis hepatis), and osteolytic bone lesions. The skin lesions are usually purplish nodules that may be mistaken for Kaposi’s sarcoma, but the histology is distinct—​acute neutrophilic inflammation and capillary proliferation, and clusters of bacilli revealed by modi- fied silver staining. The organism may be cultured from blood. A similar syndrome in HIV-​positive patients can be caused by the agent of trench fever, Bartonella quintana. Bacillary angiomatosis usually responds to treatment with a macrolide antibiotic. Cats and cat fleas form a reservoir for B. henselae, and patients who de- velop bacillary angiomatosis frequently have a history of contact with cats. Disseminated fungal infections In regions where invasive fungal infections are endemic (such as Histoplasma capsulatum in the Mississippi river region, Coccidioides immitis in the southern United States of America, and Talaromyces marneffei in South East Asia) or where there is a relevant travel history, disseminated fungal infection should be considered in HIV-​positive patients presenting with fever, weight loss, anaemia, pulmonary infiltrates, lymphadenopathy, and hepatosplenomegaly. Papular skin lesions might be seen in disseminated histoplasmosis and T.  marneffei infection. Similar lesions resembling giant molluscum (see next) might occur with disseminated cryptococcosis. Blood or bone marrow cultures or direct identification by the use of special stains on tissue obtained from skin lesions, bone marrow, or liver are required for diag- nosis. Initial therapy is generally with intravenous amphotericin; itraconazole (for histoplasmosis and T. marneffei) or fluconazole (for coccidioidomycosis) might be adequate for subsequent main- tenance treatment. Leishmaniasis (See Chapter 8.8.12.) HIV-​associated disseminated leishmaniasis is mostly reported from the Mediterranean littoral, South America, and Africa. It is caused by dissemination of leishmania spp., protozoan para- sites transmitted by sandflies. A high index of clinical suspicion is required, because although the classic features are fever, weight loss, anaemia, and hepatosplenomegaly, a high proportion of pa- tients have fever alone. The disease may present months or years after exposure in an endemic country. Leishmania can be trans- mitted by shared needles in injecting drug users. Most cases can be diagnosed by bone marrow examination or splenic aspirate; serology may be helpful. Treatment is with lipid formulations of amphotericin B. Haematological conditions Thrombocytopenia is relatively common (5–​15%) in HIV infec- tion and may be how the disease first presents. It is associated with antiplatelet antibodies. Symptomatic thrombocytopenia is un- common but more likely in the later stages of HIV infection. Life-​ threatening bleeding is rare. Thrombocytopenia is not a marker for HIV progression and spontaneous remissions are frequent. Antiretroviral treatment is first-​line when the CD4 count is low; zi- dovudine is known to increase platelet production. When specific treatment for thrombocytopenia is required, the principles and response are similar to those that apply in the treatment of HIV-​ negative immune thrombocytopenia, and include the use of pred- nisolone, intravenous immunoglobulin, and splenectomy. Anaemia is common in patients with advanced HIV infection, and is frequently related to medications (such as zidovudine). Human (B19) parvovirus infection is a reversible cause of chronic anaemia in HIV infection. Bone marrow biopsy typically shows an absence of erythroid development with occasional giant pronormoblasts, and B19 parvovirus is detected by PCR. The anaemia might respond to treatment with intravenous immunoglobulin. Mild neutropenia is common in HIV-​positive patients at all stages of infection, and may be partly responsible for the increased risk of pyogenic bacterial infections; however, profound neutropenia (below 0.5 × 109/​litre) is rare. Antineutrophil antibodies might be present. Drugs (such as co-​trimoxazole, ganciclovir, and antiretro- virals) may increase the incidence and severity of neutropenia. In selected HIV-​positive patients with refractory or life-​threatening bacterial or fungal infection and severe neutropenia, the addition of recombinant human granulocyte colony-​stimulating factor to the treatment regimen might improve the outcome. Skin conditions in advanced HIV In the later stages of HIV infection, several infections have atyp- ical cutaneous manifestations. All have become rare in settings where antiretroviral treatment is available to prevent advanced immunosuppression. These conditions include giant molluscum contagiosum, characterized by large, flesh-​coloured, non​tender umbilicated lesions often affecting the face in homosexual men. In advanced HIV disease, genital herpes simplex infection may cause painful chronic genital or anal ulcers that can become resistant to aciclovir and related compounds; intravenous foscarnet or cidofovir are effective. Aciclovir-​resistant varicella zoster virus also occurs in AIDS; and reactivation of varicella zoster virus can take an un- usual form, with a subacute course and dissemination causing scat- tered vesicular lesions in the absence of dermatomal zoster. CMV is a cause of chronic perianal ulceration that can be treated with ganciclovir. Atypical cutaneous presentations of syphilis can occur at any stage of HIV infection. HIV-​associated nephropathy (HIVAN) HIV can directly infect glomerular and tubular epithelial cells and renal disease is relatively common in HIV-​infected patients, most commonly caused by a collapsing focal segmental glomerulosclerosis, also known as HIV-​associated nephropathy (HIVAN). This typic- ally presents as a nephrotic syndrome, but with minimal oedema. It appears to be more common in Africans and African Americans than in the white population. Renal function usually improves with

8.5.23  HIV/AIDS 919 the use of antiretroviral medication, but some patients progress to chronic renal failure and require renal replacement therapy or trans- plantation. Other renal diseases are described in HIV including membranoproliferative glomerulonephritis associated with hepa- titis C, immune complex glomerulonephritis with IgA deposits, and membranous nephropathy. Drug toxicity is an important cause of renal impairment in HIV patients, including drugs used to treat op- portunistic infections (e.g. cidofovir, amphotericin B, pentamidine). Antiretroviral treatment (e.g. tenofovir) may cause Fanconi’s syn- drome due to tubular damage; atazanavir and indinavir may induce renal calculi and a nephropathy). Renal impairment caused by con- current conditions such as diabetes or hypertension can also arise in patients with HIV infection. HIV and hepatitis virus coinfections Because of common risk factors for blood-​borne virus infections, there are increasing numbers of individuals with HIV who are coinfected with either hepatitis C (HCV) or hepatitis B (HBV) virus, or both. Over the past few years, new data have become available on the size of this problem and some management strategies have emerged. HIV/​HCV coinfection HCV coinfection occurs in up to a third of those with HIV. The group with the highest prevalence is the haemophiliac population, but the group most at risk now is injecting drug users (IDU). Anywhere be- tween 50 and 75% of IDU with HIV are coinfected. More recently, there is an awareness that growing numbers of men who have sex with men are acquiring HCV sexually. HCV/​HIV coinfection increases the risk of liver disease pro- gression compared to HCV infection by itself. In addition, the treatment of HIV with antiretrovirals might carry an increased risk of hepatotoxicity in those with coinfection. The impact of HCV on HIV progression is less clear but it might blunt CD4 re- covery with ART. Treatment aimed at clearing HCV in those with coinfection ini- tially used combinations of pegylated interferon and ribavarin, but were less likely to lead to a sustained virological response compared to treating those with HCV infection alone. However, the advent of directly acting antiviral drugs (DAA) for HCV in- fection has revolutionized the management of coinfected patients (see Chapter  8.5.22). In HIV-​negative patients with HCV, these new drugs have produced cures of greater than 90% and similar results are seen in those with HIV coinfection. The optimum DAA combination for those with coinfection is yet to be determined and will depend on the HCV genotype. It is important to be aware of drug interactions with ART and, in some circumstances, the ART might need to be altered to get the best chance of clearing the HCV infection. HIV/​HBV coinfection The scale of the HIV epidemic in Africa and, now, in Asia means that up to 90% of those with HIV will have evidence of past or current HBV infection. Estimates vary, but up to 10% of those with HIV may be HBV carriers. Unlike with HCV, there is no evidence that liver disease due to HBV is worse in HIV coinfected individuals. Although HBV DNA levels are higher in HIV coinfection, there is evidence that there is less liver injury. New HBV infections in HIV-​positive individuals are less likely to cause acute hepatitis and jaundice, but are more likely to result in chronic HBV carriage. HBV infection does not seem to affect the progression of HIV disease. Treatment of HBV coinfection is less well defined than treatment of HCV coinfection. Although pegylated interferon might be useful, there are no large trials of its efficacy in HIV. Lamivudine mono- therapy is more likely to lead to resistant HBV with the ‘YMDD’ mu- tant in the presence of HIV. Drugs currently used to treat HBV in the absence of HIV coinfection also have anti-​HIV activity, particu- larly lamivudine, emtricitabine, and tenofovir. Caution must be used in treating HBV with these agents in coinfected patients as mono- therapy with these drugs will lead to resistant HIV. A recently intro- duced drug to treat HBV, entecavir, was thought not to have activity against HIV but had now been shown to have an anti-​HIV effect and this can also lead to HIV resistance. By contrast, if HBV coinfected patients require antiretroviral therapy, a combination of lamivudine and tenofovir, or emtricitabine and tenofovir, is recommended as part of the antiretroviral regimen to decrease HBV replication and avoid the onset of resistance. Management of HIV infection and prognosis The advent of antiretroviral therapy (ART) in the mid-​1990s led to marked reductions in morbidity and mortality attributable to HIV and its complications. A decline in the incidence of opportunistic in- fections, notably pneumocystis pneumonia, disseminated M. avium complex (MAC) infection, CMV retinitis, cerebral toxoplasmosis and cryptococcal meningitis, and associated mortality was reported from the United States of America and Europe after the introduc- tion of treatment based on a minimum of three antiretroviral drugs. HIV infection in adults in resource-​rich countries is now almost exclusively managed on an outpatient basis. Antiretroviral treat- ment has transformed HIV/​AIDS from a uniformly fatal disease to a long-​term condition. Although the prognosis is variable and influenced by adherence to ART, a large-​scale study of mortality in HIV-​infected adults in Europe has shown that in patients who are stable on treatment the standardized mortality ratio (SMR) ap- proaches normal (1.05) and nearly half of patients have SMR less than 2, which is lower than for type 1 diabetes. In industrialized countries, ART has also influenced the proportion of deaths from non-​AIDS causes, which now exceed AIDS-​related deaths and typ- ically include non-​AIDS-​defining cancers, cardiovascular disease, and liver disease. The recent changes in WHO and many national ART guidelines recommending immediate initiation of ART for all HIV+ individuals irrespective of CD4 count will, in the long term, enhance clinical outcomes further. This carries a burden of clinical management and cost of drug provision for the lifetime of all HIV+ individuals. However, these costs are offset by the significantly im- proved clinical benefit and also by the reduction in the risk of on- ward viral transmission. Initial assessment and management Ideally, HIV infection should be identified at the asymptomatic stage. At the time of diagnosis, patients should undergo a base- line assessment that includes taking a detailed history, including a sexual history, and determination of risk factors for HIV

920 section 8  Infectious diseases infection. An assessment of cardiovascular risk factors should be made, including smoking and family history. A detailed phys- ical examination should be performed with attention to the skin and mucous membranes, blood pressure, and body mass index, fundoscopy, and should include a search for lymphadenopathy and signs of liver disease. Initial investigations include full blood count, biochemical screen including liver profile, bone profile, and estimated glomerular filtration rate, lipids, chest radiography, and serological screening for infections that may require add- itional treatment (hepatitis B and C, syphilis) or which can reacti- vate during immunosuppression (CMV and toxoplasma and TB). Baseline investigations include CD4 lymphocyte (T-​helper cell) count (lymphocyte subsets profile), quantitative estimation of HIV RNA in the blood plasma (viral load), and HIV genotypic resist- ance testing; and should include tissue typing for HLA-​B*5701, a marker for abacavir hypersensitivity. In women, cervical cytology screening is indicated at annual intervals. All HIV+ individuals should be referred for appropriate TB screening. Following initial assessment, hepatitis B immunization can be provided to susceptible individuals, and pneumococcal vaccine can be offered. Consideration should also be given to HPV vaccin- ation in women and in men who have sex with men. Psychological support and counselling are often needed. There should be a dis- cussion about who should be informed about their HIV status, including the primary care physician, and family members and friends for support. The issue of disclosure to sexual partners should also be raised, with advice on reducing risk of transmission (including the importance of barrier methods such as condoms). Antiretroviral treatment of the HIV-​infected partner reduces risk of transmission to their HIV-​negative sexual partner, although safe sex counselling should still be provided. The CD4 lymphocyte count and HIV viral load are the two laboratory markers that have the best prognostic value. The CD4 count is a reliable indicator of HIV-​related immune im- pairment. CD4 counts, normal at or above 900/​mm3, vary con- siderably, even in the absence of HIV infection. A fall in the CD4 lymphocyte count to below 200/​mm3 is associated with a risk of opportunistic infections of about 80% over 3 years without anti- retroviral treatment. However, progression is variable and a mi- nority remain well for several years with stable low CD4 counts. This variability is explained partly by differences in HIV viral load. The level of CD4 lymphopenia generally determines the spectrum of potential infections (Table 8.5.23.1). For instance, whereas oral and oesophageal candidiasis and pneumocystis Table 8.5.23.1  Principal complications of untreated HIV infection Infections Neoplasms Direct HIV effects Early/​intermediate HIV infection (CD4 >200/​mm3) Herpes zoster Non-​Hodgkin’s lymphomaa Persistent generalized lymphadenopathy Oral hairy leucoplakia Cervical intraepithelial neoplasia Atopy; eczema Oral candidiasis; candidal vaginitis Anal intraepithelial neoplasia Recurrent aphthous ulcers (oral and gastrointestinal tract) Pulmonary tuberculosisa Immune thrombocytopenia Bacterial pneumonia, especially pneumococcal Neutropenia Bacteraemia, especially pneumococcal and salmonella Neuropathy (mononeuritis multiplex; Guillian–​Barré syndrome) Bacillary angiomatosis HIV-​associated nephropathy (HIVAN) Lymphocytic interstitial pneumonitis (LIP) Late HIV infection (CD4 <200/​mm3) Pneumocystis pneumoniaa Kaposi’s sarcomaa HIV enteropathy Candidal oesophagitisa Primary cerebral lymphomaa Peripheral neuropathy (distal, axonal) Cerebral toxoplasmosisa Hodgkin’s lymphoma Autonomic neuropathy Cryptococcal meningitisa Conjunctival carcinoma Myelopathy Chronic cryptosporidial diarrhoeaa ? Cervical carcinomaa HIV dementiaa Chronic isosporiasisa, microsporidiosis ? Anal carcinoma Wasting syndromea Chronic HSVa ulceration Cardiomyopathy Extrapulmonary tuberculosisa Disseminated M. avium complex (MAC)a CMV (retinitis and disseminated)a Progressive multifocal leucoencephalopathya Recurrent bacterial pneumoniaa Recurrent bacteraemia, especially salmonellaa Disseminated histoplasmosisa, and P. marneffei a AIDS-​defining conditions; incomplete list. ? Signifies suspected but unproven association. Many of the early/​intermediate manifestations also occur in late-​stage HIV disease; non-​Hodgkin’s lymphoma is more common during the later stages.

8.5.23  HIV/AIDS 921 pneumonia can occur at CD4 counts of 100 to 200/​mm3, dissem- inated MAC infection and CMV retinitis are rarely seen until the CD4 count is below 50/​mm3. The prognostic value of measuring HIV RNA in plasma was re- ported from the United States of America in 1996. In HIV-​positive men, in a subgroup of the Multicenter AIDS Cohort Study, only 8% with less than 5000 copies of HIV RNA/​ml progressed to AIDS over 5 years, whereas 62% with viral loads above 35 000 developed AIDS. For a given level of CD4 lymphocytes, variations in viral load broadly predict the risk of progression. The most useful prognostic information was therefore derived from the CD4 count and viral load taken together (Fig. 8.5.23.14). Based on the findings from the START trial it is now recommended that ART is initiated for all HIV+ individuals irrespective of CD4 count or viral load as long-​ term clinical benefit has been shown. In industrialized countries, HIV viral load measurements are widely available. Techniques include reverse transcription followed by amplification by the polymerase chain reaction (RT-​PCR), branched DNA (bDNA) signal amplification, and nucleic acid sequence-​based amplification. Highly sensitive tests with very low detection limits (20 copies/​ml) are generally used. Development of point-​of-​care viral load measurements are currently in pro- gress and development of technology that can be transferred to resource-​limited settings is under investigation. Such use of viral load measurement for monitoring will make CD4 count moni- toring for stable virally suppressed patients on ART irrelevant, although initial CD4 count testing at the time of HIV diagnosis remains an important tool to direct OI prophylaxis assess the po- tential risk of IRIS for those starting therapy and plan preventive vaccination courses. The treatment of HIV-​2 infections is comparatively complex, largely because of a paucity of data. The rate of progression is highly variable and not all who are infected progress to disease. There are no data on when to start treatment in this group but there is con- sensus that those with symptomatic disease should be treated with antiretrovirals, as should pregnant women. Additionally, lower than normal CD4 counts or detectable HIV-​2 viraemia might be indi- cations to start treatment. However, measuring HIV-​2 RNA in the blood is not an assay that is widely available and commercial tests to quantitate the RNA are not available. Antiretroviral therapy Nucleoside analogues Knowledge of the viral lifecycle (Fig. 8.5.23.3) led to the devel- opment of several antiretroviral compounds with clinically useful activity against HIV (Table 8.5.23.2). The forerunner of these was zidovudine (AZT or ZDV), first shown to be active against HIV in vitro in 1985. Zidovudine, a nucleoside analogue that inhibits HIV reverse transcriptase, slowed down the rate of disease progres- sion over a 12-​month period in patients with AIDS and improved short-​term survival, well-​being, body weight, and neurological features. However, clinical progression associated with viral re- sistance to the drug was observed after a year or two of therapy. When early treatment with zidovudine was compared to deferred zidovudine, there was no difference in survival or disease progres- sion after 3 years. The clinical failure of monotherapy prompted combination therapy in an attempt to reduce the development of drug resistance. Double nucleoside combinations proved superior to zidovudine monotherapy, especially in patients without prior exposure to zi- dovudine. Treatment with at least three drugs is more effective and has become the standard of care. In general, two nucleoside drugs are used with either a non​nucleoside reverse transcriptase inhibitor, an integrase inhibitor, or a protease inhibitor. A nucleotide agent, tenofovir, has similar properties and is usually grouped in the same category as the nucleoside analogues. Non​nucleoside reverse transcriptase inhibitors The prototype of the class is nevirapine, a potent and selective in- hibitor of HIV reverse transcriptase. When nevirapine is given alone, resistance develops rapidly; this drug is of limited effect- iveness in double therapy or when added to failing regimens. However, in antiretroviral-​naive patients without AIDS (CD4 200–​600/​mm3), over 50% of patients treated with nevirapine plus two nucleosides (zidovudine and didanosine) had undetectable 0.8 0.6 1.0 0.4 0.2 0.0 Proportions of AIDS-free patients 0 2 4 6 8 10 0 2 4 6 8 10 Years after measurements V (70) IV (20) 0.8 0.6 1.0 0.4 0.2 0.0 Proportions of AIDS-free patients Years after measurements V (141) I (110) II (180) III (237) IV (202) 0 to 200 CD4+ cells/mm3 (n = 100) 500 CD4+ cells/mm3 (n = 870) (b) (a) Fig. 8.5.23.14  Curves showing AIDS-​free survival with time among groups with different baseline CD4 lymphocyte counts, according to HIV-​1 RNA category.
The five categories were (copies/​ml): I, 500 or less; II, 501–​3000; III, 3001–​10 000;
IV, 10 001–​30 000; and V, above 30 000. (Sample sizes are shown in brackets.)

922 section 8  Infectious diseases plasma HIV RNA after 1 year of therapy, compared with 12% for zidovudine/​didanosine only. Efavirenz and etravirine are other non​nucleoside reverse transcriptase inhibitors (NNRTIs) with similar properties to nevirapine. The most recently licensed NNRTI, rilpivirine, has been shown to be as effective as efavirenz in treatment-​naive patients who have HIV viral loads of less than 100 000 copies/​ml but was not non​inferior at higher viral loads. NNRTIs are not active against HIV-​2. Protease inhibitors The HIV-​encoded protease (or proteinase) is required for the pro- duction of mature infectious viral particles. This enzyme cleaves several structural proteins and enzymes from the polyprotein pre- cursors produced by translation of the gag and gag–​pol genes. Inhibitors of HIV protease act synergistically with nucleoside drugs and are potent inhibitors of HIV replication. In early studies, indinavir, in combination with two nucleoside analogues (zidovudine/​lamivudine or stavudine/​lamivudine) pro- duced good results in a large controlled trial with clinical endpoints (ACTG 320 clinical trial). Similar results were subsequently re- ported for combination therapy with other protease inhibitors (PIs). The PIs in current use are generally ‘ritonavir-​boosted’, that is, they are used in combination with low-​dose ritonavir to improve pharmacokinetics (via cytochrome P450 interactions) of the prin- cipal PIs (especially lopinavir, atazanvir, and fosamprenavir). A re- cently developed drug, cobicistat, interacts with the cyp450 system in a manner analogous to ritonavir and can be used to boost PIs. Cobicistat has no direct antiviral activity. PIs have a higher threshold for development of resistance muta- tions compared to NNRTIs, so that when resistance does occur it is more likely to be due to poor absorption and suboptimal blood levels. Nevertheless, PI mutations do occur and can be a problem in drug-​experienced patients. Newer PIs, such as tipranavir and darunavir, are active against some of the PI-​resistant isolates and have an increasing role in therapy, including for salvage therapy. Entry inhibitors HIV entry inhibitors are a relatively new class of antiretroviral drugs that target viral entry into cells. This class contains two sub- groups, fusion inhibitors and coreceptor antagonists. The fusion in- hibitor enfuvirtide (T-​20) stops the HIV glycoprotein gp41 from effecting fusion of the viral and cellular membranes, and thereby prevents HIV entry into host cells. This drug is licensed for use in treatment-​experienced patients in combination with other drugs. It must be given by subcutaneous injection and is associated with a high rate of injection site reactions. Coreceptor antagonists act as functional antagonists of the chemokine receptor CCR5 and are active against the R5-​tropic subgroup of HIV-​1 viruses. Maraviroc is the first to be licensed from this subgroup, but others, such as cenicriviroc, are in clin- ical trials. These drugs are not effective against strains of virus using CXCR4 (more common in late disease), so tropism assays to determine the type of coreceptor usage of a patient’s virus are needed before these drugs are used. The place of these agents in HIV therapy is yet to be determined. Integrase inhibitors This class of drugs inhibits an essential enzyme that catalyses the integration of HIV proviral DNA into the host cell genome. The en- zyme, integrase, is also involved in viral assembly and is not a feature of host cells. The first drug to be licensed in this group, raltegravir, is a potent inhibitor of HIV replication which currently should only be used in combination with other active antiretrovirals in pa- tients. More recently developed integrase inhibitors, dolutegravir Table 8.5.23.2  Principal antiretroviral agents Nucleoside reverse transcriptase inhibitors Non​nucleoside reverse transcriptase inhibitors Protease inhibitors Entry inhibitors Zidovudine (AZT/​ZDV) Nevirapine Lopinavira Fusion inhibitor Lamivudine (3TC) Efavirenz Ritonavir Enfuvirtide Emtricitabine (FTC) Etravirine Atazanavira Rilpivirine Abacavir (ABC) Lersivirineb Saquinavira (obsolete) CCR5 antagonistsb Didanosine (ddI) (obsolete) Fosamprenavira Maraviroc Stavudine (d4T) (rarely used) Indinavira (obsolete) Cenicrivirocb Nelfinavir (obsolete) Nucleotide reverse transcriptase inhibitor Tipranavira Integrase inhibitors Tenofovir (TDF) Tenofovir (TAF) Darunavira Raltegravir Elvitegravirb Dolutegravirb Other compounds (not shown) are at earlier phases of development and evaluation. a Given with low-​dose ritonavir for pharmacokinetic enhancement. b Experimental, in advanced clinical trials. Development discontinued due to poor results in recent clinical trials.

8.5.23  HIV/AIDS 923 and elvitegravir, have a higher resistance threshold and can be given once daily. Integrase inhibitors, because they are well tolerated and have few drug interactions, are rapidly becoming the preferred agents in HIV infection. Other drugs There is a need for new drug classes for use after development of drug resistance, allowing additional options for switching after treat- ment failure or drug intolerance, and also to provide compounds that avoid the long-​term toxicities associated with current antiretro- virals. Immunotherapy with interleukin-​2 (IL-​2), which raises CD4 lymphocyte counts and is given by subcutaneous injection, has been shown to be clinically ineffective. Recent studies have involved the use of depot injections of long-​ acting integrase inhibitors. Such developments might be useful for those unable or unwilling to take tablets long term. The role of these preparations for treatment and prevention are being investigated. HIV-​2 Treatment Although HIV-​2 can cause disease, there are fewer data on how to treat this virus. HIV-​2 is intrinsically resistant to NNRTI and to the fusion inhibitor, enfuvirtide. Protease inhibitors are variable in their potency. Generally, lopinavir or darunavir are preferred to the others available. Nucleoside reverse transcriptase inhibitors and integrase inhibitors have good activity. Advice should be sought from an ex- pert in treating HIV-​2. When to start antiretroviral treatment The optimum time to start antiretroviral therapy is now known. A major trial (Strategic Timing of Anti-​Retroviral Treatment, START) compared clinical outcomes for individuals with HIV and CD4 counts greater than 500 cells/​cc3 who were randomly allocated to either start immediate ART or defer according to local guidelines (CD4 <350 cells for most sites). This trial reported a significant benefit of starting im- mediate ART greater than 500 cells and this has now become incorp- orated into revised WHO and many national guidelines. The decision to start treatment should also take into account how quickly ART should be implemented and will include assessment of age and pres- ence of coinfection with TB, hepatitis B or C. Patients who are clinic- ally well after starting ART should be seen for follow-​up at intervals of 3–​6 months for regular viral load measurements. What to start with Antiretroviral therapy consists of at least three drugs from two dif- ferent drug classes, usually a backbone of two nucleosides with ei- ther a non​nucleoside reverse transcriptase inhibitor, an integrase inhibitor, or a protease inhibitor (see Table 8.5.23.3). For improved pharmacokinetics, the protease inhibitor is usually combined (‘boosted’) with a second protease inhibitor (i.e. low-​dose ritonavir or a newer drug, cobicistat). Several initial regimens have equivalent efficacy. Current recommendations are based on viral efficacy, toler- ability, and cost with a preference given to regimens that are taken only once a day. The most important cause of treatment failure is inadequate treat- ment, which may relate to failure to take the drugs regularly (i.e. non-​ adherence, lack of availability of drugs, or poor absorption). Before starting treatment it is, therefore, important to discuss the patient’s views about taking medication regularly. Simplified regimens with combination single tablet, once daily formulations have helped with adherence (see next). Several other factors also influence the selec- tion of the initial regimen, including potential drug interactions (e.g. with antituberculosis treatment), toxicity, ease of administration, presence of renal or hepatic dysfunction, female gender. HIV viral load and CD4 count should be checked within 2 months. The aim of initial treatment is to achieve a sustained reduction in viral load to undetectable levels (<20 copies/​ml) within 2–​3 months of starting treatment. Patient adherence A substantial proportion of all patients, including those with HIV, struggle to follow treatment recommendations. Reasons for non-​ adherence include poor communication, the complexity of drug re- gimens and number of tablets, disruption of life (including timing and food restrictions), side effects, concerns about long-​term effects, and lack of confidence in non​curative treatments of indefinite dur- ation. Adherence to treatment requires a high level of understanding and motivation in the patient. This is of particular concern in HIV therapy because of the risk of developing drug-​resistance mutations during suboptimal therapy. The recent development of simplified regimens (one or two tablets taken once or twice daily) has helped. Patients can be helped to be adherent by skilled support from trained professionals such as counsellors or pharmacists. Changing therapy The principal reasons for changing antiretroviral treatment are treatment failure, toxicity, and poor adherence. There is no agreed definition for treatment failure. Patients whose viral load is not sup- pressed to less than 40 copies/​ml within 3 months, or was initially suppressed and subsequently rises, should be considered for chan- ging to a completely new regimen of at least three drugs. This should be guided by a resistance test (see next). Continuing viral replica- tion in the presence of antiretroviral treatment should be avoided because of progressive accumulation of resistance mutations which can compromise future treatment options. If adherence is poor or likely to be the cause of treatment failure, changing to a combination that is simpler to take should be considered (e.g. based on once or twice daily dosage and low pill burden). Poor absorption of protease inhibitors might sometimes cause treatment failure related to low blood levels, without development of resistance; measurement of blood levels can be useful in this context. Table 8.5.23.3  Initial antiretroviral regimens Examples Comment TDF/​FTC + integrase inhibitor Preferred regimen TDF/​FTC + PI with RTV Preferred regimen TDF/​FTC + EFV Alternative if no contraindications to EFV 3TC/​ABC + PI with RTV ABC contraindicated if HLA-​B57.01 3TC/​ABC + integrase inhibitor 3TC/​ABC + EFV Only appropriate if viral load <100 000 3TC, lamivudine; ABC, abacavir; FTC, emtricitabine; PI, protease inhibitor; TDF, tenofovir; RTV, ritonavir; EFV, efavirenz. TDF/​FTC + integrase inhibitor. TDF/​FTC + PI with RTV. See British HIV Association guidelines: http://​www.bhiva.org for latest UK guidelines.

924 section 8  Infectious diseases If treatment needs to be changed because of drug toxicity (e.g. a se- vere rash), a single drug substitution can be made if the responsible agent is identified. ‘Salvage’ therapy Salvage therapy is generally defined as treatment following ex- posure to multiple antiretroviral drugs. In this situation, numerous drug-​resistance mutations are usually present and the likelihood of achieving sustained viral suppression below the detection level is much lower than for patients who have limited or no previous antiretroviral exposure. This is especially true if drugs from all three major classes have previously been used. Studies using clinical endpoints suggest that declines in viral load correlate with improve- ments in clinical outcome, even if suppression to below the detec- tion limit is not achieved. Several factors may be considered when selecting a treatment regimen in these circumstances, including the history of drug classes to which the patient has not been exposed and the results of tests for viral resistance. It might be possible to recycle some drugs with less likelihood of resistance, or to include new drugs active against resistant isolates (e.g. darunavir) or new classes of drugs (e.g. maraviroc). When initiating salvage therapy it is important to use at least two new drugs to which the patient has not been exposed in order to reduce the risk of further resistance developing. Drug resistance Viral resistance is a major factor in treatment failure. Resistant mu- tants can arise spontaneously even in the absence of antiretroviral therapy; however, the selection of drug-​resistance mutants occurs rapidly when HIV replicates in the presence of subtherapeutic levels of antiretroviral drugs, and is eliminated when HIV replication is completely suppressed by a potent drug combination. Extensive genotypic variation of HIV occurs because of very high viral turnover and transcription errors by the reverse transcriptase enzyme, so that all possible single point mutations are likely to occur over time. Although mutations causing resistance to single agents may arise before antiretroviral treatment is started, on statistical grounds it is unlikely that specific combinations of multiple muta- tions will be present. Control of viral replication with a highly potent treatment regimen limits the appearance of resistant HIV mutants. Genotypic and phenotypic assays have been developed to test for drug resistance in HIV isolates. Genotypic assays that identify codon mutations correlating with in vivo resistance to antiretrovirals are relatively easy to perform, inexpensive, and most widely used. Phenotypic assays that measure the ability of the virus to grow in increasing concentrations of drugs are time-​consuming and expen- sive, but provide more direct evidence of resistance to a particular drug. Resistance assays are widely used in the selection of drug re- gimens and investigation of treatment failure. Interpretation of re- sistance patterns is increasingly difficult as the number of drugs and mutations involved increases. Resistance mutations to antiretroviral agents in newly acquired HIV, indicating transmitted drug resistance, are identified in ap- proximately 10% of recent seroconverters in Europe. However, the presence of a mutation does not necessarily denote clinical resist- ance. In the absence of therapy, wild-​type virus predominates, and resistance mutations may be undetectable though present in small copy numbers. This can lead to treatment failures with the resistant mutants increasing as wild-​type virus is eradicated by drugs. For this reason, baseline resistance testing at diagnosis is now advocated in an attempt to identify resistance mutations at the outset. Drug toxicity and interactions Adverse reactions to antiretroviral agents are relatively common and may lead to the patient stopping their therapy (Table 8.5.23.4). Minor gastrointestinal disturbances (nausea, vomiting, and diar- rhoea), rashes, and headache are common, but some adverse re- actions are serious. Drug interactions must be considered when prescribing antiretroviral drugs, especially in advanced HIV disease. Antiretroviral agents can interact with each other and with other drugs. For example, phenytoin drastically reduces plasma levels of efavirenz. Ritonavir, a potent inhibitor of cytochrome P450, is espe- cially prone to raising blood levels of other drugs and should not be given with most antiarrhythmics, anxiolytics, and antihistamines. Caution is required with several analgesics, anticonvulsants, and other categories of medication. Metabolic complications Metabolic problems, especially mitochondrial toxicity and disturb- ances of lipid and glucose metabolism, have emerged as important adverse effects of antiretroviral therapy. Mitochondrial toxicity is as- sociated with nucleoside drugs (especially didanosine and stavudine, which are now rarely prescribed) and might result in neuropathy, myopathy, pancreatitis, hepatic steatosis, hyperlactataemia, and lactic acidosis. Lactic acidosis causes non​specific symptoms, including malaise, gastrointestinal disturbance, and liver function abnormalities, and can progress to death, particularly if antiretro- virals are not stopped. There is no evidence that routine moni- toring of lactate levels is helpful. Nucleoside drugs are thought to cause mitochondrial dysfunction by inhibiting mitochondrial DNA polymerase-​γ. In addition, lipid abnormalities are commonly seen in the face of antiretroviral therapy, leading to raised triglycerides and cholesterol. Hyperlipidaemia has been particularly associated with the use of protease inhibitors, as has insulin resistance. A syndrome of lipodystrophy (progressive loss of fat from face and limbs) is associated with thymidine analogue nucleoside drugs, especially stavudine, and to a lesser extent zidovudine. Truncal fat accumulation, has also been associated with protease inhibitors. Cardiovascular disease After the introduction of antiretroviral treatment, early reports sug- gested a possible increase in cardiovascular disease in patient with HIV. Several variables potentially affect the risk of myocardial in- farction and other cardiovascular events in this patient group. The Data Collection on Adverse Events of Anti-​HIV Drugs (D:A:D.) study group has shown an association with antiretrovirals and car- diovascular events. The relative risk of myocardial infarction may be increased by about 10% when other factors, such as lipid levels, are taken into account. The INITIO trial showed an increased incidence of the metabolic syndrome with antiretroviral treatment, with an as- sociated increased risk of cardiovascular disease. Although there is still some controversy, it seems that the nucleoside analogue abacavir is more commonly associated with an increased cardiovascular risk than other antiretrovirals, though the mechanism remains unclear. A variety of studies have tried to quantitate the risk of car- diovascular disease in HIV and to analyse the pathogenesis. Investigation of the intima media thickness in the carotid ar- tery shows increased thickening in those with HIV compared to

8.5.23  HIV/AIDS 925 non​infected matched controls. Assessments of coronary calcifi- cation and plaque disease again point to slightly worse measures in HIV. Whether these observations correlate with outcome is un- certain. More recently attention has focused on the role of chronic inflammation due to HIV infection causing arterial endothelial dysfunction. Studies have shown elevated levels of C-​reactive pro- tein and proinflammatory cytokines, such as IL-​6, in HIV. There may also be abnormalities in coagulation that contribute to an Table 8.5.23.4  Principal toxicities of antiretroviral drugs Nucleoside reverse transcriptase inhibitors (NRTI) Class effects GI disturbances, raised liver enzymes, hepatic steatosis, lactic acidosis Zidovudine (AZT) Headache, nausea (usually resolve within 2–​4 weeks) Anaemia (avoid if anaemic at baseline) Macrocytosis (benign) Nail pigmentation Myopathy (rare on lower dosages 500–​600 mg/​day) Lipodystrophy with facial wasting (long-​term effect, unknown incidence) Abacavir (ABC) Hypersensitivity 5%; may be fatal if rechallenged (closely associated with HLA-​B*57.01) Didanosine (ddI) Pancreatitis, peripheral neuropathy—​no longer used Stavudine (d4T) Lipodystrophy with facial wasting; peripheral neuropathy; rarely used Lamivudine (3TC), Emtricitabine (FTC) No major toxicities Nucleotide RTI Tenofovir dioproxil fumarate (TDF) Renal failure (case reports, rare, incidence unknown) renal protein and phosphate leak; osteoporosis Tenofovir alafenamide (TAF) Diarrhoea; rash Reduced incidence of renal or bone adverse effects compared to TDF Non​nucleoside RTI (NNRTI) Efavirenz Neuropsychiatric disturbances (8%)—​vivid dreams, impaired concentration, mood changes (usually transient, <4 weeks duration); rash Nevirapine Rash (20%, severe 6%); rarely Stevens–​Johnson syndrome; hepatitis (esp. in women with CD4 >250/​mm3 or men with CD4 >400/​ mm3—​avoid) abnormal LFT Etravirine Rash with eosinophilia; toxic epidermal necrolysis Rilpivirine Insomnia, Neuropsychiatric disturbances; rash; osteonecrosis Protease inhibitors (PI) Class effects GI disturbances; hyperlipidaemia, truncal fat accumulation, diabetes, bleeding in haemophiliacs, raised liver enzymes Lopinavir Diarrhoea Ritonavir Circumoral and peripheral parasthesiae (unusual in low dosage) Saquinavir Rash, peripheral neuropathy; no longer used Nelfinavir Diarrhoea; no longer used Indinavir Renal calculi, haemolysis; no longer used Atazanavir Hyperbilirubinaemia, jaundice Tipranavir Rash (caution in sulphonamide allergy), liver dysfunction Darunavir Diarrhoea, rash (caution in sulphonamide allergy) Entry inhibitors Fusion inhibitors Enfuvirtide Injection site reactions (painful, erythematous nodules); headache, dizziness, nausea, eosinophilia CCR5 antagonists Maraviroc Cough, muscle and joint pain, diarrhoea, sleep disturbance, raised liver enzymes (and possibly hepatitis) Integrase inhibitors Raltegravir Nausea, diarrhoea, headache, insomnia; raised creatine kinase (CK) in some patients Dolutegravir Hypersensitivity reactions; diarrhoea, raised CK Elvitegravir Rash, diarrhoea, dyspepsia

926 section 8  Infectious diseases increased cardiovascular risk. Levels of D-​dimer tend to be higher in HIV infection compared to controls. The absolute risk is very small when compared to the risks associ- ated with smoking, diabetes mellitus, and male gender. Nevertheless, increasing attention is being paid to modifying cardiovascular risk factors in those on HIV therapy, such as smoking cessation pro- grammes, treatment of hypertension, and managing hyperlipid- aemia. Statins are slightly less effective lipid-​lowering agents in those with HIV and there are no data at present to show that statins are clinically effective in HIV in preventing cardiovascular events. It should be noted that some statins, such as simvastatin, have sig- nificant interactions with some antiretrovirals. Current guidelines suggest avoiding the use of abacavir if there are significant cardio- vascular risk factors present in an individual. In addition, it may be prudent to change from a protease inhibitor to an integrase in- hibitor or to a non​nucleoside reverse transcriptase inhibitor, such as rilpivirine, in patients with hyperlipidaemia and risk factors. Recent attention has also focused on changes in bone metab- olism. HIV-​infected patients have an increased risk of osteoporosis and bone fractures, which are often multifactorial. Antiretroviral therapy seems to be a factor with tenofovir DF being particularly implicated as it can lead to urinary phosphate wasting. The use of tenofovir alafenamide might reduce this risk. Proinflammatory cytokines might also play a role in a manner analogous to the car- diovascular risks but other contributing causes include smoking, vitamin D deficiency, low testosterone levels in males, low oestrogen levels in postmenopausal women, and increasing age. Hepatitis C infection is associated with osteoporosis and may contribute signifi- cantly in coinfected patients. Patients on HIV therapy also have an increased incidence of avascular necrosis (osteonecrosis) of the fem- oral head. Although guidelines have been produced for screening for bone disease in HIV, the merits of intervening outside of the ex- isting guidance for non-​HIV-​infected individuals are not clear. Treatment interruptions In general, once treatment is started it is continued indefinitely. For individuals who do choose to interrupt treatment, the virus will re- bound in plasma within 2-6 weeks. There has been recent interest in whether interrupting treatment (in supervised or structured treat- ment interruptions) might be beneficial. In theory, it was postulated that ART interruptions might enhance immune responses, reduce long-​term drug toxicity, or reduce resistant virus by allowing re- population with wild-​type virus. A large trial (SMART) was stopped early because of a paradoxical result. The study randomized patients with stable disease on therapy to continuing therapy or to stopping (and restarting if the CD4 count fell below 250). Not only were there more HIV complications in the stopping group, but this group, sur- prisingly, also had a higher incidence of cardiovascular disease. It is postulated that the increased cardiovascular risk is related to endothelial inflammation secondary to uncontrolled viraemia. It is thus unlikely that treatment interruption will be a sensible man- agement strategy and patients should be counselled about the need for long-​term treatment. Interruption of ART is a common occur- rence for multiple reasons: drug stock-outs, patient travel, inter- current illness, toxicities and treatment fatigue. Whilst the SMART trial identified a significant risk to stopping ART amongst individ- uals with low nadir CD4 counts when ART was initiated in chronic disease, the SPARTAC trial, where short course ART was used in primary HIV infection and restarted later by CD4 threshold, did not see any similar mortality or morbidity. It can be hypothesized that ART interruption, whilst not recommended, might be safer in indi- viduals with preserved immune function. Similarly, in the context of the HIV remission/​cure trial setting with very careful viral load monitoring, this approach might be necessary to test ultimately if an intervention has led to viral control off therapy. Immune reconstitution inflammatory syndrome (IRIS) Since the introduction of antiretroviral therapy, there have been reports of unusual symptoms and signs appearing in patients some weeks to months after starting therapy. Because these clinical problems arise in the face of increasing CD4 counts, the syndrome has been called the immune reconstitution inflammatory syndrome (IRIS) or immune reconstitution disease. In the absence of HIV, paradoxical clinical re- sponses have been described in tuberculosis and in leprosy. In the set- ting of HIV, IRIS often takes the form of an exacerbation of a previously treated opportunist infection or an unusual clinical presentation of an opportunist infection that was subclinical at the time ART was started. The incidence of IRIS is difficult to determine, particularly as there is no currently agreed definition, but it might occur in up to 20% of patients starting ART. Usually, IRIS starts within a few months of starting ART and is temporally related to a rise in CD4 count. A pro- posed definition includes (1) new or worsening symptoms of an in- fection or inflammation after starting antiretrovirals, (2) symptoms not explained by a new infection or the expected course of an infec- tion previously diagnosed, and (3) a decrease in viral load by at least one log10. The pathogenesis of IRIS is poorly understood, but many patients have been found to have raised IL-​6 levels, possibly related to a brisk Th-​1 lymphocyte response. The most common opportunistic infections complicated by IRIS are tuberculosis, M. avium (MAC) infections, pneumocystis pneu- monia, cryptococcal meningitis, and cytomegalovirus. IRIS compli- cating tuberculosis is probably the most common problem. Patients can develop fever or lymphadenopathy or might present with pleural effusions. Bone and joint involvement also occurs. IRIS is possibly more common in those presenting with extrapulmonary tubercu- losis and can be fatal in tuberculous meningitis. IRIS can occur in those receiving treatment for MAC and in one series complicated 30% of cases. The usual problem is lymph node enlargement, which can be massive and might mimic lymphoma. Some cases are complicated by hypercalcaemia. Cryptococcal meningitis, when complicated by IRIS, might present as an apparent relapse with fever, headache, and signs of meningeal irritation. Rapidly expanding cerebral cryptococcomas may lead to fatal increases in intracranial pressure. In parts of the world where this fungus is a common problem, screening patients for cryptococcal antigen (CRAG) is done before ART are started. Cytomegalovirus infections can also be complicated by IRIS with a worsening of signs of retinitis or a more benign vitritis. Rarely, pa- tients have presented with a uveitis some years after starting ART. Some of the common features in the aforementioned conditions are that affected patients often started ART at very low CD4 counts and with very high HIV viral loads. There is no consensus on the best management of IRIS, but there is no rationale for stopping ART and most cases are self-​limiting. Steroids and non​steroidal anti-​ inflammatory drugs are frequently used, but the use of steroids has only recently been shown to help in TB-associated IRIS.

8.5.23  HIV/AIDS 927 HIV and ageing There are two aspects to the issue of HIV and ageing. The first, and obvious, thing is that with the remarkable success of ART, people with HIV are now living longer and acquiring the non​communicable diseases and comorbidities of the general ageing population. This has implications for non​specialists, particularly geratologists, who will need to familiarize themselves with HIV and, in particular, the current therapies. The question is whether these comorbidities occur earlier in those with HIV; that is, does HIV cause premature ageing? The recognized increased cardiovascular risks, neurocognitive dis- orders, and metabolic changes, including osteoporosis, might suggest that this is the case. However, it is difficult to untangle the effects of ART from the effects of long-​term HIV infection. There is some evi- dence to suggest that chronic HIV infection, even when controlled by ART, leads to increased endothelial inflammation and that this might be responsible for some of cardiovascular complications (and others) seen in HIV. In addition, HIV infection appears to accelerate the ex- haustion (and hence, ageing) of the immune system. Children and HIV Paediatric HIV is even more an epidemic of sub-​Saharan Africa than is adult infection: approximately 90% of children and 67% of adults with HIV live in sub-​Saharan Africa. Furthermore, the shape of the paediatric epidemic is changing, as access to antiretroviral therapy increases and prevention of mother-​to-​child transmission (PMTCT) programmes decrease transmissions. The combined impact of these changes is that, globally, the overall numbers of children living with HIV is still high (currently estimated at 1.8 m, UNAIDS 2015 Report), and the adolescent epidemic, which previously did not exist, is a growing challenge, bringing with it major issues around ART non​adherence. In Europe and North America, however, numbers of newly HIV-​infected children have dwindled to a trickle. Most paediatric infections result from the mother-​to-​child trans- mission (MTCT) of HIV, although some children may be infected by blood products or sexual abuse. The risk of MTCT is increased during advanced maternal HIV disease or acute maternal infection during pregnancy, by vaginal delivery, and by breastfeeding (see ‘Mother-​to-​child transmission’, next). Diagnosis is important as early as possible because without antiretroviral therapy (ART) ap- proximately 50% of HIV-​infected children progress to AIDS during the first year of life; however, HIV antibody detection is not helpful towards diagnosis before 18 months of age, because over this period uninfected children may have maternal HIV antibody. Techniques for virus detection (e.g. HIV DNA by PCR) allow confirmation of HIV infection irrespective of the presence of maternal antibody. Point-​of-​care testing (by HIV DNA PCR) can successfully diagnose HIV infection in in utero infected infants within hours of birth. The natural history is very different in resource-​rich versus re- source-​limited countries. In the latter, HIV-​infected children without antiretroviral therapy have a mortality rate of 45 to 59% at 2 years. In Europe and the United States of America, about 20% of untreated children would develop AIDS or die in infancy; by 5 years, 40% of children would have developed AIDS and 25% would have died. Without any intervention, the most common AIDS diagnosis in infancy is pneumocystis pneumonia, typically presenting at 10 to 14 weeks of age. HIV encephalopathy is also common in untreated HIV-​infected infants, with severe developmental delay occurring in about 10% and more subtle delays in an additional 40%. Although progression to HIV disease is generally much more rapid in infected children than adults, there is a substantial subset of 5–​10% of ART-​naïve HIV-​infected children who maintain CD4 counts that are normal-​for-​age for HIV-​uninfected children. Unlike ‘elite con- troller’ adults in whom viral replication is suppressed by the HIV-​spe- cific immune response to very low levels, viral loads in the paediatric non​progressors remain at high levels (104–​105 copies/​ml plasma). This scenario is reminiscent of the natural hosts of SIV infection, such as the sooty mangabey, who are disease-​free despite persistent high viral loads of c.105 copies/​ml plasma. However, while this is of scientific importance and interest to help direct vaccine development, the current recommen- dation is to offer ART to all children irrespective of CD4 count and viral load. In particular the high level of HIV viral load observed in these young people confers a significant risk of onward viral transmission. In older children, clinical conditions suggestive of HIV infection include persistent oral candida, parotid swelling, and recurrent or frequent serious bacterial infections including pneumonia, menin- gitis, and sepsis. Failure to thrive, diarrhoea, fever, lymphadenop- athy, and hepatosplenomegaly are more common in HIV-​infected infants but are non​specific and less predictive. HIV dementia and other neurological and developmental problems are associated with a poor prognosis. HIV-​related lymphocytic interstitial pneumonitis (LIP) typically occurs in children and is characterized by progres- sive, widespread, reticulonodular shadowing on chest radiography. LIP develops insidiously and may initially be asymptomatic; chronic lung disease develops with cough, breathlessness, hypoxia, clubbing, and secondary bacterial infections, and bronchiectasis occurring in severe cases. LIP is often associated with other lymphoproliferative manifestations (such as parotitis) and relatively well-​preserved im- mune function, and may be treated with oral prednisolone. The management of HIV-​infected children has changed dramat- ically in recent years. As in adult infection, there has been a move to initiate ART earlier and earlier in the course of infection, but the very high mortality in HIV-​infected infants and difficulty to pre- dict sudden declines in infants on the basis of clinical evaluation and measurements of CD4 count and viral load has meant that ART initiation in all infected children aged less than 1 year, irre- spective of CD4 count or viral load, has been practised based on WHO guidelines since 2008. In 2013, WHO guidelines moved fur- ther to recommend that all HIV-​infected children aged less than 5yrs should receive ART irrespective, while in children aged over 5 years the practice has been to start ART based on the same absolute CD4 count and clinical criteria as used in adults. From 2016, recom- mendations now are that all HIV-​infected children should receive ART, irrespective of CD4 count or clinical status. Principles of antiretroviral treatment are similar in children and adults. Particular challenges to the effective use of ART that are spe- cific to children include the need to adjust drug dose as the child grows (to avoid underdosing and drug resistance); the lack of paedi- atric formulations suitable for infants, fewer drug choices, limited paediatric toxicity data, and non​specific presentation of drug toxicity in children; reliance of children on caregivers who themselves might have HIV and be ill; and, in adolescents, problems of adherence and coming to terms with an HIV diagnosis. The issue of disclosure, the process by which the child or adolescent learns of the HIV diagnosis,

928 section 8  Infectious diseases is especially challenging, not only because the optimal approach and timing needs to be carefully tailored to each individual setting, but also because there may be some sense of guilt on the parental side. An additional problem in the use of ART in infants, where infected mothers have received single-​dose nevirapine (or other antiretro- viral therapy during pregnancy to reduce MTCT), is that the trans- mitted virus in such a setting is usually nevirapine resistant. This should in future not be a problem as current recommendation for all HIV infected pregnant women is to start lifelong ART. However, it is important to note the potential for drug-​resistant virus among multiparous HIV-positive women who might have acquired drug-​ resistant HIV through previous pregnancies. Prevention of opportunistic infections (See Table 8.5.23.5.) The risk of developing an opportunistic infection rises greatly once the peripheral CD4 lymphocyte count falls consistently below 200/​mm3. It is standard practice to introduce low-​dose co-​trimoxazole prophylaxis for pneumocystis pneumonia at this stage. This also reduces the risk of cerebral toxoplasmosis and may prevent bacterial pneumonia. Studies in Africa, in both children and adults, have shown that co-​trimoxazole prophylaxis is associated with decreased mortality. The risk of developing active tuberculosis in HIV-​positive American intravenous drug users with positive tuberculin skin tests has been shown to be about 8% per year and can be reduced by taking isoniazid for a year. In developing countries, in particular, the risk of active tuberculosis in HIV-​positive individuals is high and isoniazid alone or in combination with rifampicin can reduce the risk, but there is a challenge in implementation, which includes addressing the need to exclude active TB. Bacillus Calmette–​Guérin (BCG) vaccination does not appear to be protective in HIV. Primary prophylaxis might prevent other conditions, such as CMV retinitis, cryptococcal meningitis, and histoplasmosis, but be- cause of the relatively low incidence and lack of predictors of risk for these conditions, it is not cost-​effective. Before the advent of antiretroviral therapy, after treatment of an opportunistic infection the predisposition to the infection usually remained. Thus, in early Table 8.5.23.5  Prophylaxis of major opportunistic infections in HIV Infection Indications Regimens Comments First line Alternatives Pneumococcal pneumonia All HIV-​positive patients Pneumococcal vaccine None Clinical effectiveness unproved; antibody response greater if CD4 >350/​mm3 P. jirovecii pneumonia CD4 <200/​mm3; or symptomatic HIV; or following PCP Co-​trimoxazole 480–​960 mg daily (or 960 mg, 3 times per week) Dapsone; dapsone with pyrimethamine; monthly nebulized pentamidine; atovaquone May be stopped if CD4 is sustained

200/​mm3 on anti-​HIV treatment Cerebral toxoplasmosis CD4 <100/​mm3 plus toxoplasma IgG-​positive following treatment of cerebral toxoplasmosis As above Dapsone with pyrimethamine Primary prophylaxis usually incidental to that for PCP prophylaxis; pentamidine not protective Sulfadiazine 0.5–​1 g, 4 times daily with pyrimethamine 25–​75 mg/​day, and folinic acid; protects against P. jirovecii as well Clindamycin with pyrimethamine, and folinic acid May be stopped if CD4 is sustained 200/​mm3 on anti-​HIV treatment Tuberculosisa Tuberculin reaction 5 mm induration with no previous BCG; or high-​risk exposure to tuberculosisa Isoniazid 300 mg/​day with pyridoxine 50 mg/​day for 6–​12 months Rifampicin with isoniazid for 3 months Rifampicin should not be given with protease inhibitors or nevirapine M. avium
complex (MAC) CD4 <50/​mm3 following treatment of disseminated MAC Clarithromycin 500 mg, twice daily, or azithromycin 1200 mg/​week Rifabutin; rifabutin with azithromycin Primary prophylaxis not recommended Clarithromycin 500 mg, twice daily, with ethambutol 15 mg/​kg per day with or without rifabutin 300 mg/​day Azithromycin with ethambutol, with or without rifabutin May be stopped if CD4 is sustained 200/​mm3 on anti-​HIV treatment Cytomegalovirus (CMV) CD4 <50/​mm3 and CMV antibody-​positive Valganciclovir 900 mg daily None Primary prophylaxis not recommended Following CMV retinitis or other CMV disease Valganciclovir 900 mg daily; or ganciclovir 5–​6 mg/​kg IV on
5–​7 days/​week Foscarnet IV; cidofovir IV; ganciclovir intraocular implant May be stopped if CD4 is sustained 200/​mm3 on anti-​HIV treatment Cryptococcal meningitis CD4 <50/​mm3 following treatment of cryptococcal meningitis Fluconazole 100–​200 mg/​day orally Itraconazole orally Primary prophylaxis recommended in endemic settings Fluconazole 200 mg/​day orally Itraconazole orally; amphotericin B IV weekly or 3 times/​weeky Fluconazole superior to itraconazole for secondary prophylaxis May be stopped if CD4 is sustained 200/​mm3 on anti-​HIV treatment IV, intravenous; PCP, pneumocystis pneumonia. a In circumstances of contact with MDR-​TB, specialist advice about prophylaxis should be sought.

8.5.23  HIV/AIDS 929 studies, following an episode of pneumocystis pneumonia, patients had a 50% chance of a further episode within a year. Secondary prophylaxis with co-​trimoxazole proved effective. Secondary prophylaxis for pneumocystis and other opportunistic infections, including MAC and CMV, is discontinued if there is a good response to antiretroviral treatment, with CD4 counts sustained above 200/​ mm3 and low plasma levels of HIV RNA. Simple measures, other than drugs, can reduce the risk of some infections. Avoiding undercooked eggs and poultry may reduce the risk of disseminated salmonella infection and adequate boiling of drinking water can prevent cryptosporidiosis. Stopping cigarette smoking reduces the risk of bacterial chest infections. Prevention of HIV transmission Sexual transmission Sexual transmission accounts for most new cases of HIV infection. Education to alter behaviour and reduce the risk of HIV infection is a key component of HIV control programmes. Condom promo- tion in Thailand has made an impact on HIV transmission rates. The presence of other sexually transmitted infections, especially those causing genital ulcers, facilitates HIV transmission. Accordingly, studies in Tanzania and elsewhere have demonstrated that pro- grammes to prevent and treat sexually transmitted infections reduce the incidence of new HIV infections. Herpes simplex virus type 2 (HSV-​2) is of particular importance in facilitating HIV-​1 transmis- sion, because of its high prevalence worldwide, including developing countries. Aciclovir suppression of HSV-​2 infection has been shown to reduce genital shedding of HIV-​1 and plasma HIV viral load, but field studies have not demonstrated a consequent reduction in risk of HIV acquisition. This may be a consequence of poor HSV sup- pressive therapy as well as the overriding driver or transmission of HIV viral load. These studies were undertaken with suboptimal HSV suppression and not in the era of universal ART. The risk of HIV transmission is related to the HIV viral load, and is reduced dramatically by antiretroviral treatment. A systematic re- view in 2009 showed no transmissions in over 5000 sero-​discordant heterosexual couples when the index patient’s viral load was less than 400 copies/​ml. HIV may be detected in the semen of patients with undetectable viral load and so it should not be assumed that the risk of transmission is zero. These findings have led to a de- bate on the potential value of large-​scale antiretroviral treatment as a strategy for controlling HIV transmission in populations with high prevalences of HIV. Subsequently two large randomized con- trolled trial (RCT) have shown a very dramatic reduction in the risk of sexual transmission of HIV where the HIV-positive partner in a sero-​different relationship receives suppressive ART. The HPTN052 and PARTNER study both identified a reduction in viral transmis- sion when the HIV-positive partner was on suppressive ART of up to 96% and subsequent follow-​up study of couple out to 5 years after the trial reported have shown a sustained reduction in transmission risk of 93% overall. The approach at a population level to implement ‘treatment as prevention’ has been a focus especially among those at highest risk of transmission. With the adoption of the most recent ART guidelines recommending ART initiation for all HIV-positive individuals irrespective of CD4 count, the secondary impact on re- duced viral transmission at a population level is estimated to be very significant. However, the key barrier to population level impact of such a wide spread intervention is knowledge of HIV status. At pre- sent slightly over 50% of all people living with HIV in highest burden settings in sub-​Saharan Africa are aware of their HIV status and in the developed world this remains around 20%. Until HIV testing can diagnose more than 90% of all people living with HIV, ART ini- tiation will have a limited impact of stopping the epidemic. In 2014 UNAIDS and partners launched an initiative to support a target glo- bally that 90% of all people living with HIV should know their status with repeat annual testing for those testing HIV negative, 90% of all diagnosed HIV-positive individuals should start ART and of these 90% should have an undetectable plasma viral load (90:90:90). The foreskin in males, rich in Langerhans cells, is an important portal of entry for HIV infection. Randomized trials in Africa have confirmed that the risk of acquiring HIV for heterosexual men is re- duced by over half in circumcised men compared to uncircumcised men. Adult male circumcision, although not fully protective, may therefore be a valuable addition to HIV prevention programmes in resource-​limited countries. A program in high burden settings to encourage voluntary medical male circumcision for HIV-​negative men has been supported in many African countries although uptake and resources are limited. Male circumcision will not affect men who have sex with men due to the nature of sexual exposure. Large studies to assess the efficacy of vaginal microbicides in the prevention of sexually transmitted infections and HIV have been disappointing. However, a controlled study to assess the feasibility of using antiretroviral drugs for pre-​exposure prophylaxis (PrEP) of sexually acquired HIV yielded promising results using tenofovir and emtricitabine in a daily single tablet. Four RCTs of combined tenofovir and emtricitabine as daily oral prescription have shown effectiveness of between 44 and 75%. The cost-​effectiveness and ap- propriate use of PrEP remain to be determined and are influenced mostly by the cost of drug. New studies have explored alternative agents and routes for prevention that may avoid the need for daily oral dosing; these include monthly injectable agents as prevention, an integrase inhibitor injection (Cabotegravir) as well as infusions of monoclonal antibodies (AMP study HPTN085) or antiretroviral impregnated vaginal rings (Dapivirine). Mother-​to-​child transmission In the absence of intervention, mother-​to-​child transmission (MTCT) rates are approximately 25% in non​breastfed infants (7% in utero and 18% intra-​partum) and 40% in breastfed children. The numbers of children infected via MTCT, currently estimated at c.240 000 new infections per year (10–​12% of total new infections) have dropped substantially since the advent of effective prevention to mother-​to-​child transmission (PMTCT) programmes. PMTCT started with the ACTG 076 study in 1994 reporting a 67% reduction in MTCT as a result of AZT monotherapy given through pregnancy and intra-​partum, and to the infant for 6 weeks. More recently, all HIV-​infected mothers receive ART throughout pregnancy and MTCT rates are down to 1–​2% or lower. If viraemia is success- fully suppressed by ART through pregnancy to undetectable levels (<50 copies/​ml plasma) transmission rates are zero. The current 1–​ 2% MTCT rate results either from a failure of HIV testing of the mother, or ART non​adherence, or acute maternal infection during pregnancy. HIV antibody tests are negative during peak viraemia (c.107 HIV copies/​ml) and MTCT rates in these settings are 20%.

930 section 8  Infectious diseases In the absence of ART, post-​partum transmission through breast- feeding carries the risk of MTCT as described. In resource-​limited settings, prior to ART being indicated for all HIV-​infected mothers, breastfeeding for 6 months was still recommended because the sub- stantial nutritional and immunological benefits outweighed even the increased risk of HIV infection in the infants. Blood products Screening of blood products began as soon as testing for HIV be- came available, and heat treatment for factor VIII concentrate was also introduced. These measures dramatically reduced the risk of virus transmission by blood and blood products in industrialized countries. However, there may still be a problem in resource-​limited countries where screening is not efficient, or where the background seroprevalence of potential donors is so high that HIV-​infected blood may be screened as negative when donated by an individual in the ‘window period’ immediately after initial infection (see ‘Diagnosis of HIV infection’, earlier). Injecting drug use Needle exchange programmes and the prescription of controlled drugs to registered addicts can reduce the incidence of new HIV infections in injecting drug users. Major problems still exist in countries such as India and Russia, where injecting drug use is more common and education about the risk and the availability of clean needles is very limited. Occupational exposure and postexposure prophylaxis Based on data from more than 3000 occupational exposures to HIV, the average risk of HIV infection after needlestick injury or other percutaneous exposure was calculated to be 0.3% (about 1 in 325). The risk following mucous membrane exposure has been es- timated to be around 0.1%. The risk of transmission is greatest for deep injuries, if there is visible blood on the device, during proced- ures involving direct cannulation of blood vessels or if the source patient has advanced HIV disease. A small retrospective case-​con- trol study demonstrated an 80% reduction in the likelihood of seroconversion in healthcare workers who took zidovudine soon after percutaneous exposure to HIV. In view of the greater activity of antiretroviral drug combinations but without direct evidence, it is currently recommended that high-​risk occupational exposures to HIV are treated as soon as possible with two nucleoside inhibi- tors and an integrase inhibitor (such as tenofovir, emtricitabine, and raltegravir) for 1 month. Nevirapine is not recommended in postexposure prophylaxis regimens because of a relatively high rate of adverse reactions. A careful risk assessment should be done, and if a significant risk of HIV transmission is identified, antiretroviral therapy should be offered and started promptly to maximize the chance of success. Following possible sexual exposure to HIV, antiretroviral therapy may reduce the risk of seroconversion, but there are no random- ized studies to confirm this. A comparative study in men who have sex with men in Brazil reported that individuals who took antiretro- viral therapy after sexual intercourse were less likely to acquire HIV infection (0.6% vs. 4.2%). Unprotected receptive anal intercourse (including sexual assault) is associated with the greatest risk (esti- mated up to 3%). After possible sexual exposure to HIV, a risk as- sessment is recommended and antiretroviral therapy should be offered if a significant risk is identified. Treatment should be started as soon as possible and is unlikely to be effective if started more than 72 h after exposure. PEP is no longer recommended for individuals where the exposure is with a known HIV-positive partner on fully suppressive ART as the risk of HIV acquisition remains very low. Vaccine development The high degree of viral variation and immune escape present dif- ficulties for the development of an effective preventive HIV vac- cine. Nonetheless, group-​specific neutralizing antibodies have been identified. In particular, there is evidence that broad CD8+ T-​cell responses directed against the relatively invariant, internal p24 Gag ‘capsid’ protein can be successful in achieving durable immune con- trol of HIV at very low or undetectable (below 50 HIV RNA copies/​ ml plasma) levels. Current vaccine efforts are therefore focused prin- cipally on inducing broad CD4+ and CD8+ T-​cell responses against HIV. These responses, however, would be expected to control rather than eliminate the virus altogether, and might lead to disease modi- fication rather than complete prevention. Although a vaccine cap- able of inducing broadly neutralizing antibodies against the range of HIV variants would eliminate the virus, no antigen capable of doing so has been identified to date. So far, non​infectious killed whole virus or recombinant subunit vaccines have not been successful in protecting chimpanzees from HIV infection, or macaques from SIV infection and disease. Certain live attenuated strains of SIV, with deletion mutations in nef and other regulatory genes, initially appeared to protect adult monkeys from challenge with virulent SIV strains, but subsequently were re- ported to cause AIDS in neonatal macaques. Human testing of candidate HIV vaccines, including a vaccine made from recombinant fragments of gp120, the surface glyco- protein of HIV that binds to host cell CD4 receptors, has so far not been successful. Large phase III trials in Thailand and the United States of America involving over 5000 uninfected high-​risk volun- teers showed no protection by a vaccine using recombinant gp120 (VaxGen) that had produced good neutralizing antibodies in pilot studies. Several new approaches are being examined, which may prove more effective in inducing protective humoral and killer T-​cell-​ mediated immunity. These include DNA vaccines, consisting of pieces of HIV DNA incorporated into harmless plasmid DNA from bacteria, and the use of live vectors (e.g. poxviruses such as canarypox and modified vaccinia) to deliver portions of the HIV envelope. A common approach now is to use a ‘prime-​boost’ strategy whereby a DNA vaccine dose is given, followed by a boosting with the DNA incorporated in a vector, such as modified vaccinia. One of the most potent vaccines uses a replication-​in- competent adenovirus type 5 as the vector. However, a phase IIb ef- ficacy trial (STEP/​HVTN 502) using the adenovirus type 5 vector with gag, pol, and nef genes was stopped prematurely. Not only was no efficacy shown, but there was evidence that those already immune to human adenovirus from natural infection were more likely to become infected by HIV. The reasons for this are not clear, but the National Institutes of Health has stopped or paused other trials using adenovirus vectors as a result. Trials with canarypox and other vectors continue. This approach, using DNA vaccines to stimulate CD8+ re- sponses, is also being evaluated for therapeutic vaccination in

8.5.23  HIV/AIDS 931 HIV-​positive patients with suppressed viraemia who are being treated with antiretroviral agents, to determine if vaccination will allow interruption of treatment without loss of virological control. Effective vaccination is likely to hold the greatest promise for controlling HIV infection in the future, but experience to date would indicate that researchers continue to face a formidable challenge. HIV cure While ART has dramatically altered the clinical course of dis- ease and markedly improved survival, ART alone is unable to cure HIV infection. This is a function of an inaccessible pool of latently infected cells, where viral DNA is incorporated into host cell genome. These latently infected cells, termed the HIV reservoir, represent the current barrier to a cure for HIV. There is no evidence that ART alone can ever achieve eradication or ‘cure’ of HIV. Although HIV may be undetectable in plasma for many years, this long-​lived reservoir of infectious virus can be recovered from latently infected (resting) memory CD4 lympho- cytes. The half-​life of this cell population is long, about 6 months, and it is not known whether HIV can ever be eradicated from this infected cell line. Other compartments exist that are relatively in- accessible to drugs—​for example, in the central nervous system, retina, and testes—​and unless viral replication can be success- fully prevented at such sites there is also the risk of reinfection of compartments previously cleared by therapy. One pool of latently HIV-​infected cells is the gut associated lymphoid tissue which, irrespective of the route of viral transmission, are the first cells infected and remain the largest source of reservoir cells. Recent data suggest that despite plasma viral levels remaining below the limit of detection (<20 copies HIV RNA/​ml) on ART, there can be very low level ongoing viral replication (to 1 copy/​ml) and for this reason despite years of successful viral suppression cessation of ART is invariably accompanied by viral rebound in the vast majority of individuals. People rapidly initiating ART around the time of acute infection have a much smaller HIV reservoir than individuals starting treatment after years of uncontrolled viral replication. In addition, early treatment, before any significant immune damage has occurred enables normalization of immune function. Among such treated acute HIV-​infected individuals there appears to be a greater chance of spontaneous viral control after stopping therapy; so-​called viral remission or posttreatment control. Even so, stopping therapy is not recommended given that in most individuals viral recrudescence occurs within 4–​8 weeks, and its associated risk of onward transmission and potential in- crease in inflammatory mediated complications. The Berlin patient There is to date only one man who has been successfully cured of HIV infection; Timothy Brown, the so-​called Berlin patient. Having received successful ART for many years with an undetect- able HIV plasma viral load he was diagnosed with acute myeloid leukaemia. As treatment for the leukaemia he underwent total body irradiation (including head), chemotherapy, and bone marrow transplantation with homozygous delta-​32 base pair deletion in the CCR5 gene from the donor bone marrow. He then experienced graft vs. host disease, relapse of the acute myeloid leukaemia, and the entire process was repeated using a second transplantation from the same Δ32 donor. Subsequently he stopped ART and re- mained with an undetectable viral load throughout follow up (Fig. 8.5.23.15). Despite multiple large blood sampling, tissue biop- sies (including brain) there has been no evidence of HIV DNA or RNA from any site. Therefore, by all available assays, he is cured of HIV infection. 107 800 100 200 300 400 500 600 101 102 103 104 HIV-1 RNA (cop/ml) CD4+ T-cells (µl) June ‘06 HIV DNA env HIV DNA LTR ART AML diagnosis AML relapse 2nd allogenetic transplantation 1st allogenetic transplantation 100% chimerism Limit of detection Oct ‘06 Feb ‘07 Mar ‘07 Jul ‘07 Jan ‘08 Mar ‘08 Jan ‘11 105 106 ART CD4+ T cell count HIV-1 RNA Fig. 8.5.23.15  The Berlin patient. Adapted from Hütter G et al. (2009). Long-​Term Control of HIV by CCR5 Delta32/​Delta32 Stem-​Cell Transplantation. NEJM, 360, 692–​8.

932 section 8  Infectious diseases Several other bone marrow transplant recipient cases have at- tempted to replicate this initial cure case. None, however, have been successful and viral recrudescence after ART cessation has in most cases been rapid. What represent the key differences between these cases and Timothy Brown is unclear but might be the graft-​versus-​ host disease experienced by Timothy Brown that was not a key fea- ture of other cases. Data from primate experiments point to the fact that for the Berlin patient, either the genetic mutation of the bone marrow donor or the graft-​versus-​host disease ‘played a significant role’, in the ultimate cure. Viral remission Since a sterilizing cure for HIV (as seen with Timothy Brown) seems highly unlikely and certainly not scaleable, the concept of a ‘functional cure’ for HIV, such as used in describing cancer, has been sought. In this scenario, while it is anticipated that detection of viral genome may still be the case, the patient remains aviraemic off ART with preserved immune function (in terms of normal CD4 count, CD4:CD8 ratio). Such cases of viral remission have been reported among very early treated infected infants and treated acute infections where therapy was subsequently stopped. In both groups, viral detection in terms of HIV reservoir (total HIV DNA) from blood and tissues, as well as very sensitive low copy viraemia from plasma, is still possible but using standard diagnostic viral load tests these remain below the limit of detection off ART. Such cases remain rare but suggest that very early ART may limit the size of the HIV reservoir to such a low level and enable normalization of the immune system that allows spontaneous control of viral rep- lication off therapy. Interventions to activate and eliminate viral reservoir towards HIV remission CD4 bearing cells containing incorporated HIV DNA are the key barrier to a cure for HIV. While ART is highly effective at blocking ongoing viral replication and further infection of uninfected cells, it is unable to remove viral DNA that is present within quiescent resting CD4 cells. These cells, termed the reservoir of HIV latent infection, or their progenitor cells potentially survive the lifetime of an infected individual and for most individuals, despite years of viral suppression, represent the source of rebounding virus when therapy is stopped. Removal or reduction in the frequency of such HIV-​infected viral reservoir cells is the goal of any novel ‘cure’ intervention. A concept termed ‘Kick and Kill’ is currently under investigation in small ‘pilot proof of concept’ studies. Here agents that reverse viral latency, which include histone deacetylase inhibi- tors, immune activators, or monoclonal antibodies act to ‘kick’ viral replication and the ‘kill’ aspect of the approach is usually under- taken by targeted primed HIV-​specific immune responses, driven by therapeutic vaccination (Fig. 8.5.23.16). FURTHER READING Abdool Karim SS, et al. (2010). Timing of initiation of antiretroviral drugs during tuberculosis therapy. N Engl J Med, 362, 697–​706. Altfeld M, Goulder P (2007). ‘Unleashed’ natural killers hinder HIV. Nat Genet, 39, 708–​10. Appay V, Kelleher AD (2016). Immune activation and immune ageing in HIV infection. Curr Opin HIV AIDS, 11, 242–​9. Anti-CD3 + anti-CD28 co-stimulation IL-2 IL-7 Prostratin HDAC inhibitors Other molecules Integrated provirus Translation and virion assembly mRNA splicing and nuclear export Transcription HIV antigen loading into MHV class I Budding and maturation HIV Env HIV-specific cytotoxic CD8+ T cell Activating latent virus is a necessary step in many HIV cure strategies Cytotoxic molecules induce cell lysis Productively infected CD4+ T cell Latently infected CD4+ T cell Fig. 8.5.23.16  HIV cure ‘kick and kill’—​various strategies might be invoked to activate latently infected T cells so that drugs and vaccine-​induced CD8+ cytotoxic T cells can act to eliminate the infected cell and virus. Adapted with permission of Future Medicine Ltd from Marsden MD and Zack JA (2009). Establishment and maintenance of HIV latency: model systems and opportunities for intervention. Future Virology, 5(1), 97–​109; permission conveyed through Copyright Clearance Center, Inc.

8.5.24 HIV in low- and middle- income countries 93

8.5.24 HIV in low- and middle- income countries 933

8.5.24  HIV in low- and middle-income countries 933 Bartlett JG, et al. (2012). Medical management of HIV infection, 16th edition. Johns Hopkins Medicine, Health Publishing Business Group, Baltimore, MD. Cohen MS, et al. (2011). Prevention of HIV-​1 infection with early anti- retroviral therapy. N Engl J Med, 365, 493–​505. Desai M, et al. (2015). Risk of cardiovascular events associated with current exposure to HIV antiretroviral therapies in a US veteran population. Clin Infect Dis, 61, 445–​52. Ford N, et al. (2015). World Health Organization guidelines on post­ exposure prophylaxis for HIV: recommendations for a public health approach. Clin Infect Dis, 60(S3), S161–4. GBD 2015 HIV Collaborators (2016). Estimates of global, regional and national incidence, prevalence and mortality of HIV, 1980–​2015; the Global Burden of Disease Study. Lancet HIV, 3, e361–​387. Goulder PJR, Lewin SR, Leitman EM (2016). Paediatric HIV infec- tion: the potential for cure. Nat Rev Immunol, 16, 259–​71. Haynes BF (2015). New approaches to HIV vaccine development. Curr Opin Immunol, 35, 39–​47. He W, et al. (2008). Duffy antigen receptor for chemokines mediates trans-​infection of HIV-​1 from red blood cells to target cells and af- fects HIV-​AIDS susceptibility. Cell Host Microbe, 4, 52–​62. Heaton RK, et al. (2015). Neurocognitive change in the era of HIV combination antiretroviral therapy:  the longitudinal CHARTER study. Clin Infect Dis, 60, 473–​80. Insight START Study Group (2015). Initiation of early antiretroviral therapy in early asymptomatic HIV infection. N Engl J Med, 373, 795–​807. Kooij KW, et al. (2015). Low bone mineral density in patients with well-​suppressed HIV infection:  association with body weight, smoking, and prior advanced HIV infection. J Infect Dis, 211, 539–​48. May MT, et al. (2014). Impact on life expectancy of HIV-​1 positive individuals of CD4+ cell count and viral load response to antiretro- viral therapy. AIDS, 28, 1193–​202. Mayer KH, et al. (2012). Raltegravir, tenofovir DF and emtricitabine for postexposure prophylaxis to prevent sexual transmission of HIV: safety, tolerability and adherence. J Acquir Immune Defic Syndr 59, 354–​9. McCormack S, et al. (2016). Pre-​exposure prophylaxis to prevent the acquisition of HIV-​1 infection (PROUD): effectiveness results from the pilot phase of a pragmatic open-​label randomised trial. Lancet, 387, 53–​60. Meintjes G, et al. (2018). Prednisone for the prevention of paradoxical tuberculosis-associated IRIS. N Engl J Med, 379, 1915–25. Muenchhoff M, et al. (2016). Non-​progressing HIV-​infected children share fundamental immunological features of non-​pathogenic SIV infection. Sci Transl Med, 8, 358ra125. Persaud D, et al. (2013). Absence of detectable HIV-​1 viremia after ces- sation in an infant. N Eng J Med, 369, 1828–​35. Prendergast A, et al. (2007). International perspectives, progress, and future challenges of paediatric HIV infection. Lancet, 370, 68–​80. Robertson J, et  al. (2006). Immune reconstitution syndrome in HIV: validating a case definition and identifying clinical predictors in persons initiating antiretroviral therapy. Clin Infect Dis, 42, 1639–​46. Sabin CA, et al. (2016). Is there continued evidence for an association between abacavir usage and myocardial infarction in individuals with HIV? A cohort collaboration. BMC Med, 14, 61. Smit M, Brinkman K, Geerlings S (2015). Future challenges for clinical care of an ageing population infected with HIV: a modelling study. Lancet Inf Dis, 15, 810–​8. The SPARTAC trial investigators (2013). Short course antiretroviral therapy in primary HIV infection. N Engl J Med, 368, 207–​17. The Strategies for Management of Antiretroviral Therapy (SMART) Study Group (2006). CD4+ count–​guided interruption of antiretro- viral treatment. N Eng J Med, 355, 283–​96. Online resources British HIV Association. http://​www.bhiva.org Joint United Nations Programme on HIV/​AIDS. http://​www.unaids.org NAM. Aidsmap. http://​www.aidsmap.com. University of California (San Francisco). HIV InSite Gateway. http://​ hivinsite.ucsf.edu/​. 8.5.24  HIV in low-​ and
middle-​income countries Alison D. Grant and Kevin M. De Cock ESSENTIALS The HIV pandemic has disproportionately affected people in low-​ and middle-​income countries. In many countries in sub-​Saharan Africa, HIV infection is established in the general population: in southern Africa, which is particularly severely affected, adult HIV prevalence has reached 30% in some areas. Local epidemiology depends on the balance between incidence (due to sexual contact, mother-​to-​child transmission, or exposure to blood or blood products) and mortality, and the effect of antiretroviral therapy on both mortality and transmis- sion. The main route of transmission is sex between men and women. Clinical features—​the manifestations of HIV disease vary by geographical region, reflecting increased frequency of exposure in low-​ and middle-​income countries to common pathogens such as tuberculosis, non​typhoid salmonellae, and Streptococcus pneumoniae. People with advanced immunosuppression are also at risk of disease due to geographically-restricted opportunistic patho- gens (e.g. leishmania and Talaromyces marneffei). Diagnosis and management—​diagnosis of HIV-​related disease can be difficult where there is limited access to laboratory diagnos- tics, and presumptive therapy for opportunistic infections, based on the most likely aetiologies, might be necessary. Based on trial data showing that antiretroviral therapy confers clinical benefits for people with CD4 counts above 500 cells/​μl, and that people on anti- retroviral therapy with undetectable viral loads are extremely unlikely to transmit HIV, since 2015 the World Health Organization has re- commended antiretroviral therapy for all HIV-​positive people re- gardless of disease stage, using standardized drug regimens, and viral load monitoring for those on treatment. The UNAIDS-​promoted 90:90:90 targets (diagnose 90% of people living with HIV; assure 90% of diagnosed persons receive antiretroviral therapy; achieve viral suppression in 90% of those treated) are now widely accepted. Prognosis—​life expectancy for HIV-​positive people in low-​ and middle-​income countries has been greatly improved by antiretroviral

934 section 8  Infectious diseases therapy, but remains shorter than in high-​income countries. For people initiating HIV care with advanced disease, early mortality re- mains high. More work is needed to improve coverage of HIV testing among asymptomatic people, along with effective linkage to and re- tention in care. Prevention—​this requires political commitment to creating an en- vironment that supports education about HIV, prevents stigma and discrimination, and protects the rights of key populations such as sex workers, men who have sex with men, transgender people, and people who inject drugs. Despite overall reduced HIV incidence since the late 1990s, the goal of preventing HIV transmission re- mains elusive in many settings, with HIV incidence rates highest among key populations; gay men and other adolescents, and young women. Major recent advances in HIV prevention science have been demonstration of the powerful preventive effect of anti- retroviral therapy through viral load suppression in HIV-​positive individuals, thus reducing infectiousness, and in the form of pre-​ exposure prophylaxis in HIV-​negative people. Universal testing of pregnant women linked to lifelong antiretroviral therapy can al- most eliminate mother-​to-​child transmission. Prevention interventions for general populations should include information and education; promotion of partner reduction and of condoms, which are highly protective against sexual transmission if used correctly and consistently; and encouragement of universal knowledge of HIV serostatus. Targeted interventions should be fo- cused on groups and situations in which HIV transmission is most intense, guided by local epidemiology. Male circumcision has a pro- tective efficacy of almost 60% against heterosexual acquisition of HIV infection in men, but other methods of prevention must still be promoted among circumcised men. No vaccine is available. Epidemiology The 2018 report from the Joint United Nations Programme on HIV/​ AIDS (UNAIDS) and the World Health Organization (WHO) esti- mated that, in 2017, 36.9 million (range 31.1–43.9 million) people were living with human immunodeficiency virus (HIV) infection worldwide. This global pandemic comprises a mosaic of local epi- demics, each with its own characteristics. Variation, both between regions and between groups of individuals affected within one re- gion, is one of the pandemic’s striking features. Broadly, there are two patterns:  generalized epidemics, established in the general population in most countries in sub-​Saharan Africa; and concen- trated epidemics, in specific populations in most other regions. Local epidemiology depends on the relative contribution of the three major routes of HIV transmission to incidence: sexual contact (heterosexual and homosexual); mother-​to-​child; and exposure to blood or blood products; mortality; and the effect of antiretroviral therapy (ART) on both incidence and mortality. Within sub-​Saharan Africa, there have been substantial regional differences in the evolu- tion of the epidemic. The main route of transmission is sex between men and women. Among key populations at increased risk are gay men and other men who have sex with men (MSM), among whom HIV epidemics are expanding in many countries despite falling in- cidence overall. Other key populations include people who inject drugs, and transgender people and, depending on local contexts, ­migrants, prisoners, and specific occupational groups, such as truck drivers and fisher folk. The HIV epidemic has been most severe in southern Africa; adult HIV prevalence in some parts of southern Africa is almost 30%. The reason for regional differences is not fully understood, but likely contributors include behavioural factors such as young age of sexual debut in women, age-​disparate relationships, and transactional sex; biological risk factors including herpes simplex virus type 2 (HSV-​2) infection, and lack of male circumcision; and structural factors such as labour migration. Global HIV incidence peaked in the mid-1990s, and has sub- sequently declined, but the decline is too slow to meet global tar- gets. In 2017, there were an estimated 1.8 million new infections, of which 980 000 were in sub-Saharan Africa. The reduction in HIV incidence is attributed to a range of reasons including maturation of the epidemic; successful prevention campaigns, particularly the prevention of mother-​to-​child transmission (estimated to have prevented 1.4 million infections between 2010 and 2017); and be- haviour change; high mortality among HIV-​positive people likely also contributed. HIV prevalence among young people has fallen in many, but not all, high-​prevalence countries, reflecting this reduc- tion in HIV incidence. Adolescent girls and young women remain at disproportionately high risk of acquiring HIV in high prevalence settings such as eastern and southern Africa. In 2017, global deaths due to HIV were estimated at 940 000, and in sub-​Saharan Africa at about 570 000; these have fallen since 2006, as ART coverage has increased. The total number of people living with HIV is increasing, reflecting ongoing incidence combined with reduced mortality as ART coverage increases. Prevention of new infections remains the key to controlling the epidemic. Understanding the local epi- demiology, and the performance of treatment and prevention pro- grammes, is essential to guide prevention and control efforts. Prevention Prevention of HIV infection requires political commitment to create an environment that supports education and openness about HIV; prevents punitive laws, stigma, and discrimination; and protects the rights of key populations most at risk for HIV. Involvement of civil society and those living with HIV is especially important. A series of randomized controlled trials, including HPTN 052, START, and several trials of pre-​exposure prophylaxis (PrEP), have fundamentally changed the prevention and treatment landscape. HPTN 052 showed that ART reduced heterosexual transmission from an HIV-​positive individual to his/​her sex partner by 96%. START demonstrated a 57% reduction in severe disease or death in HIV-positive people initiating ART at a CD4 count above 500 cells/​µl as opposed to deferring therapy. Several studies in different populations at risk have shown that antiretroviral drugs in the form of PrEP are effective in protecting HIV-​negative persons from ac- quiring HIV, although adherence has been a challenge. In 2014 UNAIDS introduced the concept of ‘90:90:90’, aiming to diagnose 90% of persons living with HIV; assuring that 90% of those receive ART; and ensuring 90% of those on ART are vir- ally suppressed. Most recent guidelines from WHO recommend PrEP for groups in which annual HIV incidence is 3% or higher. ‘Combination prevention’ incorporates HIV testing, ART, and PrEP

8.5.24  HIV in low- and middle-income countries 935 as appropriate, behaviour change including partner reduction and correct and consistent use of condoms, male circumcision for men at heterosexual risk, harm reduction for people who inject drugs, and structural interventions. Prevention of sexual transmission The traditional approach has been that of ‘ABC’, standing for Abstinence, Being faithful, and using Condoms if neither abstinent nor monogamous. While abstinence is an appropriate recom- mendation for the youngest age group, there is no evidence that abstinence promotion or education are effective as broader strat- egies. Reduction in the number of sexual partners and avoidance of concordant partnerships are important. Correct and consistent use of condoms is highly protective against acquiring HIV infection, but is difficult to sustain in long-​term relationships. Since HIV-​ positive people aware of their HIV status tend to alter their behav- iour to prevent transmission to others, promoting knowledge of HIV status is important. Concomitant sexually transmitted infec- tions can increase infectiousness as well as susceptibility to HIV, so screening and treatment for sexually transmitted infections should be enhanced. HPTN 052, a randomized trial of immediate ART for the HIV-​ positive partner in discordant couples where the HIV-​positive partner had a CD4 count of 350–​550 cells/​µl, found a 96% reduc- tion in HIV transmission to the HIV-​negative partner. START was a randomized trial comparing immediate vs. deferred ART among HIV-​positive people with CD4 greater than 500  cells/​µl, and showed a 57% reduction in severe events or death with early treat- ment. Therefore, not only does early ART have prevention benefit, it also benefits individual health. In consequence, and analogous to how we approach syphilis or tuberculosis control, diagnosing and treating persons with HIV are now key priorities and cen- tral to management and prevention of HIV/​AIDS. Studies are in progress to assess the impact of early ART on HIV incidence at population level. Different studies have confirmed the efficacy of oral tenofovir alone or in combination with emtricitabine as PrEP in preventing HIV acquisition among HIV-​negative MSM, people who inject drugs, discordant couples, and young heterosexuals. The effective- ness of PrEP is associated with adherence; lack of adherence being the likely explanation for negative findings in two studies among African women. The first study of a tenofovir-​based vaginal gel using a pericoital dosing schedule provided 39% protection from HIV ac- quisition. As with oral PrEP, efficacy requires adherence. Extensive research is underway and planned of different dosing schedules, oral and topical PrEP regimens using different drugs, longer-​acting injectable agents and sustained release delivery systems, and rectal preparations. Although genital herpes is a risk factor for HIV transmission and acquisition, clinical trials of herpes suppressive therapy have not shown that this intervention reduces HIV incidence. Currently there is no vaccine against HSV-​2. Voluntary medical male circumcision has a protective efficacy of almost 60% against heterosexual acquisition of HIV infection in men. The once-​only nature of this intervention, its independence from adherence, and its sustained efficacy make it an important public health intervention. Prevention benefit to women is in- direct, there being no evidence of reduced HIV transmission from HIV-​positive circumcised men. Medical devices have been intro- duced which can replace surgery for male circumcision, although their uptake has been limited. Complication rates from surgery or devices have been low, and include infection, haemorrhage, and penile deformity. There have been occasional reports of tetanus, usually following unsterile dressing of the surgical wound. Uptake of voluntary medical male circumcision has been greater in East than in southern Africa. Key populations may benefit from special services tailored to their needs in specific venues or using outreach approaches, avoiding stigmatization and healthcare worker disapproval that all too often characterize public healthcare settings. A particular challenge is HIV prevention in adolescents and especially girls and young women, particularly in sub-​Saharan Africa. Social grant programmes incorporating cash transfers aim to reduce pressure for transactional sex, older sex partners, teenage preg- nancy and early marriage, and school drop-​out. While increased education generally reduces high-​risk behaviour in young women, the impact of such programmes on HIV incidence has been mixed. For sex workers and their clients, correct and consistent condom use, and prompt diagnosis and treatment of other sexually trans- mitted infections must be promoted. Important interventions for MSM include HIV counselling and testing, access to correct and consistent condom use and lubricants, and addressing drug use that might lead to unsafe behaviour. The quest continues for an effective HIV vaccine. Prevention of transmission by injecting drug use The public health approach emphasizes harm reduction, whose es- sentials include information and education; access to HIV testing, ART, and PrEP; needle and syringe programmes; treatment for drug dependence including opioid substitution therapy; clin- ical services for associated conditions such as viral hepatitis and skin infections; and interventions to prevent sexual transmission of HIV. Prevention of transmission through blood, blood products, and nosocomial exposures Although eliminated in high-​income countries, transmission of HIV by blood transfusion remains a possibility in many countries. Basic measures to prevent transfusion-​transmitted HIV include appropriate management of conditions predisposing to the need for transfusion (such as childbirth and malaria), and avoidance of all but essential transfusions. Family and paid donors should be avoided in favour of regular, low-​risk donors. All blood destined for transfusion should be screened for HIV, syphilis, and hepatitis B and C and, as far as possible, obtained from centralized services that can assure safe blood. Preventive measures against nosocomial transmission include universal precautions, which treat all body fluids as potentially infectious. Exposure-​prone procedures might require other pro- tection in addition to gloves such as masks, gowns, and goggles. Injection safety requires absolute avoidance of re-​use of nee- dles and syringes, and assurance of their safe use and disposal. Healthcare institutions require policies concerning availability of postexposure antiretroviral prophylaxis following occupational exposure.

936 section 8  Infectious diseases Prevention of mother-​to-​child transmission In industrialized countries, combination ART for pregnant women, elective caesarean section if necessary, and avoidance of breast- feeding have rendered perinatal HIV transmission very rare. A four-​ pronged integrated approach in low-​ and middle-​income countries, promoted by United Nations agencies, includes primary prevention of HIV infection in girls and young women, prevention of unin- tended pregnancy in HIV-​positive women, interventions to prevent transmission of HIV from positive women to their offspring, and diagnosis and care of HIV-​positive infants and their mothers. HIV testing should be recommended to all pregnant women. Until 2012, recommendations for preventing mother-​to-​child transmission offered complex alternatives for maternal and infant prophylaxis, so-​called Options A and B. A proposal from Malawi that rapidly became adopted, ‘Option B+’, recommended immediate and lifelong combination ART for all pregnant women, offering simplicity, protection against transmission during breastfeeding, and protection during subsequent pregnancies. WHO guidelines re- commending ART for all HIV-​positive persons also cover pregnant women, who should initiate ART upon diagnosis during pregnancy or breastfeeding and continue it lifelong. Infants born to HIV-​ positive women should receive 6–​12 weeks of prophylaxis using AZT and/​or nevirapine according to WHO guidelines. In countries where HIV-positive mothers are recommended to breastfeed; exclu- sive breastfeeding for six months is recommended with introduction of complementary food thereafter; breastfeeding should continue to 12 months, with gradual weaning over one month. Clinical features Acute HIV disease Symptoms associated with primary HIV infection are described in Chapter 8.5.23. Although rarely specifically diagnosed, HIV pri- mary infection is important to consider in the differential diagnosis of an acute febrile illness, particularly in high-​prevalence settings. In a study of women in Kenya, the most common symptoms reported by those newly infected were fever, headache, fatigue, and arthralgia, whereas the most strongly associated clinical features were lymph- adenopathy, vomiting, diarrhoea, and fever. Progression from HIV infection to symptomatic disease Data from representative cohorts of people with well-​defined dates of seroconversion show that the progression of HIV disease from primary infection to the stage of advanced immunosuppression in low-​ and middle-​income countries is little different from that ob- served in the pre-​ART era in high-​income countries. Once people reach the stage of advanced immunosuppression, survival is likely to be shorter than in high-​income countries if they do not have ac- cess to ART and interventions to prevent and treat opportunistic infections. Symptomatic HIV disease HIV-​positive people experience much higher incidence of diseases caused by pathogens common in low-​ and middle-​income countries, such as tuberculosis, pneumococcal disease, and non​typhoidal sal- monella, compared to their HIV-​negative counterparts. Tuberculosis is often the first manifestation of HIV disease, although by the time they present with tuberculosis, about half of HIV-​positive people will already have a CD4 count below 200 cells/​µl. Other early pre- senting symptoms of HIV disease are skin conditions such as gen- eralized pruriginous dermatitis (prurigo) and herpes zoster, both of which have a high positive predictive value for underlying HIV in- fection among populations with high HIV prevalence. Advanced HIV disease When HIV-​positive people reach the stage of advanced immuno- suppression, the spectrum of disease varies by geographical re- gion. Tuberculosis, bacterial infections due to pathogens such as Streptococcus pneumoniae, and cryptococcal disease are common worldwide. Disease due to non​typhoidal salmonella species might be more common where sanitation is poor. Pneumocystis pneu- monia is relatively common in Asia and South Africa but in many countries in sub-​Saharan Africa it is less common as a cause of severe respiratory symptoms than bacterial infections and tuber- culosis, perhaps because disease due to these more common patho- gens occurs earlier in the course of HIV disease. The frequency of cerebral toxoplasmosis varies by region, influenced particularly by consumption of undercooked meat. Exposure to some opportun- istic infections is geographically limited. Talaromycosis (formerly penicilliosis), caused by Taralomyces marneffei, is largely confined to Southeast Asia and southern China (Chapter 8.7.6), and exposure to Trypanosoma cruzi, which can reactivate, most often causing cere- bral lesions, is largely restricted to the Americas. Diseases charac- teristic of very advanced immunosuppression, such as those due to cytomegalovirus and Mycobacterium avium intracellulare, have been rare in many low-​ and middle-​income countries, probably be- cause survival with advanced disease in the absence of ART is short. Despite increasing ART coverage, many HIV-​positive people still present with advanced disease, and the spectrum of disease among HIV-​positive people admitted to hospital continues to reflect ad- vanced immunosuppression, with tuberculosis and severe bacterial infections the leading causes of admission and in-​hospital mortality. Tuberculosis (Chapter 8.6.25) Tuberculosis is the most important cause of HIV-​related severe morbidity and mortality in low-​ and middle-​income countries. It results both from reactivation of latent infection as well as rapid pro- gression following new or re-​infection. Molecular epidemiological studies show that new infections are an important mechanism of recurrence, which is common in HIV-​positive people. The diagnosis of tuberculosis is more challenging in low-​ and middle-​income countries. HIV-​positive people with tuberculosis are less likely to have symptoms typical of pulmonary disease and to have smear-​negative sputum, making the diagnosis harder to con- firm, especially in settings where the main diagnostic test is sputum microscopy. This is a particular problem for people with advanced immunosuppression, where a delay in initiating tuberculosis treat- ment may be fatal. Sputum culture is more sensitive than micros- copy, particularly if liquid culture media are used, but in many low-​ and middle-​income countries facilities for mycobacterial cul- ture are very limited. An automated, nucleic acid amplification-​based assay (Xpert MTB/​RIF, described in detail in Chapter  8.6.25), which is more

8.5.24  HIV in low- and middle-income countries 937 sensitive than microscopy and provides rapid diagnosis of rifam- picin resistance, is recommended by WHO for HIV-​positive people with symptoms suggesting tuberculosis. However, implementation of Xpert MTB/​RIF has not improved outcomes for patients with drug-​susceptible tuberculosis. In South Africa, where Xpert MTB/​ RIF has replaced microscopy as the first test for tuberculosis, the expected increase in tuberculosis case notifications has not been seen in practice, probably because Xpert has provided bacterio- logical confirmation among people who were previously treated for tuberculosis on clinical criteria. Xpert MTB/​RIF is increasingly used as the initial test for all people with symptoms suggesting tu- berculosis; however, logistical barriers and cost limit its use in primary care settings. A low-​cost lateral flow assay which detects lipoarabinomannan (LAM), a component of the mycobacterial cell wall, in urine has a role in the diagnosis of tuberculosis among hos- pitalized HIV-​positive people with advanced immunosuppression. Its low sensitivity among other patient groups limits its wider use. A sensitive, low-​cost test for tuberculosis which can be used in pri- mary care settings remains an urgent priority. A particular challenge is posed by drug-​resistant tuberculosis. Extensively drug-​resistant tuberculosis (defined as resistance to at least rifampicin, isoniazid, any quinolone, and one of the injectable agents: amikacin, capreomycin, or kanamycin) has been identified in every world region. The seriousness of this issue was highlighted by a nosocomially-​transmitted outbreak of extensively drug-​resistant tuberculosis in South Africa, identified in 2005–​6 in an HIV care fa- cility, with high case fatality. The problem of drug-​resistant tubercu- losis is made worse by the paucity of facilities for drug-​susceptibility testing in most countries that carry the highest burden of tubercu- losis, making rapid appropriate treatment, and interruption of trans- mission, challenging. Xpert MTB/​RIF allows rapid identification of rifampicin resistance, and if widely used will identify more people with drug-​resistant tuberculosis, but outcomes for patients will only improve if they can access, and complete, effective treatment. Major gaps persist between the estimated number of people who develop drug-​resistant tuberculosis and those who start treatment, and treatment outcomes remain poor. There remains an urgent need for more accessible diagnostic tests for drug resistance, more effective and more tolerable treatment for people with drug-​resistant tuber- culosis, better linkage to HIV care for HIV-​positive people with tu- berculosis, and better infection control in health facilities to prevent nosocomial transmission to both patients and staff. The WHO strategy to reduce the burden of tuberculosis com- prises the so-​called ‘three Is’: intensified case-​finding, isoniazid pre- ventive therapy, and infection control. As part of intensified case finding, HIV-​positive people should be screened for tuberculosis at every clinical encounter, based on reporting any of cough, weight loss, night sweats, or fever. Screening is particularly important for people newly diagnosed HIV positive, among whom the prevalence of active tuberculosis is particularly high. The four-​symptom screen has high negative predictive value for active tuberculosis. Those without symptoms might benefit from isoniazid preventive therapy for six months, or potentially lifelong if a test for latent tuberculosis infection is positive. ART reduces the risk of a new tuberculosis epi- sode, although the risk remains high, particularly among those with low CD4 counts. All people with newly diagnosed tuberculosis should know their HIV status, because in some settings more than 70% will be HIV-positive, and should receive ART and co-​trimoxazole prophy- laxis. For patients whose HIV-positive status is diagnosed at the time of a tuberculosis episode, WHO guidelines recommend that tuber- culosis treatment should be started first, followed by ART as soon as possible afterwards, regardless of the CD4 count. Randomized controlled trials suggest that people with newly diagnosed HIV-​ associated tuberculosis and CD4 less than 50 cells/​µl are most likely to benefit from early initiation of ART, within two weeks of initiation of tuberculosis treatment. Among individuals with tuberculous meningitis, data from one trial showed no survival benefit from im- mediate vs. deferred ART, and further data are needed to determine the optimum timing of ART initiation for these patients. Interaction between HIV infection and
‘tropical’ diseases Malaria (Chapter 8.8.2) In areas of year-​round (stable or holoendemic) malarial transmis- sion, studies from Uganda and Malawi suggest that HIV infection impairs acquired immunity to falciparum malaria, resulting in in- creased frequency of malarial parasitaemia and clinical malaria among adults and older children proportional to the degree of im- munosuppression, but no increase in severe or complicated malaria. However, HIV-​positive infants in a holoendemic area of Kenya were at increased risk of severe anaemia and hospitalization for malaria. In studies from South Africa of non​immune adults and older children resident in areas of intermittent (low or unstable) malaria transmis- sion, HIV-​positivity was associated with an increased risk of severe and fatal falciparum malaria, inversely proportional to their CD4 counts. In HIV-​positive pregnant women, the beneficial effects of parity on severity of malaria are attenuated, and their peripheral and placental parasitaemia, and risk of suffering an episode of malaria or anaemia during pregnancy, are increased. Among HIV-​positive pregnant women, malaria is associated with an increased risk of low birth weight, preterm birth, intrauterine growth retardation, and postnatal infant mortality. ART and co-​trimoxazole reduce the risk of febrile malarial episodes. HIV-​positive people in malaria-​ endemic areas should sleep under insecticide-​treated bed nets, and non​immune people travelling to malarial areas should use bednets and take antimalarial chemoprophylaxis. In malaria-​endemic areas, HIV-​positive pregnant women should take either continuous co-​ trimoxazole or intermittent preventive therapy to prevent adverse malaria outcomes in infants. In malaria-​endemic areas, HIV-​positive people who develop fever should be investigated for other causes in addition to malaria; those taking co-​trimoxazole prophylaxis should not be given pyrimethamine with sulfadoxine as malaria treatment or seasonal malaria chemoprophylaxis. ART might interact with antimalarial drugs (see http://​www.hiv-​druginteractions.org). In particular, artesunate plus amodiaquine is more likely to cause se- vere neutropaenia in HIV-​positive people, particularly if they are taking zidovudine or co-​trimoxazole, and these combinations should be avoided if possible. Amodiaquine should also be avoided among people taking efavirenz because of increased risk of hepatotoxicity. Leishmaniasis (Chapter 8.8.13) The HIV epidemic led to localized increases in visceral leishmaniasis, predominantly in people with CD4 counts below 200 cells/​µl, particu- larly among injecting drug users around the Mediterranean; in the

938 section 8  Infectious diseases northeast of Africa (Ethiopia and Sudan); Brazil; and India. Wider use of ART has resulted in declining numbers of coinfected cases in some areas. In HIV-​positive people, visceral leishmaniasis most often pre- sents classically with fever, hepatosplenomegaly, and pancytopenia, although presentations range from asymptomatic to multiorgan in- volvement. The treatment of choice is liposomal amphotericin B, but treatment outcomes are less good for HIV-​positive compared to HIV-​negative people. Amphotericin B deoxycholate or sodium stibogluconate are much less satisfactory. Observational studies sug- gest that combination treatment, such as with liposomal amphotericin B and miltefosine, might be effective; a trial comparing this combin- ation to liposomal amphotericin B alone is in progress among HIV-​ positive people in Ethiopia. ART is important to reduce mortality and risk of relapse, and expert opinion supports early ART initiation. Relapse remains common, and secondary prophylaxis is needed, al- though there is no trial-​based evidence to support choice of regimen. Cutaneous leishmaniasis can present with atypical skin lesions, which might be disseminated and can recur after treatment. Trypanosomiasis (Chapters 8.8.11, 8.8.12) Asymptomatic infection with Trypanosoma cruzi can reactivate to cause Chagas disease in the context of advanced HIV disease, most often resulting in meningoencephalitis or cerebral mass lesions. Myocarditis is common at autopsy, although rarely apparent clin- ically. Screening of HIV-​positive individuals from endemic areas is recommended: early ART is important to prevent reactivation of chronic disease. There is no evidence of an interaction between human African trypanosomiasis and HIV infection, although re- ports suggest high mortality among HIV-​positive patients treated for central nervous system disease. Helminths There is little evidence of interaction between intestinal nematodes and HIV infection; the expected association with Strongyloides ster- coralis hyperinfection has not been observed, although it is common with another retroviral infection, human T-​lymphotropic virus (HTLV-​1) (Chapter 8.5.25). HIV infection does not affect the man- agement of onchocerciasis, although skin disease may be more se- vere. Data concerning an association between Wuchereria bancrofti infection and HIV infection are inconsistent. There is no consistent association between Schistosoma mansoni infection and HIV; how- ever female genital schistosomiasis is reported to be associated with HIV infection and could facilitate HIV transmission. Atypical forms of neurocysticercosis, such as giant brain cyst and spinal epidural lesions, are reported. Fungi People with advanced HIV disease are at risk of systemic fungal in- fections. Cryptococcal disease and histoplasmosis (which, based on clinical features, might be mistaken for tuberculosis) are found worldwide. Others have restricted geographical distribution, such as talaromycosis in Southeast Asia and China. Paracoccidioidomycosis (Paracoccidioides brasiliensis infection—​Chapter 8.7.4), is the most common invasive fungal infection in South America, but reports of coinfection with HIV are less common. There is no evidence of important interactions between HIV in- fection and typhoid fever, melioidosis, amoebiasis, or giardiasis. Leprosy might be unmasked by ART as an immune reconstitution phenomenon. Clinical staging of HIV disease Given limited laboratory facilities in some settings, HIV viral load es- timation and CD4 counts are not always available. A system designed to estimate HIV disease stage based on clinical symptoms, modified by CD4 count if available, was published by WHO in 1990 and re- vised in 2006 (Table 8.5.24.1). This has been widely used in resource-​ constrained settings to guide when to start ART. As countries move towards ‘test and start’, clinical staging will be less important, but re- mains a guide to prognosis where CD4 counts are not available. The ‘cascade of care’ The cascade of care concept has gained popularity as a way to illustrate, at population level, the proportion of HIV-​positive people at different stages of accessing care and treatment (Fig. 8.5.24.1). This concept is Table 8.5.24.1  WHO clinical staging system for adults and adolescents with confirmed HIV infection WHO clinical stage Defining conditions 1 Asymptomatic Persistent generalized lymphadenopathy 2 Unexplained moderate weight loss (<10%) Recurrent respiratory tract infections Herpes zoster Angular cheilitis Recurrent oral ulceration Papular pruritic eruption Seborrhoeic dermatitis Fungal nail infections 3 Unexplained severe (>10%) weight loss Unexplained chronic diarrhoea (>1 month) Unexplained persistent fever (>1 month) Persistent oral candidiasis Oral hairy leukoplakia Pulmonary tuberculosis Severe bacterial infections Acute necrotizing ulcerative stomatitis, gingivitis, or
  periodontitis Unexplained anaemia (<8 g/​dl), neutropaenia (<0.5 × 109/​l)
  or thrombocytopaenia (<50 × 109/​l) 4 HIV wasting syndrome Pneumocystis pneumonia Recurrent severe bacterial pneumonia Chronic herpes simplex infection (>1-​month duration) Oesophageal candidiasis Extrapulmonary tuberculosis Kaposi’s sarcoma Cytomegalovirus infection Central nervous system toxoplasmosis HIV encephalopathy Extrapulmonary cryptococcosis Disseminated non​tuberculosis mycobacterial infection Progressive multifocal leukoencephalopathy Chronic cryptosporidiosis Chronic isosporiasis Disseminated mycosis (extrapulmonary histoplasmosis
  or coccidioidomycosis) Recurrent septicaemia Lymphoma (cerebral or B-​cell non-​Hodgkin) Invasive cervical carcinoma Atypical disseminated leishmaniasis Symptomatic HIV-​associated nephropathy or
  cardiomyopathy

8.5.24  HIV in low- and middle-income countries 939 not new, and similar ideas have been described previously for other diseases such as tuberculosis. Nonetheless, cascade of care diagrams are valuable to illustrate how the ultimate goal of maximizing the pro- portion of HIV-​positive people whose viral load is suppressed on ART is dependent on preceding steps in the care pathway. Cascade analyses can highlight where, for a given population, where there are bottle- necks, and thus where efforts are most needed. UNAIDS’ target of ’90:90:90’ by 2020, considered in the context of the cascade of care, would result in 73% of HIV-​positive people having an undetectable viral load. Mathematical modelling has sug- gested this would result in substantial epidemic impact at a popula- tion level, but more ambitious targets will be needed subsequently. HIV testing HIV testing is the point of entry to prevention, care, and support services. Healthcare workers should recommend HIV testing; spe- cific recommendations for provider-​initiated testing and counsel- ling in different epidemiological settings are listed in Table 8.5.24.2. Rapid diagnostic tests for HIV, allowing point-​of-​care testing with same-​day results, have made it much easier for people to gain knowledge of their HIV status in healthcare facilities at all levels, in community-​based sites, or at home. HIV self-​testing, whereby an individual performs their own test on a suitable specimen, is be- coming more widely available; people with reactive results on self-​ testing always need retesting using a recommended algorithm. Testing strategies should be guided by local HIV prevalence; WHO provides comprehensive guidance on testing systems and ap- propriate algorithms. Quality management systems are essential to ensure that correct results are delivered. People who test positive for the first time should always have a second test, based on a different specimen, before initiating HIV treatment, to minimize the risk that a false positive test result leads to inappropriate treatment. People with previously-confirmed HIV infection who are taking ART should not be retested for HIV antibodies; they might have false-​ negative results, particularly with tests based on oral fluid specimens. People with confirmed HIV-​positive test results need to be linked to treatment and care services, to promote timely initiation of ART and avoid the morbidity and mortality associated with advanced disease. In some studies promoting HIV testing in community set- tings, subsequent linkage to care has been limited. Reasons include structural barriers such as distance to clinic and social barriers such as stigma; further work is needed to find effective ways to overcome context-​specific barriers and optimize linkage, especially for men, who access care at routine health services less often than do women. Antiretroviral treatment The roll-​out of ART has been an extraordinary public health success. By 2017, 21.7 million people were estimated to have started ART, representing more than half of those estimated to be living with HIV worldwide. Guidelines have evolved to recommend immediate ART initiation, reflecting increasing evidence supporting the clin- ical benefits of treatment at earlier stages of HIV disease, along with the development of treatment regimens which are far more tolerable and less demanding, generally allowing once-​daily dosing. WHO now recommends ART for all HIV-​positive people, regard- less of their clinical stage or CD4 count, with particular priority for those in WHO clinical stage 3 and 4, and those with a CD4 count of 350 cells/​µl or less. The approach to ART delivery in low-​ and middle-​income coun- tries has taken a public health rather than an individualized ap- proach. The aim is to maximize the survival of all HIV-​positive people in the population by using ART regimens which are stand- ardized, rather than tailored to the individual; by simplifying man- agement so that HIV care can be undertaken by other healthcare 2015 2016 2017 50M 40M 30M 20M 10M People living with HIV People living with HIV who know their status People receiving antiretroviral therapy People living with HIV who have suppressed viral loads 0 Number Fig. 8.5.24.1  Global HIV treatment cascade, 2015-2017. The pale section at the top of the bar shows the gap between the number reached and the number needed to achieve the 90:90:90 target. Courtesy of UNAIDS (http://aidsinfo.unaids.org/).

940 section 8  Infectious diseases workers where there are few doctors; and using clinical and basic laboratory monitoring so that ART can be delivered even if CD4 counts and HIV viral load measurements cannot be done. However, without viral load measurements, it is much harder to know if treat- ment is effective or not, and scale-​up of viral load monitoring is a priority. Dried blood spots can be used to transport specimens for viral load measurements from remote clinics. Antiretroviral agents are described in detail in Chapter 8.5.23. The recommended first-​line regimen for treatment of adults (including pregnant or breastfeeding women) or adolescents with HIV-​1 in- fection comprises two nucleoside reverse transcriptase inhibitors (NRTIs) and one non​nucleoside reverse transcriptase inhibitor (NNRTI) or an integrase inhibitor (Table 8.5.24.3). For HIV-​2 in- fections, NNRTIs are ineffective, and a boosted protease inhibitor-​ based regimen would be the preferred first-​line regimen. Second-​line therapy is based on a boosted protease inhibitor in combination with two previously unused NRTIs. The increasing number of people taking ART includes people with a diverse range of care needs. This has led to the concept of ‘differ- entiated care’, meaning there may be particular priorities for certain categories of patient. Specific categories identified include people starting ART when they are well, who may need extra support with adherence and retention; people presenting with advanced disease (CD4 count below 200 cells/​µl and/​or WHO stage 3 or 4) who are at high risk of morbidity and mortality and may need a more intensive package of care; and among people taking ART, distinguishing be- tween those who are stable, whose care could potentially be decen- tralized to community-​based models, and those who are ‘unstable’, needing additional support, closer monitoring, or a change in treat- ment regimen. Conventionally, ART initiation has been considered to be non-​ urgent in most circumstances, and much emphasis was placed on counselling around adherence and treatment preparedness prior to ART initiation. While treatment literacy is important, it is also im- portant that ART initiation is not unduly delayed, particularly for people with advanced disease. The package of care for people with advanced disease should also include screening for tuberculosis and cryptococcal disease, and treatment if necessary; and co-​trimoxazole and isoniazid preventive therapies, as indicated. Immune reconstitution inflammatory syndrome (IRIS) (see also Chapter 8.5.23), either ‘paradoxical’, that is, worsening of an existing HIV-​associated disease, or ‘unmasking’ of a previously undiagnosed disease after the start of ART, occurs in about 16% patients, and is associated with a low baseline CD4 count, and (for paradoxical IRIS) with earlier ART initiation. Case fatality for patients with IRIS is rela- tively low (4.5% overall) but appears higher (around 21%) in patients with cryptococcal meningitis; in these patients ART initiation should be deferred until there is evidence of a response to antifungal therapy (usually 2–​6 weeks, depending on the antifungal regimen used). Supporting adherence to treatment and retention in care is critical to assuring that individuals and populations gain maximum benefit from ART. Interventions may include adherence clubs supporting community-​based care; medication adherence training, peer coun- sellor interventions, mobile-​phone based text messaging, and re- minder devices. Prevention of HIV-​related disease In addition to the provision of ART, other interventions are effective in preventing illness among HIV-​positive people. Co-​trimoxazole prophylaxis reduces morbidity and mortality among HIV-​posi- tive children and adults. WHO guidelines recommend starting co-​ trimoxazole for adults in WHO stage 3 or 4, or with tuberculosis, irrespective of CD4 count; or if the CD4 count is below 350 cells/​ µl, irrespective of WHO stage. Co-​trimoxazole can be discontinued among adults who are clinically stable on ART with immune re- covery and/​or virological suppression. Where malaria and /​ or se- vere bacterial infections are common, WHO recommends provision of co-​trimoxazole to all, continued indefinitely. The benefits of long-​ term co-​trimoxazole use, particularly among people stable on ART with virological suppression, might need to be balanced against the potential for promoting antibiotic resistance. HIV-​positive people should be screened for symptoms of active tuberculosis (cough, Table 8.5.24.2  WHO recommendations for provider-​initiated counselling and testing Type of HIV epidemic Provider-​initiated testing and counselling should be recommended for: All All clients attending services for malnutrition, sexually transmitted infections, viral hepatitis, and tuberculosis (confirmed or under investigation), and services for key populations Generalized All clients in all services, including those listed above; also children under 5 years; immunization and antenatal care services Concentrated All clients (adults, adolescents, and children) in clinical settings who present with symptoms or medical conditions that could indicate HIV infection, including tuberculosis, whether confirmed or under investigation Table 8.5.24.3  First-​line antiretroviral therapy recommendations for adults: WHO recommendations Preferred regimen Tenofovir plus lamivudine (or emtricitabine) plus efavirenz Alternative regimens Zidovudine plus lamivudine plus efavirenz (or nevirapine) Tenofovir plus lamivudine (or emtricitabine) plus dolutegravira Tenofovir plus lamivudine (or emtricitabine) plus efavirenz 400 mga Tenofovir plus lamivudine (or emtricitabine) plus nevirapine Special circumstances Regimens containing abacavir and boosted protease inhibitors a Efficacy (and, for dolutegravir, safety) data for pregnant/​breastfeeding women and people receiving treatment for tuberculosis are pending.

8.5.25 HTLV- 1, HTLV- 2, and associated diseases 9

8.5.25 HTLV- 1, HTLV- 2, and associated diseases 941

8.5.25  HTLV-1, HTLV-2, and associated diseases 941 fever, weight loss, night sweats) at each clinical encounter, and those who do not have active tuberculosis should be offered isoniazid pre- ventive therapy. Routine screening for cryptococcal antigen prior to ART, followed by pre-​emptive antifungal treatment if crypto- coccal antigen positive, should be considered for people with CD4 counts less than 100 cells/​µl where the prevalence of cryptococcal antigenaemia in the population is greater than 3%. Interventions to prevent malaria are described earlier. Appropriate vaccines include those against hepatitis B, pneumococcal disease, influenza, and tet- anus. Nutritional support should be provided for the malnourished. To reduce infective diarrhoea, household-​based water treatment methods are recommended, along with proper disposal of faeces and hand washing with soap. KDC’s work towards this publication was made possible by support from the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR) to the U.S. Centers for Disease Control and Prevention (CDC), Division of Global HIV & TB (DGHT). The opinions and conclusions in this chapter are those of the authors and do not necessarily represent the official position of the funding agencies. FURTHER READING World Health Organization (2018). Latent TB Infection: Updated and consolidated guidelines for programmatic management. https://www. who.int/tb/publications/2018/latent-tuberculosis-infection/en/ World Health Organization (2015). Consolidated guidelines on HIV testing services. 5Cs: consent, confidentiality, counselling, correct results and connection. http://​apps.who.int/​iris/​bitstream/​10665/​179870/​1/​ 9789241508926_​eng.pdf?ua=1&ua=1 World Health Organization (2016). Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Recommendations for a public health approach, 2nd edition. http://​ apps.who.int/​iris/​bitstream/​10665/​208825/​1/​9789241549684_​ eng.pdf 8.5.25  HTLV-​1, HTLV-​2, and associated diseases Kristien Verdonck and Eduardo Gotuzzo ESSENTIALS Human T-cell leukaemia virus (HTLV)-​1 and HTLV-​2 belong to the genus Deltaretrovirus of the family Retroviridae. They only infect humans, produce a lifelong infection, and can be transmitted from mother to child, through sexual intercourse, and via cellular blood components and organ transplantation. Both viruses are present in all continents and have a heterogeneous distribution. HTLV-​1-​endemic foci (general population prevalence >1%) are found in Japan, the Caribbean, South America, Africa, and Australo-​Melanesia. There are endemic foci of HTLV-​2 among native Amerindians and Central African populations. HTLV-​1 and 2 also occur among people who inject drugs. It is unclear why some infected people develop associated diseases while others remain asymptomatic. Clinical features—​(1) HTLV-​1—​up to 10% of carriers develop clin- ical manifestations, including adult T-​cell leukaemia/​lymphoma, HTLV-​associated myelopathy/​tropical spastic paraparesis, and in- fectious diseases such as strongyloidiasis, scabies, and tuberculosis. (2) HTLV-​2—​can cause HTLV-​associated myelopathy/​tropical spastic paraparesis, arthritis, bronchitis, and pneumonia. Diagnosis and prevention—​HTLV enzyme-linked immunosorbent assays are used for screening, followed by confirmatory testing of positive results. Mother-​to-​child transmission of HTLV-​1 can be re- duced by avoiding breastfeeding; condom use protects against sexu- ally transmitted infection; screening of blood donors is performed in many countries. No vaccine is available and there are no effective antiviral drugs. Historical perspective In 1979, human T-​lymphotropic virus 1 (HTLV-​1) was isolated from a patient with a T-​cell malignancy. In the years that followed, several syndromes, previously considered idiopathic, were linked to this virus:  adult T-​cell leukaemia/​lymphoma (ATL), tropical spastic paraparesis, uveitis, and infective dermatitis. HTLV-​2 was discovered in 1982, and HTLV-​3 and HTLV-​4 in 2005. It is not yet known whether HTLV-​3 and -​4 cause human disease. Epidemiology HTLV originated several millennia ago. There are six molecular sub- types of HTLV-​1 and four of HTLV-​2, several of which have spread from Africa to the rest of the world. The sequence variation in these viruses is linked with specific populations and geographical loca- tions, but not with disease outcome. It has been estimated that more than 5–​10 million people are in- fected with HTLV-​1 worldwide and that the number of HTLV-​2-​ infected people also amounts to several millions. These estimates should be interpreted with caution because most of the HTLV prevalence studies were done in blood donors, pregnant women, and other selected population groups, and because data are lacking in large areas of the world. HTLV-​1 infection is present throughout the world, but its distri- bution is heterogeneous. The highest HTLV-​1 prevalence (up to 10% of the general population) has been found in southwestern Japan. HTLV-​1-​endemic foci (1–​10%) have also been reported in the Caribbean (Jamaica, Haiti and Martinique), South America (Peru, Colombia, and French Guyana), West and Central Africa (Guinea-​ Bissau, Côte d’Ivoire, Ghana, Nigeria, Gabon, Cameroon, the Central African Republic and the Democratic Republic of the Congo), the Middle East (Iran), and Australo-​Melanesia (Aboriginal popula- tions of Australia, Papua New Guinea, and the Solomon Islands). In Brazil, Mozambique, Iran, Taiwan, and Romania, the prevalence is 0.1 to 1%. HTLV-​1 infection is uncommon in Western Europe and the United States; here, the infection is concentrated in immigrants from endemic regions and people who inject drugs.

942 section 8  Infectious diseases For HTLV-​2, there are two endemic foci:  native Americans (prevalence 1–​58%) and Central African populations (prevalence up to 14%). The virus also occurs among people who inject drugs in all continents (prevalence up to 20%). In endemic populations, the prevalence of HTLV-​1 and HTLV-​2 tends to be higher in elderly than in young people, and is higher in women than in men. Other risk factors include prolonged breast- feeding, unsafe sex practices, blood transfusion, and injection drug use. Pathogenesis The genomes of HTLV-​1 and HTLV-​2 consist of RNA, which, during infection, is transcribed to DNA and inserted as provirus into the DNA of human cells. HTLV-​1 infects mainly CD4 and HTLV-​2 CD8 lymphocytes. HTLV-​1 produces almost no cell-​free virus particles in vivo. Instead, the virus spreads from an infected cell to another cell via a tight and organized cell–​cell contact (virological synapse). In early stages of HTLV-​1 infection, dendritic cells have a central role. During chronic infection, there is little production of mature vir- ions, and the viruses propagate via mitosis of infected lymphocytes (clonal expansion). As a consequence, HTLV-​1 has a remarkable genetic stability, which is unusual for a retrovirus. HTLV-​1 encodes several regulatory gene products, of which Tax and the HTLV-​1 bZIP factor (HBZ) are the most important. Tax and HBZ control proviral transcription, mRNA splicing and transport, and the expression of different host genes. Tax and HBZ gene prod- ucts make the infected CD4 cells proliferate and are involved in the pathogenesis of the malignant as well as the inflammatory complica- tions of HTLV-​1 infection. HTLV-​1 infection is a necessary but insufficient condition for the development of associated diseases. As most of the HTLV-​1-​infected people (about 90%) remain asymptomatic, other viral, host, genetic, or environmental factors must contribute to the risk of disease. The strongest correlate of disease risk is the proviral load. The proviral load (the proportion of peripheral blood mononuclear cells carrying integrated HTLV provirus) remains relatively stable in any given subject, but varies between subjects. A high HTLV-​1 proviral load is related to the risk of HTLV-​associated myelopathy (HAM)/​tropical spastic paraparesis (TSP), and perhaps also of ATL. The main deter- minant of an individual’s proviral load and risk of HAM/​TSP is the cytotoxic T-​lymphocyte response against HTLV-​1. Diagnosis of infection HTLV enzyme-linked immunosorbent assays are available for screening purposes. In samples with a positive result, confirmatory testing with western blot, line immunoassay, immunofluorescence, and/​or polymerase chain reaction is recommended to eliminate false-​ positive reactions and to discriminate between HTLV-​1 and HTLV-​2. Prevention and treatment of infection HTLV-​1 mother-​to-​child transmission can be reduced from 15–​ 25% to less than 5% by avoiding breastfeeding. The incidence of sexual transmission among stable partners is about 1 per 100 person-​years for HTLV-​1 and -​2. Condom use pro- tects against infection. Transfusion of HTLV-​1-​contaminated cellular blood components leads to infection in more than 40% of recipients. In many coun- tries, candidate blood donors are screened for HTLV, as are donors of solid organs. There are no vaccines and no effective antiviral drugs against HTLV-​1 and HTLV-​2. HTLV-​1 disease outcomes The lifetime risk for HTLV-​1 carriers to develop ATL is 1% to 5% (see Table 8.5.25.1). HAM/​TSP occurs in 0.3% to 4%, and for HTLV-​1-​associated diseases in general, the risk is estimated in 10%. Adult T-​cell leukaemia/​lymphoma (ATL) ATL is an aggressive malignancy of HTLV-​1-​infected CD4 lympho­cytes. Clinical features include lymphadenopathy, hepatosplenomegaly, skin lesions, and opportunistic infections. Hypercalcaemia and lytic bone lesions are found in up to 70% of patients. Peripheral blood smears might show lymphoid cells with large, multilobed nuclei (‘flower cells’). HTLV-​1-​induced ATL is classified as acute, lymphoma-​type, chronic, and smouldering, based on total lymphocyte count, pres- ence of abnormal lymphocytes in peripheral blood, calcium and lactate dehydrogenase levels, lymphadenopathy, and solid organ involvement. Despite advanced care, including intensive chemotherapy, the median survival after diagnosis of the acute and lymphoma-​type ATL is only 7–​13 months. Chronic and smouldering forms have a better prognosis, but can evolve to acute ATL. The combination of zidovudine with interferon-​α improves survival in patients with leu- kaemic subtypes of ATL. Allogeneic haematopoietic stem cell trans- plantation can be curative but is not always possible. HTLV-​associated myelopathy/​tropical spastic paraparesis (HAM/​TSP) HAM/​TSP is a chronic, debilitating condition characterized clin- ically by spastic weakness of the legs (Fig. 8.5.25.1), pain, bladder problems, sensory signs and symptoms, constipation, and/​or sexual dysfunction. The main pathological feature is an inflamma- tion of the white and grey matter of the spinal cord. Cerebrospinal fluid examination might show mild lymphocytosis and protein increase. The diagnosis of HAM/​TSP requires demonstration of HTLV-​ 1 and exclusion of other causes of myelopathy, such as spinal cord compression, vitamin B12 and folate deficiency, multiple sclerosis, amyotrophic lateral sclerosis, and lathyrism. Several treatment strategies have been proposed, including cor- ticosteroids, interferon-​α, interferon-​ß-​1a, reverse transcriptase inhibitors (e.g. zidovudine, lamivudine, and tenofovir), histone deacetylase inhibitors (valproate), NF-​κB inhibitors, fucoidan, ciclosporin, and monoclonal antibodies to CD25 or interleukin-​ 15. There are case reports of good response to zidovudine and lamivudine in combination with corticosteroids in patients with rapidly progressive disease. However, only two of these treatment strategies (interferon-​α and zidovudine and lamivudine) have been

8.5.25  HTLV-1, HTLV-2, and associated diseases 943 tested in randomized controlled studies. They showed no satisfac- tory effect on symptoms or proviral load. Infective dermatitis Infective dermatitis is a chronic, relapsing disease that affects mostly children. Clinical characteristics include a papular rash, with exud- ates and crusting, mainly on the scalp, but also on the ears, eyelid margins, paranasal skin, neck, axilla, and groin (Figs. 8.5.25.2, 8.5.25.3). Watery nasal discharge and crusting of the nostrils are fre- quent. Clinical and histopathological images may resemble atopic dermatitis. The response to corticosteroids and antibiotics is generally good, but relapses are frequent after withdrawal of treatment. Case reports suggest that HTLV-​1-​infected children with infective derma- titis have an increased risk to develop HAM/​TSP and ATL. Other diseases An increasing body of evidence shows that symptoms and signs sug- gestive of neurological disease are frequent among HTLV-​1-​infected people, also among those who do not have a formal diagnosis of HAM/​TSP. Arthropathy, uveitis, dry eye syndrome, thyroiditis, polymyositis, and alveolitis are other inflammatory conditions linked to HTLV-​1. Carriers of HTLV-​1 are also at increased risk of infectious complications, notably strongyloidiasis (Chapter 8.9.4), scabies, tuberculosis, and perhaps also leprosy, onychomycosis, paracoccidioidomycosis, periodontal disease, and bronchiectasis. These infectious complications of HTLV-​1 appear to be associated with more morbidity and mortality than was previously appreciated. When HTLV-​1 carriers live in an overcrowded and contaminated environment with inadequate sanitation, they are more likely to suffer severe infectious outcomes. This has been reported among Aboriginal people of central Australia and is likely to occur in other HTLV-​1-​endemic areas as well. HTLV-​2 disease outcomes HTLV-​2 is less pathogenic than HTLV-​1 (see Table 8.5.25.1), but has been linked with HAM/​TSP, arthritis, pneumonia, and bronchitis. A prospective study of HLTV-​2-​infected candidate blood donors in the United States of America found a twofold increase in mortality compared to uninfected control subjects. Likely future developments Developments in the near future will probably include the following: • Treatment trials for HAM/​TSP and ATL • Clearer picture of HTLV-​1, -​2, -​3, and -​4 epidemiology and dis- ease outcomes • Better understanding of pathogenesis of associated diseases (role of viral, genetic, and environmental factors) • Biomarkers to predict disease progression • Better understanding of interaction with HIV and hepatitis B and C • Research into preventive and therapeutic vaccines and antiviral therapy Table 8.5.25.1  HTLV-​1 and HTLV-​2 disease outcomes and main clinical features HTLV-​1 Malignant disease ATL Lymphadenopathy, hepatosplenomegaly, skin lesions, opportunistic infections, hypercalcaemia. Poor prognosis. Affects more men than women, mostly adults. Inflammatory syndromes HAM/​TSP Weakness of the legs with signs of pyramidal tract involvement (hyperreflexia, clonus, spasticity, Babinski’s sign), loss of vibration sense, pain, urinary problems, constipation, and sexual disorders. Progressive disease. Affects more women than men; mostly adults, sometimes children. Occurs mostly sporadically, sometimes in families. Uveitis Blurred vision with floaters, iritis, vitreous opacities, retinal vasculitis, uni-​ or bilateral. Intermediate uveitis in >50% of cases. Sometimes preceded by an episode of thyroiditis. Resolves spontaneously, but more rapidly with corticosteroids. Relapse is frequent. Affects more women than men; mostly adults, sometimes children. Arthritis Resembles rheumatoid arthritis. Infectious complications Strongyloidiasis Disseminated, life-​threatening strongyloidiasis can develop. Relapse after treatment is common. Infective dermatitis Generalized papular rash, with exudates and crusting on scalp, ear, eyelid margins, paranasal skin, neck, axilla, and groin. Watery nasal discharge, lymphadenopathy. Chronic syndrome; good response to antibiotics but frequent relapse. The syndrome has an inflammatory as well as an infectious component. Affects usually young children. Scabies Severe forms can occur, with extensive, crusted lesions, located mainly in pressure areas. Tuberculosis Increased risk of active tuberculosis. Specific clinical features remain to be clarified. Bronchiectasis Reported among indigenous people in Central Australia. High mortality. Linked to recurrent respiratory tract infections. HTLV-​2 HAM/​TSP Similar symptoms as in HTLV-​1, but milder and more slowly progressive disease. Acute bronchitis and pneumonia Specific clinical features remain to be clarified. Arthritis ATL, adult T-​cell leukaemia/​lymphoma; HAM/​TSP, HTLV-​associated myelopathy/​tropical spastic paraparesis.

944 section 8  Infectious diseases FURTHER READING Bangham CRM (2018). Human T-cell leukemia virus type 1: persist- ence and pathogenesis. Annu Rev Immunol, 36, 43–71. Biswas HH, et al.; HTLV Outcomes Study (2010). Increased all-​cause and cancer mortality in HTLV-​II infection. J Acquir Immune Defic Syndr, 54, 290–​6. de Castro-​Costa CM, et  al. (2006). Proposal for diagnostic criteria
of tropical spastic paraparesis/​HTLV-​I-​associated myelopathy (TSP/​HAM). AIDS Res Hum Retroviruses, 22, 931–​5. Gessain A, Cassar O (2012). Epidemiological aspects and world dis- tribution of HTLV-​1 infection. Front Microbiol, 3, 388. Martin F, Taylor GP (2011). Prospects for the management of human T-​cell lymphotropic virus type 1-​associated myelopathy. AIDS Rev, 13, 161–​70. Murphy EL (2016). Infection with human T-lymphotropic virus types-1 and -2 (HTLV-1 and -2): implications for blood transfusion safety. Transfus Clin Biol, 23, 13–9. Roucoux DF, Murphy EL (2004). The epidemiology and disease outcomes of human T-​lymphotropic virus type II. AIDS Rev, 6, 144–​54. Tsukasaki K, et al. (2009). Definition, prognostic factors, treatment, and response criteria of adult T-​cell leukemia-​lymphoma: a pro- posal from an international consensus meeting. J Clin Oncol, 27, 453–​9. Verdonck K, et  al. (2007). Human T-​lymphotropic virus 1:  re- cent knowledge about an ancient infection. Lancet Infect Dis, 7, 266–​81. Willems L, et al. (2017). Reducing the global burden of HTLV-1 in- fection: an agenda for research and action. Antiviral Res, 137, 41–8. Fig. 8.5.25.1  Spastic paraplegia in a South African patient with HTLV-​1 infection. Copyright D. A. Warrell. Fig. 8.5.25.2  Patient with HTLV-​1-​associated infective dermatitis. The child has a papular rash on the forehead, crusting on the scalp, and lesions in the armpits. Courtesy of Dr Francisco Bravo, Institute of Tropical Medicine Alexander von Humboldt, Lima, Peru. Fig. 8.5.25.3  Patient with HTLV-​1-​associated infective dermatitis. This disease can affect adults, although it mostly occurs in children. Typical characteristics are crusting on the scalp and lesions on the eyelid margins, in the neck, and in the armpits. Courtesy of Dr Francisco Bravo, Institute of Tropical Medicine Alexander von Humboldt, Lima, Peru.

8.5.26 Viruses and cancer 945

8.5.26 Viruses and cancer 945

8.5.26  Viruses and cancer 945 8.5.26  Viruses and cancer Robin A. Weiss ESSENTIALS Viruses are important in cancer for three main reasons: (1) As a cause of cancer—​about 15% of the worldwide cancer burden is due to vir- uses. (2) In understanding of the biology of cancer-​through the dis- covery and characterization of oncogenes and tumour suppressor genes. (3) In the treatment of cancer—​some viruses selectively rep- licate in and destroy proliferating cells, viruses as foreign antigens may aid the recognition of cancer cells by the host’s immune system (‘xenogenization’), and viruses can also be used as vectors for im- munization and for gene therapy. Viral cancers are prevented by early screening for tumours, screening for the virus in order to pre- vent transmission, and immunization as in the cases of hepatitis B virus and human papilloma virus. Viruses as aetiological agents of cancer Oncogenic viruses establish persistent infections, which usually occur decades before malignancy. Table 8.5.26.1 lists the viruses implicated in human cancer. In most, but not all cases, the viral genome is present in the malignant cells; the exceptions appear to be those that promote cancer indirectly, such as HIV and hepatitis C virus (HCV). Several of these viruses and the non​malignant dis- eases they cause are discussed in detail in the chapters devoted to individual viruses. Cancer is usually a rare outcome of virus infection, and other cofactors play a part in viral carcinogenesis. For example, Epstein–​ Barr virus (EBV) is a ubiquitous infection, yet childhood Burkitt’s lymphoma occurs only in areas of holoendemic malarial infection, whereas undifferentiated nasopharyngeal carcinoma occurs mainly in southern Chinese populations. Aflatoxin in the diet acts syner- gistically with hepatitis B virus (HBV) to induce liver cancer, and in hereditary epidermodysplasia verruciformis, ultraviolet radiation acts with human papilloma virus strains (usually HPV-​5) to cause skin cancer. The underlying cause of all forms of Kaposi’s sarcoma is Kaposi’s sarcoma herpes virus (KSHV), also known as human herpes virus 8 (HHV-​8), which also causes primary effusion lymphoma and plasmablastic multicentric Castleman’s disease. Kaposi’s sarcoma occurs much more frequently in immunodeficient patients. Its rela- tive risk in recipients of organ transplants is about 400, and in people with AIDS about 20 000. Oncogenic viruses belong to many virus families with different routes of transmission. HBV is frequently acquired perinatally or through subsequent exposure to blood. Human T-​cell lymphotropic virus type 1 (HTLV-​1) is transmitted vertically through infected cells in breast milk. Sexual transmission is common to HIV, HBV, HPV, and HTLV-​1 (with a male to female bias). Oncogenic vir- uses do not appear to be transmitted by the respiratory route or via arthropod vectors, except for some veterinary cases (e.g. bovine leucosis virus). Whereas EBV (transmitted through saliva) occurs throughout the global population, HBV, HTLV-​1, and HHV-​8 have a higher prevalence in those population groups in which the associ- ated cancers occur. Certain common human viruses are highly oncogenic in ex- perimental animals but are not linked epidemiologically to human cancer, namely the polyomaviruses BK and JC, and the adenoviruses types 2 and 12. There are claims that a simian relative of BK virus, simian vacuolating virus 40 (SV40), is linked with mesothelioma, osteosarcoma, and ependymoma in humans, but these findings re- main controversial. However, in 2008, a novel virus, MCPyV, was linked to Merkel skin cell cancer as a genuinely oncogenic human polyoma virus. Mechanisms of viral carcinogenesis Physical and chemical carcinogens are usually mutagens. They cause DNA mutations in specific genes that contribute to the eventual ma- lignant phenotype of the cancer. Oncogenes were first discovered in animal retroviruses, such as the Rous sarcoma virus of chickens, and originate from cellular genes. Most retroviruses do not carry onco- genes, but the DNA provirus integrates into chromosomal DNA and can activate adjacent cellular oncogenes. Oncogene activation by retro- viruses is comparable to activation by chromosomal translocation. The mechanism of cell transformation by HTLV-​1 is different from that of most animal retroviruses. HTLV-​1 encodes viral proteins, Tax, and HBZ, which are essential to promote full viral gene transcription Table 8.5.26.1  Viruses implicated in human cancer Virus Malignancy Human papilloma virus (HPV): types 16, 18 HPV types 5, 8 Cervical cancer c.40% head and neck squamous cancer Skin cancer in epidermodysplasia veruciformis Polyomavirus (MCPyV) Merkel cell skin cancer Hepatitis B virus (HBV) Primary liver cancer Hepatitis C virus (HCV) Primary liver cancer Epstein–​Barr virus (EBV) Burkitt’s lymphoma, immunoblastic lymphoma, c.25% Hodgkin’s disease, undifferentiated nasopharangeal carcinoma, leiomyosarcoma, c.10% breast cancer Kaposi’s sarcoma herpesvirus (KSHV or HHV-​8) Kaposi’s sarcoma, primary effusion, lymphoma, multicentric Castleman’s disease Human T-​lymphotropic virus type 1 (HTLV-​1) Adult T-​cell leukaemia

946 section 8  Infectious diseases and cell transformation. Tax acts as a transcriptional activator, by as- sociating with host nuclear proteins which activate expression of the viral genome. However, Tax also up-​regulates certain cellular genes, such as the interleukin-​2 receptor. HTLV-​1 ‘immortalizes’ CD4+ T lymphocytes in culture, rather as EBV ‘immortalizes’ B lymphocytes, but this is only one step in the pathway to malignancy. Cell transformation by DNA viruses is best understood for polyomaviruses and adenoviruses. The transforming genes of these viruses are expressed early in the infection cycle and prevent tumour suppressor protein function. Adenovirus proteins E1A and E1B and polyomavirus large T antigen bind to p53 and retinoblastoma (Rb) proteins and block their normal interaction in the cell cycle. Thus, instead of mutating these cellular tumour suppressor genes, DNA tumour viruses block the normal function of their proteins, which similarly results in unregulated cell proliferation. The Kaposi’s sar- coma herpes virus genome carries several oncogenes, including a homologue of cyclin D2 (CCND2), which inactivates Rb by a dif- ferent mechanism, phosphorylation. Most oncogenic viruses persist in the tumour cells, often by integrating into chromosomal DNA. Oncogenic herpesviruses do not integrate but are maintained episomally. Epstein–​Barr virus-​ associated nuclear antigen 1 (EBNA-​1) is required for episomal replication of EBV, and latency-​associated nuclear antigen for main- tenance of Kaposi’s sarcoma herpes virus, while other nuclear and latent membrane proteins are responsible for the transformed cell phenotype. With HBV, integrated copies are found in many liver carcinoma lines, but a requirement for integration has not been un- equivocally shown. HBV expresses transactivating functions from the X gene, so its transformation may resemble that of HTLV-​1. Indirect carcinogenic effects are those in which damage to tis- sues by viruses may allow clones of premalignant cells to prolif- erate that would not otherwise do so. HCV and possibly HBV do this by destroying normal liver cells, resulting in a much greater rate of liver cell regeneration. HIV promotes tumour development by destroying helper T-​cell immunity to other oncogenic viruses. The cancers which occur more frequently in AIDS are also seen in im- munosuppressed transplant patients (e.g. non-​Hodgkin’s lymphoma and Kaposi’s sarcoma), and usually have a viral aetiology. Treatment and prevention Oncogenesis is multifactorial, requiring several sequential events before a patient presents with a fully malignant tumour. Yet, if a virus plays a crucial role in oncogenesis, its elimination should prevent that type of cancer. Currently, there is no special approach to the treatment of cancers that have a viral aetiology. Among the lymphoid malignancies, some respond well to radiotherapy or chemotherapy, such as Hodgkin’s disease, whereas others seldom show remission, such as adult T-​cell leukaemia. Cancers that express viral antigens should be responsive to immunotherapy. For tumours in which viral proteins are required for the maintenance of the malignant state, those proteins are potential molecular targets, as drugs that block them might spare normal cellular functions. Prevention offers the greatest promise of reducing cancer mor- tality due to viruses. Prevention can be accomplished by three strat- egies: (1) early screening for tumours, (2) screening for the virus with prevention of transmission, and (3) immunization. Early screening is exemplified by cervical smears. Screening to prevent iatrogenic transmission via blood and blood products is routinely employed for potentially oncogenic viruses such as HBV, HCV, HIV, and HTLV-​1. In Kyushu, Japan, where infection was endemic, HTLV-​1 is being steadily eradicated through a policy of antenatal screening to pre- vent transmission via milk. Prevention of cancer by immunization against infection by onco- genic viruses is likely to have a major impact on world cancer mor- tality. The HBV vaccine is based on surface antigen and two HPV vaccines protective against cervical cancer were licensed in 2006. Intensive research is also being undertaken on vaccines for HIV and HCV, but there are immense obstacles to successful immunization against HIV as the virus is extraordinarily variable. Nevertheless, immunization against oncogenic viruses is becoming a most ef- fective cancer prevention strategy. Viruses as therapeutic agents Viruses may be put to use in the fight against cancer. First, some cytopathic viruses preferentially replicate in proliferating cells and destroy them, such as parvoviruses and mutant adenoviruses. Second, viruses as foreign antigens may aid the recognition of cancer cells by the host’s immune system. Although the mechanism is ill understood, ‘xenogenization’ of tumour cells by virus infection can, in some cases, enhance immune attack against non​infected cells in the same tumour. Third, viruses are used as vectors for immuniza- tion and for gene therapy, by restoring tumour suppressor functions, by enhancing immune responses through the expression of antigens or cytokines, and by locally delivering genes for enzymes that con- vert inert prodrugs into active, chemotherapeutic agents. FURTHER READING Ajila V, et al. (2015). Human papilloma virus associated squamous cell carcinoma of the head and neck. J Sex Transm Dis, 2015, 791024. Astbury K, Turner MJ (2009). Human papillomavirus vaccination in the prevention of cervical neoplasia. Int J Gynecol Cancer, 19, 1610–​13. Chang Y, Moore PS, Weiss RA (2017). Human oncogenic viruses: na- ture and discovery. Proc Trans R Soc London B, 372, 20160264. Desjardins A, et al. (2018). Recurrent glioblastoma treated with re- combinant poliovirus. N Engl J Med, 379, 150–61. De Martel C, et al. (2012). Global burden of cancers attributable to in- fections in 2008: a review and synthetic analysis. Lancet Oncol, 13, 607–​15. Goossens N, Hoshida Y (2015). Hepatitis C virus-​induced hepato­ cellular carcinoma. Clin Mol Hepatol, 21, 105–​14. Gutierrez-​Xicotencatl L, et  al. (2016). Humoral immune response against human papillomavirus as source of biomarkers for the pre- diction and detection of cervical cancer. Viral Immunol, 29, 1–​12. Matsuoka M, Jeang KT (2011). Human T-​cell leukemia virus type 1 (HTLV-​1) and leukemic transformation: viral infectivity, Tax, HBZ and therapy. Oncogene, 30, 1379–​89. Morrison BJ, et  al. (2015). Serodiagnosis for tumor viruses. Semin Oncol, 42, 191–​206. Pierangeli A, Antonelli G, Gentile G (2015). Immune deficiency asso- ciated viral oncogenesis. Clin Microbiol Infect, 21, 975–​83.

8.5.27 Orf and Milker’s nodule 947

8.5.27 Orf and Milker’s nodule 947

8.5.27  Orf and Milker’s nodule 947 Plymoth A, Viviani S, Hainaut P (2009). Control of hepatocellular carcinoma through hepatitis B vaccination in areas of high endem- icity: perspectives for global liver cancer prevention. Cancer Letters, 286, 15–​21. Schinzari V, Barnaba V Piconese S (2015). Chronic hepatitis B virus and hepatitis C virus infections and cancer synergy between viral and host factors. Clin Microbiol Infect, 21, 969–​74. Wendzicki JA, Moore PS, Chang Y (2015). Large T and small T antigens of Merkel cell polyomavirus. Curr Opin Virol, 11, 38–​43. Zur Hausen H (2009). Papillomaviruses in the causation of human cancers. Virology, 384, 260–​5. 8.5.27  Orf and Milker’s nodule Emma Aarons and David A. Warrell ESSENTIALS Orf and milker’s nodule are characteristic nodular skin lesions caused by parapox viruses of sheep and goats, or cattle, respectively. These viruses are epitheliotropic and able to suppress the host’s immune re- sponse. The infections are occupational zoonoses of people working with ruminants. A single or small number of somewhat painful pus- tules develops, usually on the hand, at the site of contact with an animal’s lesions. Fever is unusual, but local lymphadenopathy, ery- thema multiforme, or secondary infection may occur. Spontaneous resolution within 6 weeks is usual. Multiple, giant lesions may develop in the immunosuppressed. Topical cidofovir is effective in severe cases. Aetiology Orf virus, the prototype species of the Parapoxvirus genus of the Chordopoxvirinae subfamily, causes ‘scabby mouth’ (sore mouth, ecthyma contagiosum, contagious pustular dermatitis), a debilitating disease of sheep and goats. Other parapoxviruses-pseudocowpox virus and, less commonly, bovine papular stomatitis virus-cause similar dis- eases in cattle, predominantly dairy herds. Parapoxviruses have also been reported to infect various deer species, camels, and seals. The vir- ions are ovoid (approximately 220–​300 × 140–​170 nm), with a charac- teristic ball-​of-​wool appearance on transmission electron microscopy. Full genome sequences of orf virus, pseudocowpox virus, and bovine papular stomatitis virus have been published. Each of these double-​ stranded DNA viruses of 134 to 145 kbp is predicted to encode several putative virulence factors that contribute to the dermal lesions, char- acterized by capillary proliferation and dilatation. Epidemiology Orf has been recognized for more than 200 years as a disease of mainly young lambs and kids, which contract the infection from one another, or possibly from persistence of the virus in the pas- tures where the virus can remain viable for long periods in dried scabs from lesions. Painful muzzle lesions prevent lambs or kids from feeding, resulting in failure to grow properly, so the disease can have important economic impact. Pseudocowpox and, less commonly, bovine popular stomatitis cause cutaneous teat and oral infections in cattle, predominantly in dairy herds. However, in most countries bovine parapoxvirus infections are of limited economic significance. Parapoxvirus transmission to humans is by direct contact of broken skin with an animal’s lesions and possibly with fomites, hence human disease is an occupational zoonosis. The lesions on the human hand caused by different parapoxviruses are clinically indistinguishable. Therefore, their recognition as ‘orf’ or ‘milker’s nodule’ will depend only on which animal is thought to have been the source. Since human orf, in particular, is familiar to veterinarians, shepherds, farmers, abat- toir workers, and butchers and is generally self-​limiting, it often goes unreported. In the United Kingdom it is known to be preva- lent in sheep farming communities in Wales. Outbreaks of orf are associated with the end of the lambing season and with Islamic religious festivals associated with animal sacrifice. Human to human spread of parapoxvirus has not been recorded. Infection confers only partial immunity, so that repeated milder attacks are possible. Immunopathology Parapoxviruses infect skin keratinocytes and excite a brisk im- mune response locally and in lymphoid tissue, involving CD4+ and CD8+ cells, interferon, and antibody. However, the parapoxvirus genome encodes a variety of virulence and immunomodulatory factors that subvert or suppress the host’s immune response, al- lowing viral replication. These include viral IL-​10 and viral vas- cular endothelial growth factor (both believed to have been acquired from the mammalian host genome during viral evolu- tion), interferon resistance protein, chemokine-​binding protein, a granulocyte-​macrophage colony-​stimulating factor (GM-​CSF)/​ interleukin-​2 (IL-​2) inhibitory factor, and an inhibitor of apop- tosis. In vaccinology, recombinant orf virus is a promising viral vector for delivering pathogen antigens to the immune system, and inactivated virus is immunoenhancing. Clinical features In orf virus-​infected lambs and kids, papules and vesicles appear on the muzzle or nostrils (Fig. 8.5.27.1). These become dry and scabby, and then gradually heal without scarring over 4–​8 weeks, although more persistent infections may occur. Parapoxvirus infec- tion of calves may present very similarly, or as less severe inflamma- tory ring lesions. Infected cows and ewes develop lesions on their teats. In humans, after an incubation period of 2–​6 days, a somewhat painful, small, red, firm papule enlarges to form a flat-​topped haem- orrhagic pustule or bulla with prominent margin and an eroded, crusted centre, sometimes surrounded by pustular satellite lesions (Fig. 8.5.27.2). The lesion is typically 1–​3 cm in diameter, but may be as large as 5 cm. They are usually solitary or few in number and

948 section 8  Infectious diseases commonly occur on the extensor surface of a finger or hand, but also on the palm, forearm, and occasionally the face or scalp. The surrounding skin might be reddened, sometimes diffusely, and erysipelas-​like lesions have been described. Lymphangitis or re- gional lymphadenopathy may occur. Giant, multiple, fungating granulomatous or tumour-​like lesions have been reported, usu- ally in immunocompromised patients with haematological ma- lignancies. Patients with atopic eczema may develop widespread eruptions. Slight fever and malaise can occur. Complications in- clude secondary infection, and generalized papulovesicular rashes, usually classified as erythema multiforme, in as many as one-​fifth of cases (Fig. 8.5.27.3), typically developing 10–​14 days after the ini- tial lesion. Bullous pemphigoid-​like eruptions have been reported. Spontaneous recovery without residual scarring is usually complete within 6 weeks. Diagnosis The characteristic lesion in someone exposed to ruminant animals, especially to lambs or kids, allows a clinical diagnosis. The skin le- sions caused by the different parapoxviruses are indistinguishable. The diagnosis can be confirmed in the laboratory by polymerase chain reaction, by electron microscopy of a biopsy of the lesion, and fluorescent antibody staining. Skin biopsy specimens show distinctive histopathological changes. There is hyperkeratosis with cellular swelling, balloon degeneration and vacuolation in the upper epidermis, and the presence of eosino- philic B type intracytoplasmic inclusion bodies. Differential diagnosis In those at occupational risk of parapoxvirus infection, the differen- tial diagnosis includes cowpox (Chapter 8.5.4), an orthopoxvirus. Whitlows (felons), including herpetic whitlow (Fig. 8.5.27.4), Fig. 8.5.27.1  Contagious pustular dermatitis (‘orf’) in a lamb. (a) (b) Fig. 8.5.27.2  Typical parapoxvirus lesions of ‘milker’s nodule’ on a dairy farmer’s arm. (a) (b) Fig. 8.5.27.3  Generalized vesicular eruption ‘erythema multiforme’ complicating orf of the left middle finger in a veterinary student:
(a) arms, (b) mouth. Copyright Prof D. A. Warrell

8.5.28 Molluscum contagiosum 949

8.5.28 Molluscum contagiosum 949

8.5.28  Molluscum contagiosum 949 Fig. 8.5.27.4  Herpetic whitlows on adjacent fingers. Courtesy of the late Dr B. E. Juel-​Jensen. impetigo, pyogenic granuloma, cutaneous anthrax, fungal or atypical mycobacterial infection, and tumours might also cause confusion. Treatment Lesions are usually self-​limiting. Secondary infection should be treated if it occurs. Large lesions can be removed surgically, but recurrence can occur in the immunocompromised. Cidofovir (topically or intravenously) and imiquimod (topically) have been used successfully to treat giant or persistent lesions, especially in immunosuppressed patients. Prevention Live attenuated virus vaccines have been used to protect sheep and goats from orf. Farmers should avoid handling animals with obvious lesions. FURTHER READING Chakhunashvili G, et al. (2018). Parapoxvirus infections in the country of Georgia: a case series. Am J Trop Med Hyg, 98, 1870–75. Diven DG (2001). An overview of poxviruses. J Am Acad Dermatol, 44, 1–​14. Groves RW, Wilson-​Jones E, MacDonald DM (1991). Human orf and milkers’ nodule: a clinicopathologic study. J Am Acad Dermatol, 25, 706–​11. Haig DM (2006). Orf virus infection and host immunity. Curr Opin Infect Dis, 19, 127–​31. Hautaniemi M, et  al. (2010). The genome of pseudocowpoxvirus:
comparison of a reindeer isolate and a reference strain. J Gen Virol, 91, 1560–​76. Joseph RH, et al. (2015). Erythema multiforme after orf virus infec- tion: a report of two cases and literature review. Epidemiol Infect, 143, 385–​90. Torfason EG, Gunadóttir S (2002). Polymerase chain reaction for laboratory diagnosis of orf virus infections. J Clin Virol, 24, 79–​84. 8.5.28  Molluscum contagiosum David A. Warrell and Christopher P. Conlon ESSENTIALS Molluscum contagiosum is caused by a Molluscipox DNA virus which infects keratinocytes of the epidermal stratum spinosum, producing distinctive small umbilicated papules on the skin. Its genome encodes a variety of proteins that suppress the host’s immune response. In chil- dren it is spread by skin contact, producing few or many lesions, while in sexually active adults it causes anogenital lesions. Molluscum is self-​ limiting within a few years in the immunocompetent, but those with preexisting atopic eczema and immunosuppression, notably AIDS, commonly develop persistent diffuse eruptions with larger papules. Lesions can be removed mechanically or chemically. More severe in- fections can be treated with imiquimod or cidofovir. Aetiology Molluscum contagiosum, first described clinically in the early 19th century, is caused by a virus of the genus Molluscipox. This large (200–​300 nm long), brick-​shaped, double-​stranded DNA virus, is a member of the Chordopoxvirinae subfamily of the Poxviridae. It multiplies in the cytoplasm of keratinocytes of the deep epidermal stratum spinosum. Molluscum contagiosum shares unique gen- omic features with parapoxviruses such as orf (Chapter  8.5.27), including a high guanine-​cytosine content, three orthologous genes, and a paucity of nucleotide metabolism genes. Restriction endonuclease analysis of the genome has identified four types (I, II, III, and IV). Types I and IV are the most common types clin- ically but type II is more common in the setting of HIV infection. Like orf virus, Molluscum contagiosum encodes several proteins that suppress host immunity. MC054L is a human (IL-​18) binding protein homologue. MC148 antagonizes CC chemokine receptor 8. MC013L promotes viral replication by inhibiting the differen- tiation of infected keratinocytes. MC159L causes abnormal pro- liferation of epithelium by inhibiting tumour necrosis factor and apoptosis-​inducing factors. MC080R, an MHC class I homologue, might interfere with natural killer cell activity. Molluscum contagiosum has not been transmitted to laboratory animals and, at present there is no cell culture system in which to propagate the virus. It has been grown in human foreskin grafted to athymic mice. Epidemiology Molluscum contagiosum has a worldwide incidence of 2–​8%. It also affects animals:  chimpanzees, kangaroos, dogs, horses, and birds. In tropical climates, it is more common in younger children (1–​4 years), and in temperate climates, in older school-​age children (10–​12 years). The prevalence in American children is less than 5%. It is highly contagious by skin-​to-​skin contact, especially in humid and unhygienic conditions. Fomites such as shared towels, the use

950 section 8  Infectious diseases of communal bathtubs and swimming pools, contact sports such as wrestling, and tattooing, all promote infection. Lesions are spread over the body by autoinoculation. Vertical transmission has also been reported. Sexual transmission accounts for a second peak of incidence in young adults. The risk and extent of infection is in- creased in those with generalized skin diseases such as atopic ec- zema and in those with congenital or acquired immunodeficiency, caused by HIV, lymphomas, sarcoidosis, organ transplantation, and immunosuppressive therapy. In HIV seropositive people, the preva- lence of molluscum contagiosum is 5–​20%, but in those with CD4 cell counts below 100/​ml it increases to 30%. Clinical features The incubation period varies from 7 days (in newborns) to 50 days or even up to 6 months. The classic lesion is a painless, discrete, shiny, pearly, hemispherical, firm papule with a central umbilication (depression). In immunocompetent children, lesions can occur singly but are commonly multiple, fewer than 30 to several hundred (Figs. 8.5.28.1, 8.5.28.2). They grow gradually to a diameter of 3–​5 mm over 6–​12 weeks. A grey-​white creamy material might be extruded if a lesion is squeezed. Occasionally, a single lesion may grow to 1.5 cm in diameter, or a plaque of very small lesions develops (agminate form). The average duration of single lesion is about 2 months, but lesions may continue to appear for 6–​8 months. Spontaneous clear- ance is complete without scarring within 2–​4 years. In about 10% of cases, especially where there is a history of atopy, a patchy erythema or dermatitis develops around the lesions, causing itching which encourages scratching and autoinoculation. Lesions are most com- monly seen on the axilla and other flexures, trunk, neck, or face, but any part of the skin can be affected. Conjunctival inoculation may result in unilateral conjunctivitis or corneal or conjunctival nodules. Lesions are rare on the palms, soles, and buccal mucous membrane. In immunocompetent sexually active teenagers and adults, infections usually result in anogenital lesions. In patients with HIV and other types of immunosuppres- sion, molluscum can be widespread and lesions numerous, but particularly involves the face (eyelids), neck, trunk, and around and inside the mouth in men who have sex with men. Lesions often lack the classic umbilication and may become so large, atypical, and even necrotic that they are mistaken for basal cell carcinomas or other skin tumours. The disease persists and spreads, especially when HIV is advanced. The use of topical steroids and calcineurin inhibi- tors for atopic eczema can lead to a high number of lesions in the abnormal skin. Diagnosis The diagnosis is usually clinical and the lesions might best be examined with a dermascope, but histological and electron microscopic examination of a curetted papule establishes the diagnosis. The demarcated lesion shows lobules of epidermis, de- pleted of Langerhans cells, penetrating down to the dermis with a central crater opening onto the surface through a narrow pore (Fig. 8.5.28.3). It contains keratinocyte debris with numerous Fig. 8.5.28.1  Molluscum contagiosum: cluster of lesions in an immunocompetent child. Courtesy of Dr Susan M. Burge. Fig. 8.5.28.2  Molluscum contagiosum: characteristic papules with central punctum. Courtesy of Dr Susan M. Burge. Fig. 8.5.28.3  Molluscum contagiosum showing the characteristic demarcated lesion. Courtesy of K. Hollowood.

8.5.29 Newly discovered viruses 951

8.5.29 Newly discovered viruses 951

8.5.29  Newly discovered viruses 951 Henderson–​Paterson molluscum bodies. These are 35 µm in diam- eter, ovoid, eosinophilic, intracytoplasmic inclusion bodies within keratinocytes (Fig. 8.5.28.4). They stain purple with Tzanck re- agent in scrapings from the lesions. In HIV patients, histological appearances can differ. Differential diagnosis The differential diagnosis includes lepromatous leprosy, Darier’s disease (keratosis follicularis), epithelial naevi, and skin tumours such as basal cell epithelioma or trichoepithelioma. Giant lesions might be confused with keratoacanthoma, common warts, or warty dyskeratoma. In the genital area, genital warts (condylomata acuminata) may look similar. In immunosuppressed people, umbilicated cutaneous lesions of disseminated Talaromyces marnef- fei infection, histoplasmosis, paracoccidioidomycosis, or crypto- coccosis can appear identical to molluscum. Treatment (See also Section 23.) Treatment might not be necessary, depending on the site and number of lesions and the age of the patient. An enormous number of local treatments are claimed to be effective, but evidence is lacking. Mechanical methods include picking out lesions on the tip of a needle or with adhesive tape, curettage, cryotherapy with liquid nitrogen, and diathermy. Topical chemicals include tre- tinoin, podofilox, cantharidin, acetic acid, phenol, salicylic acid, silver nitrate, trichloroacetic acid, lactic acid, and benzoin. Agents can be delivered to the inside of the lesion using the sharpened end of a wooden applicator stick. In children, local anaesthetic cream should be applied beforehand. Curettage might be the most effective therapeutic approach but requires skilled staff, so in many cases it might be more appropriate to wait for spontaneous resolution. In patients with HIV, molluscum usually responds dramatically to highly active antiretroviral therapy (HAART). In severe cases, 5% imiquimod cream or cidofovir (intravenously or topically) have proved effective but in immunocompetent children, 5% imiquimod cream is not recommended. Prevention In schoolchildren, spread can be prevented by avoiding swimming pools, contact sports, and shared towels, until the lesions have resolved. FURTHER READING Brown J, et  al. (2006). Childhood molluscum contagiosum. Int J Dermatol, 45, 93–​9. De Clercq E (2003). Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clin Microbiol Rev, 16, 569–​96. Haral A, et  al. (2016). To treat molluscum contagiosum or not—​ curettage: an effective, well-​accepted treatment modality. Paediatr Dermatol, 33, 640–​5. Lee R, Schwartz RA (2010). Pediatric molluscum contagiosum: reflec- tions on the last challenging poxvirus infection, part 1. Cutis, 86, 230–​6, 287–​92. Schwartz JJ, Myskowski PL (1992). Molluscum contagiosum in pa- tients with human immunodeficiency virus infection: a review of twenty-​seven patients. J Am Acad Dermatol, 27, 583–​8. Shisler JL. (2015). Immune evasion strategies of molluscum contagiosum virus. Adv Virus Res, 92, 201–​52. Smith KJ, Skelton H (2002). Molluscum contagiosum: recent advances in pathogenic mechanisms, and new therapies. Am J Clin Dermatol, 3, 535–​45. Smith KJ, Yeager J, Skelton H (1999). Molluscum contagiosum:  its clinical, histopathologic, and immunohistochemical spectrum. Int J Dermatol, 38, 664–​72. van der Wouden JC, et al. (2006). Interventions for cutaneous mol- luscum contagiosum. Cochrane Database Syst Rev, 2, CD004767. 8.5.29  Newly discovered viruses Susannah J.A. Froude and Harriet C. Hughes ESSENTIALS Although humans are affected by an enormous range of microorgan- isms, almost all newly discovered emerging pathogens are viruses that are often zoonotic or vector-​borne. These emerging viruses often have high baseline mutation rates, allowing them to adapt relatively easily to new hosts and enabling them to take advantage of new epi- demiological opportunities provided by the changing environment. A range of apparently new human viral pathogens has been reported Fig. 8.5.28.4  Molluscum contagiosum showing keratinocyte debris with Henderson–​Paterson molluscum bodies. Courtesy of K. Hollowood.

952 section 8  Infectious diseases increasingly in international outbreak information over the last few years. How they will influence global public health remains to be seen. Emerging viruses that might be of particular global public health importance include, respiratory coronaviruses, Zika virus, and se- vere fever and thrombocytopenia syndrome virus. Other emerging viruses of importance include bocavirus, Bufavirus, PARV4, human parechovirus, Itaya, Heartland, and Bourbon virus. The human pathogenicity of other emerging viruses is less certain. Coronaviruses Severe acute respiratory syndrome (SARS) Coronaviruses (CoV) are single-​stranded RNA viruses commonly associated with respiratory illness and less often with gastrointes- tinal and neurological disease in a wide variety of mammals and birds. They have high rates of mutation and recombination and a propensity to cross host species. The severe acute respiratory syn- drome (SARS) outbreak of a new human coronavirus, SARS-​ associated coronavirus (SARS-​CoV), between November 2002 and July 2003 spread across five continents and caused over 700 human deaths. This pandemic triggered renewed interest in this area, leading to increased understanding of the origin of SARS-​CoV. In the early phase of the outbreak, the infecting SARS viruses showed closer similarities to animal viruses than later on in the pandemic. Virological studies suggest that the animal viruses crossed over to humans on more than one separate occasion, so repeated similar events should be expected in the future. Bats are increasingly rec- ognized as reservoirs of emerging viruses. The discovery of species-​ specific, SARS-​like coronaviruses in horseshoe bats with the same genome organization as human SARS coronaviruses indicates that a human SARS virus originated in one or more bat species. It is likely that an intermediate animal host is also required to allow modifica- tion of the mutating progenitor virus before transmission to humans is possible. Understanding this reservoir might help to prevent fu- ture human outbreaks of SARS-​CoV. MERS-​CoV Middle Eastern respiratory syndrome (MERS) has recently emerged as a novel pathogen that remains an endemic low level public health threat. First isolated from a patient who died in September 2012 in Saudi Arabia, it was similar to the SARS-​CoV strain involved in the 2002–​2003 epidemic and was termed the Middle East Respiratory Syndrome coronavirus (MERS-​CoV). From the initial case to the end of September 2018, there have been 2260 laboratory confirmed cases in 27 countries with a mortality rate of 36%. Death is more likely in patients with increasing age and with other comorbidities. The viral origin of MERS-​CoV is still unclear but, like SARS, it is thought to have originally infected bats. Unlike SARS, however, at some point in the distant past MERS-​CoV spread to dromedy camels that are currently thought to be the main reservoir. Camels have been found with virus identical to human strains across the Arabian Peninsula. New human coronaviruses Two new human coronaviruses have been discovered since the SARS epidemic.:  HCoV-​NL63 and HCoV-​HK. HCoV-​NL63 was first identified in a child with bronchiolitis in the Netherlands. Studies published in 2004 and 2005 found 8 to 9% of children aged under 5 years with known respiratory illness were positive for HCoV-​NL63 by polymerase chain reaction (PCR), while tests for common re- spiratory viruses were negative. Longitudinal studies showed that seroconversion usually occurred by the age of 3.5 years. Significant sequence heterogeneity exists and it is likely, therefore, that there are two closely related genotypic subgroups. In 2005, another human coronavirus was discovered, HCoV-​ HKU1. It was first described in Hong Kong in a 71-​year-​old man with pneumonia who had recently returned from China. It has since been reported in patients in Australia and the United States of America. Common clinical findings in young children included rhinorrhoea, cough, fever, and abnormal breath sounds on auscul- tation. The possibility of central nervous system infection and hepa- titis (in a liver transplant recipient) was suggested in two separate patients in one study. Genomic and phylogenetic analysis suggests that this virus is most closely related to the mouse hepatitis virus, a coronavirus studied since the 1930s. Both HCoV-​HKU1 and HCoV-​NL63 have a worldwide distribu- tion and are associated with a low mortality which suggests they are not viruses that have only recently infected humans rather they have only recently been identified. They join the two previously identified coronoviruses HCoVOC43 and HCoV229E in causing respiratory tract infection. New human polyomaviruses: KI, WU, and
Merkel cell polyomavirus The double-​stranded DNA human polyomaviruses, JC virus and BK virus, are ubiquitous worldwide and are pathogenic in immuno- compromised hosts. In 2007, two new human polyomaviruses were described, KI virus and WU virus. They share a phylogenetic rela- tionship and together may form a new subclass. They have been iso- lated primarily from respiratory secretions. KI was discovered after molecular screening of respiratory samples. WU was first detected by high-​throughput sequencing of respiratory secretions from a patient with an acute respiratory disease of unknown aetiology. Analysis of two more cohorts in different continents revealed that most patients positive for WU were aged under 3 years, and that all infected adults were immunocompromised. The clinical spectrum of the disease included upper and lower respiratory tract infection, bronchiolitis, croup, and, rarely, gastroenteritis. However, the role of these viruses as respiratory pathogens has since been questioned after further studies detected them both in asymptomatic children and those concurrently infected with other respiratory viruses. Studies to establish the role of WU and KI in immunocomprom- ised adults have been inconclusive. The relatively high seropreva- lence of these viruses in healthy blood donors suggest that a benign primary infection with these viruses occurs in childhood, followed by a period of latency and subsequent reactivation in the context of immunosuppression. In 2008, another novel polyomavirus termed ‘Merkel cell polyomavirus’ was found to be integrated within the cellular genome of cells of the rare skin cancer Merkel cell carcinoma which primarily occurs in elderly and immunosuppressed people. This is consistent with the oncogenic potential of other polyomaviruses. Merkel cell

8.5.29  Newly discovered viruses 953 polyomavirus has also been isolated in respiratory samples from symptomatic adult and paediatric patients though its precise role as a pathogen in this context is still yet to be confirmed. New human Parvoviruses: Bocavirus, Bufavirus, Human parvovirus 4 (PARV4) Parvoviruses are a family of small non​enveloped, linear single-​ stranded DNA viruses. They are divided into two subfamilies depending on their ability to cause infection in vertebrates. Identified in 1975, parvovirus B19 was previously thought to be the only parvovirus known to be pathogenic in humans as the cause of fifth disease in children (Chapter 8.5.20). However, since 2005 fur- ther genera and species of Parvovirus have been identified and asso- ciated with disease in humans. Human bocavirus Human bocavirus (HBoV) was first described in September 2005 following isolation by random PCR in pooled respiratory samples from hospitalized children in Sweden. HBoV is closely related to ca- nine minute virus and bovine parvovirus. Although Koch’s postulates have not yet been fulfilled, supportive molecular evidence demonstrated this virus in respiratory sam- ples from children with lower respiratory tract disease who tested negative for common respiratory viruses. It has been found most commonly in children aged under 3 years, particularly in preterm infants with mild to severe respiratory symptoms. Although a study conducted in the Netherlands showed no difference between the detection of HBoV in children with or without lower respiratory tract infection in paediatric intensive care, higher levels of HBoV were seen in the symptomatic patients compared to asymptomatic controls. This may reflect differences in viral load of acute infection versus asymptomatic shedding. A more recent study of patients hos- pitalized with acute lower respiratory tract infection in Argentina in 2010 found a bimodal age distribution of HBoV (<1 year and

30 years) with a significantly higher rate of coinfection (predomin- antly with respiratory syncytial virus) found in children compared to adults. Severe cases are rare although life threatening infections with HBoV1 have been reported. Related viruses HBoV2, HBoV3, and HBoV4 have more recently been identified in faecal samples of children in several countries including the United Kingdom, Pakistan, and Thailand. An asso- ciation with acute gastroenteritis has been described: in one study, HBoV2 was the third most prevalent virus seen in children with acute gastroenteritis after rotavirus and astrovirus. Of these newer viruses HBoV2 is the most commonly isolated species. Further studies are needed to determine the site of replication and potential association with clinical disease. The conditions that have been associated with bocavirus infection are gastroenteritis and flaccid paralysis. Bufavirus Metagenomic analysis of faecal samples from children with acute diarrhoea in Burkina Faso identified a highly divergent parvovirus that was named Bufavirus. Analysis of samples revealed it was pre- sent in 4% of samples. Sequencing of the coding region demon- strated less than 31% similarity with known parvoviruses: it was therefore declared a new genus with at least two species. Similar studies in Tunisia have demonstrated the presence of at least one of the genotypes in 1.6% of samples. Analysis of stool from patients of all ages with gastroenteritis in Finland revealed 1.1% had PCR de- tectable Bufavirus. Unlike the initial studies all the positive samples in the Finish study were from adults. This virus has been shown to be circulating in northern Europe as well as Africa and its causative role in gastroenteritis remains unclear. Human Parvovirus 4 (PARV4) In 2005 a novel DNA virus was identified in a high risk patient with symptoms in keeping with an HIV seroconversion illness. The virus was identified via a sequence independant PCR amplification method. This virus was termed Human Parvovirus 4 (PARV4) and subsequent studies have demonstrated its presence in a large number of intravenous drug users that are also HIV positive. Investigation of patients with HIV acquired by other routes does not demonstrate PARV4 infection. Studies on blood donors and pooled blood prod- ucts has demonstrated the presence of PARV4 which is biologically plausible as parvoviruses are resistant to viral inactivation methods used on plasma derived products. Unlike other parvoviruses, PARV4 has been shown to be a blood borne virus; studies in haemophiliacs who received blood products in the 1970s and 1980s have a high rate of PARV4 compared to their sibling controls that did not have haemophilia, or receive blood products, all of whom were seronega- tive. Autopsy samples on HIV positive patients from the Congo and Nigeria have a genetically distinct PARV4 from that which is seen in European patients, which suggests different transmission networks. Vesivirus Single-​stranded RNA vesiviruses of the Calciviridae family are common marine microorganisms, but are also known to infect land mammals. They cause a broad spectrum of disease in animals including vesicular rash, encephalitis, haemorrhagic disease, spon- taneous abortion, and hepatitis. Their effect on humans is not well established, but a recent seroprevalence study has shown that 12% of successful blood donors tested had evidence of past exposure to vesivirus. This was significantly higher (29%) in patients with hepa- titis of unknown, but suspected, infectious cause, and even higher (47%) in patients with hepatitis of unknown cause associated with blood transfusion or dialysis. Vesivirus viraemia was also shown to be present in some of those tested. Picornaviridae New parechoviruses: Human parechovirus and Ljungan virus Human parechovirus and Ljungan virus are the two species of the genus parechovirus of the family Picornaviridae. Human parechoviruses are single-​stranded RNA viruses which differ from other family members in having only three, rather than four, capsid proteins, and in exerting atypical cytopathic effects. HPeV-​ 1 and HPeV-​2 were previously designated enterovirus 22 and 23, but were reclassified in 1999. By 2015 16 human parechovirus genotypes had been described.

954 section 8  Infectious diseases HPeV infections are common, with at least 95% of the adult popu- lation positive for HPeV-​specific antibodies. Most infections are thought to predominantly affect neonates and young children and, although the clinical spectrum of disease differs between the viruses, it has been compared to infection with enterovirus. Earlier studies of HPeV-​1 suggested infection resulted in more gastrointestinal and re- spiratory illness which was often severe, and was occasionally found as a copathogen with other respiratory viruses such as respiratory syncytial virus. The role of HPeV-​1 as a respiratory pathogen has since been challenged and often infections are asymptomatic. HPeV-​ 3 has been shown to present as a sepsis-​like syndrome predominantly affecting neonates. Cerebrospinal fluid (CSF) from central nervous system infection with HPeV-​3 has a non​inflammatory profile; how- ever, virus has been amplified from the CSF, nasopharyngeal swabs, and rectal swabs. The range of neurological infection is poorly de- fined because of the benign CSF appearance. Children with HPeV-​3 positive CSF specimens in the United States of America showed a predominance of male infants presenting with sepsis-​like syndromes in a late summer/​autumn distribution in odd numbered years. This seasonal distribution was not reflected in a similar survey in the United Kingdom, in which patients presented in the spring in even-​ numbered years, and were almost always infants less than 3 months of age. The combination of prominent abdominal distension with erythematous rash has been seen in infants with confirmed HPeV in- fection during an outbreak of HPeV3 infection in New South Wales; such signs are postulated to be important clues to the diagnosis in the absence of raised C-​reactive protein or lymphocyte count. More recently described HPeV-​8 (Brazil, 2009)  and HPeV-​10 (Sri Lanka, 2010) were both found in stool specimens of children with acute gastroenteritis. It is also likely that further novel human parechoviruses will be discovered and their contribution as human pathogens investigated. Another parechovirus, Ljungan virus, has recently been postu- lated as a major aetiological agent in sudden infant death syndrome. Ljungan virus mainly affects rodents and is known to be associated with perinatal rodent death both in the wild and in laboratory mice. Interestingly, a strong epidemiological link between small rodent numbers and human intrauterine fetal death has been described in Sweden. In addition, Ljungan virus has been detected in brain, heart, and lung tissue in cases of sudden infant death syndrome. Whether true causation can be proven is yet to be established. Small rodent models for type 1 diabetes mellitus have shown high rates of Ljungan virus infection leading to the investigation of its role in the pathogenesis of diabetes mellitus. Swedish patients with diabetes mellitus have also been found to have an increased prevalence of antibodies to Ljungan virus; however, Ljungan virus RNA has not been detected in patients with diabetes. Investigations into this as- sociation are ongoing. Human cardiovirus Saffold virus Investigation of an 8-​month old girl with pyrexia of unknown origin, led to the discovery in 2007 of a novel cardiovirus of the family Picornaviradae, named Saffold virus. Several strains of Saffold virus have since been described and have been detected in faecal and respiratory specimens of children worldwide, from a patient with aseptic meningitis and from children with non-​polio acute flaccid paralysis, though causality has not yet been proven. Interestingly, Saffold virus is grouped with Theiler’s murine encephalomyelitis virus, which is known to cause a multiple sclerosis-​like syndrome in mice. Although this might be the first human cardiovirus, a specific clinical association is yet to be found. Emerging and Novel human flaviviruses Usutu virus Usutu virus, named after a river in Swaziland, was first isolated from mosquitoes in South Africa in 1959. It is a mosquito-​borne flavivirus of the Japanese encephalitis group and was isolated once from a man with fever and rash. Although a virus of tropical or subtropical Africa, the epidemiology might be changing, as demonstrated by its isolation from several bird species during a die-​off in Austria in 2001. This reflects the pattern of the emergence of West Nile virus in the United States of America in 1999, which first affected birds and subsequently humans. Neuroinvasive infection secondary to Usutu virus was reported for the first time worldwide in 2009 when Usutu virus was detected by RT-​PCR in CSF and serum samples in two im- munocompromised patients in Italy who had both received blood transfusions. Clinical symptoms in both patients included fever (>39.5°C), headache, and neurological disease. Since then there have been a total of 17 cases of neuroinvasive disease reported in Europe. The extent of the human pathogenic potential of Usutu virus remains to be seen, but there is concern that it might follow a re- current theme of flavivirus emergence in previously cooler climates following climate change. Surveillance systems already in place in areas of endemic West Nile virus could be adapted to detect more cases of Usutu virus if surveillance in wild birds and vectors indi- cated a need. Alkurma virus Alkhurma virus, a re-​emerging tick-​borne flavivirus, is related to Kysanur Forest disease and shares clinical features with dengue fever. It was first described in a butcher in Saudi Arabia in the 1990s, and over the next 10 years, had a case fatality rate of around 25%. In 2009, 4 further sporadic cases were described in Jeddah in the post-​Hajj period and all might be linked to the slaughtering/​processing of sheep. In 2010 two discrete cases were reported in travellers returning to Italy from Egypt, neither of whom had links to the slaughtering of sheep. Both patients had recalled visiting the same camel market and one recalled a tick bite. These cases have highlighted the need to fur- ther understand the epidemiology of this re-​emerging disease. Zika virus This RNA arbovius was first isolated from a Rhesus monkey in the Zika forest in Uganda in 1947 and until recently only caused spor- adic cases. It is a single-​stranded RNA virus related to Dengue and Chikungunya virus. Entomological and epidemiological studies undertaken in Africa and Asia have demonstrated ongoing viro- logical activity since its initial isolation in these regions. Infections are often asymptomatic. When symptoms occur they are generally mild and consist of fever, maculopapular rash, arthralgia, myalgis, and non​purulent conjunctivitis. Symptoms develop three to twelve days after being bitten by an infected Ades mosquito. Early in clinical

8.5.29  Newly discovered viruses 955 infection it is not possible to distinguish between infections with Zika, Dengue, or Chikungunya. (See Chapter 8.5.14.) In 2007 the largest documented outbreak occurred in French Polynesia on the island of Yap when 11% of the population were in- fected and more severe neurological features, such as Guillian–​Barré syndrome, were seen. This outbreak was noticeable not only for its size but also because it documented transmission outside the trad- itional endemic areas of Africa and Asia. Outbreaks in Polynesia have continued since then. In 2014–​2015, following an outbreak, there was noted to be an increase in fetal central nervous system malformations. Viraemic travellers have introduced Zika to countries outside its previously known range. Zika virus infection was first reported in South America in 2015 further establishing it as an emerging infectious disease. In 2015 in Brazil, the number of cases of micro- cephaly increased 20-​fold. This increase occurred less than a year after the emergence of Zika virus infection in the region. Zika virus RNA has been found in the amniotic fluid of pregnancies with microcephalus and in the blood and tissue samples of micro- cephalic neonates. Along with microcephaly, other abnormalities have been described and it is now recognized that there is a distinct congenital Zika syndrome. Zika virus has emerged beyond its geographical range and has the potential for causing outbreaks wherever the Ades mosquito is found. Its clinical features and mode of transmission is similar both to dengue and chickunguna viruses, both of which are global public health concerns. Zika has the potential to follow the same course although with the possible potential for neurological and teratogenic complications. This highlights the need for ef- fective mosquito control and for vaccine development. Novel human Bunyaviruses The genus Phleobovirus in the family Bunyaviridae consists of more than 70 antigenically different viruses divided into complexes depending on their route of spread either by sand flies, mosquitoes, or as more recently reported, by ticks. Garissa and Ngari Genetic reassortment of segmented RNA viruses such as influenza is well known to have an important role in the emergence of vir- uses with new disease potential and host range. There is less gen- etic information on bunyaviruses, but there is increasing evidence that this mechanism could account for their evolution and increase their potential to cause disease in humans. Bunyaviruss have a trisegmented negative sense RNA genome. Evidence supports gen- etic reassortment as the driving force in bunyavirus evolution, as novel reassortment viruses continue to be identified. The first association of Ngari virus with human haemorrhagic fever was discovered during an extensive investigation of a large outbreak in Kenya, Tanzania, and Somalia in 1997 to 1998. A pre- viously unidentified member of the orthobunyavirus genus (family Bunyaviridae) was found in two cases. The virus was initially named Garissa virus, but subsequent genetic analysis showed that it was not a separate orthobunyavirus but had arisen by genetic segment reassortment between two known orthobunyaviruses, Bunyamwera virus and Ngari virus. Further sequence analysis of multiple orthobunyaviruses revealed that Ngari virus is a reassort­ ment Bunyamwera virus. Itaya Orthobunyaviruses have a global distribution and novel viruses have been identified in South America that have also arisen fol- lowing genomic reassortment. Itaya virus was identified in Peru in 2015 by investigating samples from patients with febrile illnesses in 1999 and 2006. The clinical syndrome is similar to that seen with dengue virus. Genomic analysis showed that it had arisen from reassortment between Caraparu virus, a known orthobynyavirus, and an unidentified group C orthobunyavirus. Severe fever and thrombocytopenia syndrome virus Severe fever and thrombocytopenia syndrome virus (SFTSV) was the first pathogenic tick-​borne phlebovirus identified. In 2009, in rural provinces in China, an outbreak of severe fever and thrombocytopenia syndrome (SFTS) was noted. Investigations were undertaken to determine if this outbreak was due to infec- tion with the bacterium Anaplasma phagoocytophilum. In view of this, enhanced surveillance of similar clinical syndromes was undertaken in selected provinces in China. Investigations led to the identification of SFTSV, a novel phlebovirus associated with a clin- ical syndrome whose major features are fever, thrombocytopenia, gastrointestinal symptoms, leucopoenia, and an unusually high case fatality rate. Epidemiological studies showed that the virus was isolated from the blood of 171 patients out of 241 hospitalized patients who ful- filled the clinical criteria for SFTS; no viral DNA was isolated form healthy matched controls. Seroprevalance studies have been under- taken in Chinese provinces where fatalities have occurred and prevalence ranges from 0.84% to 6.37% which may indicate asymp- tomatic or mild infection can occur. When investigating the patients’ homes, mosquitoes and ticks were commonly found. SFTSV DNA has not been found in mosquitoes but is repeatedly reported from Haemaphysalis longicoruis ticks. Studies in animals have shown in- fection in a variety of small and large mammals with goats and cattle having the highest seroprevalance rates. Ribavarin can inhibit viral replication in vitro but does not affect mortality rates in vivo; treatment for this infection remains only supportive. Following initial reports in China, cases were reported in both Japan and South Korea. 70% of patients have been farmers and out- breaks show seasonal variation being most common between spring to early autumn when ticks, thought to be the vector, are prevalent. However, aerosolized disease has been postulated which highlights the need for infection control precautions in novel infections. SFTS remains a serious condition with an average case fatality rate of 12% for SFTSV infection. Heartland virus Heartland virus is an arthropod-​borne Bunyavirus and the second reported that is transmitted by ticks. In 2009 two unrelated farmers from Missouri were admitted with a febrile illness following tick bites. Both men lived on farms and recalled removing embedded ticks in the days prior to their admission. Both men presented with fever, fatigue, headache, nausea, and non-​bloody diarrhoea, with one patient also reporting a dry cough and myalgia,

956 section 8  Infectious diseases but neither had a rash. Initial blood results on both patients demon- strated leucopoenia, thrombocytopenia, mild hyponatraemia, and elevated hepatic aminotransferases. Both recovered and had problems with short term memory in the following weeks but had no long term sequalae. Since 2009, seven additional cases have been reported from the United States and two of them have been fatal. Amblyomma amer- icanum, also known as the lone star tick, is implicated as the vector as Heartland virus has been repeatedly isolated this species. The full vertebral host populations remain to be fully identified but it is known to include white-​tail deer, raccoons, moose, and coyotes. The wildlife distribution includes central and eastern United States. Lujo virus Lujo virus is a novel, genetically distinct, highly pathogenic arena virus associated with haemorrhagic fever with an exceptionally high case fatality rate of 80%. It was first isolated in South Africa in 2008 during a nosocomial outbreak of five cases following the transfer of the index case from Zambia. The technique of unbiased pyrosequencing used during the investigation of this outbreak might be useful in identifying other novel pathogens in the future. Mimivirus With a diameter of 600 nm and with a dsDNA genome of 1.2 Mb, mimivirus is the largest virus so far discovered. It was initially thought to be a Gram-​positive coccoid bacterium and is visible with the light microscope. The virus species Acanthamoeba polyphaga mimivirus is within a family of its own, the Mimiviridae. Phylogenetic analysis has shown its relationship to other large DNA viruses including the Iridoviridae and Poxviridae, though its precise position in the phylogenetic tree remains under debate. Discovered during the investigation of respiratory pathogens using an amoeba coculture system, it might have originated in marine environments. Although it replicates within amoebae, it has yet to be shown to multiply ef- fectively in mammalian cells. Mimivirus might have a role in re- spiratory disease. A pneumonic illness can be produced in mice and a laboratory technician occupationally exposed to high con- centrations of mimivirus antigens developed a subacute, spontan- eously resolving pneumonia with seroconversion to mimivirus. The prevalence of antibodies to mimivirus was 9.66% in 376 Canadian patients with community acquired pneumonia compared to 2.3% of healthy controls. Two studies of pneumonia in intensive care units have shown seroconversion to the virus in more patients with ventilator-​associated pneumonia than in controls. Seropositivity to mimivirus in ventilated patients in a prospective matched cohort study was associated with longer duration of ventilation and longer intensive care unit stay. There was no mortality difference between seropositive patients and matched seronegative controls. Mimivirus antibodies have been found to be more prevalent in populations admitted from nursing homes and in those rehospitalized after dis- charge. These seroprevalence studies must be interpreted cautiously because of possible cross-​reactivity with other pathogens. More recent studies using real-​time PCR have also been inconclusive. Although mimivirus DNA was recovered from a bronchoalveolar lavage of a patient with relapsing pneumonia in the absence of other causative pathogens, a prevalence study of 69 ventilated patients in an intensive care setting found no evidence of mimivirus infection using real-​time PCR. A study of paired serological and DNA detec- tion in lower respiratory sampeles may be useful in investigating the role of mimivirus in respiratory disease. Titi Monkey adenovirus An outbreak of fulminant pneumonia affected 34% of New World monkeys housed in a closed colony in the United States. The Virochip microarray was used to identify the causative agent, a novel adenovirus—​the Titi Monkey adenovirus. An exposed worker subsequently developed an acute respiratory tract infec- tion lasting 4 weeks and seroconverted to Titi Monkey adeno- virus. A critically ill family member of this person also developed symptoms and tested positive serologically, suggesting poten- tial for primate–​human and human–​human transmission of this novel agent. Bourbon virus Bourbon virus is a novel virus that has been isolated twice from clinical cases. A  member of the Thogotovirus genus of the orthomyxoviridae family it joins six distinct viruses, two of which (Thogoto and Dhori) are known to cause disease in humans. The initial case was reported in a previously well male patient in his fifties who lived in Bourbon County in Kansas, United States. He became unwell several days after sustaining tick bites while working on his house. His initial symptoms were nausea, weakness, and diar- rhoea and progressed to multiorgan failure and death in 11 days. Throughout his illness a diffuse maculopapular rash on his chest, abdomen, and back was noted along with persistent periodic fevers. His initial laboratory findings were leucopoenia, thrombocyto- penia, and elevated liver transaminases. Both traditional culture and electron microscopy combined with newer genetic techniques were used to identify this novel virus which shares greater than 70% overall average nucleotide sequence identity with Dhori virus. Unlike other Thogotovirus infections, no neurological symptoms were reported and unlike other orthomyxoviriae, infections no re- spiratory symptoms were noted. The precise role of Bourbon virus in his illness is unknown but the high-​level virus noted in his blood samples suggests it contributed. The precise route of infection is unknown but the infection occurring in late spring following an embedded tick supports a tick-​borne route of infection. A second case has been reported again in the United States from the state of Oklahoma; this patient made a complete recovery. FURTHER READING Abed Y, et al. (2006). Human parechovirus types 1, 2 and 3 infections in Canada. Emerg Infect Dis, 12, 969–​75. Allander T, et al. (2005). Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A, 102, 12891–​6.

8.5.29  Newly discovered viruses 957 Allander T, et al. (2007). Identification of a third human polyomavirus. J Virol, 81, 4130–​6. Arthur JL, et  al. (2009). A novel bocavirus associated with
acute gastroenteritis in Australian children. PLoS Pathog, 5, e1000391. Ashraf U, et al. (2015). Usutu virus: an emerging flavivirus in Europe. Viruses, 7, 219–​38. Babakir-​Mina M, et al. (2011). The novel KI, WU, MC polyomaviruses: possible human pathogens? New Microbiol, 34, 1–​18. Barzon L, Ongoing and emerging arbovirus threats in Europe. Journal of Clinical Virology, 107, 38–47. Bissel SJ, et al. (2015). Human parechovirus 3 meningitis and fatal leukoencephalopathy. J Neuropathol Exp Neurol, 74, 767–​77. Carletti F, et al. (2010). Alkhurma haemorrhagic fever in travellers returning from Egypt, 2010. Emerg Infect Dis, 16, 1979–​82. Charrel RN, et  al. (2005). Low diversity of Alkhurma hemor- rhagic fever virus, Saudi Arabia, 1994–​1999. Emerg Infect Dis, 11, 683–​8. Chen EC, et al. (2011). Cross-​species transmission of a novel adeno- virus associated with a fulminant pneumonia outbreak in a New World monkey colony. PLoS Pathog, 7, e1002155. Drexler JF, et  al. (2009). Novel human parechovirus from Brazil. Emerg Infect Dis, 15, 310–​13. Drexler JF, et al. (2011). Aichi virus shedding in high concentrations in patients with acute diarrhea. Emerg Infect Dis, 17, 1544–​8. ECDC (2015). Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephally and Guillian–​Barré syndrome. https://​ecdc.europa.eu/​sites/​portal/​files/​media/​en/​publications/​ Publications/​zika-​virus-​americas-​association-​with-​microcephaly-​ rapid-​risk-​assessment.pdf Esper F, et  al. (2006). Coronavirus HKU1 infection in the United States. Emerg Infect Dis, 12, 775–​9. Fang, LQ, et  al. (2015). Emerging tick-​bourne infections in main- land China: an increasing public health threat. Lanc Infect Dis, 15, 1467–​79. Fauci AS, et al. (2016). Zika virus in the Americas—​yet another arbo- virus threat. N Engl J Med, 374, 601–​4. Gaynor AM, et  al. (2007). Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS Pathog, 3, e64. Gerrard SR, et al. (2004). Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa. J Virol, 78, 8922–​6. Ghietto LM, et al. (2011). High frequency of human bocavirus 1 DNA in infants and adults with lower acute respiratory infection. J Med Microbiol. Epub ahead of print Nov 24 2011. Gong Z, et al. (2015). Probable aerosol transmission of severe fever with thrombocytopenia syndrome virus in southeastern China. Clin Microbio Infect. 21, 1115–​20. Harvala H, et  al. (2008). Epidemiology and clinical associations of human parechovirus respiratory infections. J Clin Microbiol, 46, 3446–​53. Harvala H, et al. (2011). Comparison of human parechovirus and en- terovirus detection frequencies in cerebrospinal fluid samples col- lected over a 5-​year period in Edinburgh: HPeV type 3 identified as the most common picornavirus type. J Med Virol, 83, 889–​96. Hontz RD, et al. (2015). Itaya virus, a novel Orthobunyavirus associ- ated with human febrile illness, Peru. Emerg Infect Dis, 21, 760–​4. Jones MS, et al. (2005). New DNA viruses identified in patients with acute viral infection syndrome. J Virol, 79, 8230–​6. Kapoor A, et al. (2009). A newly identified Bocavirus species in human stool. J Infect Dis, 199, 196–​200. Khatami A, et al. (2015). Sepsis-​like disease in infants due to human parechovirus type 3 during an outbreak in Australia. Clin Infect Dis, 60, 228–​36. Kim Pham NT, et al. (2010). Novel human parechovirus, Sri Lanka. Emerg Infect Dis, 16, 130–​2. Kosy, et al. (2015). Novel thogotovirus associated with febrile illness and death, United States. Emerg Infect Dis, 21, 760–​4. McMullan LK, et al. (2012). A new phlebovirus associated with severe febrile illness in Missouri. N Engl J Med, 367, 834–​41. Nguyen NL, et al. (2009). Serologic evidence of frequent human in- fection with WU and KI polyomaviruses. Emerg Infect Dis, 15, 1199–​205. Niklasson B (2011). Current views on Ljungan virus and its relation- ship to human diabetes. J Med Virol, 83, 1673. Park S, et al. (2014). Severe fever with thrombocytopenia syndrome virus, South Korea, 2013. Emerg Infect Dis, 20, 1880–​2. Pyrc K, et  al. (2007). The novel human coronaviruses NL63 and HKU1. J Virol, 81, 3051–​7. Ren L, et al. (2009). Saffold cardiovirus in children with acute gastro- enteritis, Beijing, China. Emerg Infect Dis, 15, 1509–​11. Sharp CP, et al. (2009). High frequencies of exposure to novel human parvovirus, PARV4 in haemophiliacs and injecting drug users
detected by a serological assay for PARV4 antibodies. J Infect Dis, 200, 1119–​25. Smith AW, et al. (2006). Vesivirus viremia and seroprevalence in humans. J Med Virol, 78, 693–​701. Vabret A, et al. (2005). Human coronavirus NL63, France. Emerg Infect Dis, 11, 1225–​9. van de Pol AC, et al. (2009). Human bocavirus and KI/​WU polyoma­ viruses in pediatric intensive care patients. Emerg Infect Dis, 15, 454–​7. van der Hoek L (2007). Human coronaviruses, what do they cause? Antivir Ther, 12, 651–​8. Väisänen E, et al. (2014). Bufavirus in faeces of patients with gastro- enteritis, Finland, Emerg Infect Dis, 20, 1077–​9. Wang LF, et al. (2006). Review of bats and SARS. Emerg Infect Dis, 12, 1834–​40. Wattier RL, et  al. (2008). Role of human polyomaviruses in re- spiratory tract disease in young children. Emerg Infect Dis, 14, 1766–​8. Wei J, et al. (2015). The first human infection with severe fever with thrombocytopenia syndrome virus in Shaanxi Province, China. Int J of Infect Dis, 35, 35–​27. Weissenbock H, et al. (2002). Emergence of Usutu virus, an African mosquito-​borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis, 8, 652–​6. Wikan N, et al. (2016). Zika virus: history of a newly emerging arbo- virus. Lancet Infectious Diseases, 16(7), e119–e126. Xue-​Jie Yu, et al. (2011). Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med, 364, 1523–​32. Yu XJ, et al. (2011). Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med, 364, 1523–​32. Zumla A, et al. (2015). Middle East respiratory syndrome. Lancet, 386, 995–​1007.

8.5.3 Epstein– Barr virus 754

8.5.3 Epstein– Barr virus 754

754 section 8  Infectious diseases Whitley RJ, et al. (1998). Guidelines for the treatment of CMV diseases in patients with AIDS in the era of potent antiretroviral therapy. Arch Intern Med, 158, 957–​69. Human herpesvirus 6 and 7 Hall CB, et al. (1994). Human herpesvirus-​6 infection in children: a prospective study of complications and reactivation. N Engl J Med, 331, 432–​8. Yamanishi K, et  al. (2007). Human herpesvirus-​6 and 7. In:  Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2819–​45. Lippincott, Williams and Wilkins, Philadelphia, PA. Zerr DM, et al. (2005). A population-​based study of primary human herpesvirus 6 infection. N Engl J Med, 352, 768–​76. Zerr DM, et al. (2005). Clinical outcomes of human herpesvirus 6 re- activation after hematopoietic stem cell transplantation. Clin Infect Dis, 40, 932–​40. Human herpesvirus 8 Ganem D (2007). Kaposi’s sarcoma-​associated herpesvirus. In: Knipe DM, et al. (eds). Fields virology, 5th edition, vol. 2, pp. 2847–​88. Lippincott, Williams and Wilkins, Philadelphia, PA. Hayward GS (1999). Kaposi’s sarcoma HV strains: the origins and global spread of the virus. Semin Cancer Biol, 9, 187–​99. Martin JN, et al. (1998). Sexual transmission and the natural history of human herpesvirus 8 infection. N Engl J Med, 338, 948–​54. Mesri EA, et al. (2010). Kaposi’s sarcoma and its associated herpes- virus. Nat Rev Cancer, 10, 707–​19. Cercopithecine herpesvirus 1 Cohen JI, et al. (2002). Recommendations for the prevention of and therapy for exposure to B virus. Clin Infect Dis, 35, 1191–​203. Sabin AB, Wright AM (1934). Acute ascending myelitis following a monkey bite, with the isolation of a virus capable of reproducing the disease. J Exp Med, 59, 115–​36. 8.5.3  Epstein–​Barr virus Alan B. Rickinson and M.A. Epstein ESSENTIALS Epstein–​Barr virus is a human herpesvirus with a linear double-​ stranded DNA genome that is carried asymptomatically by most people. Symptomless primary infection is usual in childhood, establishing a lifelong carrier state where the virus persists as a la- tent infection of circulating B cells. The virus replicates recurrently in oropharyngeal epithelial cells, with consequent shedding of virus in saliva transmitting infection. Infectious mononucleosis If delayed beyond childhood, primary infection causes infec- tious mononucleosis in up to at least 50% of cases. This is typically characterized by sore throat, fever, anorexia, headache, fatigue, malaise (often disproportionately severe), generalized lymphaden- opathy, splenomegaly (60%), hepatomegaly (10%), and jaundice (8%). Diagnosis can be confirmed by the Monospot test (which de- tects heterophil antibodies that are present in 85% of cases) or, more accurately, by the presence of IgM antibodies to Epstein–​Barr virus capsid antigen. Treatment is supportive unless there are complica- tions. Most cases resolve within 1 to 4 weeks; chronic or recurrent forms are described but are very rare. Primary infection in boys with the X-​linked lymphoproliferative trait, a rare congenital immunodeficiency, presents in many cases as a highly exaggerated form of infectious mononucleosis culmin- ating in a severe or fatal haemophagocytic syndrome. In other rare cases, primary infection of immunocompetent people can lead to ‘chronic active Epstein–​Barr virus infection’, a recurrent febrile condi- tion that resembles persistent infectious mononucleosis; best known among Asian children, this again can progress to severe or fatal haemophagocytosis. Cancer Epstein–​Barr virus is strongly linked to several different human can- cers. These include at least three types of B-​cell malignancy, Burkitt lymphoma, lymphoproliferative disease/​lymphoma of the im- munocompromised, and Hodgkin lymphoma. Non-​B-​cell tumours include extranodal lymphoma of T or natural killer cell origin, un- differentiated nasopharyngeal carcinoma, and a subset of gastric carcinomas. Together these constitute a global burden of 200 000 Epstein–​Barr virus-​positive cancers per year. B-​cell malignancies ‘Endemic’ Burkitt lymphoma—​all cells of this common malignancy of children in equatorial areas of Africa and New Guinea carry the Epstein–​Barr virus genome. A cofactor, hyperendemic malaria, ex- plains the unusual geographical distribution of the high-​incidence disease. Presentation is in the jaw or, less frequently, in the abdomen; peripheral lymph nodes and spleen are spared. The tumour grows re- markably quickly but is very sensitive to cyclophosphamide treatment. An essentially similar tumour, ‘Sporadic’ Burkitt lymphoma occurs worldwide but at much lower incidence and with only a minority of tumours being Epstein–​Barr virus genome-​positive. The sporadic disease presents more often in the abdomen than the jaw, and lymph nodes can be involved; the response to treatment is poorer and com- bination therapy is required, and survival after relapse is uncommon. A third form, ‘AIDS-​associated Burkitt lymphoma’, occurs at un- expectedly high frequency in individuals with HIV infection, often as one of the first AIDS-​defining symptoms and before overt im- mune impairment; some 30–​40% of such tumours are Epstein–​Barr virus-​positive. Besides Burkitt lymphoma, Epstein–​Barr virus is firmly linked to two other types of B-​cell malignancy. One is lymphoproliferative disease/​lymphoma of the immunocompromised, typically seen in transplant patients in the first year after transplantation when immune impairment is most severe, and also in late-​stage AIDS patients; such lesions consist of Epstein–​Barr virus-​positive cells growing out op- portunistically in the absence of effective immune surveillance. The other B-​cell tumour is Hodgkin lymphoma, where the virus genome is present in all the Reed–​Sternberg and mononuclear tumour cells in

8.5.3  Epstein–Barr virus 755 about 30–​40% of cases seen in the Western world and in a somewhat higher proportion of cases elsewhere. Non-​B-​cell malignancies and other conditions associated with Epstein–​Barr virus Undifferentiated nasopharyngeal carcinoma—​this epithelial tu- mour occurs in all racial groups but is most common in southern Chinese and Inuit people. All cases worldwide are Epstein–​Barr virus genome-​positive. Besides virus infection, both genetic and environ- mental (dietary and perhaps herbal remedy) cofactors are involved in tumourigenesis. Radiotherapy, now combined with chemotherapy, is the treatment of choice. A histologically similar carcinoma of the sal- ivary gland, most often observed in Inuit people, is also consistently Epstein–​Barr virus genome-​positive and is regarded as a variant of nasopharyngeal carcinoma. In addition, some 8–​10% of gastric carcinomas around the world carry the Epstein–​Barr virus genome. These Epstein–​Barr virus-​ positive tumours show different cellular genetic changes from those seen in other forms of gastric carcinoma and have a slightly better re- sponse to treatment, suggesting that they represent a distinct subset of the disease. Epstein–​Barr virus is also very strongly linked to a highly aggressive extranodal lymphoma of T-​ or NK-​cell origin, sometimes presenting as a disfiguring nasal lesion, other times as a rapidly growing NK-​ cell leukaemia. The lymphoma is 100% Epstein–​Barr virus genome-​ positive. Responses to chemotherapy are currently very poor. Besides being present in certain AIDS lymphomas, Epstein–​Barr virus is also linked to two other HIV-​associated conditions. One is a very rare tumour of smooth muscle cell origin, leiomyosarcoma of AIDS, which is Epstein–​Barr virus-​positive in all cases. The other is a non​malignant wart-​like lesion of the oral cavity, oral hairy leukoplakia, which is caused by the virus actively replicating as a lytic infection of squamous epithelial cells. Both conditions are markers of the profound immune impairment seen in late-​stage AIDS; accord- ingly, both show much reduced incidence following the introduction of highly active antiretroviral therapy. More controversially, Epstein–​Barr virus has been linked with cer- tain autoimmune diseases. In particular, there is strong serologic and epidemiologic evidence to suggest that previous exposure to Epstein–​Barr virus markedly increases the risk of developing multiple sclerosis. Although the Epstein–​Barr virus/​multiple sclerosis connec- tion is receiving much attention, the mechanism that might underpin such an association remains uncertain. Background The virus Epstein–​Barr virus (EBV) was discovered in 1964 during a sus- tained search for a viral cause of endemic Burkitt lymphoma (see ‘Endemic (‘African’) Burkitt lymphoma’, next). EBV is one of eight human herpesviruses and the only one belonging to the γ-​1 sub- family. It has a typical herpesvirus structure, with an outer envelope and a protein capsid shielding the inner core with its large double-​ stranded linear DNA genome. EBV clearly has a long coevolutionary history with our species as related γ-​1 viruses have been found in both Old World apes and monkeys and in New World monkeys. This indicates that a common ancestor of all contemporary γ-​1 viruses must have been present in early primate species before the evolu- tionary split between Old World and New World primates occurred (about 35 million years ago based on palaeontology, or 45 million years ago based on DNA sequence analysis). Viral infections, lytic and latent On transmission to a naïve host, herpesviruses typically replicate in one or more cell types as a lytic infection, producing thousands of copies of new infectious virions but killing the host cell in the process, then establish a latent (antigenically silent) infection in one particular cell type. This reservoir of latency allows the virus to escape detection and survive for years in the immune-​competent host. EBV accords to this pattern, replicating in oropharyngeal epi- thelium and probably also locally infiltrating B cells, before going latent in the recirculating memory B cell pool. In addition, however, the virus has evolved a unique set of protein-​coding latent genes which, when expressed collectively, are able to drive B-​cell growth. In the newly infected host, EBV uses this growth-​transforming ability to expand the number of infected cells invading the B-​cell system, before switching off latent gene expression in those cells and going silent in memory B cells. The same growth-​transforming ability can also be seen in vitro when the virus infects B cells and drives them into continuously growing, latent antigen-​expressing lymphoblastoid cell lines. Virus-​coded proteins and the immune response Different sets of virus-​coded proteins are expressed in lytic and latent infection. There are over 60 EBV-​coded proteins expressed during lytic cycle, and these are categorized as immediate early, early, or late antigens, according to the order in which they appear. By contrast, growth-​transforming infection involves the coordinated expression of eight unique latent proteins. Both lytic and latent proteins elicit a range of antibody and cell-​mediated immune responses that help to bring the initial infection under control; those responses then mature and persist for life. Assays for serum antibodies to repre- sentative lytic and latent proteins can therefore be used to identify individuals who have never been infected by EBV, those undergoing primary infection, and those with an established carrier state. Epidemiology The virus is widespread in all human populations. Primary infection usually occurs in early childhood, when it is almost always clinic- ally silent. This leads to a lifelong carrier state, in which the virus persists as an antigenically silent, latent infection in a small number of memory B cells. Reactivation of the virus infection is normally contained by the host’s humoral and cellular immune responses. However, low level reactivations into lytic cycle from recirculating latently infected B cells are thought to seed subclinical foci of lytic- ally infected epithelial cells in the mouth and pharynx, and perhaps also in the salivary glands. This leads to recurrent low-​level shedding of infectious virus in the buccal fluid of virus carriers, allowing oral transmission of the infection within the population. In developing countries, 99% of children are infected by the second to the fourth year of life. By contrast, in industrialized countries with higher standards of hygiene, as many as 50% of children, particularly those

756 section 8  Infectious diseases from high socioeconomic groups, enter adolescence uninfected (Fig. 8.5.3.1). Infectious mononucleosis Careful prospective studies on student populations report that up to 80% of those who first acquire the virus in the second or third decade develop some clinical symptoms of infectious mononucle- osis. However the severity of symptoms varies greatly between in- dividuals and it is likely that only a minority of the aforementioned will be sufficiently ill to consult a doctor. Given its strong associ- ation with delayed infection, mononucleosis is therefore mainly a disease of upper socioeconomic groups in Western societies, and is exceptionally rare in developing countries (Fig. 8.5.3.1). Although most cases occur in adolescents and young adults, children and older people might sometimes develop the disease. Primary in- fection in adolescence or later is likely to be acquired by kissing a virus-​shedding healthy carrier. This explains why case-​to-​case in- fection and epidemics are not seen, and why the incubation period, estimated as 30 to 50 days, is difficult to calculate. Symptomatic pri- mary EBV infection can also be acquired through latently infected B lymphocytes present in blood transfusions or organ grafts, where the donor is an EBV carrier and the recipient is EBV-​naïve. Symptoms Classical infectious mononucleosis might follow days of vague indisposition, or can start abruptly. It presents with sore throat, fever with sweating, anorexia, headache, and fatigue, with mal- aise quite out of proportion to the other complaints. Dysphagia might be noticed, and also brief orbital oedema. Erythematous and maculopapular rashes occur in a small number of untreated patients, but much more frequently in those that have been taking ampicillin for sore throat before infectious mononucleosis has been diagnosed (Figs. 8.5.3.2 and 8.5.3.3). Tonsillar and pharyn- geal oedema can rarely cause pharyngeal obstruction (Fig. 8.5.3.4) Signs The fever can rise to 40°C, but swings are not seen. There is red- ness and oedema of the pharynx, fauces, soft palate, and uvula (Fig.  8.5.3.4a), and about half the patients develop greyish ex- udates on the tonsils (Fig. 8.5.3.4b). Generalized lymphadenop- athy is almost always present, and is most marked in the cervical region; the glands are symmetrical, discrete, and slightly tender. Splenomegaly is seen in about 60% of cases and an enlarged liver in 10%. There is usually a moderate bradycardia. Besides the rash, characteristic palatal enanthematous crops of reddish petechiae (Fig. 8.5.3.4c) are found in about one-​third of patients, and jaun- dice occurs in about 8%. Clinical course Mild cases might resolve in days, but 1 to 4 weeks is more usual, followed by a period of lethargy. The duration of this convalescence is influenced by psychological factors, particularly the speed with which patients are encouraged to resume full activity. About 1 case in 2000 might continue in a truly chronic or recurrent form for several months or years (see ‘Chronic active EBV infection’, next). Most other cases of so-​called chronic infectious mononucleosis are manifestations of chronic fatigue syndrome (Chapter 26.5.4), but whether this is a true entity rather than a form of depression or a be- lief disorder is highly controversial. Credible connections with EBV have not been established. Africa SE Asia developed countries less well off developed countries affluent classes 100 75 50 25 0 Percentage of individuals infected with EBV Age (years) 70+ 60 50 40 30 25 20 15 5 10 Fig. 8.5.3.1  Comparison of the ages at which people in different populations become infected with EBV. In developing countries, almost all children have acquired the virus by 2 to 4 years of age, depending on geographical region. In developed countries with high standards of living and hygiene, the time of infection is delayed for many, more markedly among the affluent than the less well off. Among the very rich, as many as 50% may reach adolescence or young adulthood without having encountered the virus, and will undergo delayed primary infection, with a high risk that this will be accompanied by the symptoms of infectious mononucleosis. Reprinted with permission from Epstein MA (2002). Infectious mononucleosis. In: Encyclopedia of life sciences, 10, 211–​16. Copyright © 2001 John Wiley & Sons, Ltd. Fig. 8.5.3.2  Typical maculopapular erythematous rash in a patient with infectious mononucleosis who was treated with ampicillin. Copyright D. A. Warrell.

8.5.3  Epstein–Barr virus 757 Complications Minor non​specific complications can occur. Rare, more ser- ious complications include secondary bacterial throat infections, traumatic rupture of the enlarged spleen, asphyxia from pharyn- geal oedema, massive hepatic necrosis, Guillain–​Barré syndrome, and autoimmune manifestations such as thrombocytopenia and haemolytic anaemia. Differential diagnosis Classical infectious mononucleosis is diagnosed by the clinical fea- tures, combined with serological and haematological laboratory investigations (see next). An infectious mononucleosis-​like disease can occur in primary cytomegalovirus infection and in toxoplas- mosis, but in both conditions the sore throat is much less severe, and with cytomegalovirus the lymphadenopathy might be minimal or absent; an infectious mononucleosis-​like syndrome is also some- times seen with primary HIV infection. Laboratory diagnosis Several diagnostic methods have been developed and evaluated. These include (i)  the Monospot test for heterophile antibodies, (ii) multiplexed bead-​based assay (BBA), enzyme immunoassay (EIA), or immunofluorescence assay (IFA) for IgM/​IgG antibodies to EBV viral capsid antigen (VCA) and EBV nuclear antigen 1 (EBNA1) and (iii) measurement of EBV viral genome load (EBV-​ VL) either in whole blood or in peripheral blood mononuclear cells using quantitative real-​time polymerase chain reaction (QRT-​PCR) assay. The Monospot screening test detects the pres- ence of heterophile antibodies in the patient’s serum. Although these heterophile antibodies are not directed against virally en- coded proteins, they are present in up to 85% of acute infectious mononucleosis sera; cases of Monospot-​negative infectious mono- nucleosis tend to be outside the usual 15-​ to 25-​year age range, and false-​positive tests may occur in pregnancy and autoimmune dis- ease. The diagnosis of infectious mononucleosis can be confirmed by the presence of serum IgM antibodies to VCA, accompanied by rising IgG anti-​VCA antibodies, in the absence of detectable IgG antibodies to EBNA1; patients remain IgM anti-​VCA-​positive for about 2 months while the IgG anti-​EBNA1 response typically appears 3  months or more after the disease course. Eventually, the patient’s serological picture assumes that of the lifelong virus carrier state, that is IgM anti-​VCA-​negative, IgG anti-​VCA-​posi- tive, IgG anti-​EBNA1-​positive. The QRT-​PCR assay is proving useful in the early diagnosis of infectious mononucleosis where EBV IgM immunoassays (multiplexed BBA, EIA, and IFA) prove inconclusive. Assays designed with EBV genomic probes within the BALF5 gene or within the BAMH1-​W repeat region are highly sensitive. Note that the level of EBV DNA detected in infectious 100 75 50 25 0 Percentage of patients with clinical features Duration of IM (days) 28 21 14 7 Enlarged lymph glands Enlarged spleen Temperature ↑ Sore throat Spots on palate Jaundice Rash Swollen eyes Fig. 8.5.3.3  Percentage of patients with infectious mononucleosis showing various clinical features during the course of the disease, and the timing and average duration of each. Reprinted with permission from Epstein MA (2002). Infectious mononucleosis. In: Encyclopedia of life sciences, 10, 211–​16. Copyright © 2001 John Wiley & Sons, Ltd. (a) (b) (c) Fig. 8.5.3.4  Infectious mononucleosis: (a) Oedema of fauces, soft palate, uvula, and tonsils; (b) tonsillar exudates; and (c) palatal petechiae. Courtesy of the late Dr B. E. Juel-​Jensen.

758 section 8  Infectious diseases mononucleosis blood reflects the load of circulating latently in- fected B cells, not the load of free virus; levels of cell-​free EBV DNA within plasma are very low and often undetectable even in acute disease. Other diagnostic features of the disease include the presence of lymphocytosis up to 15 × 109/​litre, composed mainly of activated cytotoxic T cells (the ‘atypical mononuclear cells’ that gave mononucleosis its name), a decreased CD4/​CD8 ratio (0.3), and raised C-​reactive protein concentrations. Treatment Bed rest and aspirin or paracetamol for headache and pharyngeal discomfort are the only treatments required for most patients with infectious mononucleosis. The role of corticosteroids, other than in patients with unusually severe symptoms, is controversial. When the fever resolves the patient should be encouraged to get up and resume some activities as fast as is practicable, but violent exercise should be avoided for 3 weeks after an enlarged spleen ceases to be palpable. Only complications need active therapy; splenic rup- ture requires surgery, bacterial infections call for appropriate anti- biotics, airway obstruction must be relieved by tracheostomy, and corticosteroids should be given for life-​threatening pharyngeal oedema, and for neurological and haematological complications. Immunocompetent patients with severe infectious mononucleosis have been treated with a variety of agents targeting lytic virus rep- lication (including aciclovir, valciclovir, famciclovir, ganciclovir with or without foscarnet, and vidarabine) together with cor- ticosteroids or intravenous immunoglobulin. The utility of such treatments remains unproven. Clinical evidence does not support the use of aciclovir alone but more patients receiving a combin- ation of antivirals and immunosuppressives survived compared to those given antiviral therapy alone. Suggested experimental treatments are 5-​substituted uracil, azacytosine derivatives aimed at destroying cells entering lytic cycle and expressing EBV-​coded thymidine kinase, and peptides inhibiting EBV-​mediated mem- brane fusion. Pathogenesis Acquisition of orally transmitted virus leads to a phase of high level virus replication in the oropharynx, through lytic infection of epithelial cells and probably also intraepithelial B lymphocytes, followed by colonization of the general lymphoid system through a latent growth-​transforming infection of B lymphocytes. In infec- tious mononucleosis patients, these combined lytic and latent in- fections stimulate an exaggerated cell-​mediated immune response, characterized by mild NK-​cell activation but large expansions of ac- tivated cytotoxic CD8+ T cells; these CD8+ T cells are found not just in the circulation but also in oropharyngeal lymphoid tissues such as the tonsils, as well as more generally in lymph nodes, spleen, and liver. This exaggerated response, and the cytokine storm that accompanies it, are thought to be responsible for the sore throat, fever, malaise, lymphadenopathy, and hepatosplenomegaly. In sup- port of this idea, in the few individuals who have been identified undergoing asymptomatic primary infection, the blood picture showed no lymphocytosis. Infectious mononucleosis is therefore an immunopathological disease. It is not known why the disease occurs frequently following primary EBV infection in adolescents and young adults, yet rarely in children; however, this must relate to circumstances that influence the size of the cell-​mediated response. One possible factor is virus dose per se, with higher doses likely to be acquired by kissing in adolesecence/​young adulthood. However, the issue of acquired dose remains contentious since recent work has de- tected EBV genome loads in the blood of asymptomatic primary infections that are the equal of those seen in infectious mono- nucleosis patients. Another possible factor is the age-​dependent maturation of the human immune system, with NK-​cell control over the very early phase of viral infection becoming less effi- cient with age, thereby placing more burden on the CD8+ T-​cell response. X-​linked lymphoproliferative disease (fatal infectious mononucleosis) (OMIM 308 240) An extremely rare genetically determined susceptibility to EBV occurs in young males of certain kindreds, who develop X-​linked lymphoproliferative (XLP) disease following primary infection. This presents initially with acute mononucleosis-​like symptoms, but progresses inexorably to haemophagocytosis, which cul- minates in the necrotic destruction of vital organs, leading to multisystem failure. The mutated X-​chromosomal gene (SH2D1A) responsible for this defect encodes a protein (SAP) that is involved in the normal regulation of T cell and natural killer (NK) cell re- sponses, in particular the ability of these cells to recognize and interact with B lymphocytes. This has several immunological con- sequences but is especially damaging with respect to combating EBV, a virus that is harboured in the B-​cell system. In patients with XLP, EBV induces the same CD8+ T-​cell and NK-​cell responses as in classical infectious mononucleosis, but the responses are nei- ther able to control the B-​cell infection, nor to receive the signals that would normally control their own expansion. As a result, XLP patients develop a highly exaggerated form of infectious mono- nucleosis, with even more dramatic CD8+ T-​cell and NK-​cell ex- pansions, very high loads of inflammatory cytokines and an often fatal haemophagocytosis. Chronic active EBV infection There are very rare cases of infectious mononucleosis that fail to re- solve, and may continue for years, often developing serious compli- cations leading to death. These cases of chronic active EBV infection can occur in both sexes, show no familial linkage, and are more common in people of Asian than European descent. Symptoms and signs Persistent fever, lymphadenopathy, and hepatosplenomegaly are frequently accompanied or followed by anaemia, thrombocyto- penia, and mononuclear cell haemophagocytosis. The disease can, therefore, lead to a clinical endpoint not unlike that seen in fatal XLP, but by a different pathogenetic route (see next). Pathogenesis and treatment Chronic active EBV infection is unique in that, in most cases, the disease is a consequence of unscheduled entry of the virus into T and/​or NK cells; the circumstances which allow such atypical

8.5.3  Epstein–Barr virus 759 infection are not understood. The infected T or NK cells appear to escape normal immune controls and so proliferate, infiltrating vital organs and releasing the cytokines that are thought to initiate haemophagocytosis. Such infections also carry an oncogenic risk and, even within 1–​2 years of first presenting, some of these patients develop a monoclonal EBV-​positive T-​ or NK-​cell lymphoma. There is no satisfactory treatment for this disease, but haemato- poietic stem cell transplantation is being evaluated with encouraging early results. Endemic (‘African’) Burkitt lymphoma The classical form of this B-​cell tumour, first described by Burkitt in 1958, is found in those parts of Africa and Papua New Guinea where the temperature does not fall below 16°C, and the annual rainfall does not fall below 55 cm. Endemic Burkitt lymphoma is a disease of childhood, is extremely rare over the age of 14 years, and in endemic areas is more common than all other childhood tumours added together. The association between latent EBV infection and the cells of endemic Burkitt lymphoma is so close (virtually 100%) that it is generally accepted that the virus is essential, although it requires combination with cofactors in a complicated chain of events to lead to the malignancy. Hyperendemic malaria has been identified as an important cofactor, and its spread by anopheline mosquitoes re- quiring warmth and moisture explains the climate dependence. Symptoms and signs The endemic tumour is usually multifocal, and the symptoms de- pend entirely on the anatomical location. Jaw tumours are present in 70% of patients, are the usual presenting feature, may be multiple in all four quadrants, and are almost always accompanied by tumour foci elsewhere. The rapidly growing mass causes loosening of teeth, and exophthalmos from orbital spread. Abdominal tumours involve retroperitoneal nodes, liver, ovaries, intestines, and kidneys. Burkitt lymphoma sometimes presents in the thyroid, the adolescent fe- male breast, the testicles, and salivary glands; extradural tumours in the spine cause rapid paraplegia, and skeletal tumours also occur. Characteristically Burkitt lymphoma does not involve the spleen or peripheral lymph nodes. The tumours are firm, very rapidly growing, painless, and cause minimal constitutional disturbance. Their site determines the clinical signs. Differential diagnosis In endemic areas, Burkitt lymphoma can be diagnosed from the clinical picture. Unlike the Burkitt tumour, retinoblastoma is intraocular; rhabdomyosarcoma is extraorbital, and does not in- volve teeth; nephroblastoma is not multifocal; and neuroblastoma and ovarian tumours can be distinguished histologically. Paraplegia of tuberculous origin causes vertebral collapse, and acute transverse myelitis is preceded by pain and fever. The anatomical distribution of other lymphomas is quite different. Laboratory diagnosis Histological examination of a biopsy sample is clearly diagnostic. Antibodies to EBV antigens show a distinct pattern, with IgG anti-​VCA titres are around 10-​fold higher than in matched controls, with antibodies to EBV-​restricted early antigens and membrane antigens also detectable. Anti-​VCA titres appear to reflect tumour load, rising with disease progression and falling with posttreatment remission; this implies that, although the tumour is latently infected, a small fraction of tumour cells in vivo spontaneously enter lytic cycle and release lytic proteins. Clinical course and treatment Tumour growth is relentless, and death ensues within a few months in the absence of treatment. Surgery and radiotherapy are ineffective, but moderate courses of chemotherapy give excellent results. This reflects the marked sensi- tivity of this rapidly growing tumour to cytotoxic drugs that induce programmed cell death (apoptosis). Cyclophosphamide, the drug of choice, remains effective after relapses. Pathogenesis Burkitt lymphoma is a monoclonal malignancy of germinal centre B-​cell origin. EBV expresses a very limited range of latent genes in the tumour cells, with EBNA1 the only consistently detect- able viral antigen. When combined with a key cellular genetic change—​a chromosomal translocation leading to overexpression of the MYC oncogene—​this restricted form of virus latency ap- pears to complete the malignant conversion of the target cell, giving rise to the tumour. Cofactors such as hyperendemic mal- aria may contribute, both by chronically stimulating turnover of the target germinal centre cell population in the B-​cell system, thereby increasing the chances of rare chromosomal transloca- tions occurring, and also by disturbing the normal virus–​host balance, thereby enlarging the pool of EBV-​infected cells in the body. It is clear that the virus is a necessary, but not sufficient element in the aetiology of endemic Burkitt lymphoma. Based on in vitro studies, EBV’s role in Burkitt pathogenesis is thought to be antiapoptotic (i.e. partially reducing the sensitivity to programme cell death that is a consequence of high MYC expression), thereby allowing MYC’s other action, as a driver of cell growth, to become dominant. Sporadic Burkitt lymphoma The sporadic form of the tumour occurs in children worldwide, but generally at a much lower incidence than endemic Burkitt lymphoma. The association of these tumours with EBV varies from 10 to 15% in the Western world, where the disease is 100-​fold less frequent than in endemic areas, to more than 50% in some other countries where the incidence is intermediate between the two ex- tremes. The same restricted EBV gene expression is seen in virus-​ positive sporadic tumours as in the endemic disease and the role of EBV, when present, is thought to be similar. Symptoms and signs Unlike endemic Burkitt’s lymphoma, the sporadic form very rarely involves the jaws, and frequently presents in lymph nodes and within the abdomen. The clinical features depend on the location of the tumours.

760 section 8  Infectious diseases Diagnosis The tumour must be distinguished from other types of non-​Hodgkin lymphoma by histological examination of biopsies. Sporadic Burkitt lymphoma has the same histologic features and carries the same type of MYC translocation as the endemic tumour, though more often with additional chromosomal aberrations.. Treatment The response to chemotherapy is not usually as good as in en- demic Burkitt lymphoma. Cyclophosphamide alone is inadequate; combination therapy is required, and survival after relapse is uncommon. AIDS-​associated Burkitt lymphoma Burkitt lymphoma appeared unexpectedly as a common tumour of HIV-​positive adults during the early years of the AIDS epi- demic in the West. In many respects, the AIDS-​associated tu- mour resembles the sporadic form seen in children, except that its incidence is much higher (even greater than the endemic dis- ease) and some 30–​40% tumours are EBV-​positive. The tumour tends to arise relatively soon in the course of AIDS, following the initial period of HIV-​induced generalized persistent lymph- adenopathy and before the inset of profound T-​cell impairment. This implies that HIV predisposes to Burkitt lymphomagenesis through stimulating germinal centre activity in the B-​cell system (like hyperendemic malaria) rather than through its subse- quent destruction of the T-​cell system. Accordingly, although highly active retroviral therapy (HAART) prevents T-​cell im- pairment and progression to late-​stage AIDS, the incidence of Burkitt lymphoma among HIV-​positive cohorts has not fallen in the post-​HAART era. Note that the same appears to be true of another AIDS-​associated B-​cell lymphoma, diffuse large B-​cell lymphoma of germinal centre origin, whose incidence is also in- creased in HIV-​infected individuals, albeit not as dramatically as Burkitt lymphoma; these tumours are less well studied as a histologically distinct group but appear to be EBV-​positive in 30–​50% cases; the virus’ role in their pathogenesis remains to be determined. Lymphoproliferative disease/​lymphoma of the immunocompromised T-​cell impairment, whether congenital, induced by immunosup- pressive therapy, or caused by late-​stage HIV infection, relaxes host control over EBV, leading to increased virus replication in the oral cavity, increased numbers of circulating virus-​carrying B lymphocytes and a reappearance of EBV growth-​transforming B-​cell infections. The higher antigen load, arising as a consequence of relaxed T-​cell surveillance, then induces higher levels of anti-​EBV antibodies in serum. Such disturbance of the virus–​host balance is initially asymptomatic but over time greatly increases the risk of EBV-​associated lymphoproliferative disease. In primary immune deficiency states Besides the special case of XLP (described earlier), many other primary immune deficiency states affecting T-​cell competence are now known and their genetic basis identified. Many of these are susceptible to the damaging effects of infection with a range of persistent viruses, including herpesviruses, that are normally con- tained by immune surveillance. EBV-​driven lymphoproliferative lesions, essentially identical to those seen in posttransplant pa- tients (see next) are frequently reported in such cases, typically in those children who have survived long enough to acquire the virus. In transplant recipients Solid organ transplant recipients, who receive long-​term immuno- suppressive drugs, have an up to 100-​fold increase in their life- time risk of developing B lymphoproliferative disease/​lymphoma compared with normal immunocompetent individuals. Most of these tumours occur within the first year of transplantation when immunosuppressive therapy is most intense. A  similar picture is seen in haemopoietic stem cell transplant recipients who are at greatly increased risk in the first 6–​12 months posttransplant before their immune system is fully re-​populated. These early onset ‘posttransplant’ lymphoproliferative disorders are fre- quently oligoclonal and almost always EBV-​driven, made up of EBV-​positive B cells expressing the same range of eight growth-​ transforming latent proteins (6 EBNAs and 2 latent membrane proteins) just as seen in EBV-​positive lymphoblastoid cell lines in culture. Such lesions arise through a failure of virus-​specific immune T-​cell surveillance and so occur with highest incidence in the most heavily immunosuppressed patients, particularly those (most often children) who were uninfected by the virus pretransplant and acquired it in the peritransplant period. The virus therefore appears to be both a necessary and sufficient cause of such lymphoproliferative disease. The lesions are refractory to conventional cytotoxic drug therapy and have attracted different treatment approaches. In the solid organ transplant setting, the initial treatment is to reduce the im- munosuppressive drug dose, with or without aciclovir therapy, and in some patients a partial recovery of T-​cell competence will be sufficient to clear the EBV-​positive lesion without initiating graft-​versus-​host disease. In the stem cell transplant setting, treat- ment with Rituximab (monoclonal antibody to the B-​cell surface antigen CD20) is now the preferred treatment option while the first effective treatment to be developed, adoptive transfer of in vitro-​ expanded EBV-​specific T-​cell preparations, is now reserved for Rituximab-​resistant disease. Because solid organ transplant recipi- ents remain on low but continual immunosuppression, they also have an increased longer-​term risk of lymphoma. Some of these late tumours resemble abovementioned lymphoproliferative lesions, whereas others are monoclonal B-​cell lymphomas of more varied type, only some of which are EBV-​positive; the role of the virus in this latter context is unclear. In people with HIV In the pre-​HAART era, HIV-​infected individuals who progressed to end-​stage AIDS were at very high risk of an EBV-​driven lymphoproliferative disease essentially similar to that seen in posttransplant patients. These lesions presented extranodally at many unusual sites, most commonly in the central nervous system (CNS) where they were frequently reported as ‘CNS

8.5.3  Epstein–Barr virus 761 lymphomas’. Disease progression is rapid, with a mean survival time from diagnosis of 3–​4 months. Radio-​ and/​or chemotherapy is disappointing because patients with late-​stage HIV are often in poor general health. Hodgkin lymphoma There had long been a suspicion that EBV is involved in the pathogenesis of Hodgkin lymphoma, largely because the age and social class-​dependence of this tumour’s incidence in the devel- oped Western world resembled that of infectious mononucle- osis; both diseases show a marked peak in young adulthood. Furthermore, it became clear that infectious mononucleosis carried with it a 4-​fold increased risk of developing Hodgkin lymphoma over the next 10 years. Indeed, the increased risk was most marked within 2–​3 years of the original attack of infectious mononucleosis. Hodgkin lymphoma is a monoclonal tumour of postgerminal centre B-​cell origin, but unusual in that the malignant popu- lation (of Reed–​Sternberg and mononuclear Hodgkin cells) typically accounts for only 1–​2% of the tumour mass. Those cells are greatly outnumbered by a non​malignant infiltrate, involving many cell types, whose different composition defines the histologically classified subtypes of the disease. The EBV genome is now known to be present in all the malignant cells in some 30–​40% of Hodgkin tumours arising in the Western world. Surprisingly, it is the mixed cellularity and lymphocyte-​ depleted subtypes that show the highest levels of EBV-​positivity (75–​90%), whereas the nodular sclerosing subtype, which makes up the bulk of the young adult Hodgkin peak in Western soci- eties, is much less often EBV-​positive. Thus, in the West, EBV is more strongly associated with Hodgkin lymphoma presenting in children and in older age patients. By contrast, in developing countries which lack the young adult peak, mixed cellularity and lymphocyte-​depleted subtypes are the most common forms of the tumour independent of age, and up to 80% of all Hodgkin tumours are EBV genome-​positive. Where present, EBV ex- presses EBNA1 and the two latent membrane proteins, LMPs 1 and 2, a pattern intermediate between the extremes seen in Burkitt lymphoma (EBNA1 only) and lymphoproliferative dis- ease of the immunocompromised (all 6 EBNAs and the two LMPs). EBV’s role in Hodgkin lymphomagenesis is not fully understood but, as in Burkitt lymphoma, its main influence is thought to be through protection against apoptosis rather than through directly driving cell growth. Undifferentiated Nasopharyngeal carcinoma This tumour is restricted to the postnasal space, where it arises from squamous epithelial cells. It has a distinct histology with heavy infiltration by non​malignant T cells, and is thus some- times designated a lymphoepithelioma. The tumour is seen at low incidence worldwide but has a remarkably high incidence among people of southern Chinese origin, as well as in the Inuit and related circumpolar peoples. In high-​incidence areas, nasopharyngeal carcinoma is the most common cancer of men, and the second most common of women. The disease also oc- curs with intermediate incidence in Malays, Dyaks, Indonesians, Filipinos, and Vietnamese people, as well as in a belt stretching across North Africa, through Sudan, to the Kenyan highlands. The tumour usually occurs in middle or old age, but in North Africa it has bimodal age peaks, one involving young people up to 20 years old and a second, much later in life. Irrespective of geographical region, nasopharyngeal carcinoma cells always carry the EBV genome and express a subset of latent virus proteins, namely EBNA1, LMP1 (in some cases) and LMP2. Symptoms and signs Nasopharyngeal carcinoma causes nasal obstruction, discharge, or bleeding; deafness, tinnitus, or earache; and headache and ocular paresis from tumour spread to involve the cranial nerves. Patients may present with a single symptom caused locally by the tumour, or with several symptoms, and about one-​third complain only of cervical lymph-​node enlargement resulting from metastatic spread from an occult primary tumour. Direct spread from the primary tumour may involve the soft tissues, bone, parotid gland, buccal cavity, and oropharynx. The neoplasm may extend into the nasal fossae, the paranasal sinuses, or the orbit, and can invade the eustachian tube or the parapharyngeal space, where cranial nerves IX, X, XI, and XII can be involved. Invasion of the skull or cranial foramina may damage cranial nerves II, IV, V, and VI. Lymphatic spread causes enlarged cervical lymph nodes, and subsequently extends to the supraclavicular glands. If blood-​borne metastases occur, they are most frequent in the bones, liver, and lungs, but may be in any organ. Differential diagnosis Nasopharyngeal carcinoma must be distinguished from other tu- mours of the nasal cavities, namely adenocarcinomas, sarcomas, malignant lymphomas, and rare malignancies such as chordoma, teratoma, and melanoma. Laboratory diagnosis The diagnosis of nasopharyngeal carcinoma is made histologically on a biopsy sample of the primary tumour or an enlarged cer- vical lymph node. Serum antibody titres to EBV antigens show a characteristic reaction pattern—​IgG and IgA antibodies to VCA and diffuse early antigen are raised, with the titre correlating with the tumour burden. Uniquely, IgA antibodies to VCA and early antigen are also found in patients’ saliva. These antibody patterns often arise many months before the onset of detectable tumour growth, and have been used in a high-​incidence area of China to screen the population for incipient cases and/​or high risk individuals. Treatment Untreated nasopharyngeal carcinoma progresses inexorably to death, but it responds well to radiotherapy, which is the initial treatment of choice. In the earliest stages of the disease, radio- therapy gives 5-​year survival rates of 50% or more, and of those surviving for 5 years, 70% remain permanently free of relapse.

762 section 8  Infectious diseases Treatment schedules combining radio-​ and chemotherapy are now giving some further improvement. However, the more ad- vanced stages of nasopharyngeal carcinoma still have correspond- ingly poor prognoses. Pathogenesis EBV is now widely accepted as an essential element in the caus- ation of nasopharyngeal carcinoma. Early studies showed that 100% of undifferentiated nasopharyngeal carcinomas world- wide are EBV-​positive, and recent studies have also detected viral DNA in a subset of the more differentiated nasopharyngeal tu- mours. Thus all forms of nasopharyngeal carcinoma, irrespective of whether they originate in high-​ or low-​incidence areas, may be associated with EBV. Both the tumour cells and the EBV gen- omes within them are clonal, indicating that the malignancy arises from a single malignantly transformed EBV-​infected epithelial cell. Evidence from EBV latency genes expressed in nasopharyngeal carcinoma and premalignant lesions strongly suggest that viral gene products contribute to the abnormal epi- thelial proliferation, though the exact mechanism remains to be determined. Non​viral factors that predispose to tumour development in- clude racial and genetic predispositions; many cases among southern Chinese people show a clear familial link, and certain HLA haplotypes are associated with the disease. Epidemiological studies also suggest that environmental cofactors associated with the Chinese way of life play a role. Two likely candidates are (1) traditional herbal medicines containing tumour-​promoting phorbol ester-​type substances, taken as snuff, and (2)  trad- itional salted fish, which has been shown to contain carcinogenic nitrosamines. Salivary gland lymphoepithelioma These relatively rare tumours resemble nasopharyngeal carcinoma, both histologically and in their prevalence in circumpolar popu- lations. Although some are in reality nasopharyngeal cancers that have spread to the parotid gland from occult primaries, others are clearly of salivary gland origin. The EBV genome, which is clonal in all the malignant epithelial cells, is not found in any other type of salivary gland tumour. The association with EBV has not been suffi- ciently explored to assess its significance. Gastric carcinoma Some 8–​10 10% of gastric carcinomas worldwide are EBV genome-​positive. These include almost all of those rare gastric tumours with a lymphoepithelioma-​type morphology, and a mi- nority of the more common gastric adenocarcinomas. There is increasing evidence from molecular studies that EBV-​positive tumours constitute an aetiologically distinct subset of gastric carcinomas, with particular patterns of cellular gene expres- sion and of hypermethylated gene sets. Furthermore the viral genome is again found in monoclonal form in every cell of the EBV-​positive tumour, providing another example of a tumour arising from a single EBV-​infected cell. Viral antigen expression tends to be more limited than in nasopharyngeal carcinoma, with EBNA1 and in most cases LMP2 being detectable. As in the naso- pharyngeal tumour, EBV’s contribution to carcinogenesis remains to be determined. T-​ and NK-​cell lymphomas EBV is now also strongly linked to a particular type of T-​ or NK-​cell lymphoma that typically presents as a granulomatous, bone-​eroding, lesion. This usually occurs in the midline of the face, or as destructive lesions of the soft palate, or as multiple intranasal masses, hence the tumour’s earlier description as le- thal midline granuloma. Such tumours are more common in men than women, and in Asian and South American populations than in people of European descent. However, all cases world- wide are EBV genome-​positive and the tumour cells express a limited range of EBV latent antigens, namely EBNA1, LMP1 (in some cells), and LMP2. Other rare forms of the same lymphoma can also present in the skin, sometimes in association with hypersensitivity to mosquito bites. Yet others arise at various sites in individuals with a history of chronic active EBV infec- tion, where EBV has gained previous access to T and/​or NK-​cell lineages and has initiated oligoclonal proliferations of latently infected T or NK cells from which a monoclonal EBV-​positive tumour arises (see ‘Chronic active EBV infection’, earlier). Occasionally, cases of EBV-​positive NK-​cell leukaemia may arise ab initio. Currently, all of these malignancies are extremely difficult to treat. Other EBV-​associated conditions linked to profound immune impairment An unexpected consequence of profound T-​cell impairment, first noted in children with progressive HIV infection but now also seen occasionally in heavily immunocompromised trans- plant recipients, is the appearance of smooth muscle tumours, leiomyomas, and leiomyosarcomas that are consistently EBV genome-​positive. As yet, very little is known about how the virus accesses such an unusual cell type or how it contributes to its ma- lignant conversion. A second EBV-​related disease of the immunocompromised state, again first noted in AIDS patients but also now seen occa- sionally in the posttransplant setting, is oral hairy leukoplakia. This is a non-​malignant lesion that typically presents as painless white patches on the tongue or the lateral buccal mucosa. The le- sions are usually multiple, up to 3 cm in diameter, slightly raised, poorly demarcated, and have a hairy or corrugate surface. The differentiating squamous epithelial cells forming the outer layers of these lesions contain large amounts of actively replicating EBV, providing the only example of a disease resulting from productive infection by the virus. Though the differentiation-​dependence of this EBV replicative lesion superficially resembles that shown by

8.5.3  Epstein–Barr virus 763 papillomaviruses, there is a crucial difference in that EBV appears to be confined to the outer epithelial layers and there is no de- tectable infection, either latent or lytic, in the basal or immediate suprabasal layers. It is not clear whether oral hairy leukoplakia simply magnifies a process of EBV replication occurring sublim- inally in all virus carriers, or is an artefact of the heavily immuno- compromised state. In affected individuals, aciclovir treatment arrests virus replication and the leukoplakia lesions regress, but only for as long as the drug is continued. EBV and autoimmune conditions Recent years have seen a reawakening of interest in possible links between EBV and autoimmune disease, with the epidemiological findings strongest in the case of multiple sclerosis. Firstly a his- tory of infectious mononucleosis increases lifetime risk of mul- tiple sclerosis by 2 to 3-​fold. Secondly almost all (>99%) multiple sclerosis patients are EBV-​seropositive (i.e. already EBV-​infected) at the time of disease presentation compared to 90–​95% matched healthy controls. Thirdly, a large prospective study found that, of 305 military recruits who subsequently developed multiple sclerosis, only 10 were EBV-​seronegative at recruitment and all 10 seroconverted 2–​6 years before disease presentation. Whether such evidence truly reflects a role for EBV in the pathogenesis of multiple sclerosis, and what that role might be, remain hotly de- bated questions. Prospects for a prophylactic EBV vaccine Given the wide range of non​malignant and malignant diseases with which EBV is aetiologically linked, a vaccine able to protect against infection would have an enormous global impact. Efforts in that direction have re-​doubled following the development of successful vaccine against the oncogenic human papillomavirus types 16 and 18. Whether the papillomavirus vaccine strategy, based on the induction of virus-​neutralizing antibodies, will be effective in the context of EBV, a herpesvirus with a more com- plex biology and different transmission route, remains unclear. Human trials of a new vaccine, using a multimeric form of the major EBV envelope glycoprotein gp350 as immunogen, will soon begin to resolve that question. FURTHER READING Ascherio A, Munger KL (2010). Epstein–​Barr virus infection and multiple sclerosis:  a review. J Neuroimmune Pharmacol, 5,
271–​7. Ascherio A, Munger KL (2015). EBV and autoimmunity. Curr Top Microbiol Immunol, 390, 365–​85. Balfour HH, et  al. (2013). Behavioral, virologic, and immuno- logic factors associated with acquisition and severity of primary Epstein–​Barr virus infection in university students. J Infect Dis, 207, 80–​8. Bar RS, et al. (1974). Fatal infectious mononucleosis in a family. N Engl J Med, 290, 363–​7. Burkitt D (1958). A sarcoma involving the jaws of African children. Br J Surg, 46, 218–​3. Burkitt D (1963). A lymphoma syndrome in tropical Africa. Int Rev Exp Pathol, 2, 67–​138. Cohen JI, et al. (2011). Epstein–​Barr virus: an important vaccine target for cancer prevention. Sci Transl Med, 3, 107fs7. de Thé G, et al. (1978). Epidemiological evidence for a causal relation- ship between Epstein–​Barr virus and Burkitt’s lymphoma: results of the prospective Ugandan study. Nature, 274, 756–​61. Dharnidkana VR, et al. (eds) (2010). Post-​transplant lymphoprolifera- tive disorders. Springer-​Verlag, Berlin Heidelberg. Dunmire SK, et  al. (2015). Infectious mononucleosis. Curr Top Microbiol Immunol, 390, 211–​40. Epstein MA (2001). Historical background. Philos Trans R Soc Lond B Biol Sci, 356, 413–​20. Epstein MA, Achong BG, Barr YM (1964). Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet, i, 702–​3. Gottschalk S, Rooney CM, Heslop HE (2005). Post-​transplant lymphoproliferative disorders. Annu Rev Med, 56, 29–​44. Greenspan JS, et al. (1985). Replication of Epstein–​Barr virus within the epithelial cells of oral hairy leukoplakia, an AIDS-​associated le- sion. N Engl J Med, 313, 1564–​71. Henle G, Henle W, Diehl V (1968). The relation of Burkitt’s lymphoma tumor-​associated herpesvirus to infectious mononucleosis. Proc Natl Acad Sci (USA), 59, 94–​101. Hislop AD, et al. (2007). Cellular responses to virus infection in hu- mans:  lessons from Epstein–​Barr virus. Annu Rev Immunol, 25, 587–​617. Hjalgrim H, et al. (2003). Characteristics of Hodgkin’s lymphoma after infectious mononucleosis. N Engl J Med, 349, 1324–​32. Hoagland RK (1955). Transmission of infectious mononucleosis. Am J Med Sci, 229, 262–​72. Kanekiyo M, et  al. (2015). Rational design of an Epstein–​Barr virus vaccine targeting the receptor-​binding site. Cell, 162,
1090–​100. Kuppers R (2003). B cells under influence: transformation of B cells by Epstein–​Barr virus. Nat Rev Immunol, 3, 801–​12. Lee AW, et al. (2015). Management of nasopharyngeal carcinoma:
current practice and future perspective. J Clin Oncol, 33,
3356–​64. Levin LI, et al. (2010). Primary infection with the Epstein–​Barr virus and risk of multiple sclerosis. Ann Neurol, 67, 824–​30. Munz C (ed) (2015). Epstein–​Barr virus (volumes 1 and 2). Current Topics in Microbiology and Immunology 390, 1–​391, and 391, 1–​505. Palendira U, Rickinson AB (2015). Primary immunodeficiencies and the control of Epstein–​Barr virus infection. Ann N Y Acad Sci, 1356, 22–​44. Schlossberg D (ed) (1989). Infectious mononucleosis, 2nd edition. Springer-​Verlag, Berlin. Sprunt TP, Evans FA (1920). Mononuclear leucocytosis in reaction to acute infections (‘infectious mononucleosis’). Bulletin of the Johns Hopkins Hosp, 31, 410–​17. Taylor GS, et  al. (2015), The immunology of Epstein–​Barr virus-​ induced disease. Annu Rev Immunol, 33, 787–​821. Torre D, Tambini R (1999). Acyclovir for treatment of infectious mononucleosis: a meta-​analysis. Scand J Infect Dis, 31, 543–​7. Vouloumanou EK, et al. (2012). Current diagnosis and management of infectious mononucleosis. Curr Opin Hematol, 19, 14–​20. Young LS, Rickinson AB (2004). Epstein–​Barr virus: 40 years on. Nat Rev Cancer, 4, 757–​68.

8.5.4 Poxviruses 764

8.5.4 Poxviruses 764

764 section 8  Infectious diseases 8.5.4  Poxviruses Geoffrey L. Smith ESSENTIALS Poxviruses are large, complex DNA viruses that have played sev- eral seminal roles in medicine and biological science. Cowpox virus was introduced by Jenner as the first human vaccine in 1796; widespread vaccination with vaccinia virus led to the global eradi- cation of smallpox in 1977, the only human disease to have been eradicated. Smallpox—​is caused by variola virus, the most infamous poxvirus. A systemic infection, spread by the respiratory route, with character- istic skin blisters that had a centrifugal distribution on the body and, with variola major, produced mortality rates of 30–​40% in unvaccin- ated populations. Other poxviruses—​molluscum contagiosum virus is the only other poxvirus that infects only humans, causing benign skin tumours that may be single or multiple, typically persisting for months be- fore undergoing spontaneous regression (see Chapter  8.5.28). Several other poxviruses may cause zoonotic infections in hu- mans, including cowpox virus, vaccinia virus, monkeypox virus, orf virus, psuedocowpox virus, tanapox virus, and Yaba monkey tumour virus. The development of vaccinia virus as an expression vector pion- eered the concept of using genetically engineered viruses as live vac- cines. Vaccinia virus is also being developed as an oncolytic agent. Poxviruses remain excellent models for studying virus-​host inter- actions and virus immune evasion strategies. Introduction Poxviruses are large DNA viruses that replicate in the cell cyto- plasm. The most infamous was variola virus, which caused smallpox, a disease responsible for devastating epidemics with up to 40% mortality and that influenced human history. Smallpox was eradicated (in 1977)  by immunoprophylaxis with vaccinia virus, a related orthopoxvirus. Since then, poxvirus infections in humans have been restricted to molluscum contagiosum (Chapter  8.5.28) and rare zoonoses caused by monkeypox, cowpox, orf (Chapter 8.5.27), pseudocowpox, tanapox, and Yaba monkey tumour viruses. Classification The Poxviridae is divided into the Entomopoxvirinae and Chordopoxvirinae subfamilies whose members infect insects and chordates, respectively. The Chordopoxvirinae is subdivided into ten genera and additional unassigned viruses (Table 8.5.4.1). Viruses within different genera are antigenically distinct, while those within a genus are cross-​reactive and cross-​protective. Orthopoxviruses have been the most important for humans (Table 8.5.4.1), and four of the nine poxviruses that infect humans are orthopoxviruses: cowpox, variola, monkeypox, and vaccinia viruses. Different orthopoxviruses are distinguishable by their biological properties, such as pock type and ceiling temperature on the chorioallantoic membrane, or by the restriction pattern of genomic DNA and the genome sequences that have enabled de- velopment of species-​specific polymerase chain reaction detection methods. Vaccinia virus has no known natural animal reservoir and its origin remains a mystery. It caused human disease only as a rare complication after vaccination against smallpox. Cowpox and monkeypox viruses were named after the species from which they were isolated, but the natural reservoir of each virus is rodents. Infections in cows or monkeys, like the occasional transmission to humans, are zoonoses. Human monkeypox virus infections are often caused by handling or consumption of infected ‘bush meat’. In 2003, there was an outbreak of monkeypox in the United States of America following the importation of Gambian rodents carrying the virus and, in 2018, a small outbreak occurred in the UK, having been imported from Nigeria. Cowpox, monkeypox, and vaccinia Table 8.5.4.1  Poxvirus classification Subfamily Genus Species Entomopoxvirinae Chordopoxvirinae Orthopoxvirus a,bVaccinia virus bVariola virus bMonkeypox virus bCowpox virus Ectromelia virus Camelpox virus Taterapoxvirus Raccoonpox virus Skunkpox virus Volepox virus Avipoxvirus aFowlpox virus Canarypox virus Capripoxvirus aSheepox virus Goatpox virus Lumpy skin disease virus Cervidpoxvirus aMule deerpox virus Crocodylidpox virus aNile crocodilepox virus Leporipoxvirus aMyxoma virus Rabbit fibroma virus Molluscipoxvirus a,bMolluscum contagiosum virus Parapoxvirus b*Orf virus bPseudocowpox virus Suipoxvirus aSwinepox virus Yatapoxvirus bTanapox virus bYaba monkey tumour virus Unassigned Squirrelpox virus a denotes the prototype for each genus. b denotes those viruses that infect humans.

8.5.4  Poxviruses 765 viruses have a broad host range, while variola virus infected only humans and the lack of an animal reservoir aided the smallpox eradication campaign. Camelpox virus is another orthopoxvirus and, like variola virus, is restricted to a single host species in which it can cause serious disease. Poxvirus biology Poxviruses replicate in the cytoplasm, encode enzymes for tran- scription and DNA replication, and have large, complex vir- ions (Fig. 8.5.4.1) and double stranded DNA genomes of 134 to 360 kbp. Vaccinia virus is the most intensively studied poxvirus. It encodes about 200 genes (the exact number varying with the strain of virus) of four classes (early 1, early 2, intermediate, and late) that are expressed in a strictly regulated manner. Transcription of each class is dependent upon the prior expression of the previous class. Virus morphogenesis is complex (Fig. 8.5.4.2a) and produces two forms of infectious virion:  intracellular mature virus and extracellular enveloped virus. Some authors refer to these virions as mature virus and extracellular virus, respectively. Intracellular mature virus particles remain within the cell until cell lysis and form most of the progeny, whereas extracellular enveloped virus is released by exocytosis (Fig. 8.5.4.2b) before cell death and rep- resents a small fraction of total infectivity. The extracellular en- veloped virus possesses an additional lipid envelope with which several virus proteins are associated, giving it distinct immuno- logical and biological properties. An extracellular enveloped virus is necessary for efficient virus dissemination in vitro and within the infected host. Immunity to extracellular enveloped virus-​specific antigens, which are highly conserved among orthopoxviruses, is required for protection against disease and the B5 protein on the surface is an important target against which neutralizing Abs are directed (Putz et al., 2005). Pathogenesis Poxvirus infections cause a local skin lesion or generalized pus- tular rash. Detailed experimental analysis of human smallpox was impossible, but generalized poxvirus infections have been studied in experimental models, namely monkeypox in monkeys, rabbitpox (a neurovirulent vaccinia virus) in rabbits, ectromelia virus in mice, and myxoma virus in European rabbits. The Fig. 8.5.4.1  Electron micrograph of material from a smallpox lesion, viewed by negative contrast, showing a clump of variola virus particles. Courtesy of the late Henry Bedson. (a) (b) Fig. 8.5.4.2  Electron micrographs showing (a) a cytoplasmic vaccinia virus factory containing maturing virus particles with stages of morphogenesis numbered 1 to 4 and (b) fully enveloped virus particles, one of which (number 2) is leaving the cell by exocytosis.

766 section 8  Infectious diseases spread of variola virus in humans was probably similar to that of ectromelia virus in mice, and is characterized by sequential phases of virus infection, replication and release, accompanied by cell necrosis. Studies with vaccinia virus showed that a fusion complex on the surface of intracellular mature virus particles consisting of more than 10 virus proteins is essential for virus entry into cells. Both intracel- lular mature virus and extracellular enveloped virus (after it has shed its outer membrane) can fuse with the host cell membrane at either the cell surface or within acidified vesicles, but all routes require the intracellular mature virus fusion complex. Virus enters through skin abrasions (ectromelia and cowpox) or inhalation of airborne virus and establishes a respiratory infection (ectromelia, rabbitpox, and variola). In smallpox, the respiratory route was the most important and sometimes the only possible route of transmission from index cases to contacts; also patients became infectious only after enanthem developed. A respiratory infection was established in the epithelial cells of the alveoli and small bronchioles. Here, alveolar macrophages became infected and transmitted the virus via lymphatics to the local lymph node, where further virus replication occurred. Virus released into the blood (primary viraemia) was mostly cell-​associated and spread to other organs of the reticuloendothelial system, notably the liver, spleen, and lymph nodes. Extensive replication here released larger amounts of virus into the blood (secondary viraemia) enabling the virus to infect other organs such as the kidneys, lungs, and intestines and to reach the skin and produce the skin lesions with the characteristic centrifugal distribution (Figs. 8.5.4.3–​8.5.4.5). Lesions started with a papule that became pustular and then crusted. After 2–​3 weeks the scab was shed, leaving a scar. The incubation period of smallpox was approximately 12 days. Symptoms included headache, fever, mal- aise, vomiting, and, in severe cases, prostration, toxaemia, and hypo- tension. Delayed onset of the skin eruptions usually correlated with a grave prognosis. Haemorrhagic or flat confluent-​type smallpox had very high mortality rates. The outcome of infection depended upon the age and physio- logical and immunological status of the patient and the strain of virus. Variola major was more virulent and produced fatality rates in unvaccinated patients of between 5 and 40%, while the milder variola minor, called alastrim in the Americas, caused only 0.1–​2% mortality. Morphologically, the viruses were indistinguishable and vaccination with vaccinia virus was equally effective against both. However, alastrim virus was consistently more thermolabile and had a lower ceiling temperature of 37.5°C compared to 38.5°C for variola major, 39°C for monkeypox, 40°C for cowpox, and 41°C for vaccinia virus. The genomes of nearly 50 variola virus strains isolated from different places in the world at different times have been sequenced and compared, allowing the spread and evolution of variola virus in humans to be analysed. Comparisons of variola major and minor virus strains showed the genomes are very closely related, but there are too many minor differences to provide an understanding of why these viruses produced such different mor- tality rates in humans. Very young and old patients were most susceptible to smallpox and those aged 5–​20 years most resistant. Pregnancy and immunological Fig. 8.5.4.3  Smallpox in a 9-​month-​old boy in Pakistan, photographed on the eighth day of the rash. Courtesy of the World Health Organization. (a) (b) Fig. 8.5.4.4  Ethiopian patient, in 1968, showing classical centrifugal distribution of lesions with fewer on trunk (a) than on face (b). Copyright D. A. Warrell.

8.5.4  Poxviruses 767 deficiency, particularly in cell-​mediated immunity, increased the se- verity of infection. Pregnant women were more likely than any other group to develop haemorrhagic-​type smallpox, which was usually fatal. The greater importance of cell-​mediated immunity rather than antibody in recovery from poxvirus infections was illustrated in sev- eral ways. Firstly, in children with severe defects in cell-​mediated immunity there was a progressive and uncontrolled virus replication from the vaccination site that was usually fatal. In contrast, defects in antibody production were usually tolerated if the cell-​mediated immune response was normal. Secondly, passive administration of antivaccinia virus serum had little effect on mice infected with ectromelia virus, whereas prior infection with vaccinia virus was protective. Thirdly, in mice infected with ectromelia virus, the ef- fective mechanisms that combated infection in the liver and spleen were operative by 4–​6 days postinfection and coincided with the maximum levels of cytolytic T cells, but preceded the development of systemic antibody. The eradication of smallpox Early attempts to control smallpox relied upon variolation or in- oculation, in which material isolated from a mild case of smallpox was administered by sniffing or scratching. This was replaced by vaccination in 1798 after Jenner noticed that milkmaids, who often acquired cowpox infections on their hands from the teats of cows, were protected from smallpox. Jenner infected a boy (James Phipps) with poxvirus material (probably cowpox) derived from a cow via a milkmaid (Sarah Nelmes) and challenged him subsequently with smallpox. Protection was achieved and, due to the efficacy and greater safety of this procedure, it replaced variolation rapidly. Sometime between 1798 and the 20th century, vaccinia virus re- placed cowpox virus as the smallpox vaccine. In 1959, the World Health Assembly (WHA) adopted a recommendation to achieve the global eradication of smallpox. With fresh funding and a plentiful supply of potent freeze-​dried vaccine this goal was achieved in 1977. Two years later, the World Health Organization (WHO) certified that eradication was complete. This triumph of preventive medicine justifies the saying ‘prevention is better than cure’, but also demon- strates that prevention is best achieved by eradication. Posteradication issues Following the eradication of smallpox the WHO sought to centralize all known stocks of variola virus and these stocks are now held in maximum security laboratories (Biosafey Level 4)  in the United States and the Russian Federation. These facilities are inspected regu- larly by the WHO and any work with live virus requires prior per- mission from the WHO. In 1996 the WHA passed a resolution that these stocks of virus should be destroyed. Since then temporary re- tention of these viruses has been authorized to enable research that is essential for public health benefit to be completed. This research aims to develop (1) diagnostic tests that can swiftly identify cases of smallpox and distinguish this from infections caused by other vir- uses; (2) drugs that can treat smallpox; and (3) a safer vaccine that can be tolerated by those for whom the current vaccine is contra- indicated. Good progress towards these objectives has been achieved. Poxvirus genomes The DNA sequence of more than 100 orthopoxvirus genomes has been determined (see http://​www.poxvirus.org), including about 50 strains of variola virus and at least one strain of most orthopoxviruses. The central region (about 100 kb) of these genomes is very highly conserved and 89 of the genes within this region are present in every sequenced chordopoxvirus. These genes probably represent the core genome of an ancestral poxvirus from which the current poxviruses evolved. During their evolution poxviruses acquired additional genes that became located in the more variable terminal regions of the genome and these give each virus its characteristic host range, virulence, and tropism. These genes vary in number and type be- tween poxviruses, and encode non​essential proteins that affect virus virulence, host range, and immune modulation. A surprising feature of some orthopoxviruses is the fragmentation of several genes that are intact in other viruses, indicating that orthopoxvirus evolution has involved both gain and loss of gene function. The retention of these non​functional genes by some viruses, such as variola, suggests that they became non​functional in the relatively recent evolutionary past, and perhaps that variola virus is a ‘recent’ human pathogen that never became fully adapted to humans. In 2016, the sequence of a variola virus genome was obtained by sequencing DNA from a mum- mified child from Lithuania who died in about 1650. This sequence is evolutionarily basal to all other variola genomes, contains the same pattern of gene degradation found in 20th century variola viruses, and suggests that the genetic diversification of variola virus in man was more recent that thought hitherto (Duggan et al., 2016). Fig. 8.5.4.5  Moderately severe monkeypox in a girl of 7 years from Équateur Province, Democratic Republic of the Congo. Courtesy of the World Health Organization.

768 section 8  Infectious diseases Recreation of poxviruses by synthetic biology Since 2012 it has been possible to recreate an infectious poxvirus from cloned DNA (Domi et al., 2002). This was achieved by trans- fecting the cloned virus genome into cells that are infected with a helper poxvirus. Furthermore, advances in synthesis of DNA have enabled recreation of an entire poxvirus genome from simple chem- icals and from this genome to recreate infectious virus. Given that the genome sequence of variola virus is in the public domain, it is therefore possible to recreate an infectious variola virus from simple chemicals. Although such activity is prohibited absolutely by the WHO, the fact that this is possible means that the potential for variola virus and smallpox to reappear can never be completely eliminated. Poxvirus expression vectors Vaccinia virus recombinants expressing foreign genes were developed in 1982 and have become a widely used laboratory tool; they are also being engineered as live vaccines for infectious disease and cancer. Infection with the recombinant virus allows expression and simultan- eous delivery of the foreign antigen to the immune system. Moreover, the large capacity of vaccinia virus allows expression of multiple for- eign genes from a single virus so creating polyvalent vaccines. Safer vaccinia virus strains that do not cause vaccination complications (ec- zema vaccinatum, generalized vaccinia, progressive vaccinia, enceph- alopathy (<2 years), or encephalitis (>2 years)) are being created by genetic engineering. An alternative strategy is to use poxviruses that establish only abortive infections in human cells, such as modified vac- cinia Ankara or the avipoxviruses fowlpox virus and canarypox virus. Human monkeypox Monkeypox was discovered in captive primates in 1958, but in 1970 was isolated in the tropical rainforests of West and Central Africa from humans who had suffered generalized poxvirus rashes visibly very similar to smallpox. The virus is quite distinct from variola in biological properties such as pock morphology, ceiling temperature, and lesion morphology on rabbit skin, and its genome sequence. Moreover, although monkeypox virus produced a very similar dis- ease to smallpox in humans, person-​to-​person transmission was inefficient. Thus, human monkeypox virus infections are single or multiple sporadic cases restricted to dense tropical rainforests in Central and West Africa. Clinically, human monkeypox closely resembles ordinary, discrete-​type smallpox except that there is a pronounced lymph node enlargement (Fig. 8.5.4.5). Two clades of monkeypox virus have been identified (from Central or West Africa) that differ in their virulence in humans. The Central African strains gave mortality rates in unvaccinated children (<8 years old) between 1970 and 1986 of 11.2%. In contrast, West African strains, such as the one that caused an epidemic in the United States of America in 2003, are milder and no mortalities were reported. Prevention and treatment In endemic parts of Africa, or in the face of new epizootics, use of current smallpox vaccine has been discussed, but the prevalence of HIV/​AIDS in some areas would restrict widespread, safe use of this approach. Cidofovir treatment cured monkeys infected with monkeypox virus. Cowpox virus and pseudocowpox virus Cowpox virus has a broad host range including cattle, humans, large felines, and even elephants, but it is not enzootic in cattle and its nat- ural hosts are rodents. It is distinguishable from vaccinia virus by the pock type, ceiling temperature, genome size and sequence, and the production of cytoplasmic type A inclusion bodies. Pseudocowpox is enzootic in cattle, unlike cowpox. Historically, pseudocowpox virus was important because it was sometimes used mistakenly for vaccination and, being a parapoxvirus, was ineffective in preventing smallpox. Its misuse compromised Jenner’s correct assertion that cowpox virus was an effective smallpox vaccine. In humans, cowpox virus produces an acutely inflamed, local lesion, similar to a primary smallpox vaccination. There is usually fever, enlargement of the local lymph nodes, and pain. Unlike vac- cinia virus, which occasionally produced a generalized infection (Fig. 8.5.4.6), cowpox virus lesions are always local. Human lesions caused by pseudocowpox virus (milker’s nodules) are extremely rare and are less painful than those caused by cowpox. Tanapox virus and Yaba monkey tumour virus Tanapox virus and Yaba monkey tumour virus are the sole members of the Yatapoxvirus genus (another yatapoxvirus called Yaba-​like Fig. 8.5.4.6  Generalized vaccinia. Courtesy of the late Dr B. E. Juel-​Jensen.

8.5.5 Mumps Epidemic parotitis 769

8.5.5 Mumps: Epidemic parotitis 769

8.5.5  Mumps: Epidemic parotitis 769 disease virus, is considered a tanapox virus strain). These viruses rep- licate slowly in cell culture and cause zoonotic infections in humans. Tanapox virus was isolated in the Tana valley in Kenya (1957–​1962) from humans suffering from localized skin lesions typical of pox- viruses (Fig. 8.5.4.7). Probably the virus is transmitted from infected monkeys by biting insects, particularly during wet weather condi- tions. Usually it produces a solitary lesion that is preceded for a few days by a mild fever. The lesion takes 5 to 6 weeks to clear and is dis- tinguished from other poxvirus lesions by its failure to become pus- tular. This virus cannot be cultured on the chorioallantoic membrane. Yaba monkey tumour virus was discovered in Yaba, Lagos, Nigeria in 1957 as a virus causing cutaneous histiocytomas in rhesus mon- keys and can infect humans if injected subcutaneously or intrader- mally. The lesions are not neoplastic and are cleared by the immune response. Cutaneous poxviruses (Orf virus and molluscum contagiosum virus) See Chapters 8.5.27 and 8.5.28. FURTHER READING Andrei G, Snoeck R (2010). Cidofovir activity against poxvirus infec- tions. Viruses, 2, 2803–​30. Damon IK (2011). Status of human monkeypox clinical disease, epi- demiology and research. Vaccine, 29 Suppl 4, D54–​9. Di Giulio DB, Eckburg PB (2004). Human monkeypox: an emerging zoonosis. Lancet Infect Dis, 4, 15–​25. Domi A, Moss, B (2002). Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells. Proc Natl Acad Sci USA, 99, 12415–​20. Duggan A, et al. (2016). 17th century variola virus reveals the recent evolution of smallpox. Current Biol, 26, 3407–​12. Fauquet CM, et  al. (eds) (2005). Virus taxonomy:  eighth report of the international committee on the taxonomy of viruses. Elsevier, Amsterdam. Fenner F, et  al. (1988). Smallpox and its eradication. World Health Organization, Geneva. Fenner F, Wittek R, Dumbell KR (1989). The orthopoxviruses. Academic Press Ltd, London. Hwenda L, Larsen BI (2011). The remaining smallpox stocks:  the wrong debate? Lancet, 378, e7; author reply e7. Mercer AA, Schmidt A, Weber O (2007). Poxviruses. Berhäuser-​ Verlag, Berlin. Moss B (2007). Poxviridae: the viruses and their replication. In: Knipe DM, et  al. (eds) Field’s virology, 5th edition, 2, pp. 2905–​46. Lippincott Williams & Wilkins, Philadelphia, PA. Moss, B (2012). Poxvirus cell entry: how many proteins does it take? Viruses, 4, 688–​707. Putz M, et al. (2006). Quantification of antibody responses against multiple antigens of the two infectious forms of vaccinia virus provides a benchmark for smallpox vaccination. Nature Med, 12, 1310–​15. Williams G (2010). Angel of death:  the story of smallpox. Palgrave Macmillan, Basingstoke. 8.5.5   Mumps: Epidemic parotitis B.K. Rima ESSENTIALS Mumps is an acute, systemic, highly infectious, communicable in- fection of children and young adults, caused by a paramyxovirus (with an RNA genome). Transmission is by airborne droplet spread. After an incubation period of 14 to 18 days, typical presentation is with fever, pain near the angle of the jaw, and swelling of the parotid glands. Complications include orchitis, meningitis, and encephalitis. Diagnosis is obvious clinically in cases with a con- tact history and parotitis, but serological (mumps-​specific IgM and IgA) and RNA-​based (RT-​PCR) tests are used when this is not the case (e.g. the patient presenting with meningitis). Treatment is symptomatic. Prevention is by vaccination, often given as one component of a trivalent mumps/​measles/​rubella vaccine at 14 to 16 months of age. A follow-​up vaccination is now recommended at 4–​5 years of age. Introduction and historical perspective The primary clinical manifestation in mumps, swelling of the salivary glands, is so characteristic that the disease was recognized very early as different from other childhood illnesses. Hippocrates described the disease in the 5th century BC and also noted swelling of the testes (orchitis) as a common complication of mumps. In 1790, Hamilton noted the infection in the central nervous system and meninges. In 1934, mumps was shown to be a filterable virus by Johnson and Goodpasture, who also fulfilled Koch’s postulates by infecting volun- teers with virus propagated in monkeys. Since 1967, live attenuated vaccines have been licensed to control and prevent the infection. Fig. 8.5.4.7  Tanapox lesion on the leg of a Kenyan patient. Courtesy of the late P. E. C. Manson-​Bahr.

770 section 8  Infectious diseases Aetiology and genetics Mumps virus (MuV) can be grown in tissue cultures of chick em- bryo, monkey kidney, and most human cells. The virus can also be cultured in the yolk sac or embryonic cavity of chick embryos. Cytopathic changes (syncytium formation and cell rounding) may be seen as early as 24 h postinfection and earlier if immunofluor- escence is used. MuV is thermolabile. It can be stored for years at –​70°C, but infectivity is lost in a few days at room temperature. Treatment with ether or paraformaldehyde inactivates the virus rap- idly, but does not destroy the antigens responsible for reactivity in classical and no longer routinely performed tests such as comple- ment fixation, haemagglutination, or reactivity in the skin. MuV is an enveloped RNA virus with a genome of 15 384 nu- cleotides. Its inner core is a ribonucleoprotein complex (the nu- cleocapsid) containing the non​segmented, negative-​strand, RNA molecule encapsidated by the nucleocapsid protein (N). The nucleocapsid has the herringbone structure characteristic of paramyxoviruses (Fig. 8.5.5.1a). Attached to this are two further proteins involved in transcription and replication of the RNA genome: the phosphoprotein (P) and the large replicase protein (L). The nucleocapsid is surrounded by a lipid bilayer (Fig. 8.5.5.1a, b). On the inner leaflet is a membrane or matrix protein (M) that plays an essential role in virus budding. On the outer surface are two glycoproteins, one carrying the haemagglutinin-​neuraminidase activity (HN), the other is the fusion protein (F). A complex be- tween the HN and F proteins is responsible for the fusion of the virion membrane with that of the host cell. The function of a non-​ structural, small hydrophobic protein (SH) is unknown; it is asso- ciated with the endoplasmic reticulum in MuV-​infected cells. The SH protein sequence is hypervariable and this is used to assign MuV strains to one of 12 currently recognized genotypes. The non-​ structural V protein functions in combating the host’s innate im- mune response by targeting STAT 1 and STAT 3 for degradation. The gene order (Fig. 8.5.5.1c) leads to an expression gradient in which the abundance of mRNAs decreases with increasing distance to the promoter at the 3'-​terminal end of the genome, so that the N mRNA is more abundant than the L mRNA. Epidemiology and pathogenesis Mumps is highly infectious. Transmission depends on close per- sonal contact with a patient who is excreting virus in the saliva and spreading it in droplets. In the prevaccine era, the peak incidence was in the late winter or early spring, in 3–​7-​year cycles. Most morbidity is associated with meningitis and orchitis. Case fatality is about 2 per 1000. The incubation period lies between 14 and 18 days. In any outbreak, 30 to 40% of those infected have subclinical illness. MuV causes an infection of the upper respiratory tract that spreads to draining lymph nodes. The subsequent viraemia and infection of lymphocytes and macrophages causes spread to many organs, but because mumps is so rarely lethal, details are scant. Mumps virus can spread to most organs in the body. Lymphocytic infiltration and (b) HN RNA L P N M F (c) M N Genome 3' 5' N H SH P / V 549 224 170 375 102 436 57 582 2261 F2 F1 54 1906 3226 4481 6209 6522 15384 8428 L 391 (a) Fig. 8.5.5.1  Structure and genome organization of the mumps virus: (a) a disrupted, negatively stained, mumps virion. The viral nucleocapsid protrudes from the particle and the fringe of
viral spikes is visible (bar = 100 nm); (b) diagram of the localization of the nucleocapsid (N), phospho-​ (P), large (L), matrix (M), haemagglutinin-​neuraminidase (HN), and fusion (F) proteins in the mumps virion; and (c) structure of the genome of mumps virus indicating the localization of the genes, the nucleotide number of their starting and stopping position, and (in boxes) the number of amino acid residues in each of the viral proteins.

8.5.5  Mumps: Epidemic parotitis 771 destruction of periductal cells lead to blockage of the ducts both in salivary glands and in the seminiferous tubules of the testes. The lymphatics in the tissues surrounding and overlying the parotid glands become obstructed, producing a gel-​like oedema that may spread down over the chest wall, especially when the swelling of the salivary glands is severe. Rarely, mumps causes hydrocephalus by destruction of the lining of the aqueduct. Clinical features and diagnosis Parotitis A patient with mumps parotitis may have a fever without rigors (40–​ 40.5°C) as well as pain near the angle of the jaw. The face and neck become distorted with swelling. The skin over the gland is hot and flushed but there is no rash, unlike in the swelling of erysipelas. If the swelling is severe, the mouth cannot be opened for pain and tight- ness, and is dry because the flow of saliva is blocked. This lasts for 3 or 4 days. Sometimes, as one side clears, the parotid on the other side swells. When there is bilateral parotitis, clinical diagnosis is usu- ally obvious. One condition that must be excluded is bull neck diph- theria (Chapter 8.6.1), which can look very like mumps. Rarely, the submaxillary and sublingual salivary glands may also be affected. The symptoms are similar to those in parotitic mumps, but it is difficult or impossible to distinguish the swelling from other forms of submaxillary swellings, especially inflammation of various groups of lymph nodes and Ludwig’s angina. In mumps, the neck swelling is ill-​defined, and the angle of the jaw is impalpable. To de- termine if cervical lymph nodes are swollen from some other cause, the pharynx must be examined carefully. The fauces must be exam- ined for signs of tonsillitis that might cause cervical adenitis. The lymph nodes in contact with the submaxillary and sublingual sal- ivary glands drain the corner of the eye, the side of the nose, the cheeks, the lips, and the floor of the mouth, all of which must be explored, before a diagnosis of submaxillary or sublingual mumps can be made. Laboratory tests are needed to confirm the diagnosis. Other alternative diagnoses need to be considered. In infectious mononucleosis, the glands stand out distinctly and the parotid is not affected. In septic parotitis there is more parotid tenderness; there may be fluctuation, and pus exudes from the orifice of Stensen’s duct. Calculus causes spasmodic pain and swelling and may be detected radiographically. Recurrent parotitis and Mikulicz’s syndrome are unlikely to be confused with mumps except in the earliest stages, nor are uveoparotid fever and tumours of the gland, as they are chronic conditions. Orchitis Orchitis may occur 4 or 5 days after the onset of parotitis. Quite often it occurs without preceding parotitis. It is an acute condition, with chills, sweats, headache, and backache, and a swinging temperature as well as severe local testicular pain and tenderness. The scrotum is swollen and oedematous, and the testicles are impalpable. Usually, only one testicle is affected but sometimes both: the second testicle may become affected just as the swelling of the first is subsiding. The illness lasts 3 or 4 days before the swelling begins to subside. Orchitis is unusual before the age of puberty, though it has occurred in young boys and even in infants. In adolescent and young males it develops in 1:5 cases. Some degree of atrophy of the testicle occurs in at least one-​third of patients with orchitis. Azoospermia after mumps is rare and only temporary. The fear of sterility after mumps orchitis has been exaggerated and one can reassure the patient. Orchitis when it occurs without parotitis is difficult to distinguish from gonococcal epididymo-​orchitis, unless there has been contact with mumps. The rare case of orchitis in infancy may resemble torsion of the testis and perhaps it is safer to operate than risk a serious misdiagnosis. Meningitis and encephalitis MuV frequently invades the nervous system: changes in the elec- troencephalogram and increased levels of protein and lymphocyte levels in the cerebrospinal fluid can be shown in at least half the pa- tients. However, in most cases, neurological symptoms or signs are absent. Mumps virus was one of the most common known causes of lymphocytic meningitis. This may develop a few days after the start of parotitis, but almost as often it occurs in the absence of parotitis. Occasionally, the patient develops transient paralysis of limbs re- sulting in the occurrence of quadriplegia or single nerve paralysis in some patients. Polyneuritis, neuritis of the trigeminal or facial nerve, and retrobulbar optic neuritis have been described in mumps but all are rare. The meningitis is usually mild and self-​limiting. Mumps encephalitis is a different entity; cerebrospinal fluid is normal and contains no virus. The outlook is different. The patient is confused and may lapse into coma and remain comatose for days, weeks, or months. Almost 2% of the encephalitis cases are fatal. At autopsy there is perivascular demyelination as in other forms of postinfectious encephalitis (Chapter 24.11.2). Other complications Deafness is reported in up to 0.3% of the cases, but it is rarely per- manent and often unilateral. Women sometimes complain of ovarian pain during an attack of mumps, but it is rarely as severe as in men with orchitis. There is no evidence that it affects fertility. Mastitis occurs in 15% of the cases, both in men and women, but it is usually mild and fleeting. Mild upper abdominal pain in about 50% of the cases may be related to viral changes in the pancreas. The amount of amylase in duodenal fluid may be less than normal. This is probably caused by a blockage of the ducts in the pancreas. Although there are anecdotal reports of diabetes occurring after an attack of mumps, there is no virological or immunological evidence for a direct link though the virus is known to be able to infect the pancreatic islet B cells. Mumps in the fetus and infant Abortion may occur in women with mumps in the first trimester of pregnancy. It is not common and probably not caused by direct viral damage to the fetus. The connection between primary endocar- dial fibroelastosis and mumps remains vague. The disease’s declining incidence has been attributed to mumps vaccination. Some studies using reverse transcription polymerase chain reaction (RT-​PCR) indicate that viral RNA can be amplified from myocardial samples in a high percentage of cases. However, the latter technique is open to contamination problems and hence this link remains to be con- firmed. Mumps virus has not been isolated from heart tissue at aut- opsy and these infants have no mumps antibody in their blood. They may show a delayed hypersensitivity response to the skin test. This has not been explained, but may reflect some immune defect in the fetus which could cause myocarditis and fibroelastosis.

8.5.6 Measles 772

8.5.6 Measles 772

772 section 8  Infectious diseases In the normal infant, maternal IgG passes to the fetus and seems to protect the infant against mumps during the first year of life. The typical disease of mumps in infants is a rare clinical finding, even in populations with no previous experience of the disease. MuV may be isolated in vague respiratory infections in infants. Laboratory diagnosis In patients without parotitis, especially meningitis, and in the absence of contact history, serological tests and RT-​PCR are the only means of reaching a firm diagnosis. MuV isolation is an insensitive method and now rarely used. MuV contains several different antigenic compo- nents, which provoke distinct antibodies that are useful for laboratory confirmation. Antibody to the N protein rises in the first 2 weeks of in- fection but then declines rapidly. Antibody to the HN protein appears at the end of the first week, usually in high titre: it may persist for years and indicates past infection. Neutralizing antibodies also develop, but titres are a poor correlate of protection. Nowadays, sensitive enzyme immunoassays allow early diagnosis by detection of mumps-​specific IgM and IgA. In recent outbreaks in the United States of America and in the United Kingdom, IgM-​negative cases have been identified. IgA can be detected in saliva or mouth washings on about the fourth day after infection, and in the serum early in the disease. Measurement of antibodies in acute and convalescent sera is a reliable method for diagnosis, especially in patients who have no parotitis. RT-​PCR meth- odology targeting the detection of mumps RNA in nose and throat swabs has been developed and is replacing and adding to serology-​ based techniques in routine laboratory diagnosis and confirmation. Treatment There is no specific antiviral treatment. Symptomatic treatment in- cludes simple analgesics, but for the severe pain of orchitis, morphine (15–​30 mg) may be required for a day or two. Corticosteroids are worth trying in severe cases of parotitis, more especially in orchitis. An adult dose of 60 mg prednisolone daily for 2 or 3 days sometimes gives dramatic relief from pain, though it may not reduce the swelling. Prevention and control The mainstay of prevention is vaccination of susceptible individ- uals. Isolation is not effective as the patient has been infectious for days before parotitis occurs and subclinical cases are frequent. Attenuated live vaccine gives 95% seroconversion, and protection lasts for at least 15 years. In developed countries, mumps vaccine is currently given between 14 and 16 months of age as one compo- nent of a live attenuated trivalent mumps/​measles/​rubella (MMR) vaccine. A two-​dose schedule with follow-​up at 4–​5 years of age is now recommended. This has suppressed the incidence of mumps by more than 98% in the United States of America and in the United Kingdom. Nevertheless, both countries have had recent outbreaks of mumps in college age populations in both unvaccinated individuals as well as those with a documented vaccination history. It is not clear whether this is due to primary vaccine failure or waning immunity in the absence of frequent challenge. Identification of patients in several outbreaks who had received two doses of mumps vaccine indicates that waning immunity is the most likely explanation for recurrent outbreaks. Recommendation of a third dose of vaccine is being considered. The ability of new variant wild-​type virus strains to break thought the protective immunity established by older vac- cines which are largely based on genotype A strains, appears a less likely cause of the current outbreaks. Mumps vaccination is contra- indicated in pregnant women and patients with immunodeficiency due to immunosuppressive therapy or disease. However, HIV sero- positive children should be vaccinated with the MMR vaccine. FURTHER READING Christie AB (1980). Infectious diseases: epidemiology and clinical prac- tice, 3rd edition. Churchill Livingstone, Edinburgh. Duprex WP, Rima BK (2011). Mumps virus. In: eLS. John Wiley & Sons, Ltd, Chichester. http://​www.els.net/​. Rima BK, Duprex WP (2008). Mumps virus. In:  Mahy BWJ, van Regenmortel MHV (eds) Encyclopedia of virology, 3rd edition. Academic Press, London. Rubin SA, Sauder CJ, Carbone KM (2013). Mumps virus. In: Knipe DM, Howley PM (eds) Fields virology, 6th edition, Ch. 35, pp. 1024–​41. Wolters Kluwer, Lippinocott Williams & Wilkins, Philadelphia, PA. Wright KE (2006). Mumps. In: Newton VA, Vallely PJ (eds) Infection and hearing loss, pp. 109–​26. John Wiley and Sons Ltd, Chichester. 8.5.6  Measles Hilton C. Whittle and Peter Aaby ESSENTIALS Measles is a single-​stranded RNA virus that is spread by aerosolized droplets and is highly transmissible. It causes a spectrum of disease ranging from mild in the well-​nourished to severe in the malnour- ished or immunosuppressed: mortality is 3–​10% in Africa. Clinical features—​10 to 14 days after infection, the viral prodrome typically consists of runny nose and fever, sometimes also diarrhoea or convulsions; signs include mild conjunctivitis, red mucosae, and (on the buccal mucosa) Koplik’s spots. After 14–​18 days a morbilliform rash first appears on the forehead and neck, then spreads to involve the trunk and finally the limbs. Other manifestations include severe con- junctivitis (especially in those who are vitamin A deficient), pneumonitis and enteritis (which may cause profuse diarrhoea). Early complications include (1) pneumonia—​caused by secondary bacterial infection and responsible for most deaths; (2) stomatitis—​caused by herpes sim- plex virus and/​or candidal infection; (3) enteritis—​due to candidal or bacterial superinfection; (4) eye infection—​corneal ulceration may be caused by some combination of measles itself, herpes simplex infec- tion, vitamin A deficiency, and use of traditional eye medicines; more than half of childhood blindness in Africa is related to measles; (5) skin and other infections (e.g. pyoderma); (6) encephalitis—​occurs in 0.1–​ 0.2% of cases; probably attributable to a neuroimmunologic process;

8.5.6  Measles 773 mortality is 10–​15%, and 25% of children are left with permanent neurological disability. Late complications include malnutrition, giant cell pneumonia, and subacute sclerosing panencephalitis. Diagnosis and treatment—​diagnosis is primarily clinical, but signs might be less clear-​cut in vaccinated subjects. Detection of measles-​specific IgM antibody or detection of measles antigen in saliva or urine can clinch the diagnosis if the rash is mild or atyp- ical. Management is supportive, including administration of vitamin A, and with prompt treatment of secondary infections. Prophylactic antibiotics such as amoxyicillin to prevent pneumonia might be war- ranted in settings with limited access to clinical services. Prevention—​(1) Passive immunization—​human immunoglobulin is highly effective if given within 5  days of exposure and should be administered to those in whom vaccination is contraindicated. (2) Active immunization—​live vaccine is often given in the developed world as one component of a trivalent measles–​mumps–​rubella vac- cine at 12 to 18 months of age. However, this is not appropriate for children in developing countries, who are infected by measles at a much earlier age, where substantial successes in controlling the dis- ease has been obtained with a strategy combining (a) catch-​up—​a one-​time mass campaign covering everybody aged 9  months to 14 years, regardless of previous measles or immunization; (2) keep-​ up—​achieving a high coverage with routine measles vaccination for each birth cohort; (3) follow-​up—​subsequent mass campaigns covering all children every 3–​5 years; and (4) mop-​up—​campaigns that target children who are difficult to reach or during outbreaks. This strategy has eliminated measles from Latin America. Introduction Measles is an acute, highly transmissible RNA viral infection of humans that is spread by aerosolized droplets. It causes much death and suffering, especially among poor children in developing countries. Its severity varies according to host and socioeconomic factors, not to antigenic variation or alteration in virulence of the virus. There is no reservoir of infection other than in humans and no evidence of a carrier state and as there is an effective vaccine, global eradication is possible but daunt- ingly high vaccine coverage of more than 95% will be needed. The virus causes a generalized infection coupled with severe damage to the immune system due to destruction of T lympho- cytes, disturbance of the Th1/​Th2 cytokine balance, and im- paired antigenic presentation. The chief clinical features result from infection of the skin, mucous membranes, and respiratory tract. Death, which occurs in up to 15% of hospitalized chil- dren in Africa, results from secondary infections and immuno- suppression. Attack rates in unimmunized home contacts are very high (of the order of 90%) and long-​life immunity follows the disease but not vaccination. Supplemental immunization activities allowing repeated vaccination every 3 to 5  years in endemic countries have lowered measles deaths dramatically. Although the coverage for the first dose of measles vaccine has reached 85% (Fig. 8.5.6.1a) and measles mortality declined by 79% between 2000 and 2014, an estimated 114 900 people still die annually of measles (Fig. 8.6.5.1b). Epidemiology Measles has been the archetypical childhood infection, known and feared by all parents. Nearly everybody contracted this most infec- tious of childhood diseases. Measles was the single biggest cause of childhood deaths. In the prevaccination era, 6 million children might have died annually of measles. With advances in coverage during the last 25 years is still one of the most important of the vaccine-​ preventable infections. The severity and age of infection varies mark- edly between poor and rich countries. In the West, most children were infected between 3 and 6 years of age, when they attended nur- sery and primary schools. Mortality was low (<0.05%) and morbidity, although considerable when compared to many other common viral infections, was limited. Most cases occurred in the winter and spring, with a biannual epidemic pattern. Widespread immunization has dramatically reduced both the number of cases and complications in high-​income countries but coverage, which needs to be over 95%, has seldom been high enough to eliminate measles except in the Americas. Thus, measles still persists in most regions of the world showing marked annual variation (Fig. 8.5.6.1c). In low-​income countries, measles is still severe and behaves dif- ferently. It kills between 3 and 10% of children in the community and some 10–​20% of those admitted to hospital. Mortality from mea- sles is considerably higher in Africa (3–​10%) than in Asia or South America (1%). West Africa has the highest case fatality rates. There are many reasons for this increase in severity in the tropics: children are infected at 1–​2 years of age; severe malnutrition leads to prolonged, severe measles. Overcrowding is another strong determinant, for secondary and tertiary cases in large families are at great risk of death. Exposure to a large dose of the virus when in close contact with the index case might be the critical factor. The severity of measles depends on the severity of disease in the index case. The high mortality found in West Africa is due to this region having the largest polygamous and extended families, which in- crease the risk of intense exposure. When females stay at home and are constrained in their social contacts, mortality is higher in girls than boys. There is also a high case fatality in children with chronic disease, including kwashiorkor, tuberculosis, and HIV infection. Hospital wards, refugee camps, and clinics in developing countries have been important centres of disease transmission. Though measles can have permanent sequelae, recent research has provided limited support for the previous belief in long-​term excess morbidity and mortality after the first 6 weeks of measles infection. Long-​term consequences might also depend on intensity of ex- posure. Index cases apparently have better long-​term survival than secondary cases, suggesting a beneficial effect of mild measles infec- tion. Long-​term morbidity is most likely to be experienced by young children who have severe measles following intensive exposure. Although measles immunization has dramatically decreased the number of cases and deaths, vaccinated cases are not infrequent as immunity wanes with time. These cases are characterized by a pro- longed incubation period, a short prodrome, mild symptoms, and a favourable outcome. The mild measles of immunized cases leads to less risk of transmission or transmission of less severe disease. Immunization reduces the number of children being susceptible in the same household and hence reduces the risk of intensive ex- posure (Table 8.5.6.1).

section 8  Infectious diseases 774 However, immunization might have negative consequences on herd immunity for an increasing number of unvaccinated children, or children who have responded poorly to the vaccine will reach adulthood without having been exposed to measles. Thus, vaccin- ated people will have lower antibody levels than naturally infected people, which is particularly important because young immun- ized mothers will transfer lower antibody levels to their offspring. In West Africa, children of immunized mothers have only half the antibody levels of children of naturally infected mothers and they become susceptible as early as 3 to 5 months of age. 5 (a) 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 0.5 1 1.5 2 2.5 3 3.5 4 4.5 100 0 10 MCV Coverage* (%) No. of reported cases Millions 20 30 40 50 60 70 80 Campaigns 90 Number of cases MCV1 Coverage* MCV2 Coverage* 0 2000 2013 2012 2011 2010 2009 2008 2007 2006 Year 2005 2004 2003 2002 2001 1.6 (b) 1.4 1.2 Estimated no. of deaths (millions) 1 0.8 0.6 0.4 0.2 Estimated no. of measles deaths in absence of vaccination Estimated no. of measles deaths with vaccination 95% confidence limits for no. of measles deaths with vaccination Estimated no. of deaths averted by measles vaccination Fig. 8.5.6.1  (a) Global annual reported cases of measles and coverage of first (MCV1) and second (MCV2) doses of measles vaccines, 1980–​2014. (b) Estimated number of measles deaths and number of deaths averted by measles vaccination-​worldwide 2000–​2014. (c) Measles case distribution by month and WHO regions, 2008–​2018. (a) Source WHO/​IVB database, 2015. (b) Reproduced from CDC (2015). Progress towards regional measles elimination. MMWR, 64,1246–​51. (c) Source data from WHO measles surveillance data 2018.

8.5.6  Measles 775 It has been argued that measles vaccines only saved ‘weak’ children who were likely to die anyway. However, many epidemiological studies, including randomized trials, have shown remarkable reductions in all-​ cause mortality after standard measles vaccine. In Bangladesh, measles vaccination was associated with a 49% reduction in all-​cause mortality from the age of 9 months, even though acute measles accounted for only 10–​12% of deaths. This unexpected benefit was not related to prevention of measles. In most studies, this non​specific benefit is par- ticularly marked for girls. The World Health Organization (WHO)’s Strategic Advisory Group of Experts on Immunization (SAGE) re- viewed the potential non-​specific effects of measles vaccine in 2014 and confirmed these observations. Recent studies have also shown that the combination of measles vaccine with other vaccines or vitamin A sup- plements may negatively influence the non​specific effects on child survival. The first study from a high-​income country has also shown that in Denmark, USA, and Italy measles–​mumps–​rubella (MMR) vaccine is associated with a non​specific benefit by reducing hospital admissions for infectious diseases, particularly respiratory infections. There is also evidence that the second dose of MMR has beneficial ef- fects on prolonged hospital admissions for infections. Popular beliefs In most cultures, measles has a specific local name and is a much-​ feared disease. Popular understanding is centred around the rash, which if it stays within the body will lead to severe disease. This belief has some basis in truth for the prodrome is prolonged in se- vere cases, and a proportion of deaths reportedly occur before the appearance of the rash during very severe epidemics. Therapeutic practices, such as rubbing the skin with palm oil or kerosene, are aimed at eliciting the rash quickly. Popular beliefs can also hamper vaccine uptake, leading to local outbreaks of measles. In the United Kingdom, after the publication of a fallacious medical article in 1998, the myth has arisen that the measles–​mumps–​rubella (MMR) vaccine can cause autism. Driven by an irresponsible press and the antivaccine lobby, measles vac- cine coverage fell from 93% to 79% and has yet to fully recover. In Germany some parents believe children benefit from measles and thus shun vaccination. In the Bible Belt of the Netherlands, strong religious beliefs preclude all vaccinations. Marginalized 40000 (c) 35000 30000 25000 20000 15000 10000 5000 Month of onset Measles cases (Lab+Epi+Clinical) 2014-01 2014-02 2014-03 2014-04 2014-05 2014-06 2014-07 2014-08 2014-09 2014-10 2014-11 2014-12 2015-01 2015-02 2015-03 2015-04 2015-05 2015-06 2015-07 2015-08 2015-09 2015-10 2015-11 2015-12 2016-01 2016-02 2016-03 2016-04 2016-05 2016-06 2016-07 2016-08 2016-09 2016-10 2016-11 2016-12 2017-01 2017-02 2017-03 2017-04 2017-05 2017-06 2018-01 2018-02 2018-03 2018-04 2018-05 2018-06 2017-07 2017-08 2017-09 2017-10 2017-11 2017-12 0 WPR SEAR EUR EMR AMR AFR Fig. 8.5.6.1  Continued Table 8.5.6.1  Impact of measles immunization on the transmission and severity of measles Outcome measurements Bissau 1980–​1982 Senegal 1983–​1990 Bissau 1991 Case fatality ratio: vaccinated /​ unvaccinated (95% CI) Acute mortality within 1 month 0.39 (0.13–​1.14) 0.0 (0–​0.92) 0.30 (0.13–​0.72) Delayed mortality from 1 month to 3 years 0.44 (0.22–​0.90) Secondary attack rate ratio according to vaccinated/​ unvaccinated index cases 0.28 (0.10–​0.79) 0.36 (0.15–​0.87) Based on data from Aaby P, et al. (1986). Vaccinated children get milder measles infection: a community study from Guinea-​Bissau. J Infect Dis, 154, 858–​63, and Samb B, et al. (1997). Decline in measles case fatality ratio after the introduction of measles immunization in rural Senegal. Am J Epidemiol, 145, 51–​7.

776 section 8  Infectious diseases communities like the Roma shun medical and other authorities, and many are unvaccinated. Thus it is difficult to eliminate measles in Europe. In northern Nigeria, a predominantly Muslim area, re- ligious and political leaders have warned of the dangers of Western vaccines: coverage fell, and large outbreaks of polio and measles have ensued. The virus and its antigens Measles mainly infects humans, but like the other closely related morbilliviruses (such as rinderpest or canine distemper virus) it is able to cross species to infect other primates, but these outbreaks have not proved to be reservoirs of infection for humans. The virus contains a single strand of RNA, is highly pleomorphic, and ranges from 100 to 300 nm in diameter. It propagates by budding from the cell membrane, from which it acquires an envelope. The membrane of infected cells and the virion envelope contain two surface glyco- proteins, the haemagglutinin (H)  and fusion (F)  proteins, and a non​glycosylated matrix (M) protein, which forms the inner layer. The H protein, which allows attachment of the wild type virus to cells, via the CDw150 and nectin-​4 receptors, is the main target for neutralizing antibodies. The CDw150 receptor is expressed on im- mature lymphocytes and on effector memory T cells, and is rapidly induced on T and B cells after activation; the nectin-​4 or poliovirus receptor-​like 4 is expressed on epithelial cells. The F protein is re- sponsible for fusion and syncytium formation of infected cells. The internal components or nucleocapsid consist of RNA, the nu- cleoprotein (N), which is the major protein, the phosphoprotein (P), and the large protein (L). The F protein is remarkably stable, the H protein shows minor antigenic variation, but the N protein, which contains a variable region in the C-​terminal, is highly divergent among different strains of virus. Genetic analysis of Haemagglutinin and Nucleoprotein genes allowed molecular surveillance of the mea- sles virus to track the international spread of the virus. There is also variation in the M protein, which some claim is related to persistent infection. The virus and its antigens are shown in Fig. 8.5.6.2. Pathogenesis and the immune response The course of infection and the immune response to this invasion are shown in Fig. 8.5.6.3. The measles virus, which is thermolabile and survives best at low humidities, is spread to susceptible contacts in droplets during sneezing and coughing. First, it infects and multi- plies in lymphoid cells in the mucosa of the upper respiratory tract or the conjunctivae. Some 4–​6 days later, the virus is found in the reticuloendothelial tissue of the liver and the spleen after passage through lymph nodes and spread via the blood. Here it multiplies, causing fusion of cells to form giant cells with many nuclei. Viral antigens, which can be found by immunofluorescent techniques in and on the surface of both these cells and lymphocytes, now induce the immune response. First, natural killer cells and cytotoxic T cells mount a cell-​mediated reaction that contains the virus and limits its spread within cells. Later, B cells are primed to produce antibody. Defects in the cellular immune system, as in severe malnutrition, cancer, or primary and secondary immunodeficiencies, allow wide- spread multiplication of the virus to cause fatal giant cell pneumonia. Fusion (F) Haemagglutinin (H) Lipid bilayer RNA Large protein (L) Phosphoprotein (P) Nucleocapsid (N) Matrix (M) Fig. 8.5.6.2  The virus and its antigens. Reprinted by permission from Macmillan Publishers Ltd: Moss WJ, and Griffen DE (2006). Global measles elimination. Nat Rev Microbiol, 4, 900–​8, copyright © 2006. Measles Stage Day Infection 0 Invasion 6 12 Rash 16 21 Recovery Epithelia Lymph node Blood Induction ab c.m.i. Allergy Immune response Lymphoid tissue Secondary infections Secondary anergy Persistent immunity Epithelia Fig. 8.5.6.3  Pathogenesis of measles. + Denotes, amount of virus; ab, antibody. Reproduced with permission from Parry EHOP (ed) (1984). Principles of medicine in Africa, 2nd edition. Oxford University Press, Oxford.

8.5.6  Measles 777 Around day 8, the measles virus is carried by the blood, either free or in mononuclear cells, to the target tissues, which are epithelia of the skin, eye, lung, and gut. Again, the agent multiplies to cause a bright erythema of the mucosae and Koplik’s spots (see next), which are foci of viral multiplication. At this stage, measles virus may be cultured from nasopharyngeal secretions, and antigen can be de- tected by immunofluorescent techniques or PCR in the character- istic giant cells of the buccal mucosa, in epithelial cells, and in both B and T lymphocytes in the blood. The rash, appearing around days 14–​16, is the sign of a strong and complicated allergic reaction to the virus in epithelia. The ex- tent and severity of the rash, which reflects the clinical severity of the disease, is determined by the number of target cells infected. Histological examination shows virus in the disrupted epidermis, in the corium, and in capillary endothelium. These tissues are infil- trated by mononuclear cells together with antibody, immune com- plexes, and complement. An intact cell-​mediated immune response is essential to generate the rash and clear the virus, for if impaired, as in the case of children with leukaemia, or occasionally in severe kwashiorkor, the virus multiplies unchecked and no rash appears. Some 2 or 3 days after the start of the rash, around day 17 or 18, the virus can no longer be cultured from epithelia, for infected cells have been disrupted and the free virus neutralized by antibody. The first antibody to appear is to the nucleoprotein antigens. The second to appear, which is largely responsible for neutralization of the virus, is to the haemagglutinin. Finally, the antibody to the fusion glycoprotein appears in a low titre. This antibody stops cell-​to-​cell spread of the virus. At this stage the child is markedly immunosup- pressed and thus susceptible to secondary infections of the eyes, mouth, gut, and lungs. Latent viruses, such as herpes simplex or cytomegalovirus, may be reactivated and in turn cause further damage to the immune system. The delayed hypersensitivity reac- tion, as measured by skin tests to old tuberculin or candida antigen, is absent or severely impaired. By the third week, day 21, as the patient recovers, antibody is in full production. Levels remain elevated for the rest of the patient’s life, ei- ther because of repeated subclinical infections or because the virus persists in latent form in the spleen and other organs, so stimulating antibody. Occasionally, the virus persists in the brain in a damaging form to cause subacute sclerosing panencephalitis (see next). Immunosuppression The mechanisms of immunosuppression are complex (Fig. 8.5.6.4). The CD4 + and CD8 + cytotoxic T-​cell response, which is exuberant, may result in the destruction of infected T cells and dendritic cells thus leading to their depletion, deficient antigen processing, and generalized immunosuppression. Cross-​binding of the CD46 cel- lular receptor down-​regulates interleukin 12 (IL-​12), a crucial cytokine in the development of Th1 and delayed hypersensitivity responses. Infection of CDw150+ lymphocytes, in particular ac- tivated CD45RA-​ memory lymphocytes, results in suppression of lymphoproliferation and cell death and loss of cell-​mediated immunity. Thus, measles ultimately dampens the Th1 response, resulting in a skewing towards a Th2 cytokine response and suscep- tibility to intracellular and other pathogens for 4–​6 weeks. This im- munosuppression might be in the interest of the host by limiting further autoallergic damage of infected tissues. However, the recent claim that this immunological amnesia may last for 2–​3 years is not supported by long-​term follow-​up studies of mortality after measles in both W. Africa and Bangladesh. Pathogenesis in the underprivileged, in the malnourished, and in the HIV-​infected Measles is severe, prolonged, and carries a high case fatality rate due to secondary infections in children of the developing world, as it was formerly in the underprivileged in Europe. Two explanations are offered. Crowding leads to a high dose of measles virus and also in- creases the chances of secondary infection. The period of incubation T Cell ↑ IL-10 ↑ IL-4 ↓ IL-12 ↓ Differentiation Lymphocyte apoptosis Impaired lymphoproliferation Immunomodulatory cytokines Interleukin-12 downregulation Impaired antigen presentation T Cell T Cell Monocyte DC Fig. 8.5.6.4  Potential mechanisms of immune suppression following measles virus infection. Reprinted from Moss WJ, Ota MO, and Griffen DE (2004). Measles: immune suppression and immune responses.
Int J Biochem Cell Biol, 36, 1380–​5, copyright © 2004, with permission from Elsevier.

778 section 8  Infectious diseases has been found to be short, around 10–​12 days, in severe and fatal cases, consistent with the concept of infecting dose as a mechanism of severe disease. Alternatively, or in tandem, malnutrition dimin- ishes the immune response to the virus, allowing great proliferation of virus and subsequent damage to the host. The immune response follows, which generates a severe and widespread rash followed by prolonged immunosuppression. Secondary bacterial infections with, for example, Streptococcus pneumoniae, or latent infections such as herpes simplex or Mycobacterium tuberculosis occur in the wake of this intense damage to the immune system, often killing or maiming the child. Virus persists in lymphocytes and epithelial cells for up to 30 days after the start of the rash. Anorexia, increased catab- olism, protein loss from the gut, and further malnutrition exaggerate the problem, which is worst in the weanling child (Fig. 8.5.6.5). The death rate after measles in hospitalized infants is higher in severely malnourished and HIV-​infected children, and prolonged viral shedding occurs in these children. Thus, in regions of high prevalence, HIV-​infected children may be unrecognized trans- mitters of the virus but to date there is no evidence that this has hampered measles control. Asymptomatic HIV-​infected children respond suboptimally to vaccination; those with AIDS are even less likely to respond and may be threatened by persistent measles infec- tion (see prevention). Clinical features There is a spectrum of severity ranging from mild in the privileged and well-​nourished to severe in the blatantly malnourished or im- munosuppressed. However, the rule is not inviolate and other fac- tors such as the age and dose of infection are probably as important in determining the severity of disease. Measles, often severe, occa- sionally infects unvaccinated young adults or those who have lived in isolated communities. The clinical features of measles and some complications are shown in Fig. 8.5.6.6 and discussed next. Prodrome (days 10–​14) A diagnosis of measles is often missed at this stage, when fever coupled with a runny nose, and sometimes complicated by con- vulsions, is the main feature. Other signs are mild conjunctiv- itis, red mucosa, Koplik’s spots, and diarrhoea. Koplik’s spots Impaired immunity Secondary infection Malnutrition Acute measles infection Fig. 8.5.6.5  The complex interaction between infection, nutrition, and impaired immunity seen in measles. Reproduced with permission from Greenwood BM (1996). The host’s response to infection. In: Weatherall DJ, Ledingham JGGL, Warrell DA (eds) (1996). Oxford textbook of medicine, 3rd edition, p. 282. Oxford University Press, Oxford. Measles Incubation Prodrome Allergic Secondary infections Late complications Systemic Respiratory Skin Eye Mouth Gut Nutritional 0 10 14 19 28 4 5 6 Days after infection Weeks Tuberculosis Pneumocystis Blindness Gut infected Fever Fever Cough Cough Pneumonitis Rash Pyoderma, ulcers Conjunctivitis Keratitis, perf Kopliks Diarrhoea Herpes Anorexia Protein loss Loss of weight Marasmus Kwashiorkor Gut infected Pneumonia Infection Diarrhoea-bacterial Candida Fig. 8.5.6.6  Clinical features of measles and some of its complications. Reproduced with permission from Parry EHOP (ed) (1984). Principles of medicine in Africa, 2nd edition, Oxford University Press, Oxford.

8.5.6  Measles 779 are found in the buccal mucosa (Fig. 8.5.6.7). They are small, irregular, bright-​red spots with a minute bluish-​white speck in the centre of each of them. The prodrome is prolonged in severe cases, and reduced in individuals with modified measles due to maternal antibodies, previous immunization, or the prophylactic use of immunoglobulin. Rash (days 14–​18) The morbilliform rash first appears on the forehead and neck and then spreads, over a period of 3–​4 days, to involve the trunk and fi- nally the limbs (Fig. 8.5.6.8). In children in Africa and other parts of the developing world the rash is often red, confluent, raised (Fig. 8.5.6.9), very extensive, and sometimes accompanied by bleeding into the skin and gut. Later, the rash blackens (postmeasles ‘staining’, see Fig. 8.5.6.10), then the skin peels causing extensive desquamation (Fig. 8.5.6.11). Other epithelial surfaces are inflamed, the severity matching that of the rash. Cough may be hoarse and coupled with inspiration difficulty if the larynx and trachea are inflamed. Signs of pneumonitis are ap- parent, which in severe cases can cause cyanosis or be complicated by mediastinal and subcutaneous emphysema. Conjunctivitis, espe- cially in those who are vitamin A deficient, can be severe. Enteritis might cause profuse diarrhoea with a resulting loss of protein, and malabsorption of food and water. The mouth is painful and red, which adds to the misery of the child, who becomes anorexic and may even refuse to suck the breast. In the uncomplicated case, as is usual in the West, the convalescent period is short, usually lasting less than a week. Complications should be suspected if fever persists while the rash is fading or desquamating. Complications Early complications (days 18–​30) As a result of the widespread, severe allergic reaction to the measles virus signified by the rash, the patient is left severely immunosup- pressed and is susceptible to infection. Pneumonia This causes the most deaths (Table 8.5.6.2) and is heralded by a rise in fever, leucocytosis, and respiratory difficulties. Lobar pneumonia is usually caused by S. pneumoniae, but bronchopneumonia, which is more common, results from other bacteria, such as Staphylococcus aureus, or secondary viral infections with, for example, herpes simplex or adenovirus. A  variety of other organisms such as Gram-​negative bacteria, cytomegalovirus, fungi, M.  tuberculosis, and Pneumocystis jirovecii should be considered as potential lung pathogens in the malnourished or immunocompromised child. Stomatitis and enteritis Chronic diarrhoea and a sore mouth caused by candidal infection are common complications of measles in children in the develop­ ing world. The gut is often superinfected with Bacteroides spp., Escherichia coli, Pseudomonas spp., and S. aureus, which results in malabsorption and protein loss. Deep ulcers caused by herpes sim- plex virus erode the corners of the mouth, gums, and inner surface of the lips causing much misery, illness, and pain (Fig. 8.5.6.12). Fig. 8.5.6.7  Koplik’s spots on the buccal mucosa. Courtesy of the late Dr B. E. Juel-​Jensen. (a) (b) Fig. 8.5.6.8  (a, b) The morbilliform rash first appears on the forehead and neck and then spreads, over a period of 3–​4 days, to involve the trunk and finally the limbs. Copyright D. A. Warrell.

780 section 8  Infectious diseases Eye infections Corneal ulceration leading to impaired vision or blindness is common after measles, especially in malnourished and vitamin A deficient children (Fig. 8.5.6.13). Several studies from Africa have shown that more than half of childhood blindness is related to mea- sles. The mechanisms are still under discussion. In northern Nigeria, herpes simplex was found in 47% of active corneal ulcers after mea- sles, and measles virus in 12%: the children often had evidence of oral herpes. In a study in Tanzania, blindness precipitated by mea- sles was associated with vitamin A deficiency (50%), herpes simplex infection (21%), and the use of traditional eye medicine (17%). Skin and other infections Pyoderma is common after measles. In the malnourished patient, deep eroding ulcers can bore through the skin, even into bone. When originating in the mouth they are known as cancrum oris or noma (Fig. 8.5.6.14). Otitis media is also common. Encephalitis This is a rare, but much feared, complication found in approximately 1 to 2 per 1000 cases. The onset is usually between 4 and 7 days after the start of the rash, but, rarely, it might occur within 48 h or up to 2 weeks from the onset. In addition to seizures, there is often fever, irritability, headache, and a disturbance in consciousness that can progress to profound coma. The disorder is probably attributable to a neuroallergic process. Lymphocytes from the cerebrospinal fluid have been shown to respond to myelin basic protein, as in experi- mental allergic encephalomyelitis. The virus cannot be isolated from cerebrospinal fluid, which contains lymphocytes and raised levels of IgG but normal levels of measles antibody. Mortality and morbidity Fig. 8.5.6.9  Measles rash in an African child. Fig. 8.5.6.10  Darkening measles rash after several days (‘measles staining’). Courtesy of the late Dr B. E. Juel-​Jensen. Fig. 8.5.6.11  Desquamating measles rash in an African child. Table 8.5.6.2  Complications and mortality in inpatients with measles, northern Nigeria, July–​December 1978 No. Died Percentage dead Pneumonia 169 32 18.9 Gastroenteritis   65   9 13.8 Marasmic kwashiorkor   25   6 24.0 Laryngotracheobronchitis   21   4 19.0 Encephalitis   10   4 40.0 Reproduced with permission from Parry EHOP (ed) (1984). Principles of medicine in Africa, 2nd edition. Oxford University Press, Oxford. Fig. 8.5.6.12  Deep ulcers caused by herpes simplex virus. Copyright D. A. Warrell.

8.5.6  Measles 781 are high: 10–​15% of patients die and 25% of children are left with permanent brain damage. Treatment is supportive; dexamethasone has no convincing beneficial effect. Late complications Malnutrition This is the most frequent complication, for children of the developing world often lose a lot of weight during measles and may take many weeks to regain it. Those originally underweight, who have had severe measles, are at greatest risk, for anorexia in these children is prolonged, much protein is lost from the gut, and secondary in- fections, which lead to marasmus or marasmic kwashiorkor, are frequent. Measles has been shown to persist in the epithelia and lymphocytes of the severely malnourished for 30 or more days after the rash. Persistent infection Pneumonitis Giant cell pneumonia is found in patients with defects in cell-​ mediated immunity. Children with leukaemia or kwashiorkor are particularly vulnerable, as are those with symptomatic HIV in- fection. The lung disease might develop weeks after measles, and in most cases the rash of measles has been absent and thus the diagnosis may not be suspected. The diagnosis is made by viro- logical and/​or histological examination of lung tissue. Most of these children die. Subacute sclerosing panencephalitis Persistent measles virus infection in the brain is responsible for this rare, progressive disease of the brain, which is found in 1 in 10 000 to 100 000 children after measles. The child with subacute sclerosing panencephalitis has usually experienced normal measles, albeit at a young age, 5–​10 years earlier. The first indication is a disturbance in intellect and personality. Behavioural disorders and deterioration in school work are frequently mentioned. There then follow, over a period of weeks and months, myoclonus-​like seizures, signs of extrapyramidal and pyramidal disease, and finally a state of decere- brate rigidity followed by death. The electroencephalogram shows a characteristic regular series of high-​amplitude, spike-​like waves. Very high titres of measles complement-​fixing and haemagglutinin-​ inhibiting antibody are present both in serum and cerebrospinal fluid. Treatments for subacute sclerosing panencephalitis have in- cluded the use of transfer factor, plasmapheresis, and antiviral drugs, but to no avail. Multiple sclerosis, autism, and Crohn’s disease There is no convincing evidence that measles virus or immune re- sponses to it have a causative role in these diseases. The alleged as- sociation between the measles–​mumps–​rubella (MMR) vaccine, autism, and Crohn’s disease was based on weak science and has now been convincingly refuted by larger and stronger epidemiological studies. Subsequent molecular studies have failed to confirm the ori- ginal finding of measles virus and genomic RNA in diseased bowel. The false alarm raised by this report caused a substantial reduction in the number of children vaccinated against measles in the United Kingdom. Diagnosis This is primarily clinical, although signs might be less clear-​cut in vaccinated subjects. Thus, in areas of high vaccine coverage the detection of measles-​specific IgM antibody by enzyme-​linked im- munoassay or, better still, the detection of measles by polymerase chain reaction in blood or urine can clinch the diagnosis if the rash is mild or atypical. Subclinical measles, which boosts immunity, is common in vaccinated children after exposure to measles: the Fig. 8.5.6.13  Corneal ulceration leading to impaired vision or blindness after measles, especially in malnourished and vitamin A deficient children. Copyright D. A. Warrell. Fig. 8.5.6.14  Cancrum oris, or noma, following measles.

782 section 8  Infectious diseases diagnosis is made by detecting a fourfold or greater rise in measles antibody within 2–​6 weeks of exposure. It is not clear if such cases are infectious. Treatment of measles and its complications No effective antimeasles drug exists, yet some children do benefit from treatment in hospital. The following criteria indicate severe measles and a need for hospital admission: a widespread, confluent rash darkening to deep red or purple; signs of laryngeal obstruc- tion; subcutaneous emphysema; marked dehydration; blood in the stool or more than five stools a day; convulsion or loss of con- sciousness; severe secondary pneumonia; corneal ulceration; se- vere ulceration of the mouth and skin. These signs should be taken particularly seriously when the child is underweight or frankly malnourished. Hydrate the child orally or intravenously. Treat lobar pneu- monia with benzylpenicillin, and bronchopneumonia with amoxi- cillin. If severe, or if there is coexisting HIV infection or severe malnutrition, use combined antibiotics such as ampicillin and gen- tamicin. If staphylococcal infection is suspected use flucloxacillin plus gentamicin. Antibiotic eye ointments relieve discomfort and possibly prevent secondary infections of measles conjunctivitis. Antibiotics (topical and systemic) and vitamin A should be given routinely for the treatment of eye ulcers. If herpes simplex virus is the cause, use aciclovir topically or, when severe, systemically. Candida infections of the mouth or gut often respond dramatically to nystatin. Feeding, by tube if necessary, needs careful planning and presentation, for the anorexic infected child will be in severe negative energy balance due to a greatly increased catabolic rate. Case fatality rates are 30–​50% lower in those children in hospital treated with vitamin A. This should be given orally at the time of diagnosis and on the next day in a dose of 50 000 IU for children less than 6 months of age, 100 000 IU for children between 6 and 12 months of age and in a dose of 200 000 IU for older children. If eye signs of vitamin A deficiency are present, if the child is mal- nourished or the measles severe the initial dose should be repeated 2 to 3 weeks later. The prophylactic use of antibiotics such as amoxicillin or co-​trimoxazole to prevent secondary infections after measles is a widespread practice based on slender evidence. The only community randomized, placebo-​controlled trial was small: those children who received co-​trimoxazole had less pneumonia and conjunctivitis and had a significantly higher weight gain (see Table 8.5.6.3). Prevention Passive immunization with human immunoglobulin is highly ef- fective if given within 5 days of exposure, in a dose for children of 0.2 ml/​kg. Immunoglobulin should be given to those in whom vac- cination is contraindicated such as severely immunocompromised children with cancer, AIDS, or congenital immunodeficiencies. For children with severe malnutrition, WHO recommends measles vac- cination in the acute phase followed by a second dose on recovery as the immune response is suboptimal. This is widely practised in hos- pitals in developing countries and in refugee camps where there are practical difficulties in providing immunoglobulin. Although live vaccines are theoretically undesirable in these immunocomprom- ised children, no head-​to-​head trials of these two preventions have been conducted. The currently used vaccines are live strains, attenuated by culture in chick fibroblasts. The Edmonston–​Zagreb strain, which has been cultured in human diploid cells, is also widely used. It is more ef- fective than other vaccines in the presence of antibody, and should be used in a standard dose if vaccinating infants below 9 months of age, or if a booster dose is required. The complications of vaccin- ation are few and generally mild. Fever of moderate severity is in- frequent, and a mild rash with some signs of upper respiratory tract infection occurs rarely. Underweight children respond normally to the vaccine, as do ill children attending the outpatient department and those on the ward. As clinics and hospitals are major sites of transmission of the virus in the developing world, all susceptible children in these places should be vaccinated unless severely im- munocompromised. Asymptomatic HIV-​infected children are ini- tially protected by measles vaccine but antibody wanes more quickly than in uninfected children. WHO recommends early vaccination at 6 months of age followed by additional vaccinations at 9 months and another later in childhood. The measles vaccination policy for low income countries has seen major changes in the last 25 years. The optimal age for vaccin- ation in the developed world is between 14 and 16 months, when Table 8.5.6.3  Prophylactic antibiotic to prevent complications after measles in Guinea-​Bissau Outcome Co-​trimoxazole (n = 46) Placebo (n = 38) Adjusted odds ratio (95% CI) Pneumonia 1 (2%) 6 (16%) 0.14 (0.01–​1.50) Hospitalization 0 3 –​ Diarrhoea 3 (7%) 5 (13%) 0.17 (0.01–​1.55) Severe fever 6 (13%) 11 (29%) 0.36 (0.09–​1.43) Stomatitis 4 (9%) 7 (18%) 0.43 (0.08–​2.26) Conjunctivitis 12 (26%) 17 (45%) 0.31 (0.10–​1.03) Weight gain (g/​day) 32 15 –​ Adapted and reproduced from Garly M-​L, et al. (2006). Prophylactic antibiotics to prevent pneumonia and other complications after measles: community based randomised double blind placebo controlled trial in Guinea-​Bissau. BMJ, 333, 1245–​50, copyright © 2006, with permission from BMJ Publishing Group Ltd.

8.5.6  Measles 783 maternal antibody has disappeared and the children will have the highest antibody response. However, this recommendation could not be applied to children in developing countries, because there measles infects at a much earlier age. In 1970s, the World Health Organization recommended vaccination at 9  months of age but, by then, 5 to 15% of children may have had measles in endemic areas. This policy was not based on good evidence; it is still not known if vaccination at 9 months is better for saving children than vaccination at 7, 8, or 10 months of age, or a two-​ dose regime in infancy. Intriguingly, all studies from developing countries suggest that the benefit of measles vaccine on overall survival is greater when given early and studies from randomized trials where the children were tested for prevaccination antibody levels have indicated that children vaccinated in the presence of maternal antibody have a much stronger non​specific benefit from the vaccine. Through the 1990s it became clear that several doses of measles vaccines were needed to improve measles control. The developed countries have used two-​dose strategies with a second dose being given at school entry or to young teenagers. Latin America has obtained major successes with a combination of improved vaccin- ation coverage and regular immunization campaigns providing a second opportunity for measles vaccination. The strategy has the following elements: (1) catch-​up—​a one-​time mass campaign covering everybody between 9 months and 14 years of age regard- less of previous measles or immunization; (2) keep-​up—​achieving a high coverage for routine measles vaccination at 9 months of age for each birth cohort; (3) follow-​up—​subsequent mass cam- paigns covering all children every 3–​5 years; and (4) mop-​up—​ campaigns that target children who are difficult to reach or during outbreaks. As a result of this strategy, Latin America was declared free of internal measles transmission. Since there is no immediate risk of measles infection, the age of routine vaccination has been raised to 12  months as this is associated with higher antibody responses. The Latin American model has been transferred to other re- gions. Rebranded as SIA (supplementary immunization activ- ities), it has assured a spectacular success in reducing measles mortality in Africa. The goal of reducing global measles deaths by 90% by 2010 compared to 2000 has been met. Furthermore, WHO is now recommending a second dose of measles vaccine in the second year of life and more than half of the world’s chil- dren are now receiving a second routine measles vaccination (see Fig. 8.5.6.1a) However, these campaigns, which are donor driven, are expensive and should not be seen as a substitute for an in- adequate immunization service. Recently, following the credit crunch, international financial support for this initiative has decreased and many countries have not been able to raise suffi- cient money for SIAs. The world has not met the global targets for 2015: a higher than 90% coverage for the first routine measles vaccine in every country, a measles incidence of under five cases/​ million, 95% reduction of measles deaths and elimination of mea- sles in four WHO regions. Unfortunately, it is donor policy only to measure the coverage for the first measles vaccination by 12 months of age and there- fore some countries are no longer providing routine measles vaccination after 12 months of age. Although it is WHO policy that any unvaccinated child coming to a clinic should receive the measles vaccine, there is a drive to reduce wastage and not to open a vial of measles vaccine unless 5–​7 children are present for vaccination. Such policies make it difficult to achieve high coverage and some countries have seen a decline in the measles vaccination coverage. Elimination or eradication? Global measles eradication has yet to be made official policy but as polio eradication approaches there will be increasing interest in continuing with programmes to eradicate measles and rubella. The Americas have attained elimination (i.e. no internal transmission of the virus), and other regions are pursuing such a policy. Measles satisfies the criteria for eradication for there is no animal reservoir, it is only transmitted between humans, it is easy to diagnose, and vaccines are available. Measles elimination can be accomplished for prolonged periods in defined geographical regions provided there is sufficient funding and political will. This was obtained for the first time in the Gambia in the mid-​1960s as part of the smallpox eradication and measles vaccination campaigns. Rinderpest, a virus closely related to measles that decimated cattle and wild game populations over the centuries, was eradicated in 2010. Now a WHO panel has declared that measles can and should be eradicated by 2020. It stressed that eradication activities should be carried out as part of routine immunization services and estimated it would cost $US 7.8 billion. However, eradicating measles will be a daunting task for, despite global coverage of 85%, the infection is still rife in many countries (see Fig. 8.5.6.1c) First, it is the most infectious of diseases and will require vaccine coverage of greater than 95%. When there is little risk of infection, it will be increasingly difficult for parents to ap- preciate the necessity for vaccination especially as risk, although small, needs to be perceived. Secondly, herd immunity will be- come a problem as with less exposure to the virus, vaccine-​induced immunity will wane more rapidly. Thirdly, some countries like Pakistan or Afghanistan and many countries in Africa will be stern tests since political instability, wars, and natural disasters make it difficult to maintain sufficiently high coverage. Fourthly, but most demanding, will be to assure long-​term funding as donors have a tradition of changing priorities. The international health community is split over whether eradi- cation can be attained with the Latin American strategy using existing vaccines or whether new vaccines and delivery systems such as aerosolization are needed. New vaccines, which can be given in early infancy, or two-​dose strategies using the standard Edmonston–​Zagreb vaccine at 4 and 9 months of age, might be necessary to contain measles in the developing world. The latter strategy has the advantage that it might confer beneficial non​ specific effects on child survival in countries with high childhood mortality. In Guinea-​Bissau, per protocol analysis of a trial of two doses of Edmonston–​Zagreb measles vaccine in infancy revealed a mortality rate 30% lower than in the controls who received a single dose of measles vaccine at 9 months of age. Coverage of at least 95% of all susceptible children, including those between 3 and 9 months of age, with a vaccine that is at least 95% effective

8.5.7 Nipah and Hendra virus encephalitides 784

8.5.7 Nipah and Hendra virus encephalitides 784

784 section 8  Infectious diseases is assumed to be necessary if the virus is to be eradicated. Current vaccines do not meet these standards except when two doses have been given in national campaigns. New recombinant vaccines al- though successful in macaques have yet to be tried in humans; al- ternative routes of administration of the live vaccine given either as drops intranasally or by aerosol to the lung have proved less immunogenic than subcutaneous injection. The future programme to eradicate measles infection will face a more general challenge: should the number of doses of mea- sles vaccine and campaigns be reduced and should the vaccine be phased out once eradication has been reached? With the fear of bioterrorism, it is unlikely that all immunization will be stopped in the posteradication era; maybe an inactivated vaccine will be used instead of the live vaccine as is happing in the polio eradica- tion programme. But more important there is growing evidence, as recognized by WHO, that measles vaccine has beneficial non-​ specific effects in reducing lower respiratory infections in both low-​ and high-​income settings. Thus, there clearly is a risk that stopping live measles vaccination or reducing the number of doses administered in childhood would also reduce these bene- fits. Hence, eradication of measles infection and removal of the measles vaccine could lead to increases in morbidity and mor- tality with other pathogens. FURTHER READING Aaby P, et al. (1983). Measles mortality, state of nutrition, and family structure. A  community study from Guinea-​Bissau. J Infect Dis, 147, 693–​701. Aaby P, et al. (1995). Non-​specific beneficial effects of measles immun- ization:  analysis of mortality studies from developing countries. BMJ, 311, 481–​5. Aaby P, et al. (1996). No long-​term excess mortality after measles in- fection: a community study from Senegal. Am J Epidemiol, 143, 1035–​41. Aaby P, et al. (2003). Differences in female-​male mortality after high-​ titre measles vaccine and association with subsequent vaccination with diphtheria—​tetanus-​pertussis and inactivated poliovirus: re-​ analysis of West African studies. Lancet, 361, 2183–​88. Aaby P, et al. (2010). Non-​specific effects of standard measles vaccine at 4.5 and 9 months of age on childhood mortality: randomised con- trolled trial. BMJ, 341, c6495. de Quadros CA, et al. (1996). Measles elimination in the Americas. Evolving strategies. JAMA, 275, 224–​9. de Vries RD, et  al. (2015). Morbillivirus infections. Viruses, 7, 699–​706. Fenner F (1948). The pathogenesis of the acute exanthems: an inter- pretation based on experimental investigations with mouse-​pox (infectious ectromelia of mice). Lancet, 2, 915–​20. Fowlkes A, et al. (2011). Persistence of vaccine-​induced measles anti- body beyond age of 12 months: a comparison of response to one and two doses of Edmonston–​Zagreb measles vaccine among HIV-​infected and uninfected children in Malawi. J Infect Dis, 204, S149–​57. Garly ML, et al. (2006). Prophylactic antibiotics to prevent pneumonia and other complications after measles: community based random- ized double blind placebo controlled trial in Guinea-​Bissau. BMJ, 333, 1245–​50. Griffen DE (2010). Measles virus-​induced suppression of immune responses. Immunol Rev, 236, 176–​89. Jaye A, et al. (1998). Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children. J Clin Invest, 102, 1969–​77. Laksono BM, et al. (2016). Measles virus host invasion and patho- genesis. Viruses, 8, 210–23. Mina MJ, et al. (2015). Long term measles-​induced immunomodu­ lation increases overall childhood infectious disease mortality. Science, 348, 694–​9. Morens DM, et  al. (2011). Global Rinderpest eradication:  les- sons learned and why humans should celebrate too. J Infect Dis, 201, 502–​5. Morley D (1969). Severe measles in the tropics. Br Med J, 1, 363–​5. Moss WJ (2017). Measles. Lancet, 390, 2490–502. Muscat M (2011). Who gets measles in Europe? J Infect Dis, 204, S353–​5. Samb B, et al. (1997). Decline in measles case fatality ratio after intro- duction of measles immunization in rural Senegal. Am J Epidemiol, 145, 51–​7. Strebel PM, et al. (2011). A world without measles. J Infect Dis, 204, S1–​3. Whittle HC, et al. (1979). Severe ulcerative herpes of mouth and eye following measles. Trans R Soc Trop Med Hyg, 73, 66–​9. Whittle HC, et al. (1999). Effect of sub-​clinical infection on maintaining immunity against measles in vaccinated children in West Africa. Lancet, 353, 98–​101. 8.5.7  Nipah and Hendra virus encephalitides C.T. Tan ESSENTIALS Nipah and Hendra are two related viruses of the Paramyxoviridae family that have their reservoir in large Pteropus fruit bats. Human disease manifests most often as acute encephalitis, which can be late-​onset or relapsing, or pneumonia, with high mortality. Transmission from bats to human includes direct spread from consumption of food contaminated by infected bat secretions, and contact with infected animals; human-​to-​human spread can also occur. Introduction Nipah and Hendra viruses are two new zoonotic viruses that have emerged in recent years. Both are Paramyxoviridae family sharing many similar characteristics. Because of their homology, a new genus called Henipavirus (Hendra + Nipah) was created for these two viruses.

8.5.7  Nipah and Hendra virus encephalitides 785 Hendra virus infection Hendra virus was first isolated in an outbreak of acute respiratory illness involving horses in Queensland, Australia in 1994. A horse trainer and stable hand were also infected, manifesting with respira- tory illness from which the horse trainer died. A second human death occurred in 1995, when a farmer who had contact with ill horses about a year earlier died from encephalitis. Another two deaths involving veterinary workers occurred in the Hendra virus outbreaks in July 2008 and July 2009, also in Australia. Since then, more than 50 spillover events of Hendra virus infection have oc- curred in Queensland and New South Wales in Australia, all involving horses. Five of these involved subsequent horse-​to-​human transmission, with four deaths among a total of seven human cases. To date all cases have been in Australia. Thus, Hendra virus can cause respiratory and encephalitic illness in humans who have close contact with infected horses. There could be considerable delay before the manifestations of the encephalitic illness. The reservoir of Hendra virus is the Pteropus genus of fruit bats (see Chapter 8.5.10, Fig. 8.5.10.16) which also harbour Nipah, Menangle, Tioman, and Australian bat lyssaviruses. Treatment and prevention Treatment is primarily supportive and although ribavirin has been used in several human cases, there is no evidence of its efficacy. A vaccine for horses is now available to prevent Hendra virus infec- tion. It is hoped that this will prevent the infection among veterinary healthcare workers. Nipah virus infection In late 1998 to early 1999, there was an outbreak of viral enceph- alitis in several pig-​farming villages in peninsular Malaysia which subsequently involved abattoir workers in Singapore. More than 300 patients were affected. Isolation of virus from cerebrospinal fluid specimens of several patients indicated that this was due to the previously unknown Nipah virus. Epidemiology Human Nipah virus infection was transmitted by close contact with infected pigs. Human-​to-​human transmission was thought to be rare, although the virus could be readily isolated from patients’ respiratory secretions and urine. The bat as reservoir host As for Hendra virus, the reservoir of Nipah virus is fruit bats of the Pteropus species. Half-​eaten fruits dropped by bats near pig farms may have infected an animal that subsequently ingested them. Pigs were the amplifying hosts for the virus. There was pig-​to-​pig trans- mission which subsequently spread to humans. Pathology and pathogenesis Vasculitis of the medium-​sized to small blood vessels in brain, causing thrombosis, and vascular occlusion with areas of necrosis and ischaemia, were the major findings (Fig. 8.5.7.1). There were also viral inclusions indicating direct viral involvement of the neurons. Vasculitis was also seen in lung and kidney. Clinical manifestations During the outbreak, more than half of the patients had affected family members, suggesting a high infection rate. Some of the household members had seroconversion without clinical dis- ease, indicating subclinical infection at a ratio of asymptomatic vs. symptomatic infection of 1 to 3. The infection involved all age groups. The incubation period was less than 2 weeks in most patients. The clinical manifestations were those of an acute encephalitis with fever, headache, vomiting, and reduced level of conscious- ness. Distinctive clinical features were areflexia, hypotonia, and prominent autonomic changes such as tachycardia and hyperten- sion. Segmental myoclonus found in about one-​third of patients was characterized by focal, rhythmic jerking of muscles, com- monly involving the diaphragm and anterior muscles of the neck. Respiratory tract involvement with cough was seen at presentation in 14% of patients. There were some patients who had non​enceph- alitic infection with seroconversion and systemic symptoms but no evidence of encephalitis. The overall mortality of acute Nipah encephalitis was 40%. Severe brainstem involvement was associated with poor prognosis. Laboratory investigations Cerebrospinal fluid examination was abnormal in 75% of patients with elevated protein levels or elevated white cell counts. Glucose levels were within normal limits. These features are non​specific. IgM and IgG antibody detection in serum and cerebrospinal fluid were critical to the diagnosis of Nipah virus infection. The antibody test utilized an enzyme-​linked immunosorbent assay (ELISA) test. The rate of positive IgM was 100% by day 12 of illness. For IgG, it was 100% by 4 weeks of illness. Brain MRI in acute encephalitis showed multiple, disseminated, small discrete hyperintense lesions best seen in the fluid attenuation inversion recovery (FLAIR) sequence, particularly in the subcortical Fig. 8.5.7.1  Nipah virus encepahalitis: vasculitis of a medium-​sized cerebral blood vessel, showing thrombosis and vascular occlusion with areas of necrosis and ischaemia.

786 section 8  Infectious diseases and deep white matter (Fig. 8.5.7.2). The lesions were likely to cor- respond to the microinfarctions noted in postmortem tissues. Similar imaging changes were also seen in asymptomatic patients with Nipah virus infection. Treatment and prevention Treatment is mainly supportive with mechanical ventilatory sup- port for seriously ill patients. Ribavirin, a broad-​spectrum antiviral agent, appeared to reduce the mortality rate. Relapse and late-​onset Nipah encephalitis Close to 10% of patients suffered a second or even a third neuro- logical episode months or years after recovery from acute enceph- alitis. About 5%, who were either asymptomatic or only had mild non​encephalitic illness initially, also developed similar neurologic episodes (late-​onset Nipah encephalitis) for the first time after a de- layed period. Clinical, radiologic, and pathologic findings indicate that relapse and late-​onset Nipah encephalitis was the same disease process, which was distinct from acute Nipah virus encephalitis. The common clinical features were fever, headache, seizures, and focal neurological signs. There was an 18% mortality. MRI showed patchy areas of confluent cortical lesions. Necropsy showed focal confluent encephalitis due to a recurrent infection. Nipah encephalitis in Bangladesh, India,
and Philippines Almost yearly outbreaks of Nipah encephalitis have been reported in Bangladesh from 2001 onwards. Two other outbreaks were reported from north-​eastern India, in Siliguri district in 2001, Nadia district in 2007, and Kerala in 2018. As in Malaysia, Nipah virus caused a fatal encephalitic illness in humans in Bangladesh and India. However, the Bangladeshi and Indian outbreaks showed prominent human-​to-​human spread of infection with physicians who cared for the patients also affected. There was florid pulmonary involvement in some patients. Brain MRI in some patients showed confluent high signal lesions involving both grey and white matter, which is unlike the acute Nipah enceph- alitis in the Malaysian outbreak, suggesting some differences in the pathology from the Malaysian patients. The RNA of Nipah virus in Bangladesh and India was close to, but not identical with, that causing the outbreak in Malaysia. Pteropus bats were also the reser- voir of Nipah virus in Bangladesh. There might be a variety of modes of transmission from bats to humans in the Bangladesh and Indian outbreaks. Consumption of raw date-​palm juice and half-​eaten fruits contaminated by secretions from bats are suggested modes of transmission. In 2014, an outbreak of Nipah virus infection occurred in Mindanao, Southern Philippines, involving 17 patients, with 82% mortality among those with encephalitis. The outbreak involved two villages where there were deaths of horses, and consumption of the horse meat by the villages. The spread of infection was thought to be from bats to horse, horse to human, and human to human. The virus was found to have 99% homology to the Malaysian virus. Pteropus bats are widespread in large parts of Asia, Africa, and Australia. Nipah virus has been isolated in urine of Pteropus bats in Cambodia, and Nipah viral antigen has been found in saliva of Pteropus bats in Thailand. Serological evidence of Henipavirus infection has been reported in fruit bats from Papua New Guinea to Ghana, Africa from east to west, and Yunnan, China to Australia from north to south, indicating potential human Nipah virus infection elsewhere. In fact, a recent study in Cameroon, West Africa showed serological evidence of spill over of human Henipavirus infection, all involving individuals who butchered bats for bushmeat. Menangle and Tioman viruses These are two other newly identified paramyxoviruses harboured by Pteropis fruit bats. Menangle causes disease in pigs but neither has been implicated in human infections. FURTHER READING Chadha MS, et  al. (2006). Nipah virus-​associated encephalitis out- break, Siliguri, India. Emerg Infect Dis, 12, 235–​40. Ching PKG, et  al. (2015). Outbreak of Henipah virus infection, Philippines, 2014. Emerg Infect Dis, 21, 328–​31. Chong HT, Jahangir Hossain M, Tan CT (2008). Differences in epi- demiologic and clinical features of Nipah virus encephalitis between the Malaysian and Bangladesh outbreaks. Neurol Asia, 13, 23–​6. Chua KB, et al. (1999). Fatal encephalitis due to Nipah virus among pig-​farmers in Malaysia. Lancet, 354, 1257–​9. Goh KJ, et  al. (2000). Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. N Engl J Med, 342, 1229–​35. Hsu VP, et  al. (2004). Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis, 10, 2082–​7. Pernet O, et al. (2014). Evidence for Henipavirus spillover into human populations in Africa. Nat Commun, 5, 5324. Playford EG, et al. (2010). Human Hendra virus encephalitis associ- ated with equine outbreak, Australia, 2008. Emerg Infect Dis, 16, 219–​23. Tan CT, et al. (2002). Relapse and late-​onset Nipah encephalitis. Ann Neurol, 51, 703–​8. Fig. 8.5.7.2  Nipah virus encepahalitis: MRI FLAIR showing disseminated, small discrete hyperintense lesions.

8.5.8 Enterovirus infections 787

8.5.8 Enterovirus infections 787

8.5.8  Enterovirus infections 787 8.5.8  Enterovirus infections Philip Minor and Ulrich Desselberger ESSENTIALS Enteroviruses are single-​stranded, positive-​sense RNA viruses com- prising poliomyelitis viruses (3 types), coxsackie A viruses (23 types), coxsackie B viruses (6 types), and echoviruses (33 types). The human enteroviruses are classified into four species (A–​D) on the basis of sequence comparisons. Transmission is by the faeco-​oral route, with marked seasonal peaks of infection in areas of temperate cli- mate, but infections occurring all year round in tropical regions. Pathogenesis—​following transmission, enteroviruses undergo a first round of replication in cells of the mucosal surfaces of the gastrointestinal tract and in gut-​associated lymphoid cells, followed by viraemia, which leads to infection of distant organs (brain, spinal cord, meninges, myocardium, muscle, skin, and so on), where lesions might be produced. Shedding of virus occurs from throat and faeces for many weeks. Clinical manifestations and diagnosis Most enterovirus infections are silent or only produce minor illness, but severe major illness can develop in a few of the infected. Infection with poliovirus is normally inapparent, but a few of the infected (1% or less) develop neurological symptoms comprising aseptic meningitis, or paralytic poliomyelitis. Five to ten days after a mild upper respiratory tract infection, the disease presents with flaccid paralysis resulting from motor neuron destruction; this can affect the limbs (spinal form) or muscles supplied by the medulla oblongata or bulb (bulbar form), with potentially life-​threatening re- spiratory muscle involvement. Treatment is supportive; mortality is 2–​5% in children and 15–​30% in adults, and there is residual paralysis in 90% of survivors. Other clinical syndromes include:  (1) aseptic meningitis, the most frequent clinical presentation of enterovirus infection, caused by coxsackie viruses and echoviruses; (2) encephalitis, a rare event, possibly following aseptic meningitis; (3)  pleurodynia (Bornholm disease), presenting abruptly with fever and chest pain and usually caused by coxsackie B viruses; (4) myopericarditis; (5) herpangina; (6) exanthema, rubella-​like or hand-​foot-​and-​mouth disease; and (7) conjunctivitis. Diagnosis is by virus isolation in cell culture or by viral genome detection using RT-​PCR. Prevention Paralytic poliomyelitis has been eradicated in most countries of the world following universal mass vaccination with formaldehyde-​ inactivated poliovirus (Salk vaccine) and/​or live-​attenuated viral vaccine (Sabin vaccine). However, it persists in a few countries (e.g. Pakistan, Afghanistan, and Nigeria) from which it has been exported to otherwise polio-​free states. For example, historically strains from Nigeria have caused disease in other countries of Western Africa, Indian strains were repeatedly isolated in Angola, there was a major outbreak in Tajikistan caused by strains from Northern India, and in 2011 there were at least 18 cases in China caused by strains from Pakistan. The incident in China was particularly unexpected as the immunization programme has been well executed and was effective for many years. As long as there are pockets of infection, the world remains at risk of re-​emergence of the disease. At present there are three countries that have never eradicated polio; namely Pakistan, Afghanistan and Nigeria. India was declared polio-​free in 2014. Nigeria was on the verge of being declared polio free in 2015 after three years without a case caused by wild type virus when one was reported. No case attributable to a naturally occurring wild type 2 virus has been reported anywhere in the world since October 1999 and there has been no case attributed to a wild type 3 virus since November 2012. Progress is striking but as long as there are pockets of infection, the world remains at risk of re-​emergence of the disease. Introduction Enteroviruses are a major group of viruses causing systemic infec- tion in humans. They form two genera of the Picornaviridae family (the Enterovirus and Parechovirus genera) and occur in at least 66 serotypes in humans. They infect via the gastrointestinal tract and are mostly clinically inapparent. However, viraemia can be fol- lowed by infection of organs distant from the site of entry with often devastating effects in the form of meningitis, encephalitis, paralysis, myopericarditis, and also rashes and conjunctivitis. The viruses Viruses of the Picornaviridae are non​enveloped icosahedral particles of 27 to 30 nm in diameter and contain single-​stranded RNAs of posi- tive polarity and 7.2-​ to 8.4-​kb size as their genome. The nucleic acid is polyadenylated at the 3′ end and carries a small protein, VPg, co- valently linked at its 5′ end. The enteroviruses and parechoviruses form two of the current genera of the Picornaviridae family. In 2016 there were a further 27 genera based on the sequence of the genomes. By late 2018 there were 40 and the reader is referred to the website of the International Committee on Virus Taxonomy (ICTV) for the current score. In turn, the Picornaviridae are one of five families of the new order Picornavirales. Three serotypes of poliomyelitis virus (poliovirus), 23 types of coxsackie A virus, six types of coxsackie B virus, and 33 types of enteric cytopathic human orphan (echo) vir- uses are recognized within the Enterovirus genus. The parechoviruses comprise echoviruses 22 and 23, and 14 other human viruses, and were established as a separate genus on the basis of the highly diver- gent sequence of their genomes. Other classic features of the entero- viruses, such as their stability at acid pH (in contrast to rhinoviruses or aphthoviruses), their buoyant density in caesium chloride gradi- ents, and the nature of their broad clinical effects and persistence in the environment are also shared by the parechoviruses. The three-​dimensional structure of the poliovirus particle has been elucidated by crystallographic analysis (Fig. 8.5.8.1). The viral capsid consists of 60 protein subunits, each containing the four unglycosylated viral proteins VP1 to VP4. The capsid proteins are arranged in such a way that VP1 molecules form the apices at the fivefold symmetry axis of the icosahedron, whereas two other proteins VP2 and VP3 are arranged in the centre of the triangular face near the threefold axis of symmetry; VP4 is an internal pro- tein. All proteins interact with each other. The N-​terminus of VP4 is myristoylated.

788 section 8  Infectious diseases Viruses initiate replication by attachment to their cellular receptors, and some of these have been characterized. The poliovirus receptor (PVR or CD155) is a member of the immunoglobulin superfamily. Transgenic mice expressing the human PVR become susceptible to poliovirus infection with a subsequent pathology similar to that of infected primates. Tests using these animals have been incorporated into regulatory requirements as supplements or replacements for pri- mates for vaccine testing (see next). Other enterovirus receptors are the decay accelerating factors (DAF; receptor for various echovirus types, coxsackie B virus types, and coxsackievirus A21), implicated in the complement pathway, and the integrin VLA-​2 (receptor for echovirus types 1 and 8). Other cell surface molecules might be in- volved as coreceptors in the virus–​cell receptor interactions of many enteroviruses, as the expression of a single identified receptor is not always sufficient to make a previously resistant cell line susceptible to productive infection. It is also of interest that some strains of polio- virus, mainly of serotype 2, are able to paralyse mice after infection. The receptor involved in mice has not been identified. The positive-​sense RNA genome acts as a messenger molecule. All enterovirus RNAs have a genome linked protein (VPg) at the 5’end of an untranslated region (UTR) of approximately 750 nu- cleotides in length. The region is highly structured and contains an internal ribosomal entry site. This is important for binding of the RNA to ribosomes and subsequent translation of the RNA into pro- tein. Downstream of the 5′ UTR is a large single open reading frame containing three parts: P1, coding for structural proteins VP1 to VP4; P2, coding for proteins 2A, 2B, and 2C; and P3, coding for proteins 3A to 3D. Proteins 2A and 3C are viral proteases and pro- tein 3D is the RNA-​dependent RNA polymerase (RdRp). P2 and P3 proteins (with the exception of VPg = 3B) are only found in in- fected cells. The P1 to P3 proteins are synthesized as one large pre- cursor from which the individual proteins are produced by complex autocleavage and cleavage cascades. RNA replicates via double-​ stranded replicative intermediates. The ratio of positive-​stranded to negative-​stranded RNA molecules in infected cells is approximately 100:1. During replication, RNA recombination occurs frequently. Replication of and translation from the same RNA cannot occur at the same time. Poliovirus-​infected cells undergo a shut off of cel- lular mRNA synthesis and cellular protein translation. Naked en- terovirus RNA is infectious on transfection (poliovirus was the first RNA virus rescued this way) and can be transcribed and recovered from full-​length cDNA clones which are also infectious upon trans- fection. This technique (reverse genetics) permits biochemical ma- nipulation and structure–​function studies at the molecular level. The extensive antigenic variation of enterovirus capsid proteins allows typing into polioviruses, coxsackieviruses, and echoviruses (b) VP1 VP1 Canyon 5x 3x VP3 VP2 VP2 VP3 (a) (d) (c) Fig. 8.5.8.1  Structural features of picornaviruses. (a) Electron micrograph of negatively stained poliovirus. Magnification ×270 000. (b) Diagram of the picornavirus capsid, showing the packing arrangements of VP1, VP2, and VP3, and the interspersed canyon. VP4 is located on the interior of the capsid. The biological protomer (grey) is different from the icosahedral subunit (triangle shown at right). (c) Model of poliovirus type 1, Mahoney, based on X-​ray crystallographic structure determined at 2.9 Å. The fivefold axis of symmetry is marked (5×). Surrounding the fivefold axis are canyons, the receptor-​binding site. (d) Model of poliovirus type 1, Mahoney, produced by image reconstruction from cryoelectron microscopy data obtained at 20 Å resolution. From Racaniello VR (2007). Picornaviridae: the viruses and their replication. In: Knipe DM, et al. (eds) Fields virology, 5th edition, pp. 795–​838. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia. With permission of author and publisher.

8.5.8  Enterovirus infections 789 using type-​specific neutralizing antisera, but there is some cross-​ reactivity. The main antigenic sites are located on all three major virion proteins (VP1–​VP3), and some involve sequences from more than one protein. The molecular mechanisms for the high genomic diversity of picornaviruses are thought to be based on misincorporations of nucleotides during chain elongation (due to the high error rate of the viral RdRp) and to frequent RNA recom- bination events, which also occur in natural infections. Comparison of complete RNA genome sequences of many en- teroviruses shows a very close relationship between some entero- virus and rhinovirus sequences. Within the echoviruses, however, there is great diversity. A subdivision of human enteroviruses into four species according to genomic relatedness has been proposed: 1 Human enterovirus A:  coxsackieviruses A2 to A8, A10, A12, A14, and A16, and human enteroviruses EV71, EV 76, EV89–​91, and EV114 2 Human enterovirus B: coxsackieviruses B1 to B6, A9, all echo- viruses except types 22 and 23 (now in the Parechovirus genus), and human enteroviruses EV69, EV73 to 75, EV77–​88, EV97, EV100–​101, EV106, and EV107 3 Human enterovirus C: poliovirus types 1 to 3, coxsackieviruses A1, A11, A13, A17, A19 to A22, and A24, and human entero- viruses EV96, EV99, EV102, EV104–​105, EV109, and EV116–​118 4 Human enterovirus D: human enterovirus enteroviruses EV68, EV70, EV94, and EV111 Hepatitis A virus has previously been designated EV72, but is now the type species in a separate genus, Hepatovirus. There are an add- itional five enterovirus species which encompass the non​human ­enteroviruses and three rhinovirus species making a total of 12 species in the enterovirus genus on the basis of sequence similarity. Enteroviruses are sometimes designated by a letter indicating their species and a number indicating their identity; for example entero- virus 71 is of species A and is sometimes referred to as A71; entero- virus 68 is of species D and is sometimes referred to as D68. Pathogenesis The most widely accepted pathway of enterovirus pathogenesis is based on that developed by Bodian for poliovirus, in which the virus infects the host via the gastrointestinal tract and undergoes primary replication in lymphoid cells lining the alimentary tract (oropha- ryngeal, intestinal). A viraemic phase follows, allowing infection of distant target organs: spinal cord and brain, meninges, myocardium, skeletal muscles, skin, and mucous membranes. Other tissues (e.g. lymph nodes and brown fat tissue), can also become infected. Intensive multiplication in the central nervous system (CNS) leads to the destruction of motor neurons and results in paralysis. A slightly different and more subtle model of poliovirus pathogen- esis was proposed by Sabin, in which the virus infects the mucosal surface, thus accounting for the fact that virus can be shed in faeces long after it has become undetectable in lymphoid tissues and when neutralizing antibody is detectable in the blood. The primary repli- cation creates a viraemia which seeds distant, still unknown, sites and virus replication there results in a second viraemia, which can be detected about 1 week postinfection and can lead to systemic in- fection including CNS involvement. There are very many unknowns in polio pathogenesis. For instance, it is still not clear where the virus that is shed in the faeces is produced, nor precisely from which cells. It can be inferred that they are few in number and of limited func- tion because of the level of virus excreted and the fact that infection is almost always silent if confined to the gut. Shedding of virus occurs from the throat and faeces for many weeks and even months after infection and thus ensures transmis- sion (see next). Virus replication in sites distant from the port of entry normally terminates with the appearance of neutralizing antibody, first IgM at 1 to 2 weeks after infection and then IgG and secretory IgA. Individuals with B-​cell immunodeficiencies might develop persistent infections. Most enterovirus infections are silent or produce a ‘minor illness’ with the symptoms of a mild upper respiratory tract infection, with or without a fever. In a minority of infections (1% or less) one of the following systemic ‘major diseases’ may develop: • Paralytic poliomyelitis, aseptic meningitis (polioviruses) • Aseptic meningitis, herpangina, conjunctivitis, hand-​foot-​and-​ mouth disease (coxsackie A viruses) • Aseptic meningitis, myopericarditis, encephalitis, pleurodynia (coxsackie B viruses; EV71) • Aseptic meningitis, rashes, conjunctivitis (echoviruses) • Polio-​like illness, aseptic meningitis, hand-​foot-​and-​mouth dis- ease, epidemic conjunctivitis (EV68–​71) Symptoms of clinical illness caused by enteroviruses are summar- ized in Table 8.5.8.1 and are discussed in more detail next. Clinical symptoms Central nervous system infections (See Chapter 24.11.2.) Poliomyelitis Evidence of poliomyelitis as an ancient human disease is revealed on a funerary stele from Middle Kingdom Egypt, about 1300 bc, but there is little documentation of its occurrence until nearly the end of the 19th century when it appeared in epidemics in children (hence the alternative name ‘infantile paralysis’). The appearance of poliomyelitis coincided with the improvement in standards of public hygiene and is explained by the consequent exposure of infants to infection at a later age. Maternal antibody is capable of confining infection to the gut, where the virus can persist until the immune response develops to eliminate it. In contrast, when maternal antibody has declined in older infants, the virus can spread to sites outside the intestine, causing paralysis. Even under modern conditions of hygiene, infection with all three poliovirus types is normally inapparent and illness with neuro- logical symptoms results in about 1% of infections or less. This can present as aseptic meningitis with neck stiffness, usually followed by recovery after 10 days (abortive or non​paralytic poliomyelitis). Meningitis is also caused by several other enteroviruses (see next). The more serious presentation is paralytic poliomyelitis, appearing 5–​10  days after a mild upper respiratory tract infection (‘minor illness’) and progressing to flaccid paralysis resulting from motor

790 section 8  Infectious diseases neuron destruction (‘major illness’). This might be accompanied by spasms and lack of coordination of non​paralysed muscles. Various forms of the ‘major illness’ reflect infection of different parts of the CNS. Paralysis of limbs (Fig. 8.5.8.2) results from destruction of motor neurons in the lower part of the spinal cord (‘spinal form’), while the more life-​threatening bulbar poliomyelitis (‘bulbar form’) involves infections of the medulla oblongata or bulb. Respiratory function can be affected in both the spinal and bulbar forms of the disease; encephalitis is rare. In children under 5 years old, paralysis of one leg is most common; in children 5–​15 years of age, weak- ness of one limb or paraplegia are frequent; quadriplegia is most common in adults and is often accompanied by urinary bladder and respiratory muscle dysfunction. Muscular function in limbs might return slowly, but there is residual paralysis in 90% of survivors. Of paralytic cases, 10–​25% have bulbar symptoms with hypertension, shock, and dysphonia. Complications are nosocomial pneumonias (by staphylococci or Gram-​negative bacteria), urinary tract infec- tions, and emotional problems. The mortality from paralytic polio is 2–​5% among children and 15–​30% among adults. Muscle weakness can develop many years after the initial polio disease (postpolio syn- drome or postpolio neuromuscular atrophy). A persistent poliovirus infection as cause of this has been assumed, based on the presence of viral RNA in cerebrospinal fluid and neural tissue. However, such RNA has also been found in patients with other neurological and non​neurological diseases and is, therefore, less likely to be related to the postpolio syndrome. The alternative view is that the postpolio syndrome is anatomical in origin, such that the initial attack of polio destroys motor neurons and reduces the backup available as the patient ages. Aseptic meningitis Aseptic meningitis is the most frequent clinical presentation of en- terovirus infection and can be caused by coxsackieviruses of both groups A and B, and echoviruses, mainly of types  4, 6, 11, 14, 16, 25, 30, and 31 (see Table 8.5.8.1). The disease starts with fever, headache, neck stiffness, and photophobia. Sensory or motor deficits are un- usual, but confusion is common. The symptoms can persist for 4 to 7 days. The cerebrospinal fluid usually shows pleocytosis consisting of 10 to 500 leucocytes/​µl, mainly lymphocytes. Polymorphonuclear cells may predominate at the onset, but bacterial infection and pos- sibly abscesses should be considered if they persist. The protein concentration in cerebrospinal fluid may be normal or slightly in- creased; the glucose level is normal. Complete recovery is the usual outcome of aseptic meningitis. Table 8.5.8.1  Clinical symptoms and their possible enteroviral causes Clinical symptom
(phenotype) Causative viruses PV1–​3 CVA1–​24 CBV1–​6 EV1–​33 EV68–​116 Paralysis + + (+) (+) (+) Meningitis + + + +1 Encephalitis + + +2 Febrile illness + + + Neonatal syst. inf. + +3 Herpangina +4 (+) Exanthema + + +5 Conjunctivitis +6 (+) +7 +8 HFMD +9 +10 Pneumonia + + +

Pleurodynia (+) + (+) Myocarditis + + Hepatitis + Diarrhoea + +11 PV, poliovirus; CVA, coxsackie A virus; CBV, coxsackie B virus; EV, enterovirus; HFMD, hand-​foot-​and-​mouth disease; 1, types EV4, 6, 11, 14, 16, 25, 30, 31; 2, type EV71; 3, types CVA6, 7, 13; 4, types CVA1–​6, 8, 10, 22; 5, types EV4, 9, 16; 6, type CVA24; 7, types EV7, 11; 8, type EV70; 9, types CVA10, 16; 10, type EV71. Fig. 8.5.8.2  Acute monoplegia in a Thai child in 1979 caused by poliomyelitis. Copyright D. A. Warrell.

8.5.8  Enterovirus infections 791 Encephalitis Enterovirus encephalitis is rare but might follow aseptic meningitis. Enterovirus infection in patients with hypogammaglobulinaemia or agammaglobulinaemia can persist for years with chronic men- ingitis or encephalitis and a high mortality rate as sequelae. Enterovirus 71 infection, which is normally associated with hand-​ foot-​and-​mouth disease, has been found to cause severe meningo- encephalitis (with brain stem involvement), polio-​like acute flaccid paralysis, and a high case fatality rate in children during several recent outbreaks in Bulgaria, Taiwan, and Malaysia. In some of the fatal cases there might have been coinfections with a species B adenovirus. Enterovirus 71 occurs in three genotypes and is rapidly evolving; it is most closely related to coxsackievirus A16 which also causes hand-​foot-​and-​mouth disease. Vaccines against EV71 have been licensed in China where EV71 of the C4 genotype has caused fatalities. Neonatal infections Neonatal infection followed by severe generalized disease can be caused by coxsackie B viruses and echoviruses, mainly of types 6, 7, and 11. These viruses seem to be transmitted late in pregnancy, perinatally, or postnatally by the mother or other virus-​infected infants in neo- natal wards or special care baby units. The infants develop either heart failure due to a severe myocarditis or a meningoencephalitis; hepatitis and adrenalitis might also occur. The mortality is high. Viruses can be recovered from brain, spinal cord, myocardium, and liver at autopsy. Bornholm disease (epidemic pleurodynia) This is usually caused by coxsackie B viruses but can also be caused by echoviruses of types 1, 6, 9, 16, and 19 and by coxsackie A vir- uses of types 4, 6, 9, and 10. The disease can strike families in small outbreaks. It typically starts abruptly with fever and chest pain due to the involvement of the intercostal muscles or abdominal pain re- sulting from involvement of muscles of the abdomen. There might be severe frontal headache. The symptoms last 3 to 14 days and are followed by complete recovery. Myopericarditis Enterovirus-​induced myocarditis is mostly due to infection with cox- sackie B viruses in the young. The onset of disease is usually acute, very severe, and may be fatal in neonates; however, in adolescents and adults it is normally mild. The virus may persist after the initial infection and cause dilated cardiomyopathy. In fatal cases (usually neonates 2–​11 days after onset of disease) there is cardiac dilatation, myocyte necrosis, and an inflammatory reaction. The diagnosis is often difficult, particularly in older patients, as pericarditis, coronary artery occlusion, or heart failure may have been diagnosed initially. Typical clinical findings are often tachycardia, arrhythmias, murmurs, rubs, and cardiomegaly. Besides causing acute myocarditis, chronic enterovirus infection can lead to chronic myocarditis and dilated cardiomyopathy, possibly due to immunopathological mechanisms. In chronic disease, neither infec- tious virus nor viral antigens are normally detected in heart biopsies; however, viral RNA is regularly found in cardiac muscle suggesting that the viral genome persists. The true significance of the presence of the viral genome in such cases is still under discussion since viral RNA is also found in cardiac muscle of apparently healthy controls. The disease can be produced with coxsackie B viruses in mice. In this animal model there is also initial viraemia and replication in myocytes, but this is followed by disappearance of infectious virus and destruction of myocytes, possibly by autoimmune mechanisms. Herpangina This is caused by coxsackieviruses of types A1 to A6, A8, A10, and A22. Children and young adults between 2 and 20 years of age are mainly affected. The disease presents with acute onset of fever, sore throat, and pain on swallowing, as well as vomiting and abdominal symptoms. Small vesicular lesions or white papules surrounded by a red halo can be seen on the fauces, pharynx, palate, uvula, and tonsils (Fig. 8.5.8.3). The disease is mild and self-​limiting. Exanthemas Rubella-​like rashes can be produced by echoviruses of types 4, 9, and 16, but also coxsackieviruses A9, A16, and B5 (Fig. 8.5.8.4). They usually occur in the summer and might be accompanied by fever, malaise, cervical lymphadenopathy, and aseptic meningitis. Fig. 8.5.8.3  Herpangina due to coxsackievirus A6 infection. Courtesy of the late Dr B. E. Juel-​Jensen. Fig. 8.5.8.4  Exanthema due to coxsackievirus infection. Courtesy of the late Dr B. E. Juel-​Jensen.

792 section 8  Infectious diseases Hand-​foot-​and-​mouth disease A typical distribution of vesicular lesions in hands, feet, and mouth (but also buttocks and genitalia) is produced by infection with coxsackievirus type A16 and enterovirus 71, and less frequently with coxsackieviruses A4, A5, A9 and A10, B2, and B5 (Fig. 8.5.8.5a, b). Enterovirus 71 can produce more severe clinical symptoms (see earlier). Foot-​and-​mouth disease The aphthovirus causing foot-​and-​mouth disease in cloven-​hoofed ani- mals is endemic in Africa, Asia, and South America. Virus is secreted before blisters on the mouth and feet appear in animals. The zoonosis in humans is very rare, with about 37 recorded cases. Human infection occurs from virus entering through broken skin, drinking unpasteur- ized milk, or by inhalation of droplets. A 2-​ to 6-​day incubation period is followed by blisters of hands, feet, and mouth, fever, and sore throat; complete recovery ensues. No person-​to-​person spread is recorded. Conjunctivitis Several enterovirus types cause conjunctivitis, often affecting large numbers of people epidemically. Most notable causes are echovirus types 7 and 11, coxsackievirus A24 and B2, and enterovirus 70 that often produces a haemorrhagic conjunctivitis. Diabetes and pancreatitis Insulin-​dependent diabetes mellitus (IDDM, or type 1 diabetes) is likely to be an autoimmune disorder in which the insulin-​secreting pancreatic islet cells (β cells) are destroyed. The human disease has long been thought to be caused by infectious agents, particularly since association between enterovirus infection and the development of IDDM has been shown in animal model studies (infection of mice with coxsackie B3–​B5 viruses). However, there is also a strong gen- etic component in the development of IDDM. Gastroenteritis and diarrhoea Although enteroviruses infect via the gastrointestinal tract and readily replicate there, they very rarely cause diarrhoea. Outbreaks of diarrhoea with echovirus type 11 have been reported. In Japan, the Aichi virus, the type species of the Kobuvirus genus of the Picornaviridae family, has been identified as the cause of multiple outbreaks of gastroenteritis in humans, mostly associated with the consumption of raw oysters. This virus seems to circulate widely in populations of Japan and other south-​east Asian countries, with subclinical infections likely to be common (see Chapter 8.5.9). Chronic fatigue syndrome Chronic fatigue syndrome, also previously known under the names of myalgic encephalomyelitis (ME), Royal Free disease, Iceland dis- ease, postviral fatigue syndrome, and neuromyasthenia, can occur both sporadically and epidemically. The main clinical feature is excess fatigability of skeletal muscle, accompanied by pain. Other symptoms include headaches, inability to concentrate, paraesthesia, and impair- ment of short-​term memory. A major problem in diagnosis is a clear definition of the clinical entity. Several virus infections have seemed to precede the development of chronic fatigue syndrome; they are mainly enterovirus infections, chronic Epstein–​Barr virus infection, and also infections with Toxoplasma and Leptospira spp. The strin- gency of the association of chronic enterovirus infection with the appearance of chronic fatigue syndrome is very controversial. A re- port of a joint working group of the Royal Colleges of Physicians, Psychiatrists, and General Practitioners has concluded that persist- ence of enteroviruses is unlikely to play a role in the development of chronic fatigue syndrome. Similar conclusions have been drawn for the possibility of a causal link between chronic Epstein–​Barr virus infection and chronic fatigue syndrome (see Chapter 8.5.3). Laboratory diagnosis of enterovirus infections Virus isolation Virus isolation is an excellent procedure to diagnose enterovirus infections although its use is declining in favour of molecular methods. Virus is shed for weeks, and sometimes months, from the primary infection sites (cells lining the gut, see earlier). Starting from a few days after infection, virus can be found in concentra- tions of 105 to 106 tissue culture infectious doses 50%/​g (TCID50/​g) of faeces. Throat swabs are also a good source for virus, particularly early in infection and when there are respiratory symptoms. In cases of meningitis, enteroviruses can be propagated in cell culture from the cerebrospinal fluid, but the method is much less sensitive than genome detection (see next). Viruses are readily isolated in secondary cultures of monkey kidney cells, or in cultures of per- manent cell lines derived from human embryonic kidney, human amnion, or human fetal lung. The cytopathic effect produced by enteroviruses is non​specific. Typing of a cytopathic agent is carried out using antiserum pools (see next) or in multistep procedures. Most coxsackie A viruses (with the exception of coxsackievirus A9) do not grow well in cell culture but can be readily isolated by (b) (a) Fig. 8.5.8.5  (a, b) Hand-​foot-​and-​mouth disease due to coxsackievirus infection. Courtesy of the late Dr B. E. Juel-​Jensen.

8.5.8  Enterovirus infections 793 intracerebral, intraperitoneal, or subcutaneous infection of mice, causing flaccid paralysis and death. In contrast, coxsackie B viruses cause spastic paralysis. Polioviruses or echoviruses do not usually grow in mice although polioviruses will replicate in transgenic ani- mals that have appropriate receptors (see earlier). Serology Neutralization assays were the method of choice for typing entero- viruses for many years. Due to the large number of enterovirus types, these tests are labour intensive and not apt for rapid diagnosis. Pools of type-​specific antisera (prepared by Drs Lim, Melnick, and Benyesch, and termed LMB pools) have greatly helped to establish the epidemiology of enterovirus infections worldwide. Recurrent enterovirus infections during a lifetime often result in elevated serum antibody titres which obscure diagnostic changes. Significant antibody rises are, therefore, rarely observed in paired sera (taken at the onset of and during convalescence from disease). A coxsackie B virus-​specific IgM test (using an IgM antibody cap- ture technique) has been developed for rapid diagnosis. However, there is cross-​reactivity between the IgM responses to different en- teroviruses, including different genera of the picornaviruses, and so this test is not very specific. Prolonged presence of enterovirus-​ specific IgM has also been observed. In summary, the usefulness of serology for the diagnosis of enterovirus infection is limited. Genome detection The reverse transcription–​polymerase chain reaction (RT-​PCR) technique is widely applied to test for the presence of enterovirus genomes and is the method of choice for identifying virus infection in many countries. This technique is very sensitive and specific, par- ticularly in diagnosing CNS infections from cerebrospinal fluid spe- cimens, and has become the ‘gold standard’ of diagnosis, surpassing viral culture. Enterovirus RNAs have also been detected in myocar- dial biopsies from patients with myocarditis and dilated cardiomy- opathy, in muscle of people with inflammatory muscle disease and chronic fatigue syndrome, and in brain biopsies. The significance of these findings is not clear, as infectious virus can rarely be isolated and viral antigen cannot be detected. Highly conserved sequences of the 5′ end of enterovirus genomes have allowed the design of PCR primers detecting most enterovirus RNAs. As the EV22 genome is very different from that of the other enteroviruses (see earlier), tailor-​made primers have to be added in a multiplex RT-​PCR to in- clude detection of these viruses, which cause infections particularly in neonates and infants. A modified RT-​PCR procedure can differ- entiate between wild type and vaccine-​derived poliovirus infections. Modern sequencing techniques (Deep, Massive parallel or Next Generation sequencing) offer the possibility of sequencing without either cell culture isolation, PCR or knowledge of the sequence to be sought and are having a major impact on diagnosis of enterovirus and other infections, and environmental surveillance. Epidemiology of enterovirus infections Enteroviruses are mainly transmitted by the faeco-​oral route, due to the fact that viruses are shed in faeces for weeks or months after infection. Spread is particularly intense within families, usually starting from the primary infection of young children. In temperate climates, there are seasonal peaks (July–​September in the northern hemisphere and December–​February in the southern hemisphere), whereas in subtropical and tropical climates enterovirus infections occur all the year round. Most primary human enterovirus infec- tions occur during the first decade of life. Type-​specific surveillance in several geographical regions has shown that coxsackieviruses A9, A16, P and B4 and echovirus types 6, 9, 11, 19, 22, and 30 are most frequently found. Prevention of enterovirus infections As there are only three poliovirus types and no significant animal reservoir, it has been possible to develop very successful poliovirus vaccines. In 1954, a formalin-​inactivated poliovirus vaccine (IPV) was introduced by Dr Jonas Salk in the United States of America, and in 1962 Dr Albert Sabin introduced a vaccine consisting of live-​ attenuated strains of the three poliovirus types which could be given orally (OPV). Protection by the live-​attenuated vaccine is effected mainly at the site of entry by eliciting locally virus-​specific IgAs and IgGs. Inactivated vaccine mainly elicits serum IgGs which prevent infection of the CNS and other sites distant of the port of entry by neutralization of viraemic virus. The main characteristics of IPV and OPV are summarized in Table 8.5.8.2. Inactivated poliovirus vaccine The early IPVs developed by Salk were of relatively low potency. High potency vaccines, based on large-​scale cell culture followed by virus purification and concentration but using the same inacti- vation procedures, were developed in the Netherlands in the 1980s and form the basis of IPVs used today. Much of the developed world including Europe and the United States of America now uses only IPVs, having previously used the live-​attenuated vaccines. Other countries including Mexico and Russia have changed from using OPV to IPV, and middle income countries such as Argentina and Uruguay are proposing to do so when poliomyelitis is eradicated, since OPV was thought to be better able to eradicate poliomyelitis and was proven to be able to break epidemic transmission. However, Scandinavian countries and the Netherlands had eliminated the disease with IPVs. IPV is given by injection and is more expensive per dose than the oral vaccine, but has advantages as outlined next, mainly the avoidance of vaccine-​associated paralysis. IPV is the vaccine of choice in cases of immunodeficiency. The World Health Organization has recommended the introduction of a single dose of IPV in all countries in preparation for the cessation of OPV usage (see next). Live-​attenuated poliovirus vaccine This vaccine has several advantages compared to the inactivated vac- cine (Table 8.5.8.2) as it: • parallels the natural infection; • stimulates both local secretory IgA in the pharynx and alimentary tract, and systemic circulating virus-​specific IgG antibody; • is easy to administer as an oral vaccine; • is more cost effective; and • is proven to be capable of interrupting virus circulation and epidemics.

794 section 8  Infectious diseases The disadvantage is that in a few cases the attenuated vaccine strains have reverted to virulence in vaccine recipients or their contacts. Since the early 1980s, all cases of polio in the United States of America and Europe were found to be vaccine-​related, occurring either in vac- cine recipients or their close contacts who became infected by them, or were imported from endemic countries and were not indigenous original wild type strains. The risk of vaccine-​associated poliomyelitis is between 0.5 and 3.4 cases/​million of susceptible children immun- ized. Vaccine-​related polio is mostly caused by type 2 or type 3 viruses, probably due to the fact that the number of point mutations in type 1 vaccine virus compared to wild type virus is much higher than in type 2 and type 3 vaccine viruses. However, as the disease becomes in- creasingly rare in the countries concerned and the world at large, indi- genous cases or importation of virus are increasingly rare, and the risks of oral vaccination begin to outweigh its benefits. The major current disadvantage, however, is that in addition to poliomyelitis in vaccine recipients and their immediate contacts, the re-​introduction of polio by the use of oral vaccine has now been frequently documented in many regions where immunization programmes are imperfect and the virus is able to regain the ability to transmit from person to person and cause outbreaks. This poses a risk to the whole eradication programme. A further issue is the chronic infection of hypogammaglobulinaemic patients unable to mount a humoral response. Such individuals, if given the live vaccine, can go on to excrete virus for many years, al- though this is not common. These risks are not associated with the use of IPV, which has become the vaccine of choice in many countries. Polio eradication and surveillance For many years it was thought that the Sabin oral poliovirus vac- cines were ineffective in tropical countries, being unable to the con- trol the disease, much less eradicate it. While many reasons were put forward, the lack of impact of polio vaccination programmes was probably due to loss of vaccine potency through failure to maintain storage at cool temperatures (‘cold chain’), and also the epidemiology of poliovirus infection. In temperate countries, poliomyelitis is sea- sonal with infections peaking in the summer months. A strategy of vaccination based on immunization of young children at a set age (usually a few months) is, therefore, able to build up a highly im- mune population resistant to infection in the winter so that trans- mission of the wild type virus becomes more difficult; thus, the virus and the disease are eradicated. In tropical countries, where exposure is year round, it is a matter of chance whether a child will first be naturally infected or immunized, and virus circulation can continue. This was recognized by Sabin in 1960, but not acted upon until some 20 years later when the strategy of National Immunization Days was developed in South America. This approach involves immunizing all children below a certain age in a country within a very short period, so that all susceptible children’s intestinal tracts are occupied by vac- cine virus and are, therefore, resistant to infection by the wild type virus. Transmission of wild type virus is therefore broken, and the virus dies out. The World Health Organization (WHO) pronounced the inten- tion of eliminating poliomyelitis due to wild type virus in 1988, with a target date of 2000 for completion. While the target date was clearly missed, the Americas have been free of polio since 1992. In 2000, the Western Pacific Region was declared polio-​free by the WHO, and the European region in 2002. The enormous progress achieved between 1988 and 2004 is shown in Fig. 8.5.8.6. The scale of the undertaking is colossal, and the progress towards eradication is extraordinary. For example, in 1992 in China, all children aged 5 or less were immunized during a 1-​week period. This amounted to one-​quarter of the world’s children. However, eradication of polio has proved to be very difficult in some regions. At the time of writing, virus is still known to be Table 8.5.8.2  Characteristics of poliovirus vaccines Characteristic Live-​attenuated poliovirus vaccine (OPV) Inactivated poliovirus vaccine (IPV) Virus source Attenuated virus (Sabin strains) Virulent virus strains Primary course 3 doses at monthly intervals starting at age of 2 months (temperate climates; more doses in tropics) Three doses at 2-​month intervals Administration route Oral Parenteral (injection) Immunity produced—​systemic IgA, IgM, IgG IgM, IgG, (IgA) —​local IgA (IgA, minimal) Booster doses required

  1. at school entry Yes (every 3–​5 years or when exposed)
  2. between 15 and 19 years
  3. in adult life when exposed (last dose 10 years or more ago) Efficacy Good in temperate climates, variable in tropics Good Spread to contacts Yes No Vaccine-​associated paralysis 0.5–​3.4 cases/​million first doses in susceptible children No Production cost per dose .07 .7 Requirement on personnel Not highly trained Trained and skilled Requirement of ‘cold chain’ Yes Less than OPV Combination with other vaccines No Possible Use in immunodeficient children No Possible

8.5.8  Enterovirus infections 795 endemic in only Pakistan and Afghanistan, although some cases have been reported in Nigeria, where full access to at-risk areas is difficult (Fig. 8.5.8.7). Eradication before long is a real possibility although it is not a trivial matter. It was only in 2011 that India re- corded no cases at all. It was certified to be polio-​free in 2014. The virus has been reintroduced repeatedly into countries where it had been eradicated, particularly into Angola from India and into the Democratic Republic of Congo from Angola. It has also been intro- duced into Tajikistan (European Region) from India, and into China from Pakistan. The effect of importation from one country to others was graphically illustrated in 2004 when immunization stopped in Nigeria after it had been suggested that the vaccine contained oestro- gens to render recipients sterile. The result was the re-​emergence and re-​introduction of polio into much of Central Africa, where it had been previously considered eradicated, and outbreaks occurred in Yemen and Indonesia; it is surmised that pilgrims returning from Mecca were infected by coreligionists from Nigeria and reintroduced the virus. The situation was only brought under control by massive coordination of immunization activities throughout the region. Lingering on of wild type polio up until 2010 in India was in part due to vaccine refusals similar to those in Nigeria. The last case of polio in India was in 2011. In Pakistan the numbers increased by 62% in 2010, due to civilian turmoil, flood catastrophes, and lack of political will to eradicate. In 2014 a poliovirus strain from Pakistan was found to be circulating silently in Israel. Israel has used only IPV for nearly ten years and was initially reluctant to re-​introduce OPV to break transmission. The in- cident showed that under certain circumstances IPV will not prevent circulation, although in other circumstances such as in Scandinavia and the Netherlands it has been very effective at eradicating the virus as well as disease. Two poliovirus isolates of another Pakistan-​derived virus were made in Egypt, again without any cases but no further isolations of this strain have been reported, possibly because of the continued routine use of OPV in Egypt. Finally, an outbreak of cases caused by a Pakistan virus occurred in Syria where there has been armed conflict, and immunization is very difficult. The outbreak was brought under control. Similar concerns arise in Africa, where Nigeria remained a po- tential source of polio virus for many years and there have been out- breaks in Chad, Kenya, and other countries. The Central African Republic has no cases at the time of writing, but is a particular focus of attention because of its poor domestic circumstances and the occurrence of cases in the surrounding countries. The eradication of poliomyelitis is not trivial. Nonetheless there has been no case of poliomyelitis attributable to a naturally occurring type 2 strain of poliovirus since October 1999 and at the time of writing there had been no case of poliomyelitis caused by a wild type 3 virus since November 2012. Moreover, Nigeria has had few reported cases since 2012, and the two remaining endemic countries Pakistan and Afghanistan had fewer than 60 cases in 2015. It is very possible that poliomyelitis will be eradicated in the near future. As long as pockets of infection persist, the world will remain at risk of the re-​emergence of polio. Thus, part of the challenge is to demon- strate that the virus has in fact been eliminated, and this depends on rigorous effective surveillance. One approach is to obtain data on cases of acute flaccid paralysis of whatever cause, including the Guillain–​ Barré syndrome. All cases should be investigated to see whether they are due to poliovirus infection or not. It is considered that the back- ground rate of paralysis in the absence of poliomyelitis should be one case per 100 000 members of the population, providing a control for the adequacy of the surveillance scheme. All poliovirus isolates iden- tified in paralysis surveillance are examined to establish whether they are derived from vaccine or represent wild type strains. Surveillance of environmental samples including sewage for poliovirus also plays an increasing role in global polio eradication, and isolates have been obtained in areas where there are no recorded cases of disease as afore- mentioned in Israel and Egypt and at one stage in India. Many coun- tries perform routine environmental studies of this kind. There are possible concerns over the adequacy of either approach. Once wild type poliovirus has been eradicated, the only sources of the virus will be manufacturers of vaccines, laboratories holding stocks or potentially infected samples, and recipients of live-​ attenuated vaccine. While manufacturers and laboratory workers can be required to work under high containment level conditions to avoid escape of virulent virus, vaccinees pose a particular problem. The oral vaccine works by establishing an infection in the recipient, and there are numerous instances of outbreaks caused by vaccine-​ derived polioviruses that have recovered the ability to circulate (cir- culating vaccine-​derived viruses or cVDPVs). This has been observed in Haiti, Egypt, the Philippines, and Madagascar among many others, and might be relatively common, particularly where vaccination con- tinues with the live vaccine for a long time with poor coverage, so that vaccinated and unvaccinated individuals mix, providing the ideal conditions for the selection of transmissible virus. However, the Polio incidence: 1988 Polio incidence: 2004 Equator Equator Known or probable wild poliovirus circulation Fig. 8.5.8.6  World maps depicting the circulation of wild type poliomyelitis virus for 1988 and 2004, as reported by the World Health Organization. From Palllansch M, Roos R (2007) Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In: Knipe DM, et al. (eds) Fields virology, 5th edition, pp. 839–​93. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia. With permission of author and publisher.

796 section 8  Infectious diseases vaccine virus seems to be poorly transmissible compared to the wild type. In countries such as Cuba where it has been given only in the early part of the year as a matter of policy, virus is not detectable after 6 months. Thus, it might be possible to stop vaccinating with no fur- ther precautions, and deal with the re-​emergence of polio as cVDPVs on a case-​by-​case basis. The Global Action Plan (third draft) or GAP3 provides instructions of the containment of poliovirus in manufac- ture, diagnosis, research, and any other usage. Natural wild type 2 poliovirus has not been implicated in polio- myelitis since 1999. The only cases occurring now are due to the vac- cine directly in vaccinees or their contacts or indirectly in the form of cVDPVs. Immunization with type 2 OPV should therefore cease, but the strategies to be followed are complex, requiring that within a country or region all type 2 usage should stop together, prepar- ations should be made for possible type 2 outbreaks, particularly of cVDPVs and to protect the population from disease by the use of IPV. The withdrawal of type 2 OPV, was implemented in 2016, and will provide a model for the cessation of all OPV use and the final eradication of polio. The final stages and containment of polioviruses are the subject of the WHO document on the final strategic plan for eradication, which includes timings of the responses required and GAP3, both of which are available from the WHO website. A further concern relates to people with B-​cell immunodeficiency who can become chronically infected but be apparently healthy, sometimes for decades as described earlier. During this time the virus might adapt to an extent that neurotropism is regained, and an unvaccinated population will again be highly susceptible. The numbers and geographical distribution of such long-​term excretors are unknown but most exposed individuals do not excrete virus for very long periods, and even those that do usually cease excreting virus spontaneously, albeit after a period of a few years. On the other hand, an individual is known to have excreted type 2 polio for at least 28 years. Long-​term secretors of poliovirus could initiate a new outbreak of polio in a population in which poliovirus circulation has been considered to be eliminated. It would be unwise to stop polio vaccination with IPV until such individuals are known to no longer excrete virus. Stocks of OPV, preferably monovalent type 1, 2, and 3 poliovirus vaccines, should be kept in reserve in case there is an outbreak. New approaches to vaccine development are under con- sideration; for example, producing live attenuated strains that are considered unable to revert to virulence, or new strains of virus to be used for production of IPV that are hyperattenuated and genetically very stable and therefore safe or expression of stable empty capsids which are non infectious. These developments are ongoing. The de novo synthesis of infectious particles from poliovirus RNA transcribed from synthetic cDNA in a cell-​free HeLa cell extract has created a huge debate on the scientific value of such an experiment, concerns about poliovirus eradication, issues of national security and freedom of virological/​biological research. Since then, other viruses (ΦX174, the 1918 H1N1 influenza virus a.o.) have been synthesized de novo, permitting the effect of drastic changes in viral sequence on viral phenotypes to be examined in more detail than is possible by established methods of genetic manipulation. The use- fulness of the method to carry out broad-​based research into ques- tions of viral pathogenesis and attenuation for vaccine production (e.g. by changing codon usage) is becoming appreciated but it has also led to considerable efforts by the scientific community and in- dustry to define and assess possible misuse of this technology. Case or outbreak following importation Endemic countries Wild virus type 1 Wild virus type 3 Wild virus type 1 & 3 Excludes viruses detected from environmental surveillance and vaccine derived polioviruses. Data in WHO/HQ as of 22 Apr 2008 Fig. 8.5.8.7  Cases of wild type polio in 2011. From www.polioeradication.org, with permission from WHO.

8.5.9 Virus infections causing diarrhoea and vomit

8.5.9 Virus infections causing diarrhoea and vomiting 797

8.5.9  Virus infections causing diarrhoea and vomiting 797 FURTHER READING Cello J, Paul AV, Wimmer E (2002). Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural tem- plate. Science, 297, 1016–​18. Centers for Disease Control and Prevention (CDC) (2011). Progress toward poliomyelitis eradication—​Nigeria, January 2010–​June 2011. MMWR Morb Mortal Wkly Rep, 60, 1053–​7. Centers for Disease Control and Prevention (CDC) (2011). Update on vaccine-​derived polioviruses—​worldwide, July 2009–​March 2011. MMWR Morb Mortal Wkly Rep, 60, 846–​50. Dunn G, et al. (2015). Twenty-​eight years of poliovirus replication in an immunodeficient individual: impact on the global polio eradica- tion initiative. PLoS Pathog, 11, e1005114. Hovi T, et  al. (2011). Role of environmental poliovirus surveil- lance in global polio eradication and beyond. Epidemiol Infect, 140, 1–​13. Joint Working Group of the Royal Colleges of Physicians, Psychiatrists, and General Practitioners (1997). Chronic fatigue syndrome, pp. 58. Royal College of Physicians Publication Unit, London. Kew OM, et al. (2002). Outbreak of poliomyelitis in Hispaniola asso- ciated with circulating type 1 vaccine-​derived poliovirus. Science, 296, 356–​9. Knowles NJ, et  al. (2011). Picornaviridae. In:  King AMQ, et  al.
(eds) Virus taxonomy:  classification and nomenclature of viruses,
pp. 855–​80. Ninth report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, CA. Larson HJ, Ghinai I (2011). Lessons from polio eradication. Nature, 473, 446–​7. Martin J, et al. (2000). Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-​day period of virus excretion. J Virol, 74, 3001–​10. Melnick JL (1996). Enteroviruses:  polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In:  Fields BN, et  al. (eds) Fields virology, 3rd edition, pp. 655–​712. Lippincott-​Raven, Philadelphia, PA. Mendelsohn C, Wimmer R, Racaniello VR (1989). Cellular receptor for poliovirus: molecular cloning, nucleotide sequence and expres- sion of a new member of the immunoglobulin superfamily. Cell, 56, 855–​65. Minor PD (1990). Antigenic structure of picornaviruses. Curr Top Microbiol Immunol, 161, 122–​54. Minor PD (1996). Poliovirus. In: Nathanson N, et al. (eds) Viral patho- genesis, pp. 555–​74. Lippincott-​Raven, Philadelphia. Minor P (2005). Picornaviruses. In: Mahy BW, ter Meulen V (eds) Topley and Wilson’s microbiology and microbial infections. Virology, 10th edition, pp. 857–​87. Hodder Arnold, London. Nathanson N (2008). The pathogenesis of poliomyelitis: what we don't know. Adv Virus Res, 71, 1–​50. Offit PA (2005). The cutter incident, pp. 256. Yale University Press, New Haven. Pallnsch M, Oberste MS, Whitton LS (2013). Enteroviruses:  polio- viruses, coxsackieviruses, echoviruses, and newer enteroviruses. In:  Knipe DM, et  al. (eds) Fields virology, 6th edition, pp. 490–​ 530. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia, PA. Racaniello VR (2013). Picornaviridae:  the viruses and their repli- cation. In: Knipe DM, et al. (eds) Fields virology, 6th edition, pp. 453–​89. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia, PA. Racaniello VR, Baltimore D (1981). Cloned poliovirus complemen- tary DNA is infectious in mammalian cells. Science, 214, 916–​19. Skern T (2010). 100 years poliovirus: from discovery to eradication: a meeting report. Arch Virol, 155, 1371–​81. Wimmer E (2006). The test-​tube synthesis of a chemical called polio- virus: the simple synthesis of a virus has far-​reaching societal impli- cations. EMBO Rep, 7 Spec No, S3–​S9. Wimmer E, et  al. (2009). Synthetic viruses:  a new opportunity to understand and prevent viral disease. Nat Biotechnol, 27, 1163–​72. Wimmer E, Paul AV (2011). Synthetic viruses:  a new opportunity to understand and prevent viral disease. Annu Rev Microbiol, 65, 583–​609. World Health Organization (2008). Global polio eradication initia- tive. Annual report 2007. Impact of the intensified eradication effort. WHO, Geneva. http://​polioeradication.org/​wp-​content/​uploads/​ 2016/​07/​AnnualReport2007_​English.pdf Yamashita T, et al. (2000). Application of a reverse transcription-​PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans. J Clin Microbiol, 38, 2955–​61. Websites ICTV. Taxonomy. http://​ictvonline.org/​virusTaxonomy.asp World Health Organization. Polio eradication initiative. http://​www. emro.who.int/​entity/​polio/​ and http://​www.polioeradication.org/​ Dataandmonitoring/​Poliothisweek.aspx 8.5.9  Virus infections causing diarrhoea and vomiting Philip R. Dormitzer and Ulrich Desselberger ESSENTIALS Acute gastroenteritis is frequently caused by rotaviruses, human caliciviruses (noroviruses, sapoviruses), astroviruses and enteric adenoviruses (group F): these cause much disease worldwide and considerable mortality, mainly in developing countries. Other vir- uses found in the human gastrointestinal tract are not regularly associated with diarrhoeal disease, except in patients who are im- munosuppressed and in whom herpes simplex virus, cytomegalo- virus, and picobirnaviruses can cause diarrhoea, as can HIV itself. Epidemiology—​(1) Rotaviruses—​a major cause of endemic infantile gastroenteritis worldwide; transmission is by the faecal-​oral route; there is a strict winter peak of infections in temperate climates, but these occur year-​round in tropical and subtropical regions; many animals and birds harbour a large diversity of rotaviruses and may act as a reservoir for human infections. (2) Human caliciviruses—​ the most important cause of non​bacterial gastroenteritis outbreaks worldwide; transmission is by the faecal-​oral and emesis-​oral routes; contaminated food (oysters, green salads, fresh fruit, cold foods, and sandwiches) and water are frequently implicated in outbreaks.

798 section 8  Infectious diseases Clinical features and management—​following an incubation period of 1–​2 days, there is sudden onset of watery diarrhoea lasting between 4 and 7 days, vomiting, and varying degrees of dehydration. Other features include abdominal cramps, headache, myalgia, and fever. Treatment is supportive, mainly with oral rehydration solutions or—​in more severe cases—​intravenous rehydration. Continued feeding is recommended, with zinc supplementation in areas where micronu- trient deficiency may be present. Diagnosis—​viral infection can be demonstrated by virus-​specific enzyme-​linked immunosorbent as- says and by viral genome detection using the polymerase chain re- action (for adenoviruses) or reverse transcription-​polymerase chain reaction (RT-​PCR) (for rotaviruses, caliciviruses, and astroviruses). Prevention and control—​two live attenuated oral rotavirus vaccines have been licensed in numerous countries since 2006, one vac- cine licensed in India in 2014 has achieved WHO prequalification; and three more have national licenses in India, China, or Vietnam. In countries where universal mass vaccination of children as part of childhood vaccination schemes has been established, a significant reduction of rotavirus-​associated acute gastroenteritis has been re- corded. In Mexico, introduction of rotavirus vaccines was associated with a substantial drop in overall mortality due to diarrheal disease of any cause in children 23 months of age and younger. Vaccines against human norovirus disease are under development. Outbreak control measures focus on the interruption of person-​to-​person transmis- sion, the removal of common sources of infection (food, water, and so on), and improvement of general environmental hygiene. Introduction Acute gastroenteritis and vomiting in humans is a well-​characterized clinical entity caused by various microbial agents (viruses, bacteria, parasites, and so on). Viral gastroenteritis is a global problem, par- ticularly in infants and young children. Many viruses are found in the human gut, but not all of them produce acute gastroenteritis, and even pathogenic agents of gastroenteritis may also be shed asymptomatically (Table 8.5.9.1). Viral infections normally associated with gastroenteritis are caused by rotaviruses, human caliciviruses (noroviruses, sapoviruses), astroviruses and enteric adenoviruses (group F). Other viruses found in the human gastrointestinal tract (enteroviruses, reo- viruses, non​group F adenoviruses, toroviruses, coronaviruses, parvoviruses) are not regularly associated with diarrhoeal disease. Finally, there are viruses causing diarrhoea in immunosuppressed patients (most commonly those infected with HIV), including herpes simplex virus, cytomegalovirus, and picobirnaviruses. HIV itself can also infect the gut directly. Recently, the study of inter- actions of different microbes infecting the gut has shown that bac- terial flora can modulate the replication of enteric viruses. Such studies of the gut microbiome are attracting increased attention with the aim of better understanding these mechanisms in health and disease. Only the major virus groups regularly causing gastroenteritis in humans are described here in separate sections. Clinical symptoms, diagnosis, treatment, epidemiology, and vaccine development are reviewed under common headings. Rotaviruses Structure Rotaviruses are the major cause of infantile gastroenteritis world- wide and also of acute diarrhoea in the young of many mammalian species. They constitute the genus Rotavirus in the Reoviridae family, with a genome of 11 segments of double-​stranded RNA encoding six structural viral proteins (VP1–​VP4, VP6, VP7) and six non-​ structural proteins (NSP1–​NSP6). All genes are monocistronic, ex- cept for RNA segment 11, which encodes two proteins (NSP5 and NSP6). The icosahedral virion has three concentric protein layers and no lipid envelope (Fig. 8.5.9.1a). In electron micrographs of negatively stained specimens, virions have a characteristic appear- ance as 75-​nm wheel-​like particles (Fig. 8.5.9.2), the name of the virus being derived from Latin rota  =  wheel. The inner layer (consisting of VP2) encloses the genome seg- ments, the viral RNA-​dependent RNA polymerase (RdRp) VP1, and the capping enzyme VP3. The addition of a middle layer consisting of VP6 leads to the formation of transcriptionally ac- tive subviral particles, referred to as the double-​layered par- ticles (DLPs). VP6 is the most immunogenic rotavirus protein. Infectious virions (triple-​layered particles, TLPs) have an add- itional layer, which mediates the translocation of the DLP into the cytoplasm during cell entry. This outermost layer consists of two proteins, VP4 and VP7. VP7 forms a shell, which is shed in the low calcium environment of the cytoplasm. VP4 forms spikes, which are important for attachment and membrane penetration. Crystal structures, electron cryomicroscopy image reconstruc- tions, and functional studies have provided evidence that a fold-​ back rearrangement of the VP4 spike, which is mounted on the VP6 layer and secured by the surrounding VP7 shell, is required for membrane penetration during entry (Fig. 8.5.9.1b). To achieve Table 8.5.9.1  Virus infections of the human gut Viruses found as Genus (family) Regular cause of diarrhoea and vomiting Rotaviruses (Reoviridae)a Human caliciviruses (Caliciviridae) a Group F adenoviruses (Adenoviridae) Astroviruses (Astroviridae) Occasional cause of diarrhoea and vomiting Enteroviruses (Picornaviridae)b Reoviruses (Reoviridae) Adenoviruses other than Group F (Adenoviridae) Toroviruses (Coronaviridae) Coronaviruses (Coronaviridae) Parvoviruses (Parvoviridae) Cause of diarrhoea in immunodeficient patients Human immunodeficiency virus (Retroviridae) Herpes simplex virus (Herpesviridae) Cytomegalovirus (Herpesviridae) Picobirnaviruses (Birnaviridae) a Not all infections cause disease (see text). b Outbreaks of diarrhoea caused by echovirus type 11 infections have been reported (see Chapter 8.5.8), and Aichi virus is an endemic cause of diarrhoea in Asia and of traveller’s diarrhoea.

8.5.9  Virus infections causing diarrhoea and vomiting 799 maximal infectivity of the virion, the VP4 spike must be cleaved by intestinal trypsin or cellular proteases. Classification Most commonly, rotaviruses are classified according to the im- munological reactivity and genomic sequences of three of their structural components (VP6, VP7, and VP4), although a more recent and comprehensive classification differentiates all 11 gene segments of rotaviruses into genotypes. Specific sequences and epitopes on the middle-​shell protein VP6 allow at least 7–​8 groups (A–​G/​H, possibly I) to be distinguished. Group A rotaviruses cause the vast majority of human infections and acute gastroenteritis and have been divided into subgroups on the basis of additional de- terminants on VP6. Group B rotaviruses have caused epidemics of diarrhoea affecting adults and children, mainly in China and India. Group C rotaviruses generally cause milder diarrhoeal disease. The remaining groups are only known to infect non​human hosts. Both surface proteins, VP4 and VP7, elicit and are the targets of neutralizing antibodies. A dual-​type classification system has been devised for group A rotaviruses, which differentiates glycoprotein (G) types (VP7-​specific) and protease-​sensitive protein (P) types (VP4-​specific). For G types, serotype and genotype are equivalent; for P types serotype and genotype may be different, and genotypes are more commonly used. For example, G1P[8]‌ is G serotype and genotype 1, P genotype 8. At least 12 G types and 15 P types have been found in humans. However, at present, rotaviruses carrying a relatively restricted number of G types (G1–​G4, G9, and G12) and P types (P[4], P[6] and P[8]) cause most human disease. Rotavirus genomes undergo point mutations continuously (due to the high inherent error rate of the viral polymerase), with one of the conse- quences being antigenic drift. In addition, rotaviruses can exchange (reassort) genome segments during mixed infections, providing a further mechanism to introduce genetic diversity (antigenic shift). Zoonotic rotavirus infections of humans are well documented, and previously rare serotypic variants have become established among strains pathogenic to humans (mainly in tropical and subtropical regions). Hence, rotavirus epidemiology continues to evolve by various mechanisms, and eradication of rotaviruses is not feasible. Although the mechanisms of rotavirus genetic diversity resemble those of influenza viruses, rotaviruses do not undergo the frequent waves of global strain replacement and occasional pandemic shifts observed with influenza viruses. Fig. 8.5.9.2  Rotavirus particles in the faeces of a child admitted to hospital with acute gastroenteritis. Negative staining with aqueous 2% potassium phosphotungstate, pH 7.0. Scale bar represents 100 nm. Four different morphologies of particles are shown: (a) triple-​layered particle containing RNA; (b) triple-​layered particle without RNA (empty, core penetrated with stain); (c) double-​layered particle containing RNA; and (d) double-​layered empty particle. Courtesy of M. Jenkins, Regional Virus Laboratory, East Birmingham Hospital. From Desselberger U (1992). Reoviruses. In: Greenwood D, Slack R, Peutherer J (eds) Medical microbiology, 14th edition, p. 620. Churchill Livingstone, Edinburgh, with permission of the publisher. VP4 VP7 VP6 VP2 RNA (a) (b) VP8* VP7 VP6 VP2 VP5* (c-c) Fig. 8.5.9.1  (a) The icosahedral rotavirus virion has three layers: (1) an inner VP2 layer that contains the genome, polymerase, and capping enzyme; (2) a middle VP6 layer; and (3) an outer VP7 layer. The VP4 spike is anchored in the VP6 layer and protrudes through the VP7 layer (b). For efficient infection, VP4 must be cleaved by proteases into the VP8* fragment, which includes the receptor binding ‘head’ of the spike, and the VP5* fragment, which combines with the N-​terminal region of VP8* to form the remainder of the spike and includes hydrophobic loops involved in the disruption of a host cell membrane during entry. Panel (a): based on Dormitzer P, et al. (2004). Structural rearrangements in the membrane penetration protein of a non-​enveloped virus. Nature, 430, 1053–​58;
Yeager M, et al. (1990). Three-​dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J Cell Biol, 110, 2133–​44.
Panel (b): from Settembre EC, et al. (2011). Atomic model of an infectious rotavirus particle. EMBO J, 30, 408–​16. With permission of the authors and the publisher.

800 section 8  Infectious diseases Replication Studies of rotavirus replication have been enabled by adaptation of many rotavirus strains to growth in mammalian cell culture and the ability to assign gene functions through a classic genetic approach in which rotavirus strains exchange genome segments through reassortment during mixed infection of single cells. Recently, ­reverse genetics systems for rotaviruses based only on plasmids have been developed. The primary targets of rotavirus infection are the mature epi- thelial cells at the tips of the villi of the small intestine. The VP8* head of the VP4 spike mediates attachment to cells. Some strains bind cell surface sialic acids; most strains that infect humans bind histo-​blood group antigens. Human polymorphism in histo-​blood group antigens, therefore, influences susceptibility to some rotavirus strains. After attachment, productively entering rotavirus particles are enveloped in vesicles derived from closely fitting membrane in- vaginations that form around the bound particles. Formation of the membrane invaginations appears to be driven by interactions be- tween rotavirus surface proteins and cell membrane glycolipids. As the VP7 shell is released from the TLP, a jack-​knifing rearrangement of the VP5* fragment of the VP4 spike, analogous to the rearrange- ments of enveloped virus fusion proteins, appears to drive disrup- tion of the vesicle so that the DLP is delivered into the cytoplasm. In the cytoplasm of an infected cell, the DLP extrudes 11 dif- ferent newly synthesized mRNAs without releasing the genome segments. One viral non​structural protein, NSP3, binds to the non​polyadenylated 3′ ends of viral mRNAs, substituting for the host poly(A) binding protein in circularizing mRNA by binding the translation initiation factor eIF4G, which is bound to the 5´ end of the RNA. NSP3 also shuts off host translation by depleting eIF4G pools. New DLPs assemble in cytoplasmic inclusion bodies, termed viroplasms. Because each infectious unit corresponds to a small number of virus particles, it is likely that one of each of the 11 genome segments is packaged into each new DLP. The mechanism of this specific and highly selective packaging is likely to involve complex RNA-RNA and RNA-protein interactions involving the rotavirus non-structural protein NSP2. Viroplasms form complexes with the cellular organelle lipid droplets. DLPs released from viroplasms bind to the virally encoded glyco- protein NSP4, which is integrated into the endoplasmic reticulum membrane. When budding into the endoplasmic reticulum lumen, DLPs acquire a transient envelope, which is lost as the outermost protein layer is added to complete the formation of virions. Virions are released from infected enterocytes after transport to the cell surface by a vesicular transport pathway that bypasses the Golgi apparatus. Rotavirus replication in the gut results in very high concentra- tions of viral particles (up to 1011/​ml) in faeces at the peak of acute diarrhoea. The physical hardiness of the shed particles ensures their efficient transmission to new hosts. Pathogenesis The pathogenesis of rotavirus diarrhoea is complex. Viral infec- tion causes direct damage to the enteric epithelium, resulting in the blunting and denudation of villi. The villous damage is repaired by cells emerging and differentiating from the crypts of the gut epithelium, which shows a reactive hyperplasia. Loss of functioning absorptive cells leads to a degree of malabsorption and osmotic fluid loss. However, there also appears to be a secretory component to rotavirus diarrhoea. By raising intracellular calcium concentra- tions in infected cells, NSP4 activates a plasma membrane anion channel causing fluid secretion. There is evidence that a fragment of NSP4 is released from infected cells, acting as a viral enterotoxin to induce a secretory state of uninfected cells. The enteric nervous system also plays a role in pathogenesis. Enteric nervous system inhibitors diminish fluid secretion in the gut of rotavirus-​infected animals. Rotaviruses infect and stimulate enterochromaffin cells to release serotonin (5-​HT) which activates brain structures involved in nausea and vomiting. Immune response A primarily serotype-​specific humoral immune response is elicited after neonatal or primary rotavirus infection. However, during the first 2 years of life children are repeatedly infected with rotaviruses, leading to multiple serotype-​specific, and also partially heterotypic, protection. The presence of rotavirus-​specific secretory IgA copro-​ antibodies seems to correlate best with protection against disease, although the exact correlates of protection remain to be determined. Rotavirus-​specific cytotoxic T-​cell responses are capable of clearing infections, but appear to be less important than humoral immune responses in protecting against repeated infections. The abun- dant antibody that is produced against VP6 during infection does not neutralize extracellular virus. However, anti-​VP6 IgA, which is transported across enterocytes for secretion into the gut lumen, can inhibit viral replication heterotypically (within group A) by binding DLPs in the cytoplasm (‘intracellular neutralization’). The role of anti-​VP6 IgA in protecting against infection in humans is not yet known, but it has become apparent that heterotypic protec- tion mechanisms (after both natural infection and vaccination) play a considerable role. Small llama-​derived VP6-​specific antibody fragments inhibit rotavirus replication in vitro, presumably by a similar mechanism to that of anti-​VP6 IgA. In a double-​blind, placebo-​controlled trial in Bangladesh in male infants with severe rotavirus-​associated diarrhoea and no other pathogens detected, the addition of one of these VP6-​specific antibody fragments to standard oral rehy- dration solution treatment reduced stool output by a statistically significant 22.5%. Human caliciviruses Structure and classification These viruses were first recognized as the cause of gastroenteritis during outbreaks in Norwalk, Ohio, in the late 1960s. Norwalk virus particles are spherical and measure 27–​35 nm in diameter. Norwalk virus and Norwalk-​like viruses are all members of the Caliciviridae family. Their 7.7-​kb genome consists of single-​stranded RNA of positive polarity. Cup-​shaped depressions on the surface of virions have given the name to this viral family (Latin calix  =  goblet, cup) (Fig. 8.5.9.3c, d). The T = 3 icosahedral viral capsid is formed from a single protein, which has a shell (S) domain that makes the icosa- hedral contacts and a protruding domain (P) that makes twofold

8.5.9  Virus infections causing diarrhoea and vomiting 801 contacts. The P2 subdomain is the furthest protruding and most variable part of the virus, and it contains receptor binding sites. Phylogenetic trees of full-​length sequences of caliciviral cDNAs have led to their classification into five genera: viruses of the genera Norovirus and Sapovirus infect humans, whereas viruses of the genera Vesivirus, Lagovirus, and Nebovirus only infect animals. Until recently viruses of the Norovirus genus were often termed ‘small round structured viruses’ and those of the Sapovirus genus ‘classical caliciviruses’. Noroviruses and sapoviruses are genetically very di- verse (consisting of at least five genogroups/​clades each and 3–​>20 genetic clusters/​genotypes within them) and constantly evolve. Replication Details of the replication of human caliciviruses have been deduced from those of animal caliciviruses, because, historically, there has been no reproducible in vitro cell culture system for the human caliciviruses. Recently, replication of human noroviruses has been demonstrated in stem cell-derived human intestinal enteroid mono- layer cultures, in the presence of bile. The viruses seem to interact with species-​specific receptors, and a single protein precursor is cotranslationally and posttranslationally cleaved in a way similar to that observed for the polyprotein of picornaviruses. A reverse genetics system for murine norovirus has been developed. Murine norovirus also replicates in cell culture and in mice, thus providing a useful animal model for norovirus infection. Immune response Although calicivirus infections elicit humoral and cell-​mediated immune responses in humans (with an antibody response mainly directed to the P2 portion of the capsid antigen), they do not seem to give full protection against subsequent infection. Like human rotaviruses, noroviruses use certain histo-​blood group antigens act as receptors. Humans who are ‘secretors’ of such antigens are more susceptible to infection with some norovirus strains than ‘non​ secretors’, depending, in part, on strain-​specific receptor usage. Due to the genetic variability in human susceptibility to some norovirus strains, pre-​existing antibody may not necessarily correlate with protection from reinfection. The level of carbohydrate receptor-​ blocking antibodies in sera from previously exposed human ‘se- cretors’ does correlate with protection from severe disease upon reinfection, although correlates of protection following immuniza- tion and natural infection may differ. Astroviruses Structure and classification Astroviruses are members of the family Astroviridae. They possess a 6.8-​kb genome of single-​stranded RNA of positive polarity. So far, eight serotypes have been distinguished that correlate well with major differences in genome sequences (i.e. genotypes). Astrovirus particles have a characteristic appearance by electron microscopy (Fig. 8.5.9.3e), which has been refined by crystallographic charac- terization of the capsid spike. Replication Human astroviruses grow well in particular cell cultures. After viral absorption to unidentified cellular receptors, receptor-​mediated endocytosis, and uncoating in the cytoplasm, full-​length and subgenomic RNAs are synthesized. These direct the production of protein precursors, which are posttranslationally cleaved. Some pro- teins are translated by –​1 ribosomal frameshifting. Replication takes place purely in the cytoplasm. Enteric adenoviruses Structure and classification Adenoviruses are non​enveloped icosahedral viruses possessing a genome of linear double-​stranded DNA of approximately 35 kbp in size. Their capsid measures between 70 and 80 nm in diameter and consists of 240 hexons and 12 pentons, which form the base of each projecting fibre at a fivefold vertex of the icosahedral virus particle (Fig. 8.5.9.3b). Human adenoviruses occur in more than 50 distinct serotypes, ordered in six subgroups (A–​F). Adenoviruses of sub- group F, consisting of serotypes 40 and 41, are regularly associated with gastroenteritis. Adenoviruses of different groups (causing re- spiratory tract infections) are also found frequently in the human gut but are not regularly associated with diarrhoea. Replication Adenoviruses attach to susceptible cells via the fibre proteins and enter via receptor-​mediated endocytosis. Phased early and late gene transcription of the viral DNA in the cell nucleus is followed by trans- lation and morphogenesis in the cytoplasm, and numerous particles are released after cell death. The virally encoded early proteins E1A and E1B induce host cells to enter the S phase, prevent apoptosis, and inhibit antiviral responses. Late adenovirus gene expression blocks (a) (c) (d) (e) (g) (b) (f) Fig. 8.5.9.3  Electron micrographs of (a) rotavirus, (b) enteric adenovirus, (c) Norwalk-​like virus, (d) sapovirus, (e) astrovirus, (f) enterovirus, and (g) parvovirus. Negative staining with 3% phosphotungstate, pH 6.3; bar represents 100 nm. Courtesy of Dr J. Kurtz, Oxford Public Health Laboratory (astroviruses) and
Dr J. Gray, Clinical Microbiology and Public Health Laboratory, Cambridge
(all other viruses). Reproduced from Zuckerman A, Banatvala J, Pattison J (eds) (2000). Principles and practice of clinical virology, 4th edition, p. 236. Wiley &
Sons, Chichester, with permission of the publisher.

802 section 8  Infectious diseases RNA transcription from cellular DNA. Some adenoviruses seem to decrease the expression of major histocompatibility complex class 1 antigens on the surface of infected cells, thus reducing susceptibility to adenovirus-​specific cytotoxic T cells. There is a serotype-​specific humoral immune response providing homotypic protection. Viral gastroenteritis Clinical features The onset of acute viral gastroenteritis follows a short incubation period of 1–​2 days. It is sudden, with watery diarrhoea lasting be- tween 4 and 7 days, vomiting, and varying degrees of dehydration. Over one-​third of children with rotavirus infection have a fever of more than 39°C. Fewer children have a high fever after infection with caliciviruses, and the duration of diarrhoea after infection with caliciviruses is, as a rule, shorter (1–​2 days) than after infec- tion with rotaviruses or enteric adenoviruses (4–​7 days). Disease due to calicivirus infection may be accompanied by abdominal cramps, headache, and myalgia. In rotavirus infection all degrees of severity are seen. Inapparent infections are not infrequent, par- ticularly in neonates, in whom the infection is caused by so-​called nursery strains. It is uncertain whether the asymptomatic nature of rotavirus infection in neonates is due to infection with particular strains or depends on maturational factors, such as changes in the expression on enterocytes of potential rotavirus receptors or the presence of maternal antibodies that provide partial protection. Rotavirus infections are frequently accompanied by respiratory symptoms, but there is no strong evidence that rotavirus replicates in the respiratory tract. Viraemia may be common among patients with rotavirus gastroenteritis and associated with more severe disease. In immunodeficient children, rotavirus may replicate at extraintestinal sites, and chronic gut infections with rotaviruses, adenoviruses, noroviruses, and astroviruses have been observed, accompanied by virus shedding over weeks and even months. Diagnosis The diagnosis of rotavirus, astrovirus, and enteric adenovirus infec- tions is relatively easy for a well-​equipped clinical laboratory, as large numbers of particles are shed during the acute phase of the illness. In contrast, human caliciviruses replicate for a shorter period and are shed at lower concentrations. Diagnosis is commonly carried out by virus-​specific enzyme-​linked immunosorbent assays (ELISAs) and more recently by viral genome detection using the polymerase chain reaction (PCR) (for adenoviruses) and reverse transcription-​PCR (RT-​PCR) (for rotaviruses, caliciviruses, and astroviruses). PCRs are extremely sensitive diagnostic tools, allowing both viral detection and typing. Aliquots of PCR amplicons can also be sequenced, and the information used to establish phylogenetic trees. Such trees are becoming increasingly important not only for virus classification but also for epidemiological studies and surveillance (see next). Electron microscopy of negatively stained specimen suspensions is a ‘catch all’ method that can diagnose less common viral enteric pathogens that are not detected by standard assays, such as non-​group A rotaviruses. Besides its complexity in a diagnostic environment the main disadvan- tage of electron microscopy is its relatively low sensitivity. The mor- phological appearances of the main viruses pathogenic for humans are shown in Fig. 8.5.9.3. Treatment Treatment is mainly with oral rehydration solutions or, in more se- vere cases, intravenous rehydration. In poorly nourished popula- tions, zinc supplementation, used in addition to oral rehydration, decreases the duration of diarrhoea. The enkephalinase inhibitor racecadotril, used as a supplement to oral rehydration, has been shown to significantly decrease the duration and total fluid loss in rotavirus-​infected children. In severe and prolonged rotavirus infec- tions, particularly in immunocompromised hosts, treatment with oral immunoglobulins can decrease the duration of diarrhoea and virus shedding; however, this is not a routine treatment. Otherwise treatment is symptomatic, but the use of antimotility drugs (codeine phosphate, diphenoxylate, loperamide) in children is not advised. Specific antiviral agents have been tested in animal models of rota- virus infections but have not been developed for human treatment. Epidemiology Rotaviruses Rotavirus infections occur endemically worldwide and in 2013 caused approximately 213 000 infant and childhood deaths annually, mainly in developing countries. Therefore, development of vaccine candidates has been a major goal since the early 1980s (see next). The epidemiology of rotaviruses is complex. Besides chil- dren, elderly patients and patients with immunodeficiencies can be affected. There is a strict winter peak of rotavirus infections in temperate climates, but infections occur year-​round in trop- ical and subtropical regions. Transmission is by the faeco-​oral route. Nosocomial infections on infant hospital wards occur and are difficult to eliminate. Group A rotaviruses of different G and P types are found to cocirculate in various populations within the same geographical location, and the relative incidence of different types changes over time. Various surveys have shown that usually more than 90% of cocirculating strains are types G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8], with the G9 and G12 strains emerging relatively recently. Most mammalian as well as avian spe- cies harbour a large diversity of rotaviruses and may act as a reser- voir for human infections. An animal source is suspected for many of the more unusual human group A rotavirus isolates and pos- sibly for group B rotavirus isolates. The latter caused outbreaks in children and adults in China during the 1980s and have also been isolated from patients with diarrhoea in different regions of India and Bangladesh. Group C rotaviruses are associated with small outbreaks in humans in both developed and developing countries. Human caliciviruses Age-​related seroprevalence studies of human caliciviruses have shown that infection is much more frequent and occurs from younger ages onwards more than previously thought. Approximately 50% of children have been infected by the age of 2 years. In countries where rotavirus vaccination programmes have been established, norovirus infections are now the predominant cause of hospitalization of chil- dren with acute gastroenteritis. The rate of inapparent infection with noroviruses is high, particularly in the young. Human caliciviruses cause outbreaks of acute gastroenteritis, often due to contamination of food or water, and are now recognized as the most important cause of non​bacterial gastroenteritis outbreaks worldwide. Contaminated

8.5.9  Virus infections causing diarrhoea and vomiting 803 oysters, green salads, fresh fruit, cold foods, and sandwiches are often implicated as sources of infection. Outbreaks occur in older children and adults in recreational camps, hospitals, nursing homes, schools, cafeterias, hotels, cruise ships, at banquets, and so on. Human calicivirus outbreaks occur worldwide throughout the year, in con- trast to the regular winter peaks of rotavirus infections in temperate climates. The viruses are highly infectious (i.e. a few virus particles constitute an infectious dose), relatively resistant to inactivation, and spread rapidly. Transmission is by the faecal-​oral route and also by projectile vomiting, which scatters viruses into the environment by aerosol. There is cocirculation of highly divergent genotypes. Astroviruses Endemic infections with astroviruses occur in infants and elderly people, but they can also cause food-​borne outbreaks of diarrhoea. There are at least eight genotypes, correlating well with known sero- types, which cocirculate. Serotype 1 is most frequently found, fol- lowed by serotypes 2 to 4 at intermediate frequencies and serotypes 5–​8 at low frequencies. Seroprevalence studies have indicated that infection by more than one serotype is not unusual. Vaccine development Vaccines, when available, have been confirmed as the best individual and population-​based tools to restrict infection with epidemic vir- uses. Of the gastroenteritis-​inducing viruses, vaccine development has only been intensively directed towards rotaviruses. The first, a live attenuated, quadrivalent rhesus rotavirus (RRV)-​based human reassortant vaccine that contained strains with VP7 from each of human rotavirus serotypes G1 to G4, was licensed in 1998. Although the vaccine offered significant protection from severe, dehydrating disease, it was taken off the market by the manufacturer due to a tem- poral association of immunization with intestinal intussusception. The epidemiological findings have not lead to a satisfactory explan- ation of the association and of possible mechanisms of pathogenesis. Subsequently, additional live attenuated oral rotavirus vaccines have been developed. Rotarix® and Rotateq® have been licensed in many countries worldwide since 2006; Rotavac® was licensed in India in 2014 and received WHO prequalification in 2018. Three additional live attenuated rotavirus vaccines have received national licenses in India, China, or Vietnam. Although these are all oral, live attenuated vaccines, there are some significant differences be- tween them. Rotateq® is a pentavalent vaccine that contains genes encoding the human antigens G1 to G4 and P[8]‌ in monoreassortant viruses on a bovine rotavirus (WC3 strain, G6P7[5]) genetic back- bone. Rotarix® is a monovalent vaccine derived from an attenuated human G1P[8] strain. Rotavac®, developed by an Indian manufac- turer in collaboration with a consortium of international institutions is a monovalent vaccine based on a naturally occurring reassortant strain (116E, G9P[11]). Strain 116E carries a bovine rotavirus VP4 antigen on the background of antigens derived from human rota- viruses. 116E-​like strains were originally isolated from nosocomially infected, asymptomatic neonates in India. The pentavalent Rotateq® was designed to elicit type-​specific anti- bodies against all the rotavirus types that are recognized to cir- culate most frequently in humans. The monomeric Rotarix® and Rotavac® rely on cross-​protection between serotypes. The potential for cross-​protection is supported by two clinical observations: (1) cross-​protection against various rotavirus serotypes is accu- mulated through successive natural infections and; (2) vaccination with one rotavirus type can provide protection, even if subsequent infections are by rotaviruses of a different type. In countries where universal mass vaccination of children as part of childhood vaccination schemes has been accepted, a substantial reduction in rotavirus disease and an unexpected rise of herd im- munity have been recorded. In Mexico, overall childhood mortality from diarrhoea decreased after introduction of the vaccine. Recently, a decrease of the annual infant and childhood mortality worldwide from rotavirus-​associated diarrhoea was recorded. A relatively low risk of intussusception associated with rotavirus vaccination has been detected in some postmarketing studies, which has led the Centers for Disease Control and Prevention to recommend that a history of intussusception in an infant should be a contraindication to rotavirus vaccination. Based on the age-​dependence of the risk of intestinal intussusception, the first dose of the licensed rotavirus vaccines is to be given before 15 weeks of age. Although the efficacy of rotavirus vaccination is lower in im- poverished settings in sub-​Saharan Africa and South East Asia, in 2009 the Strategic Advisory Group of Experts of the World Health Organization (WHO) recommended worldwide use of the vaccine, since ‘vaccine efficacy estimates correlate inversely with disease in- cidence and child mortality strata’. There is ongoing postmarketing surveillance in order to estimate the global impact of the vaccine and also to monitor whether or not novel rotavirus strains may emerge. Extension of rotavirus immunization to the settings of greatest med- ical need is being advanced, in part, by the emergence of rotavirus vaccine manufacturers in developing countries. The factors involved in decreased rotavirus vaccine effectiveness in low-income coun- tries are complex; recognition of these factors will help to gradually improve rotavirus vaccine effectiveness worldwide. Next gener- ation approaches to immunization against rotavirus infection are under investigation, such as the use of virus-​like particles obtained from baculovirus-​recombinant coexpressed rotavirus proteins, en- hancement of rotavirus immunogenicity by microencapsidation, DNA-​based candidate vaccines, and possibly ‘edible vaccines’. A recombinant subunit vaccine, consisting of the VP8* head domain fused to the P2 epitope from tetanus toxin is currently in clinical trials. A bivalent, recombinant virus-​like particle vaccine against norovirus disease is in clinical trials, with a trend towards efficacy seen in a small human challenge study, and a phase 2 immunogen- icity and safety study has been completed. Outbreak control Nosocomial rotavirus outbreaks among paediatric populations (on hospital wards and in day-​care centres) are common. There have been numerous reports of outbreaks of diarrhoea and vomiting occurring in adults and children due to infections with caliciviruses acquired from banquets, travel on cruise ships, cafeterias, schools, hotels, fast-​food restaurants, and so on. Outbreak control measures should focus on the interruption of person-​to-​person transmission, and the removal of common sources of infection (such as food and water) in conjunction with

804 section 8  Infectious diseases measures to improve environmental hygiene. Measures include frequent hand washing supplemented with the use of hand sanitizers, exclusion of ill staff from food handling and some child and patient care duties, elimination of bare hand contact with prepared foods, and disinfection of contaminated equipment and environmental surfaces. FURTHER READING Abdelhakim AH, et al. (2014). Structural correlates of rotavirus cell entry. PLoS Pathog, 10, e1004355. Ahmed SM, et al. (2014). Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-​analysis. Lancet Infect Dis, 14, 725–​30. Aiyegbo MS, et al. (2013). Human rotavirus VP6-​specific antibodies mediate intracellular neutralization by binding to a quaternary structure in the transcriptional pore. PLoS One, 8, e61101. Angel J, et al. (2012). Rotavirus immune responses and correlates of protection. Curr Opin Virol, 2, 419–​25. Atmar RL, et al. (2015). Serological correlates of protection against a GII.4 norovirus. Clin Vacc Immunol, 22, 923–​9. Ball JM, et al. (1996). Age-​dependent diarrhoea induced by a rotaviral nonstructural glycoprotein. Science, 272, 101–​4. Bernstein Di, et al. (2015). Norovirus vaccine against experimental GII.4 illness:  a challenge study in healthy adults. J Infect Dis, 211, 870–​8. Bhandari N, et  al. (2014). Efficacy of a monovalent human-​bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-​ blind, placebo-​controlled trial. Lancet, 383, 2136–​43. Burnett E, Parashar U, Tate J (2018). Rotavirus vaccines: effectiveness, safety, and future directions. Pediatr Drugs, 20, 223–233. Borodavka A, et al. (2017). Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments. eLife 6, e27453. Centers for Disease Control and Prevention (CDC) (2009). Reduction in rotavirus after vaccine introduction—​United States, 2000–​2009. MMWR Morb Mortal Wkly Rep, 58, 1146–​9. Centers for Disease Control and Prevention (CDC) (2011). Addition of history of intussusception as a contraindication for rotavirus vac- cination. MMWR Morb Mortal Wkly Rep, 60, 1427. Cheung W, et  al. (2010). Rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication. J Virol, 84, 6782–​98. Clarke E, Desselberger U (2015). Correlates of protection against human rotavirus disease and the factors influencing protection in low-​income settings. Mucosal Immunol, 8, 1–​17. Clarke IN, Lambden PR (2005). Human enteric RNA vir- uses:  noroviruses and sapoviruses. In:  Mahy BWJ, ter Meulen V (eds) Topley and Wilson’s microbiology and microbial infections, 10th edition, pp. 911–​31. Hodder Arnold, London. Cortese MM, Parashar UD (2009). Prevention of rotavirus gastro- enteritis among infants and children. Recommendations of the Advisory Committee on Immunization Practices (ACIP). Morb Mort Wkly Rec MMWR, 58, 1–​25. Crawford SE, et al. (2017). Rotavirus infection. Nat Rev Dis Primers, 3, 17083. Currier RL, et al. (2015). Innate susceptibility to norovirus infections influenced by FUT2 genotype in a United States pediatric popula- tion. Clin Infect Dis 60, 1631–​8. Debbink K, et al. (2014). The state of norovirus vaccines. Clin Infect Dis, 58, 1746–​52. Desselberger U (2009). Rotaviruses. In:  Zuckerman A, et  al. (eds) Principles and practice of clinical virology, 6th edition, pp. 337–​53. Wiley-​Blackwell, Chichester. Desselberger U (2014). Rotaviruses. Virus Res, 190, 75–​96. Desselberger U (2017). Differences of rotavirus effectiveness by country: Likely causes and contributing factors. Pathogens, 6, E65. Desselberger U, Huppertz HI (2011). Immune responses to rotavirus infection and vaccination and associated correlates of protection. J Infect Dis, 203, 188–​95. Donaldson EF, et al. (2010). Viral shape-​shifting: norovirus evasion of the human immune system. Nat Rev Microbiol, 8, 231–​41. Dong J, et al. (2011). Crystal structure of the human astrovirus capsid spike. Proc Natl Acad Sci U S A, 108, 12681–​6. Dormitzer PR (2014). Rotaviruses. In:  Mandell GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases, 8th edi- tion, pp. 1854–​64. Churchill Livingston Elsevier, Philadelphia, PA. Estes MK, Greenberg HB (2013). Rotaviruses. In: Knipe DM, et al. (eds) Fields virology, 6th edition, pp. 1347–​401. Lippincott Williams & Wilkins, Philadelphia, PA. Ettayebi K, et al. (2016). Replication of human noroviruses in stem cell-derived human enteroids. Science 353, 1387–93. Firth AE, Brierley I (2012). Non-​canonical translation in RNA viruses. J Gen Virol, 93, 1385–​409. Fischer Walker CL, et al. (2013). Global burden of childhood pneu- monia and diarrhoea. Lancet, 381, 1405–​16. Franco MA, et al. (2006). Immunity and correlates of protection for rotavirus vaccines. Vaccine, 24, 2718–​31. Gastañaduy PA, et al. (2013). Effect of rotavirus vaccine on diarrhea mortality in different socioeconomic regions of Mexico. Pediatrics, 131, e1115–​20. Gray J, Desselberger U (2009). Viruses other than rotaviruses asso- ciated with acute diarrhoeal disease. In: Zuckerman A, et al. (eds) Principles and practice of clinical virology, 6th edition, pp. 355–​72. Wiley-​Blackwell, Chichester. Green KY (2013). Caliciviridae: the noroviruses. In: Knipe DM, et al. (eds) Fields virology, 6th edition, pp. 582–​608. Lippincott Williams & Wilkins, Philadelphia, PA. Groome MJ, et al. (2017). Safety and immunogenicity of a parenteral P2-VP8-P subunit rotavirus vaccine in toddlers and infants in South Africa: a randomised, double-blind, placebo-controlled trial. Lancet Infect Dis, 17, 843–53. Hagbom M, et  al. (2011). Rotavirus stimulates release of serotonin
(5-​HT) from human enterochromaffin cells and activates brain struc- tures involved in nausea and vomiting. PLoS Pathog, 7, e1002115. Hall AJ, et al. (2011). Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep, 60, 1–​18. Hu L, et al. (2012a). Rotavirus non-​structural proteins: structure and function. Curr Op Virol, 2, 380–​8. Hu L, et al. (2012b). Cell attachment protein VP8* of a human rota- virus specifically interacts with A-​type histo-​blood group antigen. Nature, 485, 256–​60. Human Microbiome Project, Consortium (2012). Structure, func- tion and diversity of the healthy human microbiome. Nature, 486, 207–​14. Imbert-​Marcille B-​M, et  al. (2014). A FUT2 gene common poly- morphism determines resistance to rotavirus A of the P genotype. J Infect Dis, 209, 1227–​30. Iturriza-​Gomara M, Desselberger U, Gray J (2003). Molecular epi- demiology of rotaviruses:  genetic mechanisms associated with

8.6 Bacteria 958

8.6 Bacteria 958

8.6 Bacteria CONTENTS 8.6.1 Diphtheria  959 Delia B. Bethell and Tran Tinh Hien 8.6.2 Streptococci and enterococci  965 Dennis L. Stevens and Sarah Hobdey 8.6.3 Pneumococcal infections  975 Anthony Scott 8.6.4 Staphylococci  991 Kyle J. Popovich, Robert A. Weinstein, and Bala Hota 8.6.5 Meningococcal infections  1010 Petter Brandtzaeg 8.6.6 Neisseria gonorrhoeae  1025 Jackie Sherrard and Magnus Unemo 8.6.7 Enterobacteria and bacterial food poisoning  1032 Hugh Pennington 8.6.8 Pseudomonas aeruginosa  1041 G.C.K.W. Koh and Sharon J. Peacock 8.6.9 Typhoid and paratyphoid fevers  1044 Christopher M. Parry and Buddha Basnyat 8.6.10 Intracellular klebsiella infections (donovanosis
and rhinoscleroma)  1051 John Richens and Nicole Stoesser 8.6.11 Anaerobic bacteria  1055 Anilrudh A. Venugopal and David W. Hecht 8.6.12 Cholera  1060 Aldo A.M. Lima and Richard L. Guerrant 8.6.13 Haemophilus influenzae  1066 Esther Robinson 8.6.14 Haemophilus ducreyi and chancroid  1071 Nigel O’Farrell 8.6.15 Bordetella infection  1073 Cameron C. Grant 8.6.16 Melioidosis and glanders  1076 Sharon J. Peacock 8.6.17 Plague: Yersinia pestis  1081 Michael Prentice 8.6.18 Other Yersinia infections: Yersiniosis  1086 Michael Prentice 8.6.19 Pasteurella  1088 Marina S. Morgan 8.6.20 Francisella tularensis infection  1091 Petra C.F. Oyston 8.6.21 Anthrax  1094 Arthur E. Brown 8.6.22 Brucellosis  1102 Juan D. Colmenero and Pilar Morata 8.6.23 Tetanus  1109 C. Louise Thwaites and Lam Minh Yen 8.6.24 Clostridium difficile  1115 David W. Eyre and Mark H. Wilcox 8.6.25 Botulism, gas gangrene, and clostridial gastrointestinal infections  1120 Dennis L. Stevens, Michael J. Aldape, and Amy E. Bryant 8.6.26 Tuberculosis  1126 Richard E. Chaisson and Jean B. Nachega 8.6.27 Disease caused by environmental mycobacteria  1150 Jakko van Ingen 8.6.28 Leprosy (Hansen’s disease)  1154 Diana N.J. Lockwood 8.6.29 Buruli ulcer: Mycobacterium ulcerans infection  1167 Bouke de Jong, Françoise Portaels, and Wayne M. Meyers 8.6.30 Actinomycoses  1170 Klaus P. Schaal 8.6.31 Nocardiosis  1176 Roderick J. Hay 8.6.32 Rat bite fevers (Streptobacillus moniliformis and
Spirillum minus infection)  1179 Andrew F. Woodhouse 8.6.33 Lyme borreliosis  1181 Gary P. Wormser, John Nowakowski, and Robert B. Nadelman 8.6.34 Relapsing fevers  1188 David A. Warrell

8.6.1 Diphtheria 959

8.6.1 Diphtheria 959

8.6.1  Diphtheria 959 8.6.35 Leptospirosis  1198 Nicholas P.J. Day 8.6.36 Non​venereal endemic treponematoses:
Yaws, endemic syphilis (bejel), and pinta  1204 Michael Marks, Oriol Mitjà, and David Mabey 8.6.37 Syphilis  1210 Phillip Read and Basil Donovan 8.6.38 Listeriosis  1223 Herbert Hof 8.6.39 Legionellosis and legionnaires’ disease  1226 Diego Viasus and Jordi Carratalà 8.6.40 Rickettsioses  1230 Karolina Griffiths, Carole Eldin, Didier Raoult, and Philippe Parola 8.6.41 Scrub typhus  1252 Daniel H. Paris and Nicholas P.J. Day 8.6.42 Coxiella burnetii infections (Q fever)  1257 Thomas J. Marrie 8.6.43 Bartonellas excluding B. bacilliformis  1262 Bruno B. Chomel, Henri-​Jean Boulouis, Matthew J. Stuckey,
and Jean-​Marc Rolain 8.6.44 Bartonella bacilliformis infection  1272 A. Llanos-​Cuentas and C. Maguiña-​Vargas 8.6.45 Chlamydial infections  1278 Patrick Horner, David Mabey, David Taylor-​Robinson, and Magnus Unemo 8.6.46 Mycoplasmas  1295 Jørgen Skov Jensen and David Taylor-​Robinson 8.6.47 A checklist of bacteria associated with infection
in humans  1307 John Paul 8.6.1  Diphtheria Delia B. Bethell and Tran Tinh Hien ESSENTIALS Diphtheria is a potentially lethal infection caused by toxin-​producing strains of Corynebacterium diphtheria, a Gram-​positive bacillus. Humans are the only known reservoir, with spread via respiratory droplets or direct contact with skin lesions. Although now rare in developed countries, this vaccine-​preventable disease remains an important problem in countries with poor or failing health systems with about a 10% mortality rate. Pathogenesis—​diphtheria develops when toxigenic bacteria lodge in the upper airway or on the skin of a susceptible individual. An intense inflammatory reaction develops, leading to a characteristic greyish-​coloured pseudomembrane that is adherent to underlying tissues. Systemic effects are caused by release of diphtheria toxin, carried by a lysogenic corynebacteriophage, a single molecule of factor A of which can kill a eukaryotic cell. Clinical features—​after an incubation period of 2–​6 days the dis- ease presents acutely in several ways, classified by the location of the pseudomembrane:  (1) anterior nasal—​usually relatively mild; (2)  tonsillar (faucial)—​the commonest form, with malaise, fever, sore throat, painful dysphagia and tender cervical lymphadenop- athy; (3) tracheolaryngeal—​with particular risk of airway obstruction; (4) malignant—​with rapid onset, circulatory shock, cyanosis, gross cervical lymphadenopathy (‘bull neck’), and very poor prognosis; (5) cutaneous—​usually mild but chronic; morphological features can be extremely variable. Later complications include (1) myocarditis—​ seen in 10% of cases; and (2) segmental demyelinative neuropathy—​ most often palatal paralysis, and more sinister paralyses of pharyngeal, laryngeal, respiratory, and limb muscles. Diagnosis—​infection may be confirmed by bacterial culture, with detection of toxin production by one of several laboratory techniques, or of the toxin-​producing gene by polymerase chain reaction. Treatment and prognosis—​aside from supportive care, this in- volves (1) antitoxin—​20 000 to 100 000 units, depending on disease severity; preferably given within 48 h of the onset of symptoms; (2) antibiotics—​benzylpenicillin (or penicillin V), or erythromycin in those allergic to penicillin; (3) maintaining the airway—​lifesaving pro- cedures such as tracheostomy may be required. Recovery is usually complete if the patient survives. Prevention—​diphtheria is completely preventable by vaccination, but immunity is not lifelong and may wane in adult life if booster doses are not given regularly. Similarly, infection does not necessarily confer complete protection and the disease may recur in previously infected individuals. Introduction Diphtheria is an acute and potentially highly lethal infection of the upper respiratory tract caused by toxigenic strains of Corynebacterium diphtheriae and C. ulcerans. Today diphtheria has been virtually eliminated from most developed countries by mass immunization, yet it remains a threat in countries with poor vaccine coverage. During the 1990s there was a huge epidemic in parts of the former Soviet Union. Smaller outbreaks have been reported in several other countries. Historical perspective Since ancient times diphtheria has been one of the most feared child- hood diseases, characterized by devastating outbreaks. Diphtheria was recognized as an infectious disease by Brentonneau in 1819. The causative bacillus was described by Löffler in 1884 and a soluble toxin was identified by Roux and Yersin in 1889. In 1890, Fränkel developed an attenuated vaccine and von Behring produced an antitoxin, the first therapeutic antiserum that was first used clinically by Roux in 1894. Before the introduction of antitoxin, mortality in some epidemics had exceeded 50%. In 1913, von Behring produced a successful vaccine and the Schick (skin) test was used to detect immunity. In the United

section 8  Infectious diseases 960 Kingdom there was an average of 50 000 cases and 4000 deaths each year from 1915 to 1942 and it was the leading cause of death among children aged 4 to 10 years. During the Second World War, more than a million cases were reported, including 50 000 deaths. In the United States of America, W. Barry Woods Jr declared in 1961 that: ‘Were it possible merely to apply what is now known about diphtheria to every part of the world, this devastating malady could be wiped from the face of the earth’. However, even in that country, epidemic out- breaks continued in major cities (e.g. the 1970 San Antonio epidemic involving 201 cases with three deaths mainly in the unimmunized poor non​white population aged less than 15 years). In the United Kingdom, mass vaccination had reduced diphtheria to approximately 8–​10 notified cases each year. In 2016 there were 7100 cases reported to WHO, with the most cases reported from India. Pathogenesis C. diphtheriae are slender pleomorphic Gram-​positive rods or clubs. There are four biotypes: gravis, intermedius, belfanti, and mitis, any of which can cause diphtheria if they produce exotoxin. Early mani- festations of diphtheria, including pseudomembrane formation, result from an inflammatory reaction to the multiplying toxigenic C. diphtheriae. Fluid and leucocytes move from dilated blood vessels to surround necrotic epithelial cells. The fluid clots to enmesh dead cells, leucocytes, diphtheria bacilli, cellular debris, and occasionally small blood vessels. The resulting pseudomembrane is therefore ad- herent to underlying tissues and bleeds when pulled away. C. diphtheriae does not usually pass beyond the pseudomembrane site; it is the toxin that causes the later complications of diphtheria. Diphtheria toxin is a 535-​amino acid residue 62-​kDa exotoxin con- sisting of three domains, A (enzymatic), B (binding), and T (trans- location). Domain B binds on the cell surface to heparin-​binding epidermal growth factor (EGF)-​like growth factor precursor and CD9 complex, allowing the lethal factor A to pass through the endosome membrane into the cytosol where it catalyses the NAD+-​dependent ADP-​ribosylation of eukaryotic elongation factor 2 preventing pro- tein synthesis leading to cell death, facilitated by apoptosis. Delivery of a single molecule of factor A to the cytosol of a eukaryotic cell will kill it. Employing this mechanism, recombinant diphtheria toxin with its B domain truncated and fused with the human interleukin (IL)-​2 receptor is marketed as denileukin diftitox (DT388-​IL2) for the treatment of cutaneous T-​cell lymphoma, chronic lymphocytic leukaemia, and non-​Hodgkin’s lymphoma. The structural gene of the toxin (TOX) is carried by a lysogenic corynebacteriophage. However, TOX gene expression is regulated by the bacterial chromosome and requires low extracellular iron concentrations. Locally the toxin causes tissue necrosis and, when absorbed into the bloodstream, systemic complications. In addition to bacterial exotoxin, cell wall components such as the O-​ and K-​ antigens are important in disease pathogenesis. Pathological changes may be seen in all human cells, but the most profound changes are seen in the myocardium, peripheral nerves, and kidneys. Common cardiac changes include fatty degeneration of cardiac muscle (myocarditis) and infiltration of the interstitium with leucocytes, which may involve the conduction fibres. Although the heart can recover completely from these effects, severe fi- brosis and scarring may lead to death in late convalescence. Mural endocarditis may cause embolism leading to cerebral infarction and hemiplegia. Valvular endocarditis is extremely uncommon. Neuritic changes may be seen in the nerves to the heart during the late para- lytic stage of the disease. Diphtheria toxin also causes demyelination and degeneration of both sensory and motor nerves. It affects the nerves to the eye, palate, pharynx, larynx, heart, and limb muscles. It is unclear whether the toxin crosses the blood–​brain barrier to cause central lesions. Epidemiology Humans are the only known reservoir for C. diphtheriae. In most cases transmission to susceptible individuals results in transient pharyngeal carriage rather than disease. Spread is via respiratory droplets or direct contact with skin lesions. Cutaneous diphtheria is more contagious than respiratory diphtheria and chronic skin infections are the main reservoir in environments of poverty and overcrowding. Patients may become carriers of the infection and continue to harbour the organism for weeks or months. The or- ganism can survive for up to 5 weeks in dust or on fomites. Today diphtheria remains an important health problem in coun- tries with poor vaccine coverage. In these areas, children generally meet C. diphtheriae early, sometimes becoming a carrier, and young children may have severe or fatal attacks of diphtheria. C. diphthe- riae tends to die out in highly immunized populations, and children may grow to adult life without encountering the bacillus. Recent serological studies in several countries indicate that up to 50% of adults are susceptible to diphtheria, and their immunity decreases significantly with increasing age. This potential risk is becoming in- creasingly important with the growth in international travel. Immunity to systemic disease depends on the presence of IgG antitoxin antibodies. Type-​specific protection against carriage and mild forms of local disease is induced by antibodies to the variable K-​antigens of the bacterial cell wall. Infection does not always confer protective immunity and outbreaks of mild disease have been re- ported even in highly vaccinated populations. In endemic countries protective immunity is boosted naturally through circulating strains of toxigenic C. diphtheriae. Diphtheria is a devastating but preventable disease. Experience suggests that declining immunity in adults poses the risk of outbreaks, but is probably not sufficient in itself to sustain a large diphtheria epi- demic unless there are large numbers of susceptible children and ado- lescents. In the newly independent states of the former Soviet Union (NIS), economic hardship, large urban migration, and low vaccin- ation coverage due to failing health systems probably contributed to the massive outbreak of the 1990s. This started in Russia but spread to all the NIS, leading to more than 150 000 cases and 5000 deaths between 1990 and 1998 and more than 2700 cases subsequently. Widespread immunization campaigns have largely controlled the epi- demic, but the risk of diphtheria remains in all countries of the former Soviet Union (e.g. there were outbreaks in Western Siberia in 2003 and the southern Urals in 2004) and rare cases of diphtheria continue to be reported in tourists and travellers to the NIS. Since 2002 C.  ulcerans infections have been more commonly re- ported than C. diphtheria in the United Kingdom and France. The pa- tients usually contracted the infection from raw milk or animals or close contact with companion animals and pets (cow, goat, cat, and dog). The

8.6.1  Diphtheria 961 increasing incidence of the diseases has resulted in the expansion of the notification criteria of the E-​CDC and US-​CDC for diphtheria to in- clude infection caused by C. diphtheria and C. ulcerans. This increase in the number of clinical cases of diphtheria also emphasized the need to maintain vaccination coverage in the population above the 95% as re- commended by the World Health Organization. In May 2010, a diphtheria outbreak was reported from Cite Soleil district, Port-​au-​Prince, Haiti, in one of the settlements housing people displaced by the January 2010 earthquake. In 2017 an outbreak of diphtheria was reported in an Amerindian tribe in Venezuela, a country that had last reported a case 25 years earlier. Clinical features Early features Diphtheria has an incubation period of 2–​6 days and presents acutely in a variety of forms, classified according to the location of the pseudomembrane. Anterior nasal This is usually unilateral and relatively mild unless it coexists with other forms. It is relatively common in infancy. There is a nasal dis- charge, initially watery, then purulent and blood-​stained. The nostril may be sore or crusted and a thin pseudomembrane can sometimes be seen within the nostril itself. Tonsillar (faucial) This is the most common form of diphtheria. Malaise, sore throat, and moderate fever develop gradually. At the onset of symptoms only a small, yellow-​grey spot of pseudomembrane may be present on one or both tonsils and is easily mistaken for other types of tonsillitis; it is associated with marked fetor. The surrounding areas are dull and inflamed. Over the next few days the pseudomembrane enlarges and may extend to cover the uvula, soft palate, oropharynx, nasopharynx, or larynx (Fig. 8.6.1.1). There is tender cervical lymphadenopathy, nausea, vomiting, and painful dysphagia. The pseudomembrane be- comes greenish-​black and eventually sloughs off. Tracheolaryngeal Some 85% of tracheolaryngeal presentations are secondary to faucial diphtheria, but occasionally there may be no pharyn- geal pseudomembrane. Initial symptoms include moderate fever, hoarseness, and a non​productive cough. Over the next day or two, as the pseudomembrane and associated oedema spread, the patient becomes increasingly dyspnoeic with severe chest recession, cyan- osis, and eventual asphyxiation unless the obstruction is relieved. Tracheostomy brings instant relief if the obstruction is confined to the larynx and upper trachea. In a minority of cases the pseudo- membrane also involves the bronchi and bronchioles and tracheos- tomy has little effect. Malignant The onset is rapid, with high fever, tachycardia, hypotension, and cyanosis. Pseudomembrane spreads from the tonsils to cover much of the nasopharynx. It has a thick edge and as this advances the earlier parts become necrotic and foul-​smelling. There is gross cer- vical lymphadenopathy. Individual lymph nodes are difficult to feel because of surrounding oedema; this is the characteristic ‘bull neck’ of malignant diphtheria (Fig. 8.6.1.2). The patient may bleed from the mouth, nose, or skin (Figs. 8.6.1.3, 8.6.1.4). Cardiac involve- ment with heart block occurs within a few days. Acute renal failure may ensue. Survival is unlikely. Cutaneous diphtheria In contrast to respiratory forms, cutaneous diphtheria is usually chronic but mild. The morphological features of individual lesions can be extremely variable as C. diphtheriae can colonize any pre-​ existing skin lesion (such as impetigo, scabies, surgical wounds, or Fig. 8.6.1.1  Severe diphtheria in Vietnamese children. Typical faucial pharyngeal pseudomembrane. Copyright Bridget Wills. Fig. 8.6.1.2  Malignant diphtheria with typical bull neck. Copyright Rachel Kneen.

section 8  Infectious diseases 962 insect bites) without altering their picture. However, the ulcera- tive form is the most frequent and typical (Fig. 8.6.1.5). Initially vesicular or pustular, and filled with straw-​coloured fluid, it soon breaks down to leave a punched-​out ulcer several millimetres to a few centimetres across. Common sites are the lower legs, feet, and hands. During the first 1–​2 weeks, it is painful and may be covered with a dark pseudomembrane which separates, revealing a haemorrhagic base, sometimes with a serous or serosanguinous exudate. The surrounding tissue is oedematous and pink or purple in colour. Spontaneous healing to leave a depressed scar usually takes 2–​3 months, and sometimes much longer. Systemic compli- cations such as myocarditis are rare. Occasionally, the affected limb becomes paralysed. Other sites A mild conjunctivitis may accompany faucial diphtheria. Occasionally, pseudomembrane forms in the lower conjunctiva and spreads over the cornea causing considerable damage. Dysphagia may indi- cate that pseudomembrane has spread from the tonsils to the oe- sophagus. Other parts of the gastrointestinal tract are not usually affected, but melaena with colicky abdominal pain is described. Diphtheria may spread by fingers from the throat to vulva or penis causing localized sores. C. diphtheriae occasionally invades the va- gina and cervix, allowing the absorption of toxin. Endocarditis is rare, but at least one reported case recovered following antimicro- bial treatment. Diphtheria caused by other corynebacteria C. ulcerans produces two toxins, one of which seems to be the same as diphtheria toxin. It may cause membranous tonsillitis, but toxic manifestations are rare. C. ulcerans has been spread to humans in cows’ milk. C. pseudodiphtheriticum is commonly present in the flora of the upper respiratory tract. It is non​toxigenic, but can cause exudative pharyngitis with a pseudomembrane identical to that produced by C. diphtheriae. More commonly it causes endocarditis in patients with anatomical abnormalities or infections of the lungs, trachea, or bronchi in immunosuppressed patients or those with pre-​existing respiratory disease. Later complications Patients surviving acute diphtheria may develop one or more later complications. These result from delayed effects of the toxin fol- lowing haematogenous spread. The risk and severity of complica- tions correlates directly with the extent of the pseudomembrane and the delay in administration of antitoxin. Cardiovascular Approximately 10% of patients with diphtheria will develop myo- carditis, usually those with clinically severe infection. There is a much greater frequency of cardiac involvement in laryngeal and malignant diphtheria than in faucial diphtheria, and where anti- toxin administration was delayed more than 48 h after onset of symptoms. Cardiac toxicity usually appears after the first week of illness, but in malignant diphtheria can occur after just a few days. Patients com- plain of upper abdominal pain and may vomit. They become very lethargic and tired. Examination reveals a rapid, thready pulse with hypotension. At this stage profound shock may lead to death. In less severe cases, congestive cardiac failure may develop with a displaced apex beat, gallop rhythm, and murmurs audible over all areas of the Fig. 8.6.1.4  Malignant diphtheria with serosanguinous oral discharge. Copyright Tran Tinh Hien. Fig. 8.6.1.5  Cutaneous diphtheria. Courtesy of the late Dr B. E. Juel-​Jensen. Fig. 8.6.1.3  Malignant diphtheria with serosanguinous nasal discharge. Copyright Rachel Kneen.

8.6.1  Diphtheria 963 heart. Profound bradycardia may result from heart block. There is hepatomegaly and oliguria. Electrocardiography (ECG) is the best way to demonstrate car- diac involvement (Fig. 8.6.1.6). The most common abnormalities are T-​wave inversion with ST-​segment changes in one or more chest leads and prolonged QTc and PR intervals. There may be right or left axis deviation, bundle branch block, or heart block. Very occasionally, atrial fibrillation or tachyarrhythmias are seen. Many more bursts of arrhythmias can be demonstrated if 24-​h ECG monitoring is performed. Numerous ectopic beats have been recorded in patients who lacked other manifestations of cardiac in- volvement. Although most patients surviving myocarditis recover completely, the presence of left bundle branch block at discharge is associated with poor long-​term outcome. Neurological Diphtheria toxin causes a segmental demyelinative neuropathy. Neurological complications usually appear weeks after the onset of the disease, when the patient appears to be recovering, and may show a temporal progression. Palatal paralysis is relatively common and may be seen from the third week onwards. The pa- tient develops a nasal voice and regurgitates fluids through the nose. This usually resolves within a week or so. From the third to the fifth week there may be blurred vision from paralysis of ac- commodation, or a transient squint from external rectus paralysis. From about the sixth or seventh week more sinister paralyses may develop involving pharyngeal, laryngeal, respiratory, and limb muscles (Fig. 8.6.1.7). The nerves to the heart may be affected causing tachycardia and dysrhythmias. In severe cases patients may become profoundly hypotonic over a few hours and can die from respiratory arrest. However, if intensive care facilities and skilled staff are available, complete recovery over the following weeks or months should ensue. Differential diagnosis Clinical diagnosis is difficult where diphtheria is rare. The dif- ferential diagnosis includes infectious mononucleosis, strepto- coccal or viral tonsillitis, peritonsillar abscess, Vincent’s angina, oral thrush, anthrax (Chapter  8.6.20, Fig. 8.6.20.2), Lassa fever (Chapter 8.5.17), and leukaemia and other blood dyscrasias. The bull neck of malignant diphtheria may be mistaken for mumps. In adults, secondary syphilis can sometimes cause a glairy (resem- bling egg white) exudate on the tonsils, and may be accompanied by rash and laryngitis. Clinical investigation Bacterial culture of C. diphtheriae is the mainstay of investigation. Material for culture should be obtained preferably from the edges of the mucosal lesions and inoculated onto appropriate selective media. Suspected colonies may be tested for toxin production by gel precipitation (Elek’s test), guinea pig inoculation, or enzyme immunoassay. Direct smears of infected areas of the throat are often used for diagnostic purposes, but are only of value in ex- perienced hands. More reliably the diphtheria toxin gene may be detected directly in clinical specimens using polymerase chain re- action techniques. Criteria for diagnosis In areas where diphtheria is relatively common and during out- breaks, the disease should be suspected in any patient with exudate in the throat. Treatment must not be delayed until the disease is con- firmed, except in cases of suspected cutaneous diphtheria without associated respiratory symptoms. Fig. 8.6.1.6  Fifteen-​year-​old girl with cardiac and neurological complications (paralysis of muscles innervated by cranial nerves
IX, X, and XII). Copyright D. A. Warrell. Fig. 8.6.1.7  Generalized muscle weakness. Copyright Rachel Kneen.

section 8  Infectious diseases 964 Other corynebacterial skin infections C. diphtheriae and some other corynebacteria are associated with cutaneous ‘desert sores’. Erythrasma is caused by C. minutissimum and, in HIV-​immunosuppressed patients, C. striatum can cause ex- uberant ulceration (Fig. 8.6.1.8). Treatment Antitoxin is the mainstay of treatment, but to be maximally ef- fective it must be given before the toxin has reached tissues such as the heart and kidneys, preferably within 48 h of the onset of symptoms, implying that it must be given empirically before bac- teriological confirmation. Dosage depends on the site of primary infection, the extent of pseudomembrane, and the delay between the onset of symptoms and antitoxin administration. Between 20 000 and 40 000 units are given for faucial diphtheria of less than 48 h duration or for cutaneous infection, 40 000 to 80 000 units for faucial diphtheria in excess of 48 h duration or for laryngeal in- fection, and 80 000 to 100 000 units for malignant diphtheria. For doses over 40 000 units, a portion is given intramuscularly followed by the bulk of the dose intravenously after an interval of 30 min to 2 h. Anaphylaxis can occur following antitoxin administration, and adrenaline (epinephrine) should always be available. Antibiotics are given to eradicate the organism and prevent further toxin production. Benzylpenicillin 150 000 to 250 000 units/​kg per day (90–​150 mg/​kg per day) is given intravenously in four to six divided doses in children aged 1 month to 12 years. In adults the dosage is 12 million to 20 million units/​day (7.2–​12 g/​ day) in four to six divided doses. Oral penicillin V is substituted when the patient is able to swallow. Erythromycin may be used for penicillin-​sensitive individuals, but it may not be as effective in eradicating carriage. Antibiotic therapy should continue for 10–​14 days. Facilities for urgent tracheostomy should always be available in case of respiratory obstruction. Indications include increasingly laboured breathing and agitation. This procedure will be lifesaving in many cases. Most tracheostomies can be closed after just a few days. Steroids may be used in conjunction with tracheostomy to reduce airway swelling, but there have been no controlled trials to support their use. Steroids are of no benefit in preventing myocar- ditis or neuritis. Patients with signs or symptoms of cardiac involvement need to be managed in intensive care units. Oxygen should be given. Temporary cardiac pacing is useful in patients with heart block, but is of doubtful value in cases of malignant diphtheria. An isopren- aline infusion may buy valuable time while the patient is transferred to a centre with facilities for pacing. Digoxin has been used in con- gestive cardiac failure. It has been suggested that carnitine may pre- vent some cases of myocarditis. There is no specific treatment for neuritis. The severest cases will need mechanical ventilation and intragastric or intravenous feeding. With skilled nursing care full recovery can be expected. Patients re- covering from clinical disease should complete active immunization during convalescence. Prevention Diphtheria toxoid is highly effective in conferring protection against clinical disease. Circulating antitoxin levels of less than 0.01 IU/​ml are considered non​protective, while levels of 0.01 IU/​ ml may confer some protection. Levels of 0.1 IU/​ml or more are considered fully protective, and levels above 1.0 IU/​ml are as- sociated with long-​term protective immunity. The potency of (a) (b) (c) Fig. 8.6.1.8  Corynebacterium striatum infection on the thigh of an African patient with HIV-​immunosuppression (a) clinical appearance of exuberant ulcerative lesion, (b) and (c) histopathological appearances of a biopsy of the lesion showing Corynebacteria (Gram-​positive short rods, banded forms that look like diplococci and clubbed forms). (a) Courtesy of C. P. Conlon, Oxford. (b) and (c) Courtesy of Kevin Hollowood, Oxford.

8.6.10 Intracellular klebsiella infections (donova

8.6.10 Intracellular klebsiella infections (donovanosis and rhinoscleroma) 1051

8.6.10  Intracellular klebsiella infections (donovanosis and rhinoscleroma) 1051 Vaccines The greatest need for typhoid vaccination is among infants, children, and young adults in endemic areas, especially where the burden of disease is high and antibiotic resistance is increasing, and among la- boratory workers handling the organisms. In practice, vaccines are given mostly to travellers to endemic areas. The currently available vaccines are the parenteral Vi vaccine, given as a single injection, and the live attenuated Ty21a vaccine, given as three or four oral doses. The Ty21a vaccine should not be given to immunosuppressed persons or those taking mefloquine or antibiotics. A new Vi conjugate vaccine has recently been approved by WHO for use as a public health tool to control typhoid in endemic areas. This new vaccine is considered to be more effective than the standard Vi vaccine and has the potential to be delivered as part of the EPI programme. Current typhoid vaccines do not protect against paratyphoid infection and the protection afforded by vaccination can be overcome by large inocula of bacteria. Efficacy figures derive largely from trials conducted in partly immune popula- tions and overestimate the benefit in persons without prior exposure. The risks of typhoid in travellers are low (3–​30 cases per 100 000) and the precise efficacy of currently recommended doses in previously un- exposed adults remains unknown. Circumstantial evidence indicates that the typhoid vaccines afford protection to travellers visiting endemic areas. Travellers without the vaccine seem more susceptible to the dis- ease, and this is true for even short-​term (less than 1 week) travellers to endemic areas. New vaccines are being developed including several variants of the Vi conjugate vaccine and single dose oral vaccines. Paratyphoid fever S. Paratyphi A is the main serovar causing paratyphoid in Asia. S. Paratyphi B and S. Paratyphoid C are sporadically reported world- wide. Paratyphoid A has recently been increasing in South Asia and China, including drug-​resistant disease. The minimum inhibitory concentrations of S. Paratyphi to the commonly used antibiotics are often higher than those of S. Typhi. Outbreaks of paratyphoid are more often food-​borne than water-​borne, probably because larger inocula are needed to establish infection. Paratyphoid has a shorter incubation period (4–​5 days). The clinical syndromes can be indis- tinguishable and Paratyphi A may be as severe as Typhi. The man- agement of paratyphoid is the same as that of typhoid. Efforts are being directed at the development of a paratyphoid vaccine. Areas of uncertainty and controversy The best recommendation for first-​line antibiotic therapy in endemic areas has been an area of uncertainty. Many practitioners have used fluoroquinolones for first-​line therapy where multidrug resistance is common. The spread of S. Typhi with low and high-​level resistance to fluoroquinolones, particularly the H58 lineage in South Asia, means that approach can no longer be universally recommended. The op- timum treatment for such infections is undefined. The extended-​ spectrum cephalosporins, such as ceftriaxone, and azithromycin are available options but the recent outbreak of ceftriaxone resistant typhoid is a major concern. In some areas isolates have regained sen- sitivity to the older agents and chloramphenicol and cotrimoxazole are being used. New laboratory breakpoints for the fluoroquinolones and azithromycin have been recently introduced. Whether isolates with extended-​spectrum cephalosporin or azithromycin resistance become common in the next decade remains to be seen. Another area of controversy is the use of vaccination as a public health tool in endemic areas. The case for vaccination is hampered by the lack of knowledge of the true burden of enteric fever in Asia and Africa, in particular the burden of severe and fatal disease. It is possible the increases in antimicrobial resistance may swing the cost–​benefit ratio in favour of vaccination. Recent Vi vaccine trials have demonstrated the potential cost-​effectiveness of vaccination. The realization that typhoid is common in children under 5 years has also focused attention on the development of vaccines appro- priate for this age group. WHO now recommends the usage of the Vi conjugate typhoid vaccine in countries with the highest burden of disease or high levels of antibiotic resistance in S. Typhi. Finally, reliable rapid diagnostics for undifferentiated febrile illness, including enteric fever, after malaria has been excluded are needed for low income countries. Targeted antibiotic therapy based on rapid diag- nostics could help decrease the rapid spread of antibiotic resistance. FURTHER READING Basnyat B, et al. (2005). Enteric fever (typhoid) fever in travellers. Clin Infect Dis, 41, 1467–​72. Crump J, et al. (2015). Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance and antimicrobial management of invasive Salmonella infcetions. Clin Microbiol Rev, 28, 901–​37. Hoffman SL, et al. (1984). Reduction of mortality in chloramphenicol-​ treated severe typhoid fever by high-​dose dexamethasone. N Engl J Med, 310, 82–​8. Mogasale, et al. (2014). Case fatality rate and length of hospital stay among patients with typhoid intestinal perforation in developing countries: a systematic literature review. PLoS One, 9, e93784. Waddington CS, et al. (2014). An out-​patient, ambulant design, con- trolled human infection model using escalating doses of Salmonella Typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis, 58, 1230–​40. Wong V, et  al. (2015). Phylogeographical analysis of the dominant multidrug-​resistant H58 clade of Salmonella Typhi identifies inter-​ and intracontinental transmission events. Nature Genet, 47, 632–​37. 8.6.10  Intracellular klebsiella
infections (donovanosis and rhinoscleroma) John Richens and Nicole Stoesser ESSENTIALS Two rare intracellular species of Klebsiella, a Gram-​negative bacillus, cause granulomatous disease in humans that is found in small en- demic foci in warm climates, linked to poverty and poor hygiene.

section 8  Infectious diseases 1052 Donovanosis—​caused by Klebsiella granulomatis (previously named Calymmatobacterium granulomatis); presumed to be sexually trans- mitted; presents with genital ulcers or growths, often accompanied by an inguinal ‘pseudobubo’ (granuloma inguinale). Diagnosed by demonstrating Donovan bodies (vacuoles containing capsulated coccoid bacteria) lying within histiocytes in material taken from a typical lesion. Treatment is with azithromycin; surgery may be needed for complications. Rhinoscleroma—​caused by Klebsiella rhinoscleromatis; transmission believed to occur from person to person; following a period of rhinitis most typically manifests with bulky growths in the upper respiratory tract. Diagnosed by demonstrating intracellular organisms in typical lesions, combined with culture. Treatment is with ciprofloxacin; sur- gical debulking of lesions and/​or reconstruction may be required. Donovanosis Introduction and epidemiology Donovanosis was first described in Calcutta by Donovan in 1905. It is an infection endemic in certain areas of India, Papua New Guinea, the Caribbean, southern Africa, and parts of South America, including Brazil. An important focus among Australian aborigines has recently been eliminated. Donovanosis seems to be retreating, raising hopes of eventual eradication. Dark-​skinned people appear to have greater susceptibility. The predilection of lesions for the anogenital region of sexually active adults and the frequent asso- ciation with other sexually transmitted infections point strongly to sexual transmission. In the past, epidemics of donovanosis in New Guinea were linked to ritual homosexual and heterosexual practices. Perinatal transmission has been observed in a few cases. Aetiology An unusual Gram-​negative bacillus can be isolated in HEp-​2 cells or human peripheral blood mononuclear cells from patients with the characteristic lesions of donovanosis. This organism will not grow on conventional solid media. Previously named Donovania and subsequently Calymmatobacterium by Aragão and Vianna in 1913, it has now been classed as Klebsiella granulomatis on the basis of close DNA homology with other Klebsiella species. K.  granulomatis shows morphological identity with Donovan bodies observed within clinical lesions of donovanosis and pa- tients with characteristic lesions have high levels of antibody that react equally with Donovan bodies and with K. granuloma- tis. K. granulomatis is pathogenic only to humans. Experimental transmission has been reported with lesion material, but to date not with a pure culture of this organism. Donovanosis shows a close macroscopic and microscopic similarity to rhinoscleroma which produces granulomatous lesions of the upper airways. These lesions contain intracellular clusters of the closely related organism Klebsiella rhinoscleromatis. Pathogenesis The organism has a special tropism for dermal macrophages. The response to infection is characterized by vigorous granulomatous inflammation that damages the skin and subcutaneous tissues. Extension of the infection is a local process of spreading ulcer- ation. The inguinal lesions are probably seeded by lymphatic spread. Haematogenous dissemination and spread to the upper genital tract of women are exceptional. Lesions in women tend to be more exten- sive and may progress rapidly during pregnancy. Clinical features After an incubation period of 1–​360 days (median 50 days), the dis- ease usually starts with a small genital papule or nodule which rapidly progresses into a painless ulcer displaying a deep red colour, contact bleeding, and a rolled edge. Hypertrophic lesions that pout outwards from the surrounding skin are frequent. Other presentations include verrucous, necrotic, or sclerotic lesions. Local lymphoedema is seen commonly in women. Chronic lesions tend to expand gradually along skin folds forming a large continuous area of ulceration with a characteristic serpiginous outline (Fig. 8.6.10.1). Inguinal lesions are common (Fig. 8.6.10.2). They start as a firm, subcutaneous swellings and often ulcerate. The term ‘pseudobubo’ tends to be applied to any inguinal lesion in donovanosis although it was originally coined to describe a subcutaneous inguinal abscess, which is a rare event. Such lesions have even given rise to suspicion of bubonic plague when Donovan bodies in the aspirate were misinterpreted. Primary lesions of the cervix simulate carcinoma of the cervix. Upper genital tract in- volvement in women might simulate pelvic inflammatory disease or malignancy and hydronephrosis can ensue. Anal lesions have been de- scribed in homosexual men. Involvement of the rectum seldom occurs. Oral lesions of donovanosis with extension to cervical nodes have been described. Haematogenous dissemination is associated with pregnancy and causes lesions of bone, liver, and spleen. Lesions in infants tend to involve the ears and nearby lymph nodes. Patients coinfected with HIV tend to have lesions that heal more slowly and are associated with more significant tissue destruction. Complications of donovanosis include extensive scar formation, lymphoedema of the genitalia, penile autoamputation, and the de- velopment of squamous carcinoma in active or healed lesions. Secondary infection with fusospirochaetal organisms can cause rapid, extensive, and sometimes fatal tissue destruction. Fig. 8.6.10.1  Characteristic serpiginous ulcer in female patient with long-​standing donovanosis.

8.6.10  Intracellular klebsiella infections (donovanosis and rhinoscleroma) 1053 Differential diagnosis and clinical investigations Klebsiella granulomatis is difficult to culture and the diagnosis is made by demonstrating Donovan bodies lying within histiocytes in material taken from a typical lesion. Donovan bodies show well with Giemsa’s, Leishman’s, and Wright’s stains but poorly with haema- toxylin and eosin. Histology typically shows a heavy plasma cell infiltrate and epithelial hyperplasia in addition to histiocytes con- taining Donovan bodies (Fig. 8.6.10.3). Differential diagnoses in- clude squamous carcinoma of cervix, vulva, or penis, secondary syphilis, and conditions that produce genital lymphoedema such as filariasis and lymphogranuloma venereum. Molecular diagnostic tests for the detection of K. granulomatis DNA have been developed but are not validated or approved for diagnostic use by the United States Food and Drug Authority (FDA). Serology has no role in diagnosis. Patients should be screened for other sexually transmitted infections. Treatment In 1913, Aragão and Vianna described the value of trivalent antimony in treating donovanosis (Fig. 8.6.10.2). The British Association for Sexual Health and HIV (BASHH) (2018), United States CDC (2015), and European guidelines (2016) recommend azithromycin 1 g orally once per week or 500 mg daily until lesions have healed (and a min- imum of three weeks [BASHH, CDC]). Alternative regimens include doxycycline (100 mg orally twice daily) or ciprofloxacin (750 mg or- ally twice daily) or erythromycin (500 mg orally four times daily) or co-​trimoxazole (960 mg orally twice daily). Combination therapy or the addition of an aminoglycoside (e.g. gentamicin 1 mg/​kg every 8 hours) can be considered if there is no improvement within a few days of treatment. Erythromycin is safe and gives good results in pregnant women. Women in labour found to have untreated lesions of the cervix should be delivered by caesarean section to reduce known risks of haematogenous dissemination and transmission to the neonate. A week of prophylactic treatment can be offered to healthy contacts to abort incubating infections. Patients with genital deformity might benefit from plastic surgical procedures. Patients with HIV should be managed with the same treatments, although the duration required might be longer. Rhinoscleroma Introduction, aetiology, and epidemiology Rhinoscleroma is a chronic granulomatous infection, predomin- antly of the upper respiratory tract. It is endemic to Mexico, Central and South America; Africa (especially Egypt and Uganda); Central and Eastern Europe (e.g. Siberia, Turkestan), the Middle East, India, China, the Philippines, Indonesia, and Papua New Guinea. The disease was first described in Austria and Germany by Hebra and Kaposi in 1870; the histological features by Johann von Mickulicz in 1877; and the causative organism, Klebsiella rhinoscleromatis, first identified by von Frisch in 1882. The term ‘scleroma respiratorium’ was proposed by Belinov in 1932 as it was found to affect both upper and lower respiratory tracts. Although limited epidemiological data are available, young adults appear to be most commonly affected. Pathogenesis Transmission is believed to occur through the inhalation of contam- inated droplets or material. The infection probably begins in areas of epithelial transition, such as the nasal vestibule. The nasal cavity is the most affected part of the body, but infection can spread to the larynx, nasopharynx, oral cavity, sinuses, soft tissues of the lips/​ nose, trachea, and bronchi. Patients with rhinoscleroma have impaired cellular immunity with a decrease in the CD4+ T-​lymphocyte count, poor activation of Fig. 8.6.10.2  Inguinal lesion: from Aragão and Vianna’s paper on the value of trivalent antimony in treating donovanosis. From Aragão H, Vianna G (1913). Resquizas sobre o Granuloma venereo. Mem Inst Oswaldo Cruz, 5, 211–​38. Fig. 8.6.10.3  Donovan bodies: Giemsa-​stained smear from donovanosis lesion demonstrating the characteristic ‘closed safety pin’ appearance of encapsulated organisms within a large histiocyte.

section 8  Infectious diseases 1054 macrophages, and inhibition of phagocytosis. A Mexican study has shown that the HLA DQA103011-​DQB10301 haplotype is associ- ated with the development of rhinoscleroma. Clinical features Rhinoscleroma typically presents in three progressive, overlapping stages, and runs a slow, fluctuating course over several years. Symptoms of the first stage mimic those of the common cold, although usually with a malodorous, purulent discharge persisting over several months. The second stage, which lasts months to years, represents the proliferative stage, during which the granulomas form. These cause deformity, and in some cases, breathing difficulties, due to occlusion of affected parts of the respiratory tract. The usual clinical presenta- tions at this stage are with nasal obstruction and bleeding and nasal deformity (splaying of the lower nose, often with a visible growth ex- tending down to the upper lip, known as Hebra nose) (Fig. 8.6.10.4). The granulomatous process can extend into and destroy neighbouring soft tissues, cartilage, bone, and skin. The third, fibrotic, stage is char- acterized by extensive scarring and stricture formation. A rare case of pneumonia and systemic sepsis has been reported. Differential diagnosis and clinical investigations Histology shows a dense infiltrate of plasma cells among which are large foamy histiocytes (Mikulicz cells) containing Gram-​negative bacteria and Russell bodies, which are thought to be aggregated, un- released immunoglobulin components within plasma cells (Fig. 8.6.10.5). The diagnosis is usually made by demonstrating intracellular organisms in Giemsa-​stained or silver-​stained sections taken from typical lesions, combined with culture for K. rhinoscleromatis. Culture is only positive in 50–​60% of cases. Differential diagnoses include fungal infections, mucocutaneous leishmaniasis, tuberculosis, leprosy, granulomatosis with polyangiitis (Wegener’s granulomatosis), and car- cinoma. Computed tomography (CT) scanning and endoscopic tech- niques provide useful ways to define the extent of the disease. Treatment Treatment with ciprofloxacin 500 mg twice daily for 4–​12 weeks ap- pears to be substantially superior to previously used antibiotic re- gimens (rifampicin, streptomycin, tetracyclines, ampicillin, and co-​trimoxazole). Long-​term follow-​up might be required to monitor for recurrent disease. Debulking operations might be needed for obstructing nasal and tracheal disease, and tracheostomy can be required as a temporary measure. Reconstructive surgery might be needed to deal with late fibrotic stenosis. FURTHER READING Borgstein J, Sada E, Cortes R (1993). Ciprofloxacin for rhinoscleroma and ozena. Lancet, 342, 122. Bowden FJ, et al. (1996). Pilot study of azithromycin in the treatment of genital donovanosis. Genitourin Med, 72, 17–​9. Canalis RF, Zamboni L (2001). An interpretation of the struc- tural changes responsible for the chronicity of rhinoscleroma. Laryngoscope, 111, 1020–​6. Carter JS, et al. (1999). Phylogenetic evidence for reclassification of Calymmatobacterium granulomatis as Klebsiella granulomatis comb. nov. Int J Syst Bacteriol, 49, 1695–​700. Centers for Disease Control and Prevention (2010). Sexually transmitted diseases treatment guidelines. MMWR, 59 (No. RR-​12), 1–​109. Mackay IM, et  al. (2010). Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmabacterium (Klebsiella) granulomatosis from genital ul- cers. Clin Infect Dis, 42 1431–​8. O’Farrell N (2002). Donovanosis. Sex Transm Infect, 78, 452–​7. O’Farrell N, Hoosen A, Kingston M (2018). 2018 UK national guideline for the management of donovanosis. Int J STD AIDS, 29, 946–8. Richens J (1991). The diagnosis and treatment of donovanosis (granu- loma inguinale). Sex Transm Infect, 67, 441–​52. Velho PE, Souza EM, Belda Jr W (2008). Donovanosis. Braz J Infect Dis, 12, 521–​5. Fig. 8.6.10.4  Rhinoscleroma with characteristic nasal splaying (Hebra nose) and obstruction of the left nostril in a 30-​year-​old man from Papua New Guinea. From Cooke R and Stewart B (2004). Colour atlas of anatomical pathology, Third edition, p. 37. Churchill Livingstone, Edinburgh, with permission. Fig. 8.6.10.5  Rhinoscleroma. Silver-​stained preparation showing bacteria. Copyright J Richens.

8.6.11 Anaerobic bacteria 1055

8.6.11 Anaerobic bacteria 1055

8.6.11  Anaerobic bacteria 1055 8.6.11  Anaerobic bacteria Anilrudh A. Venugopal and David W. Hecht ESSENTIALS Anaerobic bacteria will not grow when incubated with 10% CO2 in room air, but they vary in their tolerance of different levels of oxygen. Anaerobic bacteria are important commensal flora of the skin and oral, intestinal, and pelvic mucosae, and are classified ac- cording to their Gram-​staining characteristics and ability to pro- duce spores: (1) Gram-​positive—​cocci, non-​spore-​forming bacilli, and spore-​forming bacilli (notably the Clostridium spp.); (2) Gram-​ negative—​cocci and bacilli. Many anaerobic bacteria possess virulence factors that facilitate their pathogenicity (e.g. histolytic enzymes and various toxins). Clinical features—​anaerobes typically cause clinically significant infections when there is tissue compromise, ischaemia, or mu- cosal injury. These infections are often polymicrobial in nature and can include (1) bacteraemia; (2) central nervous system infection—​ intracranial abscesses by contiguous spread, for example, from chronic otitis media, or haematogenous spread (e.g. from tooth abscess); (3)  head and neck infections—​periodontal and pharyn- geal infections from spread of gingival disease; (4) pleuropulmonary infections (e.g. lung abscess from aspirated oropharyngeal flora); (5) intra-​abdominal infections—​often caused by mixed colonic flora that have been displaced by bowel injury; (6) gastrointestinal infec- tions; (7) genitourinary infections; (8) skin and soft tissue infections—​ ranging from cellulitis to necrotizing fasciitis; should be considered in cases of infected animal and human bites, and in intravenous drug users; diabetic foot ulcers often have polymicrobial infections that include anaerobes. Diagnosis—​a putrid odour of the affected tissue or drainage is highly suggestive of an anaerobic infection, as is the presence of gas in tissues. Care must be taken when collecting specimens for an- aerobic culture because many of the organisms are very sensitive to oxygen, and some cannot tolerate more than a few minutes at ambient oxygen levels. However, anaerobic spores are aerotolerant, can survive in harsh oxygen-​laden environments, and will germinate under appropriate conditions. Treatment and prevention—​aside from supportive care, treatment requires (1) drainage of abscesses and resection of devitalized tissue; and (2) antibiotics—​agents that are active against anaerobes include clindamycin, metronidazole, vancomycin, β-​lactam/​β-​lactamase inhibitor combinations, carbapenems, moxifloxacin, tigecycline, chloramphenicol, and even macrolides but resistance patterns have been changing and the choice of empirical therapy is best guided by knowledge of local susceptibility testing results. Prophylaxis against anaerobic bacteria significantly reduces postoperative infection rates following intra-​abdominal surgery. History In 1690 Antonie van Leeuwenhoek first described anaerobic bac- teria as ‘animalcules’ that could survive in the absence of air. This observation was overlooked until nearly 200 years later. In 1861 Louis Pasteur had observed that the bacteria near the surface of a droplet of water had stopped moving while the organisms at the centre of the droplet continued to move about. He hypothesized that oxygen in the air had caused the death of the surface bacteria. Pasteur’s early experiments with bacterial fermentation led to the development of anaerobic bacteriology. Years later in 1916 it was the invention of the anaerobic jar by James McIntosh and Paul Fildes that allowed for the repeated culture and study of anaerobes. This led to the discovery of many anaerobic bacteria responsible for various human diseases. Definition Anaerobic bacteria are organisms that cannot grow in the presence of various levels of oxygen. Room air is approximately 20% oxygen and when cultured in this environment, anaerobes will not grow on solid media. Reduced oxygen tensions are required for their growth. Anaerobes are described as strict, moderate, or facultative anaer- obes, according to their tolerance of oxygen. Strict anaerobes may grow only at oxygen levels of less than 0.5%. They are usually catalase negative and lack superoxide dismutase rendering them susceptible to toxic oxygen radicals, although this is not always the case. Moderate anaerobes also grow poorly in air and prefer media that have oxygen levels of 2–​8%. Facultative anaer- obic bacteria are organisms that can grow in various levels of oxygen including normal oxygen tensions. Taxonomy of important anaerobic organisms Table 8.6.11.1 lists the species of anaerobes that colonize human mucosal surfaces and skin or produce clinically significant disease. They are classified according to their Gram-​staining characteristics and ability to produce spores. Epidemiology Limited data about the incidences of anaerobic infections are available. Most anaerobic infections occur as contamination of endogenous flora into neighbouring sites. There are some not- able exceptions that can occur after exposure to environmental sources of anaerobes can include but are not limited to infections with Clostridium tetani, Clostridium perfringens, Clostridium bot- ulinum, Clostridium difficile (see Chapter 8.6.25), Enterotoxigenic Bacteroides fragilis. Human commensal flora Commensal bacteria are organisms that live on both mucosal sur- faces and skin of humans but under normal circumstances do not cause disease. They play an important role in normal host physi- ology by colonizing and helping to prevent infections by pathogenic organisms. By producing toxic metabolites, lowering the local pH, and depleting the area of nutrients they make the surrounding area

section 8  Infectious diseases 1056 uninhabitable for other pathogenic organisms. Anaerobes make up a large part of this commensal flora in humans, as outlined next. Skin The commensal flora of the skin consists predominantly of aer- obes, anaerobes, and yeasts. The principal anaerobes present are Gram-​positive bacilli of the genus Cutibacterium (formerly Propionibacterium) and the Gram-​positive cocci from the genus Peptostreptococcus. The three main species of Cutibacterium include C. acnes, C. granulosum, and C. avidum. They occur mainly in hair follicles and sebaceous glands. C. acnes produce free fatty acids from triglycerides, but, while this may control the growth of pathogenic bacteria on the surface of skin, it has also been associated with the development of acne. Upper respiratory tract and oral cavity The nasal cavity mucosa tends to be colonized with organisms that are similar to the organisms found on skin surfaces and the sebaceous glands. Oropharyngeal flora typically includes Peptostreptococci, Tannerella forsythia, and Fusobacterium. In the oral cavity, areas such as the tonsillar crypts, gingival crevices, and the clefts on the tongue have a more favourable atmosphere for anaerobes. Their lower oxygen levels promote colonization with Prevotella, Peptostreptococci, Fusobacterium, and other anaerobic Gram-​positive bacilli. Gastrointestinal tract The upper gastrointestinal tract from oesophagus to jejunum is relatively free of microorganisms but can become transiently col- onized with bacteria following meals or from the swallowed secre- tions of the upper airway. The terminal ileum tends to have a flora more closely resembling that of the large intestines where anaerobes can outnumber the aerobes from 100 to 1000:1. Among a diverse group of anaerobes, the Bacteroides fragilis group predominates. B. vulgatus and B. thetaiotaomicron are more common than B. fra- gilis. Another group of colonizing anaerobes found in the stool are Clostridium spp., including C. perfringens and C. novyi. There have been reports in both adults and children of colonization with C. dif- ficile where they have displayed positive stool test results but without symptoms. Genitourinary tract The kidneys, ureters, urinary bladder, and proximal part of the urethra are normally free of organisms as they are constantly flushed with urine if the anatomy of the urinary tract is normal. The distal portion of both male and female urethras have a scanty flora including aerobic skin colonizers and some anaerobic organ- isms including Bacteroides, Fusobacterium, Peptostreptococcus, and Clostridium spp. The vaginal flora can include both aerobes and anaerobes but, by adulthood, anaerobes such as Lactobacillus, Prevotella, Fusobacterium, and Peptostreptococci predominate (Fig. 8.6.11.1). Pathogenesis Several factors predispose to the pathogenesis of anaerobic infec- tions, including tissue injury and destruction, impaired blood supply, or any breakdown in the integrity of mucosa or skin. Many anaerobic bacteria possess one or more characteristics that enhance their pathogenic virulence. These can include enzyme production, toxins, polysaccharide capsules, lipopolysaccharides, and spore formation. Histolytic enzymes such as collagenases, fibrinolysins, lipases, and other enzymes are produced by Bacteroides and Prevotella. These enzymes cause tissue destruction, whereas the α-​toxin found in C. perfringens can also cause haemolysis. Porphyromonas gingivalis, Bacteroides spp., and Fusobacterium produce heparinases that can promote coagulation leading to tissue ischaemia. Porphyromonas gingivalis, B. fragilis, and Fusobacterium nucleatum produce catalase and superoxide dismutase which are believed to help the organisms tolerate higher levels of oxygen. Various organisms, including C. perfringens and C. difficile, have also been found to have enterotoxins that alter intestinal cell func- tion and cause cell death, leading to diarrhoea. Certain species of clostridium, including C. botulinum and C. tetani, produce neuro- toxins that block neuromuscular transmissions leading to par- alysis or spasms plus rigidity, respectively. Endotoxins can cause macrophage and complement activation leading to fever, hypoten- sion, and oedema from the release of cytokines. Enterotoxigenic B.  fragilis is known to produce a metalloproteinase toxin that causes cell proliferation and protein shedding resulting in a diar- rhoeal illness. Gram-​negative anaerobes, like their aerobic counterparts, have a lipopolysaccharide layer in their outer membrane that can act as an endotoxin. The lipopolysaccharide of the anaerobic Gram-​negative cell walls are typically less potent than the lipopolysaccharide of Table 8.6.11.1  Taxonomy of important anaerobic bacteria Gram-​positive anaerobes Gram-​negative anaerobes Cocci Bacilli Cocci Bacilli Non-​spore forming Spore forming Actinomyces spp. Clostridium spp. Veillonella spp. Bacteroides fragilis group Peptostreptococcus spp. Bifidobacterium spp. Other Bacteroides spp. Streptococcus spp. Eubacterium spp. Bilophila wadsworthia Finegoldia magna Eggerthella spp. Fusobacterium spp. Lactobacillus spp. Porphyromonas spp. Mobiluncus spp. Prevotella spp. Cutibacterium spp.

8.6.11  Anaerobic bacteria 1057 aerobic Enterobacteriaceae. One exception to this rule is the lipo- polysaccharide of F. nucleatum which is believed to involved in its pathogenic role in periodontal disease and Lemierre’s disease. The capsular polysaccharides associated with the B. fragilis group, Prevotella melaninogenica, and Peptostreptococcus, are associated with impaired phagocytosis by host cells and can lead to abscess formation. Finally, spore-​producing organisms like the Clostridium species can survive in harsh oxygen-​laden environments by developing spores that will later germinate when environmental conditions become favourable again. Clinical spectrum Anaerobic bacteraemia Anaerobes account for about 5% of all positive blood cultures. The predisposing factors for anaerobic bacteraemia in newborns include prematurity, prolonged labour, chorioamnionitis, and necrotizing en- terocolitis. Underlying risk factors for anaerobic bacteraemia in chil- dren and adults include malignancies, immunosuppression, hepatic failure, and diabetes mellitus. The organism isolated depends on the underlying infectious condition. In cases of gastrointestinal or nec- rotic skin infections it is usually a member of the B. fragilis group. Peptostreptococcus and Clostridium spp. are other commonly found blood isolates. The isolation of Clostridium septicum or the facultative anaerobe Streptococcus bovis in bacteraemic patients should raise the suspicion of underlying cancer, especially of colonic origin. Central nervous system infections Intracranial infections with anaerobes can arise by contiguous spread from surrounding structures (e.g. with chronic otitis media, mastoiditis, or sphenoidal sinusitis). This can lead to abscess for- mation, septic thrombophlebitis, or venous sinus infections. When frontal sinusitis spreads to involve the frontal bone, it can lead to osteomyelitis and subperiosteal abscess known as Pott’s puffy tu- mour. Intracranial abscesses can also arise as a result of haema- togenous seeding of the brain parenchyma from suppurative distant foci such as dental abscesses and alveolar infections. Abscesses that arise may be single or multiple in number and involve any portion of the brain, although the site involved depends on the mode of infec- tion. Anaerobic organisms commonly associated with infections of the central nervous system include Prevotella, Peptostreptococci, and Fusobacterium spp., although these are often polymicrobial infec- tions involving aerobes. Head and neck infections The origin of oral, head, and neck infections is often the anaerobic flora of the oral cavity. Lower oxygen tension in the gingival crev- ices promotes colonization with anaerobes. When dental hygiene is Anaerobic Infections Brain Abscess Animal and Human Bites Cellulitis Foot ulcers Bacteraemia Pelvic Abscess Endometritis Salpingitis Tubo-ovarian Abscess Septic Abortion Gingivitis Periodontal Infections Periodontal Abscess Vincent’s Angina Ludwig’s Angina Lemierre’s Syndrome Lung Abscess Empyema Aspiration Pneumonitis Necrotizing Pneumonia C. difficile associated Disease Appendicitis Peritonitis Pericolonic Abscess Intra-abdominal Abscess Chronic Otitis Media Sinusitis and Mastoiditis Commensal Flora spp. Oral Cavity and Upper Respiratory Tract Gastrointestinal Tract Genitourinary Tract Skin Bacteroides fragilis group Other Bacteroides spp. Clostridium spp. Peptostreptococci spp. Fusobacterium spp. Fusobacterium spp. Peptostreptococci spp. Clostridium spp. Other Bacteroides spp. Prevotella spp. Lactobacilli spp. Cutibacterium spp. Peptostreptococc spp. Fusobacterium spp. Tannerella forsythia Prevotella spp. Veillonella spp. Fig. 8.6.11.1  Human anaerobic commensal flora (left) and clinical spectrum of anaerobic infections (right).

section 8  Infectious diseases 1058 poor, dental plaque develops leading to gingivitis, periodontal in- fections, and abscesses. Periodontal infections result from spread of gingival disease to the surrounding tissue. As infection spreads from more superficial gingival disease to deeper infections there is a shift in the pathogenic organisms from Gram-​positive cocci and bacilli to Gram-​negative bacilli. Anaerobic pharyngeal infections may arise in relation to gingivitis. Complications might arise by con- tiguous spread of these infections along medial, lateral, or submax- illary spaces. Ludwig’s angina is described as a brawny induration of the submaxillary and sublingual spaces with cellulitis usually arising from spread of lower molar dental infections. Severe infections threaten the airway and can extend to the mediastinum. Vincent’s angina is an acute necrotizing ulcerative gingivitis manifested by in- flamed gingivae, interdental ulcerations, and halitosis. Otitis media and sinusitis occasionally involve anaerobes, although they are more common in chronic infections of these spaces. Fusobacterium necrophorum may be a cause of pharyngitis in up to 10% of cases. Lemierre’s syndrome frequently occurs in young, previously healthy patients as the result of spread of an oropharyngeal infection leading to septic thrombophlebitis of the internal jugular vein. Clinical fea- tures helpful in diagnosing this condition are recent oropharyngeal infection, clinical evidence of thrombophlebitis including ipsilat- eral neck tenderness with fevers and chills, as well as isolation of the anaerobic pathogen. Most commonly, the organism isolated is Fusobacterium necrophorum, although other organisms have been identified. Early diagnosis is required to reduce morbidity associ- ated with distant septic emboli and mortality. Pleuropulmonary infections The spectrum of anaerobic lung infections includes lung abscesses, empyema, aspiration pneumonia, and necrotizing pneumonias. The origins of these anaerobes are usually from the oropharyn- geal flora. Peptostreptococcus, Fusobacterium, pigmented Prevotella, Porphyromonas, and Bacteroides spp. are most commonly isolated, often in combination with aerobes and other microaerophilic an- aerobes such as Streptococcus spp. Patients with an anaerobic lung abscess will often complain of fevers, weight loss, and foul-​smelling or foul-​tasting sputum. This odour may be detectable on entering the patient’s room. Intra-​abdominal infections The presence of an intra-​abdominal infection can usually be diag- nosed clinically from a thorough history and physical examination. If the patient is not being taken for an immediate laparotomy, then patients with suspected intra-​abdominal infection should undergo computed tomography (CT) scanning to evaluate the abdomen. Intra-​abdominal infections are often caused by mixed colonic flora that have been displaced by surgery, penetrating trauma, intestinal malignancy, inflammatory bowel disease, or perforation of co- lonic diverticula. Intra-​abdominal abscesses, pericolonic abscesses, and peritonitis may develop. Isolates are most commonly mixed facultative and strict anaerobes, notably the facultative anaerobe Escherichia coli and the anaerobe B. fragilis group. Other commonly occurring anaerobes include Peptostreptococcus spp., Fusobacterium spp., and Clostridium spp. Gastrointestinal infections C. difficile infection is discussed in Chapter 8.6.24. Clinical symp- toms often include abdominal pain and distension, fevers, and profuse foul-​smelling watery diarrhoea. Laboratory findings in- clude leucocytosis (leukaemoid reactions) and hypoalbuminaemia. Polymerase chain reaction assays of stool samples that target the presence of the toxin B gene have sensitivities ranging from 85 to 95% and specificities of 95–​99%, which are improved compared to the toxin immunoassays. Recommendations about the use of oral metronidazole and vancomycin are discussed in detail in the adult clinical practice guidelines (see Further reading). Fidaxomicin is another treatment option that offers the advantage of lower recur- rence rates compared to oral vancomycin. It is important to perform strict contact isolation measures for patients suspected of C. difficile disease to prevent nosocomial transmission and encourage proper hand hygiene with soap and water. Enterotoxigenic Bacteroides fragilis has been associated with a diarrhoeal illness in humans. The Enterotoxigenic Bacteroides fra- gilis organism produces a metalloprotease B. fragilis toxin that is proinflammatory and causes disruption of the colonic epithelial cells. Patients often present with tenesmus, abdominal pain, and an inflammatory diarrhoea. Genitourinary infections Like the oral cavity and colon, the female genital tract has an in- creased colonization ratio of anaerobes to aerobes of nearly 10:1. Disruption of the integrity of the tissues of the female genital tract increases the risk of anaerobic infections such as periurethral and labial pyogenic infections, pelvic abscesses, postpartum endo- metritis, salpingitis, tubo-​ovarian abscess, and septic abortions. B. fragilis group, Prevotella spp. including P. bivia, P. disiens, and P. melaninogenica, Peptostreptococci, and Clostridium spp. are com- monly isolated. Actinomyces have been associated with intrauterine device-​related infections. Bacterial vaginosis (see Chapter 9.4) is an infection characterized by malodorous vaginal discharge caused by a polymicrobial infection often including Prevotella, Peptostreptococci, Mobiluncus, and the facultative anaerobe Gardnerella vaginalis. Clostridium sordellii is a toxin-​producing Gram-​positive anaerobe that has been known to cause pelvic infections. This usually presents with vague symptoms caused by deep infections following child- birth, medically induced abortions, or trauma. There is rapid clinical deterioration with profound hypotension, an intense leukaemoid re- action, and it is associated with a high mortality rate. Skin and soft tissue infections These usually arise after the integrity of the skin has been lost from injury, ischaemia, or surgery, and there is contamination by either faecal or oral secretions. These are usually polymicrobial infec- tions with aerobic and anaerobic bacteria. The spectrum of disease ranges from cellulitis to necrotizing fasciitis. Anaerobic skin in- fections should also be considered in cases of infected animal and human bites and decubitus ulcers, and with intravenous drug users. Diabetic foot ulcers are often polymicrobial infections that include anaerobes. Common isolates from soft tissue infections include B. fragilis group, Peptostreptococci, and Clostridium spp. Bone and joint infections Osteomyelitis and septic arthritis from anaerobes are rare but can result when infection spreads from surrounding soft tissue, as in the case of diabetic foot ulcers. Diagnosis is made by the careful col- lection of fluids in anaerobic containers or by bone biopsy cultures.

8.6.11  Anaerobic bacteria 1059 Fusobacterium spp. and other Bacteroides spp. have been isolated from joint infections on a few occasions. Diagnosis Clinical clues A putrid odour of the affected tissue or discharge is very suggestive of anaerobic infections. Underlying illnesses such as diabetes mellitus, abscess formation, tissue ischaemia, and necrotic tissue are predisposing factors. Another important clue is the location of the infection in relation to mucosal surfaces that are normally colonized by anaerobes such as intra-​abdominal and oral infections. Gas in tis- sues suggests anaerobic infection. It can often be detected on radio- graphs in cases of skin and soft tissue infections or with CT or MRI in deeper infections. However, gas formation in tissues is not specific to anaerobes and can be found in many aerobic infections as well. Some anaerobic infections, such as actinomycosis, might be identified on smears or tissue biopsy by the presence of filamentous Gram-​positive bacilli and ‘sulphur granules’, although these may be easily missed. Collection of specimens The greatest barrier to the diagnosis of anaerobic infections (aside from not considering anaerobes) is faulty collection and transport of specimens. Aspirated pus or excised infected tissue should be sent to the microbiology laboratory under anaerobic conditions as soon as possible. The use of swabs is discouraged because of the low yield of organisms. Gram’s stains and plating of the specimens should be done promptly with minimal exposure to air to minimize the loss of obligate anaerobes. Immediately after inoculation, the media should be kept under anaerobic conditions in either anaerobic jars or chambers at 35–​37°C. Most microbiology laboratories will not set up anaerobic cultures if the specimens have been collected or trans- ported improperly. Another method to isolate anaerobes is to inocu- late from 1–​10 ml of the suspected infected fluid in to an anaerobic blood culture bottle with proper labelling of the specimen source. Anaerobic blood cultures Recent studies have confirmed their importance in detecting both anaerobic and early facultative anaerobic bacteraemias. Paired cul- tures should be drawn, including both aerobic and anaerobic bottles, and these should be collected from peripheral sites. This is important as positive cultures for anaerobes often occur when not suspected. Treatment Susceptibility and resistance The Clinical Laboratories and Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) each respectively will publish their own breakpoints to determine the cut offs for the susceptibility of various anaerobic bacteria. The breakpoints recommended by each committee do not always correlate, and this can lead to variations in reported rates of resistance. The recommendations for testing and breakpoints is regularly reviewed by these committees. Resistance to various classes of antibiotics will vary according to the species of the anaer- obic bacteria and these are discussed in more detail next. β-​Lactam antibiotics β-​lactams like penicillin have traditionally been used to treat anaer- obic infections. Penicillin G continues to have activity against many anaerobes, but resistance has been growing particularly in the anaer- obic Gram-​negative bacilli. There are three main ways that anaerobes can develop resistance to β-​lactam antibiotics: (1) β-​lactamase en- zymes including penicillinases and cephalosporinases; (2) reduced affinity of the penicillin binding proteins present in the cell wall; and (3) alterations in the porin channels leading to decreased perme- ability of the cell walls to β-​lactams. The most common mechanism of resistance is the development of the inactivating enzymes like β-​lactamases. β-​lactam/​β-​lactamase inhibitor combinations such as ampicillin/​sulbactam, piperacillin/​tazobactam, and ticarcillin/​ clavulanate demonstrate a high degree of in vitro activity against many anaerobes including members of the B. fragilis group. Many anaerobes can produce cephalosporinases, which limits the use of cephalosporins as single agents in the treatment of anaerobic in- fections. Since cephalosporinases have little activity against second generation cephalosporins we still see moderate antianaerobic in vitro activity with cephamycins such as cefoxitin particularly in the Bacteroides fragilis group. Newer cephalosporins like ceftolozane/ tazobactam and ceftazidime/avibactam have limited anaerobic activity, including variable activity against the Bacteroides fragilis group. They should be combined with metronidazole when treating serious anaerobic infections. Carbapenems such as imipenem/​ cilastatin, meropenem, ertapenem, and doripenem typically dem- onstrate excellent in vitro activity against nearly all anaerobes. The presence of carbapenemases are less common among anaerobes. Chloramphenicol Chloramphenicol is a bacteriostatic drug that has been used to treat severe anaerobic infections. Although resistance is rare, use of the drug does carry risks of significant side effects that can include aplastic anaemia and haemolytic anaemia. The drug requires vigi- lant monitoring when used. It is rarely used in the United States. Clindamycin Clindamycin is a bacteriostatic lincosamide that has demonstrated good activity against anaerobes in the past. Resistance rates have been increasing globally in Bacteroides fragilis group anaerobes and non-​Bacteroides anaerobes. It is often used for dental infections and aspiration pneumonias but is no longer recommended as empiric therapy for anaerobic coverage in intra-​abdominal infections due to increased resistance among Enterobacteriaceae. Nitroimidazoles Metronidazole has demonstrated excellent activity against most anaerobic bacteria. The rates of resistance in the anaerobic Gram-​ negative bacilli, including the Bacteroides fragilis group, has been low. Aerobic and facultative anaerobic organisms often show high rates of resistance and, as such, microaerophilic Streptococci often demonstrate resistance. Other nitroimidazoles like tinidazole and secnidazole also have anaerobic activity but are mainly used for the treatment of bacterial vaginosis. Others Tigecycline has activity against many Gram-​positive and Gram-​ negative anaerobic bacteria including the Bacteroides fragilis group, C. perfringens and Peptostreptococcus spp. Among fluoroquinolones,

8.6.12 Cholera 1060

8.6.12 Cholera 1060

section 8  Infectious diseases 1060 moxifloxacin has demonstrated moderate to good in vitro activity against most anaerobes. There are recent reports of isolates of the Bacteroides fragilis groups that were multidrug resistant and also of an isolate of Bacteroides thetaiotaomicron that was resistant to both metronidazole and carbapenems. These increasing reports of resistance raise the con- cern for gene transfer from resistant aerobic Enterobacteriaceae to anaerobes. Updated data on anaerobic resistance in the United States is available in the CLSI M100-​S22 document that was last published in 2012. Anaerobes from the non-​Bacteroides group are generally more susceptible to antianaerobic antibiotics. Antibiotic resistance among anaerobes is less predictable than with aerobic and faculta- tive anaerobes. Institutions should perform susceptibility testing at least annually to establish patterns of resistance to be reported with the hospital’s yearly antibiogram. When susceptibility testing is per- formed, the routine antimicrobials recommended for testing both Gram-​negative and Gram-​positive anaerobic organisms are peni- cillin/​β-​lactamase inhibitor combinations, the carbapenem class of antibiotics, metronidazole, and clindamycin. Additionally, routine testing against penicillin is recommended for Gram-​positive an- aerobes. When indicated, susceptibility testing to cephalosporins, carboxy-​ or ureidopenicillins, chloramphenicol, moxifloxacin, or tetracycline can also be performed. Susceptibility of individu- ally recovered isolates should be considered if they were cultured from otherwise sterile sites, in cases of severe infections, those re- quiring long-​term antibiotics, or those not responding to initial empiric therapy. The choice of empiric antibiotics for anaerobic in- fections should be based on the hospital or regional susceptibility antibiograms if the individual isolate susceptibility is not available. Surgery Often, antimicrobial therapy alone is not sufficient to cure anaerobic infections. Since many infections are associated with abscess forma- tion or occur in areas with tissue ischaemia, surgical intervention frequently becomes imperative with drainage of abscesses and resec- tion of devitalized tissue. Surgical antimicrobial prophylaxis In cases where contamination of surgical wounds by the local flora could result in infection, it has become common practice for sur- geons to use antimicrobial prophylaxis in the perioperative period. In intra-​abdominal procedures, prophylaxis against anaerobic bac- teria significantly reduces postoperative infection rates. The regi- mens cover both aerobes and anaerobes. The choice and duration of therapy depends on the nature of the surgery and whether it is an elective or emergency procedure. These decisions are based on timing, type of clinical presentation, and intraoperative findings. The antimicrobials used include cephamycins such as cefoxitin or the addition of other more specific antianaerobic agents such as metronidazole. Ertapenem has proven to be effective in the prophy- laxis of infections for elective colorectal surgery. FURTHER READING Aldape MJ, Bryant AE, Stevens DL (2006). Clostridium sordellii in- fection: epidemiology, clinical findings and current perspectives on diagnosis and treatment. Clin Infect Dis, 43, 1436–​46. Brook I, Wexler HM, Goldstein, EJC (2013). Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Micro Rev, 26, 526–​46. CLSI (2012). Performance standards for antimicrobial susceptibility testing; twenty-​second informational supplement. CLSI document M100-​S22. Clinical and Laboratory Standards Institute, Wayne, PA. Finegold SM, George WL (eds) (1989). Anaerobic infections in humans. Academic Press, New York, NY. Hecht DW (2004). Prevalence of antibiotic resistance in anaerobic bac- teria: worrisome developments. Clin Infect Dis, 39, 92–​7. Nagy E, et al. (2011). Antimicrobial susceptibility of Bacteroides fra- gilis group isolates in Europe: 20 years’ experience. Clin Infect Dis, 17, 371–​9. Sadarangani SP, et al. (2015). Metronidazole-​ and carbapenem-​resistant Bacteroides thetaiotaomicron isolated in Rochester, Minnesota, in 2014. Antimicro Agents Chemother, 59, 4157–​61. Snydman DR, et al. (2011). Update on resistance of Bacteroides fragi- lis group and related species with special attention to carbapenems 2006–​2009. Anaerobe, 17, 147–​51. 8.6.12  Cholera Aldo A.M. Lima and Richard L. Guerrant ESSENTIALS Vibrio cholerae is a Gram-​negative organism that can be subdivided into over 200 serogroups based on the somatic O antigen, with only serogroups O1 and O139 causing epidemic and pandemic disease. Historically it has killed millions from dehydrating diarrhoea, encour- aged the birth of modern epidemiology, the sanitary revolution, and oral rehydration therapy; it persists today as a glaring reminder of poverty and inadequate water/​sanitation. Contaminated food (es- pecially undercooked seafood) is the usual route of transmission in developed countries; contaminated water and street food vendors are more common vehicles in less developed countries. Clinical features and diagnosis—​typical presentation is with sudden onset of voluminous, painless, watery diarrhoea, which can exceed 500 to 1000 ml/​h, leading to severe dehydration in a couple of hours and risk of death. Definitive diagnosis is by isolating V. cholerae from stool or rectal swab samples. Treatment—​oral rehydration therapy with sugar or starch, water, and salts must be provided in the community and at field stations, clinics, and hospitals where most patients present: this reduces the case fatality of untreated severe cholera from about 50% to 1% or less. Antibiotics can shorten the illness and decrease diarrhoeal purging: tetracycline, cotrimoxazole, ciprofloxacin, or azithromycin have been effective, but there is increasing resistance. Prevention—​effective preventive measures include (1)  ensuring a safe water supply; (2)  improving sanitation; (3)  making food safe for consumption by thorough cooking of high-​risk foods, es- pecially seafood; and (4)  health education through mass media. Newer-​generation killed oral cholera vaccines have been licensed

8.6.12  Cholera 1061 and proved to be well tolerated, protective, cost-​beneficial, and a potential tool to control cholera together with the other preventive recommendations. Introduction and historical perspective Cholera, the dreaded scourge causing death from dehydrating diar- rhoea, existed for centuries in South Asia until, in 1817, it broke out along trade routes; since then there have been seven pandemics across all six inhabited continents. The whole-​genome sequences of globally and temporally representative V.  cholerae isolates, including the Haitian outbreak in 2010, have shown that since the 1950s these isolates have spread from the Bay of Bengal in three independent waves with a common ancestor. Analysis of the whole-​ genome sequences of Vibrio cholerae strains also demonstrated that genomic changes and alterations in CTX phage, particularly in the gene encoding the B subunit of cholera toxin, were major changes in the evolution of V. cholerae. Cholera was largely responsible for encouraging the birth of modern epidemiology and for driving the sanitary revolution in Western Europe and North America in the 19th century. In the last one-​third of the 20th century, it helped drive scientific discoveries of cell signalling, intestinal ion trans- port, and oral rehydration therapy, which have brought global diar- rhoea mortality down from over 5 million/​year to below 2 million/​ year. Yet cholera persists today as a disease of poverty, along with other faecally transmitted pathogens, a sign of inadequate water and sanitation among the desperately poor and displaced around the world. Aetiology, genetics, and pathophysiology Thirty years before the causative agent Vibrio cholerae was dis- covered during the fifth pandemic in 1884 in Kolkata, India, by Robert Koch, John Snow’s classic epidemiological study of cholera in London in 1854 suggested that it was transmitted by contamin- ated drinking water. Snow even postulated that a toxin might cause the dramatic fluid loss. V. cholerae is a halophilic flagellated curved Gram-​negative organism classified by biochemical tests and fur- ther subdivided into serogroups based on the somatic O antigen. Among over 200 serogroups, only O1 and O139 cause epidemic and pandemic disease. The other strains are classified as non-​O1 and non-​O139 V.  cholerae. Serogroup O1 is further subdivided into three serotypes (Inaba, Ogawa, and Hikojima) and into two phenotypically different biogroups (Classical and El Tor). The latter is named for the Egyptian village quarantine station where it was first isolated in 1905 from Indonesian pilgrims travelling to Mecca. This strain then became the cause of the seventh pan- demic that continues around the world today. The O139 serogroup, first seen in 1992, appears to have emerged from horizontal gene transfer of a fragment of DNA that encodes O-​antigen biosynthesis from another serogroup (perhaps O22) into the seventh pandemic V. cholerae O1 El Tor strain. O139 and O1 (both Classical and El Tor biotypes) now coexist and continue to cause large outbreaks in India and Bangladesh. V. cholerae O1 (biotype El Tor) has two circular chromosomes and the entire genome sequence has been described. The large chromosome has most of the genes required for growth and pathogenicity and the small chromosome encodes components of several essential metabolic and regulatory path- ways. Critical to the pathogenicity of V. cholerae (and distinct from environmental isolates) is the acquisition of two distinct phages. The first contains a ‘pathogenicity island’ (VPI) encoding the ‘toxin coregulated pilus’. Remarkably, toxin coregulated pilus serves as both a major intestinal colonization factor and as the receptor for the second phage, CTXϕ, that encodes for cholera toxin and ac- cessory proteins (including ACE and Zot) as well as containing genes required for phage replication, integration, and regulation in the RS2 region. Genes encoding colonization factors or toxin are regulated in response to environmental conditions. The 32-​ kDa transmembrane protein ToxR binds upstream of ctxAB to increase transcription and synthesis of cholera toxin. ToxR also regulates the expression of other genes in the ToxR regulon; hence, the expression of ToxR is controlled by environmental factors. Characterization of V. cholera O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti showed that all strains pro- duced increased cholera toxin (2–​10-​fold) compared to the wild type El Tor strains and also produced more toxin coregulated pilus and ToxT. These essential virulence factors are regulated primarily by ToxT via the ToxR virulence regulon. Vibrios are acquired from contaminated water or food and they must pass though the acidic stomach before they are able to col- onize the upper small intestine. Colonization occurs with fila- mentous protein fimbriae, called toxin coregulated pili, which extend from the vibrio wall and attach to receptors on the mucosa. V. cholerae adhere to the M cells without causing tissue damage and rapidly multiply to 107 to 108 cells/​g of tissue. Attached vib- rios efficiently deliver cholera toxin directly to the epithelial cells (Fig. 8.6.12.1). The A subunit consists of two peptides linked by a disulphide bond. The larger, A1, containing the toxic activity, is endocytosed following toxin binding via its B subunit to GM1 ganglioside. The A1 subunit catalyses the covalent bonding of adenosine diphosphoribose from nicotinamide adenosine di- nucleotide to the α-​subunit of Gs, the heterotrimeric adenylyl cyclase-​stimulating G protein, thus activating adenylate cyclase to form cAMP. cAMP then acts to open the cystic fibrosis transmem- brane conductance regulator chloride channel causing increased chloride secretion by the intestinal crypt cells and a blockade of neutral sodium and chloride absorption by villous cells. This leads to voluminous fluid efflux into the small intestinal lumen which exceeds the absorptive capacity of the bowel and results in watery diarrhoea. The diarrhoeal fluid contains large amounts of sodium, chloride, bicarbonate, and potassium, but little protein or blood cells. The loss of electrolyte-​rich isotonic fluid leads to blood volume depletion with attendant low blood pressure and shock. Loss of bicarbonate and potassium leads to metabolic acidosis and potassium deficiency. Epidemiology Ever since Snow’s seminal epidemiological treatise, cholera has been described as the classic water-​borne disease. However, it is also transmitted by contaminated food, especially undercooked sea- food or food mixed with contaminated water. Contaminated food

section 8  Infectious diseases 1062 (especially undercooked seafood) is the usual vehicle for transmis- sion in developed countries, and contaminated water and street food vendors are more common vehicles in less developed coun- tries. V. cholerae is found in brackish surface water and in shellfish, and survives and multiplies in association with zooplankton and phytoplankton independently of infected human beings. There is no known other animal reservoir for V. cholerae. V. cholerae is en- demic in the Indian subcontinent and the re-​emergence of cholera in other continents is highly dependent on environmental factors. The association of the bacteria with plankton has led to the sug- gestion that ship ballast is a cause of its global spread. V. cholerae has evolved to survive in the aquatic environment and then in the host. In water, V. cholerae vibrios are free swimming or attached to plants, green algae, copepods, crustaceans, or insects. In humans, the intestinal milieu fosters the acquisition of genetic elements from the toxin coregulated pilus bacteriophage, lacking in most environ- mental strains. Toxin coregulated pilus phage encodes type IV fim- bria which serves as colonization factor and receptor for the CTX phage that carries genes encoding cholera toxin. Thus, both bac- teriophages integrate into the bacterial genome and form episomal replication intermediates. The production of cholera toxin and the biogenesis of CTX phage both depend on a type II secretion appar- atus, encoded within the bacterial genome. In Bangladesh and Peru, where the disease has been endemic and epidemic, cholera tends to occur in the warm seasons, albeit before and after the monsoon rains in Bangladesh. Most V. cholerae infections are asymptomatic (case:infection =  1:3 to 1:100) or associated with mild non​specific diarrhoea. Since a high inoculum dose is required for infection, person-​to-​person infection is rare without intervening water or food contamination. Infection and its severity also depend on the gastric acid barrier, local intestinal immunity, and blood group. Those with blood group O are at higher risk of severe El Tor cholera than are those with other blood groups. This susceptibility might explain the lower prevalence of blood group O in the Ganges Delta area. A recent study showed that Lewis blood group antigen type Le(a + b –​) are more susceptible and Le(a –​ b +) are less susceptible to V. cholerae O1 associated symp- tomatic disease. This might be important in evaluating population risk factors for cholera and in vaccine efficacy studies. In cholera-​en- demic areas, the highest attack rates are in children aged 2 to 4 years. Cholera toxin A1 GM1 Na+ Na+ H+ H+ PKA cAMP AC ATP α Golgi ERD2 A1 ARF ERAD G-Protein CFTR NHE3 NHE2 Cl— B Fig. 8.6.12.1  Pathophysiology of cholera. V. cholerae produces a major virulence factor, cholera toxin, an 84-​kDa protein consisting of a dimeric A subunit and five identical B subunits. Cholera toxin binds to a monosialoganglioside GM1 receptor at the host mucosal surface and triggers endocytosis of the holotoxin. The A1 domain of the A subunit is transported through the Golgi and endoplasmic reticulum to activate the Gsα subunit of G protein. This A1 domain interacts with ADP-​ribosylating factors (ARFs) to ADP-​ribosylate this Gsα subunit leading to activated G protein and consequent activation of adenylyl cyclase (AC). The AC cleaves ATP to cAMP which subsequently activates protein kinase A which inhibits NaCl absorption (NHE transporters) and increases chloride secretion through the cystic fibrosis transmembrane regulator (CFTR).

8.6.12  Cholera 1063 In newly invaded areas, attack rates are similar for all ages. First illnesses are often seen in adult men, presumably because of greater exposure to contaminated food and water. The current seventh pandemic began in 1961, in Sulawesi (Celebes), Indonesia. By 1966 the disease had spread to other coun- tries in eastern Asia including Bangladesh, India, the former Union of Soviet Socialist Republics, Iran, and Iraq. Cholera reached West Africa in 1970, and in 1991 it appeared in Latin America for the first time in more than a century. Until 1992 only serogroup O1 had been implicated in epidemics while other serogroups had caused only sporadic cases of diarrhoea. However, in late 1992 cholera broke out in India and Bangladesh caused by a previously unrecognized serogroup of V. cholerae, designated O139. It is unclear whether this new serogroup from Southeast Asia will spread to other regions of the world. It is estimated that 1.3–​4.0 million cholera cases and 21 000–​143 000 deaths occur every year worldwide (Fig. 8.6.12.2). Hence, cholera continues as a significant global public health problem. In 2015, a total of 172 454 cases including 1304 deaths were reported from 42 counties, resulting in a case fatality rate of 0.8%. Cases were reported from 16 countries in Africa, 13 in Asia, 6 in Europe, 6 in the Americas, and 1 in Oceania. Afghanistan, the Democratic Republic of the Congo, Haiti, Kenya, and the United Republic of Tanzania were responsible for 80% of all reported cases. From all cases reported, 41% were from Africa, 37% from Asia, and 21% from Hispanic population. Thirteen countries reported im- ported cholera cases. All cases of suspected cholera should be reported to local and national health authorities, since cholera outbreaks can become massive epidemics. These cases should be confirmed by labora- tory investigation. If a patient older than 5 years develops severe dehydration or dies from acute watery diarrhoea, or if there is a sudden increase in the daily number of patients with acute watery diarrhoea, a cholera outbreak should be suspected. Prevention and vaccines Since contaminated water and food are the main vehicles of trans- mission, effective preventive measures include ensuring a safe water supply (especially for municipal water systems), improving sani- tation, making food safe for consumption by thorough cooking of high-​risk foods (especially seafood), and providing health education through mass media (Box 8.6.12.1). Three safe and well-​tolerated oral cholera vaccines that pro- vide significant protection have been approved by World Health Organization (WHO) for use in both endemic and epidemic cholera:  Dukoral, Shanchol, and Euvichol. Dukoral is a killed Fig. 8.6.12.2  Countries and areas reporting cholera cases in 2015. From Cholera. Weekly Epidemiological Record (2016), 38(91), 433–​440, © WHO 2016. Box 8.6.12.1  Prevention of cholera • Ensure a safe water supply. • Wash hands after defecation and before food preparation. • Improve sanitation, making water and food safe for consumption. • Provide health education through mass media. • Vaccination and improvements in sanitation work synergistically.

section 8  Infectious diseases 1064 whole cell V.  cholerae plus recombinant B subunit of cholera toxin (rCTB-​WC). Sanchol (Shantha Biotechnics) was devel- oped and licensed in 2009 via a public-​private partnership in India as a new killed whole-​cell-​only oral cholera vaccine, which was modified from an earlier oral cholera vaccine produced in Vietnam. Euvichol (EuBiologics, The Republic of Korea) has the same characteristics as Shanchol and was prequalified by WHO in December 2015. All three vaccines were recommended two doses for full protection. In a trial in Kolkata, India, the two-​ dose regimen confer 65% cumulative protection at five years of follow-​up. The same regimen in a trial in Dhaka, Bangladesh was found to confer 53% protection over two years of follow-​ up. A single dose of the killed oral cholera vaccine (Shanchol) was also efficacious in older children (≥5 years of age) and in adults during the initial six months after vaccination in an urban endemic cholera in Dhaka, Bangladesh. Because vaccine efficacy is overcome by larger infectious doses, vaccine should be seen as synergistic with improvements in water, hygiene, and sanitation that reduce the numbers of vibrios ingested. Clinical features The incubation period of cholera usually ranges from 18 h to 5 days. There is a sudden onset of voluminous watery diarrhoea with occasional vomiting. Diarrhoea is severe in 5–​10% of those infected. Its most distinctive feature is the painless purging of voluminous stools resembling rice-​water with a fishy odour. The vomitus is generally a watery and alkaline fluid. Severe diarrhoea can exceed 500 to 1000 ml/​h, leading to severe dehydration in 2 h and risk of death. Dehydration can be classified based on the pres- ence and severity of clinical findings. Table 8.6.12.1 summarizes the clinical assessment of patients with mild, moderate, or severe dehydration. In all cases the key is to rapidly replace fluid deficits, correct metabolic acidosis and potassium losses, and to continue replacing ongoing fluid losses. Because cholera toxin has pro- longed effects, it is imperative to continue replacing fluid losses, for which a ‘cholera cot’ with a central hole, plastic sheet, and bucket to monitor purging can be tremendously helpful to both the patient and medical attendants. Signs of severe dehydration include absent or low-​volume peripheral pulse, undetectable blood pressure, poor skin turgor, sunken eyes, and wrinkled hands and feet. Metabolic acidosis can develop and lead to gasping (Kussmaul) breathing. Urine output is diminished or absent until dehydration is corrected. Complications generally result from inadequate fluid replace- ment, acute renal failure due to protracted hypotension, hypogly- caemia, hypokalaemia, and cramps due to electrolyte imbalance. Differential diagnosis Most cases are indistinguishable from other cases of diarrhoeal diseases, but since the treatment of any dehydrating diarrhoea is the same—​fluid replacement—​identification of the pathogen is not essential for patient management. However, if an adult patient becomes severely dehydrated and is in the right epi- demiological setting or with a history of travelling, the clinician and public health authorities should be alert to the possibility of cholera. Criteria for diagnosis Definitive diagnosis is by isolating V.  cholerae from stool or rectal swab samples on selective media. V. cholerae survives in faecal specimens if kept moist. Cary–​Blair transport medium should be used for transport to the laboratory for plating onto thiosulphate citrate bile salts sucrose (TCBS) agar that inhibits most other normal faecal flora but supports the growth of the vibrios. Specimens should also be inoculated into alkaline pep- tone water, an enrichment broth that preferentially supports the growth of vibrios. After 6–​12 h of incubation, a second TCBS plate is inoculated. These plates are incubated for 18–​24 h, and V.  cholerae colonies appear as smooth yellow colonies with slightly raised centres. V.  cholerae is a Gram-​negative polar monotrichous oxidase-​positive asporogenous curved rod that ferments glucose, sucrose, and mannitol and is positive in the ly- sine and ornithine decarboxylase tests. The organism is classified by biochemical tests and is further subdivided into serogroups based on the somatic O antigen. Presumptive identification Table 8.6.12.1  Assessment of patients with diarrhoea for dehydration Feature No dehydration Some dehydrationa Severe dehydrationa, b General appearance Well, alert Restless, irritable Lethargy or unconscious; floppy Eyes Normal Sunken* Very sunken and dry* Tears Present Absent* Absent* Mouth and tongue Moist Dry* Very dry* Thirst Drinks normally, not thirsty Thirsty, drinks eagerly Drinks poorly or not able to drink Skin pinchc Goes back quickly Goes back slowly Goes back very slowly a Two or more of these signs including one indicated by *. b Absence of radial pulse and low blood pressure are also signs of severe dehydration in adults and children older than 5 years. c The skin pinch is less useful in patients with marasmus (severe wasting), kwashiorkor (severe malnutrition with oedema), or in obese patients. From Azurin JC, et al. (1967). A long-​term carrier of cholera: cholera Dolores. Bull World Health Organ, 37, 745–​9.

8.6.12  Cholera 1065 of V. cholerae O1 or O139 can be made on the basis of typical colonies, which are oxidase-​positive and agglutinate with O1 or O139 antiserum. Rapid tests include dark-​field microscopy and rapid immuno- assays which can be useful for monitoring epidemiological patterns in remote areas where cultures are not readily available. New out- breaks must be confirmed by cultures. Polymerase chain reaction and DNA probes are available but are not practicable in many areas where cholera is common. Treatment Treatment must be provided at the community and field stations, clinics, and hospitals where most of the patients present. Oral re- hydration therapy was a major therapeutic breakthrough that has drastically decreased mortality from cholera and other dehydrating diarrhoeal diseases. The case fatality rate of untreated severe cholera approaches 50%, but with oral rehydration therapy it is decreased to 1% or less. The physiological basis for oral rehydration therapy is Na+-​coupled transport with glucose; transport from the entero- cyte to the lateral intercellular space creates a local osmotic gradient that initiates water flow (Fig. 8.6.12.3). The oral rehydration salts (ORS) formulation approved by the WHO is based on the electro- lyte composition lost in stool in patients with cholera. Table 8.6.12.2 summarizes the electrolyte concentrations from cholera stool and several oral rehydration formulations, including that approved and recommended by the WHO. Around 5–​7.5% of the bodyweight should be given as ORS with additional ORS to compensate for other losses. In patients who are severely dehydrated, having lost at least 10% (5 litres for a 50-​kg patient) of their bodyweight, volume replacement must be rapid. Lactated Ringer’s solution is an excellent commercially available intravenous fluid. Other polyelectrolyte solutions with added potas- sium can also be used. Since ORS is the best polyelectrolyte solution to compensate for the acidosis and potassium deficiency, they should be given as soon as possible after initial intravenous fluid resuscitation. A formulation of ORS that uses rice rather than glucose is better for cholera patients because it reduces the purging rate by providing polymeric glucose with lower osmolarity. ORS have been modified to prevent hypernatraemia (more common with S Ouabain Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na− K+ K+ K+ CI− CI− CI− CI− CI− CI− CI− 0 mV− S H+ H+ H+ Co2 + H2O HCO3−

8.6.13 Haemophilus influenzae 1066

8.6.13 Haemophilus influenzae 1066

section 8  Infectious diseases 1066 other diarrhoeas) by having a reduced concentration of sodium (75 mmol/​litre). This hypo-​osmolar solution is also acceptable for cholera. ORS are easily prepared by adding the following simple ingredients to 1 litre of water: 2.6 g sodium chloride, 2.9 g trisodium citrate, 1.5 g potassium chloride, and 13.5 g glucose (or 50 g boiled and cooled rice powder). Adults and children are encouraged to eat, and breastfeeding can continue as there is no scientific basis for resting the gut. Antibiotics can shorten the illness and decrease diarrhoeal purging. One-​ to 3-​day courses of tetracycline, co-​trimoxazole, or ciprofloxacin have been effective but there is increasing resistance. Azithromycin has been used more recently, but growing macrolide resistance may limit its use as well. Antibiotic sensitivity testing is, therefore, recommended during outbreaks. Antibiotics are not in- dicated for asymptomatic contacts. Prophylactic use of antibiotics increases the risk of the development of resistance and it is not indi- cated to prevent cholera. Prognosis Case fatality should be 1% or less if adequate oral rehydration therapy is used early in the illness, even at the community level. Adequate fluid and electrolyte replacement reverses or prevents complications such as acute renal failure or hypoglycaemia even in moderate or severe cholera. Cholera may well persist in its brackish marine res- ervoir, but improved water and sanitation and increasingly available vaccines promise to control this dreaded disease. Other issues (health economics, areas of uncertainty or controversy, and likely developments ahead) Areas of uncertainty or controversy include the mechanisms and importance of natural reservoirs of cultivable and even non-​ culturable vibrios and marine organisms from plankton to shellfish in the ecology of cholera. Despite the remarkable advances in under- standing the pharmacological mechanisms of cholera toxin action, reliable, effective, and inexpensive means of blocking the effects of the toxin remain elusive. With molecular genetic understanding of virulence and pro- tective immunity, likely developments in the near future include the promise of new and better vaccines, toxin-​blocking or absorption-​ enhancing drugs, and continued improvements in oral rehydration therapy, perhaps with nutrients, micronutrients, or probiotics that compete with vibrio colonization or deliver proabsorptive drugs or nutrients. FURTHER READING Ali M, et al. (2015). Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis, 9, e0003832. Arifuzzaman M, et al. (2011). Individuals with Le(a+b–​) blood group have increased susceptibility to symptomatic Vibrio cholerae O1 in- fection. PLoS Negl Trop Dis, 5, e1413. Chin CS, et  al. (2011). The origin of the Haitian cholera outbreak strain. N Engl J Med, 364, 33–​42. Ivers L (2016). Eliminating cholera transmission in Haiti. N Eng J Med, 376, 101–​3. Kim EJ, et  al. (2015). Whole-​genome sequence comparisons
reveal the evolution of Vibrio cholerae O1. Trends Microbiol, 23, 479–​89. Mutreja A, et al. (2011). Evidence for several waves of global trans- mission in the seventh cholera pandemic. Nature, 477, 462–​5. Qadri F, et al. (2015). Feasibility and effectiveness of oral cholera vac- cine in an urban endemic setting in Bangladesh: a cluster random- ised open-​label trial. Lancet, 386, 1362–​71. Qadri F, et al. (2016). Efficacy of a single-​dose, inactivated oral cholera vaccine in Bangladesh. N Eng J Med, 374, 1723–​32. Sack DA, et al. (2004). Cholera. Lancet, 363, 223–​33. Saha DS, et al. (2006). Single-​dose azithromycin for the treatment of cholera in adults. N Engl J Med, 354, 2452–​62. Son MS, et  al. (2011). Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J Clin Microbiol, 49, 3739–​49. Sur D, et al. (2009). Efficacy and safety of a modified killed-​whole-​ cell oral cholera vaccine in India: an interim analysis of a cluster-​ randomized, double-​blind, placebo-​controlled trial. Lancet, 374, 1694–​702. Sur D, et al. (2011). Efficacy of a low-​cost, inactivated whole-​cell oral cholera vaccine: results from 3 years of follow-​up of a randomized, controlled trial. PLoS Negl Trop Dis, 5, e1289. World Health Organization (2015). Cholera 2015. Weekly Epidemiol Rec, 38, 433–​40. 8.6.13  Haemophilus influenzae Esther Robinson ESSENTIALS Haemophilus influenzae is a Gram-​negative bacillus that is an ex- clusively human pathogen and commensal. There are six capsular serotypes (a–​f), of which type b (Hib) is a major cause of childhood infectious disease. Transmission occurs by close bodily contact, the main source being other children. Carriage of the organism may be followed by disease in susceptible individuals. Clinical features In infants, Hib causes symptoms ranging from a mild non​specific fe- brile illness (occult bacteraemia) to full blown sepsis with meningitis, epiglottitis, pneumonia, septic arthritis, or cellulitis. Non​typeable H. influenzae are common nasopharyngeal commensals and cause otitis media and conjunctivitis in children. In adults, non​typeable Acknowledgement: The authors and editors gratefully acknowledge the inclu- sion in this chapter of material contributed to previous editions of the Oxford Textbook of Medicine by Professor Derrick W. Crook.

1067 8.6.13  Haemophilus influenzae H. influenzae cause exacerbations of chronic bronchitis, sinusitis, and pneumonia. Other Haemophilus species, including H. parainfluenzae, are common commensals and rare causes of infective endocarditis and other infection syndromes. Diagnosis and treatment Gram staining of cerebrospinal, synovial, or pleural fluid is a key in- vestigation, but definitive diagnosis requires culture or detection of H. influenzae DNA by polymerase chain reaction methods. Treatment requires good supportive care, appropriate antibiotics and, for men- ingitis, adjunctive corticosteroids. Antibiotic resistance leads to the agent of choice for invasive Hib dis- ease being a third-​generation cephalosporin with good cerebrospinal fluid penetration (e.g. ceftriaxone or cefotaxime). Chloramphenicol with or without ampicillin remains effective in some developing countries. Antibiotic treatment of otitis media, sinusitis, and chronic bron- chitis associated with non​typeable H. influenzae is widely practised, but largely unsupported by evidence. Corticosteroids reduce mortality, severe hearing loss, and neuro- logical sequelae of Hib meningitis, except in children in low-​income countries. Prevention Conjugate Hib vaccines are given as part of the routine infant im- munization schedule and have virtually eliminated invasive Hib disease from North America, Europe, and some other countries. Vaccines for non​typeable H. influenzae are in development. Introduction Haemophilus influenzae is a human-​adapted pathogen and com- mensal with no other reservoir. It is found in the nasopharynx and also the genital and intestinal tracts. It was first isolated in 1890 by Pfeiffer, who mistakenly thought it was the cause of a concurrent influenza pandemic. Description of the organism The genus Haemophilus includes H. influenzae, H. ducreyi, which causes chancroid, the sexually transmitted infection (Chapter 8.6.14), and other human-​adapted species that are commensals: H. parain- fluenzae and H. haemolyticus. Other human commensals, and occasional pathogens, that were previously included in this genus, namely H. aphrophilus, H. para- phrophilus and H. segnis have been reclassified as Aggregatibacter spp. H.  influenzae is a small (0.2–​0.3 × 0.5–​0.8 μm), Gram-​negative, non​motile coccobacillus that grows well on rich media (e.g. choc- olate agar) incubated in 5% CO2. It is fastidious and requires the growth supplements X factor (haemin) and V factor (NAD). H. parainfluenzae requires only V factor. H. influenzae produces 2-​ to 3-​mm-​diameter grey translucent colonies after 18–​25 h incuba- tion. Precise speciation requires bacterial genome sequence analysis (e.g. 16s ribosomal DNA). A minority of H. influenzae strains have polysaccharide capsules and can be serologically classified in six serotypes (a to f). These strains are relatively non​diverse, consisting of few lineages, suggesting that the genes encoding the capsule were acquired relatively recently. Most strains are non​encapsulated and are non​typeable (NTHi) by serological methods. These strains are genetically heterogeneous. Pathogenicity H.  influenzae expresses several cell surface features essential for colonization of the nasopharynx. These are virulence factors, of which the capsule is the most important. Of the six antigenically distinct structures (types a–​f), type b accounted for virtually all in- vasive H. influenzae disease in children prior to Hib vaccination. The serotype b capsule allows the organism to resist phagocytosis by interfering with binding of serum complement. The capsule also resists desiccation, perhaps promoting host-​to-​host transmission. Serum antibody directed against serotype b capsular polysaccharide is protective. This observation stimulated the development of the highly successful H. influenzae type b (Hib) vaccine, now used rou- tinely in national childhood immunization programmes. Modern vaccines contain capsular polysaccharide covalently conjugated to a protein carrier, such as tetanus toxoid. Other cell surface structures involved in pathogenesis, particu- larly in NTHi, include lipopolysaccharide, pili, and other adhesion proteins. Epidemiology Haemophilus influenzae type b Haemophilus influenzae type b (Hib) is a major cause of childhood infectious disease. Acquisition occurs by close bodily contact, usu- ally from other children, and is usually followed by asymptomatic carriage. The organism dwells harmlessly for months in the naso- pharynx. In a few susceptible individuals, acquisition immediately precedes invasive disease. Carriage rates increase from birth until 4  years and are higher in developing countries, especially where there is crowding, day care attendance, and contact with siblings. The incidence of Hib infections varies with age. Neonates are pro- tected, after which disease peaks by 9 months of age and declines to very low levels by 4 years. Age-​specific disease incidence is inversely related to serum antibodies to Hib. Risk factors for H. influenzae in- fection include complement deficiency, hypogammaglobulinaemia, hyposplenism, sickle cell anaemia, malignancy, and HIV infection. Ethnicity is also important, with higher rates of invasive disease ob- served in Native American and Aboriginal populations. Co-infection with respiratory viruses is also an important risk factor for disease. The main diseases caused by Hib are meningitis, bacteraemia, pneumonia, epiglottitis, and arthritis. Before the introduction of im- munization, Hib was the most important cause of childhood men- ingitis in the United States of America, accounting for 80% of cases and in the United Kingdom, accounting for approximately 50% of cases (Fig. 8.6.13.1). In contrast, it was a much less prominent cause of meningitis than Neisseria meningitidis in the ‘meningitis belt’ of Africa. In Western countries, the case fatality of Hib meningitis was about 5% and long-​term morbidity (deafness and neurological and learning deficits) occurred in at least 10% of cases. Hib immunization virtually eliminates carriage and pro- duces a marked herd effect, protecting against disease. Since the

section 8  Infectious diseases 1068 implementation of Hib conjugate vaccination, the disease has al- most disappeared from North America and Europe. There has been a similar dramatic decline in the majority countries with good vac- cine coverage, including the Gambia and Uganda. A striking but temporary re-​emergence of Hib disease in the United Kingdom was attributed to the introduction, in 2000, of a combined Hib-​ acellular pertussis vaccine that induced lower Hib antibody levels (Fig. 8.6.13.2). In 2014, the World Health Organization (WHO) estimated that vaccination against Hib protected 56% of children in 100 countries. However, there were an estimated 199 000 deaths in children under age 5 from Hib that year (down from 363 000 in 2000), of which nearly 80% were attributed to pneumonia. In some parts of Asia, particularly China, Hib is far less prevalent and so the health benefits of mass vaccination may be insufficient to justify a national vaccination programme. Non​typeable H. influenzae (NTHi) Non​typeable H.  influenzae is acquired soon after birth:  20% of children are colonized in the first year of life and over 50% by 5 years. Colonization persists throughout adulthood. NTHi causes mainly non​invasive infections in children and older adults. It is an important cause of otitis media, sinusitis, bronchitis, and post-​ traumatic meningitis. Since the introduction of the conjugate pneumococcal vaccine, NTHi has become the most common cause of otitis media. 0 0.1 0.15 0.4 0.8 1.5 2.5 7 15 25 40 60 0.55 10 20 30 40 50 60 70 Age in years Cases/100 000/year H. influenzae N. meningitidis S. pneumoniae Fig. 8.6.13.1  Incidence of meningitis in the United States of America caused by H. influenzae, N. meningitidis and S. pneumoniae before the introduction of the Hib conjugate vaccine. Data derived from various sources. Hib vaccine catch-up Hib combined vaccine Hib vaccine introduced 1000 500 0 1990 1995 2000 2005 2010 2015 Year Cases Cases of invasive Haemophilus infection by year and strain, England Hib Not typed NTHi Other capsular serotypes Total Fig. 8.6.13.2  Cases of invasive Haemophilus infection by year and strain in England. Data from Public Health England/​Health Protection Agency.

1069 8.6.13  Haemophilus influenzae NTHi can cause community-​acquired pneumonia in adults, which can be severe. Bacteraemia is increasingly recognized in neo- nates and elderly populations. With the decline in Hib disease fol- lowing introduction of vaccination programmes, NTHi now cause most cases of invasive H. influenzae disease in countries with high vaccination coverage (Fig. 8.6.13.2). Antibiotic resistance H.  influenzae resistant to antibiotics was first reported in the early 1970s. Since then, the prevalence of ampicillin-​resistant β-​ lactamase-​producing strains has risen rapidly in most parts of the world and strains resistant to tetracycline, chloramphenicol, and tri- methoprim, or multiresistant to these antibiotics have emerged. In recent years, ampicillin resistance rates have remained reasonably constant in Europe at 10 to 20%. β-​Lactamase-​negative ampicillin-​resistant H.  influenzae is an emerging pathogen and high rates have been reported in certain countries, for example, Japan (c.30–​50%) and Spain (c.56%). Clonal outbreaks have been reported in Scandinavia and Korea, although prevalence elsewhere remains low. Possible explanations for this in- clude low vaccine coverage, underdosing of oral ampicillin, or fre- quent use of cephalosporins. Clinical features Hib invasive disease (See Table 8.6.13.1.) Bacteraemia and meningitis In infants, clinical features vary from a mild non​specific febrile illness, reflecting occult bacteraemia, to full blown sepsis with men- ingitis. Severe cases show typical features of meningitis including al- tered mental status, stiff neck, and sepsis. There may be disseminated intravascular coagulation with purpuric rash and septicaemic shock, reminiscent of meningococcaemia. Diagnosis is by blood culture and cerebrospinal fluid (CSF) microscopy and culture. Lumbar puncture should not be performed if there is a suspicion of raised intracranial pressure, but antibiotic treatment must not be delayed. Epiglottitis Epiglottitis is an acute, life-​threatening, medical emergency. Symptoms are of systemic sepsis (fever, tachycardia, and tachypnoea), with rapidly evolving local signs and symptoms. Sore throat, drooling, dysphagia, hoarseness, barking cough, and stridor are characteristic. Attempts to examine the throat may precipitate acute airway obstruction; if visu- alized, the epiglottis is inflamed and swollen, looking like a red cherry. Pneumonia Pneumonia is common in Hib disease. The main features are fever with signs of respiratory distress including tachypnoea, nasal flaring, and intercostal recession. Chest examination and radiography are diagnostic of pneumonia. Septic arthritis The child with septic arthritis shows features of sepsis, is unwilling to use the affected limb, and resists movement of the painful joint. Examination and culture of joint fluid are diagnostic. Cellulitis Cellulitis is rare in children, but classically peri-​orbital. Cellulitis of the neck is seen occasionally in adults. These presentations have been increasingly associated with H. influenzae type f infection, es- pecially since the introduction of Hib conjugate vaccine. Non​typeable H. influenzae NTHi causes several clinical syndromes (Tables 8.6.13.1 and 8.6.13.2). Acute otitis media is common in childhood: up to 75% of children suffer at least one episode before the age of five. It usually presents with irritability, with or without fever. The tympanic membrane is inflamed and may perforate, discharging pus. Although not used routinely, tympanocentesis is the most reliable means of aetiological diagnosis. NTHi is a common cause of conjunctivitis, especially in chil- dren. Historically, H. influenzae biotype aegyptius has also been associated with conjunctivitis. Brazilian purpuric fever, a ful- minant septicaemic illness with high case fatality, is also caused by H. aegyptius. NTHi sinusitis presents with local pain, a sense of pressure in the head, local facial oedema, and visible pus draining from the ostia of the sinuses. Diagnosis is by skull radiography, or computed tomog- raphy (CT) scan. Sinus aspiration provides an aetiological diagnosis. In adults, exacerbations of chronic bronchitis are commonly as- sociated with NTHi. H. influenzae and S. pneumoniae are cultured from sputum of up to 50% of cases although their precise aetio- logical role is uncertain. Table 8.6.13.1  Clinical manifestations of Haemophilus influenzae disease: percentage of cases in which syndrome manifests Disease Haemophilus influenzae type b (Hib) Non​typeable Haemophilus influenzae (NTHi) Meningitis 52% 34% Pneumonia 12% 21% Epiglottitis 10% Not described Isolated septicaemia 8% 28% Cellulitis 5% Not described Other 13% 17% Data derived from various sources. Table 8.6.13.2  Clinical syndromes caused by non​typeable Haemophilus influenzae Syndrome Percentage of overall disease burden Otitis media

50% of childhood cases Exacerbations of chronic bronchitis 50% of patients positive for Haemophilus during exacerbation Bacterial conjunctivitis 25% of adult and 50% of childhood cases Sinusitis 40% of childhood cases Pneumonia Up to 20% of childhood cases Neonatal sepsis 5–​7% of neonatal sepsis Data derived from various sources.

section 8  Infectious diseases 1070 NTHi can cause severe invasive disease such as neonatal sepsis, resembling group B streptococcal neonatal sepsis, and pneumonia in adults, particularly older people. It has also been implicated in meningitis associated with skull fracture. Laboratory diagnosis Culture or detection of specific DNA is essential for aetiological diagnosis. Direct examination of cerebrospinal, pleural, or synovial fluids by Gram’s stain may reveal organisms with the morphological features of H. influenzae. Blood culture using most commercial systems yields excellent growth in both anaerobic and aerobic bottles. However, growth on agar and differentiation of Haemophilus spp. requires special con- ditions (see above). Antibiotic susceptibility testing using antibiotic discs requires supplemented media to support the growth of Haemophilus spp. This may be inaccurate and should be supplemented by measure- ment of β-​lactamase activity. Chloramphenicol disc susceptibility is frequently inaccurate and should be supplemented by an assay for chloramphenicol acetyltransferase activity. Capsular type b antigen can be rapidly detected in CSF, sterile site fluid, or urine. Polymerase chain reaction (PCR) of CSF was devel- oped for diagnosing H. influenzae meningitis. Capsular typing of H. influenzae can be achieved serologically but a PCR-​based method is more reliable. Treatment Antibiotics A third-​generation cephalosporin with good CSF penetration is the first-​line antibiotic treatment for invasive Hib disease. High-​ dose ceftriaxone or cefotaxime are effective for treating H. influen- zae meningitis and septicaemia, but cefuroxime must not be used. Alternatives include chloramphenicol alone (depending on the prevalence of chloramphenicol resistance) or in combination with ampicillin. β-​Lactamase-​negative ampicillin-​resistant H.  influen- zae with reduced cephalosporin susceptibility have been described; carbapenem resistance remains rare. Antibiotics are commonly prescribed for otitis media, sinus- itis, and chronic bronchitis, but large meta-​analyses have failed to demonstrate convincing efficacy; some subgroups may benefit. Oral amoxicillin is first-​line. Amoxicillin/​clavulanate, trimetho- prim, tetracycline (adults only), and quinolones (adults only) can also be used. Local sensitivity patterns should be used to guide empiric therapy. Corticosteroid treatment in meningitis Corticosteroids significantly reduce mortality, severe hearing loss, and neurological sequelae in Hib meningitis. In community-​ acquired bacterial meningitis, corticosteroid therapy should be started with the first antibiotic dose for adults. In children, data support the use of adjunctive corticosteroids in children only in high-​income countries. Prevention and control Polysaccharide conjugate vaccines are the best preventive measure for controlling Hib disease. Highly effective vaccines contain cap- sular antigen polyribosyl-​ribitol-​phosphate (PRP) conjugated to tetanus toxoid (PRP-​T), outer membrane protein (PRP-​OMP), or mutant diphtheria toxoid (PRP-​CRM, HbOC). Three doses are given at intervals between the ages of 2 and 6 months. In many countries, a booster dose is given at age 1–​2 years. Carriage rates are decreased to very low levels, with invasive disease being virtually eliminated. Rifampicin can be used to eradicate carriage and may prevent sec- ondary cases among close contacts. This is appropriate only where they have not received Hib vaccine. Significant efforts are now being focused on producing a NTHi vaccine; however, there are major challenges still to be overcome. Other nasopharyngeal Haemophilus spp. H. parainfluenzae H. parainfluenzae is a well-​adapted commensal that colonizes vir- tually everyone soon after birth but is rarely associated with disease. It has been isolated in cases of infective endocarditis, neurosurgical meningitis, prosthetic device infection, and brain and liver ab- scesses. It is treated in the same way as H. influenzae. H. haemolyticus A common commensal, H. haemolyticus has also been occasionally associated with bacteraemia and joint infection. FURTHER READING Barbour ML, et al. (1995). The impact of conjugate vaccine on carriage of Haemophilus influenzae type b. J Infect Dis, 171, 93–​8. Kim KS (2010). Acute bacterial meningitis in infants and children. Lancet Infect Dis, 10, 32–​42. Jalalvand F, Riesbeck K (2018). Update on non-typeable Haemophilus influenzae-mediated disease and vaccine development. Expert Rev Vaccines, 18, 1–10. Morris SK, Moss WJ, Halsey N (2008). Haemophilus influenzae type b conjugate vaccine use and effectiveness. Lancet Infect Dis, 8, 435–​43. Prasad K, Karlupia N, Kumar A (2009). Treatment of bacterial menin- gitis: an overview of Cochrane systematic reviews. Respir Med, 103, 945–​50. Ulanova M, Tsang RS (2009). Invasive Haemophilus influenzae dis- ease: changing epidemiology and host-​parasite interactions in the 21st century. Infect Genet Evol, 9, 94–​605. Van Eldere J, et al. (2014). Non-​typeable Haemophilus influenzae, an under-​recognised pathogen. Lancet Infect Dis, 14, 1281–​92. Watt JP, et al. (2009). Burden of disease caused by Haemophilus influen- zae type b in children younger than 5 years: global estimates. Lancet, 374, 903–​11. Zwahlen A, et  al. (1989). The molecular basis of pathogenicity in Haemophilus influenzae:  comparative virulence of genetically-​re- lated capsular transformants and correlation with changes at the capsulation locus cap. Microb Pathog, 7, 225–​35.

8.6.14 Haemophilus ducreyi and chancroid 1071

8.6.14 Haemophilus ducreyi and chancroid 1071

1071 8.6.14  Haemophilus ducreyi and chancroid 8.6.14  Haemophilus ducreyi
and chancroid Nigel O’Farrell ESSENTIALS Haemophilus ducreyi is a Gram-​negative, facultative anaerobic bacillus that is the cause of chancroid. The condition was endemic in sub-​ Saharan Africa and the Caribbean, but the overall global incidence of the condition has decreased dramatically since the mid-​1990s. More recently the organism has been identified as a cause of paediatric skin disease in some South Pacific islands and Ghana. Clinical features—​after an incubation period of 4 to 10 days, pres- entation is with a tender genital papule that develops into a pustule and then an ulcer with a ragged undermined edge and a yellow base that bleeds readily. The usual sites of infection in men are the prepuce and coronal sulcus, and in women the labia minora and fourchette. Inguinal lymphadenopathy is found in about half the male cases. Chancroid is an important risk factor for the transmission of HIV infec- tion. HIV infection may result in atypical manifestations of chancroid. Diagnosis and treatment—​nucleic acid amplification tests are the optimal method of diagnosing H. ducreyi. Treatment is with either ciprofloxacin, erythromycin, azithromycin, or ceftriaxone. Introduction The causative organism is Haemophilus ducreyi, a Gram-​negative fac- ultative anaerobic bacillus. Chancroid has also been known as soft sore (ulcus molle) and was first differentiated from syphilis by Ricord in 1838 in France. In 1889 in Naples, Ducrey inoculated the forearms of patients with material from their own genital ulcers and main- tained serial ulcers through multiple generations. The first successful culture was undertaken by Lenglet in 1898. The Ito-​Reenstierna test was developed subsequently using a commercial antigen for intra- dermal testing and proved positive in 90% of true cases. Aetiology Recent phylogenetic studies suggest that the causative organism should be reclassified as an actinobacillus of the Pasteurellaceae. Traditional phylogenies divide genital strains into class 1 and II clades. The organism is a small, non​motile, and non-​spore-​forming Gram-​ negative rod that requires enriched media for growth. Colonies can be seen after 48 h incubation in 5% CO2 and are greyish in colour. These colonies are cohesive and can be pushed across culture media with a thin wire. Microscopic examination using Gram’s stain shows streptobacillary chaining. Epidemiology Overall, the global incidence of chancroid has decreased dramatic- ally since the mid-​1990s when it accounted for 30–​50% of genital ulcers in southern Africa. Chancroid was endemic in eastern and southern Africa and the Caribbean. In Asia, cases in India and Thailand used to be fairly common, but are now only sporadic. The decrease in chancroid may reflect changes in sexual behaviour, the increased use of and adherence to syndromic management for genital ulcers that included effective antibiotic cover for chancroid, or some other unknown factors. Sporadic outbreaks have been reported in the West. These have usually been associated with sex work and have been brought under control using intensive partner notification schemes. The male to female ratio is about 5:1. Chancroid is more common in uncircumcised than circumcised men. This may reflect inferior standards of genital hygiene and a tendency for small microabrasions to develop in the subpreputial space that might provide a portal of entry for infection. Asymptomatic carriage has been identified in women but is uncommon. Recently H. ducreyi has been implicated as a cause of skin ulceration mainly involving the legs of children in some South Pacific islands. Pathology and pathogenesis The pathogenesis of chancroid is incompletely understood. Bacterial adherence to susceptible cells appears to involve interaction be- tween a protein mediator and liopoligosaccharide with fibronectin contained in the extracellular matrix, followed by the elaboration of a heat shock protein (GroEL). A cytotoxin, similar to cytolethal distending toxin, appears to play an important role in epithelial in- jury and ulcer formation. The histological features include a superficial purulent exudate in the epidermis and a perivascular and interstitial mononuclear cell infiltrate, containing CD4+ T-​lymphocytes, in the dermis. This may partly explain the increased risk of HIV transmission among people with chancroid. H. ducreyi cutaneous ulcer strains may arise from Class I and II Clades. Clinical features The usual incubation period is 4 to 10 days (range 1–​5 days) and there are no prodromal symptoms. Lesions start as a tender papule that develops into a pustule and then an ulcer. Classically, ulcers have a ragged undermined edge with a grey or yellow base that bleeds when touched. Lesions may be single or multiple. The usual sites of infection are the prepuce, coronal sulcus, frenulum, and glans in men, and the labia minora and fourchette in women. Ulcers of the vaginal wall and cervix are uncommon. Extragenital lesions are rare but have been reported on the fingers, breasts, and inner thighs. H. ducreyi does not disseminate. Clinical variants can occur. These include giant phagedenic ul- cers, dwarf chancroid similar to herpes, follicular chancroid similar to pyogenic infection, and single painless ulcers not unlike syphilis. Painful inguinal lymphadenopathy is found in about one-​half the male cases but less so in women. These lymph glands may develop into buboes that should be managed by aspiration rather than in- cision and drainage. Fluctuant buboes may rupture spontaneously causing delayed healing.

section 8  Infectious diseases 1072 The differential diagnosis includes syphilis, genital herpes, lymphogranuloma venereum, and donovanosis. Mixed infections with other causes of genital ulceration should always be considered as coinfection with syphilis has been documented. The presence of HIV infection may result in atypical manifest- ations, for example, numerous lesions, extragenital involvement, or slow resolution after treatment. H. ducreyi lesions affecting the lower extremities in children in some South Pacific Islands can be mistaken for yaws. Laboratory diagnosis Nucleic acid amplification tests are now the optimal method of diagnosing H. ducreyi but their availability remains limited in areas where chancroid is found. Primers have been developed to amp- lify sequences from the H. ducreyi 16S ribosomal RNA gene, the rrs (16S) to rrl (23S) ribosomal intergenic spacer region, and the groEL gene. Multiplex polymerase chain reaction tests have been developed that can identify infection with H. ducreyi, Treponema pallidum, and Herpes simplex virus types 1 and 2 from genital ulcers. Whole-genomic sequencing has helped to better define the phylo- genetic tree of H.ducreyi and has shown that the cutaneous strains in children arise from class I and II clades. Antigen detection using fluorescence techniques may be useful but is expensive. Serological tests are unable to differentiate between old and new infections and have limited application. Culture was the usual method of diagnosis of chancroid until rela- tively recently but has now been overtaken by nucleic acid amplifica- tion tests. Culture media must be fresh and may need fine adjustment depending on the characteristics of local strains of H. ducreyi. Two culture media are required to achieve a reasonable sensitivity of 50–​ 80%. Media used include gonococcal agar base and Mueller–​Hinton with various additives and supplements. Vancomycin may be used to inhibit Gram-​positive bacteria. Cultures should be incubated at 33°C with 5% carbon dioxide in a humid atmosphere. Thioglycolate haemin-​based transport medium may allow storage of viable organ- isms at 4°C for 24 h or possibly longer. Most strains are β-​lactamase producers. H. ducreyi reduces nitrate to nitrite and all strains are oxi- dase positive and catalase negative. Gram-​stained smears of material from ulcers may show characteristic Gram-​negative coccobacilli in a ‘school of fish’ or ‘railroad track’ appearance. Histology shows superficial necrosis with large numbers of neu- trophils, endothelial proliferation, and infiltration with plasma cells, lymphocytes, and fibroblasts. Treatment Current treatment of chancroid comprises one of the following re- gimens: ciprofloxacin (500 mg twice daily for 3 days), erythromycin (500 mg three times daily for 7 days; can be used in pregnant women), azithromycin (a single oral dose of 1 g), or ceftriaxone (a single dose of 250 mg intramuscularly). Trimethoprim/​sulphamethoxazole is no longer recommended. Healing of ulcers is usually achieved after 7 to 14 days. Longer courses of treatment are sometimes required in HIV-​positive patients who should be followed up until healing is complete. Single-​dose treatment should probably not be given to HIV-​positive patients. In the preantibiotic era, circumcision, saline soaks, and improved hygiene were recommended. Initially organisms were sensitive to penicillin, but resistance emerged fairly rapidly. Trimethoprim/​ sulphamethoxazole then became the mainstay of treatment but re- sistance to this antibiotic emerged in the early 1990s. Chancroid and HIV Chancroid has been identified as an important risk factor for the bidirectional transmission of HIV, particularly in eastern and southern Africa. In some high-​risk groups it was undoubtedly an important factor in driving the initial spread of HIV. At the bio- logical level, the mechanism for this is likely to be that chancroid ulcers allow a route of entry and exit for HIV and are likely to bleed when subject to trauma. In addition, subpreputial lesions in men that subsequently heal might result in partial phimosis with thin- ning of the superficial mucosa. This mucosa would then be more susceptible to trauma during sexual intercourse thereby increasing the potential risk of HIV transmission through microulcerations. There are some reports that HIV-​positive men have increased num- bers of ulcers that heal slowly, although this may be related to low CD4 counts. Prevention and control In developed countries intensive partner notification and epi- demiological treatment of sexual contacts have formed the basis for managing outbreaks. In most developing countries, genital ulcers have been managed by the syndromic approach. This involves treating for the most likely causes of ulceration that in the past have been syphilis and chancroid. However, the prevalence of chancroid in previously en- demic countries has reduced considerably leaving genital herpes as the most frequent cause of genital ulceration. With this emergence of genital herpes, the case for treating empirically for chancroid has weakened and it may be that new epidemics of chancroid will emerge as treatment regimens change. FURTHER READING Bong CT, Bauer M, Spinola SM (2002). Haemophilus ducreyi: clinical features, epidemiology, and prospects for disease control. Microbes Infect, 4, 1141–​8. Gonzalez-​Beiras C, et al. (2016). Epidemiology of Haemophilus ducreyi infections. Emerg Infect Dis, 22, 1–​8. Marks M, et al. (2018). Direct whole-genome sequencing of cutaneous strains of Haemophilus ducreyi. Emerg Infect Dis, 24, 786–9. Mitja, O, et al. (2014). Haemophilus ducreyi as a cause of skin ulcers in children from a yaws-​endemic area of Papua New Guinea. Lancet Global Health, 2, e235–​41. Spinola S (2008). Chancroid. In: Holmes KK, et al. (eds) Sexually trans- mitted diseases. McGraw-​Hill, New York, NY. Spinola SM, Bauer ME, Munson RS (2002). Immunopathogenesis of Haemophilus ducreyi infection (chancroid). Infect Immun, 70, 1667–​76. Trees DL, Morse SA (1995). Chancroid and Haemophilus ducreyi: an update. Clin Microbiol Rev, 8, 357–​75.

8.6.15 Bordetella infection 1073

8.6.15 Bordetella infection 1073

8.6.15  Bordetella infection 1073 8.6.15  Bordetella infection Cameron C. Grant ESSENTIALS Bordetella are small Gram-​negative coccobacilli, of which Bordetella pertussis is the most important human pathogen. Bordetella pertussis is the cause of whooping cough, which re- mains one of the 10 leading causes of death among children less than five years old. Transmission of this highly infectious organism is primarily by aerosolized droplets. Clinical features—​presentation varies with age, immunization, and previous infection: (1) infants—​apnoea, cyanosis, and par- oxysmal cough; (2) non​immunized children—​cough, increasing in severity with distressing, repeated, forceful expirations fol- lowed by a gasping inhalation (the ‘whoop’); (3)  children
immunized in infancy—​whooping, vomiting, sputum produc- tion; (4)  adults—​cough, posttussive vomiting and absence of fever. Mild illness is common as is asymptomatic infection. Complications include pneumonia, pulmonary hypertension, seizures, and encephalopathy. Most deaths occur in those less than two months old. Diagnosis and treatment—​culture lacks sensitivity; the preferred diagnostic methods are polymerase chain reaction detection from nasopharyngeal samples and serology (IgG antibodies to pertussis toxin). Macrolide antibiotics are recommended if started within four weeks of illness onset. Prevention—​pertussis vaccines protect against disease more than infection. Preventing severe disease in young children remains the primary goal, hence schedules consist of a three-​dose infant series and subsequent booster doses. Acellular vaccines enable im- munization schedules to include adolescents and adults. Acellular pertussis vaccine given to pregnant women reduces the risk of per- tussis in young infants. Antibiotic prophylaxis is given when there is an infant at risk of exposure. Introduction Seven Bordetella species cause human infections. Bordetella per- tussis, as the principal cause of whooping cough (pertussis), is the most important. Pertussis remains one of the 10 leading global causes of death in children less than five years old. The epidemiology of B. pertussis infection and pertussis disease differ. Immunization has caused a large reduction in severe per- tussis disease but minimal change in the circulation of B. pertussis. Eradication of pertussis is not currently possible. Pertussis has always been an important cause of severe disease in early infancy. In the era before mass immunization pertussis caused more infant deaths than measles, diphtheria, poliomyelitis, and scarlet fever combined. With mass immunization severe pertussis disease incidence has decreased dramatically. However, the poten- tial persists for B. pertussis infection to cause fatal disease in young infants throughout the world. Aetiology Two of the seven Bordetella species that infect humans, B. pertussis and human-​adapted B. parapertussis, are strictly human pathogens. They evolved independently from a common B. bronchiseptica an- cestor. The B. pertussis population is continuously evolving. As a re- sult, antigenic variation occurs between B. pertussis in circulation and the B. pertussis vaccine strains and composition. Use of both whole-​cell and acellular vaccines is associated with clonal expansion of B. pertussis strains that could potentially lead to decreased vaccine efficacy. In immunocompromised hosts B. bronchiseptica causes respira- tory illnesses. B. holmesii and B. hinzii cause respiratory illnesses, bacteraemia, and endocarditis. B. trematum is occasionally isolated from ear and skin infections and B. petrii from patients with cystic fibrosis. Epidemiology The estimated number of pertussis deaths globally decreased from 390 000 deaths in 1999 to 160 700 in 2014. Underestimation and how this varies with age and surveillance in- tensity is central to understanding pertussis epidemiology. Pertussis occurs in all ages; however, incidence has always been highest in in- fants and children. It is estimated that each year 6% of adults ex- perience a B. pertussis infection. While most of these infections are asymptomatic, they remain a potential source for spread to those at risk of severe disease. The incidence of reported pertussis has increased over the past 30 years, and particularly so since the mid-​2000s. Contributory factors to these increases include heightened awareness of the dis- ease as a cause of illness across the age range, increased availability of more sensitive diagnosis tests, the more rapid wane of immunity that occurs following immunization with acellular compared to whole-​ cell vaccines, and genetic changes in the bacterium. Mortality The propensity for pertussis to kill young infants is unique among vaccine preventable diseases, with the exception of tetanus. Estimates of pertussis mortality are complicated by the relationship between pertussis and malnutrition and by the very small proportion of deaths in young children globally for which the cause is medically certified. A prolonged period of weight loss frequently complicates pertussis in the developing world. In the developed world use of complimentary notification systems is required in order to prevent underestimation of the number of pertussis deaths. Pertussis deaths occur despite intensive care. Treatment of young infants with critical pertussis illness remains challenging. Morbidity Most pertussis incidence estimates are based on passive notifica- tion which identifies only a minority of cases. The proportion of cases notified decreases with increasing age and decreasing severity. Approximately 15–​20% of acute persistent cough illnesses in school-​ age children, adolescents, and adults are caused by B.  pertussis
infection. The incidence rate, in adolescents and adults, of

section 8  Infectious diseases 1074 symptomatic cough illnesses caused by B. pertussis infection is 25 to 500 per 100 000. Prevention Neither disease nor immunization confer lifelong immunity. Pertussis vaccines protect against disease more than infection. Schedules consist of a three-​dose infant series and subsequent booster doses. In infants, one dose of pertussis vaccine provides 50% protection and two doses provide 80% protection against severe dis- ease. Pertussis remains endemic in adolescents and adults. Without boosters it is also endemic in school-​age children. Whole-​cell and acellular pertussis vaccines are combined with other antigens. Acellular vaccines contain between one and five B. pertussis antigens. The most efficacious whole-​cell and acellular vaccines induce protection against clinical disease in approximately 85% of recipients. In order to minimize the pertussis risk to infants the primary series must be completed without delay. However, without booster doses, timely completion of the primary series is insufficient to pre- vent disease in infants. Because the primary infant immunization series cannot protect the youngest infants, a dose of pertussis vac- cine given during pregnancy is now a component of the immuniza- tion schedule in several countries. Immunity induced by whole-​cell pertussis vaccines persists for approximately 5 years after completion of a primary series. Duration of protection is shorter following an acellular vaccine primary im- munization series. Protection following both disease and immuniza- tion is superior to either alone; hence those who have had pertussis should be immunized. Acellular vaccines are both safe and efficacious in adolescents and adults. In adolescents, protection against clinical pertussis wanes within two to four years of immunization. Currently immuniza- tion of pregnant women is the only adult immunization strategy for which there is evidence of prevention of pertussis in young infants. Acellular pertussis vaccine should be given during each pregnancy. Preferably it should be given early in the interval between 27 and 36 weeks gestation.
Because of the risk of nosocomial spread, healthcare workers should receive acellular pertussis vaccine booster doses. Pathogenesis/​pathology Pathogenesis remains poorly understood and the pathophysiology of paroxysmal cough and other characteristic features of the illness remain unknown. B. pertussis is highly infectious, and each primary case produces approximately 15 secondary cases. Transmission is primarily by aerosolized droplets. There is an average of two weeks between suc- cessive cases. In immunized populations the household secondary attack rate remains greater than 80%, although many such infections are asymptomatic. Bordetella spp. have multiple virulence factors including fila- mentous haemagglutinin, fimbriae, pertactin, pertussis toxin, ad- enylate cyclase, tracheal cytotoxin, and lipopolysaccharide. While their individual effects have been characterized, how they act to- gether to cause pertussis disease is not known. Several are im- mune-​modulatory. Pertussis is characterized by an inadequate immunological response. Impairment of the immune response by B. pertussis virulence factors is a potential mechanism that contrib- utes to disease severity. Clinical features Presentation varies with age, immunization, and previous infection. Mild illness which is difficult to distinguish from illnesses caused by other respiratory pathogens, is common. In infants, apnoea, cyanosis, and paroxysmal cough are key symp- toms. These can occur early in the illness before duration of cough allows for the pertussis clinical case definition to be met. Thus, per- tussis must be considered in infants presenting with an acute life-​ threatening event or apnoea. Pertussis in the non​immunized child is a coughing illness increasing in severity over several weeks with distressing repeated forceful expirations followed by a gasping inhalation. Between par- oxysms symptoms can be minimal. The contrast between parental descriptions of the previous night’s events and the normal appear- ance the following morning can deceive the assessing clinician. Following pertussis, viral respiratory tract infections can cause coughing paroxysms to recur. In school-​age children immunized in infancy, clinical symptoms which distinguish pertussis are whooping, vomiting, sputum pro- duction, and the absence of wheezing. Most B.  pertussis infections in adults are asymptomatic or are atypical with few symptoms. Persistent cough is the cardinal fea- ture of clinical pertussis in adults. Pertussis should be considered in any adult with an acute cough that persists for two weeks or more. Cough is worse at night and often paroxysmal. Symptoms that, if present, increase the likelihood that a cough illness is caused by B. pertussis infection are the presence of whooping or post-tussive vomiting. Symptoms which decrease the likelihood that a cough illness is caused by B. pertussis infection are the presence of fever and/or the absence of paroxsymal cough. Differential diagnosis Not considering pertussis in someone with an acute persistent cough is a more important cause of a missed diagnosis than is an atypical presentation. In infants, coinfection with respiratory viruses occurs not infrequently, causing more severe disease and diagnostic confusion. A careful history of coughing illnesses in other household mem- bers is critical. Successive household members are symptomatic over weeks to months rather than having almost concurrent re- spiratory illnesses. Infections with Mycoplasma pneumoniae, Chlamydia pneumo- niae, C. trachomatis, adenoviruses, and other respiratory viruses can cause illnesses which overlap clinically with pertussis. Particularly because it is also worse at night, cough from sinusitis can be con- fused with pertussis. Presentation of cough illness in adults is often delayed until symp- toms have persisted for several weeks. Other causes of chronic cough such as asthma, gastro-​oesophageal reflux, tuberculosis, and malig- nancy need to be considered.

8.6.15  Bordetella infection 1075 Clinical investigations Laboratory diagnosis of pertussis has improved with the develop- ment of polymerase chain reaction (PCR) and serological assays. B. pertussis is a small Gram-​negative coccobacillus. It is strictly aerobic and fastidious; special media such as charcoal blood agar are necessary. Culture lacks sensitivity. Careful collection and rapid transport of the nasopharyngeal sample to the laboratory is required. Immunization and antibiotic treatment reduce culture positivity. The organism is most abundant before the onset of par- oxysmal cough and is rarely recovered once cough has been present for three weeks. PCR is more sensitive than culture. Sensitivity decreases with illness duration and less so with antibiotic treatment. Real-​time PCR assays enable laboratory confirmation within hours of specimen collection. Antibodies to pertussis toxin are specific to B.  pertussis. Only measurement of IgG antibodies is recommended. In the absence of immunization in the previous two years a single antibody titre of 100 IU/​ml has been shown to be sensitive and specific for recent B. pertussis infection. A sensitive and specific oral fluid assay that measures IgG to pertussis toxin has been developed. The preferred laboratory test varies with age and cough duration. PCR is particularly useful in infants. In older children, adolescents, and adults the sensitivity of culture and PCR is lower, and, particu- larly with later presentation, serology is more useful. Criteria for diagnosis The World Health Organization surveillance case definition is a case diagnosed as pertussis by a physician; or a person with cough for two weeks with at least one of: paroxysms of coughing, inspiratory whoop, or posttussive vomiting without other apparent cause. Laboratory confirmation is by isolation of B. pertussis; or detection of genomic sequences by PCR, or positive paired serology. Pertussis should be considered in anyone with paroxysmal cough of any dur- ation, or cough with inspiratory whoop, or cough ending in apnoea, vomiting, or gagging. Treatment (See Table 8.6.15.1.) Antibiotic treatment reduces infectivity. B. pertussis cannot be isolated from most patients after five days of an appropriate anti- biotic. If started within two weeks of symptom onset, antibiotic treatment may decrease symptom severity. Antibiotics are recommended if started within four weeks of illness onset. Erythromycin, azithromycin, and clarithromycin are effective against B.  pertussis. Azithromycin is as efficacious, better tolerated, and requires a shorter treatment course than erythromycin. It should be used with caution in those with pro- longed QT syndrome and other proarrhythmic conditions. Trimethoprim-​sulfamethoxazole is an alternative but data on its efficacy is limited. Azithromycin is the preferred macrolide for infants less than one month old with clarithromycin not recommended in this age group. Use of erythromycin or azithromycin in infants less than one month old is associated with an increased risk of infantile hyper- trophic pyloric stenosis, and monitoring for this condition should be continued for one month after treatment with azithromycin or erythromycin. Prophylaxis is most important when there is an infant at risk of exposure. Interruption of household transmission is only possible if treatment is started within three weeks of symptom onset in the primary case and before any symptomatic secondary cases. Table 8.6.15.1  Choice of antibiotic agents for the treatment or prevention of pertussis1,2 Age Antibiotic Recommended Alternative Azithromycin Erythromycin Clarithromycin Trimethoprim-​ Sulfamethoxazole Younger than 1 month 10 mg/​kg/​day as a single dose daily for 5 daysa,b 40 mg/​kg/​day in four divided doses for 14 days Not recommended Contraindicated if age <2 months 1 to 5 months 10 mg/​kg/​day as a single dose daily for 5 daysa,b 40 mg/​kg/​day in four divided doses for 14 days 15 mg/​kg per day in two divided doses for 7 days 2 mo or older: TMP, 8 mg/ kg/day; SMX, 40 mg/kg/ day in 2 doses for 14 days 6 months or older and children 10 mg/​kg as a single dose on day 1 (maximum 500 mg), then 5 mg/​kg/​ day as a single dose on days 2 to 5 (maximum 250 mg/​day)a 40 mg/​kg/​day in four divided doses for 7 to 14 days (maximum 1 to 2 g/​ day) 15 mg/​kg per day in two divided doses for 7 days (maximum 1 g/​day) 2 mo or older: TMP, 8 mg/ kg/day; SMX, 40 mg/kg/ day in 2 doses for 14 days Adolescents and adults 500 mg as a single dose on day 1, then 250 mg as a single dose on days 2 to 5a 2 g/​day in four divided doses for 7 to 14 days 1 g/​day in two divided doses for 7 days M, TMP, 320 mg/​day;
SMX 1600 mg/​day in two divided doses for 14 days a TMP indicates trimethoprim; SMX, sulfamethoxazole. Azithromycin should be used with caution in people with prolonged QT interval and certain proarrhythmic conditions. b Preferred macrolide for this age because of risk of idiopathic hypertrophic pyloric stenosis associated with erythromycin.

  1. American Academy of Pediatrics. Red Book: 2018 Report of the Committee on Infectious Diseases. 31st edition. Elk Grove Village, IL. American Academy of Pediatrics;
    2018, p. 625.
  2. Tiwari T, Murphy TV, Moran J, National Immunization Program CDC. Recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: 2005
    CDC Guidelines. Morbidity & Mortality Weekly Report 2005; Recommendations & Reports, 54(RR-​14): 1–​16.

8.6.16 Melioidosis and glanders 1076

8.6.16 Melioidosis and glanders 1076

section 8  Infectious diseases 1076 There are no agents with proven efficacy for the treatment of pertussis-​induced cough. Prognosis/​outcome Pertussis in young infants is unpredictable with the potential for rapid deterioration. Complications include pneumonia, pulmonary hypertension, seizures, and encephalopathy. Pneumonia, seizures, and encephalopathy also occur in adults with pertussis. Other complications reported in adults include cough-​induced urinary incontinence and syncope, herniated inter- vertebral disc, inguinal hernia, hearing loss, angina, carotid artery dissection, and death. Areas of uncertainty or controversy and future developments Modifications to current acellular vaccine immunization schedules could potentially increase protection of young infants against per- tussis. Maternal immunization during pregnancy is one such option. Because of the potential for pregnancy immunization to alter the immune response of the infant to vaccine doses received during in- fancy, it may be necessary for booster childhood dosing to begin at a younger age. The immunization of household members may provide indirect protection of young infants against exposure to B. pertussis. Much remains to be learnt about the pathogenesis of pertussis and the pathophysiology of clinical manifestations and the optimal man- agement of young children with life-​threatening pertussis. Vaccines with greater efficacy against B. pertussis infection will be necessary for immunization to be able to reduce endemic disease incidence. Improved surveillance is required in both developed and developing countries. The need to extend the duration of immunization-​induced pro- tection will lead to further refinement of immunization schedules and of pertussis vaccines. Multicentre randomized clinical trials are necessary to provide the evidence base for the intensive care man- agement of life-​threatening pertussis. FURTHER READING Black RE, et al. (2010). Global, regional, and national causes of child mortality in 2008:  a systematic analysis. Lancet, 375, 1969–​87. Cherry JD (2014). Adult pertussis in the pre-​ and post-​vaccine eras: lifelong vaccine-​induced immunity? Expert Rev Vaccines, 13, 1073–​80. Crowcroft NS, Pebody RG (2006). Recent developments in pertussis. Lancet, 367, 1926–​36. Hewlett EL, et al. (2014). Pertussis pathogenesis—​what we know and what we don’t know. J Infect Dis, 209, 982–​5. Mattoo S, Cherry JD (2005). Molecular pathogenesis, epidemi- ology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev, 18, 326–​82. Mooi FR, Van Der Maas NA, De Melker HE (2014). Pertussis resur- gence: waning immunity and pathogen adaptation—​two sides of the same coin. Epidemiol Infect, 142, 685–​94. Moore A, et al. (2017). Clinical characteristics of pertussis-associated cough: a diagnostic systematic review and meta-analysis. Chest, 152, 353–67. Munoz FM, et  al. (2014). Safety and immunogenicity of tetanus diphtheria and acellular pertussis (Tdap) immunization during pregnancy in mothers and infants: a randomized clinical trial. JAMA, 311, 1760–9. Tiwari T, Murphy TV, Moran J, National Immunization Program CDC (2005). Recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: 2005 CDC Guidelines. Morbidity & Mortality Weekly Report 2005, Recommendations & Reports, 54, 1–​16. Yeung KHT, et al. (2017). An update of the global burden of pertussis in children younger than 5 years: a modelling study. Lancet Inf Dis, 17, 974–80. 8.6.16  Melioidosis and glanders Sharon J. Peacock ESSENTIALS Melioidosis is a serious infection caused by the soil-​dwelling Gram-​negative bacillus Burkholderia pseudomallei. It is most com- monly reported in north-​east Thailand and northern Australia, but is increasingly recognized around the world. Infection is predom- inantly acquired through bacterial inoculation, often related to oc- cupation, and mostly affects adults betweens the fourth and sixth decade who have risk factors such as diabetes mellitus and renal impairment. Clinical features—​these are very varied, ranging from a septicaemic illness (the most common presentation), often associated with con- comitant pneumonia (50%) and other features including hepatic and splenic abscesses, to a chronic illness characterized by fever, weight loss, and wasting. Case fatality is 40% in north-​east Thailand (20–​30% in children) and 14% in Australia. Diagnosis and treatment—​diagnosis requires culture of B. pseudomallei (a hazard group 3 biological agent) from any spe- cimen. Serological tests should be used with caution in those with suspected melioidosis who are culture-​negative. Aside from sup- portive care and drainage of collections of pus, prolonged anti- microbial therapy is required, with a parenteral phase of 10 to 14 days (ceftazidime or a carbapenem) followed by oral therapy for 12 to 20 weeks (trimethoprim-​sulfamethoxazole). B. pseudomallei is difficult to eradicate and recurrence occurs in 6% of cases within the first year. Glanders—​this resembles melioidosis and is caused by Burkholderia mallei, which appears to have evolved from a single clone of B. pseudomallei.

8.6.16  Melioidosis and glanders 1077 Genetics and pathogenesis The Burkholderia pseudomallei K96243 genome is composed of two chromosomes of 4.07 Mbp and 3.17 Mbp which show func- tional partitioning of genes. The large chromosome encodes many of the core functions associated with metabolism and growth, while the smaller chromosome carries more accessory functions associated with adaptation and survival in different environments. At least 6% of the genome is made up of puta- tive genomic islands that have probably been acquired via hori- zontal gene transfer. Findings from whole genome sequencing of B. pseudomallei are indicative of a high rate of genetic recombin- ation, and consistent with Australia as an early reservoir for the current global B. pseudomallei population with transmission to Southeast Asia, followed by onward transmission to South Asia and East Asia. Experimental studies indicate a role in virulence for lipopoly- saccharide, capsular polysaccharide (CPS), flagella, hemolysin- coregulation protein 1 (Hcp1), a type VI secretion system, and a type III secretion system (TTSS3) that shares homology with the inv/​spa/​prg TTSS of Salmonella typhimurium and the ipa/​mxi/​ spa TTSS of Shigella flexneri. Other candidate virulence factors include a siderophore for iron acquisition, and secreted proteins such as haemolysin, lipases, and proteases. Data from in vitro models and post-​mortem studies indicate that B.  pseudomallei is equipped for intracellular survival. The organism survives and replicates within neutrophils and monocytes, and employs mul- tiple mechanisms to escape macrophage killing and evade host immunity. Epidemiology, aetiology, and prevention The first reported case of melioidosis occurred in a 40-​year-​old morphine addict in Rangoon (Yangon), Myanmar in 1911. The incidence of recognized cases is highest in north-​east Thailand and northern Australia, but melioidosis is also known to occur in nu- merous countries across South and East Asia, in Central America, Ecuador, and Brazil, and in several countries in Africa. The route of infection is most commonly via skin inoculation or bacterial contamination of wounds, but other routes include ingestion, in- halation, and aspiration including near drowning. Activities of daily living associated with disease acquisition include exposure to soil or water, an open wound, eating food contaminated with soil or dust, drinking untreated water, and outdoor exposure to rain. Melioidosis incidence peaks between the fourth and sixth decades; children represent one-​sixth of infected individuals in north-​east Thailand. Diabetes mellitus, excess alcohol consump- tion, smoking, steroid intake, chronic renal failure, and chronic lung disease are independent risk factors. One or more risk fac- tors are present in approximately 80% of affected adults but only 30% of children (most commonly penetrating injury). Most cases in Thailand occur in rice farmers who work without protective footwear. Avoidance of contact with the environment in which B.  pseudomallei exists is likely to prove an effective preventive measure, but such strategies are not in place across rural Asia. Clinical features, differential diagnosis, and criteria for diagnosis The period between B. pseudomallei exposure and onset of clin- ical manifestations is difficult to define since most patients do not report a specific inoculation event. An incubation period of 1 to 21 days (mean 9 days) was determined for 25% of cases in Australia with a specific inoculation event, but this may not be representa- tive for cases overall. The longest recorded incubation period is 29 years. Time from onset of disease to clinical presentation is also variable; in north-​east Thailand, approximately one-​third of pa- tients have symptoms for less than 7 days, one-​half for 7 to 28 days, and the remainder have symptoms for more than 28 days. Manifestations range from a fulminant sepsis and rapid death to a chronic illness characterized by fever, weight loss, and wasting. The most frequent clinical picture is a septicaemic illness, often associ- ated with bacterial dissemination to distant sites such that concomi- tant pneumonia (Fig. 8.6.16.1) and hepatic and splenic abscesses are common. Pneumonia occurs in around 50% of patients. Infection can also occur in bone, joints, skin (superficial pustules and cuta- neous abscesses, Fig. 8.6.16.2), soft tissue (pyomyositis), testis, and prostate. A specific syndrome of meningoencephalitis with brain stem involvement and risk of respiratory arrest, flaccid paraparesis, or peripheral motor weakness occurs in 4% of cases in northern Australia. Central nervous system infections occur in around 1.5% of melioidosis patients in Thailand (Fig. 8.6.16.3), although menin- goencephalitis is not recognized in this setting. Involvement of the vascular tree is recognized but unusual. Acute parotitis accounts for one-​third of childhood cases in Thailand but is unusual in adult- hood. The number of sites involved is variable and possible combin- ations include positive blood cultures but no other focus, positive blood cultures and one or more distant foci, and negative blood cul- tures with one or more foci. Classification of patients into different categories based on these observations has been suggested, but it may be more accurate to consider disease as a continuum. A high index of suspicion is required in order to diagnose melioidosis in the non​endemic setting. Clinicians should con- sider the possibility in patients with a fever who have one or more of the following: (1) residency at any time in an endemic region or a relevant travel history; (2) an occupation or other pursuits that may have resulted in contact with soil or water containing B. pseudomallei (including military personnel who are on exer- cise or active service); and (3) the presence of risk factors such as diabetes mellitus or renal disease. The variability in clinical features is such that it is often impossible on clinical grounds to differentiate between melioidosis and other acute and chronic bacterial infections, including tuberculosis. Confirmation of the diagnosis relies on good practices for specimen collection, la- boratory culture, and identification of B. pseudomallei. Clinical investigation and confirmation of diagnosis Early discussion with the clinical microbiology laboratory is im- portant during investigation of suspected cases. This will raise

section 8  Infectious diseases 1078 awareness for the presence of a significant pathogen in a mixed culture. In addition, B. pseudomallei is classified as a hazard group 3 biological agent and safe handling requires use of a containment level 3 laboratory. Samples of blood, urine, throat swab, and re- spiratory secretions should be obtained for culture from all pa- tients, together with pus and wound swabs where relevant. All sample types should be taken where possible since site of culture positivity may not necessarily relate to clinical focus of infection (as an extreme example, it is possible for a throat swab to be positive (a) (b) Fig. 8.6.16.1  Chest radiographs of two patients with melioidosis. (a) Left upper lobe involvement with abscess formation. (b) Diffuse pulmonary involvement with marked radiological changes in the right lung field. (a) (b) Fig. 8.6.16.2  Skin and soft tissue involvement in two patients with melioidosis. Skin pustules (a) and subcutaneous abscess (b) occurring as secondary foci of infection associated with disseminated infection. Fig. 8.6.16.3  CT brain scan of a patient presenting with fever, headache, confusion, and hemiparesis. The image shows a ring-​enhancing lesion with surrounding oedema in the right frontoparietal lobe, pus from which grew B. pseudomallei.

8.6.16  Melioidosis and glanders 1079 in a patient with a splenic abscess in the absence of features of re- spiratory infection). B. pseudomallei colonization is extremely rare and isolation of even a single colony from a low-​quality sample can clinch the diagnosis. Bacterial detection and identification using the polymerase chain reaction on clinical specimens is described but is not available in routine microbiology laboratories, and is re- ported to be less sensitive than culture. Negative microbiological cultures do not rule out melioidosis since patients who have been commenced on effective antimicro- bial agents may be culture-​negative. Serodiagnostic tests should be considered for the investigation of persons with suspected meli- oidosis who are culture-​negative, but should be interpreted with caution. A rising antibody titre to B. pseudomallei in paired serum samples taken 2 weeks or more apart in an individual who does not normally reside in an area where melioidosis is endemic is highly supportive of the diagnosis of melioidosis in the presence of clin- ical features of disease. This ideal is often difficult to achieve since the potential exposure event may have occurred months or years before presentation and may not be remembered. In such cases, a single high antibody titre at presentation is indicative of exposure. Serodiagnostic tests in people who have resided in areas where melioidosis is endemic have very limited value since background seropositivity in the healthy population is high and the detection of antibodies to B. pseudomallei has a low diagnostic accuracy for active melioidosis. A small number of patients with culture-​proven melioidosis do not mount a detectable antibody response, and a negative serological result does not rule out exposure or active in- fection. The most commonly used serodiagnostic method is the in- direct haemagglutination assay (IHA). Cut-​off points ranging from an IHA titre of 1:20 to 1:80 have been used to indicate exposure. In patients with melioidosis, laboratory tests should be em- ployed to detect acute renal failure, abnormal liver function tests, and anaemia, all of which are well recognized during severe meli- oidosis. Arterial blood gases should be taken in patients with lung involvement and/​or any evidence of respiratory impairment. Serum C-​reactive protein levels do not give an accurate reflection of dis- ease severity. Chest radiographs should be obtained in all patients. Features are highly variable and include focal, multifocal, or lobar consolidation, localized patchy alveolar infiltrate, diffuse interstitial shadowing (consistent with blood-​borne spread of infection), pleural effusion, and upper lobe involvement which may include cavitation. The radiographic pattern may be indistinguishable from tubercu- losis. The development of empyema and/​or lung abscess(es) is well recognized, and repeat chest radiographs are indicated for patients with respiratory involvement. Abdominal ultrasound examination or computed tomography (CT) scan should be performed to ex- clude the presence of abscesses in liver and spleen. Clinical evidence of prostatic involvement requires appropriate imaging (transrectal ultrasonography or CT scan). The need for other imaging should be guided by clinical features and organ involvement. Management, prognosis, and outcome Appropriate antimicrobial agents should be started immediately on suspicion of the diagnosis of melioidosis. Recommendations are given in Box 8.6.16.1. Treatment is divided into intravenous and oral phases. Initial parenteral therapy is given for 10 to 14 days or until clinical response is seen (whichever is the longer). Ceftazidime or a carbapenem antibiotic is the treatment of choice. Ceftazidime is used as first-​line therapy in Thailand, with a switch to a carbapenem antibiotic in the event of treatment failure on ceftazidime. Parenteral treatment at the Royal Darwin Hospital, Australia, consists of ceftazidime or meropenem plus granulo- cyte colony stimulating factor (G-​CSF) if the patient has septic shock. The routine addition of trimethoprim/​sulfamethoxazole (TMP-​SMX) to ceftazidime or meropenem during the initial in- tensive therapy phase has been discontinued, although this drug is still used in some centres for patients with neurological or pros- tatic melioidosis in view of its excellent penetration. Intravenous amoxicillin/​clavulanate is second-​line therapy but is associated with higher rates of treatment failure and there are few indica- tions for this agent if first-​line agents are available. Oral treatment is given for 12 to 20 weeks or longer if clinically indicated and consists of TMP-​SMX. The routine addition of doxycycline to oral regimens has ceased following the outcome of a randomized controlled trial conducted in Thailand, which found equivalence between TMP-​SMX alone and TMP-​SMX plus doxycycline. First-​ line oral treatment for pregnant women and children is amoxi- cillin/​clavulanate; this is also an alternative for adults who cannot tolerate TMP-​SMX. Collections of pus should be drained wherever feasible. Patients with severe melioidosis associated with septic shock, respiratory failure, acute renal failure, and other manifestations of a severe septic illness require intensive care management, Box 8.6.16.1  Antimicrobial therapy for melioidosis Initial parenteral therapy • Ceftazidime 50 mg/​kg per dose (up to 2 g) every 6 h, or meropenem 25 mg/​kg per dose (up to 1 g) every 8 h. • Intravenous amoxicillin/​clavulanate can be used as a second-​line agent and is associated with equivalent mortality but a higher rate of treatment failure compared with ceftazidime. Dosage 20/​5 mg/​kg every 4 h. • Duration of parenteral therapy: a minimum of 10 days or until clear clinical improvement (whichever is the longer). Extend therapy to 4–​8 weeks for deep-​seated infection such as septic arthritis, osteomyelitis and central nervous system infection. Oral eradication therapy Adults • Trimethoprim/​sulfamethoxazole using a weight-​based dosing schedule: 2 × 160/​800 mg (960 mg) tablets every 12 h if more than 60 kg, 3 × 80/​400 (480 mg) tablets every 12 h if 40–​60 kg, and 1 × 160/​800 mg (960 mg) OR 2 × 80/​400 (480 mg) tablets every 12 h if less than 40 kg. Children ≤8 years and pregnant women • Amoxicillin/​clavulanate 20/​5 mg/​kg orally every 8 h. • For adult patients less than 60 kg, a dose of 1000/​250 mg three times daily is suggested. In regions where amoxicillin/​clavulanate is only avail- able in fixed 2:1 combinations, use 500/​250 mg three times daily with additional amoxicillin (500 mg three times daily). For patients more than 60 kg, use a maximum dose of 1500/​375 mg three times daily. • Duration of oral therapy: 12–​20 weeks.

section 8  Infectious diseases 1080 although many cases occur in geographical regions where such resources are scarce. Fever clearance is often slow (median fever clearance time of around 9 days), and without evidence of clinical deterioration is not normally sufficient to indicate a change in therapy. Sputum and draining abscess cultures may remain posi- tive for several weeks in a patient who is otherwise responding to treatment. The benefit of other interventions for critically ill septic patients such as goal-​directed therapy, intensive glycaemic control, and activated protein C has not been evaluated in pa- tients with melioidosis. A randomized placebo-​controlled trial of G-​CSF for severe melioidosis conducted in Thailand failed to show an outcome benefit. Several features can be used to predict risk of death. The Acute Physiology and Chronic Health Evaluation II (APACHE II) score is an independent predictor of death from melioidosis. Time to blood culture positivity has prognostic significance, with a mor- tality rate of 74% for those with a positive culture within 24 h compared with 41% in those with a positive culture after 24 h. In patients who have a positive blood culture, counts of less than one colony-​forming unit (CFU)/​ml blood have been reported to be as- sociated with a mortality of 42%, compared with a mortality of 96% in those with counts of more than 100 CFU/​ml. B. pseudomallei count in urine is also associated with mortality. Patients with meli- oidosis whose urine culture was negative for B. pseudomallei had the lowest death rate (39%). Mortality was 58% in those with posi- tive spun urine pellet only, 61% in those with between 103 CFU/​ml and 105 CFU/​ml B. pseudomallei in neat urine, and 71% in those with ≥105 CFU/​ml B. pseudomallei in neat urine. Sputum culture positive for B.  pseudomallei in patients with culture-​confirmed melioidosis is associated with a higher mortality (72%) compared with that for melioidosis patients with sputum culture-​negative for B. pseudomallei (42%). Recurrent melioidosis is not uncommon (6% in the first year and 13% over 10 years). Three-​quarters of recurrent cases are due to relapse caused by a strain that has persisted within the host following the pri- mary episode, and the remainder represent reinfection by a different strain. One-​quarter of patients with recurrence die as a direct result. The risk of nosocomial infection between patients or transmis- sion to family or other contacts has not been the subject of specific study. Several case reports have been published. Melioidosis in two infants in northern Australia was related to breastfeeding by mothers with mastitis caused by B. pseudomallei, and the wife of a Vietnam veteran with chronic prostatitis caused by B. pseudo- mallei developed an antibody response to the organism in the ab- sence of clinical manifestations of melioidosis. Person-​to-​person transmission occurred between two siblings with cystic fibrosis and may have occurred between a diabetic brother and sister living in north-​east Thailand, and a case of nosocomial infection from a suspected environmental source has been reported from an endemic area. Likely developments in the near future The overall incidence of melioidosis is likely to rise among wealthier nations within Asia as the number of susceptible elderly people increases. The number of reported cases worldwide is also likely to increase alongside the dissemination of diagnostic la- boratories. Probably the most important strategy required to reduce mortality from melioidosis in rural Asia is early recogni- tion and timely administration of antimicrobial drugs together with adequate fluid resuscitation. Further studies are required to define safe and affordable interventions that improve outcome where intensive care facilities are unavailable, such as protocols to optimize fluid management and glycaemic control in a general ward setting. Overview of glanders Burkholderia mallei, the cause of glanders, appears to have evolved through genomic downsizing from a single clone of B.  pseudomallei. Historically, this pathogen was an important cause of morbidity and mortality in horses worldwide and was occasionally transmitted to humans or other animals. In horses, donkeys, and mules it causes nodules and ulcerations in the upper respiratory tract and lungs. The cutaneous form is known as ‘farcy’. The mallein skin test is a sensitive and specific clinical test for equine glanders. No naturally acquired case has been reported in the United States of America or the United Kingdom since 1938, but it is thought to still occur in the Middle East, Africa, and Asia. Outbreaks of equine glanders reported in Bahrain since 2010 and in Lebanon since 2011 may be linked to importation of horses from elsewhere in the region, including Syria. Clinical manifest- ations of glanders in humans resemble those of melioidosis. The untreated case fatality rate is 95%. The approach to investigation, diagnosis, and management is as for melioidosis. The organism requires handling in a containment level 3 laboratory; important differentiating bacterial features between B. mallei and B. pseu- domallei are that the former is non​motile and susceptible to gen- tamicin. In vitro susceptibility is otherwise similar to that for B.  pseudomallei, and glanders should respond to the regimens used to treat melioidosis. FURTHER READING Cheng AC, et al. (2008). Consensus guidelines for dosing of amoxicillin-​ clavulanate in melioidosis. Am J Trop Med Hyg, 78, 208–​9. Chetchotisakd P, et al. (2014). Trimethoprim-​sulphamethoxazole versus trimethoprim-​sulphamethoxazole plus doxycycline as oral eradicative treatment for melioidosis (MERTH):  a multicentre, double-​blind, non-​inferiority, randomised controlled trial. Lancet, 383, 807–​14. Chierakul W, et  al. (2005). Two randomized controlled trials of ceftazidime alone versus ceftazidime in combination with trimethoprim-​sulfamethoxazole for the treatment of severe melioid- osis. Clin Infect Dis, 41, 1105–​13. Currie BJ, et al. (2010). The epidemiology and clinical spectrum of melioidosis: 540 cases from the 20-​year Darwin prospective study. PLoS Negl Trop Dis, 4, e900. Wiersinga WJ, et  al. (2018). Melioidosis. Nature Disease Primers Reviews, 4, 17107.

8.6.17 Plague Yersinia pestis 1081

8.6.17 Plague: Yersinia pestis 1081

1081 8.6.17  Plague: Yersinia pestis Michael Prentice ESSENTIALS Bubonic plague is a flea-​borne zoonosis caused by the Gram-​negative bacterium Yersinia pestis, which mainly affects small burrowing mam- mals including domestic rats. Human disease occurs in endemic countries—​currently mainly in Africa (including Madagascar)—​following bites from fleas recently hosted by a bacteraemic animal. Historical use of Y. pestis as a biological warfare agent has raised fears of its future use in bioterrorism. Clinical features—​the commonest presentation is acute painful lymphadenitis (80–​95% of suspected cases), with sudden onset of fever, chills, weakness, headache, and development of an intensely painful swollen lymph node (bubo). Primary septicaemia with no bubo occurs in 10% of cases. Spread to the lungs occurs in less than 10% of cases, resulting in pneumonia which can result in onward re- spiratory transmission by droplet infection. Overall mortality without treatment is 50–​90%. Diagnosis and treatment—​diagnosis is usually by culture from ap- propriate specimens (blood culture, bubo aspirate, sputum, cerebro- spinal fluid), but rapid confirmation can be provided by detection of Yersinia pestis F1 antigen by immunofluorescence or dipstick in clin- ical material. Aside from supportive care, early antimicrobial therapy (gentamicin, doxycycline, ciprofloxacin, levofloxacin, moxifloxacin, or streptomycin) greatly improves survival. Prevention—​is by reducing the likelihood of people being bitten by infected fleas, or being exposed to infected droplets from humans or animals with plague pneumonia. Postexposure chemoprophylaxis may be advised for those who have been in unprotected close con- tact with a person with pneumonic plague. There is no current com- mercially available vaccine. Introduction Alexandre Yersin isolated the bacterium now known as Yersinia pestis in 1894 from a patient with bubonic plague in Hong Kong, during a plague pandemic when disease spread to ports all over the world from a focus in China. Most mortality in this pandemic was seen in India and China in the late 19th and early 20th cen- tury when millions died. Experimental work in India in the early years of the 20th century confirmed the flea–​rat cycle of trans- mission, allowing rational control measures to be developed. This pandemic is called the third plague pandemic because of a retrospective association of bubonic plague with two historical disease pandemics. The second pandemic was the Black Death, which killed one-​third of the European population between 1347 and 1352. The first plague pandemic refers to an outbreak which began in the reign of the Roman Emperor Justinian in the 6th century AD. Ancient DNA studies have confirmed these histor- ically recorded pandemics were caused by Y. pestis strains closely resembling, but ancestral to, current pandemic strains. They have also shown Y. pestis was a common cause of septicaemic death across Europe and Asia over 5000 years ago in the Bronze Age, but was not at that time a primarily flea-​transmitted pathogen. Studies combining geolocation data with phylogenetic trees of current and historical Y. pestis strains (Phylogeography) suggests plague emerged in the Qinghai-​Tibet Plateau of China adjacent to intersecting ancient trade routes which later spread the disease. Aetiology Y. pestis strains form a clonal group within Y. pseudotuberculosis, an enteric pathogen of mammals spread by the faeco-​oral route (this has implications for laboratory identification, see next; see also Other Yersinia, Chapter 8.6.18). These are Gram-​negative bac- teria within the order Enterobacterales. Ancient DNA sequences (paleogenomics) and current pathogenicity studies have mapped a plausible stepwise evolutionary pathway for an ancestral Y.  pseudotuberculosis-​like organism to acquire a plague-​causing phenotype. The starting point is that Y.  pseudotuberculosis and Y. pestis both contain a virulence plasmid pYV which is essential to cause disease, and a very similar chromosome. Subsequent steps to respiratory and flea-​bite transmission include gene acquisition in the form of two plasmids not present in Y. pseudotuberculosis, pPst (pPCP1) and pFra (pMT1), and then a handful of key inactivating mutations in Y. pseudotuberculosis genes among the many pseudo- genes identified in current Y. pestis strains. Functional predictions from Y. pestis genome sequences obtained from Bronze Age human remains suggest the earliest strains dating from 1700 to 2900 BC were capable of causing pneumonic and septicaemic plague, but could not cause bubonic plague. Strains capable of causing bubonic plague efficiently transmitted by flea vectors emerged sometime between the first and second century BC. Epidemiology Between 1 January 2010 and 31 December 2015, 3248 cases of plague in humans were reported to the World Health Organization, resulting in 584 deaths (18%), a decreasing incidence compared with previous years. The six countries reporting most of the cases over this period (accounting for over 99% of the total), were, in descending order: Madagascar, Democratic Republic of the Congo, Uganda, Peru, Tanzania, and the United States. Notably, the very large enzootic focus covering the western United States contrib- uted only 39 human cases (five fatalities) over this period. Ninety-​ six per cent (96%) of cases were reported from Africa (including Madagascar). In 2017 Madagascar experienced a large outbreak of 2300 cases; 76% pneumonic, 8.6% fatal. The plague is seasonal in most endemic countries, with a well-​defined geographical distribution correlated with that of Acknowledgement: The author gratefully acknowledges the substantial contribu- tion to this chapter made by Dr Tom Butler based on previous editions. 8.6.17  Plague: Yersinia pestis

section 8  Infectious diseases 1082 the predominant flea vectors and rodent reservoirs, and their ecology. Most cases in the United States occur from May to October, when people are outdoors in contact with rodents and their fleas. Plague is a zoonosis with humans figuring as an incidental host. It is transmitted among animal reservoirs by flea bites and ingestion of animal tissues. The fleas of many major animal reservoirs such as burrowing rodents including ground squirrels and prairie dogs in the United States and tarbagans in Asia, can only contact humans in rural areas. Human infection is more frequent when disease occurs in small mammals in closer contact with humans, particularly urban and domestic rats. The oriental rat flea Xenopsylla cheopis is the most efficient vector. Risk factors for acquiring plague include contact with rodents or carnivores in endemic areas and presence of ref- uges or food sources for wild rodents near homes. Human to human transmission of pneumonic plague is limited to rare outbreaks in endemic areas. Fatal plague pharyngitis has been reported following consumption of raw camel meat in endemic areas. Although there are no reports of the use of Y. pestis as a biological weapon since World War II, the possibility of bioterrorism would nowadays be in- vestigated if any case of plague, particularly pneumonic plague, was diagnosed in a non​endemic area (e.g. Europe, Eastern United States of America). Pathogenesis/​Pathology In the arthropod-​parasitizing portion of its life cycle, Y. pestis multiplies and forms biofilm-​embedded aggregates in the flea midgut and foregut after ingestion of a blood meal containing bacteria from a mammalian host. Blocked fleas die, but make persistent efforts to feed, regurgitating oesophageal contents and inoculating Y. pestis into each bite site. Some fleas might be long-​ lived successful vectors without blockage. The ability to colonize and multiply in the flea midgut requires the phospholipase D activity encoded by the ymt gene on the pMT1 (pFra) plasmid. Experiments trying to achieve Y.  pseudotuberculosis transmis- sion by fleas have shown that as well as adding a ymt gene, mu- tation of a small number of chromosomal genes which are intact in Y. pseudotuberculosis is required to prevent rapid flea death on infection (caused by urease activity) and promote biofilm formation. These mutations are present in all current Y. pestis strains. However, the ymt gene and most of these chromosomal mutations are absent from early Bronze Age Y. pestis sequences. This suggests plague in humans began as a disease spread by the respiratory or oral route, and efficient flea transmission, which is currently the most common mode of transmission, is a more recent development. Several key features of mammalian Y. pestis infection affecting transmissibility require a plasminogen activator protease expressed on its surface, encoded by the pla gene on the small pPst (pPCP1) plasmid. Pla is essential for Y. pestis to cause primary pneumonic plague, and the pla gene is present in the earliest Y. pestis sequences from Bronze Age human remains. It is also required after intra- dermal inoculation for multiplication in lymph nodes (bubo forma- tion) and enhances systemic spread as bacteraemia following bubo formation. There is evidence of recent evolution of Pla facilitating Y. pestis transmission by fleas. All current pandemic Y. pestis strains contain the same point mutation in pla when compared to current non​pandemic Y. pestis strains which are closer to Y. pseudotuber- culosis in phylogeny, and the early Bronze Age Y. pestis sequences. This mutation optimizes protease activity in vitro, but both sequence variants confer the ability to cause primary pneumonic plague on Y. pestis when tested in animal models. However, the current pan- demic Y. pestis strain variant of Pla increases bacteraemia following primary pneumonic plague or subcutaneous inoculation compared to the ancestral form. Maintenance of flea transmission requires extreme virulence in the mammalian host. Because of the small volume of blood in a flea meal and a large minimum infectious dose for the flea, a very high level of bacteraemia (108/​ml) is required in the mammalian host to infect fleas. Few bacteria are transmitted by a flea bite and the organism has a low minimum infectious dose for mammals. In the most common current form of plague, bubonic plague, flea bites inoculate Y. pestis at approximately 26ºC. Y. pestis travels inside macrophages to the regional lymph nodes from the site of inocula- tion before switching to extracellular replication in growing necrotic foci containing large numbers of bacteria. This results in a swollen and painful lymph node termed a bubo. Extracellular survival re- quires expression of a type III secretion system (injectisome) encoded by the Yersinia virulence plasmid pCD/​pYV to inject viru- lence effectors (Yop proteins) into mammalian host immune effector cells. This forestalls the usual immune response, preventing phago- cytosis. The injectisome component LcrV (V antigen) also has an extracellular anti-​inflammatory activity, preventing recruitment of inflammatory cells and granuloma formation which would nor- mally terminate an infection. An antiphagocytic polypeptide cap- sule (fraction 1 or F1 antigen), specified by the caf1 gene on the pFra/​pMT1 plasmid, may also be important in mammalian patho- genesis in some host species. Primary pneumonic plague occurs following inhalation of in- fected respiratory droplets from another person or animal. In con- trast to bacteria inoculated by flea bite, bacteria inoculated in this way are already growing at 37ºC. Several important Y. pestis viru- lence factors are highly expressed at 37ºC but not 26ºC, so the pathogen is pre-​armed when transmitted by this route. Animal models suggest that primary pneumonic plague is a biphasic illness with extensive manipulation of the host intrinsic immune response by Y. pestis. In a mouse model, during an initial preinflammatory phase lasting about 36 hours, extensive bacterial replication occurs in the lungs with little inflammatory response due to targeting of al- veolar macrophages by type III secretion effectors and effects of Pla on neutrophil recruitment. After 36–​48 hours, a proinflammatory phase begins with upregulation of proinflammatory cytokines and influx of neutrophils to the alveolar spaces. Pla is required for this switch to inflammation to occur, but the precise mechanism is un- known. Y. pestis can resist neutrophil-​mediated killing with its type III secretion system and bacterial numbers remain high. By 72 hours after infection the continuous recruitment of neutrophils to the lungs in response to persistent replicating bacteria results in severe inflammatory damage to alveolae, with oedema and haemorrhage. The damage is thought to be caused by neutrophil-​associated highly

1083 8.6.17  Plague: Yersinia pestis reactive oxygen species and proteases. The frequently fatal necrotic pneumonia of primary pneumonic plague is therefore primarily host-​response-​mediated. Clinical features The most common presentation is acute painful lymphadenitis (80–​95% of suspected cases). There is sudden onset of fever, chills, weakness, and headache. At the same time, or shortly afterwards, patients notice the bubo, which is signalled by intense pain in one anatomical region of lymph nodes, usually the groin, axilla, or neck (Fig. 8.6.17.1). The swelling is so tender that patients avoid any mo- tion that might provoke discomfort. If the bubo is in the femoral area, the patient will flex, abduct, and externally rotate the hip to relieve pressure on that area, and will walk with a limp. With an ax- illary bubo, the patient will abduct the shoulder or hold the arm in a splint. When the bubo is in the neck, patients will tilt their neck to the opposite side. Buboes are oval swellings varying from 1 to 10 cm in length and elevate the overlying skin, which might appear stretched or ery- thematous. They can be a single smooth uniform mass, or an ir- regular cluster of several nodes with intervening and surrounding oedema. Overlying skin is warm with an underlying tender, firm non​fluctuant mass. Patients are typically prostrate and lethargic, but can show restlessness or agitation. Occasionally, they are delirious with fever, and seizures are common in children. Fever of 38.5–​40°C is usual, with pulse of 110–​140/​min. Blood pressure is characteristic- ally low, 100/​60 mm/​Hg and may be unobtainable if systemic sepsis syndrome occurs as a consequence of the host response to large amounts of circulating bacterial endotoxin. As part of this response, disseminated intravascular coagulopathy can occur involving arteri- olar thrombosis, skin and serosal haemorrhage, acral cyanosis, and tissue necrosis, as well as multiple organ failure and adult respira- tory distress syndrome. A minority of patients (10–​20%) develop systemic Y. pestis sepsis with no bubo (primary septicaemic plague) and less than 10% develop secondary pneumonic plague or menin- gitis as a consequence of bacteraemia. Prevention Plague prevention measures seek to reduce the likelihood of people being bitten by infected fleas, or exposed to infected droplets from humans or animals with plague pneumonia. In plague-​endemic areas, monitoring and control of the local plague hosts is important, as well as rat-​proofing and insecticide treatment of houses, and wearing shoes and garments to cover the legs. Because removing the flea food supply by poisoning their normal hosts can increase human contact with starving fleas, flea control by application of insecticides prior to vector control in plague outbreak areas is required. Infection control meas- ures for patients with suspected pneumonic plague centre on respira- tory isolation with droplet precautions (wearing of disposable masks by medical attendants to reduce the risk from large respiratory drop- lets) until they have received antibiotic treatment for 48 hours. For potential aerosol generating procedures (eliciting respiratory samples from suspected plague patients) WHO now recommends personal protective equipment (PPE) such as an N95 face mask, gown, gloves and face shield or goggles should be worn. Postexposure chemo- prophylaxis is advised for persons who have been in unprotected close contact (defined as coming within 2 m) with a person with pneu- monic plague who has not received antibiotic treatment for at least 48 h. Doxycycline, ciprofloxacin, levofloxacin, chloramphenicol, or cotrimoxazole can be used as prophylaxis. Standard isolation precau- tions are recommended for non​pneumonic plague patients. There is no currently commercially available plague vaccine in the Western world. A live attenuated vaccine based on a former French vaccine strain Yersinia pestis EV is licensed in China, Russia, and some other former Soviet Union countries to protect against bubonic and pneumonic plague when administered by par- enteral or oral routes. A large variety of candidate plague vaccines are under development. Subunit vaccines, mostly based on com- binations of mixed or fused immunogenic plasmid-​specified pro- tein antigens LcrV and Fraction 1, which in some animal models protect against pneumonic challenge, administered by injection with an adjuvant, have been brought through Phase II trials in dif- ferent countries. T-​cell mediated immunity is important in mouse immunity to pneumonic plague and various live vaccines likely to induce cellular immune responses to Y. pestis antigens have also been constructed. Attenuated bacterial vaccines based on heter- ologous expression of antigens including LcrV and Fraction 1 in different hosts such as the established vaccine strain Salmonella enterica Serovar typhi Ty21A, attenuated Salmonella enterica Serovar typhimurium or attenuated Yersinia pseudotuberculosis have been protective in animal trials. Viral delivery platforms de- veloped for other vaccines have been used in animals for delivery Fig. 8.6.17.1  A right femoral bubo consists of an enlarged, tender lymph node with surrounding oedema. Copyright D. A. Warrell.

section 8  Infectious diseases 1084 of FI/​LcrV and other Y. pestis antigens. An oral live recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated LcrV is under field trials in the United States for efficacy in prevention of sylvatic plague in rodents. Differential diagnosis Other infections producing acute lymphadenitis (Streptococcal lymphadenitis, cat scratch fever, and so on) do not generally share the same suddenness of onset leading to death 2 to 4 days after the onset of symptoms. The plague bubo is also distinctive in the usual absence of a detectable skin lesion or ascending lymphangitis. A mi- nority of patients show various skin lesions (pustules, eschars, or papules) presumably representing the site of flea bite in the skin area draining to the bubo (Fig. 8.6.17.2). Clinical investigation The diagnosis should be suspected in febrile patients exposed to rodents or other mammals in endemic areas. Y. pestis is on a short list of pathogens to be excluded in any unexplained outbreak of se- vere respiratory disease which could follow an aerosol release by bioterrorists. Appropriate diagnostic specimens include blood culture (usu- ally positive in bubonic plague), bubo aspirate, sputum, and cere- brospinal fluid (CSF), depending on clinical presentation, and, if necessary, post-​mortem. A bubo aspirate is obtained by inserting a 10-​ml syringe with a 21-​gauge needle containing 1 ml of sterile saline, through the skin, into the bubo. The saline is injected and reaspirated until blood-​tinged fluid appeared in the syringe. Y. pestis grows on standard laboratory media and standard trans- port media preserves viability. The organism is characterized as a slow growing, non​lactose fermenting, non​motile Gram-​negative rod, first seen at 24 hours on standard laboratory media; oxidase negative, catalase posi- tive, urease negative, indole negative. It may be misidentified as Y. pseudotuberculosis or another Gram-​negative species by routine commercial identification systems, including matrix-​assisted laser desorption ionization–​time of flight mass spectrometry (MALDI-​ TOF MS). It is important to notify the laboratory if the diagnosis is clinically suspected. In the United States Yersinia pestis is a ‘Tier 1 select agent’ under bioterrorism legislation and diagnostic cultures are strictly notified and controlled. Gram stain of smears of sputum, bubo aspirate, or CSF may show small Gram-​negative rods or cocco-​bacilli, bipolar staining can be seen with Wayson or Giemsa stains (Figs. 8.6.17.3 and 8.6.17.4). Rapid diagnosis is provided by detection of Yersinia pestis F1 antigen by im- munofluorescence in clinical material. An F1-​antibody containing dipstick has been shown to be a sensitive and specific assay in field con- ditions in Madagascar on a variety of clinical specimens (sputum, bubo Fig. 8.6.17.2  Right axillary bubo was accompanied by a purulent ulcer on the abdomen, which was the presumed site of the flea bite. Copyright Tom Butler. Fig. 8.6.17.3  Bubo aspirate shows bipolar bacilli stained with methylene blue (Wayson’s Stain). Copyright Tom Butler. Fig. 8.6.17.4  Gram stain of spinal fluid in plague meningitis shows numerous Gram-​negative bacilli. Copyright Tom Butler.

1085 aspirate, CSF). Current trials of this dipstick in Africa are ongoing. Polymerase chain reaction assays for various targets and an enzyme-​ linked immunosorbent assay (ELISA) for Yersinia pestis LcrV antigen have also been developed, but are not in routine clinical use. Criteria for diagnosis Culture of the organism, F1 antigen detection, or seroconversion (a fourfold or greater titre change) to Y. pestis F1 antigen by passive haemagglutination testing of paired serum specimens (PHA test) are all criteria for diagnosis. Specificity of the PHA test requires confirmation with the F1 antigen haemagglutination-​inhibition test. Seroconversion can occur 5 days after onset of symptoms, but is more usual between 1 and 2 weeks after onset. Treatment Streptomycin is traditionally regarded as the most effective treatment for plague at a dose of 1 g twice daily (30 mg/​kg/​per day) for 10 days, and was the first antimicrobial shown to be effective against pneu- monic plague. The more readily available aminoglycoside gentamicin is as effective as streptomycin in the treatment of human plague, when given at standard doses for severe sepsis. Seven-​day courses of intramuscular gentamicin 2.5 mg/​kg 12 hourly or oral doxycycline therapy 100 mg (adults) and 2.2 mg/​kg (children) orally 12 hourly are highly effective in adults and children with bubonic, septicaemic or pneumonic plague (tetracyclines are contraindicated in preg- nancy, breastfeeding, and children younger than 7 because of tooth discolouration). In a mouse septicaemia model, third-​generation cephalosporins and quinolones were as effective as streptomycin and tetracycline. In a mouse model of pneumonic plague, β-​lactam anti- biotics were less effective than aminoglycosides and quinolones. Oral chloramphenicol is recommended for plague meningitis at a loading dose of 25  mg/​kg followed by 60  mg/​kg per day in four divided doses, reducing to 30 mg/​kg per day orally on clinical improvement to complete a total course of 10 days. General therapeutic measures for systemic bacterial sepsis, including intravenous fluids, are appro- priate but no available trial data for the use of these in plague is avail- able. A consensus view of treatment for pneumonic plague resulting from biological weapon attack suggests streptomycin, gentamicin, tetracycline, or fluoroquinolones can be effective. The quinolones ciprofloxacin, moxifloxacin and levofloxacin have been approved by the Food and Drug Administration (FDA) for pneumonic and septicaemic plague treatment based on efficacy in a primate animal model, and partly because they also show efficacy against other po- tential bioterrorism organisms such as Bacillus anthracis. Although still very rare, natural antimicrobial resistance has been detected. A  wild-​type Y.  pestis strain resistant to multiple antimicrobials was first reported from Madagascar in 1997, and subsequently a different strain resistant to the first-​line antibiotic streptomycin was also identified. Worryingly, both plasmids respon- sible for these resistance patterns were self-​transferrable to other bacteria. Fortunately, no other Y. pestis strains with multiple anti- microbial resistance have subsequently been isolated in Madagascar. A non​pandemic strain of Y. pestis resistant to several antimicrobials was isolated from an animal in Mongolia. Prognosis Untreated bubonic plague has a mortality of 50–​90% and un- treated meningitis, pneumonia or septicaemia is fatal in most cases. Diagnosis and appropriate therapy reduces bubonic plague and septicaemia mortality to 5–​20% but delay in diagnosis and therapy can be fatal. Primary pneumonic plague mortality in the older litera- ture was reported to approach 100% untreated and be up to 50% with delayed antimicrobial therapy. However in the 2017 Madagascar pneumonic plague outbreak, the WHO reported overall case fatality rates of under 10%. Areas of uncertainty or controversy Although several FI-​LcrV subunit vaccines have been developed in different countries they have not been commercialized, partly be- cause of concerns about vaccine escape with F1-​negative strains and LcrV polymorphisms, and conflicting animal data on protection against aerosol challenge. Likely developments over the next 5–​10 years Novel vaccines in clinical use for animals and people might affect epidemiology. More detailed paleogenomic data on the Y. pestis evo- lution timeline may be available. Manipulation of the host response in animal models might suggest a strategy to reduce primary pneu- monic Y. pestis mortality. FURTHER READING Achtman M, et al. (1999). Yersinia pestis, the cause of plague, is a re- cently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A, 96, 14043–​8. Dennis DT, et al. (1999). Plague manual: epidemiology, distribution, surveillance and control. World Health Organization, Geneva. http://​ www.who.int/​csr/​resources/​publications/​plague/​WHO_​CDS_​ CSR_​EDC_​99_​2_​EN/​en/​ Hinnebusch BJ, Chouikha I, Sun YC (2016). Ecological opportunity, evolution, and the emergence of flea-​borne plague. Infect Immun, 84, 1932–​40. Kool JL (2005). Risk of person-​to-​person transmission of pneumonic plague. Clin Infect Dis, 40, 1166–​72. Mead PS (2018). Plague in Madagascar—a tragic opportunity for improving public health. N Eng J Med, 378, 106–8. Mwengee W, et al. (2006). Treatment of plague with gentamicin or doxycycline in a randomized clinical trial in Tanzania. Clin Infect Dis, 42, 614–​21. Pechous RD, et  al. (2016). Pneumonic plague:  the darker side of Yersinia pestis. Trends Microbiol, 24, 190–​7. Rasmussen S, et al. (2015). Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. Cell, 163, 571–​82. Riddell SW, Kiska DL, Mahlen S (2016). Sentinel level clinical lab- oratory guidelines for suspected agents of bioterrorism and emerg- ing infectious diseases:  Yersinia pestis. American Society for Microbiology, Washington, DC. https://​www.asm.org/​images/​ PSAB/​LRN/​Ypestis316.pdf 8.6.17  Plague: Yersinia pestis

8.6.18 Other Yersinia infections Yersiniosis 1086

8.6.18 Other Yersinia infections: Yersiniosis 1086

section 8  Infectious diseases 1086 8.6.18  Other Yersinia infections: Yersiniosis Michael Prentice ESSENTIALS Yersiniosis is caused by the enteropathogenic Gram-​negative organ- isms Yersinia enterocolitica and Yersinia pseudotuberculosis, which are worldwide zoonotic pathogens. Disease is acquired by consumption of contaminated food or water and is commonest in childhood, and in colder climates. Presentation is with diarrhoea, fever, and abdom- inal pain, which may mimic appendicitis. Late complications include reactive arthritis, erythema nodosum, and erythema multiforme. Systemic infection is more likely with Y. pseudotuberculosis and a sub- group of Y. enterocolitica, and also in patients with diabetes or iron overload. Diagnosis is by culture of the organism or convalescent ser- ology. Most cases of enteritis are self-​limiting and antimicrobials are not indicated, but septicaemia or focal infection outside the gastro- intestinal tract requires antibiotics (usually cefotaxime, ceftriaxone, or ciprofloxacin). Prevention is by standard food hygiene precautions. Introduction and historical perspective Yersinia pseudotuberculosis was first identified in 1883 and Y. entero- colitica in 1939. Water-​borne outbreaks of Y. pseudotuberculosis were recognized in Japan and Korea from the 1920s onwards. Y. enteroco- litica was rarely reported before the 1960s and the first large-​scale outbreak of human disease was reported in 1976. Aetiology Enteropathogenic Yersinia are Gram-​negative organisms of the order Enterobacterales. Whole genome sequencing of the 18 mainly environmental species in the genus Yersinia has shown that the en- teric pathogens Y. enterocolitica and Y. pseudotuberculosis evolved independently from non​pathogens rather than from a pathogenic common ancestor. However, they are both transmitted by the oral route and share many pathogenic features. Epidemiology Both enteropathogenic yersiniae are zoonotic pathogens distrib- uted worldwide but more common in temperate and cold countries. Y. enterocolitica commonly colonizes and infects domestic animals, particularly pigs. Y. pseudotuberculosis is associated with wild mam- mals such as rodents, rabbits, deer, and birds, and human infection is more rarely diagnosed. Y. enterocolitica infection is most common in children under the age of 5 years. Virulence plasmid-​negative Y. enterocolitica strains (biovar 1A or phylogroup PG1 strains) are ubiquitous in the environment and the most common Yersinia spe- cies isolated from faeces and food other than pork in most countries. In Germany 40% of blood donors have anti-​Yersinia Yop antibodies thought to relate to Y. enterocolitica infection, and it is the third most common cause of bacterial diarrhoea in Scandinavian countries and New Zealand. Seroepidemiology and culture studies suggests human disease is at least 10-​fold rarer in the United Kingdom, although animals in the United Kingdom frequently carry the organism. In the United States of America, high virulence ‘American’ strains of Y. enterocolitica (phylogroup PG2) have been displaced by European pig-​associated strains (mainly phylogroup PG3, some PG4–​6) of lower virulence in recent years. Infection with serotype O:8, phylogroup PG2 strains, previously rare in Europe, was reported in Poland from 2006 onwards. Recent outbreaks of yersiniosis involving Y. enterocolitica have been mainly pork-​meat related; for example, children in New Zealand consuming cocktail sausages, although large raw and pasteurized milk-​related outbreaks have been reported from the United States, Japan, and Canada in the past. Recent outbreaks of Y. pseudotuberculosis have followed consumption of lettuce and raw carrot (Finland), various raw vegetables (Russia), well water (Korea and Japan), raw milk (Finland), and homogenized milk (Canada). Pathology All recognized pathogenic Yersinia contain the pYV virulence plasmid. Ingested enteropathogenic Yersinia expressing invasin proteins and adhesins, YadA and Ail, adhere to and then pass through M cells over- lying Peyer’s patches. They then multiply in lymphoid tissue, remaining extracellularly located due to the activity of the pYV plasmid-​speci- fied injectisome (type III secretion system). This inactivates phago- cytic cells, including neutrophils, by injecting Yop proteins into them. Y. enterocolitica classically causes terminal ileitis with or without adja- cent mesenteric adenitis (microabscesses inside lymph nodes), while Y.  pseudotuberculosis causes mesenteric adenitis without terminal ileitis. Some strains of Y. enterocolitica (biovar 1B, phylogroup PG2, so-​called American strains, which until recently were rarely found in Europe) and Y.  pseudotuberculosis contain a high pathogenicity island and produce an additional iron-​binding siderophore. These strains are more likely to produce systemic infection and bacteraemia. Correspondingly, patients with iron overload (polytransfused, haemo- chromatosis) are at risk of serious or fatal consequences if infected by any enteropathogenic Yersinia, especially when using iron che- lators. Invasive Y. enterocolitica and Y. pseudotuberculosis strains can also penetrate from the gut to the liver and spleen without traversal of Peyer’s patches, possibly via phagocytic cells. Some strains of Y. pseudo- tuberculosis produce a superantigenic toxin, Y. pseudotuberculosis-​de- rived mitogen (YPM). Y. enterocolitica strains produce a heat-​stable enterotoxin. Y. enterocolitica strains contain several metabolic operons found in salmonella and not present in Y. pseudotuberculosis, which may contribute to the enteritis phenotype. Y. enterocolitica biovar 1A or phylogroup PG1 strains contain some virulence-​associated genes, and are pathogenic in insect models, but are equally common in asymp- tomatic patients and their pathogenicity for humans is uncertain. Prevention Standard food hygiene precautions are effective including avoiding consumption of undercooked or raw meat (e.g. pork chitterlings),

1087 8.6.18  Other Yersinia infections: Yersiniosis especially by children, and pasteurization of milk. Chlorination of water supplies is important for Y.  pseudotuberculosis control. Yersinia grow (slowly) at refrigerator temperature, and prolonged cold storage of contaminated food or blood products can greatly in- crease their contamination. Clinical features Following an incubation period of 1 to 11 days (usually 4–​6 days), enteric Yersinia infection usually presents with diarrhoea, fever, and abdominal pain. Abdominal pain in older children and adults is often central or right-​sided, simulating appendicitis (pseudoappendicitis). Diarrhoea can be minimal or absent. Y.  enterocolitica diarrhoea contains blood in 25–​50% of cases. Infection is usually self-​limiting, but bacteraemia and systemic spread can occur with subsequent focal infection in various tissues, including mycotic aneurysm. Most patients experiencing systemic enteropathogenic Yersinia sepsis have diabetes, iron overload, or immunosuppression. Contamination of blood for transfusion with Y.  enterocolitica, presumably introduced at the time of donation and multiplying on storage, is a rare but usually fatal cause of blood transfusion reactions and systemic sepsis. Immunological complications of enteric infection are common in northern Europe where HLA-​B27 is frequent. Reactive arthritis follows several weeks after diarrhoea with other complications such as erythema nodosum, erythema multiforme, vasculitis, and glom- erulonephritis. A specific Yersinia-​associated variant of erythema multiforme has been reported from Germany with localization of eruption to the neck, shoulders, and arms, accompanied by ery- thema nodosum, conjunctivitis, and arthralgia. Y. pseudotuberculo- sis strains producing superantigenic toxin YPM are associated with Far Eastern scarlet-​like fever (FESLF) in eastern Russia, a childhood illness with desquamating rash, arthralgia, and polyarthritis also seen in Japan (Izumi fever) and Korea. There is epidemiological overlap between populations exposed to Y. pseudotuberculosis and the incidence of Kawasaki disease, an idiopathic acute systemic vas- culitis of childhood. Differential diagnosis Differential diagnosis includes appendicitis, other causes of ter- minal ileitis, mesenteric adenitis (Crohn’s disease, tuberculosis), and fever with abdominal pain. Other causes of community-​acquired septicaemia should be considered for the rarer systemic infection presentation. Clinical investigation Culture of material from normally sterile sites (blood culture, lymph nodes) is carried out on standard media. Selective cefsulodin-​ irgasan-​novobiocin agar is used for faeces and other contaminated specimens. Culture-​independent diagnosis is not yet in wide use. Standard identification of cultures to species level is possible in most laboratories, but some Y. enterocolitica strains isolated from faeces lack the virulence plasmid and their pathogenicity is uncertain. Isolation of Y. enterocolitica from a sterile site (e.g. blood) suggests pathogenicity. Reference laboratories separate Y. enterocolitica into distinct biotypes and serotypes of more or less established virulence, serotype Y. pseudotuberculosis, and provide convalescent serological tests. Standard MALDI-​TOF databases can speciate Y. enterocolitica and Y. pseudotuberculosis and a research database has successfully biotyped Yersinia species. Y. enterocolitica serotypes are broadly con- sistent with genomic phylogeny, but in Y. pseudotuberculosis sero- type is poorly predictive of genetic relatedness or pathogenicity for humans. A  pan-​Yersinia multilocus sequence typing schema has been devised which provides speciation and typing across the entire Yersinia genus. Criteria for diagnosis Diagnosis is by culture of the organism from a sterile site, bioserotyping or genetic (e.g. multilocus sequence typing) typing of faecal isolates of Y. enterocolitica into a pathogenic group, con- valescent serology by agglutinating antibodies, enzyme-​linked im- munosorbent assay (ELISA), or Western blot. Y. pseudotuberculosis is rarely isolated from faeces and serology is the usual diagnostic method. Treatment Most cases of enteritis are self-​limiting and antimicrobials are not in- dicated. Septicaemia or focal infection or scarlet-​like fever (FESLF) outside the gastrointestinal tract require antibiotics. Y. enterocolit- ica strains possess two different β-​lactamases and, in the absence of controlled trial data, therapy with cefotaxime, ceftriaxone, or ciprofloxacin are most commonly recommended for acute sepsis. Gentamicin is sometimes given in addition to β-​lactams. Y. pseudo- tuberculosis sepsis can be treated by the same agents, although this organism does not produce β-​lactamase and is generally ampicillin sensitive. Acute enteritis is usually self-​limiting. Septicaemic illness has a high mortality (up to 50%), probably associated with predisposing illnesses. In northern European countries with high HLA-​B27 prevalence, Yersinia postinfection complications, including reactive arthritis, can result in chronic illness which responds poorly to antimicrobials. Areas of uncertainty or controversy Virulent plasmid-​negative phylogroup PG1, biovar 1A Y. enteroco- litica strains might have some role in diarrhoea. Likely future developments Increased use of culture-​independent diagnosis from faeces and DNA typing of isolates (e.g. multilocus sequence typing) can speed diagnosis. Because chronic oropharyngeal colonization with Y. enterocolitica is fre- quent in apparently healthy domestic animals, such as pigs, breaking the transmission chain requires selective breeding of specific pathogen-​free

8.6.19 Pasteurella 1088

8.6.19 Pasteurella 1088

section 8  Infectious diseases 1088 herds. This has been accomplished for a small number of herds in Norway, removing Yersinia, Salmonella, and Campylobacter carriage, but is not yet widespread. Sequencing of more strains of Y. enterocolit- ica (including Y. enterocolitica phylogroup PG1, biovar 1A strains) and Y. pseudotuberculosis will shed more light on pathogenic mechanisms and organism evolution. FURTHER READING Guern ASL, et al. (2016). Yersiniosis in France: overview and potential sources of infection. Int J Infect Dis, 46, 1–​7. Hall M, et al. (2015). Use of whole-​genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels. J Clin Microbiol, 53, 35–​42. Huovinen E, et  al. (2010). Symptoms and sources of Yersinia enterocolitica-​infection:  a case–​control study. BMC Infect Dis, 10, 122. Kolstoe EM, et al. (2015). Specific pathogen-​free pig herds also free from Campylobacter. Zoonoses Public Health, 62, 125–​30. Reuter S, et al. (2014). Parallel independent evolution of pathogen- icity within the genus Yersinia. Proc Natl Acad Sci U S A, 111, 6768–​73. Rimhanen-​Finne R, et  al. (2009). Yersinia pseudotuberculosis causing a large outbreak associated with carrots in Finland, 2006. Epidemiology and Infection, 137 (Special Issue 03), 342–​7. Vincent P, et al. (2007). Similarities of Kawasaki disease and Yersinia pseudotuberculosis infection epidemiology. Pediatr Infect Dis J, 26, 629–​31. 8.6.19  Pasteurella Marina S. Morgan ESSENTIALS Pasteurella multocida is an important human Gram-​negative pathogen residing primarily in the oropharynx of mammals and transmitted through bites and scratches. Presentation is typically within 12 h of the injury with rapidly spreading cellulitis or sepsis, leading to ser- ious morbidity and mortality (up to 40%) if untreated. Diagnosis is clinical: fresh bite wound cultures are unhelpful, but the organism is usually cultured in cases with established infection, especially if pre- senting within 24 hours of the injury. Treatment requires thorough wound debridement, with delayed closure if possible, along with antimicrobials to provide empirical cover against pasteurellae and all the other expected pathogens (e.g. amoxicillin-​clavulanate plus ciprofloxacin, or meropenem plus clindamycin). Prevention is by avoidance of animal bites or scratches and prompt hygienic man- agement of wounds: antibiotic prophylaxis (amoxicillin-​clavulanate or—​for the penicillin allergic—​doxycycline or azithromycin) should be reserved for high-​risk bites (e.g. cat bites) or high-​risk wounds that are difficult to debride adequately. Introduction Pasteurella multocida (literally ‘killer of many species’) is a major human pathogen and causes severe morbidity. Pasteurella septicaemia is associated with a mortality of 40% and a propen- sity for metastatic infection. Infection usually follows close animal contact or bites. The or- ganism is part of the colonizing oral flora in virtually every species from birds to elephants, water buffalo and Tasmanian devils, but found especially in cats. Historical perspective The genus Pasteurella was named in honour of Pasteur who, in 1880, discovered P. multocida to be the cause of fowl cholera. Pasteurella spp. cause haemorrhagic septicaemia, ‘shipping fever’ in cattle, and respiratory infections in goats, sheep, and rabbits. Aetiology, genetics, pathogenesis, and pathology Nearly all infected patients have a history of animal exposure. Pasteurella spp. such as P. dagmatis, P. pneumotropica, P. bettyae, P. haemolytica, and P. caballi rarely cause human infection although P. bettyae has caused infection after hamster bites. Pasteurella spp. are small, Gram-​negative coccobacilli, often with bipolar staining. Unusually for a Gram-​negative rod, P. multocida is sensitive to penicillin and fails to grow on MacConkey’s agar. Potential virulence factors include capsule lipopolysaccharide, a cytotoxin, iron acquisition proteins, and other surface structures including homologues of the Bordatella pertussis filamentous haemagglutinin. An aggressive and opportunistic pathogen, P. multocida infection can colonize the oropharynx in those working with animals, causing invasive infection in those with underlying pathology such as liver cirrhosis or bronchiectasis. P. multocida is particularly associated with infection following animal bites, licks, or scratches. Metastatic infection of bones, joints, and brain following bacteraemia is not uncommon, whereas endocarditis and mycotic aneurysms are very rare. Cat-​related trauma is particularly likely to result in pasteur- ella infection, especially septic arthritis and osteomyelitis following hand bites. Small, sharp cat teeth leave a septic focus in deeper tis- sues, under an apparently innocuous puncture wound. There are increasing numbers of reports of prosthetic joint infection following cat bites, with a propensity for knee replacements in females with rheumatoid arthritis. Necrotizing soft tissue infections such as tenosynovitis, septicaemia, and liver and brain abscesses are the more common manifestations, with very rare reports of epiglottitis, chorioamnionitis, and neonatal sepsis. Pasteruella tenosynovitis is especially severe, often resulting in amputation of digits. Epidemiology Infection can be occupationally related (e.g. in veterinary surgeons, farmers, and postmen), but more commonly follows bites from

8.6.19  Pasteurella 1089 companion animals. Animal bites account for roughly 2% of at- tendances at emergency departments in the United Kingdom, and nearly 60% of cat bites are infected with. P. multocida, usually with anaerobes. Prevention Avoidance of animal bites or scratches and prompt hygienic man- agement of wounds are key to preventing infection. Antibiotic prophylaxis should be reserved for high-​risk bites (e.g. cat bites) or high-​risk wounds that are difficult to debride ad- equately. Oral co-​amoxiclav, 625 mg three times daily for 3 to 5 days will cover Pasteurella spp. as well as the hundreds of possible oral commensals present. Patients who have undergone mastectomy and those with dia- betes, immunosuppression, cirrhosis, steroid therapy, splenectomy, or prosthetic joints are ‘high-​risk patients’ for whom prophylaxis should be seriously considered. ‘High-​risk wounds’ include punc- ture wounds, particularly to the hand or wrist, and crush wounds with devitalized tissue. Erythromycin, clindamycin, and flucloxacillin are ineffective against Pasteurella spp. and should not be used for prophylaxis or treatment in the absence of sensitivity information. Numerous re- ports of breakthrough P. multocida septicaemia and meningitis have occurred during erythromycin or flucloxacillin therapy. Alternative prophylaxis for penicillin-​allergic patients includes doxycyc- line, azithromycin, aztreonam, or linezolid. Metronidazole can be added for deep, penetrating wounds such as cat bites that cannot be debrided easily. Clinical features of Pasteurella infection The most common presentation of pasteurella infection is soft tissue infection but septic arthritis, osteomyelitis, osteomyelitis, septicaemia, and meningitis can occur, particularly in infants and immunocompromised individuals. Soft tissue infections are usu- ally cat or dog bites, or cat scratches, but might occur if an animal licks broken skin. Since Pasteurella spp. are extremely pyogenic, bite-​related or scratch-​related infections usually present 8–​12 h after the incident and rapidly spreading cellulitis is typical (Fig. 8.6.19.1). Purulent discharge occurs in 40%, lymphangitis in 20%, and re- gional lymphadenopathy in 10%. P. multocida can cause bone and joint infections such as septic arthritis (usually monoarticular and involving the knee joint) or osteomyelitis. Most cases of septic arth- ritis occur after an animal bite distal to the joint. Osteomyelitis results either from extension of soft tissue infection or via direct in- oculation of bacteria into the periosteum by the animal bite; osteo- myelitis is more frequently associated with cat bites than dog bites, presumably because of cats’ small, sharp teeth. Patients presenting with sepsis and positive blood cultures for pasteurella are less likely to have a history of a bite and are more likely to have comorbidities and need intensive care support. These patients, however, usually also have a history of contact with animals. Respiratory tract Pasteurella spp. can be commensals or cause infections such as glossitis, pharyngitis, sinusitis, otitis media, epiglottitis, bronchitis, pneumonia, and empyema. In one study of 108 patients with pleuropulmonary P. multocida infections, an underlying disease was found in 90% and mortality was 29%. P. multocida can also cause several other serious invasive in- fections such as meningitis, bacteraemia, endocarditis, and peritonitis. Intra-​abdominal infections include peritonitis and appendicitis. Chorioamnionitis is associated with neonatal sepsis. Differential diagnosis Of the hundreds of bacterial species contaminating animal bites, other major pathogens to consider include streptococci, staphylo- cocci, and especially anaerobes; the latter more common in deep penetrating wounds. Clinical investigation A history of animal bite or scratch preceding any presentation of sepsis should alert the clinician to the possibility of pasteurella infec- tion. Established infections necessitate the taking of blood cultures and culture of any discharge. The microbiology laboratory should be alerted to the possibility of pasteurella so that specimens can be cultured appropriately. Unlike most Gram-​negative rods it does not grow on MacConkey’s agar and is susceptible to penicillin. Selective media containing vancomycin, clindamycin, and amikacin have been used to isolate pasteurella. Most strains are catalase, oxidase, indole, sucrose, and decarboxylate ornithine positive. Antibiotic susceptibility testing is warranted for isolates cultured from normally sterile sites and respiratory specimens, particularly in the immunocompromised. Pasteurella is usually susceptible to penicillin, amoxicillin-​clavulanate, piperacillin-​tazobactam, doxy- cycline, fluoroquinolones, extended spectrum cephalosporins (e.g. ceftriaxone, cefpodoxime, and cefixime), and carbapenems (imipenem, meropenem, and doripenem). Fig. 8.6.19.1  Pasteurella multocida hand infection, preoperative.

section 8  Infectious diseases 1090 Typing of P. multocida has traditionally been done serologically. There are 5 capsular serogroups and 16 somatic serotypes; most human infections are caused by serotypes A, D, and F. New typing methods rely on molecular methods. Subspecies of P. multocida can be identified by polymerase chain reaction fingerprinting. Human infections have been reported with P. multocida subsp. multocida, P. multocida subsp. septica, P. multocida subsp. gallicida, P. canis, P. dagmatis, and P. stomatis. Treatment Indications for hospital admission after animal bites include sys- temic sepsis, involvement of joint or tendon, immunocompromise, bites requiring reconstructive surgery, severe cellulitis, and infection refractory to oral therapy. Hands are especially prone to infection because of the numerous small compartments and lack of soft tis- sues separating the skin from bone and joint. Inadequate debridement and incorrect antibiotic prophylaxis are major contributors to the excessive morbidity of P. multocida infection. Where adequate debridement of deep wounds, especially cat bites, is not possible, irrigation with 250 ml saline, using a 19-​ or 20-​gauge needle or plastic intravenous catheter on a 30-​ml syringe, followed by prophylactic antibiotics might be effective (Fig. 8.6.19.2). Thorough irrigation and debridement of the wound, and, where possible, delayed closure of limb bites maximizes salvage. Limbs should be elevated and immobilized. Tenosynovitis can be so ad- vanced on presentation that amputation is the only option. Pus must be drained and affected joints washed out, and the wound left open where possible. Facial bites can be closed primarily since bleeding is profuse and wounds are easily cleaned. While most pasteurellae are susceptible to penicillin, β-​ lactamase producing strains are increasingly reported, so penicillin monotherapy is not advised until susceptibility is confirmed. Broad-​ spectrum empiric antimicrobial therapy should be directed at polymicrobial infection that occurs after bite infections and a com- bination of a penicillin and a β-​lactamase inhibitor (e.g. amoxicillin-​ clavulanate) recommended in patients without penicillin allergy. Definitive treatment is based on the result of wound cultures and should be continued for 7–​10 days. Alternative treatments for established pasteurella infections for patients allergic to penicillins, or infected with β-​lactamase produ- cing strains, include oral doxycycline or intravenous aztreonam, or ciprofloxacin. Patients without a history of anaphylaxis to penicillins can be treated with ceftriaxone. For established soft tissue infection, 10–​14 days of therapy is usual, compared with 3 weeks for tenosynovitis, 4 weeks for septic arthritis, and 6 weeks for osteomyelitis. In practice, intravenous therapy until the C-​reactive protein falls to less than 50 mg/​litre is a useful ob- jective guideline for switching to oral therapy. Prognosis The prognosis of P. multocida infections depends on the site of infec- tion and comorbidities. Soft tissue infections usually resolve with ad- equate debridement, drainage, and antibiotics. Established bite-​related hand infection unfortunately often results in permanent impairment of function. Factors particularly associated with poor outcome in- clude inadequate initial antimicrobials and inadequate debridement. Pasteurella septicaemia, metastatic infection, and death been reported following inappropriate therapy with erythromycin or flucloxacillin. P. multocida prosthetic joint infection, usually associated with rheuma- toid arthritis and female gender, results in loss of the prosthesis in 70% of patients, even with early appropriate antibiotic therapy. Areas of controversy The role of antimicrobial prophylaxis following animal bites, in the absence of any other risk factor for infection, is debatable. One meta-​analysis of eight randomized trials concluded that the rela- tive risk for infection in patients given antibiotics compared with controls was 0.56 (95% confidence interval, 0.38–​0.82), whereas an- other meta-​analysis included trials with few cat bites, resulting in no evidence for the benefit of prophylaxis. While there may be cause for careful consideration of prophylaxis for other animal bites, anti- microbial prophylaxis for cat bites is usually indicated. FURTHER READING Adlam C, Rutter JM (1989). Pasteurella and pasteurellosis. Academic Press, London. Cummings P (1993). Antibiotics to prevent infection in patients with dog-​bite wounds: a meta-​analysis of randomised trials. Ann Emerg Med, 23, 535–​40. Giardano A, et al. (2015). Clinical features and outcomes of Pasteurella multocida infection. Medicine (Baltimore), 94, e1285. Heydemann J, Heydemann J, Antony S (2010). Acute infection of a total knee arthroplasty caused by Pasteurella multocida: a comprehensive Fig. 8.6.19.2  The same patient: infected area being incised and drained.

8.6.2 Streptococci and enterococci 965

8.6.2 Streptococci and enterococci 965

8.6.2  Streptococci and enterococci 965 diphtheria vaccine is reduced in children aged 7 years and older so that reactogenicity is minimized. The recommended schedule for vaccination against diphtheria varies between countries. In the United Kingdom three primary doses of adsorbed diphtheria–​tetanus–​pertussis–​haemophilus influenzae type b vaccine (DTP-​Hib) are given at 2, 3, and 4 months; a first booster dose with DTP at age 3 to 5 years, and a second booster dose with DT at school leaving. The primary course does not need to be repeated if boosters are delayed. People living in low-​endemic or non​endemic countries should receive booster doses of DT approximately every 10 years. It is now recom- mended by the World Health Organization that DT rather than T (tetanus toxoid alone) should be used when tetanus prophylaxis is needed following injury. Tetanus-​diphtheria vaccine is recom- mended for all travellers who have not received the vaccine within the last 10 years. Where diphtheria is endemic the primary course alone should be sufficient to prevent an epidemic of diphtheria, as natural mech- anisms such as frequent skin infections caused by C. diphtheriae probably contribute to maintaining immunity. One or two DT or DTP booster doses may need to be added to the routine schedule in areas at increased risk of diphtheria. Adults in developing coun- tries do not require routine immunization. Aggressive action is needed in the event of a diphtheria outbreak. Groups at risk should be immunized, there should be prompt diag- nosis and management of cases, and identification of close con- tacts should be made so that the spread of infection can be halted. A single dose of DTP should be used for children under 3 years of age, and DT for children aged over 3 years and adults. Additional doses of vaccine will be needed in non​immunized (Schick test posi- tive) people. Susceptibility to diphtheria may be assessed using the Schick test: 0.1 ml of toxin is injected into the skin of one forearm (test site) and the same quantity of a heat-​inactivated toxin injected into the other forearm (control site). A positive reaction occurs in in- dividuals without toxin-​neutralizing antibodies and consists of an area of redness appearing after 24–​36 h at the test site only and persisting for 4–​5 days. If no toxin-​neutralizing antibodies are pre- sent, there will be either no reaction at either site (negative test) or a pseudoreaction at either site due to antibodies to substances other than diphtheria toxin in the test materials. This test is no longer commonly performed due to limited availability of the test materials. FURTHER READING Celik T, et al. (2006). Prognostic significance of electrocardiographic abnormalities in diphtheritic myocarditis after hospital discharge: a long-​term follow-​up study. Ann Noninvasive Electrocardiol, 11, 28–​33. Christie AB (ed) (1987). Diphtheria. In:  Infectious diseases:  epide- miology and clinical practice, 4th edition, pp. 898–​928. Churchill Livingstone, New York, NY. Crowcroft NS, et al. (2006). Screening and toxigenic corynebacteria spread. Emerg Infect Dis, 12, 520–​1. European Centre for Disease Prevention and Control (2009). https://​ ecdc.europa.eu/​en/​diphtheria Health Protection Agency (n.d.). Diphtheria. http://​www.hpa.org.uk/​ HPA/​Topics/​InfectiousDiseases/​InfectionsAZ/​1191942152928 Hofler W (1991). Cutaneous diphtheria. Int J Dermatol, 30, 845–​7. Jayashree M, Shruthi N, Singi S (2006). Predictors of outcome in pa- tients with diphtheria receiving intensive care. Indian Pediatr, 43, 155–​60. Mikhailovich VM, et al. (1995). Application of PCR for detection of toxigenic C. diphtheriae strains isolated during the Russian diph- theria epidemic, 1990 through 1994. J Clin Microbiol, 33, 3061–​3. ProMed-​mail (2009). 30 October 2009 Diphtheria—​Haiti:  RFI 20091030.3755. http://​www.promedmail.org/​ ProMed-​mail (2010). 19 May 2010 Diphtheria—​Haiti:  (Port-​au-​ Prince) 20100519.1644. http://​www.promedmail.org/​ Rakhmanova G, et al. (1996). Diphtheria outbreak in St. Petersburg: clinical characteristics of 1,860 adult patients. Scand J Infect Dis, 28, 37–​40. Vitek CR (2006). Diphtheria. Curr Top Microbiol Immunol, 304, 71–​94. World Health Organization (2006). Diphtheria vaccine: WHO pos- ition paper. Weekly Epidemiol Rec, 81, 24–​32. WHO (2016). Immunization, Vaccines and Biologicals. Reported Cases of Selected Vaccine Preventable Diseases (VPDs). http://www.who. int/immunization/monitoring_surveillance/data/en/.Accessed April 15, 2017. Wren MW, Shetty N (2005). Infections with Corynebacterium diph- theriae: six years’ experience at an inner London teaching hospital.
Br J Biomed Sci, 62, 1–​4. 8.6.2  Streptococci and enterococci Dennis L. Stevens and Sarah Hobdey ESSENTIALS The streptococci are a diverse group of Gram-​positive pathogenic cocci that cause clinical disease in humans and domestic animals. They are traditionally classified on the basis of serological reac- tions, particularly Lancefield grouping based on cell-​wall carbo- hydrates, and haemolytic activity on blood agar. Six groups can be defined by genetic analysis: pyogenic streptococci, milleri or anginosus group, mitis group, salivarius group, mutans group, and bovis group. Group A streptococci (S. pyogenes) Group A  streptococci are carried, usually in the nose or throat, by 5–​20% of children and 0.5% of adults. More than any other human pathogen, group A  streptococci cause a wide variety of infections ranging from pharyngitis, erysipelas, cellulitis, and Acknowledgement: The author of the present chapter and the editors acknow- ledge the inclusion of much material from the chapter in previous editions by Professor S. K. Eykyn.

section 8  Infectious diseases 966 necrotizing fasciitis to the postinfectious sequelae: rheumatic fever and poststreptococcal glomerulonephritis. These microbes con- tinue to evolve, as evidenced by over 150 different genetic types and the emergence of novel infections such as streptococcal toxic shock syndrome. Group A streptococci are easy to culture in the laboratory from appropriate samples; diagnosis can also be made by detection of the group A antigen or confirmed serologically. All strains remain sensi- tive to penicillin, which is the antibiotic of choice, with erythromycin usually given to those who are penicillin allergic, although epidemics of pharyngitis caused by erythromycin-​resistant strains have been widely reported. Genetic differences and the presence of multiple virulence factors have frustrated efforts to develop effective vaccines. Group B streptococci (S. agalactiae) Group B streptococci are carried in the throat by 5–​10% of adults, as well as in the urethra, vagina, perineum, and anorectum. They cause a variety of infections: (1) neonatal infection—​including bacteraemia and meningitis; screening for vaginal carriage during the third tri- mester of pregnancy and intrapartum treatment with intravenous penicillin has reduced the incidence of early-​onset neonatal disease; (2)  post-​partum infection—​puerperal infection usually manifests
as endometritis with fever and uterine tenderness, occurring within 24–​48 h of delivery or abortion; also (3) skin and soft tissue infec- tions, urinary tract infections, and bacteraemias (especially in pa- tients with diabetes mellitus, malignancy, HIV infection, and chronic renal or liver disease). Group B streptococci are readily isolated from any clinical spe- cimen in the laboratory, and detection of group B antigen in body fluids by latex particle agglutination enables rapid diagnosis. They are sensitive to penicillin (the antibiotic of choice), erythromycin, and cephalosporins. The polysaccharide capsule of group B strepto- coccus is a major virulence factor, with at least six different serotypes identified: experimental immunization using the polysaccharide pro- vides type specific protection, but no such vaccine has yet been de- veloped for human use. Other groups of streptococci Groups C and G—​produce infections that are similar to those caused by group A streptococci but tend to be less virulent. They are im- portant causes of cellulitis, particularly recurrent cellulitis associated with saphenous vein donor site infections in patients with coronary artery bypass surgery. Streptococcus milleri or Streptococcus anginosus group—​includes S. constellatus, S. intermedius, and S. anginosus. These are found in the normal flora of the upper respiratory tract, gastrointestinal tract, and genital tract; commonly isolated from a range of pyogenic infections (e.g. dental or other abscesses), sometimes in pure culture, but often with other organisms, particularly anaerobes. Streptococcus mitis, Streptococcus salivarius, and Streptococcus mutans groups of streptococci (oral/​viridans streptococci)—​these in- clude S. pneumoniae (see Chapter 8.6.3) and those oral streptococci that are the most common causes of infective endocarditis of oral or dental origin. They occasionally cause bacteraemia in neutro- penic patients, particularly those who have received prophylaxis with fluoquinolones such as ciprofloxacin. Streptococcus bovis group—​a gastrointestinal commensal; most pa- tients with S. bovis bacteraemia will have endocarditis in association with colonic pathology or cirrhosis of the liver. Streptococcus suis—​an occupational cause of septicaemia, menin- gitis, septic arthritis, pneumonia, and endophthalmitis among those working with pigs and pork in Southeast Asia. Enterococci Part of the normal gut flora of humans and animals, these are an increasingly important cause of nosocomial infection and colon- ization, possibly the result of the large-​scale use of antibiotics such as cephalosporins and quinolones to which they are inherently resistant. Enterococcus faecium and E.  faecalis have also become vancomycin resistant, a characteristic dramatically increasing treat- ment failures, although they remain sensitive (at the time of writing) to linezolid, an oxazolidinone antimicrobial. Introduction The term streptococcus was first used by Billroth in 1874 to describe chain-​forming cocci found in infected wounds. In 1879, Pasteur also found them in the blood of women with puerperal sepsis. In 1884, Rosenbach defined these streptococci as Streptococcus pyo- genes. This organism remains one of the most important human pathogens. The genus Streptococcus contains many other species of varying degrees of pathogenicity for humans and animals. S. faeca- lis and S. faecium were split from the genus Streptococcus in 1984 and became Enterococcus spp. and numerous other species have since been included in this genus. The nutritionally variant strepto- cocci S. adjacens and S. defectivus have also been assigned to a new genus Abiotrophia, to which the newly described species A. elegans has been added. Classification Traditionally, classification of streptococci has relied on serological reactions, particularly the Lancefield grouping based on cell-​wall carbohydrates, and haemolytic activity on blood agar, which has led to rather unsatisfactory streptococcal taxonomy. Genetic analysis has now enabled the subdivision of the species of Streptococcus into six clusters or groups as follows: pyogenic streptococci, milleri or anginosus group, mitis group, salivarius group, mutans group, and bovis group. Since the medically important members of the mitis, salivarius, and mutans groups are all oral streptococci and are of clinical relevance predominantly in endocarditis, they will be con- sidered together. Pyogenic streptococci The pyogenic streptococci include the major human pathogen S.  pyogenes (Lancefield group A), group B streptococci (S.  aga- lactiae), and groups C and G streptococci. These organisms are β-​haemolytic on blood agar.

8.6.2  Streptococci and enterococci 967 S. pyogenes (b-​haemolytic group A) The prevalence and severity of streptococcal pharyngitis has re- mained constant over the centuries of recorded history, although the incidence of complications such as peritonsillar abscess and mastoiditis have declined with the advent of antibiotics. Since the beginning of the 20th century, and long before the introduction of antibiotics, the prevalence and severity of scarlet fever and rheum- atic fever following infections with S. pyogenes declined until the 1980s. In the mid-​1980s, highly virulent streptococci appeared causing very severe infections such as streptococcal toxic shock syn- drome and necrotizing fasciitis, often in otherwise healthy people. Such cases occurred not only in the United Kingdom but also in most of the developed world. S. pyogenes infection is usually com- munity-​acquired but can be acquired in hospital where the most ser- ious infections are postoperative. Carriage Although S. pyogenes is an invasive organism, it survives on epi- thelial surfaces (asymptomatic carriage) usually in the nose and throat. Carriage can also be anal, vaginal, and on the scalp. Pharyngeal carriage rates are usually much higher in children (5–​ 20%) than in adults (0.5%) and also vary with season, year, and geographical location. They are higher in crowded living condi- tions. S. pyogenes can persist for months after acute pharyngitis, though in decreased numbers. Survival in the environment is poor and S. pyogenes can only survive on skin and inanimate objects for a limited period of time. Pathogenicity, virulence, and typing S.  pyogenes is an extracellular pathogen and produces virulence factors that enable it to avoid host defences and spread in tissues. An important virulence factor is the M protein and streptococci rich in M protein resist phagocytosis by granulocytes. Immunity to S. pyogenes infection is associated with the development of op- sonic antibodies to antiphagocytic epitopes of M protein; the im- munity is usually type specific and lasts for many years. M protein was first described in the 1920s by Rebecca Lancefield; over 100 M types have now been differentiated. Lancefield also developed the supplementary T typing system which distinguishes 26 serotypes of a trypsin-​resistant surface protein (T antigen), most of which can be expressed by several different M types. Certain M types also produce a serum opacity factor (OF+). These typing systems are still widely used in epidemiological studies to distinguish between strains of S. pyogenes. However, more modern methods utilize procedures to sequence the M protein gene. Recent studies have shown consider- able genetic diversity in S. pyogenes, and horizontal transfer and re- combination of virulent genes have played a major role. This finding is likely relevant to the emergence of new unusually virulent clones of the organism. In addition to M protein, lipoteichoic acid, important in the host–​ bacterial interaction, is expressed on the surface of the organism and is the adhesin that binds the organism to fibronectin on the surface of the oral epithelial cell membranes and initiates the colonization that precedes infection. S. pyogenes has a hyaluronate capsule which, like M protein, is also antiphagocytic, and is an additional virulence factor. The extent of encapsulation varies, and colonies with prom- inent capsules are very mucoid on blood agar. Strains of S. pyogenes that are both rich in M protein and heavily encapsulated are readily transmitted from person to person and have been associated with epidemics of acute rheumatic fever. S.  pyogenes produces many extracellular substances, several of which are important in the pathogenesis of infection. The most familiar are streptolysin O, deoxyribonuclease (DNase) B, and hyaluronidase, as serum antibodies to these provide retrospective confirmation of recent streptococcal infection. Other extracellular products include DNases A, C, and D, streptolysin S, proteinase, streptokinase, and the substances previously known as erythrogenic toxins. These toxins have now been designated streptococcal pyro- genic exotoxins (SPE)-​A, -​B, -​C, and more recently several others. SPE-​A and SPE-​C are coded by a phage gene and readily transmitted to susceptible strains. These toxins, known as superantigens, have diverse effects on the host. In addition to the rash of scarlet fever, they cause fever and induce lethal shock in animals. They have pro- found effects on the immune system including increasing suscep- tibility to endotoxic shock, induction of cytokine production, and cause clonal proliferation of T lymphocytes. Recently, nicotine adenine dinucleotidase (NADase) has been found in 100% of strains of group A streptococci (GAS) associated with invasive GAS infections such as toxic shock syndrome and necrotizing fasciitis. There is evidence that the gene for NADase is found in all strains of GAS but only produced extracellularly in these invasive strains. In addition, production of NADase by M1 strains, the most common strain associated with invasive types of infections, began around 1985, just before the recognition of severe invasive GAS infections. S. pyogenes can penetrate the upper respiratory tract mucosa or a break in the skin, causing local infection or may spread along tissue planes or lymphatics. The M protein is not toxic in itself but pro- tects the streptococcus from phagocytosis, and antibodies to the M protein are opsonic. In about two-​thirds of patients with serious in- vasive disease, who might present with fever, shock, and renal im- pairment, the portal of entry is the skin and infection of soft tissue is apparent, but in others the site of infection might be deep in the fascia or muscle. Infections caused by S. pyogenes S. pyogenes causes a variety of illnesses ranging from very common infections such as pharyngitis, impetigo, and cellulitis to less common, more severe infections such as puerperal sepsis, necro- tizing fasciitis, bacteraemia, and toxic shock. S. pyogenes is also as- sociated with the non​suppurative sequelae of acute rheumatic fever and acute glomerulonephritis. Streptococcal pharyngitis Streptococcal pharyngitis or tonsillitis is one of the most common bacterial infections in children from 5 to 15 years, but all ages are susceptible. The incubation period, at least in outbreaks, is short (1–​ 3 days) and the onset of the infection is marked by the abrupt onset of sore throat and pain on swallowing with malaise, fever, and head- ache. The signs are redness and oedema of the pharynx, enlarged red tonsils (Fig. 8.6.2.1) with spots of white exudate, fever, and en- larged tender anterior cervical lymph glands. Nausea, vomiting, and abdominal pain are common in children, and in infants and pre- school children there may be few definite signs of pharyngitis but

section 8  Infectious diseases 968 fever, nasal discharge, enlarged cervical lymph glands, and otitis media occur. Direct extension of streptococcal pharyngitis can give rise to acute sinusitis or otitis media, and other suppurative complications include peritonsillar abscess (quinsy), mastoiditis, retropharyngeal abscess, and suppurative cervical lymphadenitis. Scarlet fever Scarlet fever results from infection with a strain of S. pyogenes that pro- duces SPE (erythrogenic toxin). It is usually associated with strepto- coccal pharyngitis but can follow streptococcal infections at other sites including surgical site infections. Scarlet fever rarely follows streptococcal pyoderma. Most cases occur in school-​age children and the rash must be distinguished from viral exanthems, Kawasaki’s disease, and staphylococcal toxic shock syndrome. The rash, which generally appears on the second day of clinical illness, is usually a diffuse erythema, symmetrical, and blanches on pressure. It is seen most often on the neck, chest, folds of the axilla, and groin. Occlusion of sweat glands gives the skin a ‘sandpaper’ texture, a useful sign in dark-​skinned patients. The face appears flushed with circumoral pallor. There are small red haemorrhagic spots on the palate, and the tongue is initially covered with a white fur through which red papillae appear (‘strawberry tongue’); after the rash develops, the white fur peels off leaving a raw red papillate surface (‘raspberry tongue’). The rash persists for several days and later (up to 3 weeks) peeling (des- quamation) may occur, usually on the tips of the fingers, toes, or ears, and less often over the trunk and limbs. A similar rash may develop as a reaction to streptokinase thrombolytic therapy. Streptococcal perianal infection (cellulitis) This is a superficial well-​demarcated rash spreading out from the anus in young children, usually boys, associated with itching, rectal pain on defaecation, and blood-​stained stools. S. pyogenes is iso- lated from perianal cultures and usually also from pretreatment throat swabs. Streptococcal vulvovaginitis Vulvovaginitis in prepubertal girls is often caused by S. pyogenes and presents with serosanguinous discharge and erythema of the labia and vaginal orifice. As with perianal infections, S. pyogenes is usu- ally also found in the throat. In both streptococcal perianal infec- tion and vulvovaginitis, more than one child in the family may be affected and nasopharyngeal carriage is likely in both infected and uninfected children. Streptococcal skin and soft tissue infections Pyoderma/​impetigo  Almost any purulent lesion of the skin can yield S. pyogenes, sometimes with Staphylococcus aureus. Such le- sions include impetigo, infected cuts and lacerations, insect bites, scabies, intertrigo, and ecthyma. S. pyogenes often causes secondary infection in varicella, occasionally with resultant bacteraemia. The term pyoderma is used synonymously with impetigo for discrete purulent, apparently primary infections of the skin that are preva- lent in many parts of the world, especially in children. These lesions are initially papules, then vesicular with surrounding erythema, and finally pustules with crusting exudate; they may be localized to one part of the body or generalized. Outbreaks of impetigo can occur among adults subject to skin trauma, such as rugby football players (scrumpox), and streptococcal infection of cuts on the hands and forearms are an occupational hazard for workers in the meat trade. Epidemics of impetigo can occur in day care centres, prisons, and schools. Ecthyma is an ulcerated form of impetigo in which the ul- ceration extends into the dermis. In recent times, approximately 50% of cases of impetigo are caused by Staphylococcus aureus. Invasive streptococcal infections of skin and soft tissues Erysipelas  This is an acute inflammation of the skin with lymph- atic involvement. The streptococci are localized in the dermis and hypodermis. It usually affects the face, particularly in elderly people, but may occur elsewhere. It may be bilateral (Fig. 8.6.2.2) and is sometimes recurrent. There is generally a history of sore throat, but the mode of spread to the skin is unknown. It is usually accom- panied by fever, rigors, and toxicity. The cutaneous lesion begins as Fig. 8.6.2.2  Bilateral facial erysipelas. Copyright S. J. Eykyn. Fig. 8.6.2.1  Streptococcal tonsillitis: suppurative complications. Copyright D. A. Warrell.

8.6.2  Streptococci and enterococci 969 a localized area of brilliant erythema and swelling and then spreads with rapidly advancing raised red margins that are well demarcated from adjacent normal tissue. Facial erysipelas begins over the bridge of the nose and spreads over the cheeks. Vesicles and bullae appear, which become crusted when they rupture. There is marked oedema and the eyes are often closed. When the infection resolves it is often followed by desquamation. Intense local allergic reactions to topical agents, such as cosmetics, may cause confusion. Cellulitis  Cellulitis (Fig. 8.6.2.3) is commonly caused by strepto- cocci and Staphylococcus aureus. This is an acute spreading inflam- mation of the skin and subcutaneous tissues with local pain swelling and erythema. Fever, rigors, and malaise may precede by a few hours the appearance of the skin lesion and associated lymphangitis and tender lymphadenopathy. Streptococcal cellulitis differs from ery- sipelas in that the lesion is not raised and the demarcation between affected and unaffected skin is indistinct. It can result from infection of burns, mild trauma, or surgical wounds. When this involves the leg, fungal infection of the feet is often present and predisposes to streptococcal invasion. After the first episode, there is a tendency for recurrence in the same area. Recurrences are more common in patients with chronic venous insufficiency, lymphatic obstruction, and at the saphenous vein donor site in patients following coronary bypass surgery. These latter infections are most commonly caused by group C or G streptococci. Intravenous drug users are also at risk of streptococcal cellulitis associated with skin and tissue infection and septic thrombophlebitis. (Type II) necrotizing fasciitis (streptococcal gangrene)  This in- fection, described by Meleney in 1924, involves the deep subcuta- neous tissues and fascia (and occasionally muscle as well) with extensive necrosis and gangrene of the skin and underlying struc- tures. It is generally community-​acquired, usually involving the arm or leg, but can also occur after surgery, which can sometimes be quite minor. Some people with this infection are diabetic, but the majority are previously healthy. Risk factors providing a portal of entry include surgery, trauma, childbirth, intravenous drug abuse, and chickenpox. Blunt trauma and muscle strain and the use of non-​ steroidal anti-​inflammatory agents are also risk factors. The infection begins at the site of trivial or even inapparent trauma with redness, swelling, fever, and rapidly escalating focal pain followed by purple discoloration and the development of bullae, which are often haem- orrhagic. In patients who develop infection deeply in traumatized tissue such as muscle, fever, and severe pain may be the only initial signs and symptoms of infection. Bacteraemia is often present and within days skin necrosis occurs followed by extensive sloughing. The patient is profoundly ill and the disease has a high case fatality rate of 30–​70%. Features of streptococcal toxic shock syndrome are associated in many cases. The United Kingdom media memorably dubbed S. pyogenes the ‘flesh-​eater’ in reports of a cluster of cases of necrotizing fasciitis in 1994. Treatment involves early intravenous antibiotics. The organisms are sensitive to penicillin but, paradox- ically, the drug may not be effective in high concentrations (the ‘Eagle effect’). Clindamycin has advantages over penicillin, based on animal studies and one retrospective study in humans. The efficacy of clindamycin is likely due to its ability rapidly to inhibit toxin pro- duction by Gram-​positive pathogens. Urgent surgical debridement of necrotic tissue and intensive care to support failing organs and systems (e.g. cardiovascular and renal) are extremely important. Benefits of immunoglobulin are suggestive but inconclusive. Streptococcal toxic shock syndrome This syndrome was described in 1989 in patients with severe S. pyo- genes infection and clinical features remarkably similar to those of the staphylococcal toxic shock syndrome described a decade earlier. Streptococcal toxic shock syndrome is defined as any acute S. pyogenes infection associated with the sudden onset of shock and multiorgan failure. Streptococcal toxic shock syndrome may be as- sociated with necrotizing fasciitis, myositis, pneumonia, peritonitis, or post-​partum sepsis. It can occur at all ages and many of those affected are young and previously healthy. Most cases have been community-​acquired, though it can be acquired in hospital. M1 has been the predominant serotype in many countries, though others, especially 3, 4, 6, 11, 12, and 28, have also been implicated. Most strains produce SPE-​A. Interestingly there is an amino acid hom- ology of 50% and immunological cross-​reactivity between SPE-​A and staphylococcal enterotoxins B and C, which together with staphylococcal toxic shock syndrome toxin-​1 are relevant in non-​ menstrual staphylococcal toxic shock syndrome. Diffuse scarlatina type rash is present in only 5–​10% of cases (Fig. 8.6.2.4). Streptococcal bacteraemia In parallel with the increase in serious S. pyogenes infections, there has been an increase in bacteraemic infections, both community-​ and Fig. 8.6.2.3  Cellulitis. Copyright S. J. Eykyn. Fig. 8.6.2.4  Scarlatina-​like rash of streptococcal toxic shock syndrome. Copyright D. A. Warrell.

section 8  Infectious diseases 970 hospital-​acquired (usually postoperative) (Fig. 8.6.2.5). While many patients have an underlying disease, generally malignancy, im- munosuppression, or diabetes, others are previously healthy adults between 20 and 50 years old. The portal of entry is usually the skin. The mortality is higher in patients with underlying disease, those with necrotizing fasciitis, myositis, pneumonia, or post-​partum sepsis, and the very young or old. Puerperal and neonatal infection Historically S. pyogenes has always been an important cause of puer- peral sepsis (‘childbed fever’). However, in the postantibiotic era, it was rarely encountered in obstetric practice until the 1980s when sporadic cases occurred, some with streptococcal toxic shock syn- drome, and some women have died. These infections follow abortion or delivery when streptococci (usually colonizing the patient herself) invade the endometrium, lymphatics, and bloodstream. They can be devastatingly severe and present with non​specific signs such as restlessness and gastrointestinal upset that may not immediately sug- gest sepsis. Fever may be absent resulting in further diagnostic con- fusion. The streptococcal infection involves the uterus and adnexae and sometimes distant sites such as joints as well. It can also affect the baby, causing serious neonatal infection including meningitis. Instrumentation in the presence of asymptomatic vaginal or anorectal carriage of S. pyogenes can result in severe infection. Small epidemics of puerperal sepsis have been reported where a healthcare provider has been a carrier that caused infection. In some cases, S. pyogenes can cause infection in the third trimester, before delivery. In these cases, this bacterium causes a transient bacteraemia that seeds the placenta or amnion, sometimes in association with abruptio pla- centa. These are devastating infections for the mother and the child. Other infections S. pyogenes can cause pneumonia (usually associated with viral in- fection or pulmonary disease), osteomyelitis, septic arthritis, menin- gitis, pericarditis (Fig. 8.6.2.6), endophthalmitis, and endocarditis. Laboratory diagnosis of S. pyogenes infection S. pyogenes is easy to culture in the laboratory and usually grows on blood agar in 24 h in atmospheres containing 10% CO2. Throat swabs must be taken before antibiotics are given or the chance of recovery is greatly reduced. Kits for the detection of the group A antigen directly from throat swabs are available and give few false-​positive reactions; they are seldom used in the United Kingdom but are commonly used in the United States of America. Ideally, two swabs are obtained. One is used for the rapid test and, if negative, the other is cultured ap- propriately. Even trivial skin lesions, such as impetigo or surgical site infection, are worth swabbing (if necessary with a moistened swab). Swabs from the surface of cellulitis and erysipelas rarely yield strepto- cocci, although they may be recovered from specimens obtained by aspiration approximately 20% of the time, although this is seldom carried out. Blood cultures should be done in any patient who is ill, whether febrile or not. Serological confirmation of infection with S. pyogenes when the organism has not been isolated can be obtained by the detection of raised antibodies to its extracellular products. Most laboratories tend to use two or more tests. Interpretation requires knowledge of the level of titres in the community for those without a history of recent streptococcal infection. In the United Kingdom the upper limit of titres in teenagers and young adults without such a his- tory is antistreptolysin O (ASO) 200, antideoxyribonuclease B (ADB) 240, and antihyaluronidase (AHT) 128. Management and antibiotic treatment of S. pyogenes infection Remarkably, S. pyogenes remains exquisitely sensitive to penicillin and this is the antibiotic of choice for treatment, parenterally for se- vere infections and orally otherwise. Conventionally, 10 days’ treat- ment is recommended for pharyngeal infections to eradicate the organism and prevent acute rheumatic fever. In practice, compliance with this regimen is poor as once the symptoms abate there is a natural reluctance to continue the antibiotic. Treatment of patients allergic to penicillin is usually with erythromycin or the newer macrolides (azithromycin and clarithromycin), but some 3–​5% of strains are Fig. 8.6.2.5  S. pyogenes bacteraemia 3 days after a skin graft. Copyright S. J. Eykyn. Fig. 8.6.2.6  Peeling of the skin of the soles of the feet in a patient with S. pyogenes pericarditis. Copyright S. J. Eykyn.

8.6.2  Streptococci and enterococci 971 erythromycin resistant in most of the Western world. Epidemics caused by erythromycin-​resistant strains have been described in Japan, Finland, Sweden, and the United States of America. S. pyogenes is also sensitive to cephalosporins. Topical agents such as mupirocin and fusidic acid are useful in addition to systemic antibiotic treat- ment in impetigo and other skin lesions. Patients with streptococcal toxic shock syndrome require intensive care and many require ino- tropic support, ventilation, and haemodialysis. Urgent surgical inter- vention is needed for necrotizing fasciitis and myositis. Clindamycin (in addition to penicillin) has been recommended for patients with established invasive streptococcal infections, since this drug stops the metabolic activity of the streptococci and thus halts further produc- tion of toxin. This is especially relevant in type II necrotizing fasciitis/​ myositis and streptococcal toxic shock syndrome. Recently there has been emergence of clindamycin resistance in strains causing invasive infections and scarlet fever, and linezolid or tedizolid are reasonable alternatives. Intravenous immunoglobulin has also been used in an attempt to neutralize the streptococcal toxins, but reports of its effects are inconclusive, largely because neutralizing antibodies, though pre- sent, are in low concentration and there are batch-​to-​batch variations from the same and different suppliers. Prevention of recurrent cellu- litis of the lower legs involves meticulous foot hygiene with treatment of tinea pedis (if present) and reduction in skin carriage using top- ical mupirocin. Oedematous limbs can benefit from elastic stockings. Antibiotic prophylaxis may be required in cases of frequent recur- rence refractory to these measures. Lastly, it should be remembered that S. pyogenes is readily transmitted from person to person and thus appropriate infection control precautions should be taken until swabs show that the organism has been eradicated. β-​Haemolytic group B streptococci (S. agalactiae) The group B streptococcus has been known for over a century as a cause of bovine mastitis, and in the 1930s it was recognized as a vaginal commensal, an occasional cause of puerperal fever, and an uncommon cause of invasive disease in adults. Not until the 1960s was it realized that the group B streptococcus was an important neo- natal pathogen, and some 20 years later it had replaced Escherichia coli as the predominant neonatal pathogen. Group B streptococcus can also cause a broad range of infections in non​pregnant adults including skin and soft tissue infections, bacteraemia, urinary tract infections, bone and joint infections, endocarditis, and meningitis. Carriage Group B streptococci can be recovered from various sites in healthy adults, but vaginal carriage has been most extensively investigated. Swabs from the lower vagina are more often positive than cervical swabs and carriage rates of 3% to over 40% have been reported. Higher rates have been obtained with selective media and enrich- ment techniques. Carriage also increases with sexual activity and is highest in women attending genitourinary clinics. The urethra, va- gina, perineum, and anorectal region have all been suggested as the prime site of carriage. Approximately 5–​10% of healthy adults carry group B streptococci in the throat, independent of urogenital and anorectal carriage. Pathogenicity, virulence, and typing The chief determinant of virulence appears to be the capsular polysaccharide, and most human strains carry one of six sialic acid-​containing polysaccharides that surround the cell wall. In add- ition, a protein antigen (c, X, or R) may be carried. Certain combin- ations are common; serotypes III or III/​R form one-​quarter of all isolates from superficial sites on women, but three-​quarters of all group B streptococci causing meningitis in infants. They are also the most common serotypes found in adult (non​pregnant) infections. The type polysaccharide, like the M protein of S. pyogenes, inhibits phagocytosis. Colonization of the mucous membranes of the neo- nate results from vertical transmission of the organism from the mother either in utero by the ascending route or at delivery. The rate of vertical transmission in neonates born to mothers colonized with group B streptococci is about 50%, but the incidence of symptomatic infection in neonates born to colonized mothers is only about 1–​2%. It is much higher in preterm infants. Nosocomial colonization of neonates can also occur. In most cases of adult infections (other than in pregnant women) the source of the infection is unknown. Infections caused by group B streptococci These are commonly neonatal or puerperal infections, but group B streptococci also cause infection in non​pregnant adults, particularly in those with diabetes. Neonatal infection The frequency of neonatal infection (bacteraemia, meningitis, or both) has been variously quoted as between 0.3 and 5.4 cases/​1000 live births, but these figures have wide confidence limits. Two fairly distinct clinical patterns of disease predominate, but the spectrum is wide and includes impetigo neonatorum, septic arthritis, osteomye- litis, pneumonitis, peritonitis, pyelonephritis, facial cellulitis, con- junctivitis, and endophthalmitis. Early-​onset disease  Symptoms of group B streptococcus (GBS) disease develop within the first 6 days of life with a mean of 20 hours, although they can present at birth suggesting an intrauterine onset of infection. Early-​onset disease usually presents with bacteraemia with no identifiable focus of infection, but can also be pneumonia or, infrequently, meningitis. The presenting signs include lethargy, poor feeding, jaundice, grunting respirations, pallor, and hypotension and they are common to all types of disease. Respiratory symptoms are nearly always present. The only reliable way of detecting meningitis is by lumbar puncture. Mortality rates are high in low birth weight babies. In addition to positive blood cultures, the infecting strain can be found in the mother’s vagina and cultured from ‘screening’ sites on the baby; these include ear, throat, and nasogastric aspirate. Late-​onset disease  Late-​onset GBS disease usually presents be- tween 7 days and 3 months after birth, often in previously healthy babies born after a normal labour who are admitted unwell from home. The pathogenesis is less clear than in cases of early-​onset dis- ease and only about one-​half of these cases are associated with mu- cosal colonization during delivery. Most babies have meningitis and concomitant bacteraemia and present with non​specific symptoms such as lethargy, poor feeding, irritability, and fever. Neurological sequelae are common among survivors. Late, late-​onset disease  This is also called very-​late-​onset or GBS beyond early infancy. It occurs in infants more than 3 months of age and is more common in babies born before 28 weeks’ gestation or in those with underlying immunodeficiency.

section 8  Infectious diseases 972 Puerperal infection Puerperal infection with group B streptococci usually oc- curs within 24–​48 hours of delivery or abortion. The source of the organism is always the vagina and infection is more likely when there has been premature rupture of the membranes and chorioamnionitis. Most infections are endometritis with fever and uterine tenderness sometimes associated with retained products of conception, but group B streptococci can also cause wound infection after caesarean section. Bacteraemia is common. Other bacteria, both aerobes and anaerobes, are sometimes iso- lated from the genital tract and wounds in addition to the group B streptococcus. Very rarely the streptococcus may spread to other sites in puerperal women. Infection in non​pregnant adults The prominence given to group B streptococci as neonatal and puer- peral pathogens has tended to overshadow their importance in men and non​pregnant women in whom they cause significant morbidity and mortality. The incidence is 4 to 7 per 100 000 population, al- though rates as high as 26 per 100 000 have been reported in those aged over 65 years. In view of the reductions in GBS infection seen in pregnant women and infants, infection in non​pregnant adults now account for three-​quarters of invasive disease. Most infections are community-​acquired, occur in middle-​aged and elderly people, and are as common in men as women. Risk factors for invasive infection include diabetes mellitus, malignancy, alcoholism, chronic renal or liver disease, cardiovascular disease, collagen vascular diseases, and trauma. Skin and soft tissue infections are especially common in pa- tients with diabetes. Occasional urinary tract infections occur, in men as well as women. Bacteraemic infections serve to emphasize the virulence of group B streptococci, and they have increased in incidence, or perhaps have been increasingly recognized, since the early 1990s. Other clinical manifestations include endocarditis, ver- tebral osteomyelitis, septic arthritis, endophthalmitis, and menin- gitis. As with staphylococcal infections, some bacteraemic patients have more than one metastatic focus of infection, which can lead to diagnostic confusion. Laboratory diagnosis of group B streptococcal infection Group B streptococci are readily isolated from any clinical specimen and easily identified by Lancefield grouping. The group B antigen is not shared by any other streptococcus. Importantly the antigen can be reliably detected in fluids such as blood, urine, or cerebrospinal fluid by latex particle agglutination enabling a rapid diagnosis. It is routine practice to obtain third trimester vaginal cultures during the third trimester of infection. Treatment of group B streptococcal infection Group B streptococci are sensitive to penicillin and this is the anti- biotic of choice for treatment. They are rather less sensitive to peni- cillin than S.  pyogenes with minimum inhibitory concentrations some fourfold to tenfold higher. For this reason, penicillin is some- times combined with gentamicin for meningitis and other serious infections, though this is not of proven benefit. Certainly, the max- imum recommended dose of parenteral penicillin should be given whether combined with gentamicin or not. Penicillin allergy is not likely to be an issue in neonates; adults with meningitis can be treated with chloramphenicol. Most group B streptococci are sensi- tive to erythromycin and they are sensitive to cephalosporins. Prevention of neonatal infection with group B streptococci Intrapartum antibiotic prophylaxis Risk factors for early-​onset GBS disease include: delivery at less than 37 weeks’ gestation; premature rupture of membranes; prolonger rupture of membranes (>18 h before delivery); chorioamionitis; GBS bacteriuria during pregnancy; temperature >38°C during la- bour); sustained intrapartum fetal tachycardia; previous infant with GBS disease. These factors have been used to develop guidelines for the prevention of early-​onset GBS disease. The United States Centers for Disease Control recommends screening of pregnant women by culture at 35–​37 weeks’ gestation and intrapartum antibiotic prophy- laxis for women found to be colonized with GBS. This has resulted in a dramatic decline in the incidence of early-​onset GBS disease from 1.8 to 0.28 cases per 1000 live births between 1990 and 2008. In contrast the United Kingdom Royal College of Gynaecologists advo- cates a risk factor-​based approach, as there are no clinical trial data to support routine antibiotic prophylaxis and concerns related to antibiotic use. Recent analyses, however, suggest that culture-​based screening may be more cost effective than the current risk factor-​ based strategy. Vaccination The capsular polysaccharide antigens (Ia, Ib, II, III, IV, V, VI, and VIII) and C surface proteins of GBS have long been recognized to generate protective antibody responses. Analysis of genome sequences from GBS strains has identified several antigens and pro- teins that could potentially be used as vaccine candidates and some of these are in clinical trials. β-​Haemolytic groups C and G streptococci These streptococci are sometimes referred to as ‘large colony-​forming group C and G streptococci’ to distinguish them from the small colony-​forming strains of streptococci with the same Lancefield antigens that belong to the anginosus or milleri group (see next). Groups C and G streptococci are closely related genetically. They are most conveniently regarded as ‘pyogenes-​like’, as the infections they cause are similar to those caused by S. pyogenes though these streptococci tend to be less virulent than S.  pyogenes. Infections with these streptococci are less common than S. pyogenes infections. Although poststreptococcal glomerulonephritis has been associated with pharyngitis caused by both groups C and G streptococci, acute rheumatic fever has not. Group C streptococci are less frequently encountered in human infections than group G and most group C infections are caused by Streptococcus dysgalactiae subsp. equisilmi- lis. Those caused by Streptococcus dysgalactiae subsp. zooepidemicus have an animal source. Animal infections include mastitis in cows and ‘strangles’ in horses. Risk factors for infection with group C and G streptococci include: advanced age; underlying medical condition (e.g. diabetes mellitus, cardiovascular disease, cirrhosis, alcoholism, bone and joint disease, skin conditions); immunocompromise (e.g. malignancy, immunosuppressive drugs, HIV infection); surgical procedures; animal exposure. Clinical manifestations include pha- ryngitis, cellulitis, septic arthritis, bacteraemia, endocarditis, and a wide range of other infections.

8.6.2  Streptococci and enterococci 973 Streptococci of the anginosus or milleri group This group of streptococci has been a source of considerable taxonomic confusion, partly as a result of a lack of international consensus on no- menclature but also because of a lack of reliable phenotypic differences between taxa within the group. Most clinicians are familiar with the organism they know as ‘Streptococcus milleri’. There are three species of milleri streptococci, S. anginosus, S. constellatus, and S. intermedius, but despite increasing awareness of the clinical significance of the milleri group little is known about the association between individual spe- cies and specific sites of isolation and diseases. These streptococci are found in large numbers in the normal flora of the upper respiratory tract, gastrointestinal tract, and genital tract, and are commonly iso- lated from a range of pyogenic infections, sometimes in pure culture but often with other organisms, particularly anaerobes. These infec- tions include dental abscesses, intra-​abdominal abscesses (especially of the liver), subphrenic abscesses, lung abscesses and empyema, and brain abscesses. Such is the propensity of these organisms to cause deep-​seated abscesses that isolation of a milleri streptococcus from a blood culture should prompt investigations to detect such a focus. Milleri streptococci are also commonly isolated from inflamed appen- dices and postappendicectomy wound infection. Unlike other viridans and non​haemolytic streptococci, milleri streptococci seldom cause endocarditis. They form minute colonies on blood agar and are pref- erentially anaerobic on primary isolation. They may be α-​, β-​, or non​ haemolytic. Some have the Lancefield antigens A, C, G, or F. All group F streptococci are milleri group whereas not all milleri streptococci are group F. Another useful clue to their identity in the laboratory is the distinct caramel smell of many strains on blood agar, the result of the diacetyl metabolite. Most strains are very sensitive to penicillin. Streptococci of the mitis, salivarius, and
mutans groups (oral/​viridans streptococci) This group of usually α-​haemolytic (viridans) streptococci includes S. pneumoniae and those oral streptococci (S. mitis, S. oralis, S. san- guis, S. gordonii, and, rarely, S. salivarius) that are the most common cause of infective endocarditis of oral or dental origin. These strepto- cocci occasionally cause bacteraemia in neutropenic patients, who sometimes have detectable mouth lesions, and neonatal infection, as they are found as part of the normal vaginal flora. These infec- tions should be suspected in neutropenic patients who have received prophylaxis with fluoroquinolones such as ciprofloxacin. Streptococci of the bovis group Although this group comprises at least three species, S. bovis is the main species of medical importance. S. bovis is similar to the en- terococci in that it bears the Lancefield group D antigen and is a gastrointestinal commensal, but, unlike the enterococci, it is sen- sitive to penicillin. It can be misidentified in the laboratory either as an oral streptococcus or as an enterococcus. Most patients with S. bovis bacteraemia will have endocarditis and it is seldom isolated from other sites. It is important to recognize S. bovis in a blood culture as the organism is associated with colonic pathology or liver cirrhosis, and patients should be specifically investigated for these. With the advent of more comprehensive identification methods, Streptococcus bovis biotype I—the organism commonly associated with infective endocarditis and colorectal cancer—has been renamed S. gallolyticus subspecies gallolyticus. Nutritionally variant organisms previously classified as streptococci, now Abiotrophia spp. These organisms, which occasionally cause endocarditis, require pyridoxal or thiol group supplementation for growth in the la- boratory and tend to form satellite colonies surrounding colonies of Staphylococcus aureus. Although most blood culture media will support their growth, successful subculture requires supplementa- tion or cross-​streaking of the plates with Staphylococcus aureus to provide the necessary growth factors. The Abiotrophia include three species, S. adjacens, S. defectivus, and the recently described A. ele- gans. They are less susceptible to penicillin than other streptococci. Streptococcus suis This streptococcus, which can be misidentified in the laboratory as S. bovis or an enterococcus as it reacts with group D antiserum, is an important pathogen of young pigs causing meningitis, septicaemia, arthritis, pneumonia, and endocarditis and is also carried in the pharynx of healthy pigs. S. suis type II (also referred to as group R streptococci) is not only the most invasive type in pigs, it can cause serious infection—​mainly septicaemia and meningitis, but also septic arthritis, pneumonia, and endophthalmitis—​in humans, in whom it is an occupational disease of pig farmers, abattoir workers, and fac- tory workers handling pig meat (Fig. 8.6.2.7) (see Chapter 24.11.1). The streptococcus probably enters the bloodstream via skin abrasions that are common in the abovementioned occupations. S. suis type II meningitis results in deafness in about one-​half of those affected. Enterococci Enterococci are Lancefield group D, Gram-​positive cocci that can grow and survive in extreme cultural conditions, and are also more resistant to antibiotics than streptococci. They form part of the normal gut flora of humans and animals. Overall, the most common clinical isolates of enterococci are Enterococcus faecalis, but the more antibiotic-​resistant species E. faecium is increasingly encountered in hospitals. Nosocomial isolates of enterococci dra- matically increased in the 1990s. Other species, including E. cas- seliflavus, E. durans, and E. avium, are occasionally isolated. In most cases it is unnecessary to determine the species of enterococci in a clinical laboratory but sometimes differentiation between E. faeca- lis and E. faecium is helpful (e.g. in epidemiological studies and in endocarditis because of their different antibiotic susceptibilities). Infections caused by enterococci Enterococci are an increasingly important cause of nosocomial infection and colonization, possibly as a result of the large-​scale

section 8  Infectious diseases 974 use of antibiotics such as cephalosporins and quinolones to which they are inherently resistant. They occasionally cause community-​ acquired urinary tract infections, but the most important community-​acquired infection is endocarditis, which is increasing in incidence. This infection is almost always caused by E. faeca- lis. Any patient admitted from the community with E. faecalis in blood cultures should be assumed to have endocarditis until proved otherwise. Enterococci are predominantly hospital pathogens and cause urinary infection, particularly after instrumentation, intra-​ abdominal infections, wound infections (usually with other organ- isms), infections associated with intravascular devices and dialysis, and occasionally endocarditis. Antibiotic sensitivity and treatment Enterococci are not only intrinsically resistant to many antibiotics, but they also show a remarkable ability to acquire new mechanisms of resistance. This allows them to survive in environments in which large quantities of antibiotics are used and also has important therapeutic consequences, particularly for the treatment of endocarditis and other serious infections. Fortunately, many patients from whom entero- cocci are isolated do not require antibiotic treatment. Sensitive en- terococci cannot be killed by ampicillin/​amoxicillin alone, although combination with an aminoglycoside is bactericidal (synergy); but many strains now exhibit high-​level gentamicin resistance and for them the combination is not bactericidal. E. faecium is almost always resistant to ampicillin/​amoxicillin and E.  faecalis is occasionally. The first published report of vancomycin-​resistant enterococci was in 1988 from a London hospital outbreak, though such strains had been recognized a year before in Paris. Most strains of vancomycin-​ resistant enterococci in the London outbreak were E. faecium and overall most are E. faecium. There are four recognized phenotypes of vancomycin resistance; the first isolates of vancomycin-​resistant enterococci were highly resistant to vancomycin and teicoplanin and exhibit what is known as the VanA resistance phenotype. Since then, levels of resistance to teicoplanin in this phenotype have been more varied. Most VanA enterococci are E. faecium, but this phenotype also occurs in E. faecalis and occasionally in other species. The VanB phenotype is associated with low-​level vancomycin resistance and sensitivity to teicoplanin and is found in both E. faecalis and E. fae- cium. Both VanA and VanB are acquired traits. The VanC phenotype is an intrinsic property of E. casseliflavus and E. gallinarum and these species have low-​level resistance to vancomycin but are sensitive to teicoplanin. A fourth phenotype, VanD, has been described in a single strain of E. faecium. Vancomycin-​resistant E. faecium, though not vancomycin-​resistant E.  faecalis, is sensitive to quinupristin/​ dalfopristin and all vancomycin-​resistant enterococci are sensitive to the oxazolidinone linezolid. The antibiotic susceptibilities of the enterococci outlined here serve to emphasize that these bacteria are the most antibiotic-​resistant Gram-​ positive bacteria now encountered in hospital practice. Fortunately, many, perhaps most, of the patients from whom they are isolated do not require antibiotic treatment at all, but for those who do, the ef- fective treatment of serious infection caused by enterococci and par- ticularly antibiotic-​resistant strains requires microbiological expertise. FURTHER READING Bisno AL, Brito MO, Collins CM (2003). Molecular basis of group A streptococcal virulence. Lancet, 3, 191–​200. Bisno AL, Stevens DL (2000). Streptococcus pyogenes (including strepto- coccal toxic shock syndrome and necrotizing fasciitis). In: Mandell GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases, pp. 2101–​17. Churchill Livingstone, New York, NY. Colman G, et al. (1993). The serotypes of Streptococcus pyogenes pre- sent in Britain during 1980 to 1990 and their association with dis- ease. J Med Microbiol, 39, 165–​78. Davies MR, et al. (2015). Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin ac- quisition and multidrug resistance. Nat Genet, 47, 84–7. Edwards MS, Baker CJ (2000). Streptococcus agalactiae (group B streptococcus). In:  Mandell GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases, pp. 2156–​67. Churchill Livingstone, New York, NY. Jacobs JA (1997). The ‘streptococcus milleri’ group: Streptococcus ang- inosus, Streptococcus constellatus and Streptococcus intermedius. Rev Med Microbiol, 8, 73–​80. Katz AR, Morens D (1992). Severe streptococcal infections in histor- ical perspective. Clin Infect Dis, 14, 298–​307. (a) (b) Fig. 8.6.2.7  (a) S. suis septicaemia with meningitis in a Vietnamese pig farmer. (b) S. suis pyogenic arthritis in a Thai abattoir worker. (a) Copyright D. A. Warrell. (b) Courtesy of the late Professor Prida Phuapradit.

8.6.20 Francisella tularensis infection 1091

8.6.20 Francisella tularensis infection 1091

1091 8.6.20  Francisella tularensis infection review of the literature in the last 10 years. Int J Infect Dis, 14 Suppl 3, e242–​5. Medeiros I, Saconato H (2001). Antibiotic prophylaxis for mammalian bites. Cochrane Database Syst Rev, 2, CD001738. Morgan MS (2005). The hospital management of animal bites. J Infect, 61, 1–​10. Talan DA, et al. (1999). Bacteriologic analysis of infected dog and cat bites. N Engl J Med, 340, 85–​92. Wilson BA, Ho M (2013). Pasteurella multocida: from zoonosis to cel- lular microbiology. Clin Microbiol Rev, 26, 631–​55. 8.6.20  Francisella tularensis infection Petra C.F. Oyston ESSENTIALS Fransicella tularensis is a small Gram-​negative coccobacillus that circulates in small rodents, rabbits, and hares, most frequently in Scandinavia, northern North America, Japan, and Russia. Clinical presentation depends on the route of infection. Most commonly this follows the bite of an infected arthropod vector, resulting in ulceroglandular tularaemia. The most acute and life-​threatening dis- ease, respiratory or pneumonic tularaemia, arises following inhal- ation of infectious aerosols or dusts. The organism is highly fastidious, requiring rich media for isolation and specialized reagents for posi- tive identification; most cases are diagnosed serologically. Treatment is with supportive care and antibiotics (usually ciprofloxacin, doxy- cycline, or gentamicin). There is no vaccine. Historical perspective Francisella tularensis was first isolated during an outbreak of a plague-​like disease in rodents in California in 1911. Since then it has been recognized as a zoonotic infection of humans capable of causing significant morbidity or death. Human infection occurs following contact with infected animals or invertebrate vectors. It is also called Francis’ disease, deer fly fever, rabbit fever, water-​rat trappers’ disease, wild hare disease (yato-​byo), and Ohara’s disease. It is highly infectious by the aerosol route and, as such, has been of concern as a biological threat agent. Aetiology, genetics, pathogenesis, and pathology The genus Francisella includes two species, Francisella tularensis and Francisella philomiragia. F. tularensis is a small (0.2–​0.5 µm × 0.7–​1.0 µm) Gram-​negative coccobacillus that is non​motile and an obligate aerobe. The four subspecies of F. tularensis are: F. tularensis subsp. tularensis (also called F. tularensis type A or F. neoarctica); F. tularensis subsp. holarctica (also called F. tularensis type B); F. tula- rensis subsp. novicida and F. tularensis subsp. mediastica. Molecular typing methods have identified distinct genotypes of F. tularensis subsp. tularensis that differ in their grographic location and viru- lence. Most human and animal infections are caused by F. tularensis subsp. tularensis and F. tularensis subsp. holoarctica. Human dis- ease has also been reported with F. tularensis subsp. novicida and F. philomiragia. F.  tularensis can infect a variety of hosts including humans to cause tularaemia. An intracellular pathogen, it is one of the most highly infectious bacteria known with an infectious dose in hu- mans as low as 10 bacteria by the inhalational route. It multiplies to high levels within macrophages, and mutants unable to multiply in macrophages are avirulent. F. tularensis susbp. tularensis causes more severe infections than F. tularensis susbp. holoarctica. F. tula- rensis susbp. tularensis genotype A1b are more likely to be associated with invasive disease and higher mortality than type A1a, A2, or B. F. tularensis multiplies at the site of inoculation and spreads to the regional lymph nodes and then systemically. An acute inflam- matory reaction with neutrophils, macrophages, and lymphocytes is seen at the site of inoculation, resulting in tissue necrosis and, some- times granuloma formation. F. tularensis is an intracellular pathogen that replicates primarily on host macrophages. Macrophage uptake occurs by an unusual process called ‘looping phagoyctosis’ that in- volves symmetric and spacious pseudopod loops. Once ingested, phagosome maturation and phagosome–​lysosome fusion are im- paired resulting in organism escape and multiplication in the cytosol. Similar events occur in neutrophils where the organism suppresses oxidative burst and escapes into the cytoplasm. Within the macro- phage cytosol the organisms activate a multimolecular complex, the inflammasone, which leads to the release of proinflammatory cyto- kines and trigger caspase-​1 dependent cell death. The virulence of F. tularensis has been correlated with several phenotypic character- istics including capsule formation, lipopolysaccharide, pili, produc- tion of acid phosphatases, and a siderophore. Genomic studies have identified genes in the Francisella pathogenicity island which are in- volved in intracellular survival and animal virulence. Epidemiology F. tularensis is mainly isolated in the northern hemisphere, most frequently in Scandinavia, northern America, Japan, and Russia (100–​400 cases/​year), but has never been isolated in the United Kingdom. F. tularensis subsp. tularensis accounts for 90% of infec- tions in North America whereas F. tularensis subsp. holarctica in- fections are more common in the rest of the world. The organism infects more than 100 species of wild and domestic vertebrates, such as small rodents, rabbits, hares, squirrels, hamsters, mice, and voles. Outbreaks in human populations frequently mirror outbreaks of disease occurring in wild animals. A wide range of arthropod vec- tors have been implicated in the transmission of the disease within wild animal populations and to humans. Rural populations, espe- cially those individuals who spend periods of time in endemic areas such as farmers, hunters, walkers, and forest workers, are most at risk of contracting tularaemia. Transmission to humans occurs from contact with an infected animal or biting insect. Contaminated meat and water are important environmental sources of the infection

section 8  Infectious diseases 1092 and outbreaks associated with contaminated water supplies can involve large numbers of cases. Transmission can also occur from airborne spread of contaminated materials such as dust, hay, and water. Airborne transmission was the suspected source of an out- break of pneumonic tularaemia in the United States of America and was associated with lawn mowing or brush cutting. Recent reports of tularaemia have been from Russia (following a sable bite), nor- thern Spain (possibly associated with aerosolized contaminated water), and the United States of America (Utah). Prevention Avoidance of contact with infected animals and vectors reduces the risk of infection. Hunters in particular should wear gloves when skinning dead animals, and meat should be thoroughly cooked be- fore eating. Reducing the risk of inhalation of infectious dusts (e.g. during farming activities in endemic areas) by wearing respiratory protection should be considered. No licensed vaccine is available for prevention of tularaemia. However, concern about the use of Francisella as a bioterrorism threat has led to continuing efforts to develop a vaccine. Clinical features Tularaemia in humans can occur in several forms depending on the route of infection. Although tularaemia can be a severely debilitating and even fatal disease, especially when caused by viru- lent strains, many cases of disease caused by lower virulence strains go undiagnosed owing to the non​specific nature of the symptoms. The incubation period is normally 3 to 5 days (range 1–​21 days), and patients develop influenza-​like symptoms which may be pro- tracted and relapsing if untreated. Infection through skin or mucous membranes Infection through the skin results in ulceroglandular tularaemia (Figs. 8.6.20.1, 8.6.20.2); where no ulcer is reported, this is termed glandular tularaemia. These forms of tularaemia are the most common presentations of the disease and can arise following the bite of an infected vector or through direct contact with the flesh of an infected animal. A lesion develops at the site of infection, often a single papule which turns into an ulcer surrounded by a zone of inflammation. The ulcer is relatively painless and heals within a week. Within 3 to 5 days following infection, the patient develops fever, chills, malaise, headaches, and a sore throat. The local draining lymph nodes become enlarged and painful, like a bubo. Lymphadenopathy can take a significant period to resolve even with treatment, and without treatment suppuration occurs in approximately 30% of patients. Symmetrical rashes have been attrib- uted to hypersensitivity (Fig. 8.6.20.3). Less commonly, infection can occur through the conjunctiva. This is termed oculoglandular tularaemia and arises following direct contamination of the eye (e.g. through rubbing the eyes after skin- ning an infected rabbit). The patient develops conjunctivitis in the infected eye, swollen eyelids, and a purulent discharge. Untreated, the infection can spread to the local lymph nodes, in a similar way to ulceroglandular tularaemia. Ingestion of infected meat can result in oropharyngeal (Fig. 8.6.20.4) or gastrointestinal tularaemia. Ulcers, pharyngitis, and swollen cervical lymph nodes develop, and a yellow-​white pseudomembrane might be seen in oropharyngeal tularaemia. Gastrointestinal tularaemia can range from a mild but persistent diarrhoea to an acute fatal disease with extensive ulceration of the bowel, depending on the size of the infecting dose. Any of the aforementioned infections can disseminate and pro- gress to systemic disease without the appearance of swollen lymph nodes or ulcers. This is termed typhoidal tularaemia. Severe compli- cations might also occur, such as septic shock. Infection through inhalation Inhalation of F. tularensis results in respiratory or pneumonic tu- laraemia. Pneumonia can also arise following haematogenous spread in other forms of tularaemia. Symptoms can be variable and depend on the virulence of the strain involved. Infection with the most highly virulent strains can have a case fatality rate of up to 30% if untreated, but antibiotic therapy reduces this to approxi- mately 2%. Presentation can range from a mild pneumonia to an Fig. 8.6.20.1  Hands in a case of ulcero-​(cutano-​)glandular tularaemia. Courtesy of A Berglund, Fallund, Sweden. Fig. 8.6.20.2  Inguinal lymphadenopathy in ulceroglandular tularaemia. Courtesy of A Berglund, Fallund, Sweden.

1093 acute infection with high fever, malaise, chills, cough, delirium, and pulse–​temperature dissociation. Radiological examination might reveal parenchymal infiltrates, most commonly in one lobe, and hilar lymphadenopathy can be present. Differential diagnosis Diagnosis of tularaemia is difficult due to the non​specific nature of most of the symptoms, particularly if the ulcer has already healed. A high index of clinical suspicion is therefore required. Other dis- eases which must be rapidly excluded in patients presenting with acute respiratory distress and fever or influenza-​like disease include plague and Q fever (Table 8.6.20.1). Oculoglandular tularaemia can be confused with severe infection caused by a range of viral and bac- terial conjunctival pathogens. Criteria for diagnosis Most cases of tularaemia are diagnosed on the basis of the epidemio- logical and clinical picture. The diagnosis is confirmed serologically and a range of serological tests for the detection of antibodies against F. tularensis is commercially available. The antibody response peaks at 4–​6 weeks, but can be detected from 2 weeks. Routine cultures of specimens such as blood, sputum, pleural fluid, skin lesions, and lymph nodes are frequently negative as the organism is fastidious and requires enriched media and prolonged culture. The micro- biology laboratory should be alerted to the possibility of F. tularensis as it is a biohazard group 3 pathogen and should be processed in a containment level 3 laboratory. Most strains require cystine or cyst- eine for growth and more than 2 days’ incubation at 35°C to produce colonies. Some strains grow on conventional media such as choc- olate agar, modified Thayer–​Martin medium, or buffered charcoal yeast agar. The organisms are tiny, poorly staining Gram-​negative coccobacilli and are oxidase negative, weakly catalase positive, β-​ lactamase positive, urease negative, and satellite or XV test nega- tive. Polymerase chain reaction and enzyme-​linked immunosorbent assay (ELISA) can be used to positively identify the bacteria, both following isolation and in specimens. Such direct detection of the pathogen is useful in patients who are serologically negative (e.g. in the early days of infection). Treatment Antimicrobial therapy should be administered to patients in whom tularaemia is suspected or confirmed. There are no randomized controlled trials comparing the efficacy of different drug regimens. Historically, aminoglycosides have been the drugs of choice for the treatment of tularaemia. Although it is clinically effective, strepto- mycin is rarely used now; gentamicin is a suitable alternative and is usually given for 7 to 14 days. For patients with milder disease, oral therapy with tetracyclines or fluoroquinolones has been recommended. Doxycycline is ef- fective in the treatment of tularaemia and can also be used in chil- dren and pregnant women. Ciprofloxacin has been shown to be highly effective and can be considered the current drug of choice for uncomplicated tularaemia. It has been shown to be effective in treating tularaemia in children and may be suitable for use in preg- nant women. Both tetracyclines and fluorquinolones have been as- sociated with relapse after cessation of treatment. Fig. 8.6.20.3  Hypersensitivity reaction in infection with Francisella tularensis. Courtesy of A Berglund, Fallund, Sweden. Fig. 8.6.20.4  Oral tularaemia in a case from northern Sweden. Courtesy of A Berglund, Fallund, Sweden. 8.6.20  Francisella tularensis infection

8.6.21 Anthrax 1094

8.6.21 Anthrax 1094

section 8  Infectious diseases 1094 Meningitis should be treated with an aminoglycoside in combin- ation with chloramphenicol. Supportive care should be provided as appropriate; some patients may require intensive care with respiratory support should sepsis develop. Suppurating nodes should be drained. Prognosis Tularaemia responds well to antibiotic therapy, especially if started early in infection. The mortality rate of the more acute forms of the disease is reduced from 30% to 2% if the patient receives appropriate antibiotics. Most deaths are associated with pneumonic or typhoidal forms. Relapse can occur when antibiotic therapy is withdrawn (even with aminoglycosides or fluoroquinolones). Other issues Patients are not considered an infection risk and do not require iso- lation. Tularaemia is notifiable in some countries, although not in the United Kingdom. Autopsies should only be performed by personnel wearing res- pirators if death from tularaemia is suspected. Bodies should not be embalmed before burial. Likely future developments Work is under way to identify a vaccine against tularaemia that will be suitable for licensing. It is highly likely that progress will be made in this area in the next few years, although it can take many years to obtain approval. FURTHER READING Centers for Disease Control and Prevention. Emergency preparedness and response:  tularaemia. https://​emergency.cdc.gov/​agent/​tular- emia/​index.asp Dennis DT, et al. (2001). Tularemia as a biological weapon: medical and public health management. JAMA, 285, 2763–​73. Health Protection Agency (2007). Tularemia. http://​webarchive. nationalarchives.gov.uk/​20070129155246/​http://​www.dh.gov. uk/​PolicyAndGuidance/​EmergencyPlanning/​DeliberateRelease/​ DeliberateReleaseTularemia/​fs/​en Jacobs RF, Condrey YM, Yamauchi T (1985). Tularemia in adults and children: a changing presentation. Pediatrics, 75, 818–​22. Mulligan MJ, et al. (2017). Tularemia vaccine: safety, reactogenicity, ‘take’ skin reactions and antibody responses following vaccination with a new lot of the Francisella tularensis live vaccine strain—​a phase 2 randomized clinical trial. Vaccine, 35, 4730–​7. 8.6.21  Anthrax Arthur E. Brown ESSENTIALS Anthrax is primarily a disease of herbivorous mammals, caused by the Gram-​positive rod Bacillus anthracis, which causes human infec- tion when its spores enter the body, most commonly from handling infected animals or animal products. The disease occurs in most countries of the world, but is only sporadic where the condition is controlled in livestock by vaccination programmes. Anthrax is a leading agent of biological warfare. Pathophysiology—​after entry into the body, anthrax spores are phagocytosed by macrophages and carried to regional lymph nodes, where they germinate to produce vegetative bacilli that enter the blood stream. These produce anthrax toxins, which have effects including impairment of cellular water homeostasis and of many intracellular signalling pathways. Clinical features—​anthrax occurs in four clinical forms based on the route of exposure. (1) Cutaneous—​lesions are usually found on exposed areas of skin; a small papule develops at the site of in- fection, enlarges, and ulcerates, with the painless ulcer becoming covered with a black leathery eschar surrounded by non​pitting oedema before healing in 2 to 6 weeks; associated systemic symp- toms are usually mild. (2)  Gastrointestinal—​acquired by eating Table 8.6.20.1  Differential diagnosis of tularaemia Tularaemia Differential diagnosis Ulceroglandular Pyogenic bacterial infection, orf, pasteurella infections, syphilis, chancroid, lymphogranuloma venereum, scrub typhus, streptococcal and staphylococcal cellulitis, mycobacterial infections (including tuberculosis), sporotrichosis, herpes simplex virus, anthrax Glandular Pyogenic bacterial infection, cat-​scratch disease, toxoplasmosis, mycobacterial infections, sporotrichosis, streptococcal and staphylococcal adenitis, syphilis, plague Oropharyngeal Streptococcal pharyngitis, infectious mononucleosis, adenoviral infection, diphtheria Oculoglandular Pyogenic bacterial infection, cat-​scratch disease, herpes simplex virus, syphilis, adenovirus Typhoidal Enteric fever, brucellosis, leptospirosis, malaria, Q fever, rickettsial infection, toxic shock syndrome, endocarditis Gastrointestinal Enterohaemorrhagic E. coli, GI anthrax, Clostridium perfringens, listeriosis Respiratory Q fever, other atypical bacterial pneumonias (mycoplasma, Chlamydia pneumoniae, legionnaires’ disease, psittacosis), viral pneumonia (influenza, hantavirus, respiratory syncytial virus, cytomegalovirus), tuberculosis, pneumonic plague

8.6.21  Anthrax 1095 contaminated food and comprising (a) oropharyngeal anthrax, pre- senting with fever, neck swelling, sore throat, oropharyngeal ulcer, and dysphagia, OR (b) terminal ileal/​caecal anthrax, presenting with fever, nausea, vomiting, and abdominal pain, followed by rapidly developing ascites and bloody diarrhoea. (3) Inhalation—​aerosol exposure leads to a non​specific viral-​type prodrome which pro- gresses to a fulminant stage of severe respiratory distress, cyanosis, stridor, and profuse sweating; up to half of patients develop an- thrax meningitis; shock and death typically follow in hours or days. (4) Injection—​acquired by injection of heroin contaminated with anthrax spores. Symptoms range widely, but usually include inflam- mation and oedema at the injection site (no eschar), which may be complicated by compartment syndrome, necrotizing fasciitis, and sepsis. Diagnosis—​may be very difficult in the absence of a known outbreak, particularly for inhalation anthrax where a clinical clue is widening
of the mediastinum caused by lymphadenopathy. Confirmation is by laboratory identification of B. anthracis. Serological testing can be used for retrospective diagnosis. Treatment—​this is with supportive care and antibiotics, which are effective against the multiplying (vegetative) form of B. anthracis, but not against the spore form. Mild cases of cutaneous anthrax are usu- ally treated with oral penicillin. For gastrointestinal, inhalational, and meningeal anthrax, at least two antibiotics should be given intra- venously; for example, ciprofloxacin or doxycycline along with an- other antimicrobial expected to be effective (penicillin, ampicillin, rifampin, vancomycin, chloramphenicol, imipenem, clindamycin, and clarithromycin). Prognosis—​the mortality of untreated cutaneous anthrax is 10–​20%,
but fatalities are rare with appropriate antibiotic treatment. Almost all cases of inhalation anthrax and anthrax meningitis are fatal; initiation of treatment after the start of fulminant disease is rarely effective. Prevention—​routine immunization of livestock should be instituted in endemic areas with continuing cases of animal anthrax. Carcasses of animals suspected of dying from anthrax must be disposed of ap- propriately. Anthrax vaccines should be offered to members of high-​ risk groups (those at occupational risk, laboratory workers, and some military groups). Postexposure prophylaxis should be given following suspected exposure to aerosolized anthrax spores (e.g. ciprofloxacin for 60 days). Introduction Anthrax is a zoonosis caused by Bacillus anthracis, a bacterium which can infect most species of mammals. Herbivores are particu- larly susceptible to B. anthracis, acquiring the infection most often through contact with spores in the soil. The bacteria multiply rap- idly to high concentrations in these animals which, upon death and spilling of blood and secretions, sporulate on the carcass and soil. Products from the spore-​contaminated carcass provide the mode of B. anthracis exposure to humans. Thus, it is control of the infection in animals that is crucial to preventing anthrax in humans. Effective control relies on (1) vaccination of livestock and (2) proper disposal of infected animal carcasses. Anthrax occurs among animals in most countries of the world. It is still prevalent in epidemic or endemic form in many countries of Asia, Africa, and the Middle East. In North America and Australia anthrax has become rare. At the same time anthrax has gained in importance due to its potential as a biological weapon. Appreciation of this threat has increased since 2001 and spurred development of improved methods of prevention, diagnosis, and treatment. Historical importance In agricultural settings, anthrax has been recognized for more than two thousand years. With industrialization, anthrax became a problem for workers processing animal products which originated in endemic/​epidemic countries. Today, use of B. anthracis as a bio- weapon is perceived in developed countries as its major threat to the public health. Prominent reminders are the accidental release of B. anthracis spores from a Soviet military facility in 1979 and their deliberate release by letter in the United States of America in 2001. In the late 1800s, non​agricultural exposure to B. anthracis spores carried by animal hides and wool led to cases of cutaneous and in- halation anthrax (‘woolsorters’ disease’) in industrializing countries. In Liverpool, a disinfection station was established where imported wool and other animal fibres were bathed in formaldehyde. This public health measure led to a marked decrease in cases in England, but the model was not followed elsewhere. B. anthracis played a central and interesting role in the birth of medical microbiology. In the 1870s, Robert Koch cultured the or- ganism on artificial media, described the vegetative and spore phases of its life cycle, and demonstrated disease causality by ful- filling ‘Koch’s Postulates’. Louis Pasteur added extensively to the anthrax-​based evidence for the germ theory of disease and studied the attenuation of the organism. In the early 1880s, Pasteur in France and Greenfield in England independently demonstrated that heat-​attenuated strains of B. anthracis protected sheep, goats, and cows from anthrax. This dis- ease of livestock was of enormous economic importance in Europe and in less than 20 years, millions of sheep and cattle had been given this first animal vaccine. But stable attenuation of strains so that they were both safe and immunogenic remained challenging. It was 40 years later that Max Sterne developed such a strain of B. anthracis. This live attenuated strain became and remains the basis for the an- thrax vaccines used globally to protect livestock. Aetiology B.  anthracis combines a rapidly multiplying, vegetative phase in animals which resists phagocytosis and produces a lethal toxin-​ mediated disease with a spore phase which resides dormant in the environment. The vegetative phase is a large, non​motile Gram-​ positive rod. In clinical specimens it has a large capsule and occurs singly or in short chains that appear as ‘jointed bamboo’ rods. The vegetative form sporulates when it is deprived of essential nutri- ents. The spore, the infectious form of the organism, consists of a compact core protected by multiple concentric shells (cortex, coat, exosporium; see Fig. 8.6.21.1). They resist heat, desiccation, ultra- violet light, gamma irradiation, and some disinfectants.

section 8  Infectious diseases 1096 B. anthracis is a highly clonal species with a genome consisting of a single circular chromosome (5.2 Mbp) and two plasmids, pX01 (182 Kbp) and pX02 (95 Kbp). The homogeneity of B.  anthracis strains constrained studies of transmission and epidemiology in the past. But research, accelerated after the anthrax letters in the United States (2001), led to strain-​distinguishing genetic techniques. Strains can now be typed and distinguished based on single nucleo- tide polymorphisms (SNPs) and variable number tandem repeat (VNTR) markers. Strains of B. anthracis fall into three major lineages (A, B, C) with lineage A causing more than 90% of infections globally. The SNP and VNTR techniques allow further subdivision of the B. anthracis population into 12 subgroups and more than 220 unique genotypes. Future analyses of yet unstudied isolates and strains emerging in outbreaks due to long dormant spores are expected to further ex- pand these numbers. Ecology Anthrax is a zoonosis with a life cycle passing through ani- mals (primarily herbivores) and the environment (largely soil). B. anthracis spores infect a mammal where they germinate and multiply as vegetative bacilli. If the animal dies, these bacilli spill on the ground via blood and secretions, and start to sporulate upon exposure to air. Soil containing spores becomes a reservoir for subsequent exposure of other animals. New infections occur via ingestion of vegetation or soil, inhalation of dust, or bites of flies each of which could carry spores. Animals vary greatly in susceptibility to parenterally intro- duced spores with lethal doses (LD50) ranging from less than 10 to more than 107 spores. However, the LD50 is some thousand-​fold higher, even in susceptible animals, when the spores must over- come healthy and intact skin or mucosa. On the spectrum of sus- ceptibility, herbivores are highly susceptible while humans are moderately so. The factors leading to animal outbreaks are complex and incom- pletely understood. The death of a bacteraemic animal is likely to lead to spore production when ambient temperature and humidity is high. Flies can transfer spores to nearby foliage, increasing the incidence of infection in other browsing animals (‘case multi- pliers’) or infect animals more remotely through biting (‘space multipliers’). The blood and nutrients which enter the soil from the dead animal may produce more grass in that contaminated spot and attract other browsers to this risk area. Rains, while washing spores off nearby foliage, tend to drain in ways that concentrate spores in depressions which may become sites of outbreaks (‘hot spots’) years or decades later. Epidemiology Forms of disease Anthrax in humans has traditionally been classified based on ei- ther occupation (agricultural vs. industrial) or route of infection (cutaneous, ingestion, inhalation). A more recent classification (see Table 8.6.21.1) also distinguishes deliberate release. Human cases in the agricultural setting (usually cutaneous form) result from direct contact with infected animal carcasses, generally by herders, butchers, and slaughterhouse workers. Industrial cases involve workers who have contact, either directly or via aerosol, with spore-​contaminated animal products such as hides, goat’s hair, wool, or bone. Gastrointestinal anthrax follows ingestion of B.  anthracis-​ contaminated food, often by villagers who are not aware that the animal was sick and so do not insure that meat is well cooked. Gastrointestinal cases are underdiagnosed in endemic regions. Inhalation anthrax is caused by alveolar deposition of the 1–​2 µm spores. Historically those working with herbivore hides in indus- trial mills were at risk, but naturally acquired inhalation anthrax is now rare. Fig. 8.6.21.1  Transmission electron micrograph of B. anthracis spores showing core, surrounded by cortex, coat, and exosporium with filamentous ‘hairy nap’. Courtesy of Joel Bozue, USAMRIID. Table 8.6.21.1  Classification of human anthrax based on source of infection or mode of acquisition Anthrax classification Based on source of infection Agricultural Traditional grouping Industrial Traditional grouping Laboratory Rare modern event Bioweapon—​deliberate release Potentially important modern event Based on mode of acquisition Cutaneous Most common form Gastrointestinal Underappreciated in endemic areas Inhalation Likely due to deliberate release Injection European outbreak (contaminated heroin)

8.6.21  Anthrax 1097 Burden and distribution of disease The worldwide incidence of human anthrax is not known, but is es- timated to be about 2000 cases annually, of which some 95% are cu- taneous. Based on reporting of anthrax outbreaks in animals, the World Health Organization (WHO) characterizes several countries in Africa, the Middle East, and Asia as hyperendemic or epidemic (see Fig. 8.6.21.2). Many other countries in these regions, as well as in southern Europe and the Americas, are considered endemic while most remaining countries have at least sporadic cases. The largest reported outbreak of agricultural anthrax in recent times occurred in Zimbabwe in the late 1970s during the civil war. Most of the estimated 10 000 human cases were cutaneous, while a small number were gastrointestinal. Disruption of veterinary health services, especially anthrax vaccination, led to outbreaks among cattle and other livestock, and an associated epidemic in humans. Outbreak examples An outbreak of the oropharyngeal form of anthrax occurred in Thailand in 1982 when 24 people developed anthrax after eating undercooked meat from infected cattle and water buffalo. In Switzerland in 1991, workers in one textile factory contracted an- thrax (24 cutaneous and one inhalation case) from contaminated Pakistani goat hair. An unusual outbreak of inhalation anthrax oc- curred in 1979 among residents of Sverdlovsk in the former Soviet Union. Spores accidentally released into the atmosphere from a military laboratory were carried downwind and caused at least 77 cases of human inhalation anthrax (66 deaths) and the deaths of many animals. Since 2000, there have been deaths among drug users in Europe due to B.  anthracis-​contaminated heroin. Seventy laboratory-​ confirmed cases, including at least 26 deaths, have been reported from the United Kingdom, Denmark, France, and Germany. Clinical presentations lacked a typical recognizable pattern. Genetic studies suggested that B. anthracis in these cases was from two introduc- tions of, similar strains. Deliberate release State-​sponsored biological weapons programmes often selected an- thrax as an ideal organism. It is easily obtained and cultured, spores are very stable and small enough to reach alveoli when aerosol- ized, and inhalation infections are usually fatal. In the early 1970s more than 140 countries signed or ratified the Biological Weapons Convention, agreeing to terminate offensive weapons programmes and destroy existing weapons stockpiles. Monitoring compliance of this convention remains problematic. Anthrax has also been favoured by terrorist groups. In the early 1990s, members of the Aum Shinrikyo cult dispersed aerosols of B. anthracis spores over a Japanese city. Fortunately there was no Hyperendemic/epidemic Probably free Free Unknown Endemic Sporadic Fig. 8.6.21.2  Global anthrax epidemiology by country. Status is colour coded (see key) on this map. Adapted from the World Anthrax Data Site a website at the Louisiana State University WHO Collaborating Center (data as of 2003).

section 8  Infectious diseases 1098 disease outbreak because the cult had used an avirulent (Sterne) strain. In 2001, at least five letters containing anthrax spores (the virulent Ames strain) were mailed in the United States to several government and news offices. This led to 11 cases of inhalation anthrax with five deaths, and another 11 suspected or confirmed cases of cutaneous anthrax. The outbreak made clear how effi- ciently B. anthracis spores could be aerosolized. Thus, while the threat of human anthrax due to natural exposure has lessened in most developed countries, the threat of its deliberate release has increased. Pathogenesis General Transmission of anthrax to humans is via spores entering the skin or gastrointestinal or respiratory tracts. Spores are phagocytosed by macrophages and dendritic cells, and germinate intracellularly ei- ther at the local site or in draining lymph nodes. Many genes are expressed in the resulting vegetative bacilli, including a set located on plasmids and responsible for virulence. Genes on plasmid pX02 code for a poly-​D-​glutamic acid which forms a capsule resistant to phagocytosis. Strains which lack this capsule are avirulent and form the basis of most anthrax vaccines. Genes expressed on the other plasmid (pX01) guide the syn- thesis of two exotoxins. Within hours of germination, vegetative bacilli start to synthesize the three proteins [protective factor (PA), lethal factor (LF, 85-​kDa) and oedema factor (EF, 89-​kDa)] which combine to form the anthrax toxins: lethal toxin (LT; PA + LF) and oedema toxin (ET; PA + EF). These exotoxins interfere with neutrophil and macrophage function. Thus, early on the combined effect of the capsule and exotoxins is to weaken the protective re- sponse of the innate immune system and allow rapid expansion of the B. anthracis population. The infection may become systemic with bacteraemia reaching 107–​108 bacilli/​ml. In the late stage of disease, the high levels of toxaemia produced by the large biomass of bacilli cause multiorgan damage with particular targeting by LT of the cardiovascular system (heart and vasculature) and by ET of the liver. The biology of the anthrax toxins and their cellular effects are complex. Soluble PA binds to cell surface receptors where it is cleaved by a furin protease. The larger C-​terminal fragment (PA63) remains bound and forms oligomers on cell surface lipid rafts where multiple copies of EF and/​or LF bind. The whole complex is internal- ized through endocytosis and EF/​LF are released into the cytoplasm through a channel within the PA oligomer. EF augments the conver- sion of adenosine triphosphate (ATP) to cAMP while LF inactivates key protein kinase pathways. The metabolic consequences include immune compromise, massive oedema, and the organ failure of se- vere anthrax. Organ-​specific When spores of B. anthracis are introduced cutaneously, they ger- minate, multiply, and produce exotoxins resulting in local tissue necrosis, oedema, and a paucity of leucocytes. Spread to draining lymph nodes results in haemorrhagic, oedematous, and necrotic lymphadenitis. Gastrointestinal anthrax follows ingestion of food contaminated with B. anthracis spores. Localization, germination, and multiplica- tion of bacilli in the oropharynx and regional lymph nodes causes oropharyngeal ulcers, localized oedema, and neck swelling. Spores carried further tend to localize in the ileum or caecum and cause mucosal inflammation, ulcers, and ascites. These bacteria drain to mesenteric lymph nodes causing haemorrhagic adenitis. Inhalation anthrax follows deposition of spores in alveoli, phagocytosis, transport to tracheobronchial and mediastinal lymph nodes, and intracellular germination. In the mediastinum, haemorrhage, oedema, and necrotic lymphadenitis develop. Since this is not a pneumonia, sputum examination does not reveal the organism. Injection anthrax occurs a few days after injection of heroin con- taminated with B. anthracis spores. It is characterized by massive local oedema which may be associated with local complications, dis- semination and/​or sepsis. All primary forms of anthrax can be complicated by septicaemia and, less often, haemorrhagic meningitis. These complications are especially frequent with inhalation anthrax and should be assumed for treatment decisions. Criteria for diagnosis The diagnosis of anthrax may be suspected on clinical and epi- demiological grounds, and is confirmed by laboratory identification of B. anthracis. Clinical specimens containing large Gram-​positive rods, singly and in short chains of 2–​4 cells, should be interpreted as possible Bacillus species. Demonstration of encapsulation of these bacilli by India ink, Giemsa’s, or polychrome methylene blue stains leads to a presumptive identification of B. anthracis. Culture isolates have classic morphological characteristics:  Gram-​positive, broad spore-​forming rods; intracellular oval spores which do not swell the vegetative cell; non​motile; ‘ground-​glass’ colonies which are (nearly always) non​haemolytic on sheep blood agar. Standard con- firmatory tests include lysis by specific γ-bacteriophage and direct immunofluorescent assays for cell wall or capsular antigens on the vegetative cells. Serological testing is not helpful at the onset of symptoms but can be used for retrospective diagnosis. Specific antibodies are detect- able by enzyme-​linked immunosorbent assay (ELISA), with testing of paired samples preferred. Polymerase chain reaction tests for the PA gene has been used but is not yet standardized. Delayed-​type hypersensitivity assessed by antigen skin test (anthraxin) was devel- oped in Russia for detection of prior infection or vaccination. Other technologies include immunohistochemistry for PA and capsule antigens, and genetic sequencing. While culture loses much utility after the start of antibiotics, toxin-​based assays may remain positive for up to another six days. Clinical features Cutaneous anthrax Anthrax acquired its name from the Greek description of the skin lesion’s characteristic eschar as being the colour of coal (Greek

8.6.21  Anthrax 1099 anthrakos = coal). These cutaneous lesions (Fig. 8.6.21.3) are usu- ally found on exposed areas of skin, such as the face, neck, arms, or hands, and may be single or multiple depending on the extent of exposure. The incubation period is usually 2–​5 days. The lesion starts as a small papule at the site of infection and then enlarges and ulcerates. The depressed ulcer becomes covered with a black lea- thery eschar surrounded by non​pitting oedema that is occasionally massive (‘malignant oedema’). Established lesions are characteris- tically painless and may be hypoaesthetic. (Pain suggests secondary infection.) Small satellite vesicles, containing many organisms and few white cells, may surround the original lesion. Regional lymph- adenitis is common while systemic symptoms are usually mild. Lesions heal slowly (2–​6 weeks) without scarring. In 10–​20% of un- treated patients the disease becomes systemic, with bacteraemia and toxaemia. Gastrointestinal anthrax Gastrointestinal anthrax is acquired by eating contaminated and inadequately cooked meat from an infected animal, and thus often occurs in social or familial clusters. The disease has an incubation period of 2–​5 days and occurs in two forms. Oropharyngeal anthrax follows deposition of bacteria in the oro- pharynx. Patients present with fever, neck swelling, sore throat, and dysphagia. The neck swelling is caused by enlargement of the jugular lymph nodes together with subcutaneous oedema as in diphtheria. Enlarged nodes and local oedema may obstruct the airway. The mu- cosal lesion starts as an area of inflammation, progressing through necrosis and ulceration. A pseudomembrane (but no eschar) forms over the ulcer (see Fig. 8.6.21.4). Subcutaneous oedema may ex- tend to the anterior chest wall and axilla, with inflammation of the overlying skin. In the more common form of gastrointestinal anthrax, B. anthracis organisms are deposited in the terminal ileum or caecum, occasion- ally in more proximal parts of the gastrointestinal tract. Disease onset is non​specific with fever, nausea, vomiting, and abdominal pain, followed by rapidly developing ascites and bloody diarrhoea. Haematemesis, melaena, haematochezia, and/​or profuse watery diarrhoea may occur. Presentation may be that of an acute abdomen. In severe cases, toxaemia, shock, and death follow. Early diagnosis is difficult, except when suspected due to the epidemiological setting, and the disease is likely underreported. Inhalation anthrax Inhalation anthrax results from the deposition of aerosolized spores in pulmonary alveoli. The incubation period ranges from 1 to 5 days, with modelling suggesting it is inversely related to (a) (b) (c) Fig. 8.6.21.3  Cutaneous anthrax. (a) Early ulcerated skin lesion with forming eschar. (b) Large lesion with extensive eschar on a Nigerian patient who carried an infected carcass on his shoulder. (c) Ulcer with satellite lesions on a Thai patient. Copyright D. A. Warrell. Fig. 8.6.21.4  Oropharyngeal anthrax in a Thai man showing extensive lesion in posterior pharynx with pseudomembrane. From Sirisanthana T, et al. (1984). Outbreak of oral-​pharyngeal anthrax: an unusual manifestation of human infection with Bacillus anthracis. Am J Trop Med Hyg, 33, 144–​50.

section 8  Infectious diseases 1100 dose. The prodrome is non​specific with malaise, myalgia, fever, and non​productive cough—​similar to that of viral respiratory dis- eases. Some patients improve transiently after 2 to 4 days of symp- toms. A fulminant stage then follows, bringing severe respiratory distress, cyanosis, stridor, and profuse sweating. Subcutaneous oedema of the chest and neck may develop. A characteristic radio- graphic finding is mediastinal widening with clear lung fields. By advanced imaging techniques (CT or MRI), nearly all patients have mediastinal lymphadenopathy as well as pleural effusions (Fig. 8.6.21.5). Inhalation anthrax becomes systemic and commonly involves the central nervous system—​about half of patients develop men- ingitis. Shock and death typically occur less than 24 h into the ful- minant phase. Autopsies of persons dying of inhalation anthrax revealed serosanguinous pleural effusions and haemorrhagic oe- dema in the mediastinum where lymph nodes had haemorrhagic necrosis. About half of cases had cerebral oedema and about 20% had ascites. Injection anthrax Injection anthrax is caused by the injection of heroin which is con- taminated with spores of B. anthracis. Clinical presentations are di- verse and inconsistent, and thus quite challenging. Most common are erythema and oedema near the injection site, occurring within 1–​3 days of injection. Eschars are not found. Infection of the arm can lead to compartment syndrome or necrotizing fasciitis; pain may be very severe. The infection often becomes systemic with toxaemia, sepsis, and a rapid death. Meningeal anthrax Meningo-​encephalitis, associated with B.  anthracis bacteraemia, may complicate any primary form of anthrax. (Anthrax menin- gitis without a known primary site occurs but rarely.) Within a few days of the primary lesion the patient suddenly develops confusion, loss of consciousness, and/​or focal neurological signs. The cerebro- spinal fluid contains a high concentration of organisms and may be haemorrhagic. Differential diagnosis The differential diagnosis of anthrax varies by type. For cutaneous anthrax, the differential diagnosis includes staphylococcal or streptococcal skin infections, ulceroglandular tularaemia, bubonic plague, bites of brown recluse spiders, orf, rickettsial pox, and scrub typhus. For oropharyngeal anthrax, the differential diagnosis in- cludes diphtheria and peritonsillar abscess. For inhalation anthrax, the differential diagnosis includes pneumonic plague, histoplas- mosis, sarcoidosis, tuberculosis, and lymphoma. Clinical investigations Only one anthrax vaccine has been tested in a controlled, human efficacy trial. This was in the 1960s with a product similar to today’s Anthrax Vaccine Adsorbed. The trial showed the vaccine to be effi- cacious. Vaccines since have been studied for safety and immuno- genicity in humans, and efficacy in animals. For practical and ethical reasons human efficacy studies are unlikely. There is a similar challenge for the evaluation of passive immune therapies. Prior to the development of penicillin, anthrax antiserum was given as treatment for anthrax. Since 2001, human polyclonal anthrax immune globulin (collected from human vaccinees) has been stockpiled in the United States and used in a handful of cases. A monoclonal anti-​PA antibody has been developed and also stock- piled. Neither of these antitoxin therapies has been evaluated in con- trolled, human efficacy trials. Treatment Antibiotics are effective against the multiplying (vegetative) form of B. anthracis, but not against the spore form. At least two should be used in combination for all severe anthrax disease. To counter the toxaemia, it is recommended that the combination include an in- hibitor of protein synthesis. Severe disease will also require intensive supportive care. Cutaneous anthrax has been treated successfully with penicillin through much of the world. But while penicillin sensitivity is the norm, the organism possesses an inducible β-​lactamase. Thus, al- ternatives to penicillin-​based drugs should be used (1) in severe or systemic disease; (2) in outbreaks where the drug has been given to at-​risk animals; and (3)  when deliberate release is suspected. Given those caveats, cases of non​systemic cutaneous anthrax can be treated with penicillin for 7–​10 days (orally with penicillin VK 250 mg every 6 h or parenterally with penicillin G 2 million units every 6 h). Ciprofloxacin, erythromycin, doxycycline, or chloram- phenicol can be used in penicillin-​sensitive patients. Antibiotics decrease the likelihood of systemic disease and thus mortality, but the time to resolution of skin lesions is unchanged. Skin lesions Fig. 8.6.21.5  CT image of an American adult with inhalation anthrax showing mediastinal enlargement secondary to lymphadenopathy. Note small bilateral pleural effusions and nearly clear lung fields. From Jernigan JA, et al. (2001). Bioterrorism-​related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis, 7, 933–​44.

8.6.21  Anthrax 1101 should be covered with sterile dressings and used dressings should be decontaminated. In gastrointestinal, inhalational, and meningeal anthrax, at least two high-​dose intravenous antibiotics should be given. If naturally acquired, penicillin G (4 million units every 4 h) has been the drug of choice; ciprofloxacin (400 mg every 12 h) or doxycycline (100 mg every 12 h) are currently recommended in the United States. All cases of systemic anthrax should be considered as high risk for developing meningitis. Thus, only drugs with good penetration of the central nervous system should be used. Anthrax caused by a deliberate release will generally be ac- quired by inhalation. In this setting, drug resistance due to gen- etic modification is of concern and drug sensitivity testing of the organism is imperative. Treatment should begin intravenously with ciprofloxacin or doxycycline, along with one or two other antimicrobials expected to be effective. Rifampicin, vancomycin, chloramphenicol, imipenem, clindamycin, and clarithromycin are candidates. Prognosis The mortality of untreated cutaneous anthrax is 10–​20%. With appropriate antibiotic treatment, fatalities become rare (<1%). Mortality of oropharyngeal anthrax is about 15% in treated patients; mortality of the more common form of gastrointestinal anthrax is uncertain, but estimated at about 40%. Nearly all cases of inhalation anthrax have been fatal. An excep- tion to this general experience occurred with the anthrax letter cases (United States in 2001) in which mortality was about 50%. Survival in that setting was found to be associated with three factors: initi- ation of multidrug treatment during the prodromal stage, repeated drainage of pleural effusions and extensive supportive measures. Despite these improvements, inhalation anthrax which progresses to the fulminant phase is nearly 100% fatal, as it is for those who de- velop meningo-​encephalitis. Prevention Control of anthrax in animals decreases the likelihood of human exposure and is the key public health measure for preventing human disease. Immunization of livestock should be instituted in endemic areas with continuing cases of animal anthrax. The basis for nearly all animal (non​human) vaccines against anthrax is a live attenu- ated (non​encapsulated) strain of B. anthracis developed in South Africa by Max Sterne in the 1930s. This was a huge public health advance and converted a scourge of man and animals into an occa- sional problem. Cases of animal and human anthrax should be reported to the appropriate authorities. Carcasses of animals, domestic or wild, sus- pected of dying from anthrax should be incinerated along with the nearby vegetation, in a manner that also sterilizes the underlying soil. If this is not possible, the carcass should be buried intact to a depth of six feet or more to decrease the likelihood of scavenger ani- mals digging it up and opening the body. Gastrointestinal anthrax of humans can be prevented by avoidance, ideally, of meat from sick animals or at least proper cooking when contamination is suspected. Anthrax vaccines should be offered to members of high-​risk occu- pational groups, such as laboratory workers and some veterinary and military groups. Current anthrax vaccines for humans are all produced from at- tenuated strains of B. anthracis and do not use live bacilli. The United Kingdom and the US vaccines are made from cell-​free culture fil- trates and induce antitoxin immunity. PA is the sufficient and seem- ingly necessary antigen in these vaccines, and hence its name. The licensed vaccine in the United States is Anthrax Vaccine Adsorbed (AVA); it is given intramuscularly at 0, 1, 6, 12, and 18 months. More than 95% of vaccinees are seropositive after the first three doses. The licensed vaccine in the United Kingdom is Anthrax Vaccine Precipitated (AVP); it is given intramuscularly at 0, 3, 6, and 26 weeks. The Russian vaccine is a suspension of live spores of attenuated strain STI-​1 and was first licensed in 1953. It is given either by scarification through a drop of vaccine or subcuta- neously at 0 and 3 weeks. The Chinese vaccine is a spore suspension of attenuated strain A16R and has been in use since the 1960s. It is given by scarification and boosted at 6 to 12 months. All these vac- cines require booster doses (usually annually) to maintain protective immunity. Drawbacks of the current cell-​free vaccines (AVA and AVP) are the incomplete characterization of the vaccine and the complex, lengthy immunization schedules. These drawbacks, along with the increased possibility of a deliberate release, have renewed efforts to validate shorter, simpler immunization regimens, and to develop alternative vaccines. A recombinant PA vaccine has progressed to clinical testing. Additional antigens and adjuvants are being ex- plored, as are new approaches which include live vaccines, DNA, and carrier vectors. Postexposure prophylaxis is given to those with suspected ex- posure to aerosolized anthrax spores (deliberate release). Oral ciprofloxacin, levofloracin, doxycycline, and penicillin G procaine have been approved for this indication in the United States. For preg- nant and lactating women and for children, amoxicillin is recom- mended. A lengthy (60-​day) course of antibiotics is used because they are not effective until spores germinate and the organism be- comes metabolically active. Effective antibiotics limit multiplication of B. anthracis, but success here removes the stimulus for protective immune responses. Therefore, disease may occur after cessation of antibiotics, when postexposure prophylaxis compliance is poor, or if the B. anthracis strain is drug resistant. For these reasons, concur- rent vaccination (3 SC doses of AVA given at 2-​week intervals) is recommended along with antibiotics as postexposure prophylaxis in the United States. Special circumstances The WHO has estimated that 50 kg of B. anthracis spores released over a city of 5 million people would infect 250 000 people, killing 40% of them. Numbers would be influenced by the quality of the aerosol, dispersal method, and weather conditions. Cases would be largely inhalation and the intensive medical care required would overwhelm the medical surge capacity of most cities. Antibiotics and vaccine for postexposure prophylaxis would be

8.6.22 Brucellosis 1102

8.6.22 Brucellosis 1102

section 8  Infectious diseases 1102 needed in great quantities. These realities will challenge prepared- ness planners. Challenges and future developments Methods for detection of spores in the environment, especially in the atmosphere, will improve. But specificity of these assays will remain challenging, since true-​positives will be rare and false-​positives will be costly and socially disruptive. The mechanisms by which anthrax toxins compromise immune responses and cause organ failure will become more clear and re- sult in improved therapies. New vaccines and simpler immunizing schedules will become available. Concern will remain that the increasing capacity to genetically modify B. anthracis strains may lead to deliberate releases with organisms that are resistant to antibiotics and/​or have altered vac- cine target sites. FURTHER READING Berger T, et al. (2014). Injectional anthrax—​new presentation of an old disease. Euro Surveill, 19, pii: 20877. Beyer W and Turnbull PCB (2009). Anthrax in animals. Molecular Aspects of Medicine, 30, 481–9. Brachman PS, et al. (1962). Field evaluation of a human anthrax vac- cine. Am J Public Health, 52, 632–​45. CDC (2010). Use of anthrax vaccine in the United States. MMWR Morb Mortal Wkly Rep, 59, 1–​30. Davies JCA (1982). A major epidemic of anthrax in Zimbabwe, part 1. Cent Afr J Med, 28, 291–​8. Friedlander AM, et  al. (2017). Anthrax vaccines. In:  Plotkin SA, et  al. (eds) Vaccines, pp. 134–​48. 7th edition. Elsevier Saunders, Philadelphia, PA. Ganz HH, et al. (2014). Interactions between Bacillus anthracis and plants may promote anthrax transmission. PLoS Negl Trop Dis, 8, e2903. Hendricks KA, et al. (2014). CDC expert panel meetings on preven- tion and treatment of anthrax in adults. Emerg Infect Dis, 20, http://​ dx.doi.org/​10.3201/​eid2002.130687 (Internet; cited 29 June 2018). Holty J-​EC, et al. (2006). Systematic review: a century of inhalation an- thrax cases from 1900 to 2005. Ann Intern Med, 144, 270–​80. Inglesby TV, et al. (2002). Anthrax as a biological weapon, 2002: up- dated recommendations for management. JAMA, 287, 2236–​52. Keim P, et al. (2015). Whole genome analysis of injectional anthrax identifies two disease clusters spanning more than 13 years. E Bio Medicine, 2, 1613–8. Liu S, et al. (2014). Anthrax lethal and edema toxins in anthrax patho- genesis. Trends in Microbiol, 22, 317–​25. Meselson M, et al. (1994). The Sverdlovsk anthrax outbreak of 1979. Science, 266, 1202–​8. Plotkin SA, et al. (1960). An epidemic of inhalation anthrax, the first in the twentieth century: I. Clinical features. Am J Med, 29, 992–​1001. Rasko DA, et al. (2011). Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation. Proc Natl Acad Sci USA, 108, 5027–​32. Sirisanthana T, et al. (1984). Outbreak of oral-​pharyngeal anthrax: an unusual manifestation of human infection with Bacillus anthracis. Am J Trop Med Hyg, 39, 144–​50. Turnbull PCB, Shadomy SV (2011). Anthrax from 5000 BC to AD 2010. In: Bergman NH (ed) Bacillus anthracis and anthrax, pp. 1–​15. John Wiley & Sons, London. Van Ert ML, et  al. (2007). Global genetic population structure of Bacillus anthracis. PLoS One, 2, e461. 8.6.22  Brucellosis Juan D. Colmenero and Pilar Morata ESSENTIALS Brucellosis is a worldwide zoonotic disease. It remains endemic in the Mediterranean basin, Northern Africa, the Middle East, Western Europe, Central and South America, sub-​Saharan Africa, the Indian subcontinent, and Central Asia. There are three species especially pathogens for humans; Brucella melitensis (most commonly asso- ciated with goats, sheep, and camels), B. abortus (cattle) and B. suis (pigs). Brucellosis is usually transmitted by direct contact with infected animals, by ingestion of untreated dairy products, and less frequently by inhalation (laboratory workers) or inoculation (veterinary). Clinical features—​symptoms are very non​specific and hetero- geneous, hence epidemiological information collected in the
clinical history is very important. In most of cases the infection manifests as a febrile syndrome with no apparent focus, comprising chills, profuse sweating, asthenia, arthralgia, and myalgia. Between 20 and 40% of patients develop focal complications, which can af- fect any organ or system, especially the locomotor and genitourinary system. Diagnosis and treatment—​definite diagnosis always requires la- boratory confirmation, either by isolating the organism from blood, body fluids or tissues, or by demonstration of high titres of specific antibodies or seroconversion. In addition to adequate symptomatic and supportive measures, treatment is based on the administration of doxycycline plus an aminoglycoside or rifampicin. Doxycycline plus streptomycin/​gentamicin regimen is more effective and results in fewer therapeutic failures and relapses. Prevention—​human brucellosis can be prevented by eradicating the disease in animals by vaccination. Other preventive measures include occupational hygiene (especially in exposed professionals such as farmers, shepherds, abattoir workers, butchers, and labora- tory workers), avoiding keeping farm animals in close proximity to houses, and avoiding the consumption of unpasteurized raw milk or its by-​products. Aetiological agent The Brucella genus belongs to the Alphaproteobacteria family. Six species are recognized within the genus Brucella:  B.  abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae. This clas- sification is mainly based on the difference in host preference

8.6.22  Brucellosis 1103 and in pathogenicity. They are small non​encapsulated non​motile non​sporulating intracellular facultative Gram-​negative aerobic ba- cilli. Several new species have recently been described, including at least two species in marine mammals (B. ceti in dolphins, porpoises, and whales, and B. pinnipedialis in seals) and an additional species, B. microti, in the common vole (Microtus arvalis). Epidemiology Brucellosis is a worldwide zoonotic disease. It remains a serious public health problem in many low-​ and middle-​income coun- tries. In the past 15 years, the epidemiology of human brucellosis has evolved. Brucellosis continues to be endemic in some coun- tries of the Mediterranean basin, Northern Africa, the Middle East, Western Europe, Central and South America, sub-​Saharan Africa, the Indian subcontinent, and Central Asia. The incidence of brucel- losis varies widely between and within countries. While some areas, such as Peru, Kuwait, and parts of Saudi Arabia, have a very high incidence, the low incidence reported in other known brucellosis endemic areas may reflect low levels of surveillance and reporting. Demographic, occupational, and socioeconomic factors may also play a role in these differences. Brucella spp. can infect a wide variety of both domesticated and feral animals. B. melitensis (goats, sheep, and camels) is the most pathogenic to humans, followed at some distance by B.  abortus (cattle), and B. suis (pigs). Exceptionally, B. canis can be pathogenic for humans but no human cases have been reported due to B. ovis or B. neotomae. The pathogenicity for humans of the different marine Brucella species (B. ceti and B. pinnipediae) found in cetaceans and pinnipeds remains to be established. The control and eradication of animal brucellosis is very diffi- cult because infection is often not apparent in cattle, sheep, goats, or other ruminants, as well as ancient practices such as nomadism and the involvement of different wild animals sharing a habitat with herds. This helps to explain the re-​emergence of brucellosis. Brucella spp. can survive for long periods in dust, dung, water, slurry, aborted animal fetuses, soil, meat, and dairy products. The precise duration of survival is dependent on many variables such as the nature of the substrate, number of organisms, temperature, pH, and sunlight. Brucellosis can be transmitted to humans through consumption of unpasteurized dairy products or through direct or indirect contact with infected animals, placentas, aborted fetuses, or a contaminated environment, as well as by inhalation, conjunc- tival contamination, skin cuts or abrasions, and accidental self-​ inoculation with live vaccines. Certain professions, such as farmers, shepherds, abattoir workers, veterinarians, and laboratory workers, are at particular risk. Human-​ to-​human transmission by blood transfusion, tissue transplantation, or sexual contact has been reported occasionally but is nevertheless exceptional. Pathogenesis Brucella species can survive and replicate inside macrophages and non​professional phagocytic cells, thus explaining the tendency of brucellosis to have a prolonged clinical course and to relapse. Soon after Brucella penetrates skin or mucosa there is migration of ac- tivated macrophages to the site of invasion. Brucella later passes through the lymphatic vessels to regional lymph nodes, then via the blood stream to all organs of the body, particularly those rich in reticuloendothelial tissue. Organ localization is associated with inflammatory cellular infiltrates with or without granuloma forma- tion, necrosis, or even abscess formation. Experimental data in murine models suggest that brucella may use different strategies to evade the host immune response, including evasion of intracellular destruction by restricting fusion of type IV secretion system-​dependent brucella-​containing vacuoles with lysosomal compartments, inhibition of apoptosis of infected mononuclear cells, and prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells. Inhibition of phagosome-​lysosome fusion has been proposed as the main mech- anism for intracellular survival of brucella, and lipopolysaccharide O-​antigen as the major virulence factor that governs the early be- haviour of bacteria inside macrophages. The initial response is neither antigen nor organism specific (in- nate immunity), involving γδ T-​cell (Vγ9Vδ2), natural killer, and CD4 and CD8 T-​cell activation. Lipopolysaccharide on the surface of brucella is recognized by these cells, which activate macrophages and facilitate phagocytosis. Activated γδ T cells may provide the initial γ-​interferon (INFγ), tumour necrosis factor-​α (TNFα), and other cytokine secretions which become cytotoxic for brucella-​ infected monocytes and the bacteria, impairing their intracellular survival. Most brucella are rapidly eliminated by phagolysosome fu- sion. Killing inside macrophages is initiated by cytokines secreted by T-​helper cells. Macrophages activate TNFα secretion, initiating a complex cascade of host defence mechanisms, resulting in hydro- lytic enzymes and the peroxide–​halide system (‘oxidative-​burst’ or ‘oxygen-​based killing’). Some brucella survive in compartments which are rapidly acidified. Brucella resists lysosome-​mediated killing and phagosome acidification. The mechanism of trafficking of the brucella-​containing phagosome within macrophages and the lack of fusion with the lysosome is not understood. Brucella multi- plies in the macrophage endoplasmic reticulum without affecting host-​cell integrity. The organisms are released by cell lysis and ne- crosis. In the early stage of infection, brucella activates the cAMP/​ PKA pathway, which regulates a variety of mechanisms favouring infection. In the first week of infection, antilipopolysaccharide IgM appears in the serum, followed one week later by IgG and IgA which peak during the fourth week. Antilipopolysaccharide anti- bodies have a limited role in host protection, but are important for diagnosis. Clinical features The incubation period of brucellosis ranges between seven days and three months, although incubation periods of as long as 10 months have been reported, thereby explaining how the symp- toms can begin at a very distant place from where the infection was acquired. This is an important fact to consider in the case of febrile syndromes in travellers returning from countries where brucel- losis is endemic. Epidemiological data of patients with brucellosis are shown in Table 8.6.22.1. The distribution by age and gender is shown in Fig. 8.6.22.1.

section 8  Infectious diseases 1104 Brucellosis is a systemic infection with a wide clinical spectrum, ranging from asymptomatic forms to severe cases causing death. Symptoms can start suddenly or gradually. In most cases the infec- tion manifests as a febrile syndrome with no apparent focus, with chills, profuse sweating, asthenia, malaise, arthralgia, and myalgia. Table 8.6.22.2 shows the main symptoms and signs of brucellosis at the time of diagnosis. Brucellosis lacks a typical pattern of fever. Usually patients have a predominantly evening fever which is indistinguishable from other febrile syndromes. Because the clinical picture is very non​specific, the differential diagnosis of brucellosis is very broad. Therefore, epidemiological data collected in the clinical history are very im- portant. Classifying brucellosis as acute or chronic is of no clinical interest and provides no useful information concerning diagnosis or treatment. Brucellosis can affect virtually any organ or system, causing focal forms with long clinical courses, which are considered true com- plications of the infection. In the absence of focal complications, physical examination of patients with brucellosis is non​specific. Approximately 30% of patients have hepatomegaly and 20% spleno- megaly; less than 5% have maculopapular rash. Notable discrepancies exist in the incidence rate and clinical spectrum of focal forms. Some authors consider these discrepan- cies to be a consequence of the lesser virulence of some Brucella spp., especially B. abortus. However, this fact does not explain the low incidence of focal forms in some reports referring to B. meli- tensis. These discrepancies seem to be mainly related to the retro- spective character of the reports and the lack of uniform definitions and diagnosis of focal forms. Around 20–​40% of patients with brucellosis develop a focal com- plication in the clinical course of their disease. These focal forms of brucellosis have been described in almost all organs and sys- tems, with the osteoarticular and genitourinary forms being more common and those affecting the heart and the central nervous system more severe. Table 8.6.22.3 shows the focal complications of a large cohort of adult patients with brucellosis in the Regional University Hospital in Malaga, Spain. Osteoarticular complications represent between 60 and 70% of the focal complications of brucellosis. The disease can affect the musculoskeletal system at virtually any site, but in adults the axial skeleton is the most frequently involved. Sacroiliitis and vertebral osteomyelitis (spondylodiscitis) account for more than 80% of cases with osteoarticular involvement. This tendency for axial in- volvement in adults may be related to closure of the metaphysis, change in bone vascularization, and redistribution of the bone marrow from the long to the axial bones, which occurs from the second decade of life. In vertebral osteomyelitis the lumbar segment is the most fre- quently involved, followed by the thoracic, and finally the cervical segments. Multiple level involvement is rare, accounting for less than 5% of cases. Although brucellar spondylodiscitis is considered a mild form of infectious vertebral osteomyelitis (Fig. 8.6.22.2), in our own experience a considerable percentage of patients required surgical treatment because of the development of paravertebral and/​or epidural masses (Figs. 8.6.22.3 and 8.6.22.4), psoas abscess Table 8.6.22.1  Epidemiological data for brucellosis in Spaina Number of cases Percentage Gender Male Female 655 293 69.1 30.9 Origin Urban Rural 356 592 37.6 62.4 Possible source of infection Habitual or occasional contact with goats or sheep Habitual or occasional contact with goat or sheep products Consumption of unpasteurized dairy products Multiple sources of infection Other sources of infection Unknown source of infection 124 72 241 382 51 88 13.1 6.6 25.5 40.3 5.4 9.3 a Based upon 948 patients with B. melitensis infection seen at the Regional University Hospital of Malaga between 1982 and 2013. 0 20 40 60 80 100 120 140 160 < 20 21–30 31–40 41–50 51–60 61–70 71–80 >80 year Age distribution by sex Male Female Fig. 8.6.22.1  Age and gender distribution of patients with brucellosis. Table 8.6.22.2  Summary of symptoms and signs at the time of diagnosisa Symptoms Number
of cases Percentage Fever 932 98.3 Chills 802 84.6 Sweating 790 83.3 Constitutional symptomsb 678 71.5 Arthralgia 456 48.1 Myalgia 380 40.1 Spinal pain 179 18.9 Testicular pain (referred to 655 males) 49 7.5 Signs Hepatomegaly 319 33.6 Splenomegaly 183 19.3 Lymphadenopathy 65 6.9 Skin rash 24 2.5 a Based upon 948 patients with B. melitensis infection seen at the Regional University Hospital of Malaga between 1982 and 2013. b Constitutional symptoms = two or more of the following: anorexia, asthenia, malaise.

8.6.22  Brucellosis 1105 (Fig. 8.6.22.5), spinal cord or radicular compression, or instability of the column. Sacroiliitis, together with vertebral osteomyelitis, is the most frequent osteoarticular involvement in adult patients. It almost in- variably occurs unilaterally, and in those cases of bilateral involve- ment it is usually asymmetric, which helps to differentiate it from non​infectious sacroiliitis. Peripheral arthritis is a frequent form of osteoarticular brucellosis. The knee and ankle are the most fre- quently affected joints, two-​thirds of which were monoarthritis and one-​third oligoarthritis. The age of patients with peripheral involve- ment or sacroiliitis is significantly lower than in patients with ver- tebral osteomyelitis. Large bone osteomyelitis, relatively frequent a few decades ago, is now exceptional. After osteoarticular complications, the most common focal complications of brucellosis are those affecting the genitourinary system. Epididymo-​orchitis is, without doubt, the most fre- quent genitourinary complication, affecting 2–​20% of males with brucellosis. Pathogens of the genus Brucella possess great tropism for the genitourinary system of their usual hosts (cows, goats, sheep, and pigs), which has been associated with the high con- centration of erythritol in the testicular and placental tissues of these animals. This carbohydrate is a known stimulus for growth of Brucella spp. Although erythritol is not present in the male re- productive system, the high concentration of other carbohydrates could account for the incidence of human epididymo-​orchitis. Although the prognosis of brucellar epididymo-​orchitis is usually good, delay in diagnosis or inappropriate management may result in serious complications, such as testicular abscess, that may then require orchiectomy. Although Brucella spp. can be isolated in the urine of patients with acute infection, renal involvement is unusual. On rare occasions, renal involvement severely compromises renal function and even the life of the patient. The pathogenic mechanism of renal involve- ment is multiple. In most cases, lesions are secondary to interstitial nephritis caused by direct invasion of the bacteria. In other cases, the renal lesion has a glomerular predilection as a consequence of the deposition of circulating immune complexes. In these cases, the coexistence of an underlying endocarditis is frequent. Neurologic complications, although infrequent (2–​5% in the lar- gest series of brucellosis reported) are of marked clinical importance due to their severity and important sequelae. The clinical spectrum of neurobrucellosis is variable. Different clinical pictures have been described, including meningitis, meningoencephalitis, intracerebral haemorrhage, benign intracranial hypertension, optic neuritis, arachnoiditis, polyradiculoneuritis, and myelitis. The lesions are located mainly in the meninges, where a diffuse in- flammatory infiltrate can be observed extending to the perineurium of the nerve sheaths and to the vessel walls. These pathologic findings explain the wide clinical spectrum of neurobrucellosis. Inflammatory changes in the cerebrospinal fluid are a constant feature of neurobrucellosis. Most patients have lymphocytic pleo- cytosis (between 20 and 500 cells/​ml), elevated total protein, and hypoglycorrhachia. Brucella can be isolated from cerebrospinal fluid in 35–​50% of cases of meningitis or meningoencephalitis. Cardiac involvement in brucellosis is rare. The prevalence of car- diovascular involvement in large series of adult brucellosis ranges from 1 to 2%. Endocarditis is the most common cardiovascular com- plication, and a large proportion of cases (over 50% in some series) involve a previously healthy native valve. The aortic valve is involved in more than 75% of cases. Although a complete cure has occasion- ally been achieved with medical treatment alone, most patients require surgical treatment because of haemodynamic instability. Pericardial and myocardial involvement is relatively common in pa- tients with Brucella endocarditis, though this can also happen as an isolated event, in which case the prognosis is more favourable. Liver involvement is very frequent in brucellosis, although it is usually limited to soft, painless hepatomegaly, or slight increases in levels of aminotransferases. These clinical and biochemical abnor- malities are completely reversed with adequate treatment. We do not believe they represent a true complication of the disease. The con- cept of hepatic complication should be reserved for those cases with clinical expression (right upper quadrant pain and/​or jaundice) in the presence of severe disturbances of the biochemical liver param- eters or liver abscess. Defined in these terms, 2–​4% of adult patients have hepatic complications. Table 8.6.22.3  Description of focal complications in patients with active brucellosisa Location Number of cases Percentage Osteoarticular 208 21.9 Genitourinary 47 4.9 Neurologic 19 2.0 Hepatic 17 1.8 Cardiovascular 12 1.3 Other focal complications 20 2.1 More than one focal complication 26 2.7 Total 323 34.1 a Based upon 948 patients with B. melitensis infection seen at the Regional University Hospital of Malaga between 1982 and 2013. Fig. 8.6.22.2  Early brucellar lumbar vertebral osteomyelitis. Decreased height of the intervertebral disc with anterior superior epiphysitis.

section 8  Infectious diseases 1106 Among the hepatosplenic focal forms of brucellosis, the presence of chronic hepatosplenic abscesses is an uncommon, but severe, complication. Chronic hepatosplenic abscess appears to be a clin- ical entity with its own characteristics, which clearly differentiate it from splenic infarctions with abscesses, occasionally associated with endocarditis, and from small, asymptomatic abscesses detected in acute bacteraemic forms of brucellosis. The diagnostic yield of abdominal CT in hepatosplenic abscess is very high (Fig. 8.6.22.6), not only because it enables 100% of the lesions to be detected, but also because it defines with much greater precision than ultrasound the extent of the lesion, some of which present such a characteristic morphology that their finding in endemic areas should lead us to consider the diagnosis of brucellosis. Fig. 8.6.22.3  Brucella melitensis vertebral osteomyelitis. A, T1-​weighted MR sagittal image showing decreased signal with poor definition of the upper end plate. B, T2-​weighted MR showing increased disk signal and osteolysis, corresponding to a more evolved vertebral osteomyelitis. Fig. 8.6.22.4  Psoas abscess secondary to lumbar brucellar vertebral osteomyelitis. Fig. 8.6.22.5  T1-​weighted MR sagittal image after gadolinium administration showing an epidural abscess in a patient with Brucella melitensis vertebral osteomyelitis.

8.6.22  Brucellosis 1107 Other digestive system complications of brucellosis are excep- tional, although cholecystitis, ileitis, colitis, and pancreatitis have been reported. Diagnosis Due to its heterogeneous and poorly specific clinical symptom- atology, the diagnosis of brucellosis always requires laboratory con- firmation, either by isolating the pathogen or by demonstration of high titres of specific antibodies or seroconversion. The definitive diagnosis of brucellosis requires the isolation of the organism from the blood, body fluids, or tissues. Peripheral blood is the clinical sample most commonly used for isolation of Brucella spp. In acute forms produced by B. melitensis the yield of blood cultures is usually high, reaching 70–​80% of cases. This is not- ably reduced, however, in cases of long illness, in patients with focal complications and in patients with infections caused by B. abortus and B.  suis. Occupational acquisition has occurred in laboratory workers handling blood cultures containing brucella. If the infection is suspected, cultures should be handled in a safety hood to reduce this risk. The semi-​automated methods (BACTEC 9204 or Bac/​Alert) considerably shorten the time taken for detection; the presence of brucella can be detected between the third and seventh day of incubation. Recently, some authors have proposed polymerase chain reaction (PCR)-​based assays for the direct detection of Brucella organisms in blood and other clinical samples. Some studies have shown that real-​time PCR assays are far more sensitive than conventional cul- tures, and this, coupled with its speed and reduction in risk to la- boratory workers, could make this technique a very useful tool not only for the initial diagnosis of brucellosis, but also for the diagnosis of focal complications and the differentiation between active and past brucellosis. Despite the apparent advantages that PCR-​based methods have over conventional microbiological tests for the diag- nosis of human brucellosis, their application in clinical practice re- mains very limited. More experience is still needed before deciding whether this can replace the traditional cultures. Despite the important advances made in the diagnosis of human brucellosis following the introduction of new semi-​automated methods for blood culture processing, diagnosis of this disease is still based mostly on the demonstration of specific antibodies by means of different serological techniques. This is mainly because the greatest incidence of brucellosis is found in underdeveloped coun- tries with poor technical resources, as well as the fact that it tends to occur in rural communities. The major brucella antigen useful for diagnosing human brucel- losis is the smooth lipopolysaccharide of the outer cell membrane. Several serological tests are available for the diagnosis of human brucellosis, including the Rose Bengal test (RBT), standard agglu- tination, Coombs anti-​Brucella test, immunocapture-​agglutination test, and ELISA. All of these have good sensitivity, but lack the de- sired specificity in highly endemic areas, in occupationally exposed patients, or those with a recent history of brucellosis. Moreover, all these serological tests can produce cross-​reactions with other bac- teria, including Yersinia enterocolitica O:9, Escherichia coli O:157, Francisella tularensis, Salmonella urbana O:30 and Vibrio cholera. The strategy most widely used for the serological diagnosis of bru- cellosis consists of a combination of a rapid screening test, such as the RBT, and a confirmation test, such as standard agglutination, Coombs anti-​Brucella test, immunocapture-​agglutination test, or ELISA. The RBT is a simple and rapid plate agglutination test that uses a suspension of B. abortus in an acid buffer. It can detect agglutinating and non​agglutinating antibodies and avoids the prozone phenom- enon. The sensitivity of the RBT is greater than 95%, regardless of the stage of the disease. The serum (tube) agglutination test (SAT) is a very useful test for the diagnosis of human brucellosis when it is performed with a standardized antigen preparation. The RBT can be used qualitatively or quantitatively. The quantitative RBT test demonstrates sensitivity and specificity equivalent to that achievable by performing SAT. The immunocapture-​agglutination test (Brucellacapt®) has re- cently been introduced in the diagnosis of human brucellosis. A good correlation between the results of Brucellacapt and Coombs antibrucella test has been reported, though the immunocapture-​ agglutination test is technically easier to perform and to interpret. In our experience, RBT titres ≥1/​4, SAT ≥1/​160, or immuno-​ agglutination test ≥1/​320 in patients with a compatible clinical pic- ture can be considered diagnostic of active brucellosis. In highly endemic areas, in patients with a history of brucellosis or with focal complications, the serological diagnosis often requires the com- bined use of RBT, SAT, and the immunocapture-​agglutination test. Treatment Brucella species are sensitive in vitro to many antimicrobial drugs. However, the results of routine susceptibility tests do not always cor- relate with clinical efficacy. Tetracyclines, aminoglycosides, and ri- fampicin are the mainstay of treatment of brucellosis. Trimethoprim/​ sulfamethoxazole and quinolones are also useful alternatives. Acute uncomplicated brucellosis almost invariably responds well to appropriate antimicrobial treatment. Unfortunately, the results achieved with monotherapy in the treatment of brucellosis have been suboptimal. For this reason, the usual treatment is based on a combination of two antimicrobial drugs. Fig. 8.6.22.6  Abdominal CT with oral and intravenous contrast showing a brucellar splenic abscess.

section 8  Infectious diseases 1108 Currently, the optimal treatment of uncomplicated brucellosis should be based on a six-​week regimen of doxycycline (100 mg/​12 h, orally) combined with streptomycin for 2–​3 weeks. With this thera- peutic schedule, the time to disappearance of fever is usually less than a week. Although gentamicin, in a dose of 5 mg/​kg/​day intra- venously or intramuscularly, administered for 7–​10 days in combin- ation with doxycycline yielded good results in one study, experience with this regimen is too limited to justify its use over doxycycline plus streptomycin. The relapse and treatment failure rates with doxy- cycline/​streptomycin are around 5–​7% and 1–​2%, respectively. The need for parenteral administration of aminoglycosides may compli- cate the use of this regimen. The combination of doxycycline plus rifampicin (600–​900 mg/​ day orally), with both drugs administered for six weeks, is a good alternative. All other regimens/​combinations should be considered second-​line. The data available allow us to conclude that the regimen of doxy- cycline plus streptomycin is more effective than doxycycline plus ri- fampicin. A possible explanation of treatment failures and relapses with this latter regimen could be related to the fact that rifampicin might enhance the plasma clearance of doxycycline, thus resulting in subtherapeutic levels. In any case, the fact that doxycycline plus rifampicin is an oral regimen might allow for better implementation in clinical practice in areas with a less well-​developed health infra- structure. Nevertheless, it should be borne in mind that the use of this therapeutic schedule also poses problems in developing coun- tries due to its potential to induce resistance to rifampicin in other infections, mainly tuberculosis. Most authors continue antibiotic therapy for 45  days. Shorter treatment periods yield inferior results, while longer periods do not offer clear advantages. Some osteoarticular focal forms, such as sacroiliitis, do not appear to require longer treatment. In contrast, orchiepididymitis, vertebral osteomyelitis, endocarditis, and other local complications of brucel- losis require prolonged therapy, such as the continuation of doxy- cycline for eight weeks. The treatment of central nervous system complications of brucel- losis poses a special problem because of the need to achieve suitable concentrations of drugs in the cerebrospinal fluid. Since doxycycline and aminoglycosides have a low blood/​brain barrier penetration it is recommended that drugs which achieve this concentration, such as rifampicin or co-​trimoxazole, be added to the standard regimen of doxycycline plus streptomycin. The optimal duration of treatment for neurobrucellosis has not been determined, though it should not be less than 8–​12 weeks and possibly longer, depending on the clin- ical response. The optimal therapy for brucellosis during pregnancy has not been determined with certainty. Co-​trimoxazole plus rifampicin for at least 45 days has been used in individual cases with reported success. Despite the long duration of brucellosis treatment, the side effects of the standard medication are mild or moderate, and only rarely require treatment withdrawal. Prevention Brucellosis is a zoonosis with a strong correlation between animal and human disease. Prevention of human brucellosis is therefore based on elimination of the disease from animals. This goal is very difficult to achieve in countries with limited resources. The most successful method for prevention and control of brucellosis in animals is through herd management, hygiene, and vaccination. Although there is no completely effective and safe vaccine, the at- tenuated strains of B. melitensis strain Rev.1 for sheep and goats and B. abortus strain 19 or RB51 for cattle, have proven to be superior to all others. Vaccine efficacy is limited and offers an animal protection rate of 65–​90%. Given that at the present time there is no effective vaccine for human use and bearing in mind that the main source of infection is through direct or indirect exposure to infected animals or their products and ingestion of contaminated food products, the pre- vention of human brucellosis should be based on the following two pillars; first, occupational hygiene, especially in exposed profes- sionals such as farmers, stockmen, shepherds, abattoir workers, but- chers, dairymen and laboratory workers; and second, food hygiene, avoiding consumption of unpasteurized raw milk or its by-​products. FURTHER READING Ariza J, et al. (2007). Perspectives for the treatment of brucellosis in the 21st century: the Ioannina recommendations. PLoS Med, 4, e317. Colmenero JD, et al. (1996). Complications associated with Brucella melitensis infection: a study of 530 cases. Medicine (Baltimore), 75, 195–​212. Colmenero JD, et al. (2007). Clinical findings, diagnostic approach, and outcome of Brucella melitensis epididymo-​orchitis. Diagn Microbiol Infect Dis, 57, 367–​72. Colmenero JD, et al. (2008). Clinical findings, therapeutic approach, and outcome of brucellar vertebral osteomyelitis. Clin Infect Dis, 46, 426–​33. Corbel MJ (1997). Brucellosis:  an overview. Emerg Infect Dis, 3, 213–​21. Corbel MJ, et al. (2006). Brucellosis in humans and animals. WHO Press, World Health Organization, Geneva. Dean AS, et al. (2012). Global burden of human brucellosis: a system- atic review of disease frequency. PLoS Negl Trop Dis, 6, e1865. Erdem H, et  al. (2013). Diagnosis of chronic brucellar meningitis and meningoencephalitis: the results of the Istanbul-​2 study. Clin Microbiol Infect, 19, E80–​6. Franc KA, et al. (2018). Brucellosis remains a neglected disease in the developing world: a call for interdisciplinary action. BMC Public Health, 18, 125. Foster JT, et al. (2012). Genotyping of Brucella species using clade specific SNPs. BMC Microbiology, 12, 110. Ko J, et  al. (2003). Molecular host-​pathogen interaction in brucel- losis: current understanding and future approaches to vaccine de- velopment for mice and humans. Clin Microbiol Rev, 16, 65–​78. Madkour MM (ed) (2001). Madkour’s brucellosis, 2nd edition. Springer, Berlin. Meng F, et  al. (2018). Rifampicin versus streptomycin for brucellosis treatment in humans: A meta-analysis of randomized controlled trials.
PLoS One, 13, e0191993. Orduña A, et al. (2000). Evaluation of an immunocapture-​agglutination test (Brucellacapt) for serodiagnosis of human brucellosis. J Clin Microbiol, 38, 4000–​5. Pappas G, et  al. (2006). The new global map of human brucelosis. Lancet Infect Dis, 6, 91–​9.

8.6.23 Tetanus 1109

8.6.23 Tetanus 1109

8.6.23  Tetanus 1109 Porte F, et al. (2003). Role of the Brucella suis lipopolysaccharide o antigen in phagosomal genesis and in inhibition of phagosome-​ lysosome fusion in murine macrophages. Infect Immun, 71, 1481–​90. Queipo-​Ortuño MI, et al. (2008). Usefulness of a quantitative real-​time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis. Clin Microbiol Infect, 14, 1128–​34. Ruiz-​Mesa JD, et al. (2005). Rose Bengal test: diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency depart- ments in endemic areas. Clin Microbiol Infect, 11, 221–​5. Solís García del Pozo J, et  al. (2012). Systematic review and meta-​ analysis of randomized clinical trials in the treatment of human bru- cellosis. PLoS One, 7, e32090. 8.6.23  Tetanus C. Louise Thwaites and Lam Minh Yen ESSENTIALS Tetanus is a disease characterized by muscle spasms caused by a toxin produced by the bacterium Clostridium tetani. Without treatment mortality is high due to muscle spasms which prevent
respiration or due cardiovascular system instability secondary to autonomic nervous system dysfunction. Tetanus is prevented by good wound hygiene and/​or vaccination and, although rare in developed countries, the disease remains a significant problem in many countries where facilities for treatment are often poor and mortality remains high. Historical perspective Tychon the soldier [was hit by] an arrow in his back  . . .  [He] sounded like someone gnashing his teeth in a fury of rage . . . He was arched back in opisthotonos, his jaws locked together against his will. A friend forced some wine between his teeth, but Tychon could not swallow, and the liquid was expelled in spurts from his nostrils. Hippocrates (c.425 BC) Tetanus has been a well-​recognized complication of battle injuries, known since ancient times. In 1884, working at the University of Gottingen, 22-​year-​old Arthur Nicolaier demonstrated that soil contamination of wounds caused tetanus and isolated the causa- tive bacterium Clostridium tetani. The toxin it produced was dis- covered in 1890 by Faber, the same year that Behring and Kitasato produced the first antitoxin. However, it wasn’t until the First World War that there was widespread use of antitoxin, when heavy contamination of wounds resulted in large numbers of tetanus cases and mortality rates approaching 100%. The introduction of antitoxin was associated with a significant reduction in tetanus infection rates. Working at the Pasteur Institute in Paris, the French veterinarian Gaston Ramon developed a method to detoxify tetanus toxin using formaldehyde and in 1926 he and Zoller performed the first suc- cessful vaccination of humans. Tetanus vaccination was introduced to the United Kingdom’s Armed forces in 1938. In the Second World War, following compulsory vaccination of US forces, only 12 tetanus infections were reported out of 2.7 million hospitalized patients. Routine vaccination was introduced in infant immunization pro- grammes in the United Kingdom during the 1950s, becoming part of the national schedule in 1961. By 1970 people sustaining tetanus-​ prone wounds were also offered vaccination. As a result of these and similar sustained vaccination programmes, in high-​income coun- tries tetanus is rare but in countries with less developed vaccination schemes, tetanus continues to be a major problem. In 1988 the World Health Organization launched a major ini- tiative to eliminate neonatal tetanus, prompted by an estimated 800 000 neonatal deaths a year due to the disease. The initiative mainly employed a policy of maternal vaccination, thus maternal tetanus elimination was added to its aims in 1995 and a more inclu- sive vaccination strategy was employed. To date 37 out of 59 coun- tries targeted have achieved this goal. Aetiology, genetics, pathogenesis, and pathology Clostridium tetani is a Gram-​positive spore-​forming anaerobic ba- cillus able to infect and cause disease in both humans and animals. The bacterium is highly sensitive to oxygen but can survive in the environment as an extremely resistant metabolically inactive spore. Under suitable anaerobic conditions the spore germinates and the bacteria multiply, releasing a highly potent neurotoxin, tetanus toxin, which is responsible for the clinical features of tetanus. Aetiology Spores of C. tetani can be isolated from the environment throughout the world, particularly in soil, but are also found in human and animal faeces and even street dust. Germinant receptors on the spores recognize specific nutrients and mediate germination of the spores, an irreversible process that involves the loss of the peptido- glycan cortex and rehydration of the core with corresponding loss of resistant properties. An obligate anaerobe, C. tetani multiplies in low-​oxygen tensions. During the exponential phase of vegetative growth tetanus toxin is released. Structurally similar to botulinum toxins, tetanus toxin is one of the most potent toxins known, with a median human lethal dose (LD 50) of only 2.5 ng/​kg. Genetics and pathophysiology C.  tetani was first sequenced in 2003 but few strains have been sequenced and, unlike C. botulinum, there appears to be much less genetic diversity. Tetanus toxin (also known as tetanospasmin or tet- anus neurotoxin) is a 150 kDa protein encoded on a plasmid. Non-​ pathogenic strains of C. tetani exist which do not produce tetanus toxin. Plasmid identification has been used clinically to support the diagnosis of tetanus following isolation of C. tetani. Amino acid sequences of this toxin appear highly-​conserved in the five plas- mids sequenced to date. The toxin consists of a heavy and a light chain joined by a disulphide bond. The heavy chain mediates toxin entry into motor nerves, either locally or after circulation in the

section 8  Infectious diseases 1110 bloodstream. Its motor specificity is due to binding specific domains within the motor nerve membrane. The toxin is transported retro- gradely in the motor neurons to the central nervous system unlike the structurally similar botulinum toxins which remain in the per- iphery, hence the different clinical effects. The tetanus toxin enters the presynaptic neuron, and the light chain is translocated into the cytosol where it acts as a metalloprotease cleaving the SNARE pro- tein, vesicle associated monophosphate 2 (VAMP2), necessary for presynaptic vesicle docking and neurotransmitter release. This effect is limited to presynaptic inhibitory neurons, reducing central inhib- ition of motor neurons, and resulting in the increased muscle tone and spasms characteristic of tetanus. Epidemiology In countries with good immunization programmes and high levels of hygiene, tetanus has largely been eliminated, but in poorer regions tetanus remains a common cause of morbidity and mortality. The World Health Organization’s maternal and neonatal tetanus elim- ination initiative has made a significant impact on the number of cases of tetanus affecting women and newborn infants, but still an estimated 49 000 infants continue to die every year from the disease. The burden in older children and adults is unknown but is likely to be substantial as the major focus of preventative programmes has been immunizing pregnant women and infants, leaving other age-​ groups vulnerable to infection. Tetanus is caused by wound contamination with C. tetani. In neo- natal tetanus the entry site is usually the umbilical stump although traditional practices such as ear-​piercing have also been linked. In children and older infection often enters through very minor cuts and abrasions. However deeper wounds are often associated with worse prognosis. Global incidence of tetanus In 1988 an estimated annual total of 787 000 neonatal deaths due to tetanus prompted the World Health Assembly to call for the elimination of neonatal tetanus by 1995. Slow progress re- sulted in this goal being postponed several times. Nevertheless, remarkable progress has been made and 37 out of the 59 targeted countries have now reached this target: China eliminating the dis- ease in 2012 and India in June 2015 (see map, Fig. 8.6.23.1). The Fig. 8.6.23.1  MAP of maternal and neonatal tetanus (MNT) elimination. © World Health Organization 2017.

8.6.23  Tetanus 1111 proportion of global neonatal deaths due to tetanus has fallen from 14% in 1993 to 1.79% in 2008. Three main strategies have been employed to attain these results:  immunization, improved sur- veillance, and improved birth hygiene. Although most effort has focused on increasing immunization coverage, improved birth hy- giene has also an important effect, and indeed much of the success of China’s elimination campaign has been attributed to achieving a vast increase in numbers of women delivering in healthcare facil- ities. It should however be noted that, as elimination is defined as less than one case per 1000 live births in every district of a country, it is still possible for neonatal cases to be seen after ‘elimination’ has occurred. The true worldwide incidence of tetanus occurring in age-​ groups beyond the neonatal period is unknown as most countries do not collect accurate data. However, the current focus on infant immunization schedules means that although infants and children are often protected against tetanus, lack of subsequent booster dosing means significant numbers of older children and adults are unprotected. In some countries males may receive booster doses with military service, but generally men are more vulnerable as they are not covered by maternal and neonatal tetanus elimin- ation schemes. Outbreaks of tetanus after natural disasters such as the Haiti earthquake in 2010 and Tsunami of 2005 have been reported. In such situations contamination of wounds in unvac- cinated individuals is compounded by poor access to healthcare in disaster areas. Risk groups in high-​income countries In developed countries tetanus is rare but has not and can never be eradicated. Elderly people born before routine national immuniza- tion are particularly vulnerable. People who inject drugs are also a high-​risk group due to the increased chance of sustaining a contam- inated wound. In addition to poor hygiene, mixing or cutting drugs may increase the chance of an anaerobic focus of infection. Heroin may be cut with quinine, which is acidic and leads to local tissue necrosis and has been reported to be associated with particularly severe cases of tetanus. Clinical features Skeletal muscle spasm is the characteristic feature of tetanus, par- ticularly the facial muscles (causing ‘lockjaw’ and ‘risus sardonicus’, Fig. 8.6.23.2) and extensor spinae (opisthotonus, Fig. 8.6.23.3). Severe spasms can impede respiration which, if not controlled, is usually the primary cause of death in tetanus. Dysfunction of the autonomic nervous system also occurs and is clinically apparent as fluctuating blood pressure and variable heart rate. Presentation Symptoms of tetanus evolve gradually over a period of days and weeks or, in very severe cases, hours. The time course of tetanus is divided into specific periods. The incubation period is the period from inoculation to the first symptom (therefore may be un- known if no entry site is found) and is usually around 7–​14 days (slightly shorter in neonates and inoculation is assumed to occur at birth, thus is equal to age at first symptom). The period of onset is the period from the first symptom to the first spasm and is usu- ally 2–​5 days. Both these periods can be shorter in severe disease which tends to progress more rapidly. The initial symptoms on admission by 2422 patients (excluding neonates) admitted to the Hospital for Tropical Diseases Ho Chi Minh City are shown in Table 8.6.23.1. As tetanus develops muscle tone gradually increases until spasms occur. In the face trismus (‘lockjaw’) is common and the characteristic risus sardonicus is seen due to spasm of facial muscles. Involvement of the erector spinae group of muscles re- sults in opisthotonus. Sometimes tetanus is confined to a local Fig. 8.6.23.2  Risus sardonicus. Courtesy of the late Professor Sornchai Looareesuwan. Fig. 8.6.23.3  Opisthotonus. Copyright D. A. Warrell.

section 8  Infectious diseases 1112 group of muscles producing only local muscle spasm. If this occurs in the head, it is termed ‘cephalic tetanus’ (Fig. 8.6.23.4). Unlike local tetanus elsewhere, this form is potentially dan- gerous if laryngeal muscle spasms cause airway obstruction leading to asphyxiation. This occurs commonly in generalized tetanus and is a life-​threatening emergency. Respiratory tract secretions are increased in tetanus due, perhaps, to a combin- ation of autonomic nervous system stimulation and pharyngeal and laryngeal muscle spasms that prevent swallowing. Spasm and hypertonus of the respiratory muscles is a serious occurrence that, without artificial respiratory support, is a common cause of death in tetanus. Neonatal tetanus has a similar clinical picture (Fig. 8.6.23.5), with infants usually presenting with difficulty feeding followed by frank spasms. The World Health Organization (WHO) case-​ definition of neonatal tetanus is ‘an illness occurring in a child who has the normal ability to suck and cry in the first two days of life but who loses this ability between days 3 and 28 of life and becomes rigid and has spasms’. Natural history In centres with facilities to control muscle spasm and provide mechanical ventilation, autonomic system effects are responsible for a second group of major complications. The syndrome of auto- nomic instability usually takes the form of labile hypertension and tachycardia associated with increased circulating catecholamines. However, it may manifest as more sustained hypertension and tachycardia or, less commonly but more seriously, with periods of hypotension and bradyarrythmias. It is associated with acute renal failure and acute respiratory distress syndrome (ARDS). More recently, facilities for ventilation and improved neonatal critical care monitoring has meant that autonomic nervous system dysfunction has also been described in neonates. Although there are fewer published descriptions, it is likely this follows a similar course to that in adults. Patients with severe tetanus typically re- quire long periods (several weeks) of mechanical ventilation and long periods in hospital. As a consequence, secondary hospital-​ acquired infection is common, further impacting on morbidity and mortality. A variety of scores have been used to describe clinical severity and prognosis in tetanus. The Ablett score is the most commonly used severity score and is shown in Table 8.6.23.2. Scores used by Philips and Patel have been used to predict severity, but the more Table 8.6.23.1  Features on admission at hospital for tropical diseases Symptom Percentage of admissions Trismus 98 Dysphagia 83 Back pain 94 Muscle stiffness 95 Muscle spasms 46 Difficulty breathing   7 Fever   8 Fig. 8.6.23.4  Cephalic tetanus. Courtesy of Dr Pedro Pardal, Belém, Brazil. (a) (b) Fig. 8.6.23.5  Neonatal tetanus. Copyright D. A. Warrell.

8.6.23  Tetanus 1113 recent Tetanus Severity Score showed superior predictive value in Vietnamese patients. These scores are all simple to use and are cal- culated from baseline data and features of the history. In neonatal tetanus, birth weight and age at presentation are the most important predictors of outcome. Diagnosis Tetanus is a clinical diagnosis. The presence of generalized muscle rigidity and trismus are characteristic of tetanus. In neonatal tet- anus the ability to suck and cry before the onset of symptoms is an important feature differentiating it from other conditions affecting feeding. In most cases C. tetani is not isolated from wounds and its cul- ture is not required for diagnosis. In vaccinated individuals, the per- sistent presence of toxigenic C. tetani has been described without associated clinical disease. Nevertheless, in unvaccinated popula- tions, culture of C. tetani from a wound is supportive of a diagnosis of tetanus. More recently polymerase chain reaction (PCR) has been used to detect toxin from cultures of C. tetani, confirming that the isolated bacteria is of a pathogenic species. Although tetanus has occasionally been reported in the presence of ‘protective’ levels of antibody, serum concentrations of antibody greater than 0.1 IU/​ml measured by enzyme-​linked immuno- sorbent assay (ELISA) are usually taken to exclude a diagnosis of tetanus. Differential diagnosis The differential diagnosis includes local causes of trismus and pharyngeal muscular spasms such as oropharyngeal infections, tumours, or temporomandibular joint pathology. Headaches and nuchal rigidity occur in meningitis, but this occurs without generalized muscle tone abnormality and conscious level may be reduced. Rabies, like tetanus, may result from an infected animal bite, but its incubation period is usually much longer. Hydrophobic spasms can resemble tetanic spasms, particularly in the case of cephalic tetanus. Strychnine is a competitive antagonist of the inhibitory neuro- transmitter glycine that causes hyperreflexia and severe muscle spasms leading to convulsions. It may be very hard to distinguish this from tetanus, but the diagnosis may be suspected by a history of ingestion and confirmed by toxicological tests of urine, serum, or gastric contents. Dystonic reactions to antidopaminergic drugs may cause muscle tone abnormality but may also be associated with ab- normal eye or tongue movements which do not occur in tetanus. Anticholinergics and withdrawal of the precipitating drug can eliminate these. In children and neonates, hypoglycaemia, hypocalcaemia, and meningoencephalis can present with some of the features of tet- anus, however a more careful history and clinical examination should differentiate these. Treatment The three principals of management are removal of toxin, spasm control, and control of autonomic disturbance. Wounds should be cleaned adequately and, if necessary surgi- cally debrided. Antibiotics are given to prevent any further bac- terial multiplication—​either penicillin or metronidazole. In a series of 45 clinical isolates of toxin producing C. tetani, all were sensi- tive to penicillin and metronidazole. An open, non​randomized controlled trial of 173 patients by Ahmadsyah showed reduced mortality in those treated with metronidazole compared to peni- cillin, however a subsequent randomized controlled trial testing two penicillin preparations against metronidazole in 161 patients showed no difference. Penicillin is a non​competitive inhibitor of GABA-​A receptors and in large doses is known to cause seizures and thus could potentiate the effects of tetanus toxin. In view of this and trial evidence, metronidazole is usually the recommended antibiotic. Antitoxin should be given to neutralize any unbound tetanus toxin and, if given early enough, may limit the severity of tetanus. Recent debate has continued about appropriate dose and route of antitoxin. Currently doses of 3000–​6000 IU of human tetanus immunoglobulin (HIG) intramuscularly are recommended, or 500–​1000 IU/​kg equine antitoxin. However, these doses were based on observational studies from the 1960s and some recent recommendations suggest lower doses are safe. The use of equine antitoxin is associated with the risk of anaphylactic reactions or serum sickness, although with modern preparations these are less common. Data from some randomized controlled trials and case series suggest that intrathecal administration of antitoxin may confer further benefit; however, many of these trials are open to bias and the results of two meta-​analyses give conflicting results. As tetanus tends to occur in the world’s poorest regions, there is usually a limited range of treatment options. Chlorpromazine, Table 8.6.23.2  Ablett score for tetanus Grade Clinical features I • Mild to moderate trismus (little or no dysphagia) • General spasticity • No respiratory embarrassment • No spasms II • Moderate trismus • Well-​marked rigidity • Mild to moderate but short spasms • Moderate respiratory embarrassment with an increased respiratory rate greater than 30 breaths/​min • Mild dysphagia III • Severe trismus • Generalized spasticity • Reflex prolonged spasms • Increased respiratory rate greater than 40 breaths/​min • Apnoeic spells • Severe dysphagia • Tachycardia greater than 120 beats/​min IV • Grade III and violent autonomic disturbances involving the cardiovascular system • Severe hypertension and tachycardia alternating with relative hypotension and bradycardia, either of which may be persistent

section 8  Infectious diseases 1114 phenobarbitone, or diazepam are the most commonly available drugs. Many areas lack facilities for mechanical ventilation so muscle-​relaxant effects must be titrated against respiratory suppres- sion. In most countries benzodiazepines are the first line agents. Diazepam is often the most common choice due to its widespread availability and low cost, but other preparations such as midazolam are more suited to long-​term administration. High dose regimens may be required with doses up to 100 mg/​hr diazepam reported. In 1966 Hendrickse reported one of the few randomized trials com- paring diazepam, chlorpromazine and phenobarbitone with chlor- promazine and phenobarbitone alone in 104 neonates and 45 older children. Mortality in the neonates was identical but in the older children the death rate was almost halved in those treated with diazepam, although numbers were too small to reach statistical significance. The use of many other muscle relaxants in tetanus has been de- scribed in case reports. Intrathecal baclofen has been the subject of over 30 case reports. As a GABA-​B agonist it has been reported to control spasms in tetanus. However, it requires intrathecal adminis- tration (usually via continuous infusion) and is also associated with respiratory depression, therefore it cannot be advised in settings without mechanical ventilation. In a series of 22 patients treated in Portugal, intrathecal baclofen was successful in controlling spasms in all but one patient. However, 19 patients required mechanical ventilation and one patient suffered meningitis due to infection of the spinal catheter. Magnesium sulphate has been used to treat both spasms and autonomic dysfunction in tetanus. It is a calcium antagonist causing presynaptic neuromuscular blockade as well as vasodila- tation and reduced catecholamine release. In a case series of 40 patients in Sri Lanka, early use of magnesium sulphate was asso- ciated with the successful avoidance of mechanical ventilation in 57% of patients. Three randomized controlled trials of magnesium sulphate in tetanus have been performed subsequently. In two trials, comparing magnesium with diazepam in a total of 78 pa- tients with moderate and severe tetanus, improved spasm control was found in magnesium-​treated patients. In one of these trials, magnesium sulphate was associated with less respiratory depres- sion than diazepam, but mechanical ventilation was still required in 4/​18 patients and mortality rates were high (9/​18 in magne- sium vs. 10/​18 with diazepam). One large double-​blind random- ized controlled trial compared adjunctive magnesium sulphate in 197 patients with severe tetanus and found that although magne- sium did not reduce the requirement for mechanical ventilation, requirement for other muscle relaxants was reduced. This trial also looked at the effects of magnesium on autonomic dysfunction and found improved cardiovascular stability with reduced levels of circulating catecholamines. In this trial blinding procedures meant that doses could not be titrated against clinical effect and it is possible that lower concentrations may have been achieved. In contrast, trials with less blinding may have achieved higher serum concentrations of magnesium. Control of autonomic dysfunction in tetanus is notoriously dif- ficult due to often rapid fluctuations in blood pressure. For this reason, short-​acting calcium antagonists and β-​blockers are pre- ferred but often episodes of hypotension occur. Careful fluid man- agement is necessary to optimize intravascular volume and cardiac output in this situation. Vasopressor and inotrope infusions may also be required. As tetanus does not confer natural immunity it is necessary to fully vaccinate patients with a primary course of tetanus toxoid to confer protection in future. Prevention Tetanus is prevented by immunization and good hygiene. As tetanus is a non​communicable disease, there is no herd immunity effect and unvaccinated individuals are equally vulnerable irrespective of the community around them. Tetanus vaccines are made from tetanus toxoid adsorbed onto aluminium to increase immunogen- icity. They are available as single dose tetanus toxoid or combined with diphtheria toxoid, either high dose diphtheria toxoid (DT) for use in children under 7 years, or low dose diphtheria toxoid (dT) for use in older people. Combinations of DT or dT are available with whole cell or acellular pertussis and Haemophilus influenza B, hepatitis B, and polio. Tetanus toxoid alone or in combination is safe and effective with infrequent and mild adverse effects. It can be used in pregnancy and immunodeficiency; however, in these cases immune responses may be diminished. Current WHO recommendations are for six-​dose schedules: a pri- mary course of three doses in infancy followed by boosters between 4 and 7 years and 12 and 15 years and in adult life (e.g. first preg- nancy). Some 86% of the world’s children now receive a three-​dose primary course of diptheria, tetanus toxoid, and pertussis (DTP3). However, it is likely that the primary vaccination series produces protective antibody for 6–​10 years, but without boosting, individ- uals are vulnerable to tetanus. In the United Kingdom a total of five doses is recommended, unless others are deemed necessary after sustaining tetanus-​prone wounds. In non​immunized adolescents and adults or those with un- known/​incomplete vaccination history, a three-​dose primary course is recommended. The first two doses should be given at least four weeks apart and the third at least 6 months after the second. In these people a total of five doses is expected to confer lifelong protection. This schedule is also recommended for pregnant women with the first two doses given in pregnancy (usually dT). The time course of antibody response to vaccination means that in unimmunized individuals a single tetanus dose will not protect against tetanus in those sustaining tetanus-​prone wounds and additional protection is given by giving antitoxin (see Table 8.6.23.3). People already vaccinated may have a more rapid antibody response. In one study booster doses in previ- ously vaccinated people, protective levels were achieved within 2  days. However, in high-​risk injuries, antitoxin is still re- commended. As tetanus vaccine is an inactivated vaccine, the coadministration of vaccine and immunoglobulin is theoretic- ally possible. In vivo studies are limited to previously vaccinated populations and while there may be some short-​term attenuation of immune response, it appears that long-​term immunity in al- ready immune individuals is not affected. Rapid diagnostic tests are now available for the detection protective levels of antibodies and have been found useful to identify people requiring tetanus boosters in emergency departments.

8.6.24 Clostridium difficile 1115

8.6.24 Clostridium difficile 1115

8.6.24  Clostridium difficile 1115 Areas of uncertainty, controversy, and future developments Tetanus continues to be a significant problem in much of the world. Considerable progress has been made towards its elimination; however, access to vaccination programmes in many communi- ties is still limited by humanitarian, sociological, or geographical factors. While in many countries maternal and infant vaccination schemes have achieved good coverage, the provision of subsequent boosters necessary for long-​term protection has been given scant attention, leaving older individuals susceptible to disease. Funding and implementing large scale booster programmes remains a major challenge for the global health community. While this is happening simple and affordable treatments are still needed that will reduce the need for expensive (and often unavail- able) intensive care unit management. FURTHER READING Borrow R, Balmer P, Roper M (2006). The immunological basis for immunisation series. Module 3: tetanus update 2006 (Vaccines and biologicals). World Health Organization, Geneva. Brüggemann H, et  al. (2015). Genomics of clostridium tetani. Res Microbiol, 166, 326–​31. Department of Health. Immunisation against infectious disease: ­chapter 30. Tetanus. https://​www.gov.uk/​government/​publications/​ tetanus-​the-​green-​book-​chapter-​30 Health Protection Agency (2013). HPA expert working group interim guidance on the use of tetanus immunoglobulin for the treatment of Tetanus (pp. 1–​2). https://​www.gov.uk/​government/​uploads/​ system/​uploads/​attachment_​data/​file/​400084/​expert_​working_​ group_​interim_​guidance_​on_​the_​use_​of_​tetanus_​immuno- globulin_​for_​the_​treatment_​of_​Tetanus.pdf Patel JC, Mehta BC (1999). Tetanus: study of 8,697 cases. Indian J Med Sci, 53, 393–​401. Rodrigo C, Fernando D, Rajapakse S (2014). Pharmacological man- agement of tetanus :  an evidence-​based review. Critical Care, 18, 217. Thwaites CL, Beeching NJ, Newton CR (2015). Maternal and neonatal tetanus. Lancet, 385, 362–​70. 8.6.24  Clostridium difficile David W. Eyre and Mark H. Wilcox ESSENTIALS Clostridium difficile is a Gram-​positive spore-​forming anaerobic bacillus that is ubiquitous in nature, and particularly common in healthcare
environments. Its spores are part of the colonic flora in about 2–​3%
of healthy adults, with colonization rates increasing, typically up to
10–​20%, during hospitalization. Disease occurs when the organism shifts from quiescent spores to replicating vegetative cells with toxin
(A and B) production; this happens when there is inhibition of the resi- dent colonic flora (gut microbiome) by prescribed antibiotics, although cases can occur when no such precipitant is identified. C. difficile in- fection is now recognized as the most important bacterial enteric pathogen in wealthier countries, epidemics, and outbreaks of which are common, most notoriously now due to the ribotype 027 (NAP-​1) strain that is associated with more severe disease and poor outcomes. Clinical features—​these range from trivial diarrhoea that subsides rap- idly when antibiotics are stopped to fulminant pseudomembranous col- itis, which may progress to toxic megacolon; most cases have watery and voluminous diarrhoea, possibly accompanied by abdominal cramping. Diagnosis and treatment—​the condition should be suspected in any patient who has unexplained diarrhoea, particularly in as- sociation with antibiotic use. Diagnosis is established ideally by demonstrating C. difficile toxin in stool, although there has been a recent vogue for polymerase chain reaction-​based detection of toxin gene, despite this being less specific. Treatment is by stopping the implicated antibiotic, supportive care, avoiding antiperistaltic agents, and giving oral vancomycin or fidaxomicin; metronidazole is less ef- fective. Bezlotoxumab (given in addition to antibiotic) has recently been approved to reduce recurrences in high risk patients. Prevention—​the most important issues are controlling anti- biotic prescribing to reduce exposure overall, and particularly to fluoroquinolones, and infection control in healthcare facilities, including prompt diagnosis and isolation of patients with diarrhoea to limit spore dissemination. Table 8.6.23.3  Recommendations for wound prophylaxis Wound type Active immunization Passive immunization Clean wound Only if vaccination history incomplete (i.e. give booster if not up to date or initiate primary course as described in text) No Low risk tetanus-​prone

  1. Wounds or burns: • Requiring surgical intervention or when treatment delayed >6 h • With significant degree of devitalized tissue • Containing foreign bodies • Individuals with systemic sepsis
  2. Puncture-​type injury, particularly in contact with soil and/​or manure
  3. Open fractures Only if vaccination history incomplete (i.e. give booster if not up to date or initiate primary course as described in text) If vaccination history incomplete, one dose human immune globulin (in different site to vaccination) High-​risk tetanus-​prone As above but with heavy contamination with material likely to contain tetanus spores and/​or extensive devitalized tissue Only if vaccination history incomplete (i.e. give booster if not up to date or initiate primary course as described in text) All one dose human immune globulin (in different site to vaccination, if given)

section 8  Infectious diseases 1116 Introduction Clostridium difficile (recently renamed as Clostridioides difficile) is an anaerobic spore-​forming Gram-​positive bacillus found in healthcare facilities and widely in the environment, which can colonize and prolif- erate in the human gut, especially following changes in gut microbiome after antibiotic use. The key components of the gut microbiome re- sponsible for preventing C. difficile growth are unclear, but recently secondary bile acids and Clostridium scindens have been identified as possible inhibitors. Pathogenic C. difficile strains produce exotoxins that cause acute colonic mucosal inflammation. Clinical features range from asymptomatic colonization through severe diarrhoea to ful- minant pseudomembranous colitis and occasionally toxic megacolon. C. difficile is non​invasive and so extraintestinal disease is very rare. Antibiotic-​associated diarrhoea and enterocolitis have been rec- ognized throughout the antibiotic era. C difficile was initially dis- covered in 1935 as part of a survey of intestinal bacteria of newborn children, but it was not until the late 1970s that it was established as a significant human pathogen. Within 30 years of this discovery, C. dif- ficile was established as a prominent healthcare-​associated infection in developed countries, primarily as epidemic strains caused mul- tiple outbreaks associated with poor outcomes. C. difficile infection (CDI) has therefore become a major priority for patients, healthcare professionals, and policymakers. There is also considerable activity to develop new treatment and preventative options for CDI. Aetiology, pathogenesis, and pathology C. difficile is acquired by faeco-​oral transmission, usually via inges- tion of spores, given their resistance to gastric acid and ability to survive the aerobic environment outside the normal host (see Box 8.6.24.1). Stimulated by primary bile acids, spores germinate to vege- tative forms, leading either to asymptomatic colonization or disease. Asymptomatic C. difficile colonization, about 3% in healthy adults, rising to 10–​20% in hospital inpatients, is relatively uncommon rela- tive to the rates of antitoxin antibodies (up to 70%) found in adults. Presumably, therefore, most encounters lead to excretion of the bac- terium without symptoms, at least in younger individuals. Infants are frequently colonized, up to 35% in the first 1–​2 years of life, falling to about 5–​15% by 1–​8 years and then to adult levels thereafter. It is un- known why children so uncommonly experience CDI despite such high carriage rates; lack of receptors for C. difficile toxins has been suggested but not proven. Subsequent development of antitoxin anti- bodies is an important host defence in exposed individuals. The normal gut microbiome is a key barrier to infection. Perturbation of gut bacteria, in most cases by antibiotics, allows C. difficile to colonize and/​or to proliferate and cause disease. In asymptomatically colonized individuals, the composition of the gut microbiome is similar to C. difficile culture-​negative patients; how- ever, in CDI marked changes in the microbiome are seen, with a significant reduction in the diversity present and marked changes in the dominant bacteria. Toxins A and B are the two principal C. difficile virulence deter- minants. The genes for these toxins are part of a conserved pathogen- icity locus, PaLoc, which is present in the chromosome of toxigenic strains, but absent in avirulent non​toxigenic strains. Toxins A and B enter host cells via receptor-​mediated endocytosis, and irrevers- ibly inactivate Ras family small GTPases. This leads to disruption of control of the actin cytoskeleton, membrane blebbing, and eventual apoptosis of the cell. There is continuing uncertainty surrounding the relative importance of toxins A and B in human disease. Animal experiments have provided conflicting data, but this likely part re- flects interspecies differences in the role of toxins. Blocking toxin A in addition to toxin B alone has recently been shown in humans to confer no additional benefit, suggesting that the latter is more im- portant in CDI. Most strains causing disease in humans carry both toxins, but A-​B + strains can cause the typical range of disease from asymptomatic colonization to severe colitis. The epidemic ribotype 027 (also known as NAP1/​BI) strain carries an additional binary toxin, which also disrupts the cell actin cytoskel- eton. This strain also contains a truncation in the tcdC gene originally hypothesized to be a negative regulator with the truncation resulting in increased toxin production; however, this explanation for the increased virulence of C. difficile ribotype 027 has been recently questioned. Epidemiology Widely used surveillance definitions classify CDI into healthcare-​ and community-​associated on the basis of the time since last healthcare exposure. Rates of healthcare-​associated CDI are typically 4–​10/​10 000 bed-​days in endemic settings, whereas rates in com- munity patients without healthcare exposure in the last 12 weeks are 8–​25 cases per 100 000 person-​years. CDI rates vary markedly across countries, with ascertainment bias secondary to differences in re- questing/​testing rates or testing methodology important issues. Prior antibiotic exposure is a major risk factor for CDI, with second-​ and third-​generation cephalosporins, clindamycin, and fluoroquinolones most frequently implicated. There is large vari- ation in the risks reported for different antibiotics, at least in part due to variations in antibiotic resistance patterns in locally circulating C. difficile strains. There is also a distinction to be drawn between selecting for C. difficile and induction of toxin production and thus CDI. Furthermore, differing levels of exposure to C. difficile will af- fect the recorded CDI rates associated with a particular antibiotic. Repeated or prolonged exposure to antibiotics increases CDI risk, and the last antibiotic before symptom onset may be a relatively low risk agent, the issue here being cumulative damage to the normally protective gut microbiome. The exact temporal relationship between antibiotic exposure and C. difficile acquisition is not fully defined. Antibiotic exposure in the last 90 days increases risk of CDI, but exposure in the last 30 days is associated with greater risk. However, as disruption of gut flora persists beyond 90 days, patients might re- main at risk of CDI for much longer. Box 8.6.24.1  Consequences of C. difficile acquisition • Ingestion followed by excretion • Asymptomatic colonization • Diarrhoea/​recurrent infection • Colitis • Pseudomembranous colitis • Pan-​colitis, toxic megacolon • Death

8.6.24  Clostridium difficile 1117 Multiple comorbidities and age >65  years (which is pos- sibly a proxy for the former) are important risk factors for CDI. Immunosuppressed patients, and those with renal failure or in- flammatory bowel disease have higher CDI rates, but there might be confounding here due to higher diarrhoea rates and/​or anti- biotic use. Gastric acid suppressive medication, proton pump in- hibitors in particular, is a possible risk factor that is frequently cited, although recent doubt about this association due to con- founding has been reported. Transmission Molecular typing studies demonstrate infected, and to a lesser extent colonized, patients contaminate their surroundings with C. difficile. As most CDI cases occur in healthcare facilities or are admitted to them, C. difficile is widely present in hospitals. Significant healthcare CDI outbreaks occur, and serially screened hospital inpatients ac- quire C. difficile colonization and infection at rates proportional to their length of stay. These observations led to the view that most CDI was acquired from other cases in hospitals. However, whole genome sequencing of strains from CDI cases has recently demon- strated that only a third of these are sufficiently genetically similar to isolates from any previous case to support the latter as the source of infection. Overall, less than a fifth of cases were genetically related to a previous case, and had shared some form of hospital contact. These findings are contingent on appropriate infection prevention and control measures being in place (detailed next). Finding that cases are not the source for most infections has led to a search for alternative sources. Longitudinal studies show long-​term C. difficile carriage is unlikely to be a significant source; carriage of toxigenic strains is protective against subsequent CDI, and most cases acquire C. difficile shortly before symptom onset. Asymptomatic patients and children are a possible reservoir. C. diffi- cile has also been recovered from domestic and production animals, retail meat and ready-​to-​eat food, and water supplies. Recovery from foods is usually uncommon; it is therefore difficult to study such likely sporadic contamination episodes. Different C. difficile strains have different environmental niches, such that no one source is likely to explain all CDI. For example, ribotype 027 has caused significant healthcare CDI outbreaks, but unlike many toxigenic strains, has not been found in healthy children. Similarly, ribotype 078 is strongly linked to pig farming and is an increasing cause of CDI. Clinical features The clinical features of CDI range from mild, self-​limiting diar- rhoea to fulminant colitis (Fig. 8.6.24.1). The principal symptom in CDI is watery voluminous diarrhoea with ≥3 unformed stools in 24 hours, but overt gastrointestinal bleeding is rare. Diarrhoea is accompanied by evidence of colonic inflammation including ab- dominal cramps, pain, fever, nausea, anorexia, and leucocytosis. Markers of severe disease include total peripheral white blood cell count less than 15 × 109/​litre, acute kidney injury with a rise in serum creatinine to 1.5-​fold or less above baseline, and hypoalbuminaemia; in very severe CDIs, hypotension, elevated serum lactate, ileus, and toxic megacolon (severe dilation of the colon) can occur. Sigmoidoscopy is an uncommon route to diagnosis, but character- istically findings range from mild patchy friable erythema to severe pseudomembranous colitis (raised yellow plaques that coalescence to form confluent pseudomembranes, which might bleed when disturbed) (Fig. 8.6.24.2). Fig. 8.6.24.1  Plain abdominal X-​ray showing distal colitis with proximal dilated loops of bowel. The descending colon lacks normal haustrations, resulting in a ‘lead pipe’ colon (arrows); the distal transverse colon shows mucosal thickening referred to as ‘thumb-​printing’ (arrows). Fig. 8.6.24.2  Sigmoidoscopic appearances in pseudomembranous colitis: friable white/​yellow plaques.

section 8  Infectious diseases 1118 Complications include recurrence, sepsis, intestinal perfor- ation, requirement for colectomy and death. All-​cause mortality at 14–​30  days following CDI is around 16%, varying by strain type, and is highest in CDIs due to ribotypes 027 and 078 and related strains. Differential diagnosis The main differential is antibiotic-​associated diarrhoea, which can also be due to microbiome disturbance or increased peristalsis; antibiotic-​associated diarrhoea generally resolves with reduc- tion in antibiotic dose or cessation. Rates of antibiotic-​associated diarrhoea range from 5 to 40% depending on the agent used. CDI only accounts for a small subset of patients with diarrhoea fol- lowing antibiotics, and only 5–​15% of tests submitted for C. difficile testing are positive. In hospitalized patients, diarrhoea might also be caused by other drugs, including laxatives and cytotoxics, and by enteral feeding. Other potential causes of inflammatory diarrhoea include enteric pathogens (salmonella, shigella, Campylobacter jejuni, and so on), ischaemic colitis, and inflammatory bowel disease. Concurrent CDI can exacerbate inflammatory bowel disease, while asymptomatic colonization with C. difficile might also occur alongside concurrent colitis and potentially obscure the cor- rect diagnosis. Rare cases of antibiotic-​associated colitis include Staphylococcus aureus, Klebsiella oxytoca, Clostridium perfringens, and Candida spp. Clinical investigations CDI should be strongly suspected in any patient who has diarrhoea in association with current or recent antibiotic use, and considered in unexplained acute diarrhoea even without prior antibiotic ex- posure. About one-​third of community-​associated CDI cases have no recent history of prescribed antibiotic exposure. Diagnosis of infection depends on characteristic symptoms plus the presence of free faecal C. difficile toxin (or less commonly pseudomembranous colitis at endoscopy or histologically). An important caveat is that in life threatening CDI there can be an ileus and so lack of diarrhoea. An unexplained high white blood cell count is another potential in- dicator of CDI. Available routine tests for CDI can detect the presence of bacteria directly (e.g. by culture or via C. difficile glutamate dehydrogenase), its toxin (e.g. by cytotoxin assay or more commonly by enzyme im- munoassay, EIA), or bacteria with the potential to produce toxin (e.g. toxigenic culture or nucleic acid amplification tests, NAATs, for toxin genes). Notably, there are two reference standard tests, one for detection of bacteria with potential to produce toxin (cytotoxigenic culture), and the other for toxin detection (cell cytotoxicity assay, CTA). Only tests that detect the presence of toxin in faeces (CTA or toxin EIA) have been shown to correlate with clinical outcome. All-​cause mortality is elevated in patients with positive faecal toxin assays, whereas patients with a positive NAAT, but negative toxin as- says have similar outcomes to those with negative NAAT and nega- tive toxin results. Therefore, isolated use of NAAT testing can lead to overdiagnosis of infection. Faecal culture is rarely performed for routine diagnosis of C. difficile, but might be required for surveil- lance (genotyping) studies. No ideal single test exists for detection of C. difficile toxin. CTA is technically demanding and slow (24–​48 hours to result); while toxin EIAs are rapid, sensitivity is typically only 80% and false-​positive re- sults occur (in typical use, about one to two out of every 10 positives). This has led to the development of two-​step testing strategies, adopted in UK, European, and also partly in recent US guidelines. A highly sen- sitive initial screen (e.g. glutamate dehydrogenase or NAAT), is used as a prompt rule out with high negative predictive value. Confirmatory testing is then undertaken with EIA or CTA to improve specificity. However, given the imperfect sensitivity of EIA, clinical judgement is still required, as it may be appropriate to treat a patient for CDI with a negative EIA result if there is a strong clinical suspicion of infection. Addition of a third test using a NAAT is an option but requires careful interpretation to avoid overdiagnosis; this might identify patients with diarrhoea of another aetiology concurrently carrying a toxigenic strain of C difficile (who can represent a cross infection risk), or possible false-​ negative toxin test results. Repeat testing is generally discouraged when a negative is obtained first (given the high negative predictive value of first step screening tests). Routine repeat testing after positive CDI tests has no value, as toxin can be detected in faeces for several weeks following clinical recovery. If symptoms recur then repeat testing should be carried out to establish whether CDI is the cause. Treatment Treatment involves, where possible, stopping any causative anti- biotic, supportive care, and specific therapy with oral vancomycin or fidaxomicin (metronidazole is less effective—below). Bezlotoxumab, a monoclonal anti-toxin B antibody that is given in addition to CDI standard antibiotic treatment, has recently been approved to reduce recurrences in high risk patients. In patients with mild disease simply stopping provocative antibiotics might resolve symptoms; however, given the potential for severe disease, usual practice is to treat all pa- tients with CDI. Older US and European guidelines have advocated 10–​14 days treatment with oral metronidazole (500 mg three times daily) for patients with mild-​moderate disease and oral vancomycin (125 mg four times daily) for patients with severe disease. However, two recent randomized controlled trials (RCTs) have demonstrated vancomycin is superior to metronidazole for all CDI, clinical reso- lution occurred in 81% and 73% respectively (p = 0.02), with the ad- vantage for vancomycin more marked in severe disease 79% versus 66% (p = 0.06), as previously reported. Rates of clinical cure with fidaxomicin were non​inferior to vancomycin in two major RCTs, and fidaxomicin was associated with reduced risk of recurrence compared with vancomycin (15% vs. 25%, p = 0.005). Concurrent, non-​CDI therapy antibiotics should be avoided where possible, as these are associated with longer durations of diarrhoea, reduced clinical cure, and increased recurrence rates. Antiperistaltic agents, such as loperamide, should be avoided in acute disease. Patients with severe disease where oral or nasogastric antibiotics may not reach the diseased site (e.g. with ileus), should be managed with intravenous metronidazole ± rectal vancomycin. Early surgical opinion should be sought for patients with very severe disease, as

8.6.24  Clostridium difficile 1119 fulminant CDI might require subtotal colectomy. More recently, a diverting loop ileostomy and colonic lavage has been associated with reduced morbidity and mortality. The risk of recurrent CDI (within 4–​8 weeks) increases with each subsequent episode from around 25–​45% to 60%. Fidaxomicin might be appropriate for the treatment of first episodes of CDI in patients at increased risk of recurrence (e.g. receiving concomi- tant antibiotics, with severe infection, older people with multiple comorbidities) or those with a first recurrent episode. There are no widely accepted prediction scores in use to identify either those at risk of severe CDI or recurrent infection. Other treatments used for recurrent CDI include tapered/​pulsed doses of vancomycin, and sequential therapy with vancomycin followed by rifaximin. There is weak quality evidence that pooled intravenous immunoglobulin might be effective in patients with recurrent CDI, presumably by augmenting the host antitoxin antibody response. Faecal microbiota transplantation is very effective in patients with multiple recurrences; resolution following a single duodenal infu- sion of donor faeces was 81% compared with 31% with vancomycin treatment in an RCT (p <0.001). There remain many unanswered questions about faecal microbiota transplantation including the op- timal volume of donor faeces, the screening repertoire for donors and recipients, and the route of administration. More widespread use of faecal microbiota transplantation is currently limited by con- cerns about the long-​term safety of donor faeces, including potential transmission of infectious agents to the recipient, and alteration of the gut microbiome given its far-​reaching (although currently poorly understood) effects on human health and disease. Encapsulated faeces and defined populations of bacteria might overcome some of the aforementioned issues. Prevention Community and hospital antimicrobial stewardship, in particular restricting use of high risk agents such as fluoroquinolones, cephalo- sporins, and clindamycin reduces the risk of CDI. The most effective class of antimicrobial to restrict likely depends on the resistance profile of circulating C. difficile strains. Clindamycin restriction was effective at controlling an institutional outbreak caused by a clindamycin-​resistant C. difficile clon e. Fluoroquinolone restric- tion has played a major role in reductions in CDI incidence in the United Kingdom in the last decade, largely driven by reductions in ribotype 027 and other fluoroquinolone-​resistant lineages. Rapid identification, isolation and testing of potential cases, and prompt treatment reduces the potential for healthcare-​ associated transmission. Contact precautions and hand washing with soap and water (C. difficile spores are resistant to alcohol gels that are widely used for hand hygiene) should be implemented on suspicion of CDI. These should be continued until at least reso- lution of diarrhoea, and longer if resources allow (as excretion of viable C. difficile can continue beyond the end of symptoms). Environmental cleaning around cases should be with chlorine-​ releasing agents with activity against C. difficile spores; vaporized hydrogen peroxide is used in some settings. Coordinated infec- tion control programmes are required, complemented by active monitoring of CDI incidence, and staff, patient, and visitor edu- cation programmes. Areas of uncertainty, controversy, and future developments Preventing CDI is a major focus for researchers. Although all based on C. difficile toxins A and B as immunogens, three distinct vaccines have completed phase 2 clinical trials; one has been terminated mid- phase 3 for unclear reasons. Other approaches to prevention include an orally delivered β-​lactamase that aims to degrade β-​lactams on the large intestine, and so avoid the deleterious effects of these anti- biotics on the gut microbiome. A charcoal-​based absorbent is also being investigated as a way of blocking the harmful effects of anti- biotics on gut bacteria. The diagnosis of CDI could be improved if rapid highly sensitive toxin tests were available. The absence of commercially available antitoxin antibody tests is a hindrance to identifying (those with low levels) who are at increased risk of CDI. Similarly, biomarkers that can accurately predict CDI severity and outcome would be valuable adjuncts to targeting therapies optimally. It remains unclear if C. difficile could have a pathogenic role in infants; high colonization rates tend to obscure any possible such role. Studies to date have typically failed to examine sufficiently wide repertoires of alternative potential pathogens in infants harbouring C. difficile. Whole genome sequencing has already proved a valuable tool for deepening our understanding of CDI epidemiology. The growing suspicion that foods might be an important source of C. difficile will require large prospective studies, noting that contamination is likely to be sporadic, and highly discriminatory fingerprinting to determine if such theories are true. FURTHER READING Bartlett JG (2006). Narrative review: the new epidemic of Clostridium difficile-​associated enteric disease. Ann Intern Med, 145, 758–​64. Cohen SH, et al. (2010). Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol, 31, 431–​55 (2010). Davies KA, et al. (2014). Underdiagnosis of Clostridium difficile across Europe:  the European, multicentre, prospective, biannual, point-​ prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis, 14, 1208–​19. Debast SB, et al. (2014). European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance docu- ment for Clostridium difficile infection. Clin Microbiol Infect, 20 (Suppl 2), 1–​26. Eyre DW, et al. (2013). Diverse sources of C. difficile infection iden- tified on whole-​genome sequencing. N Engl J Med, 369, 1195–​205. Johnson S, et  al. (2014). Vancomycin, metronidazole, or tolevamer
for clostridium difficile infection:  results from two multinational, randomized, controlled trials. Clin Infect Dis, 59, 345–​54. Loo VG, et al. (2011). Host and pathogen factors for Clostridium diffi- cile infection and colonization. N Engl J Med, 365, 1693–​703. Louie TJ, et al.; OPT-​80-​003 Clinical Study Group (2011). Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med, 364, 422–​31. McFarland LV, et al. (1989). Nosocomial acquisition of Clostridium difficile infection. N Engl J Med, 320, 204–​10.

8.6.25 Botulism, gas gangrene, and clostridial gas

8.6.25 Botulism, gas gangrene, and clostridial gastrointestinal infections 1120

section 8  Infectious diseases 1120 Pépin J, et al. (2004). Clostridium difficile-​associated diarrhea in a re- gion of Quebec from 1991 to 2003: a changing pattern of disease severity. Can Med Assoc J, 171, 466–​72. Planche TD, et al. (2013). Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C. difficile infection. Lancet Infect Dis, 13, 936–​45. van Nood E, et al. (2013). Duodenal infusion of donor feces for recur- rent Clostridium difficile. N Engl J Med, 368, 407–​15. Walker AS, et al. (2013). Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection. Clin Infect Dis, 56, 1589–​600. Wilcox MH (2012). Progress with a difficult infection. Lancet Infect Dis, 12, 256–​7. Wilcox MH (2014). The trials and tribulations of treating Clostridium difficile infection—​one step backwards, one step forwards, but still progress. Clin Infect Dis, 59, 355–​7. Wilcox MH, et al. (2017). Bezlotoxumab for prevention of Clostridium difficile infection recurrence. N Engl J Med, 376, 305–17. 8.6.25  Botulism, gas gangrene, and clostridial gastrointestinal infections Dennis L. Stevens, Michael J. Aldape,
and Amy E. Bryant ESSENTIALS Botulism Human botulism is caused by seven serological types of C. botulinum, which is ubiquitously distributed in the soil. Poisoning usually results from ingestion of preformed toxin in food, although this is rapidly in- activated at ordinary cooking temperatures, but it can also result from contaminated wounds. C.  botulinum toxin binds irreversibly to the neuromuscular junction and is the most lethal known microbial toxin. Clinical features, diagnosis, and treatment—​there are five forms of clinical botulism: (1) food-​borne botulism—​ingestion of food contam- inated by preformed toxin; (2) wound botulism—​infection of a wound with C. botulinum and in vivo toxin production (3) infant botulism—​ ingestion of clostridial spores that colonize the gastrointestinal tract and release toxin; (4) adult enteric infectious botulism—​similar to in- fant botulism; (5) inhalational botulism—​considered a potential agent of bioterrorism. Clinical presentation is with symptoms suggesting gastrointestinal tract illness, followed by neurological symptoms including diplopia, blurred vision, dizziness, and difficulty with speech or swallowing, leading on to generalized flaccid paralysis. The diag- nosis can be confirmed by testing for botulinum toxin in the patient’s serum, urine, or stomach contents, or in the suspect food. Treatment requires (1)  supportive care—​this, including mechanical ventilation, may be needed for many months until new synapses have developed; (2) antitoxin—​this reduces case fatality and shortens the illness. Gas gangrene Gas gangrene is caused by C.  perfringens (most commonly), C. histolyticum, C. novyii, C. sordellii, and C. septicum, which occur nat- urally in soil and in the gastrointestinal tracts of humans and animals. Common causes of the condition are severe trauma that interrupts the blood supply to the soft tissues (gunshot wounds, penetrating or crushing injuries) with contamination by dirt, vegetation, or clothing containing vegetative forms of clostridia or spores. Skin popping of black tar heroin is another recently recognized cause. The clostridia responsible elaborate a wide range of toxins with varying effects: the principle toxin of C. perfringens is α-​toxin, a phospholipase C that cleaves phosphatidylcholine in eukaryotic cell membranes and acti- vates neutrophils, platelets, and endothelial cells, causing obstruction of local blood flow. Clinical features—​severe and sudden pain is the most characteristic symptom. Infection progresses rapidly with local ecchymosis, blis- tering, massive swelling, and crepitus indicating gas in the tissue as progressive necrotizing soft tissue infection destroys muscle, fascia, fat, and skin. Without rapid and appropriate treatment, bacteraemia, hypotension, and multiple organ failure ensue. Diagnosis and treatment—​diagnosis must be made on clinical grounds, although Gram stain of the wound discharge or tissue sample may be helpful. Treatment requires (1) early recognition and aggressive surgical debridement of devitalized tissue; (2)  antimicrobials—​most commonly penicillin and clindamycin (which suppresses α-​toxin pro- duction); (3)  anti-​α-​toxin serum. The benefit of hyperbaric oxygen has not been proven in controlled trials. In an experimental model of gas gangrene, hyperbaric oxygen did not improve the efficacy of clindamycin or penicillin. Prevention—​prophylactic antibiotic treatment reduces the risk, but this depends upon factors including the time interval between the injury and surgical debridement. Particular forms of gas gangrene—​(1) C.  septicum can grow at ambient oxygen tensions, causing ‘spontaneous gas gangrene’ in normal tissues, most commonly when bacteria spread from a co- lonic adenocarcinoma to uninjured muscle. (2) C.  sordellii causes haemoconcentration, leukaemoid reaction without fever, and grad- ually progressive shock that is fatal in 75–​80% of patients. Women infected during parturition, after medical abortion, or following gy- naecological surgery almost always die. C. perfringens gastrointestinal infections Food poisoning—​if foods, such as meat and heavy gravy, infected with type A strains of C. perfringens are allowed to sit at room temperature, bacilli can multiply greatly. If the food is then inadequately heated before consumption, preformed heat-​labile enterotoxin, combined with toxin produced in the gut, causes self-​limiting abdominal pain and diarrhoea, usually without fever or vomiting. Necrotizing enterocolitis—​C. perfringens type C β-​toxin causes fulmi­ nating enterocolitis that destroys intestinal mucosa. Epidemic out- breaks occurred in postwar Germany (Darmbrand) and Papua New Guinea (enteritis necroticans, ‘pig bel’) following ingestion of contamin- ated food, or dramatic change from vegetarian to meat diets. Treatment consists of supportive care and antibiotics (usually benzylpenicillin). Complications (e.g. intestinal perforation), may require surgery, in which case mortality is high. A toxoid vaccine is protective and should be con- sidered in areas of Papua New Guinea where the disease still occurs. Acknowledgement:  The authors acknowledge inclusion of material from the chapter in a previous edition by Dr H. E. Larson.

8.6.25  Botulism, gas gangrene, and clostridial gastrointestinal infections 1121 Botulism Definition Botulism is an acute symmetrical descending paralysis caused by a neurotoxin produced by Clostridium botulinum. Food contamin- ated by C. botulinum spores and elaborated toxin produces illness when ingested. Wound infections with C. botulinum or intestinal tract colonization in infants and adults occasionally cause botu- lism. Although the illness is most commonly described in humans, botulism can occur in wild ducks feeding off the bottoms of alkaline lakes in the western United States of America. The illness is called ‘limber neck’. Occurrence C. botulinum is ubiquitously distributed in the soil. The surfaces of potatoes, vegetables, and other foods are easily contaminated with spores, which survive brief heating at 100°C. Autoclaving or use of pressure cookers that are appropriately adjusted are very effective at killing spores. In the 1920s in the United States of America, pres- sure cookers calibrated at sea level which were then used at several thousand feet above sea level in the western states were the cause of outbreaks of botulism among families that home-​canned food. The anaerobic conditions created by canning, smoking, or fermenta- tion facilitate clostridial growth and toxin release. Canned food with neutral pH, such as canned corn, is particularly prone to promoting the growth of clostridia. Spores germinate in sausage or cheese kept for extended periods at room temperature. An 18th-​century report associated paralytic illness with eating sausages, hence botulus, a Latin word for ‘sausage’. Cases have been associated with fermented milk in Africa, cheese sauce on baked potatoes in North America, fermented stew in Japan, and imported fish in the United Kingdom. Although past outbreaks typically involved small groups of people, home-​canned peppers served in a restaurant caused two large outbreaks in the United States of America. Outbreaks caused by commercially processed foods are infrequent, but contamination of hazelnut purée added to commercially produced yoghurt caused 27 cases of botulism in Wales and north-​west England in 1989, the largest recorded outbreak in the United Kingdom. Most of the contaminated cartons could not be accounted for, suggesting that the attack rate varied or that mild symptoms were not diagnosed as botulism. Commercially prepared chopped garlic in soybean oil caused 36 cases dispersed over eight provinces and states in North America. Some outbreaks involved only single contaminated items, such as in the Loch Maree episode in 1922 where eight people died after eating duck paste, the 1978 outbreak in Birmingham involving four people who ate tinned Alaskan salmon, and one case in 1989 fol- lowing a meal on a commercial airliner. Uneviscerated fresh fish have been associated with botulism, usually where there have been deficiencies in refrigeration. Purified botulinum toxin has recently come into therapeutic use. Toxin injections produce temporary muscle weakness and are ef- fective in the treatment of strabismus, blepharospasm, and torti- collis, and are also used for cosmetic purposes. Treatment doses are considered too small to elicit systemic symptoms. Under experi- mental conditions, aerosolized botulinum toxin causes illness in monkeys, and the toxin has been utilized as an agent for biological warfare or terrorist activity. For example, botulinum toxin was loaded into Scud missile warheads by Iraq during the first Gulf War and stockpiled by the Aum Shinrikyo cult in Japan. The toxin There are seven serological types of botulinum toxin (A–​G). Types A, B, and E account for nearly all human cases. Serotypes implicated in outbreaks of botulism parallel the geographical distribution of soil spores. Type E is nearly always associated with fish, but outbreaks caused by fish products can also involve types A and B. C. botulinum toxin is heat labile and rapidly inactivated at or- dinary cooking temperatures. It is a protein neurotoxin, and a dose as small as 0.1 µg is sufficient to cause death in humans. The 150-​ kDa molecule is composed of two peptide chains connected by di- sulphide bonds. One chain binds to and penetrates the neuron, and the other cleaves a protein essential for neurotransmitter release, reducing acetylcholine availability for impulse transmission. Toxin types A, C, and E hydrolyse a protein in the presynaptic membrane while types B, D, F, and G hydrolyse a protein in the synaptic vesicle. Pathogenesis Botulinum toxin is absorbed directly across mucous membranes. Locally acting toxin may produce some symptoms but cranial nerve paralysis results from blood stream distribution. Cranial nerves are preferentially affected because botulinum toxin binds more rapidly to sites where the cycles of depolarization and repolarization are fre- quent. Binding is irreversible and the toxin cannot thereafter be neu- tralized by antitoxin. Recovery occurs when nerve terminals sprout from the axon to form new motor endplates. Botulinum toxin blocks impulse transmission mediated by acetyl- choline at neuromuscular junctions, at autonomic ganglia, and at parasympathetic nerve terminals. Nerve stimulus transmission is blocked because the toxin prevents release of acetylcholine from the presynaptic membrane. Impulse conduction within peripheral nerves and muscle contraction are not affected. The synthesis of acetylcholine and impulse transmission within terminal nerve fibrils remain intact. On the other hand, the miniature endplate potentials spontaneously generated by release of acetylcholine in a resting nerve decrease and eventually disappear in the presence of toxin. If a poisoned nerve is stimulated repetitively, temporary summation of acetylcholine release occurs producing an augmented response. History The symptoms of botulism vary from mild fatigue to severe weak- ness and collapse leading to death within a day. Initially, nausea, vomiting, abdominal bloating, and dryness in the mouth and throat may suggest gastrointestinal tract illness. Diplopia, blurred vision, dizziness, unsteadiness on standing, and difficulty with speech or swallowing are common early neurological symptoms. Subsequently, there is progression to weakness or paralysis in the limbs, and generalized weakness and lassitude. The dryness of the mouth and throat may become so severe as to cause pain. Eventually there may be difficulty holding up the head, constipation, urinary hesitancy, and problems in breathing. The incubation period is between 12 and 72 h. Patients with short incubation periods are likely to have ingested large amounts of toxin. However, individuals are known to have ingested large amounts of contaminated food without developing symptoms.

section 8  Infectious diseases 1122 Physical examination Negative findings in botulism are pertinent. Higher mental functions are preserved, although sometimes patients are drowsy. Sensation is intact. Fever is unusual. The mouth is dry and the tongue is fur- rowed. Lateral rectus weakness in the eyes produces internal stra- bismus. Failure of accommodation is common and the pupils may be fixed in mid position or dilated and unresponsive to light. Ptosis, weakness of other extraocular muscles, and inability to protrude the tongue or to raise the shoulders are other early findings. Weakness in the limbs is of the flaccid, lower motor neuron type, and deep tendon reflexes are initially preserved. Facial muscles may be spared; gag and corneal reflexes are not lost. Weakness of the respiratory muscles develops early in relation to other findings and deterioration can be rapid. Paralysis descends symmetrically from cranial nerves to upper extremities to respira- tory muscles to the lower extremities in a proximal to distal pattern. Hypotension without compensatory tachycardia, intestinal ileus, and urinary retention are evidence of the widespread autonomic paralysis. Symptoms and signs can be confined to the autonomic nervous system. Diagnosis The diagnosis in the first case of an outbreak can be missed because cranial nerve symptoms and signs are ignored in what is apparently a gastrointestinal disturbance. The differential diagnosis usually lies between botulism and the descending form of acute inflammatory polyneuropathy or Guillain–​Barré syndrome. There can be simi- larities in the clinical presentation and progression of symptoms in the two diseases. Patients with botulism have normal cerebro- spinal fluid findings and respiratory weakness and failure develop early, before the presence of severe limb weakness. Patients with the Guillain–​Barré syndrome have marked limb weakness before the development of respiratory failure. Sensation and mental status are preserved in botulism. Other diagnoses that may be considered include diphtheria, in- toxication with atropine or organophosphorus compounds, myas- thenia gravis, cerebrovascular disease involving the brainstem and producing bulbar palsy, paralytic rabies, tick paralysis, and neuro- toxic snake bite. Botulism is distinguished from polymyositis and periodic paralysis by its rapid progression and cranial nerve ab- normalities. Sometimes patients with other types of poisoning are thought to have botulism, most often with an outbreak of staphylo- coccal food poisoning. Individuals with carbon monoxide poi- soning have been mistakenly been thought to be poisoned by food, but they invariably have headaches and altered consciousness. Poisoning from chemicals or fish produces rapid onset of symptoms. Mushroom poisoning is characterized by severe abdominal pain. Appropriate samples for microbiological investigation should also be collected: suspected food sample; faeces, rectal washout, vomitus, and gastric contents in suspected food-​borne or infant botulism; pus or tissue in wound botulism. These should be inoculated into a cooked meat broth or other anaerobic medium. The diagnosis of botulism can be confirmed by testing for botu- linum toxin in the patient’s serum, urine, stomach contents, or in the suspect food; 10 ml of serum should be taken before antotoxin treat- ment. Mice are inoculated intraperitoneally with 0.5 ml of sample, with and without mixing with polyvalent botulinum antitoxin, and observed for signs of botulism. Electromyography can be helpful in confirming a diagnosis of botulism. Single or low-​frequency stimuli evoke muscle action potentials that are reduced in amplitude; tetanic or rapid stimuli produce an enhanced response. Nerve conduction velocities are normal. This result readily differentiates botulism from the Guillain–​Barré syndrome. Patients with myasthenia gravis usu- ally have muscle action potentials of normal or minimally decreased amplitude. Treatment The priorities in management are assessment of respiratory func- tion followed by administration of antitoxin. Respiration should be monitored closely with a view to elective intubation since deterior- ation can occur rapidly. Prolonged respiratory support may be re- quired. Profound hypotension can be secondary to hypoxaemia, acidosis, and accumulated fluid deficits, or can be a feature of the autonomic paralysis. Treat autonomic paralysis by expanding the intravascular volume using whole blood, protein, and/​or saline while monitoring central venous pressure or by infusing a low dose of dopamine. Trivalent (types A, B, and E) antitoxin reduces case fatality and shortens the course of the illness. To be useful it must be given early, before free circulating toxin has bound to its peripheral targets and before the diagnosis can be confirmed by animal tests. Heptavalent equine antitoxin is available from the Health Protection Agency in the United Kingdom and through the State Departments of Health in the United States; one-​half of the dose is given intramuscularly and one-​half intravenously. An intradermal 0.1-​ml test dose is given, but most serum reactions are not predicted by this test. As the anti- toxin is derived from horse serum, serum sickness and anaphylaxis may occur. A pentavalent ovine antitoxin is available for military use only. One study of 132 patients with type A food-​borne botulism re- ported reduced mortality in those treated with antitoxin compared with those who were not. Earlier administration appears to reduce the duration of symptoms and duration of mechanical ventilation. Human-​derived botulinum immunoglobulin (called BIG-​IV or BabyBIG) is available for use in infants less than one year of age who are diagnosed with infant botulism. BG-​IV should be administered as early as possible in the illness. Many years ago, it was shown that patients dying of botulism car- ried bacilli in their intestine. The discovery that clinical disease can result from toxin formed within the gastrointestinal tract of infants and adults makes antimicrobial treatment theoretically appealing. Gastric lavage, repeated high enemas, and cathartics have been utilized in an attempt to remove unabsorbed toxin. Drugs capable of reversing neuromuscular blockade have been used to treat pa- tients with botulism, but without any noticeable effect on respiratory muscle weakness or tidal volume. The mortality from botulism in the early part of the 20th century was 60–​70%, but this improved to 23% for cases reported between 1960 and 1970 since the use of respiratory support. In a single large outbreak in 1977 there were no deaths among 59 cases. Recovery from botulism depends on the formation of new neuromuscular junctions; clinical improvement thus takes weeks to months. One severe case required respiratory support for 173 days with eventual recovery. Very prolonged fatigue and dyspnoea on exertion can be due to factors other than the neuromuscular blockade.

8.6.25  Botulism, gas gangrene, and clostridial gastrointestinal infections 1123 Wound botulism Symptoms and signs of botulism can develop in people with injuries. Recognition may be complicated by the presence of fever from wound infection or gas gangrene, or by the absence of gastrointes- tinal symptoms. The diagnosis is confirmed by electromyography; botulinum toxin is detected in serum in only about one-​half of the reported cases. The incubation period averages 7 days with a range of 4 to 17 days. Clinical findings and management are the same as for patients with food-​borne botulism. Since 1991, wound botulism has increasingly become a complication of injection drug abuse; small abscesses at injection sites yield C. botulinum. An epidemic of wound botulism in the United States of America has been associated with the injection of black tar heroin. C. botulinum can be recovered from wounds in the absence of clinical botulism. Infant botulism Sporadically, cases of botulism are recognized in infants less than 6 months of age. Previously healthy babies develop constipation, which progresses over 3–​10 days to poor feeding, irritability, a hoarse cry, and weakness in head control. Examination shows a generally weak, hypotonic, afebrile infant. Abnormalities in eye movements and pupillary reactions are sometimes present and deep tendon re- flexes are reduced or absent. There is considerable range in severity; respiratory failure can develop but most recover completely. The diagnosis can be confirmed by finding C. botulinum and toxin in the faeces, and by electromyography. Botulinum toxin is not present in the serum. The disease is thought to follow ingestion of C. botulinum spores, which multiply in the infant’s gastrointestinal tract and produce toxin. Excretion of C. botulinum and toxin may continue for as long as 3 months. Honey has been a source of spores in some cases. Other than supportive measures, no consistent pat- tern in treatment using antitoxin, antibiotics, cathartics, or enemas has been established. Gas gangrene Definition Gas gangrene is a rapidly developing and spreading infection of muscle caused by toxin-​producing clostridial species. Gas gan- grene is accompanied by bacteraemia, hypotension, and multiorgan failure, and is invariably fatal if untreated. Aetiology Clostridia are mainly saprophytes, occurring naturally in soil and in the gastrointestinal tracts of humans and animals. Most cases of gas gangrene are caused by Clostridium perfringens type A, but some are due to C. novyi and a few to C. septicum, C. histolyticum, C. sor- dellii, and C. fallax; not uncommonly more than one species is iso- lated. Oxygen inhibits growth of most, although C. septicum is quite aerotolerant. Gas gangrene has been a major cause of wound infection on the battlefield, although recently civilian and iatrogenic traumas have become more common. Disease development requires an anaer- obic environment and contamination of the wound with spores or vegetative organisms usually through soil contact. However, proximity to faecal sources of bacteria is also a risk factor for cases occurring after hip surgery, adrenaline injections into the buttock, or amputation of the leg for ischaemic vascular disease. Wound contamination with dirt, shrapnel, or bits of clothing reduces local oxygen concentrations. Traumatic gas gangrene develops in deep wounds involving large muscle masses in the shoulder, hip, thigh, and calf, and particularly in those situations where damage to major arteries has occurred. Thus, gunshot wounds, crush injuries, and open fractures account for most of the cases. High-​velocity bul- lets of large calibre are commonly used in contemporary times in civilian and military firearms and these produce extensive tissue damage. Necrotic tissue, foreign bodies, and ischaemia in a wound reduce the locally available oxygen and favour outgrowth of vegeta- tive cells and spores. The incidence of gas gangrene after trauma reflects the speed at which injured people can be evacuated and receive appropriate treat- ment. During the Vietnam and Falklands conflicts there were very few cases of gas gangrene among American and British wounded cared for by highly organized surgical teams. This reduction was likely due to more timely cleansing of wounds, maintaining blood flow by vascular surgery, and the use of antibiotics. In comparison, when a jet airliner crashed in the Florida everglades, 8 of the 77 in- jured survivors developed the disease. In modern times, natural disasters such as tornados and earthquakes have been associated with gas gangrene caused by C. perfringens. In these cases, patients who were traumatized suffered severe injuries, but medical care was not available for 3–​5 days. Gas gangrene occurred in 20% of such patients with open wounds in an earthquake in Wenchwan, China in 2006. Clearly, broken bones, penetrating injuries, contamination with soil, and delayed medical care contributed. Non​traumatic or ‘spontaneous gas gangrene’ occurs without a preceding injury. Classically, it presents as a primary infection of the perineum or scrotum or in a limb secondary to seeding from clostridial colonization of a colonic neoplasm. These cases are most commonly caused by the more aerotolerant C. septicum where pro- duction of superoxide dismutase permits the organisms to survive in the presence of small amounts of oxygen. Recently, C. novyi, C. sordellii, and C. perfringens have been associ- ated with necrotizing soft tissue infections at injection sites in drug addicts. Outbreaks of these infections were reported in Scotland, Ireland, England, and the United States of America in 2000 and were characterized by extensive soft tissue necrosis, hypotension, severe constitutional toxicity, and a high case fatality rate. C. sordellii infections have been described in women following natural childbirth or therapeutic abortion, and in men, women, and children following a variety of traumatic and surgical procedures. These infections are perhaps the most aggressive of all clostridial in- fections, in part because of a unique syndrome of absence of fever, profound hypotension, diffuse capillary leak, haemoconcentration, and leukaemoid reaction resulting in 70% mortality within 2 to 4 days of hospital admission. The toxins responsible for this remark- able infection have not been fully elucidated. Toxins The clinical and histological manifestations of gas gangrene are attributable to the production of potent bacterial exotoxins. The clostridia responsible for gas gangrene elaborate a wide range of toxins. More than 12 have been described for C. septicum, C. novyi, and C. perfringens. The principal toxin of C. perfringens is α-​toxin,

section 8  Infectious diseases 1124 a phospholipase C. This toxin cleaves phosphatidylcholine found in cell membrane of eukaryotic cells, releasing diacylglycerol and phosphorylcholine. In small doses, this toxin can hyperactivate a variety of cells including neutrophils, platelets, endothelial cells, and macrophages; in high doses it is cytotoxic. Interestingly, this toxin can cause the rapid and irreversible cessation of blood flow to normal tissue. This perfusion deficit is the consequence of toxin-​ induced platelet/​neutrophil aggregates that irreversibly occlude small to medium-​sized vessels. Experimentally, active or passive immunization against α-​toxin is protective against active infection. A second toxin, θ-​toxin, is a cholesterol-​dependent thiol-​activated cytolysin that lyses red blood cells and other cells by its ability to form pores in cell membranes. Electron microscopy of θ-​toxin-​ treated cells shows arc and ring structures of 7.5–​18 nm appearing in the plasma membrane as early as 1 h postexposure. These plasma membrane defects increase with time and can be visualized adjacent to toxin molecules that have been labelled with ferritin. α-​Toxin and θ-​toxin are not readily detected in the tissues or serum of patients with gas gangrene, possibly because the toxin binds rapidly and irre- versibly to lipid moieties in the cytoplasmic membranes. History The incubation period of gas gangrene is usually less than 4 days, often less than 24 h, and occasionally as short as 1–​6 h. Pain is the most characteristic symptom. Patients describe this as severe or excruciating and sudden in onset. Evolution of symptoms and signs can be very rapid. Toxicity may prevent the patient from giving an adequate history. Physical examination Early on it may be difficult to account for the patient’s pain by ob- jective physical findings. Swelling, bluish discoloration, or darkening of the skin occurs at the affected site. The traumatic or surgical wounds become oedematous and a thin, serous discharge emerges from the site. Pain steadily increases in severity; the overlying skin becomes stretched and develops a brown or ‘bronzed’ discoloration. Haemorrhagic vesicles and finally areas of frank necrosis appear. A sweet odour from the wound has been described. Gas is not in- variably present early in the course, but radiographs may detect gas earlier than can physical examination. Later, crepitus and exquisite tenderness are present in the wound. Profound constitutional changes occur. Patients become sweaty and febrile, and though alert and oriented, are very distressed. The pulse is elevated out of proportion to the fever. Death can occur within 48 h. At operation, infected muscle appears dark red with purple discoloration; frank gangrene and liquefaction may be seen. Involved muscle does not bleed when cut or contract when directly stimulated. Rapidly progressing necrotizing infections of the soft tissue may be monomicrobial, caused by clostridia, Streptococcus pyogenes, Staphylococcus aureus, Vibrio vulnificus, or Aeromonas hydroph- ila. Alternatively, necrotizing infections may be polymicrobial and caused by mixed aerobic and anaerobic microbes. Clostridia and polymicrobial infections are usually associated with gas in the tissue, whereas the others are not. Polymicrobial necrotizing in- fections occur most commonly following gastrointestinal surgery, penetrating injury to the abdomen, surgical incisions in the vaginal mucosa (episiotomy), or in diabetic patients with peripheral vascular disease. All of these necrotizing infections may destroy fascia, but frequently also destroy muscle, subcutaneous tissue, and skin. Diagnosis The diagnosis of gas gangrene must be made on clinical grounds and prompt recognition and treatment improve the prognosis. Sudden deterioration in a postoperative patient or following trauma requires examination of the wound and surrounding tissue. Cases of primary gas gangrene and cases following elective surgery may have a higher fatality because recognition is delayed. Gram’s staining of the wound discharge, of an aspirate, or of a needle biopsy may aid diagnosis. In gas gangrene there are many large plump Gram-​positive bacilli, usually without spores. Few, if any, polymorphonuclear leucocytes are present in the tissues or exudates, likely due to toxin-​induced inhibition of cellular extravasation. CT scanning can detect gas deep in muscle, but the absence of gas does not exclude the diagnosis. Culture of clostridia does not confirm a diagnosis of gas gangrene as simple colonization without clinical disease occurs in up to 30% of traumatic wounds. Treatment Surgical removal of all affected muscle is essential to eliminate the conditions that allow the organism to grow. High-​velocity missiles distribute energy radially from their path, producing more extensive tissue damage than missiles at low speeds or with a small mass. Thus, wounds should be excised widely by resection back to healthy, viable muscle and skin. Closure should be delayed for 5 to 6 days until it is certain that the wound is free of infection. Administration of appropriate antimicrobial agents is also re- quired. Penicillin has been the drug of choice based on in vitro sus- ceptibility testing, but experimental evidence has demonstrated that clindamycin or tetracycline is superior to penicillin. This improved efficacy is most likely because these two protein synthesis inhibitors prevent the production of toxins. This has led to the use of penicillin and clindamycin as combination therapy. Ceftriaxone or erythro- mycin are alternative choices for severely penicillin-​allergic patients. Hyperbaric oxygen therapy (typically 100% oxygen at 303 kPa for 60–​120 min, 2–​3 times daily) has been used to treat gas gangrene; however, an effect on mortality has never been shown by controlled trials and comparable survival rates have been achieved without using it. Experimental studies have demonstrated that hyperbaric oxygen alone was neither effective in an animal model of C. perfrin- gens gas gangrene nor did it improve the efficacy of clindamycin or penicillin. Therapeutic administration of gas gangrene antitoxin made from horse serum is controversial. Use during the Second World War reduced mortality, but serum sickness and other allergic reactions occurred. It is no longer produced in the United States of America. In recent studies, active immunization of animals with a truncated, non​toxic form (C-​domain) of the α-​toxin was 100% protective against active muscle infection with C. perfringens. Prevention Prophylactic antibiotic treatment reduces the risk of gas gangrene, but this depends on the time interval between the injury and surgical debridement, the associated vascular deficit, the presence of foreign body, the presence of a compound or open fracture, and the dur- ation of antibiotic administration. Patients have clearly developed

8.6.25  Botulism, gas gangrene, and clostridial gastrointestinal infections 1125 gas gangrene after prophylactic administration of β-​lactam anti- biotics. Gas gangrene can develop from wounds contaminated with either vegetative organisms or spores. Antibiotics may be more effective in the former case since spores, until they germinate, are not affected by antibiotics. Metronidazole or clindamycin may be useful in patients who are hypersensitive to β-​lactam antibiotics. Experimentally, active immunization against the α-​toxin provides impressive protection against C. perfringens gas gangrene, but no ac- tive or passive vaccine is currently available. Clostridial infections of the gastrointestinal tract Necrotizing enterocolitis Definition Necrotizing enterocolitis is a fulminating clinical illness charac- terized by extensive necrosis of the intestinal mucosa and wall. Terms such as darmbrand (Germany), enteritis necroticans, pig bel (Papua New Guinea), or gas gangrene of the bowel describe geographic variants. Cases occur sporadically in adults or as epi- demics in all ages. Necrotizing enterocolitis occurs in infants, and some of these cases have demonstrated clostridia in the wall of the intestine. Aetiology Gram’s staining of the necrotic mucosa and the bowel wall shows many Gram-​positive bacilli that are typically identified as C. per- fringens (C. welchii). Sporadic cases usually yield C. perfringens type A. However, in the German and especially in the Papua New Guinea outbreaks, there is substantial evidence implicating C. per- fringens type C. Type C produces large amounts of β-​toxin, which has lethal and necrotizing effects. Papua New Guinea highlanders have a high prevalence of antibodies to β-​toxin; antibodies are rare in people who live where the disease is uncommon. Patients with pig bel have rising levels of antibodies to β-​toxin, and spe- cific passive or active immunization prevents disease. It is not clear whether exogenous human infection with these organisms occurs or whether the lesions are produced by the overgrowth of en- dogenous clostridia. Sweet potato, a local dietary staple, contains an inhibitor of trypsin. Combined with a low-​protein diet this may impair the ability of the intestine to inactivate endogenously pro- duced β-​toxin. However, the methods used for roasting the pigs offer many opportunities for clostridial contamination. History and physical examination Sporadic cases in patients over 50  years of age or among those recovering from gastric surgery are regularly reported from Scandinavia, Europe, the United States of America, Australia, and the Middle East. Alternatively, epidemic outbreaks as described in postwar Germany and among the highlanders of Papua New Guinea follow ingestion of contaminated food or a dramatic change in eating habits. Severe intermittent abdominal pain is the first symptom and pain rapidly becomes continuous. Bloody diarrhoea and vomiting are common. Patients quickly develop tachycardia, followed by hypotension and evidence of multiorgan failure. On examination there is fever with abdominal distension, localized or diffuse ten- derness, and reduced bowel sounds. A tender mass may be palpated. Following resolution of infection, malabsorption and partial small bowel obstruction may develop because of intestinal scarring. Treatment and prevention Patients with suspected pig bel should be treated with nasogastric suction and intravenous fluids. Pyrantel is given by mouth and the bowel rested by fasting. Benzylpenicillin, 1 MU, is given intraven- ously every 4 h and the patient observed for complications requiring surgery. Mild cases recover without surgical intervention, but if sur- gical indications are present the mortality ranges from 35 to 100%, in part due to perforation of the intestine. As pig bel continues to be a common disease in Papua New Guinea, consideration should be given to the use of a C. perfringens type C toxoid vaccine in local areas. Two doses spaced 3–​4 months apart are preventive. Clostridium perfringens food poisoning Occurrence and clinical findings In the United Kingdom and the United States of America, food poi- soning caused by C. perfringens is the third most common type of food-​borne illness. Meat and poultry are responsible for at least 90% of the outbreaks, which occur where food is prepared in large quan- tities. Two-​thirds of the reported outbreaks are in schools, hospitals, factories, restaurants, or catering establishments, and in a typical outbreak 35–​40 people are affected. An estimated 12 000 cases were associated with a single outbreak in 1969. The circumstances surrounding an outbreak repeat themselves with monotonous regularity. A meat dish is prepared by stewing, braising, boiling, or steaming and this is allowed to stand at ambient temperatures for a period of 4–​24 h. The food is served cold or after rewarming. Six to twelve hours after eating the meal, people com- plain of cramping abdominal pain and then diarrhoea. Vomiting is unusual and fever inconsequential. Twelve to twenty-​four hours later, the diarrhoea and pain have subsided. Fatal cases occur rarely; at autopsy they show severe enterocolitis. Undoubtedly many cases of C. perfringens food poisoning occur at home but are not reported. Antibodies to the toxin mediating the symptoms are very common and it is likely that nearly everyone has experienced this disease once or more in their lifetime. Aetiology C. perfringens is a ubiquitous sporulating anaerobe with an unpar- alleled virtuosity for production of biologically significant toxins. The clinical effects of infection with any particular strain may de- pend largely on its toxin-​producing capacity. Strains associated with food poisoning have several special characteristics. They are type A, although their production of α-​toxin is variable; the organ- isms are often heat resistant to 100°C. Eighty-​six per cent (86%) of food-​poisoning strains produce a specific heat-​labile enterotoxin. Toxin production in vitro is closely associated with sporulation ra- ther than with the multiplication of vegetative cells. In vivo, toxin probably acts by damaging enterocyte membranes. Free enterotoxin has been detected in diarrhoeal stool after C. perfringens food poi- soning. Antibody to enterotoxin increases after such episodes, and ingestion of 8–​12 mg enterotoxin by volunteers produces abdominal pain and diarrhoea. C. perfringens is a normal human faecal organism, is regularly found in the intestinal tract of domestic animals, often contaminates

8.6.26 Tuberculosis 1126

8.6.26 Tuberculosis 1126

section 8  Infectious diseases 1126 raw meat, and can be carried by flies. The distribution of enterotoxin-​ producing strains may be more restricted. However, surface con- tamination of meat with C. perfringens is common and subsequent rolling or grinding distributes these organisms throughout. Spores germinate and multiply to 106 to 107 cells/​g in the anaerobic envir- onment created when meat or meat gravy cools slowly or stands at ambient temperature. Reheating may not kill these cells and, when ingested, they multiply still further, sporulate, and release their toxin. Enterotoxin-​producing strains of C. perfringens may sometimes cause diarrhoea by means of overgrowth in the gut. Patients, usually elderly, can experience diarrhoea without known contact with con- taminated food. The diarrhoea may be short-​lived or persist inter- mittently for several months. Colony counts of 108 to 1010/​g of faeces are associated with the presence of high titres of free toxin. Previous antimicrobial treatment may encourage the overgrowth and the same strain has been found to cross-​infect patients. FURTHER READING Botulism Arnon SS, et al. (2006). Human botulism immune globulin for the treatment of infant botulism. N Engl J Med, 354, 462–​71. Cherington M (2004). Botulism: update and review. Semin Neurol, 24, 155–​63. Chertow DS, et al. (2006). Botulism in 4 adults following cosmetic in- jections with an unlicensed, highly concentrated botulinum prepar- ation. JAMA, 296, 2476–​79. Fox CK, Keet CA, Strober JB (2005). Recent advances in infant botu- lism. Pediatr Neurol, 32, 149–​54. Lalli G, et al. (2003). The journey of tetanus and botulinum neuro- toxins in neurons. Trends Microbiol, 11, 431–​7. Sobel J (2009). Diagnosis and treatment of botulism: a century later, clinical suspicion remains the cornerstone. Clin Infect Dis, 48, 1674–​5. Gas gangrene Aldape MJ, Bryant AE, Stevens DL (2006). Clostridium sordellii in- fection: epidemiology, clinical findings and current perspectives on diagnosis and treatment. Clin Infect Dis, 43, 1436–​46. Bryant AE, Stevens DL (1996). Phospholipase C and perfringolysin O from Clostridium perfringens upregulate ELAM-​1 and ICAM-​1 ex- pression, and induce IL-​8 synthesis in cultured human umbilical vein endothelial cells. Infect Immun, 64, 358–​62. Bryant AE, et al. (1993). Clostridium perfringens invasiveness is en- hanced by effects of theta toxin upon PMNL structure and function. FEMS Immunol Med Microbiol, 7, 321–​36. Bryant AE, et al. (2000). Clostridial gas gangrene I: cellular and molecular mechanisms of microvascular dysfunction. J Infect Dis, 182, 799–​807. Bryant AE, et al. (2000). Clostridial gas gangrene II: phospholipase C-​ induced activation of platelet gpIIbIIIa mediates vascular occlusion and myonecrosis in C. perfringens gas gangrene. J Infect Dis, 182, 808–​15. Bryant AE, et  al. (2006). Clostridium perfringens phospholipase C-​ induced platelet/​leukocyte interactions impede neutrophils diape- desis. J Med Microbiol, 55, 495–​504. Centers for Disease Control (2000). Update: Clostridium novyi and un- explained illness among injecting-​drug users. MMWR Morb Mortal Wkly Rep, 49, 543–​5. Cohen AL, et  al. (2007). Toxic shock associated with Clostridium
sordellii and Clostridium perfringens after medical and spontaneous abortion. Obstet. Gynecol, 110, 1027–​33. Darke SG, King AM, Slack WK (1977). Gas gangrene and related in- fection: classification, clinical features and aetiology, management and mortality: a report of 88 cases. Br J Surg, 64, 104–​12. Maclennan JD (1962). The histotoxic clostridial infections of man. Bacteriol Rev, 26, 177–​276. Shouler PJ (1983). The management of missile injuries. J R Nav Med Serv, 69, 80–​4. Stevens DL, Bryant AE (2005). Clostridial gas gangrene: clinical cor- relations, microbial virulence factors, and molecular mechanisms
of pathogenesis. In: Proft T (ed) Microbial toxins: molecular and cellular biology, pp. 313–​35. Horizon Bioscience, Norfolk, UK. Stevens DL, et al. (1993). Evaluation of therapy with hyperbaric oxygen for experimental infection with Clostridium perfringens. Clin Infect Dis, 17, 231–​7. Stevens DL, et al. (2004). Immunization with the C-​domain of alpha-​ toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens. J Infect Dis, 190, 767–​73. Stevens DL, Bryant AE (2017). Necrotising soft tissue infections. N Engl J Med, 377, 2253–65. Gastrointestinal infections Abrahao C, et al. (2001). Similar frequency of detection of Clostridium perfringens enterotoxin and Clostridium difficile toxins in patients with antibiotic-​associated diarrhea. Eur J Clin Microbiol Infect Dis, 20, 676–​7. Alfa MJ, et al. (2002). An outbreak of necrotizing enterocolitis asso- ciated with a novel clostridium species in a neonatal intensive care unit. Clin Infect Dis, 35, S101–​5. Bos J, et al. (2005). Fatal necrotizing colitis following a foodborne out- break of enterotoxigenic Clostridium perfringens type A infection. Clin Infect Dis, 40, e78–​83. Fisher DJ, et  al. (2005). Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal dis- ease isolates carrying a plasmid enterotoxin gene. Mol Microbiol,
56, 747–​62. Lawrence GW, et al. (1990). Impact of active immunisation against en- teritis necroticans in Papua New Guinea. Lancet, 336, 1165–​7. Li DY, et al. (2004). Enteritis necroticans with recurrent enterocutaneous fistulae caused by Clostridium perfringens in a child with cyclic neu- tropenia. J Pediatr Gastroenterol Nutr, 38, 213–​15. Obladen M (2009). Necrotizing enterocolitis—​150 years of fruitless search for the cause. Neonatology, 96, 203–​10. Sobel J, et  al. (2005). Necrotizing enterocolitis associated with Clostridium perfringens type A in previously healthy north American adults. J Am Coll Surg, 201, 48–​56. 8.6.26  Tuberculosis Richard E. Chaisson and Jean B. Nachega ESSENTIALS Tuberculosis is caused by organisms of the Mycobacterium tuber- culosis complex, including M.  tuberculosis (the most important), M. bovis, and M. africanum. It has been present since antiquity and

8.6.26  Tuberculosis 1127 is the leading infectious cause of death ahead of HIV infection. An estimated 2 billion people worldwide carry latent infection, when M. tuberculosis persists within cells and granulomas, with the poten- tial to reactivate to cause disease decades later. Tubercle bacilli are transmitted between people by aerosols gen- erated when an infectious person coughs. Proximity to an infectious person determines the risk of infection. Host immunity and factors affecting it—​most importantly HIV infection but also diabetes, cig- arette smoking, and alcohol and drug abuse—​determine the risk of active disease following infection. Clinical presentation of active tuberculosis is highly variable, depending on the site, extent of disease, and the immune status of the host. Disease is generally classified as pulmonary or extrapulmonary, with considerable clinical heterogeneity within each group. Clinical features—​pulmonary tuberculosis Following deposition of tubercle bacilli in the alveoli of the lungs, they are ingested by alveolar macrophages, multiply intracellularly, and eventually cause cell lysis with release of organisms. Over a period of weeks, infection spreads to regional lymph nodes, else- where in the lungs, and systemically. Infected people who success- fully contain viable bacilli in granulomas may retain a latent infection, with lifetime risk of reactivation of about 10%. Active pulmonary tuberculosis—​this is usually a subacute respiratory illness, the most frequent symptoms of which are cough, fever, night sweats, and malaise. The cough is initially non​productive, but often progresses to sputum production and occasionally haemoptysis. Loss of appetite and excessive weight loss are common. Clinical features—​extrapulmonary tuberculosis This can be generalized or confined to a single organ, and is found in 15–​20% of all cases of tuberculosis in otherwise immunocompe- tent adults, more than 25% of cases under 15 years of age, and in more than 50% of HIV-​related cases. Children under 2 years of age have high rates of miliary or disseminated tuberculosis and menin- geal disease. Infection spreads from the lungs by lymphatic and haema- togenous routes. The tissues and organs most likely to be affected are the pleura, lymph nodes, kidneys, and other genitourinary or- gans, bone, and central nervous system. Tuberculosis bacteraemia is unusual, but seen most often in patients with HIV infection and low CD4 lymphocyte counts. Pleural tuberculosis—​this is usually the result of relatively small numbers of tubercle bacilli invading the pleura from adjacent lung tissue, in which case the duration of symptoms is generally brief, with patients complaining of symptoms including fever, chest pain, and non​productive cough. Pleural tuberculosis involving larger numbers of bacilli produces frank empyema and is more common in older patients. Lymphatic tuberculosis—​classic scrofula of the cervical or supraclavicular lymph node chains is the most common presen- tation, but multiple lymph node groups can be involved in HIV-​ infected patients. Genitourinary tuberculosis—​the most common manifestation is renal tuberculosis, resulting from haematogenous seeding of the renal cortex during primary infection; this is frequently asymptom- atic, but might be evident as sterile pyuria. Bone and joint tuberculosis—​the most common form is vertebral tuberculosis (Pott’s disease), resulting from haematogenous seeding of the anterior portion of vertebral bodies during primary infection; presentation is typically with back pain; constitutional symptoms are not prominent in most cases. Tuberculous meningitis—​meningeal and leptomeningeal bacterial replication results in a robust inflammatory reaction that increases cerebrospinal fluid pressure and can cause cranial neuropathies. Common symptoms are headache, stiff neck, meningism, and an al- tered mental status, including irritability, clouded thinking, and mal- aise. The condition is not common, but usually fatal if untreated. Miliary/​disseminated tuberculosis—​these describe widespread in- fection with absent or minimal host immune responses, usually arising as a result of primary infection, and seen more frequently in children and immunocompromised adults. Typical presentation is with fever and other constitutional symptoms over a period of several weeks. Diagnosis Tuberculin skin testing—​intracutaneous injection of purified proteins of M. tuberculosis provokes a delayed hypersensitivity reaction which produces a zone of induration in those who are infected, but cannot distinguish disease from latent infection and may be falsely positive from Bacille Calmette–​Guérin vaccination or non​tuberculous myco- bacterial infections. Interferon-​γ release-​based assays—​these detect in vitro responses to M. tuberculosis antigens. These appear to be more specific than tuberculin skin testing because false-​positive reactions due to sen- sitization from Bacille Calmette–​Guérin vaccination are less likely to occur. They may also be more sensitive, and are appealing because they do not require patients to return for reading of induration. Detection of tubercle bacilli—​microscopical staining of acid-​fast bacilli in sputum or other tissue is the method most widely used to diagnose tuberculosis because it is inexpensive, rapid, and techno- logically undemanding. However, a relatively large number of bacilli are needed for a positive test, and up to 50% of patients with sputum cultures positive for M. tuberculosis have negative acid-​fast smears. Culture of M. tuberculosis is the gold standard for confirming the diag- nosis, but takes 10–​40 days, depending on the method used. Nucleic acid amplification assays and other rapid diagnostic methods allow faster detection of both the presence of mycobacteria and assess- ment of drug resistance: these have promise in resource-​limited set- tings, but further validation in endemic countries is needed. Nucleic acid amplification—​several new commercial assays that amplify M. tuberculosis DNA can result in rapid diagnosis of tuber- culosis (<1 day). Some tests also can detect drug-​resistant mutations, providing timely detection of multidrug-​resistant tuberculosis. Particular issues—​(1) Pulmonary tuberculosis—​this can involve any portion of the lungs, hence radiographic findings are usually only suggestive, not diagnostic. (2) Pleural tuberculosis—​diagnosis can be inferred from pulmonary findings when pulmonary parenchymal in- volvement is manifest, otherwise analysis of pleural fluid is essential. (3) Lymphatic tuberculosis—​swelling of involved nodes accompanied by a positive tuberculin skin test and typical biopsy findings are strongly suggestive of tuberculosis and warrant presumptive therapy. (4) Tuberculous meningitis—​diagnosis requires a high degree of sus- picion; presumptive therapy is frequently necessary.

section 8  Infectious diseases 1128 Treatment Drug-​susceptible tuberculosis—​combination therapy with isoniazid and rifampin (and other antituberculosis drugs in the first 8 weeks) is highly effective. Treatment is usually once daily but can be given as infrequently as twice per week, with two major interventions to im- prove adherence and prevent bad outcomes being directly observed therapy and the use of fixed-​dose combination tablets. Modern ‘short course’ combination chemotherapy is curative in 6 months, except for bone and central nervous system tuberculosis, which re- quire 12 months. Second-​line agents are reserved for treatment of drug-​resistant tuberculosis and are generally less potent, more toxic, and less readily available. Drug-​resistant tuberculosis—​this significant challenge arises both through infection with drug-​resistant strains (primary or ‘new’ drug resistance) and by selection for drug-​resistant strains due to inef- fective therapy (secondary or ‘previously treated’ drug resistance). Multidrug-​resistant tuberculosis is defined as resistance to at least ri- fampicin and isoniazid. Extensively drug-​resistant disease, which has been reported in more than 70 countries, is defined as multidrug re- sistant plus resistance to fluoroquinolones and at least one injectable second-​line agent (capreomycin, amikacin, or kanamycin). Patients with drug-​resistant tuberculosis should be managed by a physician who is a tuberculosis expert because of the complexity of their regi- mens and their high risk of failure of death. Prevention Strategies to control tuberculosis include: (1) Identification and treat- ment of infectious tuberculosis cases, which rapidly eliminates in- fectiousness. (2) Treatment of latent tuberculosis infection—​the use of preventive therapy in high-​risk individuals known or strongly suspected to be latently infected with M.  tuberculosis can benefit not only the individual patient who does not fall ill with tubercu- losis, but also potential contacts of that patient, who might become secondarily infected were disease to develop. (3) Prevention of ex- posure to infectious particles in air, especially in hospitals and other institutions—​infected patients must be identified and managed in respiratory isolation. (4)  Vaccination—​the attenuated live vaccine, Bacille Calmette–​Guérin, is widely administered throughout the world, but remains controversial. Proponents argue that it provides about 50% protection against active tuberculosis disease and also diminishes haematogenous dissemination of primary tuberculosis infection, thereby reducing the incidence of miliary tuberculosis and tuberculous meningitis in children. Introduction Tuberculosis (TB) is one of the most important diseases in the his- tory of humanity, and remains an extraordinary burden on human health today. Archaeological evidence demonstrates that tubercu- losis was present in antiquity, and large epidemics of the disease emerged in Europe in the Middle Ages. While contemporary phys- icians consider tuberculosis to be one of the classic infectious dis- eases, recognition of the clinical manifestations of the disease has evolved over the past two millennia. The Greek term phthisis was used by Hippocrates to describe the wasting disease later known as tuberculosis. While the Greeks recognized various clinical manifestations of tuberculosis, understanding of the connection between the forms was limited. In the Middle Ages, the study of anatomy and the correlation of pathological findings with clinical syndromes led to a better understanding of the disease. The term ‘tuberculosis’ was used first only in the early 19th century, derived from the tubercles characterized in the study of pathological fea- tures of the disease. The impact of tuberculosis on the human population cannot be overstated, as the disease has killed hundreds of millions of people over the centuries and has had economic and social effects perhaps unparalleled in the history of medicine. Between 1700 and 1950, tuberculosis was a great killer in the developed world, earning the sobriquet ‘the captain of the men of death’ from John Bunyan, and ‘the White Plague’ from René and Jean Dubos. The inspiration that artists have drawn from tuberculosis, portrayed in literature, opera, and art, testifies not only to the importance of the disease within their contemporary societies, but also to the extent to which tuber- culosis affected artists themselves. The annals of art are filled with those who succumbed to tuberculosis including Keats, Chopin, the Brontë sisters, Stevenson, Poe, and many, many others. The conquest of tuberculosis through the development of vac- cines, drugs, and diagnostics was a principal goal of biomedical re- search in the 19th and 20th centuries. The first description of the tubercle bacillus as the cause of tuberculosis by Robert Koch in 1882 was a scientific landmark. The postulates established by Koch for determining the microbial aetiology of disease have continuing in- fluence today, and molecular correlates of those derived by Koch fur- ther strengthen the ingenuity of his thesis. Koch also developed the microscopic and culture methods for detecting tubercle bacilli, still widely used today. Calmette and Guérin developed an effective vac- cine for tuberculosis in the early 20th century, but use of the vaccine was not broad enough to control the disease and it may no longer be effective. The discovery of streptomycin by Schatz and Waksman in 1943 was a major triumph; both Koch and Waksman received the Nobel Prize for their work. The development of additional anti- microbial agents against tuberculosis in the 1950s, 1960s, and 1970s, and the evaluation of chemotherapy in elegant studies conducted by the British Medical Research Council, the United States Public Health Service, and the United States Veterans Administration led to a marked apathy about tuberculosis in the closing decades of the 20th century. Despite the availability of curative chemotherapy for more than half a century, however, tuberculosis continues to kill more than 1.5 million people/​year, and causes an enormous amount of suf- fering and disability. In 1994, the World Health Assembly declared that tuberculosis was a global health crisis, and the situation has only grown more serious since then. Epidemics of HIV-​related tu- berculosis and multidrug-​resistant disease have expanded in re- cent years, and global control of tuberculosis remains a formidable challenge. The unique biological properties of the causative organism, Mycobacterium tuberculosis complex, allow for a long incubation period between the time of infection and the development of symp- toms. Latent tuberculosis infection can persist for decades before causing disease, or can persist for the lifetime of an infected person without ever causing clinically evident illness. Because latent infec- tion creates a large reservoir of carriers of the infection, disease elim- ination is difficult to envisage.

8.6.26  Tuberculosis 1129 Aetiology Tuberculosis is a granulomatous disease caused by organisms of the M. tuberculosis complex, including M. tuberculosis, M. bovis, and M. africanum, of which M. tuberculosis is the most important. M. tu- berculosis and the other mycobacteria are small rod-​shaped or curved bacilli in the order Actinomycetales, family Mycobacteriaceae, with a unique thick cell wall composed of glycolipids and lipids. The lipid-​rich coat of the mycobacteria renders these organisms resistant to acid decolorization following carbol-​fuchsin staining, hence the term ‘acid-​fast bacilli’. Classification of the mycobac- teria was based for many years on the staining and growth prop- erties described by Runyon, but this unwieldy system has been largely replaced with modern techniques that identify mycobac- teria by specific DNA sequences and, to a lesser extent, biochem- ical assays. Mycobacteria are frequently considered according to the diseases they cause more than their behaviour in the labora- tory: M. tuberculosis complex causes tuberculosis; M. leprae causes leprosy; and the non​tuberculous mycobacteria, including rapid growers, are associated with a variety of manifestations, particularly in immunocompromised hosts. The organisms of the M. tuberculosis complex are remarkably slow growing, with a generation time between 20 and 24 h. The exceed- ingly slow intrinsic reproductive rate of M. tuberculosis contributes both to its behaviour as a pathogen and to difficulties in recovering the organism in cultures. Moreover, M. tuberculosis is able to persist in a latent form within cells and granulomas for many years, and can reactivate to cause disease decades after infection is acquired. Tubercle bacilli are not known to form spores, but both typical bacilli and non​staining forms of the bacteria persist in cells and tissues, as evidenced by detection of DNA, years after infection is acquired, and retain the capacity to replicate and produce clinical illness. These unique biological characteristics make the tubercle bacillus exceedingly difficult to combat and control. Epidemiology Global incidence Despite the widely held belief that tuberculosis was waning during the 1980s, global tuberculosis incidence has been steady or increasing for several decades. In Western Europe and North America, the incidence of tuberculosis peaked in the 1700s and 1800s, and then declined over a period of years before the devel- opment of chemotherapy. Improvements in hygiene and nutrition, along with reductions in household crowding, were credited with these trends. Following the introduction of curative treatment for tuberculosis in the era following the Second World War the inci- dence of disease fell even further, and tuberculosis deaths were greatly decreased. The success in controlling tuberculosis experi- enced in the western nations was not replicated in developing coun- tries, and increasing epidemics of the disease have been occurring in these areas. In addition, progress in tuberculosis control in the western nations ironically led to neglect of public health pro- grammes that were responsible for reductions in morbidity. As a consequence of inattention to control, the United States of America experienced a resurgence of tuberculosis between 1985 and 1992, with a 21% increase in the annual number of reported cases during that time. In the United Kingdom, tuberculosis incidence has lev- elled off in recent years, with an annual incidence of 11 cases per 100 000 people since 1991. Worldwide, tuberculosis continues to kill more than 1.5 million people per year, making it the leading infectious cause of death ahead of HIV infection. Tuberculosis is a leading cause of death in AIDS, and HIV-​related tuberculosis deaths are attributed to AIDS not tuberculosis. The World Health Organization (WHO) estimates that 1.5 bil- lion people, or one-quarter of the world’s population, are infected with M. tuberculosis. From this seedbed of latent infection, about 10.5  million people became ill with TB and 1.7  million died in 2016, and most of the cases were detected in developing coun- tries. The global distribution of tuberculosis case rates is shown in Fig. 8.6.26.1. Disease due to M. tuberculosis is most common in developing nations, both in absolute numbers and incidence of new cases. Twenty-​two countries account for 80% of all cases of tuberculosis; India and China are responsible for 40% of cases. In general, the highest incidence of disease is found in the coun- tries of sub-​Saharan Africa where HIV infection has contributed to extraordinary increases in case rates. The greatest number of cases arise in the populous nations of Asia, which have moderately high rates of disease per capita. The global incidence of tuberculosis is decreasing slightly, though population growth is resulting in higher numbers of cases each year. Declines in incidence in the devel- oped world have been offset by increasing rates in the HIV-​ravaged countries of Africa and by escalating incidence in Eastern Europe in the aftermath of the collapse of communism and its public health infrastructure. Effect of age Tuberculosis typically affects young adults, with peak incidence in those aged 25 to 44 years. The dynamics of tuberculosis within a particular country or region, however, reflect both historical trends in tuberculosis transmission and current risk factors and practices of disease control. For example, in Western Europe tuberculosis is seen in two demographic groups:  elderly native Europeans who were presumably infected many years ago and who experience reactivation of latent infections as they age or become immuno- compromised, and younger immigrants from high-​incidence countries in the developing world. Interestingly, increasing age is not a risk factor for developing active tuberculosis per se; among ageing populations infected with M.  tuberculosis earlier in life, the risk of developing disease decreases over time. In the United States of America tuberculosis is seen in young adults who have immigrated from endemic areas and in those with HIV infection, whereas reactivation tuberculosis in older people is increasingly uncommon. In the developing world, tuberculosis most com- monly occurs in young adults, with rapidly escalating rates in those with HIV infection. In all countries where tuberculosis is preva- lent, children who acquire tuberculosis from adults account for up to 10% of all cases. Interestingly, children between the ages of 5 and 15 years have extremely low rates of tuberculosis, even in areas with a high disease burden. Infection and disease The epidemiology of tuberculosis can be considered as a func- tion of two distinct but related phenomena:  the likelihood of

section 8  Infectious diseases 1130 becoming infected with M. tuberculosis and the probability of developing disease once infection has occurred. Risk factors for becoming infected relate to exposure to infectious cases. Throughout the world, living with someone who has infectious tuberculosis is the most important risk factor for acquiring in- fection. The longer the duration of undiagnosed tuberculosis, the greater the severity of disease, and the more intimate the con- tact, the greater the chance of becoming infected. Exposure to infectious cases in other environments, including healthcare fa- cilities, prisons, and the workplace, is another important route of infection. In areas of the world where tuberculosis is relatively widespread, exposure in the community is commonplace and probably unavoidable. In low prevalence countries, community exposure is most likely to occur in distinct pockets of increased incidence, such as poorer areas of large cities or neighbourhoods with high HIV prevalence. Effect of host immunity After M. tuberculosis infection is acquired, the risk of developing disease is dependent on host immunity. As discussed next, several conditions have been identified that increase the risk of active disease in a person with latent tuberculosis infection, most notably HIV infection. Reactivation from latent tuberculosis infection is an important mechanism for the development of adult tuberculosis. However, studies using DNA fingerprinting techniques show that a significant proportion of tuberculosis cases thought to be due to reactivation are actually recently acquired due to reinfection or new infection, particularly in high HIV prevalence settings. Effect of M. tuberculosis strain Interestingly, strain differences in M. tuberculosis have not been associated with the risk of disease, although inoculum size is as- sociated with probability of becoming ill. For example, household contacts of heavily sputum acid-​fast bacilli smear-​positive cases of tuberculosis who become infected have a higher incidence of active disease than contacts of acid-​fast bacilli smear-​negative cases who become infected. On the other hand, while there is some evidence that specific strains of M. tuberculosis may more successfully infect contacts than other strains, the risk of disease in those infected with these transmissible strains is not elevated. Susceptibility Tuberculosis is a disease traditionally associated with specific population groups, notably the poor, alcohol and drug abusers, and, more recently, those with HIV infection. The increased in- cidence of tuberculosis in impoverished populations is probably Fig. 8.6.26.1  WHO-​estimated global tuberculosis incidence rates in 2016. From ‘Global tuberculosis report 2017’. Geneva: World Health Organization; 2017.

8.6.26  Tuberculosis 1131 multifactorial, involving increased risk of infection (e.g. due to crowded living conditions and a higher background prevalence of disease in the community) and increased risk of developing dis- ease after infection (e.g. due to malnutrition). Similar reasons may explain the higher rates of tuberculosis seen in cigarette smokers and alcohol and drug abusers, with suppression of host cellular immunity either directly or indirectly caused by substance abuse. The more recent association of tuberculosis and HIV infection is clearly related to development of cellular immunodeficiency in those with HIV, but in many settings those at highest risk for HIV infection are also more likely to be latently infected with M. tuber- culosis than others. Effect of the HIV epidemic The impact of HIV infection on the epidemiology of tubercu- losis is striking. As will be discussed next, HIV infection is the most potent known biological risk factor for tuberculosis. The relative risk of tuberculosis in an HIV-​infected person is 200 to 1000 times greater than in someone without HIV infection. The risk of tuberculosis increases shortly after HIV seroconversion, doubling within the first year. As a result of the extraordinary risk conferred from HIV infection, most tuberculosis patients in many sub-​Saharan countries are HIV seropositive. The incidence of active tuberculosis in HIV-​infected patients not receiving anti- retroviral therapy in the United States of America, with latent tuberculosis infection defined by a positive tuberculin skin test, is about 10% per year. Even when antiretroviral therapy is pro- vided to individuals with HIV infection, the risk of tuberculosis remains substantially higher than in HIV-​uninfected people from the same population. Of note, an annual incidence rate of about 10% is described in HIV-​infected patients in South Africa regard- less of tuberculin skin test status. In addition, HIV infection is the unifying theme in many nosocomial outbreaks of tuberculosis, as infection is spread among immunocompromised patients re- ceiving medical care at the same facility. It is increasingly apparent that control of tuberculosis will not be possible globally without control of HIV infection. Effect of drug resistance Another very important trend in tuberculosis epidemiology is the growing problem of drug-​resistant tuberculosis. Drug-​resistant tu- berculosis is reported as two types: ‘new’ and ‘previously treated’. New drug resistance is caused by transmission of a resistant strain of M. tuberculosis and previously treated implying the possibility of acquired drug resistance during previous treatment (e.g. non-​ adherence or inadequate treatment regimen), though recurrence of tuberculosis with drug resistance might also indicate previous in- appropriate treatment of an unrecognized resistant strain. A global survey of resistance performed by the WHO and the International Union Against Tuberculosis and Lung Disease found that the me- dian prevalence of primary drug resistance was 10%, and the median prevalence of acquired resistance was 36%. Moreover, ‘hot spots’ of drug-​resistant tuberculosis were identified on all continents. The most notable of these are in the former Soviet nations where multidrug-​resistant (MDR) tuberculosis, defined as resistance to at least rifampicin and isoniazid, is identified in 10 to 20% of all cases. Multidrug-​resistant tuberculosis treatment is exceedingly difficult, since the drugs used are less effective, costlier, and poorly toler- ated due to drug-​related side effects. Furthermore, failure to con- trol the spread of drug-​resistant tuberculosis has led to outbreaks of extensively drug-​resistant (XDR) tuberculosis, which is defined as MDR tuberculosis plus resistance to fluoroquinolones and at least one injectable second-​line agent (capreomycin, amikacin, or kana- mycin). XDR tuberculosis been associated with high rates of mor- tality in HIV-​infected individuals in South Africa and is reported in more than 70 countries globally. Drug-​resistant tuberculosis (MDR or XDR) will likely continue without effective implemen- tation of measures to rapidly diagnose drug resistance and treat it appropriately. Pathogenesis The development of active tuberculosis, like all infectious dis- eases, is a function of the quantity and virulence of the invading organism and the relative resistance or susceptibility of the host to the pathogen. Indeed, one lineage of tuberculosis known as the W/​Beijing family of strains is predominant in Southeast Asia, but widely distributed in India and South Africa. W/​Beijing strains of M. tuberculosis have been associated with outbreaks of drug-​ sensitive and drug-​resistant tuberculosis and may be more virulent than other strains. Genetic host factors also play a key role in innate non​immune resistance to M. tuberculosis. For example, the human gene SLC11A1, which has been mapped to chromosome 2q, may help determine susceptibility to tuberculosis, according to a study in Africa. But like many infectious diseases, it is likely that resist- ance to tuberculosis is polygenic. Transmission Tubercle bacilli are transmitted between people by aerosols gener- ated when an infectious person coughs or otherwise expels infec- tious pulmonary or laryngeal secretions into the air. M. tuberculosis bacilli excreted by this action are contained within droplet nuclei, extremely small particles (less than 1 µm) that remain airborne for long periods and are disseminated by diffusion and convection until they are deposited on surfaces, diluted, or inactivated by ultra- violet radiation. Individuals breathing air into which droplet nu- clei have been excreted are at risk of acquiring tubercle bacilli by inhaling these nuclei and having them deposited in their alveoli, where a productive infection may occur. Transmission of tubercu- lous infection by other routes, such as inoculation in laboratories and aerosolization of bacilli from tissues in hospitals, has been docu- mented, but these are an insignificant means of spread. M. bovis can be acquired from contaminated milk from tuberculous cows, but modern animal husbandry practices and the pasteurization of milk have substantially reduced this mode of infection throughout most of the world. Natural history of tuberculosis in humans People who are in contact with someone with infectious tuberculosis may acquire infection, as described earlier (see Fig. 8.6.26.2). Factors that affect the likelihood of infection being transmitted include the severity of the disease in the index case (e.g. extent of radiographic abnormalities, cavitation, frequency of cough), the duration and closeness of exposure and environmental factors such as humidity, ventilation, and ambient ultraviolet light. Several studies in diverse

section 8  Infectious diseases 1132 locations and circumstances have shown that approximately 20–​ 30% of close contacts of an untreated tuberculosis patient become infected with M. tuberculosis, as demonstrated by the development of a reactive tuberculin skin test. Immune response Deposition of tubercle bacilli in the alveoli results in a series of pro- tective responses by the cellular immune system that forestall the development of disease in most infected people. Alveolar macro- phages ingest tubercle bacilli, which then multiply intracellularly and eventually cause cell lysis with release of organisms. Killing of M. tuberculosis within macrophages is prevented by inhibition of phagolysosome formation by the tubercle bacilli through a process that is not understood. Additional alveolar macrophages engulf progeny bacilli, resulting in further intracellular growth and cell death. Over a period of weeks as tubercle bacilli proliferate within macrophages and are released, infection spreads to regional lymph nodes, elsewhere in the lungs, and systemically. Foci of tubercle bacilli can be established in multiple organs, including the lymph nodes, brain, kidneys, and bones. In most people, specific im- munity is developed after several weeks and consists of activated T lymphocytes mediating a Th1 type response. Macrophages act as antigen-​presenting cells, interacting with CD4 lymphocytes primed for M. tuberculosis antigens. Activated CD4 lymphocytes produce both IL-​2, which promotes activation of additional T lymphocytes, and interferon-​γ, which binds with receptors on macrophages and promotes intracellular killing of organisms. Tumour necrosis factor-​α production is induced in macrophages, and this too pro- motes killing of intracellular bacilli. The specific role of CD8 cells in the control of tuberculosis has not been fully elaborated, although there is evidence that cytotoxic T lymphocytes may play a role in containing a tuberculous infection. In addition, CD8 lymphocytes also produce interferon-​γ and participate in granuloma formation. Recent evidence also supports a role of innate immunity in combat- ting tuberculosis infection. The classic immunological response to infection with tubercle bacilli is the walling off of viable bacilli in granulomas. Granulomas are collections of cells surrounding a focus of M. tuberculosis, usu- ally within macrophages but sometimes extracellularly, that serve to contain the infection. Granulomas consist of macrophages, CD4 and CD8 lymphocytes, fibroblasts, giant cells, and epithelioid cells that produce an extracellular matrix of collagenous and fibrotic materials which are continually remodelled and can become cal- cified. A calcified granuloma at the initial site of infection in the lung is referred to as a Ghon complex, while the combination of a Ghon complex and a calcified regional lymph node is called Ranke’s complex. The development of the cellular immune response to M. tu- berculosis is accompanied by the development of delayed-​type hypersensitivity to specific antigens from tubercle bacilli. While delayed-​type hypersensitivity is distinct from the cell-​mediated immunity that provides protection from disease, this sensitivity to tubercle-​derived proteins has proved enormously useful for diagnosing tuberculosis infection. The use of purified protein de- rivatives of tuberculin is the basis for estimating the prevalence of latent tuberculosis infection in populations, is essential in studying the natural history of tuberculosis infection, and is fre- quently helpful in evaluating patients with suspected tuberculosis disease. The difference between delayed-​type hypersensitivity and immunity to tuberculosis is underscored by the observation that 80–​90% of patients with active disease, and therefore clearly not immune, have positive tuberculin tests. For most people acquiring a new tuberculous infection, the de- velopment of cell-​mediated immunity to the organism is protective and holds the bacilli in check, though viability is often maintained. A small proportion of them will be unable to contain the infection and will progress to active tuberculosis disease, often referred to as primary tuberculosis. Factors associated with early progression of infection to disease include immunosuppression, particularly with HIV infection, a higher inoculum of organisms, malnutrition, and, perhaps, concomitant illness. While rates of active disease in chil- dren > 2 years of age who are contacts of cases are no higher than for older contacts, young children with primary tuberculosis do de- velop more severe forms of tuberculosis than adults, including dis- seminated disease and tuberculous meningitis. Reactivation Those who successfully contain the organism have a latent tuber- culosis infection that may reactivate later in life. Based on studies of latent tuberculosis infection acquired in childhood or adoles- cence, the lifetime risk of reactivation of M. tuberculosis is about 10%. Table 8.6.26.1 lists conditions that are associated with an increased risk of reactivating latent tuberculosis infection. The most potent of these is HIV infection, which increases the rate of reactivation by as much as 1000-​fold. Immunosuppression from malignancy, cytotoxic therapy, corticosteroids, and other agents that alter cellular immune responses also increase the likelihood that latent tuberculosis infection will reactivate. Other important factors that increase the risk of tuberculosis include diabetes and end-​stage renal disease, injection drug use (independent of HIV infection), low body weight, gastrointestinal surgery, and silicosis. Cigarette smoking is associated with increased tuberculosis inci- dence, as is alcohol abuse. Recently, the use of inhibitors of tumour necrosis factor-​α for the treatment of rheumatoid arthritis or in- flammatory bowel disease has been associated with increased risk of tuberculosis. Rates of tuberculosis are usually higher in older people than in younger adults in developed countries, but this might represent a higher prevalence of latent infection in older co- horts, rather than immunological senescence. Exposure (to infectious case) No infection c.70% Infection c.30% Inadequate host defences adequate Containment 90–95% Early progression (primary TB ≤2 years) 5–10% Late progression (reactivation TB) 5% Inadequate host defences adequate Continued containment 85–90% Fig. 8.6.26.2  Natural history of tuberculosis.

8.6.26  Tuberculosis 1133 Clinical features Classification of tuberculosis infection and disease Infection with M. tuberculosis can result in clinical manifestations ranging from asymptomatic carriage of tubercle bacilli to life-​ threatening pneumonia. Asymptomatic individuals with evidence of M. tuberculosis infection by tuberculin skin test or interferon-γ release assay are considered latently infected. In recent years the classification of the different stages of M. tuberculosis in humans has evolved as our understanding of the natural history of M. tu- berculosis has changed, and individuals can no longer be categor- ized simply as latently infected or actively diseased. Rather, the clinical manifestations of M. tuberculosis infection can be viewed as a spectrum, ranging from complete elimination of infection by host immune responses to truly latent infection with bacilli present but controlled by the host, to varying stages of subclinical infection with active bacterial replication but no symptoms, to active disease. Clinically, patients must still be considered to have either latent in- fection or active disease, but the status of latent infection can be further characterized by imaging studies, and research is underway evaluating the potential of gene expression signatures as predictors of subsequent disease. Current management of latent infection is based on an assessment of the risk of progression to active disease, as shown in Table 8.6.26.1. Clinical presentation of active tuberculosis This is highly variable, depending on the site and extent of disease and the immune status of the host. Historically, active tuberculosis has been classified as ‘primary’ or ‘post-​primary’ on the basis of both the presumed duration of infection and the clinical features of the disease. Recent studies using molecular epidemiological techniques, however, suggest that this classification may be unreliable. For ex- ample, the ‘classic’ presentation of reactivation tuberculosis has been seen in patients whose infection is clearly newly acquired, such as in nosocomial outbreaks where DNA fingerprinting confirms recent transmission. For practical purposes, tuberculosis is generally div- ided into pulmonary and extrapulmonary forms, with considerable clinical heterogeneity within these categories. Pulmonary tuberculosis Pulmonary tuberculosis is usually a subacute respiratory infec- tion with prominent constitutional symptoms. The most frequent symptoms of pulmonary tuberculosis are cough, fever, night sweats, and malaise. Cough in pulmonary tuberculosis is initially non-​ productive, but often progresses to sputum production and, in some instances, haemoptysis. The sputum is generally yellow in colour, and is neither malodorous nor thick. Haemoptysis can be seen in patients with untreated tuberculosis, but is also a feature of treated tuberculosis; damage from prior tuberculosis might result in bron- chiectasis or residual cavities that can either become superinfected or erode into blood vessels or airways, producing haemoptysis. Extremely advanced tuberculosis can also present with bloody sputum. Rarely, the bleeding is massive leading to shock, asphyxia, and death. Chest pain is not a prominent symptom in pulmonary tubercu- losis, although musculoskeletal pain from coughing might be noted. In patients with tuberculous pleurisy, however, chest pain may be present, particularly on inspiration. Radicular pain across the chest may be associated with spinal tuberculosis. Dyspnoea alone may be a sign of extensive parenchymal destruction, large pleural effusions, endobronchial obstruction, or pneumothorax. Patients with tuberculosis also experience loss of appetite and weight loss or cachexia, often out of proportion to their diminished intake of food. Increased tumour necrosis factor-​α is hypothesized to be the cause of cachexia in tuberculosis. Other symptoms with mild severity such as emotional liability, irritability, depression, and headache are frequent. The duration of symptoms varies greatly, but most patients will report weeks to months of feeling ill before presentation. In surveys of populations with high rates of disease and poor access to medical care, a history of cough for more than 3 weeks was strongly asso- ciated with a diagnosis of active tuberculosis, but in HIV-​infected patients any duration of cough predicts elevated risk for disease. Untreated tuberculosis is associated with high mortality, but many patients have persistent symptoms for years. A study of untreated pulmonary tuberculosis in the pretherapy era found that after 5 years 50% of patients had died, 25% had spontaneously healed, and 25% were chronically ill with pulmonary disease. A subset of patients has rapidly progressive disease, the so-​called ‘galloping consumption’ of old. Nowadays this is most often seen in patients with HIV infection or other forms of severe immunosuppression. These patients have an escalating course of severe pulmonary symptoms over a period of several weeks, often in the setting of disseminated disease. Failure promptly to diagnose and treat these patients results in death. Physical findings in pulmonary tuberculosis are limited and not generally helpful in making a diagnosis. Fever is an irregular and unreliable feature, and while most patients complain of fevers before Table 8.6.26.1  Incidence of active tuberculosis in people with a positive tuberculin skin test, by selected risk factors Risk factor Number of tuberculosis cases/​100 person-​years Recent tuberculosis infection:   Infection <1 year past 2–​8   Infection 1–​7 years past 0.2   HIV infection 3.5–​14 Injection drug use   HIV seropositive 4–​10   HIV seronegative 1 Silicosis 3–​7 Radiographic findings consistent with
prior tuberculosis 0.2–​0.4 Weight deviation from standard:   Underweight by ≥15% 0.26   Underweight by 10–​14% 0.20   Underweight by 5–​9% 0.22   Weight within 5% of standard 0.11   Overweight by ≥5% 0.07 Diabetes mellitus 0.3 Renal failure 0.4–​0.9 None of the above factors 0.01–​0.1

section 8  Infectious diseases 1134 presentation, only one-​half to three-​quarters of patients with con- firmed tuberculosis have a documented fever. Examination of the chest may reveal dullness to percussion and crepitations, although these findings are highly variable and non​specific. Signs of consoli- dation are usually absent. The classic post-​tussive crepitations de- scribed in the last century are not often present and are not specific to tuberculosis. Patients with disseminated tuberculosis may have lymphadenopathy, hepatomegaly, or evidence of central nervous system involvement, but these are not generally seen in typical pul- monary tuberculosis. Finger clubbing and cyanosis are findings as- sociated with prolonged and advanced pulmonary disease. Thus, the diagnosis of tuberculosis almost always rests on the patient’s history and epidemiological characteristics, in conjunction with laboratory studies described next. The most important step in making a timely diagnosis of tuberculosis is to think of it in the first place. Radiological evaluations play a critical role in the diagnosis of pulmonary tuberculosis. Disease due to M. tuberculosis can involve any portion of the lungs, and radiographic findings are usually only suggestive, not diagnostic, of tuberculosis. The typical radiological manifestations of pulmonary tuberculosis are upper lobe infiltrates that may show cavitation. M. tuberculosis exhibits a unique predi- lection for the upper zones of the lungs for reasons that are not well understood. Latent infection characteristically reactivates in the ap- ical segments of the upper lobes, or the superior segments of the lower lobes. The infiltrates are often fibronodular and irregular, and can be diffuse and associated with volume loss. Cavities, when pre- sent, are rarely symmetrical and do not usually have air–​fluid levels, such as those seen in pyogenic lung abscesses. Several examples of the radiographic appearance of pulmonary tuberculosis are seen in Fig. 8.6.26.3. (a) (b) (c) (d) Fig. 8.6.26.3  Radiographic appearance of pulmonary tuberculosis. (a) Extensive tuberculosis with right upper lobe volume loss and multiple small cavities. This patient was the source of at least 14 secondary cases in contacts. (b) A 69-​year-​old man with right pleural tuberculosis. (c) Diffuse pulmonary nodules in an HIV-​infected man with pulmonary tuberculosis. (d) Cavitary upper lobe disease in an HIV-​infected woman.

8.6.26  Tuberculosis 1135 The classic radiographic presentation described here is neither pathognomonic nor highly sensitive for pulmonary tuberculosis. Several other lung infections, notably the pulmonary mycoses, can present with similar findings. More importantly, one-​third to one-​ half of patients with pulmonary tuberculosis lack the classic radio- graphic findings described. Lower lung zone infiltrates, mid-​lung focal infiltrates, pulmonary nodules, and infiltrates with mediastinal or hilar adenopathy are also seen. HIV-​infected tuberculosis pa- tients, in particular, most often present with these ‘atypical’ findings, and up to 5% of them might have a normal chest radiograph in the setting of sputum cultures that yield M. tuberculosis. The lack of typ- ical radiographic features should not, therefore, deter the clinician from considering the diagnosis in a patient with a clinical history compatible with and symptoms of tuberculosis. CT is increasingly used to evaluate pulmonary disorders, including tuberculosis. While the classic findings described earlier do not usually require confirmation with a more sensitive test, CT scanning is sometimes used to evaluate radiographic findings that are not readily explained after an initial assessment. CT scans of the chest in patients with tuberculosis may reveal a greater extent of involvement than conventional radiographs, including multiple nodules, small cavities, and multilobar infiltrates. However, CT scanning can only suggest the possibility of tuberculosis in a patient with other signs and symptoms consistent with the diagnosis, and further evaluation is still required. The laboratory diagnosis of pulmonary tuberculosis relies on the microbiological evaluation of sputum or other respiratory tract spe- cimens. A  definitive diagnosis requires growth of M. tuberculosis from respiratory secretions, while a probable diagnosis can be based on typical clinical and radiographic findings with either acid-​fast bacilli-​positive sputum or other specimens, or typical histopatho- logical findings on biopsy material. These latter approaches, how- ever, have a variable lack of specificity depending on the prevalence of disease due to non​tuberculosis mycobacteria in the population. Throughout most of the world, sputum acid-​fast staining is the sole test used to confirm the diagnosis of pulmonary tuberculosis. In the settings where it is utilized, the positive predictive value of the sputum acid-​fast smear is very high, as the likelihood of non-​ tuberculous mycobacterial disease is quite low. In industrialized countries, disease due to the non​tuberculous mycobacteria is rela- tively more common and reliance on smears without cultures is potentially misleading. Despite the best efforts of clinicians, a con- firmed diagnosis of tuberculosis cannot be established in some pa- tients who have the disease, and a response to presumptive therapy forms the basis for establishing the diagnosis. Further details on the microbiological approach to diagnosis are provided next. Extrapulmonary tuberculosis In the United States of America extrapulmonary tuberculosis is de- fined as disease outside the lung parenchyma; in the United Kingdom it is defined as disease outside the lungs and pleura. This seemingly subtle distinction has considerable epidemiological impact, how- ever, as pleural tuberculosis is the most common extrapulmonary site of disease in the United States of America. During the initial seeding of infection with M. tuberculosis, de- scribed earlier, haematogenous dissemination of bacilli to several organs can occur. These localized infections, as in the lung, can progress into primary tuberculosis or become walled off in small granulomas where bacteria may remain dormant if they are not killed by cell-​mediated immune responses. Extrapulmonary tuber- culosis, therefore, can either be a presentation of primary or reacti- vation tuberculosis. Extrapulmonary tuberculosis may be generalized or con- fined to a single organ. In otherwise immunocompetent adults, extrapulmonary tuberculosis is found in 15–​20% of all tuberculosis cases. In young children and immunosuppressed adults, rates of extrapulmonary disease are substantially higher, appearing in more than one-​half of HIV-​related tuberculosis cases and one-​quarter of tuberculosis cases under 15 years of age. Children less than 2 years old have high rates of miliary and meningeal disease. The organs most frequently involved in extrapulmonary tuber- culosis are listed in Table 8.6.26.2. To some extent the frequency with which specific organs are involved reflects the pathophysiology of the disease. Infection spreads from the lungs, the primary site of inoculation, by lymphatic and haematogenous routes. The tissues and organs most likely to be affected are the pleura, lymph nodes, kidneys and other genitourinary organs, bone, and central nervous system. Although infection is transiently spread in the blood, tu- berculosis bacteraemia is unusual and is seen most often in patients with HIV infection and low CD4 lymphocyte counts. The clinical presentation of extrapulmonary tuberculosis depends largely on the organ involved. Both pulmonary and extrapulmonary disease are found in up to 50% of patients with HIV-​related tubercu- losis, so it is important to consider the possibility of extrapulmonary pathology when pulmonary tuberculosis is diagnosed in an HIV-​ infected patient (and vice versa). Pulmonary involvement is seen in up to one-​quarter of patients with tuberculous meningitis and to lesser degrees with other sites of disease. Pleural tuberculosis This is the result of two distinct pathophysiological sequences, which present in strikingly different manners. Most pleural tu- berculosis is associated with primary infection and is the result of seeding of the visceral pleura with relatively small numbers of tu- bercle bacilli via direct extension from adjacent lung tissue. A large proportion of patients with this form of tuberculous pleurisy will have obvious pulmonary disease, although findings can be subtle. The duration of symptoms is generally brief (e.g. several weeks, and patients complain of fever, chest pain, and non​productive cough). Other constitutional and respiratory symptoms might be present. Unlike pneumococcal pneumonia, which presents abruptly, tuber- culous pleurisy starts more insidiously. Table 8.6.26.2  Common sites of extrapulmonary tuberculosis Site Percentage of
extrapulmonary cases Pleura 20–​25 Lymphatics 20–​40 Genitourinary 5–​18 Bone/​joint 10 Central nervous system 5–​7 Abdominal 4 Disseminated 7–​11

section 8  Infectious diseases 1136 The second form of pleural tuberculosis occurs when larger numbers of bacilli invade the pleural space and multiply, producing frank empyema. Tuberculous empyema is seen in older patients, almost all of whom have extensive pulmonary disease. Patients present with prolonged symptoms of cough, chest pain, fever, cach- exia, and night sweats. Pneumothorax is a common complication of tuberculous empyema and may be associated with a more rapid disease course. The radiographic picture in tuberculous pleurisy reflects the underlying pathophysiology of the disease. Patients with the pri- mary type of pleurisy tend to have small unilateral effusions, and up to one-​half have visible parenchymal lesions on plain radiographs. In patients with tuberculous empyema, the effusions are larger and more likely to be loculated, and adjacent pulmonary involvement is often evident. The diagnosis of pleural tuberculosis can be approached along several lines. When pulmonary parenchymal involvement is mani- fest, sputum smears and cultures have a high yield, and the diagnosis of pleural disease can be inferred from the pulmonary findings. When pulmonary findings are minimal or the initial test results unrevealing, analysis of pleural fluid is essential. Acid-​fast stains of pleural fluid are usually negative in patients with primary tuber- culous pleurisy as the number of organisms in the pleural space is small. Repeated sampling will show organisms in less than one-​half of cases. Similarly, culture results might be negative. The pleural fluid is usually serous and exudative, with a protein concentration that is more than 50% of the serum level, normal or low glucose, and a slightly acidic pH. The pleural fluid white blood cell count is usually in the range of 1000 to 10 000 per µl with a lymphocytic predominance. Lactate dehydrogenase levels are generally elevated, as are adenosine deaminase levels. All of these tests are non​specific and cannot reliably distinguish tuberculosis pleurisy from other pleural diseases. Pleural biopsy is frequently useful in establishing a diagnosis of tuberculous pleurisy. Percutaneous biopsy of the pleura reveals granulomatous inflammation in up to 80% of patients, and cultures obtained at the time of biopsy are positive in over one-​half of pa- tients. If a first attempt fails to provide a diagnosis, a second biopsy might be successful. More recently, thoracoscopy has been utilized to improve the yield of biopsy by visualizing biopsy targets rather than blindly sampling with a percutaneous pleural needle. Lymphatic tuberculosis This can occur in any location, but classic scrofula involving the cervical or supraclavicular chains is the most common presenta- tion. Mediastinal and hilar lymphatic tuberculosis is a feature both of primary and disseminated disease, but discovery of these lesions is usually incidental. The pathophysiology of lymphatic tubercu- losis is thought to result from drainage of bacilli in the lungs into supraclavicular and posterior cervical lymph node chains. In con- trast, lymphatic disease caused by non​tuberculous mycobacteria usually involves anterior cervical, preauricular, or submandibular lymph nodes, suggesting acquisition through the oropharynx. In patients with HIV infection, multiple lymph node groups can be in- volved including axillary, inguinal, mesenteric, and retroperitoneal. Symptoms in lymphatic tuberculosis are generally limited, un- less the disease is disseminated. Painless swelling of a lymph node is the most common presentation. Constitutional symptoms are not prominent in most cases. Examination of the area may reveal several enlarged lymph nodes, as only about 20% of patients have disease of a solitary node. The diagnosis of lymphatic tuberculosis usually depends on cultures from affected nodes. Biopsies may show granulomatous changes and acid-​fast bacilli. Such findings are non​specific, how- ever, and cannot distinguish tuberculous from non​tuberculosis lymphadenitis. As discussed elsewhere, the presence of a positive tu- berculin skin test in the setting of typical biopsy findings is strongly suggestive of tuberculosis; in the setting of suspected lymphatic tu- berculosis, these findings warrant presumptive therapy. Genitourinary tuberculosis This encompasses a broad array of clinical entities, ranging from disease of the kidneys to endometrial, prostatic, and epididymal disease. The most common of these is renal tuberculosis, which re- sults from haematogenous seeding of the renal cortex during the primary infection. The pathogenesis of other genitourinary sites is either from downstream extension of renal infection over time or from haematogenous seeding at the time of the initial acquisition of M. tuberculosis. Renal tuberculosis is probably underdiagnosed because it is fre- quently asymptomatic. Many cases of genitourinary tuberculosis are diagnosed as a result of routine urinalyses that detect sterile py- uria. The development of symptoms reflects a more advanced stage of disease, associated with considerable tissue destruction. When genitourinary tuberculosis is symptomatic, the most common symptoms are localized and include urinary symptoms and flank pain. In men, tuberculosis can cause prostatitis and epididymitis, both of which can present with pain resulting from swelling. In women, genital tract tuberculosis may be symptomatic when it in- volves the ovaries and Fallopian tubes; pelvic pain is also a feature of endometrial tuberculosis. Menstrual abnormalities and infertility may be the only signs of genital disease, however. The diagnosis of genitourinary tuberculosis depends on the ana- tomical site of the disease. Renal tuberculosis, as noted, is suggested by sterile pyuria, and the diagnosis rests on isolation of organisms in the urine. Early morning urine samples are more likely to grow M. tuberculosis than spot samples obtained at other times. In pa- tients with symptoms of upper urinary tract illness, radiological studies are often helpful. The kidneys may appear calcified on ab- dominal radiographs. Intravenous pyelography may show distorted or dilated calyces or renal pelvis, papillary necrosis, cavitation, or abscesses of the renal parenchyma, or intrarenal or ureteral obstruc- tions. Use of renal ultrasonography or CT scanning may be more sensitive for identifying the abnormalities of renal tuberculosis, but contrast radiography is the technique with which the greatest ex- perience has accrued. When tuberculosis of the bladder is suspected, cystoscopy with biopsy may lead to the identification of granulomas before identification of organisms by culture. Diagnosis of prostatic, testicular, or epididymal tuberculosis is usually accomplished with cultures obtained by fine needle aspiration or transurethral resec- tion of the prostate. Cervical and endometrial tuberculosis can be diagnosed by biopsy with culture. Tuberculous meningitis This is the most common central nervous system manifestation of tuberculosis (see Chapter 24.11.1). It is much more likely to occur

8.6.26  Tuberculosis 1137 in children under the age of 15 years and in HIV-​infected patients than in immunocompetent adults. Although meningitis accounts for only a small fraction of all cases of tuberculosis, it is a devastating form of the disease that is uniformly fatal if left untreated. The pathogenesis of meningeal tuberculosis varies with the age and immunological status of the patient. Reactivation of micro- scopic granulomas in the meninges was found by Rich to cause diffuse meningeal infection. These foci of infection are probably implanted at the time of primary bacillaemia. When these lesions rupture into the subarachnoid space they invoke an inflammatory response leading to tuberculous meningitis. Meningeal disease can also complicate miliary disease, especially in children. Likewise, adults can acquire meningeal disease during bacillaemia of miliary disease, but this is not the usual pathogenesis of meningeal infection. Rarely, invasion into the spinal canal from a paraspinous or vertebral focus can also be the source of central nervous system involvement. The clinical features of tuberculous meningitis are the conse- quence of the pathophysiological process underlying the disease. Meningeal and leptomeningeal bacterial replication results in a ro- bust inflammatory reaction, often localized to the base of the brain. The number of bacilli present is usually limited, and the severity of illness is a function of the host response. Meningeal inflammation causes increases in cerebrospinal fluid pressure and can also cause cranial neuropathies. Patients complain of headache, neck stiff- ness, meningism, and an altered mental status, including irritability, clouded thinking, and malaise; as the disease progresses, symptoms worsen considerably. The clinical spectrum of tuberculous meningitis has historically been categorized in three stages, defined by the British Medical Research Council in 1948. Stage 1 consists of a prodrome lasting for 1 to 3 months. Non​specific symptoms such as fever, malaise, and headache predominate. In this stage, patients are conscious and ra- tional, but may have signs of meningism. Focal neurological signs are absent and there are no signs of hydrocephalus. In stage 2 dis- ease, single cranial nerve abnormalities such as ptosis or facial par- alysis appear, and paresis and focal seizures might occur. Kernig’s and Brudzinski’s signs have been noted as well as hyperactive deep tendon reflexes. Prominent signs include alterations in mentation, behavioural change, impaired cognitive ability, and increasing stupor. Headache and fever are also common features of this stage of disease. In stage 3, patients are comatose (Glasgow coma scale 8 or below) or stuporous and often have multiple cranial nerve palsies and hemi- plegia or paraplegia. By this stage, hydrocephalus is common and chronic inflammation in the enclosed space of the skull may result in significant intracranial hypertension. Seizures may be a prominent feature. Fever, headache, altered level of consciousness, and meningism are present in most patients in most large studies, although no one single sign or symptom has any reliable degree of sensitivity or spe- cificity. Children can be especially difficult to diagnose as symptoms such as fever, vomiting, drowsiness, or irritability are commonly seen in many minor viral illnesses. Transient tuberculous meningitis that presents as an aseptic men- ingitis and resolves without treatment has been described. Benign presentations of meningeal tuberculosis are uncommon in clinical practice, and when the diagnosis is made, treatment is mandatory, even in the patient with seemingly trivial symptoms. The diagnosis of tuberculous meningitis is often difficult and re- quires a high degree of suspicion. In the setting of disseminated disease, signs of tuberculosis in other organs, particularly the lungs, are often present. Between 25 and 50% of patients with meningitis in most series also have radiographic evidence of pulmonary tu- berculosis, either active or healed. The critical features of tuber- culous meningitis, however, are found in the cerebrospinal fluid. Patients with tuberculous meningitis usually have elevated cere- brospinal fluid pressure. An exudative fluid with a mononuclear cell pleocytosis is characteristic. Cerebrospinal fluid is usually clear and the protein is generally in the range of 100–​500 mg/​dl. Hypoglycorrhachia is typical, with cerebrospinal fluid glucose less than 50% of the serum value. The white blood cell count rarely ex- ceeds 1000 per µl, and cell counts below 500 are typical. In early meningitis, the cells may be predominantly neutrophils, but mono- nuclear cells predominate in most instances. Acid-​fast stains of concentrated cerebrospinal fluid are only positive in one-​third or fewer of patients, and cultures are positive in only one-​half, although repeated sampling increases the yield. The disastrous consequences of failing to diagnose tuberculous meningitis, coupled with the low yield of cerebrospinal fluid acid-​ fast stains and cultures, has prompted the development of additional tests for establishing a diagnosis. Adenosine deaminase was initially reported to be exceptionally accurate for tuberculous meningitis. Subsequent experience, however, has found it to be insufficiently specific to distinguish tuberculosis from a variety of other acute and chronic meningitides. Several other tests based on identification of mycobacterial antigens or specific antibodies have been evaluated, but none has been found to be reliable. Nucleic acid amplification tests such as polymerase chain reaction (PCR) have great appeal, but the sensitivity and specificity of available assays are only moderately good. Thus, the diagnosis of tuberculous meningitis often rests on the astute judgement of a clinician with a high degree of suspicion based on epidemiological and clinical clues. Presumptive therapy is frequently necessary. Central nervous system tuberculomas These are an unusual manifestation and are seen in a small pro- portion of patients with tuberculous meningitis. Tuberculomas are the result of enlarging tubercles that extend into brain paren- chyma rather than into the subarachnoid space. Patients with HIV infection appear to have an increased risk of central nervous system tuberculomas, but the disease is far less common than toxoplas- mosis, even in areas where tuberculosis is highly prevalent. Central nervous system tuberculomas may appear with clinical features of meningitis or of intracranial mass lesions. In the absence of menin- geal involvement, seizures or headaches may be the only symptoms. The diagnosis is suggested by brain imaging, with MRI scanning being more sensitive than CT scanning. Biopsy of the lesion is re- quired for diagnosis, and material should be submitted for histo- pathological staining and culture. Bone and joint tuberculosis These can affect several areas, but vertebral tuberculosis (Pott’s dis- ease) is the most common form, accounting for almost one-​half of cases. Haematogenous seeding of the anterior portion of vertebral bone during initial infection sets the stage for later development of Pott’s disease. Infection grows initially within the anterior vertebral

section 8  Infectious diseases 1138 body, then may spread to the disc space and to paraspinous tissues. Destruction of the vertebral body causes wedging and eventual col- lapse. Patients usually complain of back pain, with constitutional symptoms less prominent. Neurological impairment is a late com- plication, but delays in diagnosis are common and many patients ex- perience neurological sequelae. Imaging studies of the spine usually reveal anterior wedging, collapse of vertebrae, and paraspinous ab- scesses. The diagnosis is established with bone biopsy or curettage, or by culture of the drainage from a paraspinous abscess. Abdominal tuberculosis Tuberculosis in the abdomen is relatively uncommon and can take two forms: (1) tuberculous enteritis and (2) peritoneal tuberculosis. The former was much more common in the era of unpasteurized milk and was due to M. bovis. Tuberculosis can occur in the intestines by a variety of means; swallowed sputum, haematogenous spread, or the ingestion of contaminated milk or food. The most common presentation is with non​specific abdominal pain. Diarrhoea occurs as a result of either bowel wall inflammation or partial obstruction, and sometimes rectal bleeding occurs. The ileo-​caecal region is most commonly af- fected and can result in a mass in the right iliac fossa. Tuberculous enteritis can mimic Crohn’s disease clinically, endoscopically, and radiographically. Bacilli are rarely seen in biopsies, so culture of tissue is essential in suspected cases. Peritoneal tuberculosis probably results from haematogenous spread from a pulmonary focus or, sometimes, spread from adja- cent enteric infection. Abdominal pain and fever, in association with other systemic symptoms of tuberculosis, are common but the main clinical presentation is with ascites. The ascitic fluid is lymphocytic with a high protein content, although the latter might not be seen in patients with cirrhosis (who are at increased risk of peritoneal tuber- culosis). Bacilli are rarely seen in the ascitic fluid so culture is essen- tial. Diagnosis is best made by biopsy of peritoneal tubercles under direct vision, either by laparoscopy or by mini-​laparotomy. One of the main differential diagnoses is ovarian cancer and it should be recognized that serum CA-​125 can be elevated in tuberculous peritonitis. Miliary tuberculosis and disseminated tuberculosis These are terms used interchangeably to describe widespread in- fection and the absence of minimal host immune responses. The term ‘miliary tuberculosis’ is derived from the classic radiographic appearance of haematogenous tuberculosis, in which tiny pul- monary infiltrates with the appearance of millet seeds are distrib- uted throughout the lungs. Miliary tuberculosis is a more common consequence of primary tuberculosis infection than reactivation, and is seen more frequently in children and immunocompromised adults. Primary miliary tuberculosis presents with fever and other constitutional symptoms over a period of several weeks. Clinical evaluation may reveal lymphadenopathy or splenomegaly and chor- oidal tubercles on retinoscopy. Laboratory tests might show only an- aemia. The chest radiograph is initially normal but later develops the typical miliary pattern. Involvement of multiple organ systems is the rule; usually liver, spleen, lymph nodes, central nervous system, and urinary tract. Patients with reactivation of latent infection who present with miliary disease may have a more fulminant course, al- though progression to severe disease without treatment is the rule in all patients. The diagnosis is made on tissue biopsy and culture, as sputum smears are usually negative, reflecting the small numbers of bacilli typically present in respiratory secretions. Other forms of extrapulmonary tuberculosis are less common than those listed earlier, and the diagnosis is based on a combination of clinical suspicion and the results of biopsies and cultures. Ocular, adrenal, and cutaneous tuberculosis are all rarely encountered in the modern era, even in immunocompromised patients. Diagnostic testing Evaluation of patients for M. tuberculosis infection or disease relies on both non​specific and specific tests. Imaging studies, body fluid chemistries and cell counts, and histochemical staining, as just de- scribed, are useful and important tests for the diagnosis of tuber- culosis. Specific studies for identifying mycobacterial infections include the tuberculin skin test, interferon-γ release assays, acid-​ fast microscopy, nucleic acid amplification tests, and mycobacterial culture. Tuberculin skin testing Tuberculin skin testing involves the intradermal injection of puri- fied proteins of M. tuberculosis (purified protein derivative, or PPD tuberculin) that provokes a cell-​mediated delayed-​type hypersen- sitivity reaction which produces a zone of induration. Tuberculin originated with Robert Koch who prepared a tubercle sensitin that he thought would cure tuberculosis. Administration of Koch’s tu- berculin, of course, did not cure the disease, and hypersensitivity reactions to the agent were sometimes severe or fatal, bringing Koch great discredit. Fortunately, it was recognized that because tuberculin induced reactions in people who were infected with tu- berculosis the substance might prove a better diagnostic test than treatment. Current tuberculin preparations are composed of pro- teins derived from culture filtrates and stabilized with a detergent to prevent precipitation. The standard dose of tuberculin is 5 tu- berculin units (TU) of PPD-​S, equivalent to 0.1 mg tuberculin in a volume of 0.1 ml. In recent years, a worldwide shortage of tuberculin has limited use of this technique. Tuberculin testing is used to identify people with M. tuberculosis infection, and the test cannot distinguish those who have disease from those with latent infection. Induration is the key feature of a tuberculin response, and the result of tuberculin testing is categor- ized according to the amount of induration measured. Because tuberculin contains antigens also found in non​tuberculous myco- bacteria, such as Bacille Calmette–​Guérin (BCG), false-​positive reactions occur. Tuberculin skin testing should be done by the Mantoux method of intradermal injection, as this is the only technique that has been standardized and extensively validated. Multipuncture devices should not be used. The amount of induration should be measured 2 to 7 days after the injection; measurements performed precisely 48 to 72 h later are not essential. The transverse diameter of induration should be measured in millimetres using a ruler. Criteria for the interpretation of tuberculin skin tests vary ac- cording to clinical and epidemiological circumstances. Cut-​off points for positive tests developed by the American Thoracic Society and the Centers for Disease Control and Prevention

8.6.26  Tuberculosis 1139 (CDC) are listed in Table 8.6.26.3. A cut-​off point of 5 mm indur- ation is used for individuals who are at high risk of tuberculosis infection, or at high risk of disease if infected. Such people include the close contacts of infectious patients and patients with radio- graphic abnormalities consistent with tuberculosis. The rationale for the 5-​mm cut-​off in these patients is that the prior probability of infection is high. A 5-​mm cut-​off is also used for HIV-​infected patients and those immunocompromised by corticosteroids or other agents. Failure to diagnose tuberculosis infection in these people could be calamitous, so a lower threshold is used to maxi- mize sensitivity. The use of control antigens such as candida or tetanus toxoid to aid the interpretation of tuberculin tests in HIV-​infected patients has been shown to be of no value and is not recommended. A cut-​off point of 10 mm induration is used for people from popu- lations with a high prevalence of tuberculosis or for individuals with conditions that increase the risk of developing active disease if in- fected. This would include immigrants from endemic areas, resi- dents of some inner cities, and healthcare workers, as well as patients with diabetes, renal disease, silicosis, and other medical conditions associated with an elevated risk of reactivation of latent tuberculosis. Finally, a cut-​off of 15 mm is used in people who have no risk fac- tors for tuberculosis infection or disease. In most instances, these patients would not be tested. Tuberculin testing does have limitations in both sensitivity and specificity. False-​negative tuberculin tests result from both errors in ap- plying and interpreting the test and from anergy. Errors in in- jection of tuberculin are common, and inter-​reader variability in measuring results is high. Fortunately, if there is doubt about the interpretation of a skin test, multiple readers can measure the result over a period of days, or the test can be repeated and re- interpreted. Specific anergy to tuberculin is seen in several situ- ations. Approximately 10–​20% of patients with culture-​confirmed pulmonary tuberculosis fail to respond to tuberculin as a result of anergy. These patients often will mount a response after their disease has been treated. HIV-​infected patients have a high preva- lence of anergy, both to tuberculin and other antigens. Only 10–​ 40% of patients with low CD4 counts and confirmed tuberculosis respond to tuberculin. Transient anergy is associated with acute viral infections such as measles, live virus vaccinations, and other acute medical illnesses. Interferon-​γ release assays Tuberculin skin testing is frustratingly crude and somewhat cum- bersome, but despite its limitations has proved superior to nu- merous more ‘modern’ assays including antibody tests and other in vitro immunodiagnostics. Recently, however, the use of assays to detect interferon-​γ production by sensitized T cells in response to challenge with specific antigens from the RD1 region of the M. tu- berculosis genome has shown promise as an alternative to tuberculin testing. Two commercial interferon-​γ release assays, one an enzyme-​ linked immunospot (T-​SPOT-​TB) and one an enzyme-​linked im- munosorbent assay (ELISA) (Quantiferon TB Gold-​In Tube or Quantiferon Plus) are now approved in several countries for in vitro diagnosis of tuberculosis infection. These assays are more than 90% sensitive for active tuberculosis and more specific than tuberculin testing in BCG-​vaccinated individuals, correlate better than tuber- culin skin testing with exposure to a point source of infection, and may not be compromised by immunosuppression related to HIV in- fection. In some studies, these assays have greater sensitivity than tuberculin skin testing and almost always have better specificity. In evaluating individuals with latent tuberculosis infection, however, the lack of a gold standard of diagnosis makes comparisons diffi- cult. However, emerging evidence suggests that interferon-​γ release assays may be more accurate than tuberculin testing in predicting which people are at greatest risk of developing subsequent active tuberculosis disease. Thus, the assays have enormous potential and Table 8.6.26.3  Criteria for tuberculin positivity, by risk group Reaction ≥5 mm induration Reaction ≥10 mm induration Reaction ≥15 mm induration HIV-​positive persons Recent immigrants (i.e. within the last 5 years) from high-​prevalence countries or regions Persons with no risk factors for tuberculosis Recent contacts of infectious tuberculosis patients Injection drug users Persons with fibrotic changes on chest radiograph consistent with prior tuberculosis Residents and employees of the following high-​risk congregate settings: Prisons and jails Nursing homes and other long-​term facilities for older people Hospitals and other healthcare facilities Residential facilities for patients with AIDS Homeless shelters Patients with organ transplants and other immunosuppressed patients (receiving the equivalent of ≥15 mg/​day prednisone for 1 month or more) Persons with the following clinical conditions that place them at high risk: Silicosis Diabetes mellitus Chronic renal failure Some haematological disorders (e.g. leukaemias and lymphomas) Other specific malignancies (e.g. carcinoma of the head or neck and lung) Weight loss of ≥10% of ideal body weight Gastrectomy Jejunoileal bypass Others Mycobacteriology laboratory personnel Children <4 years of age or infants, children, and adolescents exposed to adults at high risk

section 8  Infectious diseases 1140 might contribute to improved detection of both active and latent tu- berculosis infections. One serious limitation of interferon-​γ release assays is high rates of reversion of positive tests in individuals undergoing serial testing, including healthcare workers. Several studies have shown that among healthcare workers in low incidence areas, between 40 and 70% of initially positive individuals become negative when re-​tested 6–​18 months later. Management of positive tests in people at low risk of infection, such as healthcare workers in settings with little or no exposure to the disease, is challenging and underscores the wisdom of the 2000 ATS/​CDC guidelines for tuberculin testing: a decision to test is a decision to treat. Only people felt to be at risk for M. tuberculosis infection and who should be treated, if positive, should be tested. Microscopic staining Microscopic staining of acid-​fast bacilli is the method most widely used to diagnose tuberculosis throughout the world. Acid-​fast staining is inexpensive, rapid, and technologically undemanding, making it an attractive technique for identifying mycobacterial in- fections. The waxy glycolipid matrix of the mycobacterial cell wall is resistant to acid–​alcohol decolorization after staining with carbol-​ fuchsin dyes, and red bacilli are visible after counterstaining. Both the Ziehl-​Neelsen method (which requires heat fixation) and the Kinyoun method utilize methylene blue or malachite green counter- stains, and have similar sensitivities for identifying acid-​fast bacilli in clinical specimens. The major limitation of acid-​fast staining is that a relatively large number of bacilli must be present to be seen microscopically. Acid-​fast smears are generally negative when there are fewer than 10 000 bacilli/​ml of sputum, and many microscope fields need to be examined to identify bacilli even when there are 10 000 to 50 000 bacilli/​ml. Thus, up to 50% of patients with sputum cultures positive for M. tuberculosis have negative acid-​fast smears. In settings where the sputum smear is the only test done to confirm tuberculosis, many smear-​negative cases go undetected. This is a serious problem for patients without cavitary tuberculosis, who tend to have fewer bacilli in their sputum, including many HIV-​infected tuberculosis patients in developing countries. Several techniques can be used to improve the yield of sputum smears. The most important method is enrichment of the spe- cimen through concentration of the sputum. Centrifugation of sputum allows examination of the bacilli-​rich pellet, which improves the sensitivity of smears substantially. Treatment of sputum with mucolytic agents is also helpful in identifying or- ganisms by both smear and culture. Use of fluorochrome proced- ures to identify mycobacteria is more sensitive, but less specific, than acid-​fast stains. Auramine O or auramine-​rhodamine dyes are used on concentrated smears and examined under a fluor- escence microscope. This technique allows much more rapid screening of slides than the traditional methods, but confirm- ation of positive results with Ziehl-​Neelsen or Kinyoun staining is essential, as false-​positive fluorochrome results are not un- common. Fluorescence microscopy has been limited historically to well-​equipped reference laboratories, but the introduction of light-​emitting diode (LED)-​based fluorescent microscopes has substantially lowered the cost of this technology and increased availability in resource-​limited areas. The proper collection of specimens is also important for opti- mizing the results of microscopy and culture. Early morning sputum specimens tend to have a higher yield than specimens collected at other times, and overnight sputum collections have provided even greater sensitivity. Morning gastric aspirates have a moderate yield for acid-​fast bacilli in children, who generally have a difficult time producing sputum. Sputum induction with hypertonic saline is useful in evaluating patients with minimal or no sputum produc- tion, and the use of fibreoptic bronchoscopy is often advocated for patients with negative sputum smears. Examination of multiple specimens increases the sensitivity of sputum microscopy for acid-​fast bacilli. The first smear identifies 70–​80% of patients, the second another 10–​15%, and the third an- other 5–​10%. Review of additional specimens has little value. In addition to the modest sensitivity of acid-​fast staining, the specificity of this technique can also present problems. The mor- phological properties of the mycobacteria are sufficiently similar to make distinguishing M. tuberculosis from non​tuberculous myco- bacteria impossible on the basis of acid-​fast smears. This is not a serious problem where tuberculosis is common and non​tuberculous mycobacterial infections are unusual. However, in many industrial- ized countries, disease due to the non​tuberculous mycobacteria is relatively common, and distinguishing these types of infections has important therapeutic and public health implications. Thus, while sputum microscopy is useful because of its rapidity and low cost, it should be supplemented with culture or other more sensitive and specific tests whenever feasible. Culture, nucleic acid amplification, and susceptibility testing Culture of M. tuberculosis This is the gold standard for confirming the diagnosis of tubercu- losis. A variety of media are available that support the growth of mycobacteria, including egg-​based and potato-​based solid media and several broth-​based media. The intrinsic growth rate of M. tu- berculosis makes the recovery of the organism in culture a slow pro- cess. In traditional egg-​based media such as Lowenstein–​Jensen, growth of colonies of M. tuberculosis takes between 3 and 6 weeks, and 7H11 agar requires an average of 3 to 4 weeks to show colonies. Obviously, the slow pace of these traditional culture systems inter- feres with optimal patient management, and more rapid techniques are required. Several faster (not rapid) systems for detection of mycobacteria in culture have been commercially developed. The Mycobacterial Growth Indicator Tube (MGIT) is a broth-​based system that uses fluorescence detection to monitor growth. Both manual and automated systems are available. Once growth is detected, staining to identify acid-​fast organisms and species identifica- tion need to be performed. The time to detection of mycobac- teria using MGIT is considerably faster than conventional solid media, and the yield can be appreciably higher. Contamination of cultures with bacteria and fungi is common, and laboratory cross-​contamination remains a concern. Nevertheless, the use of MGIT can increase case detection rates and speed the time to de- tection of tuberculosis. Many clinical laboratories use more than one culture system for mycobacteria, both to increase the overall recovery rate and to

8.6.26  Tuberculosis 1141 provide quality control. In addition, if one culture becomes contam- inated, alternative cultures can still be utilized. Preparation of specimens for mycobacterial culture This follows the same steps as outlined for acid-​fast smears. In addition, specimens being submitted for culture also require de- contamination to prevent overgrowth by more rapidly multiplying bacteria. Sodium hydroxide (NaOH) and N-​acetyl-​l-​cysteine are commonly used together for mucolysis and decontamination. By necessity, decontamination also inactivates more than 50% of mycobacteria in a specimen, thereby reducing the potential yield of the culture. Failure to decontaminate, however, leads to bacterial overgrowth and uninterpretable results. Lack of growth as a result of overdecontamination and bacterial overgrowth resulting from underdecontamination underscore the importance and utility of obtaining multiple specimens for culture, when possible. As with sputum smears, the yield of mycobacterial culture increases with evaluation of additional specimens. Speciation After mycobacterial growth has been identified, speciation of the organism is required. Conventional techniques for identification of mycobacterial species involve characterization of colony morph- ology, pigmentation, rate of growth, and biochemical tests. Niacin reduction, nitrate reduction, and lack of catalase activity at elevated temperatures are all characteristic of M. tuberculosis. Species iden- tification using these methods is time consuming and tedious, and further delays the diagnosis of tuberculosis. The use of nucleic acid probes has dramatically simplified speciation of mycobacteria in recent years. DNA probes that react with specific mycobacterial rRNA sequences to form DNA–​RNA hybrids that can be readily detected by chemoluminescence are commercially available for M. tuberculosis, M. avium complex, M. kansasii, and M. gordonae. These probes can be performed within hours of detection of myco- bacterial growth, and significantly accelerate the diagnosis of specific pathogens. The sensitivity of these probes is approximately 90–​95%, depending on the species, with specificities approaching 100%. Cultures that fail to respond to any of the DNA–​RNA probes are al- most always due to another mycobacterial species, but final identifica- tion depends on the laborious biochemical techniques of old. Nucleic acid amplification The difficulties of identifying mycobacteria in patient specimens ac- centuate the need for rapid and sensitive diagnostic methods for tu- berculosis. Recently, several commercial nucleic acid amplification tests have been introduced, including assays based on reverse tran- scription (RT)-​PCR, transcription-​mediated amplification, ligase chain reaction, and strand displacement amplification. All of these techniques use specific M. tuberculosis DNA sequences as targets for nucleic acid amplification. The great advantage of these assays is that they can provide results within 1 day of the collection of specimens. Their disadvantage is that they are uniformly less sensitive than cul- ture, particularly in sputum smear-​negative patients. Early studies also suggested that specificity was excellent overall but was reduced in smear-​positive samples; further refinements in these assays have resulted in improved sensitivity and specificity, and their diagnostic role in both smear-​negative sputum and extrapulmonary disease has grown accordingly. Recent advances in DNA amplification have made rapid detection of M. tuberculosis gene sequences more easily performed, marking a potentially revolutionary change in the diagnosis of tuberculosis. Line probe assays, using solid-​phase PCR techniques to identify signature sequences from M. tuberculosis can identify up to 99% of sputum smear-​positive specimens and between 50 and 70% of smear-​negative specimens. The turn-​around-​time for these assays is 4–​8 hours, and most results can be returned, in theory, within a few days, dramatically accelerating the diagnosis. In addition, as dis- cussed next, line probe assays can also be used to detect resistance mutations associated with isoniazid, rifampicin, ethambutol, and several second-​line antituberculosis drug resistance. Unfortunately, rapid genetic testing for pyrazinamide resistance, a key predictor of response to treatment of MDR and XDR tuberculosis, is not yet possible with these assays. Another new addition to the diagnostic armamentarium is the Xpert MTB/​RIF® assay. This commercial kit uses molecular bea- cons to identify both M. tuberculosis gene sequences and specific mutations responsible for more than 95% of all rifampicin resist- ance. It detects M. tuberculosis complex and rifampicin resistance by PCR amplification of rpoB gene sequences. The test kit is con- tained in a small cartridge into which sputum is placed, and the entire process is automated within the cartridge and a tabletop machine, with results returned in 90–​120 minutes. The sensitivity of the assay is more than 98% for smear-​positive sputum and 60–​ 70% for smear-​negative samples, with a sensitivity of more than 95% for rifampicin-​resistance. The ability to provide a diagnosis of tuberculosis and determine rifampicin-​susceptibility within 2 h is of enormous importance, and this assay is now being rolled out through much of the world. Conventional drug suscepti- bility testing is still required for patients found to have rifam- picin resistance, but from a clinical perspective the diagnosis of rifampicin-​resistant tuberculosis is tantamount to MDR tubercu- losis in most settings, and treatment decisions can be based on the Xpert ­result. A new version of the Xpert platform, Xpert Ultra, has a new cartridge design that has greatly improved sensitivity and retains good specificity. Antigen testing A rapid, point-​of-​care test that allows diagnosis of tuberculosis at the bedside is the Holy Grail of tuberculosis control, but such a tool has been challenging to develop. One approach evaluated in re- cent years is testing urine for the M. tuberculosis cell wall antigen lipoarabanomannin, using lateral flow technology similar to home pregnancy tests. Several early studies found lipoarabanomannin present in urine of a high proportion of hospitalized HIV-​infected patients with tuberculosis, but subsequent studies found very low yields (<15%) in ambulatory patients with or without HIV infec- tion, though specificity is high. The WHO currently recommends that bedside urine lipoarabanomannin testing be used in hospital- ized patients with HIV and CD4 cell counts less than 100 suspected of having tuberculosis. Here, sensitivity might be as high as 50–​60%, and when combined with Xpert testing can result in a diagnosis in up to 90% of patients. Drug susceptibility testing Susceptibility testing of M. tuberculosis isolates is essential for both clinical management and public health purposes. Susceptibility tests

section 8  Infectious diseases 1142 for the first-​line antituberculosis drugs should be performed on at least one culture at the time of diagnosis for all patients. If the initial isolate is susceptible to the first-​line agents and treatment proceeds without incident, additional susceptibility tests are not required. Susceptibility testing should be performed for patients who relapse with tuberculosis and for patients who are treatment failures after 3–​4 months of therapy. Conventional susceptibility testing for M. tuberculosis uses standard concentrations of antituberculosis drugs to measure in- hibition of bacterial growth in culture. Drugs tested routinely include isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin. Testing of second-​line antituberculosis drugs is only done when resistance to the first-​line agents is documented or strongly suspected. Susceptibility testing is generally performed on subcultures of the primary isolate, though direct inoculation of sputum or other spe- cimens can be performed in the case of a strongly positive acid-​fast bacilli smear. The standard method for measuring susceptibility to antituberculosis drugs is the proportions method. The organism is grown on agar plates in the presence of known concentrations of specific drugs. Growth on the plates is then compared with growth on control plates. By convention, if the test plate shows a colony count that is more than 1% of the control value, the isolate is re- sistant. Laboratories will report the isolate as being susceptible or resistant to the concentration of the drug used in the assay. Another method for susceptibility testing is to use the MGIT system, in which culture bottles contain antituberculosis drugs. Growth indices are compared to control cultures to determine sus- ceptibility. The MGIT system provides results more quickly than the proportions method, is automated, but is more expensive. Recently, the microscopic examination of growth in wells that are filled with liquid culture medium (MODS) has been reported to enable de- tection within about 10 days and permit rapid assessment of drug resistance. This technique has some promise in resource-​limited settings, but it is labour intensive and needs further validation in endemic countries. As noted earlier, the use of molecular methods to determine tuberculosis drug susceptibility is a major advance. Specific mu- tations in M. tuberculosis have been identified which confer resist- ance to antituberculosis drugs. For example, mutations in a small region of the rpoB gene of M. tuberculosis are responsible for more than 90% of all rifampicin resistance. Sequencing of this portion of the genome using a variety of techniques has been shown to be feasible in research laboratories. Rapid identification of rifam- picin resistance by molecular methods (line probe assay) would be of enormous clinical benefit, as almost all rifampicin-​resistant M. tuberculosis isolates are also resistant to isoniazid and are, by definition, multidrug resistant. Thus, early detection of resistance mutations would allow early initiation of appropriate treatment and infection control measures. A point-​of-​care nucleic acid amplifica- tion test, such as the Xpert MTB-​RIF, can detect at least rifampicin resistance, and a new cartridge design that detects mutations asso- ciated with resistance to other drugs has recently been introduced. Among culture-​positive patients, a single, Xpert MTB/​RIF test done on sputum directly had 98.2% and 72.5% sensitivity in smear-​ positive and negative tuberculosis, respectively; and a 99.2% speci- ficity in patients without tuberculosis. Molecular diagnosis of other types of resistance has been bolstered by whole genome sequencing, which allows a clearer understanding of associations between gen- etic polymorphisms and phenotypic resistance. Treatment of active tuberculosis The treatment of tuberculosis requires the use of a combination of antimycobacterial drugs active against the strain of M. tuberculosis causing the patient’s disease. The use of multiple agents is necessitated by the emergence of drug resistance when single agents are used. Mutations that confer resistance to antimycobacterial drugs arise spontaneously in wild-​type populations of M. tuberculosis in frequen- cies ranging from 1 in 105 to 1 in 108 bacilli. In the presence of large numbers of organisms, such as are present during active pulmonary disease, a single agent will kill susceptible bacilli, but naturally drug-​ resistant mutants will survive and eventually emerge to cause drug-​ resistant disease. Since the mechanisms of resistance are genetically distinct and arise independently, multiple drug resistance within a single organism is exceedingly rare in nature. The use of two or more agents with different mechanisms of action assures that populations of drug-​resistant bacilli are not selected for during therapy. Antituberculosis drugs These are divided into first-​line and second-​line agents. The first-​ line agents are widely available and used routinely in the treatment of tuberculosis, while the second-​line agents are generally less po- tent, more toxic, and less readily available. Exceptions are the newer fluoroquinolones, such as moxifloxacin and levofloxacin, which ap- pear to have good activity against M. tuberculosis. The addition of moxifloxacin to standard treatment has not been shown to shorten the duration of tuberculosis treatment, however. Second-​line drugs are reserved for the treatment of drug-​resistant tuberculosis. Table 8.6.26.4 lists the first-​line antituberculosis drugs, their activity in the treatment of tuberculosis, and common toxicities. Regimens currently used for the treatment of tuberculosis have been developed on the basis of trials conducted by the British Table 8.6.26.4  Drugs for the treatment of tuberculosis Agent Activity Toxicity Isoniazid Bactericidal Liver, peripheral nerve, hypersensitivity Rifampicin Bactericidal and sterilizing Liver, gastrointestinal, discoloration of body fluids, nausea, haematological Rifapentine Bactericidal and sterilizing Liver, gastrointestinal, discoloration of body fluids, nausea, haematological Pyrazinamide Sterilizing Liver, hyperuricaemia, gout, malaise, gastrointestinal Ethambutol Bacteriostatic (dose-​dependent) Liver, optic neuritis, skin

8.6.26  Tuberculosis 1143 Medical Research Council since the late 1970s. By combining drugs that target both rapidly growing bacillary populations and slow-​growing or semi-​dormant organisms within cells, modern short-​course chemotherapy can successfully cure drug-​susceptible pulmonary tuberculosis in 6 months. The regimens recommended for treatment of drug-​susceptible tuberculosis are shown in Table 8.6.26.5. Treatment of extrapulmonary tuberculosis is generally for the same duration as for pulmonary disease, with the exceptions of bone and joint and central nervous system tuberculosis, which are treated for 12 months. The dynamics of mycobacterial growth are such that treatment need be administered only once daily, and can be given as infre- quently as once a week in some circumstances. The long generation time of M. tuberculosis and a postantibiotic effect of antituberculosis drugs make more frequent drug dosing unnecessary. The dosages for drugs are listed in Table 8.6.26.6 according to the frequency with which they are administered. Isoniazid remains a key component of treatment because of its high bactericidal activity. Rifampicin is essential for short-​ course therapy because it is active against all populations of ba- cilli, both within and outside of cells. Pyrazinamide is uniquely active during the first 2 months of therapy, but appears to have no activity thereafter. The addition of pyrazinamide to the treatment regimen allows the duration to be reduced from 9 to 6 months, however. Ethambutol has bacteriostatic activity at lower doses and bactericidal activity at high doses. This agent is primarily given to prevent the emergence of drug resistance, as it appears to add little activity to combination regimens against drug-​susceptible tuberculosis. Although antituberculosis therapy is remarkably well tolerated and almost always given on an ambulatory basis, important drug toxicities do exist. The most serious adverse drug reaction during tuberculosis treatment is liver toxicity, which may occur in up to 5 to 10% of treated patients. Isoniazid, rifampicin, and pyrazinamide are all associated with liver toxicity and use of these agents to- gether increases the risk of a reaction. Isoniazid is the agent most frequently implicated when reactions occur. Isoniazid can produce an idiosyncratic hepatocellular injury, manifested by elevated liver enzymes and clinical hepatitis. Elevation of transaminases does not always portend the development of hepatitis, but may serve as an important signal to anticipate clinical toxicity. The development of signs and symptoms of hepatitis, such as abdominal pain, nausea, vomiting, or jaundice, requires immediate discontinuation of iso- niazid, as continuing treatment can result in death from hepatic Table 8.6.26.5  2016 American Thoracic Society/​Centers for Disease Control and Prevention/​Infectious Disease Society of America/​Treatment Guidelines for drug-​susceptible tuberculosis in children and adults Regimen Intensive Phase Continuation Phase Range of total doses Comment Drugs Interval and doses (minimum duration) Drugs Interval and doses (minimum duration) 1 INH RIF PZA EMB Seven days per week for
56 doses (8 weeks), or five days per week for 40 doses (8 weeks) INH RIF Seven days per week for
126 doses (18 weeks), or five days per week for
90 doses (18 weeks) 182 to 130 This is the preferred regimen for patients with newly diagnosed pulmonary tuberculosis. 2 INH RIF PZA EMB Seven days per week for
56 doses (8 weeks), or five days per week for 40 doses (8 weeks) INH RIF Three times weekly for
54 doses (18 weeks) 110 to 94 Preferred alternative regimen in situations in which more frequent DOT during continuation phase is difficult to achieve. 3 INH RIF PZA EMB Three times weekly for 24 doses (8 weeks) INH RIF Three times weekly for
54 doses (18 weeks) 78 Use regimen with caution in patients with HIV and/​or cavitary disease. Missed doses can lead to treatment failure, relapse, and acquired drug resistance. 4 INH RIF PZA EMB Seven days per week for
14 doses then twice weekly for 12 doses5 INH RIF Twice weekly for 36 doses
(18 weeks) 62 Do not use twice weekly regimens in HIV-​ infected patients or patients with smear-​ positive and/​or cavitary disease. If doses are missed then therapy is equivalent to once weekly, which is inferior. Reproduced from Nahid P et al. (2016). Official American Thoracic Society/​Centers for Disease Control and Prevention/​Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-​Susceptible Tuberculosis. Clin Infect Dis 63(7), e147–​95 with permission from Oxford University Press. Table 8.6.26.6  Dosage recommendation for the initial treatment of tuberculosis in children and adults Drugs Daily dose Twice-​weekly dose Thrice-​weekly dose Children Adults Children Adults Children Adults Isoniazid (mg/​kg) 10–​20 (max. 300 mg) 5 (max. 300 mg) 20–​40 (max. 900 mg) 15 (max. 900 mg) 20–​40 (max. 900 mg) 15 (max. 900 mg) Rifampicin (mg/​kg) 10–​20 (max. 600 mg) 10 (max. 600 mg) 10–​20 (max. 600 mg) 10 (max. 600 mg) 10–​20 (max. 600 mg) 10 (max. 600 mg) Pyrazinamide (mg/​kg) 15–​30 (max. 2 g) 15–​30 (max. 2 g) 50–​70 (max. 4 g) 50–​70 (max. 3.5 g) 50–​60 (max. 3.5 g) 50–​60 (max. 3.5 g) Ethambutol (mg/​kg) 15–​25 (max. 1.5 g) 15–​25 (max. 1.5 g) 50 (max. 4 g) 50 (max. 4 g) 25–​30 25–​30 Streptomycin (mg/​kg) 20–​40 (max. 1.0 g) 15 (max. 1.0 g) 25–​30 (max. 1.5 g) 25–​30 (max. 1.5 g) 25–​?30 (max. 1.5 g) 25–​30 (max. 1.5 g)

section 8  Infectious diseases 1144 failure. Risk factors for developing isoniazid hepatotoxicity include increasing age, chronic liver disease, alcohol abuse, daily dosing of isoniazid, and use of other hepatotoxic drugs, including rifampicin and pyrazinamide. In addition, individuals with a slow isoniazid acetylation genotype are significantly more likely to develop hep- atotoxicity from the drug than intermediate or rapid acetylators. Isoniazid interferes with metabolism of pyridoxine (vitamin B6) which can result in a sensory neuropathy. Coadministration of pyridoxine with isoniazid abrogates this effect without comprom- ising the antimicrobial activity. Rifampicin also causes hepatotoxicity, although the character- istic picture of liver disturbances due to rifampicin is cholestasis. However, the incidence of hepatotoxicity when rifampicin is given with isoniazid is substantially greater than when isoniazid is given alone. Rifampicin predictably causes a discoloration of body fluids, resulting in orange-​tinted tears, sweat, and urine. Haematological toxicity from rifampicin includes thrombocyto- penia and anaemia. Higher doses of rifampicin may produce a hypersensitivity reaction, with fever, rash, and joint swelling. It is for this reason that doses of rifampicin are not escalated during intermittent therapy, whereas the intermittent dosages of the other drugs are increased to deliver weekly doses that are equiva- lent to daily dosing. Pyrazinamide is often associated with arthralgias, and may pre- cipitate gout. Pyrazinamide inhibits renal tubular uric acid excre- tion, resulting in increased serum uric acid levels. Frank gouty arthritis is relatively uncommon with pyrazinamide use, and its fre- quency is reduced with intermittent dosing. Routine use of allopur- inol to prevent gout is not recommended. The major toxicity of ethambutol is optic neuritis, which is common at doses above 30 mg/​kg daily and unusual at doses below 25 mg/​kg daily. Patients receiving ethambutol should have baseline tests of visual acuity and colour discrimination, with monthly moni- toring while on treatment. Ethambutol use is discouraged in chil- dren under 8 years old because of their inability reliably to report visual disturbances. However, the incidence of optic neuritis with the doses of ethambutol typically used is so low that its use in young children is only relatively contraindicated. Monitoring of therapy Patients receiving therapy for tuberculosis require regular moni- toring to assess adherence with therapy, clinical response, and adverse reactions. In the initial phase of therapy, monitoring by a nurse or other trained clinician at least weekly is recommended, and supervision of every dose of medication is suggested by the WHO and other authorities (see next). Patients should be ob- served for clinical responses, including fever defervescence, im- provement in cough and appetite, and weight gain. Improvement in these symptoms and signs can take several weeks, but usually occurs within 3 weeks after starting treatment. Failure to improve suggests that the patient is not adhering to treatment, has drug-​ resistant tuberculosis, or has another illness in addition to, or in- stead of, tuberculosis. Treatment response should also be documented with repeated sputum smears and cultures and a follow-​up chest radiograph after 2 to 3 months (for pulmonary tuberculosis). All patients should have a repeat sputum smear and culture after 2 months of therapy; those who are smear or culture positive at 2 months should have another at 3 months. Failure to convert sputum smears and cultures to negative with 3 months of therapy is associated with a high risk of treatment failure; patients who are still smear or culture posi- tive at 4  months of treatment are considered treatment failures and should be evaluated for drug-​resistant disease. A culture at the end of therapy is recommended to document cure, while an end of therapy radiograph is not necessary. Because mycobacterial DNA can remain in pulmonary secretions long after the disease is effect- ively treated, monitoring with nucleic acid amplification tests is not recommended. Monitoring for drug toxicity is also required throughout therapy. At least monthly monitoring for symptoms and signs of liver toxicity is essential, and patients should be advised to stop therapy and seek care if evidence of hepatitis is noted. Routine liver enzyme moni- toring is recommended primarily for patients with underlying liver disease or baseline abnormalities in liver enzymes. Patients with symptoms of hepatitis, of course, should have liver studies obtained. As already noted here, monthly visual assessment is also recom- mended when ethambutol is given. Adherence to therapy and directly observed therapy Since the 1960s experts in tuberculosis have noted that the suc- cess of treatment depends largely on adherence to therapy. Poor adherence to therapy is responsible for treatment failures, early relapses, and the emergence of drug-​resistant disease. Two major interventions to improve adherence and prevent poor outcomes are directly observed therapy (DOT) and the use of fixed-​dose combination tablets. DOT was first promoted in the 1950s in India, and experience with DOT grew over the ensuing years. Intermittent dosing of tuberculosis therapy, along with the rela- tively short course of treatment, make supervision of treatment feasible in many settings. Ecological and programmatic studies of DOT programmes have shown that the introduction of DOT im- proves cure rates for tuberculosis, reduces non​adherence, and re- duces the emergence of drug-​resistant disease. Two observational studies have shown better survival of HIV-​infected tuberculosis patients who receive DOT. On the other hand, two randomized trials of DOT in developing countries have not found improved treatment completion rates compared with self-​administered treatment. These trials have been criticized for demonstrating only that DOT can be done badly, but the lack of randomized studies documenting that DOT per se leads to improved outcomes is of some concern. The data from observational studies are compelling, however, and DOT has been shown to be cost-​effective in resource-​limited settings and, therefore, is strongly encouraged by many experts and pro- fessional organizations. Use of wireless technologies, including secure video links, is a modern ­alternative to in-person supervi- sion of therapy. The use of fixed-​dose combination tablets is intended to reduce the risk of selecting for drug resistance, as opposed to improving adherence generally. By combining two, three, or four medica- tions in the same tablet, depending on the regimen being used, the opportunity for patients to receive partial treatment that would select for drug resistance is avoided. The bioequivalence of fixed-​dose combinations to individual medications has been established for some, but not all, of the combination products on the market.

8.6.26  Tuberculosis 1145 The catastrophic state of global tuberculosis control in the 1990s led the WHO to promulgate the directly observed therapy, short-​ course (DOTS) strategy. This strategy is a series of policies related to national tuberculosis control practices. The five elements of the DOTS strategy are: 1 Governmental commitment to tuberculosis control 2 A reliable supply of tuberculosis drugs 3 Diagnosis of tuberculosis cases microscopically 4 A registration system for tracking the outcomes of treatment 5 Supervision (DOT) of at least the first 8 weeks of treatment The DOTS strategy has been extremely successful in focusing attention on serious problems in tuberculosis treatment and con- trol, and implementation of the programme in several countries has produced remarkable improvements in clinical outcomes for patients with tuberculosis. There is strong evidence that the use of the DOTS strategy results in lower rates of drug-​resistant tu- berculosis. However, further expansion of the DOTS strategy and improvements in tuberculosis treatment programmes are clearly needed. Treatment of multidrug-​resistant tuberculosis This is beyond the scope of this chapter. Patients with drug-​ resistant tuberculosis should be managed by a physician who is a tuberculosis expert. Effective treatment and cure of multidrug-​ resistant tuberculosis (MDR-​TB) requires use of a combination of drugs that include second-​line drugs which are less effective than first-​line agents, have a greater toxicity, or demonstrate both disadvantages. In recent years, a shorter course of treatment with the so-​called ‘Bangladesh regimen’ of seven antituberculosis drugs has shown high MDR tuberculosis cure rates with just nine months of treatment. In addition, two new drugs, bedaquiline and delamanid, have been shown to speed the time to culture conversion and cure when given in combination with other second-​line drugs and will be a significant advance to further improve outcomes of such patients. Evidence from clinical trials and programmatic data from South Africa show that the use of bedaquiline to treat MDR-TB greatly improves survival and clin- ical outcomes. In 2018, the WHO recommended routine use of bedaquiline for treating MDR-TB and the elimination of inject- able agents. Supervised therapy is considered mandatory for pa- tients with resistant tuberculosis. Physician mistakes remain one of the leading causes of the emergence of multidrug-​resistant and extensively drug-​resistant tuberculosis (XDR-​TB), and the identi- fication of a drug-​resistant isolate of M. tuberculosis should result in immediate expert consultation. It is also clear that addressing drug-​resistant tuberculosis cannot be accomplished without ad- dressing the overall tuberculosis control effort. Treatment of tuberculosis in HIV-​infected people The United States (ATS/​CDC/​Infectious Disease Society of America) recommendations for the treatment of tuberculosis in HIV-​infected adults are, with a few exceptions, the same as those for HIV-​uninfected adults (i.e. standard 6-​month rifampicin-​ based therapy). The development of acquired rifampicin resist- ance has been noted among HIV-​infected patients with advanced immune suppression treated with twice weekly rifampicin-​based or rifabutin-​based regimens. Consequently, patients with HIV infection and tuberculosis should receive daily treatment. DOT and other adherence-​promoting strategies are especially important for patients with HIV-​related tuberculosis. A series of randomized clinical trials has shown that treatment outcomes, both survival and progression of HIV disease, are better among tuberculosis patients coinfected with HIV who start antiretroviral therapy within two to eight weeks of the initiation of tuberculosis treatment. For those in- dividuals with CD4 + T-​cell counts less than 50 cells/​mm3, survival is improved if treatment is initiated within two weeks, while for those with CD4 + counts more than 50 cells/​mm3 outcomes are better if antiretroviral therapy is started within eight to 12 weeks after tuber- culosis treatment begins. Drug interactions There are three possible complications that arise when tubercu- losis treatment and antiretroviral drugs are coadministered: shared side effects and toxicity, drug interactions arising from the in- duction of metabolism (cytochrome P450 enzymes) and efflux pumps by rifampicin, and the immune reconstitution inflamma- tory syndrome. Rifamycins induce the activity of cytochrome P450 enzymes that are important in drug metabolism. Several key anti- retroviral drug classes, protease inhibitors, non​nucleoside reverse transcriptase inhibitors and integrase inhibitors, are substrates of cytochrome P450 enzymes. Protease inhibitors are also substrates of P-​glycoprotein, which is also induced by rifamycins. The avail- able rifamycins differ in potency as P450 enzyme inducers, with ri- fampicin and rifapentine being the most potent and rifabutin the least. Coadministration with rifampicin reduces the concentrations of non​nucleoside reverse transcriptase inhibitors and integrase inhibitors to a moderate extent, but dramatically reduces the con- centrations of protease inhibitors. Rifabutin does not significantly affect the concentrations of ritonavir-​boosted protease inhibitors and is recommended when protease inhibitors have to be used. However, the use of rifabutin in low resource settings is currently limited due to its very high cost and the widespread use of fixed-​ dose combination antituberculosis drugs that include rifampicin. Patients diagnosed with tuberculosis while receiving antiretroviral therapy should continue the regimen without change with the ex- ception of those patients receiving a protease-​based regimen (e.g. lopinavir/​ritonavir) who should have their dose slowly doubled to 800/​200 mg twice daily during a 2-​week period. Such increase should continue for the duration of tuberculosis treatment and for an additional 2 weeks after the conclusion. While the patient is receiving the increased dose of lopinavir/​ritonavir, transamin- ases should be monitored on a regular basis, since however, that protease-​based regimen boosting has been associated with higher rates of toxicity. Immune reconstitution inflammatory syndrome Between 8 and 45% of patients with HIV infection commencing antiretroviral therapy while being treated for tuberculosis develop paradoxical deterioration of tuberculosis, the so-​called immune re- constitution inflammatory syndrome (IRIS). Paradoxical deterior- ation was well known in the pre-​HIV era, but occurs much more frequently in HIV-​infected patients starting antiretroviral therapy. The pathogenesis of IRIS is not completely understood. The most common manifestations of tuberculosis-​related IRIS are focal in- flammatory exacerbations of tuberculosis (lymphadenitis, serositis,

section 8  Infectious diseases 1146 or abscesses, new infiltrates), ‘unmasking’ of tuberculosis or other subclinical diseases after antiretroviral therapy initiation, and so on. It typically occurs within 2–​4 weeks after antiretroviral initi- ation. Risk factors associated with an increased risk of IRIS include shorter intervals between antituberculosis therapy and antiretro- viral therapy initiation, low baseline CD4 counts and high baseline viral load, and vigorous CD4/​viral load response to antiretroviral therapy. However, new or worsening clinical features should be at- tributed to IRIS only after a thorough evaluation has excluded other possible causes, notably poor adherence to antituberculosis therapy, MDR tuberculosis, new opportunistic diseases, and systemic drug hypersensitivity reactions. The benefit of adjunctive corticosteroids in the management of patients with IRIS is suggested by results of at least one randomized controlled trial which showed that the use of 1.25 mg/​kg prednisone for 2 weeks followed by 0.75 mg over two weeks in non​severe IRIS was been associated with decreased hospi- talization and morbidity Adjunctive steroid treatment Corticosteroids are frequently advocated with tuberculosis treat- ment to reduce inflammation, but evidence for this practice is often lacking, particularly in HIV infection. Dexamethasone reduced mortality in a large study of Vietnamese adults with tuberculous meningitis, though rates of neurologic sequelae among survivors were unchanged. The HIV-​infected subgroup of the latter study appeared to gain a similar benefit, but this failed to achieve statis- tical significance. A Ugandan study of adjunctive prednisolone in HIV-​infected patients with pleural tuberculosis found faster reso- lution with prednisolone, but no mortality benefit. Of great concern, however, was their finding of excess cases of Kaposi’s sarcoma in the prednisolone arm. This sobering result is a reminder that the additive immunosuppressant effect of glucocorticoids can have severe con- sequences in HIV infection.. The recent multicentre Investigation of the Management of Pericarditis (IMPI) trial of the management of tuberculosis pericarditis enrolled African adults with definite and probable tuberculosis pericarditis and randomized them to re- ceive adjunctive prednisolone or placebo and to receive M indicus pranii vaccine or placebo in a 2-​by-​2 factorial design, together with standard TB treatment. Neither intervention had a significant effect on the primary composite endpoint of death, recurrent pericar- dial effusion with tamponade requiring pericardiocentesis, or con- strictive pericarditis. However, prednisolone reduced pericardial constriction by almost 50%, a finding that has critical implications in sub-​Saharan Africa where access to cardiothoracic surgery for peri- cardiectomy is limited. Patients assigned to adjunctive glucocortic- oids had an increased risk of developing cancer, notably Kaposi’s sarcoma, particularly if they also received M. indicus pranii vaccine. In general, glucocorticoids should only be used for patients with tu- berculous meningitis or in patients without HIV infection who have pericardial tuberculosis considered at high risk of developing con- strictive pericarditis. Treatment of latent tuberculosis infection Isoniazid chemoprophylaxis Prevention of tuberculosis with isoniazid therapy was first docu- mented in children in the mid-​1950s. Subsequently, several controlled trials of isoniazid chemoprophylaxis were under- taken, and its efficacy firmly established. A meta-​analysis of 11 placebo-​controlled trials of isoniazid, involving more than 70 000 persons, found that treatment reduced tuberculosis incidence by 63%. Among patients who adhered to more than 80% of the iso- niazid regimen, protection was 81%. These studies also showed that isoniazid chemoprophylaxis reduced tuberculosis deaths by 72%. The efficacy of isoniazid therapy to prevent tuberculosis in high-​risk persons is incontrovertible. Enthusiasm for isoniazid chemoprophylaxis was considerably dampened in the late 1960s and early 1970s when drug-​related hepatotoxicity, including deaths, was observed. Several studies based on decision analysis or modelling suggested that the risks of chemoprophylaxis might outweigh the benefits, and use of pre- ventive therapy was curtailed or ignored in many settings. Because the risk of isoniazid-​related hepatotoxicity increases with age, use of chemoprophylaxis in people older than 35 years was particularly discouraged. Preventive therapy in high-​risk individuals The resurgence of tuberculosis in the developed world, particularly HIV-​related tuberculosis, and the uncontrolled global epidemic have renewed interest in the use of preventive therapy in high-​risk individuals known or strongly suspected to be latently infected with M. tuberculosis. The ATS/​CDC guidelines on screening for latent tu- berculosis that stress the importance of targeting efforts on popu- lations and patients who would benefit from treatment to prevent active disease. In the past, screening for tuberculosis infection has been unfocused and often directed at patients who, if found to be in- fected, would have little risk of progressing to active disease. Current guidelines propose that only people with a high risk of disease or high prior probability of latent tuberculosis be tested, and that treat- ment be offered to infected individuals regardless of age. Individuals who should be targeted for tuberculin testing are those listed in the first two columns of Table 8.6.26.3 (i.e. those in whom a positive test is considered equal to or exceeding 5 or equal to or exceeding 10 mm induration). People without risk factors for tuberculosis (those in whom a positive test is equal to or exceeding 15 mm) should not be tested. Treatment regimens for latent tuberculosis are listed in Table 8.6.26.7, along with the rating given to the regimen by the ATS and CDC. Isoniazid remains a favoured drug for tuberculosis pre- ventive therapy because of its well-​documented efficacy, low cost, Table 8.6.26.7  Treatment regimens for latent tuberculosis Drug regimen Duration (months) Interval Rating (HIV−) Rating (HIV+) Isoniazid 9 Daily A II A II Isoniazid 9 Twice weekly B II B II Isoniazid 6 Daily B I C I Isoniazid 6 Twice weekly B II C II Rifampicin Rifapentine and Isoniazid 4 3 Daily Once weekly B II A I B III B I A, strongly recommended; B, recommended; C, optional; I, randomized trials; II, data from other scientific studies; III, expert opinion.

8.6.26  Tuberculosis 1147 and relatively low toxicity. The optimal duration of isoniazid therapy for latent tuberculosis has been the subject of extensive debate in re- cent years. The International Union Against Tuberculosis and Lung Disease conducted a landmark trial in Eastern Europe in the 1970s and 1980s that compared no treatment to 3, 6, or 12 months of iso- niazid in adults with fibrotic changes on radiographs. The results showed that, compared to placebo, 12 months of isoniazid reduced the incidence of tuberculosis by 75%, compared to 66% for 6 months and 20% for 3  months. In addition, patients who completed the 12 months of therapy and were judged to be compliant experienced a 92% reduction in tuberculosis risk, compared to a 69% decrease for compliant patients completing a 6-​month regimen. A meta-​analysis by the Cochrane Collaborative found that 12 months of isoniazid was more effective than 6 months for prevention of tuberculosis. An analysis of varying durations of isoniazid therapy in Alaskan natives revealed that the effectiveness of isoniazid therapy was optimal after 9 months, and that further treatment conferred no additional benefit. Several studies of isoniazid in HIV-​infected patients in Africa, how- ever, have found that prolonged treatment for three or more years is more efficacious than shorter durations, presumably by preventing disease due to reinfection in these highly susceptible patients. Several recent studies have demonstrated that shorter durations of preventive therapy using the combination of rifapentine and iso- niazid given once a week under direct observation is an acceptable alternative to longer courses of isoniazid alone. A large study spon- sored by the CDC’s TB Trials Consortium found that the rifapentine/​ isoniazid 3-​month regimen was not only non-​inferior to isoniazid for 9 months in high-​risk individuals, but almost reached superiority and was better tolerated, with significantly less hepatotoxicity. Another study in HIV-​infected adults in South Africa found rifapentine/​iso- niazid to be of similar efficacy to isoniazid alone for 6 months. A re- cent study by the AIDS Clinical Trials Group found that one month of daily rifapentine and isoniazid was noninferior to nine months of isoniazid in HIV-infected adults and adolescents. A one-month regimen has obvious clinical and public health advantages. Although isoniazid is a well-​tolerated drug, serious hepatotoxicity can occur in a small proportion of patients. Isoniazid may result in asymptomatic elevations in hepatic aminotransferase levels, but this does not always signal impending clinical toxicity. Hepatotoxicity is of concern when symptoms of hepatitis develop, including pain, nausea, vomiting, and jaundice. Continuing isoniazid in the pres- ence of symptoms can lead to death from fulminant hepatic necrosis and liver failure, with a case fatality rate of 10–​15%. Studies in the 1960s and 1970s found evidence of hepatotoxicity in 1–​5% of re- cipients of isoniazid, with higher rates among older patients. More recent experience with isoniazid therapy that is closely monitored shows a risk of hepatotoxicity in the range of 0.1–​0.3%. Thus, appro- priate patient screening and follow-​up makes the use of isoniazid for treating latent infection markedly safer. The use of rifampicin alone or rifapentine and isoniazid is better tolerated than isoniazid alone, but safety monitoring is still required. Other regimens In addition to the 3-​month rifapentine/​isoniazid treatment de- scribed earlier, other alternative regimens are sometimes used in selected situations. A  3-​month regimen of rifampicin alone was found to reduce the incidence of tuberculosis by about 65% in men with silicosis, and was more effective than 6 months of isoniazid. The combination of rifampicin and isoniazid given for three to four months is widely used for treatment of latent tuberculosis in chil- dren and improves completion rates. This regimen has also been found to be equally effective as isoniazid in studies in adults. The use of rifampicin does pose the risk of important drug inter- actions. For example, reduction in methadone concentrations caused by rifampicin can precipitate narcotic withdrawal. Moreover, rifam- picin can lower levels of protease inhibitors and non-​nucleoside reverse transcriptase inhibitors used to treat HIV infection. The efficacy of the oral contraceptive pill is also reduced. If multidrug-​ resistant tuberculosis is suspected, preventive therapy with etham- butol or pyrazinamide and a fluoroquinolone (e.g. moxifloxacin) for 6–​12 months may be used, but clinical data are absent. Candidates for treatment of latent tuberculosis are listed in Table 8.6.26.3. Criteria for treatment include a positive tuberculin test according to the categories in Table 8.6.26.3, elevated risk for developing active tuberculosis if untreated, and exclusion of ac- tive tuberculosis by clinical evaluation and chest radiograph. In addition, HIV-​infected and other severely immunocompromised persons who are contacts to an infectious tuberculosis patient should be treated for latent tuberculosis regardless of tuberculin skin test results. Monitoring treatment Patients receiving treatment for latent tuberculosis should be moni- tored for drug toxicity, as well as to promote adherence to therapy. As in treatment of active tuberculosis, patients receiving isoniazid should be warned about signs and symptoms of hepatotoxicity and advised to discontinue therapy and seek care if any of these occur. Patients with, or at risk of, chronic liver disease should have baseline liver enzymes obtained, with monthly monitoring if the results are abnormal. All patients should be clinically evaluated at least monthly to assess adherence and toxicity. Treatment using other preventive regimens and treatment of patients with mild transaminase eleva- tions (three times upper limits of normal or less) can proceed with regular clinical and laboratory monitoring. Higher elevations of transaminases, or the development of symptoms or signs of hepatitis should be managed with discontinuation of therapy at least tempor- arily. Patients who complete therapy for latent tuberculosis do not need periodic monitoring for tuberculosis subsequently. Prevention of tuberculosis Strategies to control tuberculosis are aimed at the prevention of the spread of M. tuberculosis infection and the development of clinical tu- berculosis. The principal approaches employed toward this end are: • identification and treatment of infectious tuberculosis cases • treatment of latent tuberculosis infection • prevention of exposure to infectious particles in air, especially in hospitals and other institutions • vaccination Identification and treatment of infectious tuberculosis cases Case identification and treatment reduces transmission by ren- dering patients with communicable tuberculosis non​infectious. Patients with pulmonary tuberculosis produce infectious aerosols

section 8  Infectious diseases 1148 that can transmit tubercle bacilli to contacts breathing the same air. When cases are identified and treated, infectiousness is rapidly eliminated. The duration of treatment required to prevent further transmission of infection is not known precisely, but experimental, clinical, and microbiological data suggest that the level of infec- tiousness is reduced enormously within several days of beginning effective treatment. The number of secondary infections gener- ated by an infectious tuberculosis patient varies greatly depending on the duration of illness, the extent of pulmonary pathology, the amount of patient coughing, and the environment into which the patient expels infectious aerosols. Early diagnosis and treatment re- duces the number of secondary infections, while delays can result in ongoing transmission to large numbers of contacts. Failure to retain patients in treatment until they are cured also contributes to spread of infection. Treatment of latent tuberculosis infection This is discussed earlier on in this chapter. The benefit of treating latent infection is not only to the individual patient who does not fall ill with tuberculosis, but also accrues to the potential contacts of that patient, who might become secondarily infected were disease to develop. Targeting of high-​risk groups for screening and treatment of latent tuberculosis thereby reduces tuberculosis incidence within communities. Groups that should be targeted for screening are listed in the first two columns of Table 8.6.26.3. Prevention of exposure especially in hospitals and other institutions Control of exposure to infectious aerosols can have a major im- pact on the spread of tuberculosis. In the late 1980s and early 1990s, transmission of tuberculosis, including multidrug-​resistant tuber- culosis, was widespread in hospitals, homeless shelters, and cor- rectional facilities in New York City. More recently, the outbreak of XDR tuberculosis in the KwaZulu-​Natal province of South Africa is a tragic reminder of the importance of infection control measures in institutions. The congregation of large numbers of highly sus- ceptible people, especially HIV-​infected persons, in closed environ- ments with untreated tuberculosis patients has resulted in numerous microepidemics of both drug-​susceptible and drug-​resistant tuber- culosis. Reversal of the resurgence of tuberculosis in New York at that time was attributable in large part to strengthening of infection control practices. Identification and isolation of infected patients Tuberculosis infection control involves prompt identification and isolation of patients with suspected tuberculosis. The decision to isolate a patient in a hospital setting is a function of epidemiological and clinical factors. Patients with known tuberculosis risk factors who present with symptoms and signs characteristic of pulmonary tuberculosis should be placed in respiratory isolation. Local epi- demiological data should influence isolation practices. In settings where tuberculosis is prevalent, all HIV-​infected patients with pneu- monia may require isolation, whereas isolation can be more selective and based on individual patient features in low prevalence settings. Respiratory isolation requires placement of the patient in a room with negative air pressure relative to adjoining areas, ventilation to the room should provide at least six complete air changes per hour, and air should not be recirculated without filtering or irradiation. Patients should be instructed to cover their coughs at all times, and should wear surgical face masks when outside the room to reduce aerosol generation. Anyone entering the patient’s room should wear an appropriate face mask or respirator to prevent inhalation of droplet nuclei with tubercle bacilli. A considerable amount of debate has oc- curred in recent years in the United States of America regarding what constitutes appropriate protection for healthcare workers exposed to infectious tuberculosis. This debate is influenced as much by phil- osophy as by science, and will not be detailed here. Use of surgical masks for the protection against tuberculosis is clearly inappropriate, even though these masks are useful when placed on patients to pre- vent creation of infectious aerosols. Tightly fitting face masks that filter out more than 99.7% of particles less than 0.5 µm in size (high-​ efficiency particle air filters) are effective. Other devices, including positive air pressure respirators, are also effective. Use of ultraviolet germicidal irradiation can be useful for redu- cing the number of infectious particles in ambient air in settings where ventilation alone is not sufficient. Ultraviolet light must be concentrated in areas of rooms where exposure to people will not occur, such as upper air zones, in order to prevent skin and ocular toxicity. Areas where ultraviolet lights are often used include bron- choscopy suites, inside air circulation ducts, in emergency rooms, and in homeless shelters. Criteria for discontinuation of respiratory isolation are listed in Box 8.6.26.1. Guidelines for taking patients out of isolation in the hospital are strict and are intended to protect other vulner- able patients and hospital staff from any exposure to the disease. Respiratory isolation is not usually required or practical in the home setting, and patients with infectious tuberculosis do not need to be hospitalized solely for respiratory isolation. It is assumed that con- tacts in the home environment will already have had significant ex- posure to tuberculosis by the time a diagnosis is made, and isolation of the patient affords no measurable benefit. Exceptions to this may include patients living in congregate living facilities or other spe- cial situations. The primary protective measures for contacts of cases are a clinical evaluation to identify and evaluate symptoms of tuber- culosis and tuberculin skin testing with treatment of latent infec- tion, if present. Instituting infection control measures is likely to be challenging in developing countries where the healthcare system is already overburdened and where facilities often lack negative pres- sure isolation rooms and air filtration systems. In such settings, work practice and administrative control measures have been emphasized and are considered to be more effective and less expensive. These measures consist of policies and procedures intended to promptly identify infectious tuberculosis cases so that additional precautions and healthcare steps can be taken. Box 8.6.26.1  Criteria for discontinuing respiratory isolation for tuberculosis in hospital inpatients • Alternative diagnosis established • Infectious tuberculosis ruled out • Tuberculosis diagnosed and: -​ Treatment given for at least 14 days and -​ Clinical response to therapy documented, including improvement in fever and cough and -​ Acid-​fast smears of sputum negative or -​ Patient discharged to home

8.6.26  Tuberculosis 1149 BCG vaccination Vaccination against tuberculosis with the Bacille Calmette–​Guérin (BCG) vaccine is widely administered throughout the world but is a practice mired in controversy. BCG is an attenuated live bacterial vaccine developed in the early 20th century by Calmette and Guérin at the Institut Pasteur in France. After a series of uncontrolled and anecdotal assessments of the vaccine, a series of controlled trials of BCG was begun in the 1930s and continued through to the 1990s. The efficacy of BCG has varied greatly in these studies, ranging from more than 80% protection to complete lack of protection, with pos- sibly increased risk in vaccine recipients. Meta-​analyses of BCG trials find a protective benefit of BCG based on historical trials, but recent studies have not demonstrated efficacy. There is evidence that BCG diminishes haematogenous dissem- ination of primary tuberculosis infection and thereby reduces the incidence of miliary tuberculosis and tuberculous meningitis in children. It is primarily for this reason that BCG is included in the Expanded Programme on Immunization of the WHO. The current efficacy of BCG for preventing pulmonary tubercu- losis is debated on the basis of several recent trials which have failed to show protection. Several hypotheses have been proposed for the variation in efficacy reported in various studies, including differences in susceptibility within populations, environmental exposure to mycobacteria which masks vaccine effect, and attenuation of vaccine immunogenicity. This last explanation is very compelling and fits well with clinical trial data. Unlike most vaccines, BCG is not stand- ardized and there is no seedlot of vaccine from which new batches are derived. BCG is grown in several laboratories around the world and has not been re-​passaged in animals since it was derived from cattle a century ago. Multiple commercial and non​commercial BCG prod- ucts are in use presently, and comparative genomic analysis dem- onstrates considerable genetic heterogeneity in these strains, with many gene deletions and polymorphisms. One analysis of BCG trials found that protective efficacy was reduced in studies using multiply-​ passaged vaccine strains. The evidence supports the hypothesis that BCG has become further attenuated over time and no longer pro- motes immunity to M. tuberculosis infection and disease in adults. This position has not been universally accepted, however, and BCG remains one of the most widely administered vaccines in the world, largely for its perceived effects on paediatric tuberculosis. Areas for further research Effective global tuberculosis control will require a coordinated set of clinical and public health strategies that are based on a thorough understanding of the epidemiology, pathogenesis, and therapy of in- fection with M. tuberculosis. The WHO’s END TB strategy, which focuses on finding and effectively treating cases, has been aug- mented with additional strategies for intensified case-​finding, use of preventive therapy and infection control, particularly in countries with large HIV epidemics. Use of improved methods for the diag- nosis at point of care and treatment of tuberculosis infection and disease, particularly drug-​resistant tuberculosis, is urgently needed. Effective regimens for the treatment of multidrug-​resistant and ex- tensively drug-​resistant tuberculosis as well as shortening the total duration of drug-​susceptible tuberculosis (e.g. from 6 to 4 months), with both existing and new agents, need to be developed. A better understanding of the pathogenesis of and natural immunity to tu- berculosis may contribute to the development of a more effective vaccine. A recent trial of a subunit vaccine and adjuvant, which showed >50% efficacy in preventing tuberculosis disease in adults with latent infection, gives hope for future contributions to epidemic control from vaccines. The sequencing of the genome of M. tubercu- losis promises to open the door to a new generation of research on tuberculosis and its control. Scientific progress alone, however, will be insufficient to combat tuberculosis worldwide. The willingness of societies and nations to pay for the deployment of the fruits of bio- medical research, both past and future, to combat the disease where it is prevalent will be required for the conquest of tuberculosis. FURTHER READING Abdool Karim SS, et al. (2010). Timing of initiation of antiretroviral drugs during tuberculosis therapy. N Engl J Med, 362, 697–​706. Blanc FX, et al. (2011). Earlier versus later start of antiretroviral therapy in HIV-​infected adults with tuberculosis. CAMELIA (ANRS 1295–​ CIPRA KH001) study team. N Engl J Med, 365, 1471–​81. Boehme CC, et al. (2010). Rapid molecular detection of tuberculosis and rifampin resistant. N Engl J Med, 363, 1005–​15. Davies PDO, Barnes P, Gordon SB (eds) (2008). Clinical tuberculosis, 4th edition. Hodder Arnold, London. Dheda K, Barry CE 3rd, Maartens G (2016). Tuberculosis. Lancet, 387, 1211–​26. Diacon AH, et al. (2014). Multidrug-​resistant tuberculosis and culture conversion with bedaquiline. N Engl J Med, 371, 723–​32. Dorman SE, et  al. (2014). Interferon-​γ release assays and tuberculin skin testing for diagnosis of latent tuberculosis infection in healthcare workers in the United States. Am J Respir Crit Care Med, 189, 77–​87. Fox W, Ellard GA, Mitchison DA (1999). Studies on the treatment of tuberculosis undertaken by the British Medical Research Council tuberculosis units, 1946–​1986, with relevant subsequent publica- tions. Int J Tuberc Lung Dis, 3(10 Suppl 2), S231–​79. Gler MT, et al. (2012). Delamanid for multidrug-​resistant pulmonary tuberculosis. N Engl J Med, 366, 2151–​60. Mayosi BM, et al. (2014). Prednisolone and mycobacterium indicus pranii in tuberculous pericarditis. N Engl J Med, 371, 1121–​30. Meintjes GM, et al. (2018). Prednisone for the prevention of paradox- ical tuberculosis-associated IRIS. N Engl J Med, 379, 1915–25. Murphy RA, et  al. (2012). Coadministration of lopinavir/​ritonavir and rifampicin in HIV and tuberculosis co-​infected adults in South Africa. PLoS One, 7, e44793 Nahid P, et  al. (2016). Official American Thoracic Society/​Centers for Disease Control and Prevention/​Infectious Diseases Society of America clinical practice guidelines: treatment of drug-​susceptible tuberculosis. Clin Infect Dis, 63, e147–​95. Schnippel K, et al. (2018). Effect of bedaquiline on mortality in South African patients with drug-resistant tuberculosis: a retrospective cohort study. Lancet Respir Med, 6, 699–706. Sterling TR, et al. (2011). Three months of rifapentine and isoniazid for latent tuberculosis infection. N Engl J Med, 365, 2155–66. Uthman OA, et al. (2015). Optimal timing of antiretroviral therapy initi- ation for HIV-​infected adults with newly diagnosed pulmonary tubercu- losis: a systematic review and meta-​analysis. Ann Intern Med, 163, 32–​9. Van Der Meeren O, et al. (2018). Phase 2b Controlled Trial of M72/ AS01(E) Vaccine to Prevent Tuberculosis. N Engl J Med, 379, 1621–34. Xie YL, et  al. (2017). Evaluation of a Rapid Molecular Drug- Susceptibility Test for Tuberculosis. N Engl J Med, 377, 1043–54.

8.6.27 Disease caused by environmental mycobacteri

8.6.27 Disease caused by environmental mycobacteria 1150

section 8  Infectious diseases 1150 8.6.27  Disease caused by environmental mycobacteria Jakko van Ingen ESSENTIALS Introduction—​there are over 130 species of mycobacteria; species other than M.  tuberculosis complex and M.  leprae are collectively referred to as the non​tuberculous or environmental mycobacteria. Non​tuberculous mycobacteria are divided into two groups, the slow growers, and the rapid growers. The most common organisms causing human disease are the slow-​growing species M. avium com- plex and M. kansasii and, less commonly, M. marinum, M. xenopi, M. simiae, M. malmoense, and M. ulcerans. The rapid growers that are human pathogens are M. abscessus, M. fortuitum, and M. chelonae. Ecology and epidemiology—​non​tuberculous mycobacteria are ubi- quitous in the environment and have been isolated from water, soil, domestic and wild animals, milk, and food products. Transmission to humans is though inhalation, ingestion, or traumatic inoculation. The prevalence of non​tuberculous mycobacteria infections is likely to have been underestimated, and appears to be increasing in developed countries. Clinical features—​four clinical syndromes have been described: (1) pulmonary disease; (2)  lymphadenitis; (3)  postinoculation mycobacteriosis; (4) disseminated disease. Cervical lymphadenitis is the most common presentation in children, whereas chronic pul- monary disease is more frequent in adults. Diagnosis—​microscopic examination using acid fast stains and culture on appropriate media remain the cornerstone of diagnosis. The use of molecular techniques such as line probe assays and 16S ribosomal DNA sequencing have enabled more accurate speci- ation of non​tuberculous mycobacteria. Treatment—​this depends on the site and severity of the infection, the presence of predisposing conditions, and the species of myco- bacterium. Therapy of disease due to slow growers is usually based on multidrug regimens containing clarithromycin or azithromycin; that for rapid growers is designed based on drug susceptibility testing results. Introduction Owing to the advent of molecular tools for identification, the genus Mycobacterium is now known to host over 140 species. The spe- cies other than the causative agents of tuberculosis and leprosy (Hansen’s disease) are collectively referred to as non​tuberculous mycobacteria (NTM) or environmental mycobacteria. The latter nomenclature reflects the habitats of these mycobacteria and the source of human infections. The environmental mycobacteria are subdivided into slow and rapid growers, according to their rate of growth on subculture. A small subset of the environmental mycobacteria is capable of causing opportunistic infections in humans; most of these are slow growers. The bacteria of the M. avium complex (MAC, a complex that includes M. avium, M. intracellulare, M. chimaera and several rarely isolated species) are the most frequent causative agents of human infections, followed by M. kansasii, M. ulcerans, M. mari- num, M. malmoense, M. xenopi, and M. simiae. Among the rapid growers, only the M. abscessus group, M. chelonae, and M. fortuitum are commonly associated with human infections. The relative fre- quency of disease caused by these species differs by geographical region. The principal pathogenic environmental mycobacteria and the diseases associated with these species are listed in Table 8.6.27.1. Environmental mycobacteria cause two named diseases with characteristic features: fish tank (or: swimming pool) granuloma caused by M. marinum, and Buruli ulcer caused by M. ulcerans. Disease due to other environmental mycobacteria is much less spe- cific, often resembles tuberculosis, and requires identification of the causative organism for diagnosis. Ecology and epidemiology The environmental mycobacteria are particularly associated with soil and water. They have been isolated from various natural waters, varying from swamps to oceans, as well as from treated tap water. NTM have also been isolated from domestic and wild animals, milk, and food products. Transmission to humans is by aerosol inhalation, ingestion, or traumatic inoculation. Skin test surveys have revealed that human infection is widespread and common, though overt disease is rare. Infection by environmental mycobac- teria may give rise to false-​positive tuberculin skin test results and may affect the efficacy of Bacillus Calmette–​Guérin (BCG) vac- cination. This might explain, in part, the diversity of protection by BCG seen in various trials. Table 8.6.27.1  Principal pathogenic environmental mycobacteria and associated diseases Slow growers M. avium complex Pulmonary disease, lymphadenitis, disseminated disease M. kansasii Pulmonary disease M. xenopi Pulmonary disease, spondylodiscitis in HIV-​infected patients M. malmoense Pulmonary disease, lymphadenitis M. simiae Pulmonary disease M. szulgai Pulmonary disease M. marinum Cause of fish tank granuloma or swimming pool granuloma M. ulcerans Cause of Buruli ulcer disease M. haemophilum Lymphadenitis, skin disease in transplant recipients M. terrae complex Tenosynovitis M. gordonae Common in the environment, rare cause of disease Rapid growers M. abscessus Pulmonary disease, disseminated skin disease M. chelonae Pulmonary disease, disseminated skin disease
(both rare) M. fortuitum Pulmonary disease, postinoculation localized skin infections

8.6.27  Disease caused by environmental mycobacteria 1151 The incidence of overt disease likely results from an interplay be- tween host susceptibility, virulence, and load of the various environ- mental mycobacteria in the local environments and opportunities for infection. Human transmission of overt disease is highly excep- tional; only among cystic fibrosis patients, there is now evidence of transmission of M. abscessus. The frequency of disease caused by different species of NTM is un- known; this is because, unlike tuberculosis, reporting of cases is not mandatory. Clinical and laboratory studies from the United States, Canada, western Europe, and Australia indicate that the burden of NTM has been underestimated and is increasing in developed coun- tries. This may be a result of increased clinical attention, increased use of computed tomography scanning, improved laboratory tech- niques for detection, and a growing number of people at increased risk because of immunosuppressive drug use, chronic pulmonary diseases, and HIV infection. Clinical features The NTM cause four main types of disease: pulmonary, lymphaden- itis, postinoculation, and disseminated. Pulmonary disease Chronic pulmonary disease Chronic pulmonary infections are the most frequent disease mani- festation of NTM. Estimates of the incidence of pulmonary disease caused by environmental mycobacteria differ from 1 per 100 000 population per year in Denmark to 4.3 per 100 000 population per year in Ontario, Canada. In many regions, the incidence of environ- mental mycobacterial disease in the middle-​aged and elderly white population exceeds that of tuberculosis. Two distinct disease entities exist; the cavitary disease type, radiologically similar to tuberculosis (see Fig. 8.6.27.1), affects pa- tients with pre-​existent pulmonary diseases, especially chronic ob- structive pulmonary disease. As a result, it is more common among men and usually appears in their late 50s or 60s. The nodular-​ bronchiectatic disease type (see Fig. 8.6.27.2) is a more subtle dis- ease that mostly affects the lingula and middle lobe. This disease type is more common among female lifetime non​smokers with no significant pulmonary history. The symptoms of cough, malaise, weight loss, and reduced exer- cise tolerance develop over months or even years. Especially for the cavitary disease type, clinical distinction from tuberculosis is diffi- cult, though its course is more prolonged. Diagnosis relies on iso- lation and accurate identification of the causative agents. Because these are environmental organisms, a single culture yielding envir- onmental mycobacteria is insufficient for diagnosis. Positive cul- tures from non​sterile samples such as those from the respiratory tract can result from accidental presence after environmental ex- posure or contamination during sample acquisition or handling. Hence, clinical and radiological as well as microbiological (i.e. multiple positive cultures yielding the same species) signs of in- fection must be obtained and other disease rigorously excluded to make a diagnosis of true environmental mycobacterial disease. Especially in the nodular-​bronchiectatic disease type, cultures of bronchial washings and CT imaging are often required for diag- nosis and follow-​up. Acute pulmonary disease Environmental mycobacteria, especially MAC, can cause a hyper- sensitivity pneumonitis. Exposure is often from indoor spas, hence the name ‘hot tub lung’. This acute or subacute disease results from either inflammation after antigen exposure, or true infection, or both. Dyspnoea, cough, and fever are the most common symp- toms. Occasionally, hypoxemic respiratory failure may occur and require intervention. CT reveals diffuse infiltrates with prominent nodularity of all lung fields. The optimal treatment remains contro- versial and corticosteroids, antimycobacterial treatment, or both Fig. 8.6.27.1  Chest radiograph of a patient with right upper lobe cavitary M. avium disease. Fig. 8.6.27.2  CT image of nodular-​bronchiectatic M. intracellulare pulmonary disease.

section 8  Infectious diseases 1152 can be successful. Interrupting exposure to the mycobacteria is the most important intervention. Lymphadenitis Lymphadenitis is the second most frequent environmental myco- bacterial disease. It predominantly, though not exclusively, affects immunocompetent children under the age of 8 years. Cervicofacial lymph nodes are most frequently affected, although infection of axillar and inguinal lymph nodes has been reported. Disease that involves the abdominal lymph nodes is observed in HIV-​infected patients. In these patients, as well as in otherwise immunocom- promised patients, lymphadenitis can be a sign of disseminated dis- ease (see next). Lymphadenitis is generally caused by slow-​growing environ- mental mycobacteria, mostly M. avium complex, M. haemophilum, M. malmoense, and M. kansasii. The different species seem to af- fect children of different ages, with M. avium affecting the youngest. The risk is reduced by neonatal BCG vaccination. Surgical treatment is curative and lymph node excision is preferred over incision and drainage, which may lead to sinus formation. A 3-​month regimen of rifabutin and clarithromycin or a wait-​and-​see policy can be suc- cessful in selected cases. Postinoculation mycobacterioses Postinoculation mycobacterioses affect the organs that have imme- diate interactions with the environment (i.e. the skin and the eyes). It remains unknown whether the mycobacteria are permanent mem- bers of the human skin microbiome. Skin disease caused by NTM need not be a postinoculation disease; it may be a sign of dissemin- ated disease (see next). Localized skin infections NTM cause two named postinoculation skin diseases with charac- teristic clinical features: Buruli ulcer disease is a severe skin infection by M. ulcerans, presenting as nodular or, in later stages, ulcerative lesions and is endemic to parts of West Africa, Australia, and Latin America, with minor pockets in East Asia. The source of M. ulcerans infection remains controversial, although water insects may be vec- tors. This disease is covered in Chapter 8.6.29. The swimming pool granuloma or fish tank granuloma is a local- ized nodular or pustular, sometimes ulcerative, skin lesion resulting from local infection of an existing skin abrasion by M. marinum. The infection is acquired during swimming or fish tank cleaning activities. There may be ‘sporotrichoid’ spread of lesions along the draining lymphatics. The disease can be self-​limiting, but chemo- therapy accelerates resolution. Local spread of the infection can occur and lead to tenosynovitis, osteomyelitis, or even disseminated disease. Most other cases of postinoculation environmental mycobacterioses are caused by rapid-​growing M. fortuitum and M. chelonae. These in- clude injection site abscesses and footbath-​associated furunculosis. These diseases present as sporadic cases, though miniepidemics may be noted as a result of reusing of contaminated drug vials or nee- dles or suboptimal hygiene measures in nail salons or other spas. Injection site abscesses can take months to develop and are either lo- calized abscesses or multiple abscesses with spreading cellulitis. The latter occur in patients who inject frequently (e.g. insulin-​dependent diabetics). Surgical excision or drainage cures localized disease; 2–​4 months of antibiotic treatment can be warranted for multiple or spreading lesions. Tenosynovitis caused by environmental mycobacteria is rare (Fig. 8.6.27.3); gardeners seem to be at increased risk and inocula- tion occurs in wounds from thorns or other plant material. Bacteria of the M. terrae–​M. nonchromogenicum complex are the most fre- quent causative agents and related to wound contamination with soil. In rare cases, MAC, M.  kansasii, M.  malmoense, and rapid growers have been isolated. Eye infections Trauma to the cornea can lead to infection by rapid-​growing M.  fortuitum, M.  abscessus or M.  chelonae. These localized in- fections respond well to topical treatment with combinations of macrolides, quinolones, and aminoglycosides based on in vitro suscpetibilities. Corneal grafting and systemic therapy may be warranted in severe cases. Accidental inoculation may occur during surgery with contam- inated materials and can lead to severe infections. Osteomyelitis of the sternum and endocarditis with septicaemia has been reported after cardiac surgery. Causative agents are mainly rapid growers. Disseminated disease Prior to the HIV pandemic, disseminated infections by envir- onmental mycobacteria were rare and restricted to patients with congenital immune deficiencies. Disseminated disease caused by M. avium (or, less frequently, M. genavense or M. simiae) was an im- portant and frequently lethal clinical entity during the early phase of the HIV pandemic, before the advent of effective antiretroviral therapy (ART). This was particularly true for countries with a low tuberculosis burden. Disseminated M. avium infection was far less frequent in HIV-​infected patients Africa. Dissemination of the causative mycobacteria was thought to start from the intestines, as many patients were known to harbour M. avium in their faeces before the onset of disseminated disease. Since the introduction of ART, disseminated environmental myco- bacterial disease has become infrequent in HIV-​infected patients. At the same time, notification of this disease has not diminished, as more cases are now diagnosed in patients who are treated with immunosuppressive drugs, mostly after solid organ transplantation or in patients with haem- atological malignancies. In these ‘new’ patient categories, the dominant causative agents are M. avium, M. genavense, M. haemophilum, and Fig. 8.6.27.3  Erythematous swelling in tenosynovitis caused by M. malmoense.

8.6.27  Disease caused by environmental mycobacteria 1153 M. chelonae. Disseminated disease presents with two distinct clinical syndromes. M. avium and the difficult to culture M. genavense cause a non​specific disease with symptoms of fever, weight loss, night sweats, malaise, and anaemia (or, in M. genavense disease, pancytopenia); diar- rhoea, abdominal lymph node enlargement, and abdominal pain are frequent, especially in patients with HIV infection. The diagnosis is usually made by culture of bone marrow, liver, or other biopsies, or by blood culture. M. haemophilum and the rapid growers cause a dissem- inated disease with subcutaneous abscesses, nodular lesions, or skin ul- ceration. Their localization to the skin has been related to these species’ preferences for lower temperatures. Diagnosis is usually made by cul- ture and histological examination of biopsies of lesions, or blood cul- tures. Disease caused by M. haemophilum can be difficult to diagnose as the bacteria need an external iron source (e.g. blood, hence its name) for in vitro growth. More recently a multicountry outbreak of M. chimaera, linked to heater-cooler units during cardiac surgery, has resulted in dis- seminated disease, including endocarditis. Diagnosis Microscopic examination using acid fast stains and culture on ap- propriate media remain the cornerstone of diagnosis. Specimens may be stained with the Ziehl–​Neelsen stain or one of its modifica- tions (e.g. Kinyoun stain, and appear pink as a result of staining with carbol-​fuschin). Microscopy is relatively insensitive as it requires at least 10 000 organisms per ml of sputum for smear positivity. The sensitivity of microscopy can be improved by use of a fluorochrome stain such as auramine-​O or auramine-​rhodamine and examination by fluorescence microscopy. Mycobacterial culture is more sensitive but more time-​consuming than microscopy as it requires specialized equipment and a contain- ment level 3 facility. Non​sterile specimens such as sputum should be decontaminated before culture in order to eliminate more common bacteria or fungi that would overwhelm growth of mycobac- teria. Sterile samples such as serous fluids, blood, or cerebrospinal fluid can be inoculated directly on to appropriate solid media (e.g. Lowenstein Jensen medium) or liquid media (e.g. Mycobacterium growth indicator tube, MGIT). Once cultures have grown, speciation is preferentially performed by molecular tools such as line probe assays or DNA sequencing, which has enabled more accurate speciation of NTM. Susceptibility testing of NTM is done in specialist reference labora- tories; broth microdilution in cation-​adjusted Mueller Hinton me- dium is the recommended method. Only results of drugs with clear in vitro–​in vivo correlations should be reported. These correlations are most clear for macrolides, aminoglycosides, fluoroquinolones, and co-​trimoxazole, although the latter is only relevant for rapid growers. Treatment The choice of therapy depends on the causative agents and their in vitro susceptibility, the predisposing conditions and their prog- nosis, and the site of disease as well as its severity. In general, there is a lack of evidence for the efficacy of regimens as very few clinical trials have been performed. For skin disease caused by M. marinum, drug susceptibility tests have a limited role as the disease usually responds to monotherapy with doxycycline, minocycline, or trimethoprim-​sulfamethoxazole, or combinations of clarithromycin and ethambutol, or rifampicin and ethambutol. Multidrug therapy may be indicated in severe, spreading disease. Surgical excision, curettage, or drainage cures lo- calized skin disease caused by rapid growers (see earlier) and surgical excision is the treatment of choice for lymphadenitis and even single nodular pulmonary lesions. For extrapulmonary disease by rapid growers where chemotherapy is needed, results of drug susceptibility tests should guide the selection of a regimen. A minimum of two active drugs is needed, based on the severity of disease and a treat- ment duration of 4–​6 months may be indicated; timing of clinical improvement guides the treatment duration. For extrapulmonary and disseminated disease caused by slow-​growing species, mainly M. avium complex, treatment regimens should include a macrolide (clarithromycin, azithromycin), a rifamycin (rifampicin, rifabutin), and ethambutol. Pulmonary disease by environmental mycobacteria is difficult to treat; the long treatment duration and drug toxicities are a sig- nificant burden for patients. For disease caused by slow growers, mainly MAC, drug susceptibility results are only helpful for the macrolides. In case of macrolide susceptibility, most clinicians have adopted the use of macrolides, combined with rifampicin and ethambutol, despite limited evidence for additional efficacy of macrolides (Table 8.6.27.2). These regimens should be used for a total duration of 24 months or up to 1 year after culture conversion. The notable exception is M. kansasii, for which short (9 month) regimens of rifampicin and ethambutol are highly effective. The role of quinolones in pulmonary disease by slow growers seems limited. For pulmonary disease by rapid growers, mostly the M. absces- sus group and M. fortuitum, drug susceptibility results guide the selection of drug regimens. For M.  abscessus group infections, a macrolide combined with amikacin and either cefoxitin or imipenem, is often used, with addition of tigecycline especially in cystic fibrosis patients. After an intensive phase of 2–​4 months, a switch is often made to 2–​3 oral agents like macrolides (des- pite frequent inducible resistance), clofazimine, doxycycline, and fluoroquinolones, sometimes with inhaled amikacin. There is very Table 8.6.27.2  Recommended regimens for treatment of pulmonary infections caused by the more usually encountered slow-​growing environmental mycobacteria in HIV-​negative patients Species Regimen Areas of uncertainty M. avium complex 18–​24 months of rifampicin, ethambutol, and a macrolide Role of clofazimine, role of aminoglycosides in severe disease M. kansasii 12 months isoniazid, rifampicin, and ethambutol Role and duration of rifampicin, ethambutol, and macrolide regimen M. xenopi 24 months of rifampicin, ethambutol, and a macrolide Role of quinolones M. malmoense 24 months of rifampicin, ethambutol, and a macrolide Role of quinolones M. simiae 18–​24 months of co-​trimoxazole, moxifloxacin, and a macrolide Role of drug susceptibility testing, no evidence-​based treatment regimen

8.6.28 Leprosy (Hansen’s disease) 1154

8.6.28 Leprosy (Hansen’s disease) 1154

section 8  Infectious diseases 1154 little evidence to support these regimens and expert consultation should be sought. For M. chelonae, tobramycin or imipenem can be combined with a macrolide, with macrolide-​fluoroquinolone combinations for subsequent oral therapy. Macrolides may not be effective against M. fortuitum owing to natural resistance and a multidrug regimen that combines a quinolone with trimethoprim-​ sulfamethoxazole, doxycycline, an aminoglycoside, or imipenem can be used. Treatment duration in pulmonary disease by rapid growers is not well studied; for M. abscessus it equals that of MAC, for other species it may be shorter based on clinical and bacterio- logical response. Cure rates of pulmonary disease by environmental mycobacteria are limited, in the 50–​70% range; M. kansasii disease has more fa- vourable outcome. Adjunctive surgical resection of the affected areas of the lung improves outcomes in selected cases. To achieve success in the treatment of environmental mycobac- terial disease, optimal treatment of the underlying and predisposing conditions is vital. FURTHER READING Bryant JM, et  al. (2016). Emergence and spread of a human-​ transmissible multidrug-​resistant nontuberculous mycobacterium. Science, 354, 751–​7. Falkinham JO III (2009). Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment. J Appl Microbiol, 107, 356–​67. Floto RA, et al. (2016). US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the man- agement of non-​tuberculous mycobacteria in individuals with cystic fibrosis. Thorax, 71 (Suppl 1), 1–​22. Griffith DE, et al. (2007). An official ATS/​IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial dis- eases. Am J Respir Crit Care Med, 175, 367–​416. Lindeboom JA, et al. (2007). Surgical excision versus antibiotic treat- ment for nontuberculous mycobacterial cervicofacial lymph- adenitis in children: a multicenter, randomized, controlled trial. Clin Infect Dis, 44, 1057–​64. Research Committee of the British Thoracic Society (2008). Clarithromycin vs ciprofloxacin as adjuncts to rifampicin and
ethambutol in treating opportunist mycobacterial lung diseases and an assessment of Mycobacterium vaccae immunotherapy. Thorax, 63, 627–​34. Wolinsky E (1979). Nontuberculous mycobacteria and associated diseases. Am Rev Respir Dis, 119, 107–​59. 8.6.28  Leprosy (Hansen’s disease) Diana N.J. Lockwood ESSENTIALS Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae, an acid-​fast intracellular organism not yet cultivated in vitro. It is an important public health problem worldwide, with an estimated 4 million people disabled by the disease. Transmission of M. leprae is only partially understood, but untreated lepromatous patients discharge abundant organisms from their nasal mucosa into the environment. Clinical features These are determined by the degree of cell-​mediated immunity towards M. leprae, with tuberculoid (paucibacillary) and leproma- tous leprosy (multibacillary) being the two poles of a spectrum: (1) tuberculoid—​well-​expressed cell-​mediated immunity effectively controls bacillary multiplication with the formation of organized epithelioid-​cell granulomas; (2) lepromatous—​there is cellular anergy towards M. leprae with abundant bacillary multiplication. Between these two poles is a continuum, varying from the patient with mod- erate cell-​mediated immunity (borderline tuberculoid), through borderline, to the patient with little cellular response, borderline lepromatous. Presenting symptoms—​most commonly (1) anaesthesia—​ranging from a small area of numbness on the skin due to involvement of a dermal nerve, to peripheral neuropathy with affected nerves tender and thickened; (2)  skin lesions—​most commonly macules
or plaques; tuberculoid patients have few, hypopigmented lesions that are anaesthetic; lepromatous patients have numerous, some- times confluent lesions. Other manifestations—​these include (1) type 1 (reversal reactions)—​ occur in borderline patients; characterized by acute neuritis and/​or acutely inflamed skin lesions; often occur in the first 2 months after starting treatment; (2) type 2 (erythema nodosum leprosum reac- tions)—​occur in up to 50% of patients with lepromatous leprosy; (3) neuritis—​silent neuropathy is an important form of nerve damage, causing lifelong morbidity; (4) eye disease—​blindness occurs in at least 2.5% of patients. Diagnosis This is made by recognition of typical skin lesions or thickened per- ipheral nerves, supported by the finding of acid-​fast bacilli on slit skin smears that should be taken from at least four sites (earlobes, and edges of active lesions). Treatment There are six main principles of treatment: (1) stop the infection with chemotherapy—​first-​line antileprosy drugs are rifampicin, clofazimine, and dapsone, given in combination and duration as determined by whether disease is paucibacillary or multibacillary; these are highly effective in killing bacilli but may not halt nerve damage; (2) treat new nerve damage—​a 6-​month course of ster- oids should be given to those with nerve damage for less than 6 months; (3)  treat reactions—​steroids are likely to be required; (4) educate the patient about leprosy; (5) prevent disability; and (6) support the patient socially and psychologically—​patients with leprosy the world over are frequently stigmatized; words such as ‘leper’ should be avoided; the disease can be referred to as ‘Hansen’s disease’. Prevention Vaccination with Bacille Calmette–​Guérin (BCG) can provide some protection against leprosy (20–​80% in different trials).

8.6.28  Leprosy (Hansen’s disease) 1155 Aetiology Leprosy is caused by Mycobacterium leprae, an acid-​fast intracel- lular organism not yet cultivated in vitro. It was first identified in the nodules of patients with lepromatous leprosy by Hansen in 1873. M. leprae preferentially parasitizes skin macrophages and periph- eral nerve Schwann cells. A second agent of leprosy, M. lepromatosis, which diverged form M. leprae 10 million years ago has also been recognized as causing leprosy in about 40 patients and the main focus of origin is Mexico. The importance of this organism will be determined over the next few years. In vivo cultivation of M. leprae M. leprae can be grown in the mouse footpad, but growth is slow, taking over 6 months to produce significant yields. The nine-​ banded armadillo is susceptible to M. leprae infection and develops lepromatous disease. The armadillo and mouse models of M. lep- rae infection have been useful for producing M. leprae for bio- logical studies and studying drug sensitivity patterns, respectively. Biological characteristics M. leprae is a stable hardy organism that withstands drying for up to 5 months. It has a doubling time of 12 days (compared with 20 min for Escherichia coli). The optimum growth temperature is 27 to 30° C, consistent with the clinical observation of maximal M. leprae growth at cool superficial sites (skin, nasal mucosa, and peripheral nerves). M. leprae isolates from different parts of the world have similar biological characteristics. M. leprae possesses a complex cell wall comprising lipids and carbohydrates. It synthesizes a species-​ specific phenolic glycolipid and lipoarabinomannan. Antibody and T-​cell screening have identified numerous protein antigens and pep- tides that are important immune targets. M. leprae genome M. leprae has a 3.27-​Mb genome that displays extreme reductive evolution. Less than one-​half of the genome contains functional genes and many pseudogenes are present. One hundred and sixty-​ five genes are unique to M. leprae and functions can be attributed to 29 of them. These unique proteins are being identified and analysed to aid in development of new diagnostic tests. Comparison of bio- synthetic pathways with Mycobacterium tuberculosis is giving new insights into M. leprae metabolism. For lipolysis M. leprae has only two genes (M. tuberculosis has 22); M. leprae has also lost many genes for carbon catabolism and many carbon sources (e.g. acetate and galactose) are unavailable to it. This gene loss leaves M. leprae unable to respond to different environments and underlies the impossibility of growing the organism in vitro. Using comparative genomics and analysis of single nucleotide polymorphisms it has been shown that all extant cases of leprosy can be attributed to a single clone which then disseminated worldwide. Leprosy probably originated in India or eastern Africa and spread with successive human migrations. Epidemiology Leprosy continues to be an important public health problem worldwide. In 2014, 213 889 new cases were detected and registered. The highest numbers of cases were in India, Brazil, Indonesia, Ethiopia, and Bangladesh. India accounts for 58% of the global dis- ease burden. From 1990, the World Health Organization (WHO) led a leprosy elimination campaign and this defined elimination as less than 1 case per 10 000 population. Prevalence figures are highly influenced by operational activities such as reducing the length of treatment. The global focus is now on detecting new cases and pro- viding sustainable care for leprosy patients. An estimated 4 million people are disabled by leprosy. Leprosy has not always been a trop- ical disease; it was widespread in medieval Europe and was endemic in Norway until the early 20th century. In North America, small foci of infection still exist in Texas and Louisiana. Nearly all new patients now seen in Europe and North America have acquired their infec- tion abroad. Risk factors Leprosy is a chronic disease with a long incubation period. An average incubation time of 2 to 5 years has been calculated for tu- berculoid cases and 8 to 12 years for lepromatous cases. American servicemen who developed leprosy after serving in the tropics pre- sented up to 20 years after their presumed exposure. Most leprosy patients do not have known contact with a leprosy patient. Age, sex, and household contact are important determinants of leprosy risk; incidence reaches a peak at 10 to 14 years; the excess of male cases is attributed to women’s reluctance to present to health workers with skin lesions. Poor nutritional status is cited as predisposing to leprosy, but no good evidence substantiates this. Improved socioeconomic conditions, extended schooling, and good housing conditions reduce the risk of leprosy. Subclinical infection with M. leprae is probably common but the development of established disease is rare. Little work has been done on the early events in infec- tion with M. leprae because there is no simple test that can establish whether an individual has encountered M. leprae and mounted a protective immune response. HIV and leprosy It was predicted that HIV infection would produce anergic, leproma- tous leprosy, However HIV/​leprosy coinfected patients have disease types across the leprosy spectrum with typical leprosy skin lesions and nerve involvement. Their skin lesions have typical leprosy hist- ology with granuloma formation even in the presence of low cir- culating CD4 counts. Patients coinfected with HIV and leprosy are at higher risk of developing leprosy reactions and nerve damage. Leprosy might also present as an immune reconstitution syndrome in patients who have recently started on highly active antiretroviral therapy and have rising CD4 counts. These patients have borderline leprosy which is very immunologically active with inflamed skin le- sions and reactions. Transmission The transmission of M.  leprae is only partially understood. Untreated lepromatous patients discharge abundant organisms from their nasal mucosa into the environment. Studies in Indonesia and Ethiopia using polymerase chain reaction primers to detect M. leprae DNA in nasal swabs have shown that up to 5% of the population in leprosy endemic areas carry M. leprae DNA in their

section 8  Infectious diseases 1156 noses. The organism is then inhaled, multiplies on the inferior tur- binates, and has a brief bacteraemic phase before binding to and entering Schwann cells and macrophages. The combination of an environmentally well-​adapted organism, high carriage rates, and a long incubation period means that, even with effective antibiotics, transmission will continue for a long time. Pathogenesis Leprosy is a bacterial infection in which the clinical features are determined by the host’s immune response (Table 8.6.28.1). Immune response to M. leprae and the leprosy spectrum The Ridley–​Jopling classification (Fig. 8.6.28.1) places patients on a spectrum of disease according to their clinical features, bacterial load, and histological and immunological responses. The two poles of the spectrum are tuberculoid (TT; paucibacillary) and leproma- tous leprosy (LL; multibacillary). At the tuberculoid pole, well-​ expressed cell-​mediated immunity effectively controls bacillary multiplication with the formation of organized epithelioid-​cell granulomas; at the lepromatous pole there is cellular anergy to- wards M. leprae with abundant bacillary multiplication. Between these two poles is a continuum, varying from the patient with moderate cell-​mediated immunity (borderline tuberculoid, BT) through borderline (BB) to the patient with little cellular response, borderline lepromatous (BL). The polar groups (TT, LL) are stable, but within the central groups (BT, BB, BL) the disease tends to downgrade to the lepromatous pole in the absence of treatment, and upgrading towards the tuberculoid pole can occur during or after treatment. Both T cells and macrophages play important roles in the pro- cessing, recognition, and response to M. leprae antigens. In tuber- culoid leprosy, in vitro tests of T-​cell function, such as lymphocyte transformation tests, show a strong response to M.  leprae pro- tein antigens with the production of Th1-​type cytokines such as interferon-​γ and interleukin 2 (IL-​2). Skin tests with lepromin, a heat-​killed M.  leprae preparation, are strongly positive. Staining of skin biopsies from tuberculoid lesions with T-​cell markers shows highly organized granulomas composed predominantly of CD4 cells and macrophages with a peripheral mantle of CD8 cells. This strong cell-​mediated immune response clears bacilli but with concomitant local tissue destruction, especially in nerves. Patients with lepromatous leprosy have no cell-​mediated im- munity to M. leprae with a failure of the T-​cell and macrophage response. Tests for lepromin are negative. This anergy is spe- cific for M. leprae. Patients with lepromatous disease respond to other mycobacteria such as M. tuberculosis, both in vitro and in skin tests. Identification of cell types in lepromatous granulomas shows a disorganized mixture of macrophages and T cells, mainly CD8 cells. The T-​cell failure may be due to clonal anergy or active suppression. Defects in cytokine production have been demon- strated; intralesional injections of recombinant IL-​2 reconstitute the local immune response with elimination of M. leprae from macrophages. There is low production of Th2-​type cytokines. Macrophage defects described in lepromatous disease include defective antigen presentation and recognition, defective IL-​ 1 production, a failure of macrophages to kill M. leprae, and a macrophage suppression of the T-​cell response. Patients with lep- romatous leprosy produce a range of autoantibodies that are both organ specific (against thyroid, nerve, testis, and gastric mucosa) and non​specific, such as rheumatoid factors, anti-​DNA, cryo- globulins, and cardiolipin. Table 8.6.28.1  Major clinical features of the disease spectrum in leprosy Clinical features Classification Tuberculoid (TT) Borderline tuberculoid (BT) Borderline (BB) Borderline lepromatous (BL) Lepromatous (LL) Paucibacillary Multibacillary Skin Infiltrated lesions Defined plaques, healing centres Irregular plaques with partially raised edges Polymorphic,
‘punched-​out centres’ Papules, nodules Diffuse thickening Macular lesions Single, small Several, any size, ‘geographical’ Multiple, all sizes,
bizarre Innumerable, small Innumerable, confluent Nerve Peripheral nerve Solitary enlarged nerves Several nerves, asymmetrical Many nerves, asymmetrical pattern Late neural thickening, asymmetrical, anaesthesia, and paresis Slow symmetrical loss, glove and stocking anaesthesia Microbiology Bacterial index 0–​1 0–​2 2–​3 1–​4 4–​6 Histology Lymphocytes + ++ ± ++ ± Macrophages –​ –​ ± –​ –​ Epithelioid cells ++ ± –​ –​ –​ Antibody, anti-​M. leprae –​/​+ –​/​++ + ++ ++ +, present, ++, present strongly, –​, absent.

8.6.28  Leprosy (Hansen’s disease) 1157 Bacterial load In lepromatous leprosy, bacilli spread haematogenously to cool superficial sites including eyes, upper respiratory mucosa, testes, small muscles, and bones of the hands, feet, and face as well as to peripheral nerves and skin. The heavy bacterial load causes struc- tural damage at all these sites. In tuberculoid leprosy, bacilli are not readily found. Nerve damage Neural inflammation is pathognomonic of leprosy. Nerve damage occurs in small nerve fibres, both sensory and autonomic, in the skin, and in peripheral nerve trunks. Nerve damage occurs before diagnosis, during treatment, and after treatment. In lepromatous in- fection, almost all the cutaneous nerves and peripheral nerve trunks are involved. Bacilli are found in Schwann, perineural, and endothe- lial cells. Extensive demyelination occurs and later wallerian degen- eration. Despite large numbers of organisms in the nerve there is only a small inflammatory response, but ultimately the nerve becomes fibrotic and is hyalinized. At the tuberculoid end of the spectrum nerve damage is secondary to a granulomatous response to M. lep- rae antigens. Perineural inflammation and epithelioid granulomas destroy the Schwann cells and axons. In borderline leprosy the com- bination of M. leprae antigens and a cell-​mediated immune response results in small granulomas abutting strands of normal-​looking but heavily bacillated Schwann cells giving rise to the widespread nerve damage in borderline leprosy. The persistence of M. leprae antigens in Schwann cells means that immune-​mediated nerve damage can occur after successful antibacterial treatment. Leprosy reactions Leprosy reactions are episodes of inflammation that occur across the Ridley–​Jopling spectrum. Type 1 (reversal reactions) occur in bor- derline patients (BT, BB, BL) and are delayed hypersensitivity reac- tions caused by increased recognition of M. leprae antigens in skin and nerve sites. They are characterized by an increase in lympho- cytes (CD4 and IL-​2-​producing cells) within lesions, severe oedema with disruption of the granuloma, and giant cell formation. There is local production of Th1-​type cytokines such as interferon-​γ and tumour necrosis factor-​α. Type 2 reactions, erythema nodosum leprosum (ENL), are partly due to immune complex deposition and occur in patients with borderline lepromatous and lepromatous leprosy who pro- duce antibodies and have a large antigen load. There is vasculitis with lesional immunoglobulin deposition, complement activation, and polymorphs and circulating immune complexes. There is also enhanced T-​cell activity with increased CD8 cells, increased circu- lating IL-​2 receptors, and high levels of circulating tumour necrosis factor-​α. After reaction, lepromatous patients revert to a state of immunological unresponsiveness. Clinical features of leprosy Patients commonly present with skin lesions, weakness, or numb- ness due to a peripheral nerve lesion, or a burn or ulcer on an anaes- thetic hand or foot. Borderline patients may present in reaction with nerve pain, sudden palsy, multiple new skin lesions, pain in the eye, or a systemic febrile illness. The cardinal signs are: • typical skin lesions, anaesthetic at the tuberculoid end of the spectrum • thickened peripheral nerves • acid-​fast bacilli on skin smears or biopsy Early lesions The most common early lesion is an area of numbness on the skin or a visible skin lesion. The classic early skin lesion is indeterminate leprosy, which is commonly found on the face, extensor surface of the limbs, buttocks, or trunk. Indeterminate lesions consist of one or more slightly hypopigmented or erythematous macules, a few centimetres in diameter, with poorly defined margins. Hair growth and nerve function are unimpaired. A biopsy may show the perineurovascular infiltrate and only scanty acid-​fast bacilli. The in- determinate phase may last for months or years before resolving or developing into one of the determinate types of leprosy. Skin The most common skin lesions are macules or plaques; papules and nodules are more rare. Lesions can be found anywhere although rarely in the axillae, perineum, or hairy scalp. Skin lesions should be assessed for inflammation, colour, and sensation. Tuberculoid patients have few granulomatous hypopigmented lesions while lep- romatous patients have numerous, sometimes confluent lesions. The few tuberculoid lesions are usually asymmetrical; more numerous lesions are likely to be distributed symmetrically. Anaesthesia Anaesthesia may occur in skin lesions when dermal nerves are in- volved or in the distribution of a large peripheral nerve. In skin lesions the small dermal sensory and autonomic nerve fibres supplying dermal and subcutaneous structures are damaged causing local sensory loss and loss of sweating within that area. Peripheral neuropathy Peripheral nerve trunks are vulnerable at sites where they are super- ficial or are in fibro-​osseous tunnels. At these points a small in- crease in nerve diameter raises intraneural pressure, causing neural Cell mediated Immunity TT BT BB BL LL Bacterial load 0 3 6 Type 1 reactions ENL reactions Fig. 8.6.28.1  Ridley–​Jopling spectrum of bacterial load, cell-​mediated immunity, and reactions.

section 8  Infectious diseases 1158 compression and ischaemia. Damage to peripheral nerve trunks produces characteristic signs with dermatomal sensory loss and dysfunction of muscles supplied by that peripheral nerve. The pre- dilection sites for peripheral nerve involvement are ulnar nerve (at the elbow) (Fig. 8.6.28.2), median nerve (at the wrist), radial nerve, radial cutaneous nerve (at the wrist), common peroneal nerve (at the knee), posterior tibial and sural nerves at the ankle, facial nerve as it crosses the zygomatic arch, and great auricular nerve in the pos- terior triangle of the neck (Fig. 8.6.28.3). All these nerves should be examined for enlargement and tenderness. Peripheral nerve func- tion should be assessed by testing the motor function of the small muscles of the hands and feet using the Medical Research Council (MRC) grading scale. Sensory function is best assessed using graded nylon monofilaments (Semmes–​Weinstein) as in diabetic screening. Patients should be asked about symptoms of neuropathy. Tuberculoid leprosy (TT) Infection is localized and asymmetrical. A typical tuberculoid skin lesion is a macule or plaque, single, erythematous, or purple, with raised and clear-​cut edges sloping towards a flattened hypopigmented centre. The surface is anaesthetized, dry, and hairless. Sensory im- pairment can be difficult to demonstrate on the face where there are abundant nerve endings. If peripheral nerve trunk involvement is present, only one nerve trunk is enlarged. No M. leprae are found in skin smears. True tuberculoid leprosy has a good prognosis, many infections resolve without treatment, and peripheral nerve trunk damage is limited. Borderline tuberculoid leprosy (BT) The skin lesions are similar to tuberculoid leprosy and there may be few or many lesions (Figs. 8.6.28.4, 8.6.28.5). The margins are less well defined and there may be satellite lesions. Damage to periph- eral nerves is widespread and severe, usually with several thickened nerve trunks. It is important to recognize borderline tuberculoid lep- rosy because these patients are at risk of reversal reactions leading to rapid deterioration in nerve function with consequent deformities. Borderline leprosy (BB) Borderline disease is the most unstable part of the spectrum and patients usually downgrade towards lepromatous leprosy if they are not treated or upgrade towards tuberculoid leprosy as part of a reversal reaction. There are numerous skin lesions which may be macules, papules, or plaques, and they vary in size, shape, and dis- tribution. The edges of the lesions may have streaming, irregular borders. Annular lesions with a broad irregular edge and a sharply defined punched-​out centre are characteristic of borderline disease (Fig. 8.6.28.6). Nerve damage is variable. Borderline lepromatous leprosy (BL) This is characterized by widespread variable asymmetrical skin le- sions. There may be erythematous or hyperpigmented papules, succulent nodules or plaques, and sensation in the lesions may be Fig. 8.6.28.2  The effects of ulnar and median nerve paralysis
with wasting of the small muscles of the hand and evidence of neuropathic damage. Copyright D. A. Warrell. Fig. 8.6.28.3  Thickening of greater auricular nerve. Copyright D. A. Warrell. Fig. 8.6.28.4  BT leprosy. This Ethiopian woman was several hypopigmented patches. Testing for anaesthesia will confirm the diagnosis of BT leprosy.

8.6.28  Leprosy (Hansen’s disease) 1159 normal (Fig. 8.6.28.7). Peripheral nerve involvement is widespread. While patients with borderline lepromatous leprosy do not have the extreme consequences of bacillary multiplication that are seen in lepromatous disease, they might experience either or both reversal and ENL reactions. Lepromatous leprosy (LL) The patient with untreated polar lepromatous leprosy might be carrying 1011 leprosy bacilli. The onset of disease is frequently in- sidious, the earliest lesions being ill-​defined, shiny, hypopigmented, or erythematous macules. Gradually the skin becomes infiltrated and thickened and nodules develop (Fig. 8.6.28.8); facial skin thickening causes the characteristic leonine facies (Fig. 8.6.28.9). Hair is lost, especially the lateral third of the eyebrows (madarosis). Dermal nerves are destroyed leading to a progressive glove and stocking anaesthesia. Position sense is preserved. Sweating is lost, which is uncomfortable in the tropics as compensatory sweating occurs in the remaining intact areas. Damage to per- ipheral nerves is symmetrical and occurs late in the disease. Infiltration of the corneal nerves causes anaesthesia of the cornea, which predisposes to injury, secondary infection, and blindness (Fig. 8.6.28.10). Nasal symptoms can often be elicited early in the disease. Septal perforation can occur. There might be papules on the lips and nodules on the palate, uvula, tongue, and gums (Fig. 8.6.28.11). Bone involvement is common, with absorption of the terminal phalanges and pencilling of the heads and shafts of the metatarsals. Fig. 8.6.28.5  Active tuberculoid lesions showing the sharp outer edge, thin raised erythematous dry rim, and the broad hypopigmented dry centre. The ‘satellite’ lesion at the lower outer edge indicates that this is borderline tuberculoid leprosy. Biopsies and smears should be taken from the raised active rim. Copyright D. A. Warrell. Fig. 8.6.28.6  Multiple, asymmetrical erythematous lesions in BB leprosy. Sensation was intact inside the lesions. Fig. 8.6.28.7  BL leprosy with multiple erythematous lesions. No anaesthesia was present. Fig. 8.6.28.8  Advanced nodular lepromatous leprosy. This Indian patient presented with ulcerating nodules all over his body.

section 8  Infectious diseases 1160 Testicular atrophy results from diffuse infiltration compounded by acute orchitis that can occur during ENL reactions. The conse- quent loss of testosterone leads to azoospermia and gynaecomastia (Fig. 8.6.28.11). The extremities become oedematous. The skin of the legs becomes ichthyotic and ulcerates easily. Other forms of leprosy There are several variant forms of leprosy. Pure neural leprosy, when patients have no skin lesions, has been reported from India and Brazil where it is the presenting form for up to 10% of patients. There is asymmetrical involvement of peripheral nerve trunks and no visible skin lesions. On nerve biopsy all types of leprosy have been found. Histoid lesions are distinctive nodules occurring in leproma- tous patients who have relapsed due to dapsone resistance or noncompliance with chemotherapy. Lucio’s leprosy is a form of lepromatous leprosy found only in Latin Americans; it is characterized by a uniform diffuse shiny skin infiltration. Eye disease in leprosy Blindness due to leprosy, which occurs in at least 2.5% of patients, is a devastating complication for a patient with anaesthesia of the hands and feet. Eye damage results from both nerve damage and bacillary invasion. Lagophthalmos results from paresis of the orbicularis oculi due to involvement of the zygomatic and temporal branches of the facial (VIIth) nerve. These superficial branches are frequently involved in borderline tuberculoid cases, particularly if there are facial skin lesions. In lepromatous dis- ease, lagophthalmos occurs later and is usually bilateral. Damage to the ophthalmic branch of the trigeminal (Vth) nerve causes anaesthesia of the cornea and conjunctiva resulting in drying of the cornea and making the cornea susceptible to trauma and ulceration (Fig. 8.6.28.12). Lepromatous infiltration in cor- neal nerves produces punctate keratitis and corneal lepromas. Invasion of the iris and ciliary body makes them extremely sus- ceptible to reactions. Leprosy reactions Type 1 (reversal reactions) These are characterized by acute neuritis and/​or acutely inflamed skin lesions. Nerves become tender with new loss of sensation or motor weakness. Existing skin lesions become erythema- tous or oedematous (Figs. 8.6.28.13 and 8.6.28.14); new lesions Fig. 8.6.28.9  Lepromatous leprosy. Copyright D. A. Warrell. Fig. 8.6.28.10  Active, untreated lepromatous leprosy, showing generalized infiltration of the skin, swelling of fingers and lips, and thinning of eyebrows and eyelashes. The residual annular lesions visible in both pectoral regions indicate that this patient has ‘downgraded’ from borderline. Fig. 8.6.28.11  Complications of lepromatous leprosy. Gynaecomastia is visible in this man, secondary to testicular involvement in lepromatous leprosy. Multiple nodules are present, many dark brown, due to clofazimine pigmentation. He also has new erythematous lesions of ENL.

8.6.28  Leprosy (Hansen’s disease) 1161 might appear (Fig. 8.6.28.15). Occasionally oedema of the hands, face, or feet is the presenting symptom, but constitutional symp- toms are unusual. Type 1 reactions occur in borderline patients; 35% of borderline lepromatous patients will experience a type 1 reaction. Patients often present with a skin lesion in reaction since a previously quiescent lesion has become active and visible. The peak time for reactions in the first 2 months after starting treatment and in the puerperium. Late reactions can occur years after finishing multidrug treatment. Some patient experience re- peated reactions (Fig. 8.6.28.15). Type 2 (ENL reactions) These occur in lepromatous and borderline lepromatous pa- tients. Up to 50% of lepromatous patients will experience ENL reactions and 5–​10% of borderline lepromatous patients. Attacks are acute and may recur over several years. ENL mani- fests most commonly as painful red nodules on the face (Fig. 8.6.28.16) and extensor surfaces of limbs (Fig. 8.6.28.17). The lesions can be superficial or deep, with suppuration or brawny induration when chronic. Acute lesions crop and desquamate, fading over several days. ENL is a systemic disorder producing fever and malaise and may be accompanied by uveitis, dactylitis (Fig. 8.6.28.18), arthritis, neuritis, lymphadenitis, and orchitis. Recent studies have established the importance of pain as a marker of severity. ENL is often not recognized as a complication of leprosy outside endemic areas. Fig. 8.6.28.12  Corneal damage to eye secondary to lagophthalmos caused by involvement of the zygomatic branch of the facial nerve. Fig. 8.6.28.14  Reversal-​reaction plaque on the left cheek and ear. The edge of this borderline tuberculoid lesion has become very sharply defined, more raised, and erythematous, dry, and scaly. Treatment with corticosteroids is imperative as the patient is at grave risk of rapidly developing lagophthalmos due to associated involvement of branches of the facial nerve. Fig. 8.6.28.15  Type 1 (reversal) reaction: this BL patient
developed new, sharp-​edged, well-​defined, erythematous
plaques with desquamating surfaces about 6 months after starting chemotherapy. Fig. 8.6.28.13  Severe reversal (type 1) reaction. This Indian
woman has erythematous, oedematous, and desquamating reactional lesions.

section 8  Infectious diseases 1162 Neuritis Silent neuropathy is an important form of nerve damage and presents as a functional neural deficit without a manifest acute or subacute neuritis (Figs. 8.6.28.2, 8.6.28.3, 8.6.28.19 and 8.6.28.20). An Indian study following a cohort of 2608 patients found that 75% of those developing deformity had no history of reactions. In Ethiopian and Bangladeshi cohort studies, silent neuritis accounted for most neuritis. This emphasizes the import- ance of regular nerve function testing so that new deficits can be detected. Fig. 8.6.28.16  Erythema nodosum leprosum (ENL) on the forehead of a patient with early lepromatous leprosy. The papules (and nodules) are firm and tender, with rather indefinite edges. In dark-​skinned patients the ENL lesions are often easier to feel than to see, especially over the extensor surfaces of the arms and thighs. Copyright D. A. Warrell. Fig. 8.6.28.17  Erythema nodosum leprosum (ENL) of the shins. Copyright D. A. Warrell. Fig. 8.6.28.18  Dactylitisas part of an ENL reaction. Copyright D. A. Warrell. Fig. 8.6.28.19  Peripheral nerve thickening in leprosy. This young man had marked thickening of his great auricular nerve. Fig. 8.6.28.20  This foot shows thick, dry cracked skin together with neuropathic damage in an anaesthetic foot. The toes are clawed, the foot arch has collapsed and there is evidence of a Charcot ankle joint.

8.6.28  Leprosy (Hansen’s disease) 1163 Diagnosis The diagnosis is made on the clinical findings of one or more of the cardinal signs of leprosy and supported by the finding of acid-​fast bacilli on slit skin smears. The whole body should be inspected in a good light otherwise lesions may be missed, particularly on the but- tocks. Skin lesions should be tested for anaesthesia to light touch, pin prick, and temperature. The peripheral nerves should be pal- pated systematically, examining for thickening and tenderness, and peripheral nerve function should be assessed. Histological exam- ination of a biopsy taken from the active edge of a lesion is helpful to support the diagnosis and confirm the classification. The path- ologist should be asked to examine for neural inflammation which will differentiate leprosy from other granulomatous conditions. Serology is not usually helpful diagnostically because antibodies to the species-​specific glycolipid PGL-​1 are present in 90% of un- treated lepromatous patients but only 40–​50% of paucibacillary pa- tients and 5 to 10% of healthy controls. Polymerase chain reaction for detecting M. leprae DNA in skin and nerve biopsies can be a useful confimatory test. Outside leprosy endemic areas, doctors frequently fail to con- sider the diagnosis of leprosy. Of new patients seen from 1995 to 1999 at The Hospital for Tropical Diseases, London, diagnosis had been delayed in over 80% of cases. Patients had been mis- diagnosed by dermatologists, neurologists, orthopaedic sur- geons, and rheumatologists. A common problem was failure to consider leprosy as a cause of peripheral neuropathy in patients from leprosy endemic countries. These delays had serious con- sequences for patients; over one-​half of them had nerve damage and disability. Slit skin smears The bacterial load is assessed by making a small incision through the epidermis, scraping dermal material, and smearing evenly onto a glass slide. At least four sites should be sampled (earlobes and edges of active lesions). The smears are then stained and acid-​ fast bacilli are counted. Scoring is done on a logarithmic scale per high-​power field. A score of 1 + indicates 1 to 10 bacilli in 100 fields, 6 + over 1000 per field. Smears are useful for confirming the diagnosis and should be done annually to monitor response to treatment. Differential diagnosis Doctors should be aware of the normal range of skin colour and texture in their local population, and also of the common endemic skin diseases, such as onchocerciasis, that may coexist or mimic leprosy. Skin The variety of leprosy skin lesions means that a potentially wide range of skin conditions come into the differential diagnosis. At the tuberculoid end of the spectrum, anaesthesia differentiates leprosy from fungal infections, vitiligo, and eczema. At the lepromatous end the presence of acid-​fast bacilli in smears differentiates leprosy nodules from onchocerciasis, Kaposi’s sarcoma, and post-​kala-​azar dermal leishmaniasis (Fig. 8.6.28.21). Nerves Peripheral nerve thickening is rarely seen except in leprosy. Hereditary sensory motor neuropathy type III is associated with palpable peripheral nerve hypertrophy. Amyloidosis, which can also complicate leprosy, causes thickening of peripheral nerves. Charcot–​ Marie–​Tooth disease is an inherited neuropathy that causes distal atrophy and weakness. The causes of other polyneuropathies such as HIV, diabetes, alcoholism, vasculitis, and heavy metal poisoning should all be considered where appropriate. Treatment There are six main principles of treatment: 1 Stop the infection with chemotherapy. 2 Treat new nerve damage. 3 Treat reactions. 4 Educate the patient about leprosy. 5 Prevent disability. 6 Support the patient socially and psychologically. These objectives need the patient’s cooperation and confidence and can be achieved through the leprosy outpatient clinic with ap- propriate support and patient education. On the first visit there should be a careful assessment of skin and mucosal involvement and accurate evaluation of nerve and eye function. Each patient should be classified using the Ridley–​Jopling classification and assessed for evidence of a reaction of new nerve damage. Fig. 8.6.28.21  African woman with facial epidermoid cysts superficially resembling lepromatous leprosy. Copyright D. A. Warrell.

section 8  Infectious diseases 1164 Chemotherapy All patients with leprosy should be given an appropriate multidrug combination. The first-​line antileprosy drugs are rifampicin, clofazimine, and dapsone. The drug combination and duration are determined by the classification of the patient. The WHO has recommended a simple classification for use in the field de- termined only by the number of skin lesions. Patients are clas- sified as paucibacillary if they have up to five skin lesions and as multibacillary if they have six or more skin lesions. In the specialist clinic setting, where skin smears and skin biopsies can be combined with clinical data, patients can be classified into paucibacillary (skin smear-​negative TT and BT) and multibacillary (skin smear-​positive BT, all BB, BL, and LL). Table 8.6.28.2 gives the drug combinations, doses, and duration of treatment. Patients with multibacillary dis- ease and an initial bacterial index greater than 4 can be treated for 24 months. Rifampicin is a potent bactericide for M. leprae. Four days after a single 600-​mg dose, bacilli from a previously untreated patient with multibacillary disease were no longer viable in a mouse footpad test. It acts by inhibiting DNA-​dependent RNA polymerase. Because M. leprae can develop resistance to rifampicin as a one-​step pro- cess, this drug should always be given in combination with other antileprotics. Dapsone (DDS, 4,4-​diaminodiphenylsulphone) is weakly bac- tericidal. Oral absorption is good and it has a long half-​life, averaging 28 h. It commonly causes mild haemolysis, but rarely an- aemia. Glucose-​6-​phosphate dehydrogenase deficiency is seldom a problem. The ‘DDS syndrome’, which is occasionally seen in leprosy, begins 6 weeks after starting dapsone and manifests as exfoliative dermatitis associated with lymphadenopathy, hepatosplenomegaly, fever, and hepatitis. Clofazimine is a red fat-​soluble crystalline dye. The mechanism of its weakly bactericidal action against M.  leprae remains un- known. The most troublesome side effect is skin discoloration, ran- ging from red to purple-​black, the degree depending on the drug dose and extent of leprous infiltration (Fig. 8.6.28.22(a) and (b)). The pigmentation usually fades within 6 to 12 months of stop- ping clofazimine, although traces of discoloration might remain for up to 4 years. Urine, sputum, and sweat may become pink. Clofazimine also produces a characteristic ichthyosis on the shins and forearms. Other drugs bactericidal for M. leprae include the fluoroquinolones pefloxacin and ofloxacin, minocycline, and clarithromycin. These agents are now established second-​line drugs. Minocycline causes a black pigmentation of skin lesions and so might not be an appropriate substitute for clofazimine if pigmentation is to be avoided. A single-​dose triple-​drug combination (rifampicin, ofloxacin, and minocycline) has been tested in India for patients with single skin lesions and improved 98% of patients. This regimen can also be used in patients who experience adverse effects of dapsone or clofazimine, even in patients with a high BI. Although the study had major flaws and single-​dose treatment is less effective than the conventional 6-​month treatment for paucibacillary leprosy, it is an operationally attractive field regimen or for use in patients with peripatetic lifestyles. Table 8.6.28.2  WHO recommended multidrug therapy regimens Type of leprosy a Drug treatment Monthly supervised Daily self-​administered Duration of treatment Paucibacillary Rifampicin 600 mg Dapsone 100 mg 6 months Multibacillary Rifampicin 600 mg Clofazimine 50 mg 12 months Clofazimine 300 mg Dapsone 100 mg a WHO classification for field use when slit skin smears are not available: paucibacillary—​up to five skin lesions; multibacillary—​more than six skin lesions. (a) (b) Fig. 8.6.28.22  Clofazamine pigmentation in Ethiopian (a) and Peruvian (b) patients. Copyright D. A. Warrell.

8.6.28  Leprosy (Hansen’s disease) 1165 The principal outcome of treatment is improvement of skin le- sions; nerve damage might also improve but to a lesser extent. At the end of a 6-​month treatment of borderline disease there may still be signs of inflammation, which should not be mistaken for active infection. Relapse is uncommon with a cumulative relapse rate of 1.07% for paucibacillary leprosy and 0.77% for multibacillary lep- rosy at 9 years after completion of multidrug therapy. M. leprae is such a slow-​growing organism that relapse only occurs after many years. M. leprae isolates from relapsed patients who have received multidrug therapy are fully drug sensitive and patients can be re- treated with the same regimen. The distinction between relapse and reaction may be difficult. Since the introduction of multidrug therapy more than 16 mil- lion patients have been treated successfully. Clinical improvement has been rapid and toxicity rare. Monthly supervision of the ri- fampicin component has been crucial to success. Other benefits are reduced deformity rates and increased compliance in control schemes. Reactions may develop months or years after stopping chemotherapy, especially in patients with borderline lepromatous or lepromatous leprosy. It is, therefore, vital when discharging patients to warn them to return should new symptoms appear, especially in hands, feet, or eyes. Patients with reactions or physical or psycho- logical complications will need long-​term care. Treatment of new nerve damage Patients with nerve damage present for less than 6 months (assessed by patient history or testing) should receive a 6-​month course of steroids starting at a dose of 40 mg prednisolone per day. A random- ized controlled trial has shown that nerve damage present for more than 6 months is not improved by steroid treatment. Management of reactions Awareness of the early symptoms of reversal reactions by both pa- tient and physician is important because, if left untreated, severe nerve damage may develop. The peak time for reversal reactions is in the first 2 months of treatment. Patients should be warned about reactions because the sudden appearance of reactional lesions after starting treatment is distressing and undermines confidence. The treatment of reactions is aimed at controlling acute inflammation, easing pain, reversing nerve and eye damage, and reassuring the pa- tient. Multidrug therapy should be continued. Type 1 (reversal) reactions Simple anti-​inflammatory drugs are rarely sufficient to control symptoms. If there is any evidence of neuritis (nerve tenderness, new anaesthesia, and/​or motor loss), corticosteroid treatment should be started. Prednisolone should be given, starting at 40–​60 mg/​day, re- ducing to 40 mg after a few days, and then by 5 mg every 2–​4 weeks. Patients with borderline tuberculoid leprosy in reaction commonly need 4 months of steroids while borderline lepromatous reactions may need 6 months or more. Type 2 (ENL) reactions This is a difficult condition to treat and frequently requires treat- ment with high-​dose steroids (80 mg/​day, tapered down rapidly) or thalidomide. Since ENL frequently recurs, steroid dependency can easily develop. Thalidomide (400 mg/​day) is superior to steroids in controlling ENL and is the drug of choice for young men with severe ENL (Fig. 8.6.28.16). Women with severe ENL might benefit from thalidomide treatment. This is a difficult decision for the woman and her physician and needs careful discussion of the benefits and risks (phocomelia when thalidomide is taken in the first trimester). Women should use double contraception and report immediately if menstruation is delayed. Unfortunately, the problems with thalido- mide mean that it is unavailable in several leprosy endemic countries despite its undoubted value. A study in Ethiopia showed that pa- tients had a mortality rate of 10% while having ENL, mainly caused by steroid side effects. Clofazimine has a useful anti-​inflammatory effect in ENL but takes 6 weeks to become effective and can be used at 300 mg/​day for several months. Low-​grade chronic erythema nodosum with iritis or neuritis will require long-​term suppression, preferably with thalidomide or clofazimine. Acute iridocyclitis is treated with 1% hydrocortisone eye drops given 4 hourly and 1% at- ropine drops twice daily. Neuritis Silent neuritis should be treated similarly to reversal reactions with prednisolone at a dose of 40 mg/​day which should be reduced slowly over a period of months. Education of patients Stigmatization due to leprosy occurs worldwide. Patients are fright- ened of social ostracization, physical rejection, and the development of deformities. It is often useful to ask them about their fears so that these can be addressed. They should be reassured that having started treatment they are not infectious to family or friends and can have a sex life. The importance of compliance with antibiotic therapy needs to be emphasized. The patient needs a careful explanation of the diagnosis, aetiology, and prognosis. Prevention of disability The morbidity and disability associated with leprosy is secondary to nerve damage. A major goal in prevention of disability is to create patient self-​awareness so that damage is minimized. Monitoring sen- sation and muscle power in patient’s hands, feet, and eyes should be part of the routine follow-​up so that new nerve damage is detected early. The patient with an anaesthetized hand or foot needs to under- stand the importance of daily self-​care, especially protection when doing potentially dangerous tasks and inspection for trauma. It is helpful to identify for each patient potentially dangerous situations, such as cooking, car repairs, or smoking. Soaking dry hands and feet followed by rubbing with oil keeps the skin moist and supple. An anaesthetized foot needs the protection of an appropriate shoe. For anaesthesia alone, a well-​fitting ‘trainer’ with firm soles and shock-​absorbing inners will provide adequate protection. Once there is deformity, such as clawing, shoes must be made specially to ensure protection of pressure points and even weight distribution. The patient should be taught to question the cause of an injury so that the risk can be avoided in the future. Plantar ulceration occurs secondary to increased pressure over bony prominences. Ulceration

section 8  Infectious diseases 1166 is treated by rest. Unlike ulcers in the feet of patients with diabetes or ischaemia, ulcers in leprosy heal if they are protected from weight-​ bearing. No weight-​bearing is permitted until the ulcer has healed. Appropriate footwear should be provided to prevent recurrence. Physiotherapy exercises should be taught to maximize function of weak muscles and prevent contracture. Contractures of hands and feet, foot drop, lagophthalmos, entropion, and ectropion are amen- able to surgery. Social, psychological, and economic
rehabilitation The social and cultural background of the patient determine the na- ture of many of the problems that may be encountered. The patient may have difficulty in coming to terms with leprosy. The community might reject the patient. Education, gainful employment, confidence from family, friends, and doctor, and plastic surgery to correct stig- matizing deformity all have a role to play. Prognosis Most patients, especially those who have no nerve damage at the time of diagnosis, do well on multidrug treatment with resolution of skin lesions. Left untreated, borderline patients will downgrade towards the lepromatous end of the spectrum and lepromatous pa- tients will have the consequences of bacillary invasion. Borderline patients are at risk of developing type 1 reactions, which can result in devastating nerve damage. Treatment of the neuritis is currently unsatisfactory and patients with neuritis might develop permanent nerve damage despite corticosteroid treatment. It is not possible to predict which patients will develop reactions or nerve damage. Nerve damage and its complications can be severely disabling, espe- cially when all four limbs and both eyes are affected. Leprosy in women Women with leprosy are in double jeopardy; not only might they de- velop postpartum nerve damage but also they are at particular risk of social ostracization with rejection by spouses and family. Pregnancy and leprosy There is little good evidence that pregnancy causes new disease or relapse. However, there is a clear temporal association between parturition and the development of type 1 reactions and neuritis when cell-​mediated immunity returns to prepregnancy levels. In an Ethiopian study, 42% of pregnancies in borderline lepromatous patients were complicated by a type 1 reaction in the postpartum period. In the same cohort, patients with lepromatous leprosy ex- perienced ENL reactions throughout pregnancy and lactation. ENL in pregnancy is associated with early loss of nerve function compared with non​pregnant individuals. Pregnant and newly de- livered women should have regular neurological examination and steroid treatment instituted for neuritis. Rifampicin, dapsone, and clofazimine are safe during pregnancy. Clofazimine crosses the placenta and babies may be born with mild clofazimine pigmenta- tion. Reactions can be managed with the steroid regimens given here, but with a more rapid reduction in dose. Women should be warned before becoming pregnant of the risk that their condition may de- teriorate after delivery. Ideally pregnancies should be planned when leprosy is well controlled. Prevention and control Leprosy control is now becoming more integrated into general services. Different models of providing leprosy control are used depending on the local facilities. In some endemic countries largely vertical programmes are being retained; in others such as Brazil lep- rosy services are provided within dermatological services. Effective treatment is not merely restricted to chemotherapy but also involves good case management with effective monitoring and supervision and prevention of disabilities. Treating patients with leprosy is a long-​term enterprise involving patients, their families, and health workers. Vaccines against leprosy The substantial cross-​reactivity between Bacille Calmette–​Guérin (BCG) and M. leprae has been exploited in attempts to develop a vaccine against leprosy. Trials of BCG as a vaccine against leprosy in Uganda, New Guinea, Burma, and South India showed it to confer statistically significant but variable protection, ranging from 80% in Uganda to 20% in Burma and this protective effect has been con- firmed in a meta-​analysis. A case-​control study in Venezuela showed BCG vaccination to give 56% protection to the household contacts of patients with leprosy. Combining BCG and killed M. leprae has been tried, but in both a large population-​based trial in Malawi and an immunoprophylactic trial in Venezuela there was no advantage for BCG plus M. leprae over BCG alone. Areas of uncertainty and controversy The optimum duration of treatment is a controversial area. The dur- ation of treatment for multibacillary (MB) patients was reduced from 24 months to 12 months without good evidence. However, this occurred after the definition of MB patients was broadened and in India up to 60% of MB patients are smear-​negative borderline tu- berculoid patients. The concern relates to patients with a high initial bacterial load. Data from India show that patients with a high ini- tial bacterial load (bacterial index >4) treated with 2 years of rifam- picin, clofazimine, and dapsone had a relapse rate of 8/​100 person years, whereas patients treated to smear negativity had a relapse rate of 2/​100 person years. The dilemma is that since skin smears are abandoned in many programmes those patients in need of longer treatment courses cannot be identified. A new treatment, uniform multidrug treatment (U-​MDT), in which all leprosy patients are given 6-​months of rifampicin, dapsone, and clofazimine is being tried. The problem is that this regimen adds in clofazimine for many patients who do not need it and will probably be inadequate for the small number of lepromatous leprosy patients with high bacterial loads who also maintain the infection in the community. These

8.6.29 Buruli ulcer Mycobacterium ulcerans infecti

8.6.29 Buruli ulcer: Mycobacterium ulcerans infection 1167

1167 8.6.29  Buruli ulcer: Mycobacterium ulcerans infection arguments illustrate the difficulty in providing sound evidence for policy decisions when a decade-​long wait to establish relapse rates is needed. Areas where further research is needed The epidemiology of leprosy still poses unanswered questions. Why are 64% of all patients with leprosy in India? Is this due to living conditions, genetic susceptibility, or particular environmental con- ditions in India? Early detection of cases is vital at both an individual and a popu- lation level. It is now recognized that substantial nerve damage oc- curs before diagnosis. A test for early infection might help detect individual cases before nerve damage is established and before the spread of infection. Leprosy-​specific peptides for skin tests have been generated and are being evaluated. The medical management of reactions and nerve damage is cur- rently limited to steroids. These are not effective for about 30% of patients. Trials to determine the effectiveness of established and out-​of-​patent immunosuppressants, such as azathioprine and ciclosporin, are taking place. The WHO started the 1990s with the bold slogan of ‘Eliminating leprosy as a public health problem by 2000’. This initiative gal- vanized leprosy control programmes worldwide, but the unique biology of M. leprae and its interaction with the human host ren- dered this target unattainable. However, there is a strong perception that leprosy has been eliminated and this has hindered research and planning. The WHO policy for 2011–​2015 focuses on sustaining leprosy work. Leprosy is a bacterial disease with challenging im- munological complications and will be a global and individual problem for many decades. It is unlikely to be eradicated until there is considerable improvement in general health, wealth, living con- ditions, and education. FURTHER READING Britton WJ, Lockwood DN (2004). Leprosy. Lancet, 363, 1209–​19. Fine PE (2007). Leprosy: what is being ‘eliminated’? Bull World Health Organ, 85, 2. International Federation of Anti-​Leprosy Associations (ILEP) (n.d.). Working for a world without leprosy. http://​www.ilep.org.uk Lockwood DNJ, Lambert S (2010). HIV and leprosy? Where are we at? Lepr Rev, 81, 167–​75. Monot M, et al. (2005). On the origin of leprosy. Science, 308, 1040–​2. Rodrigues L, Lockwood DNJ (2011). Leprosy now: epidemiology, pro- gress, challenges, and research gaps. Lancet Infect Dis, 11, 464–​70. Scollard DM (2008). The biology of nerve injury in leprosy. Lepr Rev, 79, 242–​53. Setia MS, et al. (2006). The role of BCG in prevention of leprosy: a meta-​analysis. Lancet Infect Dis, 6, 162–​70. Ustianowski AP, et al. (2006). Interactions between HIV infection and leprosy: a paradox. Lancet Infect Dis, 6, 350–​60. Van Brakel WH, et al. (2005). The INFIR Cohort Study: assessment of sensory and motor neuropathy in leprosy at baseline. Lepr Rev, 76, 277–​95. World Health Organization (2006). Global strategy for further reducing the leprosy burden and sustaining leprosy control activities 2006–​2010. World Health Organization, Geneva. 8.6.29  Buruli ulcer: Mycobacterium ulcerans infection Bouke de Jong, Françoise Portaels, and
Wayne M. Meyers ESSENTIALS Buruli ulcer is caused by Mycobacterium ulcerans, which secretes a cytotoxic and immunosuppressive toxin, mycolactone. The disease is characterized by necrosis of skin, subcutaneous tissue, and bone, and is re-​emerging as a potentially disabling affliction of inhabitants of tropical wetlands. Major foci are in West and Central Africa, with an increasing focus in Australia, Mexico, South America, and Southeast Asia. It is not contagious; environmental sources include water, vege- tation, and insects, with humans probably becoming infected by traumatic introduction of the bacillus into the skin from the overlying M. ulcerans-​contaminated surface in most instances. Clinical presen- tation may be as a cutaneous nodule, undermined ulcer, plaque, or widely disseminated oedematous lesion. Clinical diagnosis is often accurate by experienced clinicians, and smears for acid-​fast bacilli, culture, polymerase chain reaction assays, and histopathology are confirmatory. Treatment was formerly by wide surgical excision and skin grafting, yet antibiotics (rifampicin with streptomycin given for 8 weeks) have now been found effective, including an all-oral regimen. Introduction Buruli ulcer is an indolent necrotizing infection of the skin, sub- cutaneous tissue, and bone caused by Mycobacterium ulcerans. In 1962 Clancey and Dodge described many patients from Buruli County, Uganda, with cutaneous ulcers reminiscent of those Cook described in 1897 from the same area, and named the disease Buruli ulcer. Since the World Health Organization (WHO) Buruli ulcer initiative there has been increased attention to efforts for the treatment and control of Buruli ulcer. Aetiology MacCallum and colleagues first isolated the causative agent in 1948 from patients in Australia. M. ulcerans, a slow-​growing acid-​ fast bacillus, grows optimally at 32° C and produces mycolactone, a cytotoxic and immunosuppressive polyketide assembled by plasmid-​encoded synthases of the aetiological agent. This toxin is the primary virulence factor of M. ulcerans. Data from whole genome sequencing define three major lineages of M.  ulcer- ans that infect humans. Lineage 1 contains the South American strains, lineage 2 the Asian strains and lineage 3 the African and Australian subgroups. ​Single nucleotide polymorphism analysis through whole genome sequencing provides insights into the population structure and evolution of M. ulcerans across Africa. Portaels et al. were the first to culture M. ulcerans from the envir- onment, in 2008.

section 8  Infectious diseases 1168 Epidemiology and transmission Buruli ulcer is the third most common human mycobacterial in- fection worldwide, after tuberculosis (Chapter 8.6.26) and leprosy (Chapter 8.6.28). It occurs in humid, rural tropical, subtropical re- gions and temperate regions, and most endemic foci of Buruli ulcer are near rural freshwater wetlands, especially ponds and swamps. Presently, major endemic areas are Benin, Cameroon, Democratic Republic of Congo, Gabon, Ghana, Ivory Coast, and adjacent coun- tries. Increasing case clusters have also been reported in Australia (Far North Queensland and Victoria, where it is known as Daintree or Bairnsdale ulcer) and sporadic cases in Mexico, South America, Malaysia, Japan, China, and Papua New Guinea. Documented environmental sources of M. ulcerans DNA include irrigation systems, water bugs living among aquatic plant roots in swamps, terrestrial vegetation, and mosquitoes. In Australia, koalas, possums, potoroos, rats, some domestic animals (dogs, horses, and one cat) and imported alpacas acquire the infection naturally. The mode of transmission is not fully understood, although dis- ease is known to be linked to contaminated water. Outbreaks of dis- ease often follow environmental changes that promote flooding or alter water courses, such as deforestation or construction of dams and irrigation systems. Increased farming activities near wet- lands and global climatic changes may contribute to the rapid re-​ emergence of Buruli ulcer. In West Africa the peak age of onset is 5 to 15 years, although the disease can affect any age group. In Australia the median age at presentation is 55–​65 years. Transmission is probably via skin trauma, although insects may play a role. The trauma may be slight (e.g. hypodermic injection) or severe (e.g. land mine wound or snake bite). Biting insects (e.g. mosquitoes, water bugs) may serve as vectors. In Australia, risk for Buruli ulcer in humans is associated with the frequency of de- tection of M. ulcerans in the local possum faeces, as well as mos- quitoes, suggesting a possible role in transmission. Possums tend to be symptomatic with M. ulcerans ulcerations and are the likely reservoir in South Eastern Australia, with transmission possibly caused by mosquitoes that developed in water sources contamin- ated with possum faeces, or rested on M. ulcerans-​contaminated vegetation. Possums may be useful sentinels to predict spread of Buruli ulcer in humans in South Eastern Australia, though no such link has been established in Far North Queensland. Extensive sur- veillance of rodents in Africa has failed to identify a similar res- ervoir to date. Mosquitoes do not seem to play a major role in ecology of M. ulcerans in Africa. Aerosols arising from ponds and swamp surfaces may disseminate M. ulcerans. Rare instances of person-​to-​person transmission of Buruli ulcer are anecdotal, and to our knowledge, none of these events has been established as M. ulcerans infection. Pathogenesis Predisposing host factors are poorly understood. Putatively, severity and course of infection are related to pathogen virulence, mode of infection, inoculum size, host genetic factors, and immunological response of the host. A T-​helper 1 cell (Th1) response tends to lo- calize and heal infections while a T-​helper 2 cell (Th2) type response is associated with dissemination. Once introduced, the small amount of mycolactone produced by inoculated M. ulcerans causes tissue necrosis and apoptosis, suppressing local immune responses, and ensuring survival of the bacillus in necrotic tissue. Mycolactone tar- gets subcutaneous fat cells, permitting necrosis to spread just super- ficial to fascial planes. M. ulcerans may invade lymphatic and blood vessels, causing metastatic spread of the mycobacterium despite its preference for cooler temperatures (32°C). Clinical features Except for those with massive lesions, patients are usually surprisingly well without systemic symptoms or abnormal laboratory findings. Meyers et al. have published a model for the natural history and inter- relationships of the forms of Buruli ulcer disease. Buruli ulcer may be localized or disseminated, and can be classified in three categories based on the extent of the ulceration, per WHO guidelines. Localized disease Typically, the initial cutaneous lesion is a single, firm, painless, non​tender, movable, subcutaneous nodule up to 3 cm in diameter. Limbs are preferred sites, often around joints. The natural history of the disease is markedly variable, but nodules usually ulcerate within l to 3 months of inoculation. A whitish necrotic slough de- velops in the ulcer base with induration and hyperpigmentation of surrounding skin. Ulcer borders are undermined, sometimes extending widely (major ulcerative disease) (Fig. 8.6.29.1). Some small (1–​2 cm in diameter) ulcerated lesions with shallow under- mining self-​heal (minor ulcerative disease). Without treatment, major ulcerative lesions tend to become inactive, after months or years, and heal by scarring. Scars are depressed and stellate, often causing disfiguring and crippling cicatricial contractures. Fig. 8.6.29.1  Pristine Buruli ulcer on the left deltoid area in a 12-​year-​ old Congolese boy who had received a hypodermic injection at this site 3 months previously. Note central necrotic slough in the base of the ulcer and undermined edges.

1169 Disseminated disease Disseminated disease may develop from nodules, arise from local- ized major ulcerative lesions, or disseminate directly and rapidly from the site of inoculation, causing indurated plaques covering even an entire limb or vast areas of the trunk. Without treatment, such lesions will eventually slough, leaving a large ulcer with con- tinuing extension of disease at the borders. Eyes, breasts, and geni- talia may be damaged or destroyed. While metastatic spread may arise from localized disease, patients with the high bacterial loads and disseminated disease are most prone to metastatic lesions. Spread may be to distant skin sites or bone, especially bones of the limbs. In Africa, M. ulcerans osteomye- litis develops in approximately 10% of patients and often leads to amputations or other disabilities. Differential clinical diagnosis Clinical diagnosis is sometimes perplexing. Differential diagnoses include bacterial, mycotic, and parasitic infections, inflammatory lesions, and tumours. Ulcers resembling Buruli ulcer include trop- ical phagedenic ulcer (malodorous and not undermined), venous stasis ulcer (not undermined), and venomous snake bite or spider bite (history helpful). Pathology Optimal biopsy specimens contain the necrotic base of ulcers and undermined edge of lesions including subcutaneous tissue and fa- scia. Histopathological sections reveal a contiguous coagulation necrosis (non​caseating) of the deep dermis, panniculus, and fa- scia. Vasculitis and mineralization are common. Clumps of extra- cellular acid-​fast bacilli are most plentiful in the base of the ulcer; however, intracellular M. ulcerans may be seen in inflammatory cells at the edge of necrotic foci. Necrosis extends well beyond the loca- tion of bacilli. Local and regional lymph nodes are often invaded and sometimes necrotic. In bone, the marrow is necrotic and con- tains acid-​fast bacilli, and trabeculae are eroded. These features are distinct from those of osteomyelitis of all other known aetiologies. Development of delayed-​type hypersensitivity granulomas heralds healing by fibrosis. Laboratory diagnosis Fine needle aspirates are often employed for laboratory studies on closed lesions. Smears stained by the Ziehl-​Neelsen method from the ulcer base reveal acid-​fast bacilli in clumps in around 30–​60% of polymerase chain reaction confirmed Buruli ulcer le- sions. M. ulcerans is a slow-​growing organism that can be cultured in vitro at 29–​33°C, albeit with low rate of cultivability (20–​60%). Polymerase chain reaction provides specific identification of M. ulcerans, with IS2404 as the most sensitive target given the pres- ence of over 200 copies per genome. Histopathological changes are characteristic. Treatment The former recommended treatment for most patients was wide surgical excision followed by skin grafting. Heating the lesion at 40°C can be a useful adjunct. Today, antimicrobial therapy (ri- fampicin 10 mg/​kg by mouth plus streptomycin 15 mg/​kg by intra- muscular injection, or rifampicin plus clarithromycin) without surgery is recommended and heals most nodular and minor ul- cerative disease, and some advanced lesions. Controlled trials have established efficacy. Physiotherapy is essential to prevent contrac- ture deformities. Prevention and control Bacille Calmette-​Guérin (BCG) vaccination provides short-​lived protection. Practical control measures for inhabitants of endemic areas are usually ineffective; however, use of a protected water supply is important. Tourists should avoid wetlands in endemic countries. Socioeconomic impact Patients are often stigmatized by disability or cosmetic damage, and may require welfare services for life, but such services are often locally limited or unavailable. They also often require protracted hospital stays, taxing overburdened services. Given certain similarities between Buruli ulcer and leprosy, combined control and prevention strategies could be put in place in countries endemic for both diseases. FURTHER READING Alffenaar JW, et  al. (2010). Pharmacokinetics of rifampin and clarithromycin in patients treated for Mycobacterium ulcerans in- fection. Antimicrob Agents Chemother, 54, 3878–​83. Buultjens AH, et  al. (2018). Comparative genomics shows that Mycobacterium ulcerans migration and expansion preceded the rise of Buruli Ulcer in Southeastern Australia. Appl Environ Microbiol, 84, e02612–17. Carson C, et  al. (2014). Potential wildlife sentinels for monitoring the endemic spread of human Buruli ulcer in South-​East Australia. PLoS Negl Trop Dis, 8, e2668. Converse PJ, et al. (2011). Treating Mycobacterium ulcerans disease (Buruli ulcer): from surgery to antibiotics, is the pill mightier than the knife? Future Microbiol, 6, 1185–​98. Doig KD, et al. (2012). On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer. BMC Genomics, 13, 258. Fyfe JA, et al. (2010). A major role for mammals in the ecology of Mycobacterium ulcerans. PLoS Negl Trop Dis, 4, e791. Kiszewski AE, et al. (2006). The local immune response in ulcerative lesions of Buruli disease. Clin Exp Immunol, 143, 445–​51. Lavender CJ, et  al. (2011). Risk of Buruli ulcer and detection of Mycobacterium ulcerans in mosquitoes in southeastern Australia. PLoS Negl Trop Dis, 5, e1305. 8.6.29  Buruli ulcer: Mycobacterium ulcerans infection

8.6.3 Pneumococcal infections 975

8.6.3 Pneumococcal infections 975

8.6.3  Pneumococcal infections 975 Murray BE (1990). The life and times of the Enterococcus. Clin Microbiol Rev, 3, 46–​65. Royal College of Obstetricians and Gynaecologists (2003). Green Top Guideline 36. Prevention of early onset neonatal group B streptococcal diseases. http://​www.rcog.org.uk/​files/​rcog-​ corp/​uploaded-​files/​GT36GroupBStrep2003.pdf Stevens DL (1992). Invasive group A streptococcus infections. Clin Infect Dis, 14, 2–​13. Stevens DL (1995). Streptococcal toxic shock syndrome: spectrum of disease, pathogenesis and new concepts of treatment. Emerg Infect Dis, 1, 69–​78. Stevens DL (2004). Streptococcal infections. In: Goldman L, Ausiello D (eds) Cecil textbook of medicine, 22nd edition, pp. 1782–​7. Saunders, Philadelphia, PA. Stevens DL, et  al. (2000). Molecular epidemiology of nga and NAD glucohydrolase/​ADP-​ribosyltransferase activity among Streptococcus pyogenes causing streptococcal toxic shock syndrome. J Infect Dis, 182, 1117–​28. Stevens DL, et al. (2005). Practice guidelines for the diagnosis and management of skin and soft-​tissue infections. Clin Infect Dis, 41, 1373–​406. Stevens DL, Bryant AE (2017). Necrotising soft tissue infections.
N Engl J Med, 377, 2253–65. Verani JR, McGee L, Schrag SJ, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC) (2010). Prevention of peri- natal group B streptococcal disease—​revised guidelines from CDC. MMWR Recomm Rep, 59(RR-​10), 1–​36. Woodford N (1998). Glycopeptide-​resistant enterococci: a decade of experience. J Med Microbiol, 47, 849–​62. 8.6.3  Pneumococcal infections Anthony Scott ESSENTIALS Streptococcus pneumoniae is an encapsulated Gram-​positive bac- terium that lives almost exclusively in the human nasopharynx. Each pneumococcus expresses one of more than 90 immunologically dis- tinguishable capsular polysaccharides that are the principal target of systemic human immunity and define its serotype. Epidemiology Pneumococci are transmitted through contact with infected nasal se- cretions or by airborne dissemination, and most preschool children carry them in their nasopharynx. The risk of acquisition is increased by contact with other children, crowded environments, and cold weather. The incidence of pneumococcal disease is highest in young children and elderly people, and also increased in males, certain in- digenous populations, smokers, alcoholics, and patients with chronic medical illnesses or immune susceptibility, including HIV infection, sickle cell disease, and splenectomy. Clinical features Pneumonia—​before introduction of Pneumococcal conjugate vac- cines, pneumococci were the most common cause of severe community-​acquired pneumonia at all ages in the developed and developing world, though their role is now diminishing. Typical pres- entation of pneumococcal lobar pneumonia is with abrupt onset of fever, followed by cough, difficulty breathing, pleuritic chest pain, haemoptysis, and purulent sputum. Physical signs include high pyr- exia, raised respiratory rate, cyanosis, and chest features of lobar con- solidation; namely reduced chest movement, dullness on percussion, fine crepitations, and bronchial breathing over the affected area. The chest radiograph shows a lobar opacity, often with a pleural effusion. Other diseases—​pneumococci cause significant morbidity in adults and children through meningitis and septicaemia, and they can also cause bronchopneumonia and multiple disease syndromes sim- ultaneously (e.g. meningitis and pneumonia). In children, the most common pneumococcal disease is otitis media. Other less common presentations include sinusitis, pleural empyema, pericarditis, endo- carditis, septic arthritis, osteomyelitis, peritonitis, and conjunctivitis. Diagnosis S. pneumoniae is a fastidious organism that grows successfully on blood agar, producing α-​haemolysis. Blood culture is the principal aetiological tool to diagnose pneumococcal pneumonia, but cul- tures are positive in only 15–​30% of cases. The capsular serotype is identified by a positive Quellung reaction with specific rabbit anti- sera. In addition: (1) pneumococci can be observed on microscopy as Gram-​positive diplococci in sputum or, in cases of meningitis, in cerebrospinal fluid, and can be cultured from both specimens; (2) a urinary antigen test for the common pneumococcal constituent C-​polysaccharide is sensitive and specific for pneumococcal pneu- monia in adults, but not in children; (3) polymerase chain reaction is useful in cerebrospinal fluid, especially when the patient is partially treated and cultures are sterile. Treatment and prognosis Most pneumococci are sensitive to β-​lactam antibiotics, but some are resistant. (1) Pneumonia—​when caused by sensitive or inter- mediately resistant pneumococci, this should be treated with high-​dose oral amoxicillin or intravenous cefotaxime, the latter being effective against pneumococci with cephalosporin min- imum inhibitory concentrations up to 1–​2 µg/​ml. Macrolides and newer fluoroquinolones can be used to treat infections that are fully resistant to β-​lactam antibiotics. (2) Meningitis—​when caused by susceptible pneumococci, ceftriaxone is effective; vancomycin should be added as empirical meningitis therapy in areas with penicillin-​resistant pneumococci; dexamethasone is an effective adjunctive treatment for pneumococcal meningitis where HIV prevalence is low. The case fatality of pneumococcal pneumonia is 5%, but in bacter- aemic pneumonia and pneumococcal meningitis it is 30%. Prevention A single dose of 23-​valent capsular polysaccharide vaccine prevents invasive pneumococcal disease in elderly or high-​risk populations. In infants and young children, 10-​ or 13-​valent pneumococcal conju- gate vaccine is highly effective in preventing invasive pneumococcal disease as well as pneumococcal pneumonia, meningitis, and otitis media. It is given routinely as two or three doses in infancy, with a booster dose at 12–​15 months of age. Immunization of children re- duces pneumococcal transmission and prevents pneumococcal dis- ease in older family members.

section 8  Infectious diseases 976 Introduction Streptococcus pneumoniae (the pneumococcus) is a ubiquitous yet potentially fatal human pathogen. Its only viable habitat is the human nasopharynx. Throughout the world, most children and a significant minority of adults carry it at any one time. Most people are exposed to the pneumococcus several times a year but only rarely does this result in illness. When it invades it causes a diverse range of dis- ease syndromes of which pneumonia, meningitis, and septicaemia have high case fatalities, and yet its most common disease mani- festation, otitis media, is relatively benign. In old age it affects the healthy, but throughout life it is a burden to those with chronic med- ical illnesses. Pneumococcal disease is common in temperate and tropical climates; however, because the pneumococcus is fastidious in culture, it is rarely identified, and the disease burden is frequently underestimated. Its differentiation into more than 90 serotypes indi- cates the complexity of its immunological interaction with humans and its need for adaptability in this long-​enduring host–​pathogen relationship. Microbiologists and physicians have regarded the pneumococcus as a formidable opponent for over 135 years. They have fought it with antibiotics and, more recently, with efficacious vaccines, each of which renders its nasopharyngeal niche a hostile home. Yet, through its capacity to combine DNA from other bacteria into its own chromosome, it has evolved and survived. It has all the fascination of the esoteric, yet a busy doctor will not pass a week in practice without seeing a case. Historical perspective S. pneumoniae is a Gram-​positive bacterium first isolated in 1881 by Sternberg in the United States of America and, simultaneously, by Pasteur in France through experiments inoculating rabbits with human saliva. Serotypes of pneumococcus are defined by the rabbit immune response to its variable capsular polysaccharide. In 1910, Neufeld and Händel described two serotypes; there are now over 90 serotypes. Convalescent sera from surviving pneumonia patients were shown to be protective against pneumococcal disease in rabbit models in 1891. The protective substance was identified as homolo- gous anticapsular antibody and this underpinned the development of serum therapy in the early years of the 20th century. Although successful, serum therapy required determination of the serotype of the infecting pneumococcus from sputum or lung aspirate cultures, leading to a delay in treatment. With the introduction of sulphona- mide antibiotics in 1938, serum therapy was abandoned. Antibiotic chemotherapy has been the mainstay of management of pneumococcal disease ever since, but the rapid evolution of re- sistant strains in the 1990s reactivated interest in vaccine develop- ment. Efficacy of a polyvalent capsular polysaccharide vaccine was demonstrated against pneumococcal pneumonia in South African miners in 1976. It was poorly immunogenic in infants, among whom most episodes of pneumococcal disease occur, but conjugation of the polysaccharide to immunogenic proteins overcame this limi- tation and a pneumococcal conjugate vaccine (PCV) against seven serotypes was introduced into the childhood immunization pro- gramme in the United States of America in 2000. In 1928, Griffith inoculated rabbits with a suspension of live avirulent unencapsulated pneumococci and heat-​killed serotype 3 pneumococci. The rabbits subsequently succumbed to serotype 3 septicaemia. The avirulent isolate was derived from a serotype 2 strain suggesting that it acquired the type 3 capsule, and virulence, from the heat-​killed organisms. In 1944, Avery isolated and puri- fied the ‘active principle’ that brought about this transformation and characterized it chemically as DNA. The sequence of the pneumo- coccal genome was first described in 2000 and thousands of strains have now been fully annotated. These sequences have been used to identify conserved surface-​expressed proteins that may serve as antigens in non​capsular vaccines. Epidemiology Incidence The risk of pneumococcal disease is highest in infancy and declines throughout the first 5 years of life. The lowest risk is in older children and young adults and from the age of 50 years onwards disease risk rises progressively (Fig. 8.6.3.1). Among children younger than 5 years, the incidence of culture-​ proven pneumococcal disease was 70–​100 per 100 000 population in the United States of America before vaccine introduction; in Africa it was 110–​430 per 100 000 population. In developed countries, the in- cidence was 15–​20 per 100 000 population among adults of all ages and at least 50 per 100 000 population among adults aged 65 or over. In the prevaccine era, Native Australians and Alaskans and White Mountain Apaches had incidence rates of 200–​1000 per 100 000 popu- lation among children, 50–​180 per 100 000 population among adults 18–​59 years old, and 120–​170 per 100 000 population among older adults. The epidemiology of pneumococcal disease is markedly different in the meningitis belt of West Africa. Here, infants and working-​ age adults are at highest risk, the disease is strongly associated with the dry season, and the case fatality rate is greater than 60% among adults aged 40 years or more. Serotype 1 accounts for more than half of all infections and the burden of meningitis caused by pneumo- coccus, with an incidence of 8–​12 per 100 000 population, rivals that caused by Neisseria menigitidis. The total burden of pneumococcal disease is frequently under- estimated because it is difficult to detect. Studies of ‘invasive pneumococcal disease’, which rely on cultures of S.  pneumoniae from specimens of blood, cerebrospinal fluid, and pleural fluid, fail to identify most of the cases of pneumococcal pneumonia that are not bacteraemic. Lung aspirates obtained by percutaneous fine needle puncture significantly increase the yield of pneumococci from pneumonia cases at all ages but are now rarely undertaken. A World Health Organization (WHO) model of the incidence of pneumo- coccal pneumonia, meningitis, and other serious manifestations esti- mated the annual global burden of disease in children aged less than 5 years as 14.5 million cases in 2000 leading to 826 000 deaths; the es- timate for 2015 was 317 300 deaths. Most of these deaths take place in Africa and Asia. The global burden of disease in adults is not known. Carriage, transmission, and serotypes Viable S.  pneumoniae have been described in collections of dust and in epizootics of some mammals, but the principal habitat and

8.6.3  Pneumococcal infections 977 critical ecological niche of the pneumococcus is the human naso- pharynx (Fig. 8.6.3.2). Infants can acquire infection within hours of birth and most infants in developing countries become infected in the first 3 months of life. In The Gambia, more than 90% of children aged less than 5 years old are colonized by the pneumococcus at any one time. In the United Kingdom, carriage prevalence among chil- dren is approximately 50%. Adults also carry S. pneumoniae in the nasopharynx but at lower prevalence. Although the pneumococcus has evolved more than 90 capsular serotypes, prior to vaccine introduction 58 to 66% of disease in chil- dren was caused by just seven (serotypes 1, 5, 6A, 6B, 14, 19F, and 23F), depending on region. The ratio of the incidence of invasive disease to the incidence of nasopharyngeal acquisition provides an index of the invasiveness of pneumococcal serotypes and can be used to group them. In the prevaccine era, serotypes 1, 5, 12F, and 46 were found among series of invasive isolates but were rarely isolated in the nasopharynx. These are labelled ‘adult’ types because they are associ- ated with disease in adults. Other serotypes, including 10A, 11A, 15B, 15C, 16F, and 33F, were found among series of colonizing isolates but were uncommon causes of disease. A third group, which includes serotypes 6A, 6B, 14, 19A, 19F, and 23F, were found very commonly in the nasopharynx but also caused invasive disease. These are la- belled ‘paediatric’ types because they caused most of the invasive disease episodes among young children before vaccine introduction. Serotypes that are highly prevalent in the nasopharynx tend to be less invasive than others but when they do cause disease the case fatality 300 White Black Other Race 400 200 100 0 Age group, y Incidence, cases per 100 000 <2 2–4 5–17 18–34 35–49 50–64 65–79 ≥80 Fig. 8.6.3.1  Incidence of invasive pneumococcal disease in the United States of America between 1995 and 1998 by age and race. The data are taken from the Active Bacterial Core Surveillance of the Centers for Disease Control and Prevention. From Robinson KA, et al. (2001). Epidemiology of invasive Streptococcus pneumoniae infections in the United States, 1995–​1998: opportunities for prevention in the conjugate vaccine era. JAMA, 285, 1729–​35. Fig. 8.6.3.2  Electron micrographs of pneumococci (strains TIGR4 and G54) adherent to D562 human pharyngeal epithelial cells in culture. From Kimaro Mlacha SZC, et al. (2013). Phenotypic, genomic, and transcriptional characterization of Streptococcus pneumoniae interacting with human pharyngeal cells. BMC Genomics, 14, 383.

section 8  Infectious diseases 978 ratios are higher. Success in colonization is a function of evading the host defences (neutrophil-​mediated killing) and outcompeting other serotypes for the ecological niche of the nasopharynx. The duration of carriage can be as short as 3 h or as long as 3 years; in most instances it is between 1 and 6 months. It declines with the age of the host, probably as a result of CD4+ T-​cell acquired im- munity mediated by IL-​17A. For some serotypes, circulating anticapsular IgG appears to reduce the acquisition of carriage and vaccine-​induced anticapsular antibodies are also highly effective in reducing carriage prevalence. The pneumococcus is transmitted by carriers, particularly pre- school children, through direct contact with nasal secretions or by infected fomites. The rapid spread of pneumococcal pneumonia in outbreaks in adults suggests that airborne dissemination, facilitated by cough, is another mechanism of spread. Risk factors Risk of pneumococcal disease is a function of exposure to the bac- terium, leading to colonization, and of host resistance to invasion. Exposure is increased in crowded environments at home and in in- stitutions (e.g. military barracks, homeless shelters, jails, miners’ compounds) and by contact with preschool children. In temperate climates pneumococcal disease follows a consistent seasonal variation with winter peaks and summer troughs. Risk is especially high at New Year when families gather and generations intermingle (Fig. 8.6.3.3). Throughout life, males have an incidence of pneumococcal disease 1.2 to 1.5 times greater than females. Chronic medical conditions predispose to pneumococcal invasion (see Box 8.6.3.1). Alcoholism is consistently associated with pneumococcal disease and may act directly on macrophage function or, like seizure disorders, by com- promising laryngeal defences leading to aspiration. Untreated HIV infection increases the risk of invasive pneumococcal disease by ap- proximately 50-​fold. Where HIV prevalence is greater than 2%, as in much of Africa, most cases of pneumococcal disease occur among HIV-​positive patients. Recurrent pneumococcal disease is especially common in this group. Respiratory viral infections, especially influenza, increase the risk of invasive pneumococcal disease. Influenza enhances the acquisition of pneumococci in the nasopharynx, in animal models, and increases the density of colonization in children. The virus facilitates pneumo- coccal binding by damaging respiratory epithelial cells and synergy between the two pathogens enhances the action of their separate neuraminidases and stimulates type 1 interferons, impairing macro- phage function. Antibiotic resistance Epidemiology Laboratory isolates of penicillin-​resistant pneumococci were first reported in 1967 and by the early 1970s they were being isolated in clinical specimens in Australia and Papua New Guinea. During the 1990s, penicillin resistance spread widely throughout the world reaching a prevalence of 35% in several countries (e.g. France, South Africa, Japan, Hong Kong). In Europe pneumococci are classified as sensitive (minimum inhibitory concentration (MIC) of 0.06 µg/​ml or below) or resistant to benzylpenicillin (MIC 2 µg/​ml and above). For pneumonia treatment, the classification between these cut-​offs depends on the dose given; for example, when treating with high doses of benzylpenicillin (2.4 g every 4 hours), MICs up to 2 µg/​ml are considered susceptible. Because of the difficulty in achieving high concentrations of penicillin in cerebrospinal fluid, 90 80 70 60 50 40 30 20 10 0 Rate (cases/100 000 population/year) 1998 1997 1996 Jan Jan Apr Jul Oct Jan Apr Jul Oct Jan Apr Jul Oct Fig. 8.6.3.3  Annualized weekly incidence of pneumococcal disease among adults in the United States of America between 1996 and 1998 showing a consistent increase in incidence in the winter and a sharp increase in incidence during the Christmas/​New Year holiday season. From Dowell SF, et al. (2003). Seasonal patterns of invasive pneumococcal disease. Emerg Infect Dis, 9, 573–​9.

8.6.3  Pneumococcal infections 979 pneumococci isolated in a case of meningitis are classified as fully resistant if the MIC is greater than 0.06 µg/​ml. In the United States of America, the classification of isolates from non​meningitis inva- sive infections (sensitive ≤2 µg/​ml; intermediate = 4 µg/​ml; resistant ≥8 g/​ml) identifies a higher proportion of isolates as sensitive and therefore treatable with benzylpenicillin. This reflects the higher doses of penicillin used in the United States of America as well as results from epidemiological surveillance. For meningitis, the sen- sitive/​resistant cut-​off is the same as in Europe. Resistance to other antibiotic classes, including macrolides, also increased during the 1990s. The use of long-​acting macrolides appears to be responsible for the observed increase in resistance to erythromycin. Multidrug-​resistant pneumococci were first observed in South Africa in 1978. Their presence suggests that the use of one antibiotic can select for resistance against another. Penicillin-​resistant strains are transmitted more successfully than penicillin-​susceptible strains but multiresistant strains spread most successfully within popu- lations. Much of this spread is driven by the expansion of a small number of clones. In the United States of America, 78% of all re- sistant isolates are represented by just 12 clonal groups. In the United Kingdom, antimicrobial prescriptions have fallen since 1995 and at the same time there has been a reduction in both penicillin-​resistant and macrolide-​resistant pneumococci. Introduction of 7-​valent PCV in the United States of America in 2000 resulted in a marked decrease in the incidence of disease with penicillin-​resistant strains in children, followed by a gradual rise be- tween 2005 to 2010; introduction of PCV13 then led to a further decline in the incidence of resistant invasive infections in children. Pneumococcal resistance to fluoroquinolones has increased in prevalence but remains less than 5%. Resistance mechanisms Pneumococcal resistance to penicillin is entirely due to the accumu- lation of genetic variations among the six penicillin-​binding proteins (PBPs) that normally catalyse cross-​linkage of the bacterial cell wall. By binding to PBPs, β-​lactam antibiotics inhibit cell wall synthesis and promote cell lysis. Resistance to penicillin occurs when a PBP variant arises which has low binding affinity for β-​lactams. Sensitive pneumococci have MICs for benzylpenicillin and cefotaxime that are approximately 0.02 µg/​ml. Mutations in PBP 2b or 2x genes lead to a 2-​ to 30-​fold increase in MIC. However, isolates that are fully resistant to penicillin usually contain alterations at three PBP genes, 2b, 2x, and 1a. High-​level resistance to cefotaxime may be observed with a combination of changes in only two (PBP 2x and 1a). Resistant strains come about through horizontal transfer and re- combination into chromosomal DNA of large sequence blocks of mosaic genes acquired from other streptococci. This transfer can in- clude capsular and resistance genes simultaneously. Pneumococci colonizing the nasopharynx are then exposed to antibiotics, which are commonly prescribed for community-​acquired respiratory tract infections, and this selects resistant strains. Pneumococcal genes cat, erm(B), and tet(M), which confer re- sistance to chloramphenicol, macrolides, and tetracyclines, respect- ively, have been found together on DNA elements (conjugative transposons) that spread between pneumococci without involving recombination, thus facilitating multidrug resistance. Resistance to fluoroquinolones and trimethoprim/​sulfamethoxazole is acquired by point mutations in topoisomerase genes and folate synthesis genes, respectively. Exposure to low levels of antibiotic selects single gene mutants, which then acquire higher resistance through add- itional mutations. Pathogenesis Pneumococci exist in two morphologically distinct phenotypes; in the opaque phase they have abundant capsular expression and in the transparent phase they have little (Fig. 8.6.3.4). In the nasopharynx, transparent-​phase pneumococci predominate as abundant capsule prevents attachment of pneumococcal cell Box 8.6.3.1  Risk factors for pneumococcal disease Social and demographic • Older age • Male sex • Black race • Indigenous populations • Lower level of education • Unemployment • Excess alcohol use • Occupational exposure to metal fumes (Welding) Exposure to pneumococci • Contact with preschool children • Day care attendancea • Crowding in the home • Crowded adult environments (homeless shelters, military, or occupa- tional barracks) • Institutionalized care • Winter season • Hospital admission Respiratory tract damage • Currently smoking • Passive smoking • Indoor air pollution • Chronic obstructive pulmonary disease • Recent viral respiratory tract infection Preexisting medical conditions • Chronic renal failure • Congestive heart failure • Cirrhosis • Cerebrovascular disease • Dementia • Seizure disorder • Asthma • Diabetes • Malignancies of the lung Immune susceptibility • HIV • Hypogammaglobulinaemia • Sickle cell disease • Asplenia/​splenectomy • Pregnancy • Not breastfeedinga • Previous pneumococcal disease a These apply only to infants or children.

section 8  Infectious diseases 980 wall structures to epithelial cells. This attachment is mediated by binding of pneumococcal phosphorylcholine and choline-​binding protein A (CbpA) to human platelet activating factor receptors and polymeric immunoglobulin receptors. Once attached, pneumo- cocci can cause disease by local spread to the middle ear or sinuses, by aerosol inhalation to the lung, or by blood stream invasion to the meninges, joint spaces, or heart valves. Blood stream invasion begins with endocytosis across the mucosal barrier although the components of this pathway are not well understood. In the blood stream pneumococci are found in the opaque phase since capsule is effective in evading opsonophagocytosis. Pneumococci colonizing the nasopharynx cannot bind to the ciliated epithelium of the bronchi and therefore make their way to the lung in aerosols. However, if the ciliated epithelium is damaged, by antecedent viral infection or by cigarette smoke, it reveals a basement membrane to which pneumococci can ad- here easily. Pneumolysin released from pneumococci causes further epithelial damage by direct cytotoxicity and encour- ages inflammation of the larger bronchioles, which leads to bronchopneumonia. In the alveoli, pneumococci multiply in serous fluid and spread from one alveolus to another through the pores of Kohn. They ad- here to the alveolar type 2 cells, through expression of CbpA, and stimulate production of the inflammatory mediators tumour ne- crosis factor-​α, nitric oxide, and interleukins IL-​1, IL-​6, and IL-​10, which initiates oedema. This creates the first pathological phase of pulmonary consolidation—​engorgement. In the second phase—​red hepatization—​erythrocytes leak into the alveolar spaces, reducing the compliance of the lung and leading to a liver-​like appearance of the gross lung specimen (Fig. 8.6.3.5). Fibrin deposition creates a mesh of erythrocytes, leucocytes, and damaged epithelial cells and the lymphatics become dilated with cells and fibrin. Without ventilation, perfusion declines, and the lung becomes maximally consolidated. CbpA binding stimulates epithelial cells to release chemokines that attract leucocytes to the lung which initiates the third phase of consolidation—​grey hepatization. Neutrophils trap pneumo- cocci against the alveolar wall and engulf them by surface phago- cytosis. C-​reactive protein enhances this process by binding to choline residues on pneumococcal surfaces. The chemokines activate complement that, together with anticapsular antibody, facilitates opsonophagocytosis. Thereafter, lung inflammation be- gins to decline simultaneously with neutrophil apoptosis, fever declines, and macrophages are recruited to the lung to absorb the debris. Over a period of weeks this leads to complete resolution of the pathology. Fig. 8.6.3.4  Immunoelectron microscopy of pneumococcal capsules showing an increased zone of capsular material in opaque (a) and (b) compared to transparent (c) and (d) variants of type 6B pneumococcal strain P324. Reproduced from Kim JO, et al. (1999). Relationship between cell surface carbohydrates and intrastrain variation on opsonophagocytosis of Streptococcus pneumoniae. Infect Immun, 67, 2327–​33.

8.6.3  Pneumococcal infections 981 Immunity to pneumococcal disease Historically, the role of anticapsular antibody in recovery from pneu- monia and the efficacy of serotype-​specific serum therapy suggested that anticapsular IgG was the primary mechanism of immunity to pneumococcal disease. The age groups at highest risk of pneumo- coccal disease, infants, and elderly people, have little anticapsular antibody or have antibody that lacks avidity. The genetic diver- sity of capsular expression into more than 90 variants suggests the antigen is under considerable immune selection and the success of polysaccharide antigens as vaccines further reinforces the import- ance of anticapsular immunity. Capsular polysaccharides are complex molecules with repeating epitopes that create cross-​linkage of antigen receptors on B lympho- cytes and which stimulate antibodies of the IgM and IgG2 isotypes. Antibody production can occur in the absence of T lymphocytes (T-​ independent) but it does not induce memory responses and can lead to antigen tolerance following repeated stimulation. Pneumococcal capsular and cell wall components both activate the human com- plement system leading to deposition of C3b and C3d on capsular polysaccharide. In the presence of both anticapsular antibody and complement, encapsulated pneumococci are opsonized and taken up by phagocytes expressing the receptor Fcγ-​RIIa (CD32). In contrast to natural immunity, conjugates of highly immuno- genic proteins (e.g. diphtheria toxoid) with polysaccharides induce T-​cell-​dependent immunity with a predominance of IgG1 antibody and a memory response. These responses are inducible even in very young infants. Anticapsular antibody responses are measured by IgG enzyme-​linked immunosorbent assay (ELISA) and a serum concentration of 0.35 µg/​ml correlates with vaccine-​induced protec- tion in infancy. In addition to adaptive immunity, innate mechanisms (e.g. lipoteichoic acid stimulation of Toll-​like receptor (TLR) 2, or pneumolysin stimulation of TLR4) in response to pneumococcal virulence factors (Fig. 8.6.3.6) appear to be important in shaping the inflammatory response to pneumococcal disease and in determining host survival. Furthermore, several lines of evidence suggest that (a) (b) Fig. 8.6.3.5  (a and b) Red hepatization in fatal pneumococcal pneumonia. Copyright D. A. Warrell. ATP-binding cassette transporter Pneumolysin PsaA PiaA PiuA Metal- binding proteins Choline- binding proteins LPXTG-anchored neuraminindase proteins Hyl Sortases PavA Eno LytA Capsule Cell wall Cell membrane PspA PspC Fig. 8.6.3.6  Pneumococcal virulence factors. Important pneumococcal virulence factors include: the capsule; the cell wall; choline-​binding proteins; pneumococcal surface proteins A and C (PspA and PspC); the LPXTG-​anchored neuraminidase proteins; hyaluronate lyase (Hyl); pneumococcal adhesion and virulence A (PavA); enolase (Eno); pneumolysin; autolysin A (LytA); and the metal-​binding proteins pneumococcal surface antigen A (PsaA), pneumococcal iron acquisition A (PiaA) and pneumococcal iron uptake A (PiuA). Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Microbiology. Kadioglu A, et al. (2008). The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol, 6, 288–​301, copyright © 2008.

section 8  Infectious diseases 982 CD4 T cells play an important role in nasopharyngeal immunity that could be exploited by vaccines consisting of pneumococcal proteins or even whole cell killed pneumococci. Prevention Pneumococcal polysaccharide vaccine In 1976, Austrian reported a trial of a 13-​valent vaccine among South African gold miners in which the efficacy against putative pneumococcal pneumonia was 78%. This led to the commercial- ization of a pneumococcal polysaccharide vaccine, initially with 14 serotypes and later extended to 23 serotypes. Trials of these vaccines in older people or in high-​risk populations do not pro- vide consistent evidence of protection against pneumococcal pneumonia. Observational studies using case–​control designs or the indirect cohort method have more consistently indicated pro- tection against bacteraemic pneumococcal disease. The evidence of effect is greater for older people than for those with chronic disease. On the basis of meta-​analyses of the observational studies, pneumococcal polysaccharide vaccination is recommended in the United Kingdom for all adults aged 65 years or more and for all persons aged 5 or more years who belong to an at-​risk group (e.g. with asplenia, splenectomy, chronic respiratory disease, chronic heart, liver, or renal disease, diabetes, or immunosuppression, including HIV infection at all stages). Among patients having planned splenectomy, vaccination should take place well before the operation. For all adults with HIV, PCV is recommended; pneumococcal polysaccharide vaccine is recommended for HIV adults who are elderly or have other comorbidities, although the use of pneumococcal polysaccharide vaccine remains controver- sial. In a study of the vaccine in HIV-​positive individuals from Uganda, the vaccinated group had an elevated risk of pneumonia. The vaccine is not recommended for HIV-​positive populations in the developing world. Pneumococcal conjugate vaccine A 7-​valent PCV, consisting of separate protein–​polysaccharide con- jugates for serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F, was licensed in the United States of America in 2000 following successful trials in Californian infants and Native American children. The seven serotypes in the vaccine accounted for 83% of invasive disease in American children less than 2 years old. The efficacy against inva- sive pneumococcal disease caused by these serotypes was 97% after a four-​dose schedule given at 2, 4, 6, and 15 months of age. The vaccine was also shown to protect against pneumococcal meningitis, bacter- aemia, pneumonia, and otitis media. PCV reduces nasopharyngeal colonization by pneumococci of the serotypes included in the vaccine and increases colonization by other serotypes commensurately. In routine immunization, this has produced two effects. First, the transmission of vaccine-​serotype pneumococci has declined providing ‘herd protection’ for older children and adults whose pneumococcal disease rates have fallen substantially (Fig. 8.6.3.7). Second, the incidence of disease caused by serotypes not included in the vaccine has increased slightly. So far this ‘serotype replacement disease’ has been much smaller in mag- nitude than the substantial reductions in vaccine-​serotype disease. However, to mitigate the effects of serotype replacement disease new vaccines with 10 serotypes (including 1, 5 and 7F) or 13 sero- types (also including 3, 6A and 19A) were developed and licensed. A 13-​valent PCV was introduced into childhood immunization pro- grammes in the United Kingdom and United States in 2010. Vaccine trials in children in South Africa and The Gambia have shown that 9-​valent PCV, which includes the common African sero- types 1 and 5, can protect against invasive pneumococcal disease among HIV-​infected children and can reduce childhood mortality and admissions to hospital with pneumonia among young children. 1998 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 1999 2000 2001 2002 2003 2004 2005 2006 2007 90 (a) (b) 80 70 60 50 40 30 20 10 0 40 35 30 25 20 15 10 5 0 Year Year Cases/100 000 population Cases/100 000 population Serotype group PCV7 type non-PCV7 type 19A Fig. 8.6.3.7  Changes in invasive pneumococcal disease incidence by serotype group among American children aged less than 5 years (a) and adults aged ≥65 years (b), 1998–​2007, illustrating herd protection in adults and serotype replacement disease in both groups. From Pilishvili T et al. (2011) Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J Infect Dis, 201, 32–​41, by permission of Oxford University Press.

8.6.3  Pneumococcal infections 983 In 2007, WHO recommended introduction of PCV into childhood immunization programmes in developing countries and, by 2017, over 50 developing countries had introduced PCV with financial support from GAVI, The Vaccine Alliance. Among HIV-​infected Malawian adults, with a previous history of invasive pneumococcal disease, 7-​valent PCV reduced recurrent invasive pneumococcal disease by 74%. Pneumococcal disease is a significant problem among African adults, including both HIV sero- positive adults in Southern Africa and otherwise healthy adults in the meningitis belt of West Africa. However arguments in favour of adult vaccination have not yet been translated into health policy. The protective efficacy of 13-​valent PCV against pneumococcal pneumonia has been evaluated among Dutch adults aged 65 years or older. The serotype responsible for the pneumonia episode was determined by detecting capsular polysaccharides in urine using an immunodiagnostic assay based on monoclonal antibodies. For dis- eases caused by serotypes in the vaccine, the efficacy was 46% against pneumonia and 75% against invasive disease. The vaccine was sub- sequently recommended for use in adults aged 65 years or older in the United States of America but not in the United Kingdom, where disease is effectively controlled by indirect protection through the childhood immunization programme. In the United Kingdom, where herd protection is now well es- tablished, the recommended immunization schedule for infants is two doses of 13-​valent PCV given at 12 weeks and 12 months of age. For children 2 to 5 years of age who belong to an at-​risk group, the recommendation is for a single dose of PCV followed 2 months later by a single dose of pneumococcal polysaccharide vaccine. Other forms of prevention For children who are at high risk of invasive pneumococcal disease, including those with sickle cell disease or nephrotic syndrome, or following splenectomy, daily prophylaxis with oral penicillin re- duces risk by over 80%. It should be continued until at least 5 years of age. In developing countries, simple measures such as reducing indoor smoke from cooking stoves and improving nutrition are likely to be effective in prevention. Zinc supplementation can reduce the inci- dence of pneumonia in children by 40%. In Pakistan, a community intervention to promote hand washing reduced pneumonia inci- dence in children by one-​half. Diagnosis Culture of S. pneumoniae A specific diagnosis of pneumococcal disease is made by culture of S. pneumoniae from a normally sterile site in a patient with a compatible illness. Pneumococci are fastidious organisms but they grow readily on 5% blood agar incubated in 5% CO2. Colonies are small and grey with a draughtsman like central indentation and are surrounded by a greenish zone of α-​haemolysis. Species iden- tity is confirmed by sensitivity to optochin (ethylhydroxycupreine), bile solubility, and serotyping. The capsular type of S. pneumoniae is differentiated by a change in the refractive index around the cell seen on microscopy in the presence of specific rabbit antisera, the (Neufeld) Quellung reaction (Fig. 8.6.3.8). In pneumococcal meningitis, cerebrospinal fluid frequently yields a positive culture. Pneumococci are also cultured from pleural and joint fluid in thoracic empyema and septic arthritis, respectively. Diagnosis of pneumococcal pneumonia by culture is, however, highly insensitive. Blood culture is a poor diagnostic test for several reasons; infection can be confined to the lungs; episodes of bacter- aemia are only intermittent; the density of bacteraemia is too low, especially in children; or the patient can have taken antibiotics that inhibit growth. Sputum culture lacks specificity, since the pharynx is colonized by pneumococci even in healthy individuals, and also has relatively poor sensitivity. Most cases of pneumococcal pneumonia are, therefore, not formally diagnosed. A measure of this insensi- tivity is obtained from the Gambian trial of PCV where 15 cases of radiographic pneumonia were prevented by vaccination for every two cases of detectable bacteraemic disease prevented. Antigen detection Patients with pneumococcal pneumonia excrete C-​polysaccharide, a universal component of pneumococcal cell walls, and capsular polysaccharides. Detection of C-​polysaccharide in urine has been commercialized in a rapid immunochromatographic test that has a sensitivity of approximately 80% for bacteraemic pneumococcal pneumonia in adults and is highly specific. In children it lacks spe- cificity as positive results may be obtained from healthy individuals who are merely colonized with pneumococci. A sensitive and spe- cific Luminex-​based serotype-​specific antigen detection assay has been developed for analysis of urine in adults but, at present, this is restricted to the serotypes contained in the 13-​valent PCV. Testing for C-​polysaccharide antigen in cerebrospinal fluid is a useful ad- junct to the diagnosis of meningitis. Polymerase chain reaction Primers targeting genes encoding the pneumococcal proteins pneumolysin, autolysin, pneumococcal surface adhesin A, and PBPs have been used for polymerase chain reaction (PCR) diagnosis. These Fig. 8.6.3.8  The (Neufeld) Quellung reaction. Pneumococci show an apparent increase in the thickness of capsule when mixed with homologous anticapsular antibodies. The negative control is shown on the left and the positive reaction on the right. From Werno AM, Murdoch DR (2008). Medical microbiology: laboratory diagnosis of invasive pneumococcal disease. Clin Infect Dis, 46, 926–​32, by permission of Oxford University Press.

section 8  Infectious diseases 984 assays have the same limitations as culture-​based detection. PCR of respiratory specimens does not distinguish colonization from lung infection, and PCR of blood has poor sensitivity for pneumococcal pneumonia. Conversely, PCR of cerebrospinal fluid is sensitive and specific and has proven useful in the investigation of epidemic men- ingitis. Quantitative real-​time PCR for the autolysin gene, lytA, has shown high specificity and good sensitivity in validation studies in adults and children in high-​income settings although it lacks speci- ficity in children in low-​income settings where carriage prevalence and carriage densities are high in health children. Clinical features S.  pneumoniae causes pneumonia, meningitis, septicaemia, otitis media, endocarditis, peritonitis, sinusitis, conjunctivitis, and puru- lent infections of the pleura, joints, and bone. These conditions do not necessarily occur in isolation; pneumococcal meningitis is quite frequently accompanied by pneumonia. Pneumococcal pneumonia Symptoms Typically, the illness starts suddenly although there may be an ante- cedent upper respiratory tract infection. Fever is usually the first symptom and it is frequently accompanied by rigors. The patient feels weak and anorexic and may have severe headache and myalgia. Cough develops within 24–​72 h and becomes a prominent symptom. At first the cough is non​productive but it becomes productive of blood-​tinged (‘rusty’) sputum and later of purulent sputum. Stabbing pleuritic chest pain also develops during the course of the illness. The patient might try to obtain relief by splinting the af- fected side of the chest or lying on the affected side. Involvement of the diaphragmatic pleura leads to misleading abdominal pain or referred pain in the shoulder. Among young children, a history of cough and difficulty breathing should raise suspicion of pneumonia. Elderly and immunocompromised patients might present with general malaise and delirium, with few respiratory symptoms and no fever. Prior antibiotic treatment also modifies the classic presentation. Physical signs On general examination, adults have tachycardia and pyrexia, with a rectal temperature as high as 40°C. With early presentation the respiratory system can appear normal, but pneumonia patients go on to develop rapid and difficult breathing (of which flaring of the alae nasi is a subtle early sign), cyanosis, and signs of lobar consoli- dation including reduced chest movement, dullness on percussion, fine crepitations, and, occasionally, bronchial breathing over the af- fected area. A pleural rub is sometimes audible. Abdominal distension, upper abdominal tenderness, and guarding suggest involvement of the diaphragmatic pleura. Mild jaundice occurs in a minority. Concomitant herpes labialis (‘cold sores’) is common. Delirium is a sign of severity and is frequently observed in elderly patients. In infants, the signs of pneumonia are non​specific; most will have a raised respiratory rate and nasal flaring but only a minority will have crepitations. In developing countries, most cases of pneumonia are diagnosed and treated by non​medical health workers. To facili- tate diagnosis and promote early treatment, the WHO has designed a simple diagnostic algorithm as part of its Integrated Management of Childhood Illness (IMCI), which defines pneumonia on the basis of respiratory rate and lower chest wall indrawing (Table 8.6.3.1). Severe pneumonia, requiring admission to hospital, is indicated by hypoxia or generic ‘danger signs’ (Fig. 8.6.3.9). Investigations The pathological process of pneumonia is confirmed by the chest radiograph. This typically shows a homogenous area of opacification confined within the lobar structure (Fig. 8.6.3.10). The lower lobes are affected more frequently than the upper lobes. The area of path- ology may be localized to a single lobule or extend over several lobes; early in the presentation there may be no abnormality at all. Table 8.6.3.1  The WHO classification of pneumonia in children in developing countries Sign or symptom Classification Treatment Cough or difficulty in breathing with: • Oxygen saturation <90% or central cyanosis • Severe respiratory distress (e.g. grunting, very
severe chest indrawing) • Signs of pneumonia with a general danger sign (inability to breastfeed or drink, lethargy or
reduced level of consciousness, convulsions) Severe pneumonia •​ Admit to hospital •​ Give oxygen if saturation <90% •​ Manage airway as appropriate •​ Give recommended antibiotic •​ Treat high fever if present Fast breathing: •​ ≥50 breaths/​min in a child aged 2–​11 months •​ ≥40 breaths/​min in a child aged 1–​5 years Chest indrawing Pneumonia •​ Home care •​ Give appropriate antibiotic •​ Advise the mother when to return immediately if symptoms of severe pneumonia •​ Follow-​up after 3 days No signs of pneumonia or severe pneumonia No pneumonia: cough or cold •​ Home care •​ Soothe the throat and relieve cough with safe remedy •​ Advise the mother when to return •​ Follow-​up after 5 days if not improving •​ If coughing for more than 14 days, refer to chronic cough Adapted from World Health Organization (2013). Pocket book of hospital care for children: guidelines for the management of common illnesses with limited resources. World Health Organization, Geneva, copyright © 2013.

8.6.3  Pneumococcal infections 985 In adults this might also reveal an underlying risk factor (e.g. lung cancer). In children, widespread patchy opacification (broncho- pneumonia) is common. Lateral radiographs add to the sensitivity of posteroanterior projections particularly for lower lobe disease hidden beneath the dome of the diaphragm. In adults, pneumococcal aetiology is defined by the C-​ polysaccharide antigen test in urine. Many patients are severely dehydrated on admission and cannot readily produce a urine spe- cimen. Blood culture is positive for S. pneumoniae in about 10 to 30% of adults and about 15% of children. Genuine sputum samples should be differentiated from upper respiratory tract secretions by a high ratio of pus cells to epithelial cells on microscopy. The appear- ance of large numbers of Gram-​positive diplococci on microscopy together with culture of S. pneumoniae is diagnostic. However, be- cause prior antibiotic use is common, sputum microscopy is positive in only about one-​quarter of patients and sputum culture is positive in only one-​half. Young children cannot normally produce a sputum specimen. Differential diagnosis The abrupt onset of symptoms often leads the patient to seek care before focal signs become established and it is not possible to differ- entiate pneumonia from other causes of acute febrile illness. In trop- ical countries, malaria is the main differential at this stage. When localizing symptoms and signs are established, pneumonia must be distinguished from pulmonary infarction. Both conditions lead to chest pain and haemoptysis and are accompanied by tachycardia. Pyrexia and rigors favour a diagnosis of pneumonia, while a very sudden history of chest pain and frank haemoptysis favour pul- monary embolism. Pulmonary oedema (secondary to heart failure), pulmonary atelectasis, pleurisy, lung abscess, tuberculosis, and acute bronchitis should also be considered in the differential diagnosis. Outside the chest, subdiaphragmatic lesions such as cholecystitis, a subphrenic abscess, or an amoebic liver abscess can mimic the clin- ical picture of lower lobe pneumonia. Bacterial pneumonia is differentiated from viral or mycoplasma pneumonia by its abrupt onset, severity of symptoms and systemic illness, raised peripheral white blood cell count, and C-​reactive protein level exceeding 125 mg/​litre in serum. Confusion, signs of multiorgan involvement, lymphopenia, or a low serum sodium should raise the possibility of legionnaires’ disease. Tuberculosis occasionally presents with an acute pneumonia in adults. In HIV-​ infected patients, the differential diagnosis also includes infection by Pneumocystis jirovecii, mycobacteria, and cytomegalovirus. Treatment Management of pneumonia first requires an assessment of severity to determine whether the patient should be treated at home, ad- mitted to hospital, or admitted to the intensive care unit. The British Thoracic Society (BTS) recommendations define pneumonia as se- vere if there are three or more CURB-​65 features: Confusion, Urea exceeding 7 mmol/​litre (Fig. 8.6.3.11), Respiratory rate equal to or exceeding 30 breaths/​min, abnormal Blood pressure, either systolic (<90 mm Hg) or diastolic (≤60 mm Hg) hypotension, and age equal to or exceeding 65 years. Additional features that might influence this assessment include the presence of coexisting disease, hypox- aemia (Pao2 less than 8 kPa or Sao2 less than 94%), and bilateral or multilobe involvement on the chest radiograph. Bacteraemia is itself an indicator of severity and increased risk of death. Supportive care includes analgesia for chest pain, ample hydration and nutrition, ad- vice to stop smoking, and oxygen for inpatients with hypoxaemia. Empirical guidelines for pneumonia treatment focus on treatment of pneumococcal pneumonia. High-​dose penicillin or amoxicillin Fig. 8.6.3.9  Kenyan child with very severe pneumonia, as defined by the WHO, receiving high-​flow oxygen therapy. Taken in the clinical service of Kilifi District Hospital; supplied by Dr Mike English. The patient’s parents gave written consent for the taking of this photograph and for its use for educational purposes. Fig. 8.6.3.10  Chest radiograph of an adult with clinical signs of left lower lobe pneumonia illustrating a well-​demarcated area of alveolar consolidation.

section 8  Infectious diseases 986 therapy will provide serum concentrations sufficiently high to treat pneumonia that is caused by pneumococci with MICs up to 2 µg/​ml. Based on efficacy, cost, and acceptability, the optimum antibiotic is amoxicillin. In the UK oral amoxicillin 500 mg three times daily for 5 days is recommended for non​severe cases of community-​acquired pneumonia treated at home. Doxycycline 100  mg daily (initial 200 mg loading dose) or clarithromycin 500 mg twice daily are ac- ceptable alternatives. A fluoroquinolone with enhanced pneumo- coccal activity (e.g. levofloxacin, moxifloxacin) can be considered in outbreaks of resistant pneumococcal disease or in patients unre- sponsive to first-​line antibiotics. Cases of pneumonia with high severity (CURB65 score 3–​4) should be treated empirically with a broad-​spectrum intra- venous antibiotic such as co-​amoxiclav (1.2 g three times daily), cefuroxime (1.5 g three times daily), cefotaxime (1 g three times daily), or ceftriaxone (2 g once daily) for 10 days. Cefotaxime or ceftriaxone are most active against pneumococci and should be ef- fective against pneumonia caused by pneumococci with high ceph- alosporin MICs of 1–​2 µg/​ml. Empiric therapy with clarithromycin (500 mg twice daily) should also be given to cover other causes of pneumonia. After 3 days of intravenous antibiotics, clinically stable patients can be safely switched to oral therapy. Pneumonia patients admitted to hospital with pneumonia of moderate se- verity (CURB65 score 2) should be treated with oral amoxicillin (500–​1000 mg three times per day) plus clarithromycin 500 mg twice daily. Course and prognosis Historically, untreated patients who survived long enough to make specific anticapsular polysaccharide antibody recovered spontan- eously by crisis, or by a more gradual lysis, 7 to 10 days after the onset of illness. However, mortality from pneumococcal pneu- monia in the preantibiotic era was 20–​40%. With antibiotic treat- ment, mortality is about 5% overall but is 30% among the subset of patients with bacteraemia. Mortality is higher among elderly and very young patients, and among those with an underlying illness such as cirrhosis, alcoholism, or heart disease. Most deaths occur within the first few days of admission to hospital. The causes of death are difficult to establish but include shock, cardiac arrhyth- mias, and respiratory failure. Pneumococcal pleural effusion and empyema A large pleural effusion or an empyema develops during treat- ment in 2 to 5% of patients with established pneumococcal pneumonia. Symptoms Some patients with pneumococcal empyema give a history of re- cent lung infection but others develop the disease without any previous illness. Hectic fever, rigors, sweats, malaise, anorexia, and marked weight loss are characteristic symptoms, often going back several weeks. Patients with a large pleural collection are breathless and may complain of dull pain on the affected side. A productive cough is unusual unless a bronchopleural fistula has developed. Physical signs General examination reveals pyrexia, tachycardia, and evidence of recent weight loss. Examination of the chest usually shows the char- acteristic signs of a pleural effusion: diminished chest movement, stony dullness on percussion, and diminished breath sounds over the accumulated fluid. The chest wall overlying an empyema may be tender. Investigations The effusion will usually be visible on the chest radiograph but loculated effusions may require localization by ultrasonography. On aspiration, turbid fluid or thick pus is obtained which contains pneumococci and degenerate white cells. If antibiotics have been given it might not be possible to culture pneumococci, but the fluid contains detectable pneumococcal antigens. The peripheral white blood cell count is raised predominantly with neutrophils. Differential diagnosis The principal differential diagnosis is pulmonary tuberculosis, and pleural biopsy may be required if the pleural fluid is sterile. The ab- sence of copious, purulent sputum differentiates pleural empyema from a lung abscess. Treatment Successful treatment requires both intravenous antibiotics and pleural drainage. Appropriate antibiotic treatment for pneumo- coccal empyema follows the recommendations for pneumococcal pneumonia, with intravenous amoxicillin or a cephalosporin. (a) (b) Fig. 8.6.3.11  Urea frost in two patients with uraemia complicating pneumococcal pneumonia. Copyright D. A. Warrell.

8.6.3  Pneumococcal infections 987 Because of the frequent coexistence of penicillin-​resistant aerobes and anaerobes, a β-​lactamase inhibitor or metronidazole should also be given. Antibiotics should be continued for 4 to 6 weeks. Course and prognosis If untreated, an empyema might rupture through the chest wall (em- pyema necessitatis) or into a bronchus causing a bronchopleural fis- tula. Even when pus is aspirated and healing achieved, subsequent fibrosis and calcification may seriously restrict expansion of the underlying lung. Pneumococcal meningitis Pneumococci colonizing the nasopharynx can gain access to the subarachnoid space either by direct spread (from paranasal sinusitis or otitis media), following damage to the base of the skull, or, more commonly, via the bloodstream where they cross the blood–​brain barrier at the choroid plexus and cerebral capillaries. Symptoms Adults with pneumococcal meningitis usually have fever, head- ache, neck stiffness, and impaired consciousness. At presentation, one-​half of all patients have been ill for less than 24 h. Nausea and photophobia are common and seizures occur before diagnosis in 5 to 10% of patients. Among elderly patients, confusion may be the only symptom. Deterioration in the psychological or neurological state of an elderly patient with community-​acquired pneumonia should be investigated with lumbar puncture. The presentation of meningitis in infants can be subtle, beginning with inability to feed and followed by irritability or lethargy. Physical signs Patients with pneumococcal meningitis are pyrexial and toxaemic. Classic signs such as nuchal rigidity, Kernig’s sign, and Brudzinski’s sign are absent in many patients with pyogenic meningitis. Bulging of the anterior fontanelle may be present in infants. Consciousness is often impaired, varying from drowsiness and confusion to deep coma. Raised intracranial pressure due to cerebral oedema or a cere- bral abscess may be indicated by bradycardia and hypertension, but papilloedema is rarely seen. A cranial CT or MRI is mandatory be- fore lumbar puncture in the presence of signs of cerebral or cranial nerve damage including a dilated pupil, ocular palsies, hemiparesis, history of focal seizures, decreased or rapidly falling level of con- sciousness, irregular respiration, tonic seizures, and decerebrate or decorticate posturing. An associated pneumococcal condition, such as otitis media or pneumonia, might be detected. Investigations Lumbar puncture should be undertaken whenever meningitis is suspected. In pneumococcal meningitis the cerebrospinal fluid is usually turbid and the leucocyte count is equal to or exceeds 1000 × 106/​litre. Most of the leucocytes are neutrophils. A few pa- tients have a low leucocyte count (<100 × 106/​litre) and in patients who present very early the leucocyte count can be normal; a repeat lumbar puncture several hours later will confirm the diagnosis. In pneumococcal meningitis, the concentration of protein in cere- brospinal fluid is increased and the ratio of glucose concentrations in cerebrospinal fluid and plasma is usually less than one-​third. In untreated cases, culture of cerebrospinal fluid is usually positive and pneumococci are visible following Gram’s staining. In patients who have received less than 48 h of antibiotic therapy the leucocyte count remains high and pneumococcal antigen may be detectable in cerebrospinal fluid. In developing countries, particularly in Asia, the use of immunochromatographic tests for C-​polysaccharide antigen in cerebrospinal fluid has increased the number of cases of pneumococcal meningitis diagnosed. Culture of blood also fre- quently reveals the pneumococcus. The peripheral white cell count is usually elevated. Differential diagnosis Pneumococcal meningitis cannot be differentiated clinically from other forms of meningitis and the aetiology must be defined by in- vestigation of the cerebrospinal fluid. An associated ear infection or pneumonia, or a history of head trauma favours pneumococcal infection. Conversely, rashes are rarely found in pneumococcal meningitis and petechiae or purpura on skin or mucosae strongly suggest meningococcal disease. Widespread use of pneumococcal conjugate vaccines has reduced the likelihood of S. pneumoniae aeti- ology in children with meninigitis. Treatment Standard empirical therapy for meningitis in adults is cefotaxime (300 mg/​kg per day divided into three or four doses) or ceftriaxone (100 mg/​kg per day divided into two doses) for 10 to 14 days. In parts of the world where strains with intermediate or full resistance to cefotaxime or ceftriaxone have emerged, vancomycin (60 mg/​ kg per day divided into four doses) should be added to the empiric therapy. Meropenem is a useful alternative to cefotaxime and is ac- tive against pneumococci of intermediate but not full cefotaxime resistance. Imipenem increases susceptibility to seizures. Penicillin or ampicillin are effective therapy for culture-​proven pneumococcal meningitis caused by penicillin-​sensitive strains, but intermediately resistant strains are not adequately treated by these drugs. In children in developing countries, outside of epidemics, the WHO recommends treatment with ceftriaxone 100 mg/​kg once daily for 5 to 7 days with a maximum daily dose of 2 g. A multicountry trial of 5 days versus 10 days of ceftriaxone therapy in children has provided empiric support for short-​course therapy for 5 days, pro- vided there is good clinical evidence of response. Treatment with antibiotics should be started as soon as a clin- ical diagnosis of bacterial meningitis is made. Delay in treatment until after hospitalization is associated with increased mortality. Other supportive therapies include adequate oxygenation, mainten- ance of normal blood pressure, prevention of hypoglycaemia and hyponatraemia, and control of seizures (which may be covert and unsuspected in an unconscious patient) with anticonvulsants. The use of dexamethasone in bacterial meningitis in adults has been controversial for many years. In a Cochrane review of five ran- domized controlled trials, the summary mortality reduction attribut- able to dexamethasone adjunctive treatment was 43%; the effect was greatest in meningitis caused by S. pneumoniae. The summary find- ings were influenced to a large extent by a single study of European patients. In a study of HIV-​infected adults in Africa, among whom case fatality rates were very high, dexamethasone was not beneficial, suggesting that dexamethasone might only be useful in populations with low HIV prevalence. The recommended dose is 10 mg every

section 8  Infectious diseases 988 6 h for 4 days. Meningeal inflammation facilitates diffusion of vanco- mycin into the cerebrospinal fluid and the anti-​inflammatory action of dexamethasone might lead to suboptimal antibiotic concentra- tions. Patients on treatment for cephalosporin-​resistant pneumo- coccal meningitis should therefore be monitored both clinically and by repeat lumbar puncture. In children, dexamethasone is highly protective against hearing loss in H. influenzae meningitis but also provides some protection in pneumococcal meningitis if given with or before administration of antibiotics. For children in developing countries, however, the evidence suggests there is no benefit to ad- junctive dexamethasone. Course and prognosis The prognosis of patients with pneumococcal meningitis is poor. Most patients develop complications of which the most important are seizures, brain infarction, brain swelling, hydrocephalus, and cranial nerve palsies. Subdural collections are commonly seen on brain imaging and may require needle puncture to exclude subdural empyema, especially if there is persistence of fever, irritability, neck stiffness, or continued cerebrospinal fluid leucocytosis detected by repeat lumbar puncture. Over one-​third of patients also develop systemic complications such as shock, cardiorespiratory failure, and disseminated intra- vascular coagulation, and these are frequently the final cause of death among older patients. Among children, supportive therapy to sustain adequate blood pressure is important to maintain cerebral blood flow against the resistance of raised intracranial pressure. The mortality from pneumococcal meningitis in industri- alized countries varies between 10 and 40%, being lower in children than in adults. In developing countries, the mortality range is higher (30–​60%). Features on admission that are asso- ciated with a poor outcome include advanced age, seizures, cra- nial nerve palsies, deep coma, low cerebrospinal fluid leucocyte count (below 1000×106/​litre), low glucose concentration in cere- brospinal fluid, and associated pneumonia. Death is almost in- evitable in patients who are in deep coma at the time they are admitted to hospital. Survivors are frequently affected by neuro- logical sequelae:  hearing loss occurs in one in five; cerebral damage is common leading to hemiparesis, ataxia, and aphasia; and cranial nerve palsies, particularly of the oculomotor nerve, occur in a small percentage. Among those who appear to make a good recovery from pneumococcal meningitis, one-​quarter have residual cognitive slowness. Otitis media In children aged below 2 years, otitis media is one of the most common reasons for seeking medical advice. The pneumococcus causes a significant fraction of all cases. Following conjugate pneumococcal vaccine introduction, the incidence of pneumo- coccal otitis media has fallen in several countries. Beyond child- hood, otitis media is uncommon. Symptoms Acute otitis media starts suddenly, although there may be a history of a recent upper respiratory tract infection. Fever, crying, and ex- treme irritability are the usual features in young children, in whom febrile convulsions may also occur. Fever and severe pain in the ear are the usual presenting complaints in older children and adults, and patients may also complain of deafness and tinnitus. Physical signs On otoscopic examination of the affected ear, the tympanic mem- brane is red and swollen and lacks the normal light reflection. It may bulge outwards into the external ear and there may be an air–​fluid level indicating a middle ear effusion. Pus or blood in the external auditory canal suggests a perforation that is confirmed by observing a ragged hole in the tympanic membrane. The affected ear is usually partially deaf. In children, meningism may be present; if so, menin- gitis must be excluded by lumbar puncture. Investigations Fine needle puncture of the tympanic membrane (tympanocentesis) and aspiration of middle ear fluid is used with variable frequency in different countries but is of most value where antibiotic resist- ance is prevalent. In complicated cases or in those not responding to initial antibiotics, culture of middle ear fluid may guide therapy. A tympanogram can identify increased middle ear pressure and ac- cumulation of fluid. Treatment Most episodes of otitis media are diagnosed clinically without micro- biological confirmation. Randomized controlled trials show that ot- itis media resolves in most otherwise healthy children whether or not they take antibiotics. This evidence underpins a policy of ob- servation without treatment. Immediate antibiotics are indicated in young infants (<6 months) and in older children (>2 years) with a clear bacteriological diagnosis of otitis media or symptoms of se- verity. The antibiotic of choice for pneumococcal otitis media is oral amoxicillin (90 mg/​kg per day) for 5 days. For penicillin-​resistant pneumococcal infection the appropriate antibiotic is intravenous ceftriaxone for 3 days. Course and prognosis Pneumococcal otitis media normally resolves rapidly and com- pletely. However, rupture of the drum can lead to partial conductive deafness and pneumococcal otitis media can give rise to a chronic discharging ear requiring prolonged or complicated treatment. The infection can spread to cause acute mastoiditis, meningitis, or a cere- bral abscess. Other clinical syndromes The pneumococcus is an important cause of bacterial sinusitis resulting from direct spread from the nasopharynx. Sinusitis that does not resolve within 5 to 7 days may require treatment with an antibiotic effective against pneumococcus. A mild form of pneumococcal bacteraemia, variously labelled as ‘occult bacteraemia’ or ‘walk-​in bacteraemia’, was encountered rela- tively commonly in children before the introduction of PCV. Significant bloodstream infection is less common but can lead to septicaemia or purulent localization in meninges, vertebrae, joints, orbits, or testes. Pneumococcal conjunctivitis has been observed in outbreaks among college students and has two un- usual features:  (1) the causative strain is unencapsulated and

8.6.3  Pneumococcal infections 989 (2) the attack rates are high, suggesting little pre-​existing im- munity. Pneumococci can also cause endophthalmitis, and are the commonest pathogen observed in case series of bacterial keratitis. Septicaemia Acute septicaemia is a less common form of pneumococcal infec- tion and is encountered most frequently in immunocomprom- ised patients or those without a spleen. Sudden fever, peripheral circulatory collapse, and bleeding (purpura fulminans) are the usual presenting features of this condition, which is indistinguish- able from other forms of overwhelming bacterial septicaemia. Leucopenia is usually found. Bleeding is due to disseminated intra- vascular coagulation. The mortality from septicaemia is very high, even when treatment is started promptly. The pneumococcus has been rarely associated with toxic shock syndrome and with haemo- lytic uraemic syndrome. Endocarditis and pericarditis Cardiac manifestations of pneumococcal infection are well de- scribed but where there is good access to antibiotics they are now rare, occurring in less than 1% of all pneumococcal infections. Acute endocarditis may complicate pneumococcal septicaemia to affect healthy heart valves, especially the aortic valve, which may rupture and cause severe aortic incompetence leading to valve replacement in those who survive the initial episode. Pneumococci may spread directly from the lower lobes of the lung to produce pericarditis which is clinically silent in some pa- tients or may be manifest only as a transient pericardial rub or an abnormal electrocardiogram. Patients with a pericardial empyema usually complain of dull or pleuritic central chest pain and give a history of persistent fever, malaise, anorexia, and weight loss over several days or weeks. Many patients with a pneumococcal pericar- dial empyema are critically ill by the time they reach hospital and have pericardial tamponade: a rapid small-​volume pulse, pulsus paradoxus, a low blood pressure, elevation of the jugular venous pressure with a further increase during inspiration, peripheral oe- dema, and ascites. The heart sounds are usually faint and a chest radiograph may show globular enlargement of the heart together with evidence of an associated lung infection. Ultrasound can help to define the best sites for diagnostic and therapeutic drainage. The electrocardiogram shows low-​voltage potentials and ST elevation or depression may be present. Pneumococcal aetiology can be con- firmed by culture or antigen detection of drained pus. Mortality is high, and among patients who survive the initial episode con- strictive pericarditis may develop within weeks or months of their acute illness. Peritonitis Pneumococcal peritonitis is an uncommon condition that is en- countered among three risk groups: (1) patients with cirrhosis of the liver or nephrotic syndrome; (2) patients with gastrointestinal disease (e.g. appendicitis), intra-​abdominal surgery, or peritoneal dialysis; and (3) otherwise healthy young girls, possibly as a com- plication of pelvic infection. The condition is characterized by sudden fever and abdominal pain and tenderness. The ascitic fluid is turbid and contains neutrophils and pneumococci. The prognosis of pneumococcal peritonitis is determined principally by the severity of the underlying illness. Future developments Childhood pneumococcal disease has been controlled in high-​ income settings and this is likely to be replicated across low-​income settings. Nonetheless, much pneumococcal disease remains. In many countries, particularly in Asia, the current PCV formulations do not target most disease-​causing serotypes. Among older adults in industrialized countries the incidence of pneumococcal infections caused by non​vaccine serotypes is significant and rising. Given that conjugate vaccines can prevent pneumonia in older adults, a conju- gate vaccine comprising the commoner serotypes not included in the current infant PCV formulations would make a significant con- tribution to health. Conjugate vaccines are expensive to produce and several lines of research are examining alternative methods to sustain vac- cine control in low-​income countries. These include novel in- expensive manufacturing technologies, strategies to sustain indirect protection with fewer doses of vaccine, and serotype-​ independent third generation vaccines or with fractional doses of vaccine. Several vaccines, consisting of either combinations of surface-​expressed proteins and virulence factors, or inactivated whole pneumococcal cells, are in phase I/​II studies. The perturbation of pneumococcal ecology by conjugate vac- cines has highlighted our ignorance of the natural habitat of Streptococcus pneumoniae. Current studies of colonizing pneumo- cocci are investigating inter-​ and intraspecies competition, bio- film development, interactions with respiratory viruses, and the mechanism of clearance induced by PCV. Studies in genomics, transcription and phylogenetic, are tackling the same problems at pathogen-​population level. As persistent colonizers of children, pneumococci are exposed repeatedly to antibiotics, encouraging the development and dis- semination of antibiotic resistance. Resistance to new agents such as fluoroquinolones is established in adult disease and has been re- ported among paediatric cases in South Africa. The problem is only likely to worsen unless antibiotic usage in low-​income countries can be brought under control through judicious and rational pre- scribing. At the same time, the clinical relevance of in vitro resist- ance to antibiotics, especially intermediate β-​lactam resistance in the treatment of pneumonia, remains an interesting area requiring further investigation. FURTHER READING Austrian R, Gold J (1964). Pneumococcal bacteremia with especial reference to bacteremic pneumococcal pneumonia. Ann Intern Med, 60, 759–​76. Austrian R, et al. (1976). Prevention of pneumococcal pneumonia by vaccination. Trans Assoc Am Physicians, 89, 184–​94. Bentley SD, et al. (2006). Genetic analysis of the capsular biosyn- thetic locus from all 90 pneumococcal serotypes. PLoS Genet, 2, e31, 1–​8.

section 8  Infectious diseases 990 Berkley JA, et al. (2005). Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med, 352, 39–​47. Black S, et al. (2000). Efficacy, safety and immunogenicity of hepta- valent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group. Pediatr Infect Dis J, 19, 187–​95. Bonten MJ, et  al. (2015). Polysaccharide conjugate vaccine
against pneumococcal pneumonia in adults. N Engl J Med, 372, 1114–​25. Briles DE, et al. (2000). Immunization of humans with recombinant pneumococcal surface protein A (rPspA) elicits antibodies that passively protect mice from fatal infection with Streptococcus pneumoniae bearing heterologous PspA. J Infect Dis, 182, 1694–​701. Brouwer MC, et al. (2015). Corticosteroids for acute bacterial menin- gitis. Cochrane Database Syst Rev, 5, CD004405. Castelblanco RL, et al. (2014). Epidemiology of bacterial meningitis in the USA from 1997 to 2010: a population-​based observational study. Lancet Infect Dis, 14, 813–​9. Cobey S, Lipsitch M (2012). Niche and neutral effects of acquired im- munity permit coexistence of pneumococcal serotypes. Science, 335, 1376–​80. Croucher NJ, et al. (2011). Rapid pneumococcal evolution in response to clinical interventions. Science, 331, 430–​4. Cutts FT, et al. (2005). Efficacy of nine-​valent pneumococcal conju- gate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-​blind, placebo-​controlled trial. Lancet, 365, 1139–​46. Dowell SF, et al. (2003). Seasonal patterns of invasive pneumococcal disease. Emerg Infect Dis, 9, 573–​9. Dowson CG, et al. (1989). Horizontal transfer of penicillin-​binding protein genes in penicillin-​resistant clinical isolates of Streptococcus pneumoniae. Proc Natl Acad Sci U S A, 86, 8842–​6. French N, et al. (2010). A trial of a 7-​valent pneumococcal conjugate vaccine in HIV-​infected adults. N Engl J Med, 362, 812–​22. Geno KA et al. (2015). Pneumococcal capsules and their types: past, present, and future. Clin Microbiol Rev, 28, 871–​99. Gessner BD, et al. (2010). African meningitis belt pneumococcal dis- ease epidemiology indicates a need for an effective serotype 1 con- taining vaccine, including for older children and adults. BMC Infect Dis, 10, 22. Ginsburg AS, et al. (2012). Issues and challenges in the develop- ment of pneumococcal protein vaccines. Expert Rev Vaccines, 11, 279–​85. Gordon SB, et  al. (2000). Bacterial meningitis in Malawian adults: pneumococcal disease is common, severe, and seasonal. Clin Infect Dis, 31, 53–​7. Griffin MR et  al. (2013). U.S.  hospitalizations for pneumonia after a decade of pneumococcal vaccination. N Engl J Med, 369,
155–​63 Heffron R (1939). Pneumonia with special reference to pneumo- coccus lobar pneumonia. The Commonwealth Fund, New York, NY. (Second printing 1979, Harvard University Press, Cambridge, MA) Johnson HL, et al. (2010). Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five:  the pneumococcal global serotype project. PLoS Med, 7, e1000348. Kadioglu A, et al. (2008). The role of Streptococcus pneumoniae viru- lence factors in host respiratory colonization and disease. Nat Rev Microbiol, 6, 288–​301. Klugman KP, et al. (2003). A trial of a 9-​valent pneumococcal con- jugate vaccine in children with and those without HIV infection.
N Engl J Med, 349, 1341–​8. Lim WS, et al. (2009). British Thoracic Society guidelines for the man- agement of community acquired pneumonia in adults: update 2009. Thorax, 64, Suppl 3, iii, 1–​55. Malley R, et  al. (2001). Intranasal immunization with killed unencapsulated whole cells prevents colonization and invasive dis- ease by capsulated pneumococci. Infect Immun, 69, 4870–​3. Moberley S et al. (2013). Vaccines for preventing pneumococcal infec- tion in adults. Cochrane Database Syst Rev, 1, CD000422. Musher DM (1992). Infections caused by Streptococcus pneumo- niae: clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis, 14, 801–​7. Pallares R, et al. (1995). Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain. N Engl J Med, 333, 474–​80. Palmu AA, et al. (2013). Effectiveness of the ten-​valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-​CV10) against invasive pneumococcal disease: a cluster randomised trial. Lancet, 381, 214–​22. Pilishvili T, et al. (2010). Sustained reductions in invasive pneumo- coccal disease in the era of conjugate vaccine. J Infect Dis, 201, 32–​41. Roca A, et  al. (2011). Effects of community-​wide vaccination with PCV-​7 on pneumococcal nasopharyngeal carriage in the Gambia: a cluster-​randomized trial. PLoS Med, 8, e1001107. Scott JA, et al. (2000). Aetiology, outcome, and risk factors for mor- tality among adults with acute pneumonia in Kenya. Lancet, 355, 1225–​30. Tettelin H, et al. (2001). Complete genome sequence of a virulent iso- late of Streptococcus pneumoniae. Science, 293, 498–​506. Trzcinski K, Thompson CM, Lipsitch M (2004). Single-​step cap- sular transformation and acquisition of penicillin resistance in Streptococcus pneumoniae. J Bacteriol, 186, 3447–​52. Tuomanen EI, et  al. (eds) (2004). The pneumococcus. ASM Press, Washington, DC. van der Poll T, Opal SM (2009). Pathogenesis, treatment, and preven- tion of pneumococcal pneumonia. Lancet, 374, 1543–​56. von Gottberg A et  al. (2014). Effects of vaccination on invasive pneumococcal disease in South Africa. N Engl J Med, 371, 1889–​99. Wahl B, et  al. (2018). Burden of Streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of
conjugate vaccines: global, regional, and national estimates for 2000-2015. Lancet Global Hlth, 6, e744–757. Waight PA, et al. (2015). Effect of the 13-​valent pneumococcal con- jugate vaccine on invasive pneumococcal disease in England and Wales 4 years after its introduction: an observational cohort study. Lancet Infect Dis, 15, 535–​43. Watera C, et al. (2004). 23-​Valent pneumococcal polysaccharide vac- cine in HIV-​infected Ugandan adults: 6-​year follow-​up of a clinical trial cohort. AIDS, 18, 1210–​3. Watson DA, et al. (1993). A brief history of the pneumococcus in bio- medical research: a panoply of scientific discovery. Clin Infect Dis, 17, 913–​24. Weinberger DM, et al. (2011). Serotype replacement in disease after pneumococcal vaccination. Lancet, 378, 1962–​73. Weiser JN, et al. (1994). Phase variation in pneumococcal opacity: re- lationship between colonial morphology and nasopharyngeal col- onization. Infect Immun, 62, 2582–​9.

8.6.30 Actinomycoses 1170

8.6.30 Actinomycoses 1170

section 8  Infectious diseases 1170 Meyers WM (1995). Mycobacterial infections of the skin. In: Doerr W, Seifert G (eds) Tropical pathology, pp. 291–​377. Springer, Berlin. Meyers WM, et al. (2011). Mycobacterium ulcerans infection (Buruli ulcer). In: Guerrant RL, et al. (eds) Tropical infectious diseases, 3rd edition, pp. 248–​52, Elsevier Saunders, Edinburgh. Nienhuis WA, et al. (2010). Antimicrobial treatment for early, limited Mycobacterium ulcerans infection:  a randomised controlled trial. Lancet, 375, 664–​72. Portaels F, et  al. (2008). First cultivation and characterization of Mycobacterium ulcerans from the environment. PLoS Negl Trop Dis, 2, e178. Portaels F, et al. (2009). Buruli ulcer. Clin Dermatol, 27, 291–​305. Portaels F, ed. (2014). Laboratory diagnosis of Buruli ulcer: a manual for health-​care providers. World Health Organization, Geneva. Silva M, et al. (2009). Pathogenetic mechanisms of the intracellular parasite Mycobacterium ulcerans leading to Buruli ulcer. Lancet Infect Dis, 9, 699–​710. Vandelannoote K, et al. (2014). Insertion sequence element-​single nu- cleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa. Appl Environ Microbiol, 80, 1197–​209. Vandelannoote K, et al. (2017). Multiple introductions and recent spread of the emerging human pathogen Mycobacterium ulcerans across Africa. Genome Biol Evol, 9, 414–26. Walsh DS, et al. (2009). Buruli ulcer (Mycobacterium ulcerans infec- tion): a re-​emerging disease. Clin Microbiol Newsletter, 31, 119–​28. Walsh DS, et al. (2015). Leprosy and Buruli ulcer: similarities suggest combining control and prevention of disability strategies in coun- tries endemic for both diseases. Lepr Rev, 86, 1–​5. World Health Organization (2012). Treatment of Mycobacterium ulcer- ans disease (Buruli ulcer): guidance for health workers. World Health Organization, Geneva. Zogo B, et al. (2015). A Field Study in Benin to Investigate the Role of Mosquitoes and other flying insects in the ecology of Mycobacterium ulcerans. PLoS Negl Trop Dis, 9, e0003941. 8.6.30  Actinomycoses Klaus P. Schaal ESSENTIALS Human actinomycoses are always synergistic polymicrobial infections in which fermentative actinomycetes—​predominantly Actinomyces israelii, A. gerencseriae, or Propionibacterium propionicum—​are the principal pathogens, usually needing the assistance of so-​called concomitant microbes to produce disease. Nearly all of the mem- bers of the mixed actinomycotic microflora belong to the indi- genous microbial community of human mucous membranes, hence actinomycoses present as sporadic endogenous infections which are not transmissible. Clinical features—​the initial actinomycotic lesion usually develops in tissue adjacent to a mucous membrane as a subacute to chronic process that is granulomatous as well as suppurative, typically giving rise to multiple abscesses and draining sinus tracts that are preferen- tially located in the cervicofacial region, thorax, or abdomen. These characteristically progress slowly, penetrate tissues without regard to natural organ borders, and spread haematogenously, with symp- toms remitting and exacerbating with and without antimicrobial treatment. Diagnosis—​this can be difficult as clinical symptoms, radiographic, or histopathological signs, and the results of serological tests may all be misleading. The finding of so-​called sulphur granules is pathog- nomonic: these are macroscopically visible, yellowish, or reddish to brownish particles that exhibit a cauliflower-​like appearance under the microscope at low magnifications, and which may be found as free structures in pus or embedded in affected tissue. Reliable diag- nosis chiefly rests on bacteriological culture but the introduction of MALDI-​TOF technology has allowed better identification in some instances. Treatment and prognosis—​antibacterial drugs used for treatment should be active against both the causative actinomycetes and all concomitant bacteria. For cervicofacial actinomycoses, the rare cutaneous processes, and most thoracic forms of the disease, this requirement is best fulfilled by amoxicillin plus clavulanic acid in medium to high doses: abdominal cases and the presence of un- usually resistant concomitant bacteria may require the addition of further antimicrobials (e.g. an aminoglycoside plus either metronida- zole or clindamycin). The prognosis of cervicofacial and cutaneous actinomycoses is good provided that treatment is adequate; thoracic and abdominal forms are more serious, with grave prognosis without proper treatment. Definition Actinomycoses are sporadically occurring endogenous polymicrobial inflammatory processes in which fermentative (facultatively an- aerobic or capnophilic) actinomycetes of the genera Actinomyces and Propionibacterium, but rarely also Bifidobacterium, may act as the principal pathogens. Clinically, the subacute to chronic, granu- lomatous as well as suppurative disease tends to progress slowly and usually gives rise to multiple abscesses and draining sinus tracts. Because the term ‘actinomycosis’ denotes a polyaetiological inflam- matory syndrome rather than a condition attributable to a single actinomycete species, it should only be used in the plural. Aetiology of human actinomycoses Actinomyces israelii and A. gerencseriae are by far the most frequent and most characteristic pathogens aetiologically involved in the human form of the disease. A. gerencseriae emerged from the former sero-​ and biovariety 2 of A. israelii in 1990. A third species of fila- mentous fermentative Gram-​positive bacteria, Propionibacterium propionicum (formerly Actinomyces propionicus, Arachnia propi- onica), is a much less common cause of actinomycotic infections (Table 8.6.30.1). Several other fermentative actinomycetes have occasionally been isolated from actinomycosis-​like lesions (Table 8.6.30.1). In a given

8.6.30  Actinomycoses 1171 case, however, it is often difficult to decide whether these organisms are primary pathogens or merely contaminants, especially when the specimen has had contact with mucosal secretions or when two different actinomycete species have been isolated from the same specimen (Table 8.6.30.1). Nevertheless, A. naeslundii, A. odonto- lyticus, A.  viscosus, A.  meyeri, and Bifidobacterium dentium (for- merly Actinomyces eriksonii) have all been reported to be capable of producing human infections clinically identical to those caused by A. israelii, A. gerencseriae, or P. propionicum, while A. bovis, the classic agent of bovine actinomycosis, has never been recovered with certainty from human infective processes (Table 8.6.30.1). Fermentative actinomycetes previously termed A.  naeslundii and A. viscosus underwent considerable taxonomic and nomenclatural changes recently (Henssge et al., 2009). According to these changes, organisms now named A. viscosus only occur in animals, particu- larly in hamsters. Human isolates of the former species A. naeslun- dii and A. viscosus have been assigned to A. naeslundii sensu stricto and the new species A. oris and A. johnsonii, but it is difficult to dis- criminate between these three species by routinely used diagnostic procedures. Epidemiology Actinomycoses are not transmissible and cannot be brought under control by vaccination or by measures that prevent spread. Sporadically, they occur worldwide. In Germany, the incidence of the disease was estimated to range from 1 in 40 000 (acute and chronic cases together) to 1 in 80 000 (chronic cases alone) per year, but appears to be decreasing in recent years. Men are affected 2–​4 times more frequently by cervicofacial actinomycoses than are women. However, the male to female ratio appears to vary with age. Although actinomycoses may be found in patients of any age, men are predominantly affected between their 20th and 50th years and women in the second to fourth decade of their lives. Before puberty and in old age, actinomycoses occur spor- adically in patients of both sexes without the pronounced predispos- ition of men. Pathogenesis and pathology Most of the fermentative actinomycetes pathogenic to humans are found regularly and abundantly in the mouths of healthy adults. However, these microbes occur only sporadically or in low numbers in the digestive, respiratory, and genital tracts, as well as in the mouths of babies before teething and of adults without any natural teeth or tooth implants. Therefore, these actinomy- cetes may be considered facultatively pathogenic commensals of the human mucous membranes, which, apart from the very rare actinomycotic wound infections following human bite or fist fight traumata, produce disease exclusively as endogenous pathogens. For active invasion of the tissue, the classic pathogenic fermen- tative actinomycetes apparently require a negative redox potential, which may result either from insufficient blood supply (caused by circulatory or vascular diseases, crush injuries, or foreign bodies) or from the reducing and necrotizing capacity of other microbes in the lesion. Defective functions of the immune system do not spe- cifically predispose to actinomycotic infections. Synergistic polymicrobial infection True actinomycoses are essentially always synergistic mixed infections, in which the actinomycetes act as the specific com- ponent, the so-​called guiding organisms that decide on the characteristic course and the late symptoms of the disease. The so-​called concomitant microbes (Table 8.6.30.2), which may vary considerably in composition (about 100 aerobic and anaer- obic species) and number (up to 10 per case) of species from case to case, are often responsible for the clinical picture at the begin- ning of the infection and for certain complications; they are also part of the resident or transient surface microflora of the mucous membranes of humans. Particularly pronounced synergistic interactions appear to exist between pathogenic fermentative actinomycetes, espe- cially Actinomyces israelii and A.  gerencseriae, and Actinobacillus actinomycetemcomitans, which has recently been reclassified as Table 8.6.30.1  Fermentative actinomycetes isolated from human cervicofacial actinomycotic lesions at the Hygiene-​Institute of the University of Cologne and the Institute for Medical Microbiology and Immunology of the University of Bonn, Germany, between
1985 and 1999 Species identified Number Percentage of cases One species per specimen: A. israelii 421 55.3 A. gerencseriae 111 14.6 A. naeslundii/​A. oris/​A johnsonii 122 16.0 A. odontolyticus 19 2.5 A. meyeri 5 0.7 A. georgiae 1 0.1 A. neuii subsp. neuii 1 0.1 P. propionicum 7 0.9 Bifidobacterium dentium 3 0.4 Corynebacterium matruchotii 12 1.6 Rothia dentocariosa 5 0.7 Not identified to species level 54 7.1 Two species per specimen: A. israelii + A. naeslundii/​A. oris/​A johnsonii 11 0.8 A. israelii + A. meyeri 2 0.1 A. israelii + A. odontolyticus 1 0.1 A. israelii + P. propionicum 2 0.1 P. propionicum + A. naeslundii/​A. oris/​A johnsonii 2 0.1 P. propionicum + A. neuii 1 0.1 Total number of isolates 761 100.0 Modified from Pulverer G, Schütt-​Gerowitt H, Schaal KP (2003). Human cervicofacial actinomycoses: microbiological data of 1997 cases. Clin Infect Dis, 37, 490–​7.

section 8  Infectious diseases 1172 Aggregatibacter actinomycetemcomitans. The latter organism, the species designation of which refers to its characteristic association with actinomycetes (Latin actinomycetem comitans = accompanying an actinomycete), may even sustain the inflammatory process under similar clinical symptoms after chemotherapeutic elimination of the causative actinomycete. Histopathology Initially an inflammatory granulation tissue develops, which usually breaks down to form either an acute abscess or chronic multiple ab- scesses with proliferation of connective tissue. The pathognomonic sulphur granules are formed primarily in the infected tissue, but may also appear as free structures in abscess content or sinus dis- charge. They are then of the highest diagnostic importance. Sulphur granules, which were originally designated Drusen in Harz’s first description of Actinomyces bovis in 1877, are macro- scopically visible (up to 1 mm in diameter) yellowish or reddish to brownish particles that exhibit a cauliflower-​like appearance under the microscope at low magnifications. They consist of a conglom- erate of filamentous actinomycete microcolonies formed in vivo and surrounded by tissue reaction material, especially polymorpho- nuclear granulocytes (Fig. 8.6.30.1). At high magnification, a Gram-​ stained smear of the completely crushed granule reveals the presence of clusters of Gram-​positive interwoven branching filaments with radially arranged peripheral hyphae and of a variety of other Gram-​ positive and Gram-​negative rods and cocci, which represent the concomitant flora (Fig. 8.6.30.2). A  club-​shaped layer of hyaline material may be seen on the tips of peripheral filaments, which can aid in the differentiation of actinomycotic sulphur granules from Table 8.6.30.2  Concomitant actinomycotic flora isolated from cervicofacial actinomycotic lesions at the Hygiene-​Institute of the University of Cologne and the Institute for Medical Microbiology and Immunology of the University of Bonn, Germany, between 1972 and 1999 Species/​group identified Number Percentage of cases Aerobically growing organisms Coagulase-​negative staphylococci 781 39.1 Staphylococcus aureus 99 5.0 α-​Haemolytic streptococci 206 10.3 β-​Haemolytic streptococci 85 4.3 Other aerobically growing bacteria 104 5.2 Candida spp. 22 1.1 No aerobic growth 943 47.2 Anaerobes and capnophils Aggregatibacter (Actinobacillus) actinomycetemcomitans 283 14.2 ‘Microaerophilic’ and anaerobic streptococci 992 49.7 Bacteroides ureolyticus/​Campylobacter gracilis/​ Capnocytophaga spp./​Eikenella corrodens 370 18.5 Black-​pigmented Bacteroidaceae 501 25.1 Other Bacteroides spp. and Prevotella spp. 419 21.0 Fusobacterium spp. 753 37.7 Leptotrichia buccalis 160 8.0 Propionibacterium spp.a 549 27.5 Other anaerobic bacteria 72 3.6 Total number of cases examined 1997 100.0 a Other than P. propionicum. Modified from Pulverer G, Schütt-​Gerowitt H, Schaal KP (2003). Human cervicofacial actinomycoses: microbiological data of 1997 cases. Clin Infect Dis, 37, 490–​7. Fig. 8.6.30.1  Actinomycotic sulphur granule. Particle embedded in 1% methylene blue solution, after gently pressing on the coverslip (original diameter 0.8 mm). Note the spherical segment-​like structures which represent actinomycete colonies formed in vivo and which are coloured brown because the blue dye has been reduced to its leuco base in the anaerobic centre of the particle. The blue-​coloured structures surrounding the colonies are polymorphonuclear granulocytes. Magnification × 60. Fig. 8.6.30.2  Gram-​stained smear prepared from a crushed sulphur granule. The causative actinomycetes appear as Gram-​positive irregularly curved branching filaments which are partially arranged in nest-​like structures. In addition, various other bacteria, particularly Gram-​negative rods and Gram-​positive cocci, can be seen representing the concomitant flora. Magnification ×1200.

8.6.30  Actinomycoses 1173 macroscopically similar particles of various other microbial and non​microbial origins. Clinical manifestations The primary actinomycotic lesion usually develops in tissue adjacent to a mucous membrane at sites such as the cervicofacial, thoracic, and abdominal areas. The infection tends to progress slowly and to penetrate without regard to natural organ borders, or to spread haematogenously even to distant sites. Remission and exacerbation of symptoms with and without antimicrobial treatment is charac- teristic. As in other endogenous microbial diseases, the incubation period of actinomycoses is not defined. Cervicofacial actinomycoses In the most cases, actinomycotic lesions primarily involve the face or neck. Conditions predisposing to these cervicofacial infections include tooth extractions, fractures of the jaw, periodontal ab- scesses, foreign bodies penetrating the mucosal barrier (bone splin- ters, fish bones, awns of cereals), or suppurating tonsillar crypts. Initially, the cervicofacial actinomycoses present either as an acute, usually odontogenic, abscess or cellulitis of the floor of the mouth, or as a slowly developing chronic hard painless reddish or livid swelling. Small acute actinomycotic abscesses may heal after surgical drainage alone. More often, however, the acute initial stage is followed by a subacute to chronic course if no specific antimicro- bial treatment is given, thereby imitating the primarily chronic form, which is characterized by regression and cicatrization of cen- tral suppurative foci while the infection progresses peripherally pro- ducing hard painless livid infiltrations. These may lead to multiple new areas of liquefaction, fistulae (Fig. 8.6.30.3), which often dis- charge pus containing sulphur granules, and multilocular cavities with poor healing and a tendency to recur after temporary regres- sions of the inflammatory symptoms. With inappropriate or no treatment, cervicofacial actinomycoses extend slowly, even across organ borders, and may become life-​threatening by invasion of the cranial cavity, the medias- tinum, or the bloodstream. In contrast, the so-​called (peri)apical actinomycosis which is clinically indistinguishable from common apical periodontitis and which has been accused of being respon- sible for lack of healing after endodontic treatment or tooth implant surgery, essentially always remains localized, responds to usual peri- odontitis treatment, and represents no serious threat to the patient’s health or life. This condition should, therefore, not be termed ‘actinomycosis’, but possibly ‘actinomycete periodontitis’. Thoracic actinomycoses Thoracic manifestations, which are much less common than the cervicofacial form (Table 8.6.30.3), usually develop after aspiration or inhalation of material from the mouth (dental plaque or calculus, tonsillar crypt contents) or a foreign body that contains or is con- taminated with the causative agents. Occasionally, this form of dis- ease may result from extension of an actinomycotic process of the neck, from an abdominal infection perforating the diaphragm, or from a distant focus by haematogenous spread. Primary pulmonary actinomycoses present as bronchopneumonic infiltrations that may imitate tuberculosis or bronchial carcinoma radiographically, appearing as single dense or multiple spotted shadows in which cavitations may develop (Fig. 8.6.30.4). If not diagnosed and treated properly, pulmonary infections may extend through to the pleural cavity producing empyema, to the pericar- dium, or to the chest wall; they may even appear as a paravertebral (psoas) abscess tracking down to the groin. Detailed aetiology, patho- genesis, and clinical relevance of a condition termed ‘endobronchial actinomycosis’ remain to be definitely clarified. Abdominal actinomycoses Actinomycoses of the abdomen and pelvis are rare (Table 8.6.30.3). They originate either from acute perforating gastrointestinal dis- eases (appendicitis, diverticulitis, various ulcerative diseases), from surgical or accidental trauma including injuries caused by ingested bone splinters or fish bones, or from inflammations of the female internal genital organs. Women with have intrauterine contraceptive devices or vaginal pessaries for long periods often show a characteristic colonization of the cervical canal and the uterine cavity, but particularly of the thread of the intrauterine contraceptive device, by various fermen- tative actinomycetes and other anaerobes resembling the synergistic Fig. 8.6.30.3  Primary chronic cervicofacial actinomycosis with several draining sinus tracts in a 42-​year-​old man. Table 8.6.30.3  Localization of human actinomycotic infections Body site involved Number Percentage of cases Cervicofacial area 3197 97.9 Thoracic organs 41 1.3 Abdominal organs including small pelvis 20 0.6 Extremities 4 0.1 Central nervous system 4 0.1 Total number of cases 3266 100.0 Modified from Schaal KP, Pulverer G (1984). Epidemiologic, etiologic, diagnostic,
and therapeutic aspects of endogenous actinomycete infections. In: Ortiz-​Ortiz L,
Bojalil LF, Yakoleff V (eds) Biological, biochemical, and biomedical aspects of actinomycetes, pp. 13–​32. Academic Press, Orlando.

section 8  Infectious diseases 1174 actinomycotic flora. However, this colonization only rarely results in an invasive actinomycotic process. Most abdominal actinomycoses present as slowly growing tu- mours, which, in the absence of sinus tracts discharging pus with sulphur granules, are difficult to differentiate from malignant neo- plasms such as colonic, rectal, ovarian, or cervical carcinomas. By direct extension, any abdominal tissue or organ may be involved including muscle, liver, spleen, kidney, fallopian tubes, ovaries, testes, bladder, or rectum. This can present as a ‘frozen’ pelvis. Haematogenous liver abscesses have been seen, especially associated with genital actinomycoses. Actinomycotic infections of the central nervous system Actinomycoses of the brain and the spinal cord are very rare. They may arise from direct extension of cervicofacial infections. Haematogenous spread is also possible, particularly from primary lesions in the lungs or abdomen. The spinal canal may be directly involved from these sites. Brain abscess is much more common than meningitis. Actinomycoses of the bone In contrast to bovine actinomycosis which usually affects the skel- eton, bone involvement in humans is very rare. It usually develops by direct extension from soft tissue infection resulting in a peri- ostitis with new bone formation visible by radiography. If the bone itself is invaded, localized areas of bone destruction surrounded by increased bone density usually develop. Mandible, ribs, and spine are most frequently involved. Osteonecrosis of the jaw, seen with bisphosphonates, and radionecrosis may predispose to actinomycoses. Actinomycotic endocarditis Endocarditis due to fermentative actinomycetes has occasionally been described. However, detailed bacteriological information on this condition is not yet available so that it remains to be seen whether it may rightly be termed actinomycosis or has merely to be considered an aetiological variant of the common form of endo- carditis caused by indigenous oral microbes. Cutaneous actinomycoses Actinomycotic lesions of the skin are extremely rare. Usually, they originate from wounds that were contaminated with saliva or dental plaque following human bites or fist fights, but they may also re- sult from haematogenous spread. Symptoms are similar to those of cervicofacial actinomycoses. Diagnosis Clinical symptoms are often misleading, especially in the early stages of the disease, histopathological appearances are unreliable, and diagnosis chiefly rests on bacteriological methods. Radiography In cervicofacial cases, radiography is useful only for detecting bone involvement. A pulmonary infiltrate associated with a pro- liferative lesion or destruction of ribs is highly suggestive of either actinomycosis or a tumour. Radiography may also help to locate the abdominal processes and to identify the involvement of organs such as liver, kidney, urinary bladder, or ureter. In general, however, radiographic changes are not diagnostic. Laboratory diagnosis Clinical chemistry and haematology Small, localized actinomycotic lesions are not usually associated with abnormalities. In advanced cases, however, especially those in the thoracic or the abdominal area, a raised erythrocyte sedimen- tation rate and pronounced leucocytosis may be seen. When the central nervous system is involved, a polymorphonuclear or mono- nuclear pleocytosis is commonly found. The protein content of the cerebrospinal fluid is frequently elevated and the sugar content moderately depressed. Bacteriology Pus specimens containing sulphur granules and occasionally looking like semolina should prompt the clinician to ask, and the bacteriologist to look specifically for, actinomycetes using suitable cultural techniques and other methods. Pus, sinus discharge, bronchial secretions, granulation tissue, or biopsy materials are suitable specimens. Precautions must be taken to prevent contamination of the specimen by the indigenous mucosal flora. In cases of cervicofacial actinomycoses, pus should therefore be obtained only by transcutaneous puncture of the ab- scesses or by transcutaneous needle biopsy after thorough skin disinfection. When abscesses have already been incised, a suffi- cient amount of pus should be collected instead of using only a swab. Because sputum always contains oral actinomycetes, bron- chial secretions should be obtained by transtracheal aspiration, or Fig. 8.6.30.4  Chest radiograph of pulmonary actinomycosis of the right upper lobe in a 62-​year-​old man. Initially, the disease was mistaken for bronchial carcinoma. It was diagnosed only after a huge subcutaneous abscess had developed covering the whole right shoulder blade.

8.6.30  Actinomycoses 1175 material should be collected by transthoracic percutaneous needle biopsy. Percutaneous puncture of suspected abscesses, possibly under radiological control, is often the only way of obtaining suit- able specimens for diagnosing abdominal actinomycoses. The transport of specimens to the bacteriological laboratory should be as fast as possible, preferably by messenger. Alternatively, a reducing transport medium such as one of the modifications of Stuart’s medium should be used. The specimen should arrive in the laboratory within 24 h, although it has occasionally proved possible to isolate actinomycetes from samples that took 7 days or more to get to the diagnostic laboratory by post. A quick and comparatively reliable tentative diagnosis is possible microscopically when sulphur granules are present (Fig. 8.6.30.1). The demonstration of concomitant bacteria in Gram-​stained smears prepared from crushed granule material (Fig. 8.6.30.2) allows the differentiation of actinomycotic granules from similar particles produced by Nocardia spp., Actinomadura spp., or Streptomyces spp. Use of transparent culture media and careful microscopic exam- ination of the cultures, preferably on Fortner plates, after at least 2, 7, and 14 days of incubation enables a specialized laboratory to detect possible actinomycete colonies and to subculture them for identification. Isolation and definite identification to the spe- cies level may require a further 1 to 2 weeks. Techniques such as the application of gene probes or the polymerase chain reaction for detecting and identifying fermentative actinomycetes directly in clinical samples are not yet widely used; however, sequencing of the 16S rRNA gene often helps to identify actinomycete cultures to spe- cies level although this technique is intrinsically a phylogenetic and taxonomic rather than a diagnostic tool. The use of MALDI-​TOF (matrix-​assisted laser desorption ionization time-​of-​flight) mass spectrometry to identify actinomycetes is promising and might allow identification of branching bacteria that have been misiden- tified previously. Serological diagnosis None of the routine serological methods has yet provided satis- factory results because sensitivity and specificity have been found to be too low. Treatment As the aetiology of human actinomycoses is always polymicrobial, the antibacterial drugs used for treatment should in principle cover both the causative actinomycetes and all of the concomi- tant bacteria. This usually requires the administration of drug combinations in which aminopenicillins currently represent the therapeutic basis because they are slightly more active against the pathogenic actinomycetes than is penicillin G and because they are able to inhibit Aggregatibacter (Actinobacillus) actinomycetem- comitans which is usually resistant to narrow-​spectrum penicil- lins. However, the presence of concomitant β-​lactamase producers such as Bacteroides fragilis, B. thetaiotaomicron, or Staphylococcus aureus (β-​lactamase producing) may impair the therapeutic effi- cacy of aminopenicillins and that of many other β-​lactams so that the combination with a β-​lactamase inhibitor is advisable or even necessary. For cervicofacial actinomycoses, amoxicillin plus clavulanic acid has proved to be the treatment of choice. Three doses of 2.0 g amoxi- cillin plus 0.2 g clavulanic acid every day for 1 week and three doses of 1.1 g of the combination for an additional 7 days usually result in complete cure. Thoracic actinomycoses mostly respond to the same regimen. However, it is advisable to maintain doses of 2.2 g 3 times a day for 2 weeks, and to continue treatment for 3 to 4 weeks. Advanced pulmonary cases may require the addition of 2 g ampi- cillin three times a day in order to increase the tissue concentration of aminopenicillin and, depending on the composition of the con- comitant flora, the use of an antimicrobial specifically active against resistant Enterobacteriaceae; the application of drugs such as metro- nidazole or clindamycin against strict anaerobes is only necessary as an adjunct to the aminopenicillins in chronic cases with reduced blood supply. Since in abdominal actinomycoses Enterobacteriaceae and β-​ lactamase producing Bacteroides spp. are usually present and the correct diagnosis is mostly established late, suitable antimicrobial combinations for these cases are amoxicillin plus clavulanic acid plus metronidazole plus tobramycin (gentamicin) or ampicillin plus clindamycin plus an aminoglycoside. Meropenem might also be a good choice, but this drug has not yet been widely used for treating actinomycotic infections. Neither clindamycin nor metronidazole should be used alone. Clindamycin is almost completely ineffective against Aggregatibacter (Actinobacillus) actinomycetemcomitans and metronidazole shows no activity at all against pathogenic actinomycetes. The use of fur- ther combinations, including additional aminoglycosides, ceph- alosporins, or β-​lactamase-​stable penicillins, may be necessary depending on the presence of unusual aerobic organisms. In pa- tients allergic to penicillin, tetracyclines or possibly cephalosporins may be tried instead of aminopenicillins. Incision of abscesses and drainage of pus might still be necessary as an adjunct to the anti- microbial chemotherapy and may help to accelerate recovery and to decrease the risk of relapses. Prognosis The prognosis of cervicofacial and cutaneous actinomycotic infec- tions is good provided that the diagnosis is established early and antimicrobial treatment is adequate. However, thoracic, abdominal, and systemic manifestations remain serious conditions that require all possible diagnostic and therapeutic efforts. Without proper treat- ment, the prognosis is grave. Other diseases caused by fermentative actinomycetes Fermentative actinomycetes play some part in dental caries and periodontal disease, but are clearly not the most important mi- crobes contributing to these important health problems. Lacrimal canaliculitis with and without conjunctivitis is commonly caused by fermentative actinomycetes, in particular P. propionicum, but less frequently also by Actinomyces israelii, A. gerencseriae, A. naeslundii, A. oris, or A. odontolyticus. The concomitant flora, when present, is usually less complex than that of typical actinomycoses. Removal of

8.6.31 Nocardiosis 1176

8.6.31 Nocardiosis 1176

section 8  Infectious diseases 1176 the lacrimal concretions that are usually present and local applica- tion of antimicrobials always result in prompt cure. Trueperella pyogenes (formerly Arcanobacterium, Actinomyces, or Corynebacterium pyogenes, respectively) and Arcanobacterium haemolyticum (formerly Corynebacterium haemolyticum) cause acute pharyngitis, urethritis, cutaneous or subcutaneous suppur- ations, or bacteraemia. The recently described species A. graevenit- zii, A. europaeus, A. radingae, A. turicensis, A. funkei, A. cardiffensis, A. hongkongensis, A. oricola, A. urogenitalis, A. dentalis, A. mas- siliensis, A.  timonensis, and A.  hominis, as well as Trueperella (Arcanobacterium, Actinomyces) bernardiae, Actinobaculum schaa- lii, and Varibaculum cambriense have been isolated from various clinical sources including abscesses and blood cultures, and may also be associated with mixed bacterial flora. A. neuii subsp. neuii and A. neuii subsp. anitratus are most frequently involved aetio- logically in abscesses and infected atheromas, but may also cause infections of skin structures, endophthalmitis, and bacteraemias including endocarditis. A.  turicensis and possibly A.  urogenitalis seem to be particularly common in genital infections, while A. rad- ingae was found only in patients with skin-​related pathologies and A. nasicola was isolated from pus from the nasal antrum. A. euro- paeus, A. turicensis, and A. urogenitalis as well as Actinobaculum schaalii, A. urinale, and A. massiliense were detected in predomin- antly elderly patients with urinary tract or bloodstream infections, and A. radicidentis was isolated from infected root canals of teeth. FURTHER READING Bonnefond S, et al. (2016). Clinical features of actinomycosis: a retro- spective, multicentre study of 28 cases of miscellaneous presenta- tions. Medicine (Baltimore), 95, e3923. Hall V (2011). Genus V. Varibaculum Hall, Collins, Lawson, Hutson, Falsen, Inganäs and Duerden 2003, 644VP. In:  Whitman WB,
et al. (eds) Bergey’s manual of systematic bacteriology, 2nd edition, Vol. 5: Actinobacteria, pp. 139–​40. Springer-​Verlag, Dordrecht. Henssge U, et  al. (2009). Emended description of Actinomyces naeslundii and description of Actinomyces oris sp. nov and Actinomyces johnsonii sp. nov., previously identified as Actinomyces naeslundii genospecies 1, 2 and WVA 963. Int J Syst Evol Microbiol, 59, 509–​16. Lawson PA (2011). Genus II. Actinobaculum Lawson, Falsen, Åkervall, Vandamme and Collins 1997, 902VP. In: Whitman WB, et al. (eds) Bergey’s manual of systematic bacteriology, 2nd edition, Vol. 5: Actinobacteria, pp. 109–​14. Springer-​Verlag, Dordrecht. McNeil MM, Schaal KP (1998). Actinomycoses. In: Yu VL, Merigan TCJr, Barriere SL (eds) Antimicrobial therapy and vaccines, pp. 14–​22. Williams and Wilkins, Baltimore, MD. Ng LSY, et  al. (2012). Comparison of phenotypic methods and matrix-​assisted laser desorption ionisation time of flight mass spectrometry for the identification of aero-​tolerate Actinomyces spp. Isolated from soft-​tissue infections. Eur J Clin Microbiol Infect Dis, 31, 1739–​52. Pulverer G, Schütt-​Gerowitt H, Schaal KP (2003). Human cervicofacial actinomycoses: microbiological data of 1997 cases. Clin Infect Dis, 37, 490–​7. Schaal KP, Lee HJ (1992). Actinomycete infections in humans: a re- view. Gene, 115, 201–​11. Schaal KP, Pulverer G (1984). Epidemiologic, etiologic, diagnostic, and therapeutic aspects of endogenous actinomycete infections. In: Ortiz-​Ortiz L, Bojalil LF, Yakoleff V (eds) Biological, biochemical, and biomedical aspects of actinomycetes, pp. 13–​32. Academic Press, Orlando, FL. Schaal KP, Yassin AF (2011). Family I. Actinomycetaceae Buchanan 1918, 403, emend. Stackebrandt, Rainey and Ward-​Rainey 1997, 484. In: Whitman WB, et al. (eds) Bergey’s manual of systematic bac- teriology, 2nd edition, Vol. 5: Actinobacteria, pp. 36–​42. Springer-​ Verlag, Dordrecht. Schaal KP, Yassin AF (2011). Genus I. Actinomyces Harz 1877, 133AL, emend. Georg, Pine and Gerencser 1969, 292VP. In: Whitman WB, et al. (eds) Bergey’s manual of systematic bacteriology, 2nd edition, Vol. 5: Actinobacteria, pp. 42–​109. Springer-​Verlag, Dordrecht. Schaal KP, Yassin AF, Stackebrandt E (2006). The family Actinomycetaceae: the genera Actinomyces, Actinobaculum, Arcanobacterium, Varibaculum, and Mobiluncus. In:  Balows A, et  al. (eds) The prokaryotes. A  handbook on the biology of bacteria:  ecophysiology, isolation, identification, applications,
2nd edition, Vol. 1, pp. 850–​905. Springer, Berlin. von Graevenitz A (2011). Actinomyces neuii: review of an unusual in- fectious agent. Infection, 39, 97–​100. Yassin AF, et  al. (2011). Comparative chemotaxonomic and phylo- genetic studies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. Int J Syst Evol Microbiol, 61, 1265–​74. Yassin AF, Schaal KP (2011). Genus III. Arcanobacterium Collins, Jones and Schofield 1983, 438VP. In: Whitman WB, et al. (eds) Bergey’s manual of systematic bacteriology, 2nd edition, vol. 5: Actinobacteria, pp. 114–​26. Springer-​Verlag, Dordrecht. Zelyas N, et al. (2016). Infections caused by Actinomyces neuii: a case series and review of an unusual bacterium. Can J Infect Dis Med Microbiol, 2016, 6017605. 8.6.31  Nocardiosis Roderick J. Hay ESSENTIALS Nocardia species—​Nocardia asteroides, N.  brasiliensis, and N. otidiscaviarum—​are Gram-​positive, filamentous, partially acid-​fast bacteria. They are occasionally detectable in environmental sources such as soil, but they rarely cause infections in humans, although they can give rise to a variety of different diseases. In healthy individ- uals, most commonly in the tropics, they can present with cutaneous abscesses or subcutaneous infections (actinomycetoma) in which the organisms are present as clusters of filaments or grains. In im- munocompromised patients they cause a disseminated or localized deep infection, with particular sites affected being the lungs or brain. Diagnosis of Nocardia infection depends on culture, although histo- pathology is very useful in Nocardia actinomycetomasiii. Antibiotic treatment is typically with a sulphonamide (often as co-​trimoxazole for lung infections), but combinations of drugs are usually given be- cause the responsiveness of Nocardia species is very variable.

8.6.31  Nocardiosis 1177 Introduction Nocardiosis (nocardiasis) is the infection caused by Nocardia species; over 80 different species are now known, many of which cause human infection. Nocardia farcinica, N.  brasiliensis, and the N. nova, N. abscessus, and N. transvalensis complexes are the most common. N. farcinica appears to be more virulent than the others as it is more likely to present with disseminated disease and is more resistant to antimicrobials. Molecular studies indicate that N.  brasiliensis, N.  otitidiscavarium, and N.  transvalensis exhibit diverse characteristics and it is anticipated that new species will continue to emerge. Nocardia asteroides once the name of the most common Nocardia is still a cause of disease but many of the isolates previously identified N. asteroides have been transferred to the newer species. Nocardiosis most commonly affects the lungs (39% of cases) but may be systemic (≥2 sites involved, 32%), involve the central nervous system (9%); cutaneous (8%), or occur at a single extrapulomonary site (e.g. eyes, bone; 12%). The nocardia are Gram-​positive filamentous branching bacteria that ramify in infected tissues. They can also break up into bacil- lary forms and, in some conditions, aggregate into grains typical of mycetomas. These organisms are aerobic and partially acid fast. They grow readily on ordinary laboratory media. Pathogenesis Nocardia are found in soil, particularly where there is decaying vegetation, and in aquatic environments. They can also be isolated from the air and, in most cases, systemic infection is by the airborne route; rarely nocardiosis can be acquired after inoculation into the skin. The characteristic histopathological response to infection is the production of polymorphonuclear leucocyte abscesses without ex- tensive fibrosis. Caseation and palisading granulomas are not gener- ally seen. Dissemination to other organs such as brain and skin can occur. By contrast, in primary cutaneous infections the lesion is usu- ally localized to an abscess containing filaments at the site of inocu- lation and is accompanied by local lymphadenopathy. Mycetoma grain formation may occur in some of these infections that follow inoculation. It is not known why, in some patients, transcutaneous infection with nocardia results in the development of a mycetoma whereas in others a subcutaneous abscess containing filaments is formed. The formation of mycetomas appears to be more common with N. brasiliensis infections. Epidemiology In the early 1970s the incidence of nocardiosis was estimated to be 500 to 1000 cases per year; this is likely to be an underestimate as nocardiosis is not a notifiable disease. The current incidence is likely to be much higher as the number of immunocompromised indi- viduals at risk for nocardiosis (e.g. transplant recipients) increases. Otherwise healthy patients may be infected by Nocardia, although the frequency of subclinical exposure and sensitization in normal populations is unknown. However, most patients with systemic nocardiosis are immunocompromised, most commonly with a con- dition that affects the expression of T-​lymphocyte-​mediated im- mune responses. Underlying conditions include: • malignancies, including cancer and lymphoma • HIV infection and other immunodeficiency states such as chronic granulomatous disease • solid organ transplantation • other conditions that require high doses of corticosteroids, such as collagen vascular disease and rheumatoid arthritis • pre-​existing pulmonary disease; alveolar proteinosis, in particular, seems to predispose to nocardiosis • tumour necrosis factor α inhibitors Inhalation of the organism is thought to be the most common route of infection and is supported by evidence that most infections involve the lung. Other modes of entry include ingestion of con- taminated food, direct inoculation into the skin (causing cutaneous disease), and nosocomial transmission (e.g. a report of a cluster of postoperative sternal wound infection caused by N. farcinica). The usual site of primary infection is the lung and the disease may remain restricted to this site. It may also be disseminated to other organs, particularly to the brain and skin. Nocardiosis can occur at any age, although it is rare, particularly in childhood. Clinical features Primary cutaneous nocardiosis This is an uncommon infection that appears to follow traumatic inoculation of organisms into a superficial abrasion. The usual primary lesion is a small nodule, ulcer, or abscess at the site of in- oculation. There may be a small chain of secondary nodules (as in sporotrichosis, see Chapter 8.7.1) along the course of a lymphatic and local lymphadenopathy is common (Fig. 8.6.31.1). Some such cases resolve spontaneously. This form of disease can be caused by a variety of different species Nocardia mycetoma This is discussed in Chapter 8.7.1; N. brasiliensis is the usual cause. Fig. 8.6.31.1  Extensive chronic nocardiosis at site of injury in a
27-​year-​old Peruvian man, Instituto de Medicina Tropical ‘Alexander
von Humboldt’, Universidad Peruana Cayetano Heredia, Lima, Peru. Copyright D. A. Warrell.

section 8  Infectious diseases 1178 Pulmonary nocardiosis Pulmonary infection is seen in about 75% of cases of systemic nocardiosis, even where there are disseminated lesions elsewhere. Symptoms of pulmonary nocardiosis are variable with cough, fever, and leucocytosis. In otherwise healthy individuals the changes and signs may be very similar to pulmonary tuberculosis, whereas in the immunocompromised patient the lesions present as rapidly developing single or multiple lung lesions. In HIV-​infected patients symptoms are often minimal, even in the presence of extensive dis- ease. These changes are reflected by the course of the disease. In some patients progression is rapid, in others it is chronic. Chest radiographs may show segmental or lobar infiltrates, cavi- tation, nodules, or diffuse miliary infiltrates; endobronchial infec- tion has been recorded. Calcification is not common. The infection may spread locally to involve adjacent structures such as the pleural space and diaphragm or may spread to other sites. Very occasionally, Nocardia spp. can be isolated from sputum of otherwise healthy pa- tients. Whether this reflects the process of asymptomatic sensitiza- tion is not known. Disseminated nocardiosis Haematogenous spread is seen in the immunocompromised pa- tient and may occur without evidence of pulmonary infection. The most common site for dissemination is the brain where it presents with localized abscesses without meningeal involvement. The signs are those due to an intracerebral space-​occupying lesion. Spread to other sites is less common, although dissemination to skin, liver, kidneys, and bone may occur. The acute disseminated forms and those with involvement of the central nervous system have the worst prognosis. Continued therapy with corticosteroids also appears to have bad prognostic signifi- cance. Infection in HIV-​infected patients may not be recognized before death. Rapid diagnosis is therefore a key to successful man- agement. By contrast, pulmonary infection in otherwise healthy pa- tients is usually a chronic process and has to be distinguished from tuberculosis. Laboratory diagnosis The infection is often recognized initially by direct microscopy of pus, bronchial washings, or tissue. In Gram’s stains the organisms can be shown as fine branching filaments, although distinction from other bacteria may be difficult if short rod-​like forms predominate. A modified acid-​fast stain using weak acid can be used to demon- strate filaments. Nocardia grow on ordinary media aerobically but require pro- longed incubation as colonies may take 5 to 21 days to appear. The laboratory should be informed if nocardiosis is suspected as cul- tures will need prolonged incubation. Growth may be enhanced by the use of selective media such as buffered charcoal yeast ex- tract and Thayer–​Martin medium. Nocardia have variable colonial morphology varying from chalky white to yellow, orange, or brown colonies. Speciation of nocardia using conventional phenotypic methods is difficult and time-​consuming. Polymerase chain reaction for identification of nocardia species is more rapid and accurate than the phenotypic tests but is not available in routine diagnostic laboratories. Antimicrobial susceptibility testing should be performed for all clinically significant isolates. The optimal method is the broth microdilution method, but minimum inhibitory concentrations may be difficult to interpret. Histopathological examination is useful in some cases. Filaments stain with modified acid-​fast stains using an aqueous solution of a weak acid for decolourization, but can also be highlighted with the methenamine–​silver stain (Grocott’s modification). The branching nature of the organism is best appreciated in histopathological ma- terial. Other pathogens such as Pneumocystis spp. may also be pre- sent in histopathological material. Serological tests (usually counterimmunoelectrophoresis or en- zyme immunoassay) can be obtained in reference centres and are generally used to monitor the progress of therapy rather than estab- lish the diagnosis. Therapy The mainstays of therapy are sulphonamides, and the first choice is often co-​trimoxazole, particularly in pulmonary forms, al- though it still unclear how helpful the trimethoprim component is, particularly in intracerebral infections. In many cases, drainage of abscesses may hasten recovery. Some species of Nocardia often do not respond as well to particular antibiotics; N. otitidiscavi- arum, for instance, is generally resistant to cotrimazole. Much of the recommended drug therapy is derived from the personal ex- periences of a few cases. However, wherever possible sensitivity testing is advised. Other drugs that have been used include amikacin, ampicillin, cefotaxime, imipenem, linezolid, moxifloxacin, and minocycline. Experience of other drugs is similarly limited. It is the general prac- tice to use two antibiotics eg cotrimoxazole and cefotaxime for nocardiosis. Clustering of cases may occur occasionally, suggesting exposure to a common source of infection. In two such episodes there had been extensive construction work in the vicinity of the hospital in- volved. At present, no methods of prevention are known, although the existence of more than two cases in a single or adjacent wards should alert clinicians to the possibility of environmentally acquired infection. FURTHER READING Boiron P, et al. (1992). Review of nocardial infections in France, 1987–​ 1990. Eur J Clin Microbiol Infect Dis, 11, 709–​14. Brown-​Elliott BA, Conville P, Wallace RJ (2015). Current status of Nocardia taxonomy and recommended identification methods. Clin Microbiol Newsletter, 37, 25–​32. Filice GA (2005). Nocardiosis in persons with human immunodefi- ciency virus infection, transplant recipients, and large, geographic- ally defined populations. J Lab Clin Med, 145, 156–​62. Georghiou PR, Blacklock ZM (1992). Infection with Nocardia spe- cies in Queensland: a review of 102 clinical isolates. Med J Aust, 156, 692–​7.

1179 8.6.32  Rat bite fevers (Streptobacillus moniliformis and Spirillum minus infection) Hay RJ (1983). Nocardial infections of the skin. J Hyg (Lond), 91, 385–​91. Houang ET, et al. (1980). Nocardia asteroides infection: a transmissible disease. J Hosp Infect, 1, 31–​6. Kilincer C, et al. (2006). Nocardial brain abscess: review of clinical management. J Clin Neurosci, 13, 481–​5. Sakai C, Takagi T, Satoh Y (1999). Nocardia asteroides pneumonia, subcutaneous abscess and meningitis in a patient with advanced malignant lymphoma: successful treatment based on in vitro anti- microbial susceptibility. Intern Med, 38, 683–​6. Wilson JW (2012). Nocardiosis: updates and clinical overview. Mayo Clin Proc, 87, 403–​7. 8.6.32  Rat bite fevers (Streptobacillus moniliformis and Spirillum minus infection) Andrew F. Woodhouse ESSENTIALS Rat bite fever is usually attributable to Streptobacillus moniliformis in the Americas, Europe, and Australasia, and to Spirillum minus in Asia. Bites are increasingly common among children with pet rats, and pet shop and laboratory workers. Both bacteria are com- mensals of rats, some other rodents, and their predators. After an incubation period less than 1 week, S. moniliformis causes sudden high fever, rigors, myalgia, petechial rash, and migratory reactive or septic polyarthritis with synovial effusions. Complications can in- clude fulminant septicaemia, endocarditis, pneumonia, and meta- static abscesses. S. minus infection (sodoku) has a longer incubation period with similarly high fever but concomitant exacerbation of the bite wound, local lymphadenopathy, papular rash, and arth- ralgia without effusions. In both diseases, fever subsides after a few days but may relapse repeatedly over months. Untreated, mor- tality is about 10% for S. moniliformis and 2 to 10% for S. minus. S. moniliformis can be cultured (with some difficulty) and the diag- nosis confirmed by molecular methods. S. minus infection cannot be confirmed by culture or serology, but the organism may be visualized by microscopy in involved tissue and sometimes blood. Penicillin is the treatment of choice for both infections. Prevention is by controlling peri-​domestic rats and avoiding bites by pet or laboratory rodents. Introduction The rat bite fevers (RBF) are zoonotic infections of humans caused by one of two distinct bacteria—​Streptobacillus moniliformis or Spirillum minus. The illnesses caused by these organisms have overlapping features but are also distinct in several ways. Febrile illness developing after rat bites has been recognized for centuries and the risk of infection after a bite is reported to be as high as 10%. Despite this these infections remain rare and underdiagnosed due to the non​specific nature of their clinical presentation. This is compounded by the difficulty of microbiological confirmation of infection. Rats are one of the most populous mammalian species on earth and human interaction with wild rats is increasing, particularly where rat infestation occurs in deprived urban and rural commu- nities. In addition, the popularity of rats as pets and their use as laboratory animals has led to more human-​animal interaction and greater likelihood of bites, scratches, and other forms of contact in more developed and affluent settings. Antibiotic treatment is generally effective and while untreated disease can be self-​limiting, reported mortality of up to 10% for both forms of RBF is described. Important complications include septicaemia with multiorgan failure, endocarditis, pericarditis, meningitis, deep organ abscess development, and osteoarticular infections. Aetiology Streptobacillus moniliformis is the cause of streptobacillary RBF and Haverhill fever. It is a non​motile, filamentous Gram-​negative bacillus. It is fastidious and microaerophilic, requiring blood, serum, or ascites enriched tryptone soy agar or broth to optimize growth. Spirillum minus is the other causative agent of rat bite fever, re- sponsible for the febrile syndrome known in Japan as Sodoku (de- rived from So = rat and doku = poison). S. minus is a tightly coiled Gram-​negative rod, motile by virtue of terminal flagellae. It is a spir- illum and has not been successfully cultured on artificial media but can be transferred to mice or guinea pigs by inoculation of infected tissue. The inability to grow the organism in vitro makes confirma- tory diagnosis difficult. Streptobacillus moniliformis is found in the oropharynx of most wild rats and a significant proportion of pet and laboratory rats. It has also been isolated from other rodents and animals that prey on rats, broadening the potential pool of infective sources. Spirillum minus is also a commensal of rats, estimated to be found in 25% of animals and has been found in blood and oral and respiratory tissue and secretions. Transmission of both organisms to humans occurs most often via a contaminated bite wound, but transmission without a bite is possible. Infection can occur via scratches or mucosal contact after handling either live or dead animals. Human in- fection is usually associated with rat contact, but other rodents and animals have been occasionally implicated as sources of S. moniliformis infection. Ingestion of food or fluids (e.g. water or unpasteurized milk) contaminated with S. moniliformis can lead to the form of RBF known as ‘Haverhill fever’, named after the town in Massachusetts where an outbreak of the disease was first described in 1926.

section 8  Infectious diseases 1180 Epidemiology Streptobacillus moniliformis infection occurs worldwide causing streptobacillary rat bite fever. Based on published series and case re- ports it is a more common cause of RBF than Spirillum minus in Europe, Australasia, and the Americas. Rat bite fever due to S. minus is mostly found in Asia, particularly Japan, although several case reports exist from other continents suggesting the distribution is probably worldwide. Understanding the extent of the problem of these zoonotic infections is limited by a lack of systematic reporting and data collection. In regions where rats infest human habitats, rat bites might occur at night during sleep and might not always be appreciated, with children at particular risk. Owners of pet rats, pet shop, and labora- tory workers are groups recognized to be at increasing risk of rat bite fever. Clinical features An abrupt onset systemic illness is the hallmark of the rat bite fevers. Considering the diagnosis when evaluating patients with fever and rash, and the features outlined next, is important for early recog- nition and treatment. Asking appropriate questions about potential exposures to wild or domesticated rats, directly or indirectly can be key. A history of a definite bite might not be forthcoming, but this does not rule out the diagnosis. Clinical features of Streptobacillus moniliformis infection Streptobacillary RBF presents after a short incubation, typically three to ten days after exposure. If a bite has occurred, there is usu- ally only minor evidence of the wound. Illness is sudden in onset with high fever and rigors, headache, nausea, vomiting, and my- algia. Rash occurs within the first week of illness in up to 75% of patients. The rash is varied and can be maculopapular, vesicular (sometimes with haemorrhage), petechial, or pustular. Usually sym- metrical, it involves the limbs and often the palms and soles, which is a helpful clinical feature to aid early diagnosis. Desquamation oc- curs in about a fifth of cases. Joint involvement is common early after onset of fever with about half of patients developing a migra- tory polyarthralgia or arthritis involving large and small joints. The arthritis is often reactive but true septic arthritis can occur. Haverhill fever, (erythema arthriticum epidemicum) is similar to the illness described earlier, but vomiting and pharyngitis are more prominent symptoms in this form of RBF compared to that fol- lowing bite or mucosal inoculation. Clinical features of Spirillum minus infection In spirillary rat bite fever, the bite wound is a prominent feature of the early illness. After initial healing the area becomes swollen and painful with induration and purple discoloration after an incuba- tion period of between one and four weeks. This is associated with regional lymphadenopathy and onset of systemic symptoms with fever, headache, and malaise. The bite lesion becomes ulcerated and a generalized rash appears during the first week of symptoms, usually macular and violaceous or red-​brown and involving the face and trunk as well as limbs. Arthritis and myalgia are uncommon, in contrast to streptobacillary RBF. Complications Both forms of RBF can be complicated by endocarditis but it is rare and is almost always seen in people with pre-​existing valve dis- orders. A range of other complicating infections have been described including meningitis, brain and epidural abscess, spondylodiscitis, pneumonia, myopericarditis and focal abscesses, mostly associated with S. moniliformis infection. Relapsing fever If untreated both types of RBF can cause a relapsing fever. After the initial fevers resolve over a week or so, relapse may occur over intervals of days to months with S. minus infections tending to have longer fever-​free periods. Cases of recurrent fevers over years have been reported. Differential diagnosis The non​specific presentation of rat bite fevers means that a broad differential diagnosis must be considered, particularly if a history of rat exposure is not obtained. Fever with rash and joint involvement could suggest streptococcal, staphylococcal, or meningococcal in- fection. Rickettsial infection might be a consideration depending on region of residence or travel history. The rash appearance and dis- tribution might suggest secondary syphilis. Leptospirosis should be considered, particularly with a history of exposure to rats or poten- tially contaminated water. Several viruses including coxackieviruses, parvovirus B19, and even Epstein–​Barr virus can present with similar features. Clinical investigations/​laboratory features A neutrophil leukocytosis is usually present acutely but there are no specific haematological or biochemical abnormalities typ- ical of the RBFs. False positive non​treponemal syphilis serology is sometimes seen. Microbiological investigation Discussion with the microbiology laboratory is important if the diagnosis of RBF is suspected to optimize processing of samples. Microscopy S. moniliformis might be seen on direct gram stain of infected pus or joint fluid as a pleomorphic Gram-​negative bacillus. If cultured, its pleomorphic appearance with lateral bulbar swellings on Gram stain is highly suggestive of the diagnosis. S. minus might appear as a spirochaete-​like organism with Giemsa or Wright’s stain, or dark-​field microscopy of aspirates or smears of infected tissue or fluid. Culture Streptobacillus moniliformis can be cultured from pus and other in- fected body fluids and tissue but requires enriched culture media to

8.6.33  Lyme borreliosis 1181 optimize growth. Direct isolation from blood cultures is possible but growth is inhibited by sodium polyanethol sulphonate, an anticoagu- lant found in most commercially available blood culture media, which reduces the yield. Identification by traditional biochemical methods can be difficult but polymerase chain reaction (PCR) amp- lification of 16S ribosomal RNA sequences has been shown to be a reliable means of identifying the organism once cultured. More recently matrix-​assisted laser desorption ionization–​time of flight (MALDI-​TOF) mass spectrometry analysis has been shown to suc- cessfully identify S. moniliformis. Spirillum minus is not able to be cultured. Treatment Many cases of RBF probably go undiagnosed due to the use of effective empiric antibiotics in patients presenting with non-​ specific fever syndromes with sepsis and rash or joint involve- ment. In the context of a suggestive illness with a supportive exposure history, starting treatment while awaiting confirmation is appropriate. Uncomplicated rat bite fever Both S. moniliformis and S. minus are susceptible to penicillin which remains the treatment of choice for suspected or confirmed RBF. There is no trial evidence to guide treatment recommendations. For uncomplicated RBF initial intravenous (IV) treatment for 5–​7 days with benzyl penicillin is suggested with a switch to oral treatment to complete 14 days therapy. Recommended doses of IV penicillin G have historically been relatively low with total daily doses of 1.2–​2.4 million units given in divided doses 4–​6 hourly. In practice higher doses of penicillin are likely to be used initially, particularly while invasive infection and complications such as endocarditis are excluded. Oral follow on with penicillin V (500 mg four times daily) or amoxicillin (500 mg three times daily) is suitable in uncompli- cated disease. Most β-​lactams are effective and ceftriaxone (1 gram daily IV) can make outpatient based intravenous antibiotic treat- ment a practical option. Alternatives for penicillin allergic patients include tetracycline (500 mg four times daily) or doxycycline (100 mg twice daily). Macrolides, lincosamides, and glycopeptides also have good ac- tivity, with fluoroquinolones and aminoglycosides less active in vitro. Complicated infection. Recommendations for antibiotic treatment of more complicated forms of RBF are largely pragmatic, depending on the site of in- fection, and are based on the small numbers of cases published. Treatment of endocarditis due to either organism should be with higher doses of penicillin, up to 20 million units per day intraven- ously, for four to six weeks. Combination with streptomycin for the first two weeks of treatment has been recommended but there are no data to support this recommendation. Central nervous system infections will also require high dose treatment and some forms of infection (e.g. septic arthritis, spinal epidural abscess, or deep tissue collections) might need surgical intervention in addition to antibiotic therapy to ensure resolution. Prognosis/​outcome Untreated infection can be self-​limiting but mortality rates of 10% are recognized for both organisms with a higher likelihood of death in complicated disease such as endocarditis (50%). In general, both forms of rat bite fever respond well to antibiotic treatment. Arthralgia can be a persistent and troublesome symptom after streptobacillary disease has been treated and may take many months to settle. Prevention Preventive measures to reduce the frequency of rat bite fevers include control of wild rat populations and limiting opportunities for direct contact between feral rodents and humans. In the case of domesticated or laboratory rats, handlers and owners should use ap- propriate care when handling animals. Hand to mouth or eye contact should be avoided and hand washing encouraged following contact. Children handling pet rats should be supervised and educated about appropriate hygiene measures. Any rat bites should have the wound thoroughly cleaned and consideration given to tetanus prophylaxis. The role of pre-​emptive antibiotic therapy following rat bites to prevent RBF is unproven, but several guidelines and authors advocate short courses of oral antibiotic (e.g. three days of amoxi- cillin/​clavulanate or doxycycline to cover the agents of RBF and other potential contaminating bacteria). FURTHER READING Adam JK, et al. (2014). Notes from the field: fatal rat-​bite fever in a child—​San Diego County, California, 2013. MMWR Morb Mortal Wkly Rep, 63, 1210–​1. Addidle M, et al. (2012). Epidural abscess caused by Streptobacillus moniliformis. J Clin Microbiol, 50, 3122–​4. Eisenberg T, et al. (2015). Phenotypic and genotypic characteristics of members of the genus Streptobacillus. PLoS One, 10, e0134312. Eisenberg T, et al. (2016). Approved and novel strategies in diagnostics of rat bite fever and other Streptobacillus infections in humans and animals. Virulence, 7, 630–48. Elliot SP (2007). Rat bite fever and Streptobacillus moniliformis. Clin Microbiol Rev, 20, 13–​22. Gaastra W, et al. (2009). Rat bite fever. Vet Microbiol, 133, 211–​28. Rupp ME (1992). Streptobacillus moniliformis endocarditis:  case
report and review. Clin Infect Dis, 14, 769–​72. 8.6.33  Lyme borreliosis Gary P. Wormser, John Nowakowski, and
Robert B. Nadelman ESSENTIALS Lyme borreliosis is a zoonotic bacterial infection caused by Borrelia burgdorferi sensu lato, a spirochaetal agent transmitted

section 8  Infectious diseases 1182 by certain species of Ixodes ticks. Small rodents and birds serve as reservoirs. It is the most common vector-​borne infection in the United States of America and an important infection in many countries throughout the temperate regions of Europe and nor- thern Asia, where a wider variety of borrelia species account
for differences in clinical manifestations in Eurasia compared with the United States. Clinical features—​the most common and earliest clinical mani- festation is erythema migrans, a distinctive cutaneous lesion that occurs at the site of deposition of the spirochaete by the vector tick, beginning 7–​14 days later as a red macule or papule, with the rash then expanding over days to weeks, with or without central clearing. This may be associated with ‘viral’ symptoms, fever, and regional lymphadenopathy. Later manifestations include (1) card- itis—​usually manifested by fluctuating degrees of atrioventricular block; (2) neurological involvement—​including cranial neuropathy (typically cranial nerve VII palsy), radiculopathy, and meningitis; (3)  arthritis—​typically migratory monoarthritis or asymmetric oligoarthritis; (4) acrodermatitis chronica atrophicans—​a swollen, bluish-​red appearing skin lesion in which the involved skin ultim- ately atrophies. Diagnosis—​the diagnosis of erythema migrans is purely clinical in geographical areas endemic for Lyme borreliosis:  serological testing is not recommended because it is insufficiently sensitive on acute phase serum samples. In patients with suspected later clin- ical manifestations, serological testing is essential because clinical findings alone lack sufficient specificity. Polymerase chain reaction testing of joint fluid and/​or cerebrospinal fluid can be helpful in some cases. Treatment—​most people treated for Lyme borreliosis re- spond well to a 2-​week course of antibiotic therapy (preferred oral regimen usually amoxicillin, doxycycline, or cefuroxime). Symptomatic treatment is recommended for patients who have or develop subjective complaints of unclear aetiology despite successful resolution of the objective manifestation of Lyme borreliosis following antibiotic therapy, since randomized double-​ blind placebo-​controlled trials have shown that additional anti- biotic treatment is not helpful. Prevention—​measures include avoiding exposure to ticks by limiting outdoor activities in tick-​infested locations, using tick repellents, tucking in clothing to decrease exposed skin surfaces, and frequent inspection of the skin for early detection and removal of ticks. Introduction Lyme borreliosis (also called Lyme disease) is named after Lyme, Connecticut, United States of America. It is caused by the spirochaete Borrelia burgdorferi sensu lato which is transmitted to humans by the usually asymptomatic bite of certain ticks of the genus Ixodes (Fig. 8.6.33.1). Borrelia burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) causes the disease in North America, while in Europe, several species of Borrelia in addition to B. burgdorferi (a) (b) (c) Fig. 8.6.33.1  (a) Adult female (right) and nymphal (left) Ixodes scapularis ticks. Adult female (b) and nymph (c) of Ixodes ricinus, the vector tick in Europe.

8.6.33  Lyme borreliosis 1183 cause this infection, including B. garinii which is probably the most common cause of classic Lyme neuroborreliosis (Bannwarth’s syn- drome) and B. afzelii the most common cause of acrodermatitis chronica atrophicans, a late cutaneous complication. The entire chromosome and associated plasmids of multiple different strains of B. burgdorferi have been completely sequenced. Representative strains of other pathogenic species, such as B. afzelii and B. garinii, have also been sequenced. Epidemiology In North America more than 25 000 new cases of Lyme borreliosis are reported each year, making it the most common vector-​borne disease. It occurs in north-​eastern, mid-​Atlantic, north-​central, and far western regions of the United States of America and in limited foci in Canada (mainly in eastern Ontario). Elsewhere, it occurs in much of the temperate regions of Europe and northern Asia. Ticks acquire this borrelial infection in a complex tick–​vertebrate trans- mission cycle. The white-​footed mouse is the most important res- ervoir for B. burgdorferi in North America. White-​tailed deer, an important host for adult Ixodes ticks, are not a competent reservoir for Lyme borrelia. In Europe a wide variety of small rodents and birds serve as reservoirs. Migrating birds might play a role in the spread of B. burgdorferi to new geographical locations. Lyme borreliosis occurs slightly more frequently in males than in females. There is a bimodal age distribution with the highest rates in children between 5 and 9 years old and in adults 55–​59 years old. Clinical manifestations The somewhat different manifestations of Lyme borreliosis in Eurasia compared with North America (Table 8.6.33.1) may be explained by the wider variety of borrelia species causing infec- tion in Eurasia. Clinical features are generally similar in adults and children. Erythema migrans Erythema migrans (Figs. 8.6.33.2, 8.6.33.3), the clinical hallmark of Lyme borreliosis, is recognized in approximately 90% of patients with objective clinical manifestations of B. burgdorferi infection. Typically, erythema migrans begins as a red macule or papule at the site of a tick bite that occurred 7–​14 days earlier. The rash expands over days to weeks. Central clearing might or might not be present. Secondary cutaneous lesions can develop because of haematogenous spread of spirochaetes to other cutaneous sites. Erythema migrans must be distinguished from local tick bite reactions, tinea, insect and spider bites, bacterial cellulitis, and plant dermatitis. Lesions eventu- ally resolve spontaneously, but might recur if antimicrobial therapy is not given. Systemic symptoms, such as fatigue, myalgia, arthralgia, head- ache, fever, and/​or chills, and stiff neck, are less common in pa- tients with erythema migrans caused by B. afzelii compared to either B. burgdorferi or B. garinii. Prominent respiratory and/​or gastrointestinal symptoms are so infrequent that their presence should suggest an alternative diagnosis or coinfection with an- other tick-​borne pathogen. Aside from the erythema migrans skin lesion itself, the most common objective physical findings are regional lymphadenopathy and fever. Occasional cases of a viral-​like illness without erythema migrans have been attributed to Lyme borreliosis. Carditis Typically, cardiac disease develops within weeks to months after infection, sometimes together with erythema migrans. It is usu- ally manifested by fluctuating degrees of atrioventricular block that might cause the patient to complain of dizziness, palpi- tations, dyspnoea, chest pain, or syncope. Myocarditis can be present but pericarditis with effusion is rarely observed, and endocarditis is absent. The incidence of cardiac manifestations (as measured by electrocardiogram confirmed heart block) has been observed to be low in both the United States of America (<1%) and Europe (<4%). Neurological disease The incidence of neurological Lyme disease in Europe might be higher than in the United States of America. One explanation could be the greater neurotropism of B. garinii (a genospecies which has not been isolated in North America). The principal early neuro- logical manifestations are cranial neuropathy (typically peripheral seventh nerve palsy, which can be bilateral), radiculopathy, and meningitis, which might occur alone or together. Erythema migrans Table 8.6.33.1  Lyme borreliosis in North America compared to Eurasia North American Lyme borreliosis Eurasian Lyme borreliosis Vector Ixodes(dammini) scapularis or Ixodes pacificus Ixodes ricinus or Ixodes persulcatus Aetiological agent B. burgdorferi sensu stricto B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. spielmanii Clinical features Erythema migrans is the most common manifestation Erythema migrans is the most common manifestation Systemic symptoms frequently present in patients with erythema migrans (up to 80%) Systemic symptoms infrequently present in patients with erythema migrans (<35%) Other skin manifestations such as borrelial lymphocytoma and acrodermatitis chronica atrophicans are much less common than
in Europe Other skin manifestations such as borrelial lymphocytoma and acrodermatitis chronica atrophicans are much more common than in North America (acrodermatitis chronica atrophicans is much less common in children compared with adults) Cranial nerve palsy (usually 7th) with or without meningitis is the most common neurological manifestation Painful meningoradiculoneuritis with or without cranial palsy is the most common neurological manifestation

section 8  Infectious diseases 1184 might be present concomitantly. Late neurological manifestations are uncommon and include peripheral neuropathy, encephalopathy, and encephalomyelitis. Antibiotics appear to hasten the resolution of meningitis but most studies are uncontrolled. The rate of resolution of motor dysfunc- tion, which is fully reversible in the vast majority of cases, is not en- hanced by antimicrobial therapy. Symptoms of encephalopathy and peripheral neuropathy improve or do not progress after treatment with antibiotics. Rheumatological disease Lyme arthritis occurs in both North America and Europe. In a study of 55 untreated patients with erythema migrans diagnosed in the United States of America between 1977 and 1979 and followed for a mean duration of 6 years, objective arthritis developed in more than one-​half, occurring within 1 year for 90%. Most of these pa- tients developed intermittent attacks of migratory monoarthritis or asymmetric oligoarthritis, lasting a mean of 3 months per episode (range 3 days to 11.5 months). The knee was affected at some point in almost all patients, but other large and (less often) small joints could be affected. Temporomandibular joint involvement occurred in 11 (39%) of 28 patients with arthritis in one series. Although large effusions can occur, joint pain and erythema are often minimal. Baker’s cysts might develop. Typically, synovial fluid analysis reveals a modestly elevated white cell count (median 24 250 white cells/​mm3 in one study) with a polymorphonuclear predominance and a normal glucose level. Synovitis lasting 1 year or more might ensue for a minority of United States patients, sometimes associated with joint destruction. Although B. burgdorferi DNA can be detected by polymerase chain reaction (PCR) in the synovial fluid of up to 85% of untreated patients with Lyme arthritis, B. burgdorferi has rarely been successfully cultured from joint fluid. Acrodermatitis chronica atrophicans This cutaneous manifestation of late Lyme disease develops insidi- ously on a distal extremity, mainly in elderly women. It is a swollen bluish-​red appearing skin lesion in which the involved skin ultim- ately atrophies. One-​third of patients have an associated (usually sen- sory) polyneuropathy. B. burgdorferi has been recovered from a skin biopsy specimen of an acrodermatitis chronica atrophicans lesion of more than 10 years duration. Since the usual causative agent B. afzelii does not occur in the United States of America, acrodermatitis chronica atrophicans is essentially a European disease. Miscellaneous clinical manifestations Borrelia lymphocytoma, principally caused by B. afzelii and B. garinii, is a tumour-​like nodule which typically appears on the pinna of the earlobe or on the nipple or areola of the breast. Lesions will eventually resolve spontaneously but disappear within a few weeks after antibiotic therapy. This lesion is extremely rare in North America. Direct involvement of the eye (e.g. uveitis, keratitis, vitritis, optic neuritis) has been attributed to B. burgdorferi infection. However, (a) (b) Fig. 8.6.33.2  Erythema migrans skin lesions from patients who were culture-​positive for borrelia: (a) skin lesion with target-​like appearance; (b) skin lesion with more homogeneous appearance. (a) (b) Fig. 8.6.33.3  English patient with typical erythema migrans. Copyright D. A. Warrell.

8.6.33  Lyme borreliosis 1185 since ophthalmological disorders have almost never been associated with the isolation of B. burgdorferi in culture, the actual pathogen- esis in these cases is uncertain. Conjunctivitis, originally described in 11% of patients with erythema migrans, was rare (<5%) in re- cent studies of culture-​positive patients and might be unrelated to borrelia infection. Case reports have suggested that adverse outcomes might be associated with pregnancies complicated by maternal Lyme borreliosis. However, prospective and epidemiological studies suggest that the risk of transplacental transmission of B. burg- dorferi is probably minimal when appropriate antibiotics (Tables 8.6.33.2, 8.6.33.3) are given to pregnant women with Lyme borreliosis. There are no published data to support a congenital Lyme borreliosis syndrome. Laboratory diagnosis Where Lyme borreliosis is endemic, the diagnosis of erythema migrans is purely clinical. Laboratory testing is neither necessary nor recommended. In patients with suspected extracutaneous Lyme borreliosis, serological testing is essential to support the diagnosis. Culture of B. burgdorferi has been a highly insensitive diagnostic technique for this group of patients, presumably because of inaccessibility of tissues containing the microorganism. PCR testing of joint fluid and sometimes of cerebrospinal fluid might aid in diagnosis, provided appropriate care is taken in performing the assay accurately. A two-​step approach to serological diagnosis is used in both the United States of America and Europe to increase the accuracy of a positive test. A positive or equivocal first-​step test (usually an enzyme-​linked immunosorbent assay (ELISA) or an indirect im- munofluorescence assay) is followed on the same serum sample by a second-​stage test (immunoblot). Two-​step testing, however, is not indicated for those with little or no clinical evidence of Lyme borreliosis because of a low positive predictive value. Since IgM and IgG antibodies to B. burgdorferi can persist in serum for years after clinical recovery, serology has no role in measuring response to treatment. Patients with extracutaneous Lyme borreliosis almost always have diagnostic serum antibodies at time of presentation. In some patients with early neuroborreliosis, however, antibodies to Lyme borrelia might be present in cerebrospinal fluid before they are de- tected in serum. Coinfection Ixodes scapularis ticks (Fig.  8.6.33.1a) are the vectors for sev- eral other infections that can be transmitted separately or Table 8.6.33.2  Recommended antimicrobial regimens for treatment of patients with Lyme borreliosis Drug Dosage for adults Dosage for children Preferred oral regimens Amoxicillin 500 mg three times dailya 50 mg/​kg per day in three divided doses (maximum 500 mg per dose)a Doxycycline 100 mg twice dailyb <8 years: not recommended ≥8 years: 4 mg/​kg per day in two divided doses (maximum 100 mg/​dose) Cefuroxime axetil 500 mg twice daily 30 mg/​kg per day in two divided doses (maximum 500 mg per dose) Alternative oral regimens The following dosing regimens are specifically for patients with erythema migrans or borrelial lymphocytoma: Selected macrolidesc Azithromycin 500 mg orally daily for 7–​10 days, clarithromycin 500 mg orally twice daily for 14–​21 days
(if not pregnant), or erythromycin 500 mg orally four times per day for 14–​21 days Azithromycin 10 mg/​kg daily (maximum of 500 mg per day), clarithromycin 7.5 mg/​kg twice daily (maximum of 500 mg per dose), or erythromycin 12.5 mg/​kg four times daily (maximum of 500 mg per dose) Preferred parenteral regimen Ceftriaxone 2 g intravenously once daily 50–​75 mg/​kg intravenously once daily (maximum 2 g) Alternative parenteral regimens Cefotaxime 2 g intravenously every 8 hd 150–​200 mg/​kg per day intravenously in 3 or 4 divided doses (maximum 6 g per day)d Penicillin G 3–​4 million units intravenously every 4 hd 200 000–​400 000 units/​kg per day divided into six doses given every 4 hd (not to exceed 18–​24 million units/​day) a Although higher dosage given twice daily might be equally as effective, in view of the absence of data on efficacy, twice daily administration is not recommended. b Tetracyclines are relatively contraindicated in pregnant or lactating women and in children less than 8 years of age. c Due to their lower efficacy, macrolides are reserved for patients who are unable to take or who are intolerant of tetracyclines, penicillins, and cephalosporins. Patients treated with macrolides should be closely followed to ensure resolution of the clinical manifestations. d Dosage should be reduced for patients with impaired renal function. Modified from Wormser GP, et al. (2006). The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis and babesiosis. Clinical practices guidelines by the Infectious Diseases Society of America. Clin Infect Dis, 43, 1089–​134.

section 8  Infectious diseases 1186 simultaneously with B. burgdorferi, such as Babesia microti and the rickettsial agent Anaplasma phagocytophilum that causes human granulocytic anaplasmosis (formerly known as human granulocytic ehrlichiosis). In Europe, species of Babesia and Anaplasma are present in Ixodes ricinus ticks (Fig. 8.6.33.1b, c), which are also vectors of the flavivirus causing tick-​borne enceph- alitis. Coinfection may alter the clinical presentation and response to treatment of Lyme borreliosis. Reinfections Reinfection with Lyme borrelia can often be recognized clinically by the development of a repeat episode of erythema migrans occurring at a different skin site during the months when the vector tick is plentiful in the environment. The clinical manifestations of reinfec- tion in Lyme borreliosis patients who have erythema migrans are indistinguishable from initial infection. Table 8.6.33.3  Recommended therapy for patients with Lyme borreliosisa Indication Treatment Duration (days) Range (days) Tick bite in the USA Doxycycline 200 mg (4 mg/​kg in children ≥8 years of age)
and/​or observation Single doseb Erythema migrans Oral regimenc, d 14 10–​21e Early neurological disease Meningitis or radiculopathy Parenteral regimen or oral regimen with doxycyclinec, f 14 10–​28 Cranial nerve palsyg Oral regimenc 14 14–​21 Cardiac disease Oral regimenc, h or 14 14–​21 Parenteral regimenc, h 14 14–​21 Borrelial lymphocytoma Oral regimenc, d 14 14–​21 Late disease Arthritis without neurological disease Oral regimenc 28 28 Recurrent arthritis after oral regimen Oral regimenc, i 28 28 Parenteral regimenc, i 14 14–​28 Antibiotic-​refractory arthritisj Symptomatic therapyk Central or peripheral nervous system disease Parenteral regimenc 14 14–​28 Acrodermatitis chronica atrophicans Oral regimenc 21 14–​28 Post-​treatment Lyme disease syndrome Consider and evaluate other potential causes of symptoms,
if none found then symptomatic therapy a Regardless of the clinical manifestation of Lyme disease, complete response to treatment might be delayed beyond the treatment duration. Relapse can occur with any of these regimens; patients with objective signs of relapse might need a second course of treatment. b A single dose of doxycycline can be offered to adult patients and to children ≥8 years of age in the United States of America only when all of the following circumstances exist: (a) the attached tick can be reliably identified as an adult or nymphal I. scapularis tick that is estimated to have been attached for ≥36 h based on the degree of engorgement of the tick with blood or on certainty about the time of exposure to the tick; (b) prophylaxis can be started within 72 h of the time that the tick was removed; (c) ecological information indicates that the local rate of infection of these ticks with B. burgdorferi is ≥20%; and (d) doxycycline is not contraindicated. For patients who do not fulfil these criteria, observation is recommended. c See Table 8.6.33.2. d For adult patients intolerant of amoxicillin, doxycycline, and cefuroxime axetil, a macrolide can be given (Table 8.6.33.2). Patients treated with macrolides should be closely followed to ensure resolution of the clinical manifestations. e If doxycycline is used, 10 days of therapy is effective; the efficacy of 10-​day regimens with the other first-​line agents is unknown. f Data from European studies of neuroborreliosis indicate that oral doxycycline and parenteral antibiotic therapy are equally effective in Lyme meningitis. Similar studies have not been conducted in the United States of America. For non​pregnant adult patients, the recommended dosage of doxycycline, 200 mg/​day orally (or intravenously if unable to take oral medications) in one dose or in two divided doses, may be adequate. For children ≥8 years of age the recommended dosage of doxycycline for this indication is 4 mg/​kg per day in one dose or in two divided doses (maximum daily dosage of 200 mg). g Most patients can be treated successfully with an oral regimen. Parenteral antibiotic therapy is recommended for patients with both clinical and laboratory evidence of coexistent meningitis who cannot be treated with doxycycline. Systematic studies of oral antibiotic therapy in patients with cranial nerve palsy have only evaluated doxycycline. Other oral agents such as amoxicillin or cefuroxime axetil might be effective in patients who should not receive or cannot tolerate doxycycline, but clinical trials with these antibiotics are lacking. Most of the experience in the use of oral antibiotic therapy is for patients with seventh cranial nerve palsy. Whether oral therapy would be as effective for patients with other cranial neuropathies is unknown. The decision between oral and parenteral antimicrobial therapy for patients with other cranial neuropathies should be individualized. h A parenteral antibiotic regimen is recommended at the start of therapy for patients who have been hospitalized for cardiac monitoring; an oral regimen can be substituted to complete a course of therapy or to treat ambulatory patients. A temporary pacemaker is sometimes required for patients with advanced heart block. i A second course of oral antibiotic therapy is preferred for the patient whose arthritis has substantively improved but has not yet completely resolved. Consideration of retreatment
of such patients is often postponed for several months because of the anticipated slow resolution of inflammation after antibiotic treatment. During this interval use of non​steroidal anti-​inflammatory agents (NSAIDs) might be beneficial. Parenteral antibiotic therapy is reserved for those patients whose arthritis failed to improve at all or worsened. j Antibiotic-​refractory Lyme arthritis is operationally defined as persistent synovitis for at least 2 months after completion of a 1-​month course of intravenous ceftriaxone (or at least 1 month after completion of two 4-​week courses of an oral antibiotic regimen for patients unable to tolerate cephalosporins); in addition, PCR on synovial fluid (and synovial tissue if available) is negative for B. burgdorferi nucleic acids. k Symptomatic therapy might consist of NSAIDs, intra-​articular injections of corticosteroids, or other medications. If persistent synovitis is associated with significant pain or if it limits function, arthroscopic synovectomy should be considered. Modified from Wormser GP, et al. (2006). The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis and babesiosis. Clinical practices guidelines by the Infectious Diseases Society of America. Clin Infect Dis, 43, 1089–​134.

8.6.33  Lyme borreliosis 1187 Treatment Although most manifestations of Lyme borreliosis resolve spontan- eously, antibiotics might speed the resolution of some and will al- most certainly prevent the progression of disease. An approach to treatment is summarized in Tables 8.6.33.2 and 8.6.33.3. Presently available fluoroquinolones, sulphonamides, first-​generation ceph- alosporins, rifampicin, and aminoglycosides have no appreciable activity against B. burgdorferi and should not be used. There is no evidence to support combination antimicrobial therapy, prolonged (more than 1 month) or repeated courses of antibiotics, and ‘pulse’ or intermittent antibiotic therapy. Within 24 h after initiation of antibiotics, approximately 15% of patients with erythema migrans develop transient intensification of signs (e.g. rash and fever) and symptoms (e.g. arthralgias) consistent with a Jarisch–​Herxheimer reaction. Treatment is symptomatic. Most people treated for Lyme borreliosis have an excellent prognosis. Although a minority of patients treated for erythema migrans in recent series continue to have a variety of mild non-​ specific complaints following antibiotic therapy, the development of objective extracutaneous disease after treatment is extremely rare. When such complaints are disabling and last for 6 months or more they have been referred to as post-​treatment Lyme disease syndrome. Randomized double-​blind placebo-​controlled antibiotic treatment trials of patients with post-​treatment Lyme disease syn- drome have failed to show evidence that the benefit of additional antibiotic therapy outweighs the complications of such treatment. Symptomatic therapy is recommended. Patients with neurological disease tend to do well, but may sometimes have residual deficits (e.g. mild seventh nerve palsy) after treatment. In patients with arthritis, clinical recovery oc- curs typically with oral antibiotic therapy (often in conjunction with a non​steroidal anti-​inflammatory medication (NSAID)). Occasionally patients with Lyme arthritis with subtle signs of neuroborreliosis who are treated with oral antibiotics will develop overt late neuroborreliosis and require parenteral therapy. A small number of American patients with Lyme arthritis continue to have synovial inflammation for months or even several years after the apparent eradication of B. burgdorferi from the joint following antibiotic therapy (based on negative PCR testing). Such patients have improved after intra-​articular corticosteroid injections, use of NSAIDS or disease-​modifying antirheumatic drugs, such as hydroxychloroquine or methotrexate, or synovectomy. An im- munological mechanism rather than active infection appears to be responsible for the continued inflammatory response in these patients. In North America predominantly, but also in Europe, sev- eral patients with a variety of symptoms of uncertain aetiology, including pain and fatigue syndromes, have been labelled as having ‘chronic Lyme disease’, irrespective of tick exposure in an endemic area for Lyme borreliosis or credible clinical or labora- tory evidence of infection due to Lyme borrelia. There is no sci- entific evidence that such patients have active infection due to borreliae. Prevention Preventive measures include avoiding exposure by limiting out- door activities in tick-​infested locations, using tick repellents, tucking in clothing to decrease exposed skin surfaces, bathing within 2 hours after tick exposure, and frequent skin inspec- tions for early detection and removal of ticks. Use of acaricides on property and construction of deer fences have also been proposed. Antibiotic prophylaxis with single-​dose doxycycline given after recognized I. scapularis tick bites has been shown to be 87% effective in reducing further the low (less than 5%) risk of ac- quiring Lyme borreliosis after tick bites in the United States of America. Vaccination with a single recombinant outer surface protein A (OspA) preparation has been found to be safe and ef- fective for preventing Lyme borreliosis in the United States of America, but this vaccine is no longer available. Canine vaccines for prevention of Lyme borreliosis, however, are widely used in North America. FURTHER READING Aguero-​Rosenfeld M, et al. (2005). Diagnosis of Lyme borreliosis. Clin Microbiol Rev, 18, 484–​509. British Infection Association (2011). The epidemiology, prevention, investigation and treatment of Lyme borreliosis in United Kingdom patients: a position statement by the British Infection Association. J Infect, 62, 329–​38. Cerar D, et al. (2011). Subjective symptoms after treatment of early Lyme disease. Am J Med, 123, 79–​86. Eikeland R, et al. (2011). European neuroborreliosis: quality of life 30 months after treatment. Acta Neurol Scand, 124, 349–​54. Feder HM Jr, et al. (2007). A critical appraisal of ‘chronic Lyme disease’. N Engl J Med, 357, 1422–​30. Halperin JJ, et al. (2007). Practice parameter: treatment of nervous system Lyme disease (an evidence-​based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology, 69, 91–​102. Klempner MS, et al. (2001). Two controlled trials of antibiotic treat- ment in patients with persistent symptoms and a history of Lyme disease. N Engl J Med, 345, 85–​92. Ljostad U, et  al. (2008). Oral doxycycline versus intravenous ceftriaxone for European Lyme neuroborreliosis: a multicentre, non-​inferiority, double-​blind, randomized trial. Lancet Neurol, 7, 690–​5. Mygland A, et al. (2010). EFNS guidelines on the diagnosis and man- agement of European Lyme neuroborreliosis. Eur J Neurol, 17, 8–​16, e1–​4. Stanek G, et  al. (2011). Lyme borreliosis:  clinical case definitions for diagnosis and management in Europe. Clin Microbiol Infect, 17, 69–​79. Wormser GP, et  al. (2006). The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis:  clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis, 43, 1089–​134.

8.6.32 Rat bite fevers (Streptobacillus moniliform

8.6.32 Rat bite fevers (Streptobacillus moniliformis and Spirillum minus infection) 1179

8.6.33 Lyme borreliosis 1181

8.6.33 Lyme borreliosis 1181

SECTION 1 Patients and their treatment Section editors: John D. Firth, Christopher P. Conlon, and Timothy M. Cox 1.1 On being a patient  3 Christopher Booth 1.2 A young person’s experience of
chronic disease  6 Anonymous 1.3 What patients wish you understood  8 Rosamund Snow 1.4 Why do patients attend and what do they want from the consultation?  14 Des Spence 1.5 Medical ethics  20 Mike Parker, Mehrunisha Suleman, and Tony Hope 1.6 Clinical decision-​making  26 Timothy E.A. Peto and Philippa Peto

8.6.34 Relapsing fevers 1188

8.6.34 Relapsing fevers 1188

section 8  Infectious diseases 1188 8.6.34  Relapsing fevers David A. Warrell ESSENTIALS Louse-​borne relapsing fever and tick-​borne relapsing fever are char- acterized by repeated episodes of high fever separated by afebrile periods. They are caused by Borrelia spirochaetes distinct from those responsible for Lyme borrelioses. Untreated patients may suffer as many as five (louse-​borne relapsing fever) or ten (tick-​borne re- lapsing fever) febrile relapses of decreasing severity. B. myamotoi is much less likely to relapse. Humans are the sole reservoir of epidemic louse-​borne relapsing fever caused by B. recurrentis and transmitted by body lice (Pediculus humanus corporis). Endemic tick-​borne relapsing fevers are caused by at least 17 different Borrelia species and have their own par- ticular species of soft Ornithodoros, or, in the case of B. myamotoi and B. lonestari, hard Ixodes or Ablyomma tick vectors that also act as res- ervoirs. Transmission transplacentally, or by needlestick, blood trans- fusion, or laboratory accident is also possible. Louse-​borne relapsing fever is a classic historical epidemic disease of war, famine, and refugees, now largely confined to mountainous areas of the Horn of Africa and possibly Peru but still retaining its pandemic potential. Imported cases are increasingly recognized in refugees from the Horn of Africa. Tick-​borne relapsing fever has expanding endemicity in sub-​Sahelian West Africa and is common in Rwanda and Tanzania. It occurs sporadically in parts of North America, Europe, the Middle East, and central Asia. The most distinctive feature of these infections, the relapse phe- nomenon, is explained by antigenic variation of borrelial outer-​ membrane lipoprotein (vmp). Starting 2–​18  days after infection, there is acute fever, chills, headache, pain, and prostration. Petechial rash (thrombocytopenia), bleeding, jaundice, hepatosplenomegaly, and liver dysfunction are common. In some forms of tick-​borne re- lapsing fever, there are neurological manifestations:  lymphocytic meningitis, VII and other cranial nerve lesions, myelitis, radiculitis, and uveitis during relapses. Dangerous complications are hyperpyrexia, shock, myocar- ditis causing acute pulmonary oedema, acute respiratory distress syndrome, cerebral or massive external bleeding, ruptured spleen, hepatic failure, Jarisch–​Herxheimer reactions, and typhoid or other complicating bacterial infections. Pregnant women are at high risk of aborting and perinatal mortality is high. Diagnosis by microscopy of blood films is more difficult in tick-​ borne relapsing fever than louse-​borne relapsing fever. Serology and polymerase chain reaction are used increasingly. The most important differential diagnosis in residents and travellers from tropical en- demic areas is falciparum malaria. Untreated mortality, exceeding 40% in some epidemics, can be re- duced to less than 5% by treatment with antibiotics such as penicillin, tetracycline, erythromycin, and chloramphenicol, but elimination of spirochaetaemia is often accompanied by a potentially fatal Jarisch–​ Herxheimer reactions. Prevention of louse-​borne relapsing fever is by eliminating lousiness by sterilizing clothing, using insecticides and repellents, and improving hygiene. Improved house construction, control of peridomestic rodents, use of residual insecticides, protection of sleepers with impregnated bed nets, and a postexposure course of doxycyline can reduce the risk of tick-​borne relapsing fever. Historical background A disease characterized by repeated episodes of several days of high fever separated by afebrile periods of about a week was first described by Rutty in Dublin in 1770, but Craigie in Edinburgh coined the name ‘relapsing fever’ and distinguished it from ty- phus in 1843. Obermeier saw spirochaetes, now recognized as Borrelia recurrentis (Fig. 8.6.34.1), in the blood of febrile pa- tients in Berlin in 1866. Transmission by human body lice was proved by Mackie in 1907. David Livingstone described fatal tick-​borne fever in Angola in 1857. Its cause was discovered by Ross and Milne in 1904 in Uganda and, independently, by Dutton and Todd in 1905 in Eastern Congo. Both of them were infected while carrying out autopsies and Dutton died of pneu- monia while debilitated after relapses of B. duttonii infection (Fig. 8.6.34.2). Aetiology The bacteria that cause relapsing fevers are large, loosely coiled, motile spirochaetes (genus Borrelia, family Spirochaetaceae), 8–​20 µm long and 0.2–​0.6 µm thick, with between 3 and 15 coils and, in some strains, 15–​30 axial filaments or flagella. They divide by transverse binary fission. Borrelia can be cultured on chick chorioallantoic membrane and maintained in rodents and Fig. 8.6.34.1  Borrelia recurrentis spirochaetes in a Giemsa-​stained thin blood film. Copyright D. A. Warrell.

8.6.34  Relapsing fevers 1189 ticks. In vitro culture of Borrelia species, including B. recurrentis, B. duttonii, and B. crocidurae, is now possible using Barbour–​ Stoenner–​Kelly medium and Kelly-​Pettenkofer medium for B. myamotoi sensu lato. Rapidly increasing amounts of genomic data are available. Sequencing of flagellin and rrs genes suggests that there are three phylogenetic clusters of Borrelia: (1) Lyme borreliae (B. burg- dorferi sensu stricto, B. garinii, and B. afzelii (see Chapter 8.6.33 on Lyme disease); (2)  New World tick-​borne relapsing fever borreliae (B. parkeri, B. turicatae, B. hermsii, and so on); and (3)  Old World tick-​borne relapsing fever and louse-​borne re- lapsing fever borreliae (B. crocidurae, B. duttonii, B. hispanica, B. recurrentis, and so on). Molecular phylogenetic studies have shown close identity of B. recurrentis and B. duttonii suggesting only clonal difference and that B. recurrentis adapted rapidly to louse-​transmission with genome reduction and low genetic vari- ability. The three clades of B. myamotoi from Asia, Europe, and North America produce identical clinical effects. Epidemiology Louse-​borne (epidemic) relapsing fever (LBRF) The vector of B. recurrentis is the human body louse Pediculus humanus corporis and, perhaps, the head louse P. humanus capi- tis. Body lice, unlike head lice, retreat from the skin after feeding and hide and lay their eggs in clothing seams. More than 20 000 lice have been recovered from the clothes of one person. Lice are obligate blood-​sucking human ectoparasites that ingest borreliae while feeding. Under conditions of crowding and poor hygiene they can move from person to person. When the host’s body sur- face temperature deviates far from 37°C as a result of fever, cli- matic exposure, or death, or when infested clothing is discarded, the louse is forced to find a new host who can then be infected. Transmission of B. recurrentis is by scratching, which crushes lice so that their coelomic fluid is inoculated through broken skin or intact mucous membranes such as the conjunctiva, or inoculates infected louse faeces. Blood transfusion, needlestick injuries, and contamination of broken skin by a patient’s blood can also result in infection. Unlike ticks, lice cannot infect their progeny and are therefore not reservoirs and, since there is no known animal res- ervoir, the infection must persist in humans between epidemics in mild or asymptomatic forms. Wars, famines, and other disasters that generate large numbers of refugees and prisoners favour the spread of lice and epidemic louse-​ borne infections such as relapsing fever and typhus. The yellow plague in Europe in ad 550, which halved the world’s population, and the famine fevers of the 17th and 18th centuries in Ireland and elsewhere were probably LBRF. In the 20th century, a pandemic raged in North Africa, the Middle East, and Africa from 1903 to 1936, causing an estimated 50  million cases with 10% mortality. A second epidemic in 1943–​1946 created 10 million cases. An en- demic focus persists in the Horn of Africa. In cold, wet weather, im- poverished people with louse-​infested clothes crowd together for shelter. In the Ethiopian highlands there are annual epidemics of thousands of cases coinciding with the small (belg) and big (kiremt) rains, but in the south the disease was perennial before its recent de- cline. Outbreaks have also occurred in Somalia. In Rumbek County, South Sudan in 1999–​2000, there were 20 000 cases with some 2000 deaths, 580 in January 1999 alone. In Ancash in the Peruvian Andes at altitudes above 3800 m, a cluster of 60 clinical cases was reported in 1983; 36 of the patients had B. recurrentis in their blood films. Serological evidence of B. recurrentis infection has been found in homeless people in Marseille. Recently, many cases have been iden- tified in refugees to Europe from Ethiopia, Eritrea, and Somalia, and non-endemic countries such as Mali, arriving in Germany (45 cases in 2017 alone), Italy, Switzerland, and the Netherlands. Tick-​borne (endemic) relapsing fever (TBRF) Spirochaetes have been identified in a larval Amblyomma tick in fossilized amber from the Dominican Republic, dated at 15–​20 Fig. 8.6.34.2  Temperature chart of J. Everett Dutton who, with J L Todd, discovered the transmission of TBRF in the Congo. Dutton contracted TBRF at the beginning of November 1904. He had relapses of fever and spirochaetaemia on 7 and 16 December 1904 and 8 January 1905. His death on 27 February 1905 is attributable to pneumonia while he was debilitated by relapsing fever. From Dutton JE, Todd JL (1905). The nature of human tick-​fever in the eastern part of the Congo Free State with notes on the distribution and bionomics of the tick. Liverpool School of Tropical Medicine Memoir XVII.

section 8  Infectious diseases 1190 mya, and today, TBRF is endemic in most temperate and trop- ical countries except in South and Far East Asia, Australasia, the Pacific, Arctic, and Antarctic regions. In different parts of the world, particular species of borreliae and soft ticks (genus Ornithodoros, family Argasidae) are ecologically intimate, forming Borrelia–​tick complexes (Table 8.6.34.1). At least two borreliae associated with human disease are transmitted by hard ticks (Ixodidae): B. lonestari by Amblyomma americanum (United States) and B. miyamotoi sensu lato by Ixodes spp. (Japan, Europe, Russia, United States). At least 17 Borrelia species are now known to cause human TBRF. Ornithodoros tick vectors occur in dry savannah areas and scrub, caves, piles of timber and dead trees, or in holes in walls, roof spaces, and beneath the floors of log cabins, anywhere in- habited by small rodents. Unlike LBRF, TBRFs are zoonoses, ex- cept B. duttonii infection that is transmitted exclusively between humans. Vertebrate reservoir species include rodents (rats, mice, gerbils, squirrels, and chipmunks), insectivores, lagomorphs, bats, small carnivores, dogs, and birds. Soft ticks attack at night, remaining attached for less than 30 min before retreating to their hiding places. Spirochaetes ingested while the tick sucks blood from an infected animal or human invade the tick’s salivary and coxal glands and genital apparatus. Infection is transmitted to a new host either by a bite, introducing infected saliva, or by contaminating mucosal membranes with infected coxal fluid. Borreliae are not excreted in tick faeces. Ticks remain infected for life, even after being starved of blood for as long as 7 years. Spirochaetes can be transmitted venereally from male to female ticks and by females (but perhaps not those of the O. moubata complex) transovarially to their progeny. Borreliae may also be transmitted by hard ticks (Ixodidae). Borrelia miyamotoi sensu lato was first found in I. persulcatus ticks in Hokkaido, Japan in the early 1990s and, since 2011 has been recog- nized as a human pathogen in the relapsing fever group of Borrelia. It is prevalent in I. persulcatus in Russia, I. ricinus in Western Europe, and I. scapularis, I. pacificus and I. dammini in the United States. These ticks are also vectors of Lyme disease group Borrelia. B. lone- stari is transmitted by Amblyomma americanum in the United States and may be a human pathogen. TBRF, like LBRF, may be transmitted by blood transfusion, needlestick injuries, laboratory accidents, and transplacentally. In Europe, TBRF is caused by B. hispanica, especially in Spain, Portugal, and Greece, while B. crocidurae and at least three other Borrelia spp. are present in Turkey and adjacent territories. In Israel, the incidence of B. persica infection among military personnel is 6.4/​100 000 per year. In the West African savannah region, B. cro- cidurae is the most prevalent bacterial infection, creating a medical problem second only to malaria. Its prevalence is 1% among chil- dren in western Senegal and it is increasing and spreading during the persisting drought (1970–​2009). It is a common infection in Rwanda where, in one health centre alone, 1650 proven cases are treated each year (6% of all patients). In parts of East Africa, es- pecially in Tanzania, B. duttonii is an important cause of abortion, perinatal mortality, and childhood infection. In North America, isolated sporadic outbreaks of B. hermsii, B. turicatae, and B. park- eri infection occur in mountainous areas of British Columbia, Arizona (especially along the North Rim of the Grand Canyon), California (south of Lake Tahoe), Colorado, Montana, New Mexico, and Washington (Browne Mountain). Since the mid-​1980s, more than 300 cases of TBRF have been identified in the United States of Table 8.6.34.1  Borrelia–​tick complexes causing tick-​borne relapsing fevers

  1. New World Borrelia spp. with Argasid (soft) ticks genus Ornithodoros B. hermsii O. hermsii Canada, central and western USA, Mexico B. turicatae O. turicata South-​western USA, Mexico B. parkeri O. parkeri Western USA, Baja California B. mazzotti O. talaje Mexico, Central America B. venezuelensis O. (venezuelensis) rudis Central America, Colombia, Venezuela, Argentina, Bolivia, Paraguay
  2. Old World Borrelia spp. with Argasid (soft) ticks genus Ornithodoros B. duttonii O. moubata Sub-​Saharan Africa, Madagascar B. crocidurae O. (erraticus) sonrai North, West, and East Africa, Middle East B. graingeri O. graingeri East Africa B. sp. nov. O. porcinus East Africa B. tillae O. zumpti South Africa B. persica O. tholozani Middle East, central Asia from Uzbekistan to western China B. hispanica O. erraticus Iberian Peninsula, Greece, Cyprus, North Africa B. sp. nov. O. erraticus Southern Spain B. latyschevii O. tartakowskyi Eastern Europe, Iran, Iraq, Afghanistan, central Asia B. caucasica O. (verrucosus) asperus Eastern Europe, Iraq
  3. Borrelia spp. with Ixodid (hard) ticks general Ixodes and Amblyomma B. myamotoi sensu lato Ixodes persulcatus, I. ricinus, I. scapularis, I. pacificus, I. dammini Japan, Russia, Europe, NE United States B. lonestari Amblyomma americanum South and Southeastern United States

8.6.34  Relapsing fevers 1191 America. In Western countries, TBRF has been reported more than 20 times in returned travellers, usually from West Africa. A new species of TBRF spirochaete, B. kalaharica, has been described in a traveller from South Africa. This species may also be transmitted by Ornithodoros savingyi in Nigeria. Immunopathology and the relapse phenomenon Symptomatic attacks of relapsing fever are terminated when specific bactericidal IgM antibodies generated by the B1b cell subset lyse spirochaetes in the blood, independently of complement and T cells. However, some spirochaetes persist between the relapses, extracellu- larly in various organs including spleen, liver, kidneys, eye, and espe- cially in the brain and cerebrospinal fluid. Relapse of spirochaetaemia and symptoms is explained by antigenic variation, which has been investigated in the greatest detail in B. hermsii. Silent gene sequences from an archive stored in extra chromosomal plasmids are trans- posed to one end of an expression linear plasmid where their recom- bination leads to synthesis of a new variable major outer membrane lipoprotein (vmp). This new coat allows the borreliae to escape from the host’s humoural immune response until antibodies are gener- ated against the new serotypic vmp antigen; this explains the relapse phenomenon and the successive appearance of borreliae expressing different vmps during the course of an untreated infection. Borreliae also possess defences against the host’s innate immunity. B. herm- sii surface protein BhCRASP-​1 binds factor H (FH), an inhibitor of the alternative pathway of complement activation, so protecting the pathogen against opsonophagocytosis by inhibiting C3b binding. Plasminogen is also bound and activated to plasmin by BhCRASP-​1, stimulating fibrinolysis that frees spirochaetes to spread in the blood stream. Another protective mechanism is rosetting of erythrocytes around spirochaetes. This shields them, by masking or steric hin- drance, from host antibody and may cause microcirculatory ob- struction that is damaging to the host and reminiscent of cerebral malaria. Antigenic variation might also generate isogenic serotypes with properties that promote the spirochaete’s survival in vector and reservoir species (e.g. invasiveness for vertebrates’ cerebral vascular endothelium). These same vmps are the principal tumour necrosis factor-​α (TNFα)-​inducing factors in LBRF. Pathophysiology Physiological disturbances during the spontaneous crisis and the Jarisch–​Herxheimer reaction (JHR) induced by antimicrobial treat- ment in LBRF are typical of an endotoxin reaction. Outer mem- brane vmps of B. recurrentis stimulate monocytes to produce TNFα through NF-​κB. In patients treated with antibiotics, symptoms of the severe JHR are associated with a transient marked elevation in plasma concentrations of TNFα, interleukin (IL)-​6, IL-​8, and IL-​1β (Fig. 8.6.34.3). The stimulus for cytokine release is the phagocyt- osis of spirochaetes made susceptible by the action of penicillin. Benzyl penicillin attaches to penicillin-​binding protein I in B. herm- sii spirochaetes. Large surface blebs are produced and the dam- aged spirochaetes are phagocytosed rapidly by neutrophils in the blood and by the spleen. Complement may enhance phagocytosis of spirochaetes, especially in the non​immune host, but the com- plement system is not essential for elimination of spirochaetes whether or not specific immunoglobulins are present. In vitro, sur- face contact with spirochaetes induces mononuclear leucocytes to produce inflammatory cytokines and thromboplastin, which could be responsible for the fever and disseminated intravascular coagu- lation in LBRF. Kinins may be released during the JHR of syphilis and LBRF. The marked peripheral leucopenia that develops during the reaction reflects sequestration, perhaps in the pulmonary blood vessels, rather than leucocyte destruction. Spirochaetes may be found in those organs that bear the brunt of the infection such as liver, spleen (Fig. 8.6.34.4), myocardium (Fig. 8.6.34.5), and brain (Fig. 8.6.34.6), but it is unclear how their pathological effects are produced. The petechial rash results from thrombocytopenia not vasculitis. The cardiorespiratory and metabolic disturbances in relapsing fever are principally the result of persistent high fever, accentuated by the JHR or spontaneous crisis. Pathology Most spirochaetes are confined to the lumen of blood vessels, but tangled masses are also found in the characteristic splenic miliary abscesses (Fig. 8.6.34.4) and infarcts as well as within the central nervous system adjacent to haemorrhages. Some strains of TBRF borreliae can invade the central nervous system, aqueous humour, and other tissues. In LBRF, a perivascular histiocytic interstitial Rigors Penicillin Plasma cytokine 100 000 10 000 1000 ng/l 100 10 5–1.00124824 0 300 600 Spirochaete count/mm3 blood Spirochaetes IL-1 IL-8 TNF IL-6 Fig. 8.6.34.3  Typical Jarisch–​Herxheimer reactions in a patient with LBRF treated with intravenous penicillin. Following penicillin, the number of spirochaetes (dashed red line referring to right hand axis) fell abruptly and circulating levels of TNFα, IL-​6, IL-​8, and IL-​1β started to rise after about 1 h, peaking at 4 h. As cytokine levels were increasing, this patient experienced sustained rigors which subsided before peak levels were achieved.

section 8  Infectious diseases 1192 myocarditis, found in most cases, may be responsible for conduction defects, arrhythmias, and myocardial failure resulting in sudden death (Fig. 8.6.34.5). Splenic rupture with massive haemorrhage, cerebral haemorrhage (Fig. 8.6.34.6), and hepatic failure are other causes of death. The liver shows hepatitis with patchy midzonal haemorrhages and necrosis. There is meningitis and perisplenitis. Most serosal cavities and surfaces of viscera are studded with pe- techial haemorrhages (Figs. 8.6.34.5, 8.6.34.6) and there may be Fig. 8.6.34.6  Cerebral haemorrhage. Copyright D. A. Warrell. Fig. 8.6.34.5  Epicardial and endocardial haemorrhages. Copyright D. A. Warrell. (a) (b) Fig. 8.6.34.4  Spleen in LBRF: (a) Section of spleen at autopsy; (b) Warthin Starry stain showing Borrelia recurrentis (arrows). (a) Copyright D. A. Warrell; (b), courtesy of Dr Ken Fleming.

8.6.34  Relapsing fevers 1193 massive pulmonary haemorrhage (Fig. 8.6.34.7). Thrombi are occa- sionally found occluding small vessels, but the peripheral gangrene sometimes found in patients recovering from louse-​borne typhus (see Chapter 8.6.40) does not occur in LBRF. Clinical features Louse-​borne relapsing fever Adults Prisoners and poor, malnourished street-​dwellers are the most likely to become infected, especially young men. After an incubation period of 4–​18 (average 7) days, the illness starts sud- denly with rigors and a fever that mounts to nearly 40°C in a few days. Early symptoms are headache, dizziness, nightmares, gener- alized aches and pains (especially affecting the lower back, knees, and elbows), anorexia, nausea, vomiting, and diarrhoea. Later there is upper abdominal pain, cough, and epistaxis. Patients are usually prostrated (Fig. 8.6.34.8) and most are confused. Hepatic tenderness is the commonest sign (about 60%). The liver is en- larged in approximately 50% of patients. Splenic tenderness and enlargement are slightly less common. Jaundice has been reported in 10–​80% of patients. A petechial or ecchymotic rash is seen in 10–​60% of patients (Figs. 8.6.34.9, 8.6.34.10); the lesions occur par- ticularly on the trunk. Other sites of spontaneous bleeding include Fig. 8.6.34.8  Patients presenting with relapsing fever at a clinic in Addis Ababa. Most are febrile, confused, and prostrated. Copyright D. A. Warrell. Fig. 8.6.34.7  Pulmonary haemorrhage. Copyright D. A. Warrell.

section 8  Infectious diseases 1194 the conjunctivae (Fig. 8.6.34.11), nose in 25% (Fig. 8.6.34.9), and less commonly the lungs (Fig. 8.6.34.7), gastrointestinal tract, and retina. Many patients have tender muscles. Meningism oc- curs in about 40% of patients; other neurological features include cranial nerve lesions, monoplegias, flaccid paraplegia, and focal convulsions attributable, perhaps, to cerebral haemorrhages. In untreated people, the first attack of fever resolves by crisis in 4–​10 (average 5) days, followed by an afebrile remission of 5 to 9 days, and then a series of up to five relapses of diminishing severity, occasionally complicated by epistaxis. Petechial rashes are absent during relapses. Pregnant women are especially susceptible to severe disease and abortions are frequent. Children In children older than 5 years, clinical features resemble those in adults but are generally less severe and the case fatality is lower. Fever, chills, headache, abdominal pain and tenderness, vomiting, cough, musculoskeletal pains, tachycardia, and petechial rash are common. In younger children, hepatosplenomegaly, cough, and signs of consolidation may be more common. Reported case fatal- ities in children range from 1.9 to 5.5%. Tick-​borne relapsing fever Adults After an incubation period of 2–​18  days, the illness starts with sudden fever, chills, headache, muscle and joint pains, extreme fatigue, prostration, and drenching sweats. These symptoms are similar to those in LBRF but the initial fever usually lasts about 3 days only to recur about 7–​15 days later. Epistaxis, abdominal pain, diarrhoea, cough, and erythematous or petechial rashes may follow. Jaundice is less common than in LBRF. Several cases of acute respiratory distress syndrome (ARDS) have been described in the United States of America. Neurological disturbances are more common than in LBRF, varying in incidence with the borrelia spe- cies involved, from less than 5% in patients with B. hispanica and B. persica infections to as high as 40% in patients with B. duttonii. However, one careful study in northern Tanzania found no focal neurological abnormalities in patients with B. duttonii TBRF. The neurological features that have been described are reminiscent of Lyme neuroborreliosis and include paraesthesias, visual symptoms, lymphocytic meningitis, cranial nerve palsies (especially VII, VII, V and VI), encephalitis, radiculomyelitis, sciatica, delirium, and hallucinations. Untreated patients may have up to 13 relapses (Fig. 8.6.34.2), becoming sequentially less severe. Ocular complications usually occur during the third and fourth relapses. They include conjunctival injection, eye pain, photophobia, eyelid oedema, kera- titis, various degrees of anterior and posterior uveitis, optic neuritis, and blindness. Spirochaetaemia is higher in pregnant than in non​pregnant women and abortion and perinatal mortality are common. In Fig. 8.6.34.9  Ethiopian patient with LBRF showing petechiae on the shoulder and epistaxis. Copyright D. A. Warrell. Fig. 8.6.34.10  Ethiopian patient with severe LBRF complicated by typhoid, showing jaundice, petechial haemorrhages, and emaciation. Copyright D. A. Warrell. Fig. 8.6.34.11  Subconjunctival haemorrhage in a patient with LBRF. Copyright D. A. Warrell.

8.6.34  Relapsing fevers 1195 Tabora, Tanzania, parturition was precipitated in 58% of infected pregnant women. Perinatal mortality was 436/​1000 births, its risk related to low birthweight and gestational age. Total fetal wastage was 475/​1000. Children In endemic areas of B. duttonii TBRF in East Africa, most cases are in children, many of them under 5 years old, and pregnant women, implying that older non​pregnant people may acquire some im- munity. Fever, splenomegaly, convulsions sometimes recurrent, meningism, petechiae, and jaundice are described. Neonates with congenital infection have fever, inability to suck, jaundice, and fea- tures of septicaemia. Reported case fatalities in children less than 1 year old are 2.3 to 73%, compared to 1.6 to 19% in older children. Hard-​tick-​transmitted Borrelia The index cases of B. myamotoi sensu latu infection in the United States and Europe were elderly immunosuppressed patients who presented with meningoencephalitis associated with spirochaetes in the cerebrospinal fluid (CSF) that resolved with antibiotic treat- ment. However, immunocompetent patients rarely develop menin- gitis but present, after an incubation period of 12–​16 days, with fever that recurred in only 10% of them, chills, headache, myalgia, arth- ralgia, nausea, and fatigue. Fewer than 10% of cases have erythema migrans-​like rashes. Their systemic symptoms were more severe than in patients with Lyme disease. Unlike patients with classic re- lapsing fevers, there is no dramatic febrile crisis, either spontaneous or induced by treatment and there is no evidence that B. myamotoi sensu lato possesses evasive antigenic variation that would allow re- lapsing attacks. In south-​eastern and eastern United States, patients have been de- scribed with a disease termed Southern tick-​associated rash illness or Masters’ disease. Atypical erythema migrans is associated with systemic symptoms after bites by A. americanum hard ticks. B. lone- stari has been implicated but causation is not yet proven. Severe disease Severe manifestations include hyperpyrexia, myocarditis with acute pulmonary oedema, ARDS, hepatic failure, ruptured spleen, and haemostatic failure attributable to thrombocytopenia, liver damage, and disseminated intravascular coagulation leading to cerebral, massive gastrointestinal, pulmonary, or peripartum haemorrhage. Dysentery, salmonellosis, typhoid, typhus, tuberculosis, bacterial pneumonia, and malaria are infections that can complicate relapsing fever, increasing the risk of death. The spontaneous crisis and Jarisch–​Herxheimer reaction Whether or not treatment is given, attacks of relapsing fever usually end dramatically. On about the fifth day of the untreated illness, or about 1 to 2 h after antibiotic treatment, the patient becomes restless and apprehensive and suddenly begins to have distressingly intense rigors that last between 10 and 30 min. The ensuing phenomena have features of a classic endotoxin reaction. During the initial chill phase, temperature, respiratory and pulse rates, and blood pressure rise sharply. Delirium, gastrointestinal symptoms, cough, and limb pains are associated. Some patients die of hyperpyrexia at the peak of fever. The flush phase, which lasts many hours, is characterized by profuse sweating, a fall in blood pressure, and a slow decline in temperature. Deaths during this phase follow intractable hypo- tension, sudden postural hypotension prompted by the patient’s standing up, or the development of acute pulmonary oedema attrib- utable to myocarditis. The incidence of JHRs is highest in adults with LBRF treated with intravenous tetracycline (approaching 100% in some studies). It is lower when low-​dose or slow-​release penicillin is used and in children. JHR is less commonly observed in TBRF but can be severe and even fatal. The classic JHR occurs in secondary syphilis in which the spirochaetes are in the tissues and the reaction is less frequent, more insidious, and much less severe than in relapsing fevers. Milder reac- tions have been described in Lyme disease and leptospirosis (treated with penicillin), sodoku (treated with arsenicals), Brucella melitensis (treated with tetracycline), and even in typhoid and meningococcal infections. Laboratory findings Spirochaete densities may exceed 500 000/​mm3 of blood. There is a moderate normochromic anaemia and a neutrophil leucocyt- osis with marked leucopenia during the spontaneous crisis and JHR. Thrombocytopenia is usual and there is a mild coagulopathy with evidence of increased fibrinolysis. Biochemical evidence of hepatocellular damage (raised levels of aminotransferases, alkaline phosphatase, direct and total bilirubin, low albumin) and mild renal impairment are common. The cerebrospinal fluid shows a lympho- cyte or neutrophil pleocytosis without visible spirochaetes. There is electrocardiogram (ECG) evidence of myocarditis with prolongation of the QTc interval, T-​wave abnormalities, and ST-​ segment depression with transient acute right heart strain after the JHR. Chest radiographs may show pulmonary oedema or pneu- monic consolidation. Patients with B. myamotoi sensu lato infections may have leuco- penia, thrombocytopenia, and elevated transaminase levels. Diagnosis Thick and thin blood films should be taken while patients are fe- brile. Spirochaetes are demonstrated by Giemsa’s, Wright’s, Field’s, or Diff-​Quick staining (Fig. 8.6.34.1), dark-​field examination, or a quantitative buffy coat technique (acridine orange). The sensitivity of thick films is 20 times greater than thin films. Misidentification of Plasmodium vivax microgametes as spirochaetes has led to the diagnosis of ‘pseudoborreliosis’. In TBRF, spirochaetes may be difficult or impossible to find even at the height of a relapse and, increasingly, polymerase chain reaction (PCR) and serology are being used. Lyme disease Borreliae may produce cross-​reacting antibodies due to expression of conserved antigenic epitopes, but an ELISA using the glycerophosphodiester phosphodiesterase (GlpQ) gene product can distinguish relapsing fevers from Lyme disease. In LBRF, the higher and more persistent spirochaetaemia is more easily detected. Borreliae can be isolated in mice and cul- tured in vitro. The serum of patients with relapsing fever may give positive re- actions with proteus OXK, OX19, and OX2 and false-​positive sero- logical responses for syphilis in 5 to 10% of cases.

section 8  Infectious diseases 1196 B. myamotoi sensu lato is best detected by PCR in a pretreatment blood sample, complemented by enzyme immunoassay (EIA) using a recombinant GlpQ protein. Spirochaetes may sometimes be seen in Giemsa-​stained thick blood films and in CSF and in infected se- vere combined immunodeficient mice. Differential diagnosis In a febrile patient with jaundice, petechial rash, bleeding, hepatosplenomegaly, thrombocytopenia, coagulopathy, and ele- vated serum aminotransferases, the most frequent and urgent dif- ferential diagnosis is falciparum malaria. Yellow fever and other viral haemorrhagic fevers such as Rift Valley Fever in the Horn of Africa, viral hepatitis, rickettsial infections (especially louse-​borne typhus which shares LBRF’s epidemiological predispositions), and leptospirosis might also cause confusion. Trench fever (Bartonella quintana) transmitted by lice, and sodoku (Spirillum minus) fol- lowing a rat bite can also cause episodic recurrent fever. Although the diagnosis of relapsing fever can often be confirmed quickly by examining a blood smear, the possibility of complicating bacterial infection, particularly typhoid, or coinfection with malaria should never be forgotten. In the north-​eastern United States, B. myamotoi sensu lato in- fection is sympatric with Lyme disease and must be distinguished from other deer-​tick-​transmitted infections such as Babesia microti, Anaplasma phagocytophyllum and Powassan/​deer tick fever virus diseases. Prognosis During major LBRF epidemics, overall case fatalities of 40% or higher have been reported, but in treated cases they are less than 5%. TBRF is less dangerous and deaths during relapses are most unusual but have been reported. In both LBRF and TBRF, pregnant women and infants are at greatest risk of dying. Treatment Antibiotics LBRF LBRF is readily cured without relapses by a single oral dose of 500 mg tetracycline or 500 mg erythromycin stearate. However, since few pa- tients with severe LBRF are able to swallow tablets without vomiting them up, a more reliable treatment is a single intravenous dose of 250 mg tetracycline hydrochloride or, for pregnant women and chil- dren, a single intravenous dose of 300 mg erythromycin lactobionate (children 10 mg/​kg body weight). In mixed epidemics of LBRF and louse-​borne typhus, a single oral dose of 100 mg doxycycline proved effective. Benzyl penicillin (300 000 units), procaine penicillin with benzyl penicillin (600 000 units), and procaine penicillin with aluminium monostearate (600 000 units), all by intramuscular injection, are often effective but might fail to prevent relapses. Long-​acting pre- parations clear spirochaetaemia slowly and the JHR is protracted. Some experienced clinicians prefer to use a low initial dose of peni- cillin (adult dose, 100 000–​400 000 units by intramuscular injec- tion) in severe cases and pregnant women because they believe that the incidence and severity of the JHRs will be less. Chloramphenicol is effective in a single dose of 500 mg by mouth or intravenous injection in adults. A meta-​analysis of trials of chemotherapy of LBRF concluded that no clear superiority of any drug had been confirmed and that azithromycin should be tried in future. TBRF, including B. myamotoi sensu lato infection Although TBRF is usually milder than LBRF, it is more difficult to treat because spirochaetes persist in tissues, such as the central ner- vous system and eye, and produce relapses. Oral tetracycline, 500 mg every 6 h for 10 days is, however, effective. Oral erythromycin can be given to pregnant women (500 mg every 6 h for 10 days) and children (125–​250 mg every 6 h for 10 days). In patients unable to swallow tablets, treatment can be initiated with 250 mg intravenous tetra- cycline hydrochloride or with 300 mg erythromycin lactobionate. Chloramphenicol is effective in a dose of 500 mg every 6 h for 10 days in adults, and 250 mg every 6 h for 10 days in older children. JHR Antimicrobials have reduced the mortality of relapsing fevers from 30 to 70% to less than 5%. However, drugs such as tetracycline, which rapidly eliminate spirochaetes from the blood and prevent relapses, usually induce a severe JHR that may occasionally prove fatal. Clearly, in a disease with such a high natural mortality, treat- ment cannot be withheld, especially as severe spontaneous crises, which might also prove fatal, occur in a large proportion of LBRF cases after the fifth day of fever. There is no evidence, however, that the shorter and more intense reaction following tetracycline is more dangerous than the more prolonged but apparently milder reaction following slow-​release penicillin. Neither hydrocortisone in doses up to 20 mg/​kg nor paracetamol prevent the JHR but they reduce peak temperatures, hasten the fall in temperature, and lessen the fall in blood pressure during the flush phase. Pretreatment with oral prednisolone can prevent the JHR of early syphilis, but in LBRF nei- ther an oral dose of 3 mg/​kg prednisolone given 18 h beforehand nor an infusion of 3.75 mg/​kg β-methasone prevented the reaction to tetracycline treatment. However, meptazinol, an opioid antagonist/​ agonist, diminishes the reaction when given in a dose of 100 mg by intravenous injection. The discovery of an explosive release of TNFα, IL-​6, and IL-​8 just before the start of the JHR prompted the testing of a polyclonal ovine Fab anti-​TNFα antibody. When infused for 30 min before treatment with intramuscular penicillin, this anti- body suppressed the JHR. Supportive treatment Patients must be nursed flat in bed for at least 24 h after treatment to prevent postural hypotensive collapse and the precipitation of fatal cardiac arrhythmias. Hyperpyrexia should be prevented with anti- pyretics, vigorous fanning, and tepid sponging. Although patients with acute LBRF have an expanded plasma volume, most are dehy- drated and relatively hypovolaemic. Adults may need 4 litres or more of isotonic saline intravenously during the first 24 h. Infusion should be controlled by monitoring jugular venous or central venous pres- sures. Acute myocardial failure may develop, particularly during the

8.6.34  Relapsing fevers 1197 flush phase of the JHR or spontaneous crisis. This is signalled by a rise in central venous pressure above 15 cmH2O; 1 mg digoxin given intravenously over 5–​10 min has proved effective in this emergency. Because of the intense vasodilatation, diuretics may accentuate the circulatory failure by causing relative hypovolaemia. Oxygen should be given during the reaction, particularly in severe cases. Vitamin K should be given to all patients with prolonged prothrombin times. Heparin is not effective in controlling coagulopathy and should not be used. Complicating infections (typhoid, salmonellosis, bacillary dys- entery, tuberculosis, typhus, malaria) must be treated appropriately. Prevention and control No vaccines are available. LBRF: delousing Infested clothing should be deloused using heat (>60°C), chlorine bleach, or insecticide (10% dichlorodiphenyltrichloroethane (DDT), 1% malathion, 2% temephos, 1% propoxur, or 0.5% permethrin), and patients should be bathed with soap and 1% Lysol (cresol). Lice are abundant in hair, which should be washed or shaved off. Breaking transmission from lice to the susceptible population is es- sential for the control of an epidemic. Eugenol and β-​caryophyllene from clove essential oil repel Pediculus humanus corporis. TBRF: tick control Tick infestation of dwellings can be reduced by improved house construction (e.g. rodent-​proofing of cabins on the North Rim of the Grand Canyon), control of peridomestic rodent hosts, and use of residual insecticides (pyrethroids, benzene hexachloride, λ-​ cyhalothrin, malathion, or DDT). Travellers should avoid sleeping in places where ticks and rodents are abundant, such as poorly maintained log cabins, should apply repellents to their skin (di- ethyl toluamide (DEET)), and should sleep under insecticide-​ impregnated bed nets. Postexposure prophylaxis with doxycycline (200 mg followed by 100 mg on the next 4 days) proved effective against B. persica in Israel. FURTHER READING Antinori S, et al. (2016). Louse-​borne relapsing fever among East African refugees in Europe. Travel Med Infect Dis, pii: S1477-​8939(16)00006-​5. Barbour AG (2014). Phylogeny of a relapsing fever Borrelia species trans- mitted by the hard tick Ixodes scapularis. Infect Genet Evol, 27, 551–​8. Barbour AG, Hayes SF (1986). Biology of Borrelia species. Microbiol Rev, 50, 381–​400. Boutellis A, Abi-​Rached L, Raoult D (2014). The origin and distribu- tion of human lice in the world. Infect Genet Evol, 23, 209–​17. Bryceson ADM, et al. (1970). Louse-​borne relapsing fever: a clinical and laboratory study of 62 cases in Ethiopia and a reconsideration of the literature. QJM, 39, 129–​70. Cadavid D, Barbour AG (1998). Neuroborreliosis during relapsing fever: review of the clinical manifestations, pathology, and treatment of infections in humans and experimental animals. Clin Infect Dis, 26, 151–​64. Cutler SJ, et al. (2017). Diagnosing Borreliosis. Vector Borne Zoonotic Dis, 17, 2–11. Fekade D, et al. (1996). Prevention of Jarisch–​Herxheimer reactions by treatment with antibodies against tumor necrosis factor alpha. N Engl J Med, 335, 311–​5. Felsenfeld O (1971). Borrelia: strains, vectors, human and animal bor- reliosis. Green, St Louis, . Guerrier G, Doherty T (2011) Comparison of antibiotic regimens for treating louse-​borne relapsing fever: a meta-​analysis. Trans R Soc Trop Med Hyg, 105, 483–​90. Hasin T, et al. (2006). Postexposure treatment with doxycycline for the prevention of tick-​borne relapsing fever. N Engl J Med, 355, 148–​55. Krause PJ, et al. (2015). Borrelia miyamotoi infection in nature and in humans. Clin Microbiol Infect, 21, 631–​9. LaRocca TJ, Benach JL (2008). The important and diverse roles of anti- bodies in the host response to borrelia infections. Curr Top Microbiol Immunol, 319, 63–​103. Lescot M, et al. (2008). The genome of Borrelia recurrentis, the agent of deadly louse-​borne relapsing fever, is a degraded subset of tick-​ borne Borrelia duttonii. PLoS Genet, 4, e1000185. Marosevic D, et al. (2017). First insights in the variability of Borrelia recurrentis genomes. PLoS Negl Trop Dis, 11, e0005865. Moran-​Gilad J, et al. (2013). Postexposure prophylaxis of tick-​borne relapsing fever:  lessons learned from recent outbreaks in Israel. Vector Borne Zoonotic Dis, 13, 791–​7. Negussie Y, et al. (1992). Detection of plasma tumor necrosis factor, interleukins 6, and 8 during the Jarisch–​Herxheimer reaction of re- lapsing fever. J Exp Med, 175, 1207–​12. Parry EH, et al. (1970). Some effects of louse-​borne relapsing fever on the function of the heart. Am J Med, 49, 472–​9. Perine PL, Teklu B (1983). Antibiotic treatment of louse-​borne re- lapsing fever in Ethiopia: a report of 377 cases. Am J Trop Med Hyg, 32, 1096–​100. Platonov AE, et al. (2011). Humans infected with relapsing fever spiro- chete Borrelia miyamotoi, Russia. Emerg Infect Dis, 17, 1816–​23. Schwan TG, et al. (2012). Endemic foci of the tick-​borne relapsing fever spirochete Borrelia crocidurae in Mali, West Africa, and the potential for human infection. PLoS Negl Trop Dis, 6, e1924. Talagrand-Reboul E, et al. (2018). Relapsing Fevers: Neglected Tick- Borne Diseases. Front Cell Infect Microbiol, 8, 98. Telford SR 3rd, et al. (2015). Borrelia miyamotoi disease: neither Lyme disease nor relapsing fever. Clin Lab Med. 35(4):867–​82. Trape JF et al. (2013). The epidemiology and geographic distribution of relapsing fever borreliosis in West and North Africa, with a review of the Ornithodoros erraticus complex (Acari: Ixodida). PLoS One, 8, e78473. Vidal V, et al. (1998). Variable major lipoprotein is a principal TNF-​ inducing factor of louse-​borne relapsing fever. Nat Med, 4, 1416–​20. Vuyyuru R, et al. (2011). Characteristics of Borrelia hermsii infection in human hematopoietic stem cell-​engrafted mice mirror those of human relapsing fever. Proc Natl Acad Sci U S A, 108, 20707–​12. Warrell DA, et al. (1970). Cardiorespiratory disturbances associated with infective fever in man: studies of Ethiopian louse-​borne re- lapsing fever. Clin Sci, 39, 123–​45. Warrell DA, et  al. (1971). Physiologic changes during the Jarisch–​ Herxheimer reaction in early syphilis. A comparison with louse-​ borne relapsing fever. Am J Med, 51, 176–​85. Warrell DA, et  al. (1983). Pathophysiology and immunology of the Jarisch–​Herxheimer-​like reaction in louse-​borne relapsing fever: comparison of tetracycline and slow-​release penicillin. J Infect Dis, 147, 898–​909. Wright DJ1, Maria B (2011). Ich bin ein Berliner. The contributions of early British colonial and German scientists to the elucidation of the nature of spirochaetes. Clin Microbiol Infect, 17, 484–​6.

8.6.35 Leptospirosis 1198

8.6.35 Leptospirosis 1198

section 8  Infectious diseases 1198 8.6.35  Leptospirosis Nicholas P.J. Day ESSENTIALS Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira, is a bacterial zoonosis with a worldwide impact on human and animal health. For human infection rodents are the most important reservoir; infection follows exposure to contaminated water, soil, or urine, the organism entering through skin abrasions or mucosal surfaces. Clinical features—​subclinical infection is common, but symptom- atic disease usually begins with abrupt onset of fever, chills, headache, and myalgia. Conjunctival suffusion, uncommon in other causes of febrile illness, is a useful diagnostic sign. Most cases of leptospirosis are self-​limiting, but in a minority (<10%) severe disease may follow with complications including jaundice and renal failure (‘Weil’s dis- ease’), vascular collapse, and haemorrhagic manifestations including pulmonary haemorrhage. Diagnosis—​most cases are undiagnosed due to the poor avail- ability of diagnostic tests in regions where transmission occurs. The gold standard remains the serological microscopic agglutination test, but increasingly new serological and molecular tests, including rapid diagnostic tests, are becoming available. Treatment and prognosis—​whether treatment of mild leptospirosis with antibiotics prevents more severe disease remains controversial, most experts recommend empirical treatment if leptospirosis is sus- pected. Mild disease can be treated with doxycycline or azithromycin. Severe disease should be treated with parenteral antibiotics (ß-​lactams or doxycycline). Reported mortality in hospitalized cases of leptospir- osis ranges from 4% to 52%. Introduction Leptospirosis is a widespread zoonotic infection caused by patho- genic spirochetes of the genus Leptospira. It has a major impact on both human and animal health. A variety of domestic and wild ani- mals are reservoirs for leptospires, which are maintained in the renal tubules of infected animals and shed in the urine. Humans are inci- dentally infected following direct or indirect exposure to the urine of infected animals. Infection in humans ranges from asymptom- atic through a relatively benign febrile illness to a severe potentially fatal illness associated with jaundice, renal failure, and pulmonary haemorrhage. Severe icteric leptospirosis is known as Weil’s disease after Adolf Weil, who first described the combination of abrupt high fever, jaundice, renal failure, and splenomegaly in 1886. Aetiology • Leptospirosis is caused by infection with pathogenic spirochetal bacteria of the genus Leptospira. • The genus Leptospira contains 22 species, ten of which are pathogenic. • Over 200 serovars of pathogenic Leptospira have been described, many of which are broadly associated with a particular animal host. Leptospires are highly motile, aerobic, spiral-​shaped spirochetes with a typical length of 6–​20 µm, width of 0.1 µm, helical amplitude of 0.1–​0.15 µm and wavelength of 0.5 µm. Their corkscrew motility is driven by two endoflagella, one at each end of the cell. It is thought that the positioning and action of these endoflagella is respon- sible for the hooks that occur at one or both ends of the cell, giving leptospires their characteristic ‘question mark’ appearance that in- formed the name given in the original 1907 description—​Spirocheta interrogans (Fig. 8.6.35.1). The genus Leptospira currently contains 22 species, ten of which are pathogenic (Leptospira interrogans, L.  kirschneri, L.  noguchii, L.  alexanderi, L.  weilii, L.  alstonii, L.  borgpetersenii, L.  santaro­ sai, L. kmetyi and L. mayottensis). Five species are considered to be of unclear or intermediate pathogenicity (L.  inadai, L.  fainei, L. broomii, L. licerasiae, and L wolffii), and the remaining seven are non​pathogenic free living saprophytic species (L. biflexa, L. meyeri, L. wolbachii, L. vanthielii, L. terpstrae, L. yanagawae and L. idonii). L. interrogans, L. borgpetersenii an L. kirschneri are the main patho- genic species of leptospirosis in humans and animals worldwide. There are several genetic typing schemes for identifying par- ticular strains of leptospire, including multilocus sequence typing and multiple-​locus variable-​number tandem repeat analysis. An older classification system based on serology is used in par- allel with the newer molecular classification, and forms the basis of the microscopic agglutination test which remains the gold standard for serological diagnosis of leptospirosis. Serovars are defined by cross-​agglutination absorption testing with rabbit antiserum, and approximately 250 serovars of pathogenic leptospires grouped into 24 serogroups have been described. Many of these have regional and animal host associations and for this reason the serovar classi- fication remains epidemiologically useful. For example, serovars of the serogroup Icterohaemorrhagiae are associated with rats (Rattus species), and several serovars are associated with domestic livestock animals, such as Hardjo (with cattle and sheep) and Pomona (with pigs). Several serovars are found in multiple Leptospira species, so by convention the two classification systems are used together (e.g. Leptospira borgpetersenii serovar Hardjo). Epidemics may be caused by a particular ecologically successful pathogenic clone, such as the 1995–​2005 epidemic in Thailand where most isolates were L. interrogans serovar Autumnalis of multilocus sequence typing sequence type 34. Whole genome sequencing is currently revolutionizing our under- standing of Leptospira. Pathogenic species evolved from saprophytic species through a process of gene loss (often metabolic genes neces- sary for living free in the environment) and the gain through hori- zontal transfer of genes putatively associated with adaptation to the mammalian host. Genome comparison also allows identification of genes putatively associated with disease pathogenesis. Epidemiology • Leptospirosis is a worldwide zoonosis, though more common in tropical regions.

8.6.35  Leptospirosis 1199 • Humans are incidental hosts, infected through direct or environ- mental exposure to the urine of infected reservoir animals. Leptospirosis is the most widespread human zoonosis, occurring in both temperate and tropical regions. Its incidence is around 10 times higher in tropical regions than temperate; in the tropics lepto- spirosis is mainly a disease of poverty, associated with poor sani- tation, rodent-​infested slums, occupational exposure, and flooding. Information on the epidemiology of human leptospirosis and its associated global health burden is limited, as because of difficulties in diagnosis and the poor health systems in many endemic areas it is a relatively neglected and underreported disease. Based on available data from health databases and published studies on morbidity and mortality, it has been estimated recently that roughly one million cases of leptospirosis occur annually causing around 60 000 deaths. These are likely to be underestimates. The highest burdens of mor- bidity and mortality were seen in South and Southeast Asia-Oceania, the Caribbean, Andean, Central, and tropical Latin America, and East sub-​Saharan Africa (Fig 8.6.35.2). A wide variety of mammals are reservoir hosts of pathogenic leptospires, with humans infected incidentally following direct or environmental exposure to infected animals or their urine. Hence the epidemiology of human leptospirosis is driven by the epidemi- ology of animal infection and the manner in which humans are ex- posed to these animals and their urine. Animal infection Leptospirosis is a ubiquitous global disease of animals, particularly mammals. It is found in both wild and domestic animals, and as a source of human disease and cause of economic loss is an excellent example of the ‘One Health’ concept. Animals commonly infected include rodents, cattle, swine, dogs, horses, sheep, and goats. Cats are rarely infected. Animals can be maintenance hosts or, like humans, incidentally infected. Initial infection is through the mucous mem- branes (eyes, mouth, genitals), and during an initial bacteraemic phase leptospires spread haematogenously to the renal tubules. Certain serovars are well adapted as parasites in particular host ani- mals, with infection in the renal tubules lasting for many years and causing little in the way of clinical illness while maintaining the in- fection in the environment through urinary excretion. Symptomatic disease in animals can be severe; mortality in dogs is estimated at approximately 10%. Spontaneous abortion is a common outcome of leptospirosis in cattle, swine, sheep, and goats, leading to major economic consequences. Human infection Human infection results from exposure to animal urine, contam- inated water or soil, or infected animal tissue (see Box 8.6.35.1). Fig. 8.6.35.1  Leptospire seen under darkfield microscopy. Courtesy of Vanaporn Wuthiekanun. Fig. 8.6.35.2  Estimated annual morbidity of leptospirosis by country or territory. Annual disease incidence is represented as an exponential colour gradient from white (0–​3), yellow (7–​10), orange (20–​25) to red (over 100), in cases per 100 000 population. Circles and triangles indicate the countries of origin for published and grey literature quality-​assured studies, respectively. From Costa F et al. (2015) Global Morbidity and Mortality of Leptospirosis: A Systematic Review. PLoS Negl Trop Dis 9(9), e0003898.

section 8  Infectious diseases 1200 Portals of entry include cuts or abraded skin, and mucous mem- branes such as the eyes. It is unclear whether Leptospira can pene- trate intact skin. Rarely, infection might be acquired by eating food contaminated with urine or via aerosols. Human infection does involve a period of leptospire shedding in the urine, so human to human transmission is possible but very rare. Sexual transmission has been reported, as has transplacental infection during active maternal infection which often leads to abortion, stillbirth, or neo- natal infection. In tropical regions endemic leptospirosis is mainly a disease of poverty, associated with low quality rodent-​infested urban slum housing with poor sanitation, with occupational exposure such as subsistence farming, and with environments susceptible to flooding. Outbreaks affecting thousands of people and causing hundreds of deaths are common, often associated with increased rainfall or flooding—​which presumably increase the chances of exposure to contaminated water. Pathogenesis/​Pathology • Leptospirosis has features of both an acute bacteraemic infection and a systemic vasculitis. • As accidental hosts, the human innate immune system is not well adapted to protection from leptospirosis and consequently bac- terial loads in blood are high. • Leptospires spread haematogenously to the liver, kidney, and lungs, which are the major target organs in severe leptospirosis. • The pathological consequences of infection are probably mediated by a combination of a direct toxic effect of the leptospires and the resulting immune response. • Activation of the inflammasome plays a major role in the patho- genesis of severe leptospirosis. Leptospires penetrate tissue barriers through abraded skin, the conjunctivae, or the oral cavity and can be found in the blood stream within 48 hours of initial exposure. Unlike other pathogenic spiro- chetes, such as Treponema pallidum and Borrelia burgdorferi, they form no infected lesions at the site of entry. Bacteraemia lasts from 2 to 9 days, and ends with the appearance of agglutinating antibodies. The concentration of leptospires in the blood can be as high as 106/​ml, which is similar to that seen in the B. recurrentis spirochetaemia of relapsing fever and several orders of magnitude higher than seen in bloodstream infections caused by Enterobacteraceae such as E. coli. Leptospires subsequently disseminate haematogenously to target organs including the liver, lung, and kidney, leading in severe cases to multiorgan dysfunction and death. Pathogen-​associated molecular patterns including lipopolysac- charides and outer membrane proteins activate the innate immune response through TLR2-​ and TLR4-​dependent pathways. The rela- tive inability of human TLR2 to recognize leptospiral lipopolysac- charides (as opposed to lipopolysaccharides from Enterobacteraceae) might be responsible for the relatively high levels of bacteraemia seen in leptospirosis. This hypothesis is supported by the superior ability of murine TLR4 to recognize leptospiral lipopolysacchar- ides; the mouse is a natural reservoir of leptospirosis and resistant to fatal infection. In severe leptospirosis, patients experience a ‘cyto- kine storm’ with very high levels of pro-​inflammatory cytokines such as IL-​6 and TNFα. IL-​6 and the anti-​inflammatory cytokine IL-​10 are independent predictors of fatal outcome, suggesting that an initial protective Th-​1 response is counteracted by overproduc- tion of IL-​10. Histopathologically, leptospires are seen in large and medium-​ sized blood vessels and in the capillaries and interstitial spaces of affected organs. A  diffuse systemic vasculitis is suggested by the presence of polymorphonuclear cells adherent to the endothelium and signs of endothelial cell damage. Disorders of coagulation are common in severe leptospirosis. Thrombocytopenia is common, and prothrombin time and activated partial thromboplastin time are frequently prolonged. Fibrinogen, D-​dimer, thrombin-​antithrombin III complexes, and prothrombin fragment 1 + 2 are often elevated. In one study from Thailand al- most half of severe cases met the criteria for overt disseminated intravascular coagulation. Whereas multiple organs and systems are affected in leptospirosis, with myocarditis, meningoencephalitis, and uveitis all occurring in severe disease, the most important target organs are the liver, kidney, and lung. Liver Autopsy studies show congested hepatic sinusoids and distention of the space between the sinusoidal endothelium and hepatocytes (the space of Disse). In a hamster model of leptospirosis leptospires were observed infiltrating Disse’s space and migrating between hepato- cytes, detaching the intercellular junctions and disrupting the bile canaliculi. Jaundice likely results from the consequent leakage of bile from bile canaliculi into sinusoidal blood vessels. Kidney The kidney of reservoir animals plays a key role in the leptospiral life cycle, with the proximal renal tubular lumen the major site of colonization. In humans leptospiral lipoproteins, such as LipL32, are recognized by TLR2 on tubular epithelial cells triggering an inflammatory response leading to interstitial nephritis. Autopsy studies show damage to the tubular epithelium and luminal dis- tension with hyaline casts and cellular debris. While most severe in the proximal convoluted tubule, tubular damage is more ex- tensive and less focal than seen in acute tubular necrosis from other causes. The pattern of tubular damage seen in leptospir- osis and its effects on sodium, potassium and water handling might explain the polyuria seen in mild leptospirosis, and the non​oliguric potassium wasting renal failure often seen in more severe disease. Box 8.6.35.1  Risk factors for acquiring leptospirosis • Occupational exposure—​farmers, ranchers, abattoir workers, veterin- arians, loggers, sewer workers, rice farmers, pet traders, rat catchers/​ merchants, military personnel, laboratory workers • Recreational activities—​freshwater swimming (e.g. triathlons), canoeing, kayaking, trail biking • Household exposure—​rodent infestation, pet dogs, domesticated livestock • Other—​Walking barefoot through surface water, particularly during floods, skin lesions, contact with wild rodents, accidental laboratory exposure

8.6.35  Leptospirosis 1201 Lungs Autopsy specimens are usually congested, with focal or mas- sive haemorrhage occurring in both the alveolar septa and intra-​alveolar spaces. Using immunohistochemistry leptospiral antigen has been detected in macrophages in both pulmonary septa and alveoli, suggesting that leptospires exert a local direct destructive action. Pathogenesis might also involve an immune component; in a guinea pig model, which closely replicates the pulmonary haemorrhage seen in humans, extensive deposition of immunoglobulin and complement was seen along the alveolar basement membrane. The coagulation abnormalities ubiqui- tous in severe leptospirosis might also contribute to pulmonary haemorrhage. Clinical features • The clinical course of human leptospirosis is very variable. Most cases are mild or subclinical, but some are severe and potentially fatal. • Conjunctival suffusion in febrile patients strongly suggests a diag- nosis of leptospirosis. • Jaundice is a common feature of severe leptospirosis and is often associated with renal failure (Weil’s disease). • Pulmonary haemorrhage is the most serious complication of leptospirosis and is associated with a high risk of death. Leptospirosis is usually described as a biphasic illness, with an acute bacteraemic phase lasting 2–​9 days, then defervescence and several days of improvement, followed by an ‘immune’ phase with renewed fever and the onset of complications. Clinically, however, the two phases often merge, particularly in severe disease. Symptoms After an incubation period of 3–​26 days (average 10 days), the illness usually presents non​specifically with an abrupt onset of fever, rigors, myalgias, and headache. Nausea, vomiting, and diarrhoea occur in around 50% of cases, and a non​productive cough occurs in around a third. Less common symptoms include arthralgias, bone pain, sore throat, and abdominal pain. Signs Conjunctival suffusion is uncommon in other infections but pre- sent in 55% of leptospirosis patients in one case series, and is hence an important clue to a diagnosis of leptospirosis. Subconjunctival haemorrhages can also occur (Fig. 8.6.35.3). Other clinical signs of bleeding such as petechiae, ecchymoses, and epistaxis are relatively common. Muscle tenderness (characteristically involving the calves and lower back), muscle rigidity, splenomegaly, hepatomegaly, lymphadenopathy, pharyngitis, abnormal respiratory signs, or an erythematous skin rash may be present. Aseptic meningitis is common, present in 50–​85% of patients if cerebrospinal fluid (CSF) is examined after seven days of illness. This might be due to the host immune response, though in one series Leptospira DNA was detectable by polymerase chain reaction (PCR) in the CSF of 90% of serologically confirmed cases with CSF abnormalities. Complications Weil’s disease, a severe, potentially fatal illness characterized by jaundice and renal failure, occurs in less than 10% of symptomatic leptospirosis cases. The renal failure is often non​oliguric and asso- ciated with a marked hypokalaemia. The hepatic involvement signi- fied by jaundice is generally reversible and not a cause of death. Leptospirosis-​associated severe pulmonary haemorrhage syn- drome is the most lethal complication of leptospirosis, associated with massive haemoptysis and pathophysiological features of acute respiratory distress syndrome (Fig. 8.6.35.4). It can occur either with or without jaundice and renal failure and is associated with a very high mortality (71% in one case series). Acalculous chole- cystitis and pancreatitis have been described and can cause severe abdominal pain. Fig. 8.6.35.3  Jaundice, haemorrhage, and conjunctival suffusion in acute leptospirosis. Fig. 8.6.35.4  Chest radiograph of a European traveller with leptospirosis-​associated severe pulmonary haemorrhage syndrome acquired in Sabah (Malaysia). Copyright D. A. Warrell.

section 8  Infectious diseases 1202 Other complications include uveitis, optic neuritis, periph- eral neuropathy, myocarditis, rhabdomyolysis, and gastrointes- tinal bleeding with haematemesis and/​or melaena. Vasculitis with necrosis of the extremities can be seen in severe cases. An acute haemolytic anaemia might also complicate leptospirosis, particu- larly in patients with G6PD deficiency. Differential diagnosis Undifferentiated fever Leptospirosis is a common cause of undifferentiated fever, particu- larly in tropical areas, and is often difficult to distinguish clinically from other sympatric causes. Conjunctival suffusion (as opposed to conjunctivitis), when present, is a useful almost pathognomonic sign as it rarely occurs in other infections. Common causes of fever that can present in a very similar manner to leptospirosis include malaria, dengue, scrub typhus, murine typhus, spotted fever group rickettsioses, chikungunya, Zika, ehrlichiosis, and enteric fever. As in temperate regions, cosmopol- itan viral infections such as infectious mononucleosis and influenza can be indistinguishable clinically from leptospirosis. In non​tropical areas where leptospirosis is relatively uncommon suspicion that it may be the cause of undifferentiated fever is usually prompted by a history of exposure to freshwater. Severe disease Hepatitis A  and E infection, malaria, and viral haemorrhagic fevers should be considered in cases of febrile jaundice. Severe malaria can present as jaundice with renal failure, and hantavirus infection is a cause of hepatorenal syndrome and pulmonary haemorrhage. Clinical investigations Routine tests Routine blood tests are usually non​specific, with hyponatraemia, mild to moderately raised transaminases, mildly raised white blood cell count, and thrombocytopenia all common. An elevated creatine kinase occurs in around 50% of patients. In severe cases bilirubin and creatinine can be elevated, often markedly so. The bilirubin is usually conjugated, though a mixed conjugated/​unconjugated bilirubinaemia can occur in cases complicated by haemolytic anaemia. Urinalysis often shows proteinuria, white cells, granular casts, and occasionally microscopic haematuria. The cerebrospinal fluid (CSF) may have elevated lymphocytes and/​or neutrophils with minimal to moderately elevated protein concentrations and a normal or occa- sionally low glucose. A chest X-​ray might demonstrate pulmonary involvement with consolidation or a ground glass appearance, which can indicate pul- monary haemorrhage or acute respiratory distress syndrome. Specific diagnostic tests Leptospirosis can be diagnosed by direct detection of the organism or its constituents in body fluids, by culture isolation of the organism, or by detection of specific antibodies. There is no gold standard diag- nostic test for leptospirosis. The microscopic agglutination test and bacterial culture are both relatively insensitive, even when combined (55.5% sensitivity, though 98.8% specific). The diagnostic utility of each method depends on the timing and nature of the test sample, with culture and molecular testing on blood most sensitive in the first week of illness and serological methods and urine culture more sensitive from the end of the first week onwards. Serological tests The microscopic agglutination test is the most commonly used diagnostic test, and when applied to paired acute and convalescent samples is considered the reference standard. However, it is labour-​ intensive and complex to perform—​hence only available at reference centres—​and serovar dependent. Recently developed rapid IgM ELISAs and lateral flow diagnostic tests are increasingly available but perform variably in the field, particularly in endemic areas. Molecular tests Molecular tests based on real-​time PCR and loop-​mediated iso- thermal amplification show considerable promise for rapid, accurate diagnosis in the acute phase of the illness, but are not yet widely available. Gene targets used include housekeeping genes (rrs, gyrB or secY) and pathogen specific genes (lipL32, lig, or lfb1). Whole genome sequencing of CSF has been used to make a diagnosis of leptospirosis. Culture Leptospires can be cultured using special medium from clinical spe- cimens including blood, urine, and CSF. Growth is usually observed in one to two weeks, but may take up to three months. A recently developed solid agar (LVW media) facilitates more rapid growth, isolation of single colonies, and simplified antimicrobial sensitivity testing. Treatment Antimicrobial therapy See Box 8.6.35.2 for suggested antibiotic treatment regimens. Mild disease Although most cases of leptospirosis are mild and self-​limiting, ap- propriate antibiotic therapy should be given empirically to all symp- tomatic patients suspected of having leptospirosis with the intention of reducing the duration of illness and the shedding of leptospires in the urine. Early antibiotic treatment may prevent progression to severe disease. In one retrospective case-​control study from New Caledonia, risk factors for the development of severe leptospirosis included a delay in the initiation of antibiotics of more than 2 days following the start of symptoms. Although rapid diagnostic tests are improving in both diagnostic accuracy and availability, even if a rapid test is negative treatment should still be commenced. Most treatment will be started before a definitive diagnosis is made, so the empirical regimen chosen should cover other possible diagnoses. In patients in or returning from the tropics malaria should be excluded and empirical treatment should

8.6.35  Leptospirosis 1203 cover rickettsial diseases. Fluoroquinolones are not effective in scrub typhus so should not be used. In vitro studies have shown that leptospires are susceptible to tetracyclines, macrolides, ß-​lactams, fluoroquinolones, and streptomycin. In 1984 a small randomized double-​blind study of 29 patients showed that doxycycline 100 mg bd reduced the duration of illness by 2 days when compared to placebo, and also decreased symptom severity and prevented leptospiruria. In a 2007 study in Thailand 296 patients with suspected leptospirosis or scrub typhus were random- ized to receive doxycycline (200 mg initially followed by 100 mg or- ally every 12 hours for seven days) or azithromycin (2 g on the first day followed by 1 g daily for two more days). There was no difference in fever clearance times, but oral azithromycin was better tolerated than doxycycline. Severe disease In the 1980s there were two small placebo-​controlled trials of intra- venous penicillin in severe leptospirosis. One from the Philippines recruited 41 patients and demonstrated a reduction in fever duration, abnormal renal function, and hospitalization in the penicillin group. The second randomized 79 patients in Barbados and showed no difference in clinical outcome. Leptospiruria was prevented in the penicillin group in both studies. Studies from Thailand have shown comparable efficacy for paren- teral penicillin, ceftriaxone, cefotaxime, and doxycycline for treat- ment of severe leptospirosis. In one study of 173 patients with severe leptospirosis, patients were randomized to penicillin G (1.5 million units IV qds for 7 days) or ceftriaxone (1 g IV od for 7 days). In a second study, 540 patients with suspected severe leptospirosis (264 serologically confirmed) were randomized to cefotaxime (1 g IV gds for 7 days), penicillin G (1.5 million units IV qds for seven days), or doxycycline (200 mg IV initially followed by 100 mg IV every bd for 7 days). In both studies, all regimens had similar efficacy for leptospirosis. Ideally an adequately placebo-​controlled trial should be con- ducted, but this is unlikely to happen because of ethical consid- erations. In one retrospective intensive care unit (ICU) series of leptospirosis cases prior treatment with ceftriaxone was associated with lower mortality, suggesting treatment may have a positive effect on disease progression. Leptospirosis appears to be much less prone to a Jarisch-​Herxeimer reaction to treatment than other spirochetal diseases, and antibiotics should not be withheld out of fear of this complication, which, if it exists, is rare and mild. Role of corticosteroids Intravenous corticosteroid therapy has been proposed in severe leptospirosis, particularly where there is pulmonary involvement. Some reports have suggested a possible benefit to use of steroids as an adjunct to antibiotic therapy, but there is currently insufficient evidence to support their routine use. Supportive therapy Management of patients with acute kidney injury and/​or acute respiratory distress syndrome should in general follow that of other cases of severe sepsis with these complications. Continuous haemofiltration has been shown to be more effective than peri- toneal dialysis in treating infection-​associated hypercatabolic renal failure. Peritoneal dialysis, however, may be the only option in resource-​limited settings. There is some non​randomized evidence that early initiation of dialysis without waiting for optimization of fluid status is associated with significantly lower mortality in lepto- spirosis patients with both acute respiratory distress syndrome and renal failure. In acute respiratory distress syndrome low net fluid in- take and lung-​protective ventilation practices to prevent pulmonary haemorrhage have been recommended, though there is no random- ized leptospirosis-​specific evidence to support this. Hypokalaemia is a common feature of leptospirosis-​associated non​oliguric renal failure, and should be corrected. Prognosis/​outcome Most cases of leptospirosis are mild and self-​limited. In a recent systematic review of 35 studies reporting untreated mortality rates, mortality rates for untreated anicteric patients with leptospirosis were low (median 0%, range 0–​1.7%). High case fatality rates were Box 8.6.35.2  Antimicrobial treatment of leptospirosis Outpatients with mild disease Adults Either Doxycycline 100 mg orally twice daily for 7 days Or Azithromycin 500 mg orally once daily for 3 days Children ≥8 years of age Either Doxycycline 2 mg/​kg orally per day in two equally divided doses, not to exceed 200 mg daily, for 7 days Or Azithromycin 10 mg/​kg orally on day one (maximum dose 500 mg/​ day) followed by 5 mg/​kg/​day orally once daily for two further days (max- imum dose 250 mg/​day) Children less than 8 years and pregnant women Either Azithromycin orally for 3 days Or Amoxicillin orally 25–​50 mg/​kg per day in three equally divided doses, maximum 500 mg per dose, for 7 days Severe disease Adults Either Penicillin 1.5 million units intravenously (IV) 6 hourly for 7 days Or Doxycycline 100 mg IV twice daily for 7 days Or Ceftriaxone 1–​2 g IV once daily for 7 days Or Cefotaxime 1 g IV 6 hourly for 7 days Children ≥8 years of age Either penicillin 250 000 to 400 000 units/​kg IV per day in 4 to 6 divided doses (maximum 6 to 12 million units daily) for 7 days Or Doxycycline 4 mg/​kg IV per day in two equally divided doses (max- imum dose 200 mg/​day) for 7 days Or Ceftriaxone 80–​100 mg/​kg IV once daily; maximum dose 2 g daily for 7 days Or Cefotaxime 100–​150 mg/​kg IV per day in 3 to 4 equally divided doses Children less than 8 years old and pregnant women Doxycycline should probably be avoided in children less than 8 years of age unless there are no other treatment options. For children less than 8 years of age with severe disease and β-​lactam hypersensitivity: Azithromycin 10 mg/​kg IV on day one; maximum dose 500 mg/​day, followed by 5 mg/​kg/​day IV once daily for two further days (maximum dose 250 mg/​day) Pregnant women with severe leptospirosis can be treated with IV peni- cillin, ceftriaxone, cefotaxime, or azithromycin. Doxycycline should be avoided.

8.6.36 Nonvenereal endemic treponematoses Yaws, en

8.6.36 Nonvenereal endemic treponematoses: Yaws, endemic syphilis (bejel), and pinta 1204

section 8  Infectious diseases 1204 associated with jaundice (median reported mortality 19.1%, range 0–​39.7%), renal failure (12.1%, range 0–​25%) and age over 60 years (60%, range 33.3–​60%). In other series of treated hospitalized cases mortality ranges from 4% to 52%. In a retrospective series of treated cases pulmonary involvement and central nervous system disease were associated with poor outcome. Where renal failure complicates leptospirosis, recovery of renal function after the acute period is generally rapid and complete, though recent reports from Sri Lanka and Taiwan suggest that leptospirosis might in some cases lead to chronic kidney disease. Prevention Vaccination Several human vaccines have been developed over the years, and although effective in certain epidemiological circumstances, all are serovar-​specific and none are currently widely available. Vaccination of domestic and farm animals provides variable levels of protection but is not widely practised. Exposure avoidance Control measures for preventing human leptospirosis include ro- dent control, protection of food from contamination with animal urine, and avoiding skin and mucous membrane contact with po- tential sources of infection such as flood water and animal farm water runoff. Antimicrobial prophylaxis Oral antimicrobial prophylaxis can be given to individuals at high risk of exposure. In a randomized placebo-​controlled study of 940 soldiers deployed for jungle training in Panama, significantly fewer cases of leptospirosis were observed in those who received weekly prophylaxis with doxycycline 200 mg prophylaxis compared with placebo (1 versus 20 cases). In a second study in the highly endemic flood-​prone Andaman Islands 782 individuals were randomized to weekly doxycycline 200 mg or placebo. Clinical infection rates were lower among those who received doxycycline (3.1% vs. 6.8%), but there was no difference in seroconversion rates. FURTHER READING Adler B (ed) (2015). Leptospira and leptospirosis. Springer, Berlin Heidelberg. Bharti AR, et al.; Peru-​United States Leptospirosis Consortium (2003). Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis, 3, 757–​71. Boonsilp S, et al. (2011). Molecular detection and speciation of patho- genic Leptospira spp. in blood from patients with culture-​negative leptospirosis. BMC Infect Dis, 11, 338. Brett-​Major DM, Coldren R (2012). Antibiotics for leptospirosis. Cochrane Database Syst Rev, 2, CD008264. Brett-​Major DM, Lipnick RJ (2009). Antibiotic prophylaxis for lepto- spirosis. Cochrane Database Syst Rev, 3, CD007342. Costa F, et al. (2015). Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis, 9, e0003898. Fouts DE, et al. (2016). What makes a bacterial species pathogenic? Comparative genomic analysis of the genus Leptospira. PLoS Negl Trop Dis, 10, e0004403. Limmathurotsakul D, et al. (2012). Fool’s gold: why imperfect refer- ence tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis. Clin Infect Dis, 55, 322–​31. McBride AJ, et  al. (2005). Leptospirosis. Curr Opin Infect Dis, 18, 376–​86. Taylor AJ, Paris DH, Newton PN (2015). A systematic review of the mortality from untreated leptospirosis. PLoS Negl Trop Dis, 9, e0003866. Thaipadungpanit J, et al. (2011). Diagnostic accuracy of real-​time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-​control study. PLoS One, 6, e16236. Thaipadungpanit J, et  al. (2007). A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Negl Trop Dis, 1, e56. 8.6.36  Non​venereal endemic treponematoses: Yaws, endemic syphilis (bejel), and pinta Michael Marks, Oriol Mitjà, and David Mabey ESSENTIALS The endemic treponematoses are chronic, granulomatous dis- eases caused by morphologically and serologically identical spirochaetes of the genus Treponema. They are spread by intimate but non​sexual contact and possibly by fomites, mainly among children. Treponema pallidum subsp. pertenue causing yaws (fram- boesia), T. pallidum subsp. endemicum causing endemic syphilis (bejel) and T. carateum causing pinta (carate) are distinguishable from T. pallidum subsp. pallidum, causing venereal syphilis, by their epidemiology and pathological effects and genomic structure (e.g. the arp gene). Despite the successful WHO/​UNICEF mass penicillin treatment campaign (1952–​1964), there has been a resurgence of yaws, mainly in West Africa, Southeast Asia, and the Pacific. Children living in rural areas in warm, humid climates in tropical countries are most affected by yaws. About 10% of untreated cases develop late, disfiguring, or crippling lesions of skin, bone, and cartilage. In 2012 azithromycin was demonstrated to be a highly effective treatment for yaws and mass treatment with azithromycin is now the mainstay of a new WHO yaws eradication campaign. Endemic syphilis occurs in arid areas of the Sahel and Arabian peninsula. It presents with buccal mucocutaneous lesions trans- mitted via contaminated fomites. Late systemic effects are much less common than in venereal syphilis. Pinta causes hypo-​ or hyperpigmented skin lesions and was previously reported to be endemic in Central and South America. Single-​dose benzathine-​ penicillin is effective treatment. Prevention is by improving hygiene and eliminating the reservoir of infection by mass treatment.

8.6.36  Nonvenereal endemic treponematoses: Yaws, endemic syphilis (bejel), and pinta 1205 Introduction The human treponematoses consist of venereal syphilis, caused by Treponema pallidum subsp. pallidum, and the three non​venereal endemic treponematoses; yaws (caused by T.p. subsp pertenue), bejel or endemic syphilis (caused by T.p. subsp. endemicum) and pinta (caused by T. carateum). Differences in the epidemiological and clinical features of the human treponematoses aid in diagnosis (Table 8.6.36.1) although newer molecular tools are being devel- oped to allow differentiation of the causative agents. All four diseases are caused by morphologically indistinguishable gram-​negative spirochetes, which cannot be cultured in vitro or differen- tiated by conventional testing. Sequencing of a limited number of trepo- nemal strains has shown the genome sequence of T.p. subsp. pallidum and T.p. subsp. pertenue to be more than 99.8% identical, with evidence of recombination between syphilis and yaws strains. Most of the identi- fied differences are restricted to six genomic regions, which are thought to contribute to the differences in pathogenicity between species. Clinically, the endemic treponematoses are characterized by multistage infection predominantly involving the skin, bones, and cartilage. The diseases vary in the incidence and severity of late stage disease. As with syphilis, penicillin has been the mainstay of treat- ment, but azithromycin has recently been shown to be effective in the treatment of yaws, prompting renewed efforts to eradicate it. Yaws Epidemiology Yaws is found in warm, humid environments, and predominantly affects children aged 2–​15  years living in remote, rural popu- lations. Even in countries where yaws is endemic the disease is extremely focal. It is spread by direct skin-​to-​skin, non​sexual, contact often after a cut or abrasion in the lower legs. Children born to mothers with yaws are generally unaffected, and most of the evidence indicates that the disease is not acquired congeni- tally. For every clinical case of yaws, there may be as many as six to ten latent cases in the community. Treponemal infections ex- tremely closely related to yaws and syphilis have been identified in non​human primates, but there is limited direct evidence for zoonotic infection. Yaws is the most prevalent of the endemic treponemal diseases. In the mid-​twentieth century, as many as 50 million individuals were thought to be infected by yaws. Between 1952 and 1964 a joint WHO and UNICEF programme for the control of endemic treponemal dis- eases was conducted. Approximately 300 million individuals were treated worldwide with injectable penicillin and it is believed that this reduced the global burden of diseases by as much as 98%. Following the cessation of these control programmes the disease rebounded in several countries in the 1970s and a further World Health Assembly resolution for the eradication of yaws was passed in 1978. In some countries this led to renewed control efforts but, despite these efforts, the disease was not eradicated. The number of reported cases has continued to climb in recent years but accurate prevalence and inci- dence data are lacking from most endemic countries. Yaws is currently thought to be endemic in 15 countries, mostly in West Africa, Southeast Asia, and the Pacific (Fig. 8.6.36.1). Papua New Guinea, the Solomon Islands and Ghana have each re- ported more than 15 000 suspected cases of yaws in recent years; in another eight countries transmission occurs mainly in hard-​to-​ reach populations. A further 79 countries were previously reported to have been endemic for yaws but there are limited data on the current epidemiology of yaws from most of these countries. Yaws was previously reported to be endemic in South America and the Caribbean, but control programmes in the mid-​20th century are thought to have successfully eliminated yaws from most countries in the region except Guyana. India interrupted transmission in Table 8.6.36.1  Clinical and epidemiological features of the human treponematoses Feature Venereal syphilis Yaws Endemic syphilis Pinta Organism T. pallidum subsp. pallidum T. pallidum subsp. pertenue T. pallidum subsp. endemicum T. carateum Age of infection (years) 20–​40 5–​15 2–​10 10–​30 Occurrence Worldwide Africa, South America, Oceania, Asia Africa, Middle East Central and South America Climate All Warm, humid Dry, arid Warm, rural Direct transmission:   Venereal Common No Rare No   Non​venereal Rare Common Rare Common   Congenital Yes No Unknown No Indirect transmission:   Contaminated utensils Rare Rare Common No Reservoir of infection Adults Infectious and latent cases;
possibly non​human primates Infectious and latent cases Ratio infectious:latent cases 1:3 1:5–​8 1:2 Unknown Late complications:   Skin + + + +   Bone, cartilage + + + No   Neurological + No ? No   Cardiovascular + No ? No

section 8  Infectious diseases 1206 2004 and declared elimination in 2006, following a sustained pro- gramme which began in 1996. Pathogenesis Knowledge of the pathogenesis of the treponematoses has been predominantly derived from animal models. Bacteria are acquired through breaches in the skin or mucous membranes. Following the initial infection, treponemes disseminate to lymph nodes where they multiply rapidly. The immune response is responsible for much of the pathology associated with the treponematoses and is medi- ated by both cellular and humoral immune responses. There is no naturally acquired immunity to treponemal infections and, fol- lowing successful treatment, individuals in endemic communities are at risk of reinfection. Clinical features Primary yaws The initial lesion of primary yaws is a papule which appears at the site of inoculation after approximately 21 days (range 9–​90 days). This lesion, often referred to as a ‘Mother Yaw’, may then evolve ei- ther into an exudative papilloma, 2–​5 cm in size, or degenerate to form a single, crusted, non​tender ulcer (Fig. 8.6.36.2). Lower limbs are the most frequent site for lesions of primary yaws, but other parts of the body can also be affected. Genital lesions are extremely uncommon. In the absence of treatment primary lesions may heal spontaneously over a period of 3–​6 months with the formation of a pigmented scar. On occasion primary lesions may still be present in patients who develop secondary manifestations of yaws. Secondary yaws The secondary manifestations of yaws result from haematogenous and lymphatic dissemination of treponemes, and typically occur 1–​ 2 months (up to 24 months) after the initial infection. Secondary yaws predominantly affects the skin and bones and may be accom- panied by general malaise and lymphadenopathy. A variety of skin manifestations have been described in sec- ondary yaws. These include disseminated papillomatous and ul- cerative lesions, scaly macular lesions, and hyperkeratotic lesions of the palms and soles. The latter may crack and become secondarily infected giving rise to severe pain and an abnormal gait, so called crab yaws (Fig. 8.6.36.3a). Involvement of the mucous membranes is uncommon in secondary yaws. Alongside the skin, involvement Countries reporting yaws (1982—2018) Countries reporting bejel (1982—2018) Countries reporting pinta (1982—2018) Fig. 8.6.36.1  Current known distribution of the endemic treponemal diseases. (a) (b) Fig. 8.6.36.2  Lesions of primary yaws. (a) Ulcerative lesion of primary yaws. (b) Papilloma of primary yaws. Images courtesy of O Mitjà.

8.6.36  Nonvenereal endemic treponematoses: Yaws, endemic syphilis (bejel), and pinta 1207 of the bones is one of the cardinal features of secondary yaws. The most common manifestation is osteoperiostitis. In most patients multiple bones are involved, most commonly the fingers (resulting in dactylitis), or the long bones (forearm, fibula and tibia) which results in bony swelling and pain (Fig. 8.6.36.3b). Latent yaws Untreated patients may develop latent infection, with positive ser- ology but no clinical signs. Latent cases can relapse, usually in the first 5 years (rarely up to 10 years) after infection. Relapsing lesions tend to occur around the axillae, anus, and mouth. At present no diagnostic test can distinguish between true latent infection and serofast status following successful treatment. Tertiary yaws Destructive lesions of tertiary yaws were previously reported to af- fect up to 10% of untreated patients. For reasons that are unclear these late stage manifestations are now seen less frequently. The skin, cartilage, and bones are most commonly affected. The lesions of late yaws contain relatively few treponemes and are not infectious. Nodular lesions may occur near joints and ulcerate, causing tissue necrosis. Destructive lesions of the face are some of the most marked manifestations of tertiary yaws. Gangosa, a destructive os- teitis of the palate and nasopharynx, results in mutilating facial ul- ceration. Goundou, which was rarely reported even when yaws was hyperendemic, is characterized by exostoses of the maxillary bones. Yaws is not thought to cause cardiovascular or neurological disease. Attenuated disease In some countries the clinical manifestations of yaws appear to be less florid than has been previously described. In many Pacific coun- tries the destructive lesions of tertiary yaws are now rarely seen. There is no agreed definition of attenuated yaws. Improvements in living standards, use of treponemocidal antibiotics for other infec- tions and mutations in T. p subsp. pertenue have all been proposed as explanations for why the features of the diseases may be less severe than previously. Bejel Epidemiology Bejel (endemic syphilis) is found predominantly in children aged 2–​15 years living in dry, arid environments. The disease has been reported in the Arabian peninsula and in the Sahel region of Africa (Fig. 8.6.36.1). A limited number of case reports suggest ongoing transmission in isolated, rural populations but there is limited sys- tematic data on the current distribution of bejel. The disease was previously reported to be present in several countries in northern Europe, the Balkans, Russia, and the eastern Mediterranean. Social and environmental improvements are thought to have contributed to a natural decline in the number of cases, while mass treatment campaigns are thought to have contributed to local elimination in some countries. Alongside direct inoculation via skin-​to-​skin con- tact, indirect inoculation in to mucous membranes via shared uten- sils has been reported to occur in bejel. Clinical features Primary lesions of bejel are rare, and if present often go undetected as small painless ulcers of the oropharynx and nasopharynx (Fig. 8.6.36.4a) and may only be noted when secondary lesions develop, which typically occurs 3–​6 months after initial infection. Secondary bejel is characterized by widespread lesions of the mu- cous membranes which are frequently accompanied by regional lymphadenopathy, condylomata lata, and a diffuse maculopapular rash (Fig. 8.6.36.4b). Clinically it can be difficult to distinguish sec- ondary bejel from venereal syphilis. Painful osteitis and periostitis, similar to that seen in yaws, may also be seen in secondary bejel. The late stages of bejel are characterized by destructive gumma- tous nodules that affect the skin and can progress to form infiltrated, (a) (b) (c) Fig. 8.6.36.3  Lesions of late yaws. (a) Hyperkeratotic plantar lesion of secondary yaws. (b) Dactylitis of secondary yaws. (c) Gangosa seen in tertiary yaws. Images A and C reprinted from Handbook of endemic treponematoses: yaws, endemic syphilis and pinta, Perine PL et al., Copyright © World Health Organization 1984; B courtesy of O Mitjà.

section 8  Infectious diseases 1208 pigmented lesions. Gummata of the nasopharnyx may result in a de- structive rhinopharyngitis (gangosa) which is also seen in tertiary yaws. As with the other endemic treponemal diseases, cardiovas- cular and neurological manifestations are not seen in late bejel. Pinta Epidemiology Unlike the other endemic treponemal diseases pinta predominantly affects young adults. The disease is restricted to Latin America, in particular Mexico and Colombia (Fig. 8.6.36.1). There is limited re- cent data on the prevalence of pinta in any of the countries where it was previously reported, although there are believed to be remaining foci of infection among tribes living in the Amazon. Clinical features Pinta is the most benign of the endemic treponematoses. Disease manifestations are limited to the skin and the destructive late stage manifestations of yaws and bejel are not seen. The initial lesions of primary pinta form as papules or ery- thematous plaques. These lesions may become pigmented and hyperkeratotic and are frequently accompanied by regional lymph- adenopathy (Fig.  8.6.36.5a). Exposed skin, most commonly the arms and legs, are the most frequent sites involved. Constitutional symptoms are not a feature of pinta. The early lesions of pinta normally resolve spontaneously but are followed after several months by the appearance of multiple smaller lesions (‘pintids’). These secondary lesions are characterized by al- terations in skin pigmentation. As treponemes are present in these lesions for many years these patients remain infectious. Late stage pinta is characterized by abnormally pigmented le- sions, which may contain areas of both hypo-​ and hyperpigmenta- tion. These changes may be accompanied by both skin atrophy and hyperkeratosis (Fig. 8.6.36.5b). Lesions of the bones and cartilage are not seen in pinta. Differential diagnosis The differential diagnosis of the endemic treponematoses varies between the different diseases and the stages of each disease. Venereal syphilis is a key differential diagnosis for all three en- demic treponematoses. The early lesions of yaws must be distin- guished from other ulcerative skin diseases including tropical ulcers, cutaneous leishmaniasis, and pyoderma. The mucous membrane lesions of bejel may be mistaken for oral herpes sim- plex, aphthous ulceration, or syphilis. Dactylitis due to yaws must be distinguished from that of sickle cell disease. The late stage manifestations of both diseases maybe confused with syphilis, fungal, and mycobacterial infections, psoriasis, and eczema. The lesions of early pinta may be confused with eczema, psoriasis, tinea versicolor, pellagra, syphilis, and leprosy, while the dyschromic late stage lesions may be confused with vitiligo, leprosy, and fungal infections. Of particular importance, several studies have recently identi- fied Haemophilus ducreyi as a common cause of non​genital skin lesions in children in yaws endemic communitites. These lesions are found in individuals who are both sero-​negative and sero-​ positive for yaws and clinical differentiation has not been shown to be reliable for distinguishing between ulcers caused by T.p. subsp. pertenue from those caused by H. ducreyi. (a) (b) Fig. 8.6.36.4  Lesions of primary and secondary bejel. (a) Oral ulcer of primary bejel. (b) Chronic skin lesion of seconday bejel. Images reprinted from Handbook of endemic treponematoses: yaws, endemic syphilis and pinta, Perine PL et al., Copyright © World Health Organization 1984.

8.6.36  Nonvenereal endemic treponematoses: Yaws, endemic syphilis (bejel), and pinta 1209 Diagnosis The diagnosis of the endemic treponematoses is based on a com- bination of the clinical and epidemiological features combined with microbiological evidence of infection, most frequently serology. In endemic settings the diagnosis may be relatively straightforward, although the epidemiological and serological overlap with venereal syphilis can be a diagnostic challenge. Diagnostic quandaries arise when individuals who have emigrated from an endemic area are found to have reactive serology. Clinicians need to consider the possibility that the reactive serology reflects either a previous infection with an en- demic treponemal infection or venereal syphilis. The social and medical history of the patient should be carefully reviewed. If there is clinical doubt, then treatment with benzathine-​penicillin should be offered. Serology Serology remains the mainstay of diagnosis for all of the human treponematoses. Traditional serological testing combines a specific-​ treponemal assay with a less specific non​treponemal assay. No cur- rently available serological test can distinguish between the different human treponematoses. The most commonly used treponemal tests include the T. pallidum haemagglutination and the T. pallidum par- ticle agglutination assays. These tests are highly specific but remain positive for life following infection. Non​treponemal tests include the venereal disease research laboratory (VDRL) and rapid plasma re- agin (RPR) tests. These detect several antigens including cardiolipin, lecithin, and cholesterol. Although nonspecific, VDRL/​RPR titres best reflect disease activity. Titres fall after treatment and may be- come zero, especially after treatment of early infection. There is limited access to routine diagnostic testing in many of the remote communities where the endemic treponematoses are most common. A rapid diagnostic test combining both a treponemal and a non​treponemal component, originally developed for the diagnosis of venereal syphilis, has been validated for the diagnosis of yaws and is likely to also be of value for the diagnosis of bejel and pinta. Microscopy Treponemes can be demonstrated in exudates from early lesions by darkfield examination, and they can also be found in biopsy speci- mens processed with silver or immunoperoxidase stains. However, these techniques are not routinely available in settings where these diseases are prevalent. Molecular techniques Polymerase chain reaction (PCR)-​based assays have become rou- tinely available for the diagnosis of syphilis in high-​income coun- tries. Commercial PCR assays cannot distinguish subspecies of pathogenic treponemes, but real-​time PCR assays are now available at research laboratories which can achieve this. Susceptibility in vitro and in vivo Studies to examine the in vitro and in vivo susceptibility of T. pall­ idum subspecies to antimicrobial agents are all laborious and tech- nically challenging since the bacteria cannot be grown in culture media. T. pallidum pertenue has been found to be sensitive to peni- cillin, chloramphenicol, tetracycline, and erythromycin, while it is insensitive to streptomycin and rifampicin. Treatment Long-​acting injectable benzathine-​penicillin has been the standard of care for the endemic treponematoses for more than 50  years. (a) (b) Fig. 8.6.36.5  Lesions of primary and secondary pinta. (a) Erythematous plaque of early pinta. (b) Hyperpigmented lesion of late pinta. Images reprinted from Handbook of endemic treponematoses: yaws, endemic syphilis and pinta, Perine PL et al., Copyright © World Health Organization 1984.

8.6.37 Syphilis 1210

8.6.37 Syphilis 1210

section 8  Infectious diseases 1210 Lower doses are used compared to syphilis, with a recommended dose of 0.6 MU for children under 10 years and 1.2 MU for older children and adults. Treatment failure has been rarely reported fol- lowing treatment of yaws with benzathine-​penicillin. The inability to distinguish treatment failure from reinfection makes these re- ports difficult to interpret and there is no good evidence of resist- ance to benzathine-​penicillin developing in any of the endemic treponematoses. A landmark randomized controlled trial conducted in Papua New Guinea showed that a single oral dose of azithromycin (30 mg/​kg, max 2 g) was non​inferior to penicillin for the treatment of both primary and secondary yaws. Azithromycin is now the preferred first-​line treatment for yaws because of ease of administration. Azithromycin has not been used in the treatment of bejel or pinta but is likely to be efficacious. Resistance to azithromycin in T. pall­ idum is mediated by point mutations in the 23s rRNA gene and is common in venereal syphilis, and has now been documented in yaws as well. Following treatment treponemes disappear from lesions within 8–​10 hours, skin lesions begin to heal within 2–​4 weeks, and non​treponemal test titres decrease 4-​fold or more within 6–​12 months. If treated early bony lesions may resolve but late stage scarring manifestations are irreversible. Treatment failure, either based on clinical or serological findings, is generally considered to be an indication for re-​treatment Second-​line agents For patients over the age of 10 years who are allergic to drugs of choice, oral tetracycline (500 mg q6h) or doxycycline (100 mg q12h) for 14 days are considered the best alternative agents. Adjunctive therapy Simple dry dressing is useful for keeping ulcerated lesions clean and protected from trauma. Pain control with non​opioid analgesics (e.g. paracetamol) is usually sufficient for managing mild discomfort related to arthralgias, osteoperiostitis, or palmoplantar keratosis. Prevention and control Social and economic development has led to the natural decline of the endemic treponematoses in many countries worldwide. As with syphilis, there is limited evidence for naturally acquired im- munity and individuals living in endemic communities can be- come repeatedly re-​infected following treatment. Most cases of yaws occur in children and adults appear relatively unaffected even in hyperendemic communities, which might suggest immunity can eventually develop. Experimental studies have suggested that in- dividuals may develop some degree of protection from reinfection with homologous strains but not from infection with alternative treponemal species or subspecies. There is no currently available vaccine available for any of these human treponematoses. Yaws has been the focus of several previous large-​scale control and eradication programmes. The previous eradication programme in the 1950s to 1960s resulted in a substantial reduction in the burden of disease worldwide, but focal pockets of the disease have remained, and disease prevalence has rebounded in some countries. More re- cently both Ecuador and India have achieved local elimination of yaws through government led mass treatment and case finding pro- grammes, demonstrating that eradication should be feasible. The demonstration that azithromycin was an effective agent for the treatment of yaws has led to renewed interest in yaws eradication. A WHO eradication strategy was launched in 2012 based on com- munity mass treatment with single-​dose oral azithromycin and sub- sequent clinical case detection to direct further rounds of targeted treatment. Pilot studies conducted in Ghana, Papua New Guinea, and Vanuatu demonstrated that this approach was highly effective at redu- cing the community prevalence of both clinical and latent yaws. These findings have prompted optimism that it might finally be possible to eradicate yaws worldwide. Several key areas will need to be addressed if the current eradication campaign is to be successful. In particular there is a need for a significant improvement in the accuracy of information about the current epidemiology of yaws, and for studies to understand the optimal mass treatment strategy required to achieve eradication. At present there is no specific strategy for the eradication of either bejel or pinta. It is likely that the tools developed as part of yaws eradi- cation efforts will be applicable in the control of both bejel and pinta. FURTHER READING Ayove T, et al. (2014). Sensitivity and specificity of a rapid point-​of-​ care test for active yaws: a comparative study. Lancet Glob Health, 2, e415–​e421. Cejková D, et al. (2012). Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence. PLoS Negl Trop Dis, 6, e1471. Giacani L, Lukehart SA (2014). The endemic treponematoses. Clin Microbiol Rev, 27, 89–​115. Marks M, et al. (2014). Haemophilus ducreyi associated with skin ul- cers among children, Solomon Islands. Emerg Infect Dis, 20, 1705–​7. Marks M, et al. (2015). Challenges and key research questions for yaws eradication. Lancet Infect Dis, 15, 1220–​5. Mitjà O, Asiedu K, Mabey D (2013). Yaws. Lancet, 381, 763–​73. Mitjà O, et  al. (2012). Single-​dose azithromycin versus benzathine benzylpenicillin for treatment of yaws in children in Papua New Guinea: an open-​label, non​inferiority, randomised trial. Lancet, 379, 342–​7. Mitjà O, et al. (2015). Mass treatment with single-​dose azithromycin for yaws. N Engl J Med, 372, 703–​10. Mitjà O, et al. (2018). Re-emergence of yaws after single mass azithromycin treatment followed by targeted treatment: a longitudinal study. Lancet, 391, 1599–607. Perine PL, et al. (1984). Handbook of endemic treponematoses: yaws, endemic syphilis and pinta. World Health Organization, Geneva. 8.6.37  Syphilis Phillip Read and Basil Donovan ESSENTIALS Syphilis results from infection with the spirochaete Treponema pallidum subsp. pallidum, for which humans are the only known nat- ural host. In adults it is transmitted primarily by sexual contact. The organism gains entry into the body through small breaks in the skin

8.6.37  Syphilis 1211 or the intact mucosal surfaces of the genitals, mouth, or anus, and is able to invade and survive in a wide variety of tissues. Since the availability of penicillin, syphilis has become primarily (>90%) a disease of less affluent countries or of minority subpopulations in more affluent countries with poor access to healthcare. It is also a disease of people with rapid rates of partner change (e.g. men who have sex with men and commercial sex workers). Clinical features Syphilis can manifest in three stages: (1) primary syphilis, which occurs within a few weeks to months after infection; (2) secondary syphilis, which presents after a few months, up to a year; and (3) tertiary syphilis, which presents years to decades after primary infection. These stages can overlap, and there are frequently asymptomatic periods. Primary syphilis—​this appears 9–​90 days after the organism gains entry via direct inoculation through the thin skin or mucosa of the anogenital tract or mouth during sexual exposure. The resulting le- sion is typically a painless ulcer or ‘chancre’, sometimes indurated, that appears at the site of inoculation and is associated with regional lymphadenopathy; chancres can be multiple and atypical. Secondary syphilis—​occurs 3–​6 weeks after the appearance of the chancre, with manifestations including fever, malaise, mucocutaneous lesions (rash, condyloma lata, mucous patches), generalized lymph- adenopathy, and (uncommonly) visceral disease. Invasion of the central nervous system is common, but usually asymptomatic. Latent syphilis—​the lesions of both primary and secondary syphilis may wax and wane, but they eventually resolve; there are no signs or symptoms of active syphilis, but serological tests are positive for T. pallidum. Tertiary syphilis—​affects around one-​third of infected people fol- lowing a variable period of latent infection, with manifestations including: (1) neurosyphilis—​which can present as: (a) aseptic meningitis, with variable features (e.g. focal neuro- logical deficits, cranial nerve palsies, hydrocephalus, or psy- chiatric symptoms); (b) meningovascular disease, with endarteritis leading to cere- bral infarction; (c) general paresis, involving changes in the parenchyma of the central nervous system that lead to the gradual onset of cog- nitive impairment, depression, and personality changes, later progressing to dementia, delirium, seizures, and delusions; (d) tabes dorsalis, with initial symptoms and signs including lightening pains and parasthesias, visceral crises, abnormal deep tendon reflexes, incontinence, ataxia with a wide-​ based gait, and pupillary abnormalities. (2) gummatous syphilis—​destructive granulomatous lesions most commonly present on skin, mucosal surfaces, or in bone. (3) cardiovascular syphilis—​most commonly asymptomatic aortitis, aortic incompetence, aortic aneurysm, and coronary ostial stenosis. Congenital syphilis—​most pregnant women with early syphilis will transmit the condition to the fetus via the placenta, with congenital syphilis often resulting in fetal loss, stillbirth, or neonatal or childhood disease. Diagnosis and treatment Diagnosis—​the transient nature of the lesions and the spirochetaemia limit the role of direct detection of T. pallidum, hence diagnosis usu- ally relies on serology, with tests being: (1) non​specific (or non​treponemal or reagin)—​for example, rapid plasma reagin and venereal disease research laboratory tests; de- tect phospholipid cardiolipin as an antigen; generally sensitive in early infection but tend to decline over the next several years without treatment; able to quantify disease activity and hence used for follow-​up after treatment. (2) specific (or treponemal)—​for example, T. pallidum haemagglutin- ation assay; use T. pallidum as the antigen; may become positive shortly before the non​specific tests; typically remain reactive for life after successful treatment and therefore have no role in as- sessing stage of infection, ‘cure’, or reinfection. Treatment—​parenteral penicillin G remains the preferred treatment for syphilis, with doxycycline providing an oral alternative. Successful treatment of early disease relies on demonstrating a fourfold de- crease in reagin (rapid plasma reagin or venereal disease research laboratory) titres over the next 6–​12 months. Sexual contacts of early syphilis should be treated presumptively, regardless of their test re- sults, if the contact was within 90 days, usually with a single dose of benzathine penicillin G. Prevention The chance of acquiring syphilis following one act of intercourse with an infected person is 1–​2%, which should be reduced by the use of condoms. Early treatment of disease decreases the duration of infect- ivity and thereby minimizes transmission to others, hence those at high risk of syphilis should be encouraged to undergo regular syphilis screening (as well as testing for HIV and other sexually transmissible infections). Prevention of congenital infection and serious outcomes such as stillbirth and neonatal death rely on routine antenatal screening early in the pregnancy, with prompt treatment of infected mothers. Women in high-​incidence settings should be rescreened later in pregnancy. Introduction and historical perspective In the 1490s, an epidemic of a new and virulent sexually transmis- sible disease appeared in Europe following the return of Christopher Columbus and his fleet from the Americas. This led many to believe that syphilis originated in the New World. There is now molecular phylogenetic evidence for this ‘Columbian hypothesis’. Syphilis spread rapidly through Europe where it was known by a variety of names including morbus gallicus (the French disease), lues venereum (venereal disease), and the great pox. The alternative theory proposes that syphilis was simply another variant of a preexisting treponemal infection that had adapted to sexual and congenital transmission and produced greater morbidity (the Unitarian hypothesis). A variety of yaws-​like diseases that pre- dominantly affected children were present in Europe and Africa at the time, and a few persisted in Europe into the 20th century.

section 8  Infectious diseases 1212 Syphilus (the original spelling) was an afflicted shepherd in a poem by Girolamo Fracastoro published in 1530. The original text described the symptoms of syphilis, hypothesized about its origins, and mentioned the use of early remedies such as guaiacum, a com- pound derived from a Central American tree, and mercury. Following an experiment by John Hunter in 1767 it was thought that gonorrhoea and syphilis were different manifestations of the same disease until Philippe Ricord, in 1838, clarified the differences between the two infections. Soon after, the three stages of syphilis were categorized and congenital and neurological syphilis were described. Because of the toxicity and dubious benefit of the treatments available at the time, a prospective cohort study into the natural history of syphilis was conducted in Oslo between 1890 and 1920. This study followed 1978 initially symptomatic patients and demon- strated that approximately one-​third developed late complications. Many of these complications proved fatal. Rapid advances in knowledge occurred around the beginning of the 20th century. In 1905, Schaudinn and Hoffman demonstrated spirochaetes in secondary syphilitic lesions. One year later, Von Wasserman devised the first serological test for syphilis. In 1910, Paul Erlich announced results for his compound 606 (salvarsan), a form of arsenic that showed activity against syphilis. The discovery of penicillin by Fleming, its development for thera- peutic use by Florey, Chain, and Heatley and the first clinical trial in 1943 by Mahoney revolutionized the treatment of syphilis. During the immediate postwar period the use of penicillin eclipsed other forms of therapy and by the mid-​1950s the incidence of syphilis had fallen markedly throughout the industrialized world. Aetiology, genetics, pathogenesis, and pathology Treponema pallidum subsp. pallidum, a spiral-​shaped bacterium, is a member of the order Spirochaetales and the cause of adult-​acquired and congenital syphilis. Humans are the only known natural host for all T. pallidum subspecies, although an unclassified and morpho- logically indistinguishable simian pathogen, the Fribourg–​Blanc treponeme, was isolated from a baboon in Guinea in 1962. The in- ability to culture T. pallidum in vitro has retarded study of its biology. T. pallidum subsp. pallidum is closely related to other pathogenic treponemes that cause non​venereal disease: T. pallidum subsp. car­ ateum (pinta), T. pallidum subsp. pertenue (yaws), and T. pallidum subsp. endemicum (endemic syphilis or bejel) (see Chapter 8.6.36). Subspecies pertenue and endemicum and the Fribourg–​Blanc trepo- neme have recently been demonstrated to be genetically distinct from subspecies pallidum, consistent with the lack of cross-​immunity. The spirochaete is 6–​20 μm long and only 0.10–​0.18 μm thick, making it invisible to ordinary light microscopy. Using dark-​field micros- copy, T. pallidum has 6–​20 characteristic tightly wound spirals and it moves with corkscrew motility or by bending in the middle and popping back into place with a spring. Other non​pathogenic trepo- nemes tend to have fewer coils or a jerkier motion. Commensal spe- cies of treponema (T. denticola and T. oralis) can mimic T. pallidum, limiting the usefulness of dark-​field microscopy of oral and anal lesions. The T. pallidum DNA genome was first published in 1998. It is small, with a single circular chromosome of 1 138 006 base pairs containing 1041 predicted protein coding sequences, consistent with its limited metabolic capabilities. The organism obtains most of its essential nutrients from the host environment, making it an obligate parasite. In vivo, T. pallidum has been grown in rabbits and reproduces itself slowly, doubling every 30–​33 h. T. pallidum is able to survive better with low levels of oxygen (3–​5%) and is sensitive to heat. T. pallidum is able rapidly to invade and survive in a wide var- iety of tissues after gaining entry into the body through small breaks in the skin or the intact mucosal surfaces of the genitals, mouth, or anus. The organism has a reputation as a ‘stealth’ pathogen be- cause its paucity of surface proteins and lipopolysaccharides helps it to evade the host immune response. T. pallidum induces humoral, cell-​mediated, and local innate responses that appear to confer im- munity to exogenous infection in the chronically infected person (chancre immunity). However, patients treated for early syphilis can rapidly become reinfected. From the site of inoculation, T. pallidum replicates locally and spreads to regional lymph nodes, then into the blood stream from where it can traverse junctions between vascular endothelial cells. Indeed T. pallidum DNA has been isolated from blood in one-​third of cases with primary syphilis. Lymphocyte, (CD4+) macrophage, and plasma cell infiltrates accompanied by vasculopathic changes, endarteritis, and periarteritis, underlie the histology of syphilitic le- sions of all stages. Silver staining of tissues might demonstrate the presence of spirochaetes, usually in the dermal–​epidermal junction. In secondary syphilis, treponemes are found in many sites including visceral organs, the central nervous system, and the skin. T. pallidum can remain clinically dormant in the aortic wall, pro- ducing an endarteritis in the vasa vasorum and varying degrees of thickening, scarring, and destruction of the arterial wall. This pro- cess results in the development of arterial plaques and calcification of the vessels found in cardiovascular syphilis. In meningitis, perivascular infiltration of lymphocytes and plasma cells causes the meninges to become inflamed. In meningovascular syphilis, thickening of the intima, fibrous changes in the adventitia, and vascular narrowing cause changes in brain blood vessels with resultant infarction and cranial nerve palsies. The gummas of late syphilis are chronic granulomatous lesions consistent with a hypersensitivity response with few treponemes present. Histologically, central necrosis, peripheral lymphocytosis, perivasculitis, and obliterating endarteritis are seen. Epidemiology Since the availability of penicillin, syphilis has become pri- marily (>90%) a disease of less affluent countries or of minority subpopulations in more affluent countries with poor access to healthcare. It is also a disease of populations with rapid rates of partner change, such as men who have sex with men and commer- cial sex workers. The World Health Organization estimated that in 2008 syphilis continued to infect about 10.6 million people a year globally. The greatest number of new infections, 3.5 million, is found in Southeast and East Asia. Sub-​Saharan Africa accounted for 3.4 million new cases and the Americas and Caribbean contributed 2.8 million. In Africa, between 4 and 17% of women are seropositive for syphilis in

8.6.37  Syphilis 1213 antenatal clinics, and many are coinfected with HIV. About a million pregnancies a year are seriously complicated or aborted by syphilis. The incidence of syphilis in China has risen following the political and social changes that occurred in the last part of the 20th cen- tury. In the former Soviet Union, the incidence of syphilis among the young sexually active population rose rapidly after 1991 with the degradation of the public health system. Since the beginning of the 21st century, syphilis rates in the United Kingdom, Europe, Asia, North America, Australia, and other developed countries have risen in men who have sex with men, espe- cially in those who are HIV positive. Serosorting, the phenomenon of homosexual men of similar HIV status seeking each other for un- safe sex, has been a factor. Oral sex, considered to be relatively safe in terms of transmission of HIV infection, readily transmits syphilis. Prevention Syphilis in adults is transmitted primarily by sexual contact. The chance of acquiring the infection is estimated to be between 1 and 2% following one act of intercourse with an infected person. Syphilis is found in up to 60% of sexual partners of infected individuals. As T. pallidum is present in mucosal or cutaneous lesions, infected adults are more likely to transmit the disease during primary or secondary stages. The use of condoms to prevent syphilis has not been evaluated in controlled trials. Intuitively, condoms should have some effect in reducing transmission, but they do not provide 100% protection as they do not cover all areas of anogenital skin during intercourse. Condoms should also be used for oral sex. The early recognition and treatment of syphilis decreases the dur- ation of infectivity thereby minimizing transmission to others. As symptoms are not always present, those who are at higher risk of syphilis such as men who have sex with men and commercial sex workers are encouraged to undergo regular syphilis screening, as well as testing for HIV and other sexually transmissible infections. In order to attract those most at risk of syphilis, health services need to be accessible and culturally appropriate, confidential, and provide free or affordable diagnosis and treatment. Presumptive treatment of sexual partners and early recognition and treatment of those who may be core transmitters in a sexual network is essential in any syphilis control programme. Pilot studies have shown encouraging results of syphilis chemoprophylaxis when given to high-​risk men who have sex with men. Some authorities now use the internet and dating applications to encourage men who have sex with men to have regular syphilis tests. The internet can also help these men to inform their sexual partners in a non​threatening and confidential way. Syphilis can be transmitted via donated blood or organs, although this is rare. Thus, serological screening of donors is routine in most settings. Mother-​to-​child transmission of syphilis usually occurs in utero. Prevention of congenital infection and serious outcomes such as stillbirth and neonatal death rely on screening and treating for syph- ilis in the mother early in the pregnancy. In high-​incidence popu- lations, rescreening around week 28 to 32 of the pregnancy and again at delivery is also recommended. With timely treatment of the mother, congenital syphilis is almost entirely preventable. Provision of comprehensive antenatal healthcare with affordable testing for syphilis should form part of a comprehensive syphilis control pro- gramme. Community education should encourage women to at- tend for healthcare early in pregnancy when treatment can be given with best effect. In high-​prevalence areas, if a mother first presents at term, routine treatment of the neonate with a single dose of benzathine penicillin 50 000 units/​kg is sometimes recommended if the mother has not been tested or adequate maternal treatment cannot be confirmed. Clinical features Clinical staging of syphilis is important to guide the process of con- tact tracing or partner notification. Primary, secondary, and early latent syphilis are collectively called ‘infectious syphilis’. Late latent and tertiary syphilis are generally regarded as no longer infectious for sexual partners. However, pregnant women may pose an occa- sional risk to their offspring. Treatment for later stages of syphilis is typically longer than for early syphilis. Primary syphilis The chancre, or ulcer of primary syphilis, develops at the site of in- oculation within 9 to 90 days (median 3 weeks) of infection, initially as a red macule that soon becomes papular before it ulcerates. The typical ulcer is painless, has fluid or grey slough in its centre, and a well-​defined rolled edge. Mature ulcers can have a palpable indur- ated plaque deep to the lesion. However, chancres can occasionally be painful or multiple, and clinically indistinguishable from other causes of genital ulcers (Fig. 8.6.37.1). Mixed aetiologies are always possible (Fig. 8.6.37.2). Common sites for chancres in men include the distal penis, while in women the posterior fourchette, labia, and vulva are the most commonly diagnosed sites (Fig. 8.6.37.3a). The anus, mouth, and Fig. 8.6.37.1  Multiple painful chronic chancres in a man with HIV infection. Courtesy of Dr David Bradford.

section 8  Infectious diseases 1214 lips are all possible sites for chancres, as well as other extragenital sites. If a chancre is small or hidden in the anal canal, vagina, cervix, or mouth, it usually passes unnoticed (Fig. 8.6.37.4). Most patients subsequently diagnosed with secondary syphilis do not recall the lesions of primary syphilis. Painless and typically rubbery, small lymph nodes are often felt in the affected region within a week of the development of the chancre (Fig.  8.6.37.3b). The chancre usually heals spontaneously in 3 to 6 weeks, but it may occasionally recur (‘chancre redux’). Secondary syphilis This disseminated stage of the infection typically occurs between 3 and 6 weeks following the appearance of the chancre, and the two stages might overlap. However, up to 60% of patients do not recall any signs or symptoms of secondary syphilis at all. The symptoms and signs of secondary syphilis are often de- scribed as protean, as listed in Table 8.6.37.1. Without treatment they resolve spontaneously only to reappear, usually in a milder form, in almost one-​quarter (24%) of patients in the following 12 to 24 months (Fig. 8.6.37.5). Latent syphilis Latent syphilis is present when there are no signs or symptoms of active syphilis but serological tests are positive for T. pallidum. Latent syphilis is arbitrarily divided into early latent syphilis, when the asymptomatic infection has been present for less than 1 or 2 years, and late latent syphilis after this time. In practice, asymptomatic people diagnosed through screening are often deemed to have la- tent syphilis of unknown duration. As a precaution, such patients are treated with the longer courses of antibiotics that are used for late infections. Before the antibiotic era, approximately two-​thirds of adults re- mained in the latent phase throughout their lifetime and showed no signs of tertiary syphilis. These days many common antibiotics have some activity against T. pallidum, so it is likely that antibiotics used for other conditions are also altering the natural history of, if not ac- cidentally curing, latent syphilis. Tertiary syphilis Tertiary syphilis occurred in 15–​40% of those who remain un- treated in the Oslo study, with some modest differences between Fig. 8.6.37.2  Chancre against a background of primary genital herpes. Fig. 8.6.37.4  An asymptomatic chancre on the anterior lip of the cervix in the same woman as Fig. 8.6.37.3a. (b) (a) Fig. 8.6.37.3  (a) A periurethral chancre in a woman who presented with a painless lump. (b) Chancre on thigh and inguinal lymphadenopathy of primary syphilis. Copyright D. A. Warrell.

8.6.37  Syphilis 1215 the sexes (Table 8.6.37.2). Partly as a result of the wide availability of antibiotics used for other purposes, gummatous and cardiovas- cular syphilis are now relatively rare compared to neurosyphilis; most oral antibiotics are unlikely to achieve cidal levels in the cen- tral nervous system. Neurosyphilis The diagnosis of neurosyphilis frequently raises clinical dilemmas because of the non​specific nature of its clinical presentations and the absence of definitive tests. Neurosyphilis can manifest as aseptic (basilar pattern) meningitis or meningovascular disease as early as the secondary stage or up to several years after infection. As well as the usual symptoms of meningitis, syphilitic meningitis can also present with focal neurological deficits such as hemiparesis, aphasia, seizures, or psychiatric symptoms. Cranial nerve palsies accompany syphilitic meningitis in about 40% and hydrocephalus in 35% of patients. Meningovascular syphilis stems from endarteritis leading to in- farction, most commonly 5–​12 years after infection. While any artery may be affected, the middle cerebral is the most frequently involved. Gradual onset and less extensive damage results from smaller arteries being involved than is usual in thrombotic stroke. Psychological changes can mimic the early stages of parenchymal disease. Confusing the diagnosis, up to 25% of individuals with early syphilis have T. pallidum in the cerebrospinal fluid demonstrated by rabbit inoculation or polymerase chain reaction (PCR). This largely asymptomatic phenomenon is known as neuroinvasion and it is be- lieved that most, but not all, will spontaneously clear T. pallidum from the cerebrospinal fluid. Studies in the preantibiotic era dem- onstrated that the degree of cerebrospinal fluid abnormalities (white cell count, raised protein, and reactive cerebrospinal fluid (CSF)-​ Venereal Disease Research Laboratory (VDRL) test) in asymp- tomatic neurosyphilis predicted later progression to symptomatic neurosyphilis. Rarely, after 20–​25 years, syphilitic meningitis or meningovascular disease can involve the spinal cord resulting in (often asymmetric) paresis, incontinence, hyperreflexia, extensor plantar reflexes, and loss of position, and vibration sense. Table 8.6.37.1  Clinical manifestations of secondary syphilis Features Frequency Rash Erythematous or coppery colour Non​pruritic or mildly pruritic Macular or maculopapular (50%) progressing to papular, papulosquamous, psoriasiform, annular
(dark-​skinned people), pustular, or follicular Usually symmetrical, round to oval lesions, 5–​20 mm across (Fig. 7.6.36.5a, b) Trunk, palms, soles, and body flexures are most commonly involved Occasionally papules around the forehead hairline (‘corona veneris’) Over 70% Condyloma lata Pale elevated moist plaques in warmer flexural areas such as perineum, perianal area, groin, axilla, perioral area, and nasolabial folds (Fig. 7.6.36.5c) Appear later than rash 15–​50% Mucous patches Superficial erosions, papules, or plaques of mucosa of the oropharynx or anogenital area Involvement of the pharynx may result in hoarseness or sore throat 4–​17% Constitutional Low-​grade fever, malaise, headache, myalgias, arthralgias, anorexia, and nausea Occasionally severe Common, but variable Lymphadenopathy Generalized, non​tender, and characteristically rubbery and discrete (Fig. 7.6.36.3b) Over 60% Hepatitis Mildly elevated transaminases Usually not clinically important Up to 10% Ocular Iritis or uveitis Occasional Alopecia Follicular disease can lead to patchy ‘moth-​eaten’ alopecia of the scalp or, rarely, loss of the outer part of the
eyebrows or beard Occasional Central nervous system Asymptomatic neuroinvasion occurs in up to 25% Symptomatic meningitis or meningovascular disease may be more common in HIV infection Ocular and auditory cranial nerves most commonly involved Up to 2% Kidney Asymptomatic proteinuria Nephritic syndrome Rapidly progressive glomerulonephritis Rare Heart Myocarditis Ventricular arrhythmia Rare Parotitis Rare Gastritis Gastritis and stomach ulcers resulting in nausea and abdominal pain Rare Periostitis, arthritis, or bursitis Localized Rare Malignant syphilis
(‘lues maligna’) Rapidly progressive variant with marked constitutional symptoms and disfiguring crusted necrotic ulcers Possibly more common with HIV infection and in alcohol dependant patients Rare

section 8  Infectious diseases 1216 General paresis involves changes in the central nervous system parenchyma, characterized by fibrosis and atrophy, and occurs much later, approximately 15–​25 years after the initial infection. Parenchymatous central nervous system lesions can present with, usually gradual, onset cognitive impairment, depression, and per- sonality changes, later progressing to dementia, delirium, seizures, and delusions. Neurological signs can include irregular, often large, pupils that become unresponsive to light yet still accommodate (Argyll Robertson pupils), dysarthria, facial or hand tremor, loss of facial expression, hypotonia, and hyperreflexia or loss of reflexes. Tabes dorsalis involves parenchymatous changes in the dorsal root tracts and posterior columns of the spinal cord 15–​35 years after primary infection. Initial symptoms and signs can include lightening pains and paraesthesias, visceral crises, abnormal deep tendon reflexes, incontinence, ataxia with a wide-​based gait, and papillary abnormalities. Rarely, gummas involve the cerebrum or the spinal cord. (a) (c) (b) Fig. 8.6.37.5  (a, b) Rash of secondary syphilis on palms and scalp. (c) Papular lesions of secondary syphilis. Copyright D. A. Warrell. Table 8.6.37.2  Frequency of late complications of syphilis from the Oslo study in the preantibiotic era Form of tertiary syphilis Men (%) Women (%) Benign late (gummatous) syphilis 14.4 16.7 Cardiovascular syphilis 13.6   7.6 Neurosyphilis   9.4   5.0

8.6.37  Syphilis 1217 Gummatous (late benign) syphilis Gummas are destructive granulomatous lesions that most com- monly present on skin (70%), on mucosal surfaces (10%), or in bone (10%). They can occur a few years or decades after primary infection. On the skin gummas start as painless nodules that pro- gressively become necrotic, leaving punched-​out ulcers. The face, legs (Fig. 8.6.37.6), buttocks, trunk, and scalp are common sites. Gummatous involvement of the oropharynx can lead to perfor- ations and severe scarring of the palate, pharynx, or nasal septum. Tongue involvement can lead to glossitis, swelling, and leucoplakia. Fractures can occur with gummas of bone. Other organs occasionally affected include the liver, central ner- vous system, eyes, stomach, lungs, and testes. Cardiovascular syphilis Now rare, cardiovascular syphilis develops decades after primary in- fection. The most common forms are asymptomatic aortitis, aortic incompetence, (usually proximal) aortic aneurysm, and coronary ostial stenosis. Congenital syphilis Mother-​to-​child transmission occurs via the placenta at any stage of gestation. Because the transmission is haematogenous there is no primary lesion (chancre) and it is a disseminated infection from the outset. If the mother has early syphilis, transmission is almost certain; infectivity progressively declines to below 10% in late latent infection. Fetal wastage from syphilis might manifest as first or second trimester abortion or still birth with a large pale, fibrosed pla- centa or a macerated fetus. Of the infected babies that sur- vive, only 30% have specific symptoms in the neonatal period, though almost all will exhibit symptoms by 3 months. Many of the early (at birth or in the first 2 years) lesions resemble sec- ondary syphilis in the adult (Fig. 8.6.37.7). Failure to thrive in the first few months can be the first sign. Affected infants tend to be small or premature, irritable, snuffly, and cry feebly. The skin is often dry and wrinkled. Generalized rubbery lymphadenop- athy is common, often accompanied by hepatosplenomegaly and haematological abnormalities. Early deaths can be due to diffuse pulmonary infiltration. Painful osteochondritis or epiphysitis of the long bones, and sometimes periostitis, can occur in the first 6 months with charac- teristic radiological appearances. Late (after 2  years, but rarely beyond 30  years) congenital syphilis is analogous to tertiary syphilis in adults. However, gum- matous disease might be more common while cardiovascular disease is rare compared to adult syphilis. Interstitial keratitis is the most common form of late congenital syphilis. From the fifth year of life onward, the child might develop bilateral eye pain and photophobia, and scleral vascularization. Gumma can lead to perforation of the palate. Periostitis might lead to deformity of the tibia (sabre tibia), the skull (Parrot’s nodes), the scaphoid, and the clavicle. The stigmata of congenital syphilis are permanent deformities or scars left by early or late disease. Sometimes stigmata can help to explain unexpected positive serological tests in adults, as the pa- tient may be unaware of their prior infection. The bony deformities tend to persist, while T. pallidum can also invade tooth buds af- fecting the permanent teeth (Hutchinson’s teeth) but not the milk teeth. The molars might be deformed with dwarfed cusps, while the incisors might be small, peg-​shaped, and notched at the tip. Previous interstitial keratitis can be demonstrable for life on slit-​ lamp examination. Differential diagnosis Syphilis is often described as the great imitator due to the vast number of illnesses that it mimics. Screening for syphilis was once considered routine for medical and psychiatric hospital admissions. A high index of suspicion is required for the diagnosis. Primary syphilis Chancres can resemble anogenital ulcers from any cause, and more than one condition may be present (Fig. 8.6.37.2). Other causes of genital ulcers include herpes simplex virus infections, chancroid, lymphogranuloma venereum, and donovanosis. An anal chancre can be painful and clinical indistinguishable from an ordinary anal fissure. Liberal use of the laboratory to exclude other causes of ulcers Fig. 8.6.37.6  Ulcerating nodular lesions of gummatous syphilis in a man with HIV infection. Initially thought to be Kaposi’s sarcoma, the diagnosis was made by biopsy. Courtesy of Professor David Cooper. Fig. 8.6.37.7  Bullous syphilis lesions in a neonate.

section 8  Infectious diseases 1218 is essential. Alternatively, in resource-​poor environments that must rely on syndromic management of genital ulcers, antibiotic combin- ations need to cover all the common causes of genital ulcers in that region. Secondary syphilis The rash of secondary syphilis can resemble a drug eruption, pityr- iasis rosea, tinea versicolour, seborrhoeic dermatitis, erythema multiforme, scabies, lichen planus, psoriasis, fungal infections, and leprosy. Other infections causing generalized rashes include primary HIV infection, measles, rubella, and meningococcemia. Condyloma lata can be confused with genital warts. Syphilitic alopecia can re- semble alopecia areata or fungal scalp infections. Generalized lymphadenopathy, sore throat, and fever are also seen in infectious mononucleosis, rubella, toxoplasmosis, lymphoma, acute hepatitis, and, most importantly, primary HIV infection. Symptoms of meningitis might also be present in HIV infection, bacterial meningitis, enterovirus infections, and primary herpes simplex virus infection. Latent syphilis Childhood treponemal infections such as yaws and pinta, as well as prior congenital syphilis, can be serologically indistinguishable from adult-​acquired syphilis and the specific tests are likely to re- main positive for life. People from endemic treponemal areas or who may be at risk of congenital infection with positive syphilis ser- ology should be examined for stigmata of these conditions. False-​ positive non​specific tests (VDRL and rapid plasma reagin (RPR)) occur in 1–​2% of the population (see ‘Non​specific serological tests’). Confirmation with a specific treponemal test is essential for asymptomatic people. Neurosyphilis Symptoms and signs of meningovascular syphilis are similar to those in other causes of stroke or cerebrovascular accidents due to haemorrhagic or thrombotic mechanisms. Gummas in the brain can be mistaken for tumours and abscesses, particularly in HIV infection. General paresis should be considered in the differential diagnosis of dementia, psychosis, seizures, delirium, and person- ality changes. Gummatous syphilis Other granulomatous diseases such as sarcoidosis, tuberculosis, and neoplastic lesions can be confused with gummatous syphilis. Cardiovascular syphilis Signs and symptoms of cardiovascular syphilis are similar to those of atherosclerotic disease and aortic aneurysms are more commonly due to hypertension. Other causes of aortic regurgi- tation without stenosis include Marfan’s syndrome and infective endocarditis. Clinical investigation Direct detection of the organism As with all bacterial infections, ideally T. pallidum should be directly detected (Table 8.6.37.3) because a serological response may take days to weeks to evolve. However, the transient nature of the lesions and the spirochaetaemia limit the role of direct detection, leading to reliance on serology or, in resource-​poor environments, syndromic management. The role of PCR testing of the CSF has yet to be de- termined because asymptomatic and transient neuroinvasion by T. pallidum correlates poorly with standard criteria for diagnosing neurosyphilis. The role of PCR in the diagnosis of early syphilitic lesions is well established. PCR testing is increasingly available in many settings, and can be performed on primary and secondary lesions. The sen- sitivity for primary lesions ranges between 78 and 90%, and it may precede seroconversion. Typical PCR targets are the polA gene, or the tpp47 gene; both appear equally sensitive and specific. Table 8.6.37.3  Methods of direct detection of T. pallidum Method Brief description Role Animal inoculation Fresh (or flash-​frozen to less than −78°C) lesion material or cerebrospinal fluid is usually inoculated by intratesticular or intradermal means into rabbits. The animals are then monitored for the development of skin lesions, orchitis, or serological response. The most sensitive test (approaching 100%), but only used as a gold standard to evaluate other tests in the research setting Dark-​field microscopy Fresh serous fluid with motile organisms is collected by gentle pressure on the lesions and pressed under a coverslip. An on-​site microscope with a reflecting dark-​field condenser and a skilled microscopist are required. Diagnostic criteria include morphology and motion of the organisms. Only appropriate for specialist services. Not for oral or anal lesions. Patient must attend service in person. Immediate result DFA test Specimen collected as for dark-​field microscopy, air dried on a glass slide, and stained with labelled anti-​T. pallidum globulins immediately before fluorescence microscopy. Specimen does not need to be fresh, so can be transported to laboratory. High sensitivity (>90%) if the specimen is well collected. Suitable for oral lesions DFAT test DFA test adapted to histology specimens, usually transported in 10% buffered formalin. Skin, brain, placenta, umbilical cord, or gastrointestinal biopsy specimens can be tested PCR test Suspected chancres or lightly abraded lesion swabs in PCR transport medium. Possibly cerebrospinal fluid and placental blood. Very sensitive for moist primary and secondary lesions, but only available in referral laboratories DFA, direct fluorescent antibody; DFAT, direct fluorescent antibody tissue; PCR, polymerase chain reaction.

8.6.37  Syphilis 1219 Serology Broadly there are two types of serological tests for syphilis, non​specific (or non​treponemal or reagin) tests and specific (or treponemal) tests. Although less sensitive for some stages of syphilis as well as being less specific, non​specific tests require less expertise, are cheaper, and are more indicative of active infection so they were often initially favoured for screening. Specific tests usually remain positive after treatment, limiting their ability to detect reinfection (Fig. 8.6.37.8). However, specific tests have now become first line screening assays in most settings, and are followed by reflex non​ specific testing if the specific test is reactive, Non​specific serological tests The RPR and VDRL tests are flocculation tests targeting the phospholipid cardiolipin as an antigen. They are relatively sensi- tive in early infection (77 to 88% for primary syphilis and 100% for secondary syphilis), but tend to decline over the next several years without treatment (Fig. 8.6.37.8). Non​specific tests are used for follow-​up after treatment because they are readily able to quantify disease activity. In general, a fourfold change in titre is taken as evi- dence of cure, relapse, or reinfection. Broadly equivalent, RPR tests can be read macroscopically while VDRL tests require a microscope. The toluidine red unheated serum test (TRUST) is a variant of the RPR test. The VDRL test is the only syphilis test recommended for CSF evaluation. Such antilipoidal antibodies may be produced by other forms of acute or chronic tissue damage, so confirmation with a specific test is needed if there is no other sign of syphilis. Acute false-​positive results are associated with acute infections such as hepatitis, herpes virus infections, measles, and malaria, as well as immun- izations and pregnancy. Chronic (exceeding 6  months) false-​ positive reactions are associated with connective tissue disorders, immunoglobulin abnormalities, drug injecting, ageing, malaria, and malignancy. Specific serological tests These tests use T. pallidum as the antigen and may become posi- tive shortly before the non​specific tests. The specific tests typically (more than 85%) remain reactive for life after successful treatment and they do not provide meaningful quantitative results, so they have no role in assessing stage of infection, ‘cure’, or reinfection. They are technically more difficult and expensive than non​specific tests, so they may not be available for confirmation in resource-​poor environments. Examples of specific tests include the T. pallidum haemagglutination assay (TPHA), the T. pallidum particle agglu- tination assay (TPPA), and the microhaemagglutination assay for antibodies to T. pallidum (MHA-​TP). The fluorescent treponemal antibody absorption (FTA-​ABS) test is often used as a confirmatory test and may be the first serological test to become positive in pri- mary syphilis, so it may be added to the non​specific test to investi- gate a genital ulcer. Newer multiantigen enzyme immunoassays (EIAs) for IgG antibodies against T. pallidum are becoming increasingly common in high-​volume laboratories because they are more objective and can be automated. The EIAs appear to have com- parable sensitivity (70–​90% for primary and 100% for secondary syphilis) and specificity to the other specific tests. There is con- siderable overlap in the causes of false-​positive specific and non​ specific tests. As IgM antibodies are large and considered unable to cross the placenta, FTA-​ABS, and EIA versions of the IgM test have been used on neonates to assess possible congenital infection. The use of IgM tests is not established, and a negative IgM test does not exclude con- genital syphilis. Available only in reference laboratories, western blot can detect IgG or IgM antibodies and appears to be at least as sensitive as other specific tests for syphilis. The IgM western blot looks promising as an aid to diagnosing congenital syphilis with a specificity over 90% and a sensitivity over 83%. Several rapid point-​of-​care tests have been developed for syph- ilis. The majority are qualitative, and rely on detection of specific serological markers. Sensitivity compared to conventional EIA or TPPA testing is between 80 and 100%. Some point-​of-​care tests also include a qualitative assessment of non​specific reactivity to help distinguish active from prior infection, and perform rela- tively well (sensitivity 80–​90%) when the RPR titre is greater than 1:4. These tests have a particular role in developing country settings. Criteria for diagnosis Primary syphilis The direct detection of T. pallidum (Table 8.6.37.3) from an ulcer confirms a diagnosis of primary syphilis. Alternatively, a clinic- ally suspicious ulcer and any positive serological test are accepted as a confirmed diagnosis, although more than one serological test is normally ordered if resources permit. If initially seronegative, patients with suspicious ulcers should have repeat serology in 2–​4 weeks. Secondary syphilis Treponemes may be demonstrated in moist mucocutaneous lesions of secondary syphilis, although suggestive symptoms or signs (Table 8.6.37.1) plus a positive RPR test are sufficient for the diagnosis. The non​specific tests are normally reactive at high titres. 0 1 2 3 64 32 16 8 4 2 Years Primary Secondary Latent/tertiary Treatment Titre RPR TPHA FTA (Abs) EIA Fig. 8.6.37.8  Serological response to syphilis and its treatment.

section 8  Infectious diseases 1220 Latent syphilis The diagnosis of latent syphilis requires two positive serological tests, at least one of them a specific test. Recent symptoms sug- gestive of primary or secondary (Table 8.6.37.1) syphilis, or a history of a negative test in the last 1 or 2 years, indicates early latent syphilis. The sexual risk history should be consistent with this clinical staging. Generally, non​specific reactive test titres are higher in early latent than in late latent infection. In many cases a diagnosis of late latent syphilis or latent syphilis of unknown dur- ation is a diagnosis of last resort after risk and symptom history, clinical examination, and serological picture are judged together. Past childhood treponemal infection remains a possible explan- ation in some cases. Neurosyphilis Neurosyphilis is defined as a reactive CSF-​VDRL or a CSF mono- nuclear pleocytosis of more than 5 cells/​µl, or both. While a re- active CSF-​VDRL is very specific it has limited sensitivity (up to 70%) in detecting neurosyphilis. CSF protein concentration may be elevated. Because HIV can cause a CSF pleocytosis anyway, against a background of HIV infection a cut-​off of more than 20 cells/​µl has sometimes been used for a neurosyphilis diagnosis. A bloody tap might confound the diagnosis, while symptoms or signs of neurosyphilis add confidence to the diagnosis. CT or MRI of the brain or spinal cord might demonstrate lesions compatible with tertiary syphilis. Gummatous syphilis Gumma can be diagnosed clinically (Fig. 8.6.37.6) with reactive ser- ology but, as clinical experience is limited, histological confirmation is usual. Cardiovascular syphilis Aortic valve disease or proximal aortic aneurysm with reactive syphilis serology strongly suggest cardiovascular syphilis. However, aneurysm and calcification of the aortic wall are found in other con- ditions such as hypertension. Coronary angiography demonstrates ostial stenosis. Congenital syphilis The diagnosis of congenital syphilis is often problematic, and many neonates are treated before it can be confirmed because of the high risk of serious disease. Direct detection of T. pallidum from the placenta or nasal discharge or skin lesions of a newborn infant is definitive but rarely achievable. Usually the diagnosis relies on clinical signs (if present) and serology. Positive serological tests in the neonate may reflect passive antibody transfer from the mother, but a positive non​specific test is useful if present in higher titres than the mother. An alternative approach to the management of a normal-​looking baby of an infected mother is to perform serial quantitative non​specific serology and treat if the titre rises. More experience is needed with PCR and western blot IgM antibody testing. As clinically indicated, long-​bone and chest radiology, lumbar puncture, cranial ultrasonography, and ophthalmic examination may contribute to the diagnosis where available. Treatment Choice of antibiotics Parenteral penicillin G is the original, and remains, the preferred treatment for syphilis globally. In most parts of the world this takes the form of long-​acting benzathine penicillin G injections, although some prefer daily injections with procaine penicillin, sometimes boosted with probenecid, because treponemicidal CSF levels might be achieved (Table 8.6.37.4). However, daily injections raise adher- ence and resource issues. No penicillin regime has demonstrated su- periority in controlled trials. Table 8.6.37.4  Treatment of syphilis Form of syphilis US Centers for Disease Control and Prevention (CDC) Notable variations Adult, early Benzathine penicillin G 2.4 million units (equivalent to 1.8 g) intramuscularly in one dose UK guidelines offer as an alternative: procaine penicillin G 750 mg (600 000 U) intramuscularly once a day for 10 days; or if patient averse to injections, amoxicillin 500 mg plus probenecid 500 mg orally four times a day for 14 days; or if allergic to penicillin, doxycycline 100 mg orally twice a day for 14 days Adult, late; excluding neurosyphilis Benzathine penicillin G 2.4 million units intramuscularly weekly for three doses UK alternative: procaine penicillin 750 mg intramuscularly once a day for 14 days; or if patient averse to injections, amoxicillin 2 g plus probenecid 500 mg orally four times a day for 28 days; or if allergic to penicillin, doxycycline 100 mg orally twice a day for 28 days Neurosyphilis Aqueous crystalline penicillin G 18–​ 24 million units intravenously per day (as 3–​4 million units every 4 h or as a continuous infusion) for 10–​14 days UK alternative: procaine penicillin 2.4 g intramuscularly once a day plus probenecid 500 mg orally four times a day for 14 days; or if patient averse to injections, amoxicillin 2 g plus probenecid 500 mg orally four times a day for 28 days; or if penicillin allergic, doxycycline 200 mg orally twice a day for 28 days Syphilis in a pregnant woman As per stage of adult syphilis Desensitize if allergic to penicillin (see CDC guidelines); doxycycline is contraindicated in pregnancy Congenital and childhood syphilis Aqueous crystalline penicillin G 100 000–​150 000 units/​kg intravenously in divided doses for 10 days UK and CDC alternative if active disease: procaine penicillin 50 000 units/​kg intramuscularly once a day for 10 days; older children with primary or secondary syphilis, benzathine penicillin G 50 000 units/​kg (up to 2.4 million units) in one dose; three doses if late infection or unknown duration of infection; child-​protection assessment is essential CDC, Centers for Disease Control and Prevention (United States of America).

8.6.37  Syphilis 1221 Injectable ceftriaxone has been used with short-​term success al- though the exact dose, frequency, and length of course are uncer- tain. Oral doxycycline provides an alternative for those with an allergy to penicillin, when there is no access to clean needles, or when the patient is averse to injections. Neither of these agents has been well studied. Macrolides such as erythromycin and azithromycin have also been used, but the rapid emergence of high-​level resistance on four con- tinents, attributable to a single mutation in the T. pallidum genome, makes this group of antibiotics inappropriate in most settings. Some physicians use oral corticosteroids to reduce the adverse ef- fects of the Jarisch–​Herxheimer reaction in neurological and cardio- vascular syphilis although there is no systematic evidence to support this practice. Contacts Sexual contacts of early syphilis should be treated presumptively re- gardless of their test results if the contact was within 90 days, usu- ally with a single dose of benzathine penicillin G. Contacts beyond 90 days can be treated according to the clinical picture and serology results unless follow-​up is uncertain. Follow-​up The goals of treatment are to cure symptoms and signs of infection if present, to render the patient non​infectious, and to prevent late complications occurring or progressing. In primary or secondary syphilis, lesions and constitutional symptoms should be well on the way to resolving within days. However, some symptoms of early neurosyphilis may persist for several months. Antibiotic therapy halts further damage in cardiovascular, neurological, and gumma- tous syphilis but is usually unable to repair tissue damage that has already occurred. Follow-​up serology can be performed at 3, 6, and 12 months from treatment. Defining successful treatment of early syphilis relies on demonstrating a fourfold decrease in reagin (RPR or VDRL) titres over the next 6–​12 months. If there is ongoing risk, reinfection may be impossible to separate from relapse; both are defined as a fourfold rise in reagin titres on at least two occasions. In late infections, where reagin titres are typically low, no drop in titre may be demonstrable. Frequently, a persistently low or non​reactive reagin test, an absence of current symptoms, and a history of adequate treatment must be accepted as a cure. Regular reagin testing is recommended for those at ongoing risk of reinfection. Jarisch–​Herxheimer reaction The Jarisch–​Herxheimer reaction can occur in up to 50% of those with primary syphilis and more than 70% of patients with sec- ondary syphilis, but it is uncommon in late syphilis. This transient influenza-​like reaction occurs between 4 and 24 h (median 8 h) after the first dose of antibiotics and lasts for several hours with malaise, low-​grade fever, flushing, and tachycardia. Early and late lesions might transiently flare, secondary rashes might appear for the first time (and be mistaken for penicillin allergy), and cranial nerve and cardiovascular symptoms might worsen. In rare cases, premature labour and fetal distress have been induced. The reaction can result from the release of endotoxin-​like substances from killed T. pallidum. Patients should be warned in advance and advised to stay home for the first night with paracetamol at hand. Prognosis Since the advent of penicillin, the late complications of tertiary syphilis are relatively rare and adult mortality is almost never seen. However, in resource-​poor environments or where antenatal screening is not routine, fetal wastage and serious congenital disease remain common. With treatment all mucocutaneous syphilis lesions rapidly re- solve, sometimes leaving an atrophic scar. Deformities of bones and teeth generally persist for life. The symptoms of early neuro- syphilis usually resolve although this can take several months. The symptoms and signs of late neurosyphilis generally persist but they should not progress. Follow-​up CSF examination should docu- ment a declining pleocytosis (if present initially) by 6 months, al- though the CSF-​VDRL might take longer to normalize. Areas of controversy The role of lumbar puncture Lumbar puncture is indicated if neurological or ophthalmic symp- toms or signs are present. The role of lumbar puncture in the diag- nosis, treatment, and follow-​up of other patients with syphilis has been debated. Resource and patient consent issues can be difficult. Many experts believe that if using a treatment regimen that is likely to enter the CSF such as daily procaine penicillin, then a lumbar puncture before treatment does not alter management of the case and can be omitted. Lumbar puncture can then be limited to cases where investigation might alter management, such as in cases of difficult differential diagnosis or possible relapse. HIV infection HIV and syphilis are both transmitted sexually so it is not sur- prising that both infections often coexist. Additionally, syphilis lesions can facilitate both the transmission and acquisition of HIV infection. Early syphilis can lead to a moderate decline in periph- eral CD4 cell counts and an elevation of plasma HIV-​1 viral load in HIV-​infected people; both phenomena resolve with syphilis treatment. No unique clinical syphilis syndromes have been reported in people with concurrent HIV infection. Limited, largely anecdotal, evidence suggests some more aggressive clinical manifestations of syphilis in HIV infection, but this is the exception rather than the rule. Early neurosyphilis might be more common but the diagnosis is compounded by the HIV infection itself causing neurological symptoms and CSF abnormalities. Higher CSF lymphocyte counts (20 rather than the usual 5 cells/​µl) have been used to diagnose neurosyphilis because HIV commonly causes a CSF pleocytosis without syphilis. Higher serum RPR titres (≥1:32) and a lower peripheral CD4 cell count (<350 cells/​µl) have been shown to be predictive of neurosyphilis, making these tests relative indica- tions for lumbar puncture particularly if both are present. Some

section 8  Infectious diseases 1222 have argued for routine lumbar puncture for HIV-​infected people with syphilis, while others, noting that neurosyphilis remains un- common even in this group, advocate limiting lumbar puncture to people with neurological or ocular symptoms, treatment failure, or late latent syphilis. Despite some early reports of HIV-​coinfected patients with nega- tive serological tests in early syphilis, larger studies have failed to show any significant difference and standard syphilis testing proced- ures are recommended in HIV infection. In general, authorities such as the United States Centers for Disease Control and Prevention recommend routine treatment with benzathine penicillin G (Table 8.6.37.4) in HIV infection. A higher rate of serological treatment failure (defined as a four- fold decrease in RPR titre at 6–​12 months) has been documented in HIV infection (c.20%) compared to HIV-​uninfected people (5%). However, the clinical significance of this finding is unknown, and reinfection is always a possibility. Likely developments in the near future Nucleic acid amplification tests such as PCR will aid the aetiological diagnosis of genital ulcers if they can be made widely available in multiplex form (i.e. testing for all serious causes of genital ulcers in a single test). PCR technology, including multi-locus sequence typing, has also led to the delineation of different subtypes of syphilis, and this intraspecies variation might go some way to explaining the dif- ferent phenotypes exhibited in clinical practice. PCR testing may also improve the diagnosis of congenital syphilis. At a more mundane level, improving the availability of low-​skill temperature-​stable rapid syphilis tests that do not require refriger- ation or even electricity could achieve a great deal in resource-​poor environments where syphilis is most common. Early experience with immunochromatographic test strips coated with T. pallidum antigens that give results in 8–​20 minutes have been encouraging. The sequencing of the genome might eventually enable the devel- opment of a vaccine, but funding for syphilis research would need to be dramatically increased. The role of chemoprophylaxis, typically using doxycycline, as a prevention intervention during a syphilis epidemic has been in- vestigated in a number of studies and has shown promising results among high risk men-who-have sex with men in Western countries. Further research is required to determine if this is a useful and safe intervention at a population level. Finally, the impact of HIV pre-exposure prophylaxis on con- sistent condom use among men who have sex with men and the con- sequent effect on STI transmission, including syphilis, needs careful monitoring. FURTHER READING Bristow C, et al. (2015). A review of recent advances in rapid point-​of-​ care tests for syphilis. Sex Health, 12, 119–​25. British Association for Sexual Health and HIV (2015). UK national guidelines on the management of syphilis. https://​www.bashh.org/​ guidelines Cates W Jr, Rothenberg RB, Blount JH (1996). Syphilis control: the historical context and epidemiological basis for interrupting sexual transmission of Treponema pallidum. Sex Transm Dis, 23, 68–​75. Centers for Disease Control and Prevention (2015). Sexually trans- mitted diseases treatment guidelines 2015. MMWR Recommen­ dations and Reports, 64, 1–​140. Centurion-​Lara A, et al. (2006). Molecular differentiation of Treponema pallidum subspecies. J Clin Microbiol, 44, 3377–​80. Chakraborty R, Luck S (2007). Managing congenital syphilis again? The more things change  . . . . Curr Opin Infect Dis, 20, 247–​52. Chow EPF, et  al. (2011). HIV and syphilis co-​infection increasing among men who have sex with men in China: a systematic review and meta-​analysis. PLoS One, 6, e22768. Clark EG, Danbolt N (1964). The Oslo study of the natural course of untreated syphilis: an epidemiologic investigation based on a re-​ study of the Boeck-​Bruusgaard material. Med Clin North Am, 48, 613–​23. Drummond F, et al. (2010). The intersection between HIV and syph- ilis in men who have sex with men: some fresh perspectives. HIV Therapy, 4, 661–​73. Fraser CM, et al. (1998). The genome sequence of Treponema pallidum, the syphilis spirochete. Science, 281, 375–​88. Gayet-​Ageron A, et al. (2013). Sensitivity, specificity and likelihood ratios of PCR in the diagnosis of syphilis: a systematic review and meta-​analysis. Sex Transm Infect, 89, 251–​6. Gray RT, et al. (2010). Frequent testing of highly sexually active
gay men is required to control syphilis. Sex Transm Dis, 37, 298–​305. Harper KN, et al. (2008). On the origin of the treponematoses: a phylo- genetic approach. PLoS Negl Trop Dis, 2, e148. Hart G (1986). Syphilis tests in diagnostic and therapeutic decision making. Ann Intern Med, 104, 368–​76. Holmes KK (2006). Azithromycin versus penicillin for early syphilis. N Engl J Med, 354, 205. Larsen SA, Steiner BM, Rudolf AH (1995). Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev, 8, 1–​21. Lin CC, et al. (2006). China’s syphilis epidemic: a systematic review of seroprevalence studies. Sex Transm Dis, 33, 726–​36. Marra CM (2004). Neurosyphilis. Curr Neurol Neurosci Rep, 4, 435–​40. Molina J-M, et al. (2018). Post-exposure prophylaxis with doxycycline to prevent sexually transmitted infections in men who have sex with men: an open-label randomised substudy of the ARNS IPERGAY trial. Lancet Infect Dis, 18, 308–17. Morton RS, Rashid S (2001). ‘The syphilis enigma’: the riddle solved? Sex Transm Infect, 77, 322–​4. Peeling RW, Ye H (2004). Diagnostic tools for preventing and managing maternal and congenital syphilis: an overview. Bull World Health Organ, 82, 439–​46. Read P, Fairley CK, Chow E (2015). Increasing trends of syphilis among men who have sex with men in high income countries. Sex Health, 12, 155–​63. Salazar JC, Hazlett KRO, Radolf JD (2002). The immune response to infection with Treponema pallidum, the stealth pathogen. Microbes Infect, 4, 1133–​40. Walker GJA (2001). Antibiotics for syphilis diagnosed during preg- nancy. Cochrane Database Syst Rev, 3, CD001143. World Health Organization (2007). The global elimination of congenital syphilis: rationale and strategy for action. WHO, Geneva. Zetola NM, Klausner JD (2007). Syphilis and HIV: an update. Clin Infect Dis, 44, 1222–​8.

8.6.38 Listeriosis 1223

8.6.38 Listeriosis 1223

8.6.38  Listeriosis 1223 8.6.38  Listeriosis Herbert Hof ESSENTIALS Listeriosis is caused by the Gram-​positive bacillus Listeria monocytogenes, whose natural habitat is the soil. Consumption of soft cheeses, other dairy products, meat products, seafood, and vegetables is the principal route of infection. Patients at particular risk include those who are immunocompromised, very young, or very old. Pregnant women are also at risk, although they develop only mild disease, but the bacteria can be transmitted to the child either in utero or during birth, causing serious systemic disease. Clinical features and diagnosis—​the disease varies from a mild, influenza-​like illness to fatal septicaemia and meningoencephal- itis. Purulent, localized infections of any organ are sometimes seen. Serology is not helpful for diagnosis, which is confirmed by culture from blood, cerebrospinal fluid, or organ biopsies using enrichment and selective methods or by nucleic acid amplification techniques. Typing of isolates is essential for epidemiologic purposes. Treatment, prognosis, and prevention—​aside from supportive care, the usual treatment of choice is high-​dose intravenous ampicillin, which must be administered for at least 2 weeks. The prognosis is poor, with mortality of up to 30%. Prevention depends upon those that are vulnerable avoiding high-​risk foods. There is no vaccine. Introduction Exposure of humans to Listeria monocytogenes is quite frequent, but infections are rare. Only a small proportion of exposed individuals are likely to become ill but for them, despite precise diagnosis and adequate therapy, the prognosis remains poor. Historical perspective In the 1920s, L. monocytogenes was shown to be capable of inducing systemic infections in experimental animals. About 40 years later, it became obvious that epidemics might occur in humans but it took a further 30 years before listeriosis was shown to be a food-​borne dis- ease in most instances. Today, listeria is an exciting research tool for studying the biology of intracellular microorganisms that trigger a cell-​mediated immune response. Aetiology, genetics, pathogenesis, and pathology Among the various listeria species, L. monocytogenes is the major pathogen for humans (as well as for animals). L.  monocytogenes has specific attributes for invading and surviving and replicating in host cells. Surface proteins such as internalins are critical for the adhesion to specific receptors on host cells. A pathogenicity is- land on the chromosome encoding for haemolysin (listeriolysin), phospholipases, and an actin polymerizing protein is crucial for intracellular survival, traffic in the cytoplasm, and cell-​to-​cell spread. By this means, listeria can cross anatomical barriers such as the in- testinal mucosa, the blood–​brain barrier, and the placenta. Humoral defence mechanisms are largely ineffective in coping with these bac- teria. Rather, a cell-​mediated immune response is required to over- come a listeria infection. Over the course of infection granulomas develop in infected organs, indicating a vigorous immune response. During the acute stage, when a massive multiplication of bacteria takes place intracellularly as well as extracellularly, a purulent in- flammatory reaction is seen at the site of infection. Epidemiology Listeria species are widespread in nature and their natural habitat is the soil; they have been isolated from dust, foodstuffs, animal feed, water, sewage, and numerous animals. Infections of humans are mainly due to only a few special clones. Various food items of both plant and animal origin, contaminated either during growth or during processing, can give rise to an infection. Consumption of soft cheeses such as Brie, Camembert, and blue-​vein types, other dairy products, meat products (e.g. sausages and delicatessen meat), sea- food (graved salmon), and vegetables (lettuce) is the principal route of infection. However, tomatoes, apples, and carrots are practically free of listeria. The ability of listeria to multiply at temperatures from 0 to 40°C is of particular concern if infected foods are stored in the refrigerator and consumed without further cooking. A study of food-​borne illness in the United States of America be- tween 2000 and 2008 estimated an annual incidence of 1600 cases of listeriosis, with a 16% mortality. In 2009 the estimated incidence of laboratory-​confirmed listeriosis was 0.34 per 100 000 population. An increase in listeriosis has also been reported in several European countries, and decreased salt concentrations in prepared foods have been proposed as a possible cause. Transmission from infected animals to humans is unusual, but occupational infections in vet- erinary surgeons or farm workers are reported. Human-​to-​human transmission occurs only during pregnancy, when the bacteria col- onizing the mother infect the fetus in utero or the neonate in the birth canal. In most cases disease occurs sporadically, but small epi- demics are occasionally observed due to commercially distributed, highly contaminated food items. Predisposing conditions include advanced age, pregnancy, glucocorticoid therapy, haematological or solid organ tumours, organ transplantation (especially renal), HIV infection, diabetes mellitus, endstage renal disease, chronic liver disease, collagen vascular diseases, and iron overload. Hospital-​ acquired listeriosis has been reported and has been associated with consumption of contaminated food. Nosocomial transmission be- tween neonates has been associated with poor hand hygiene, close contact between infected patients and their mothers, skin care prod- ucts, and instruments such as rectal thermometers or stethoscopes. Prevention A vaccine against L.  monocytogenes has not yet been developed. Most infections are food-​borne; food items are contaminated either intrinsically or during processing or during storage in the refriger- ator. The abovementioned foods often contaminated with Listeria

section 8  Infectious diseases 1224 should be avoided by people at special risk, and they should also not eat some preprepared food items unless they are thoroughly re- heated to piping hot temperatures. Food items that commonly carry listeria, such as salads and mushrooms, should be kept separately in the refrigerator from those likely to be free of these bacteria, such as cold meats and other ready-​to-​eat food, otherwise there will be cross-​contamination. Improvement in the microbiological safety of food production processes and the continued education of the public will further re- duce the risk of infection. Inactivation of L. monocytogenes by radiation is readily achieved without changing the appearance or taste of foods. A combination of ultrasound treatment and rinsing of food stuff with 2% organic acids for 5 min is also able to reduce the contamination with these pathogens. Clinical features Listeriosis is generally an opportunistic infection of elderly people, with men more frequently affected than women. Furthermore, im- munocompromised patients such as those with haematological or solid organ tumours, organ transplant recipients, patients with se- vere underlying illness such as diabetes mellitus, endstage kidney disease, liver cirrhosis or iron overload, and pregnant women and newborn babies are at increased risk. Occasionally people without these risk factors can be infected as well. In a few cases, a mild gastroenteritis precedes the systemic infection. The clinical presen- tation varies from a mild influenza-​like illness to fatal septicaemia and meningoencephalitis. Purulent localized infections of any organ occasionally occur. Recognized syndromes include maternofetal and neonatal listeri- osis, septicaemia, meningoencephalitis, cerebritis, and localized in- fections of various solid organs. Outbreaks of gastroenteritis with fever, diarrhoea, nausea, vomiting, and arthromyalgia have been described even in immunocompetent adults who have ingested con- taminated food. The diagnosis is usually missed because diarrhoeal stools are not cultured selectively for listeria. Septicaemia occurs mainly in adult patients with malignancies, in transplant recipients, and in immunosuppressed and elderly people. Most present with fever, hypotension, and shock. Many patients also develop meningitis. Meningitis may start abruptly but, in adults, can also develop insidiously, with progressive neurological signs espe- cially meningism. Fever may not be marked, particularly in elderly or immunosuppressed people. A purulent reaction is seen in the cerebrospinal fluid with most of the Gram-​positive bacteria lying extracellularly. Cerebritis in combination with meningitis, or separately, is in- creasingly recognized, particularly in immunosuppressed patient. Headache, fever, and varying degrees of paralysis and cerebral dis- orders such as dizziness or loss of consciousness may be observed. Rhombencephalitis begins with a headache, fever, nausea, and vomiting followed after several days with asymmetrical progressive cranial nerve palsies and decreased consciousness. Infection of the cerebellum might be followed by ataxia and problems of coordin- ation. MRI or CT may show areas of uptake without ring enhance- ment. Sometimes a brain abscess is diagnosed. In such cases the cerebrospinal fluid may show few, if any, inflammatory cells, and protein and sugar concentrations are normal. Intracerebral foci may be sealed off, so that bacteria or even bacterial DNA are not detected in cerebrospinal fluid or blood, leading to a missed diagnosis. Localized infections are rare, occurring mainly in immuno- suppressed people. They include soft-​tissue abscesses, osteo- myelitis, septic arthritis, cholecystitis, peritonitis, endocarditis, endophthalmitis, and pneumonia. They usually result from seeding during an initial bacteraemic phase, but focal skin and eye infection can also result from direct occupational exposure. In materno-​fetal listeriosis, the mother may develop fever, head- ache, myalgia, and low back pain due to the bacteraemic phase of the disease. Transplacental infection causes placentitis, amnionitis, and, depending on the time until delivery, spontaneous septic abortion or premature labour with delivery of a severely infected baby. Neonatal listeriosis of early onset results from intrauterine infec- tion and has a high mortality. The amniotic fluid is greenish and the baby septic and jaundiced, with signs of purulent conjunctivitis, bronchopneumonia, meningitis, and/​or encephalitis. Granulomas affect many organs, hence the term ‘granulomatosis infantisepticum’. Late-​onset disease, developing several days to weeks after birth in a baby who was initially healthy, presents with meningitis. The in- fection may have been acquired from the mother’s genital tract or through cross-​infection as a nosocomial infection. Differential diagnosis Since various organs may be affected, listeriosis may mimic several quite different local or systemic infectious diseases. The septic mani- festations are non​specific and, particularly in immunocompromised patients and elderly people, one should think of listeriosis. Listeria meningitis develops insidiously in most instances, in contrast to other bacterial disorders. In particular, listeria encephalitis is difficult to recognize initially because it can resemble, for instance, a cerebro- vascular accident. This infection should be considered in any patient with an acute brain stem or cerebellar disorder associated with fever, particularly if there are no risk factors for cerebrovascular disease. Bacteraemia accompanied by fever and, eventually, shock can mimic Gram-​negative sepsis. Encephalitis, which may develop slowly, can be confused in elderly people with cerebrovascular dis- ease or even with brain metastases. Criteria for diagnosis Listeria are non​sporing, facultatively anaerobic, Gram-​positive rods. Enrichment and selective methods are now well established for the isolation of these non​fastidious bacteria from the environment, food, or human specimen. Blood, cerebrospinal fluid, meconium, amniotic fluid, placental tissue, lochia, and swabs from purulent dis- charge from various organs can yield the pathogens. Gram-​positive rods may be seen in a stained smear. Sometimes they are very short and thus can be mistaken for streptococci. A predominance of monocytes among the inflammatory cells, which might lead to early suspicion of listeria, is not regularly seen. Differentiation of the various species is generally possible by means of commercially avail- able biochemical tests or massspectrometry. Serovars 1/​2a, 1/​2b, and 4b are the most prominent among human isolates. Several genetic

8.6.38  Listeriosis 1225 typing methods are used to trace food sources, distinguish relapses from reinfections, and investigate outbreaks. Overall more than 17 different Listeria spp. have been described, most of which originate from the environment. L. monocytogenes is the major pathogen, although occasional human infections with L. ivanovii and L. seeligeri have been re- ported. L. welshimeri, L. innocua, L. marthii, and L. grayi are not known to cause disease. The crucial difference is that pathogenic isolates display various virulence factors not present in non​patho- genic ones. In various isolates of L. monocytogenes, these proper- ties can be differentially expressed, so that the pathogenicity will vary from strain to strain. Nucleic acid amplification techniques have also been used to de- tect the bacteria or, for example, in patients pretreated with anti- biotics. However, serology is non​specific and does not aid diagnosis. Treatment Practically all strains of L. monocytogenes are susceptible to a large range of common antibiotics including ampicillin, gentamicin (which acts synergistically in vitro with ampicillin), co-​trimoxazole, erythro- mycin, tetracycline, chloramphenicol, vancomycin, and rifampicin. On the other hand, L. monocytogenes is inherently resistant in vitro to the cephalosporins and fosfomycin. It is also resistant to nalidixic acid but susceptible to the newer quinolones such as moxifloxacin. It should be borne in mind, however, that many of the bacteria reside intracellularly where they are protected from some of the ac- tive antimicrobial agents. Paradoxically, fosfomycin is active against intracellular Listeriae because in this particular environment, with low availability of essential nutrients such as glucose, the bacteria have to activate influx pumps which also transport fosfomycin into the bacterial cell, where it can perform its antibacterial activity. The clinical benefit of this phenomenon is not yet clear. There are no controlled trials of antibiotic treatment for listeri- osis. According to clinical experience, high-​dose intravenous ampi- cillin (i.e. 4 × 2–​3 g/​day) in combination with gentamicin (360 mg, or in a dose adjusted with the help of serum concentration meas- urements, once daily in a 60-​min infusion) remains the treatment of choice for adults. This combination should be given for 2 weeks at least. If necessary, ampicillin alone can be continued for another week or even longer (e.g. in case of endocarditis, until clinical reso- lution). For children, a daily dose of 200–​300 mg/​kg ampicillin, perhaps combined with 3–​5 mg/​kg gentamicin, is recommended. Gentamicin is best avoided in pregnancy, when ampicillin may be used alone, or erythromycin (2 g/​day intravenously for 2–​3 weeks) if the patient is allergic to penicillin. Recently, it has been ques- tioned whether the combination is better than ampicillin alone. Intravenous co-​trimoxazole (daily dose 20 mg/​kg trimethoprim

8.6.39 Legionellosis and Legionnaires’ disease 122

8.6.39 Legionellosis and Legionnaires’ disease 1226

section 8  Infectious diseases 1226 Although the therapeutic value of moxifloxacin has not yet been assessed in human listeriosis, it can be deduced from cell culture ex- periments as well as from animal experiments that this quinolone, which is highly active in vitro, is able to penetrate into host cells and effectively kill intracellular L. monocytogenes, so that rapid cure may be achieved. FURTHER READING Becattini S, Pamer EG (2017). Multifaceted defense against Listeria monocytogenes in the gastro-intestinal lumen. Pathogens, 7, E1. Gellin BG, Broome CV (1989). Listeriosis. JAMA, 261, 1313–​18. Hamon M, Bierne H, Cossart P (2006). Listeria monocytogenes:  a multifaceted model. Nat Rev Microbiol, 4, 423–​34. Hof H (2013). Chemotherapy of Listeria infections. GMS Infectious Diseases 1:  06. http://​www.egms.de/​static/​de/​journals/​id/​2013-​1/​ id000006.shtml Hof H, Lampidis R (2001). Retrospective evidence for nosocomial Listeria infection. J Hosp Infect, 48, 321–​2. Hof H, Nichterlein T, Kretschmar M (1997). Management of listeri- osis. Clin Microbiol Rev, 10, 345–​57. Lamont RF, et al. (2011). Listeriosis in human pregnancy: a systematic review. J Perinat Med, 39, 227–​36. Liu D (2006). Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen. J Med Microbiol, 55, 645–​59. Mitjà O, et al. (2009). Predictors of mortality and impact of amino­ glycosides on outcome in listeriosis in a retrospective cohort study. J Antimicrob Chemother, 64, 416–​23. Schlech WF (2000). Foodborne listeriosis. Clin Infect Dis, 31, 770–​5. Orsi RH, Wiedemann M (2016). Characteristics and distribution of Listeria spp., including Listeria species newly described since 2009. Appl Microbiol Biotechnol, 100, 5273–87. Pagliano P, Arslan F, Ascione T (2017). Epidemiology and treatment
of the commonest form of listeriosis: meningitis and bacteremia. Infez Med, 3, 210–16. 8.6.39  Legionellosis and
legionnaires’ disease Diego Viasus and Jordi Carratalà ESSENTIALS Legionellaceae are Gram-​negative bacilli, of which Legionella pneumophila is the principal cause of human infections. Their natural habitats are freshwater streams, lakes, thermal springs, moist soil, and mud, but the principal source for large outbreaks of legionellosis is cooling systems used for air conditioning and other cooling equip- ment. Legionella spp. are principally transmitted to humans through contaminated water aerosols. Middle-​aged men, smokers, regular al- cohol drinkers, and those with comorbidity are most at risk. Clinical features and diagnosis—​(1) Legionnaires’ disease (pneu- monia)—​typically presents with high fever, shivers, headache, and muscle pains; respiratory symptoms are sometimes minimal; confusion and diarrhoea may dominate the clinical picture. The radiographic and non​specific laboratory findings overlap with typical and atypical pulmonary pathogens. (2)  ‘Pontiac fever’—​an acute non​pneumonic form that presents as a self-​limiting, influenza-​like illness. Detection of urinary antigen has become the mainstay for diagnosis. Treatment, prognosis, and prevention—​aside from supportive care, the first-​choice antibiotics are macrolides (mainly azithromycin) and/​ or fluoroquinolones (especially levofloxacin). Case fatality is 5–​15% in previously well adults, but much higher in those who are immuno- compromised or develop respiratory failure. Prognosis is improved by early administration of effective anti-​legionella antibiotic therapy. Prevention is by the correct design, maintenance, and monitoring of water systems. Notification of a case allows a public health investiga- tion into the likely source and the detection, prompt treatment, and/​ or prevention of additional cases. Introduction and historical perspective Legionella spp. was first recognized as a pathogen in 1977, when L. pneumophila was identified as the agent responsible for an out- break of severe pneumonia among delegates to the 1976 American Legion Convention at a Philadelphia hotel. Several new serogroups of L. pneumophila and other Legionella spp. have since been discovered. Legionellosis refers to the two main clinical syndromes caused by bacteria of the genus Legionella. Pontiac fever is an acute, self-​ limited, non​pneumonic, influenza-​like condition caused typically by L.  pneumophila serogroup 1; legionnaires’ disease is an acute pneumonia syndrome caused by Legionella spp. Aetiology The family Legionellaceae consists of the single genus Legionella. Legionella are aerobic, non​sporing, Gram-​negative bacteria. The genus Legionella consists of 58 species encompassing at least 70 serogroups (Table 8.6.39.1). The bacterium displays dramatic pleo- morphism, presenting coccoid, bacillary, and/​or long filamentous Table 8.6.39.1  Some Legionella species L. adelaidensis L. longbeachae L. anisa L. lytica L. bozemanii L. micdadei L. brunensis L. moravica L. cherril L. nautarum L. dumoffi L. parisiensis L. fairfieldensis L. pneumophila L. feeleii L. quateirensis L. gormanii L. rubrilucens L. gratiana L. santicrusis L. hackeliae L. spiritensis L. israeliensis L. taurinensis L. jordanis L. waltersii L. lansingensis L. yabuuchiae

8.6.39  Legionellosis and legionnaires’ disease 1227 forms depending on the temperature, available nutrients, and type of medium. They grow on various solid-​selective and non​selective media. Iron, L-​cysteine, α-​ketoglutarate, and charcoal-​containing yeast extract agar buffered with an organic buffer (BCYE) is the preferred growth medium for clinical isolation. The identifica- tion of legionella at the species level requires more sophisticated testing:  growth requirements, agglutination and fluorescent anti- body technique, fatty acid, carbohydrate or ubiquinone analysis, protein profiling, and various molecular techniques. Legionella species are found worldwide. Legionella bacteria have been isolated from aqueous environments such as rivers, ponds, lakes, and thermal pools. The organisms are able to survive in moist environments for long periods of time and can withstand temperat- ures of 0–​68ºC. Legionella is primarily adapted for survival and repli- cates within numerous protozoan genera, including Acanthamoeba, Naegleria, Hartmanella, and Tetrahymena, and secondarily as a free-​ living or biofilm-​associated aquatic bacterium. This relationship increases the resistance of L. pneumophila to biocides, antibiotics, acids, and osmotic and thermal stress. L. pneumophila is the principal cause of human infection. Serogroups 1, 4, and 6 of L. pneumophila are the most frequently isolated. Seventeen other species have been implicated in human infections, including L. micdadei, L. bozemanii, L. dumoffi and L. longbeachae (30–​55% of legionnaires’ disease cases in Australia and New Zealand). Epidemiology The exact incidence of legionnaires’ disease worldwide is difficult to establish, mainly because countries differ in terms of levels of awareness, diagnostic methods, and reporting. In any case, legion- naires’ disease is substantially underdiagnosed and underreported. Population-​based incidence data suggest that more than 10 000 cases of legionnaires’ disease occur annually among adults in the United States. A significant increase in the incidence of legionellosis in the United States has been documented in recent years. According to the European Working Group for Legionella Infections, the overall incidence for Europe was 8.2 cases per million population, with wide variations between countries (range 0–​21.4 per million). Legionellosis incidence also shows marked seasonality, with most cases being reported in summer or fall. Legionella spp. are increasingly recognized as a cause of both spor- adic and epidemic community-​acquired pneumonia requiring hos- pitalization, being responsible for between 2% and 16% of all cases. The disease is rare in children, and most cases occur in those older than 50 years. Infection is more common in men. Hospital-​acquired legionellosis is uncommon but can occur when hospital water supplies are contaminated with the organism. Nosocomial infection mainly af- fects highly susceptible or immunosuppressed patient populations. Although Legionella spp. have been detected in virtually all sources of fresh water supplies, natural water supplies are rarely identified as sources of human infection. Aerosols from artificial reservoirs of water are most often implicated in community-​acquired outbreaks, and cooling towers continue to be the most frequently suspected sources. Other well recognized sources include whirlpool spas or warm-​water baths, decorative fountains, automatic car washes, and potting compost (L. longbeachae in Australia). Legionnaires’ disease may be associated with a history of recent travel. The most common source of infection is contaminated water in hotel fountains and showers, but travel-​associated legionnaires’ disease has also been linked to cruise ships. Pathogenesis Legionella spp. are principally transmitted to humans through in- halation. Aspiration and instillation of contaminated water are also possible routes of transmission, but there is no evidence of person-​ to-​person transmission. Legionnaires’ disease due to L. longbeachae is thought to have a different route of transmission—​exposure to potting compost or soil, or gardening activities. Although many people are exposed to Legionella spp., very few develop legionnaires’ disease. Susceptibility to disease is associated with older age, smoking, chronic cardiovascular or respiratory dis- ease, alcoholism, diabetes, and immunosuppression. L. pneumophila possesses many of the traditional bacterial de- terminants that are important for pathogenicity in other bacteria, including lipopolysaccharide (LPS), flagella, pili, a type II secretion system (T2SS) termed Lsp, and outer membrane proteins. The mip gene was the first L.  pneumophila virulence-​associated gene de- tected. It is required for efficient host cell infection and is conserved throughout the genus. In addition, L. pneumophila has a type IV secretion system (Dot/​Icm T4SS) that translocates several effector proteins, including many proteins with eukaryotic similarity that act on diverse host cell pathways, such as the establishment of the L. pneumophila-​containing vacuoles, bacterial entry, inhibition of host cell apoptosis and the egress of the bacteria from the host cell. In human macrophages, L. pneumophila multiplies intracellularly by avoiding phagosome-​lysosome fusion. Legionella spp. inhibit the bactericidal activity of the phagocyte, permitting prolonged intra- cellular survival within the phagosome. Additional virulence factors include cytotoxins, compounds associated with iron uptake, and β-lactamases. Clinical features and differential diagnosis Legionnaires’ disease does not have specific, defining clinical features because it presents as a range of clinical symptoms and signs (Table 8.6.39.2). Legionnaires’ disease most commonly presents acutely and resembles pneumococcal or other bacterial pneumonias. The incubation period is between two and 14 days. Table 8.6.39.2  Clinical features and laboratory findings associated with legionnaires’ disease Incubation period 2–​14 days Prodromal illness Headache, myalgia, asthenia, and anorexia Physical examination Fever, relative bradycardia, cough, purulent sputum, shortness of breath, rales, altered mental status Radiographic findings Progressive asymmetrical patchy infiltrates, cavitation, or abscess formation is rare, pleural effusion Laboratory findings Increased serum transaminase levels, microscopic haematuria, leucocytosis with relative lymphopenia, hypophosphatemia, increased serum ferritin and creatine phosphokinase levels, hyponatraemia Others Loose stools or watery diarrhoea

section 8  Infectious diseases 1228 A  prodromal illness can occur, with symptoms such as head- ache, myalgia, asthenia, and anorexia. Physical manifestations of Legionella pneumonia are not specific: fever (often up to 39oC); cough with purulent sputum in around 50% of patients; shortness of breath; and rales, sometimes accompanied by pleural effusion. Relative bradycardia has been associated with Legionella pneu- monia and is often documented in elderly patients with advanced infection. The radiographic and non​specific laboratory findings in le- gionnaires’ disease overlap with typical and atypical pulmonary pathogens. Chest radiographic findings in legionnaires’ disease are non​specific. Although practically all radiological manifestations have been described, the most characteristic findings are rapidly progressive asymmetrical patchy infiltrates. The progression of in- filtrates on chest radiograph is common, despite appropriate anti- biotic therapy within the first week. Cavitation or abscess formation is rare and most commonly reported in immunosuppressed patients receiving corticosteroids. Pleural effusion is present in up to a third of patients, but empyema is rare. Like other atypical pulmonary infections, legionnaires’ disease is associated with extrapulmonary manifestations. Legionella most frequently presents with pneumonia and encephalopathy, mainly mental confusion. The hepatic manifestations of legionnaires’ dis- ease are mildly and transiently increased serum transaminase levels. Other extrapulmonary manifestations include loose stools or watery diarrhoea and microscopic haematuria. Characteristic laboratory findings are leucocytosis with relative lymphopenia, hyponatraemia, hypophosphatemia, increased serum ferritin, and raised creatine phosphokinase levels. Although some presenting clinical features suggest legionella pneumonia, it is difficult to express them in a reliable scoring system. Combining positive and negative signs, symptoms, and laboratory features is the basis of a syndromic diagnosis using a weighted point system. However, the scores available do not dif- ferentiate reliably between Legionella pneumonia and pneumo- coccal pneumonia. As with any pneumonia, the most important complication is re- spiratory failure. Up to 20% of cases might require mechanical ven- tilation. Acute kidney injury can also occur but is usually reversible. Rarely, pericarditis and myocarditis have been described, as has meningitis. Pontiac fever Pontiac fever is the benign, acute non​pneumonic form of legionella infection which presents as a self-​limited influenza-​like illness. The incubation period is from 1 to 3 days. There are no specific clinical findings or laboratory tests for its diagnosis and no agreed clinical case definition. Antimicrobial treatment is usually not needed, and symptoms usually subside within 4 to 5 days. Laboratory diagnosis Laboratory-​confirmed cases of legionellosis are based on culture of the organism from respiratory specimens, a fourfold rise in serum antibody level against L. pneumophila serogroup 1, or detection of L. pneumophila serogroup 1 antigen in urine (Table 8.6.39.3). Culture and isolation remain the gold standard for detection of legionella and diagnosis of legionnaires’ disease. Enriched and permissive agar such as BCYE is needed for Legionellae cul- ture. Legionellae can be isolated from a variety of sample types, although lower respiratory tract secretions are the samples of choice. The cultures usually take 3–​5 days to become positive. The major limitation of the sputum culture is that fewer than half of patients with legionnaires’ disease produce sputum. However, a positive isolate implies disease as colonization is thought not to occur. Isolation of Legionella allows microbiological identification and subtyping by DNA studies to determine the environmental source of infection. The Legionella urinary antigen test is a rapid, relatively low cost, uncomplicated procedure that detects antigens of L. pneu­ mophila serogroup 1 in urine and has become the mainstay of diagnosis at many centres. Legionella antigenuria can be de- tected as early as one day after onset of symptoms and usually disappears within two months. The rapidity of diagnosis is an important advantage of the urinary antigen test, since it means that cases can be detected early and therapeutic decisions taken promptly. The test’s major disadvantage is its inability to detect organisms other than L.  pneumophila serogroup 1 reliably; a negative urine antigen test does not necessarily exclude legion- ella infection. Serologic comparison of acute and convalescent serum specimens demonstrating a fourfold rise in titre (≥1:128) of the immunoglobulin G antibody is highly specific when the L. pneumophila serogroup 1 antigen is used but less specific for other Legionella antigens. In any case, the clinical utility of serologic diagnosis is limited because four to eight weeks are required to mount a full antibody response. Furthermore, underlying medical conditions or immunosuppres- sion may occasionally prevent or delay increases in titre. Serological assays face further challenges such as cross-​reactivity, which may complicate the interpretation of results. In practice, serology is not used for clinical diagnosis but might sometimes be useful as an epi- demiological tool: a single high titre is suggestive but not confirma- tory of infection. Direct fluorescent antibody staining can identify Legionella antigens in respiratory specimens and tissues and might be useful for bronchoalveolar lavage (BAL) specimens. This technique has the advantage of providing a result within two to four hours but it is technically demanding and must be performed by skilled laboratory staff. A positive direct fluorescent antibody result in the absence of other supporting evidence is currently not accepted as sufficient for diagnosis of Legionella infection. Table 8.6.39.3  Laboratory diagnosis Test Comments Culture Detects most species, including fastidious organisms, variable sensitivity, usually takes 3–​5 days to become positive Direct fluorescent assay Variable sensitivity, result within 2–​4 hours, technically demanding, not accepted as sufficient for diagnosis Urinary antigen test Low cost, uncomplicated procedure; easy to collect samples; only detects antigens of L. pneumophila serogroup 1, sensitivity nearly 70% Serology testing Around 4–​8 weeks are required for antibody response, cross-​reactivity, useful as an epidemiological tool Nucleic acid-​based detection Requires trained personal and sophisticated machines, detects all species, sensitivity nearly 90%

8.6.39  Legionellosis and legionnaires’ disease 1229 Nucleic acid-​based tests for Legionella detection, diagnosis, and typing are becoming more widespread. Both retrospective and prospective clinical and epidemiological studies have valid- ated detection of the mip gene particularly in a variety of speci- mens, including water from cooling towers, rivers, and hot tubs as well as sputum, serum, and urine. Legionella nucleic acid-​based detection offers significant advantages over serology and culture in terms of sensitivity, and is the only approach currently suitable for diagnosis of legionnaires’ disease due to non-​pneumophila Legionella species in a time frame that could positively influence patient management. In addition, these techniques establish a direct link between environmental and clinical isolates by means of subtyping. One inherent complication with all nucleic acid amp- lification methods is the difficulty in assessing bacterial viability. In addition, this technology still requires specially trained staff and sophisticated machines. Treatment Delay in the initiation of appropriate antibiotic therapy for Legionella pneumonia significantly increases mortality. Therefore, it is recom- mended that anti-​legionella agents be included early in the empiric therapy of severe community-​acquired pneumonia and in those who are immunocompromised. Although there have been no pro- spective randomized trials addressing the subject, most authorities have recommended therapy with fluoroquinolones or macrolides, mainly levofloxacin (500 mg/​ 24 hours) or azithromycin (500 mg/​ 24 hours). Doxycycline can also be considered. In this regard, studies of animal models of Legionella pneumophila have demon- strated the superiority of most fluoroquinolones and azithromycin over erythromycin. Similarly, observational prospective studies comparing levofloxacin versus older macrolides (erythromycin or clarithromycin) in the treatment of legionnaires’ disease have shown that the use of levofloxacin is associated with a shorter time to reach clinical stability, shorter hospital stay, and fewer complications. One of the key factors is the ability of the antibiotics to reach therapeutic concentrations within alveolar macrophages where the legionella bacterium multiplies. Combined therapy, adding rifampicin to macrolides or quin- olones, has been used in severe or life-​threatening legionella pneu- monia. Nevertheless, there is no convincing evidence of any added benefit. In addition, the use of combinations increases the risk of toxicity and drug interactions. Clinicians should be alert to the risk of prolongation of the QT interval on the electrocardiogram (ECG). The total duration of quinolone treatment should be 10–​14 days, al- though a 21-​day course has been recommended for immunosup- pressed patients. The recommended duration of treatment with azithromycin is 3–​5 days for mild cases, and 5–​10 days for immuno- suppressed patients. In severe cases or in the immunocompromised, therapy should continue until the fever subsides and clinical signs improve. Prognosis Because legionnaires’ disease occurs primarily in older adults, prognosis depends largely on the host comorbidities. Prognosis is also directly related to the early administration of effective anti-​ legionella therapy. Although legionnaires’ disease has an overall mortality rate of 5–​15%, it is lower when early diagnosis allows prompt treatment, but higher in immunosuppressed individuals (up to 70%) and in those requiring intensive care unit admission. Long-​term sequelae might occur, such as tiredness or poor concen- tration, but these are not well characterized. Prevention As person-​to-​person spread does not occur, the main aspect of le- gionella control involves good environmental health measures, such as water testing and environmental surveillance, coupled to ensuring safe water supplies. There are World Health Organization (WHO) guidelines for the latter. Prompt diagnosis of severe pneu- monias and investigation of clusters of cases can pinpoint sources of infection and appropriate interventions. Areas of uncertainly and future developments More research is currently needed to estimate the incidence of legionnaires’ disease and to quantify the new risk factors, the as- sociated morbidity and mortality (especially long-​term survival or sequelae) and the economic burden. Prospective random- ized controlled trials should be conducted to assess the merits of different classes of antibiotics and determine whether mono or combination therapy is preferable. Such trials could, poten- tially, be done in the setting of an outbreak. Further research is needed to improve the diagnostic accuracy of tests. The valid- ation of molecular assays might help to achieve the diagnostic accuracy and rapidity required to guide disease management, the study of environmental distributions of strains and their relation to infection, and the exploration of genotypic factors in disease causation. FURTHER READING Bartram J, et al. (2007). Legionella and the prevention of legionellosis. World Health Organization Press, Geneva. Cunha BA (2010). Legionnaires’ disease: clinical differentiation from typical and other atypical pneumonias. Infect Dis Clin N Am, 24, 73–​105. Cunha BA, Burillo A, Bouza E (2016). Legionnaires’ disease. Lancet, 387, 376–​85. Fernández-​Sabe N, et  al. (2003). Clinical diagnosis of Legionella pneumonia revisited:  evaluation of the Community-​Based Pneumonia Incidence Study Group scoring system. Clin Infect Dis, 37, 483–​9. Guyard C, Low DE (2011). Legionella infections and travel associated legionellosis. Travel Med Infect Dis, 9, 176–​86. Mercante JW, Winchell JM (2015). Current and emerging Legionella diagnostics for laboratory and outbreak investigations. Clin Microbiol Rev, 28, 95–​133. Newton HJ, et al. (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev, 23, 274–​98. Pedro-​Botet L, Yu VL. (2006). Legionella: macrolides or quinolones?. Clin Microbiol Infect, 12 Suppl 3, 25–​30. Phin N, et  al. (2014). Epidemiology and clinical management of Legionnaires’ disease. Lancet Infect Dis, 14, 1011–​21.

8.6.4 Staphylococci 991

8.6.4 Staphylococci 991

8.6.4  Staphylococci 991 Weisfelt M, et al. (2006). Pneumococcal meningitis in adults: new approaches to management and prevention. Lancet Neurol, 5, 332–​42. Werno AM, Murdoch DR (2008). Laboratory diagnosis of invasive pneumococcal disease. Clin Inf Dis, 46, 926–​32. White B (1938). The biology of pneumococcus. The Commonwealth Fund, New York, NY. (Second printing 1979, Harvard University Press, Cambridge, MA) World Health Organization (2013). Pocket book of hospital care for children:  guidelines for the management of common illnesses with limited resources, 2nd edition. World Health Organization,
Geneva. 8.6.4  Staphylococci Kyle J. Popovich, Robert A. Weinstein, and Bala Hota ESSENTIALS Staphylococci are Gram-​positive cocci that form clusters, but can occur singly, in pairs, chains, or tetrads. They are classically distin- guished from other Gram-​positive cocci by presence of catalase, an enzyme that degrades hydrogen peroxide (H2O2). S. aureus is dis- tinguished from other coagulase-​negative staphylococci, which are generally less virulent, by the presence of coagulase, an enzyme that coagulates plasma. Many toxins and regulatory elements enhance virulence in staphylococci. Epidemiology Colonization—​staphylococci are skin commensals. About 20% of adults are persistently colonized by S.  aureus, 60% are intermit- tently colonized, and 20% are never colonized. High-​risk groups for S. aureus colonization include infants, insulin-​dependent diabetics, intravenous drug users, HIV-​infected patients, and renal dialysis patients. Methicillin-​resistant S. aureus (MRSA)—​risk factors for MRSA colon- ization and infection among hospitalized patients include antibiotic exposure, surgery, nursing home residence, or high MRSA ‘colon- ization pressure’ (i.e. frequent exposure to colonized or infected patients). However, MRSA is no longer only a hospital-​related infec- tion, with community-​associated MRSA affecting individuals without healthcare exposures. Clinical features S.  aureus infection—​clinical syndromes can be divided into three groups: (1) Illness due to release of toxins, leading to disease at sites often remote from infection—​including (a) staphylococcal scalded skin syndrome—​release of epidermolytic toxins leads to bullae and desquamation; (b)  food-​borne illness due to preformed toxin—​a heat-​stable superantigen toxin produces sudden vomiting and diarrhoea; (c)  toxic shock syndrome—​superantigen toxins cause multisystem organ dysfunction; may be menstrual (e.g. tampon-​ associated) or non​menstrual. (2) Illness due to local tissue destruction and abscess formation—​including (a) impetigo, folliculitis, and cel- lulitis; (b)  furuncles and carbuncles; (c)  mastitis; (d)  pyomyositis; (e) septic bursitis; (f) septic arthritis; (g) osteomyelitis; (h) epidural ab- scess; (i) pneumonia; (j) urinary tract infection. (3) Hematogenous infection—​including bacteraemia and endocarditis. Coagulase-​negative staphylococci—​most infections with these skin commensals are the consequence of medical interventions leading to foreign bodies (e.g. prosthetic joints or heart valves), indwelling intravascular catheters or grafts, or peritoneal catheters. Conditions include endocarditis (5–​8% of native valve infections, c.40% of pros- thetic valve infections), intravascular catheter infections (6–​27% of vascular catheter infections), prosthetic joint infections (up to 38% of arthroplasty infections), peritoneal dialysis, catheter infections, and postoperative ocular infections. Diagnosis Diagnosis relies on characteristic clinical and epidemiological fea- tures, supported by positive cultures from the relevant clinical site, with identification (when appropriate) of exotoxin-​positive strains. Outbreak and epidemiological investigations use molecular fingerprinting techniques to assess relatedness of staphylococci. Treatment Aside from supportive care, the mainstays of therapy are (1) prompt drainage of infected foci; and (2)  antimicrobials—​(a) coagulase-​ negative staphylococci—​vancomycin is the mainstay of therapy because of the high rates of methicillin resistance; (b) S. aureus—​ antimicrobial choice should be based on the local prevalence of MRSA and the clinical severity of illness; a bactericidal agent, prefer- ably a β-​lactam, is used whenever possible; oral agents active against MRSA include clindamycin, trimethoprim/​sulfamethoxazole, doxy- cycline, minocycline, linezolid; glycopeptides (i.e. vancomycin or teicoplainin) have been the usual therapy of severe infections due to MRSA, but reduced susceptibility to vancomycin has been reported. Prevention Prevention of illness due to S. aureus, particularly MRSA, relies on proactive infection control measures, including (1) surveillance for MRSA colonization; (2)  imposed grouping (cohorting) of infected and colonized patients; (3)  barrier precautions (e.g. gowning and gloving by healthcare staff); (4) improved hand hygiene; (5) cleaning patients (e.g. with chlorhexidine); (6)  improved environmental cleaning; (7) antimicrobial stewardship. Better strategies for treatment and salvage of infected cath- eters or methods for treatment of biofilm may improve treatment of coagulase-​negative staphylococcal infections. No vaccines are available. Introduction and historical perspective Staphylococci are named for their microscopic appearance, the name coming from Greek words meaning ‘bunch of grapes’ and ‘berry’. First described in 1880 by Ogston as an important cause of abscesses in humans, staphylococci are among the most common causes of bacterial colonization and infection in the community and in hospitals.

section 8  Infectious diseases 992 Staphylococcus aureus, the pre-​eminent human staphylococcus, has adapted efficiently to improvements in therapeutics. In the 1940s, shortly after the introduction of penicillin, penicillin-​resistant S. aureus was noted in the United Kingdom and the United States of America, and by the end of the decade 50% of isolates were resistant. From 1940 to 1960, a particularly invasive clone of penicillin-​resistant S. aureus, ‘phage type 80/​81’, caused pandemic hospital infections. Following the introduction of methicillin, that strain faded from concern only to be replaced in subsequent dec- ades with endemic healthcare-​associated methicillin-​resistant S. au- reus (MRSA) that frequently was resistant to multiple antimicrobial classes. Most recently, reminiscent of the 1940 to 1960 experience, invasive strains of community-​associated MRSA (CA-​MRSA) have emerged rapidly in some communities among otherwise healthy in- dividuals. Coagulase-​negative staphylococci infections, in contrast, are infecting implanted devices and occurring in association with healthcare, thereby filling a niche created by medical success. Microbiology and molecular genetics Staphylococci stain purple (‘positive’) with Gram’s stain and form grape-​like clusters, but can occur singly, in pairs, in chains, or in tetrads. Of 32 staphylococcal species, 16 colonize or infect humans. Classically, staphylococci are distinguished from other Gram-​ positive cocci by the presence of catalase, an enzyme that degrades H2O2. S. aureus is distinguished from other staphylococci by the presence of coagulase, an enzyme that coagulates plasma. Most la- boratories use latex agglutination tests to detect coagulase; other as- says include the tube coagulase and free coagulase tests. Outbreak and epidemiological investigations use molecular ‘finger­ printing’ techniques to assess relatedness of staphylococci, that is, bacteriophage typing, pulsed-​field gel electrophoresis, multilocus sequence typing, or more recently, whole bacterial genome sequencing. In epidemiologic evaluations of MRSA, for example, limitations of methods such as pulsed-​field gel electrophoresis have been the in- ability to differentiate between endemic MRSA strains. However, more recently whole genome sequencing which is highly discrim- inatory has been used as an epidemiologic tool in healthcare settings to improve our understanding of transmission of MRSA strains from one individual to another. In a study of an MRSA outbreak in a spe- cial care baby unit in the United Kingdom, whole genome sequencing allowed investigators to identify a staff member with MRSA carriage who likely allowed the outbreak to continue despite implementation of infection control measures. Whole genome sequencing was also ap- plied in an adult intensive care unit to investigate acquisitions of S. au- reus strains among patients. In this study, results of whole genome sequencing suggested that only 18.9% of S.  aureus acquisitions could be explained by transmissions from other colonized patients. Validation of this work by integrating robust epidemiologic data with whole genome sequencing results is warranted. Future applications of this technology might be valuable for evaluating and directing infec- tion prevention strategies in outpatient and inpatient settings. Pathogenesis The infectiveness of staphylococci depends in part on bacterial fac- tors that promote growth, colonization, invasiveness (i.e. regulation and virulence determinants), and antibiotic resistance and in part on host susceptibility (e.g. presence of diabetes mellitus). Likely there is an important interplay of microbial, host, and epidemiologic factors that influence severity of illness for S. aureus infections. Regulation and virulence determinants Regulation determinants ‘autoregulate’ staphylococci based on en- vironmental conditions or host factors. The major S. aureus regu- latory gene is the accessory gene regulator (agr) that facilitates intercell communication. This and other systems might have roles in tissue destruction (through exoprotein production) and endocar- ditis (through adhesin regulation). Virulence determinants (e.g. peptidoglycan, lipoteichoic acids, protein toxins, and biofilm) enhance bacterial pathogenicity but can also activate patient protective mechanisms. Peptidoglycan, an important component of Gram-​positive bacterial walls, and lipoteichoic acids, bound to the plasma membrane, are implicated in triggering the inflammatory response in humans that can enhance bacterial killing. Exoproteins and ‘superantigens’ (i.e. antigens that lead to non​specific immune activation) can be released by S. aureus to cause a severe immune response or disease remote from infection, while local toxins, for example, Panton–​Valentine leucocidin, may increase bacterial invasiveness. Biofilm, an extracellular complex of polysaccharides, enhances binding to foreign objects (e.g. intravas- cular catheters) and serves as a bacterial sanctuary from host de- fences and antimicrobials. Genome sequencing has been used to identify bacterial toxins that might be associated with worse clinical outcomes among in- dividuals with S. aureus infections. After adjusting for host factors, colonization with MRSA strains that carried the staphylococcal en- terotoxin P (sep) gene is a risk factor for subsequent development of MRSA infection. This highlights how integrating host and virulence data can improve our understanding of the pathogenesis of S. aureus infections. Antimicrobial resistance S. aureus resistance to β-​lactams is mediated by β-​lactamases (peni- cillin resistance) or, more commonly, by altered enzymes respon- sible for cell wall formation (methicillin resistance). Penicillinases propagate by plasmids or phage transfer; methicillin resistance re- sults from spread of a genomic island of DNA called the staphylo- coccal chromosomal cassette (SCC). The SCC carries the mecA gene (termed SCCmec). The product of mecA is penicillin-​binding pro- tein 2a (PBP2a), which has low affinity for methicillin and enables cell wall synthesis in spite of active antibiotics. SCCmec type IV pri- marily is associated with CA-​MRSA, while types I, II, and III are associated primarily with hospital strains. Glycopeptides (i.e. vancomycin or teicoplanin) have been the usual therapy of severe infections due to MRSA. However, vancomycin re- sistance is emerging among MRSA. Two resistance patterns exist: (1) vancomycin-​ (or glycopeptide-​) intermediate S.  aureus (VISA or GISA) and (2) vancomycin-​resistant S. aureus (VRSA). The VISA phenotype has vancomycin minimum inhibitory concentrations of 4–​8 μg/​ml, and is thought to arise from thickening of the cell wall, changes in agr function, and changes in cell metabolism that arise from subinhibitory exposure to vancomycin. VRSA have higher min- imum inhibitory concentrations (≥16 μg/​ml) due to a gene (vanA) that has been passed from vancomycin-​resistant Enterococcus faecalis to S. aureus. Clinical isolates of VRSA (13 so far) have been reported

8.6.4  Staphylococci 993 in the United States of America. Although new agents (linezolid and daptomycin) exist for therapy of MRSA and could be used for VISA/​ VRSA, fledgling resistance has been reported. Outbreaks of linezolid-​resistant MRSA have been identified, with the cfr gene believed to mediate resistance. One outbreak was felt to be associated with high usage of linezolid; the outbreak was con- trolled with antibiotic stewardship and enhanced infection control measures. Resistance to antimicrobials in the macrolide–​lincosamide–​ streptogramin (MLS) group is not predictably concordant. Clindamycin resistance can be inducible, producing misleading sus- ceptibility phenotypes in automated testing that are erythromycin resistant and, seemingly but erroneously, clindamycin susceptible, or constitutive (readily detected resistance to erythromycin and clindamycin). The double-​disc diffusion test, or D test, will detect inducible clindamycin resistance. Clindamycin therapy is unreliable in organisms with either inducible or constitutive resistance. Among the coagulase-​negative staphylococci, 80% of isolates are resistant to methicillin due to the action of mecA. Laboratory testing of coagulase-​negative staphylococci is complicated by heterotypic expression of methicillin resistance, which can lead to deceptively low methicillin minimum inhibitory concentrations. Polymerase chain reaction (PCR) testing for mecA or slide agglutination testing for PBP2a will reveal resistance; methicillin or oxacillin will not ef- fectively treat such strains. Epidemiology: S. aureus Colonization Among staphylococci, as a general rule, colonization precedes infec- tion. S. aureus colonizes multiple sites but predominately the anterior nares. Among adults, 20% are persistently colonized by S. aureus, 60% are intermittently colonized, and 20% are never colonized. Methicillin-​susceptible S. aureus (MSSA) colonization prevalence rates are about 30% in the community; the prevalence of nasal col- onization with MRSA in the general community has increased from 0.8% in 2001–​2002 to 1.5% in 2003–​2004. Colonization outside the nares (e.g. throat, axilla, inguinal area, and peri-​rectal area) can also occur with some individuals being colonized at multiple body sites. Colonization outside of the nares can have important implications for infection control, decolonization, and infection prevention. High-​risk groups for S. aureus colonization include infants, insulin-​ dependent diabetics, intravenous drug users, HIV-​positive patients, and patients undergoing either haemodialysis or peritoneal dialysis. Host factors promoting colonization may be antibiotic treatment and polymorphisms in host genes. Health care-​associated MRSA Health care-​associated MRSA infection causes significant morbidity and mortality, and has been associated with 29% longer stays and 36% greater hospital charges for patients with MRSA compared to MSSA bacteraemia. Among hospitalized patients, risk factors for MRSA colonization and infection include antibiotic exposure, sur- gery, nursing home residence, or high MRSA ‘colonization pressure’ (i.e. frequent exposure to colonized or infected patients). There is a large ‘resistance iceberg’ for MRSA; the ratio of infected-​ to-​colonized patients might reach 1:3, which complicates control measures. The hands of healthcare workers probably represent a major vector for MRSA cross-​transmission. Another mechanism of staphylococcal transmission is bacterial shedding from nares of col- onized patients or staff, which can be enhanced by rhinitis. Spread via contaminated environmental surfaces might account for an add- itional 10–​15% of MRSA transmissions in healthcare settings. Community-​acquired MRSA (CA-​MRSA) MRSA are no longer exclusively nosocomial pathogens. They have been affecting people without exposure to healthcare. Although CA-​MRSA colonization rates have lagged behind those of MSSA, infection rates for those colonized with CA-​MRSA are up to 10 times higher than rates for those colonized with MSSA. Worldwide, CA-​MRSA infections have been mainly due to only a few pulsed-​field gel electrophoresis types (e.g. USA300 strain). In the United States, USA300 is the predominant CA-​MRSA strain and in some community settings is considered an endemic pathogen. CA-​ MRSA strains are now a common cause of skin and soft tissue infec- tions in ambulatory clinics and emergency rooms in the United States. In contrast to MRSA strains traditionally associated with hospitals, CA-​MRSA strains are often characterized by an increased suscep- tibility to non-​β-​lactam antibiotics. However, a multidrug resistant strain of USA300 has been reported in the United States so continued monitoring of the local antibiogram is essential to inform empiric therapeutic decisions. In addition, the epidemiology of CA-​MRSA has evolved with CA-​MRSA strains now accounting for a significant proportion of healthcare-​associated and nosocomial bacteraemias in several United States hospitals. Risk factors for infection or coloniza- tion with CA-​MRSA include African American race, HIV infection, drug use, tattooing, and situations and environments associated with increased person-​to-​person contact such as military service, jails, homosexual contacts, sports activity, and children’s day care. Potential reservoirs and sources for CA-​MRSA include ani- mals (e.g. pigs, cattle, horses, chickens, and companion animals), prompting the terminology ‘livestock-​associated MRSA’. One study found that a novel strain of MRSA in the Netherlands was associ- ated with pig or cattle farmers. Another MRSA strain with an altered mecA gene has been identified in Europe among humans and dairy cows. The extent of transmission occurring between humans and animals, and how this contributes to spread of MRSA in the com- munity among humans, is unclear. Secular trends and morbidity Overall trends in hospitalizations for S.  aureus infections sug- gest an increasing burden of illness. Trends fostering increases in- clude ageing of populations in western societies with increased comorbidities and use of prosthetic devices, such as joint replace- ments; the emergence of CA-​MRSA, which is occurring in add- ition to, not in place of, community-​associated MSSA; and use of broad-​spectrum antibiotics. In the United States of America, it has been estimated that about 9 of every 1000 hospitalizations might be due to S. aureus, and about 43% of S. aureus admissions are due to MRSA. Mortality rates among patients infected with S. aureus are 15–​34% in various studies. Clinical factors enhancing the likelihood of death include pneumonia, older age, diabetes, inadequate therapy, and failure to drain infected foci. With the spread of CA-​MRSA into hospitals, the epidemiology and control of nosocomial MRSA may change. Recent studies in the United States have documented a decline in hospital-​associated and healthcare-​associated invasive MRSA

section 8  Infectious diseases 994 infections over the past ten years. This decline is felt to reflect im- proved recognition of healthcare-​associated infections as well as the institution of various infection control programmes in intensive care units. In contrast, the incidence of invasive CA-​MRSA infections has remained relatively stable, suggesting that infection prevention strategies may need to be expanded to outpatient and community settings. In a population based matched cohort study in the United Kingdom from 1995-2015, investigators observed that a docu- mented penicillin allergy was associated with an increased risk of MRSA and Clostridium difficile infection, possibly due to in- creased exposure to alternative antibiotics (e.g., macrolides, fluoroquinolones, clindamycin). The proportion of these pa- tients who actually had true penicillin allergies is not known. Nevertheless, this study highlights the importance of moving to- ward a more thorough evaluation of individuals who self-report penicillin allergies and how this effort could improve antibiotic stewardship efforts. Prevention: S. aureus General interventions Prevention of illness due to S. aureus, particularly MRSA, relies on proactive infection control measures. These include surveillance for MRSA colonization to detect the resistance iceberg, barrier precau- tions (use of gowning and gloving) for care of infected and colonized patients, imposed grouping (cohorting) of infected and colonized patients, isolation wards, improved hand hygiene, antimicrobial stewardship, cleaning patients with chlorhexidine, improved envir- onmental cleaning, and use of intensive care unit ‘monitors’ to pro- mote adherence to infection control measures. MRSA Studies of MRSA control suggest that multiple simultaneous inter- ventions can reduce colonization and infection rates. Highly pro- moted among packages or bundles of interventions are hospital admission surveillance nasal cultures for MRSA colonization. These are recommended in high-​risk units or when other con- trol measures fail to reduce MRSA infection rates. The strongest support for decolonization comes from outbreak investigations, particularly in neonatal units, and from quasi experimental before-​ after trials. The relative roles of MRSA active surveillance, decolonization, and routine chlorhexidine bathing have been evaluated recently in more rigorous trials. A multicentre cluster-​randomized study of daily bathing with chlorhexidine-​impregnated washcloths in comparison to non-​antimicrobial washcloths observed signifi- cant decreases in acquisition of multidrug resistant organisms and overall hospital-​acquired bloodstream infections in the chlorhexidine bathing group. While the overall rate of MRSA acquisition was lower during the chlorhexidine study period, the decline did not reach statistical significance. A separate 43-​ hospital study examined three infection control strategies for preventing infections in the intensive care unit—​(1) active de- tection and isolation of MRSA carriers, (2) active detection and isolation with targeted decolonization of MRSA carriers (using intranasal mupirocin for 5 days and daily chlorhexidine bathing), and (3) no active detection and isolation but implementation of universal decolonization (intranasal mupirocin for 5  days and daily chlorhexidine). Universal decolonization was associated with the greatest reduction in rates of MRSA clinical isolates as well as the largest decline in bloodstream infection from any pathogen. A subsequent decision-​analysis model suggested that universal decolonization was more likely than the other two ap- proached to reduce infections and healthcare costs. Surveillance for development of resistance to decolonizing agents, especially mupirocin, would be important should this approach become widespread. Editorialists commenting on this article question the further need for active detection and isolation for controlling MRSA in an en- demic setting—​an approach currently used in many hospitals—​as well as advocate adopting more ‘horizontal’ infection control ap- proaches such as universal decolonization rather than ‘vertical’ or pathogen specific strategies. As another example of a horizontal infection control approach, a longitudinal study in Australia of the incidence of hospital-​onset S. aureus bacteraemias from 2002–​2013 observed a significant de- cline in the rate of both MRSA and MSSA during this time frame. The authors attribute this nationwide decline in the rate of S. aureus bacteraemia to the several infection prevention interventions that were instituted at the local and national level, and that the focus of efforts was on reducing all healthcare-​associated infections, not just those due to MRSA. CA-​MRSA Control of CA-​MRSA presents distinct challenges. The feasibility of contact precautions or isolation of infected persons in the com- munity might be limited. Additionally, the role of fomites in trans- mission of CA-​MRSA is unknown, and community environmental decontamination may be difficult. Current guidelines for people with CA-​MRSA infections and their community contacts include proper dressings for infected areas, hand hygiene, washing clothes contaminated with infected secretions, and avoiding contact sports while lesions exist. If infection is recurrent or spreading in specific settings, such as families, decolonizaton of carriers and potential family members may be warranted in conjunction with thorough environmental cleaning. Agents useful for decolonization Potential agents used for staphylococcal decolonization include topical agents (mupirocin, chlorhexidine, tea tree oil) or short courses of systemic antimicrobials. Mupirocin 2% is effective for decolonization but recolonization can occur and resistance can develop.. Tea tree oil, from the Ti (or Tea) tree (Melaleuca alterni- folia, Myrtaceae), has been effective for some colonized patients. Chlorhexidine gluconate has potent antibacterial effects for de- colonizing skin or as a nasal gel. Failure to control spread of spe- cific clones of MRSA due to efflux of chlorhexidine from resistant bacteria has been reported, but, resistant strains have been very rare in systematic studies. Some favour combining agents (e.g. mupirocin with chlorhexidine bathing with an oral MRSA anti- biotic) to target both nasal and extranasal colonization and prevent recurrent MRSA infections. Baths containing dilute bleach solu- tions have been advocated by paediatricians for interrupting cycles of MRSA skin infection in infants, and assiduous application of

8.6.4  Staphylococci 995 approved detergents/​disinfectants or bleach can decontaminate the environment. Iodophors may be another option for the nasal treatment component of MRSA decolonization regimens; clinical trials of this alternative are currently under way. Clinical features: S. aureus Risk factors for infection Groups commonly at risk of colonization and infection include AIDS patients, intravenous drug users, and patients with diabetes mellitus. Multiple risk factors for S. aureus infection often co- exist. For example, haemodialysis and peritoneal dialysis patients are at increased colonization risk and have high-​risk foreign bodies. Conditions that predispose specifically to tissue invasion include skin trauma, haematomas, burns, or chronic diseases (e.g. dermatitis or psoriasis); surgical wounds; indwelling vas- cular catheters; and postviral sequelae such as influenza-​related mucosal damage. Rarer conditions associated with increased risks of staphylococcal infection include Chédiak–​Higashi syn- drome and Job’s syndrome. Clinical syndromes S.  aureus infection syndromes can be divided into three groups: (1) illness due to release of toxins, leading to disease at sites often remote from infection; (2)  illness due to local tissue destruction and abscess formation; and (3) haematogenous infection. Therapy for these syndromes is based on the use of active drugs at appro- priate dosages with appropriate concern for common side effects and toxicities. Toxin-​related syndromes Staphylococcal scalded skin syndrome In 1878, staphylococcal scalded skin syndrome, or Ritter’s dis- ease, was described in 297 children by the German physician Ritter von Rittershain. After release of epidermolytic toxins by S. aureus, patients develop bullae and desquamation. Though clin- ically impressive (Fig.  8.6.4.1a), this superficial desquamation can be distinguished clinically and histologically from deeper ex- foliative illnesses such as toxic epidermal necrolysis. In staphylo- coccal scalded skin syndrome, skin separation occurs within the epidermis, at the stratum granulosum, while in toxic epidermal necrolysis, separation occurs deeper, at the dermal–​epidermal junction, leading to more severe skin loss. The absence of mucosal disease in staphylococcal scalded skin syndrome also distinguishes these syndromes. Staphylococcal scalded skin syndrome occurs more commonly in children (Fig. 8.6.4.1b). Disease may be generalized or local- ized (i.e. bullous impetigo), and the burden of S. aureus may be low. Nasal or mucosal colonization may cause disease. When cases occur in epidemics, such as in neonatal units, patients and healthcare workers should be screened for carriage. Diagnosis re- lies on the characteristic clinical and epidemiological features and is supported by identification of exotoxin-​positive strains colon- izing or infecting clinical sites. Treatment involves topical or sys- temic antibiotics for infected sites and supportive care for areas of skin/​soft tissue destruction. Food-​borne illness due to preformed toxin S. aureus can produce a heat-​stable superantigen toxin that can per- sist even after cooking has eradicated the organism. Ingestion of toxin in contaminated, often unrefrigerated, food can result in epi- demic gastrointestinal disease. There is a short incubation of only 2–​6 h, followed by sudden vomiting (82%), diarrhoea (68%), and occasionally fever (16%). The differential diagnosis includes other short-​incubation toxin-​mediated gastrointestinal pathogens such as Bacillus cereus and toxins (Chapter 8.6.7). Treatment involves sup- portive care, particularly rehydration. The illness is typically self-​ limited, lasting less than 12 h. Toxic shock syndrome Staphylococcal toxic shock syndrome is caused by systemic superantigen toxins released by S. aureus, resulting in multisystem organ dysfunction. Staphylococcal toxic shock is clinically similar to streptococcal toxic shock (high fever, mental confusion, erythroderma, diarrhoea, hypotension, and renal failure), but streptococcal toxic shock is typically associated with invasive infec- tion such as necrotizing fasciitis while staphylococcal toxic shock (a) (b) Fig. 8.6.4.1  Staphylococcal scalded skin syndrome: (a) in an adult;
(b) in a child. (a) copyright Professor S. J. Eykyn; (b) copyright Professor W. C. Noble.

section 8  Infectious diseases 996 can be precipitated by clinically minor infections that are over- shadowed by the systemic effects of the toxin. Staphylococcal toxic shock occurs in two major forms, menstrual (e.g. tampon-​associated) and non​menstrual. In women with vaginal colonization by S. aureus, it is presumably the favourable micro- environment during menses that leads to increased production of toxin (TSST-​1). Management of staphylococcal toxic shock relies on systemic antimicrobial therapy (Table 8.6.4.1), supportive care, and prompt drainage of infected/​colonized foci. Common adjunctive ther- apies such as intravenous immunoglobulin to bind free toxin and antibacterials (especially clindamycin and potentially linezolid) with activity at the ribosome, which decreases bacterial protein (toxin) synthesis, have a theoretical rationale and some support from animal models; however, clinical data are limited. Illness due to local tissue invasion/​destruction S. aureus and β-​haemolytic streptococci cause approximately 80% of soft tissue infections. S. aureus is the aetiological agent of 37–​ 65% of native monoarticular joint infections in healthy adults and of 75% of joint infections in rheumatoid arthritis. Osteomyelitis, either of haematogenous or contiguous origin, is caused by S. au- reus or coagulase-​negative staphylococci in more than 50% of cases. Any local infection can lead to secondary bacteraemia and haema- togenous seeding of distant sites. Impetigo, folliculitis, and cellulitis The most superficial S. aureus infections are impetigo, folliculitis, and cellulitis. Impetigo is limited to the epidermis, folliculitis to the hair follicles, and cellulitis to the dermis and/​or the subcutaneous fat. Impetigo can appear as small round honey-​crusted lesions on the skin, primarily on exposed areas (Fig. 8.6.4.2). Impetigo typic- ally is caused by streptococci; in the United Kingdom, S. aureus is an infrequent cause. However, bullous impetigo is a clinical variant (caused by S. aureus phage type 71), reported in up to 10% of impe- tigo cases. Initially, the lesions can be vesicles that enlarge into bullae containing clear or yellow fluid. Cellulitis is typically due to streptococci, but when associated with penetrating trauma, furuncles, or carbuncles S. aureus should be considered. Diagnosis depends on the clinical appearance and the presence of purulence that can be cultured. However, aspirates of cellulitic areas are positive in less than one-​third of cases and bac- teraemia is rare. Treatment of impetigo (Table 8.6.4.2) should reflect local anti- biotic resistance patterns. Topical therapy (e.g. mupirocin or retapamulin) might be effective for limited disease, though EMRSA-​ 16, one of two predominant MRSA types in the United Kingdom, often shows high-​level mupirocin resistance. In settings of high topical fusidic acid use, resistance in S. aureus isolates has been re- ported. Systemic therapy should be used in patients with impetigo who have many lesions or who fail topical therapy. In areas where CA-​MRSA prevalence exceeds 10%, initial therapy should be dir- ected by local susceptibility patterns. Suspicion of more invasive infection, such as necrotizing fas- ciitis, should be high in cases of soft tissue infections with dis- proportionate pain, bullae, haemorrhagic or necrotic lesions, cutaneous anaesthesia, rapid progression of lesions, gas in the tissues, presence of risk factors, and when laboratory tests show elevated creatine kinase, acidosis, leucocytosis, or C-​reactive pro- tein exceeding 13 mg/​litre. Necrotizing infections should prompt inpatient antibiotic therapy assuming MRSA and urgent surgical consultation. Skin abscess furuncles and carbuncles Skin abscess is an infection within the dermis and deeper skin tis- sues. Furuncles and carbuncles are deep suppurative infections that Table 8.6.4.1  Therapy of toxic shock due to S. aureus Drug Dosage Duration/​comment For penicillin-​susceptible S. aureus: Duration based on focus of infection Adequate drainage is critical Data to support adjunctive use of immunoglobulin and/​ or clindamycin are needed Penicillina 2–​4 MU IV every 4 h Ampicillin 1–​2 g IV every 4–​6 h Ampicillin + sulbactam 1.5–​3 g IV every 6 h For methicillin-​susceptible S. aureus: Oxacillin/​flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Clindamycinb 600 mg IV every 8 h Daptomycin 6 mg/​kg IV every 24 h Teicoplanin At least 400 mg IV BID Linezolidb 600 mg IV every 12 h Quinupristin/​dalfopristin 7.5 mg/​kg every 12 h Intravenous immunoglobulin Dosage not standardized BID, twice daily; IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. b These agents may be useful for reduction of protein synthesis and toxin production, but require further study. Fig. 8.6.4.2  Staphylococcal impetigo. Copyright Dr Renwick Vickers.

8.6.4  Staphylococci 997 occur in the dermis and originate at hair follicles. Infection can be limited to small lesions that appear as painful nodules, sometimes with necrotic centres (Fig. 8.6.4.3a). Confluence of furuncles leads to the formation of carbuncles (Fig. 8.6.4.3b). Several members of a family may be affected. Mild lesions cause limited systemic com- plaints, whereas fever, malaise, or symptoms and signs of sepsis can occur with extensive disease. Skin abscesses and furunculosis are caused increasingly by CA-​ MRSA. MRSA is now the most common pathogen identified in patients to presenting to emergency rooms with skin infections in the United States. Furthermore, most of these purulent skin infec- tions are due to the epidemic strain of CA-​MRSA, USA300. The Panton–​Valentine leucocidin toxin, which is associated with most USA300 strains, has been postulated to contribute to the increased virulence observed in some of these cases. However, others have suggested other virulence factors (e.g. α-toxin, ACME) are involved and the Panton–​Valentine leucocidin toxin is merely an epidemio- logic marker of the CA-​MRSA strain. CA-​MRSA has been associ- ated with more fulminant infections, including necrotizing fasciitis, pneumonia, a sepsis-​like picture, or even Waterhouse–​Friderichsen syndrome occur. Drainage, spontaneously or surgically, is the mainstay of therapy. Early furuncles may be treated by application of moist heat to stimu- late drainage. While it was origianlly felt that for small abscesses in immunocompetent hosts, incision and drainage alone might be sufficient, more recent literature has suggested added benefit from post-drainage antibiotics. Lesions on the face, lesions with cellulitis (especially exceeding 5 cm in diameter), or the presence of systemic symptoms and/​or signs (fever, chills, or haemodynamic changes) should lead to use of antistaphylococcal antibiotics (Table 8.6.4.3) in addition to drainage. Oral agents are sufficient in most cases, but in severe infections or for bacteraemia parenteral agents should be used. One designation that has been suggested to guide empiric therapy in the outpatient setting is whether cellulitis is purulent or non​purulent. Purulent cellulitis is purulent drainage associated with cellulitis; this clinical presentation is less consistent with infection solely due to β-​haemolytic streptococci and instead should prompt empiric coverage for CA-​MRSA. In contrast, non​purulent cellulitis (i.e. no associated purulent drainage or abscess) should prompt em- piric therapy for β-​haemolytic streptococci with CA-​MRSA potentially being less of a concern if the patient is not systemically ill. However, in cases of non​purulent ceullitis where the patient does not respond to β-​lactam antibiotics or is systemically ill, empiric CA-​MRSA coverage can be added. A recent multicentre randomized controlled trial in the United States compared trimethoprim-​sulfamethoxazole versus clindamycin for the treatment of uncomplicated skin infections (cellulitis, abscess, or both) among children and adults. This study found no significant difference between treatment arms for uncom- plicated skin infections; further research is needed on the optimal therapy for individuals with underlying chronic illness or who are more severely ill. Systemic antibiotics given for skin and skin struc- ture infections might also reduce S. aureus colonization, reducing the risk of recurrent skin infections. However, some studies noted an increased risk of adverse effects with clindamycin in comparison to trimethoprim-sulfamethoxazole when used in this way. Table 8.6.4.2  Therapy of impetigo and mild soft tissue lesions caused by S. aureus Therapy Drug Dosage Duration Topical Mupirocin 2% ointment BID 5 days Retapamulin 1% ointment BID Fusidic acid 2% cream TID Oral For methicillin-​susceptible S. aureus: Dicloxacillin or Flucloxacillina 250 mg PO QID 7 days Cefalexin 500 mg PO QID For methicillin-​resistant S. aureus (or β-​lactam allergy): Clindamycin (Erys, Clins,
or D-​test negative) 300–​450 mg
PO QID 7 days Trimethoprim/​ sulfamethoxazole 1–​2 double-​strengthb tablets PO BID Doxycycline 100 mg PO BID Minocycline 100 mg PO BID Linezolid 600 mg po BID BID, twice daily; Clins, clindamycin-​sensitive; D, double-​disc diffusion; Erys, erythromycin-​sensitive; PO, by mouth; QID, four times daily; TID, three times daily. a First-​line agents. b 160 mg trimethoprim and 800 mg sulfamethoxazole in a double-​strength tablet. Pustule Cellulitis (a) (b) Fig. 8.6.4.3  (a) Pustule/​early furuncle with surrounding cellulitis due to S. aureus. (b) Coalescent furuncles (i.e. carbuncle, that required incision and drainage).

section 8  Infectious diseases 998 Mastitis Mastitis is most commonly caused by S. aureus, occurs in 1–​3% of nursing mothers typically within 3 weeks of birth, and may lead to breast abscesses. Infection can appear as a painful nodule or a draining abscess. Therapy (Table 8.6.4.3) should include topical moist heat, oral antimicrobials with efficacy against S. aureus (and MRSA in endemic areas), and abscess incision and drainage. Pyomyositis Pyomyositis, or primary bacterial abscess of skeletal muscle, is most common in the tropics where ‘tropical pyomyositis’ can account for 1–​4% of hospital admissions (Chapter 24.24.6). In non​tropical areas the syndrome is uncommon. S. aureus is the cause in about 95% of tropical cases and about 70% of other cases. Associations are with muscle trauma (20–​50% of cases), HIV infection, and possibly Toxocara canis infection. Symptoms develop subacutely over 2–​3 weeks with variable de- grees of fever, muscle pain, swelling, and induration. Large lower extremity and trunk muscles are most commonly affected. Regional lymphadenopathy is typically absent. Diagnosis relies on clinical sus- picion, helpful radiographic findings (i.e. gas or soft tissue swelling on plain radiographs, abscess or muscle enlargement on ultrasound examination, inflammation, oedema, or focal abscess in muscles Table 8.6.4.3  Therapy of cellulitis, abscess, mastitis, furunculosis, and pyomyositis caused by S. aureus Therapy Drug Dosage Duration/​comment Oral For methicillin-​susceptible S. aureus: Flucloxacillin or dicloxacillina 500 mg PO QID 5 days for cellulitis For deeper infection duration depends on proper drainage when necessary and clinical response With incision and drainage, lesions with <5 cm of cellulitis in immunocompetent patients may be cured without systemic antibiotics For deeper infection duration depends on proper drainage when necessary and clinical response Early change to oral therapy may be employed in stabilizing, non​bacteraemic patients May have a future role Cefalexin 500 mg PO QID For methicillin-​resistant S. aureus (or β-​lactam allergy): Clindamycin (Erys, Clins, or D-​test negative) 300–​450 mg PO QID Trimethoprim/​sulfamethoxazole 1–​2 double-​strengthb
tablets PO BID Doxycycline 100 mg PO BID Minocycline 100 mg PO BID Linezolid 600 mg PO BID Tedizolid 200 mg po qday Erythromycinc 250 mg PO every 6 h or 500 mg PO every 12 h Parenteral For methicillin-​susceptible S. aureus: Oxacillin/​flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg IV every 12 h Erythromycinc 250 mg IV every 6 h or 500 mg IV every 12 h Clindamycin (Erys, Clins, or D-​test negative) 600 mg IV every 8 h Linezolid 600 mg IV every 12 h Tedizolid 200 mg IV every 24 h Daptomycin 4 mg/​kg IV every 24 h Quinupristin/​dalfopristin 7.5 mg/​kg every 12 h Tigecycline 100 mg initially, then 50 mg IV every 12 h Ceftaroline 600 mg IV q12 h Telavancin 10 mg/​kg IV every 24 h Dalbavancin 1000 mg IV followed by 500 mg IV one week later Oritavancin 1200 mg IV as a single dose Note: Dosing recommendations assume normal renal and hepatic function. Trimethoprim-​sulfamethoxazole and tetracyclines are felt to have poor β-​haemolytic streptococci coverage; if coverage for β-​haemolytic streptococci is needed, one should consider adding a β-​lactam to doxycycline or trimethoprim-​sulfamethoxazole therapy. a First-​line agent. b 160 mg trimethoprim and 800 mg sulfamethoxazole in a double-​strength tablet. c In many areas high rates of resistance should prevent empiric use of erythromycin.

8.6.4  Staphylococci 999 on MRI or computed tomography (CT)), and the results of aspir- ating the lesion. Antibacterial therapy for S. aureus (Table 8.6.4.3) and open or radiographically assisted percutaneous drainage of abscesses are essential parts of therapy. Septic bursitis Infection can occur in any of the approximately 160 bursae found in humans, but septic bursitis usually affects prepatellar or olecranon bursae, usually as a result of trauma. It is due to S. aureus in more than 80% of cases but is accompanied by bacteraemia in 8% or less. Diagnosis relies on clinical recognition of the characteristic findings of fever and pain, swelling, redness, and warmth in the area of an affected bursa. Leucocytes and S. aureus are found if there is enough bursal fluid to aspirate. Treatment of septic bursitis includes appropriate antimicrobials (Table 8.6.4.4) and, if possible, drainage. Treatment failures have been described when erythromycin is used as the sole agent. Localized infection with no systemic signs may be treated with oral therapy, since high antimicrobials levels are achieved in bursal fluid. Adequate drainage is important. Patients with systemic signs or symptoms or who are immunocompromised should receive paren- teral therapy. Patients who present within 7 days of developing symptoms might be treated successfully with antibiotics and aspiration every 1 to 3 days. In this situation, bursal fluid might become sterile within 4 days and therapy should be continued for an additional 5 days. Surgical intervention is needed only for patients whose fluid remains infected or cannot be aspirated because the bursa is deep, who have foreign or necrotic material in the bursal space, or who need explor- ation or removal of the bursa because of recurrences. Septic arthritis S. aureus is the most common cause of non​prosthetic monoarticular septic arthritis. The typical pathogenesis is haematogenous seeding, but traumatic direct inoculation can occur. Important differential diagnoses include gonococcal infection in adolescents and adults and urosepsis pathogens and crystal-​induced arthropathies in older patients. Because joint destruction is rapid, prompt diagnosis through joint aspiration is essential. The mainstays of therapy are antimicrobials (Table 8.6.4.4) and prompt joint drainage by serial aspiration; arthroscopy (preferred for knee, shoulder, and ankle) with irrigation, lysis of adhesions, and removal of purulent material; or open drainage (useful for hip or shoulder infections to protect blood supply to femoral or humeral heads, and in instances where repeated aspirates or arthroscopy fail). S. aureus can be a cause of infected prosthetic joints, which can have a more indolent atypical presentation. Osteomyelitis S.  aureus osteomyelitis results from bacteraemia or contiguous spread from a soft tissue focus or chronic ulcer. Risk groups are pa- tients with diabetes mellitus, those with vascular disease or at risk Table 8.6.4.4  Therapy of septic bursitis and septic arthritis caused by S. aureus Therapy Drug Dosage Duration/​comment Oral For methicillin-​susceptible S. aureus: Flucloxacillin or dicloxacillina 500 mg PO QID For septic bursitis, continue therapy for 5 days after aspirates become sterile (with early change to oral therapy in non-​ bacteraemic patients). For septic arthritis, therapy should be continued for 4 weeks Cefalexin 500 mg PO QID For methicillin-​resistant S. aureus (or β-​lactam allergy): Clindamycin (Erys, Clins, or D-​test negative) 300–​450 mg PO QID Trimethoprim/​sulfamethoxazole 1–​2 double-​strengthb tablets PO BID Doxycycline 100 mg PO BID Minocycline 100 mg PO BID Ciprofloxacin or levofloxacin 500 mg PO BID or 500 mg PO once daily With Rifampin 300 mg PO every 12 h Linezolid 600 mg PO BID Erythromycinc 250 mg PO every 6 h or 500 mg PO every 12 h Parenteral For methicillin-​susceptible S. aureus: Oxacillin/​flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Linezolid 600 mg IV every 12 h BID, twice daily; Clins, clindamycin-​sensitive; D, double-​disc diffusion; Erys, erythromycin-​sensitive; PO, by mouth; QID, four times daily; TID three times daily; IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. b 160 mg trimethoprim and 800 mg sulfamethoxazole in a double-​strength tablet. c In many areas high rates of resistance should prevent empiric use of erythromycin.

section 8  Infectious diseases 1000 for haematogenous infection (i.e. haemodialysis), children, and eld- erly people. Diagnosis usually depends on radiographic studies. Plain radiographs may show evidence of periosteal reaction. However, the most sensitive test for osteomyelitis is MRI, which will dem- onstrate changes within bone and bone marrow. The most specific test is CT, which will reveal the presence of periosteal reaction or other bony changes not evident on plain radiographs. ‘Probing to bone’ in the case of a chronic ulcer is highly sensitive for a diag- nosis of osteomyelitis. The microbiological diagnosis of osteo- myelitis relies on positive blood or bone cultures; superficial wound or sinus track culture results are not reliable and might be misleading. Therapy for osteomyelitis includes drainage of pus (acute osteo- myelitis) or debridement of areas of avascular or ‘dead’ bone (se- questra in chronic osteomyelitis) and antibacterials with activity against the culture-​proven pathogen(s). The duration of therapy sufficient to eradicate the organism and prevent relapse is based on common experience and usually is 4–​6 weeks. Children with acute haematogenous S. aureus osteomyelitis can be treated with surgical drainage of purulent collections and short-​course intra- venous therapy (e.g. 1 week) followed by oral therapy for 4–​6 weeks as outpatients. Initial choice for therapy is based on the presence of MSSA or MRSA (Table 8.6.4.5); copathogens may require broader therapy. An open-​label study showed that for diabetic foot infec- tions, linezolid performed as well as ampicillin–​sulbactam for in- fected ulcers or osteomyelitis. A recent multicentre study in France examined the efficacy of a 6-​week course of antibiotics in comparison to 12 weeks of therapy for the management of pyogenic vertebral osteomyelitis. The au- thors observed that 6 weeks of therapy was non​inferior to the longer course of therapy. However, older age and infection with S. aureus were both significant risk factors for treatment failure, independent of duration of therapy. The optimal duration of therapy for MRSA osteomyelitis is unclear although some suggest that longer courses of at least 8 weeks be used. Epidural abscess Epidural abscesses occur adjacent to vertebral osteomyelitis and are medical/​surgical emergencies (Fig. 8.6.4.4). Enlarging epidural sites can compress the spinal cord or reduce vascular supply through thrombophlebitis. About 50% of cases follow haematogenous spread from known or occult trauma or from parenteral use of illicit drugs, while about 30% result from contiguous spread. S. aureus accounts Table 8.6.4.5  Therapy of osteomyelitis caused by S. aureus Therapy Drug Dosage Duration Parenteral For methicillin-​susceptible S. aureus: 4–​6 weeks IV Oxacillin/​ flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy):b Vancomycina 15 mg/​kg IV every 12 h Linezolid 600 mg IV every 12 h IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. b In 2011, the Infectious Disease Society of American came out with first guidelines for treatment of MRSA. Although the optimal duration of treatment of osteomyelitis due to MRSA has not been established, the guidelines suggest at least 8 weeks of therapy. Vertebral osteomyelitis and discitis Epidural abscess and cord compression Fig. 8.6.4.4  Epidural abscess and vertebral osteomyelitis due to S. aureus.

8.6.4  Staphylococci 1001 for more than 60% of cases. Risks for MRSA infection include recent healthcare exposure or rising CA-​MRSA rates. Symptoms and physical findings progress at variable rates, some- times rapidly, through four stages:  (1) back pain at the infected level, (2) pain radiating in the distribution of affected nerve roots, (3) motor weakness (including bladder and bowel dysfunction) and sensory deficit at the appropriate level, and (4) paralysis. The triad of back pain, fever, and neurological findings is highly suggestive of epidural abscess. MRI or CT scanning is most useful for evaluating epidural abscesses (Fig. 8.6.4.4). For diagnosis and therapy, a space-​ occupying lesion in the epidural space requires surgical evalu- ation and emergency laminectomy/​decompression or drainage by interventional radiography. Preoperative neurological status predicts outcome. Broad empirical antimicrobial therapy should include coverage for MRSA (Table 8.6.4.6) and Gram-​negative bacilli. If MSSA infection is diagnosed, β-​lactams are preferred over glycopeptides. Pneumonia S. aureus pneumonia can result from haematogenous spread or direct inoculation following mucosal damage. S. aureus causes less than 10% of cases of community-​acquired pneumonia but causes approximately 20–​30% of cases of nosocomial pneu- monia. Case fatality of S. aureus pneumonia ranges from 8% to more than 30%. Risks for a more severe course include MRSA, acute respiratory distress syndrome, comorbidities, and renal dysfunction. S. aureus is a cause of postviral, particularly postinfluenza, pneu- monia. Patients may report a biphasic illness. CA-​MRSA can cause a necrotizing pneumonia with more severe course. Additionally, S. aureus pneumonia might be associated with complications such as empyema, lung abscesses, and bronchopleural fistulae. Lung ab- scess must be differentiated radiographically from pneumatocele, a common and relatively benign complication of staphylococcal pneumonia. Diagnostic studies for patients with pneumonia in the pres- ence of staphylococcal bacteraemia or embolic-​appearing le- sions on chest imaging (Fig. 8.6.4.5) should seek an intravascular source (e.g. endocarditis or infectious thrombophlebitis). Therapy (Table 8.6.4.7) should include use of an active drug for at least 8 days in less complicated cases or longer if pulmonary involve- ment is secondary to an intravascular infection, presence of MRSA, or complications such as emboli or empyema. Surgical drainage is indicated for empyema. Daptomycin should be avoided because of its poorer activity in pulmonary infections. Linezolid might emerge as a drug of choice for MRSA pneumonia based on its greater penetration due to smaller molecule size and putative clinical benefit. A recent randomized controlled multicentre study examining vancomycin in comparison with linezolid for the treatment of hospital-​acquired or healthcare-​associated MRSA pneumonia found that the clinical response at the end of the study in the per-​protocol patients was significantly better with linezolid than vancomycin. However, 60-​day mortality was similar between the two arms. Furthermore, the rate of clinical success at the end of study was only 57.6% in the linezolid treated patients and 46.6% in the vancomycin-​treated patients, underscoring the potential severity of MRSA pneumonia and the need for further research to improve therapy in this area. A recent meta-analysis of 22 studies evaluated the utility of MRSA nasal screening results in predicting MRSA pneumonia. Screening had a high specificity Table 8.6.4.6  Therapy of epidural abscess caused by S. aureus Therapy Drug Dosage Duration Parenteral For methicillin-​susceptible S. aureus: ≥6 weeks IV Oxacillin/​flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Linezolid 600 mg IV every 12 h Daptomycin 6 mg/​kg IV every 24 h IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. a b c Fig. 8.6.4.5  Pneumonia due to S. aureus, from septic pulmonary emboli. Note presence of (a) empyema, (b) nodular (including pleural-​based) infiltrate, and (c) early cavitation of abscess. Table 8.6.4.7  Therapy of pneumonia due to S. aureus Drug Dosage Duration/​comment For methicillin-​susceptible S. aureus: 7–​14 days for uncomplicated infection Requires longer courses if empyema, lung abscess, or bacteraemia present Oxacillin/​flucloxacillina 1–​2 g IV every 4 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Linezolid 600 mg IV every 12 h IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent.

section 8  Infectious diseases 1002 (90.3%) and high negative predictive value (96.5%) for excluding MRSA pneumonia. Urinary tract infections S. aureus urinary tract infections (UTIs) result from ascending in- fection in catheterized patients or haematogenous seeding, which might lead to renal carbuncles (abscesses). Staphylococcal UTIs should prompt consideration of sources of bacteraemia such as endovascular infection. Clinically, patients with renal abscesses have fever and flank pain, but urinary complaints may be ab- sent, and urinalyses and urine cultures might be negative. Renal ultrasonography or CT can show a range of findings from ‘lobar nephronia’ (renal phlegmon) to large multilocular abscesses. Treatment might require percutaneous or open drainage; anti- microbial therapy (Table 8.6.4.8) should reflect results of cultures. Haematogenous infections Bacteraemia S. aureus is among the most common causes of bacteraemia in hos- pitals and the community. It causes 18–​27% of endocarditis cases (Fig. 8.6.4.6), is responsible for 13% of nosocomial bloodstream infections, and causes up to 78% of cases of intravascular catheter-​ related thrombophlebitis. Rates of community-​associated S. aureus bacteraemia in the United States of America are estimated at 17/​ 100 000 people, similar to rates of invasive Streptococcus pneumo- niae infection, with mortality of 10–​20%, depending on underlying illnesses. In Oxfordshire, England, the incidence of nosocomial MRSA bacteraemia increased from 50/​100 000 admissions in 1997 to 300/​100 000 admissions in 2004, increasing the overall burden of S. aureus disease. S. aureus in blood should always be considered a true pathogen. Bacteraemia has traditionally been categorized as ‘healthcare-​ associated’ (i.e. onset more than 2 days after admission) and ‘community-​associated’ (i.e. onset within 2 days of admission). Bacteraemia presenting within 2 days of hospitalization in indi- viduals with prior healthcare exposures (e.g. haemodialysis, re- cent prior hospitalization or surgery, or residence in a long-​term care facility) has been categorized as ‘healthcare-​associated, community-​onset’. Complications of bacteraemia include endo- carditis (itself a major cause of bacteraemia) and ‘metastatic’ seeding of distant sites, especially joints, bone, kidney, and skin (Fig. 8.6.4.7). An estimated 13% of nosocomial bacteraemias with S. aureus include endocarditis. Table 8.6.4.8  Therapy of urinary tract infection due to S. aureus Drug Dosage Duration/​comment For methicillin-​susceptible S. aureus: 7 days for ascending infection ≥ 14 days for renal abscess, bacteraemia, or complicated infection (duration is based on resolution of infected foci and/​or use of drainage) Oxacillin/​flucloxacillina 1–​2 g IV every 4 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus
(or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Linezolid 600 mg IV every 12 h IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. (a) (b) Fig. 8.6.4.6  S. aureus bacteraemia and infective endocarditis. (a) Meningococcal-​like rash in a patient with S. aureus endocarditis of a bicuspid aortic valve and aortic root abscess. (b) Splenic abscess complicating S. aureus endocarditis. Copyright Professor S. J. Eykyn. Fig. 8.6.4.7  Seeding of MRSA to the skin in a Vietnamese patient. Copyright D. A. Warrell.

8.6.4  Staphylococci 1003 If MSSA is isolated from cultures, a penicillinase-​resistant penicillin (flucloxacillin or nafcillin) should be used instead of vancomycin as these agents have better activity against MSSA. A  cephalosporin, cefazolin, has also been widely used to treat MSSA infections because of its convenient dosing. There are limited data comparing these agents for treatment of MSSA infections. Recently, a retrospective case–​control study comparing cefazolin to nafcillin for the treatment of MSSA bacteraemia observed that they had similar treatment failure rates, with cefazolin having fewer ad- verse drug effects. However, a limitation of this study is that there were few endocarditis cases and no meningitis cases, limiting the generalizability of these findings. One retrospective cohort study that compared treatment outcomes between cefazolin and oxacillin for the treatment of MSSA bacteraemia did not observe a higher rate of treatment failure with cefazolin, even for individuals with MSSA bacteraemia associated with endocarditis or deep-​seated in- fection. In addition, rather than using vancomycin for therapy, pa- tients with MSSA bacteraemia and a reported penicillin allergy may benefit from an evaluation of the allergy and potentially treatment with cefazolin. However, there are data suggesting that some MSSA strains might have a bacterial ‘inoculum effect’; that is more antibiotic might be needed to treat a heavy bacterial load, as might be seen in deep- seated infections. Patients infected with strains demonstrating this effect might have worse outcomes when treated with cefazolin. However, this might be mitigated by possible geographic variation in the prevalence of such strains. Based on preliminary in vitro and clinical evidence, combin- ation therapy (vancomycin plus a beta-lactam antibiotic) has been used for initial treatment of S. aureus bacteraemia and might be associated with fewer treatment failures for MRSA, and earlier de- finitive therapy for MSSA, in comparison to therapy with vanco- mycin alone. The principles of therapy for S.  aureus bacteraemia include evaluation for endocarditis; use of a parenteral agent; removal of infected foci (i.e. catheters or abscesses); and use of a bactericidal agent, preferably a β-​lactam, whenever possible. Occasionally, uncomplicated bacteraemia with drainage of infected foci and no embolic sites might respond to only 14 days of therapy (Table 8.6.4.9); however, more often, prolonged bacteraemia, residual disease, undrained foci of infection, infected clots, or endocarditis all warrant longer therapy (at least 4 weeks). Several studies have shown that involvement of infectious diseases spe- cialists in the care of patients with S. aureus bacteraemia is associ- ated with better management and improved outcomes, including in-hospital mortality. A recent review of treatment modalities for S. aureus bacter- aemia suggests that given the significant potential complications associated with MRSA bacteraemia, modifications to therapy should be considered earlier into therapy—​as early as three to four days—​if blood cultures have not yet cleared. This approach warrants controlled evaluation. The review also reiterates the im- portance of ensuring adequate source control as the cornerstone of therapy for MRSA bacteraemia. A multicenter, randomized, double-blind, placebo-controlled trial in the UK of 758 adults with S. aureus bacteraemia evaluated the benefit of adding rifampicin to standard antimicrobial therapy and found that rifampicin pro- vided no overall benefit. A multicenter, randomized, double-blind, placebo-controlled trial in the UK of 758 adults with S. aureus bacteremia evaluated the benefit of adding rifampicin to standard antimicrobial therapy and observed that adjunctive rifampicin provided no overall benefit. Several studies have now found that involvement of infectious disease providers in the care of patients with S. aureus bacter- emia is associated with improved management (e.g., obtaining an echocardiogram and repeating blood cultures) as well as with improved outcomes, including in-hospital mortality. In addition, Infectious Disease consultation can help improve antibiotic ad- justments, including de-escalation, and can optimize duration of therapy. Endocarditis (Chapter 16.9.2) Many features of endocarditis are non​specific (fever, tachycardia, arthralgias and myalgias, wasting, and back pain). Finding a new cardiac (especially diastolic) murmur or septic emboli provides strong supportive evidence. Other suggestive findings include petechiae, Janeway’s lesions, mycotic aneurysms of arterial ves- sels (with resultant pain, vascular leak, or adjacent deep venous thrombosis), discitis or osteomyelitis (particularly vertebral dis- ease), and neurological complications such as septic infarcts or mycotic cerebrovascular aneurysms. Conduction abnormalities (e.g. AV delay), might be noted in the presence of myocardial ab- scess. In the setting of right-​sided endocarditis, septic pulmonary emboli are common. The presence of multiple positive blood cultures is a neces- sary criterion for diagnosis of endocarditis in the untreated pa- tient. Diagnosis is aided by specific criteria (e.g. modified Duke’s criteria). Transthoracic echocardiography is indicated as a non-​ invasive method to evaluate the presence of cardiac vegetations in those with a low pretest probability of disease; individuals with non​diagnostic studies or worsening clinical course should Table 8.6.4.9  Therapy of bacteraemia, without endocarditis,
due to S. aureus Drug Dosage Duration/​ comment For methicillin-​susceptible S. aureus: 14 days with removable focus of infection Longer course of therapy for complicated infection Oxacillin/​flucloxacillina 1–​2 g IV every 4–​6 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Daptomycina 6 mg/​kg IV every 24 h Teicoplanin At least 400 mg IV BID Linezolid 600 mg IV every 12 h Quinupristin/​dalfopristin 7.5 mg/​kg every 12 h Sodium fusidate 500 mg IV every 8 h Dalbavancin, oritavancin, telavancin May have future role BID, twice daily; IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent.

section 8  Infectious diseases 1004 undergo transoesophageal echocardiogram. Patients with high clinical risk, despite non​diagnostic transoesophageal studies, should be restudied after 7–​10 days. Recent evaluations have sug- gested that patients who fit a low-​risk profile (e.g. no cardiac de- vice or prosthetic heart valve, quick clearance of blood cultures, and nosocomial onset of bacteraemia), can be evaluated ad- equately with transthoracic echocardiography, without recourse to transoesophageal echocardiogram. Therapy for staphylococcal endocarditis requires a bactericidal antibiotic (Tables 8.6.4.10–​8.6.4.12). In general, therapy should last for 4 weeks (in uncomplicated disease) to 6 weeks or more (in the setting of metastatic infection, perivalvular abscess, or other complications). Combination therapies (agents given with either vancomycin or β-​lactams) have not been demonstrated to improve outcomes in native valve endocarditis but are com- monly used. For example, the addition of gentamicin for 3–​5 days shortens the duration of bacteraemia by about 1 day, but does not influence outcome. Addition of rifampicin for bacteraemic pa- tients with putative failure of therapy (e.g. bacteraemia or fever persisting for more than 4–​5 days) is a common strategy, but the recent ARREST trial casts doubt on this strategy. Rifampicin is still recommended as part of the standard treatment of prosthetic valve endocarditis. The average time to clearance of S. aureus from the bloodstream is 5 days of β-​lactam or 1 week of vancomycin therapy. Prolonged bac- teraemia should prompt a closer evaluation of antibiotic minimum inhibitory concentrations (especially for vancomycin), a search for sequestered sites of infection or undrained foci, or a myocardial or valvular abscess. The 2011 Infectious Disease Society of America guidelines for the treatment of MRSA suggest a vancomycin trough concen- tration of 15–​20 µg/​ml for serious infections (e.g. bacteraemia, Table 8.6.4.11  Therapy of native valve right-​sided endocarditis due to S. aureus Drug Dosage Duration/​comment β-​Lactams As for left-​sided disease (Table 7.6.4.10) 4–​6 weeks after negative cultures Vancomycina As for left-​sided disease (Table 7.6.4.10) Daptomycina 6 mg/​kg IV every 24 h Above therapies can be used with: Gentamicinb 1 mg/​kg IV every 8 h 3–​5 days at start of therapy, or combined therapy with β-​lactam for MSSA infection Ciprofloxacin/​ rifampicinb 750 mg/​300 mg PO BID For use in patients with tricuspid valve endocarditis who can not/​will not be admitted for intravenous therapy BID, twice daily; IV, intravenously; PO, by mouth. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. b Use is indicated in only limited circumstances. Gentamicin therapy is optional and has not been shown to improve clinical outcomes. Table 8.6.4.12  Therapy of prosthetic valve endocarditis due to S. aureus Drug Dosage Duration/​ comment For methicillin-​susceptible S. aureus: Oxacillin/​flucloxacillina 2 g IV every 4 h ≥6 weeks with Rifampicin 300 mg PO/​IV every 8 h ≥6 weeks and Gentamicin 1 mg/​kg IV every 8 h 3–​5 days at start of therapy Cefazolin(second choice for MSSA) 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h with Rifampicin 300 mg PO/​IV every 8 h ≥6 weeks and Gentamicin 1 mg/​kg IV every 8 h 3–​5 days at start of therapy IV, intravenously; PO, by mouth. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. Table 8.6.4.10  Therapy of native valve left-​sided endocarditis due to S. aureus Drug Dosage Duration/​ comment For methicillin-​susceptible S. aureus: 4–​6 weeks after negative cultures Oxacillin/​flucloxacillina 2 g IV every 4 h Cefazolin 1–​2 g IV every 8 h For methicillin-​resistant S. aureus (or β-​lactam allergy): Vancomycina 15 mg/​kg IV every 12 h Teicoplanina At least 400 mg IV BID Linezolid 600 mg IV every 12 h Quinupristin/​dalfopristin 7.5 mg/​kg every 12 h Daptomycin 6 mg/​kg IV every 24 h Sodium fusidate 500 mg IV every 8 h Trimethoprim/​ sulfamethoxazolec 320 mg/​1600 mg IV every 12 h Above therapies can be used with: Gentamicinb (3–​5 days at start of therapy) 1 mg/​kg IV every 8 h BID, twice daily; IV, intravenously. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent. b Gentamicin therapy is optional, and has not been demonstrated to change clinical outcomes. c A recent study that compared trimethoprim-​sulfamethoxazole to vancomycin for the treatment of severe infections due to MRSA observed that therapy with trimethoprim-​ sulfamethoxazole was associated with treatment failure; patients with left-​sided endocarditis, meningitis, chronic haemodialysis, and prolonged neutropenia were excluded. This study used a fixed dose of trimethoprim-​sulfamethoxazole for enrolled patients, which may be a limitation of the study.

8.6.4  Staphylococci 1005 infective endocarditis, osteomyelitis, meningitis, pneumonia, and severe skin and soft tissue infections such as necrotizing fasciitis). Part of the impetus for higher trough levels comes from studies that have suggested that there is an increased chance of treatment failure with vancomycin as the minimum inhibitory concentra- tion for vancomycin increases to the upper limit of the susceptible range. However, in a study of an Australian cohort of patients with S. aureus bacteraemia, elevated vancomycin minimum inhibitory concentration was associated with increased 30-​day mortality but methicillin resistance and specific antibiotic selection were not, suggesting that vancomycin itself may be a marker for more difficult-​to-​treat strains but not the driver per se of worse out- comes in these patients. Increasing vancomycin dosing has not been demonstrated clearly to improve outcomes, although consensus supports in- creased trough levels of 15–​20 µg/​ml (requiring close monitoring of renal function) for serious infections. Although close moni- toring of renal function is recommended, the data establishing the causal link between serum vancomycin concentration and nephrotoxicity are limited since concomitant nephrotoxic agents might play a role in toxicity. Indications for surgical valve replace- ment include new congestive heart failure (associated with higher mortality), failure to clear the bloodstream, recurrent emboli, and myocardial or valvular abscess. As daptomycin has concentration-​dependent bactericidal activity against Gram-​positive organisms, some have suggested higher doses (e.g. 8 mg/​kg per day or greater) might be effective for treatment of complicated Gram-​positive infections. However, further evalu- ation of this is needed, in particular the safety and tolerability of higher doses. Clinical syndromes: Coagulase-​negative staphylococci Coagulase-​negative staphylococci are generally less virulent than S.  aureus. Most infections with these organisms are the consequence of medical progress, related to foreign bodies (e.g. prosthetic joints or heart valves, indwelling intravascular cath- eters or grafts, or peritoneal catheters), and occur in associ- ation with healthcare. Syndromes caused by coagulase-​negative staphylococci include endocarditis (5–​8% of native valve in- fections, c.40% of prosthetic valve infections), intravascular catheter infections (6–​27% of vascular catheter infections), prosthetic joint infections (up to 38% of arthroplasty infec- tions), peritoneal dialysis catheter infections, and postoperative ocular infections. Production of biofilm by coagulase-​negative staphylococci aids infection of both intravascular and peri- toneal catheters. Therapy for infections with coagulase-​negative staphylococci and side effects and toxicities are outlined in Tables 8.6.4.13 and 8.6.4.14. Bacteraemia and infected vascular catheters Clinical features and diagnosis Coagulase-​negative staphylococci are the most commonly re- ported bacteria in positive blood cultures; however, unlike S. aureus, coagulase-​negative staphylococci are frequently blood culture con- taminants. Typical rates of blood culture contamination by skin flora are approximately 2–​3%; higher rates might be a sign of poor phle- botomy technique. Infected intravascular catheters are common sources of coagulase-​ negative staphylococcal bloodstream infections. However, given the association of S.  epidermidis and contaminated blood cultures, a careful physical examination for signs of catheter infection is crit- ical to determine whether a single positive blood culture represents true infection and/​or an infected catheter. Suggestive findings in- clude fever, erythema at or pus expressible from the site of catheter insertion, or tenderness. Methods to enhance the identification of true bloodstream infec- tion as opposed to contamination include proper skin preparation and obtaining at least two sets of blood cultures from sites separated by location and time. The use of quantitative catheter tip cultures (more than 15 colonies) or differential time to positivity (more than Table 8.6.4.13  Therapy for coagulase-​negative staphylococcal infections Indication Drug Dosage Duration Bacteraemia (with prompt catheter removal) Vancomycina 15 mg/​kg IV every 12 h 10–​14 days Oxacillin/​flucloxacillin (methicillin-​ susceptible S. epidermidis) 1–​2 g IV every 4 h Bacteraemia (with attempted catheter salvage) Vancomycin catheter lock (for catheter salvage) 1–​5 mg/​ml vancomycin, mixed with 50–​100 U heparin or normal saline, to fill catheter lumen (total 2–​5 ml of solution) when catheter not in use 14 days Vancomycina 15 mg/​kg IV every 12 h 10–​14 days Oxacillin/​flucloxacillin (methicillin-​ susceptible S. epidermidis) 1–​2 g IV every 4 h Prosthetic
valve endocarditis Vancomycina 15 mg/​kg IV every 12 h ≥6 weeks with   Rifampicina 300 mg PO/​ IV every 8 h and   Gentamicin 1 mg/​kg IV every 8 h Oxacillin/​flucloxacillin (methicillin-​ susceptible S. epidermidis) 1–​2 g IV every 4 h Peritoneal dialysis-​ associated peritonitis Vancomycina 30–​50 mg vancomycin per litre of dialysate given intraperitoneally 10–​21 days Or Vancomycin 1 g IV once, then based on levels (keep trough

10–​15 mcg/​ml) 10–​21 days IV, intravenously; PO, by mouth. Note: Dosing recommendations assume normal renal and hepatic function. a First-​line agent.

section 8  Infectious diseases 1006 Table 8.6.4.14  Information on indications and toxicity for selected drugs Drug class Indications/​use Side effects/​toxicities Semisynthetic penicillins Flucloxacillin Oxacillin Drugs of choice in penicillin-​resistant MSSA infection Interstitial nephritis (which limits methicillin use in adults) Nafcillin Not effective in MRSA infection Neutropenia (nafcillin) Dicloxacillin CA-​MRSA may equal or exceed 50% prevalence in some areas Elevated transaminases (oxacillin, nafcillin) Range of prevalence of nosocomial MRSA is 2–​70% Adequate incision and drainage of infected foci is critical First-​generation cephalosporins Cefazolin Cefalexin Alternative agents for penicillin-​resistant, MSSA infection 15% cross-​reaction for penicillin-​allergic patients Not effective in MRSA infection Hypersensitivity CA-​MRSA may equal or exceed 50% prevalence in some areas Eosinophilia Range of prevalence of nosocomial MRSA is 2–​70% Adequate incision and drainage of infected foci is critical Penicillins and aminopenicillins Penicillin Ampicillin Amoxicillin Ampicillin + sulbactam Amoxicillin + clavulanate Penicillin is the drug of choice in known penicillin-​sensitive S. aureus infection Hypersensitivity Duration of therapy and indications similar to those of oxacillin Glycopeptides Vancomycin Teicoplanin Dalbavancin Oritavancin Telavancin Indicated for MRSA infections or MSSA infections in penicillin-​allergic patients 3–​11% of patients given vancomycin may develop anaphylactoid reaction (i.e. ‘red man’ or ‘red-​neck’ syndrome) due to overly rapid infusion Indicated for coagulase-​negative staphylococcal infections Nephrotoxicity with vancomycin (0–​7% alone, 14–​20 + % in conjunction with aminoglycoside) and teicoplanin (5%) MRSA that are vancomycin susceptible but have increased MIC may require higher doses Neutropenia with vancomycin (1–​2%) Vancomycin trough levels should be 10–​15 mg/​litre and monitored
closely in the setting of renal dysfunction; ≥15 if vancomycin
MIC >1 mcg/​ml Erythematous rash with teicoplanin (7%) Teicoplanin levels should be >10 mg/​litre in bacteraemia and >20 mg/​litre in endocarditis Lincosamide Clindamycin Indicated for non​severe MRSA infections that are erythromycin and clindamycin susceptible or that are erythromycin resistant and double-​ disc diffusion (D) test is negative 20% of patients develop diarrhoea An option for non​severe MSSA infections in penicillin-​allergic patients Increased risk of Clostridium difficile-​associated diarrhoea (10%) Tetracyclines Doxycycline Minocycline Tigecycline Not recommended in children aged <8 years Photosensitivity Bacteriostatic, not recommended for bacteraemia or severe infections Eosinophilia Recent review in osteomyelitis demonstrated success rate in over 80%; retained foreign body in osteomyelitis may lead to failure SLE-​like reaction with minocycline Likely need additional agent for treatment of long duration (i.e. rifampicin or fluoroquinolone) to prevent emergence of resistance Pseudotumour cerebri or vestibular toxicity Potency/​activity of drugs: tigecycline > minocycline > doxycycline > tetracycline Antianabolic Dihydrofolate reductase inhibitors Trimethoprim/​ sulfamethoxazole Higher failure rate as compared with vancomycin in MSSA
endocarditis seen in one study Hypersensitivity, may progress to erythema multiforme and/​or Stevens–​Johnson syndrome MRSA endocarditis success equivalent to vancomycin Macrocytic anaemia TMP/​SMX resistance may be common among nosocomial MRSA
(up to 50%) but is generally uncommon among CA-​MRSA (<10%) Photosensitivity Methaemoglobinaemia (rare) (continued)

8.6.4  Staphylococci 1007 2 h) for peripheral compared to catheter-​drawn blood cultures helps assess whether a catheter is infected. Management of bacteraemia and catheter infection An approach for management of presumed infected catheters is to remove the catheter when the index of suspicion is high and/​ or the patient is unstable, with insertion of a new catheter at an uninvolved site. When likelihood of infection is unclear and the patient is stable, the catheter can be changed over a guidewire and the tip cultured. Positive tip cultures should prompt removal of the replacement catheter and new catheter insertion at a different site. A negative culture might allow the replacement catheter to remain Drug class Indications/​use Side effects/​toxicities Fluoroquinolones Ciprofloxacin Levofloxacin Moxifloxacin Ofloxacin Should not be used as monotherapy due to rapid emergence of resistance Neurological (0.9–​11% delirium and/​or seizures) May possibly be used with other agents (e.g. TMP/​SMX, rifampicin) Arthropathy, tendinitis, tendon rupture Ciprofloxacin or levofloxacin in combination with rifampicin may
be an option for patients with uncomplicated tricuspid valve endocarditis who cannot/​will not be admitted; or those with
skin/​soft tissue infection with CA-​MRSA Hypoglycaemia Rifamycins Rifampicin Part of combination treatment of prosthetic valve endocarditis, or in setting of endovascular infection with a foreign body Gastrointestinal complaints Should be used with another agent given rapid acquisition of resistance Hepatitis Myeloid suppression Acute tubular necrosis or acute interstitial nephritis SLE-​like syndrome Macrolides Erythromycin Clarithromycin Azithromycin May be used in penicillin-​allergic patients for skin/​soft tissue infections Gastrointestinal complaints (prokinetic) Should be used with caution based on local susceptibility to erythromycin in S. aureus and emergence of resistance QT prolongation in conjunction with other medications Oxazolidinones Linezolid Comparable indications to vancomycin; of use in therapy for MRSA
or VISA/​VRSA Myelosuppression Data suggest better efficacy than vancomycin for pneumonia and
skin/​soft tissue infections with MRSA Serotonin syndrome Has been used for bacteraemia in small open-​label trials Peripheral neuropathy Bacteriostatic Lactic acidosis (due to mitochondrial toxicity) Limited clinical experience Lipopeptides Daptomycin Bactericidal Myopathy, especially with higher doses or in the setting of renal insufficiency. Cases of eosinophilic pneumonia reported May have use in VISA/​VRSA Resistance has been noted to develop on therapy Not indicated for treatment of pneumonia ‘Non​inferior’ to vancomycin for right-​sided endocarditis and uncomplicated bacteraemia with S. aureus and possibly better for MRSA Streptogramins Quinupristin/​dalfopristin May have use in soft tissue infections, bacteraemia, or osteomyelitis in settings where other agents are not available/​useful Phlebitis (30%)—​limits general usefulness May have use in MRSA or VISA/​VRSA infections Arthralgias (9.1%) Presence of inducible or constitutive clindamycin resistance (i.e. MLS resistance) may indicate elevated MICs for quinupristin/​dalfopristin Myalgias (6.6%) Sodium fusidate Topical therapy for impetigo Thrombophlebitis (parenteral use) May be used parenterally in therapy of MRSA bacteraemia or endocarditis, depending on susceptibility Reversible jaundice (parenteral use) Should not be used in newborns Thrombocytopenia (parenteral use) CA, community-​acquired; MIC, minimum inhibitory concentration; MLS, macrolide–​lincosamide–​streptogramin, MRSA, methicillin-​resistant S. aureus; MSSA, methicillin-​susceptible S. aureus; SLE, systemic lupus erythematosus; TMP/​SMX, trimethoprim/​sulfamethoxazole, VISA/​VRSA, vancomycin-​intermediate/​vancomycin-​resistant S. aureus. Table 8.6.4.14  Continued

section 8  Infectious diseases 1008 in place, although its risk of subsequent infection is increased by the exchange process. Parenteral vancomycin is the mainstay of therapy for vascular catheters infected by methicillin-​resistant coagulase-​negative staphylococci, and should be continued for 7–​14 days unless there is metastatic seeding requiring longer treatment. Antibiotic lock therapy (Table 8.6.4.13) might be useful in carefully selected pa- tients for ‘line salvage’. The presence of tenderness along the course of a tunnelled catheter is highly predictive of failure of medical man- agement and should lead to catheter removal. Endocarditis Multiple positive blood cultures with coagulase-​negative staphylo- cocci might indicate the presence of infective endocarditis. More than 80% of patients with prosthetic valve infection have persistent fever, deep valve involvement (e.g. infection of the sewing ring or valve dysfunction, dehiscence, or abscess), and/​or cardiac con- duction abnormalities. Infections within the first 6–​12 months fol- lowing surgery typically reflect acquisition of the organism in the perioperative period and may have a higher likelihood of compli- cated infection. Diagnosis of prosthetic valve infection should be sought aggressively when multiple positive cultures with coagulase-​ negative staphylococci have been obtained postoperatively soon after cardiac surgery. Physical examination usually shows fever and a new or worsening murmur or valve dysfunction. Evaluation includes serial blood cultures to document degree and persistence of bacteraemia, electrocardiography to search for conduction delay, and echocardiography or angiography for documentation of valve function. Therapy for prosthetic valve endocarditis should include parenteral vancomycin (for methicillin-​resistant strains), gentamicin, and/​or rifampicin (Table 8.6.4.13). Peritoneal dialysis-​associated peritonitis Peritoneal dialysis catheter infection is characterized by abdom- inal pain, cloudy exchange fluid, and peritoneal fluid containing predominantly polymorphonuclear leucocytes (>100 leucocytes/​ mm3). To improve diagnostic yield of peritoneal dialysate fluid cultures, 2–​3 ml of fluid can be inoculated into thioglycolate broth or blood culture bottles. Therapy for catheter-​associated S.  epidermidis peritonitis depends on susceptibility results. For susceptible organisms, β-​lactams, trimethoprim/​sulfamethoxazole, and vancomycin have all been effective, and both parenteral and oral antibiotics have been used. However, if methicillin-​resistant S. epidermidis is suspected, vancomycin therapy (Table 8.6.4.13) with moni- toring of serum levels may be indicated. Therapy can consist of either systemic or intraperitoneal antimicrobial administration. Intraperitoneal therapy is advantageous because it allows con- tinued ambulatory care and therapy directly to the site of infection. Catheter salvage is frequently possible, but relapses might require catheter removal. Other organisms S. saprophyticus is a common cause of UTIs (20% of UTIs in women 16–​35 years old). S. lugdunensis S.  lugdunensis is a coagulase-​negative staphylococcus that can be a skin commensal but has also been reported to cause clin- ical disease such as soft tissue infections, endocarditis, including native valves, and bloodstream infection. The true incidence of infections due to S.  lugdundensis is not clear given the lack of speciation of most coagulase-​negative staphylococci in many laboratories; however, it should be considered a true pathogen if isolated, rather than a contaminant. S.  lugdunensis infec- tions have been characterized by a clinical course more like that of S.  aureus, with valve destruction a prominent part of the illness. In contrast to other forms of coagulase-​negative staphylo- coccus, S. lugdunensis typically retains susceptibility to a range of antibiotics. Likely developments in the near future Future directions in the management of S. aureus infections in- clude vaccine development, new antimicrobials, enhanced under- standing of epidemiology and control of nosocomial-​associated and CA-​MRSA, and evaluation and control of the emergence of VISA/​VRSA. A bivalent vaccine containing S. aureus polysacchar- ides 5 and 8 briefly reduced risk of bacteraemia in haemodialysis recipients in a prospective study published in 2002. A novel vac- cine candidate against S. aureus was examined recently among in- dividuals undergoing cardiothoracic surgery. This vaccine did not reduce the rate of postoperative S. aureus infections in comparison to placebo and was associated with increased mortality among in- dividuals who did develop S. aureus infections; the study was halted by the independent data monitoring committee. An additional target for vaccine synthesis is the Panton–​Valentine leucocidin toxin, which might provide protection against CA-​ MRSA. Another preventive measure might be screening for nasal or skin colonization with MRSA, with subsequent decolonization of colonized persons. However, populations that require screening (i.e. universal or targeted screening), actions to pursue among the col- onized, and efficacy and costs of such a programme are all variables that require further clarification. The promise of such a strategy might be control of MRSA and reduction of the costs and morbidity associated with MRSA infection. New glycopeptides (telavancin, oritavancin, and dalbavancin), new cephalosporins with activity against MRSA (ceftobiprole and ceftaroline), and existing agents with evolving indications (daptomycin, linezolid) might improve treatment options for MRSA and VISA/​VRSA. Ceftaroline is approved in the United States for the treatment of complicated skin and soft structure in- fections as well as community-​acquired pneumonia and may be a valuable treatment option for MRSA isolates with reduced sus- ceptibility to linezolid, daptomycin, and vancomycin. Despite its recent US FDA approval in 2010, resistance to ceftaroline has been reported. In the United States, the molecular mechanism of this resistance was determined using whole genome sequencing, highlighting ways this technology can be utilized to further study multidrug resistant organisms. Both oritavancin and dalbavancin are notable for their long half-​ life, allowing for less frequent dosing. One dose of oritavancin was found to be non​inferior to a 7–​10-​day course of antibiotics for the treatment of acute skin and soft tissue infections due to Gram-​positive

8.6.4  Staphylococci 1009 organisms. Dalbavancin can be dosed weekly and has been found to be non​inferior to a regimen of vancomycin and linezolid for the treatment of acute bacterial skin and soft tissue infections. In 2014 in the United States, a new oxazolidinone drug, tedizolid, was ap- proved for the treatment of acute bacterial skin and skin structure infections; this drug has activity against MRSA. As with linezolid, tedizolid can be given orally or parenterally. In addition, tedizolid is given once daily and is active against linezolid-​resistant S. aureus strains. It remains unclear if the risk of myelosuppression that is seen with linezolid will be lower with tedizolid in comparison to those who did not. Systemic antibiotics, e.g., clindamycin, given for skin and skin structure infections may also reduce S. aureus colonization, leading to downstream benefits of fewer recurrent skin infections. However, some studies have noted increased adverse effects of clindamycin in comparison to trimethoprim-sulfamethoxazole (e.g., diar- rhea, nausea) when used for treatment of skin and skin structure infections. Pneumonia section A recent meta-analysis of 22 studies evaluated the utility of MRSA nasal screening results in predicting MRSA pneumonia. They observed that MRSA nares screening had a high specificity (90.3%) and high negative predictive value (96.5%) for exclud­ ing MRSA pneumonia. Such a tool could guide antibiotic pre- scribing, e.g., no need for empiric MRSA coverage if nasal screen is negative. New drug In 2017, the FDA approved a new fluoroquinolone antibiotic, delafloaxin, for oral and parenteral therapy of adults with skin and skin structure infections, including those due to MRSA. Better strategies for treatment and salvage of infected catheters with catheter coating (e.g. with chlorhexidine) or methods for treat- ment of biofilm might improve treatment of coagulase-​negative staphylococci. FURTHER READING Baddour LM, et  al. (2005). Infective endocarditis:  diagnosis, anti- microbial therapy, and management of complications:  a state- ment for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: endorsed by the Infectious Diseases Society of America. Circulation, 111, e394–​434. Bai AD, et  al. (2015). Impact of infectious disease consultation on quality of care, mortality, and length of stay in Staphylococcus aureus bacteremia: results from a large multicenter cohort study. Clin Infect Dis, 60, 1451–61. Blumenthal KG, et al. (2015). Improving clinical outcomes in patients with methicillin-​sensitive Staphylococcus aureus bacteremia and reported penicillin allergy. Clin Infect Dis, 61, 741–​9. Blumenthal KG, et al. (2018). Risk of meticillin resistant Staphylococcus aureus and Clostridium difficile in patients with a documented penicillin allergy: population based matched cohort study. BMJ, 361, k2400. Climo MW, et  al. (2013). Effect of daily chlorhexidine bathing on hospital-​acquired infection. N Engl J Med, 368, 533–​42. Dantes R, et  al. (2013). National burden of invasive methicillin-​ resistant Staphylococcus aureus infections, United States, 2011. JAMA Intern Med, 173, 1970–​8. Darouiche RO (2006). Spinal epidural abscess. N Engl J Med, 355, 2012–​20. Daum RS, et al. (2017). A pacebo-controlled trial of antibiotics for smaller skin abscesses. N Engl J Med, 376, 2545–55. Drees M, Boucher H (2006). New agents for Staphylococcus aureus endocarditis. Curr Opin Infect Dis, 19, 544–​50. Edmond MB, Wenzel RP (2013). Screening inpatients for MRSA—​ case closed. N Engl J Med, 368, 2314–​5. Elliott TS, et  al. (2004). Guidelines for the antibiotic treatment of endocarditis in adults: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother, 54, 971–​81. Fowler VG Jr, et al. (2005). Staphylococcus aureus endocarditis: a con- sequence of medical progress. JAMA, 293, 3012–​21. Fowler VG, et  al. (2013). Effect of an investigational vaccine for preventing Staphylococcus aureus infections after cardiothoracic surgery: a randomized trial. JAMA, 309, 1368–​78. Gemmell CG, et al. (2006). Guidelines for the prophylaxis and treat- ment of methicillin-​resistant Staphylococcus aureus (MRSA) infec- tions in the UK. J Antimicrob Chemother, 57, 589–​608. Grundmann H, et al. (2006). Emergence and resurgence of meticillin-​ resistant Staphylococcus aureus as a public-​health threat. Lancet, 368, 874–​85. Heldman AW, et al. (1996). Oral antibiotic treatment of right-​sided staphylococcal endocarditis in injection drug users:  prospective randomized comparison with parenteral therapy. Am J Med, 101, 68–​76. Hogan PG, et al. (2018). Impact of systemic antibiotics on Staphylococcus aureus colonization and recurrent skin infection. Clin Inf Dis, 66, 191–7. Holland TL, Arnold C, Fowler VG, Jr (2014). Clinical management of Staphylococcus aureus bacteremia: a review. JAMA, 312, 1330–​41. Holland TL, Fowler VG, Jr (2011). Vancomycin minimum inhibitory concentration and outcome in patients with Staphylococcus aureus bacteremia: pearl or pellet? J Infect Dis, 204, 329–​31. Holmes NE, et al. (2011). Antibiotic choice may not explain poorer outcomes in patients with Staphylococcus aureus bacteremia and high vancomycin minimum inhibitory concentrations. J Infect Dis, 204, 340–​7. Huang SS, Datta R, Platt R (2006). Risk of acquiring antibiotic-​ resistant bacteria from prior room occupants. Arch Int Med, 166, 1945–​51. Huang SS, et al. (2013). Targeted versus universal decolonization to prevent ICU infection. N Engl J Med, 368, 2255–​65. Klevens RM, et al. (2006). Changes in the epidemiology of methicillin-​ resistant Staphylococcus aureus in intensive care units in US hos- pitals, 1992–​2003. Clin Infect Dis, 42, 389–​91.

8.6.40 Rickettsioses 1230

8.6.40 Rickettsioses 1230

section 8  Infectious diseases 1230 8.6.40  Rickettsioses Karolina Griffiths, Carole Eldin, Didier Raoult, and Philippe Parola ESSENTIALS Rickettsioses are zoonoses caused by obligate Gram-​negative intra- cellular bacteria of the order Rickettsiales, comprising (1) rickettsioses due to bacteria of the genus Rickettsia, including spotted fever groups and typhus groups (Rickettsiaceae), (2)  ehrlichioses and anaplasmoses due to bacteria of the Anaplasmataceae, and (3) scrub typhus due to Orientia tsutsugamushi (see Chapter 8.6.41). Epidemiology, clinical features, and prognosis of
particular rickettsioses Tick-​borne spotted fever group—​around 25 species or subspecies of spotted fever group rickettsiae can infect humans following trans- mission from their natural vertebrate hosts by ixodid (hard) ticks. Many species are restricted to certain geographical areas, although research continues to demonstrate the presence of these emerging pathogens in previously undocumented areas and tick species. Clinical presentation is typically with fever, headache, muscle pain, rash, local lymphadenopathy, and—​for some diseases—​a typical in- oculation eschar (the ‘tache noire’) at the tick bite site. These signs vary depending on the rickettsia involved and might allow distinction between different rickettsioses occurring at the same geographical location. Diseases range in severity from mild to severe. Murine (endemic) typhus is caused by Rickettsia typhi, whose nat- ural host is rodents, and is spread by the rat flea. Human infection usually results from contamination of disrupted skin or inhalation of flea faeces containing the organism. Disease is generally mild and self-​limiting with non​specific features. Epidemic typhus—​caused by R. prowazekii, for whom humans are the major host, and transmitted by body lice, hence the disease is a par- ticular problem during times of war, conflict, famine, and natural catas- trophes. Following a non​specific prodrome, presentation is with fever, headache, myalgia, and a macular, maculopapular, or petechial rash. Mortality ranges from 4% (recent series) to 60% (without antibiotics). Other rickettsioses—​include (1)  flea-​borne spotted fever—​cat flea typhus; (2)  rickettsialpox—​transmitted from mice by house mouse mites. Diagnosis and treatment of rickettsioses Diagnosis is by direct evidence of infection by culture or polymerase chain reaction, or by serological testing. Polymerase chain reaction of skin swabs can be particularly useful. Identification of the tick vector is important. Aside from supportive care, doxycycline remains the drug of choice for immediate empirical treatment of all rickettsioses on clinical suspicion, with some of these infections having high mortality if untreated. Identification of new Rickettsia species in ticks by molecular tools and their potential role in pathogenicity in hu- mans remains an increasing area of research. Human ehrlichioses and anaplasmosis These diseases are tick-​borne zoonoses, whose causative agents are maintained through enzootic cycles between ticks and animals. Three main species cause human diseases: (1) Ehrlichia chaffeensis—​causes human monocytic ehrlichiosis; (2)  Anaplasma phagocytophilum—​ causes human anaplasmosis; and (3) E. ewingii—​causes granulocytic ehrlichiosis. These all present as undifferentiated seasonal febrile illnesses, ranging in severity from mild to severe, with multisystem organ failure. Diagnosis is by direct evidence of infection by culture or polymerase chain reaction, or (most commonly) by serological testing. Doxycycline is the antibiotic of choice. New subspecies causing human infection continue to be identified. Prevention Prevention of rickettsioses in general is by (1) avoiding arthropod bites—​by applying topical N,N-​diethyl-​m-​toluamide repellent to ex- posed skin, and treatment of clothing with permethrin; and (2) those staying in infested areas checking their bodies routinely for the presence of arthropods, and promptly removing ticks. In addition, (3) epidemic typhus—​louse eradication is the most important pre- ventive measure. No vaccines are available. Introduction Rickettsioses are mild to life-​threatening zoonoses caused by ob- ligate intracellular bacteria of the order Rickettsiales (family Rickettsiaceae). Arthropods, including ticks, fleas, and mites, are implicated as their vectors, reservoirs, or amplifiers. With an increasing number of new pathogens and recognition of new pathogenicity and affected geographical areas over the past few dec- ades, there is a better understanding of the scope and importance of these pathogens, particularly as a paradigm to understanding emerging and remerging infections. The taxonomy has under- gone numerous changes, with now three main groups classified as rickettsioses according to morphological, antigenic and metabolic characteristics: (1) Rickettsioses due to the bacteria of the genus Rickettsia, including spotted fever group (SFG), typhus groups (Rickettsiaceae); (2) Ehrlichioses and Anaplasmoses due to bacteria of the Anaplasmataceae; and (3) scrub typhus due to Orientia tsu­ tsugamushi (Chapter 8.6.41.) (See Fig. 8.6.40.1.) Phlyogenomic studies of complete genome sequences have pro- vided further insight into the genus Rickettsia, which can be clas- sified into four groups: (1) The spotted fever group (SFG), mainly associated with ticks, such as Rickettsia conorii, the causative agent of Mediterranean spotted fever and Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, but also associated with fleas (Ricketttsia felis) and mites (R. akari). (2) Typhus groups are asso- ciated with human body lice, R. prowazekii, the agent of epidemic typhus and fleas, which transmit R. typhi, the cause of murine ty- phus. Groups; (3) Rickettsia belli; and (4) Rickettsia canadensis are currently of unknown pathogenicity. Rickettsioses (human infections attributable to Rickettsia spp.) Bacteriology Rickettsioses are short Gram-​negative rods that retain basic fuchsin when stained by Gimenez’s method. Rickettsiae are 0.3–​0.5 by

8.6.40  Rickettsioses 1231 0.8–​2.0 µm in size. Their cytoplasm contains ribosomes and strands of DNA, limited by a typical Gram-​negative trilamellar structure consisting of a bilayer inner membrane, a peptidoglycan layer, and a bilayer outer membrane. Within host cells they are surrounded by an electron-​lucent slime layer. SFG rickettsiae have an optimal growth temperature of 32°C, their G + C content is 32–​33, and they can polymerize actin and thus move into the nuclei of host cells causing spotted fevers in humans. One exception is Rickettsia felis that is grown at a lower temperature (28°C). Typhus-​group rickett- siae have an optimal growth temperature of 35° C and a G + C con- tent of 29. They do not enter host cell nuclei but are confined to host cell cytoplasm, causing typhus in humans. Rickettsiae are rapidly inactivated at 56° C. They grow in eukaryotic cells where they live freely and divide by binary fission in the cytoplasm. They must be grown in tissue culture (L929 or Vero cells) or in yolk sacs of developing chicken embryos. Growth in cell monolayers is shown by plaque formation, representing disruption of massively in- fected cells. SFG rickettsiae form plaques of 2–​3 mm diameter after 5 to 8 days, whereas typhus-​group rickettsiae form plaques 1 mm in diam- eter after 8 to 10 days. Rickettsia spp demonstrate large variations in antigenic heterogeneity, influenced by the diversity in their ecological distribution. This results in the induction of different specific immune responses. The major rickettsial antigens are lipopolysaccharides, lipo- proteins, outer membrane proteins of the surface cell antigen (SCA) family, and heat shock proteins. Other antigens include a 17-​kDa lipo- protein, and autotransporter family SCA proteins include the 120-​ kDa S-​layer protein (OmpB or Sca5), OmpA (SGF only), and Sca4. Fourteen genes that may encode SCA proteins have been identified in sequenced rickettsial genomes, of which sca1 is present in all species. Vectors Vectors of rickettsial agents include mainly ticks (order Ixodidea), lice (order Phtiraptera), and fleas (Siphonaptera). Mosquitoes have also been recently suggested as vectors for R. felis. Transmission of the bacteria can either occur vertically or via co –​feeding (the pres- ence of several arthropods on the same host feeding alongside each other). Arthropods can act as a bacterial reservoir when efficient transstadial or transovarial transmission occurs. Ticks are the most important vectors and reservoirs of rickettsiae worldwide. Table 8.6.40.1 demonstrates the most important recog- nized tick vectors for each type of rickettsiae, with some rickettsiae specific to certain ticks and others carried by numerous arthropods. The geographical distribution of Rickettsiae and that of their specific vectors are closely related. R.  akari is transmitted by the mouse mite Lyponyssoides san­ guineus. However, increasing numbers of rickettsiae have also been found in other mites, including R. felis and another genotype similar to R. australis. Taxonomy and genomics Traditional bacteriological identification methods cannot be applied to rickettsiae because they are strictly intracellular. New specific and sensitive molecular tools have revolutionized the identification of new rickettsia, demonstrating the diversity in previously uniden- tified geographical areas and vectors. Molecular techniques and phylogenomic analysis have enabled the reorganization and clari- fication of the genus Rickettsia. Genotyping methods continue to identify new species in this genus (Fig. 8.6.40.2). Several dozen strains remain to be characterized. In 2001, the first genome of a tick-​transmitted rickettsia (R. conorii strain Seven) was fully sequenced, revealing several characteristics that are unique among bacterial genomes, including long, irregu- larly distributed, palindromic repeat fragments. Forty-​five strains of rickettsiae have been fully sequenced, exhibiting large variations in gene content and size. Their genome size remains small, due to genome reduction through gene loss; reduced during specialization Rickettsioses (Order Rickettsiales) Ehrlichiosis and anaplasmosis Ehrlichia chaffeensis Anaplasma phagocytophilum Ehrlichia ewingii Genus Rickettsiae Typhus group Epidemic typhus (R. prowazekii) Murine typhus (R. typhi) Rickettsia bellii group Spotted Fever Group, including (amongst others): TICK BORNE (See Table 1) including: ( Mediterranean Spotted Fever (R. conorii) Rocky Mountain Spotted Fever (R. rickettsii) R. felis infections
RICKETTSIALPOX MITES (R. akari) Rickettsia canadensis Scrub typhus (O. tsutsugamushi) Fig. 8.6.40.1  Overview of the rickettsioses covered in this chapter.

section 8  Infectious diseases 1232 Table 8.6.40.1  Characteristics of tick-​borne rickettsioses identified in human infections in 2015 Rickettsia sp. Recognized or potential tick vector(s) First identification in ticks Disease (first
clinical
description) First microbiological documentation
of human cases Selected clinical and epidemiological characteristics Confirmed pathogens Rickettsia aeschlimannii Amblyomma variegatum, Rhipicephalus annulatus, R. evertsi evertsi, H. marginatum marginatum H. marginatum rufipes, H. truncatum, H. anatolicum excavatum, I. ricinus, R. sanguineus, R. turanicus, R. bursa, H. punctate, H. detritum, H. aeguptium, H. dromedary, R. appendiculatus 1997 Spotted fever
(2002) 2002 First case described in a French patient returning from Morocco. Few human cases described in patients from Morocco, Algeria, and South Africa. Symptoms include eschar and maculopapular rash. Detected throughout countries in Europe, detected in ticks from migratory bird species. No autochthonous cases reported in Europe. No human cases identified in Asia, identified in ticks in Kazakhstan and Israel. Detected in 8 sub-​Saharan countries. Rickettsia africae Amblyomma hebraeum, A. variegatum, A. compressum, A. lepidum, A. loculosum, Rhipicephalus annulatus, R. evertsi, R. decoloratus, R. sanguineus, R. geugyu, Hyalomma impeltatum, H. aegyptium, Hy. dromedarii 1990 African tick-​bite
fever (1934) 1992 Disease distribution includes sub-​Saharan Africa, North and Central America, the Caribbean Pacific Islands. No human cases reported in Asia, or North Africa (identified in dromedary ticks in Algeria and Egypt). Outbreaks and clustered cases common (74%), Sudden onset of symptoms occur 5–​7 days after the tick bite, with fever (88%), headache, myalgia, and fatigue. eschars, often multiple, are reported inconsistently (50–​100%). maculopapular (49%) or vesicular (50%) rash, and lymphadenopathy (43%) are also reported. No fatal cases reported. Rickettsia australis Ixodes holocyclus, I. tasmani, I. cornuatus 1974 Queensland tick
typhus (1946) 1946 Disease occurs in predominantly rural settings. Cases occur from June to November. Vesicular rash (100%), eschar (65%), and lymphadenopathy (71%). Two fatal cases have been described. Rickettsia strain Atlantic rainforest (or strain Bahia) Amblyomma ovale, A. aureolatum, A. dubitatum, R. sanguineu, ? Unnamed () 2010 A novel strain closely related to R. parkeri, R, africae, and R. sibirica. Disease clinically similar to R. parkeri. Two human cases identified in Brazil. Also detected in ticks in Columbia and Argentina. Rickettsia conorii caspia R. sanguineus, R. pumilio, 1992 Astrakhan fever
(1970s) 1991 Endemic to Astrakhan region and Caspian Sea. Also detected in Kosovo and France, and a returning traveller from Chad. Disease occurs in predominantly rural settings. Associated with eschar (23%), maculopapular rash (94%), and conjunctivitis (34%). Rickettsia conorii conorii R. sanguineus, R. evertsi evertsi, R. simus, R. mushamae, Haemaphysalis puntaleachi, H leachi, R. bursa 1932 Mediterranean
spotted fever
(1910) 1932 Endemic in southern Europe. Disease occurs in urban and rural settings. Cases reported across North Africa and nine sub-​Saharan African countries. Incubation period of 6 days, sudden onset. Symptoms include fever, flu-​like symptoms, shingles, and rash. Cases generally sporadic. Atypical and life-​ threatening presentations recently reported. Rickettsia conorii indica Rhipicephalus sanguineus 1950 Indian tick typhus 2001 Prevalent in India and Pakistan. Solitary case reports in Laos, Sri Lanka, and Sicliy, Italy. Compared to Mediterranean spotted fever, rash usually purpuric. Eschar rarely found. Mild to severe. Reported in Spain and Italy. Rickettsia conorii israelensis R. sanguineus 1974 Israeli spotted fever (1940) 1971 Reported in Israel, Italy, and Portugal, Tunisa. Compared to Mediterranean spotted fever, eschars are rare (7%) and more frequent gastrointestinal symptoms reported. Mild to severe illness. 29% mortality among Portugal cohort. Rickettsia heilongjiangensis D. silvarum, H. concinna, H. japonica douglasi, H. flava 1982 Far Eastern spotted fever (1992) 1992, 1996 Russia, China, South Korea, Japan. Rash, eschar, and lymphadenopathy. Peak in Russia in July in patients aged 50+. Genetically related strain identified in severe case in Thailand. No fatal cases reported. Rickettsia helvetica Ixodes ricinus, I. ovatus, I. persulcatus, I. monospinus 1979 Unnamed (1999) 1999 Prevalent in Europe. Solitary cases identified in Laos and Thailand. Isolated in ticks in over 24 European countries, in Japan, Turkey, Algeria, and Morocoo. Although implicated in perimyocarditis and sarcoidosis, the validity of these associations has been debated or not accepted by rickettsiologists. Rash and eschar seem to occur rarely.

8.6.40  Rickettsioses 1233 (continued) Rickettsia sp. Recognized or potential tick vector(s) First identification in ticks Disease (first
clinical
description) First microbiological documentation
of human cases Selected clinical and epidemiological characteristics Rickettsia honei Bothriocroton hydrosauri Ixodes sp. 1962 Flinders island spotted fever (1991) 1992 In Asia, Australia, and Pacific. Disease occurs in predominantly rural settings. Peak in December and January. Mild disease, symptoms include fever, rash (85%), eschar (25%), and lymphadenopathy (55%). Rickettsia honei strain marmionii Haemaphysalis novaeguineae Ixodes holocyclus 2003–​2005 Australian spotted fever (2005) 2003–​2005 Four states in Australia (South Australia, Victoria, Tasmania, Queensland). Between February and June. Six confirmed cases including one with eschar and two with a maculopapular rash. Similar clinical presentation to Flinders Island spotted fever. ‘Rickettsia kellyi’ Unknown Not done Unnamed (2006) 2006 A single case in a 1-​year-​old boy with fever and maculopapular rash. Rickettsia japonica I. ovatus, D. taiwanensis Haemaphysalis longicornis, H. flava, H. hystricis, H. cornigera, H. formosensis 1996 Oriental or Japanese spotted fever (1984) 1985 Typical spotted fever in southwestern Japan, recently identified in South Korea and Thailand and Shenyang, China. Disease occurs in predominantly rural settings. Agricultural activities, bamboo cutting. April to October. Fever, headache, eschar (91%) and rash (100%). May be severe. One fatal case reported. Rickettsia massiliae Rhipicephalus sanguineus, R. turanicus, R. muhsamae, R. lunulatus, R. sulcatus, R. bursa, R. pusillus, I. ricinus, H. paraleachi, R, senegalensis, R. guilhoni, R. 1992 Unnamed (2005) 2005 The strain was obtained from the blood of a patient from Sicily in 1985, stored, and definitively identified in 2005. A second case was identified in 2008 in southern France, with fever, chorioretinitis, and rash. A third case, a traveller in Spain returning from Buenoes Aires was identified, symptoms included fever, purpuric rash, eschar on the right leg. The 4th case was a 13-​year-​old boy in Italy with fever, scalp eschar, and alopecia. One other potential case with positive serology was detected in a man bitten in England. Also detected in ticks in across Europe and in Israel, Morocco, Algeria, Tunisia, Uganda, Ethiopia, Arizona, and California. Rickettsia monacensis Ixodes ricinus, I. persulcatus, I. sinensis 1998 Spotted fever (2006) 2006 Two cases in tick-​bitten patients from Spain with fever and a maculopapular rash and one patient in Sardinia, Italy with an eschar. Detected in ticks across Europe, Morocco, Algeria, Tunisia, Korea, and China. Rickettsia parkeri Amblyomma maculatum A. americanum, A. triste, D. variabilis 1939 Unnamed (2004) 2004 Over 25 cases reported in the literature from North and Central America and South America (Argentina and traveller from Uruguay). Symptoms include fever, multiple eschars, and macuolopapular rash. ‘Rickettsia philipii’ (364D) 1966 2008 Several cases of a mild illness with eschar reported in California. Rickettsia raoultii Dermacentor reticulatus, D. silvarum, D. marginatus, R. pumilio, I. ricinus, D. nuttalli, D. niveus, H. ornithophila, H. shimoga, H. lagrangei, A. testudinarium, 1999 SENLAT/​Tick-​borne lymphadenopathy (2006) 2006 Human cases identified in France, Slovakia, Poland, and China. Detected in ticks acroos Europe, Algeria, Morocco, Far East Russia, Kazakhstan, China, Mongolia, and similar strains across Asia. Eschar on the scalp with cervical lymphadenopathy. Rickettsia rickettsii D. andersoni, D. variabilis R. sanguineus Amblyomma cajennense A. aureolatum D. nitens, A. americanum, A. imitator, Haemaphysalis leporispalustris, 1906 Rocky Mountain spotted fever (1899) 1906 Has the reputation of being the most severe tick-​borne spotted fever rickettsiosis. Case fatality rate of untreated infections can be >20%. However, case fatality has decreased dramatically in recent years in the USA, but fatal cases are still reported in South America. Peak occurrence during spring and summer. Eschars rarely reported. Broadly distributed in the western hemisphere and associated with several species of tick vectors. Fatal report of coinfection with Streptococcus pyogenes. Rickettsia sibirica mongolitimonae Hyalomma asiaticum H. truncatum, H. anatolicum excavatum, Rhipicephalus pusillus 1991 Lymphangitis associated rickettsiosis (1996) 1996 First isolated from ticks collected in Mongolia. Identified in Europe (France, Portugal, Greece, Spain) in the spring and summer, and Africa (Egypt, Algeria, South Africa). Detected in ticks in Senegal and Israel. No cases in Asia. 29 cases in the literature. Symptoms include fever (100%), headache (86%), myalgia (90%), single or multiple eschars (92%), macuolopapular rash (77%), and lymphangitis (43%). Usually mild, although severe cases with septic shock have been described.

section 8  Infectious diseases 1234 Table 8.6.40.1  (Continued) Rickettsia sp. Recognized or potential tick vector(s) First identification in ticks Disease (first
clinical
description) First microbiological documentation
of human cases Selected clinical and epidemiological characteristics Rickettsia sibirica sibirica Dermacentor nuttalli, D. marginatus, D. silvarum, D. reticulates, D. sinicus, Haemaphysalis concinna, H. yeni, Ixodes persulcatus. Unknown Siberian tick typhus (1934) 1946 Probably most prevalent rickettsiosis in Asia. Identified in Siberia, Russia, China, Mongolia, Kazakhstan, and South Korea. Cases occur during spring and summer. Increasing reports of cases. Cases generally associated with rash (100%), eschar (77%), and lymphadenopathy. Usually mild. Severe case of subspecies sibirica BJ-​90 with multiorgan dynsfunction reported in China in 2013. Dermacentor sinicus 1974 North Asian tick typhus (1977) 1984 Rickettsia slovaca Dermacentor marginatus Dermacentor reticulatus 1968 SENLAT, Tick-​borne lymphadenopathy
(1997) Dermacentor-​ borne necrosis and lymphadenopathy (DEBONEL)(1997) 1997 2003 Present throughout mainland Europe. Cases present March to May, September—​November (increased activity of Dermacentor ticks). Detected in ticks in North Africa, Russia, Georgia, and China. One possible human case in United Kingdom. More frequent in women and children. Syndrome characterized by scalp eschars (64%) and neck lymphadenopathy (69–​ 100%) (SENLAT) Fever and rash rare. Other symptoms include asthenia, headache, facial oedema. Alopecia around the eschar and chronic asthenia may occur. Rickettsia tamurae Amblyomma testudinarium 2006 Unnamed 2011 Two human cases reported in Japan and Laos. Reservoirs include pigs and wild boars. ‘Candidatus Rickettsia tarasevichiae’ Ixodes persulcatus 2003 Unnamed 2013 Five human cases reported in China, symptoms included fever, asthenia, anorexia, nausea, and headache. Eschar present in three patients. One fatal case. Previously identified in Russian ticks. Potential pathogens Rickettsia amblyommii Amblyomma americanum, A. cajennense, A. coelebs. Various other Amblyomma species 1974 Unnamed (1993) 1993 Possible cause of mild spotted fever rickettsiosis in the USA. Rickettsia also recently identified in ticks in Central and South America. Rickettsia asiatica Ixodes ovatus, I. pomerantzevi 2006 Unknown Identified in sika deer in Japan. Rickettsia bellii Various species of Amblyomma D. occidentalis, D. variabilis, D. parumapertus, D. albipictus, H. leporispalustris, Argas cooleyi, Ornithodoros concanensis, H. juxtakochi, I. loricatus 1966 –​ –​ Distinct group of rickettsial diseases. Largest known number of tick hosts. Detected in ticks in the North, Central, and South America. No known cases in humans. Rabbits and guinea pigs develop eschars after inoculation. Rickettsia canadensis Haemaphysalis leporispalustris 1967 –​ –​ Possible Rocky Mountain spotted fever-​like disease described in California and Texas. Suspected cause of acute cerebral vasculitis in Ohio. Febrile response in guinea pigs. Rickettsia hoogstraalii Carios capensis, Haemaphysalis punctata, H. sulcata, Argas persicus, Ornithodoros moubata 2006 -​ -​ Initially identified in 2006 in soft ticks in Japan. Isolated in ticks from sheep and goats in Croatia, Cyprus, Spain, Ethiopia, Turkey, the United States, and the west Indian Ocean.

8.6.40  Rickettsioses 1235 for intracellular conditions. The variation in size ranges from 1.11 to 1.2 Mb, with a mean gene count of 1236.54 +-​281.22 and a mean GC content of 31.5 +-​ 1.52. This genome reduction, or degradation, is the driving force behind the adaptation of intracellular bacteria to life within a eukaryotic cell. Genome reduction by different methods, including reversible split genes and the creation of ‘pseudogenes’ and gene remnants, contributed to the specialization necessary to restrict potential hosts. A difference of 250 lost genes has been demonstrated between the group of rickettsioses associated with ticks (including R. conorii, Ri rickettsia, R africae and R massiliae) when separating from R. felis and R. akari. Comparisons between genomes have demonstrated that genome reduction is related to in- creased pathogenicity. Numerous studies have explored the genomic difference between pathogenic and less virulent strains, indicating that it is the likely loss of transcriptional regulating genes that cause pathogenicity, as opposed to the acquisition of other virulent genes. Further phylogenomic analysis has allowed further description of the differences and mix of rickettsial gene content between species. The core genome contains 566 genes. These primarily code for translation, ribosomal structure, the biosynthesis of cell wall/​membrane and ribo- somes, replication, and energy production and conversion. However, conservation of non​coding sequences has been docu- mented, with a potential, but yet undefined role. Although it was previously thought that Rickettsiae lack plas- mids, they have been identified in at least 10 species. The presence of plasmids might indicate horizontal gene transfer between rickett- siae. Furthermore, recently 165 rickettsial genes in R. felis have been identified as potentially originating from other bacteria, including R. belli, R typhi, Legionella sp. and Francisella sp. Chimeric genes between R. felis and R. typhi have also been identified. We are pre- sented with an increasingly diversified genetic depiction of rickett- siae and this will continue to change with future research. Pathophysiology There is a large variation between the pathogenicity of different Rickettsia spp, with no current identification of the precise mo- lecular basis. When transmitted to a susceptible human host, patho- genic tick-​borne SFG rickettsiae localize and multiply in endothelial R. rickettsii str. Sheila Smith R. rickettsii str. lowa R. rickettsii str. H1p2 R. philipii str. 364D R. peacockii str. Rustic R. montanensis str. OSU 85-930 R. sibirica str. 246 R. sibirica subsp mongolitimonae R. africae ESF 5 R. parkert str. Portsmouth R. conorii str. Malish 7 R. slovaca str. 13-B R. slovaca str. D-CWPP R. honei R. heilongiiangensis 054 R. japonica YH R. massiliae MTU5 R. massiliae AZT80 R. aeschlimannii R. raoulrii R. helvetica C9P9 R. asiatica R. tamurae R. endosymbiont of Ixodes scapularis R. monacensis IrR/Munich R. akari str. Harford R. felis URRWXCal2 R. hoogstralii R. prowazekii str. Madrid E R. prowazekii str. BuV67 CWPP R. prowazekii str. Chernikova R. typhi str. TH1527 R. typhi str. Wilmington R. typhi str. B9991CWPP R. canadensis str. CA410 R. canadensis str. McKiel R. bellii OSU 85 389 R. bellii RML369 C R. atusralis str. Cutlack R. rhipicephalistr. 3 7 fernale6 CWPP Candidatus R. amblyommii str. GAT 30V Rocky mountain spotted fever Avirulent Unknown pathogenesis Unnamed rickettsiosis Unknown pathogenesis Unknown pathogenesis Siberian tick typhus Lymogabgutus associated rickettsiosis African tick bite fever R. parkeri ricketisiosis Mediterranean spotted fever Tick-borne lymphadenitis Flinders Island spotted fever Far-easern tick-borne rickettsiosis Oriental spotted fever Unknown pathogenesis Unnamed rickettsiosis Unknown pathogenesis Unknown pathogenesis Spotted fever rickettsiosis SENLAT Unnamed rickettsiosis Unknown pathogenesis Spotted fever rickettsiosis Unknown pathogenesis Spotted fever rickettsiosis Rickettsialpox Queensland tick typhus Spotted fever rickettsiosis Unknown pathogenesis Epidemic typhus Murin typhus Unknown pathogenesis Unknown pathogenesis R. rickettsii R. massiliae R. helevetica R. akari R. prowazekii R. canadensis R. bellii TG SFG Fig. 8.6.40.2  Phylogenetic tree of Rickettsia species and pathogenic potential. Reprinted from Infection, Genetics and Evolution, Vol 25, Merhej V et al., Genotyping, evolution and epidemiological findings of Rickettsia species, Pages 122–​37, Copyright © 2014, with permission from Elsevier.

section 8  Infectious diseases 1236 cells of small to medium-​sized blood vessels, causing a vasculitis that is responsible for the clinical and laboratory abnormalities that occur in tick-​borne rickettsioses. The severity of the Rickettsia is probably determined by the degree of growth on the endothelial cells, with the exception of R. akari (the Rickettsialpox agent) that mainly attacks macrophages. There are, therefore, numerous steps involved in the rickettsia-​host cell interaction and it is the molecular characteristics and expression of particular rickettsial gene products that contribute to differences in pathogenicity among species. Since Rickettsiae are obligate intracellular bacteria, the first steps involve adherence and invasion of the host cells. Expression of OmpA, a SFG rickettsia ‘outer membrane protein’ or surface antigen protein, allows adhesion and entry into host endothelial cells. TG rickettsia do not possess this OmpA, although an ORF remnant remains in R.  prowazekii. OmpB, a major surface antigen protein common to the genus Rickettsia, other outer membrane proteins, and new adhesins also contribute to adherence and invasion. After phago- cytosis and internalization, the phagocytic vacuole is rapidly lysed and rickettsiae escape phagocytic digestion to multiply freely in the host’s cytoplasm and nucleus (SFG species). The rickettsial infection not only causes host endothelial cell damage, but also initiates further endothelial activation as a form of procoagulant and proinflamattory cellular response, strongly cor- relating to the severity of the infection. R. rickettsia and R. conorii infection can cause surface platelet adhesion, increased expression of tissue factor, IL-​1A, cell adhesion molecules and plasminogen activator inhibitor-​1, and the release of von Willebrand factor. R. prowazekii infection can induce prostaglandin secretion. The inoculation eschar or ‘tache noire’, is the site of bacterial entry which has developed into a local inflammatory and necrotic skin le- sion. This eschar represents the first interaction between the human host and bacterium. There is an association between the eschar and the presence of many toxin-​antitoxin systems in the Rickettsia genome. This might be explained by the toxic effect of these sys- tems increasing the local reaction and constraining the spread of the Rickettsia, corresponding to the inverse correlation between toxin-​ antitoxin systems and human host mortality. Rickettsioses as an emerging infection Unlike fleas, lice, and mites, which have a global distribution, ticks are highly dependent on their biotopes and have reduced mobility resulting in more regional distributions of rickettsial infections. The spread of arthropods is often linked to host animal migration, including cattle and migratory birds. There are numerous factors that contribute to the increasing iden- tification of rickettsioses in previously undocumented areas. This includes increasing international travel, with tick-​borne spotted fever a more common cause than typhoid or dengue in travellers returning from sub-​Saharan Africa. Other causes include changes to land rehabilitation practices, such as forestry. Of particular im- portance to the changing epidemiology of rickettsioses, is the role of global climate change causing longer seasonal warm temperat- ures. Warmer climates can influence the rickettsial transmission by arthropods, due to increased aggressiveness and number of attacks by the brown dog tick Rh. sanguineus seen with higher tem- peratures. This has been noted clinically, with higher numbers of Mediterranean spotted fever cases and R. massilliae infections docu- mented during a warm period unusual for the time of year. Tick-​borne SFG rickettsioses Epidemiology Ixodid (hard) ticks were first implicated as vectors of SFG rickettsioses in 1906, when the Rocky Mountain wood tick (Dermacentor ander­ soni) was shown to transmit R. rickettsii, the agent of Rocky Mountain spotted fever in the United States of America. In the 1930s, the role of the brown dog tick (Rhipicephalus sanguineus) in transmitting R. conorii, the causative agent of Mediterranean spotted fever, was described. However, between 1984 and 2015 at least 18 additional rickettsial species or subspecies causing tick-​borne rickettsioses around the world were identified. Numerous agents are often ini- tially isolated from ticks, often years or decades before a definitive association with human disease is established. Keys to the epidemi- ology of tick-​borne diseases are the ecological characteristics of their tick vectors. The life cycles of most tick-​borne rickettsiae are poorly understood. In their natural vertebrate hosts, infection can result in a rickettsaemia that allows non​infected ticks to become infected and for the natural cycle to be perpetuated. Ticks can also acquire rickettsiae through transovarial passage. Because ixodid ticks feed only once at each life stage, the rickettsiae acquired can only be transmitted to another host when the tick has moulted to its next developmental stage (transstadial passage) and takes its next blood meal. When rickettsiae are efficiently transmitted both transstadially and transovarially, the tick serves as a reservoir and the distribution of the rickettsiosis and its tick host will be identical. However, transmission of R. rickettsii by Dermacentor andersoni diminishes the ticks’ survival and reproductive capacity of their filial progenies. R. rickettsii has been shown to be lethal for most experimentally and transovarially infected Dermacentor andersoni. Similarly deleterious effects have been reported in Rhipicephalus san­ guineus group ticks experimentally infected by R. conorii conorii. This has been suggested as a potential reason to explain a low prevalence of Rh. sanguineus infected with R. conorii in nature (usually <1%). However, naturally infected colonies of ticks have been maintained in laboratory conditions over several generations. External factors such as temperature can have an essential role in the survival of Rh. sanguineus naturally infected with R. conorii compared with uninfected, in liaison with the long-​recognized phenomenon known as reactivation—​that is, the change in temperature and physiology of the tick host induces the rickettsia to emerge from dormancy and attain infectivity with bad effects on ticks. Clinical features Symptoms of tick-​borne SFG rickettsioses begin 4 to 10 days after the bite and typically include fever, headache, muscle pain, rash, local lymphadenopathy, and, for some diseases, a typical inoculation eschar (the ‘tache noire’) at the site of the tick bite (Fig. 8.6.40.3). These signs vary depending on the rickettsia involved and might allow distinction between different rickettsioses occurring at the same geographical location (Table 8.6.40.1). For example, there is no eschar in Rocky Mountain spotted fever, whereas they do occur in R. parkeri infections. European Dermacentor ticks that bite hu- mans are most active during early spring, autumn, and occasionally winter, and are well known to bite on the scalp. Since R. slovaca is transmitted by Dermacentor ticks, the inoculation eschar of R. slo­ vaca infection is characteristically located on the scalp during these seasons (Fig. 8.6.40.4).

8.6.40  Rickettsioses 1237 SFG rickettsioses range in severity from mild to severe and fatal disease. Common non​specific laboratory abnormalities in rickettsioses include mild leucopenia, anaemia, and thrombocyto- penia. Hyponatraemia, hypoalbuminaemia, and hepatic and renal abnormalities can also occur. Elevated C-​reactive protein and lactate dehydrogenase are also common. Agents and diseases around the world Since 2008, five further species or subspecies of SFG rickettsiae have been confirmed to infect humans, bringing the total to 25, with a further five potential pathogens. Table 8.6.40.1 demon- strates the characteristics of tick-​borne rickettsiae identified in human infections by 2015. Newly identified rickettsia in humans since 2008 include Rickettsia sp. Strain Atlantic rainforest or strain Bahia, Candidatus Rickettsia tamaresvichiae, Rickettsia tamurae, and Rickettsia 364D. Rickettsia helvetica has been confirmed as a pathogen. Newly identified potential pathogens include Rickettsia asiatica and Rickettsia hoogstraalii. Geographical distributions have been updated in Figs. 8.6.40.5–​8.6.40.12. There are more rickettsiae ‘of unknown pathogenicity’ or ‘sus- pected to be pathogens’ to be identified as emerging pathogens in the near future. Mediterranean spotted fever This is one of the oldest known vector-​borne diseases. Rickettsia conorii conorii is the agent of Mediterannean spotted fever, which is endemic to southern Europe. Foci of cases are sometimes seen in Northern and Central Europe. Increasing numbers of cases are reported in Algeria, Tunisia, and Morocco. Rickettsia conorii conorii has been identified in nine sub-​Saharan African countries. The main vector is Rhicephalus sanguineus, the brown dog tick. Studies on nat- urally infected Rhicephalus sanguineus have demonstrated very high transovarial transmission rates (100%) over several generations. The animal reservoir has yet to be conclusively demonstrated, with high seroprevalence rates noted in dogs and hedgehogs. The usually low affinity of Rhipicephalus sanguineus for people increases in warmer temperatures and, in Europe, most cases occur in the summer. Classical risk factors include advanced age, immunodepression, chronic alcoholism, glucose-​6-​phosphate dehydrogenase deficiency, previous inappropriate antibiotic use, and delayed treatment. The most common presentation includes fever (94–​100%), flu-​ like symptoms (78%), asthenia (64%), single eschar (53–​57%), and maculopapular or petechial rash (87–​96%); the rash can spread to the palms and soles. A large case series in Portugal reported the most common symptoms as fever, myalgia, arthralgia, and headache. Clusters of cases and multiple eschars have been reported. These are novel findings, since the probability of receiving multiple concomi- tant tick bites is thought to be small. Furthermore, life-​threatening complications and atypical presentations have been reported. These include cardiac complications such as myocarditis and atrial fibril- lation. Rare complications include ocular and neurological symp- toms, pancreatic involvement, splenic rupture, acute renal failure, and haemophagocytic syndrome. The case fatality rate has been reported as 2.5%. Similar mortality rates of 3.6% were seen in case series in Algeria and Portugal, al- though previous mortality rates have been reported of up to 13%. Predictors of mortality included hyperbilirubinaemia, acute renal failure, and the absence of rash. Rocky Mountain spotted fever The agent of Rocky Mountain spotted fever is Rickettsia ricketsii. It is the most commonly reported SFG rickettsioses reported in the United States of America, but confirmed cases have also been re- ported across North and South America, including Canada, Mexico, Panama, Costa Rica, Argentina, Brazil (Brazilian spotted fever) and Columbia. The incidence of Rocky Mountain spotted fever in the United States was reported at seven cases per million in 2007, rising sharply to 14.3 cases per million to 2012, although this is likely ex- plained by cross-​reactivity of serological tests, highlighting the im- portance of polymerase chain reaction (PCR) to clearly identify the rickettsial species and improve disease surveillance. Symptoms include sudden onset fever, headache, nausea and vomiting, anorexia, and generalized myalgia. A maculopapular rash appears on the second to fourth day of illness, becoming petechial (a) (b) (d) (c) Fig. 8.6.40.3  Inoculation eschar, the hallmark of SFG rickettsiosis which may be absent or uncommon in some specific diseases, such as Rocky Mountain spotted fever, or associated with a lymphangitis, as in the case of R. sibirica mongolitimonae (a) and R. africae infection (b), or a rash, as in R. africae (c) and R. heilongjiangensis infection (d). (a) From Fournier PE, et al. (2000). Rickettsia mongolotimonae: a rare pathogen in France. Emerg Infect Dis, 6, 290–​2, with permission; (b) copyright DA Warrell; (c) copyright Dr Ed Dunbar, Manchester; (d) from Mediannikov O, et al. (2004). Acute tick-​borne rickettsiosis, caused by Rickettsia heilongjiangensis variant in the Russian Far East. Emerg Infect Dis, 10, 810–​17, with permission. (a) (b) Fig. 8.6.40.4  Patients with R. slovaca infection. Inoculation lesion on the scalp (a), residual alopecia (b). From Gouriet F, Rolain JM, Raoult D (2006). Rickettsia slovaca infection, France. Emerg Infect Dis, 12, 521–​3, with permission.

section 8  Infectious diseases 1238 or purpuric in 50–​60% of patients. The illness can rapidly progress, causing a severe vasculitis. The case fatality rate 2008–​2012 in the United States was low (0.4%, rising to 10% in Arizona, particularly among affected tribal communities), in comparison to previous re- ports of untreated case fatality rates around 20–​25%. A more severe clinical picture is seen in South America, with severe and fatal cases of R.  rickettsii infection reported. Higher case fatality rates have been reported in populations living in endemic regions in Brazil and paediatric populations in Mexico. Numerous host factors can result in increased severity, for example, increasing age, male gender, and presence of G6PD deficiency. Severe complications include renal failure, pulmonary oedema, cerebral oedema, and disseminated intravascular coagulopathy. African tick bite fever The pathogen Rickettsiae africae has been identified across most of the African continent and causes African tick bite fever, where it is most commonly transmitted through Amblyomma ticks, mainly A. hebraeum and A. variegatum. Human cases have also been identified in the Caribbean and West Indies. These ticks demonstrate aggressive behaviour and in areas of high endem- icity have been found to have high infection rates, reaching 100%. Naturally infected A.  variegatum can have a 100% transovarial transmission rate. The disease occurs in predominantly rural settings and is com- monly associated with international travellers returning from safari, hunting, camping, or adventure races. It has been identified as the second most common illness, after malaria, in travellers from sub-​ Saharan Africa. African tick-​bite fever is characterized by the occur- rence of multiple inoculation eschars in clusters of cases, explained by simultaneous mass attacks by infected Amblyomma hebraeum ticks at a particular geographical location, for example, groups of tourists on safaris. Clinical symptoms include sudden onset of fever, fatigue, myalagia, and a headache 5–​7 days following a tick bite. Inoculation eschars are reported in 50–​100% of cases. Other symptoms include generalized maculopapular or papulovesicular rash and regional lymphadenopathy. More serious complications can occur, especially in older people. Complications include myocarditis and neuropathy, but no fatal cases have been reported. Infection caused by R. felis Epidemiology R. felis was probably first detected in cat fleas (Ctenocephalides felis) in 1918 and rediscovered in 1990. R. felis was initially characterized by molecular biology techniques and named the ELB agent for the EL Laboratory (Soquel, California, United States of America). In 1994, ELB agent DNA fragments were detected in blood samples from a Texan patient that had been kept since 1991. In 1994 and 1995, iso- lation of the ELB agent was reported and the name R. felis was pro- posed, but it was not cultivated definitively at low temperature and fully characterized until 2001 in Marseille, France. Infections caused by R. felis have been initially called flea-​borne spotted fever or cat flea typhus. R. rickettsii R. parkeri R. massiliae “Rickettsia philipii” (364D) “Candidatus R. amblyommii” “Candidatus R. andeanae” R. bellii R. canadensis “Candidatus R. cooleyi” R. montanensis R. peacockii R. rhipicephali Fig. 8.6.40.5  Tick-​borne rickettsiae in North America (except Mexico). Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702. R. rickettsii R. africae R. bellii “Candidatus R. amblyommii” Fig. 8.6.40.6  Tick-​borne rickettsiae in Mexico and Central America (except Mexico). Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O,
Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702.

8.6.40  Rickettsioses 1239 R. africae R. conorii conorii R. conorii israelensis R. conorii caspia R. conorii indica R. sibirica mongolitimonae R. aeschlimannii R. slovaca R. raoultii R. massiliae R. monacensis R. hoogstraalii R. helvetica R. rhipicephali Rickettsia sp. DmS1 «Candidatus R. barbariae» Rickettsia sp. AvBat «Candidatus R. kuligani» «Candidatus R. siciliensis» «Candidatus R. rioja» «Candidatus R. vini» Rickettsia sp. strain Davousti «Candidatus R. kotlanii» Fig. 8.6.40.8  Tick-​borne rickettsiae in Europe. Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702. R. rhipicephali R. bellii R. monteiroi “Candidatus R. amblyommii” “Candidatus R. andeanae” R. rickettsii R. parkeri R. massiliae Rickettsia strain Atlantic rainforest (or strain Bahia) Fig. 8.6.40.7  Tick-​borne rickettsiae in South America. Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Modified from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702.

section 8  Infectious diseases 1240 Human cases have been identified worldwide, in Europe, Northern Africa, Asia, and Central and South America. More re- cently, cases in Australia have also been reported. Numerous fleas have been associated with R. felis, including C. felis, C. canis, Pulex irritans, Archeopsylla erinacei, and Anomiopsyllus nudata. The sole confirmed biological vector for R. felis is the cat flea (Ctenocephalides felis), however within the past few years new potential vectors have been identified. In 2012, molecular tools enabled its identification in mosquitoes, including Aedes albopictus in Libreville, Gabon, and Anopheles gambiae from the Côte d’Ivoire. Furthermore, the iden- tification of R. felis by molecular techniques in febrile patients in regions endemic for malaria suggests that mosquitoes may be com- petent vectors for this infection. Studies have demonstrated this bac- terium being found in up to 15% of patients with ‘fever of unknown origin’ in sub-​Saharan Africa. These new findings and the sharing of a common vector for Plasmodium highlights the importance of this emerging infection, with suggestions that R. felis infections should be considered in all malaria endemic regions. The potential of An. gambiae as vectors of R. felis was further demonstrated, with a recent study demonstrating rickettsemias in mice bitten by R. felis infected An. gambiae. Further study is needed on the life cycle of R. felis, its potential reservoir hosts, and the role of humans as these reservoirs. R. conorii conorii R. conorii israelensis R. aeschlimannii R. massiliae R. monacensis R. helvetica R. slovaca R. raoultii R. sibirica mongolitimonae R. africae Rickettsia sp. R. hoogstraalii Fig. 8.6.40.9  Tick-​borne rickettsiae in North Africa. Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Modified from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702. R. africae R. conorii conorii R. conorii caspia R. massiliae R. sibirica mongolitimonae R. aeschlimannii R. rhipicephali Rickettsia hoogstraalii Rickettsia sp. Strain Davousti «Candidatus R. liberiensis» Rickettsia sp. Strain Uilenbergi Fig. 8.6.40.10  Tick-​borne rickettsiae in Africa. Pathogenic rickettsiae are
indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach.
Clin Microbiol Rev, 26(4), 657–​702.

8.6.40  Rickettsioses 1241 Clinical features The principal symptoms reported in the literature are non​specific, commonly headache, fever, and a maculopapular rash. Single eschars can also be reported, making it difficult to distinguish from tick-​borne spotted fevers. Other reports include di- gestive symptoms and pneumonia. Neurological involvement has also been reported, including acute polyneuropathy and cases in Sweden include subacute meningitis, Bell’s palsy, and deafness. Rickettsialpox Epidemiology Rickettsialpox is a cosmopolitan mite-​borne spotted fever rickettsiosis caused by R. akari. Originally described in New York R. sibirica sibirica R. heilongjiangensis R. japonica R. sibirica mongolitimonae R. conorii indica R. conorii conorii R. conorii israelensis R. aeschlimannii R. raoultii R. slovaca R. helvetica R. massiliae R. monacensis R. honei R. africae «Candidatus R. kellyi » R. tamurae « Candidatus R. tarasevichiae » R. asiatica Rickettsiasp. Rickettsia sp. IG-1 R. hoogstraalii «Candidatus R. principis » R. bellii Rickettsia sp. Rickettsia sp. TwKM02 Fig. 8.6.40.11  Tick-​borne rickettsiae in Asia. Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Modified from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702. Rickettsia gravesii Rickettsia honei strain “marmionii” Rickettsia honei Rickettsia australis Rickettsia argasii Rickettsia africae Fig. 8.6.40.12  Tick-​borne rickettsiae in Australasia. Pathogenic rickettsiae are indicated by coloured symbols and rickettsiae of possible/​unknown pathogenicity by white symbols. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702.

section 8  Infectious diseases 1242 in 1946, it is still reported mainly in the United States of America. Only three confirmed cases are reported from Europe:  Ukraine, Croatia, and the Netherlands. Low R. akari antibody titres in sero- logical surveys and case reports have been documented in Albania, France, Germany, and Italy, but no confirmed cases. Recently, rickettsialpox emerged in Turkey. The disease is probably ubiquitous but underdiagnosed, particularly in the tropics. House mouse mites (Liponyssoides sanguineus) are the primary reservoir host of R. akari, haematophagous arthropods that main- tain R. akari in house mice (Mus musculus). It transmits the disease when nymphs or adults bite people exposed by contact with house mice. This mite has been harvested from various other rodents in the United States of America, Eurasia, Africa, and Korea. Clinical features The first identified case in New York was an 11-​year-​old boy who presented with a high fever, a papulovesicular lesion on his back, and axillary lymphadenopathy and, over the next few days, developed a diffuse rash and fever (40.5°C) despite penicillin therapy. He made a complete recovery. During the next few months more than 100 more cases were recognized and the causative agent, named R. akari from the Greek word for mite, was described. Rickettsialpox is often described as chickenpox-​like because the rash is often vesicular. In 83–​100% of cases, a primary eschar ap- pears at the site of a mite bite, starting as a painless vesicle that rup- tures and a dark-​brown or black crust develops over the lesion. The exanthem consists of 2–​10-​mm-​diameter discrete erythematous maculopapules distributed over the extremities, abdomen, back, chest, and face, but only rarely on palms and soles. After 2–​3 days, some lesions become indurated and small vesicles develop. Around a quarter of patients have vesicular buccal mucous membrane le- sions. Symptoms 2–​7 days after the appearance of the primary lesion include fever, sweating, lassitude, myalgia, and headache, which per- sist for 7–​10 days in the absence of antibiotic treatment. Although generally described as benign and self-​limiting, neurological symp- toms such as photophobia, vertigo, pain on movement of the eyes, and nuchal rigidity may be severe enough to warrant lumbar punc- ture. R. akari has been isolated from eschar biopsy specimens from patients with rickettsialpox. Typhus group Murine typhus Epidemiology Murine or endemic typhus was probably first reported by Bravo in Mexico in 1570, making it one of the oldest recognized arthropod-​ borne zoonoses. The first case was described clinically in grain silo workers in Australia and the disease distinguished from epidemic typhus in the 1920s. The causative organism was named R. mooseri and thereafter R. typhi. Its main vector is the rat flea (Xenopsylla che­ opis) while rodents, mainly Rattus norvegicus and Rattus rattus, are its reservoirs. Other fleas or arthropods can also transmit R. typhi, including cat fleas (C. felis), mouse fleas (L. segnis), lice, mites, and ticks, and other rodents and wild and domestic mammals may be hosts. The classic cycle of infection is flea-​borne between rats. R. typhi is only rarely transmitted transovarially in fleas. Rats are not fatally infected and rickettsaemia persists from day 7 to day 12 after inoculation. Fleas are infected for life, but their lifespan is not shortened. Rickettsiae are excreted in their faeces, where they re- main viable for several years. Most people are thought to become infected when flea faeces containing R. typhi contaminate disrupted skin or are inhaled into the respiratory tract. Rarely, infections can result from flea bites. Murine typhus is distributed worldwide but is often unrecognized, especially in tropical countries. Cases are regularly documented in the United States of America (recently in 2013 in Texas), Mexico, and Europe, and it recently re-​emerged in Japan. Ideas of prevalence are based principally on serosurveys and on cases in travellers from China, Indonesia, India, Morocco, Canary Islands, Africa, Malaysia, Thailand, and Vietnam. Serosurveys suggest that the disease is more prevalent in coastal areas of tropical countries, where rats are par- ticularly common. Epidemiological shifts have been described in the United States, such as in Southern California with a shift from urban Los Angeles to suburban cases due to opossums carrying R. typhi in- fected cat fleas. Murine typhus has also been reported from Tunisia, Brazil, and on the Thailand–Myanmar border. Recent studies have identified populations at risk in urban areas including the home- less, such as in Marseille, France, and immigrant workers, such as in Singapore. Murine typhus should be considered as an important differential diagnosis for fever of unknown origin. Furthermore, murine typhus should be considered in returning travellers with fever of unknown origin, especially those with unspecific symptoms and those returning from tropical or subtropical countries in Africa or Southeast Asia. Clinical features Murine typhus is a mild disease with non​specific features. The incu- bation period is 7–​14 days. Adults typically present with the classical triad of fever, headache, and rash, but this has been reported in less than 15% of cases in children. Fever and headache are more common than the rash, which can be of variable frequency (20–​80%) and often transient or difficult to observe. Among 83 patients in Crete, 49 (59%) presented with rash and 17 additional patients (20%) de- veloped rash subsequently. Fever (100%), headache (88%), and chills (87%) were also common. Nausea, abdominal pain, diarrhoea, jaundice, cough, confusion, and seizures have been reported and can lead to misdiagnosis. Less than 50% of patients report exposure to fleas or rats. In untreated patients, symptoms last for 7–​14 days, after which there is usually a rapid return to health. Neurological complications are rare, but have been reported, including aseptic meningitis and abducens nerve palsy, and are usually reversible with correct antibiotic treatment. Epidemic typhus Epidemiology Epidemic typhus is caused by R. prowazekii, a typhus-​group rick- ettsia. It is suspected to have been responsible for the ‘Great Plague’ of Athens in the 5th century bc. In 1909, Charles Nicolle discovered the role of lice in the transmission of typhus and later performed the first successful cultures in animals. He was rewarded with a Nobel Prize. The vectors of epidemic typhus, body lice (Pediculus humanus humanus or P. humanus corporis), are a problem particularly during times of war, conflict, famine, and natural catastrophes. They live in clothes and thrive in cold weather when clothes might be washed

8.6.40  Rickettsioses 1243 infrequently and general hygiene declines. After the Second World War, foci persisted in the cooler mountainous countries in Africa, but epidemic typhus was considered a disease of the past. However, in recent years, intermittent outbreaks have occurred in Africa (Ethiopia, Nigeria, Burundi), Mexico, Central America, South America, Eastern Europe, Afghanistan, northern India, and China. The most recent outbreak, the largest since the Second World War, occurred during the civil war in Burundi in the 1990s. Travellers are rarely infected, but those who visit or work with homeless popula- tions or refugee camps might be infected. R. prowazekii is transmitted to people when infected louse feeding sites are contaminated by their faeces, or when the conjunctivae and other mucous membranes are exposed to crushed bodies or faeces of infected lice. Transmission might also result from the inhalation of infected faeces, which is thought to be the main route of infection in health workers. People who survive epidemic typhus remain infected with R. prowazekii for life; when stressed, they might experience a recrudescence (Brill–​ Zinsser disease), and can be the source of a new epidemic if they become infested with body lice. Sporadic cases continue to be re- ported in the United States. Humans were long considered the sole reservoir of R. prowazekii but its discovery in flying squirrels and their ectoparasites in North America indicates an alternative res- ervoir. Sylvatic (flying squirrel) typhus has not yet been associated with human fatalities, but North American flying squirrel strains of R. prowazekii appear similar to those isolated from patients during louse-​borne outbreaks. A non​human typhus reservoir has also been reported in Ethiopia, where 10 isolates of R. prowazekii were obtained from hyalomma ticks recovered from livestock. In addition, evidence suggests an association of typhus-​group rick- ettsiae with ticks. There have been concerns about the use of R. prowazekii as a potential Category B bioterrorism agent. First developed by the Soviets in the 1930s, it has numerous qualities optimal for use as an aerosol weapon, including high infectivity and stability, high virulence, and the potential to engineer complete antimicro- bial resistance, while initially presenting only non​specific symp- toms. Physicians should be aware of the virulent Rickettsiae as potential infections and inefficacy of usual empirical antibiotic treatment. Clinical features After an incubation period of 10–​14 days, patients develop malaise and vague symptoms before the sudden development of fever (all cases), headache (all cases), and myalgia (70–​100%). In Burundi, a crouching attitude was observed, attributable to myalgia. Other common features are nausea or vomiting, coughing, and abnormal- ities of central nervous system function ranging from confusion to stupor and coma. Diarrhoea, pulmonary involvement, myocarditis, splenomegaly, and conjunctivitis can also occur. Most patients de- velop a macular, maculopapular, or petechial rash that classically begins on the trunk and spreads to the limbs (Fig. 8.6.40.13). It is difficult to detect in pigmented skins. Gangrene of the distal extrem- ities can occur in severe cases as mentioned in Thucydides’ descrip- tion of the Great Plague of Athens. Case fatality ranges between 4% in the antibiotic era up to 60% before antibiotics were available, with clinical severity dependant on age over 60 years and the patients’ nutritional state. Brill–​Zinsser disease can appear many years after the acute disease. It is less severe, and the rash is less frequent. Investigation and specific diagnosis Fig. 8.6.40.14 demonstrates the investigations that should be per- formed in the work up of a patient with clinical suspicion of rickett- sial infection. Serology Serological tests are the most frequently used and widely available methods for diagnosis. The Weil–​Felix test, the oldest test, is based on the detection of antibodies to various proteus antigens that cross-​ react with rickettsiae. Although it lacks specificity and sensitivity, it continues to be used in many developing countries. However, im- munofluorescence assay (IFA) is currently considered the reference method. Acute-​phase and convalescent-​phase serum specimens must be collected, several weeks apart. One limitation of serology is cross-​reactivity between antigens of pathogens within the same genus, and other genera. Furthermore, seroconversion is usually only detected at day 7–​15 post onset, extending to 25–​28 days for in- fection with R. africae. Most commercially available IFAs offer a very limited selection of antigens. IFA can be adequate to diagnose the class of infection (e.g. SFG rickettsiosis), but is unlikely to provide a specific aetiological agent unless more sophisticated assays are per- formed. Serology should be considered an initial, but not the sole, method for recognizing and diagnosing ‘emerging rickettsioses’. In the Unité des Rickettsies, Marseille, when cross-​reactions are noted between several rickettsial antigens, a rickettsia is considered to be causal when titres of IgG or IgM antibody against this antigen are (a) (c) (b) Fig. 8.6.40.13  (a) Rash in a patient with epidemic typhus due to R. prowazekii imported from Algeria to France. (b) Rash of epidemic typhus in an Ethiopian patient. (c) Peripheral gangrene in an Ethiopian patient with epidemic typhus. (a) From Niang M, Brouqui P, Raoult D (1999). Epidemic typhus imported from Algeria. Emerg Infect Dis, 5, 716–​18, with permission; (b) courtesy of the late
Dr P. L. Perine; (c) copyright D. A. Warrell.

section 8  Infectious diseases 1244 at least two serial dilutions higher than those against other rickett- sial antigens. When differences in titres between several antigens are lower than two dilutions, western blot assays and, if necessary, cross-​absorption studies are used. Other diagnostic tests have been developed, such as using monoclonal antibodies. Culture Rickettsial isolation in culture is the definitive diagnostic method, but can be performed only in P3 facilities that can maintain living host cells or cell cultures. The centrifugation shell-​vial technique using HEL fibroblasts has proved effective. Rickettsiae can be isolated from buffy coat preparations of heparinized or ethylenediaminetetraacetic acid (EDTA)-​anticoagulated whole blood, skin biopsies, and from arthropods. Culture results can be positive at 48–​72 hours post inoculation. However, the sensitivity of culture dramatically diminishes in pa- tients receiving antibiotic treatment; therefore, samples should be taken before treatment and inoculated as soon as possible. Histochemical and immunohistochemical procedures Rickettsiae can been detected in tissue specimens by various histochemical methods, including Giemsa or Gimenez staining. Immunohistochemical methods are superior for SFG rickettsiae in formalin-​fixed paraffin-​embedded skin biopsies, particularly es- chars (Fig. 8.6.40.15). Most available assays are SFG specific but not species specific. Molecular tools PCR and sequencing methods are sensitive and rapid tools for detecting and identifying rickettsiae in blood, skin biopsies, and from vectors. Primers amplifying sequences of several genes have been used. Real-​time quantitative PCR assays have been developed, as in the case of epidemic typhus. This could aid surveillance in public health programmes, especially for countries where human cases are underdiagnosed. Skin/​eschar swabs Performing PCR on an eschar swab is a new non​invasive ap- proach that has demonstrated significant results and played a role in improving the diagnosis of these emerging infections (Fig. 8.6.40.16). This approach allows more widespread documen- tation, as swabs can more easily be taken than skin biopsies, espe- cially in low-​resource settings, and sent to reference laboratories for quantitative PCR testing. This simple technique involves re- moving the crust of an eschar and rotating a sterile swab vigorously at the base of the eschar. Wetting a compress with humidified sterile water and placing on the eschar beforehand, increases the material collected. Murine typhus has also been identified by PCR on the biopsy of a petechial skin lesion. Vector identification Identifying the vector as part of the standard investigation process can help clarify the diagnosis. When possible, the tick, louse, or flea should be collected and analysed, both morphological identification under the microscope and using MALDI-​TOF. Arthropods are used as epidemiological tools to detect the presence of a pathogen in a specific geographical area. Knowledge of the correct species of the vector can help guide possible causes of infection in symptomatic patients. A recent study has proposed the dual identification, of both the tick species and pathogen infection status on collected ticks from patients using tick hemolymph protein mixture. This highly accurate, quick, and economical method is useful at the clinical level, with PCR Fig. 8.6.40.14  Investigations for clinical suspicion of rickettsioses: serology screening is often performed in local centralized laboratories. Specific PCRs are usually performed in National Reference Laboratories. Ticks should be sent in dry sterile pots to the National Reference Laboratory. All specimens should be accompanied with a request form with specific clinical details to aid diagnostic testing. Fig. 8.6.40.15  Inoculation eschar from a patient with African tick-​ bite fever showing numerous dermal inflammatory infiltrates mainly composed of polymorphonuclear leucocytes. Immunoperoxidase staining with an anti-​CD15 antibody; original magnification ×100. From Lepidi H, Fournier PE, Raoult D (2006). Histologic features and immunodetection of African tick-​bite fever eschar. Emerg Infect Dis, 12, 1332–​7, with permission.

8.6.40  Rickettsioses 1245 no entomological knowledge necessary for identification and might also be useful to help monitor tick-​borne diseases. Treatment and prognosis Early empirical antibiotic is the rule for any clinically suspected rickettsiosis, before confirmation of the diagnosis. SFG rickettsioses Doxycycline (200 mg/​day) is the treatment of choice for all SFG rickettsioses (Table 8.6.40.2), including Rocky Mountain spotted fever or other severe rickettsial disease in young chil- dren. Duration of antibiotic therapy for SFG rickettsioses is gov- erned more by clinical response than a statutory number of days. However, for most of these infections, therapy should continue for at least 3 days after the patient’s fever has subsided. A single dose of 200 mg doxycycline has proved adequate for Mediterranean spotted fever, but patients with severe SFG rickettsioses should be given doxycycline intravenously for up to 24 h after they become afebrile; however, this is not available in all countries. Macrolides can also be used as an alternative in the case of pregnant women, where doxycycline is contraindicated. Josamycin (1 g tds) may also be used in certain situations with SFG rickettsioses without severe disease, including pregnant women with Mediterranean spotted fever; however, strict follow-​up is essential. Other macrolides that can be used include clarithromycin (15 mg/​kg/​day) and azithromycin (10 mg/​kg/​day); however, erythromycin was found to be inferior to doxycycline and is not advised. Chloramphenicol is an alternative, but its use is limited by perceived side effects and it should only be considered as empirical treatment of severe cases if it is the only available drug, as in developing countries. Fluroquinolones are no longer recommended, as they are asso- ciated with worse outcomes in cell culture models and human R. conorii infections. Many classes of broad spectrum antibiotics including penicillins, cephalosporins, and aminoglycosides are ineffective against rickettsial diseases. Murine typhus Doxycycline is the drug of choice for non​pregnant adults and chil- dren. The optimal duration of therapy has not been assessed in clin- ical studies but 7 to 15 days, or for at least 48 h after the patient has become afebrile, has been recommended. A single dose of 200 mg doxycycline also proved adequate. Response to doxycycline is rapid with defervescence in 2 to 3 days. Chloramphenicol is an alternative, Table 8.6.40.2  Treatment options for SFG rickettsioses Preferred treatment for adults/​children Alternatives Tick-​borne rickettsioses Spotted fever group with inoculation eschar Mediterranean spotted fever
(Rickettsia conorii conorii) Doxycycline 200 mg two oral doses in a single day (preferred) or 200 mg single dose or 100 mg twice daily for 2–​5 days For children <45 kg: Doxycycline 2.2 mg/​kg every 12 h Josamycin 1 g every 8 h for 5 daysa (preferred for pregnant women, single dose of Doxycycline if severe forms) Clarithromycin 15 mg/​kg/​day Rocky Mountain spotted fever
(Rickettsia rickettsii) Doxycycline 100 mg every 12 h for at least 3 days after the fever subsides, standard duration of treatment 7–​14 days For children <45 kg: Doxycycline 2.2 mg/​kg every 12 h Chloramphenicol 60–​75 mg/​kg/​day in four divided doses (12.5–​25 mg/​kg every 6 h for 5–​10 days in children), may be considered if mild illness in pregnant women (otherwise doxycycline first line) All other SFG rickettsioses (conorii subspecies, sibirica sibirica, sibirica monolitimonae, australis africae,
and slovaca) Doxycycline 200 mg single dose or 100 mg twice daily for 2–​5 days Severe forms of spotted fever group Doxycycline IV (not available in all countries) Typhus group Murine typhus (Rickettsiea typhi) Doxycycline 100 mg twice daily for 3 days after resolution of symptoms Louse-​borne Epidemic typhus
(Rickettsia prowazekii) Doxcycline 200 mg for 5 days or 2–​4 days after defervescence Chloramphenicol a Erythromycin has been found to be inferior to doxycycline (Munoz Espin Arch Dis Child 1986), however other macrolides have also been used to treat 12–​27% of MSF patients, such as clarithromycin (15 mg/​kg/​day) and azithromycin (10 mg/​kg/​day). Fig. 8.6.40.16  Swabbing a skin eschar in the diagnosis of Rickettsia sibirica mongolitimonae infection by PCR. Reprinted from Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O,
Kernif T, et al. (2013). Update on tick-​borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev, 26(4), 657–​702.

section 8  Infectious diseases 1246 with the reservations discussed earlier, but relapses have been re- ported. Fluoroquinolones proved effective in vitro against R. typhi, but the few clinical studies produced contradictory results. Other antibiotics effective against R. typhi in vitro, including rifampicin, thiamphenicol, macrolides, erythromycin, clarithromycin, josamycin, and telithromycin, have no clinical application, and amoxicillin, gen- tamicin, and trimethoprim/​sulphamethoxazole are ineffective. Epidemic typhus Tetracycline and chloramphenicol are effective. Chloramphenicol is still widely used as empirical treatment of fever in tropical developing countries since its broad spectrum includes other ser- ious infections, such as meningococcaemia and typhoid fever, that can initially mimic epidemic typhus. Most patients improve mark- edly within 48 hours of starting treatment with either of these anti- biotics. However, many physicians prefer to use tetracycline for all typhus diseases, as it is cheaper and safer. A single dose of 200 mg doxycycline, the reference treatment, is extremely efficient. Few or no relapses are observed with this treatment, which should be pre- scribed for any suspected case, including children, as no risk of tooth staining has been demonstrated with this regimen. Ciprofloxacin should be avoided. Management should also include de-​lousing to prevent further human transmission. Human ehrlichioses and anaplasmosis According to the current classification, the family Anaplasmataceae comprises the genera Anaplasma, Ehrlichia, Aegyptianella, Neorickettsia, and Wolbachia, as well as two candidate genera, ‘Candidatus Neoehrlichia’ and ‘Candidatus Xenohaliotis’ These diseases, long thought to be of purely veterinary import- ance, are caused by bacteria of the family Anaplasmataceae. Ten species are now implicated in human diseases (Table 8.6.40.3) with the three main infections:  Ehrlichia chaffeensis causing human monocytic ehrlichiosis, Anaplasma phagocytophilum causing human anaplasmosis, and E. ewingii causing granulocytic ehrlichiosis. These diseases are tick-​borne zoonoses whose causa- tive agents are maintained through enzootic cycles between ticks and animals. Table 8.6.40.3  Human ehrlichioses and anaplasmosis in human infections in 2015 Genus Pathogen Disease Target cell Geographical region Vector Clinical symptoms/​ investigations Ehrlichia Ehrlichia chaffeensis Human Monocytotropic ehrlichiosis (HME) Monocyte South-​eastern/​central, mid-​ Atlantic USA Amblyomma americanum Fever, malaise, fatigue, headache, nausea, and vomiting. Thrombo-​cytopenia, lymphopenia E. ewingii Granulocytic ehrlichiosis Neutrophil South-​eastern/​central USA Amblyomma americanum E. canis HME Monocyte Reports from Venezuala Unconfirmed E. ruminantium Unconfirmed Monocyte Possible cases in South Africa Unconfirmed E. muris-​like agent HME-​like Monocyte Upper Midwestern states USA, patients exposed to ticks in Minnesota or Winsconsin, (2009) Ixodes scapularis Panola Mountain Ehrlichia Unconfirmed Atlanta, Georgia USA Amblyomma americanum No fever, myalgia Anaplasma Anaplasma phagocytophilum Human granulocytic anaplasmosis Neutrophils NE and Midwest USA, Europe Asia Ixodes persulcatus, Malaise, fever, myalgia, and headache. Thrombocytopenia, leucopenia elevated hepatic transaminase levels Anaplasma ovis Unnamed One case in Cyprus Fever, lymphadenopathy, and hepatosplenomegaly ‘Anaplasma capra’ Unnamed 28 patients in China, concurrently in local goats Ixodes persulcatus Fever, malaise, headache. Rash more common. Eschar in 3 patients Anaplasma platys Unnamed Platelets Possible case of a veterinarian after exposure in Grenada and South Africa Rhipicephalus sanguineus Migraines and seizures Neorickettsia Candidatus Neorickettsia sennetsu Infectious mononucleosis like syndrome Monocyte/​ macrophage Europe and China Ixodes ovatus, I. ricinus, I. persulcatus, I. frontalis Fever, headache, and malaise

8.6.40  Rickettsioses 1247 Bacteriology, taxonomy, and genomics The family Anaplasmataceae consists of intracellular alphaproteobacteria including human and mammal pathogens, whose host cells are of bone marrow or haematopoietic origin including erythrocytes, monocytes, or macrophages, neutrophils, and platelets (Fig. 8.6.40.17). Members of this family share a high degree of nucleotide sequence similarity in several chromosomal genes, such as rrs, groESL operon, gltA, RpoB, and Ank. The organ- isms grow within cytoplasmic vacuoles containing one to many in- dividual organisms, which resemble mulberries when observed by light microscopy, and have been called ‘morulae’ (Fig. 8.6.40.18). Anaplasma marginale, a cattle pathogen, was the first discovered, by Theiler in 1910. Since then, others have been described in ani- mals and humans. In 2001, improvements in molecular phylogen- etic methods modified the taxonomy of the Anaplasmataceae, based on comparison of sequences obtained from rrs (16s rRNA encoding gene) and the groESL operon. Analyses of other gene sequences and the complete genome sequencing of several species of the family (A. phagocytophilum, E. chaffeensis, E. ruminantium, N. sen­ netsu, and W. pipientis) have confirmed the new organization of the family Anaplasmataceae. Ehrlichia and anaplasma display a unique large expansion of immunodominant outer membrane proteins, facilitating antigenic variation. Unlike Rickettsiaceae, pathogenic Anaplasmataceae are capable of making all major vitamins, cofac- tors, and nucleotides, which could be beneficial to the invertebrate vector or the vertebrate host. Ehrlichia and anaplasma lack genes for biosynthesis of the lipopolysaccharide and peptidoglycan activating host leucocytes. Human monocytic ehrlichiosis Epidemiology The first human case of monocytic ehrlichiosis was identified in 1986, when intracytoplasmic inclusions were seen in monocytes in the peripheral blood smear of a severely ill man bitten by ticks in Arkansas, United States. This case was first assumed to be due to E. canis, the agent of monocytic canine ehrlichiosis, but E. chaffeensis was later isolated. E. chaffeensis is maintained in nature as a complex zoonosis, involving many vertebrate reservoirs for the bacterium and blood-​ meal sources for the tick vectors. The Lone Star tick (Amblyomma americanum) is its primary vector. All stages of this tick bite people. It is distributed in south, central, south-​eastern, and mid-​Atlantic areas of the United States of America, in meadows, woodlands, and hardwood forests. Primary hosts include many wild and domestic mammals, although deer are considered to be the definitive host. E. chaffeensis has been detected by PCR in other American ticks, but their role as vectors has not been demonstrated. There is no evidence of transovarial transmission, so ticks are not considered to be res- ervoirs. So far, the white-​tailed deer (Odocoileus virginianus) is the principal reservoir of E. chaffeensis, but domestic dogs (with mild Anaplasma capra KM206273 Anaplasma centrale Aomori AF283007 Anaplasma sp E1 JN558820 Anaplasma sp Rongchang EU709493 Anaplasma centrale SS40C-L AB211164 Anaplasma sp Kamoshika17 AB509223 Anaplasma marginale M60313 Anaplasma centrale AF318944 Anaplasma ovis AY262124 Anaplasma bovis U03775 Anaplasma platys M82801 Anaplasma phagocytophilum AY527213 Anaplasma phagocytophilum JFBJ01000002 Anaplasma phagocytophilum U02521 Ehrlichia chaffeensis M73222 Candidatus Neoehrlichia mikurensis AB084582 Candidatus Xenohaliotis californiensis AF133090 Wolbachia pipientis X61768 Neorickettsia sennetsu M73219 Rickettsia rickettsii L36217 Orientia tsutsugamushi D38623 Escherichia coli X80725 0.05 100 54 58 89 40 92 97 91 98 76 100 72 95 99 100 52 43 Fig. 8.6.40.17  Current phylogeny and taxonomic classification of genera in the family Anaplasmataceae. Reprinted from The Lancet Infectious Diseases, Vol 15(6), Li H et al., Human infection with a novel tick-​borne Anaplasma species in China: a surveillance study, Pages 663–​670, Copyright © 2015, with permission from Elsevier.

section 8  Infectious diseases 1248 to inapparent disease), red foxes, and domestic goats are potential reservoirs. Between 1999 and 2004, more than 1300 cases were reported to the Centers for Disease Control and Prevention (CDC). Cases con- tinued to increase, with 4613 cases reported by the national surveil- lance system between 2008 and 2012. Changes in the host–​vector ecology have influenced the emergence of monocytic ehrlichiosis, including increasing population densities and geographical distri- bution of Amblyomma americanum, increases in vertebrate host populations (wild turkeys, white-​tailed deer) for this tick, the in- creases in reservoir host population for E. chaffeensis (e.g. white-​ tailed deer), the increasing proportion of people older than 60 years of age, as well as available diagnostic procedures and improved sur- veillance and reporting. Most cases of monocytic ehrlichiosis occur in the south, central, and south-​eastern regions of the United States of America, where Amblyomma americanum reaches its highest prevalence. Monocytic ehrlichiosis is a seasonal disease whose incidence correlates with the activity of both nymphs and adult ticks. Most cases occur from May to July. Incidence based on active surveillance is 10 times higher than the highest rates reported using passive surveillance. Monocytic ehrlichiosis seems to be prevalent in Brazil and has been reported from other parts of the world including Latin America, Europe, Africa, and Asia. These diagnoses were based on serological studies, so infection by closely related organism cannot be completely ruled out. Gene fragments closely related to those of E. chaffeensis have been detected by PCR in ticks and rodents trapped in continental Asia but, so far, the disease has been clearly identified only in the United States of America. Clinical diagnosis Tick bite or tick exposure is reported in 70–​90% of patients with monocytic ehrlichiosis. It is more common in males and can affect individuals of all ages, including children and elderly people. The incubation period is 1–​2 weeks (median 9 days). It presents as an undifferentiated febrile illness ranging in severity from a mild dis- ease to multisystem organ failure. More than one-​half of patients must be hospitalized and case fatality was reported at 1% (rising to 3% for adults >70 years and 4% for children <5 years). Spatio-​ temporal analysis identified poverty status, relative humidity, the di- urnal temperature range as risk factors for monocytic ehrlichiosis, demonstrating the potential implications climate change might have on this tick-​borne disease. Asymptomatic infection might also occur and, since Amblyomma americanum is the vector of other tick-​borne agents, coinfection is possible. Clinical features include fever (98%), headache (77%), myalgias (65%), vomiting (36%), rash (35%), cough (25%), and neurological findings with impaired consciousness (20%). The maculopapular, or diffusely erythematous rash involves trunk, extremities, and, less commonly, the face and can be petechial in later stages. Malaise (30–​ 80%), lymphadenopathy, gastrointestinal symptoms, pharyngitis, and, less frequently, conjunctivitis, dysuria, and peripheral oedema can also occur. Leucopenia, thrombocytopenia, and elevated hep- atic transaminase levels are the most common laboratory findings. Severe complications can include central nervous system and renal involvement, adult respiratory distress syndrome, and disseminated intravascular coagulation. E. ewingii granulocytic ehrlichiosis E. ewingii has been known since 1992 as the agent of canine gran- ulocytic ehrlichiosis, first described in a dog in Arkansas in 1971. The disease was described subsequently in several other states in the south-​eastern and south-​central United States of America, where the recognized vector is the Lone Star tick, Amblyomma america­ num. E. ewingii can also infect white-​tailed and South Carolina deer. Human infections with E. ewingii were first reported in 1999, when blood samples collected from 413 patients with possible ehrlichiosis in Missouri between 1994 and 1998 were analysed retrospectively. The CDC in the USA report only 55 confirmed cases of E. ewingii infection between 2008 and 2012, despite the proportions of E. ewingii and E. chaffeensis infections reported in animals being similar. It has been suggested that a milder human infection might be caused by E. ewingii, therefore resulting in fewer presentations to medical centres. No fatal infections with E. ewingii have been re- ported. As no antigens for E. ewingii were available between 2008 and 2012, all diagnoses have been confirmed by PCR. Sixty-​nine per cent (69%) of cases were reported in the states of Delaware and Missouri. An underlying immunosuppressive condition was noted in 26% with a hospitalization rate of 77%. Clinical signs include fever, headache, and thrombocytopenia, with or without leucopenia; a rash is rare. Transmission of E. ewingii infection via a platelet transfusion in an immunocompromised pa- tient has also been reported. (a) (d) (b) (c) Fig. 8.6.40.18  Anaplasma phagocytophilum (a) in human peripheral blood band neutrophil (Wright’s stain, original magnification ×1000), (b) in THP-​1 myelomonocytic cell culture (LeukoStat stain, original magnification ×400), (c) in neutrophils infiltrating human spleen (immunohistochemistry with haematoxylin counterstain, original magnification ×100), and (d) ultrastructure by transmission electron microscopy in HL-​60 cell culture (original magnification ×21 960). Courtesy of V Popov. From Dumler JS, et al. (2005). Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis, 1, 1828–​34, with permission.

8.6.40  Rickettsioses 1249 Human granulocytic anaplasmosis History Human granulocytic anaplasmosis was first identified in 1990 in a patient in Wisconsin, United States, who died with a severe febrile illness two weeks after a tick bite. Clusters of small bacteria, as- sumed to be phagocytosed Gram-​positive cocci, were seen inside neutrophils in the peripheral blood, but a careful review suggested the possibility of human ehrlichiosis. Over the ensuing two years, 13 cases with similar intraneutrophilic inclusions were identified. In 1994, through application of broad-​range molecular amplification and DNA sequencing, the causative agent was recognized as distinct from E. chaffeensis. First known as the ‘HGE agent’, the disease was renamed human granulocytic anaplasmosis (HGA). Vectors Ixodes ticks are the recognized vectors. A. phagocytophilum is main- tained in a transmission cycle with Ixodes persulcatus complex ticks, including I.  scapularis in the eastern United States of America, I. pacificus in the western United States of America, and I. ricinus in Europe. A role for I. persulcatus in Eastern Europe and Asia is also suggested. Tick infection is established after an infectious blood meal. The bacterium is transmitted in ticks transstadially but not transovarially, and so ticks are not reservoirs. The major mamma- lian reservoir for A. phagocytophilum in the eastern United States of America is the white-​footed mouse Peromyscus leucopus. Other small mammals and the white-​tailed deer, Odocoileus virginianus, can also be infected. Other reservoirs might include ruminants and other mammals. In Europe, horses, cattle, sheep, goats, dog, cats, and small mammals, particularly rodents, might be reservoirs. Epidemiology In the United States, the dynamics of HGA follows that of its vectors. Surveillance demonstrates increasing ranges for white-​tailed deer and I. scapularis, particularly to the south-​east. Case reports of HGA increased during 2008–​2012, with 2867 confirmed cases (mainly by PCR) and 4982 probable cases compared to previous summaries (Fig. 8.6.40.18). This increase might be explained by a change in re- porting practices and increased clinician awareness. The national reported incidence rate was 6.3 cases per million persons-​years, the main affected states being Minnesota, Wisconsin, and Rhode Island. Since 1997, the agent and disease have been recognized across 15 countries in Europe, where more than 60 cases have been docu- mented. Sporadic human cases have also been confirmed in China and the presence of A. phagocytophilum antibodies in humans pre- senting with rickettsia like infections in Japan. Seroepidemiological studies confirm that human A. phagocytophilum infection is highly prevalent in both the United States of America and in Europe. Clinical diagnosis HGA presents most commonly as an undifferentiated febrile illness occurring in spring or summer, with the highest number of cases seen May to August. The incidence rate increases with age, particu- larly aged 60 years and over. The most frequent symptoms are malaise (94%), fever (92%), myalgia (77%), and headache (75%). A minority of patients have arthralgia, gastrointestinal symptoms (nausea, vomiting, diar- rhoea), respiratory symptoms (cough, pulmonary infiltrates, acute respiratory distress syndrome), and liver or central nervous system disturbances. Rash was observed in 6%, but no specific rash has been described in HGA. Coinfections with other Ixodes-​borne agents such as Lyme borreliosis and babesiosis have been reported. Frequent laboratory abnormalities identified in up to 329 patients included thrombocytopenia (71%), leucopenia (49%), anaemia (37%), and elevated hepatic transaminase levels (71%). The case fa- tality rate in the United States between 2008 and 2012 was 0.3%, with no deaths in patients under the age of 50. Emerging Ehrlichioses/​Anaplasmoses In 2010, a Cypriot patient presented with fever, lymphadenop- athy, and hepatosplenomegaly following a tick bite. The identi- fied pathogen was Anaplasma ovis, known to infect goats and sheep worldwide, yet no other human cases have been reported. Recently, a new tick-​borne pathogen of human anaplasmosis has been identified in northern China, with the suggested nomenclature ‘Anaplasma capra’. This pathogen was initially discovered in goats and the authors, after active surveillance, identified 28 infected pa- tients living in areas where this pathogen had previously been iden- tified. Similar clinical features were identified, with an acute onset of fever, headache, and malaise. However, rash seemed more common than with HGA, and other differences included rarer occurrences of leucopenia, thrombocytopenia, and abnormal hepatic trans- aminases. Interestingly, an eschar was reported in three patients with no molecular biological or serological evidence of involvement of a Rickettsia species. Furthermore, the visualization of morulae in peripheral blood samples was less common and there was low-​to-​ undetectable seroreactivity to the A. phagocytophilum antigen. As it is thought that HGA infections are generally underreported, it is plausible to think other undiscovered pathogens in the Anaplasma genus might be causing human disease. Physicians, particularly in the United States and China, need to be aware of these emerging pathogens. Furthermore, we are likely to see the emergence of these pathogens in humans in other parts of the globe where Ixodes ticks are present; for example, A. phagocytophilum has been reported in ticks in Brazil, Russia, Japan, and Korea. Diagnosis Laboratory confirmation of human ehrlichioses and anaplasmosis is based on several tests that are not yet widely available for routine use. PCR on whole blood samples is highly sensitive in severe cases, but the sensitivity may be low (70%). A. phagocytophilum can be cultured in special conditions. Indirect immunofluorescence serology is the most widely available technique and is the recommended technique using paired acute and convalescent sera. However, limitations in- clude delay in seroconversion and possible false-​positive detection due to cross-​reacting bacteria. Laboratory criteria for diagnosis have been defined (see Boxes 8.6.40.1 and 8.6.40.2). Treatment Tetracyclines are the reference drugs in treating human ehrlichioses and anaplasmosis for patients of all ages. Doxycyline is the anti- biotic of choice, the recommend dosage is 100 mg for adults and 2.2 mg/​kg for children 8 years or older every 12 hours. It is recom- mended that the treatment be continued for 7–​10 days, or for at least 3–​5 days after defervescence. Most patients become afebrile within 1–​3  days following treatment, and alternate diagnoses

section 8  Infectious diseases 1250 should be considered if such a response is not achieved. E. chaf­ feensis is susceptible in vitro to rifampicin (without in vivo evi- dence) but resistant to aminoglycosides, macrolides, and ketolides, co-​trimoxazole, penicillin, cephalosporin, chloramphenicol, and quinolones. Rifampicin (300 mg bd for adults, 10 mg/​kg, max 300 mg/​dose for children) is only recommended in case of allergy, preg- nancy, or under 8 years of age. Prevention Currently, no vaccines are available for rickettsial infection. Prevention is based first on avoiding arthropod bites. The best method for avoiding tick, flea, and chigger bites is topical N,N-​ diethyl-​m-​toluamide repellent applied to exposed skin, and treat- ment of clothing (including army uniforms) with permethrin, which kills arthropods on contact. Those staying in infested area should routinely check their bodies for the presence of arthropods. Prompt tick removal using blunt rounded forceps is essential for the prevention of tick-​borne illnesses. In the case of epidemic typhus, louse eradication (e.g. in refugee camps) is the most important pre- ventive measure and is essential in the control of outbreaks. Since body lice live only in clothing, the simplest method of delousing is to remove and then destroy or wash and boil all clothing. Dusting of all clothing with insecticides kills body lice and reduces the risk of reinfestation. Weekly doxycycline, 200 mg, prevents scrub typhus and a single 200 mg oral dose of doxycycline seemed effective in the epidemic typhus outbreak in Burundi, but the efficacy against rickettsial infections of doxycycline (100 mg daily), used for malaria chemoprophylaxis, is untested. Likely future developments Although they are among the oldest known vector-​borne diseases, many new rickettsioses have emerged in recent years. What are the factors influencing their emergence and recognition? People are undertaking more outdoor activities and international ‘adven- ture’ tourism is developing in rural and remote areas, resulting in increased contact with arthropods and arthropod-​borne rickettsial pathogens. The role of the primary physician, including careful his- tory taking and physical and laboratory examinations, has been em- phasized; essential for the description of emerging SFG rickettsioses, such as Flinders Island spotted fever, Japanese spotted fever, and Astrakhan fever. Increasing clinical awareness and reporting of cases plays a crucial part in developing our understanding of the disease epidemiology. Considering not only the clinical symptoms but also the season, environmental risk factors, and geographical exposition in travellers will help skilful clinicians identify these in- fections. Knowledge on the appropriate diagnostic tests to perform is essential. Molecular techniques and non​invasive swabbing of es- chars have facilitated epidemiological studies of emerging human rickettsioses all over the world and, with the help of improved cul- ture systems, have incriminated new species as causes of human dis- eases. Further developments for the use of appropriate diagnostics are necessary, including the more widespread use of real-​time PCR assays and developments in convalescent serology. Box 8.6.40.1  Case definitions of Ehrlichioses Clinical evidence: Any reported fever and one or more of the following: headache, myalgia, anaemia, leucopenia, thromobocytopenia, or any hepatic transaminase elevation. 1) Human ehrlichiosis caused by Ehrlichia chaffeensis. Laboratory confirmed: • Serological evidence of a fourfold change in immunoglobulin G (IgG)-​specific antibody titre to E. chaffeensis antigen by indirect immunofluorescence assay (IFA) between paired serum samples (one taken in first week of illness and a second 2–​4 weeks later), OR • Detection of E. chaffeensis DNA in a clinical specimen via amplifi- cation of a specific target by polymerase chain reaction (PCR) assay OR • Demonstration of ehrlichial antigen in a biopsy/​autopsy sample by immunohistochemical methods OR • Isolation of E. chaffeensis from a clinical specimen in cell culture Laboratory supportive: • Serological evidence of elevated IgG or IgM antibody reactive with E. chaffeensis antigen by IFA, enzyme-​linked immunosorbent assay (ELISA), dot-​ELISA, or assays in other formats OR • Identification of morulae in the cytoplasm of monocytes or macro- phages by microscopic examination 2) Human ehrlichiosis caused by E. ewingii: E. ewingii DNA detected in a clinical specimen via amplification of a specific target by poly- merase chain reaction (PCR) assay. 3) Human anaplasmosis caused by Anaplasma phagocytophilum
(see separate case definition). 4) Human ehrlichiosis/​anaplasmosis—​undetermined. http://​c.ymcdn.com/​sites/​www.cste.org/​resource/​resmgr/​ps/​07-​id-​03.pdf From:  http://​wwwn.cdc.gov/​nndss/​conditions/​ehrlichiosis-​and-​anaplasmosis/​ case-​definition/​2008/​ Box 8.6.40.2  Case definition of human anaplasmosis by Anaplasma phagocytophilum—​laboratory criteria for diagnosis Supportive: Serological evidence of elevated IgG or IgM antibody reactive with A. phagocytophilum antigen by IFA, enzyme-​linked immunosorbent assay (ELISA), dot-​ELISA, or assays in other formats (CDC uses an IFA IgG cutoff of ≥1:64 and does not use IgM test results independently as diag- nostic support criteria), OR Identification of morulae in the cytoplasm of neutrophils or eosinophils by microscopic examination Confirmed: • Serological evidence of a fourfold change in IgG-​specific antibody titre to A. phagocytophilum antigen by indirect immunofluorescence assay (IFA) in paired serum samples (one taken in first week of illness and a second 2–​4 weeks later) OR • Detection of A. phagocytophilum DNA in a clinical specimen via amp- lification of a specific target by polymerase chain reaction (PCR) assay OR • Demonstration of anaplasmal antigen in a biopsy/​autopsy sample by immunohistochemical methods OR • Isolation of A. phagocytophilum from a clinical specimen in cell culture From:  http://​wwwn.cdc.gov/​nndss/​conditions/​ehrlichiosis-​and-​anaplasmosis/​ case-​definition/​2008/​

8.6.40  Rickettsioses 1251 Increasing cases of monocytic ehrlichiosis are being reported out- side the United States of America and numerous rickettsia, ehrlichia, or anaplasma species have been identified in arthropods, particularly ticks, throughout the world, although their pathogenicity for people has yet to be demonstrated. More studies throughout the world may lead to the continuing description of emerging rickettsioses, con- comitantly developing our knowledge on a variety of vector-​borne diseases, veterinary medicine, and travel medicine, demonstrating the spotlight needed on this emerging infectious disease paradigm. FURTHER READING Alvarez-​Hernandez G, et  al. (2015). Clinical profile and predictors of fatal Rocky Mountain spotted fever in children from Sonora, Mexico. The Pediatric Infectious Disease Journal, 34, 125–​30. Aung AK, et  al. (2014). Review article:  Rickettsial infections in Southeast Asia: implications for local populace and febrile returned travelers. Am J Trop Med Hyg, 91, 451–​60. Badiaga S, Brouqui P (2012). Human louse-​transmitted infectious dis- eases. Clin Microbiol Infect, 18, 332–​7. Bechah Y, et al. (2008). Review: epidemic typhus. Lancet Infect Dis, 8, 417–​26. Blanton LS, et al. (2015). Reemergence of murine typhus in Galveston, Texas, USA, 2013. Emerg Infect Dis, 21, 484–​6. Botelho-​Nevers E, et  al. (2012). Treatment of Rickettsia spp. infec- tions: a review. Expert Review of Anti-​infective Therapy, 10, 1425–​37. Brouqui P, et al. (2004). Guidelines for the diagnosis of tick-​borne bac- terial diseases in Europe. Clin Microbiol Infect, 10, 1108–​32. Chapman AS, et al. (2009). Cluster of sylvatic epidemic typhus cases associated with flying squirrels, 2004–​2006. Emerg Infect Dis, 15, 1005–​11. Chen S, et al. (1994). Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol, 32, 589–​95. Dahlgren FS, et al. (2015). Human granulocytic anaplasmosis in the United States from 2008 to 2012: a summary of national surveillance data. Am J Trop Med Hyg, 93, 66–​72. Dieme C, et al. (2015). Transmission potential of Rickettsia felis infec- tion by Anopheles gambiae mosquitoes. Proc Natl Acad Sci U S A, 112, 8088–​93. Drexler NA, et  al. (2015). National surveillance of spotted fever group rickettsioses in the United States, 2008–​2012. Am J Trop Med Hygiene. Freedman DO, et al. (2006). Spectrum of disease and relation to place of exposure among ill returned travelers. N Engl J Med, 354, 119–​30. Garcia-​Alvarez L, Palomar AM, Oteo JA (2013). Prevention and prophylaxis of tick bites and tick-​borne related diseases. Am J Infect Dis, 9, 104–​16. Georgiades K, Raoult D (2011). Genomes of the most dangerous epi- demic bacteria have a virulence repertoire characterized by fewer genes but more toxin-​antitoxin modules. PLoS One, 6, 1–​10. Gikas A, et al. (2009). Murine typhus in children: clinical and labora- tory features from 41 cases in Crete, Greece. Clin Microbiol Infect, 15 Suppl 2, 211–​2. Heitman KN, et al. (2016). Increasing incidence of ehrlichiosis in the United States: a summary of national surveillance of Ehrlichia chaf­ feensis and Ehrlichia ewingii infections in the United States, 2008–​ 2012. Am J Trop Med Hyg, 94, 52–​60. Hernández-​López A, et al. (2013). To tree or not to tree? Genome-​wide quantification of recombination and reticulate evolution during the diversification of strict intracellular bacteria. Genome Biol Evol, 5, 2305–​17. Jia N, et al. (2013). Human infection with Candidatus Rickettsia tarase­ vichiae. N Engl J Med, 369, 1178–​80. Kernif T, et  al. (2016). Emerging tick-​borne bacterial pathogens. Micrbiol Spectr, 4, doi: 10.1128/​microbiolspec.EI10-​0012-​2016. Labruna MB, et al. (2010). Genetic identification of Rickettsial isolates
from fatal cases of Brazilian spotted fever and comparison with Rickettsia rickettsii isolates from the American continents, American Society for Microbiology. Li H, et al. (2015). Articles: human infection with a novel tick-​borne Anaplasma species in China: a surveillance study. Lancet Infect Dis, 15, 663–​70. Lindblom A, Severinson K, Nilsson K (2010). Rickettsia felis infection in Sweden: Report of two cases with subacute meningitis and review of the literature. Scand J Infect Dis, 42, 906–​9. Lopez-​Velez R, et al. (2015). Novel Candidatus rickettsia species detected in nostril tick from human, Gabon, 2014. Emerg Infect Dis, 21, 325–​7. Mediannikov O, et al. (2013). Common epidemiology of Rickettsia felis infection and malaria, Africa. Emerg Infect Dis, 19, 1775–​83. Medina-​Sanchez A, et al. (2005). Detection of a typhus group Rickettsia in Amblyomma ticks in the state of Nuevo Leon, Mexico. Ann N Y Acad Sci, 1063, 327–​32. Merhej V, et  al. (2014). Review:  genotyping, evolution and epi- demiological findings of Rickettsia species. Infect Genet Evol, 25, 122–​37. Nilsson K, et al. (2014). Bell’s palsy and sudden deafness associated with Rickettsia spp. infection in Sweden: a retrospective and pro- spective serological survey including PCR findings. Eur J Neurol, 21, 206–​14. Parola P (2011). Rickettsia felis: from a rare disease in the USA to a common cause of fever in sub-​Saharan Africa. Clin Microbiol Infect, 17, 996–​1000. Parola P, et  al. (2013). Update on tick-​borne rickettsioses around the world:  a geographic approach. Clin Microbiol Rev, 26,
657–​702. Portillo A, et al. (2012). Rickettsioses in Europe. Elsevier Masson SAS, France. Raghavan RK, et  al. (2014). Bayesian spatio-​temporal analysis and geospatial risk factors of human monocytic ehrlichiosis. PLoS One, 9, e100850. Raoult D (1991). Antibiotic treatment of rickettsiosis, recent advances and current concepts. Kluwer Academic Publishers, Dordrecht. Regan JJ, et al. (2015). Risk factors for fatal outcome from rocky moun- tain spotted fever in a highly endemic area—​Arizona, 2002–​2011. Clin Infect Dis, 60, 1659–​66. Renvoisé A, et al. (2012). A case of rickettsialpox in Northern Europe. Int J Infect Dis, 16, e221–​2. Renvoisé A, Joliot AY, Raoult D (2009). Rickettsia felis infection in man, France. Emerg Infect Dis, 15, 1126–​7. Walker D (2016). Changing dynamics of human-​rickettsial inter- actions. Am J Trop Med Hyg, 94, 3–​4. Walter G, et al. (2012). Murine typhus in returned travelers: a report of thirty-​two cases. Am J Trop Med Hyg, 86, 1049–​53. Yoshikawa Y, et al. (2014). Anaplasma phagocytophilum antibodies in humans, Japan, 2010–​2011. Emerg Infect Dis, 20, 508–​9. Yssouf A, et  al. (2015). Detection of Rickettsia spp. in ticks by MALDI-​TOF MS. PLoS Negl Trop Dis, 9, e0003473. Zhang J, et al. (2014). First report of Rickettsia felis in China. BMC Infect Dis, 14, 682.

8.6.41 Scrub typhus 1252

8.6.41 Scrub typhus 1252

section 8  Infectious diseases 1252 8.6.41  Scrub typhus Daniel H. Paris and Nicholas P.J. Day ESSENTIALS Orientia spp. are obligate intracellular Gram-​negative bacteria that cause scrub typhus, historically known as ‘tsutsugamushi disease’, a febrile illness characterized by early non​specific ‘flu-​like’ symptoms, and sometimes a diffuse, macular, or maculopapular rash and/​or a necrotic lesion eschar at the inoculation site. Leptotrombidium mites transmit Orientia spp. to humans via the bite of the larval stage, while all mite stages act as bacterial reservoirs through ver- tical transovarial and transstadial transmission. Scrub typhus is a leading cause of treatable undifferentiated febrile illness in many regions of Asia, and unfortunately remains an underappreciated neglected disease, mainly due to diagnostic difficulties and lack of awareness among medical staff. Complications include meningo-​ encephalitis, respiratory and renal failure, and severe multiorgan failure. Scrub typhus can be treated effectively with tetracyclines, macrolides, and chloramphenicol. Humans are dead-​end hosts and do not participate in the Orientia life cycle, hence treatment does not affect overall disease incidence. Currently there is no vac- cine available as the heterogeneity of Orientia strains, the weak and transient cross-​protection among divergent isolates, and the ap- parent loss of heterologous protection within months after natural infection pose major hurdles in the development of diagnostics and vaccines. Introduction Historically, the Rickettsia were classified into three major serological groups, but recent discoveries of novel Rickettsia spp. have challenged this classification. Based on genetic classification, the Rickettsia genus is now divided into four major groups: ‘typhus group’ (R. prowazekii, R. typhi); ‘spotted fever group’ (R. rickettsii, R. conorii and many others); ‘ancestral group’ (R. bellii and R. canadensis) and the ‘transitional group’ (R. akari, R. australis and R. felis). In 1996 O. tsutsugamushi was moved into its own Orientia genus on the basis of genotyping— ​this genus currently comprises the two human-​pathogenic species O. tsutsugamushi and O. chuto sp. nov. Although scrub typhus probably is the world’s most important rickettsial illness in terms of disease burden, the available know- ledge and literature about Orientia spp. remains rather limited. In Japanese, the jungle mite is called ‘tsutsuga-​mushi’ (tsutsuga = dan- gerous and mushi = insect). The earliest clinical accounts compatible with scrub typhus were found in the ‘Zhouhofang’, a Chinese clinical manual produced in 313 BC, and the term ‘tsutsugamushi disease’ was first applied to mite-​associated fevers in the Niigata prefecture in Japan in 1810. Since then, Orientia spp. have been found and de- scribed across Asia and the Pacific region, but also recently in Africa, Europe, and South America, following a tropical to subtropical dis- tribution (Fig. 8.6.41.1). Aetiology and epidemiology Orientia are Gram-​negative, non​flagellated, small coccobacilli found within the cytoplasm of host cells. They are transmitted to humans Fig. 8.6.41.1  World distribution of Orientia spp. Orientia spp. were thought to be geographically restricted to the Asia-​Pacific region. However, recent identification of Orientia spp. in febrile patients from the Arabian Peninsula and Chile (culture and sequencing), in rodents from Southern France and Senegal (Orientia-​specific PCR), and serologically in Kenya, Congo and Cameroon suggest that scrub typhus could be more widely distributed in the tropical/​subtropical belt around the world than previously assumed.

8.6.41  Scrub typhus 1253 by the bite of larval trombiculid mites, called ‘chiggers’ (family Trombiculidae, genus Leptotrombidium), within which the bacteria are maintained transovarially and transstadially over multiple gen- erations. Incidence is seasonal, with an increase in cases just before and during the rainy season in Southeast Asia and at harvest time in Japan. The transmitting trombiculid mites can be found from sea level to mountainous heights in Borneo (Sabah, Sarawak) and India (Kashmir, Himachal Pradesh, Sikkim and Arunachal Pradesh), in alpine conditions in the Pakistan Himalayas, in rain forests, shrubby fringes between fields and forest, abandoned paddy fields, rubber plantations, beaches, riverbanks, semiarid deserts, and commonly in areas with secondary vegetative growth (Fig. 8.6.41.2). Pathogenesis/​Pathology The outer membrane proteins of Orientia attach to host cells via syndecan and fibronectin receptors that engage integrins and trigger bacterial endocytosis and internalization. The intracellular pre- ferred location of Orientia is in the glycogen and ATP-​rich peri- nuclear region of the cytoplasm, to which the bacteria are thought to translocate via microtubules. Infected cells express and secrete in- flammatory and chemotactic cytokines, which involve activation of NF-​κB and the MAPK pathways. The systemic vasculopathy of scrub typhus involves prominent perivascular cuffing with mononuclear cells and fibrinoid necrosis of the vascular wall. These dense infiltrates contain Orientia in monocytes, lymphocytes, and macrophages (Fig. 8.6.41.3). Scrub typhus infection is typically associated with prominent systemic mononuclear cell activation, and strong pro-​inflammatory co- agulation activation in vivo. Skin biopsies of scrub typhus eschars show Orientia to be mainly within dermal dendritic cells and tissue monocytes and rarely within endothelial cells. Orientia-​infected cells that have the capacity to re-​circulate in lymphatic or blood vasculature (Trojan horse phenomenon) enable the pathogen to escape from the eschar via lymph nodes to the systematic circula- tion and reach parenchymal organs. This period of dissemination usually occurs between 3 and 14 days after the onset of fever, and during this ‘rickettsaemic window’ Orientia can be detected in the blood (i.e. using polymerase chain reaction (PCR) assays). The natural immune response to scrub typhus is challenged by the great immunogenic diversity of Orientia strains, usually re- sulting in weak and transient cross-​protection to infection with different isolates, and waning of heterologous protection within months. This short-​lived immune protection is associated with high re-​infection rates, especially in people living in endemic areas. The mechanisms of protective immunity against Orientia are under in- vestigation, but remain poorly understood. Validated experimental models to study the pathogenesis of scrub typhus are lacking, al- though infected Rhesus macaques have similar clinical and patho- physiological features as humans. Clinical features The clinical presentation starts with a non​specific ‘flu-​like’ syn- drome, including fever, fatigue, frontal headaches, myalgia, cough, (d) (e) (a) (b) (c) Fig. 8.6.41.2  Vegetation associated with scrub typhus transmission. Rodents infested with trombiculid mites excavate burrows along the dried-​mud walkways in disused paddy fields (a). Mite islands—​locations with high mite densities inhabiting the soil—​typically found along waterways, amidst bamboo groves, disused paddy fields, and in hilly coffee plantations (b, c, and d). Aerial view of Chiang Rai (north Thailand) shows the disused paddy fields with streams, jungle, plantations, and shrubby vegetation which represent high-​risk areas for acquiring scrub typhus (e). (a) (c) (d) (b) Fig. 8.6.41.3  Orientia tsutsugamushi in cell culture and skin biopsies. Obligate intracellular Orientia spp. require cells for in vitro propagation, and typically locate to the glycogen and ATP-​rich perinuclear region (immunofluorescence and immune histochemistry staining, (a) and (c), magnification ×650). Eschar biopsies from scrub typhus patients reveal the typical perivascular cuff formation with high densities of bacteria, monocytes, lymphocytes, and antigen-​presenting cells (CD14-​pos. monocytes (b) and T lymphocytes (d) stained red, Orientia in green, magnification ×400). Images provided by DHP, panels (b) and (d) were published in PLoS Negl Trop Dis, 2012 Jan; 6(1):e1466. doi: 10.1371/​journal.pntd.0001466.

section 8  Infectious diseases 1254 and restlessness/​insomnia. The presence of an eschar is a valuable diagnostic clue, but like the rash, is not always present. An eschar is a necrotic and painless inoculation lesion following a mite bite, typic- ally found in areas associated with compression; restrictive clothing like shirt cuffs, bra, underwear, sarong, or in intertriginous areas, like axillae, under the breasts, groin, or buttock regions. The finding of this important diagnostic clue is often missed, as eschars are com- pletely painless—​patients are often unaware of their presence—​and the affected body regions often difficult to examine due to cultural sensitivities. The diffuse, macular, and/​or maculopapular rash can present within 3–​10 days following onset of disease (Fig. 8.6.41.4). Lymphadenopathy is a common feature, more so than in other rick- ettsial infections, such as murine typhus. Reversible partial hearing loss has been described and appears to be a specific feature of scrub typhus. The underlying pathophysiology of this symptom is cur- rently not understood. Complications in scrub typhus include gastrointestinal symp- toms, respiratory and renal failure, encephalitis, and very rarely disseminated intravascular coagulation. The major serious compli- cations are central nervous system infection, acute respiratory dis- tress syndrome, and multiple organ dysfunction syndrome, which are associated with mortality rates reaching 20%, even if treated. Untreated scrub typhus mortality rates average around 6–​8%, but can reach 40% in certain geographical regions, depending on the strain of Orientia and the immune competence of the patient. Diagnosis The Weil–​Felix test historically separated scrub typhus from the other forms of typhus and was based on the detection of cross-​reactive anti-​Orientia antibodies to Proteus mirabilis—​specifically the OX-​ K (Kingsbury) strain. The Weil–​Felix test is unreliable, with poor diagnostic accuracy and has been replaced by the newer indirect immunofluorescent assay (IFA) and indirect immunoperoxidase test (IIP). These assays use cell-​culture-​derived Orientia antigens to detect and titrate specific antibodies in paired admission and conva- lescent samples. However, these tests are rarely standardized across laboratories, require considerable expertise, and are usually not available in rural tropical areas where they are most needed. Recently, anti-​ Orientia IgM and IgG-​based rapid diagnostic tests and Enzyme-​Linked Immunosorbent Assays (ELISAs) have become available, and are replacing the suboptimal IFAs or IIPs. Both assays use cell culture-​derived O.  tsutsugamushi antigens or recombinant proteins to detect Orientia-​specific antibodies and are inexpensive, sensitive, specific, and reproducible; rapid diagnostic tests are either immunochromatographic or semi-​ quantitative immuno dot blot assays, whereas ELISAs facilitate higher throughput of serum samples, and enable performing mul- tiple tests at one time. PCR methods detect different target genes of Orientia spp. and with their high sensitivities and specificities, these assays have be- come a central diagnostic pillar in scrub typhus, as they enable earlier diagnosis during the bacteraemic dissemination phase be- fore specific antibodies are sufficiently produced for serology. Target genes include the 47kDa, 56kDa, 16S rRNA, and groEL genes. Orientia spp. can be cultured from blood, though this requires special tissue culture techniques and Bio-​Safety Level 3 facilities, and can take several weeks. Samples taken from necrotic eschars or eschar crust can be useful for both PCR-​based or immuno­ histochemical diagnosis which, due to their high bacterial loads and isolation from blood circulation, can be used even after initi- ation of treatment. Differential diagnosis The following infectious diseases can cause ‘typhus-​like illnesses’, and can present with similar clinical features: scrub typhus, murine typhus, dengue, leptospirosis, typhoid, melioidosis, malaria, and chikungunya fever. Typhus (Rickettsia and Orientia infections)—​distinguished molecularly using PCR with genotyping or serologically by specific cross-​adsorption tests and Western blotting in paired acute and con- valescent samples (IFA, IIP, ELISA). Malaria—​direct pathogen detection via stained blood films, antigen detection assays Arbovirus infections (e.g. dengue, Chikungunya)—​diagnosis using combined antigen and antibody-​based detection algorithms (non​structural proteins and IgM detection). The dengue rash is more erythematous and homogenous than in scrub typhus and is often accompanied by pronounced thrombocytopenia. Leptospirosis—​diagnosed by PCR (whole blood), serology incl. microscopic agglutination testing and/​or culture (blood, urine, cere- brospinal fluid (CSF)—​recently possible on solid agar). Relapsing fever (lice or tick transmitted)—​direct demonstration of Borrelia spirochetes in blood smears, and/​or via serology and/​ or PCR. Meningococcal disease—​conventional blood and CSF cultures. Typhoid—​conventional blood and bone marrow cultures, and recently by rapid diagnostic testing of blood culture fluid. Viral fevers—​with maculopapular skin rash, as in Epstein–​Barr virus, infectious mononucleosis, or primary HIV infections, are dis- tinguished serologically or via PCR assays. (a) (d) (e) (f) (b) (c) Fig. 8.6.41.4  Inoculation eschar lesions and rash in scrub typhus. Eschars in the umbilicus (a), at a T-​shirt cuff line (b), the belt/​sarong line (c), and buttocks (f). Eschars and rash are not always present. The rash is typically erythematous, macular, or maculopapular, and can be very faint or difficult to discern, especially in deep skin tones (d and e). Images (a), (d), (e), and (f) were generously provided by Dr Rattanaphone Phetsouvane, from Laos and images (b) and (c) by Dr Hugh Kingston, from Cambodia and Bangladesh, respectively.

8.6.41  Scrub typhus 1255 Treatment Scrub typhus is very responsive to treatment with appropriate anti- biotics (doxycycline, azithromycin, chloramphenicol), and empirical treatment should be considered early if this diagnosis is suspected. Doxycycline: Unless contraindicated, doxycycline is the standard treatment with an adult oral dose of 100 mg twice daily for 7 days. Tetracycline 500 mg q6 h for 7 days can also be used. In uncom- plicated scrub typhus, studies have shown that shorter regimens of 3 days performed equally well as 7 days. No failures or relapses and similar fever clearance times were observed in either treatment group in a Korean study that compared a 3-​day course of doxycyc- line (100 mg every 12 hours) to a 7-​day regimen of tetracycline (500 mg every 6 hours). Similarly, a Malaysian study showed that a single dose of doxycycline 200 mg was equivalent to one week of tetracyc- line 500 mg every 6 hours for treating patients with scrub typhus—​ no relapses occurred in the 2-​week follow up period. Azithromycin: An alternative drug with comparable efficacy is azithromycin (1000 mg or 500 mg on the first day, followed by 500 or 250 mg daily for another 2 days—​3-​day regimen). Azithromycin has also been shown to be as effective as doxycycline, when used as a single-​dose treatment in uncomplicated disease. Azithromycin is particularly useful if tetracyclines are contraindicated, such as in pregnancy, although doxycycline can be considered safe until the 25th week of pregnancy. Trials of shorter courses are underway for both doxycycline and azithromycin-​based regimens. Azithromycin has been suggested as alternative treatment for doxycycline-​resistant scrub typhus in northern Thailand, although robust data on the na- ture of resistance and treatment options is lacking. Chloramphenicol is an excellent drug, and a good alternative to doxycycline, although its haematological side effects (1:21 600) and the very rare occurrence of grey baby syndrome in premature infants (circulatory collapse) have led to significant reduction of its use (500 mg q6 h in adults or 50–​75 mg/​kg/​day in children for 7 days). Combinations and drug–​drug interactions for dual treatment: The idea of achieving shorter fever clearance times using combin- ation therapy is attractive, especially in pregnancy with the aim of reducing adverse pregnancy outcomes. However, rifampicin co-treatment with doxycycline, azithromycin, or chloramphenicol might decrease the levels and effects of these drugs; hence dose ad- justment might be required. These combinations should be used with caution until reliable data become available. Combinations of doxycycline plus azithromycin or chloramphenicol are beneficial and no negative interactions are described to date. Prognosis/​Outcome Severe scrub typhus patients typically present with multiple organ dysfunction syndrome in approximately a third of hospitalized pa- tients, and high APACHE-​II admission scores, as recently reported in a large study in India. However, despite the high frequency of multiple organ dysfunction syndrome, the mortality remains rela- tively low with overall case fatality rate at approximately 9%. In pa- tients with central nervous system (CNS)-​related complications a higher mortality rate of 18% was observed in a large recent study in Laos. This study showed that Rickettsia spp., Orientia spp. and Leptospira spp. infections are important causes of CNS infections in Laos, observed significantly more frequently than conventional bac- terial aetiologies (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus suis). Scrub typhus in pregnancy is potentially worse than malaria with a high rate of poor neonatal outcome, with stillbirth, prematurity, and low birth weight occurring in just over 40% of pregnancies. Despite these data, the current evidence to support the use of doxy- cycline or azithromycin in pregnancy for scrub typhus is weak, and the optimum antimicrobial treatment for undifferentiated fevers in pregnancy remains unknown. In summary, treatment delays, diagnostic limitations, and lack of awareness are important reversible contributing factors to the cur- rently poor outcome of scrub typhus. Antibiotics effective against scrub typhus, such as doxycycline, are often not routinely given as part of empirical treatment strategies. Hence, particularly as diagnosis is difficult, scrub typhus is often undertreated, contributing to the dis- ease burden associated with this important but neglected disease. Entomology Leptotrombidium mites are the main reservoir and vectors of Orientia spp. They are usually characterized by the form of their dorsal shield structure called ‘scutum’ and the pattern of setae and sensillae hairs attached to it (Fig. 8.6.41.5). The trombiculid life cycle consists of (a) (c) (d) (b) Fig. 8.6.41.5  Morphological characteristics of chigger mites. Chigger mites are small—​a typical trombiculid mite larva is approx. 0.2 mm and has three pairs of legs, whereas adult mites have four pairs (a, scale 100 um). Morphological mite identification requires description of mouthparts (chelicerae), palpal and tarsal claws, segmentation of the legs (b, scale bar 20 um), and the characteristic dorsal shield-​like plate termed scutum (c and d, scale bars 20 um). The scutum of Aschoschoengastia sp. have a rectangular shape with fine setae hairs at the outer edges, and two hairy clubbed sensillae, the feeler hairs (c). The scutum of Gahrlipia sp., subgenus Walchia have a shield-​shaped scutum and also clubbed sensillae (d). The bases of the sensillae are often mistaken for the eyes—​these are actually pale discs located next to the scutum (arrows). Images taken with the support of Dr Sungsit Sungvornyothin, Medical Entomology Dept., Mahidol University.

section 8  Infectious diseases 1256 four stages: eggs, larvae, nymphs, and adults; only the larval forms (‘chiggers’) feed on vertebrate hosts, whereas nymphs and adult mites live in the soil and feed on the eggs of insects. Chiggers penetrate the skin with chelicerae and use their enzyme-​rich saliva to digest a tube-​like structure (termed ‘stylostome’) through the epidermal layer and inoculate Orientia, as high pathogen concentrations are found in the saliva. Mites of all stages can harbour Orientia, which are maintained through the various stages in the mite life cycle, and vertically via transovarial transmission over many generations. O. tsutsugamushi infections in mites can alter the sex ratio in some mite species, resulting in most progeny being female. Although mites transmit O. tsutsugamushi to vertebrate hosts such as rodents very effectively, only a small proportion of uninfected mites acquire Orientia during feeding on infected animals. Free-​living mites are typically collected using black plates and black cloths placed on the ground and/​or on grass, on to which they are attracted. Mite larvae (chiggers) are best collected by trapping of rodents and collected dir- ectly from ears and genital areas—​their preferred sites for attach- ment and feeding (Fig. 8.6.41.6). Areas of uncertainty, controversy, and
future developments Drug resistance Scrub typhus patients usually become afebrile within 48 hr of starting appropriate treatment, but in 1996 both chloramphenicol and doxycycline resistance were reported in Chiangrai in nor- thern Thailand; only 40% of patients cleared their fever within 72 hours, compared to 100% in patients from Mae Sod on the Thai Myanmar border; median fever clearance times in Chiangrai were 80 h (range 15–​190 h) compared to 30 h in Mae Sod (range 4–​58 h). The underlying nature of these possibly resistant infections has not been investigated further. Considering the current case fa- tality rate of 13% in hospitalized patients in this region, it is of great clinical relevance to determine if the infecting Orientia strains are truly antibiotic resistant, or whether there are other explanations for this poor clinical response. The genome of O. tsutsugamushi The genome of O. tsutsugamushi is the largest in the order Rickettsiales with a single chromosome of approx. 2.0 Mb in size. It is eccentric, as it contains the highest number of repetitive sequences of any bacterial organisms known to date; 47% of the genome are repeats derived from integrative, conjugative, and transposable elements. Both massive gene amplification and degradation have generated a huge number of repeated genes with intensive genome shuffling, but the proliferation of mobile elements and the selective pressures influencing them remain unexplained. The adaptation of rickettsia to an obligate intracellular lifestyle is associated with an increased reliance on host cellular functions. As the bacteria discard many of their enzymes over time (reduc- tive genome evolution), an increasing supplementation with host cell metabolites and substrates takes place. Large-​scale comparative genomic analyses suggest that gene loss has been a driving force for obligate intracellular bacterial genomes (and not acquisition of viru- lence factors) to adapt to particular host-​associations in eukaryotic cells. Rickettsia and Orientia genomes have revealed interesting con- troversies between reductive evolutionary forces on metabolic genes observed in all species, but proliferation of mobile genetic elements in only some. These evolutionary effects highlight the influence of chance, adaptation, and host cell exploitation during the evolution of intracellular bacteria, but the underlying mechanisms remain poorly understood. FURTHER READING Cross R, et al. (2016). Revisiting doxycycline in pregnancy and early childhood—​time to rebuild its reputation? Expert Opin Drug Saf, 15, 367–​82. Dittrich S, et al. (2015). Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study. Lancet Glob Health, 3, e104–​12. Kim YS, et  al. (2004). A comparative trial of a single dose of azithromycin versus doxycycline for the treatment of mild scrub ty- phus. Clin Infect Dis, 39, 1329–​35. Koh GC, et al. (2010). Diagnosis of scrub typhus. Am J Trop Med Hyg, 82, 368–​70. McGready R, et al. (2014). Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis, 8, e3327. Panpanich R, Garner P (2002). Antibiotics for treating scrub typhus. Cochrane Database Syst Rev, 3, CD002150. Paris DH, et al. (2012). Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium. PLoS Negl Trop Dis, 6, e1466. Paris DH, et al. (2013). Unresolved problems related to scrub typhus: a seriously neglected life-​threatening disease. Am J Trop Med Hyg, 89, 301–​7. Peter JV, et al. (2015). Severe scrub typhus infection: clinical features, diagnostic challenges and management. World J Crit Care Med, 4, 244–​50. (c) (d) (a) (b) Fig. 8.6.41.6  Methods for capturing soil and rodent-​borne mites in the field. Ongoing epidemiological field surveys serve to characterize mites transmitting scrub typhus to humans. Black plastic plates of approx. 30 cm length are placed on the ground or black cloth is placed on shrubby grass for a few minutes and free-​living mites crawl onto these (a) and (b). Mites are easily identified on the black background and are collected for microscopic morphological and molecular identification. The mite life cycle takes place in the upper soil layers, and topsoil can be collected for subsequent isolation of mites using a Berlese Funnel (c). In (d), a freshly captured rodent has chigger mites in its ears (orange or cream-​coloured spots) and distributed along the edge of the ear (arrows).

8.6.42 Coxiella burnetii infections (Q fever) 1257

8.6.42 Coxiella burnetii infections (Q fever) 1257

1257 8.6.42  Coxiella burnetii infections (Q fever) Strickman D, et  al. (1995). In vitro effectiveness of azithromycin against doxycycline-​resistant and -​susceptible strains of Rickettsia tsutsugamushi, etiologic agent of scrub typhus. Antimicrob Agents Chemother, 39, 2406–​10. Watt G, et al. (1996). Scrub typhus infections poorly responsive to anti- biotics in northern Thailand. Lancet, 348, 86–​9. 8.6.42  Coxiella burnetii
infections (Q fever) Thomas J. Marrie ESSENTIALS Q fever is a zoonosis caused by Coxiella burnetii, an intracellular Gram-​negative spore-​forming bacterium, the common animal res- ervoirs of which are cattle, sheep, and goats. Rats and mice have been implicated as reservoirs in some areas. In French Guiana the three-​toed sloth is the reservoir. C. burnetti is trophic for the endo- metrium and mammary glands of female animals, and during preg- nancy the organism reaches very high concentrations in the placenta such that at the time of parturition organisms are aerosolized and contamination of the environment occurs. Inhalation of even one microorganism can result in infection. Clinical features—​there are two main forms of the disease:
(1) acute—​can present as inapparent infection, self-​limited febrile illness, pneumonia, and hepatitis, or less commonly with a variety of organ-​specific manifestations such as encephalitis, pericarditis, and pancreatitis; Q fever in pregnancy is associated with a high rate of abortion or neonatal death. (2)  Chronic—​most often ‘culture-​ negative’ endocarditis or infection of aortic aneurysms, but occa- sionally osteomyelitis. Diagnosis, treatment, and prevention—​diagnosis is confirmed by serological testing: in acute disease antibodies to phase II antigen are higher than those to phase I, whereas the reverse is true in chronic dis- ease. Acute Q fever is treated with doxycyline or a quinolone; chronic disease with long-​term doxycycline and hydroxychloroquine; and Q fever in pregnancy with co-​trimoxazole for the duration of the preg- nancy and—​for those post-​partum women with a chronic Q fever serological profile—​1 year of doxycycline and hydroxyochloroquine after delivery. Vaccination should be offered to those whose occupa- tion places them at high risk for C. burnetii infection. History In August 1935, Dr Edward Holbrook Derrick, Director of the Laboratory of Microbiology and Pathology of the Queensland Health Department in Brisbane, Australia, was asked to investigate an outbreak of undiagnosed febrile illness among workers at the Cannon Hill abattoir. Derrick realized that he was dealing with a type of fever that had not been previously described—​he named it Q (for query) fever. Two years later, Sir Frank Macfarlane Burnet in Australia and Herald Rea Cox in the United States of America iso- lated the microorganism responsible for Q fever. Coxiella burnetii This microorganism, the sole species of its genus, has a Gram-​ negative cell wall and measures 0.3 × 0.7 µm (Fig. 8.6.42.1). It is an obligate phagolysosomal parasite of eukaryotes that sporulates, stains well with Gimenez’s stain, and multiplies by transverse binary fission. C. burnetii undergoes phase variation akin to the smooth to rough transition in some enteric Gram-​negative bacilli. In na- ture and laboratory animals it exists in the phase I state. Repeated passage of phase I virulent organisms in embryonated chicken eggs leads to the conversion from phase I virulent to phase II aviru- lent forms. Antibodies to phase I antigens usually predominate in chronic Q fever, while phase II antibodies are higher than phase I antibodies in acute Q fever. The genome of C. burnetii strain Nine Mile Phase I has 1 995 275 base pairs. There are many genes with potential roles in adhesion, invasion, intracellular trafficking, host-​ cell modulation, and detoxification. C. burnetii can now be grown in a cell-​free medium, an advance that should lead to further insight into this complex microorganism. Immune control of C. burnetii is T-​cell dependent but it does not eliminate C. burnetii from infected humans. In 80–​90% of bone marrow aspirates from those who have recovered from Q fever, poly- merase chain reaction (PCR) assays for C. burnetii DNA are posi- tive. The use of microarrays allows insight into the complexity of the host-​microorganism interaction in illnesses such as Q fever. In one such experiment 335 genes in the C. burnetii-​infected human monocytic leukaemia cell line THP-​1 were up-​ or down-​regulated at least twofold. Fig. 8.6.42.1  Transmission electron micrograph showing C. burnetii cells within a macrophage in the heart valve of a patient with Q fever endocarditis. The dark material in the centre of each cell is condensed DNA. Magnification ×15 000.

section 8  Infectious diseases 1258 Recently adipose tissue has been shown to be the reservoir for C. burnetii during bacterial latency. C. burnetii has survived for 586 days in tick faeces at room tem- perature, 160 days or more in water, 30–​40 days in dried cheese made from contaminated milk, and up to 150 days in soil. Epidemiology Q fever is a zoonosis. There is an extensive wildlife and arthropod (mainly ticks) reservoir of C. burnetii. This reservoir might differ from area to area; for example in Spain, European rabbits serve this function while in Nova Scotia Canada, cats are the prime reservoirs; rats in Netherlands, three-​toed sloth in French Guinea and mice in China are the reservoirs in those countries. Domestic animals are infected through inhaling contaminated aerosols or by ingesting in- fected material. These animals rarely become ill, but abortion and stillbirths may occur. C. burnetii localizes in the uterus and mam- mary glands of infected animals. During pregnancy there is reacti- vation of C. burnetii and it multiplies in the placenta, reaching 109 infective doses per gram of tissue. The organisms are shed into the environment at the time of parturition. Humans becomes infected after inhaling organisms aerosolized at the time of parturition, or later when organisms in dust are stirred up on a windy day. Infections have occurred up to 18 km downwind from a source. Infected cattle, sheep, goats, and cats are the animals primarily responsible for trans- mitting C. burnetii to humans. There have been several outbreaks of Q fever in hospitals and research institutes due to the transportation of infected sheep to research laboratories. Some studies have sug- gested that ingestion of contaminated raw milk is a risk factor for the acquisition of Q fever. Percutaneous infection, such as when an infected tick is crushed between the fingers, can occur but is rare. Transmission via a contaminated blood transfusion has rarely oc- curred. Vertical transmission from mother to child has been infre- quently reported. A 2007 review documents 74 cases of Q fever in pregnant women. The authors found that Q fever was present in 1 in 540 pregnancies in an area of endemic Q fever in southern France. Person-​to-​person transmission has been documented on a few oc- casions, including sexual transmission. To date, 45 countries on five continents have reported cases of Q fever. Q fever is estimated to cost $A1 million in Australia each year and results in the loss of more than 1700 weeks of work. There are several studies where young age seems to be pro- tective of infection with C. burnetii. In a large outbreak of Q fever in Switzerland, symptomatic infection was five times more likely to occur in those over 15 years of age compared with those younger than 15. In many outbreaks of Q fever, men were affected more com- monly than women. It had been assumed that this was due to the fact that certain occupations in which men predominate were more likely to be associated with Q fever. However, in France, despite similar exposures, the male to female ratio is 2.45:1. The explanation for this gender difference is that female sex hormones are protective against Q fever infection. Currently Q fever is common in several European countries with recent outbreaks in Germany and the Netherlands. There are a con- siderable number of sporadic cases of Q fever in England, France, and Spain. The outbreak in the Netherlands is the largest to date, with over 4000 cases from 2007 to 2010, and many lessons have been learned from it. In 2007 a total of 168 individual human Q fever cases were notified, occurring after visits to dairy farms with abortion problems. The outbreak was concentrated around a single village, where a case-​control study found that contact with ma- nure, hay, and straw were risk factors. Moreover, people living in the eastern part of the village close to ruminant farms, one of which was a dairy goat farm with a recent history of abortion problems, were at higher risk than people living in other parts of the village. In 2008, 1000 human cases were notified, with average age 51 years (range 7–​87 years), and 21% were hospitalized. In April 2009 a fur- ther sharp increase in human cases was observed, resulting in the total number of 2355. Pneumonia accounted for 68% of the cases and hepatitis for 15%. The gender ratio was 1 female to 1.7 males. In general, 59% of the notified human cases in 2009 lived within a 5-​km zone around the notified dairy goat, dairy sheep farm, while 12% of the Dutch population lived within such as zones. Genotyping of C. burnetii isolates found that one unique genotype predominated in dairy goat herds and one sheep herd, and this genotype was similar to the human isolates. Overall 10 different genotypes were identi- fied. Systematic culling of gestating ewes and goats on infected farms as well as other measures stopped the outbreak. More than 50 000 animals on 88 farms were culled. The incidence of Q fever in the United States is 0.38 cases per mil- lion with a 2% fatality rate. Fatal cases were underreported by case reports by a factor of 14 and by 5.2 for death certificates. Ingestion of raw milk was twice as common among cases as compared with the national average. The incidence in Germany is 1.4 cases per mil- lion. In contrast the rate per 100 000 is 2.5 in France, 2.8 in Australia, 0.02 in South Korea and in one city in French Guiana it is 37–​150 per 100 000. Clinical features Humans are the only species known consistently to develop illness following infection with C. burnetii. There is an incubation period of about 2 weeks (range 2–​29 days) following inhalation of C. burnetii. A dose–​response effect has been demonstrated experi- mentally and clinically. C. burnetii is one of the most infectious agents known; a single microorganism is able to initiate infection in humans. The resulting illness can be divided into acute and chronic varieties. Acute Q fever Self-​limiting febrile illness The most common manifestation of acute Q fever is a self-​limiting febrile illness that is dismissed as a ‘cold’. Serosurveys reveal that in most endemic areas 5–​10% of the population have antibodies to C. burnetii but never remember the illness that resulted in seroconversion. Q fever pneumonia This is the most commonly recognized manifestation of acute Q fever in some geographical locations. There is often a seasonal dis- tribution, most of the cases occurring between February and May (consistent with the birthing season in the small ruminant reser- voirs). The onset is non​specific with fever, fatigue, and headache. The headache may be very severe, occasionally so severe that it

1259 prompts a lumbar puncture. A dry cough of mild to moderate in- tensity is present in 24–​90% of patients. About one-​third of pa- tients have pleuritic chest pain. Nausea, vomiting, and diarrhoea occur in 10–​30% of patients. Most cases of C. burnetii pneumonia are mild; however, about 10% are severe enough to require ad- mission to hospital and, rarely, assisted ventilation is necessary. Death is rare in Q fever pneumonia and is usually due to comorbid illness. The white blood cell count is usually normal, but is elevated in one-​third of patients. Liver enzyme levels may be mildly ele- vated at two to three times normal. Alkaline phosphatase is raised in up to 70% of patients and 28% are hyponatraemic. Reactive thrombocytosis is surprisingly common and microscopic haema- turia is a common finding. The chest radiographic manifestations of Q fever pneumonia are usually indistinguishable from those of other bacterial pneu- monias (Fig. 8.6.42.2); however, rounded opacities are suggestive of this infection (Fig. 8.6.42.3). Some investigators have reported delayed clearing of the pneumonia; however, in our experience resolution is usually complete within 3 weeks. Hepatitis The liver is probably involved in all patients with acute Q fever. There are three clinical pictures: • Pyrexia of unknown origin with mild to moderate elevation of liver function tests. In some patients, corticosteroid therapy is necessary because patients remain febrile despite appropriate antibiotic therapy. • A hepatitis-​like picture: liver biopsy shows distinctive dough­ nut granulomas consisting of a granuloma with a central lipid vacuole and fibrin deposits (Fig. 8.6.42.4). Prolonged fever unresponsive to antibiotics is common in these patients (Fig. 8.6.42.5). • ‘Incidental hepatitis’. In this setting liver enzymes are elevated but other features dominate the clinical picture—​such as pneumonia. Thus, the liver is said to be incidentally involved. Q fever in pregnancy Acute Q fever occasionally complicates pregnancy. In 23 pub- lished cases 35% had premature birth, and 43% ended in abortion or neonatal death. In a serosurvey of 4588 pregnant women in Halifax, Nova Scotia, Canada, women seropositive for C. burnetii were three times more likely to have a current or previous neo- natal death. A study of 1174 pregnant women during the recent outbreak of Q fever in the Netherlands found that antibodies against phase II C. burnetii were not significantly associated with preterm delivery, low birth weight babies, and several other out- comes. Adverse outcomes attributable to Q fever were not seen in a recent study from Germany of 11 women with this infection during pregnancy. Whether these differences are due to proper- ties of the infecting strain is not known. In several animal species (other than humans) Q fever is associated with abortions and still- births. Clearly, more information is need on Q fever in pregnancy in humans. Fig. 8.6.42.2  Serial chest radiographs of a 35-​year-​old patient with Q fever pneumonia. The first radiograph (1 August 1989) shows a round opacity in the right upper lobe, which increases in size over the next 6 days. The pneumonia has completely cleared by 19 September 1989. 8.6.42  Coxiella burnetii infections (Q fever)

section 8  Infectious diseases 1260 Neurological manifestations Encephalitis, encephalomyelitis, toxic confusional states, optic neuritis, and demyelinating polyradiculoneuritis are uncommon manifestations of Q fever. Rare manifestations These include myocarditis, pericarditis including constrictive peri- carditis, bone marrow necrosis, rhabdomyolysis, glomerulonephritis, lymphadenopathy, pancreatitis, splenic rupture, acalculous chole- cystitis, mesenteric panniculitis, erythema nodosum, epididymitis, orchitis, priapism, and erythema annulare centrifugum. Chronic fa- tigue may be a sequel of Q fever in some patients. Chronic Q fever The usual manifestation of chronic Q fever is that of culture-​negative endocarditis. Some 70% of these patients have fever and nearly all have abnormal native or prosthetic heart valves. Hepatomegaly and/​or splenomegaly occur in about one-​half of these patients and one-​third have finger clubbing. A  purpuric rash due to immune complex-​induced leucocytoclastic vasculitis and arterial embolism occurs in about 20% of patients. Hyperglobulinaemia (up to 60 g/​ litre) is common and is a useful clue to chronic Q fever in a patient with the clinical picture of culture-​negative endocarditis. Other manifestations of chronic Q fever include osteomyelitis, infection of aortic aneurysm, and infection of vascular prosthetic grafts. The strains of C. burnetii that cause chronic Q fever do not differ from those that cause acute Q fever. Peripheral blood lympho- cytes from patients with Q fever endocarditis are unresponsive to C. burnetii antigens in vitro, while responding normally to other antigens. Positron emission scanning has emerged as a useful tool in the diagnosis and management of patients with Q fever. The site of in- fection, heart valve, aorta, bone, or joint can be visualized. Diagnosis A strong clinical suspicion based on the epidemiology and clinical features as outlined earlier is the cornerstone of the diagnosis of Q fever. This suspicion is confirmed by determining a fourfold or Fig. 8.6.42.3  Portable anteroposterior chest radiograph of a 72-​ year-​old man with Q fever pneumonia. This radiographic picture is indistinguishable from pneumonia due to any other microbial agent. Fig. 8.6.42.4  Liver biopsy showing characteristic doughnut granuloma. The hole in the centre of the granuloma is lipid that has been dissolved during the fixation process.

1261 greater increase in antibody titre between acute and 2-​ to 3-​week convalescent serum samples. A variety of serological tests are avail- able including complement fixation, microimmunofluorescence (IFA), and enzyme immunoassay. The immunofluorescence anti- body test is the best test. In acute Q fever the antibody titre to phase II antigen is higher than that to phase I antigen, while the reverse occurs in chronic Q fever. In chronic Q fever, antibody phase I titres are extremely high, in the order of 1:8192 and higher. In acute Q fever, antibody titres to phase I antigen are rarely in excess of 1:512 (usually 1:8 to 1:32), while peak antibody titres to phase II antigen are between 1:1024 and 1:2048. The micro- organism can be isolated in embryonated eggs or in tissue culture; however, a biosafety level 3 laboratory is required. The PCR can be used to amplify C. burnetii DNA from tissues or other biological specimens. Good laboratory practice, with known positive and negative con- trols is extremely important in the diagnosis of Q fever. Three dif- ferent laboratories (in France, the United Kingdom, and Australia) tested the same serum samples using an IFA test. However, the antigen used in the test differed in each laboratory—​Nine Mile strain in France; Nine Mile strain clone 4 as phase II antigen and Henzerling strain as phase 1 antigen in Australia; patient Lane strain ST 12 group for phase 1 and II antigens in the United Kingdom. Concordance was only 35%. The Australian and United Kingdom re- sults had the greatest concordance and French and United Kingdom results the lowest. Serological testing revealed no chronic serological profiles when tested in either France or Australia but 10 when tested in the United Kingdom. Serological results from a patient with treated Q fever endocarditis suggested treated (France), chronic (United Kingdom), and borderline chronic (Australia) infection. How the antigens were prepared can also make a difference in the test results, and this paper does not indicate whether the strains were grown in tissue culture or egg yolk sac. Treatment Acute Q fever is treated with a 2-​week course of tetracycline or doxycycline. Quinolones can also be used. Any patients who develop acute Q fever and have lesions of their native valves (e.g. congenital bicuspid aortic valve), prosthetic valves, or pros- thetic intravascular material should have serological monitoring every 4 months for 2 years, and if the phase I IgG titre exceeds 1:800 further investigation is warranted. Some authorities recommend Fig. 8.6.42.5  Fever in Q fever hepatitis. This patient had been febrile for 6 weeks before he was transferred to this hospital and had liver biopsy done and diagnosis of Q fever hepatitis made. 8.6.42  Coxiella burnetii infections (Q fever)

8.6.43 Bartonellas excluding B. bacilliformis 1262

8.6.43 Bartonellas excluding B. bacilliformis 1262

section 8  Infectious diseases 1262 that patients with valvulopathy who have acute Q fever should re- ceive 12 months of doxycycline and hydroxychloroquine to pre- vent chronic Q fever. The duration of treatment for chronic Q fever is determined by monitoring the serum antibody titres to C. burnetii, although some authorities recommend lifelong therapy for chronic Q fever. In general, antibiotics can be discontinued when the IgA anti- body titre to phase I antigen is less than 1:200. The treatment of choice for chronic Q fever is doxycycline 100 mg twice daily and hydroxychloroquine 200 mg three times daily to maintain a plasma level of between 0.8 and 1.2 µg/​ml. This regimen is given for 18 months. Photosensitivity to doxycycline is a potential ad- verse reaction and patients should be warned to take preventive measures. In addition, an ophthalmologist must examine the optic fundus every 6 months for chloroquine accumulation. We have used rifampicin 300 mg twice a day and ciprofloxacin 750 mg twice a day to treat patients with chronic Q fever. Rifampicin and doxy- cycline or tetracycline and trimethoprim/​sulfamethoxazole have also been used to treat chronic Q fever. Antibody titres should be measured every 3 months for the first 2 years. A progressive de- cline in antibody titre reflects the successful treatment of chronic fever. Cardiac valve replacement may be necessary as part of the management of chronic Q fever. Many patients with granulomatous hepatitis due to Q fever have a prolonged febrile illness that does not respond to antibiotics. For these individuals treatment with prednisone 0.5 mg/​kg has resulted in defervescence within 2 to 15 days. Once defervescence has oc- curred the dose of steroids is tapered over the next month. Q fever occurring during pregnancy should be treated with co-​ trimoxazole for the duration of the pregnancy. In one retrospective study this approach reduced obstetrical complications from 81 to 44%. There were no intrauterine fetal deaths in the co-​trimoxazole-​ treated group. Those with a chronic Q fever serological profile should be treated with doxycycline and hydroxychloroquine for 1 year fol- lowing delivery. Prevention A formalin-​inactivated C. burnetii whole-​cell vaccine is protective against infection and has a low rate of side effects; 1% of vaccinees developed an abscess at the inoculation site and another 1% had a lump at this site 2 months after vaccination. The vaccine should be offered to those whose occupation places them at high risk for C. burnetii infection. Good animal husbandry practices are important in preventing widespread contamination of the environment by C. burnetii. Prevention of zoonotic spread is best accomplished by isolating aborting animals for up to 14  days, raising feeding troughs to prevent contamination of feed by excreta, destroying aborted materials by burning and burying fetal membranes and stillborn animals, and wearing masks and gloves when handling aborted materials. Only seronegative pregnant animals should be brought into the facilities where research is to be done. In addition, only seronegative animals should be used in petting zoos. Blood donation should be suspended in outbreak areas for up to 4 weeks following cessation of the outbreak. FURTHER READING Angelakis E, et al. (2014). Emergence of Q fever arthritis in France.
J Clin, 52, 1064–​7. Carcopino X, et al. (2007). Managing Q fever during pregnancy: the bene- fits of long-​term cotrimoxazole therapy. Clin Infect Dis, 45, 548–​55. Eldin C, et al. (2017). From Q fever to C. burnetii infection: a pradigm change. Clin Microbiol Rev, 30, 115–90. Raoult D, Tissot-​Dupont H, Foucault C (2000). Q fever 1985–​1998: clinical and epidemiological features of 1,383 infections. Medicine (Baltimore), 79, 110–​23. Raoult D, et  al. (1999). Treatment of Q fever endocarditis:  com- parison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine. Arch Intern Med, 159, 167–​73. Roest HIJ, et  al. (2011). The Q fever epidemic in the Netherlands:
history, onset, response and reflection. Epidemiol Infect, 139, 1–​12. Schneeberger PM, et al. (2014). Q fever in the Netherlands 2007–​2010. What we have learned from the largest outbreak ever. Med Mal Infect, 44, 339–​53. 8.6.43  Bartonellas excluding B. bacilliformis Bruno B. Chomel, Henri-​Jean Boulouis,
Matthew J. Stuckey, and Jean-​Marc Rolain ESSENTIALS Bartonellae are Gram-​negative bacilli or coccobacilli belonging to the α-​2 subgroup of Proteobacteria. A given Bartonella species usu- ally persists within a given mammalian host, with transmission be- tween hosts by haematophagous arthropods. A single species, such as B. henselae or B. quintana, can cause acute or chronic infection, with vascular, proliferative, or suppurative features depending on the host’s immune response. Many new Bartonella species isolated from various mammals have been identified as zoonotic. Cat-​scratch disease—​caused by B. henselae and commonly asso- ciated with a cat scratch, presents with a discrete papule or vesicle typically developing at the site within a week, followed by regional lymphadenopathy, sometimes with fever and constitutional symp- toms. Disseminated infection can cause neuro-​retinitis and (rarely) encephalopathy. Trench fever/​Urban trench fever—​caused by B. quintana; trans- mitted by the body louse and typically presents as an acute febrile illness with recurring (quintan) fever, often accompanied by severe headache and shin pain. Bacillary angiomatosis—​caused by B. henselae or B. quintana, par- ticularly in immunocompromised patients (mainly those with HIV infection), and presents with the gradual appearance of numerous vascular tumours of the skin and subcutaneous tissues. Bacillary peliosis—​reported mainly in immunocompromised pa- tients infected with B.  henselae, it causes vascular proliferation in solid internal organs with reticuloendothelial elements. In peliosis

1263 8.6.43  Bartonellas excluding B. bacilliformis hepatis there are multiple randomly-​distributed blood-​filled cavities throughout the liver. Bacteraemia and endocarditis—​frequently ‘culture-​negative’ and most commonly caused by B. quintana or B. henselae, but also by many other Bartonella species. Diagnosis of Bartonella infections is difficult because of the fas- tidious nature of these bacteria and their non​specific clinical mani- festations. Diagnostic techniques include culture from blood and other tissues, detection of the organisms in lymph nodes or other organs by immunofluorescence, polymerase chain reaction amplifi- cation of Bartonella genes, and serology. Bartonella bacteria are susceptible to a wide range of anti- biotics in vitro, but there is poor correlation with in vivo efficacy. General recommendations are as follows: (1) cat-​scratch disease—​ symptomatic treatment only, with azithromycin in severe or com- plicated cases; (2) trench fever/​urban trench fever—​combination of doxycycline with gentamicin; (3) bacillary angiomatosis or peliosis—​ erythromycin; (4) endocarditis—​gentamicin with ceftriaxone, with or without doxycycline. Bartonella quintana infections can be prevented by delousing, changing, and/​or washing clothes. Pet cats and pet cat environment (bedding, and so on) should be treated regularly with flea control products to prevent B. henselae infection. Immunocompromised pa- tients should avoid cat scratches and exposure to cat fleas. Introduction Until the early 1990s, the genus Bartonella was composed of two spe- cies, B. bacilliformis, the agent of Carrión’s disease (Chapter 8.6.44) and B. quintana, the agent of trench fever. In 1993, Brenner et al. proposed to unify Rochalimaea spp. within the Bartonella genus in the family of Bartonellaceae based on comparison of the 16SrDNA gene sequences. Similarly, Birtles et  al. proposed the unification of the genus Grahamella within the genus Bartonella. Since then, many new species or subspecies of Bartonella have been isolated or detected from a wide range of terrestrial and flying mammals. More than 30 species have been described and many Candidatus Bartonella spp. still await to be either described (i.e. B. washoen­ sis) or isolated (i.e. B. merieuxii). Among these, at least 16 species or subspecies are zoonotic (capable of infecting both animals and people). Bartonella quintana infection in humans was first described during the First World War when more than a million soldiers were infected and got trench fever, mainly among the Russian, German, and British troops. Cat-​scratch disease (CSD) was initially identified in 1931 in France by Debré et al. and clinically described in 1950. However, its agent, B. henselae, was not isolated until 1992 and its role in bacillary angiomatosis was demonstrated using molecular methods. In the 1980s, Afipia felis had been proposed as the aetio- logical agent of CSD, but it was later proven that this bacterium was an environmental contaminant. Aetiology and genetics The bacteria of the genus Bartonella are short, pleomorphic, fas- tidious aerobes that are oxidase and catalase negative. They are closely related phylogenetically to the genera Brucella, Agrobacterium and Rhizobium (Fig. 8.6.43.1). The 1.6 Mb genome of B. quintana was found to be a derivative of the 1.9 Mb genome of B. henselae. Prophages and horizontally acquired genomic islands have been identified in B. henselae, but are absent from B. quintana. Type IV secretion system located on plasmids in Bartonella might act as a powerful system to transfer genes laterally between bacteria living in a sympatric lifestyle in amoeba. As Bartonella have no major distinguishing phenotypic characteristics, identi- fication and phylogenetic classification are mainly based on gen- etic techniques. Many DNA regions and encoding gene sequences have been used, including the 16S rDNA gene, 16S-​23S rRNA intergenic spacer region (ITS), citrate synthase gene (gltA), heat shock protein gene (groEL), RNA polymerase β-​subunit gene (rpoB), genes encoding the PAP31 and 35-​kDa proteins and cell division protein gene (ftsZ). A  phylogenetic neighbour-​joining tree resulting from comparison of sequences of the concatenated genes of Bartonella species is shown in Fig. 8.6.43.2. As suggested by La Scola et al., a new Bartonella isolate can be considered a new species if a 327-​bp gltA fragment shares less than 96% sequence similarity with the existing species and if an 825-​bp rpoB frag- ment shares less than 94% sequence similarity with the validated species. Epidemiology Worldwide, the most common Bartonella infection is CSD, caused by B. henselae. Human cases have been reported from several continents, including Europe, North and South America, North and South Africa, Asia, and Australia, and from most countries where presence of this infection was investigated. Domestic cats are the natural reservoir of B. henselae (but also B. clarridgeiae and B.  koehlerae) and infection is highly prevalent in cats in- fested by fleas living in warm and humid climates. Transmission from cat to human mainly occurs by direct inoculation via a cat scratch. The role of cat bite as a source of infection is still questioned and transmission to humans by cat flea or tick bite seems to be quite uncommon (less than 2% of cases). Flea faeces are likely the infective material that is inoculated through a cat scratch. Other Bartonella species have also been detected in cat fleas (B. clarridgeiae, B. koehlerae, and B. quintana), in rabbit fleas (B. alsatica) (Table 8.6.43.1), in various rodent fleas, sev- eral hard tick species, and recently in chiggers. B. tamiae was de- tected in chigger mites (genera Leptotrombidium, Schoengastia, and Blankarrtia) collected on wild rodents in Thailand and bed- bugs in Rwanda were polymerase chain reaction (PCR)-​positive for B. quintana. Finally, biting flies seem to play an important role in the transmission of Bartonella species among ruminants (B. schoenbuschensis, B. chomelii, B. bovis, B. capreoli, candidatus B. melophagi) and bat flies are increasingly associated with bat-​ borne Bartonella species. B. quintana infections are transmitted by the body louse, Pediculus humanus. Outbreaks of trench fever are linked mainly with poor socioeconomic conditions and wars, which predispose to body louse infestation. B. quintana infections decreased after the World War I and re-​emerged during the Second World War. More

section 8  Infectious diseases 1264 recently, there have been sporadic outbreaks of urban trench fever in Europe and the United States of America in homeless popula- tions and alcoholics. B. quintana has also been detected in head lice, especially in Africa. The epidemiology of the many other Bartonella species is still not well understood. They usually cause asymptomatic bacteraemia in reservoir hosts:  B. henselae and B. clarridgeiae in cats, B. bovis in cattle, B. alsatica in rabbits, and B. tribocorum in rats. However, endocarditis cases have also been reported in dogs, cats, and cattle (Fig 8.6.43.3). Clinical features and pathology A remarkable feature of Bartonella is the ability of a single species to cause either acute or chronic infection with either vascular prolifera- tive or suppurative features. The pathological response to infection with Bartonella varies substantially with the host’s immune status. There have been few clinical studies employing a standard case def- inition, culture confirmation, and rigidly defined disease outcomes in patients with similar immunocompetence. 1.6 Mb Bq Bh Bm Bs MI Sm At Bj Rhp Cc Rp Rc Wp 1.9 3.3 3.3 7.6 6.7 5.6 9.1 5.5 4.0 1.1 Arthropod/ mammal associated Aquatic Plant associated Mammal/arthropod associated Arthropod/ associated 1.3 1.3 Size in Mb <0.2 0.5 1 2 3 4 5 6 7 8 9 Fig. 8.6.43.1  Variation in genome sizes and lifestyles in the α-​proteobacteria. The phylogenetic relationships are shown for the 13 α-​proteobacterial species for which complete genome sequence data are currently available. Genome structures, genome sizes, and host organisms are depicted graphically. Colours along branches indicate net loss (blue) and gain (red) of genes. Roots indicate nitrogen-​fixing species that belong to the rhizobacteria. The size of the filled circles corresponds to the relative sizes of the individual replicons, with red indicating the main chromosomes in each species. Agrobacterium tumefaciens has a linear chromosome, represented by a short line. At, Agrobacterium tumefaciens; Bh, Bartonella henselae; Bj, Bradyrhizobium japonicum; Bm, Brucella melitensis; Bq, Bartonella quintana; Bs, Brucella suis; Cc, Caulobacter crescentus; Ml, Mesorhizobium loti; Sm, Sinorhizobium meliloti; Rc, Rickettsia conorii; Rhp, Rhodopseudomonas palustris; Rp, Rickettsia prowazekii; Wp, Wolbachia pipientis. Reprinted by permission from Macmillan Publishers Ltd: Batut J. et al. (2004). The evolution of chronic infection strategies in the α-​proteobacteria. Nature Reviews Microbiology 2: 933–​945, copyright © 2004.

1265 8.6.43  Bartonellas excluding B. bacilliformis B vinsonii berkhoffii B vinsonii vinsonii B vinsonii arupensis B alsatica B taylorii B rattimassiliensis B graharnii B elizabethae B tribocorum B quintana B phoceensis B henselae B koehlerae B doshiae B bovis B schoenbuchensis B birtlesii B bacilliformis B clarridgeiae dog318006 B rochalimae dog131 fox008 63 100 84 53 52 92 100 100 88 97 100 100 99 99 71 100 100 99 51 Fig. 8.6.43.2  Neighbour-​joining tree of Bartonella species based on the combined gltA, rpoB, ftsZ, and
ITS sequence alignments. Reprinted from Henn JB et al. (2009). Infective endocarditis in a dog and the phylogenetic relationship of the associated “Bartonella rochalimae” strain with isolates from dogs, grey foxes, and a human. J Clin Microbiol 47: 787–​90. Table 8.6.43.1  Species of Bartonella reported to date with epidemiological and clinical data Bartonella spp. Reservoir host Vector detection in arthropods Disease in humans First cultivation B. bacilliformis Human Sand fly (Lutzomyia spp.) CSD, END 1919 B. talpae Moles Unknown Unknown 1911 B. peromysci Unknown Unknown Unknown 1942 B. vinsonii subsp. vinsonii Canadian voles (Microtus sp.) Unknown, Ear mites? Unknown 1946 B. quintana Human, cats Human body louse (Pediculus humanus corporis) and fleas TF, BA, BAC, END 1961 B. henselae Cats, (dogs?) Cat Flea (Ctenocephalides felis) CSD, BA, BAC, LMF, END, PH, RET 1990 B. elizabethae Rodents Fleas END (1 case) 1993 B. grahamii Voles, rodents Fleas? RET (1 case) 1995 B. taylorii Rats Fleas? Unknown 1995 B. doshiae Voles Fleas? Unknown 1995 B. clarridgeiae Cats, (dogs?) Cat flea Ctenocephalides felis Unknown 1995 B. vinsonii subsp. berkhoffii Dogs, coyotes, grey foxes Fleas, ticks? END 1995 B. vinsonii subsp. arupensis Rodents, cattle Deer ticks? Fleas? BAC (1 case) 1999 B. tribocorum Rats Unknown Unknown 1998 B. koehlerae Cats Fleas END (1 case) 1999 (continued)

1266 section 8  Infectious diseases Trench fever Trench fever is also known as quintan fever or 5-​day fever (because of its tendency to relapse on the fifth day), and Wolhynia fever (be- cause the disease was first observed by German medical officers on the East German front in Wolhynia). After the bite of the body louse the incubation period ranges generally from 15 to 25 days. However, in volunteers inoculated with a large volume of crushed infected lice, incubation was less than nine days. The illness varies widely from asymptomatic to severe. The classic clinical presenta- tion among troops was an acute febrile illness, often accompanied by severe headache and shin pain. The interval between attacks of pyrexia ranges from 4 to 8 days, but is usually 5 days. Trench fever often results in prolonged disability, but mortality is rare. The first 4 to 6 weeks of the illness are the most severe and, in a few cases, Bartonella spp. Reservoir host Vector detection in arthropods Disease in humans First cultivation B. alsatica Rabbit (Oryctolagus cuniculus) Fleas, ticks? END (2 cases), LMF (1 case) 1999 B. bovis (weissii) Cow (Bos taurus), cats? Biting flies Unknown 1999 B. washoensis Rodents, dogs Fleas MYOC (1 case) 2000 B. birtlesii Rats Unknown Unknown 2000 B. schoenbuchensis Roe deer (Capreolus capreolus) Deer keds (Lipoptena cervi, L. mazamae) BAC 2001 B. capreoli roe deer Deer keds (Lipoptena cervi) Unknown 2002 B. chomelii Cows (Bos taurus) Biting flies Unknown 2004 B. rattimassiliensis Rats Unknown Unknown 2004 B. phoceensis Rats Unknown Unknown 2004 B. australis Grey kangaroos (Macropus giganteus) Unknown Unknown 2007 B. tamiae Rodents? Unknown fatigue, myalgia, headache, rash 2008 B. rattaustraliani Rattus tunneyi Unknown Unknown 2009 Bartonella queenslandensis Melomys rat Unknown Unknown 2009 B. coopersplainsensis Rattus leucopus Unknown Unknown 2009 B. japonica Apodemus argenteus Unknown Unknown 2010 B. silvatica Apodemus speciosus Unknown Unknown 2010 B. jaculi greater Egyptian jerboa (Jaculus orientalis) Unknown Unknown 2013 B. calloscuiri plantain squirrel (Callosciurus notatus) Unknown Unknown 2013 B. pachyuromidis fat-​tailed gerbil (Pachyuromys duprasi) Unknown Unknown 2013 B. acomydis golden spiny mouse (Acomys russatus) Unknown Unknown 2013 B. senegalensis Unknown Ornithodoros sonrai Unknown 2013 B. florenciae Shrew (Crocidura russula) Unknown Unknown 2013 B. mayotimonensis Daubenton’s bat (Myotis daubentonii) Bat flies END (2009) (1 case) 2014 B. naantaliensis Daubenton’s bat (Myotis daubentonii) Bat flies Unknown 2014 B. dromedarii camels (Camelus dromedarius) Hyalomma ticks? Unknown 2014 B. ancashensis Humans Sandflies? Verruga peruana 2015 B. apis Honey bees (Apis mellifera) symbiont Unknown 2016 B. koehlerae subsp. boulouisii Puma (felis concolor) Fleas? Unknown 2016 B. koehlerae subsp. bothieri Bobcat (Lynx rufus) Fleas? Unknown 2016 Candidatus B. melophagi Sheep (Ovis aries) Sheep ked (Melophagus ovinus) Pericarditis Fatigue, muscle
pain (2009) 2007 Candidatus B. thailandensis Red spiny rat Maxomys surifer Unknown Unknown 2009 Candidatus B. antechini Mardos/​Yellow-​footed antechinus (Antechinus flavipes) Fleas (Acanthopsylla jordani) and ticks (Ixodes antechini) Unknown 2011 Candidatus B. merieuxii Canids (dogs, Jackals) Fleas? Unknown 2012 Candidatus B. hemsundetiensis Daubenton’s bat (Myotis daubentonii) Bat flies Unknown 2015 BA, bacillary angiomatosis; BAC, bacteraemia; CSD, cat-​scratch disease; END, endocarditis; LMF, lymphadenopathy; MYOC, myocarditis; PH: peliosis hepatis; RET, retinitis; TF, trench fever. Table 8.6.43.1  Continued

1267 8.6.43  Bartonellas excluding B. bacilliformis chronic fever, anaemia, loss of weight, and neuropsychiatric symp- toms develop over time. Cat-​scratch disease (CSD) CSD is a common infection that is seasonal throughout the world. Cats are the main reservoir of B. henselae, and the bacterium is trans- mitted between cats by the cat flea Ctenocephalides felis. Depending on the clinical manifestations, CSD has been characterized in two forms: (1) classic typical clinical CSD with lymphadenopathy and a history of a cat scratch and/​or bite, and (2) atypical CSD. Classic CSD usually occurs in children and young adults but can also affect elderly people. Most patients with typical CSD remain afebrile. The main clinical manifestations in an immunocompetent host appear approximately two weeks after inoculation, although B. henselae DNA can be isolated from the peripheral blood of patients as long as four months after infection. One-​third of the patients present with a history of fever lasting from 0 to 70 days (mean 14.8 days) with a maximum temperature between 37.9°C and 42.0°C. The localiza- tion of lymphadenopathy is mainly axillary, cervical, or submaxil- lary, that is, the lymph nodes that usually drain the area where the cat scratch occurs (Fig. 8.6.43.4). Lymphadenopathy can sometimes last for months, and in a few cases can be prolonged for as long as 12 to 24 months. General symptoms including malaise, headache, convulsion, sore throat, otalgia, vomiting, diarrhoea, anorexia, and tiredness can persist for long durations. B. henselae has also been identified in skin biopsy specimens of patients with CSD at the pri- mary site of inoculation. Atypical CSD occurs in a minority of cases, most of whom have se- vere systemic symptoms indicating disseminated infection. Patients with atypical CSD can have prolonged fever (>2 weeks), myalgia, arthralgia/​arthropathy, malaise, fatigue, weight loss, splenomegaly, neuroretinitis, encephalopathy, and Parinaud’s oculoglandular syndrome. This syndrome appears to be the most common ocular complication of CSD, affecting approximately 5% of symptomatic patients. Bacteria from an infected cat are inoculated indirectly into the eye rather than by direct contact through a scratch. Two-​ thirds of patients with neuroretinitis have evidence of past infection with B. henselae. Other Bartonella species causing retinitis include B. quintana, B. grahamii, B. clarridgeiae, and B. elizabethae. Retinitis is typically stellar; other ocular lesions include optic disc oedema and macular star formation, loss of vision with central scotoma, and glaucoma (Fig. 8.6.43.5). The onset of neurological complications varies from a few days to 2 months after the onset of lymphadenopathy and tends to occur more often in older school-​age children. Symptoms include head- ache, malaise, lethargy lasting for one to several weeks, impaired consciousness, and acute hemiplegia. Bacillary angiomatosis Bacillary angiomatosis, also called epithelioid angiomatosis, is a vascular proliferative disease most often involving the skin, which mainly occurs in immunocompromised patients, especially HIV-​ infected individuals in an advanced stage of AIDS (<40 CD4 cells/​ ml). Without appropriate therapy, infection spreads systemic- ally, can involve virtually any organ, and is usually fatal. Rarely, it can also affect immunocompetent patients. Both B. henselae and B. quintana are considered aetiological agents. In the case of B. quintana infection, lesions are subcutaneous and/​or osteolytic, Fig. 8.6.43.3  Vegetative endocarditis on the aortic valve of a dog. Courtesy of B. Chomel. Fig. 8.6.43.4  Axillary lymphadenitis in cat-​scratch disease. Fig. 8.6.43.5  Stellar retinitis due to B. henselae. Courtesy of Dr M. J. Dolan.

section 8  Infectious diseases 1268 whereas peliosis hepatis is characteristic of B. henselae infection. Bacillary angiomatosis is manifested by the gradual appearance of numerous brown to violaceous or colourless vascular tumours of the skin and subcutaneous tissues, numbering a few to several hun- dred and varying in size from a few millimetres to several centi- metres. Three morphologically distinct cutaneous lesions have been described:  (1) pyogenic granuloma-​like lesions—​the most common type, (2) subcutaneous nodules, and (3) hyperpigmented indurated plaques. The clinical differential diagnosis includes pyogenic granuloma, haemangioma, subcutaneous tumours, and Kaposi’s sarcoma. The skin lesions are very similar to those re- ported for verruga peruana, the chronic form of Carrión’s disease. Bacillary angiomatosis lesions can also involve the bone marrow, liver, spleen, or lymph nodes. Bacillary peliosis Bacillary peliosis is a condition affecting solid internal organs with reticuloendothelial elements, especially the liver, in which bacillary peliosis causes vascular proliferation of sinusoidal hepatic capil- laries resulting in blood-​filled spaces (peliosis hepatis). The spleen, abdominal lymph nodes, and bone marrow can also be affected. The disease was first described in patients with tuberculosis and ad- vanced cancers and was associated with the use of anabolic steroids. It has also been reported in organ transplant recipients and HIV-​ infected patients with B. henselae. Bacteraemia and endocarditis Infection due to B. quintana should be suspected in homeless, indi- gent, or chronic alcoholic patients with culture-​negative endocar- ditis, especially those with a long-​standing valve lesion. B. quintana bacteraemia has also been reported in patients without endocar- ditis. Evidence of Bartonella endocarditis was found in 0.5–​12% of all patients diagnosed with endocarditis tested at reference centres in different countries in the Old World, decreasing from north to south. Among human cases of Bartonella endocarditis in Europe, 75% were associated with B. quintana and 25% with B. henselae. In North Africa, most cases were caused by B. quintana, which is also responsible for asymptomatic, prolonged, and intermittent bacter- aemia in homeless people in cities both in Europe and in the United States of America. Endocarditis caused by B. henselae should be suspected in patients with previous valve disease and culture-​negative endocarditis, espe- cially those who have contacts with cats. Endocarditis and/​or bacteraemia caused by other Bartonella species is uncommon. B. elizabethae, B. vinsonii subsp. berkhoffii, B. vinsonii subsp. arupensis, B. koehlerae and B. alsatica, B. mayotim­ onensis have been isolated or detected from heart valves of patients with culture-​negative endocarditis. One case of myocarditis has been attributed to B. washoensis. Prolonged fever Prolonged fever (>15 days) might occur in patients with atypical CSD. Prolonged fever without lymphadenopathy or fever of un- known origin has been described in several paediatric cases of CSD. Diagnosis Diagnosis is difficult because of the fastidious nature of Bartonella and the non​specific clinical manifestations. Diagnostic techniques include culture and detection of organisms in lymph nodes by im- munofluorescence, molecular techniques including PCR, and ser- ology. Table 8.6.43.2 presents the most common clinical features caused by Bartonella and the best techniques for their identification, and Fig. 8.6.43.6 presents the current diagnostic strategy. Specimen collection Various specimens, especially serum, blood, biopsy specimens, and arthropods, are useful. They should be sampled as soon as possible after the onset of disease. For serological diagnosis, serum samples should be collected early and during convalescence two to three weeks later. Serum samples can be stored easily at −20°C or below for long periods without degradation of antibodies. Blood should be sampled before antimicrobial therapy either in citrate-​containing vials for culture in cell monolayers or in ethylenediaminetetraacetic acid (EDTA) for culture on blood agar or for PCR techniques. EDTA should be avoided for cell culture since it leads to detachment of cell monolayers. Biopsies of lymph nodes, cardiac valves, vascular aneur- ysms, or grafts should be taken in two parts, one in absolute alcohol for histopathology and immunodetection and another frozen and Table 8.6.43.2  Clinical manifestations and diagnostic methods for Bartonella infections in humans Disease in humans Commonly isolated Rarely isolated Specimen Methods Cat-​scratch disease B. henselae Lymph nodes PCR, serology Endocarditis B. henselae, B. quintana B. elizabethae, B. koehlerae, B. vinsonii subsp. berkhoffii, B. vinsonii subsp. arupensis, B. alsatica, candidatus B. mayotimonensis Blood, serum, valves PCR, serology Retinitis B. henselae B. grahamii Serum, aqueous humour PCR, serology Bacillary angiomatosis B. henselae, B. quintana Blood, serum, cutaneous biopsy PCR Bacteraemia B. quintana B. henselae, B. vinsonii subsp. arupensis, B. rochalimae, B. doshiae, B. schoenbuschensis, B. tribocorum, B. tamiae, B. vinsonii subsp. vinsonii Blood, serum PCR, serology Peliosis hepatis B. henselae Blood, serum, hepatic biopsy PCR, serology Osteomyelitis B. henselae B vinsonii subsp. berkhoffii (in a cat) Blood, serum, bone biopsy PCR, serology Trench fever B. quintana Blood, serum PCR

1269 stored at −70° C in a sterile vial for culture and PCR analysis. These methods can be also used to detect Bartonella in various arthro- pods including ticks, lice, and fleas (xenodiagnosis). The arthropod should be disinfected with iodinated alcohol and then crushed in medium before being inoculated into a shell vial for culture or pro- cessing using molecular methods. Arthropods can be easily stored dry in a box and sent by mail to a reference centre for analysis. Direct diagnosis Culture The most widely used methods for isolation are direct plating of sample material onto solid media, blood culture in broth, and cocultivation in cell cultures. Bartonella can be grown on blood agar at 35°C to 37°C in a 5% CO2 atmosphere, except for B. bacilliformis which should be grown at 28–​30°C. Primary isolates in humans are typically obtained after 12 to 14 days, although an incubation period of up to 45 days can be necessary (Fig. 8.6.43.7). In animals, growth might occur in three to seven days (especially for cat and rodent isolates). Subculture in blood broth in shell vials is the most efficient culture method in human patients with endocarditis. Specimens are placed on human embryonic lung cells in shell vials and incubated at 37°C in an atmosphere of 5% CO2. Culture might be successful using blood samples, skin, lymph nodes, or other organ biopsy sam- ples. Lysis centrifugation and freezing have been shown to enhance the recovery of Bartonella from blood. However, despite improved culture methods, blood cultures might be negative if the patient has recently received antibiotics or if the organism is fastidious and/​or re- quires special culture techniques. A Bartonella-​Alphaproteobacteria growth medium has been developed and provides an improved method to isolate these fastidious microorganisms, especially from human and dog samples. MALDI-​TOF mass spectrometry is an ac- curate and reproducible tool for the rapid and inexpensive identifi- cation of Bartonella species. Immunodetection Detection of Bartonella using specific antibodies has been achieved in various situations. Demonstration of microorganisms in valve tis- sues by Warthin–​Starry staining (Fig. 8.6.43.8) is a classic criterion Serum Blood Arthropods Biopsies Samples Direct diagnosis Immunodetection PCR Culture IHC IF CA IF Elisa WB analysis PCR Serology Indirect diagnosis Fig. 8.6.43.6  Strategy for the diagnosis of Bartonella spp. infections Fig. 8.6.43.7  Colony morphology of B. henselae on Columbia 5% sheep blood agar. Fig. 8.6.43.8  Warthin–​Starry staining of a cardiac valve of a patient with B. quintana endocarditis. Arrow shows the clumps of bacilli. Magnification ×400. 8.6.43  Bartonellas excluding B. bacilliformis

section 8  Infectious diseases 1270 for the histological diagnosis of infective endocarditis. Direct im- munological detection in lymph nodes has been reported in patients with CSD, for patients with peliosis hepatis, in red blood cells of bac- teraemic homeless people, in cardiac valves, and in skin biopsies. Immunohistochemistry is a convenient tool for detecting B. quintana in tissues, but specific antibodies are often not available. Molecular biology PCR is a convenient method for detecting Bartonella either in fresh (best) or in formalin-​fixed and paraffin-​embedded tissues (not as good and reliable). The most common target genes used for the de- tection and identification of Bartonella are the citrate synthase gene (gltA), the 16S RNA gene, the 16S–​23S rRNA ITS, the 60-​kDa heat shock protein (groEL), the RNA polymerase β-​subunit gene (rpoB) and the pap31 gene. Although these methods are highly specific, their sensitivity varies according to sample type. Thus, the current strategy for the diagnosis of Bartonella infections is to use two dif- ferent target genes (e.g. ITS and gltA gene), complemented with a third gene (groEL or rpoB) if initial results are discordant. Samples should be considered positive only if at least two genes are positive and if sequences obtained give similar identification. Improvement of molecular methods might increase the test sensitivity, especially when using real-​time PCR. The diagnosis of Bartonella endocarditis by real-​time nested PCR assays performed on a LightCycler appar- atus (LCN-​PCR) using serum was proposed, which can shorten the delay in diagnosis. For the typing and characterization of B. hense­ lae isolates, multilocus sequence typing is another method that groups bacteria based on comparison of nucleic acid sequences of 450–​500 bp derived from the internal fragments of a number (typ- ically seven) of housekeeping genes. A molecular typing method based on the sequences of non​coding zones rather than sequences of housekeeping genes called multispacer typing has also been developed. Indirect diagnosis: Serology Serology is the only useful non​invasive method for the diagnosis of Bartonella infections, especially for CSD, bacteraemia, and endocar- ditis. The sensitivity of serological tests varies between laboratories, from nearly 100% to less than 30% depending on the method used for preparation of antigens. Sources of antigens for serology can be either whole-​cell lysates or outer membrane protein preparations and, more recently, recombinant proteins. The most widely used serological test for diagnosis is the indirect fluorescence assay (IFA) to detect antibodies against B. henselae whole cells. An IgG anti-​ B. henselae antibody titre ≥1:64 is considered positive for infection when patients are tested at least two to three weeks after a suspected infection. Bartonella-​associated endocarditis in humans and animals is usually associated with much higher IFA antibody titres (>1:800). False-​negative results are due to either antigenic heterogeneity among B. henselae species or to other diseases such as mycobacterial infections, lymphoma, or Kaposi’s sarcoma. Cross-​reactions have been infrequently reported either with other Bartonella species, or between Bartonella species and Coxiella burnetii or Chlamydia. Lepidi et al. have developed autoimmunohistochemistry, which is a peroxidase-​based method with the patient’s own serum as the source of antibodies directed against the aetiological microorganism, for the diagnosis of infective endocarditis. The rate of detection of bac- teria by autoimmunohistochemistry was significantly higher than that by culture but was similar to that by PCR. A more sophisticated serological method, western blot analysis after cross-​adsorption, has been shown to be a powerful tool for the identification of Bartonella to the species level in cases of endocarditis (Fig. 8.6.43.9). Treatment In vitro susceptibility to antibiotics This can be performed in either eukaryotic cells or axenic media. Bartonella species are susceptible to a wide range of antibiotics when they are grown axenically, including penicillin and cephalo- sporin compounds, aminoglycosides, chloramphenicol, tetracyc- lines, macrolide compounds, rifampicin, fluoroquinolones, and co-​trimoxazole. However, these results correlate poorly with in vivo efficacy, because most antibiotics are not bactericidal, except for aminoglycosides. This has also been reported in cell-​culture models for B. henselae in murine macrophage-​like cells and for B. quin­ tana in red blood cells. In vivo data have demonstrated the benefit of a combination of doxycycline with gentamicin in the treatment of infections, including endocarditis and bacteraemia in home- less individuals. Mutations in the 23S RNA gene and insertion of Fig. 8.6.43.9  Western blot of a patient with B. quintana endocarditis before (a) and after cross-​adsorption with B. quintana (b) or B. henselae (c). Line 1: B. quintana; line 2: B. henselae; line 3: B. elizabethae; line 4: B. vinsonii subsp. berkhoffii; line 5: B. alsatica.

1271 nine aminoacids in the L4 ribosomal protein for B. henselae and B. quintana, respectively, can be selected in vitro by erythromycin. Mutations such as the A2059G transition have been detected dir- ectly in the lymph node of a patient with CSD, suggesting that natur- ally erythromycin-​resistant strains may infect humans. Trench fever Most cases of trench fever were reported before the antibiotic era. However, successful treatment with tetracycline or chloram- phenicol was reported during the Second World War. In cases of urban trench fever, patients with chronic B. quintana bacteraemia should be treated with gentamicin (3 mg/​kg intravenously once a day) for 14 days and with doxycycline (200 mg/​day orally once a day) for 28 days. Patients with chronic bacteraemia should be carefully evaluated for endocarditis, which requires prolonged therapy under close monitoring. CSD Cases of CSD typically do not respond well to antibiotic therapy. Management consists of analgesics for pain, follow-​up, and drainage when necessary. Patients who do not improve clinically benefit from excision of affected lymph nodes and investigation for coinfection such as Mycobacterium tuberculosis and/​or lymphoma. The only double-​blind placebo-​controlled study for the treatment of CSD with azithromycin in immunocompetent patients showed only a faster reduction of their lymph node volume as compared to pla- cebo. Thus, the current recommendation for the treatment in mild to moderately ill immunocompetent patients with CSD is no anti- biotic treatment. Treatment with azithromycin could help patients with bulky lymphadenopathy or those with complicated CSD with retinitis and central nervous system disease. Endocarditis Effective antibiotic therapy for suspected Bartonella endocarditis should include an aminoglycoside (gentamicin) for at least 14 days together with ceftriaxone with or without doxycycline for 6 weeks to achieve a bactericidal effect. Valve replacement is necessary in most patients due to the extensive damage. Bacillary angiomatosis and peliosis hepatis Erythromycin is the antibiotic of choice for bacillary angiomatosis and peliosis hepatis. Treatment should be continued for at least three months for bacillary angiomatosis and four months for peliosis hepatis. Longer treatment should be given in HIV-​infected and im- munocompromised patients. An in vitro model of B. quintana cul- tured in endothelial cells has shown that erythromycin acts mainly antiangiogenically rather than as an antibiotic, explaining the often dramatic response to this antibiotic in bacillary angiomatosis. Bacillary peliosis hepatis responds to antibiotics more slowly than cutaneous bacillary angiomatosis, but hepatic lesions usually im- prove after several months of treatment. Relapses of peliosis hepatis and bacillary angiomatosis lesions in bone and skin have fre- quently been reported, mainly in severely immunocompromised HIV-​infected patients. Finally, patients who have relapses after the recommended treatment should receive secondary prophylactic antibiotic treatment with erythromycin or doxycycline as long as they are immunocompromised. Prevention B. quintana infections can be prevented by delousing, changing, and/​or washing clothes. Pet cats and pet cat environment (bedding, and so on) should be treated regularly with flea control products to prevent B.  henselae infection. Immunocompromised patients should avoid cat scratches and exposure to cat fleas. Only seronega- tive cats should be kept by immunocompromised people and regu- larly treated with flea preventatives. For other zoonotic Bartonella species, a better understanding of their epidemiology is needed to be able to apply effective prevention strategies. For instance, the modes of contamination of humans by B. alsatica, B. tamiae or B. vinsonii subsp. berkhoffii have not been elucidated. Conclusions Bacteria of the genus Bartonella are responsible for an increasing number of emerging or re-​emerging infections worldwide and can present a wide clinical spectrum, from benign and self-​limited in- fections to severe and life-​threatening diseases. Consequently, diag- nosis and treatment of these infections should be adapted to each clinical situation, to the species involved, and to whether the disease is in an acute or chronic stage. FURTHER READING Alsmark CM, et  al. (2004). The louse-​borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae. Proc Natl Acad Sci U S A, 101, 9716–​21. Angelakis E, et al. (2010). Bartonella henselae in skin biopsy specimens of patients with cat-​scratch disease. Emerg Infect Dis, 16, 1963–​5. Batut J, Andersson SG, O’Callaghan D (2004). The evolution of chronic infection strategies in the alpha-​proteobacteria. Nat Rev Microbiol, 2, 933–​45. Birtles RJ, et  al. (1995). Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov. Int J Syst Bact, 45, 1–​8. Biswas S, Raoult D, Rolain JM (2006). Molecular characterization of resistance to macrolides in Bartonella henselae. Antimicrob Agents Chemother, 50, 3192–​3. Boulouis HJ, et al. (2005). Factors associated with the rapid emergence of zoonotic Bartonella infections. Vet Res, 36, 383–​410. Breitschwerdt EB, et al. (2010). Bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Care (San Antonio), 20, 8–​30. Brenner DJ, et  al. (1993). Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales. Int J Syst Bact, 43, 777–​86. Chomel BB, et  al. (1996). Experimental transmission of Bartonella henselae by the cat flea. J Clin Microbiol, 34, 1952–​6. Chomel BB, et al. (2009). Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors. Vet Res, 40, 29. 8.6.43  Bartonellas excluding B. bacilliformis

8.6.44 Bartonella bacilliformis infection 1272

8.6.44 Bartonella bacilliformis infection 1272

section 8  Infectious diseases 1272 Debré R, et al. (1950). La maladie des griffes de chat. Bull Mem Soc Méd Hop Paris, 66, 76–​9. Dehio C (2001). Bartonella interactions with endothelial cells and erythrocytes. Trends Microbiol, 9, 279–​85. Engel P, Dehio C (2009). Genomics of host-​restricted pathogens of the genus Bartonella. Genome Dyn, 6, 158–​69. Foucault C, Brouqui P, Raoult D (2006). Bartonella quintana character- istics and clinical management. Emerg Infect Dis, 12, 217–​23. Fournier PE, et al. (2009). Rapid and cost-​effective identification of Bartonella species using mass spectrometry. J Med Microbiol, 58, 1154–​9. Henn JB, et al. (2009). Infective endocarditis in a dog and the phylo- genetic relationship of the associated “Bartonella rochalimae” strain with isolates from dogs, gray foxes, and a human. J Clin Microbiol, 47, 787–​90. Houpikian P, Raoult D (2001). 16S/​23S rRNA intergenic spacer re- gions for phylogenetic analysis, identification, and subtyping of Bartonella species. J Clin Microbiol, 39, 2768–​78. Houpikian P, Raoult D (2003). Western immunoblotting for Bartonella endocarditis. Clin Diagn Lab Immunol, 10, 95–​102. Kernif T, et al. (2010). Molecular detection of Bartonella alsatica in rabbit fleas, France. Emerg Infect Dis, 16, 2013–​4. Koehler JE, et al. (1997). Molecular epidemiology of Bartonella infec- tions in patients with bacillary angiomatosis-​peliosis. N Engl J Med, 337, 1876–​83. La Scola B, Raoult D (1999). Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-​year experience (1993 to 1998). J Clin Microbiol, 37, 1899–​905. La Scola B, et al. (2003). Gene-​sequence-​based criteria for species def- inition in bacteriology: the Bartonella paradigm. Trends Microbiol, 11, 318–​21. Lepidi H, Fournier PE, Raoult D (2000). Quantitative analysis of valvular lesions during Bartonella endocarditis. Am J Clin Pathol, 114, 880–​9. Lepidi H, et al. (2006). Autoimmunohistochemistry: a new method for the histologic diagnosis of infective endocarditis. J Infect Dis, 193, 1711–​17. Maurin M, Raoult D (1996). Bartonella (Rochalimaea) quintana infec- tions. Clin Microbiol Rev, 9, 273–​92. Maurin M, Rolain JM, Raoult D (2002). Comparison of in-​house and commercial slides for detection of immunoglobulins G and M by immunofluorescence against Bartonella henselae and Bartonella quintana. Clin Diag Lab Immunol, 9, 1004–​9. Meghari S, et  al. (2006). Anti-​angiogenic effect of erythromycin in Bartonella quintana:  in vitro model of infection. J Infect Dis, 193, 380–​6. Musso D, Drancourt M, Raoult D (1995). Lack of bactericidal effect of antibiotics except aminoglycosides on Bartonella (Rochalimaea) henselae. J Antimicrob Chemother, 36, 101–​8. Pitulle C, et al. (2002). Investigation of the phylogenetic relationships within the genus Bartonella based on comparative sequence analysis of the rnpB gene, 16S rDNA and 23S rDNA. Int J Syst Evol Microbiol, 52, 2075–​80. Raoult D, et al. (2003). Outcome and treatment of Bartonella endo- carditis. Arch Intern Med, 163, 226–​30. Rolain JM, et al. (2002). Bartonella quintana in human erythrocytes. Lancet, 360, 226–​8. Rolain JM, et  al. (2003). Molecular detection of Bartonella quin­ tana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis, 9, 338–​42. Rolain JM, et al. (2004). Recommendations for treatment of human in- fections caused by Bartonella species. Antimicrob Agents Chemother, 48, 1921–​33. Rolain JM, et al. (2006). Lymph node biopsy specimens and diagnosis of cat-​scratch disease. Emerg Infect Dis, 12, 1338–​44. Saisongkorh W, et al. (2010). Evidence of transfer by conjugation of type IV secretion system genes between Bartonella species and Rhizobium radiobacter in amoeba. PLoS One, 5, e12666. Sanguinetti-​Morelli D, et al. (2011). Seasonality of cat-​scratch disease, France, 1999–​2009. Emerg Infect Dis, 17, 705–​7. Zangwill KM (2013). Cat scratch disease and other Bartonella infec- tions. Adv Exp Med Biol, 764, 159–​66. Zeaiter Z, et al. (2002). Phylogenetic classification of Bartonella species by comparing groEL sequences. Int J Syst Evol Microbiol, 52, 165–​71. 8.6.44  Bartonella bacilliformis infection A. Llanos-​Cuentas and C. Maguiña-​Vargas ESSENTIALS Bartonellosis (Carrión´s disease, verruga peruana, Oroya fever, Guaitará fever) is caused by the Gram-​negative bacillus Bartonella bacilliformis. It is endemic in the western Andes and inter-​Andean valleys of Peru, and is still occasionally reported in Ecuador and Colombia, with infection resulting from the bite of various female sandflies. Clinical features, diagnosis, management, prognosis, and prevention—​ infection of red blood cells manifests with non​specific ‘viral-​type’ symptoms and haemolytic anaemia in the acute stage of disease. Following an asymptomatic phase, the late ‘eruptive’ stage is charac- terized by dermal nodules (‘verrugas’) that frequently heal spontan- eously. Secondary opportunistic infections are common. Diagnosis in areas where the disease occurs is usually by demonstration of bacteria in the blood film. Ciprofloxacin is the treatment of choice in most acute cases. Mortality is 1.1–​2.4% in endemic areas and around 9% in patients admitted to hospital. There is no satisfactory prevention for people living in endemic areas; tourists can take the usual precautions against being bitten by insects. Aetiological agent Barton, a Peruvian physician, described the causative organism in 1905. Bartonella bacilliformis is a small motile aerobic Gram-​ negative bacillus that stains deep red or purple with Giemsa (Fig. 8.6.44.1). This facultative intracellular haemotropic bacterium varies in morphology and quantity during various stages of the dis- ease. Although it is a pleomorphic organism, two essential types are distinguishable, bacilli, or rod-​shaped forms and coccoid forms. Rod-​shaped forms predominate in the acute stage of the disease

1273 8.6.44  Bartonella bacilliformis infection and coccoid in the convalescent stage. B. bacilliformis can infect red blood cells (Fig. 8.6.44.2), endothelial cells of capillaries, and sinus- oidal lining cells. The organism is 2–​3 µm long and 0.2–​2.5 µm thick. In cultures, 1–​10 flagella, 3–​10 µm long, may originate from one end of the organism. Bartonella can be cultured in Columbia agar sup- plemented with 10% defibrinated sheep blood at 29°C under aer- obic conditions for up to 6 weeks. Multilocus sequence typing of Peruvian isolates of B. bacilliformis showed wide genetic diversity. While seven of the eight sequence types were closely related, one exhibited profound evolutionary divergence suggesting that it might represent a new Bartonella genospecies. Epidemiology Bartonellosis has occurred since pre-​Columbian times, as proven by artistic representations in pre-​Inca pottery and lesions in an an- cient mummy. It is an endemic disease mainly in inter-​Andean val- leys in west, central, and east Andean areas of Peru (Fig. 8.6.44.3) and increasingly in Amazonian areas where alternative arthropod vectors have been suggested (i.e. ticks). In Ecuador, bartonellosis is endemic in several areas (Loja, Zamora-Chinchipe) but with spor- adic clinical cases. This has suggested the existence of attenuated or less virulent strains. Knowledge of the epidemiology of Bartonella is incomplete but studies suggest that transmission is unstable and geographically highly heterogeneous with prevalence and incidence rates that vary considerably in time and space (Fig. 8.6.44.4). The transmission is influenced by the highly variable human behaviour and the environment, heavily influenced by climatic factors that de- termine changes in the abundance and behaviour of the vectors. The epidemiological pattern is endemic plus epidemic in the new areas. Transmission is usually in rural towns and around human dwell- ings. The disease occurs between 500 and 3200 m above sea level. Transmission varies throughout the year, being greatest towards the end of the rainy season (March to May). Interepidemic periods occur every 10–​15 years. In endemic areas the infection has cluster distribution where approximately 80% of cases are concentrated in 20% of the houses. The risk of acquiring the disease is substantially higher (2.6 times) when a family member has a confirmed diagnosis of bartonellosis. The disease is most common in children under 15 years and for each year of life the risk of acquiring the disease decreases by 4%. Incidence is greatly influenced by climatic, envir- onmental, and ecological changes such as the El Niño phenomenon. At present, 11 species and subspecies of the genus Bartonella have been associated with human infections but only three have epi- demiological importance: B. bacilliformis, of which eight antigenic variants have been described in Peru; B. henselae, the major cause of cat-​scratch disease and peliosis (Chapter 8.6.42); and B. quintana (formerly Rochalimaea), the agent of trench fever (Chapter 8.6.42). Recently, a new bartonella named B. ancashensis has been isolated from two patients with the verruga form. Other bartonellas such as B. rochalimae, B. vinsonii subsp. berkhoffii, B. vinsonii subsp. aru­ pensis, B.  elizabethae, B.  koehlerae, B.  alsatica, B.  grahamii, and B. clarridgeiae occasionally cause disease in humans. In immuno- compromised people, especially those with the HIV/​AIDS, B. hense­ lae and B.  quintana cause opportunistic infections, frequently manifested as cutaneous bacillary angiomatosis, resembling verruga peruana. The genus Bartonella is a unique pathogenic bacteria known to invade red blood cells. In endemic areas, in a high transmission period (1997–​1998) the incidence rate was 12.7 person-​years and 20.5% of those patients infected with B. bacilliformis remained asymptomatic, 31.5% devel- oped the eruptive form (chronic phase) without evidence of an acute illness, and 37% developed the eruptive form preceded by some symptoms. Only 11% will developed the classic acute form, with a higher frequency in children. However, these proportions tend to shift during periods of low transmission with increase of asymptom- atic infections. Outsiders generally develop acute severe forms of the disease (Oroya fever). Large epidemics have occurred when large groups of non​residents have entered endemic areas. In 1870, an epi- demic engulfed workers building the railroad from Lima to Oroya (Fig. 8.6.44.5); it was estimated that there were 7000 deaths. Infection results from the bite of females of several species of sandflies (Lutzomyia), especially Lutzomyia verrucarum. These vectors frequent human dwellings and, because they are active during twilight hours, humans are infected around sunrise and sunset. Although the reservoir is unknown, humans are regarded as being increasingly important. Little is known about asymptom- atic carriage, but this may have a significant role in perpetuation Fig. 8.6.44.1  B. bacilliformis in blood smear stained with Giemsa. Fig. 8.6.44.2  B. bacilliformis in a red blood cell.

section 8  Infectious diseases 1274 of transmission of infection in endemic areas. To date all studies in domestic and wild animals have been negative for B. bacilliformis. Pathogenesis After inoculation of B. bacilliformis by a sandfly bite, the bac- teria multiply in endothelial cells of small vessels and phago- cytic cells near the skin. Systemic invasion and multiplication in endothelial cells and red blood cells follows. In the most serious cases, 95% of red cells are infected with numerous bac- teria. The hallmark of the disease is the severe haemolytic anaemia caused by massive infection of red blood cells and subsequent erythrophagocytosis. Several mechanisms con- tribute to anaemia: increased fragility, form and size alteration, and reduced half-​life of infected and non​infected red cells. Some inhibition of haemoglobin synthesis, probably induced by toxic factors, has also been invoked, since red cell produc- tion increases dramatically with reduction of bacteraemia. Erythrophagocytosis contributes to lymphadenopathy and hepatosplenomegaly. ‘Blockade’ of the mononuclear phagocytic system and the presence of the circulating iron leads to super- infection, usually by enterobacteria, during the anaemia stage or early recovery from it. Transient depression of cellular immunity Fig. 8.6.44.3  Endemic area for bartonellosis; near Tarma, Peru. Copyright D. A. Warrell. 2000 2006 2012 2018 Fig. 8.6.44.4  Changes of the geographical distribution of bartonellosis in Peru between 2000 to 2018.

1275 has been reported. During the anaemic phase, mild lymphopenia with a reduction of CD4, a mild increase of CD8, and decrease of the polyclonal stimulation of lymphocytes occurs. High levels of interleukin (IL)-​10 were found in the acute phase. In Gram-​negative sepsis, an uncontrolled production of IL-​10 may produce ‘immunological paralysis’ of antigen-​presenting cells. The eruptive form appears a few weeks to months after the acute illness has subsided, and in Peru is named ‘verruga peruana’ (Fig. 8.6.44.6a, b). The vascular skin lesions show endothelial pro- liferation and histiocytic hyperplasia (the cells contain degenerate organisms; see Fig. 8.6.44.7) and later show fibrosis and necrosis. Electron microscopy of verrucous tissue shows B. bacilliformis in the interstitial tissues, indicating that the presence of the bacteria is important for this unusual vascular response to occur. Verruga peruana results from persistent infection, an immune response that is probably insufficient, and a peculiar vascular reaction, which could be caused by an angiogenic bacterial factor. In endemic areas recurrences are not rare (~ 5%). Clinical features The disease has two stages, anaemic and eruptive, with an asymp- tomatic intermediate period. After an incubation period of around 60 days (range 10–​210 days), non​specific prodromal symptoms ap- pear. The onset is usually gradual with malaise, mild chills, fever, and headache. Occasionally, high fever may develop rapidly or build up over a few days. It is accompanied by sweating and rigors. Common symptoms include weakness, aching of the head, back, and extrem- ities, prostration, and depression. The classical clinical picture is dominated by severe (haemolytic) anaemia and the patient rapidly becomes pale (Fig. 8.6.44.8), dyspnoeic, and jaundiced. There might be hepatosplenomegaly, generalized lymphadenopathy, tachy- cardia, myocarditis (Fig. 8.6.44.9), purpura, hepatitis, diarrhoea, pericardial effusion, exudates, anasarca, and retinal changes (Fig. 8.6.44.10); sometimes there is generalized oedema, drowsiness, and convulsions, and exceptionally meningoencephalomyelitis. The duration of this state is variable (generally 2–​4 weeks). In pregnant women, the disease in this phase may cause abortion, fetal death, Fig. 8.6.44.5  Endemic area for bartonellosis; Rimac valley, Peru—​Puente Verrugas. Copyright D. A. Warrell. (a) (b) Fig. 8.6.44.6  Verruga peruana: miliary haemangioma-​like lesions. 8.6.44  Bartonella bacilliformis infection

section 8  Infectious diseases 1276 and transplacental transmission of the disease; maternal death is common. Between 9 to 18% patients develop the classical symptoms (fever, anaemia, and jaundice) and 82 to 91% develop fever alone. In the intermediate period, patients are asymptomatic and re- cover from the anaemia through increased bone marrow activity. This pre-​eruptive period varies from weeks to months. In the eruptive stage, many nodular lesions of varying size appear on the face, trunk, and limbs over a period of a month or more and usually persist for 3 or 4 months. There is accompanying mild arth- ralgia, myalgia, and sometimes fever. The red or purplish skin le- sions are papules a few millimetres across. Most often the eruption is miliary (miliary form) with many haemangioma-​like lesions of the dermis (Fig. 8.6.44.6a, b). Nodular lesions (nodular form) are larger but fewer and more prominent on the extensor surfaces of arms and legs (Fig. 8.6.44.11a, b). They are painless and prone to bleeding (Fig. 8.6.44.11c), secondary infection, and ulceration. The appear- ance can resemble haemangioma, cutaneous bacillary angiomatosis, granuloma pyogenicum, Kaposi’s sarcoma, fibrosarcoma, leprosy (histoid form), or yaws. Occasionally one to a few, large, deep-​seated ulcerating lesions (mular form) might develop. These tend to appear near joints where they can be painful and limit motion. Apart from skin, the mucous membranes of the mouth, conjunctiva, and nose, serous cavities, and the gastrointestinal and genitourinary tracts might be involved. The eruptive phase frequently tends to heal spon- taneously, although the course is often prolonged. A few patients de- velop a severe eruptive form, with dozens of bleeding and necrotic lesions which tend to become secondarily infected. The severe acute form can develop infectious and/​or non​infectious complications. The principal complication is superinfection leading to septicaemia, which occurs at different stages of the disease but generally in the later part of the anaemic stage and during the inter- mediate stage. Formerly, salmonella, Mycobacterium tuberculosis, and Enterobacter were the most frequent pathogens. Reactivation of toxoplasmosis, histoplasmosis, pneumocystosis, leptospirosis, ty- phus fever, and staphylococcal infections are some of the other in- fections that are now frequent. A few patients develop the following syndromes:  febrile haemorrhagic or ictero-​haemorrhagic fever; acute respiratory distress or acute neurological symptoms (convul- sions, meningeal signs, hemiparesis, anisocoria, coma). Refractory Fig. 8.6.44.7  Verruga peruana: histology. Fig. 8.6.44.8  Severe anaemia (haematocrit 9%) in a patient with acute bartonellosis. Copyright D. A. Warrell. Fig. 8.6.44.9  Cardiomegaly due to myocarditis in a patient with acute bartonellosis. Copyright D. A. Warrell. Fig. 8.6.44.10  Retinal changes in bartonellosis.

1277 haemodynamic failure, severe respiratory distress, and renal failure are some of the non​infectious complications. Diagnosis Two elements must be considered: (1) travel or residence in an en- demic area and (2) a compatible clinical picture with demonstration of the bacteria in the blood film (Fig. 8.6.44.1). Fluorescence anti- body test, indirect haemagglutination, immunoblot (94% sensitive to chronic form and 70% sensitive to acute form), enzyme-​linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are new tests that are not generally available. PCR can de- tect B.  bacilliformis-​specific DNA from blood samples as well as skin biopsies. Antibodies are non​specific due a cross-​reaction with B. henselae, B. quintana, Chlamydia psittaci, and unknown antigens. The differential diagnosis in people living in endemic areas in- cludes: leptospirosis, rickettsioses, toxoplasmosis, P. vivax malaria, and louse-​borne relapsing fever. Laboratory features Bartonella can be isolated from the blood during the anaemic stage and sometimes during the eruptive stage. The sensitivity of blood smear detection varies with the phase of the disease: 90% are positive during the acute phase, less than 10% in the verrucous phase and less than 1% in asymptomatic people. The specificity of blood smears varies from 75 to 98% and the positive predictive value is 71–​94%. The enriched media might be positive in 4 to 28 days at 28°C. As fever develops, intraerythrocytic bacteria are visible in thick and thin films stained with Giemsa’s, Wright’s, or other Romanovsky stains. Organisms can also be seen and cultivated in verrucous skin lesions. PCR is the best method to detect asymptomatic infection. The anaemia can be very severe (haematocrit <10%). It is haemo- lytic but Coombs’ test negative. The blood picture is of a macrocytic and hypochromic anaemia with polychromasia, anisocytosis, and poikilocytosis. Reticulocytosis is marked (average 11%). The marrow is hyperactive and megaloblastic with erythrophagocytosis. The white cell count is not markedly elevated unless there is a secondary infec- tion. Thrombocytopenia is quite common. After the crisis, the intra- cellular organisms become coccoid and later disappear, the white cell count rises, and there is lymphocytosis. Eosinophils, which are usu- ally absent during the acute stage, reappear in the peripheral blood. Prognosis Deaths usually occur during the anaemic phase. In the preantibiotic era, case fatality varied between 20 and 95%. At present, it varies between 1.1 and 2.4% in endemic areas and around 9% in patients admitted to hospital. During outbreaks, especially when the disease (a) (c) (b) Fig. 8.6.44.11  Verruga peruana: nodular haemangiomatous lesions. 8.6.44  Bartonella bacilliformis infection

8.6.45 Chlamydial infections 1278

8.6.45 Chlamydial infections 1278

section 8  Infectious diseases 1278 is not promptly recognized and treated, the case fatality can reach around 88%. Alterations of consciousness (excitement, stupor, and coma) and progressive or focal neurological features, biochemical evidence of hepatic dysfunction (increased transaminases and alka- line phosphatase), pulmonary complications (non​cardiogenic pul- monary oedema), severe neurological involvement, anasarca (severe hypoalbuminaemia), pregnancy, and not being indigenous are all associated with a higher mortality. Treatment Chloramphenicol, penicillin, erythromycin, co-​trimoxazole, and ciprofloxacin are effective, usually eliminating the fever in around 48 h. Because of the common association with salmonellosis, ciprofloxacin is the treatment of choice in a dose of 500 mg orally twice a day for 14 days. However, there are recent reports of increased resistance of bartonellas to quinolones. The alternative is amoxicillin plus clavulanic acid 1 g orally twice a day for 14 days, which is the treatment of choice in pregnant women and children under 14 years of age. In severe acute disease, the drugs indicated are ceftriaxone 2 g intravenously daily plus ciprofloxacin 400 mg intravenously twice a day for 14 days. Supportive treatment includes transfusion of packed red cells and empirical dexamethasone if there is severe neurological involvement. Azithromycin 500 mg orally once a day for 7 days is the drug of choice for the verrucous form. The dose in children is 10 mg/​ kg daily orally for 7 days. The alternative is rifampicin (300 mg twice a day in adults or 10 mg/​kg daily in children orally for 21–​28 days). The antibiotic treatment in the acute phase of the disease does not elim- inate the chance of developing eruptive lesions weeks or months later, suggesting persistence of bacteraemia in an unknown percentage. Prevention There is no satisfactory prevention for people who live in endemic areas. Sandflies can be eliminated temporarily by spraying inside and outside with dichlorodiphenyltrichloroethane or pyrethroids, and this strategy is recommended during outbreaks. Spraying in- secticides inside the house and mass use of long-​lasting insecticide-​ impregnated bed nets are measures that reduce both incidence and secondary attack rate of the disease. Tourists can protect themselves with insect repellents, clothes impregnated with pyrethroids, and sleeping with nets impregnated with insecticides, or by avoiding sleeping in highly endemic areas. There is no vaccine. FURTHER READING Birtles RJ, et  al. (2002). Identification of Bartonella bacilliformis genotypes and their relevance to epidemiological investigations of human Bartonellosis. J Clin Microbiol, 40, 3606–​12. Chaloner GL, et  al. (2011). Multi-​locus sequence analysis reveals profound genetic diversity among isolates of the human pathogen Bartonella bacilliformis. PLoS Negl Trop Dis, 5, e1248. Chamberlin J, et al. (2002). Epidemiology of endemic Bartonella bacil­ liformis: a prospective cohort study in a Peruvian mountain valley community. J Infect Dis, 186, 983–​90. Maguiña C (1998). Bartonellosis o Enfermedad de Carrión. A.F.A. Editores Importadores S.A. Lima, Peru. Ministry of Health of Peru (2011). Atención de la Bartonelosis o Enfermedad de Carrión en el Perú. Norma técnica No. 048-​MINSA/​ DGSP-​C.01. Sanchez Clemente N, et al. (2012). Bartonella bacilliformis: a system- atic review of the literature to guide the research agenda for elimin- ation. PLoS Negl Trop Dis. http://​www.scopus.com/​inward/​record. url?eid=2-​s2.0-​84869061206&partnerID=40&md5=be4ccb039bf6 20fbe67e6b37468c2684 8.6.45  Chlamydial infections Patrick Horner, David Mabey, David Taylor-​Robinson, and Magnus Unemo ESSENTIALS Chlamydiae are pathogenic bacteria that likely evolved from host-​ independent, Gram-​negative ancestors. The Chlamydiae have a unique biphasic developmental cycle and obligate intracellular lifestyle. Chlamydiae depend on a eukaryotic host cell for their replication which takes place in an inclusion inside the host cell, and for their dispersal, cell lysis, or extrusion subsequently occurs. Although the phylum Chlamydiae (order Chlamydiales) was ori- ginally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. The genus Chlamydia re- mains the most widely studied. The species Chlamydia trachomatis was proposed some decades ago on the basis of 16S rRNA and 23S rRNA sequences, to belong to the genus Chlamydia together with C.  muridarum and C.  suis. The other human pathogenic Chlamydiae were classified as species of a separate genus, that is, Chlamydophila pneumoniae and Chlamydophila psittaci. However, this subdivision of the family Chlamydiaceae into the two genera, Chlamydia and Chlamydophila has been controversial. Based on genomic data and the biological properties of these bacteria, it was proposed recently to classify all the 11 currently described Chlamydiaceae species within a single Chlamydia genus. This is the classification used in the present chapter. In the Chlamydia genus, C. trachomatis and C. pneumoniae are primarily human pathogens, and C. psittaci, C. abortus, and C. felis are species transmitted occa- sionally from animals. The other families, the so-​called Chlamydia-​ like organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been as- sociated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. This chapter primarily focuses on the species C.  trachomatis, which causes the disease of ocular trachoma (serovars A–​C), oculo-​ anogenital tract infection (serovars D–​K) and lymphogranuloma venereum (serovars L1–​L3). However, infections caused by C. pneumoniae and C. psittaci are also discussed.

8.6.45  Chlamydial infections 1279 Oculo-​anogenital tract infections These are caused by C. trachomatis serovars D–​K, which exist world- wide. In men they cause up to 50% of symptomatic non​gonococcal urethritis and a similar proportion of acute epididymitis. In women they cause up to 50% of (often asymptomatic) urethritis and of (mostly asymptomatic) cervicitis; further spread leads to endo- metritis, salpingitis and (occasionally) perihepatitis. Women with untreated infection have about a 17% risk of pelvic inflammatory disease, a 7% risk of salpingitis, a 0.5% risk of tubal factor infertility and 0.2% risk of ectopic pregnancy. Other diseases—​(1) Rectal and pharyngeal infections. (2)  Adult paratrachoma and otitis media. (3) Reactive arthritis—​at least one-​ third of sexually acquired reactive arthritis is initiated by genital C. trachomatis infection (see Chapter 19.6). (4) Neonatal infection—​ babies exposed to serovars D–​K at birth often develop conjunctivitis, and some develop pneumonia. Lymphogranuloma venereum The invasive ulcerative lymphogranuloma venereum is caused by C. trachomatis serovars L1, L2 (including subvariants such as L2b) or L3. Lymphogranuloma venereum has been endemic in parts of Africa, Asia, South America, and the Caribbean. The traditional clinical course comprises three stages: (a) primary—​a small painless papule occurs at the site of inoculation; followed some weeks later by (b) secondary—​inguinal and/​or femoral lymphadenopathy with systemic features; anorectal involvement is usually seen in men who have sex with men; sometimes progressing to (c)  tertiary—​severe fibrosis, which is rarely seen because of earlier broad-​spectrum antibiotic therapy. Lymphogranuloma venereum has been rare in Western Europe and North America for many years. However, since a lymphogranuloma venereum (serovar L2b) outbreak in men who have sex with men was detected in 2003 in Rotterdam, out- breaks similar to the Netherlands outbreak have been seen in sev- eral other West European countries, North America, and Australia. Lymphogranuloma venereum has now become endemic among, mostly HIV-​positive, men who have sex with men in several of those European countries. The lymphogranuloma venereum L2b strain is also found in the heterosexual population. Most of these patients have presented with proctitis or tenesmus, anorectal discharge, and discomfort, diarrhoea, or altered bowel habits. It has now also been shown that about 25% of lymphogranuloma venereum infections can be asymptomatic in some settings. Diagnosis depends on iden- tification of C.  trachomatis lymphogranuloma venereum serovars in appropriate clinical specimens using nucleic acid amplification testing and, if that is not available, serology. Recommended first-​line treatment is doxycycline (three weeks), with erythromycin as second-​ line (three weeks). Trachoma Trachoma is caused by C. trachomatis serovars A, B, Ba, and C. It is a disease of poor rural communities, mainly in Africa and Asia, where the reservoir of infection is the eye (and possibly nasopharynx) of children with active disease, with transmission from the eye of one individual to that of another via fingers, fomites, coughing and sneezing, and by eye-​seeking flies. Clinical features and diagnosis—​the active (inflammatory) stage is a follicular conjunctivitis with characteristic subconjunctival follicles that are usually seen in children in endemic areas. Repeated infec- tions lead to conjunctival scarring, with turned-​in eyelashes rub- bing against the cornea (trichiasis) which eventually causing severe damage (1.4% of global blindness, or 0.5 million cases, and 1.8 mil- lion cases of visual impairment). In endemic areas diagnosis is made on clinical grounds. Treatment and prevention—​inflammatory trachoma responds to ei- ther an appropriate course of 1% topical tetracycline ointment or a single oral dose of azithromycin up to 1 g. Community-​based mass treatment is recommended when there is high prevalence of dis- ease in children aged 1 to 9 years. Trichiasis requires surgical cor- rection. A World Health Organization initiative to eliminate blinding trachoma as a public health problem by 2020 is based on the acronym ‘SAFE’: Surgery for trichiasis; Antibiotics for treatment; Face washing; Environmental improvement to reduce fly populations that transmit the organisms. Other Chlamydiae C.  pneumoniae—​transmitted directly from person to person by droplet spread and causes respiratory tract disease (pharyngitis, bronchitis, pneumonia); it is a possible trigger for reactive arthritis and for some cases of juvenile chronic arthritis, and C. pneumoniae DNA has been detected in atheromatous arteries, but without definite evidence that it contributes to heart disease. C. psittaci—​ transmitted from psittacine birds and causes psittacosis, which can range from a mild influenza-​like illness to a fulminating toxic state with multiorgan involvement. Ornithosis refers to infection trans- ferred from non​psittacine birds. C. abortus—​causes abortion in sheep and may do so in pregnant women exposed to infected animals during the lambing season. Diagnosis and treatment Diagnosis—​depends on (1)  the use of nucleic acid amplification tests—​the ‘gold standard’ for routine diagnosis, screening, and for research into chronic or persistent disease; and—​to a much lesser extent—​(2) serology. Treatment—​Chlamydiae are particularly sensitive to tetracyc- lines (e.g. doxycycline) and macrolides (e.g. erythromycin and azithromycin). Azithromycin has gained popularity because it can be effective as a single dose: however, there is a debate as to whether it is sufficiently effective compared to doxycycline. Introduction Trachoma is recognizable from historical descriptions of blind- ness in ancient Chinese and Egyptian writings, but it was not until 1907 that L.  Halberstaedter and S.  von Prowazek first described intracytoplasmic inclusions in stained conjunctival scrapings from orangutans that had been inoculated with human trachomatous material and recognized the involvement of an infectious agent. In 1930, a chlamydial agent (Chlamydia psittaci) was first isolated from psittacosis; 27 years later the species associated with trachoma, C. trachomatis, was isolated in the yolk sac of fertile hens’ eggs. The advent of the cell-​culture technique paved the way for the isolation of oculo-​anogenital C. trachomatis by this means in 1959. C. pneumo­ niae was initially isolated in 1965 from the eye of a child participating

section 8  Infectious diseases 1280 in a trachoma vaccine study and reclassified as C. pneumoniae in 1989. Advances in immunology, molecular diagnostics, molecular biology, and more recently genomics have made it possible to ex- plore the nature, range, prevalence, and pathogenesis of clinical conditions associated with chlamydial infection. Classification The phylum Chlamydiae refers to ubiquitous pathogens infecting many eukaryotes including many species of animals, birds, insects, and amoeba. The family Chlamydiaceae contains a single genus that, based on recent genomic data and the biological properties of these bacteria, contains 11 different species. The Chlamydiaceae and Chlamydia, are the most widely studied family and genus, re- spectively, of the Chlamydiae, and until the 1990s these were the only family and genus of the order Chlamydiales. The 11 Chlamydia species include C. trachomatis causing human ocular and anogenital infections; C. pneumoniae causing mainly human respiratory tract disease, but with some strains infecting a range of animals including horses and amphibians; C. psittaci infecting birds and other animals, with occasional transmission to humans; and C.  abortus, which is endemic in ruminants causing abortion in sheep and rarely in pregnant women. Over the last 20  years research has revealed that the family Chlamydiaceae represents only the tip of the iceberg in terms of di- versity within the phylum Chlamydiae. Eight additional families of genetically obligate intracellular bacteria have since been identified. These new families are often referred to as ‘Chlamydia-​like organ- isms’ as the developmental cycle is remarkably conserved across the phylum Chlamydiae. There is evidence that several of these species may cause disease in both humans and animals. For ex- ample, Waddlia chondrophila has been linked with miscarriage and Parachlamydia acanthamoebae and Simkaniaceae negevensis with respiratory tract disease. Growth cycle, genomics, and serovars Chlamydiae likely evolved from host-​independent Gram-​negative ancestors with traditional peptidoglycan structures in their cell walls. They are bacteria specialized to exist intracellularly. The chlamydial envelope possesses bacteria-​like inner and outer mem- branes. The infectious elementary body is environmentally re- sistant, metabolically inactive, electron dense, DNA rich, and approximately 200–​400 nm in diameter. It binds to the host cell and enters by endocytosis. Fusion of the chlamydia-​containing endocytic vesicle with lysosomes is inhibited and the elementary body begins its unique bi-​phasic developmental cycle within the eukaryotic cell. After about 6–​10 h it differentiates into the larger (500–​1000 nm) non​infectious, metabolically active, more perme- able, pleomorphic reticulate body. This replicates by binary fission and by 20–​30 h has begun to reorganize into a new generation of elementary bodies (Fig. 8.6.45.1). These rapidly accumulate within the endocytic vacuole to be released from the cell (and/​or cell is lysed) between 30 and 72 h for C. trachomatis after the start of the cycle (exact time can differ dependent on species, strains, and cell type). The developmental cycle of C. pneumoniae is longer c.72 h. Chlamydiae have the ability to enter a persistent state intracellu- larly within the inclusion in response to various conditions. They can then remain dormant as abnormal reticulate bodies for consid- erable lengths of time but are able to be re-​activated. This has been widely studied in vitro. Although the evidence in vivo is limited, its occurrence has been demonstrated during cervical infection and presumptively in patients with reactive arthritis and tubal factor in- fertility. In this persistent state, chlamydial organisms have shown to be refractory to antimicrobial therapy in vitro. The genome sequences of species within the Chlamydia genus are small relative to most other bacterial genomes, and have a high level of conservation (interspecies and intraspecies), overall gene con- tent and gene synteny (order). In one study, a total of 736 protein-​ coding sequences was shown to be shared among the species of C. psittaci, C. abortus, C. pneumoniae and C. trachomatis, with the total protein-​coding sequence count of these species ranging from 874 to 1097. The minor differences are obviously sufficient to de- fine host range, tissue tropisms, and disease presentation. There was also considerable amino acid identity (average 62%) between proteins encoded by the common protein-​coding sequences from C. trachomatis and C. pneumoniae and, thus, the potential that these proteins may contain cross reactive epitopes is high. This has impli- cations for the development and interpretation of species-​specific antibody tests. The major outer membrane protein (MOMP) is immunodominant in the elementary body and contains epitopes exhibiting genus, spe- cies, and serovar specificity. The serovar-​specific epitope is the basis of the microimmunofluorescence (MIF) test by which C. trachoma­ tis has been separated into 15 serovars: A, B, Ba, and C are respon- sible mainly for endemic trachoma; D to K for oculo-​anogenital infections; and L1, L2, and L3 for the more invasive genital disease lymphogranuloma venereum (LGV). Using sequencing of the ompA gene (encoding MOMP), the corresponding genovars including intraserovar sequence variants, are nowadays usually assigned. Recent whole genome sequence analysis indicates that C. trachoma­ tis undergoes recombination (intra-​ and interserovar) in vitro and in vivo. Replacement and chimerism of large segments of the genome including ompA and accordingly encoded MOMP is most likely one of many strategies used by C. trachomatis to evolve in general and also counteract the effect of the immune system protecting the host Fig. 8.6.45.1  Elementary bodies (E) and reticulate bodies (R) of C. trachomatis, forming an inclusion in an oviduct cell; shown by transmission electron microscopy.

8.6.45  Chlamydial infections 1281 against immediate reinfection. Thus, serovar and genovar typing systems for C. trachomatis can be poor indicators of genetic related- ness within the species. Only one C. pneumoniae serovar has been identified, although minor geographical serovar variations have been described. C. psittaci was originally divided into nine serovars. Amino acid sequences of the MOMPs of all C. trachomatis sero- vars and epitope maps of different antigenic domains have been elucidated. The MOMP amino acid sequences consist of five highly conserved regions punctuated by four short extracellularly-​exposed variable sequences. Serovar-​specific epitopes have been demon- strated in variable sequence I and II, while species-​specific epitopes have been found in variable sequence IV. This understanding has formed the theoretical basis for development of C. trachomatis spe- cific MOMP peptide antibody assays. All species of Chlamydia add- itionally contain a common heat-​stable lipopolysaccharide (LPS) antigen, which is exposed on the surface of the reticulate body, but not on the elementary body. Antibodies to LPS are considered genus specific. The plasmid encoded protein Pgp3 of C. trachomatis is not present in the human pathogenic C. pneumoniae strains, as they do not contain a plasmid. Pgp3 is highly immunogenic, with most women producing serum IgG antibody following infection. Men are less likely to develop antibodies to Pgp3 following infection, the reason for which is unclear. Immune response and pathogenesis The immune response to chlamydial infections may be protective or damaging. Active trachoma is uncommon in adults in endemic areas, suggesting that protective immunity follows natural infec- tion. Similarly, genital C. trachomatis infection is most prevalent in the youngest sexually active age groups, and the chlamydial isola- tion rate for men with non​gonococcal urethritis is lower in those who have had previous episodes. The duration of ocular infection is shorter in adults than in children. It is unclear how long pro- tective immunity lasts but with genital tract infections this is be- lieved to be short lived. Several trachoma vaccine trials in the 1960s used killed whole organism vaccines, which provided some degree of protection. Primate studies suggested that vaccination could provoke more severe disease on subsequent challenge, indicating immunopathological damage by C.  trachomatis. Evidence from human studies suggests women with reinfection, which is not un- common, may be at greater risk of developing reproductive sequelae. The average duration of asymptomatic genital infection in women has been estimated to be about 16 months with about 20% and 5% still positive at two years and four years, respectively, following in- fection. The duration of infection is believed to be similar in men, but additional evidence would be valuable. Approximately 20–​25% of women and men become detection-​negative after a few weeks. This could be a consequence of ‘passive’ carriage clearing after one week, in which infection has not been established. The data in women are also compatible with a three-​rate model which in- cludes fast clearing infection as a result of a protective immune re- sponse, in addition to passive carriage and slow clearing infection. How often and how long Chlamydiae can persist in a non​replicating state in vivo (see earlier) is unknown as an answer would require invasive sampling of the upper genital tract by biopsy, in apparently healthy individuals. While recent evidence indicates that long term persistence is probably uncommon, there remains the possibility that some women with tubal factor infertility who are nucleic acid amplification test (NAAT)-negative but chlamydia-​antibody posi- tive may harbour viable organisms which could ‘reactivate’ after uterine instrumentation. Many women with urogenital tract infection, who do not prac- tice anal sex, may be coinfected in the rectum. Auto-​inoculation has been proposed as the most likely mechanism, although it is diffi- cult to exclude contamination of the swab during rectal sampling as a result of passive perineal contamination from vaginal secretions. Animal and human studies suggest that gastrointestinal C. tracho­ matis infection may occur in humans and may be less susceptible to treatment with azithromycin. Several groups have identified the DNA of C. trachomatis uro- genital serotypes in joints of patients with reactive arthritis. A recent report of C. trachomatis trachoma serotypes A–​C and not urogenital serotypes D–​K in joints is mystifying and needs substantiation given the association of reactive arthritis with urogenital infection and not trachoma. C. pneumoniae has been identified in atheromatous plaques of pa- tients with cardiovascular disease and associated serologically with disease. Experimental studies in atheroma models in mice and rab- bits have demonstrated biological plausibility, with infection accel- erating progression. Thus, it is possible that inflammation secondary to persistent infection might contribute to disease progression in hu- mans. However, placebo-​controlled antibiotic treatment trials have failed to establish any therapeutic benefit to patients with established atheromatous disease. There are several potential explanations for this including the possibility that persistent C. pneumoniae infection may be resistant to antimicrobial therapy. Chlamydial infection is associated with a T-​helper cell type 1 (Th1) or cytotoxic profile of immune response, with the production of IFN-​γ, IL-​8, IL-​1, and IL-​6 cytokines. Studies in trachoma-​endemic communities suggest that Th1 type cell-​mediated responses are important in the clearance of ocular C. trachomatis infection and are believed to also be important in resolution of genital tract in- fection. However, the specific immune responses and cytokine levels that lead to resolution of infection rather than promotion of tissue damage remain undefined. An adaptive immune memory re- sponse, as a result of previous infections, probably also exacerbates the risk of pathological outcomes in the form of fibrosis and scar- ring. Persistent chlamydial infected epithelium which results in the production of pro-​inflammatory chemokines and cytokines can also cause cell damage and fibrosis. Delayed hypersensitivity and/​ or molecular mimicry in response to specifiic chlamydial antigens, notably the chlamydial heat shock protein (hsp 60), homologous with the GroEL protein of Escherichia coli and human hsp 60, has also been proposed. However, inconsistencies in the published find- ings have made this potential mechanism increasingly controversial. It is likely that disease caused by Chlamydiae is a combination of the effects of the immunological (adaptive immunity) and the cellular (innate epithelial cell responses) systems. Recent evidence from studying trachoma suggests that other mechanisms might also be involved. A longitudinal study of patients with trachoma indicated that progression of scarring as a result of immune activity occurred in the absence of detectable reinfection. Scarring trachoma is as- sociated with non​chlamydial bacterial infection. It is possible that bacteria could continue to drive on-​going innate pro-​inflammatory

section 8  Infectious diseases 1282 responses in the conjunctiva. Another possibility is that prior re- current C. trachomatis infection modifies the conjunctival tissue, increasing susceptibility to bacteria, perhaps as a result of imprinted epigenetic changes. Whether other bacteria might also play a role in the development of tubal factor infertility or ectopic pregnancy following chlamydial pelvic inflammatory disease (PID), similar to trachoma, is unknown. Persistent chlamydial infection is only rarely detected in women with tubal factor infertility, and bacterial vagin- osis, which can colonize the endometrium, is associated with tubal factor infertility. Chlamydia genotype and disease Chlamydia trachomatis is split into two biovars: the trachoma biovar (serovars A-​K) which contains ocular and anogenital strains that are characterized by localized infections of the epithelial surface of the conjunctiva or anogenital mucosa; and the LGV biovar (serovars L1-​ L3) that contains more invasive strains which are distinguished by their ability to spread systemically through the lymphatic system, causing genital ulceration and bubonic disease. The biovars are genomically very closely related and it is unclear which genotypic differences determine variation in clinical disease. The best-​known genotype that mediates tissue tropism is poly- morphism in the trpAB operon, which enables the synthesis of tryp- tophan from indole in C. trachomatis. This is disabled in ocular but not in anogenital strains of the trachoma biovar. IFNγ, an important Th1 mediator, acts against C. trachomatis via tryptophan nutrient deprivation. Environmental tryptophan levels are therefore likely to influence whether C. trachomatis either enters into a persistent state of growth, or can be eradicated. It was recently demonstrated that in the presence of bacterial vaginosis in women, a source of indole, C. trachomatis can persist as abnormal reticulate bodies in the pres- ence of high IFNγ levels, consistent with a robust Th1 type immune response. Presumably the ocular strains lost the functional trpAB operon because of an absence of indole producing bacteria in the healthy conjunctival space. The reasons why strains of the LGV biovar are more invasive, pre- dominantly infect monocytes and macrophages and disseminate to the lymph nodes and consequently cause systemic disease remain to be clearly elucidated. The LGV strains are genomically extremely closely related (gene order and sequence) to strains of the trachoma biovar. However, the LGV strains have a slightly smaller genome which is mainly due to differences in the plasticity zone of the re- spective genomes and to the differential deletion of the chlamydial cytotoxin gene(s), which have been almost entirely removed from many LGV strains. In general, small-​scale gene variation such as single amino acid alterations, a few pseudogenes, and alterations in timing and level of gene transcription and translation might provide the lymphotropic properties. Human genotype and risk of disease Several human genetic polymorphisms have been associated with the development of disease following infection with C. trachoma­ tis. These include polymorphisms in Toll-​like receptors (TLRs) 1, 2, and 4 that are expressed in the female genital tract. Also in NLRP3 that results in altered secretion of IL-​1β, mannose-​binding lectin, human leukocyte antigen class 1 molecules (A2 and B/​C–​ trachoma) and class II molecules (DR, DQ, and DP—​tubal factor infertility), and in a range of cytokines including IL-​10 and TNFα. A recent large genome-​wide association study of polymorphisms and pathways associated with pathological sequelae of ocular C. tra­ chomatis infection identified 27 single nucleotide polymorphisms (SNPs) with strong association with scarring. Pathway analysis of genome-​wide association study data was significantly enriched for mitotic cell cycle processes, the immune response, and for multiple cell surface receptor signalling pathways. Interestingly, it failed to confirm previously identified associations of scarring with SNPs in cytokine genes such as IL-​10 and matrix metalloproteinase 9. Oculo-​anogenital tract infections Oculo-​anogenital tract infections due to C. trachomatis serovars D-​K (Table 8.6.45.1) occur worldwide and are associated with a significant health and economic burden. The World Health Organization (WHO) estimated, globally, that there were 131 mil- lion new genital infections among adults in 2012. Women with untreated infection have about a 17% risk of pelvic inflammatory disease, a 7% risk of salpingitis, a 0.5% risk of tubal factor infertility and 0.2% risk of ectopic pregnancy. Infection is usually asymp- tomatic in men and women, with undetected infection sustaining transmission in the community. The prevalence of C. trachomatis infection in population-​based studies has ranged between 0.1% and 12.1% in men, and 1.1% and 10.6% in women. The highest prevalence is in women of 15 to 24 years of age and in men of 20 to 24 years of age. In England, it has been estimated that the an- nual incidence in women aged 16–​24 years was 5.2%. In the United States of America in 2013 it is estimated that in women there were 1.3 million new cases of genital chlamydial infection. Infection is associated with young age, increasing number of sexual partners, failure to use barrier protection consistently and correctly, and lower socioeconomic status. It is likely that higher prevalence in specific sexual networks, which is related to lower socioeconomic status and social disadvantage, might result in a higher incidence. Non​gonococcal urethritis The incubation period is usually 7 to 14 days compared to 2 to 8 days for gonococcal urethritis. C.  trachomatis is detectable in the ur- ethra of up to 50% of men with symptomatic non​gonococcal ur- ethritis and in as many as 7% of those who are asymptomatic (see Chapter  9.5 on urethritis). Doxycycline 100 mg twice daily for 7 days, recommended first-​line treatment for non​gonococcal ureth- ritis, has a failure rate of 3% and azithromycin 1 g a failure rate of 10%. It is therefore likely that C. trachomatis is a cause of some cases of chronic (persistent/​recurrent) non​gonococcal urethritis. In women, chlamydial urethral infection may cause urethritis but, in contrast to men, infection and inflammation are mostly asymptomatic. The ‘urethral syndrome’ (dysuria and frequency with <105 organisms/​ml urine) is rarely of chlamydial origin. Prostatitis and epididymitis There is no evidence that C.  trachomatis causes acute symptom- atic prostatitis. Transperineal biopsies from patients with chronic non​prostatitis show chronic inflammation, but C. trachomatis has not been detected by culture or direct immunofluorescence tech- niques, although NAATs are positive in about 10%. These results,

8.6.45  Chlamydial infections 1283 combined with the failure to detect chlamydial antibody, suggest that C. trachomatis is not often implicated directly in chronic pros- tatitis. However, the observation that, in vitro, C. trachomatis can infect immortalized prostatic epithelium which results in the pro- duction of pro-​inflammatory cytokines suggests that some cases of chronic disease might be of chlamydial origin. C.  trachomatis is responsible for up to 50% of cases of acute epididymitis or epididymo-​orchitis occurring primarily in young men (≤35 years of age) in developed countries, and has been de- tected in at least one-​third of epididymal aspirates. There is usually a strong correlation between IgM and/​or IgG chlamydial anti- bodies, measured by MIF, and chlamydia-​positive epididymitis/​ epididymo-​orchitis. In developing countries, although C. tracho­ matis is important, N.  gonorrhoeae is the major cause of acute epididymitis. Historically, the assumption has been that in pa- tients older than 35 years, urinary tract pathogens are the primary cause of epididymitis/​epididymo-​orchitis. However a significant but small group of men remain at risk of sexually transmitted infections. The most recent National Survey of Sexual Attitudes and Lifestyles (NATSAL) in the United Kingdom showed that Table 8.6.45.1  Assessment of the extent to which C. trachomatis is involved in various oculo-​anogenital and associated diseases Disease Evidence that C. trachomatis is a causea Proportion of disease due to C. trachomatisb In men Acute NGU ++++ Up to 50% Persistent and recurrent NGU +++ 10–​20% (dependent on initial therapy and effectiveness of partner notification) Acute epididymo-​orchitis ++++ Up to 50% Acute and chronic prostatitis + ? Infertility + ? (only during active infection) In women Cervicitis ++++ <30% Urethritis +++ ? Vaginitis (prepuberty only) +++ ? Endometritis +++ ? Salpingitis ++++ 20–​40% Perihepatitis +++ ? Tubal factor infertility +++ 30–​45% (secondary to tubal scarring from previous infection) Ectopic pregnancy +++ ? Periappendicitis ++ ? Bartholinitis + ? Early miscarriage + ? Cervical dysplasia + ? Bacterial vaginosis –​ In men or women Conjunctivitis ++++ ? Proctitis +++ ? Arthritis (SARA) +++ About 40% Otitis media ++ ? Endocarditis ++ ? Pharyngitis + Lymphogranuloma venereum ++++ 100% (by definition) In infants Conjunctivitis ++++ ? Pneumonia ++++ ? Chronic lung disease ++ ? Gastroenteritis –​ NGU, non​gonococcal urethritis; SARA, sexually acquired reactive arthritis. a ++++, overwhelming; +++, good; ++, moderate; +, weak; –​, none. b Can vary by study population, geographic setting, and diagnostics used in studies.

section 8  Infectious diseases 1284 10–​13% of men aged 35–​64  years had at least one new sexual partner in the previous year. Thus, age alone should not be used to exclude the possibility of C. trachomatis causing epididymitis/​ epididymo-​orchitis. There is insufficient evidence that chlamydial epididymitis or chlamydial urethral infection lead to male infertility. Cervicitis, vaginitis, and bartholinitis In prepubertal children, the vaginal epithelium is columnar and susceptible to chlamydial infection. In adults, the squamous epi- thelium of the vagina is not susceptible, and the cervix is the pri- mary target for C. trachomatis, where it is an established cause of mucopurulent/​follicular cervicitis (Figs. 8.6.45.2a and b) and often asymptomatic. Women who have signs of cervicitis are at increased risk of C. trachomatis, Mycoplasma genitalium, Trichomonas vagi­ nalis, and N. gonorrhoeae infection if the latter two infections are not uncommon in the population under study. Most women with cervicitis do not have a specific pathogen detected. A significant association between cervical chlamydial infection and cervical squamous cell carcinoma, but not adenocarcinoma, has been established and it has been suggested that C. trachomatis infection may enhance the oncogenic effect of papillomaviruses. C. trachomatis has been weakly associated with bartholinitis and should be considered in the absence of other known pathogens. C. trachomatis is often detected more frequently in women with bacterial vaginosis than in those without. There is emerging evi- dence that indoles produced by bacterial vaginosis may facilitate persistence of chlamydial infection (see section ‘Chlamydia geno- type and disease’). Pelvic inflammatory disease PID comprises a spectrum of upper genital tract inflammatory dis- orders among women, which includes any combination of endo- metritis, salpingitis, tubo-​ovarian abscess and pelvic peritonitis. Chlamydial infection could be as much as 5–​6 times more likely to be the cause of PID in women aged 16–​19 years than in women aged 35–​44 years. Chlamydial infection causes around one-​third of PID cases in 16-​ to 24-​year-​old women, and about one-​fifth of cases in 16-​ to 44-​year-​olds overall. Canalicular spread of C. trachomatis to the upper genital tract leads to endometritis, which is often plasma cell-​associated and sometimes intensely lymphoid; it occurs in about 17% of women if left untreated. Further spread causes overt salpingitis (Fig. 8.6.45.3), which occurs in about 7% of women. It is women with visible evidence of salpingitis at laparosocopy who are at increased risk of reproductive sequelae. Spread to the peritoneum results in perihepatitis (the Fitz-​Hugh–​ Curtis syndrome) (Fig. 8.6.45.4), sometimes confused with acute cholecystitis in young women, in addition to peri-​appendicitis and other abdominal symptoms. This is uncommon. Surgical termination of pregnancy or insertion or removal of an intrauterine contraceptive device might predispose to dissemination of infection. PID is not easy to diagnose. Recent onset of lower abdominal pain in association with local tenderness on bimanual examination is now considered sufficient to believe that PID is possible and that early treatment is required. This is because it is now recognized that many women with salpingitis have subtle or mild symptoms and early treatment reduces the risk of developing chronic sequelae. (a) (b) Fig. 8.6.45.2  (a) Mucopurulent cervicitis; (b) follicular cervicitis. Fig. 8.6.45.3  Laparoscopic view of inflamed fallopian tube due to C. trachomatis. Courtesy of P. Greenhouse.

8.6.45  Chlamydial infections 1285 A recent multiparameter evidence synthesis study of the natural history of C. trachomatis infection estimated that about 15% of in- cident infections progress to symptomatic PID, which may remain undiagnosed, with a further 2% developing asymptomatic disease. Of the c.7% of women who develop acute salpingitis, about 5–​8% will develop tubal factor infertility and have a 2–​3% risk of ectopic pregnancy. Other consequences of PID include chronic pelvic pain lasting more than 6 months, the risk of which is difficult to estimate and may occur in about a third of women with mild or moderate PID following treatment. There is conflicting evidence of the effect of C. trachomatis on pregnancy. Some studies have shown C. trachomatis infection to be associated with low birth weight and preterm delivery, but others have failed to confirm this. However, a recent observational study from the Netherlands showed a strong association between the de- tection of C. trachomatis and preterm labour. Rectal and pharyngeal infection Rectal infections are usually asymptomatic, but anal symptoms including discharge and discomfort occur occasionally. Rectal in- fection in men is associated with unprotected receptive anal inter- course. If men are symptomatic, LGV is the most likely cause. There is increasing evidence that rectal infection in women is associated with urogenital tract infection (see earlier) in addition to receptive anal intercourse. Pharyngeal infection is usually asymptomatic. While both men and women can be infected in the throat without ano-​genital infection, this appears to be relatively uncommon. Other diseases associated with C. trachomatis Adult paratrachoma (inclusion conjunctivitis)
and otitis media Adult chlamydial ophthalmia is distinguished from trachoma by its causative C. trachomatis serovars D-​K. It commonly results from the accidental transfer of infected genital discharge to the eye. In contrast to ‘reactive’ conjunctivitis observed in multisystem disease of sexu- ally acquired reactive arthritis (see next), C. trachomatis can usually be detected in conjunctival specimens. It usually presents as a uni- lateral follicular conjunctivitis, acute or subacute in onset, with an incubation period of up to 21 days. The features are swollen eyelids, mucopurulent discharge, papillary hyperplasia, and, later, follicular hypertrophy and occasionally punctate keratitis. Up to one-​third of patients have otitis media and complain of blocked ears and hearing loss. The disease is generally benign and self-​limited. Pannus forma- tion and corneal scarring are rare and not seen if systemic treatment is given. Patients and their sexual contacts should be investigated for genital chlamydial infection and managed appropriately. Arthritis Arthritis occurring with, or soon after, non​gonococcal ureth- ritis is sexually acquired reactive arthritis. A multisystem disease including a combination of urethritis, conjunctivitis, and arthritis is seen in about one-​third of patients. Laboratory evidence of chla- mydial infection is found in at least one-​third of patients. C. tracho­ matis has also been associated in the same way with ‘seronegative’ arthritis in women. Viable C. trachomatis has not been detected in the joints of patients with sexually acquired reactive arthritis, which is probably the result of immunopathology. The role of antimicro- bial therapy is uncertain. One recent treatment trial of chronic disease using combination therapy demonstrated benefit, whereas numerous previous treatment studies using appropriate therapy provided disappointing results. Immunocompromised patients C. trachomatis has been isolated from the lower respiratory tract of a few immunocompromised adults with pneumonia, some after renal transplantation. However, other pathogens have often also been present. C.  pneumoniae is not an especially important re- spiratory tract pathogen in AIDS patients. Genital C. trachomatis infection is likely to increase viral shedding from HIV-​positive people, and enhance the susceptibility to HIV in the uninfected. Hypogammaglobulinaemic patients do not appear to be especially prone to infection with any of the chlamydial species. Neonatal infections Although intrauterine chlamydial infection can occur, the major risk of infection to the infant is from passing through an infected cervix. The proportion of neonates exposed to infection de- pends, of course, on the prevalence of maternal cervical infection, which varies widely. Conjunctivitis appears in 20–​50% of infants Fig. 8.6.45.4  Adhesions in perihepatitis (Fitz-​Hugh–​Curtis syndrome). Courtesy of P. Greenhouse

section 8  Infectious diseases 1286 exposed to C. trachomatis (serovars D–​K) infecting the cervix at birth. A mucopurulent discharge (Fig. 8.6.45.5) and occasionally pseudomembrane formation occur 1 to 3 weeks later. It usually re- solves without visual impairment. Complications tend to be in un- treated infants. Approximately one-​half of the infants who have conjunctivitis also develop pneumonia, although a history of recent conjunctiv- itis and bulging eardrums is found in only one-​half of infants with pneumonia. Chlamydial pneumonia usually begins between the 4th and 11th week of life, preceded by upper respiratory tract symp- toms. There is tachypnoea, a prominent staccato cough, but no fever, and the illness is protracted. Radiographs show hyperinflation of the lungs with bilateral diffuse symmetrical interstitial infiltration and scattered areas of atelectasis. Finding serum IgM antibody to C. trachomatis in infants with pneumonia confirms the diagnosis. Children infected during infancy are more likely to develop ob- structive lung disease and asthma than are those who have had pneumonia due to other causes. The vagina and rectum of infants also may be colonized by C. tra­ chomatis at birth in about 25% of those infected, but this has not been associated with clinical disease. The majority remained posi- tive in the rectum up to one year after birth, suggesting colonization of the gastrointestinal tract, but there is no evidence of infant chla- mydial gastroenteritis. Diagnosis The laboratory diagnosis of current C. trachomatis infection is re- commended to be performed using validated and quality assured nucleic acid amplification tests (NAATs), identifying chlamydial-​ specific nucleic acid (DNA or RNA) in clinical specimens. If chlamydial NAATs are not available or affordable, isolation of C. tra­ chomatis in cell culture or detection of chlamydial cells or antigens by direct fluorescence assays or enzyme-​linked immunosorbent as- says (ELISA) can be used for diagnosis of acute chlamydial infection. However, the modern NAATs, with their clearly superior sensitivity and other performance characteristics (e.g. high specificity, range of specimen types, automation, and independence from maintain­ ing organism viability), have made these other diagnostic methods obsolete in high-​income countries. For urogenital chlamydial detec- tion, NAATs can be used to examine non​invasively collected speci- mens such as a first catch urine samples in men, or a vulvo-​vaginal swab in women, which are the first-​choice clinical samples. Specimen type A vulvo-​vaginal swab is the preferred specimen type in women for detection using a NAAT. It is important that manufacturers’ in- structions are followed strictly with the swab being gently rotated for 10–​30 seconds inside the vagina. It can be self-​collected or clinician-​obtained. It is more sensitive than either a urine specimen or an endocervical swab and more acceptable to women than the other specimen types. Endocervical specimens require a speculum examination and it is important to ensure the columnar epithelial cells are adequately sampled. Inadequate vulvo-​vaginal swab and endocervical specimens will reduce the sensitivity of the test. First catch urine specimens from women have a good sensitivity, despite not being ideal, and can be used when a vulvo-​vaginal swab spe- cimen is not possible. A first catch urine specimen is easy to collect in men and has similar if not better sensitivity to a urethral swab (which is painful) for detection using a NAAT. A first catch urine specimen is there- fore the specimen of choice in men. Men should be instructed to not void urine at least one hour prior to producing a first catch urine specimen. There is some evidence that a self-​taken meatal swab may also be an adequate specimen type in men. A swab should be used for collecting rectal and pharyngeal spe- cimens for analysis using NAATs. These can be clinician or patient collected. Care needs to be taken when pharyngeal specimens are collected as it is important that the pharynx and not the mouth is sampled. While current NAATs are not commercially licensed for rectal and pharyngeal specimens, the published evidence suggests that test performance is also high for these specimens. When specimens are required for culture they must contain col- umnar epithelial cells. Endocervical swabs in women and urethral swabs in men are therefore the required specimen type. Nucleic acid amplification tests (NAATs) The enormous amplification of specific nucleic acid sequences with the polymerase chain reaction (PCR) assay, the strand displacement assay (SDA), and the transcription-​mediated amplification (TMA) technique has overcome the lack of specificity and relatively low sensitivity of all other C. trachomatis diagnostic tests. Furthermore, additional advantages are that many NAATs detect C. trachomatis and N. gonorrhoeae simultaneously and that NAATs can be used to test non​invasive specimens such as first catch urine samples in men or vulvo-​vaginal swabs in women (first catch urine samples in women result in a suboptimal sensitivity). The NAATs are also ideal for automation, which increases the standardization and quality as- surance, and high throughput. The currently available commercial TMA-​based C. trachomatis NAATs target 23S rRNA or 16S rRNA, which are present in many copies in each bacterial cell. Several of the commercially available C. trachomatis PCR assays, as well as the SDA-​based NAAT, detect genetic sequences in the cryptic plasmid, also present in multiple copies in each C. trachomatis cell. Plasmid-​ free C. trachomatis strains have been exceedingly rare and are most likely less virulent. Using a multiple copy target significantly in- creases the sensitivity of the NAATs. A new variant of C. trachomatis Fig. 8.6.45.5  Mucopurulent neonatal conjunctival discharge due to C. trachomatis.

8.6.45  Chlamydial infections 1287 (nvCT; serovar E) that had escaped detection by two internationally commonly used PCR-​based NAATs was found in Sweden in 2006. The nvCT has a 377 bp deletion in the cryptic plasmid, which in- cluded the genetic target sequence for both these NAATs. The nvCT caused thousands of false-​negative C. trachomatis tests in Sweden, fewer tests in other Scandinavian countries, and very few in other countries. All the main commercial NAATs used in Europe can now detect nvCT; that is, the affected NAATs have been redesigned (in- cluded a second genetic target sequence) to ensure detection of the nvCT. This underlines the importance of taking into account the structure and function of genomes when selecting appropriate target sequences for NAATs and also that dual-​target NAATs might have to be considered for many infectious diseases. The nvCT story also emphasizes the importance of detailed analysis of incidence, locally, nationally, and internationally, and timely attention to unexplained significant declines, as well as participation in appropriate external quality assurance schemes. Currently commercially available and properly validated C. tra­ chomatis NAATs are sufficiently specific to not require confirmation by another NAAT, targeting another genetic sequence, when used in high prevalence populations. However, no test is 100% sensitive or specific, and in medico-​legal cases a reactive NAAT should be con- firmed using a different NAAT target. NAAT technology is rapidly advancing with the emergence of rapid point-​of-​care NAATs and multiplex NAATs. In general, the performance characteristics of dif- ferent NAATs differ. Accordingly, care should be taken to use only those assays which have been appropriately validated using properly designed studies of sufficient size. Culture and staining of Chlamydiae The growth of Chlamydiae more than 50 years ago in cultured cells, rather than in embryonated eggs, revolutionized their detection and chlamydial research. C. pneumoniae is particularly difficult to iso- late, but will grow in selected cell lines including Hep-​2. The iso- lation of C. trachomatis involves centrifugation of specimens onto cycloheximide-​treated McCoy cell monolayers, followed by incu- bation and then staining with a fluorescent monoclonal antibody or with a vital dye, usually Giemsa, to detect inclusions. One blind passage may increase sensitivity, but cell-​culture techniques need to maintain organism viability and are slow, labour intensive, and no more than 70% sensitive compared to appropriate NAATs in many settings. Staining of epithelial cells in ocular and genital smears with vital dyes to detect chlamydial inclusions in microscopy is insensitive and often non​specific. In contrast, microscopic detection of elem- entary bodies using species-​specific fluorescent monoclonal anti- bodies is rapid, relatively sensitive, and specific for C. trachomatis oculo-​anogenital infections, in the hands of skilled observers. When NAATs are not available or affordable, this test can be suitable for testing lower number of specimens. Due to the superior sensitivity and most other performance char- acteristics of the modern C. trachomatis NAATs, almost all these earlier diagnostic methods are obsolete in high-​income countries. Point-​of-​care tests The currently available, mostly immunochromatographic, rapid point-​of-​care (POC) tests for C. trachomatis have insufficient sen- sitivity, compared to NAATs, to be used in clinical practice. Rapid POC tests using nucleic acid amplification technologies with in- creased sensitivity and high specificity have been developed. The number of such tests approved for clinical use is likely to increase over time. While modelling suggests POC NAAT tests could im- prove outcomes and reduce costs this has yet to be demonstrated in clinical practice. Serological tests NAATs detect current infection, but not past exposure. Serological tests, on the other hand, detect both current and previous exposure with varying degrees of sensitivity and specificity. Mucosal infection produces a weak antibody response in many patients, particularly in men, while invasive and/​or upper genital tract infections usually result in high antibody titres. Antibody titres, in general, decline with time since infection, but can remain detectable for many years. Serology should, therefore, not be used to detect current uncom- plicated infection. Nevertheless, when NAATs are not available, de- tection of specific antibodies to C. trachomatis might support the diagnosis of invasive disease, such as invasive LGV and neonatal pneumonia Detection of C.  trachomatis antibodies in patients is compli- cated by cross-​reaction with other chlamydial species, particularly C. pneumoniae. More recent serological assays have overcome this by using C. trachomatis specific MOMP peptides or Pgp3 antigen which is not present in C. pneumoniae. The traditional complement-​fixation test cannot distinguish be- tween the chlamydial species as it detects the genus-​specific LPS antigen. Historically most of the pertinent diagnostic information originates from use of the MIF test which measures class-​specific antibodies (IgM, IgG, IgA, or secretory) to the purified elementary bodies. A significant increase in IgM and/​or IgG titre is so unusual in uncomplicated infection that the test is of little value. In PID, espe- cially in the Fitz-​Hugh–​Curtis syndrome, antibody titres tend to be higher than in uncomplicated cervical infections. A high IgG anti- body titre (1:256 or greater), suggests causation in pelvic disease, but high titres do not always correlate with detection of Chlamydiae and are associated more with chronic or recurrent disease. Specific C. trachomatis IgM antibody in babies with pneumonia is pathog- nomonic of chlamydia-​induced disease. Furthermore, patients with rectal LGV infections develop high antibody titres and, if NAATs are not available, high antibody titres can be used as a diagnostic tool. Treatment of C. trachomatis infections Chlamydiae are intracellular and hence insensitive to aminoglycosides and other antibiotics that do not penetrate cells efficiently. They are however sensitive to many antibiotics and particularly sensitive to tetracyclines and macrolides, and also to a variety of other drugs. The rifamycins are probably more active than the tetracyclines in vitro, but the activity has been lower in vivo. Rifamycins have only rarely been used to treat refractory chlamydial infections, and they are reserved for mycobacterial infections. Antimicrobial resistance Induced resistance to antimicrobials in C. trachomatis due to muta- tions in drug target or other bacterial genes has been demonstrated in vitro. However, there is no evidence of any classical antimicrobial

section 8  Infectious diseases 1288 resistance (stable, homotypic genetic resulting in phenotypic re- sistance in bacteria) to any antimicrobials in clinical C. trachomatis strains that affects the in vivo treatment in humans. Nevertheless, vigilance is needed to detect resistant strains if and when they do emerge, which would not be possible with the routine diagnostic procedures using commercial NAATs (see earlier). There is evidence to suggest that phenotypic switching of at least part of the cell population of a C. trachomatis strain can occur. The bacterium exhibits the phenomenon of heterotypic resistance at high infectious loads in vitro and there is circumstantial evidence this may occur in vivo. That is, replication, at high loads, generates a heterogeneous population of resistant and susceptible bacteria. Subsequent subculture of a single resistant organism derived fol- lowing propagation on antimicrobial-​containing medium, results in a fully susceptible organism (i.e. the antimicrobial resistance is not genetically inherited, or the decreased biological fitness of the resistant cells results in them getting outcompeted by susceptible cells). The resistant cells might replicate more slowly, which also might make them less susceptible to antimicrobial therapy. High infectious loads are associated with disease (e.g. non​gonococcal ur- ethritis in men). It has been proposed that this might explain the reduced efficacy of azithromycin in men with non​gonococcal ur- ethritis compared to asymptomatic individuals, with a higher dose likely to increase efficacy. Antimicrobial therapy Doxycycline 100 mg twice a day for 7 days in uncomplicated ano- genital non-​LGV C. trachomatis infection including non​gonococcal urethritis is more than 97% effective. In LGV, a 21-​day treatment is recommended (see the ‘Lymphogranuloma venereum’ section). A single dose of azithromycin 1 g enhances compliance and has a similar efficacy, although efficacy is reduced in chlamydial urethritis and rectal infection. There is a danger of inducing resistance in other bacteria, for example Mycoplasma genitalium (see Chapter 8.6.46 on Mycoplasmas). It has been proposed that a longer duration of therapy and increased dose might improve efficacy of azithromycin. The macrolide erythromycin can be used to treat chlamydial infec- tions in infants and young children. The newer fluoroquinolones are among other active drugs, but they have not been appropriately evaluated in vivo and are not used regularly. Table 8.6.45.2 shows details of the doses and duration of anti- biotic treatment. Systemic treatment is given as well as, or in pref- erence to, topical treatment to eradicate nasopharyngeal carriage in trachoma, for paratrachoma and for neonatal chlamydial conjunc- tivitis, since topical treatment provides no additional benefit. Oral erythromycin should be used to treat conjunctivitis and to prevent the development of pneumonia in infants and young children due to the limited data on efficacy and optimal dose of azithromycin in this patient group. Table 8.6.45.2  Recommended first-​line treatment schedules for chlamydial infections and associated diseases (readers should refer to respective chapters for syndromic management of specific diseases and online guidelines published by BASHH, IUSTI, and CDC) Disease/​infection Antibiotic Dose schedulea Duration (days) Trachoma Azithromycin alone 20 mg/​kg (up to 1 g) Single dose Topical tetracycline 1% ointment daily 42 Oculo-​anogenital tract infectionb Doxycycline 100 mg twice daily 7 or azithromycin 1 g Single dose Rectal infection (serovars D–​K) Doxycycline 100 mg twice daily 7 Adult inclusion conjunctivitis Doxycycline 100 mg twice daily 7 or azithromycin 1 g Single dose Epididymo-​orchitis secondary to C. trachomatis Doxycycline 100 mg twice daily 14 Include ceftriaxone if N. gonorrhoeae coinfection not excluded 500 mg  intramuscularly Single dose Pelvic inflammatory disease secondary to C. trachomatis (see Chapter 9.8) Ceftriaxone 500 mg  intramuscularly Single dose then doxycycline 100 mg twice daily 14 and metronidazole 400 mg twice daily 14 Pelvic inflammatory disease secondary to C. trachomatis (see Chapter 9.8) and N. gonorrhoeae excluded Ofloxacin 400 mg twice daily 14 and metronidazole 400 mg twice daily 14 Neonatal infections Erythromycin syrup 50 mg/​kg daily in four divided doses 14 Lymphogranuloma venereum Doxycycline 100 mg twice daily 21 C. pneumoniae infections Doxycycline 100 mg twice daily 14–​21c C. psittaci infections Doxycycline 100 mg twice daily 14–​21c BASHH, British association for Sexual Health and HIV (www.bashh.org/​BASHH/​Guidelines/​Guidelines/​BASHH/​Guidelines/​Guidelines.aspx); IUSTI, International Union against Sexually Transmitted infections (www.iusti.org/​regions/​Europe/​euroguidelines.htm); CDC, Centers for Disease Control and Prevention (www.cdc.gov/​std/​tg2015/​) a All antibiotics orally unless otherwise indicated. b Doxycycline is preferred if urethritis is present in men. c. Relapse more often with short course.

8.6.45  Chlamydial infections 1289 Complicated infections In general, a longer duration of therapy is administered in com- plicated disease such as epididymo-​orchitis and PID. Treatment is usually started before a microbiological diagnosis can be established and gonorrhoea excluded. If gonorrhoea is suspected treatment regimens include cover for this infection as well. As PID is often polymicrobial, additional broad-​spectrum antibiotic cover particu- larly for anaerobes is required (see Chapter 9.8 on PID). Compliance and partner notification Compliance is improved if a brief discussion about C. trachomatis is undertaken, supplemented by a leaflet, about how it is transmitted and what can happen if it is not treated. Patients should be advised about the importance of completing the course of antimicrobial pre- scribed and to avoid sexual intercourse (including oral sex) until they and their partner(s) have completed treatment (or wait 7 days if treated with azithromycin 1 g) and their symptoms, if present, have resolved. Identification and treatment of partners is essential to re- duce the risk of reinfection and on-​going transmission from infected partner(s). This is best undertaken by a person with specific training in partner notification. In general, for symptomatic individuals the look-​back period is 4 weeks and 6 months if asymptomatic. Test of cure and repeated testing A test of cure (TOC) is not advocated to be performed in patients treated with recommended first-​line treatment. However, it is re- commended in pregnancy, in complicated infections, if symptoms persist, if second-​line or third-​line treatments have been used, and if non​compliance to therapy or re-​exposure of infection is sus- pected. It could also be considered in rectal infections, that is, when azithromycin 1 g stat has been administered for treatment of rectal infections. In these situations, TOC using NAATs should be per- formed 4–​5 weeks after completion of therapy. Accordingly, NAATs may remain positive for at least 3 weeks after treatment (and in some patients possibly up to 8 weeks due to intermittent shedding of nu- cleic acid). However, a positive test does not necessarily mean active infection as it may represent the presence of non​viable organisms. C.  trachomatis-​positive individuals are at increased risk of re-​ testing positive within the next year. There are several potential ex- planations and it is likely that more than one applies. These include reinfection, repeat infection from an untreated partner and anti- microbial failure. Accordingly, repeat testing in 3–​6 months should ideally be offered to at least young women and men (<25 years of age) who test positive for C. trachomatis. Prevention Health promotion The risk of chlamydial infection and sexually transmitted infections (STIs) in general can be reduced by consistently and correctly using condoms until all partners have had a sexual health screen, and re- ducing the number of sexual partners and avoiding overlapping sexual relationships. As the incidence of C. trachomatis infection is highest in women aged 16–​17 years, this advice needs to be pro- vided not only to sexually active adults and reinforced when and if they acquire a STI but also before individuals become sexually ac- tive. The most appropriate way to deliver this advice is through high quality sex education in schools which focuses as much on healthy relationships and self-​esteem as the negative consequences of sexual intercourse. Chlamydia screening Due to the disease burden associated with chlamydial infection, testing has been promoted and increased worldwide. Detection rates of C. trachomatis infections have increased since around 2000 in most developed countries, which is largely attributable to increased testing made possible by the introduction of highly sensitive NAATs enabling non​invasive testing. Moreover, chlamydia screening has been demonstrated in randomized controlled trials to reduce the rates of PID in women, but not prevalence. England and the United States have introduced screening programmes. England fully im- plemented the National Chlamydia Screening Programme (NCSP) in 2008 for men and women less than 25 years of age. Screening is recommended annually and on change of sexual partner. Annual screening is also recommended in the United States for sexually ac- tive women aged less than 25 years, as is screening of older women at increased risk for infection. This has resulted in a substantial in- crease in testing in both countries. Mathematical models in general have estimated that screening will reduce prevalence, that is, if the screening coverage is suffi- ciently high. In England 1.6 million tests in 16–​24 years old indi- viduals were undertaken in 2014 with an estimated 35% of young females and 14% of young males being tested. Routine surveillance data is not representative of the general population. This means that current infection in those tested is not an adequate indicator of population prevalence and exposure over time and cannot be used to evaluate effectiveness. It is, as yet, not known whether the NCSP has reduced prevalence or is cost effective. A sensitive and specific sero- logical marker of cumulative lifetime exposure would contribute to evaluation of the screening programmes. The use of Pgp3 antibody for this purpose is currently being evaluated. A recent ecological association study from the United States suggests that widespread chlamydia screening may have resulted in a decline in incidence of chlamydia infection, PID, and ectopic pregnancy. Lymphogranuloma venereum LGV is a systemic, ulcerative sexually transmitted infection caused by serovars L1, L2 (including subvariants such as L2b), and L3 of C. trachomatis. These serovars are more virulent in animal models than serovars A-​K, and more invasive in humans. Serovars A-​K are largely confined to mucosal columnar epithelial surfaces of the uro- genital tract and eye, but the LGV serovars predominantly infect monocytes and macrophages, which pass through the epithelial sur- face to regional lymph nodes and may cause ulceration and bubonic disseminated infection. Clinical features The incubation period is from less than 1 to 4 weeks. The traditional clinical course of LGV can be divided into three stages. The primary stage at the site of inoculation, the secondary stage in the regional lymph nodes and/​or the anorectum, and the tertiary stage of late sequelae affecting the genitalia and/​or rectum. The primary stage begins with a small, painless papule which might ulcerate. It occurs at the site of inoculation, usually the

section 8  Infectious diseases 1290 prepuce or glans in men; anorectal and rectosigmoid colon sites in men who have sex with men (MSM); or the vulva, vaginal wall, or occasionally the cervix in women. Extragenital primary lesions on fingers or tongue are rare. The primary lesion is self-​healing and may pass unnoticed by the patient, especially if it is in the alimentary tract of MSM. Patients with LGV presenting with buboes might not be aware of having had an ulcer. The secondary stage occurs some weeks after the primary lesion, which has usually healed. C. trachomatis-​infected cells are carried to regional or rectal lymph nodes. The inguinal form is more common in men than women, since the lymphatic drainage of the upper va- gina and cervix is to the retroperitoneal rather than the inguinal lymph nodes. LGV proctitis occurs in those who practise receptive anal intercourse, probably due to direct inoculation. The cardinal feature of the inguinal form of LGV is painful, usu- ally unilateral, inguinal, and/​or femoral lymphadenopathy (bubo) (Fig. 8.6.45.6). Adenopathy above and below the inguinal ligament gives rise to the ‘groove sign’ in 10 to 20% of patients, once believed to be pathognomonic. Enlarged lymph nodes are usually firm and often accompanied by systemic signs of fever, chills, arthralgia, and headache. Biopsy reveals small discrete areas of necrosis surrounded by proliferating epithelioid and endothelial cells. These areas of ne- crosis may enlarge to form stellate abscesses, which may coalesce and break down to form discharging sinuses, although this phe- nomenon occurs in less than one-​third of patients with inguinal dis- ease. In women, signs include a hypertrophic suppurative cervicitis, backache, and adnexal tenderness. Anorectal involvement is seen predominantly in MSM. Clinical features include a haemopurulent anal discharge, anorectal pain, and bleeding due to an acute haem- orrhagic proctitis or proctocolitis, and there may be pronounced systemic signs of fever, chills, and weight loss. In contrast to pre- vious reports from the United Kingdom, studies in the Netherlands have now shown that about 25% of LGV infections among MSM can be asymptomatic. These cases might also include early detection of LGV at a ‘presymptomatic’ stage of disease, found among people who are regularly screened for rectal C. trachomatis. Proctoscopy re- veals a granular or ulcerative proctitis from which large numbers of polymorphonuclear leucocytes are seen in rectal smears. Computer tomography or magnetic resonance imaging scans may show pro- nounced thickening of the rectal wall, with enlargement of iliac lymph nodes. Enlarged inguinal nodes may also be palpable. Extragenital infection can cause lymphadenopathy outside the inguinal region. For example, cervical adenopathy due to LGV has been reported after oral sex, and laboratory workers who de- veloped pneumonitis after accidental inhalation of LGV strains of C. trachomatis were found to have mediastinal and supraclavicular adenopathy. A follicular conjunctivitis has also been described fol- lowing direct inoculation of the eye, which may be accompanied by preauricular lymphadenopathy. Other rare manifestations of the secondary stage include acute meningoencephalitis, synovitis, and cardiac involvement. The tertiary stage appears after a latent period of several years, but all late complications are rare today because of the use of broad-​ spectrum antibiotics. Chronic untreated LGV leads to fibrosis, which may cause lymphatic obstruction and elephantiasis of the genitalia in either sex, or rectal strictures and fistulae. Rarely, it can give rise to the syndrome of esthiomene (Greek: ‘eating away’), with widespread destruction of the external genitalia (Fig. 8.6.45.7). Epidemiology LGV has been endemic in parts of Africa, Asia, South America, and the Caribbean, but before 2003 very rarely detected in Western Europe, North America, and other high-​income countries for many Fig. 8.6.45.6  Ulcer on penis and lymphadenopathy with ‘the groove sign’ due to lymphogranuloma venereum. Courtesy of C. O’Mahony. Fig. 8.6.45.7  Esthiomene: destruction of the female genitalia by lymphogranuloma venereum in a Nigerian patient. Copyright D. A. Warrell

8.6.45  Chlamydial infections 1291 years. The reported sex ratio is greater than 5:1 in favour of men, which is probably due to the easier recognition of disease in men. The global epidemiology of infection is poorly defined because LGV is often indistinguishable clinically from chancroid and other causes of genital ulceration with bubo formation, and it has been difficult to obtain laboratory confirmation in many settings world- wide. In 2003, an outbreak of LGV proctitis and proctocolitis due to a clonal expansion of a serovar L2b strain was reported among MSM in Rotterdam, the Netherlands, and since then many thousands of cases have been reported in MSM across Western Europe (including the United Kingdom), North America, and Australia. The LGV L2b strain is also found in the heterosexual population. Genomic ana- lysis of the LGV L2b strain has now shown that this is unlikely a newly emerged epidemic strain, but instead an old strain. A LGV L2b sample was originally identified in San Francisco, United States in the 1980s. Furthermore, rectal LGV has probably been present for years, remaining undetected because of the poor sensitivity of diagnostic tests prior to the introduction of NAATs and/​or lack of sampling for C. trachomatis diagnostics in many of these patients. Most infected MSM have been HIV-​positive. Many of these patients have attended healthcare departments with atypical presentations (that is without any genital ulceration or the typical inguinal buboes) including proctitis or tenesmus, anorectal discharge, and discom- fort, diarrhoea, or altered bowel habits. Due to this symptomology, LGV should be considered as a differential diagnosis in patients with proctitis or inflammatory bowel disease (IBD)-​related symptoms, especially among HIV-​positive men. Diagnosis LGV can present as a genital ulcer, inguinal lymphadenopathy (usu- ally painful) without evidence of genital ulceration, or proctitis/​ proctocolitis. The differential diagnosis of sexually acquired genital ulceration also includes chancroid, herpes, syphilis, and the exceed- ingly rare donovanosis (granuloma inguinale). Less common causes of ulceration include trauma, non​venereal infections such as cuta- neous leishmaniasis or amoebiasis, and fixed drug eruption. The differential diagnosis of inguinal adenopathy includes chancroid, herpes, and syphilis, although there is usually a genital ulcer or at least a history of an ulcer in these conditions. Chronic sinus for- mation in the inguinal region might be due to tuberculosis of the lumbar spine, and bubonic plague should be considered in endemic areas where a patient with inguinal lymphadenopathy is acutely ill. LGV proctitis should be considered as a differential diagnosis in pa- tients with inflammatory bowel disease due to ulcerative colitis or Crohn’s disease, particularly among HIV-​positive men, because clin- ical and histopathological features may be identical. Since anorectal LGV needs management different to that for non-​LGV chlamydial infections, in some European settings all MSM, irrespective of ano- rectal symptoms, who report receptive anal sex in the previous six months are tested for anorectal C. trachomatis infection with a com- mercially available NAAT. MSM who are anorectal C. trachomatis-​ positive are then recommended to be tested for LGV using a genovar L-​specific NAAT. Highly sensitive and specific laboratory diagnosis of LGV depends on identification of C. trachomatis LGV serovars (L1-​L3) in appro- priate clinical specimens using NAATs. All commercially available validated C. trachomatis NAATs detect LGV strains. However, an LGV-​specific in-​house PCR or ompA-​sequencing is required to distinguish the LGV serovars (L1-​L3) from the trachoma serovars (A-​K). If a NAAT is unavailable, serology can be used. Accordingly, due to its invasive nature LGV infections induce higher serum anti- body titres than do uncomplicated genital infections with C. tra­ chomatis serovars D-​K. The MIF test can distinguish between infections with different chlamydial species. A MIF titre exceeding 1:128 strongly suggests LGV, particularly in a patient with typical signs and symptoms, although invasive genital infection with C. tra­ chomatis serovars D-​K, as in PID, can also give rise to high anti- body titres. However, a low antibody titre does not exclude LGV. C. trachomatis can also be identified in a smear of bubo material by direct fluorescence microscopy using commercially available con- jugated monoclonal antibody, although bacterial contamination impedes detection and the sensitivity of this diagnostic method is suboptimal. C. trachomatis can be isolated in cell culture from ulcer material, bubo aspirate, or endourethral or endocervical scrapings, but the success rate is poor. Treatment Despite a paucity of strong evidence regarding the efficacy of therapy for LGV, 21 days of oral doxycycline 100 mg twice daily is the first-​ line recommendation. However, doxycycline is contraindicated in pregnancy and breastfeeding. Second-​line is erythromycin 500 mg four times daily for 21 days. Fever and bubo pain subside rapidly after antimicrobial treatment is started, but buboes may take several weeks to resolve. Azithromycin in single-​ or multiple-​dose regimens has been suggested and also successfully used in some cases, but suf- ficient evidence is lacking to currently recommend this antibiotic. Adjunctive therapy might also be needed. This includes prompt as- piration of fluctuant buboes through healthy adjacent skin and sur- gical repair, including reconstructive genital surgery, for patients with residual fibrotic lesions, strictures, fistulae, or esthiomene. Trachoma Trachoma is a chronic keratoconjunctivitis caused by the ‘ocular’ serovars A, B, Ba, and C of C. trachomatis. In the 19th century it was an important and common cause of blindness in Europe and North America, but it disappeared from more affluent parts of the world as living standards improved in the 20th century. In poor commu- nities where hygiene standards are low, there is direct transfer of chlamydial organisms from eye to eye and trachoma is endemic. As standards of hygiene improve, this mode of transmission is no longer possible, and trachoma tends to disappear. It is now a disease of poor rural communities, mainly in Africa and Asia, but it remains the leading infectious cause of blindness worldwide. A recent review by the WHO estimated that trachoma was responsible for 1.4% of global blindness, causing visual impairment in 1.8 million people and irreversible blindness in 0.5 million. Clinical features The active (inflammatory) stage of trachoma is a follicular con- junctivitis, affecting chiefly the subtarsal conjunctiva, but follicles can be seen elsewhere on the conjunctiva and at the limbus. Such subconjunctival follicles are the characteristic sign of active disease (Fig. 8.6.45.8) and are usually seen in children in endemic areas. Limbal follicles resolve leaving characteristic shallow depressions

section 8  Infectious diseases 1292 known as Herbert’s pits. New vessels (pannus) might be seen at this stage in the cornea (Fig. 8.6.45.9), usually at the superior margin, and punctate keratitis might also be a feature. Since symptoms are mild or absent at this stage, the disease might not be suspected un- less the upper eyelid is everted. C. trachomatis can often be found in active cases, although follicles can persist for some time after infec- tion has been cleared. Intense inflammation is seen in the subtarsal conjunctiva in some cases (Fig. 8.6.45.10) in which the C. tracho­ matis bacterial loads are higher. The disease can progress over many years and, with repeated infection, result in conjunctival scarring (Fig. 8.6.45.11). As the scars contract, the lid margin turns inwards (entropion), and the eyelashes rub against the cornea, a condition known as trichiasis (Fig. 8.6.45.12). This damages the cornea, even- tually rendering it opaque and causing blindness. The WHO criteria for the clinical diagnosis of active trachoma and its potentially blinding sequelae, and for grading their severity is as follows: 1 Trachomatous inflammation, follicular (TF)—​five or more follicles, each at least 0.5 mm in diameter, in the upper tarsal conjunctiva (Fig. 8.6.45.8) 2 Trachomatous inflammation, intense (TI)—​pronounced in- flammatory thickening of the tarsal conjunctiva that obscures more than one-​half of the normal deep tarsal blood vessels (Fig. 8.6.45.10) 3 Trachomatous conjunctival scarring (TS)—​easily visible scar- ring in the tarsal conjunctiva (Fig. 8.6.45.11) 4 Trachomatous trichiasis (TT)—​at least one eyelash rubbing on the eyeball, or evidence of recent removal of inturned eyelashes (Fig. 8.6.45.12) 5 Corneal opacity (CO)—​easily visible corneal opacity over the pupil, so dense that at least part of the pupil margin is blurred when viewed through the opacity Epidemiology Trachoma is a disease of poverty, which disappears as living stand- ards improve. In the past, it has been endemic in urban commu- nities such as in the East End of London, but it is now a disease of rural communities that lack access to water and sanitation, espe- cially affecting marginalized groups. The reservoir of infection in endemic areas is the eye, and possibly the nasopharynx, of children with active disease. C. trachomatis can be transferred from the eye of one individual to that of another via fingers, fomites, coughing and sneezing, and by eye-​seeking flies. Active cases tend to cluster in households with prolonged intimate contact within the family. Fig. 8.6.45.8  Everted upper eyelid showing follicular trachoma. Fig. 8.6.45.9  Extensive neovascularization of the cornea (pannus) due to trachoma. Fig. 8.6.45.10  Everted upper eyelid showing intense inflammatory trachoma. Fig. 8.6.45.11  Everted upper eyelid showing trachomatous scarring.

8.6.45  Chlamydial infections 1293 The higher prevalence of active disease and scarring in women than in men is probably due to their closer contact with children. Severe conjunctival scarring is associated with repeated exposure to reinfection. Diagnosis In trachoma-​endemic areas, the diagnosis is made on clinical grounds, following the simplified WHO grading scheme (Figs. 8.6.45.8, 8.6.45.10–8.6.45.​12). Trachomatous follicles (TF) might be con- fused with the giant papillae of vernal conjunctivitis, in which pannus may also be seen. Several viruses, notably adenoviruses, can cause a short-​lived follicular conjunctivitis. Intense cases of trachoma inflammatory (TI), in which follicles might not be visible, should be distinguished from bacterial conjunctivitis. The diagnosis of trachomatous scarring (TS) is usually obvious, as few other condi- tions cause conjunctival scarring affecting the upper lid. Laboratory diagnosis of ocular C. trachomatis infection can help to direct treat- ment to communities with the greatest need, since clinical signs can persist for years after infection has been cleared. C. trachomatis can be found by PCR or another NAAT in a high proportion of cases of active inflammation (TF or TI), but in only a minority of those with scarring disease (TS). Treatment Inflammatory trachoma (TF and TI) responds to antimicrobial treatment (Table 8.6.45.2). The WHO recommends a single oral dose of azithromycin (20 mg/​kg, to a maximum of 1 g) or, in pregnant women and children under 6 months of age, 1% top- ical tetracycline ointment, to be applied to both eyes twice daily for 6 weeks. Community-​based mass treatment is recommended where the prevalence of TF exceeds 10% in children aged 1 to 9 years. Reinfection is rapid if individual cases are treated sep- arately, and the WHO also recommends that interventions to reduce transmission, such as face washing and environmental im- provement, should be implemented where trachoma is endemic. Trichiasis requires surgical correction. Several eyelid operations have been described, but a recent randomized controlled trial showed clearly that posterior lamellar tarsal rotation is the oper- ation of choice. Prevention The WHO has launched a strategy for the global elimination of blinding trachoma as a public health problem by the year 2020, based on the acronym ‘SAFE’: Surgery for trichiasis, Antibiotics for the treatment of inflammatory disease and the elimination of the res- ervoir of infection, promotion of Face washing, and Environmental improvement to reduce fly populations and hence transmission. There is evidence that the prevalence of active trachoma has fallen in several countries in Africa and Asia in recent years following the implementation of the SAFE strategy. C. pneumoniae infections The prototype strains of C. pneumoniae were isolated in the 1960s from conjunctival samples collected from a child in Taiwan (strain TW-​183) and another in Iran (strain IOL-​207). In 1983, a third C. pneumoniae strain was isolated, this time from the throat of a pa- tient with acute respiratory (AR) disease, such as pharyngitis (strain AR-​39). This prompted the name TWAR (TW + AR) being coined for the isolates. The two original isolates (TW-​183 and IOL-​207) were serologically identical and distinct from C. trachomatis and C. psittaci. In 1989, C. pneumoniae was defined as the third species of the genus Chlamydia. Only one serovar of C. pneumoniae has been identified, although minor geographical serovar variations are described. Clinical features Respiratory tract disease After an incubation period of approximately 3 weeks, acute dis- ease often begins with pharyngitis. More than 80% of patients with lower respiratory tract disease have a sore throat. A cough may de- velop later but fever is uncommon. Bronchitis sometimes appears and in young adults about 5% of primary sinusitis is associated with C. pneumoniae. Mild respiratory tract infections are probably frequent, but pneumonia is most common. Radiographs usually reveal a unilateral pneumonia, but more severe infection causes bilateral signs. This is often difficult to distinguish clinically from Mycoplasma pneumoniae and other pneumonias. Up to one-​fifth of exacerbations of chronic obstructive pulmonary disease are associ- ated with C. pneumoniae and it has been implicated in exacerbations of both adult and childhood asthma. Arthritis An exaggerated synovial lymphocyte response to C. pneumoniae has been found in some adults with reactive arthritis and C. pneumoniae DNA and high titres of specific antibody have been detected in syn- ovial fluid from the joints of a few children with juvenile chronic arthritis, suggesting the possibility of a causal role. Atherosclerosis Finnish investigators in the 1980s observed an association between chronic coronary heart disease or acute myocardial infarction and antibody to C. pneumoniae. The idea of chronic infection was en- hanced by the detection of Chlamydiae or their DNA in at least 40% of atheromatous plaques in coronaries and other major and occasionally minor arteries, but not in normal tissue, of people as young as 15 years of age. Specific DNA was also found in peripheral Fig. 8.6.45.12  Trachomatous trichiasis.

section 8  Infectious diseases 1294 blood mononuclear cells, suggesting the possibility that they might transmit the organisms from the respiratory tract to the arterial wall. Furthermore, studies in animal models provided some support for the atheroma-​C. pneumoniae association. However, euphoria about these findings was dealt a blow by the results of three major anti- biotic trials in the United States. Subjects who received long courses of azithromycin in two trials and gatifloxacin in the other, subse- quently experienced untoward coronary events as often as those given a placebo. Admittedly this outcome was not completely unex- pected in patients with well-​established, long-​standing disease. Other diseases The existence of C. pneumoniae in peripheral monocytes means that the organisms might engage with any tissue/​organ. In hindsight, this provides some credibility for claims for a role in conditions as di- verse as Alzheimer’s disease, stroke, and multiple sclerosis, as well as chronic secretory otitis media, cystic fibrosis, sarcoidosis, and primary biliary cirrhosis. However, there is absolutely no credible evidence to suggest a causal association with any of the conditions mentioned. Epidemiology Molecular typing studies suggest that animal strains of C. pneumo­ niae are ancestral to human strains and that C. pneumoniae crossed from animals to humans as a result of at least one relatively re- cent zoonotic event. C. pneumoniae genotypes have been detected in horses, koalas, bandicoots, amphibians, and reptiles but, apart from the event mentioned earlier, there is otherwise no evidence of transfer to humans, even when there might be close contact, for ex- ample with koalas. It is thought that human strains of C. pneumoniae are transmitted directly from person to person and serological evi- dence indicates that infection is widespread and endemic in many areas. However, localized respiratory epidemics have been recorded in both military and civilian groups in Scandinavia, the United States, the United Kingdom, and elsewhere. C. pneumoniae probably causes many mild respiratory tract infections that were previously thought to be viral in origin and it is also likely that many infections labelled ‘human psittacosis’ or ‘ornithosis’ in the past were due to C. pneumoniae and not C. psittaci. C. psittaci infections The C. psittaci species forms a diverse group isolated from a variety of mammals, reptiles, and many avian species. There is a relatively low degree of homology between six serovars exhibited in DNA–​ DNA hybridization analyses, with the possibility of further differen- tiation between organisms assigned to the species. The spectrum of diseases in animals, cattle, sheep, goats, and more than 400 species of birds caused by C. psittaci includes conjunctivitis, pneumonia, enteritis, abortion, sterility, arthritis, and encephalitis, all of which result in economic loss. Organisms of all serovars, but particularly those of A, C and D, are capable of being transmitted occasionally through inhalation to humans, being a potential hazard to those who keep pet birds and those employed in the poultry industry or in slaughter houses. Infectious forms of the organisms are shed in nasal discharges or occur in faeces or feather dust from symp- tomatic as well as apparently healthy birds and may remain viable for several months. The term ‘psittacosis’ refers to both avian and human infection by C. psittaci found in psittacine birds, and ornith- osis to infection by strains from other birds. ‘Psittacosis’ is often used indiscriminately when referring to infection from psittacine and non​psittacine birds. Outbreaks and sporadic cases of psittacosis are now a rare occurrence due to quarantine of imported birds and im- proved veterinary-​hygiene measures. Clinical features After an incubation period of 1 to 2 weeks, the presentation of human infection (psittacosis) varies from a mild influenza-​like illness to a fulminating toxic state with multiple organ involvement. The disease may begin insidiously over a few days, or start abruptly with high fever, rigors, and anorexia. A headache is common, a cough, often dry, occurs in over two-​thirds of patients, and arthralgia and myalgia in over one-​third of patients. Inspiratory crepitations are more usual than classic signs of consolidation. Chest radiographs show patchy shadowing, often in the lower lobes. Homogeneous lobar shadowing is less frequent, miliary and nodular patterns even less so, and sig- nificant pleural effusions are rare. Extrapulmonary complications, mostly rare, include endocarditis, myocarditis, pericarditis, a toxic confusional state, encephalitis, meningitis, tender hepatomegaly, splenomegaly, pancreatitis, haemolysis, and disseminated intravas- cular coagulation. Fatal cases have been rare in the postantibiotic era. Improved diagnostic tests should not allow C. psittaci infections to be confused with those caused by C. pneumoniae. Other chlamydial infections C. abortus is endemic among ruminants and colonizes the placenta, causing abortion in sheep and rarely in pregnant women. They are often farmers’ wives exposed to sheep with enzootic abortion during the lambing season. C. felis is endemic among domestic cats world- wide causing feline keratoconjunctivitis, rhinitis, and pneumonitis, and it can be isolated from the genital tract of female cats. In humans it has caused follicular conjunctivitis similar to that caused by C. tra­ chomatis serovars D–​K. The so-​called Chlamydia-​like organisms are also emerging patho- gens, as many, such as Parachlamydia sp., Simkania sp., and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Diagnosis C. psittaci is more robust than other chlamydial species but, never- theless, clinical specimens should be placed in a transport medium prior to examination. Attempted isolation is a health risk and should be undertaken only by experienced workers in specially equipped laboratories. Hence, isolation in cell culture is often not done but the procedure would be along the same lines as for C. trachomatis and C. pneumoniae where cell cultures after incubation are examined, by means of a specific fluorescent monoclonal antibody for identifi- cation. Not surprisingly NAATs are far superior in performance to any other procedure for detecting C. pneumoniae and C. psittaci. In the case of the latter, some PCR-​based assays have been developed, but due to the rarity of psittacosis the performance characteristics of these assays have been rather poorly evaluated for testing clinical

8.6.46 Mycoplasmas 1295

8.6.46 Mycoplasmas 1295

8.6.46  Mycoplasmas 1295 specimens. Examination of serum samples, preferably paired, is ad- vocated for diagnosing C.  pneumoniae and C.  psittaci infections. Using a complement-​fixation test is an out-​of-​date practice and im- munofluorescence assays have been the mainstay in C. pneumoniae diagnosis but may be less reliable for C. psittaci. However, measure- ment of MOMP-​specific antibody titres in a rELISA is a reliable and more practical approach to diagnosis. Treatment The treatment of C. pneumoniae and C. psittaci infections is the same as for C. trachomatis, except that a longer duration of treatment is advisable (Table 8.6.45.2). FURTHER READING Burton MJ, et al. (2004). Cytokine and fibrogenic gene expression in the conjunctivas of subjects from a Gambian community where trachoma is endemic. Infection and Immunity, 72, 7352–​6. Campbell LA, et al. (2014). Persistent C. pneumoniae infection in ath- erosclerotic lesions: rethinking the clinical trials. Front Cell Infect Microbiol, 4, 34. de Vries HJ, et al. (2015). 2013 European guideline on the management of lymphogranuloma venereum. J Eur Acad Dermatol Venereol, 29, 1–​6. de Vrieze NH, de Vries HJ (2014). Lymphogranuloma venereum among men who have sex with men: an epidemiological and clinical review. Expert Rev Anti Infect Ther, 12, 697–​704. Gottlieb SD, et al. (2010). Summary: the natural history and immuno­ biology of Chlamydia trachomatis genital infection and implications for chlamydia control. J Infect Dis, 201(S2), 190–​204. Grayston JT, et al. (1990). A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis, 161, 618–​25. Habtamu E, et al. (2016). Posterior versus bilamellar tarsal rotation surgery for trachomatous trichiasis in Ethiopia: a randomised con- trolled trial. Lancet Glob Health, 4, e175–​84. Hadfield J, et al. (2017). Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion. Genome Res, 27, 1220–9. Harris SR, et al. (2012). Whole-​genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing. Nat Genet, 44, 413–​9. Horner PJ (2012). Azithromycin antimicrobial resistance and genital Chlamydia trachomatis infection: duration of therapy may be the key to improving efficacy. Sex Transm Infect, 88, 154–​6. Hu V, et al. (2010). Epidemiology and control of trachoma. Trop Med Int Health, 15, 673–​91. Knittler MR, Sachse K (2015). Chlamydia psittaci: update on an under- estimated zoonotic agent. Pathog Dis, 73, 1–​15. Kong FYS, et al. (2014). Azithromycin versus doxycycline for the treat- ment of genital chlamydia infection—​a meta-​analysis of random- ised controlled trials. Clin Infect Dis, 59, 193–​205. Kong FYS, et al. (2015). The efficacy of azithromycin and doxycycline for the treatment of rectal chlamydia infection: a systematic review and meta-​analysis. J Antimicrob Chemother, 70, 1290–​7. Lanjouw ES, et al. (2015). 2015 European guideline on the manage- ment of Chlamydia trachomatis infections. Int J STD AIDS, 27, 333–​48. Lanjouw E, et al. (2015). Background review for the ‘2015 European guideline on the management of Chlamydia trachomatis infections’. Int J STD AIDS, pii: 0956462415618838. Lewis ME, et  al. (2014). Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals dis- tinct growth patterns. Front Cell Infect Microbiol, 4, 71. Mabey DCW, et al. (2014). Towards a safe and effective chlamydial vac- cine: lessons from the eye. Vaccine, 32, 1572–​8. Menon, S., P. et al. (2015). Human and pathogen factors associated with chlamydia trachomatis-​related infertility in women. Clin Microbiol Rev, 28, 969–​85. Nwokolo NC, et al. (2016). 2015 UK national guideline for the management of infection with Chlamydia trachomatis. Int J STD AIDS, 27, 251–67. Price MAE, et al. (2016). The natural history of Chlamydia trachomatis infection in women: a multi-​parameter evidence synthesis. Health Technol Assess (Winchester, England), 20, 1–​250. Roberts CH, et al. (2015). Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide as- sociation study. Sci Rep, 5, 17447. Roulis EA, et al. (2013). Chlamydia pneumoniae: modern insights into an ancient pathogen. Trends Microbiol, 21, 120–​8. Sachse K, et al. (2015). Emendation of the family Chlamydiaceae: pro- posal of a single genus, Chlamydia, to include all currently recog- nized species. Syst Appl Microbiol, 38, 99–​103. Taylor-​Brown A, et al. (2015). Twenty years of research into Chlamydia-​like organisms: a revolution in our understanding of the biology and patho- genicity of members of the phylum Chlamydiae. Pathog Dis, 73, 1–​15. Unemo M, Clarke IN (2011). The Swedish new variant of Chlamydia trachomatis. Curr Opin Infect Dis, 24, 62–​9. Wang SP, et al. (1967). Trachoma vaccine studies in monkeys. Am J Ophthalmol, 63, 1615–​20. White JA. (2009). Manifestations and management of lympho­ granuloma venereum. Curr Opin Infect Dis, 22, 57–​66. 8.6.46  Mycoplasmas Jørgen Skov Jensen and David Taylor-​Robinson ESSENTIALS Mycoplasmas are the smallest self-​replicating prokaryotes. They are devoid of cell walls, with the plasticity of their outer membrane favouring pleomorphism, although some have a characteristic flask-​ shaped appearance. Mycoplasmas recovered from humans belong to the genera Mycoplasma (14 species and one candidatus species) and Ureaplasma (2 species). They are predominantly found in the re- spiratory and genital tracts, but sometimes invade the bloodstream and thus gain access to joints and other organs. Respiratory infection Clinical features—​Mycoplasma pneumoniae is the most important mycoplasmal respiratory pathogen, with presentations ranging from inapparent infection and mild, afebrile, upper respiratory tract dis- ease to severe pneumonia. It is responsible for 15–​20% of all pneu- monias in the United States of America, and is particularly common in older children and younger adults. Extrapulmonary manifestations include Stevens–​Johnson syndrome and haemolytic anaemia.

section 8  Infectious diseases 1296 Diagnosis and treatment—​diagnosis is made by nucleic acid amp- lification tests and/​or serology. Culture is slow and of limited value in clinical diagnosis. Apart from supportive care, treatment is usually with tetracyclines or macrolides, although an increasing prevalence of macrolide resistance is seen, primarily in Asia. There is no com- mercially available effective vaccine. Genitourinary and related infections Clinical features—​(1) Men—​M. genitalium causes non​gonococcal ur- ethritis in men, and Ureaplasma urealyticum but not U. parvum may play a role in some cases. (2) Women—​M. genitalium causes ureth- ritis, cervicitis, endometritis, and possibly salpingitis; M. hominis and (to a lesser extent) ureaplasmas are associated with bacterial vagin- osis, but are not a cause of the condition; M. hominis may contribute to salpingitis. (3) Pregnancy—​ureaplasma infection of amniotic fluid is associated with preterm labour; ureaplasmas may be involved in the chronic lung disease of very low birthweight babies. Diagnosis and treatment—​diagnosis of infection by ureaplasmas and M. hominis is usually by culture of swabs from the urethra or cervix/​vagina but this will not distinguish between the two urea- plasma species as do nucleic acid amplification tests; nucleic acid amplification tests are used to detect M. genitalium. Patients with non​gonococcal urethritis should ideally receive an antibiotic with activity against C.  trachomatis, ureaplasmas, and M.  genitalium. However, with an increasing prevalence of macrolide resistance in M.  genitalium, doxycycline should be first-​line treatment with moxifloxacin used for macrolide-​resistant M. genitalium. Rheumatological manifestations (1) Sexually acquired reactive arthritis is not uncommon after M.  genitalium-​positive non​gonococcal urethritis, but no causal
link has been established. (2)  Arthritis in patients with hypo­ gammaglobulinaemia is often caused by mycoplasmas (particu- larly ureaplasmas). Introduction Mycoplasmas, the trivial name for members of the class Mollicutes, are the smallest free-​living microorganisms (0.3 µm diameter). They lack the rigid cell wall of other bacteria, making them resistant to penicillins and related antimicrobials. Instead, they have a pliable trilaminar unit membrane (Fig. 8.6.46.1) enclosing the cytoplasm, DNA, RNA, and other components necessary for propagation in cell-​free media. The small size of the mycoplasma genome (as little as 580 kbp) restricts metabolic capabilities, making culture of some mycoplasmas difficult or impossible. Despite their general simi- larity, mycoplasmas are a heterogeneous group with differing host specificities, nutritional requirements, metabolic reactions, and DNA and antigenic composition. Mycoplasmas are divided into four or- ders: Mycoplasmatales, Entomoplasmatales comprising those from insects and plants, Acholeplasmatales, and the strictly anaerobic Anaeroplasmatales. The last two do not need sterol for growth. The mycoplasmas isolated commonly from humans belong to the family Mycoplasmataceae within the order Mycoplasmatales. This family includes the genus Mycoplasma, the organisms of which metabolize glucose or arginine or both, and the genus Ureaplasma, the organisms (ureaplasmas) of which uniquely hydrolyse urea. Ureaplasmas were originally termed T-​strains or T-​mycoplasmas because of the tiny (T) colonies they form on agar medium (15–​60 µm diameter), in con- trast to the larger characteristic fried-​egg-​like colonies produced by most other mycoplasmas (≥90 µm diameter) (Fig. 8.6.46.2). Historical perspective The first mycoplasma to be recognized, Mycoplasma mycoides subsp. mycoides, was isolated in 1898 from cattle with pleuro- pneumonia. As other pathogenic and saprophytic isolates accu- mulated from veterinary and human sources, they became known as pleuropneumonia-​like organisms (PPLO), a term later super- seded by mycoplasmas. The first mycoplasma of human origin, M. hominis, was recovered from a Bartholin’s gland abscess in 1937 and the first of undoubted pathogenicity, M. pneumoniae, from the respiratory tract in 1962. Ureaplasmas were first detected in the urethras of men with non​gonococcal urethritis in 1954 and M. genitalium was isolated from this site in 1981. Subsequently, in 1995, a mycoplasma of human origin named M. amphoriforme was isolated from patients with agammaglobulinaemia. Most re- cently, an uncultivated mycoplasma has been detected by mo- lecular methods in specimens from the urogenital tract of women infected with Trichomonas vaginalis. This new species is tentatively named Canditatus M. girerdii. Numerous other mycoplasmas have been isolated from various animals and have been shown to be of economic importance be- cause of the pneumonia, arthritis, keratoconjunctivitis, and mastitis they cause among livestock and poultry. This is apart from the plant diseases recognized as being due to mycoplasmas in recent years. In Fig. 8.6.46.1  Electron micrograph of M. pulmonis (murine origin), illustrating that the organism does not have a bacterial cell wall but has a trilaminar unit membrane (arrow); also note what appears to be a terminal structure (T). Magnification ×66 000.

8.6.46  Mycoplasmas 1297 humans, as in other animal species, mycoplasmas cause respiratory and genital tract diseases and escape from these sites to cause disease elsewhere (e.g. in joints or wounds). Mycoplasmas are also notorious for infecting cell cultures, par- ticularly continuous cell lines. Various mycoplasma species are responsible (e.g. M.  hyorhinis of porcine origin and M.  orale or M. fermentans of human origin). The contamination may affect al- most any property under investigation in a totally unpredictable way and may lead to misinterpretation of any result based on studies in cultured cells. Occurrence of mycoplasmas in humans Fourteen species of mycoplasmas as well as one with candidatus status, and two ureaplasma species, have been isolated from humans and are constituents of the normal flora or behave as pathogens (Tables 8.6.46.1, 8.6.46.2); in addition, several case reports have de- scribed infection with species of animal origin. Most mycoplasmas of human origin are found in the oropharynx. There is little infor- mation about the distribution or significance of M. amphoriforme, M. penetrans, M. pirum, and M. spermatophilum. Respiratory infections Relationship between mycoplasmas and
respiratory disease M. pneumoniae is the most important mycoplasma found in the re- spiratory tract (see next); most of the others behave as commensals (Table 8.6.46.1). M. fermentans has been detected in the throat more often since the use of polymerase chain reaction (PCR) (see next) and has been recovered from adults with an acute influenza-​like illness, with rare development of a fatal respiratory distress syn- drome. M. hominis is occasionally recovered from the respiratory tract. However, although it caused a mild pharyngitis in adult male volunteers inoculated orally, it is not known to do this naturally in children or adults. M. amphoriforme is a newly described species isolated from patients with chronic bronchitis, primarily those with B-​cell deficiencies, and is phylogenetically related to pathogenic species such as M. pneumoniae and M. genitalium. The clinical im- portance of M. amphoriforme in the general population is unknown. M. faucium has been found in the throat of 25% of adults by PCR, more commonly in older individuals, but is not believed to be a cause of respiratory tract disease. History of M. pneumoniae In the late 1930s, non​bacterial pneumonias or primary atypical pneumonia were distinguished from typical lobar pneumonia. Patients from whom the ‘Eaton agent’ had been isolated in embryon- ated eggs often developed cold agglutinins. This agent was presumed to be a virus until it was found to be sensitive to chlortetracycline and gold salts. Its mycoplasmal nature was established by cultivation on a cell-​free agar medium. The agent, M. pneumoniae, was estab- lished as a respiratory pathogen by studies based on isolation, ser- ology, volunteer inoculation, and vaccine protection. Clinical features of M. pneumoniae disease M. pneumoniae produces a range of effects from inapparent infec- tion and mild afebrile upper respiratory tract disease to severe pneu- monia. The most typical clinical syndrome is tracheobronchitis, often accompanied by upper respiratory tract manifestations such as acute pharyngitis. A clinical diagnosis of M. pneumoniae pneu- monia is impossible as it shares features of other non​bacterial pneumonias. Malaise and headache often precede chest symp- toms by 1 to 5 days, and pneumonia is seen radiographically be- fore physical signs such as rales are detectable. Usually, only one of the lower lobes is involved and the radiograph shows patchy opacities. Pneumonia develops in about 10% of those infected and about 20% of the pneumonia cases have bilateral opacities. Pleurisy and pleural effusions are unusual. The course of the disease is variable but often protracted. Symptoms may persist for several weeks and may relapse. The organisms can persist in respiratory secretions despite antibiotic therapy, particularly in patients with (a) (b) Fig. 8.6.46.2  (a) Fried-​egg-​like mycoplasma colonies (one ill-​ formed) and a larger bacterial colony. Transmission light microscopy, magnification ×43. (b) Section through mycoplasma colonies illustrating growth in the depth of the agar. Magnification ×78.

section 8  Infectious diseases 1298 hypogammaglobulinaemia where excretion may continue for months or years rather than weeks. Mortality is very low although a few very severe infections have been reported, usually in patients with immunodeficiency or sickle cell anaemia. In children, illness may be prolonged with paroxysmal cough followed by vomiting, simulating whooping cough. M. pneumoniae has been implicated in bronchial asthma, but this is controversial (see next). Extrapulmonary manifestations of M. pneumoniae infection Disease caused by M.  pneumoniae is usually limited to the re- spiratory tract, but various extrapulmonary complications may occur during the course of the respiratory illness or subsequently (Table 8.6.46.3). Haemolytic crisis is precipitated by cold agglutinins (anti-​I antibodies). This mycoplasma apparently alters the I antigen on erythrocytes sufficiently to stimulate an autoimmune response. A similar mechanism may be responsible for neurological and other complications. Invasion of the central nervous system cannot be discounted as M. pneumoniae has been isolated from cerebrospinal fluid in rare cases. Epidemiology of M. pneumoniae infections Pathology is age dependent. About one-​quarter of infections in chil- dren aged 5 to 15 years result in pneumonia, whereas only about 7% of infections in young adults do so. Pneumonia is less frequent thereafter, but is more severe the older the patient. M. pneumoniae causes inapparent or mild upper respiratory tract symptoms more often than severe disease. It is responsible for a minority of all upper tract infections, usually attributable to viruses or streptococci. M. pneumoniae causes many lower respiratory tract infections (e.g. about 15–​20% of all pneumonias in the United States of America). In populations such as military recruits it has been re- sponsible for up to 40% of acute pulmonary illness. M. pneumoniae infections occur globally. Infection is endemic in most areas and throughout the year, with a predilection for late summer and early autumn. Epidemic peaks have been observed about every 4 to 7 years. The incubation period ranges from 2 to 3 weeks. Spread from person to person occurs slowly, usually where there is continual or repeated close contact, as within a family. Immunopathological factors in the development of M. pneumoniae pneumonia The crucial step of adherence of M. pneumoniae organisms to re- spiratory mucosal epithelial cells, cytadsorption (Fig. 8.6.46.3), is mediated by the P1-​protein and other specialized adhesins on the mycoplasmal surface. This is often followed by cellular invasion. In animals, there is peribronchiolar and perivascular pulmonary in- filtration mostly by T lymphocytes (Fig. 8.6.46.4). The pneumonia caused by M. pneumoniae is largely an immunopathological process since immunosuppression prevents pneumonia or diminishes its se- verity. A mycoplasmal polysaccharide–​protein fraction is involved in the cell-​mediated immune response, whereas the main antigenic determinant in complement fixation and other serological reactions is a glycolipid. After the initial lymphocyte response, polymorpho- nuclear leucocytes and macrophages appear in the bronchiolar Table 8.6.46.1  Biological features, occurrence, and disease association of mycoplasmas of human origina Mycoplasma Metabolism of: Haemadsorption Frequency of detection in the: Cause of disease Respiratory tract Genitourinary tract Rectum Eye Blood M. amphoriforme Glucose Yes Rareb –​c –​c –​c –​c ?Yes M. buccale Arginine No Rare –​ –​ –​ –​ No M. faucium Arginine Yesd Common –​ –​ –​ –​ No M. fermentans Glucose, arginine No Common Rare –​ –​ Rare ?Yes M. genitalium Glucose Yes Very rare Common Common Rare ? Yes M. hominis Arginine No Rare Common Common Rare Very rare Yes M. lipophilum Arginine No Rare –​ –​ –​ –​ No M. orale Arginine Yesd Common –​ –​ –​ –​ No M. penetrans Glucose, arginine Yes –​ Rare Very rare –​ ? ? M. pirum Glucose, arginine No ? –​ Rare ? Very rare ? M. pneumoniae Glucose Yes Raree Very rare –​ –​ –​ Yes M. primatum Arginine No –​ Rare –​ –​ –​ No M. salivarium Arginine No Common Rare –​ –​ –​ Nof M. spermatophilum Arginine No –​ ?Rare ? ? ? Ureaplasma parvumg Urea Serotype 3 only Rare Common Common Rare Very rare ?Yes Ureaplasma urealyticumg Urea No Rare Common Common Rare Very rare Yes a Occasional isolations of mycoplasma species of non​human origin not included. b Except in immunocompromised patients. c No reports of detection. d With chick erythrocytes only. e Except in disease outbreaks. f Except in hypogammaglobulinaemia. g Ureaplasmas have been divided into two species formerly described as biovars.

8.6.46  Mycoplasmas 1299 exudate. The slow evolution of the primary disease contrasts with an accelerated and often more intense host response to reinfection. Children between 2 and 5 years show serological evidence of infec- tion. The pneumonia that occurs in older people is considered to be an immunological overresponse to reinfection, with lung infiltra- tion by previously sensitized lymphocytes. Chronic respiratory disease Animal mycoplasmas are frequently associated with chronic illnesses, and so the possible role of mycoplasmas has been con- sidered in human chronic respiratory disease. M. pneumoniae often persists in the respiratory tract long after clinical recovery and occasionally the disease is protracted, but there is no evidence that M. pneumoniae is a primary cause of chronic bronchitis, or that it maintains chronic disease other than by possibly causing some acute exacerbations. M. salivarium, M. orale, and perhaps other mycoplasmas present in the oropharynx of healthy people spread to the lower respira- tory tract of people with chronic bronchitis. There is no hard evi- dence that these mycoplasmas cause acute exacerbations, but they may perpetuate an episode. Specific antibody responses follow such exacerbations more frequently than at other times, suggesting that mycoplasmas multiply and contribute to the tissue damage that is primarily due to viruses and bacteria. Table 8.6.46.2  Summary of the relationship between mycoplasmas and disease. Evidence for an association (A) between the indicated mycoplasmaa and disease, and for the causation (C) of disease M. pneumoniae M. fermentans Ureaplasmas M. hominis M. genitalium A C A C A C A C A C Upper respiratory tract disease +++ +++ –​ –​ + –​ –​ Bronchitis +++ +++ –​ –​ –​ –​ Pneumonia ++++ ++++ ++ + –​ –​ –​ Asthma ++ + NE NA –​ –​ Extrapulmonary sequelae of M. pneumoniae infection (see text) +++ +++ NA NA NA NA Non​gonococcal urethritis NA –​ +++ +++ + -​ ++++ ++++ Chronic prostatitis NA NE ++ + –​ + –​ Epididymitis NA NE ++ ++ –​ +++ ++ Bartholinitis NA NE –​ + –​ NE Bacterial vaginosis NA NE ++ –​ ++++ + –​ Cervicitis NA –​ –​ –​ +++ +++ Pelvic inflammatory disease NA NE + + ++ ++ +++ +++ Infertility NA NE ++ –​ –​ ++b ++ Urinary calculi NA –​ ++ + –​ NE Pyelonephritis NA NE + –​ +++ + NE Chorioamnionitis NA + -​ +++ ++ +++ ++ NE Preterm labour/​birth NA NE +++ ++ ++ ++ + + Spontaneous abortion NA + –​ +++ + ++ + + –​ Postabortal fever NA NE ++ + ++++ +++ NE Postpartum fever NA NE ++ + ++++ +++ NE Postpartum arthritis NA NE –​ +++ +++ NE Low birthweight NA NE ++ + ++ -​ NE Neonatal chronic lung disease NA NE +++ ++ ++ + NE Rheumatoid arthritis + –​ ++ –​ + –​ –​ + –​ Juvenile chronic arthritis ++ + NE –​ –​ NE Sexually acquired reactive arthritis/​Reiter’s
disease –​ –​ ++ ++ –​ ++ ++ Arthritis in hypogammaglobulinaemia ++++ ++++ NE ++++ ++++ ++++ +++ NE Wound infections NA NE ++ + +++ +++ NE HIV infection –​ ++ –​ –​ –​ –​ NA, not appropriate to examine; NE, not examined; ++++, strong; +++, good; ++, moderate; +, weak; –​, none. a See text for M. amphoriforme and other mycoplasmas. b Tubal factor infertility.

section 8  Infectious diseases 1300 M. amphoriforme was recovered from the respiratory tract of pa- tients with chronic bronchitis, most of whom were B-​cell deficient. Recovery may depend on the eradication of this organism but its role in the general population is unknown. The role of M.  pneumoniae in asthma is controversial. Acute M. pneumoniae infection is associated with wheezing and the or- ganism has been found, mainly by PCR, more frequently in subjects with asthma than in those without. However, no causal relationship has so far been established. Genitourinary and related infections Non​gonococcal urethritis and its complications M. genitalium, (Table 8.6.46.1, Fig. 8.6.46.5a), is strongly associ- ated with acute non​gonococcal urethritis (Tables 8.6.46.2; 8.6.46.4). It has been detected almost independently of Chlamydia tracho­ matis by PCR in about 25% of cases compared with significantly fewer (about 6%) healthy controls. It also causes urethritis experi- mentally in male chimpanzees and adheres to and enters epithelial cells (Fig. 8.6.46.5b). Intracellular M. genitalium may be partially protected from antimicrobials resulting in persistent or recurrent nongonococcal urethritis. Most recently, M.  genitalium has been associated with balanoposthitis in men with acute non​gonococcal urethritis. Table 8.6.46.3  Extrapulmonary manifestations of M. pneumoniae infections System Manifestations Estimated frequency Cardiovascular Myocarditis, pericarditis <5% Dermatological Urticaria, erythema multiforme, Stevens–​Johnson syndrome, other rashes Some skin involvement in about 25% Gastrointestinal Anorexia, nausea, vomiting, and transient diarrhoea 15–​45% Hepatitis ? Pancreatitis ? Genitourinary Acute glomerulonephritis Insignificant Haematological Cold agglutinin production About 50% Haemolytic anaemia ? Thrombocytopenia ? Intravascular coagulation

50 reported cases Musculoskeletal Myalgia, arthralgia, arthritis 15–​45% Neurological Meningitis, meningoencephalitis, ascending paralysis, transient myelitis, cranial nerve palsy, poliomyelitis-​like illness <5% in a few studies based on serology Fig. 8.6.46.3  Electron micrograph of ciliated epithelial cells in the tracheal mucosa of a hamster infected with M. pneumoniae. Note cilia (c) and individual organisms (m), some with a specialized terminal structure oriented towards the membrane of the host cell (arrows). Magnification ×9880. Fig. 8.6.46.4  Pneumonia 2 weeks after intranasal inoculation of a hamster with M. pneumoniae. Note peribronchiolar and perivascular infiltration of mononuclear cells, predominantly lymphocytes. Haematoxylin and eosin, magnification ×98.

8.6.46  Mycoplasmas 1301 Although M. hominis has been isolated from about 20% of pa- tients with acute non​gonococcal urethritis, it has not been impli- cated as a cause. The role of ureaplasmas in non​gonococcal urethritis has been contentious for many years. The results of most qualitative studies have failed to demonstrate a significant difference be- tween the prevalence of ureaplasmas in men with or without acute non​gonococcal urethritis, but there are some quantitative data indicating higher titres of organisms in men with disease. There are two species of human ureaplasmas, U. urealyticum and U. par­ vum. PCR assays of clinical specimens have shown an association between U. urealyticum and acute non​gonococcal urethritis, par- ticularly when the organism was present in high titres, whereas this seems not the case for U. parvum. Intraurethral inoculation of first-​passage ureaplasma strains produced a mild urethritis and an antibody response in male chimpanzees and the disease responded to tetracycline therapy. Four investigators who inoculated them- selves intraurethrally developed urethritis. In one study, two re- ceived cloned U. urealyticum, serotype 5, isolated from a patient with acute non​gonococcal urethritis in whom no other potentially pathogenic microorganisms could be detected, although M. geni­ talium was not sought at that time. Both developed symptoms and signs of urethritis which responded to treatment with minocycline. Another volunteer experiment suggested that ureaplasmas may cause disease the first few times they gain access to the urethra but later insults result in colonization without disease, accounting per- haps for their frequent occurrence in the urethras of healthy men. At present, it is not part of standard patient management to search for ureaplasmas in men with non​gonococcal urethritis. Epididymitis and chronic prostatitis Ureaplasmas may be a rare cause of epididymitis since they have been recovered from the urethra and epididymal aspirate fluid of a patient with acute non​chlamydial, non​gonococcal epididymitis, with a specific antibody response. M. genitalium has not been sought in aspirates, but it has been found in the urethra without other known pathogens (Table 8.6.46.4). Information linking prostatic infection with acute ureaplasmal urethral infection is scanty, although ureaplasmas have been iso- lated more frequently and in greater numbers from patients with acute urethroprostatitis than from controls. Most of those with more than 103 organisms in expressed prostatic fluid responded to tetra- cycline therapy. In contrast, ureaplasmas have not been found, and M. genitalium only rarely found, in prostatic biopsy specimens from patients with chronic abacterial prostatitis. M. hominis is not associ- ated with prostatitis. Pelvic inflammatory disease (Chapter 9.8) Microorganisms in the vagina and lower cervix may ascend to and cause inflammation of the fallopian tubes and adjacent pelvic structures (Tables 8.6.46.2; 8.6.46.4). M.  hominis has been isolated from inflamed fallopian tubes, tubo-​ovarian ab- scesses, and pelvic abscesses or fluid. Laparoscopy samples have yielded M. hominis from the tubes of about 10% of women with salpingitis but not from those of healthy women. However, they may be present in mixed infections with bacterial vaginosis-​ associated bacteria as M. hominis is strongly associated with bac- terial vaginosis (Chapter 9.4). M. hominis antibody was found in approximately one-​half of salpingitis patients, but in only 10% of healthy women. Although ureaplasmas have been isolated directly from the fallopian tubes of a very small proportion of patients with acute salpingitis, from pelvic fluid, and from a tubo-​ovarian abscess, it seems that they are of little importance in acute pelvic inflamma- tory disease. Nucleic acid amplification tests (NAATs) have established that M. genitalium is involved in at least some cases of pelvic inflamma- tory disease. It is a cause of cervicitis and its presence in the cervix or upper genital tract is associated significantly with histological endometritis. It has rarely been detected in tubes but in one study of women with pelvic inflammatory disease, an antibody response was detected to M. genitalium but not to M. hominis or C. trachomatis in one-​third of the patients. Other studies have not shown this associ- ation but M. genitalium has been related serologically to tubal factor infertility. Fallopian-​tube organ culture studies have shown that gono- cocci destroy the epithelium, M. genitalium causes some damage, M. hominis organisms multiply but only produce swelling of some cilia, and ureaplasmas cause no damage. This differential effect may be a true reflection of the pathogenic potential of these microorgan- isms in vivo but, as the immune system is not operational, failure to demonstrate damage does not confirm avirulence. Inoculation of M. hominis or M. genitalium into primates caused a self-​limited acute salpingitis and parametritis with an antibody response, whereas ureaplasmas had no effect. (a) (b) Fig. 8.6.46.5  (a) Electron micrograph of M. genitalium, negatively stained to show flask-​shaped appearance and terminal specialized structure (arrow). Magnification ×90 000. (b) Electron micrograph of M. genitalium adhering to a Vero cell by the terminal structure. Magnification ×60 000. From Tully JG, et al. (1983) Mycoplasma genitalium, a new species from the human urogenital tract. Int J Syst Bacteriol, 33, 387, with permission.

section 8  Infectious diseases 1302 Effects of mycoplasmas on pregnancy Preterm birth The involvement of genital mycoplasmas is debated but ureaplasma infection of the amniotic fluid is associated with preterm labour. M. hominis probably plays a part through its involvement with bac- terial vaginosis, a known cause of preterm labour. M. hominis and ureaplasmas are unlikely to cause low birthweight in otherwise normal full-​term infants. The role of M. genitalium is controversial; it has been associated with preterm birth in some but not all studies. Postabortal and postpartum fever M. hominis has been isolated from the blood, with an antibody re- sponse, in up to 10% of women with fever after abortion, but not from those without fever. However, a pure culture of M. hominis in blood is needed before it can be accepted as a cause of fever. The role of ureaplasmas is unclear. Patients with postabortal or postpartum fever of mycoplasmal origin usually recover without antibiotic treatment. Neonatal infections Whether transmitted in utero or during birth, ureaplasmas may be isolated from the throats and tracheal aspirates of some neonates. Ureaplasma-​infected infants of very low birthweight (<1000 g) have died or have developed chronic lung disease twice as often as uninfected infants of similar birth weight or those of over 1000 g. However, the pathogenicity of ureaplasmas is uncertain since erythromycin treatment has failed to prevent disease in two trials. M. hominis has very rarely been implicated in pneumonia soon after birth but the other bacteria present could be responsible. Table 8.6.46.4  Causal relationship of M. genitalium, M. hominis, and ureaplasmas with human genitourinary, reproductive,
and perinatal disease Disease Evidence suggesting a causal relationship of: Comments on the relationship M. genitalium M. hominis Ureaplasmas Non​gonococcal urethritis Strong None Good U. urealyticum in high titres is associated with non​gonococcal urethritis but U. parvum is not. Chronic prostatitis None None Weak None of the microorganisms appears to be a cause Epididymitis Some None Some Ureaplasmas isolated from the epididymis in one case of acute disease Urinary calculi ? None Weak Experimentally, ureaplasmas cause bladder calculi in male rats but so far little evidence for a cause of natural human disease Pyelonephritis ? Weak None M. hominis possibly causes some cases of acute pyelonephritis and exacerbations Reiter’s disease/​sexually acquired reactive arthritis Some None Some M. genitalium detected in joint of one patient; ureaplasmas are related on the basis of lymphocytic response to specific antigen Bartholinitis ? Very weak None Doubtful whether M. hominis is involved Bacterial vaginosis None Weak None M. hominis and to a lesser extent ureaplasmas are associated with bacterial vaginosis, but a causal relationship is unproved Cervicitis Good None None M. genitalium is associated with non​gonococcal, non​chlamydial cervicitis Pelvic inflammatory disease Good Some Weak M. genitalium is associated serologically and has been detected in the upper genital tract of patients with endometritis and salpingitis; M. hominis probably causes a small proportion of cases, but very doubtful that ureaplasmas do Postabortal fever ? Good Weak M. hominis, and to a much lesser extent ureaplasmas, are responsible for some cases, but the proportion is unknown Postpartum fever ? Good Weak M. hominis, and to a much lesser extent ureaplasmas, are responsible for some cases, but the proportion is unknown Infertility Some None None M. genitalium is associated serologically to tubal factor infertility; ureaplasmas are associated with reduced sperm motility, but a causal relationship is unproved Preterm birth Weak Some Some/​good M. genitalium associated in a few studies, but not in others. Considerable evidence for the involvement of ureaplasmas, less so for M. hominis; both possibly as part of bacterial vaginosis Spontaneous abortion and stillbirth ? Weak Weak Maternal and fetal infections are associated with spontaneous abortion, but a causal relationship is unproved Chorioamnionitis ? Some Some An association exists with ureaplasmas, but a causal relationship is unproved Low birth weight ? None Weak An association exists with ureaplasmas in some studies, but a causal relationship is unproved Neonatal meningitis ? Some Some A rare event Neonatal lung disease ? Weak Some M. hominis has been involved in pneumonia soon after birth; ureaplasmas possibly involved in premature infants weighing less than 1000 g

8.6.46  Mycoplasmas 1303 Mycoplasmal infection should be considered in cases of neo- natal disease of the central nervous system in which the results of bacteriological staining and culture are negative. M. hominis or ureaplasmas have been found in the cerebrospinal fluid of neo- nates with meningitis or brain abscess. Joint infections Arthritis Mycoplasmas cause several animal arthritides and PCR testing showed M. fermentans and ureaplasma DNA in more than 20% of patients with rheumatoid arthritis and other chronic inflam- matory disorders, in contrast to those with non​inflammatory dis- orders. The significance of these findings is unknown. M.  hominis has been isolated from septic joints, usually hip, that have developed in mothers after childbirth. The arthritis re- sponds to tetracycline therapy and the diagnosis should be con- sidered in a post-​partum arthritis which is unaffected by β-​lactam antibiotics. Arthritis may occur soon after or concomitant with non​ gonococcal urethritis (sexually acquired reactive arthritis) or the arthritis may be associated with conjunctivitis and ureth- ritis. M.  genitalium causes uncomplicated non​gonococcal ur- ethritis and ureaplasmas do so to a lesser extent, but previous antimicrobial treatment usually prevents adequate investigation of patients with arthritis. M. genitalium has been detected in the synovial fluid of a patient with sexually acquired reactive arth- ritis and clinical experience has shown that this condition is not uncommon after M.  genitalium-​positive non​gonococcal ureth- ritis, but no causal link has been established. The latter is also true in the case of some patients whose synovial lymphocytes have been shown to proliferate in vitro in response to ureaplasmal antigens. Wound infections M. hominis has occasionally been linked to fever in patients with burns, trauma, or wound infections. It is most common in fever after surgery on the urogenital tract, but also in mediastinitis; ureaplasmas are also likely to be present, but neither these nor M.  hominis will be found unless specifically sought. Kidney transplant patients occasionally develop mixed infections with ureaplasmas and M.  hominis, which in severe cases may create fistulas. A rare wound infection is ‘seal finger’ or ‘blubber finger’, which is well known in Arctic regions where the handling of sea mammals is part of daily living. A few days after a seal bite, oedema of the affected finger develops with swelling of the interphalangeal joint adjacent to the lesion. It is extremely painful, suppuration can occur, extensive surgery may be needed, and re- sidual dysfunction is possible. The infection usually responds rapidly to tetracyclines but macrolides are inefficient. An un- named, rapidly growing new mycoplasma species can be re- covered from the lesions. Mycoplasmas in immunodeficiency states Urethritis and arthritis in patients with hypogammaglobulinaemia Prolonged urethrocystitis with persistent ureaplasmal infection has been seen in a few patients with hypogammaglobulinaemia. In addition, arthritis of mycoplasmal aetiology (Fig. 8.6.46.6a, b) should be considered in patients with this immune deficiency who develop an abacterial septic arthritis. M. pneumoniae, M. homi­ nis, M.  salivarium, and, in particular, ureaplasmas have been isolated from synovial fluids of at least 40% of these patients. In addition, vigilance should be kept for infection by mycoplasmas of non​human origin. The arthritis usually responds to tetracyc- lines or other antimicrobials to which the organisms are sensitive. Intravenous and combination therapy should be considered to avoid antimicrobial resistance developing due to suboptimal drug concentrations at the infection site. Administration of specific antiserum against the mycoplasma in question may be helpful in a few patients when antimicrobial therapy fails. Mycoplasmas in HIV-​infected patients Although M. fermentans was distributed widely in tissues taken at autopsy from some patients with AIDS, no association has been found with the stage of the disease, CD4 count, plasma HIV-​1 viral load, or rate of progression of the illness. M. penetrans, which avidly invades eukaryotic cells, was isolated from urine sediments of a few HIV-​1-​positive men who have sex with men. While it is possible that M. fermentans, M. penetrans, or other mycoplasmas might proliferate in this immunodeficiency state, there is no convincing evidence that they are important for the development of AIDS. (a) (b) Fig. 8.6.46.6  (a) Damage to the knee joint of a hypogammaglobulinaemic patient caused by U. urealyticum infection. (b) Sinus connected with the shoulder joint of a patient with hypogammaglobulinaemia; ureaplasmas were isolated repeatedly from the sinus exudate. Courtesy of A. D. B. Webster.

section 8  Infectious diseases 1304 Conditions of rare or equivocal
mycoplasmal aetiology Bacterial vaginosis (Chapter 9.4) M. hominis organisms may well have a role in the pathogenesis of bacterial vaginosis in which they occur in very large numbers, but proof is impossible due to the variety of other bacteria pre- sent in profusion. Ureaplasmas are less likely to be pathogenic and M. genitalium does not seem to be involved at all. Pyelonephritis Over 30 years ago M. hominis was isolated, sometimes in pure cul- ture, from the upper urinary tract of almost 10% of patients with acute pyelonephritis, occasionally accompanied by an antibody re- sponse, but not from patients with non​infectious urinary tract dis- eases. However, there has never been confirmation that M. hominis is a cause of acute pyelonephritis or acute exacerbations of chronic pyelonephritis. Urinary calculi Animal model and human isolation studies have suggested that ureaplasmas, which have a urease, could be involved in the devel- opment of urinary calculi, but proof is lacking. Other conditions There is no confirmation that ureaplasmas are a cause of male or female infertility, but M. genitalium may be responsible for tubal factor infertility in some instances (see earlier). There is no credible evidence that mycoplasmas are related to fibromyalgia, chronic fatigue syndrome, or the Gulf War syndrome. Laboratory diagnosis of mycoplasmal infections M. pneumoniae infection The diagnosis is made by molecular methods (NAATs), and/​ or serology and in special cases by culture. The complex culture media for M. pneumoniae isolation contain glucose, selective anti- biotics, and a pH indicator (phenol red). The fluid medium, in- oculated with the clinical specimen, is incubated at 37° C and a colour change (red to yellow) signals the fermentation of glucose (Table 8.6.46.1) with production of acid, due to multiplication of the organisms. This preliminary identification may be confirmed by subculturing to agar medium and demonstrating inhibition of colony development by specific antiserum (Fig. 8.6.46.7) or by immunofluorescence of colonies with an M. pneumoniae-​specific antibody. Culture may take as long as 5 weeks, and consequently it is of limited value in clinical diagnosis. Rapid detection of M. pneumoniae by a NAAT has become routine in most settings. Serological testing by commercially available enzyme immuno- assays specific for IgM and/​or IgG is used more routinely. IgM detection is not reliable in reinfection, which is most often the case in adults and as IgG reactivity remains for years, an increase in IgG titre is often required. The complement-​fixation test is still undertaken in some laboratories. Recent infection is indi- cated by a fourfold or greater rise in antibody, but this occurs in only about 80% of cases. A high titre (1:128 or greater) in a single serum is suggestive but not proof of infection. The complement-​ fixation test does not distinguish between M.  pneumoniae and M.  genitalium. Cold agglutinins, detected by agglutination of O Rh-​negative erythrocytes at 4° C, also correlate with specific IgM and are suggestive of a recent M. pneumoniae infection, but the test is rarely used as it is not specific. Genitourinary and other infections Swabs from the urethra or cervix/​vagina have a slightly higher sensitivity for mycoplasmal isolation than urine specimens. Ureaplasmas and M.  hominis usually show evidence of growth in culture media within 1 to 5 days. Primary isolation of M. geni­ talium in this way is difficult and can take 50 days or more, so that a NAAT is required for detection. Unfortunately, up to now, com- mercial tests have not met Food and Drug Administration (FDA) approval but CE-marked assays have been introduced in Europe, making testing available in many settings. Several of these assays combine M. genitalium detection with detection of macrolide re- sistance mediating mutations in the 23S rRNA gene. This allows treatment to be directed to the most appropriate antibiotics, such as recommended in the European guideline on M. genitalium in- fections. PCR assays have also been used to identify M. fermentans and U. urealyticum/​U. parvum. In particular, quantitative assays for U. urealyticum show some promise for clinically relevant diag- nosis in male acute non​gonococcal urethritis. M.  hominis cultured on agar medium produces colonies of ca 200 to 300 µm diameter, whereas ureaplasma colonies are tiny (15–​60 µm) (Fig. 8.6.46.8a) but can be seen more easily on medium containing manganous sulphate (Fig. 8.6.46.8b). M. hominis may grow on ordinary blood agar where it produces non​haemolytic pinpoint colonies after extended incubation. Ureaplasma colonies Fig. 8.6.46.7  Mycoplasma identification by agar growth inhibition. Colony development inhibited around a filter-​paper disc impregnated with specific antiserum. Note also antibody–​antigen precipitation at edge of inhibition zone.

8.6.46  Mycoplasmas 1305 are too small to be detected on blood agar, but occasionally a scrape from the agar surface will yield ureaplasmas when inocu- lated into ureaplasma medium. In a few research laboratories only, serological tests have been used to detect antibodies to M. hominis, M. genitalium, and the ureaplasmas. Treatment M. pneumoniae infections M. pneumoniae is sensitive to the bacteriostatic tetracyclines and macrolides. The newer macrolides, such as clarithromycin and azithromycin, are very active in vitro, but their clinical effect is not documented extensively in randomized clinical trials. The newer quinolones, such as moxifloxacin, are also highly active in vitro. They should not be used as first-​line therapy but, as they are bacteri- cidal, they may have a role in immunosuppressed patients. Recently, a rapid increase in high-​level macrolide resistance in M. pneumoniae has been reported among infected patients in Asia, but in Europe the prevalence of resistance is still below 10% in most settings. In a controlled trial, tetracycline significantly reduced the duration of fever, pulmonary infiltration, and other signs and symptoms of M. pneumoniae infection. In practice, antimicrobials may be less ef- fective because correct treatment is initiated at a late stage. Nevertheless, treatment with an antimicrobial is worthwhile. Due to the lower fre- quency of gastrointestinal side effects, newer macrolides such as azithromycin and clarithromycin are preferred when macrolides are indicated, as in children. Successful treatment of disease with bacterio- static drugs does not always correlate with eradication of organisms from the respiratory tract. Relapse can be avoided by giving antibiotics for at least 10 days. It is uncertain whether early treatment prevents complications but it should start as soon as possible, even if there is only clinical evidence and a suggestive single antibody titre. Corticosteroids in conjunction with antimicrobials appear to have been helpful in pa- tients with severe pneumonia and erythema multiforme. Genitourinary and other infections Antimicrobial susceptibility of the mycoplasma species found most commonly in the urogenital tract is presented in Table 8.6.46.5 as a combination of in vitro susceptibility data and clinical experience. Treatment must take into account the fact that several different micro- organisms may be involved and that a precise microbiological diag- nosis is not available. Patients with non​gonococcal urethritis should ideally receive an antibiotic with activity against C. trachomatis, urea- plasmas, and M. genitalium. Azithromycin is being used increasingly for chlamydial infections and is also active against a wide range of mycoplasmas, including M. genitalium and, to a lesser extent, urea- plasmas. However, high-​level macrolide resistance is common with prevalence figures of 15% where doxycycline is used for first-​line treatment of non​gonococcal urethritis and cervicitis, to over 40% in most settings where azithromycin 1 g is used as first-​line therapy. For the treatment of symptomatic non​gonococcal urethritis, recent European Guidelines on the Management of non​gonococcal ureth- ritis recommend doxycycline 100 mg bd for 7 days. If this fails, infec- tions should be treated with azithromycin 500 mg on day 1 followed by 250 mg daily on days 2–​5. If macrolide resistance is present or de- velops, moxifloxacin 400 mg daily for 7–​10 days is the only treatment option in many European countries. Strains with dual resistance to azithromycin and moxifloxacin have been reported with increasing frequency in the Asian-​Pacific region and in high-risk populations in the USA with a prevalence as high as 30% reported in one study. Such infections require treatment with alternative antibiotics such as pristinamycin which is difficult to source outside of France and which only eradicates 75% of the infections. A broad-​spectrum antibiotic should also be included in the treatment of pelvic inflammatory disease to cover C.  trachoma­ tis, M.  hominis and other bacterial vaginosis-​associated bacteria and M. genitalium. Since 20% or more of M. hominis strains are resistant to tetracyclines, other antibiotics such as clindamycin or fluoroquinolones may be needed. Prevention of infection M.  pneumoniae infection or disease may occur despite high titres of serum mycoplasmacidal antibody. The correlation be- tween the presence of IgA in respiratory secretions and resist- ance to M. pneumoniae disease endorses the importance of local (a) (b) Fig. 8.6.46.8  (a) A ureaplasma colony (15 µm diameter) (arrow) adjacent to colonies of M. hominis (130 µm diameter) grown from urethral exudate. Oblique light, magnification ×68. (b) Dark ureaplasma colonies with colonies of M. hominis on agar containing manganous sulphate. Magnification ×136.

section 8  Infectious diseases 1306 immune factors in resistance. IgA could prevent attachment of organisms to respiratory epithelial cells. Protective immunity also depends on the severity of infection. Thus, in one study, patients with non​pneumonic illness were susceptible to an epi- demic occurring 5 years later, whereas those with M. pneumo­ niae pneumonia were protected until the following epidemic 10 years later. No effective vaccine has been developed for human use. Vaccination against M. pneumoniae has been attempted. Formalin-​ inactivated vaccines prevented mycoplasmal pneumonia in only one-​ to two-​thirds of subjects, perhaps because they failed to stimulate cell-​mediated immunity and/​or local antibody. Live attenuated vaccines, containing temperature-​sensitive mutants of M.  pneumoniae, have not been considered safe for general human use. Recombinant DNA vaccines involving P1 and other proteins, or a recombinant vaccine developed by cloning part of the M. pneumoniae P1 gene into an adenovirus vector, may offer greater success in the future. No vaccine is available for M. genitalium or the other urogenital mycoplasmas, but M. genitalium infection as well as most other bacterial STIs is preventable by correct use of condoms. FURTHER READING Frølund M, et  al. (2016). Urethritis-​associated pathogens in urine from men with non​gonococcal urethritis: a case-​control study. Acta Derm Venereol, 96, 689–​94. Haggerty CL (2008). Evidence for a role of Mycoplasma genitalium in pelvic inflammatory disease. Curr Opin Infect Dis, 21, 65–​9. Herrmann R, Ruppert T (2006). Proteome of Mycoplasma pneumo­ niae. Methods Biochem Anal, 49, 39–​56. Horner P, Blee K, Adams E (2014). Time to manage Mycoplasma gen­ italium as an STI: but not with azithromycin 1g! Curr Opin Infect Dis, 27, 68–​74. Jensen JS (2006). Mycoplasma genitalium infections: diagnosis, clinical aspects, and pathogenesis. Dan Med Bull, 53, 1–​27. Jensen, JS, Bradshaw C (2015). Management of Mycoplasma genitalium infections—​can we hit a moving target? BMC Infect Dis, 15, 343. Lis R, Rowhani-​Rahbar A, Manhart LE (2015). Mycoplasma genitalium infection and female reproductive disease:  a meta-​analysis. Clin Infect Dis, 61, 418–​26. McGarrity GJ, Kotani H, Butler GH (1992). Mycoplasmas in tissue culture cells. In:  Maniloff J (ed) Mycoplasmas:  molecular biology and pathogenesis, pp. 445–​54. American Society for Microbiology, Washington, DC. Razin S, Yogev D, Naot Y (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev, 62, 1094–​156. Sutherland ER, Martin RJ (2007). Asthma and atypical bacterial infec- tion. Chest, 132, 1962–​6. Taylor-​Robinson D (1996). Mycoplasmas and their role in human
respiratory tract disease. In:  Myint S, Taylor-​Robinson D (eds) Viral and other infections of the human respiratory tract, pp. 319–​39. Chapman & Hall, London. Taylor-​Robinson D (2007). The role of mycoplasmas in pregnancy outcome. Best Pract Res Clin Obstet Gynaecol, 21, 425–​38. Taylor-​Robinson D, Gilroy CB, Jensen JS (2000). The biology of Mycoplasma genitalium. Venereology, 13, 119–​27. Taylor-​Robinson D, Jensen JS (2011). Mycoplasma genitalium:
from chrysalis to multicolored butterfly. Clin Microbiol Rev, 24, 498–​514. Taylor-​Robinson D, Keat A (2001). How can a causal role for small bacteria in chronic inflammatory arthritis be established or refuted? Ann Rheum Dis, 60, 177–​84. Waites KB, Taylor-​Robinson D (2011). Mycoplasma and ureaplasma. In: Jorgensen JH, Pfaller MA (eds). Manual of Clinical Microbio­ logy, Vol. 1, Chapter  62. American Society for Microbiology, Washington, DC. Zhang N, et al. (2014). Are Ureaplasma spp. a cause of nongonococcal urethritis? A  systematic review and meta-​analysis. PLoS One, 9, e113771. Table 8.6.46.5  Susceptibility of some genital mycoplasmas to various antibiotics. A combination of in vitro susceptibility data and clinical efficacy is given where such experience is available Antibiotics M. hominis M. fermentans U. urealyticum M. genitalium Tetracyclines Tetracycline + + + ± Doxycycline ++ ++ ++ ± Macrolides Erythromycin –​ ± + + Clarithromycin –​ ± ++ ++ a Azithromycin –​ ± + +++a Lincosamides Clindamycin +++ ++ ± ± Quinolones Ciprofloxacin + ++ + ± Ofloxacin + ++ + ± Moxifloxacin ++ +++ ++ ++b +++, extremely sensitive; ++, highly sensitive; +, moderately sensitive; ±, weakly sensitive; –​, insensitive; a, high-​level macrolide resistance may be common in settings where azithromycin 1 g single dose is commonly used. bMoxifloxacin resistance is increasingly detected.

8.6.47 A checklist of bacteria associated with inf

8.6.47 A checklist of bacteria associated with infection in humans 1307

8.6.47  A checklist of bacteria associated with infection in humans 1307 8.6.47  A checklist of bacteria associated with infection
in humans John Paul ESSENTIALS In addition to a relatively small number of well-​known pathogenic bacteria that infect otherwise healthy people (e.g. Staphylococcus aureus, Mycobacterium tuberculosis, and Streptococcus pyogenes), there is a steadily growing list of less well-​known organisms, many of which cause disease only under special circumstances. Bacteria associated with infections in humans are listed in the table that forms the bulk of this chapter, which has been de- signed to serve as a single port of call for clinicians who seek concise information on the less well-​known clinically signifi- cant bacteria. Every name in the table has been checked to see that it has ‘standing in nomenclature’:  widely used names that do not have standing in nomenclature (at the time of writing) are included, but written in inverted commas (e.g. ‘Spirillum minus’—​one of the causes of rat bite fever). For an up-​to-​date check on nomenclature, the reader is referred to
http://www.bacterio.net​. Reported antibiotic susceptibilities and treatments are listed as a rough guide only: for some organisms the only available published information consists of in vitro test results for small numbers of strains, or apparent clinical response to therapy for a single case. There is no substitute for the determination of the susceptibilities of organisms as they are cultured on a case-​ by-​case basis in tandem with the monitoring of therapeutic response. Geographical restriction and particular exposures—​some patho- genic bacteria (e.g. Burkholderia pseudomallei, the cause of meli- oidosis), are associated with special geographical areas; others are associated with particular forms of exposure (e.g. with animal bites), and Rickettsia species with tick bites. Bacterial commensals and usually harmless environmental or- ganisms as causes of disease—​given the right kind of help, bac- teria that live usually as harmless human commensals can cause disease (e.g. skin commensals such as Staphylococcus epidermidis can cause line sepsis and infect prosthetic devices; gut com- mensals such as Bacteroides species can grow in abscesses; and
oral commensals such as Streptococcus salivarius can cause endocarditis). Immunosuppressed patients, ventilated patients,
and patients undergoing continuous ambulatory peritoneal dia- lysis are vulnerable to infection by a wide range of otherwise harmless environmental organisms. Improved understanding of disease processes and discovery of ‘new’ pathogens—​a refined understanding of, say, periodontal dis- ease, has resulted in the characterization of new organisms such as Pseudoramibacter alactolyticus, Johnsonella ignava, Centipeda periodontii, and Capnocytophaga gingivalis:  some of these have subsequently been identified in systemic infections such as bacteraemia. Impact of new laboratory techniques—​these have revealed the presence of new species and new disease associations (e.g. Tropheryma whipplei) was associated with Whipple’s disease by molecular methods before the organism was cultured; molecular methods have detected oddities like Bradyrhyzobium elkanii in aortic aneurysm tissue, although its role as potential pathogen is doubtful. Changes in nomenclature—​amidst the discovery of new bacteria, taxonomic rearrangements, and changes in nomenclature pile on additional layers of confusion for the clinician. For example, it was recognized that organisms formerly known as Burkholderia
cepacia are actually a complex of several genomospecies, which have been given individual names. It is also confusing when a well-​ known genus is split to reflect the recognition that its composite species are several groups that are only distantly related (e.g. many organisms that were once known as Bacteroides species). New
organisms will continue to be described and name changes will continue to occur.

section 8  Infectious diseases 1308 Table 8.6.47.1  A check list of bacteria associated with infection in humans Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) A [Abiotrophia adiacens—​see Granulicatella adiacens] Abiotrophia A. defectiva Endophthalmitis, brain abscess, osteomyelitis, peritonitis, endocarditis Vancomycin, ceftriaxone (plus gentamicin or rifampicin) Previously known as nutritionally deficient or variant streptococci [Abiotrophia elegans—​see Granulicatella elegans] [‘Abiotrophia para-​adiacens’—​see Granulicatella notes] Achromobacter (Alcaligenes) A. aegrifaciens A. animicus A. anxifer A. denitrificans A. dolens A. insolitus A. insuavis A. mucicolens A. piechaudii A. pulmonis A. ruhlandii A. spanius A. spiritinus A. xylosoxidans Septicaemia, CAPD peritonitis, pneumonia, ear infection, pulmonary infection in cystic fibrosis, keratitis, vascular line sepsis Ureidopenicillins, ceftazidime, carbapenems [Achromobacter CDC group Vd and Achromobacter groups A, C, and D—​see Ochrobactrum] [Achromobacter groups B and E—​see Pannonibacter] Acidaminococcus A. fermentans A. intestini Abscesses, postsurgical infections Metronidazole Acidovorax (Pseudomonas) A. avenae A. delafieldii A. facilis A. temperans A. wautersii Wound infection, UTI, bacteraemia, meningitis, septic arthritis Acinetobacter A. baumannii A. beijerinckii A. bereziniae A. calcoaceticus A. guillouiae A. gyllenbergii A. haemolyticus A. johnsonii A. junii A. lwoffi A. nosocomialis A. parvus A. pittii A. radioresistens A. schindleri A. seifertii A. ursingii Septicaemia, UTI, wound infections, abscesses, endocarditis, meningitis, osteomyelitis Aminoglycosides, ureidopenicillins, ceftazidime, carbapenems, tigecycline May be multidrug-​resistant Nosocomial outbreaks reported Infections associated with debilitated patients [Actinobacillus actinomycetemcomitans—​see Aggregatibacter actinomycetemcomitans] Actinobacillus A. equuli A. lignieresii A. suis Wound infection, abscesses, endocarditis, meningitis Ampicillin (plus gentamicin for endocarditis) Associated with animal contact and bites A. hominis Septicaemia, empyema Amoxicillin–​clavulanate A. ureae (Pasteurella ureae) Meningitis, pneumonia, endocarditis, hepatitis, peritonitis Ampicillin (plus gentamicin for endocarditis), chloramphenicol Respiratory tract commensal in humans [Actinobaculum massiliense—​see Actinotignum schaali] [Actinobaculum schaali—​see Actinotignum schaali] [Actinobaculum urinale—​see Actinotignum urinale] (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1309 Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) Actinomadura A. latina A. madurae A. pelletieri A. sputi A. vinacea Actinomycetoma, Madura foot, pulmonary infection Co-​trimoxazole, dapsone Actinomyces A. cardiffensis A. dentalis A. europaeus A. funkei A. georgiae A. gerencseriae A. graevenitzii A. hominis A. hongkongensis A. israelii A. johnsonii A. massiliensis A. meyeri A. naeslundii A. neuii neuii A. neuii anitratus A. odontolyticus A. oricola A. oris A. radicidentis A. radingae A. timonensis A. turicensis A. urogenitalis A. viscosus Actinomycosis β-​Lactams Actinotignum (Actinobaculum) A. schaalii (A. massiliense) A. sanguinis A. urinale Pyelonephritis, UTI, septicaemia, superficial skin infection Penicillin, cefuroxime, nitrofurantoin, tetracycline, clindamycin Advenella A. incenata Pulmonary infection, bacteraemia Aerococcus A. sanguinicola A. urinae A. urinaehominis A. viridans Endocarditis, UTI, wounds, meningitis, abscesses, CAPD peritonitis, lymphadenitis, spondodactylitis Penicillin, vancomycin (plus gentamicin for endocarditis) Aeromonas A. allosaccharophila A. bestiarum A. diversa A. enteropelogenes A. hydrophila A. jandaei A. media A. punctata (A. caviae) A. salmonicida A. sanarellii A. schubertii A. taiwanensis A. tecta A. trota (A. tructi) A. veronii Wound infection, abscesses, septicaemia, meningitis, leech-​bite infection, alligator-​bite infection, acute diarrhoea Aminoglycosides, chloramphenicol, ceftazidime, co-​trimoxazole Infections associated with aquatic exposure. A. veroniiincludes biovars Veronii and Sobria. The taxonomic
status of some species is unclear. The status of A. allosaccharophila is controversial. A. trota may be a synonym of A. enteropelogenes Afipia A. felis Cat-​scratch disease Imipenem, aminoglycosides Cat-​scratch disease is associated also with Bartonella spp. A. broomeae Bone marrow infection, septic arthritis Imipenem, aminoglycosides Role as pathogen uncertain A. clevelandensis Bone infection Imipenem, aminoglycosides Role as pathogen uncertain A. birgiae A. massiliensis Pneumonia Imipenem, aminoglycosides Roles as pathogens uncertain (continued) Table 8.6.47.1  Continued

section 8  Infectious diseases 1310 Aggregatibacter A. actinomycetemcomitans (Actinobacillus actinomycetemcomitans, Haemophilus actinomycetemcomitans) Periodontitis, endocarditis, abscesses, pericarditis, meningitis Penicillin (plus gentamicin for endocarditis), ceftriaxone, coamoxiclav Human oral commensal. Some strains reported to be penicillin-​resistant A. aphrophilus (Haemophilus
aphrophilus, H. paraphrophilus) A. segnis (Haemophilus segnis) Sinusitis, otitis media, pneumonia, abscesses, endocarditis Ceftriaxone, cefotaxime, chloramphenicol, ampicillin, aminoglycosides Agrobacterium A. radiobacter (A. tumefaciens) Endocarditis, CAPD peritonitis, UTI, line sepsis Co-​trimoxazole, gentamicin, amikacin, piperacillin-​tazobactam The nomenclature of this taxon is unsettled. The names A. tumefaciens and A. radiobacter both have
standing in nomenclature. Transfer
of Agrobacterium to Rhizobium has been proposed [Alcaligenes denitrificans—​see Achromobacter denitrificans] Alcaligenes A. faecalis Pneumonia, otitis, UTI, osteomyelitis, bacteraemia Amoxicillin–​clavulanate, cephalosporins, fluoroquinolones [Alcaligenes xylosoxidans—​see Achromobacter xylosoxidans xylosoxidans] [Alcaligenes piechaudii—​see Achromobacter piechaudii] [Alcaligenes ruhlandii—​see Achromobacter ruhlandii] Alicyclobacillus A. consociatus From blood culture Clinical significance uncertain Alishewanella A. fetalis From fetal necropsy specimen Clinical significance uncertain Alistipes A. finegoldii (Bacteroides finegoldii) A. indistinctus A. onderdonkii A. putredinis (Bacteroides putredinis) A. shahii A. timonensis Appendicitis, peritonitis, abdominal abscess Metronidazole, ertapenem β-​Lactamase producers. Abdominal infections, found in association with other anaerobes Alloiococcus A. otitis (Alliococcus otitis) Otitis media Vancomycin Alloprevotella A. rava A. tanneri Dental plaque, atheromatous plaque Alloscardovia A. omnicolens UTI [Amycolata autotrophica—​see Pseudonocardia autotrophica] Amycolatopsis A. orientalis (Nocardia orientalis) Role as pathogen uncertain A. palatopharyngis Palatopharyngeal infection Clinical significance poorly defined Anaerobiospirillum A. succiniproducens A. thomasii Diarrhoea, bacteraemia Cefuroxime, tetracycline, chloramphenicol Infection may be related to exposure to cat or dog faeces Anaerococcus (Peptostreptococcus) A. hydrogenalis A. lactolyticus A. murdochii A. octavius A. prevotii A. senegalensis A. tetradius A. vaginalis Mixed anaerobic infections, abscesses β-​Lactams, metronidazole Anaeroglobus A. geminatus From postoperative collection Role as pathogen uncertain Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1311 Anaerorhabdus (Bacteroides) A. furcosa (A. furcosus) Lung abscess, appendix, and abdominal abscesses [‘Anguillina coli’—​see Serpulina pilosicoli] Anaplasma A. phagocytophilum Anaplasmosis Doxycycline Previously known as human granulocytic ehrlichiosis [Arachnia propionica—​see Propionibacterium propionicus] [Arcanobacterium bernardiae—​see Trueperella bernardiae] [Arcanobacterium pyogenes—​see Trueperella pyogenes] Arcanobacterium A. haemolyticum (Corynebacterium haemolyticum) Tonsillitis, cellulitis, lymphadenopathy, brain abscess, septicaemia, osteomyelitis Penicillin, erythromycin Arcobacter (Campylobacter) A. butzleri A. cryaerophilus Abdominal cramps, diarrhoea Self-​limiting Arthrobacter A. albus A. creatinolyticus A. cumminsii A. luteolus A. oxydans A. sanguinis A. scleromae A. woluwensis UTI, bacteraemia, skin infection Vancomycin, penicillins Arthrobacter sp. has been implicated in Whipple’s syndrome, a disease usually associated with Tropheryma whipplei Atopobium A. deltae A. minutum (Lactobacillus minutus) A. parvulum (Streptococcus parvulus) A. rimae (Lactobacillus rimae) A. vaginae UTI, dental abscesses, pelvic abscesses, wound infection, blood cuture of patient with Fournier’s gangrene Bacterial vaginosis Isolates from periodontal sites suggest possible role in periodontal disease [Aureobacterium—​see Microbacterium] Auritidibacter A. ignavus Otitis externa Azospirillum A. brasilense (Roseomonas fauriae) CAPD peritonitis, line sepsis Imipenem, aminoglycosides, ceftriaxone, ciprofloxacin B Bacillus B. anthracis Anthrax Penicillin, erythromycin Ciprofloxacin for postexposure prophylaxis [Bacillus brevis—​see Brevibacillus agri] [Bacillus sphaericus—​see Lysinibacillus sphaericus] B. circulans B. coagulans B. idriensis B. infantis B. megaterium B. mycoides B. thuringiensis Pneumonia, septicaemia, corneal infections, meningitis, food poisoning, eye infection, lung infection Vancomycin, clindamycin, aminoglycosides, imipenem, penicillin Other than the well-​known B. anthracis and B. cereus, Bacillus spp. are rare causes of focal and systemic sepsis. Some isolates are resistant to vancomycin. Isolates may represent specimen or laboratory contamination. B. thuringiensis is a biological insecticide which has caused corneal infection B. cereus B. cytotoxicus B. licheniformis B. pumilus B. subtilis Food poisoning, wound infection, cutaneous lesions, bacteraemia, endocarditis, eye infection Clindamycin, vancomycin, gentamicin Diarrhoea is self-​limiting. B. cereus is resistant to β-​lactams Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1312 Bacteroides B. caccae B. coagulans B. eggerthii B. finegoldii B. fragilis B. massiliensis B. nordii B. ovatus B. pyogenes (B. tectus) B. salyersae B. stercoris B. thetaiotaomicron B. uniformis B. vulgatus Abscesses, bacteraemia, bite infections, wound infections, chronic otitis media, pelvic inflammatory disease, neonatal sepsis Ureidopenicillins, carbapenems, metronidazole Resistance to metronidazole and β-​lactams has been reported. Many species previously classified as Bacteroides have been transferred to other genera: see Alistipes, Anaerorhabdus, Campylobacter, Dialister Mitsuokella, Odoribacter, Parabacteroides, Prevotella, Porphyromonas, Pseudoflavonifractor, and Tannerella Balneatrix B. alpica Pneumonia, bacteraemia, meningitis Ceftriaxone, ofloxacin, amoxicillin, netilmicin Infection associated with exposure to hot spring water Bartonella B. bacilliformis Oroya fever, verruga peruana Chloramphenicol, streptomycin B. elizabethae (Rochalimaea elizabethae) Endocarditis Gentamicin, imipenem, co-​trimoxazole B. clarridgeiae B. henselae (Rochalimaea henselae) Cat-​scratch disease, bacillary angiomatosis Aminoglycosides, doxycycline Cat-​scratch disease is associated also with Afipia felis B. quintana (Rochalimaea quintana) Trench fever, bacillary angiomatosis Aminoglycosides, doxycycline B. rochalimae​ Bacteraemia, anaemia, splenomegaly​ Levofloxacin Cause of canine endocarditis​ B. schoenbuchensis Deer ked dermatitis Evidence to associate this organism with deer ked dermatitis is circumstantial B. vinsonii arupensis Bacteraemia Ceftriaxone Zoonosis from rodents Bergeyella B. zoohelcum (Weeksella zoohelcum) Wound infection, septicaemia, meningitis Cefotaxime, penicillins, ciprofloxacin, tetracycline Associated with dog and cat bites Bifidobacterium B. adolescentis B. angulatum B. breve B. bifidumB. dentium B. longum longum B. longum infantis B. pseudocatenulatum B. scardovii Bacteraemia, abscesses, peritonitis, otitis, paronychia, UTI, dental caries Clindamycin, penicillins, cefoxitin Reported risk factors include surgery, malignancy, steroid therapy, intravenous drug use, and acupuncture. Some strains used as probiotics [Bifidobacterium inopinatum—​see Scardovia inopinata] Bilophila B. wadsworthia Appendicitis, abscesses, bacteraemia, biliary tract sepsis, mastoiditis Metronidazole, amoxicillin/​ clavulanate, ureidopenicillins, cephalosporins Bordetella B. bronchiseptica Respiratory tract infection Tetracycline, fluoroquinolones Zoonosis from dogs and other animals B. hinzii B. holmesii B. trematum Bacteraemia, otitis, wound infection B. hinzii is a pathogen of poultry B. parapertussis B. pertussis Whooping cough, respiratory tract infection Erythromycin B. parapertussis causes less severe disease The genus Borreliella has been proposed for species associated with Lyme disease Borrelia B. americana B. afzelii B. andersoni B. bavariensis B. bissettiae B. burgdorferi Lyme disease Amoxicillin, doxycycline, ceftriaxone Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1313 B. carolinensis B. garinii B. japonica B. kurtenbachii B. lusitaniae B. sinica B. spielmanii B. tanukii B. turdi B. valaisiana B. caucasica B. crocidurae B. duttonii B. graingeri B. hermsii B. hispanica B. latyschewii B. mazzottii B. parkeri B. persica B. recurrentis B. turicatae B. venezuelensis Relapsing fever Tetracycline, erythromycin, chloramphenicol, penicillin B. recurrentis is louse-​borne; other agents are tick-​borne Bosea B. massiliensis Linked with ventilator-​ associated pneumonia Doxycycline, telithromycin Amoeba-​resisting bacterium from hospital water supplies Brachyspira B. aalborgi B. pilosicoli (Serpulina pilosicoli, ‘Anguillina coli’) Intestinal spirochaetosis Of uncertain significance Bradyrhizobium B. elkanii Detected in tissue from aortic aneurysm Potential role as pathogen uncertain [Branhamella catarrhalis—​see Moraxella catarrhalis] Brevibacillus B. centrosporus Bacteraemia Vancomycin Previously confused with B. laterosporus and reported as such
in clinical literature B. massiliensis B. parabrevis Bacteraemia, abscess Vancomycin Brevibacterium B. casei B. epidermidis B. luteolum (B. lutescens) B. massiliense B. mcbrellneri B. otitidis B. paucivorans Bacteraemia, endocarditis, meningitis, chest infection, pericarditis, vascular catheter sepsis Glycopeptides Brevundimonas (Pseudomonas) B. diminuta B. vancanneytii B. vesicularis Septicaemia, endocarditis Cefazolin, ceftriaxone, piperacillin (plus gentamicin for endocarditis) Brucella B. abortus B. canis B. melitensis B. suis Brucellosis Doxycycline (plus streptomycin or rifampicin) The four species names used for clinical purposes represent biovars of a single species, B. melitensis Brucella B. ceti B. pinnipedalis Brucellosis Causes of brucellosis in marine mammals with potential to infect humans Brucella inopinata Breast implant infection Bulleidia B. extructa Necrotizing ulcerative periodontitis in HIV
patients Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1314 Burkholderia (Pseudomonas) B. ambifaria B. anthina B. cenocepacia B. cepacia (Pseudomonas cepacia) B. dolosa B. multivorans B. pseudomultivorans B. pyrrocinia B. stabilis B. vietnamiensis Lung infection in cystic fibrosis, bacteraemia, endocarditis, septic
arthritis, UTI Ureidopenicillins, ceftazidime, aztreonam, carbapenems, fluoroquinolones, co-​trimoxazole B. cepacia sensu stricto and other taxa listed are genomospecies of the B. cepacia species complex (B. cepacia sensu lato). Hard to differentiate by routine methods. Differences in disease progression in cystic fibrosis may relate to different genomospecies B. gladioli (Pseudomonas gladioli) Lung infection in cystic fibrosis Ureidopenicillins, ceftazidime, aztreonam, carbapenems, fluoroquinolones, co-​trimoxazole B. fungorum Septic arthritis, bacteraemia, meningitis Amoxicillin, cefuroxime, ceftazidime, ciprofloxacin, meropenem, co-​trimoxazole B. mallei (Pseudomonas mallei) Glanders Sulfadiazine, co-​amoxiclav, tetracycline, co-​trimoxazole B. pseudomallei (Pseudomonas pseudomallei) Melioidosis Ceftazidime, co-​trimoxazole, chloramphenicol, imipenem Buttiauxella B. agrestis B. noackiae Appendicitis, wound infection Aminoglycosides, doxycycline Cephalosporin resistance reported Butyrivibrio B. fibrisolvens Endophthalmitis Penicillin, chloramphenicol From rumina of farm animals C [Calymmatobacterium granulomatis—​see Klebsiella granulomatis] [Campylobacter butzleri—​see Arcobacter butzleri] [Campylobacter cinaedi—​see Helicobacter cinaedi] [Campylobacter fennelliae—​see Helicobacter fennelliae] [Campylobacter pyloridis—​see Helicobacter pylori] Campylobacter C. coli C. jejuni jejuni C. jejuni doylei C. mucosalis Gastroenteritis, bacteraemia Erythromycin, fluoroquinolones Infections are usually self-​limiting C. concisus C. curvus (Wolinella curva) C. gracilis (Bacteroides gracilis) C. rectus (Wolinella recta) C. showae C. sputorum C. ureolyticus (Bacteroides ureolyticus) Periodontitis, appendicitis, peritonitis, head and neck infections, abscesses Ureidopenicillins, amoxicillin/​ clavulanate, carbapenems, fluoroquinolones, metronidazole C. fetus fetus​ Fever, diarrhoea, meningoencephalitis, endocarditis, abscesses​ Erythromycin, ampicillin, chloramphenicol, gentamicin​ ​Exposure to reptiles or Asian food C. fetus testudinum Bacteraemia, wound infection, diarrhoea Cephalosporins C. fetus venerealis Bacterial vaginosis Role as human pathogen poorly defined. Reported from faeces of homosexual men C. hyointestinalis C. lari (C. laridis) C. upsalensis Diarrhoea, bacteraemia, abscess Erythromycin, ampicillin, gentamicin Zoonoses from mammals and birds Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1315 Canibacter C. oris Wound infection Ampicillin, clindamycin, ciprofloxacin, vancomycin, linezolid From dog bites Capnocytophaga C. canimorsus (CDC DF-​1) C. cynodegmi (CDC DF-​2)​ Wound infection, septicaemia, abscesses, meningitis, endocarditis​ Penicillin​ From dog bites​ C. gingivalis C. granulosa C. haemolytica C. leadbetteri C. ochracea C. sputigena Periodontitis, septicaemia, chorioamnionitis Penicillins, ciprofloxacin, tetracycline, chloramphenicol From oral flora. Infections associated with malignancy and neutropenia Cardiobacterium C. hominis C. valvarum Endocarditis, meningitis Penicillin (plus gentamicin for endocarditis) Catonella C. morbi Periodontitis, endodontic infection Role as pathogen unclear Cedecea C. davisae C. lapagei C. neteri Bacteraemia Chloramphenicol, cefamandole, gentamicin Two other species (sp. 3 and sp. 5) have been isolated from clinical specimens Cellulomonas C. denverensis C. hominis (CDC coryneform group A-​3) Bacteraemia, meningitis, pilonidal abscess, wound infection, homograft valve infection Clarithromycin, clindamycin, imipenem, minocycline, rifampicin, vancomycin [Cellulomonas cellulans—​see Cellulosomicrobium] [Cellulomonas turbata—​see Oerskovia turbata] Cellulosimicrobium C. cellulans (Cellulomonas cellulans, Oerskovi xanthineolytica) C. funkei Meningitis, pyonephrosis, CAPD peritonitis, endophthalmitis Vancomycin and gentamicin or rifampicin Centipeda C. periodontii Periodontitis Role as pathogen unclear. Shown to inhibit lymphocytes Chlamydia C. trachomatis Trachoma, genital infection, neonatal infection, lymphogranuloma venereum Erythromycin, tetracycline, azithromycin Includes 18 serovars clustered into two biovars: trachoma and lymphogranuloma venereum Chlamydophila C. abortus (Chlamydia psittaci) Abortion Associated with contact with infected ruminants C. pneumoniae (Chlamydia pneumoniae) Chest infection Tetracycline Infections in humans associated with biovars TWAR C. psittaci (Chlamydia psittaci) Psittacosis Tetracycline Zoonosis from birds Chromobacterium C. violaceum Septicaemia, osteomyelitis, abscesses, eye infection Erythromycin, tetracycline, chloramphenicol,
gentamicin Associated with exposure to soil and water Chryseobacterium (Flavobacterium) C. anthropi C. bernardetii C. gleum C. hominis C. indologenes C. nakagawai C. treverense Bacteraemia, abdominal sepsis, vascular catheter sepsis Piperacillin-​tazobactam, minocycline, fluorquinolones, rifampicin Susceptibilities vary. Often multiresistant Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1316 [Chryseobacterium meningosepticum—​see Elizabethkingia meningoseptica] [Chryseomonas luteola—​see Pseudomonas luteola] Citrobacter C. amalonaticus C. braakii C. diversus C. farmeri C. freundii C. gilenii C. koseri C. murliniae C. rodentium C. sedlakii C. werkmanii C. youngae UTI, meningitis, bacteraemia, haemolytic–​uraemic syndrome Aminoglycosides, β-​lactams Variable susceptibility. May be multiresistant. Nosocomial outbreaks of infection reported. Citrobacter spp. are part of the normal faecal flora Clostridium C. argentinense C. baratii C. beijerinckii C. bifermentans C. bolteae C. butyricum C. cadaveris C. carnis C. celatum C. clostridioforme C. cochlearium C. cocleatum C. fallax C. ghonii C. haemolyticum C. histolyticum C. indolis C. innocuum C. irregulare C. leptum C. limosum C. malenominatum C. novyi C. oroticum C. paraputrificum C. piliforme C. putrefaciens C. ramosum C. sardiniense (C. absonum) C. septicum C. sordellii C. sphenoides C. sporogenes C. subterminale C. symbiosum C. tertium Wound infection, bacteraemia, abscesses Penicillin, clindamycin, metronidazole Many Clostridium spp. have been isolated form clinical specimens. For most, their clinical significance is poorly defined. C. baratii and C. butyricum are rare causes of botulism. C. fallax, C. histolyticum, C. novyi, C. septicum, and C. sordellii are gas gangrene agents. Treatment of gas gangrene includes debridement and penicillin, clindamycin, or metronidazole. Recent studies have resulted in the proposed transfer of some of the species listed to new genera: Paraclostridium bifermentans, Paeniclostridium ghonii, Hathewaya histolytica, Hathewaya limosa, Faecalicatena orotica, Paraclostridium sordellii C. botulinum Botulism Antitoxin and respiratory support as treatment C. difficile Diarrhoea, pseudomembranous colitis Metronidazole, vancomycin Infection associated with antibiotic exposure. Recent phylogenetic studies resulted in the proposed transfer to the genus Clostridioides C. perfringens Food poisoning, necrotizing enterocolitis, gas gangrene Debridement and penicillin, clindamycin, or metronidazole for treatment of gas gangrene C. tetani Tetanus Metronidazole, penicillin Antitoxin and supportive treatment Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1317 [Clostridium glycolicum—​see Terrisporobacter glycolicus] Collinsella C. aerofaciens From faecal flora. Clinical significance is undefined [Comamonas acidovorans—​see Delftia acidovorans] Comamonas (Pseudomonas) C. kerstersii C. terrigena C. testosteroni Bacteraemia, UTI, conjunctivitis, endocarditis, wound infection, abdominal abscess, peritonitis, meningitis Ureidopenicillins, ceftazidime, ciprofloxacin, aminoglycosides, imipenem Infections in neutropenic patients. Infections associated with animal bite and exposure to tropical fish Corynebacterium C. accolens C. afermentans C. amycolatum C. aquatimens C. appendicis C. argentoratense C. atypicum C. aurimucosum (C. nigricans) C. auris C. bovis C. confusum C. coyleae C. durum C. falsenii C. freneyi C. glucuronolyticum C. imitans C. jeikeium C. kroppenstedtii C. kutscheri C. lipophiloflavum C. macginleyi C. massiliense C. matruchotii C. mucifaciens C. pilosum C. pilparense C. propinquum C. renale C. resistens C. riegelii ‘C. sanguinis’ C. singulare C. sputi C. striatum C. sundsvallense C. thomssenii C. tuberculostearicum C. tuscaniense C. urealyticum C. ureicelerivorans C. xerosis​ Septicaemia, peritonitis, UTI, eye infection, wound infection, endocarditis, osteomyelitis, septic arthritis, meningitis, abscesses Glycopeptides, β-​lactam, erythromycin, rifampicin More than 40 Corynebacterium spp. have been isolated from clinical specimens. For many of them, clinical significance and empirical therapy are poorly defined. Many isolates are susceptible to β-​lactams. Multiresistant, vancomycin-​susceptible isolates of CDC coryneform group G-​2, C. jeikeium and C. urealyticumhave been reported. Nosocomial outbreaks have been reported. Corynebacterium spp. may be specimen or laboratory contaminants. C. auriscanis C. canis C. feiburgense Wound infection ​Tetracycline ​From dog bites C. diphtheriae Diphtheria, cutaneous infection Penicillin, erythromycin Toxigenic infection requires treatment with antitoxin C. minutissimum Erythrasma, bacteraemia, endocarditis Role as an agent of erythrasma is poorly defined C. mycetoides Tropical ulcer, septicaemia Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1318 C. pseudodiphtheriticum UTI, endocarditis, lymphadenopathy, necrotizing tracheitis Penicillin C. pseudotuberculosis Lymphadenitis, pulmonary infection Penicillin, erythromycin Associated with sheep contact. May require drainage or excision C. ulcerans Diphtheria-​like disease, pharyngitis Penicillin, erythromycin Toxigenic infection requires treatment with antitoxin C. vitaeruminis Associated with aortic aneurysm Role as pathogen uncertain [Corynebacterium group A-​3—​see Cellulomonas] [Corynebacterium groups A-​4 and A-​5—​see Microbacterium] [Corynebacterium group 2—​see Arcanobacterium bernardiae] Coxiella C. burnetii Q fever Tetracycline, ciprofloxacin, co-​ trimoxazole, rifampicin Cryptobacterium C. curtum Periodontitis Cupriavidus (Ralstonia) (Wautersia) C. gilardii C. pauculus C. respiraculi C. taiwanensis Meningitis, pulmonary infection in cystic fibrosis, line sepsis Cephalosporins, imipenem, co-​trimoxazole, quinolones, amikacin D Delftia D. acidovorans (Comamonas acidovorans) Bacteraemia, endocarditis Ureidopenicillins, fluoroquinolones Dermabacter D. hominis Brain abscess, bacteraemia, wound infection Cephalosporins, glycopeptides Dermacoccus D. sp. Associated with aortic aneurysm Role as pathogen uncertain. Found on skin and mucous membranes Dermatophilus D. congolensis Cutaneous infection Penicillin Zoonosis from cattle, sheep, goats, and horses Desmospora D. activa From sputum Quinolones, carbapenems, vancomycin, linezolid, amikacin Clinical significance uncertain Desulfomicrobium D. orale Periodontitis Desulfomonas D. piger (D. pigra) Pilonidal cyst abscess, peritonitis From faecal flora Desulfovibrio D. desulfuricans D. vulgaris Bacteraemia, liver abscess Penicillin, clindamycin ‘D. fairfieldensis’ Cultured from urine of patient with UTI and meningoencephalitis Proposed name does not have standing in nomenclature Dialister D. invisus D. micraerophilus D. pneumosintes D. propionicifaciens Periodontitis, endodontic infection, bacteraemia Dichelobacter D. nodosus (Bacteroides nodosus) Pilonidal cyst, rectal fistula, wound infection Cause of ovine footrot. Isolates reported from humans may not be D. nodosus Dietzia D. aurantiaca D. cinnamea D. maris D. papillomatosis Prosthetic hip infection, bacteraemia, isolated
from CSF Vancomycin, teicoplanin, rifampicin, amoxicillin, gentamicin, clindamycin, co-​trimoxazole D. papillomatosis is associated with papillomatosis. Clinical response reported with macrolides or tetracyclines Dolosicoccus D. paucivorans Bacteraemia Cephalosporins Dolosigranulum D. pigrum Spinal cord infection, eye infection Significance as a pathogen poorly defined. Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1319 Dysgonomonas D. capnocytophagoides (CDC group DF-​3) D. gadei D. mossii Diarrhoea, bacteraemia, abscess Tetracycline, clindamycin, imipenem E Edwardsiella E. hoshinae E. ictaluri E. tarda Wound infection, abscesses, gastroenteritis β-​Lactams, aminoglycosides, fluoroquinolones Aquatic exposure, penetrating fish injury Eggerthella E. hongkongensis E. lenta (Eubacterium lentum) E. sinensis Rectal abscess, bacteraemia Penicillin, metronidazole Variable susceptibility to cefotaxime Eggerthia E. catenaformis (Lactobacillus catenaformis) Pulmonary infection . Ehrlichia E. chaffeensis E. ewingii Ehrlichiosis Tetracycline, doxycycline Antibodies to E. muris detected in healthy humans in Japan [Ehrlichia sennetsu—​see Neorickettsia sennetsu] Eikenella E. corrodens Septicaemia, endocarditis, abscesses, septic arthritis Penicillin (plus gentamicin for endocarditis) Eisenbergiella E. tayi Bacteraemia Vancomycin, metronidazole Elizabethkingia E. anophelis E. meningoseptica (Chryseobacterium meningosepticum, Flavobacterium meningosepticum) Meningitis, bacteraemia, endocarditis, necrotizing fasciitis, pneumonia Quinolones, co-​trimoxazole, minocycline, rifampicin Treatment with vancomycin is controversial Empedobacter E. brevis (Flavobacterium breve) Endophthalmitis, bacteraemia, UTI Broad spectrum cephalosporins Carbapenem-​resistant. Phylogenetic data support inclusion in the genus Moheibacter Enterobacter E. aerogenes E. amnigenus E. asburiae E. cancerogenus E. cloacae E. gergoviae E. hormaechei E. kobei E. ludwigii E. sakazakii Bacteraemia, respiratory tract infections, UTI Carbapenems, fluoroquinolones, aminoglycosides, ureidopenicillins May be multiresistant. Common cause of nosocomial infection Enterococcus E. avium E. casseliflavus (E. flavescens) E. cecorum E. dispar E. durans E. faecalis E. faecium E. gallinarum E. gilvus E. hirae E. malodoratus E. mundtii E. pallens E. pseudoavium E. raffinosus Bacteraemia, abscesses, endocarditis, meningitis, UTI, peritonitis, osteomyelitis, wound infection Penicillins, glycopeptides May be resistant to penicillins and glycopeptides. Nosocomial outbreaks reported [Enterococcus solitarius—​see Tetragenococcus solitarius] Erwinia E. persicinus UTI Cephalosporins, fluoroquinolones, aminoglycosides The causative agent of necrosis of bean pods Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1320 Erysipelothrix E. rhusiopathiae Erysipeloid, septicaemia, endocarditis Penicillin Animal contact [Escherichia adecarboxylata—​see Leclercia adecarboxylata] Escherichia E. albertii Diarrhoea Previously known as Hafnia alvei-​like strains E. coli UTI, bacteraemia, wound infection, meningitis, enteric infection, haemolytic fluoroquinolones, uraemic syndrome β-​Lactams, aminoglycosides, co-​trimoxazole Susceptibilities variable E. fergusonii Bacteraemia, wounds, UTI Chloramphenicol, gentamicin Ampicillin-​resistant E. hermanii Wounds Chloramphenicol, cephalosporins, gentamicin E. vulneris Wounds Ampicillin, cephalosporins, gentamicin Eubacterium E. brachy E. combesii E. contortum E. cylindroids E. infirmum E. limosum E. minutum E. moniliforme E. multiforme E. nitrogenes E. nodatum E. plautii E. rectale E. saburreum E. saphenum E. sulci E. tenue E. timidum E. tortuosum E. ventriosum E. yurii yurii E. yurii mararetiae E. yurii schtitka Wounds, abscesses, septicaemia, periodontitis Penicillins, clindamycin, metronidazole Ewingella E. americana Septicaemia, wounds, UTI Ureidopenicillins, aminoglycosides Exiguobacterium E. acetylicum E. aurantiacum Wound infection, bacteraemia F Facklamia F. hominis F. ignava F. languida F. sourekii UTI, bacteraemia, abscess Filifactor F. alocis F. vilosus Gingivitis, periodontitis Finegoldia F. magna (Peptostreptococcus) magnus [Flavimonas oryzihabitans—​see Pseudomonas oryzihabitans] Flavobacterium F. mizutaii (Sphingobacterium mizutae) [Flavobacterium gleum—​see Chryseobacterium gleum] [Flavobacterium indologenes—​see Chryseobacterium indologenes] Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1321 [Flavobacterium meningosepticum—​see Elizabethkingia meningoseptica] ‘Flexispira’ ‘F. rappini’ Bacteraemia, diarrhoea Not in approved lists of bacterial names. There is a growing consensus that ‘Flexispira’ actually represents several Helicobacter spp. [Fluoribacter bozemanae—​see Legionella bozemanae] [Fluoribacter dumoffii—​see Legionella dumoffii] [Fluoribacter gormanii—​see Legionella gormanii] Francisella F. hispaniensis F. philomiragia (Yersinia philomiragia) F. tularensis holarctica F. tularensis mediasiatica F. tularensis novicida F. tularensis tularensis Septicaemia, invasive systemic infection, tularaemia Fluoroquinolones, aminoglycosides, chloramphenicol, cefoxitin, streptomycin, tetracycline Fusobacterium F. gonidiaformans F. mortiferum F. naviforme F. necrogenes F. necrophorum necrophorum F. necrophorum fundiliforme F. nucleatum nucleatum F. nucleatum fusiforme F. nucleatum polymorphum F. nucleatum vincentii F. periodonticum F. russii F. ulcerans F. varium Abscesses, bacteraemia, periodontitis, endocarditis, necrobacillosis Metronidazole, penicillins, carbapenems, cephalosporins G Gardnerella G. vaginalis Intrauterine and neonatal sepsis β-​Lactams, clindamycin Associated with bacterial vaginosis Gemella G. asaccharolytica G. bergeri G. haemolysins G. morbillorum (Streptococcus morbillorum) G. parahaemolysans G. sanguinis G. taiwanensis Bacteraemia, endocarditis Penicillin or vancomycin (plus gentamicin for endocarditis) Globicatella G. sanguinis Bacteraemia, UTI, meningitis Vancomycin Gordonia (Gordona) (Rhodococcus) G. aichensis G. araii G. bronchialis G. effuse G. iterans G. otitidis G. polyisoprenivorans G. rubropertinctus G. sputi G. terrae Pulmonary infection, cholecystitis, breast abscess, sternal wound sepsis, brain abscess, bacteraemia, otitis Co-​trimoxazole, ceftriaxone, imipenem, fluoroquinolones Granulicatella G. adiacens (Abiotrophia adiacens) G. elegans (Abiotrophia elegans) Endocarditis, septic arthritis, endodontic infection Penicillin or cefazolin or vancomycin plus gentamicin (plus rifampicin) Previously known as nutritionally deficient or variant streptococci; the proposed name ‘Abiotrophia para-​ adiacens’ for strains allied to what is now known as Granulicatella adiacens does not have standing in nomenclature Grimontia G. hollisae (Vibrio hollisae) Diarrhoea β-​Lactams, quinolones Infection associated with ingestion of shellfish Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1322 H [Haemophilius aphrophilus—​see Aggregatibacter aphrophilus] [Haemophilius paraphrophilus—​see Aggregatibacter aphrophilus] [Haemophilius segnis—​see Aggregatibacter segnis] Haemophilus H. aegyptius Brazilian purpuric fever Ampicillin, cephalosporins, chloramphenicol Treated by some authors as a biotype
of H. influenzae H. parainfluenzae H. pittmaniae Sinusitis, otitis media, pneumonia, abscesses, endocarditis Cefotaxime, chloramphenicol, ampicillin, aminoglycosides The genus Aggregatibacter has been proposed to accommodate H. aphrophilus (including H. paraphrophilus as a heterotypic synonym of H. aphrophilus), H. signis, and Actinobacillus actinomycetemcomitans H. ducreyi Chancroid Macrolides, ceftriaxone, fluoroquinolones H. influenzae Bacteraemia, meningitis, epiglottitis Cephalosporins, penicillins, fluoroquinolones Many strains produce penicillinases Hafnia H. alvei H. paralvei Bacteraemia Doubtful enteropathogen. Susceptibility variable. Halomonas H. stevensii H. ‘phocaensis’ H. venusta Bacteraemia, fish bite infection Penicillins, cephalosporins, quinolones Hazenella H. coriacea Bacteraemia Helcobacillus H. massiliensis From cutaneous discharge Helcococcus H. kunzii ‘H. pyogenica’ H. sueciensis Sebaceous cyst infection, breast abscess, wound infection Penicillins, vancomycin From skin flora. The name H. pyogenica does not have standing
in nomenclature Helicobacter H. bilis (‘Flexispira rapinni’ corrig. taxon 9) Cholecystitis, bacteraemia Zoonosis from rodents H. canis Gastroenteritis Zoonosis from dogs H. cinaedi (Campylobacter cinaedi) H. fennelliae (Campylobacter
fennelliae) Proctitis in homosexual men, septicaemia Ampicillin, gentamicin Zoonoses from hamsters H. bizzozeronii H. felis H. salomonis ‘Candidatus H. bovis’ H. heilmannii (‘Gastrospirillum hominis’) H. suis Gastritis Zoonoses from domestic and farm animals. Some organisms known as ‘Flexipsira rapini’ may belong to this group of Helicobacter spp. H. canadensis H. pullorum Gastroenteritis Zoonoses from birds (or possibly rodents) H. pylori (Campylobacter pyloridis) Gastritis Omeprazole plus clarithromycin and metronidazole Numerous similar treatment combinations have been recommended ‘H. westmeadii’ Bacteraemia in AIDS Name does not have standing in nomenclature ‘H. winghamensis’ Gastroenteritis Name does not have standing in nomenclature. Possibly a zoonosis from rodents Herbaspirillum H. sp. Associated with aortic aneurism Detected by 16S gene analysis. Of doubtful clinical significance Holdemania H. filiformis From faecal flora. Clinical significance is unclear Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1323 I Ignatzschineria larvae Bacteraemia Penicillins, cephalosporins, quinolones Associated with myiasis Ignavigranum I. ruoffiae Wound infection, ear abscess Role as pathogen poorly defined Inquilinus I. limosus Pulmonary infection in cystic fibrosis, endocarditis Imipenem, quinolones, gentamicin An additional undescribed Inquilinus sp. reported from clinical material J Janibacter J. melonis Bacteraemia Vancomycin, β-​lactams, fluoroquinolones An undescribed Janibacter sp. was isolated from a leukaemia patient Johnsonella J. ignava Periodontitis K Kerstersia K. gyiorum K. similis Wound infection, abscess, otitis Variable antibiotic susceptibilities Kingella K. denitrificans K. kingae K. oralis K. potus Septic arthritis, endocarditis, bite infection Penicillins (plus gentamicin for endocarditis) [Kingella indologenes—​see Suttonella indologenes] Klebsiella K. granulomatis (Calymmatobacterium granulomatis) Donovanosis Tetracycline, co-​trimoxazole [Klebsiella ornitholytica, K. planticola, K. terrigena—​see Raoultella] K. oxytoca K. pneumoniae ssp. pneumoniae K. pneumoniae ssp. ozaenae K. quasipneumoniae K. variicola UTI, bacteraemia, wound infection, respiratory tract infection β-​Lactams, aminoglycosides, fluoroquinolones Susceptibilities vary. Nosocomial outbreaks reported K. pneumoniae ssp. rhinoscleromatis Rhinoscleroma Ciprofloxacin, rifampicin, co-​trimoxazole Kluyvera K. ascorbate K. cryocrescens K. georgiana K. intermedia (Enterobacter intermedius) Bacteraemia, UTI, mediastinitis, line sepsis Aminoglycosides, ceftazidime, imipenem, ciprofloxacin Kocuria (Micrococcus) K. kristinae K. rosea K. varians Cholecystitis, line-​related sepsis Penicillin, clindamycin, vancomycin [Koserella trabulsii—​see Yokenella regensburgei] Kurthia ‘K. bessonii’ Bacteraemia, endocarditis Penicillin Not in approved lists of bacterial names K. gibsonii K. zopfii Urethritis associated with piglet contact Isolated from faeces of patients with diarrhoea Kytococcus (Micrococcus) K schroeteri K. sedentarius Endocarditis, cerebral cyst infection Imipenem, vancomycin, rifampicin L Lactobacillus L. acidophilus L. brevis L. casei L. coleohominis L. crispatus L. fermentum L. gasseri L. iners L. jensenii Abscesses, bacteraemia, endometritis, endocarditis, lung infection, UTI Cephalosporins, vancomycin, penicillins, aminoglycosides, clindamycin Reported risk factors for infection include surgery, malignancy, diabetes, and immunodeficiency. May be vancomycin-​resistant Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1324 Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) L. leichmannii L. oris L. paracasei L. paraplantarum L. plantarum L. rhamnosus L. salivarius L. vaginalis Lactococcus (Streptococcus) L. garvieae L. lactis Bacteraemia, endocarditis, UTI Penicillin (plus gentamicin for endocarditis) Lautropia L. mirabilis Role as potential pathogen unclear. From oral flora of HIV patients and sputum of cystic fibrosis patient Leclercia L. adecarboxylata (Escherichia adecarboxylata) Bacteraemia, wound infection Variable susceptibility Legionella L. anisa L. birminghamensis L. bozemanae (L. bozemanii) L. cardiaca L. cincinnatiensis L. dumoffii L. feeleii L. gormanii L. hackeliae L. israelensis L. jordanis L. lansingensis L. longbeachae L. lytica L. maceachernii L. micdadei (L. pittsburghensis) L. nagasakiensis L. oakridgemsis L. pneumophila L. quinlivanii L. rubrilucens L. sainthelensi L. steelei L. tucsonensis L. wadsworthia L. worsleiensis Legionnaires’ disease, Pontiac fever Macrolides, fluoroquinolones, rifampicin Infections caused by species other than L. pneumophila and L. micdadei are seldom reported. L. cardiaca isolated from patient with endocarditis Leifsonia L. aquatica (Corynebacterium aquaticum) UTI, endocarditis, meningitis, CAPD peritonitis Ampicillin, chloramphenicol, gentamicin Previously confused with Aureobacterium (which has been united with Microbacterium) Leminorella L. grimontii L. richardii UTI, bacteraemia, surgical site infection, peritonitis Imipenem, chloramphenicol, tetracycline, gentamicin Leptospira L. biflexa L. borgpetersenii L. broomii L. hongkonensis L. idonii L. inadai L. interrogans L. licerasiae L. mayottensis L. kirschneri L. noguchii L. santarosai L. weilii L. wolfii Leptospirosis Penicillin, tetracycline L. interrogans is composed of severalnamed serogroups, including: australis, bataviae, canicola, copenhageni, cynopteri, hurstbridge, hardjo, grippotyphosa, icterohaemorrhagiae, panama, pomona, pyrogenes, sejroe, tarassovi (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1325 Leptotrichia L. buccalis L. goodfellowii L. shahii L. trevisanii Bacteraemia, endocarditis β-​Lactams, metronidazole Associated with dental plaque and gingivitis. ‘L. amnionii’ from amniotic fluid does not have standing in nomenclature and may belong in the genus Sneathia Leuconostoc L. citreum L. lactis L. mesenteroides ssp. cremoris L. mesenteroides ssp. dextranicum L. mesenteroides ssp. mesenteroide L. pseudomesenteroides Meningitis, bacteraemia, pulmonary infection Penicillin and gentamicin or clindamycin Vancomycin-​resistant Listeria L. ivanovii L. grayi L. monocytogenes L. seeligeri Septicaemia, meningitis, intrauterine infection, enteric infection Ampicillin and gentamicin [Listonella damsela—​see Photobacterium damselae] Luteibacter L. anthropi Bacteraemia Luteococcus L. peritonei L. sanguinis Peritonitis, bacteraemia Lysinibacillus L. meyeri L. massiliensis L. sphaericus (Bacillus sphaericus) M Massilia M. consociata M. haematophila (Naxibacter haematophilus) M. oculi M. timonae M. varians (Naxibacter varians) Bacteraemia, wound infection, eye infection Megasphaera M. elsdenii M. micronuciformis Endocarditis, abscess Metronidazole Mesorhizobium M. amorphae Pneumonia Methylobacterium M. extorquens M. mesophilicum (Pseudomonas mesophilica) Bacteraemia, CAPD peritonitis, UTI, septic arthritis Ureidopenicillins, imipenem, aminoglycosides, chloramphenicol, fluoroquinolones Detected in aortic aneurysm Microbacterium (Aureobacterium) M. arborescens M. binotii M. imperiale (CDC coryneform groups A-​4 and A-​5) M. liquefaciens (Aureobacterium liquefaciens) M. oxydans M. paraoxydans M. resistens M. trichothecenolyticum Endophthalmitis, UTI, endocarditis, soft tissue infection, hypersensitivity pneumonitis, meningitis, CAPD peritonitis, bacteraemia Glycopeptides, β-​lactams, chloramphenicol, gentamicin M. resistens is vancomycin-​resistant. Microbacterium isolates have been misidentified as ‘Corynebacterium aquaticum’ a taxon now known as Leifsonia aquatica Micrococcus M. luteus M. lytae Bacteraemia, endocarditis, septic arthritis Vancomycin, penicillin, rifampicin From skin flora. Common specimen contaminants Mitsuokella M. multocida (Bacteroides multiacidus) Role as human pathogen poorly defined Mobiluncus M. curtisii curtisii M. curtisii holmesii M. mulieris Endometritis, chorioamnionitis Ampicillin, cephalosporins, clindamycin Associated with bacterial vaginosis Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1326 Moellerella M. wisconsensis Diarrhoea Of uncertain significance Mogibacterium M. diversum M. neglectum Endodontic infection Moheibacter M. brevis (Empedobacter brevis, Flavobacterium breve) Endophthalmitis, bacteraemia, UTI Broad spectrum cephalosporins Carbapenem-​resistant Moraxella M. atlantae M. catarrhalis (Branhamella catarrhalis) M. lacunata M. nonliquefaciens M. osloensis Conjunctivitis, wound infection, endocarditis, abscesses, osteomyelitis, respiratory infections, endocarditis, bacteraemia Penicillin, cefuroxime Penicillin resistance has been reported. Some authors retain Branhamella catarrhalis [Moraxella phenylpyruvica—​see Psychrobacter phenylpyruvicus] [Moraxella urethralis—​see Oligella urethralis] Morganella M. morganii morganii M. morganii sibonii Bacteraemia, UTI, wound infection β-​Lactams, aminoglycosides Susceptibilities vary. Morganella pyschrotolerans associated with histamine fish poisoning Moryella M. indoligenes Abscess Murdochiella M. asaccharolytica Wound infection Mycobacterium M. chimaera M. chubuense M. colombiense M. conceptionense M. confluentis M. conspicuum M. cookii M. cosmeticum M. doricum M. engbaekiiM. elephantis M. europaeum M. flavescens M. florentinum M. fortuitum M. fragae M. gadium M. gastri M. genavense M. goodii M. gordonae M. haemophilum M. hassiacum M. heckeshornense M. heidelbergense M. heraklionense M. hodleri M. holsaticum M. houstonense M. immunogenum M. insubricum M. interjectum M. intracellulare M. iranicum ‘M. jacuzzii’ M. kansasii M. koreense M. kubicae M. kumamotonense M. kyorense M. lacus M. lentiflavum M. leprae M. longobardum Isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin, azithromycin, clarithromycin, quinolones, dapsone, clofazimine, imipenem, co-​trimoxazole, amikacin Many Mycobacterium spp. have been associated with infection. M. tuberculosis, M. africanum, and M. bovis are the agents of tuberculosis. M. scrofulaceum causes cervical adenitis. The agent of Buruli ulcer is M. ulcerans. M. marinum causes fish-​tank granuloma. M. lepraecauses leprosy. M. malmoense, M. szulgai, M. shimoidei, M. kansasii, and M. xenopi cause pulmonary infection. M. intracellulareand M. avium cause systemic infection mainly in immunocompromised patients. The rapid growers, M. chelonae, M. abscessus, and M. fortuitum cause local postinoculation injury and systemic infection Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1327 M. mageritense M. malmoense M. mantenii M. marinum M. marseillense M. microgenicum M. microti M. monacense M. mucogenicum M. neoaurum M. nebraskense M. neworleansense M. nonchromogenicum M. noviomagense M. novocastrense M. palustre M. paraense M. paragordonense M. parakoreense M. paraseoulense M. parascrofulaceum M. parmense M. peregrinum M. phlei M. phocaicum M. porcinum M. riyadhense M. saskatchewanense M. scrofulaceum M. seoulense M. septicum M. senuense M. setense M. sherrisii M. shimoidei M. shinjukuense M. simiae M. smegmatis M. szulgai M. terrae M. thermoresistibile M. timonense M. triplex M. triviale M. tuberculosis M. tusciae M. ulcerans M. vaccae M. vulneris M. wolinskyi M. xenopi M. yongonense Mycoplasma M. amphoriforme M. buccale M. faucium M. fermentans M. genitalium M. hominis M. lipophilum M. orale M. penetrans M. pirum M. pneumoniae M. primatum M. salivarium M. spermatophilum Respiratory infection, postpartum fever, pyelonephritis, pelvic inflammatory disease, myocarditis, pericarditis, meningitis Tetracycline, macrolides, fluoroquinolones May be resistant to macrolides. M. pneumoniaeinfection may be complicated by haemolytic anaemia, intravascular coagulation, Stevens–​ Johnson syndrome, or erythema multiforme Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1328 M. phocicerebrale (M. phocacerebrale) Seal finger Tetracycline Other Mycoplasma spp. from seals are M. phocae and M. phocirhinis Myroides (Flavobacterium) M. odoratimimus M. odoratus UTI, wound infection Minocycline May be multiresistant N Negativicoccus N. succinicivorans Bacteraemia Penicillins Isolated from skin and soft tissue samples Neisseria N. animaloris (CDC group EF-​4a) N. canis N. weaveri (CDC group M-​5, ‘Neisseria parelongata’) N. zoodegmatis (CDC group EF4-​b) Wound infections, abscesses, endocarditis, meningitis, bacteraemia Amoxicillin Zoonoses from animal bites N. bacilliformis N. cinerea N. elongata elongata N. elongata glycolytica N. elongata nitroreductens N. flavescens N. lactamica N. mucosa N. oralis N. polysaccharea N. shayeganii N. sicca N. subflava N. wadsworthii Meningitis, bacteraemia, endocarditis, osteomyelitis Penicillin, cephalosporins Bacteraemia in AIDS reported for several species. Penicillin resistance rarely reported in commensal Neisseriaspp. N. subflava includes biovars flava, perflava, and subflava N. gonorrhoeae Gonorrhoea, septicaemia, ophthalmia neonatorum Cephalosporins Susceptibility varies geographically. The name ‘Neisseria gonorrhoeae ssp. kochii’ was proposed for isolates from conjunctivitis cases in rural Egypt N. meningitidis Septicaemia, meningitis, conjunctivitis, genital infection, epiglottitis Penicillin, cefotaxime Rifampicin, ciprofloxacin, or ceftriaxone to clear carriage Neorickettsia N. sennetsu (Erhlichia sennetsu) Sennetsu fever Doxycycline Associated with eating raw fish in Asia Nocardia N abscessus N. africana N. amikacinitolerans N. anaemiae N. aobensis N. araoensis N. arthritides N. asiatica N. asteroides N. blacklockiae N. beijingensis N. brasiliensis N. brevicatena N. carnea N. concave N. cyriacigeorgica N. elegans N. exalbida N. farcinica N. higoensis N. inohanensis N. kroppenstedtii N. kruczakiae N. mexicana N. mikamii Nocardiosis (including bacteraemia, pulmonary, and soft tissue infections) Sulphonamides,
co-​trimoxazole, amikacin, imipenem Nocardia amikacinitolerans is amikacin-​resistant Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1329 N. niigatensis N. ninae N. niwae N. nova N. otitidiscaviarum N. paucivorans N. pneumoniae N. pseudobrasiliensis N. puris N. sienata N. takedensis N. thailandensis N. terpenica N. testaceus N. transvalensis N. vermiculata N. veterana N. vulneris N. wallacei N. yamanashiensis Nocardiopsis N. dassonvillei N. synnemataformans Mycetoma, cutaneous infection, pulmonary infection, conjunctivitis Fluoroquinolones, piperacillin O Ochrobactrum (Achromobacter CDC group Vd; Achromobacter groups A, C, and D) O. anthropi O. haematophilum O. intermedium O. pseudogrignonense O. pseudointermedium Bacteraemia, endophthalmitis, liver abscess Imipenem, fluoroquinolones, aminoglycosides Nosocomial infections in debilitated patients Odoribacter O. splanchinicus (Bacteroides splanchinicus) Oerskovia O. turbata (Cellulomonas turbata) Bacteraemia, endocarditis Amikacin, co-​trimoxazole, chloramphenicol Vancomycin resistance reported Oligella O. ureolytica (CDC IVe) O. urethralis (Moraxella urethralis) UTI, septicaemia Aminoglycosides, cephalosporins Associated with urinary catheters Olsenella O. uli (Lactobacillus uli) Orientia O. tsutsugamushi (Rickettsia tsutsugamushi) Scrub typhus Tetracycline, chloramphenicol P Paenibacillus P. alvei P. konsidensis P. macerans M. massiliensis P. polymyxa P. popilliae P. provencensis P. sanguinis P. sputi P. timonensis P. urinalis P. vulneris Septicaemia, meningitis, pneumonia, UTI, wound infection Vancomycin Pannonibacter P. phragmitetus (Achromobacter groups B and E) Bacteraemia Pantoea P. agglomerans (Enterobacter agglomerans) P. ananatis P. brenneri P. conspicua P. eucrina P. dispersa P. septica Bacteraemia, endocarditis, wound infection, cellulitis, alligator-​bite infection, endophthalmitis Carbapenems, fluoroquinolones, ureidopenicillins, aminoglycosides Susceptibilities vary. May be multiresistant Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1330 Parabacteroides P. distasonis P. goldsteinii (Bacteroides goldsteinii) P. merdae Abscesses Metronidazole Parachlamydia P. acanthamoebae Humidifier fever Paracoccus P. sanguinis P. yeei Bacteraemia Ampicillin, cephalosporins, ciprofloxacin Parascardovia P. denticolens (Bifidobacterium denticolens) Dental caries Parvimonas P. micra (Peptostreptococcus micros) Pasteurella P. aerogenes P. bettyae P. canis P. dagmatis P. gallinarum P. haemolytica P. multocida multocida P. multocida gallicida P. multocida septica P. pneumotropica P. stomatis Wound infection, septicaemia, abscesses, pneumonia, endocarditis, meningitis Penicillin, tetracycline, ciprofloxacin Pasteurella infections in humans relate to species usually associated with animals. There may be no history of an animal bite or contact [Pasteurella ureae—​see Actinobacillus ureae] Pediococcus P. acidilactici P. damnosus P. dextrinicus P. parvulus P. pentosaceus Bacteraemia, abscesses, pulmonary infection Imipenem, gentamicin, chloramphenicol Debilitated hospital patients. Resistant to vancomycin Peptococcus P. niger Abdominal sepsis Penicillin, clindamycin Peptoniphilus (Peptostreptococcus) P. asaccharolyticus P. coxii P. duerdenii P. gorbachii P harei P. indolyticus P. ivorii P. koenoeneniae P. lacrimalis P. olsenii P. tyrrelliae Mixed anaerobic infections, abscesses β-​Lactams, metronidazole, chloramphenicol Peptostreptococcus P. anaerobius P. stomatis ‘P. trisimilis’ Mixed anaerobic infections, abscesses, endocarditis β-​Lactams, metronidazole, chloramphenicol See also Peptoniphilus, Anaerococcus, Finegoldia Phocaeicola P. abscessus Brain abscess Photobacterium P. damselae (Listonella damsela and Vibrio damsela) Necrotizing wound infection, bacteraemia Penicillins, tetracycline, chloramphenicol Infection associated with penetrating fish injury. May require debridement Photorhabdus (Xenorhabdus) P. luminescens Bacteraemia, wound infection Cefoxitin, oxacillin, gentamicin Plesiomonas P. shigelloides Gastroenteritis, septicaemia, meningitis, endophthalmitis Ciprofloxacin, trimethoprim, cephalosporins Infections associated with contaminated food and water Porphyromonas (Bacteroides) P. asaccharolytica P. bennonis P. cangingivalis P. canoris P. cansulci P. catoniae P. circumdentaria P. crevioricanis P. endodontalis Mixed anaerobic infections at various sites, periodontitis, human and animal bites Metronidazole, ureidopenicillins, amoxicillin/​clavulanate, carbapenems, cephalosporins, chloramphenicol Members of the oral flora of humans and animals Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1331 P. gingivalis P. gingivicanis P. levii P. macacae P. somerae P. uenonis Prevotella (Bacteroides) P. amnii P. bergensis P. bivia P. buccae P. buccalis P. corporis P. dentalis P. denticola P. disiens P. enoeca P. heparinolytica P. intermedia P. loeschii P. melaninogenica P. multiformis P. multisaccharivorax P. nanceiensis P. nigrescens P. oralis P. oris P. oulorum P. pleuritidis P. timonensis P. veroralis P. zoogleoformans Abscesses, bacteraemia, wound infection, bite infections, genital tract infections, periodontitis, endodontic infection Metronidazole, amoxicillin/​ clavulanate, ureidopenicillins, carbapenems, cephalosporins, clindamycin, chloramphenicol A genus that includes the well-​known former Bacteroides melaninogenicusand allied species of anaerobes Propionibacterium P. acnes P. avidum P. granulosum P. propionicum (Arachnia propionicus) Abscesses, endocarditis, bacteraemia, septic arthritis, endophthalmitis Glycopeptides, penicillin, macrolides Associated with acne vulgaris Propionimicrobium P. lymphophilum (Propionibacterium lymphophilum) UTI Isolated from lymph nodes in Hodgkin’s disease Proteus P. mirabilis P. penneri P. vulgaris UTI, bacteraemia, wound infection, abscesses β-​Lactams, aminoglycosides, fluoroquinolones Susceptibilities vary Providencia P. alcalifaciens P. rettgeri P. rustigianii P. stuartii UTI, wound infection, bacteraemia β-​Lactams, aminoglycosides, fluoroquinolones Susceptibilities vary. P. alcalifaciens has been associated with gastroenteritis Pseudoflavonifractor P. capillosus (Bacteroides capillosus) [Pseudomonas acidivorans—​see Delftia acidivorans] Pseudomonas P. aeruginosa P. alcaligenes P. chlororaphis P. fluorescens P. mendocina P. monteilii P. mosselii P. oleovorans (P. pseudalcaligenes) P. otitidis P. pertocinogena P. putida P. stutzeri Bacteraemia, UTI, wound infection, abscesses, septic arthritis, conjunctivitis, endocarditis, meningitis, otitis Ureidopenicillins, aminoglycosides, ceftazidime, fluoroquinolones, carbapenems Nosocomial infections associated with invasive devices in debilitated patients. Nosocomial outbreaks reported. May be multiresistant Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1332 [Pseudomonas cepacia—​see Burkholderia cepacia] [Pseudomonas diminuta—​see Brevundimonas diminuta] [Pseudomonas mallei—​see Burkholderia mallei] [Pseudomonas maltophilia—​see Stenotrophomonas maltophilia] [Pseudomonas mesophilica—​see Methylobacterium mesophilicum] P. luteola (Chryseomonas luteola) Bacteraemia, endocarditis, CAPD peritonitis Ureidopenicillins, ceftazidime, ciprofloxacin, aminoglycosides P. oryzihabitans (Flavimonas oryzihabitans) Septicaemia, eye infection, CAPD peritonitis Ampicillin, tetracycline, gentamicin, cefotaxime [Pseudomonas paucimobilis—​see Sphingomonas paucimobilis] [Pseudomonas pickettii—​see Ralstonia pickettii] [Pseudomonas pseudomallei—​see Burkholderia pseudomallei] [Pseudomonas putrefaciens—​see Shewanella putrefaciens] [Pseudomonas terrigena—​see Comamonas terrigena] [Pseudomonas testosteroni—​see Comamonas testosteroni] [Pseudomonas vesicularis—​see Brevundimonas vesicularis] Pseudonocardia P. autotrophica (Amycolata autotrophica) Role as pathogen uncertain Pseudoramibacter P. alactolyticus Periodontal disease, wound infection, abscesses Penicillin, clindamycin, chloramphenicol Psychrobacter P. faecalis P. immobilis P. phenylpyruvicus (Moraxella phenylpyruvica) P. pulmonis P. sanguinis Meningitis, bacteraemia, eye infection Penicillins, aminoglycosides, chloramphenicol R Rahnella R. aquatilis UTI, septicaemia Ciprofloxacin Immunocompromised patients Ralstonia R. insidiosa R. mannitolilytica R. pickettii (Pseudomonas pickettii) R. taiwanensis Meningitis, peritonitis, bacteraemia, UTI, pulmonary infection Co-​trimoxazole, imipenem, ceftazidime, quinolones Raoultella (Klebsiella) R. ornithinolytica R. planticola R. terrigena Bacteraemia, UTI, surgical sepsis, pancreatitis Cephalosporins, carbapenems, aztreonam, quinolones, aminoglycosides β-​Lactamase producers. Associated with histamine (scombrotoxin) fish poisoning ‘Rasbo’ ‘R. bacterium’ Pneumonia, pericarditis Proposed name does not have standing in nomenclature Rhodococcus R. hoagi, R. equi, Corynebacterium equi) Bacteraemia, osteomyelitis, lung abscesses Vancomycin, erythromycin, aminoglycosides In immunocompromised patients, including AIDS Rickettsia R. africae R. akari R. australis R. conorii R. felis R. honei R. japonica ‘R. mongolotimonae’ R. prowazekii R. rickettsiae R. sibirica R. slovaca R. typhi Rickettsial spotted fever, tick typhus, tick-​bite fever, rickettsialpox Tetracycline Transmitted by arthropods. Agents of Astrakhan fever, Israeli tick typhus, and Thai tick typhus await designation of scientific names. Other Rickettsia spp. are of uncertain clinical significance Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1333 Roseomonas R. cervicalis R. gilardii ssp. gilardii R. gilardi ssp. rosea R. mucosa Bacteraemia, wound infection, peritonitis Aminoglycosides, imipenem, ciprofloxacin, ticarcillin-​clavulanate [Roseomonas fauriae—​see Azospirillum brasilense] Rothia R. dentocariosa Endocarditis, abscesses Penicillin and gentamicin R. mucilaginosa (Micrococcus mucilaginosus) (Stomatococcus mucilaginosus) Endocarditis, meningitis, neutropenic sepsis, necrotizing fasciitis Glycopeptides, imipenem, rifampicin, ceftriaxone Ruminococcus R. flavefaciens R. hansenii (Streptococcus hansenii) R. luti R. productus (Peptostreptococcus productus) Abdominal sepsis, abscesses Penicillins S Saccharopolyspora S. rosea Salmonella S. bongori​ S. choleraesuis ssp. arizonae S. choleraesuis ssp. choleraesuis S. choleraesuis ssp. diarizonae S. choleraesuis ssp. houtenae S. choleraesuis ssp. indica S. choleraesuis ssp. salamae S. enteritidis S. paratyphi S. typhi S. typhimurium Gastroenteritis, enteric fever, osteomyelitis β-​Lactams, fluoroquinolones, chloramphenicol Salmonella nomenclature is complicated by the existence of two sets of names, both of which have standing in nomenclature and which reflect two different schemes of classification. One scheme supported by phylogenetic data recognizes three distinct species, S. subterranea (an environmental organism), S. bongori (associated with reptiles and a cause of diarrhoea in humans) and S. enterica (which includes several subspecies). Users of this scheme recognize several clinically important taxa (including the agent of typhoid fever) as serovars of S enterica ssp. Enterica—​e.g. Salmonella enterica ssp. enterica serovar Typhi. As a form of shorthand, the serovars can be written thus: Salmonella Typhi, Salmonella Paratyphi, and so on. The alternative scheme recognizes S. typhi, S. paratyphi, S. enteritidis, S. typhimurium and S. choleraesuis (including several subspecies). Names from both schemes are included in the table ​S. enterica ssp. arizonae S. enterica ssp. diarizonae S. enterica ssp. enterica S. enterica ssp. houtenae S. enterica ssp. indica S. enterica ssp. salamae Scardovia S. inopinata (Bifidobacterium inopinatum) S. wiggsiae Dental caries, wound infection Selenomonas S. artemidis S. dianae S. flueggei S. infelix S. noxia S. sputigena Bacteraemia, lung abscess Clindamycin, chloramphenicol, metronidazole Malignancy and alcohol abuse reported as risk factors for infection [Serpulina—​see Brachyspira] Serratia S. ficaria S. fonticola S. grimesii S. liquefaciens Septicaemia, abscesses, burn infections, osteomyelitis Imipenem, aminoglycosides, fluoroquinolones, ureidopenicillins, ceftazidime Nosocomial outbreaks reported. May be multiresistant. At time of writing a proposal to use the name S. rubidae in place of S. rubidaea has not been validly published Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1334 S. marcescens S. odorifera S. plymuthica S. proteamaculans S. quinivorans S. rubidaea Shewanella S. algae S. putrefaciens (Alteromonas putrefaciens) (Pseudomonas putrefaciens) Abdominal sepsis, meningitis, bacteraemia Ampicillin, cefotaxime, gentamicin, chloramphenicol Debilitated patients Shigella S. boydii S. dysenteriae S. flexneri S. sonnei Enteric infection Co-​trimoxazole, fluoroquinolones Simkania S. negevensis Bronchiolitis, pneumonia Slackia S. exigua (Eubacterium exiguum) Periodontitis Sneathia S. sanguinegens (Leptotrichia sanguinegens = L. microbii) Sphingobacterium (Flavobacterium) S. multivorum S. spiritivorum S. thalpophilum Bacteraemia, pulmonary infection Co-​trimoxazole, chloramphenicol, tetracycline, cephalosporins, quinolones [Sphingobacterium mizutae—​see Flavobacterium mizutaii] Sphingomonas S. parapaucimobilis S. paucimobilis (Pseudomonas paucimobilis) S. sanguinis (S. sanguis) S. yanoikuyae Septicaemia, UTI, wound infections, CAPD peritonitis Ceftazidime, aminoglycosides Nosocomial infections Spirillum ‘S. minus’ Rat bite fever Penicillin Streptobacillus moniliformis is also a rat bite fever agent. The name ‘Spirillum minus’ does not have standing in nomenclature Staphylococcus S. argenteus S. aureus S. auricularis S. capitis capitis S. capitis ureolyticus S. caprae S. cohnii cohnii S. cohnii urealyticus S. delphini S. epidermidis S. equorum S. gallinarum S. haemolyticus S. hominis hominis S. hominis novobiosepticius S. hyicus S. intermedius S. jettensis S. lugdunensis S. massiliensis S. pasteuri S. petrasii S. pettenkoferi S. saccharolyticus S. saprophyticus S. schleiferi schleiferi S. schleiferi coagulans S. sciuri S. simulans S. vitulinus Bacteraemia, wound infection, endocarditis, catheter-​related sepsis, UTI, toxic shock syndrome, food poisoning, eye infection, osteomyelitis Glycopeptides, β-​lactams, aminoglycosides, tetracycline, macrolides, rifampicin, fluoroquinolones, daptomycin, linezolid, fusidic acid, mupirocin Staphylococci are surface commensals of humans and animals. S. aureus is also a major pathogen, causing focal and systemic sepsis, toxic shock syndrome, and food poisoning. S. epidermidis infection is often associated with foreign bodies (e.g. catheters and implants). S. saprophyticus causes UTI. S. lugdunensis is a rare cause of endocarditis. S. intermedius, S. hyicus, and others are from animals. Susceptibilities are variable but glycopeptide resistance is as yet rare Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1335 S. pulvereri S. warneri S. xylosus Stenotrophomonas S. maltophilia (Pseudomonas maltophila) (Xanthomonas maltophila) (Stenotrophomonas africana) Bacteraemia, meningitis, wound infection, UTI, pneumonia Fluoroquinolones, chloramphenicol, co-​trimoxazole Resistance to aminoglycosides, penicillins, and carbapenems reported [Stomatococcus mucilaginosus—​see Rothia mucilaginosa] Stomatobaculum S. longum Dental plaque Streptobacillus S. moniliformis ​S. hongkongensis Rat bite fever, Haverhill fever Qunisy, septic arthritis Penicillin, erythromycin ​ ‘Spirillum minus’ is also a causative agent of rat bite fever Streptococcus S. acidominimus Pneumonia, pericarditis, meningitis β-​Lactams From cattle S. agalactiae S. canis S. dysgalactiae dysgalactiae S. dysgalactiae equisimilis S. equi equi S. equi zooepidemicus S. hongkongensis S. iniae (S. shiloi) S. porcinus S. pseudoporcinus S. pyogenes S. urinalis Pharyngitis, bacteraemia, pyogenic infection, necrotizing infection, septic arthritis, UTI, glomerulonephritis, meningitis β-​Lactams, macrolides S. pyogenes (Lancefield group A), S. agalactiae (group B), and S. dysgalactiae equisimilis (groups C and G) are commensals and pathogens of humans. S. pseudoporcinus is known to colonize the female genital tract and has been reported to cause wound infection. S. iniae and S. hongkongensis are associated with fish. Others are from mammals S. anginosus S. constellatus constellatus S. constellatus pharyngis S. intermedius Abscesses, bacteraemia, endocarditis, pharyngitis β-​Lactams, macrolides Often termed ‘S. milleri’ or microaerophilic streptococci. From human oral flora S. equinus (S. bovis) S. gallolyticus ssp. gallolyticus S. gallolyticus ssp. pateurianus S. infantarius ssp. coli S. infantarius ssp. infantarius S. lutetiensis S. pasteurianus Endocarditis, CAPD peritonitis β-​Lactams (plus gentamicin for endocarditis) Intestinal streptococci from animals and humans. Some taxonomic problems relating to this group (the ‘bovis’ streptococci) await resolution S. criceti S. mutans S. ratti S. sobrinus Dental caries, endocarditis β-​Lactams From the tooth-​surface flora of humans and mammals S. cristatus S. gordonii S. massiliensis S. mitis S. oralis S. parasanguinis S. salivarius S. sanguinis S. sinensis S. tigurinus S. vestibularis Bacteraemia, endocarditis, wound infection β-​Lactams, macrolides Human oral streptococci including taxa sometimes known as the ‘viridans streptococci’. Streptococcus tigurinus has been isolated from patients with infective endocarditis, spondylodiscitis, bacteraemia, meningitis, prosthetic joint infection, and empyema S. pneumoniae S. pseudopneumoniae Pneumonia, bacteraemia, sinusitis, peritonitis, otitis, conjunctivitis β-​Lactams, macrolides, chloramphenicol Penicillin resistance locally common S. suis Meningitis β-​Lactams Associated with pig contact Streptomyces S. albus S. anulatus ‘S. paraguayensis’ S. somaliensis Actinomycetoma Dapsone, co-​trimoxazole Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

section 8  Infectious diseases 1336 S. bikiniensis S. griseus Bacteraemia, abscess, pericarditis, endocarditis Vancomycin, tetracycline, penicillin Treatment options poorly defined Succinivibrio S. dextrinosolvens Bacteraemia, pneumonia Penicillin From faecal and gingival flora Sutterella S. wadsworthensis Appendicitis, peritonitis, abscesses, osteomyelitis Amoxicillin/​clavulanate, ticarcillin/​clavulanate, meropenem, ceftriaxone One-​third of isolates reported to be metronidazole resistant Suttonella S. indologenes (Kingella indologenes) Endocarditis, eye infection Penicillin (plus gentamicin for endocarditis) T Tannerella T. forsythensis (T. forsythia, T. forsythus) Endodontic infection [Tatlockia maceachernii—​see Legionella maceachernii] [Tatlockia micdadei—​see Legionella micdadei] Tatumella T. ptyseos T. saanichensis Bacteraemia, UTI Ampicillin, tetracycline, chloramphenicol, gentamicin T. saanichensis was isolated from a patient with cystic fibrosis Terrisporobacter T. glycolicus (Clostridium glycolicum) Abdominal sepsis Tetragenococcus T. solitarius (Enterococcus solitarius) Tissierella T. praeacuta (Bacteroides praeacuta) (Clostridium hastiforme) Bacteraemia Metronidazole Trabulsiella T. guamensis Diarrhoea Co-​trimoxazole, gentamicin, chloramphenicol Role as possible pathogen uncertain Treponema T. amylovorum T. denticola T. lecithinolyticum T. maltophilum T. medium T. parvum T. pectinovorum T. putidum T. scoliodontum T. socranskii ‘T. vincentii’ Associated with periodontal disease. Role as potential pathogens unclear ‘T. carateum’ Pinta Penicillin Name does not have standing in nomenclature T. minutum ‘T. phagedenis’ ‘T. refringens’ From genital flora. Considered
non​pathogenic but have been isolated from genital lesions T. pallidum ‘T. pallidum endemicum’ Syphilis Penicillin ‘T. pallidum endemicum’ is the agent of non​venereal endemic syphilis T. pertenue (‘T. pallidum pertenue’) Yaws Penicillin Tropheryma T. whipplei (T. whippelii) Whipple’s disease Uncultured organism Trueperella (Arcanobacterium, Actinomyces) T. bernardiae UTI, septicaemia, septic arthritis β-​Lactams Previously known as CDC coryneform group 2 T. pyogenes Septic arthritis β-​Lactams Tsukamurella T. inchonensis T. paurometabola T. pulmonis T. strandjordii (T. strandjordae) T. tyrosinosolvens Septicaemia, cutaneous infections, lung infections β-​Lactam (plus aminoglycoside) Line-​associated infections in debilitated patients. T. pulmonis isolated from the sputum of a tuberculosis patient Turicella T. otitidis Otitis, cervical abscess Glycopeptides, β-​lactams U Ureaplasma U. parvum U. urealyticum Urethritis Tetracycline, erythromycin Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups) (continued)

8.6.47  A checklist of bacteria associated with infection in humans 1337 V Vagococcus V. fluvialis Ampicillin, vancomycin cefotaxime Possible role as pathogen poorly defined Varibaculum V. cambriensis Abscesses Veillonella V. atypical V. dentocariosi V. dipsar V. montpellierensis V. parvula V. rogosae Abscesses, bacteraemia Metronidazole V. dentocariosi and V. rogosae associated with tooth decay and dental plaque Vibrio V. alginolyticus Wound infection, ear infection Chloramphenicol, tetracycline Infection associated with aquatic exposure V. cholerae Cholera Tetracycline V. cincinnatiensis Bacteraemia Moxalactam, chloramphenicol, cephalosporins Risk factors for infection not defined [Vibrio damsela—​see Photobacterium damselae] V. fluvialis V. furnissii V. metschnikovii V. mimicus V. parahaemolyticus Diarrhoea, septicaemia Tetracycline, chloramphenicol Infection associated with ingestion of contaminated water or shellfish V. harveyi (V. carchariae) Wound infection Cephalosporins, chloramphenicol, gentamicin Infection associated with shark bite. May require debridement [Vibrio hollisae—​see Grimontia hollisae] V. vulnificus Wound infection, septicaemia, meningitis, endometritis Tetracycline, penicillins, gentamicin, chloramphenicol Risk factors include aquatic exposure and penetrating fish injury. May require debridement W Wautersiella W. falsenii Bacteraemia, wound infection Phylogenetic data support inclusion in the genus Moheibacter [Weeksella zoohelcum—​see Bergeyella zoohelcum] Weissella W. confusa Endocarditis Williamsia W. muralis Pulmonary infection Wohlfahrtiimonas W. chitiniclastica Bacteraemia Ceftriaxone Associated with myiasis Wolbachia W. sp. filariasis doxycycline Endosymbiont of filarial nematodes [Wolinella curva—​see Campylobacter curvus] [Wolinella recta—​see Campylobacter rectus] X Xanthomonas X. campestris Bacteraemia [Xenorhabdus luminescens—​see Photorhabdus luminescens] Y Yersinia Y. aldovae Y. bercovieri Y. enterocolitica Y. frederiksenii Y. intermedia Y. kristensenii Y. mollaretii Y. pseudotuberculosis Y. rohdei Y. similis Y. wautersii Enterocolitis, soft tissue infections, mesenteric lymphadenitis Tetracycline, chloramphenicol, aminoglycosides, fluoroquinolones, cephalosporins Medical significance of manyYersinia spp. is unclear. Antibiotic treatment is not indicated for uncomplicated enteric infection Y. pestis Plague Streptomycin, tetracycline Yokenella Y. regensburgei (Koserella trabulsii) Bacteraemia, wound infection Aminoglycosides, chloramphenicol CAPD, continual ambulatory peritoneal dialysis; CDC, Centers for Disease Control and Prevention; sp. species; ssp. subspecies; UTI, urinary tract infection. Table 8.6.47.1  Continued Nomenclature Associated infections Reported susceptibilities and treatments Notes Genus Species and subspecies (synonyms, CDC alphanumeric groups)

8.6.5 Meningococcal infections 1010

8.6.5 Meningococcal infections 1010

section 8  Infectious diseases 1010 Koser CU, et al. (2013). Rapid whole-​genome sequencing for in- vestigation of a neonatal MRSA outbreak. N Engl J Med, 366, 2267–​75. Lal Y, Assimacopoulos AP (2011). Two cases of daptomycin-​induced eosinophilic pneumonia and chronic pneumonitis. Clin Infect Dis, 50, 737–​40. Lee S, et  al. (2011). Is cefazolin inferior to nafcillin for treatment of methicillin-​susceptible Staphylococcus aureus bacteremia? Antimicrob Agents Chemother, 55, 5122–​6. Lipsky BA, Itani K, Norden C (2004). Treating foot infections in diabetic patients:  a randomized, multicenter, open-​label trial of linezolid versus ampicillin–​sulbactam/​amoxicillin-​clavulanate.
Clin Inf Dis, 38, 17–​24.] Liu C, et  al. (2011). Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-​ resistant Staphylococcus aureus infections in adults and children. Clin Inf Dis, 52, e18–​55. Markowitz N, Quinn EL, Saravolatz LD (1992). Trimethoprim-​ sulfamethoxazole compared with vancomycin for the treatment of Staphylococcus aureus infection. Ann Int Med, 117, 390–​8. Mermel LA, et  al. (2001). Guidelines for the management of intravascular catheter-​related infections. Clin Inf Dis, 32,
1249–​72. Miller WR, Seas C, Carvajal LP, et al. The Cefazolin Inoculum Effect Is Associated With Increased Mortality in Methicillin-Susceptible Staphylococcus aureus Bacteremia. Open forum infectious diseases 2018;5:ofy123. Mulligan ME, et  al. (1993). Methicillin-​resistant Staphylococcus
aureus: a consensus review of the microbiology, pathogenesis, and epidemiology with implications for prevention and management. Am J Med, 94, 313–​28. Parente DM, Cunha CB, Mylonakis E, Timbrook TT (2018). The Clinical Utility of Methicillin-Resistant Staphylococcus aureus (MRSA) Nasal Screening to Rule Out MRSA Pneumonia: A Diagnostic Meta- analysis With Antimicrobial Stewardship Implications. Clin Inf Dis, 67, 1–7. Paul M, et  al. (2015). Trimethoprim-​sulfamethoxazole versus vancomycin for severe infections caused by meticillin resistant Staphylococcus aureus:  randomised controlled trial. BMJ, 350, h2219. Rao SN, et  al. (2015). Treatment outcomes with cefazolin versus oxacillin for deep-​seated methicillin-​susceptible Staphylococcus aureus bloodstream infections. Antimicrob Agents Chemother, 59, 5232–​8. Ruhe JJ, et al. (2005). Use of long-​acting tetracyclines for methicillin-​ resistant Staphylococcus aureus infections: case series and review of the literature. Clin Inf Dis, 40, 1429–​34. Safdar N, Fine JP, Maki DG (2005). Meta-​analysis:  methods for diagnosing intravascular device-​related bloodstream infection. Ann Int Med, 142, 451–​66. Shorr AF, Kunkel MJ, Kollef M (2005). Linezolid versus vancomycin for Staphylococcus aureus bacteraemia: pooled analysis of random- ized studies. J Antimicrob Chemother, 56, 923–​9. Stevens DL, et al. (2005). Practice guidelines for the diagnosis and management of skin and soft-​tissue infections. Clin Inf Dis, 41, 1373–​406. Talan DA, et  al. (2016). Trimethoprim-Sulfamethoxazole versus Placebo for Uncomplicated Skin Abscess. N Engl J Med, 374, 823–32. Thwaites GE, et al. (2018). Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double- blind, placebo-controlled trial. Lancet, 391, 668–78. Truong J, Veillette JJ, Forland SC (2018). Outcomes of Vancomycin plus a beta-Lactam versus Vancomycin Only for Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia. Antimicro Agents Chemother, 62, e01554–17. Van Loo I, et  al. (2007). Emergence of methicillin-​resistant Staphylococcus aureus of animal origin in humans. Emerg Infect Dis, 13, 1834–​9. Wang SK, et  al. (2018). Prevalence of a Cefazolin Inoculum Effect Associated with blaZ Gene Types among Methicillin-Susceptible Staphylococcus aureus Isolates from Four Major Medical Centers in Chicago. Antimicrob Agents Chemother, 62, e00382–18. Wertheim HF, et al. (2004). Mupirocin prophylaxis against nosoco- mial Staphylococcus aureus infections in nonsurgical patients:  a randomized study. Ann Int Med, 140, 419–​25. Wertheim HF, et  al. (2004). Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-​ carriers. Lancet, 364, 703–​5. Wunderink RG, et  al. (2012). Linezolid in methicillin-​resistant Staphylococcus aureus nosocomial pneumonia: a randomized, con- trolled study. Clin Infec Dis, 54, 621–​9. Wyllie DH, Crook DW, Peto TE (2006). Mortality after Staphylococcus aureus bacteraemia in two hospitals in Oxfordshire, 1997–​2003:
cohort study. BMJ, 333, 281. Zimmermann B 3rd, Mikolich DJ, Ho G Jr (1995). Septic bursitis. Semin Arthritis Rheum, 24, 391–​410. 8.6.5  Meningococcal infections Petter Brandtzaeg ESSENTIALS Neisseria meningitidis is an obligate human Gram-​negative diplococcus. It is carried in the nasopharynx by about 3–​10% of people, with most strains being harmless and inducing immunity. Pathogenic strains usually belong to specific clones that are en- capsulated, express pili, and the major porin, PorA. Serogroups A, B, and C usually account for more than 90% of all invasive isolates. Epidemiology Asymptomatic young adolescents (Africa) and young adults (in- dustrialized countries) are the main reservoir. Meningococci are transmitted by droplets and susceptible people usually develop the first symptoms within 2 to 4 days. The incidence of disease is highest during the first 4 years of life, with a secondary lower peak in adolescents. Pathogenic strains tend to cause single
cases or small clusters in industrialized countries, whereas they cause large outbreaks in developing countries, particularly in the meningitis belt of Africa. Host factors predisposing to invasive
disease include (1) lack of protective antibodies, (2) defects in the

8.6.5  Meningococcal infections 1011 complement system, (3) HIV infection and (4) polymorphisms of complement factor H. Clinical features and prognosis Initially, N. meningitidis induces bacteraemia, with growth velocity in the circulation a major determinant of the clinical presentation and outcome. It has the propensity to invade the meninges. Low graded growth in the cerebrospinal fluid is often present in patients lacking distinct symptoms of meningitis. The two major clinical presenta- tions are meningitis and septic shock. Meningitis—​the commonest presentation (50%); preceded by low-​ grade meningococcaemia (<103/​ml). After transition to the subarach- noid space the meningococci proliferate to high levels (106–​109/​ml) in the cerebrospinal fluid. Clinically the patients develop fever, subse- quently a petechial rash (30–​80% of cases) and increasing symptoms of meningitis. If adequately treated with antibiotics, case fatality is less than 1–​2% in industrialized countries, but higher in developing countries. Brain oedema leading to herniation of the cerebellum is the main cause of death. Neurosensory hearing loss is the major complication. Septic shock—​symptoms develop in 30% of European patients. The septic shock is preceded by high grade meningococcaemia (106–​108/​ml). The massive bacterial proliferation leads to rapidly escalating endotoxin levels in plasma and large organs. Endotoxin triggers the innate immune system inappropriately. Within 12 h of initial symptoms the patient may have persistent circulatory failure and severe coagulopathy. Subsequently they develop extensive skin haemorrhages, thrombosis of the extremities causing gangrene, and impaired renal, adrenal, and pulmonary function. Mortality is high (16–​52%). Mild meningococcaemia—​in European countries, 20% of cases present with fever and usually petechial or macular rash, but without marked signs of meningitis or shock. The meningococcaemia is low grade (<104/​ml) and often transient. Occasional complica- tions include pericarditis, arthritis, ocular infection, or chronic meningococcaemia. Mortality is low (0–​5%) and usually related to late debut of shock or cerebellar herniation. Diagnosis Intra-​ and extracellular diplococci can be observed in the cerebro- spinal fluid, peripheral blood buffy coat (fulminant septicaemia), and biopsies of haemorrhagic skin lesions using Gram or acridine orange stains. N. meningitidis can be grown from blood culture and swabs from the nasopharynx/​tonsils. Polymerase chain re- action methods are increasingly used to detect and classify N. meningitidis in blood, cerebrospinal fluid, other bodily fluids, and skin biopsies. Treatment Aside from supportive care, appropriate antibiotic treatment should be started immediately in suspected cases of meningococcal in- fection: this should not be delayed while the patient is transferred to hospital, or for the results of investigations to become available. Ceftriaxone or benzylpenicillin (intravenously or intramuscularly) re- mains the drugs of choice in most countries; chloramphenicol are also effective. Prevention Vaccination—​conjugate vaccines comprising serogroup A, C, Y, and W polysaccharide are effective from 2  months of age. Two vaccines covering most serogroup B strains are marketed; one (Bexsero®) in Europe, Canada, Australia, and United States, the other (Trumenba®) in United States and Europe. A  single sero- group A conjugate vaccine (MenAfriVac®) has had a major impact on incidence and carriage in sub-​Saharan Africa. Secondary prophylaxis—​health authorities in most countries advise that close contacts of cases of meningococcal disease have eradi- cation treatment (e.g. with a single dose of ciprofloxacin 500 mg or ofloxacin 400 mg). During pregnancy a single dose of 250 mg ceftriaxone IM and for children below 12 years of age 125 mg IM is effective. Introduction Neisseria meningitidis infection remains a major public health problem worldwide by causing clusters or epidemics of meningitis and acute lethal sepsis. Case fatality has gradually declined from 70–​90% to approximately 7–​10% in industrialized countries but has remained at this level since the introduction of antimicrobial chemotherapy in 1937. The bacterium N. meningitidis is an obligate human Gram-​negative diplococcus classified as a β-​proteobacterium and is a member of the family Neisseriaceae. Meningococci are normally located in the mucous membrane of the nasopharynx and tonsils. Invasive isolates from blood and cerebrospinal fluid or as detected in tissue biopsies are encapsulated and express pili and the major porin PorA. Capsule polysaccharides that inhibit phagocytosis and bacterial adhesion are divided into at least 13 different serogroups (A, B, C, D, E, H, I, K, L, W, X, Y, and Z). Serogroups A, B, and C usually account for more than 90% of all invasive isolates. Less than 10% of clinical isolates are from serogroups X, Y, and W. The cell wall of meningococci consists of an outer lipid bilayer, containing lipopolysaccharides (LPS, endotoxin also denoted lipooligosaccharides, LOS), lipids, and outer membrane proteins, and an inner thin peptidoglycan layer. LPS is the major inflammatory (toxic) component of N. meningitidis (Fig. 8.6.5.1). Lipoproteins and fragments of peptidoglycan are weaker inflammatory molecules. They activate the innate immune system via CD14 and the Toll-​like receptors (TLR 4), (LPS), TLR 2 (lipoproteins, peptidoglycan) and TLR 9 (bacterial DNA) located on monocytes, macrophages, and to a lesser extent neutrophils (Fig. 8.6.5.2). During growth, menin- gococci release many outer membrane vesicles containing LPS and other outer membrane molecules that trigger the innate immune system in a dose-​dependent manner. Outer membrane proteins are classified according to elec- trophoretic mobility into five major classes. PorA (class 1 pro- tein) and PorB (class 2 or 3 proteins) are cation-​selective and

section 8  Infectious diseases 1012 anion-​selective porins, respectively. PorB and PorA define sero- type and serosubtype. Several surface-​exposed proteins including PorA, neisserial adhesion A, factor H binding protein, and neisserial heparin binding antigen induce bactericidal antibodies when exposed to the human immune system. Recombinant variants of these proteins are included in the new serogroup B vaccine (Bexsero® and Trumenba). Meningococci are fastidious bacteria that readily autolyse. They grow well on blood agar, supplemented chocolate agar, trypticase soy agar, Mueller–​Hinton agar, and selective GC medium. Optimal Cytoplasmic-membrane proteins Cytoplasmic-membrane Lipooligosaccharide Periplasmic space Outer membrane Pilus Capsule Phospholipid Outer-membrane proteins Fig. 8.6.5.1  Cross-​sectional view of N. meningitidis. Reproduced from Rosenstein NE, Bradley BA, Stephens DS, Popovic T, Hughes JM (2001). Meningococcal disease. N Engl J Med, 334, 1378–​88. Copyright © 2001 Massachusetts Medical Society. TIRAP IRAK-1 TRAF-6 lαβ MD-2 CD14 ↑NF κB Nuclear transcription MyD88-dependent pathway MyD88 TLR4 TIR LBP LOS sCD14 IRF-3 Tram/Trif IFNb, NO, chemokines (IP-10, MCP-5, RANTES) MyD88-independent pathway TNfα,IL-1β, MCP-1, MIP-3a, IL-6, IL-8 Fig. 8.6.5.2  Activation of Toll-​like receptor 4 by endotoxin (lipopolysaccharides or lipooligosaccharides, LOS). Reproduced with permission from Stephens DS, Greenwood B, Brandtzaeg P (2007). Epidemic meningitis, meningococcaemia, and Neisseria meningitidis, Lancet, 369, 2196–​210.

8.6.5  Meningococcal infections 1013 growth occurs at 35–​37°C in a humid atmosphere with 5–​10% carbon dioxide. The convex colonies (diameter 1–​4 mm) are trans- parent, non​pigmented, and non​haemolytic. They produce cyto- chrome oxidase and ferment glucose and maltose, but not lactose and sucrose, to acid without gas formation. Practical handling of clinical specimens Blood culture (10 ml for adults, 2–​4 ml for infants/​children) and swabs from the nasopharynx and the tonsils are collected immedi- ately. Media for blood culture and transportation of swabs should be optimal for recovery of meningococci. Cerebrospinal fluid is best cultured by direct plating of 0.1 ml on supplemented chocolate agar or a similar medium. If direct plating is impossible or delayed, the sample should be stored at + 4°C to + 20°C but preferably at refriger- ator temperature. Recovery of live meningococci may increase if some drops of the cerebrospinal fluid are stored on a sterile swab in transport medium or injected into blood culture medium and incu- bated at 35–​37°C. Direct visualization of N. meningitidis
in clinical specimens Intracellular and extracellular diplococci can be observed in the cere- brospinal fluid, peripheral blood buffy coat (fulminant septicaemia), and biopsies of haemorrhagic skin lesions using Gram’s or acridine orange stains. Polymerase chain reaction Polymerase chain reaction (PCR) is increasingly used to detect and classify N.  meningitidis in blood, cerebrospinal fluid, joint fluid, pericardial fluid, and skin biopsies. Real-​time PCR has made it possible to quantify the total number of meningococci (i.e. live plus dead bacteria), in plasma and cerebrospinal fluid. In shock plasma, nonviable meningococci outnumber those that can be cultured by a factor 1000:1. The number of N. meningitidis DNA copies is closely correlated to the LPS levels, clinical presentation, disease severity, and outcome. Blood anticoagulated with ethylenediaminetetraacetic acid is optimal for the PCR reaction but other anticoagulants (hep- arin, citrate) or even serum have been used. Epidemiology Industrialized countries Infection presents as single cases or in small clusters. The inci- dence used to be 1 to 3 per 100 000 inhabitants per year but has declined to 0.77 per 100 000 per year (2011) in Europe and 0.12 per 100 000 per year (2010) in the United States. Strains belonging to specific clonal complexes may cause a hyperendemic situation characterized by a much higher incidence than usually observed (4–​30 per 100 000 per year). This epidemiological situation may last for more than a decade in defined geographical areas before slowly declining. Serogroup A has disappeared as a cause of sig- nificant epidemics. Outbreaks in Finland in the 1970s and in New Zealand in the 1980s were exceptions. In Europe 70–​80% of the cases are presently caused by serogroup B. Immunization of in- fants with serogroup C conjugate vaccine in United Kingdom and various countries has reduced the incidence significantly. Serogroup Y strains belonging to several clonal complexes are gradually increasing in several European countries. In the United States of America serogroups B, C, and Y accounted for approxi- mately one-​third of the cases each (Fig. 8.6.5.3). Presently sero- group Y is declining in the United States. Developing countries Large-​scale epidemics are confined to developing countries, pri- marily in sub-​Saharan Africa where the incidence approaches 10–​25 per 100 000 inhabitants per year. During epidemic peaks in Africa, as many as 500–​1000 per 100 000 inhabitants may con- tract meningococcal infections. Serogroup A and to lesser extent serogroups W and C dominate the isolates of large epidemics (Fig. 8.6.5.3). Large-​scale vaccination in sub-​Saharan Africa with the newly implemented serogroup A  conjugate vaccine (MenAfriVac®) has proven very effective virtually eradicating transmission and development of new cases (2014). Two years after mass vaccination it is still very effective. Cases of serogroup C, W, and X might still occur. Meningitis belt in sub-​Saharan Africa The area stretches from the Gambia in the west to Ethiopia in the east and includes Senegal, Guinea, Mali, Burkina Faso, Ghana, Togo, Benin, Nigeria, Niger, Chad, Cameroon, the Central African Republic, and Sudan (Fig. 8.6.5.3). Mainly serogroup A strains be- longing to a few clonal complexes cause the increased attack rate. In some of these countries large-​scale epidemics occur every 8 to 12 years. Since the 1990s serogroup W has caused epidemics in West Africa. Season In temperate climates most cases occur during the winter and early spring. In the sub-​Saharan African meningitis belt the incidence in- creases from the middle of the dry season and reaches its maximum at the end of that season (harmattan). New cases decline rapidly after the start of the rainy season. Preceding infections Preceding infections (≤2 weeks) with influenza A, other airway vir- uses, and mycoplasma have been associated with meningococcal in- fections, assuming that they influenced the nasopharyngeal barrier negatively. Whether this relation is causal or merely temporal has yet to be firmly established. Age distribution Cases are seen in all age groups; however, most occur from 0 to 4 years with a smaller peak from 13 to 20 years. During epidemics the median age appears to increase. Complement-​deficient patients may contract the infection at an older age than average. Genetic diversity N. meningitidis can exchange and incorporate DNA from other Neisseria species or closely related bacteria. Meningococci are genetically more diverse than most other human pathogens. However, strains from certain clonal complexes may persist for many decades over wide areas, retaining their pathogenicity. Strains from seven clonal complexes have predominated since the late 1960s.

section 8  Infectious diseases 1014 Nasopharyngeal colonization Upper respiratory tract mucosa is the natural habitat of N. men- ingitidis. It is spread from person to person by droplets and direct mucosal contact. Most colonizing meningococci are non​pathogenic and are genetically and phenotypically different from virulent inva- sive strains. Only a minority of those colonized with virulent strains will develop invasive disease. Based on carriage and incidence data from Norway, ≤1% of those carrying hypervirulent B or C clones developed clinical disease. Colonization is asymptomatic; it induces local and systemic immune responses within 1–​2 weeks. Carriage Cross-​sectional studies in England and Norway in the 1980s and 1990s indicated that approximately 10% of the population har- boured meningococci in the upper respiratory tract. However, only 1% of the healthy normal population carried strains from typical virulent clones prevalent at the time. The acquisition rate leading to carriage appears to be independent of season. The carriage rate in England is low (2–​3%) in the first 4 years of life, rises in children aged 10 to 14 years (9–​10%), reaches a maximum among young adults of 15 to 19 years (20–​25%), and then gradually declines to less than 15% in persons above 25 years. It increases in closed or semi-​closed communities and is particularly high in mili- tary camps where strains change frequently. In university communi- ties with bar and catering facilities the carriage rate is high. Smoking increases the carriage rate. In Burkina Faso the carriage rate among more than 20 000 screened persons below 30 years was 4%. It peaked in 15–​19-​year-​old boys and in 10–​14-​year-​old girls. The most fre- quently carried strains in 2009 were Y (2.3%), X (0.44%), A (0.39%), and W (0.34%) which may change from year to year. Reservoir of virulent meningococci In industrialized countries young adults are the main reservoir. Infants and children are usually infected by a local adult carrier. Household members and kissing contacts of a patient harbour viru- lent strains more often than the average population. Healthy children and young adults carrying virulent strains are the main reservoir in sub-​Saharan Africa. Children may infect each other. Spread from patients to medical staff is uncommon. Predisposing factors for invasive disease These are summarized in Box 8.6.5.1. Lack of protective antibodies Antibodies against serogroups A, C, W, and Y capsule polysac- charides are bactericidal and confer protection at concentrations of 1–​2 µg/​ml of serum. Serogroup B polysaccharide induces a weak transient IgM but no protective IgG response. Bactericidal and opsonophagocytic antibodies recognizing surface-​exposed epitopes of the outer membrane protein, in particular PorA, are important for protection. Antibodies to newly discovered outer membrane proteins including factor H binding protein, neisserial adhesion A, neisserial heparin binding antigen (components of two serogroup B vaccines recently licensed) are bactericidal and may contribute to Fig. 8.6.5.3  Outbreaks of different serogroups of N. meningitidis since the 1960s. Purple areas indicate countries with serogroup B epidemics. Reproduced with permission from Stephens DS, Greenwood B, Brandtzaeg P (2007). Epidemic meningitis, meningococcaemia, and Neisseria meningitidis, Lancet, 369, 2196–​210 and Caugant DA (1998). Population genetics and molecular epidemiology of Neisseria meningitides. APMIS, 106, 505–​10. Box 8.6.5.1  Factors predisposing for meningococcal infections • Lack of bactericidal and/​or opsonizing antibodies • Lack of alternative pathway or late complement components • Infection with human immunodeficiency virus • Treatment with complement inhibiting drugs

8.6.5  Meningococcal infections 1015 protection. Antilipopolysaccharide antibodies, recognizing com- monly shared epitopes among virulent and non​pathogenic neisseria and closely related species, presumably play a role in protection. Defects in the complement system Reduced function of the complement system caused by defects in the alternative or terminal pathways increases the susceptibility up to 6000 times. Defects in the classic pathway appear not to predis- pose to meningococcal infection. Complement defects are rare; they play a minor role in the development of invasive serogroup A, B, and C infections in Europe. Complement defects were overrepresented in patients with the less common and presumably less virulent sero- groups X, Y, and Z in studies from the Netherlands. Defects in com- plement factors 5 and 6 are well recognized in South Africa. Defects in the terminal complement system are associated with recurrent infections, often with a relatively benign character. Other genetic predisposition for invasive infection
and outcome Genetic variants in the complement factor H region are associated with reduced, but possibly also increased, susceptibility to men- ingococcal disease. High serum levels of factor H appear to in- crease the risk of invasive meningococcal infections in England. Polymorphism in tumour necrosis factor (TNF), mannose-​binding lectin, Fcγ receptors (CD16, CD32), and Toll-​like receptor (TLR)-​4 have previously been implicated in increased susceptibility and outcome. The results have not been reproduced in larger studies. TLRs play an important role in protecting the host from intruding microorganisms. Studies from the United Kingdom and Gambia suggest that the most common polymorphism in the TLR4 gene (Asp299Gly) is not overrepresented among cases. Polymorphism in genes coding for plasminogen activator inhibitor 1 (PAI-​1), interleukin-​1 receptor antagonist (IL-​1RA) and interleukin-​1 (IL-​1) have been associated with increased disease severity but need to be confirmed in larger studies. Invasive infection Most patients appear to develop invasive disease 2–​4 days after acquiring the virulent strain in the upper respiratory tract, but some are carriers for up to 7 weeks before invasive infection de- velops. N.  meningitidis adheres to specific molecules on non-​ ciliated epithelial cells in the nasopharynx and on the tonsils (Fig. 8.6.5.4). During a period of adaptation and proliferation, meningococci presumably alter various surface structures (lipopolysaccharides, pili, outer membrane proteins) by phase variation before starting transepithelial migration. They reach submucosal tissue and, via ca- pillaries, gain access to the circulation (Fig. 8.6.5.5). The initial bacteraemic phase Bacteraemia is a prerequisite for systemic meningococcal infection. Meningococci may be eliminated from the blood by lysis induced by bactericidal antibodies and complement and by phagocytosis of op- sonized bacteria. Persistent bacteraemia allows meningeal invasion. Bacterial proliferation and the accompanying inflammatory re- sponse can occur predominantly in either the subarachnoid space, causing meningitis, or in the circulation, causing meningococcaemia with or without shock. (b) (a) (d) (c) Fig. 8.6.5.4  Attachment to and proliferation of meningococci on non​ciliated epithelial cells in nasopharynx. Reproduced with permission from Stephens DS, Greenwood B, Brandtzaeg P (2007). Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet, 369, 2196–​210.

section 8  Infectious diseases 1016 The rash Haemorrhagic skin lesions are the hallmark of systemic meningo- coccal disease, occurring in 60–​80% of all cases in industrialized coun- tries. They appear as red or bluish petechiae. These lesions are larger and more irregular in size than the petechiae of thrombocytopenic purpura. Each lesion represents a local nidus of meningococci within the endothelial cells, thrombus formation, and extravasation of erythrocytes. The petechial rash indicates meningococcaemia, not necessarily severe sepsis leading to shock. However, in fulminant meningococcal septicaemia the haemorrhagic lesions are larger (ec- chymoses) with a propensity to locate on extremities (Fig. 8.6.5.6). Some patients develop relatively large non​specific maculopapular lesions, with or without haemorrhagic lesions, at an early stage (Figs. 8.6.5.7, 8.6.5.8). The petechial lesions are difficult to discover on dark skin but may be observed in the conjunctivae (Fig. 8.6.5.9). Clinical presentations The initial symptoms of systemic meningococcal infection are attrib- utable to meningococcaemia. This may persist as a low-​grade bac- teraemia or develop into septic shock in a few hours. Development of persistent shock is the major determinant of the case fatality rate. According to a recent extensive Dutch study the disease course and outcome depended primarily on age and development of shock and less on clonal complex and serogroup of N. meningitidis. Most com- monly, the patient develops meningococcaemia without circulatory impairment which gradually evolves to meningitis within 12 to 72 hours. Less frequently, patients develop distinct meningitis and persistent shock simultaneously. Based on easily recognizable clin- ical symptoms, meningococcal infections can be classified as: (1) meningitis without shock (50%), (2)  shock without meningitis (15%), (3) meningitis and shock (15%), and (4) meningococcaemia Fig. 8.6.5.5  Events leading to the different clinical presentations of meningococcal infections. Reproduced from Rosenstein NE, Bradley BA, Stephens DS, Popovic T, Hughes JM (2001). Meningococcal disease.
N Engl J Med, 334, 1378–​88. Copyright © 2001 Massachusetts Medical Society.

8.6.5  Meningococcal infections 1017 without shock or meningitis (20%). Each clinical presentation is as- sociated with a distinct pathophysiological background and prog- nosis (Table 8.6.5.1). Distinct meningitis without persistent shock Meningism dominates the clinical presentation and the onset is often insidious. The patients, particularly children, may complain of general malaise, nausea, and headache. They vomit and become febrile. The temperature may fluctuate and can be normal at times. Many patients are initially diagnosed as ‘gastric flu’, gastroenter- itis, or upper respiratory tract infection. Gradually, the symptoms of meningitis dominate the clinical picture. The patient com- plains of headache, vomits, and develops nuchal and back rigidity, photophobia, and in more advanced cases altered consciousness; Kernig’s and Brudzinski’s signs become positive. Many patients are lethargic, and some are agitated. The blood pressure is normal or slightly elevated by stress. Occasionally it is low but can be restored to normal by infusion of a limited volume of fluid. In untreated cases brain oedema develops, the intracranial pressure rises, and the central circulation is increasingly compromised. Finally, her- niation of the cerebellum occurs with arrest of the brain circu- lation. The case fatality rate was 0–​2% in four European studies comprising 1801 patients classified as given in Table 8.6.5.1. Fig. 8.6.5.6  Massive skin haemorrhage on the extremities of a 4-​ year-​old girl with fulminant meningococcal septicaemia. The infection was caused by Neisseria meningitidis group B. The left leg had to be amputated below the knee. She needed extensive skin transplantation and several fingers had to be amputated. Fig. 8.6.5.7  Macular lesions on the legs, some with a central haemorrhagic spot in a 17-​year-​old girl with mild meningococcaemia caused by Neisseria meningitidis group C. She recovered completely after 5 days of treatment with benzylpenicillin. Fig. 8.6.5.8  Macular and haemorrhagic lesions on the legs of a 21-​year-​ old man with mild meningococcaemia caused by Neisseria meningitidis group B. He recovered completely after 5 days of penicillin treatment. Fig. 8.6.5.9  Conjunctival petechiae in an African child with meningococcal group A meningitis. Copyright D. A. Warrell. Table 8.6.5.1  Levels of N. meningitidis DNA copies, lipopolysaccharides (endotoxin), and inflammatory mediators related to the clinical presentation No shock Shocka Shock No shock Meningitisb No meningitis Meningitis No meningitis Circulation (+) ++++ ++ (+) Subarachnoid space +++++ (+) +++ (+) a Shock denotes persistent hypotension requiring treatment with volume and pressor for 24 h. b Meningitis denotes 100 × 106/​litre or more leucocytes in the cerebrospinal fluid or clinically distinct signs of meningism.

section 8  Infectious diseases 1018 Meningococcal meningitis without persistent shock accounts for more than 50% of all cases of systemic meningococcal infections in industrialized countries and an even higher proportion of cases reaching hospitals in developing countries. The combination of multiple petechiae and symptoms of meningitis supports a diag- nosis of meningococcal meningitis. Pathophysiological background N.  meningitidis multiply in a compartmentalized manner with the main proliferation occurring in the subarachnoid space. Quantitative PCR indicates that the real number of meningococci is usually less than 103/​ml in plasma and may increase to 109/​ml in the cerebrospinal fluid (Fig. 8.6.5.10). This distribution is re- flected in the levels of endotoxin and various cytokines which are low in plasma and 100–​1000 times higher in the cerebrospinal fluid. Meningococci can be cultivated from both compartments in untreated patients. Plasma proteins, mainly albumin, leak into the cerebrospinal fluid, and the influx of mainly neutrophils causes the pleocytosis. The glucose level of the cerebrospinal fluid is reduced mainly as a result of increased central glucose consumption rather than the pleocytosis. Laboratory findings The erythrocyte sedimentation rate, C-​reactive protein, and leuco- cyte count in the peripheral blood are markedly elevated with increased numbers of band forms. Sodium, potassium, calcium, and magnesium ions, pH, renal, hepatic, and coagulation param- eters are usually within normal range. Cerebrospinal fluid shows a marked pleocytosis (more than 100 × 106 leucocytes/​litre), with increased levels of protein and decreased levels of glucose. Intracellular and extracellular Gram-​negative diplococci can be detected by direct microscopy. Persistent septic shock without distinct meningitis Fulminant meningococcal septicaemia (Waterhouse–​Friderichsen syndrome) is characterized by shock and persistent circulatory failure and severe coagulopathy leading to thrombosis and extensive haemorrhage of the skin, thrombosis and gangrene of the extrem- ities, and impaired renal, adrenal, and pulmonary function. Symptoms develop very rapidly. Six to 12 hours after recognizing their first symptoms the patients are often desperately ill. Initially, they complain of ‘flu-​like’ symptoms such as fever, aching muscle, prostration, abdominal pain, and nausea. The temperature rises rap- idly, commonly to between 39.0 and 41.5°C, but occasionally lower. Diarrhoea may occur during the first few hours. The patient appears worryingly sick to relatives. The parents usually recognize cold extremities indicating impaired circulation before the skin haem- orrhagic lesions appear but misinterpret the acute symptoms as in- fluenza or acute gastroenteritis. The haemorrhagic skin lesions are first seen as bluish petechiae, which rapidly increase in size and number. They are distributed all over the body but are often more pronounced and detected earliest on the extremities. Occasionally they are seen on the conjunctivae and other mucous membranes. The circulation is severely impaired. The extremities are often cold and cyanotic with a capillary refill time of more than 3 sec- onds. The blood pressure is low despite tachycardia. The tissue perfusion remains inadequate despite extensive fluid and pressor therapy. Initially, the circulation is hyperdynamic, but gradually becomes hypodynamic from persistent vasodilatation and grad- ually reduced myocardial performance. The heart becomes dilated with a reduced ejection fraction, as observed by serial ultrasound examinations. Patients usually lack nuchal and back rigidity, and Kernig’s sign is negative. Despite impaired circulation, many patients remain Meningitis Bacteraemia Blood Bacteraemia Bacteraemia Bacteraemia <103 DNA copies/ml endotoxin <0.5 EU/ml 104 DNA copies/ml endotoxin <0.5 EU/ml 106 DNA copies/ml endotoxin 10 EU/ml 107 DNA copies/ml endotoxin 50 EU/ml Fulminant septicaemia Sepsis + meningitis Mild meningo- coccaemia Fig. 8.6.5.10  Median number of N. meningitidis (number of DNA copies) as determined by real-​time PCR and median level of endotoxin in plasma in the different clinical presentations of systemic meningococcal disease.

8.6.5  Meningococcal infections 1019 awake and alert on hospital admission, being able to communi- cate their complaints. They hyperventilate to compensate for the pronounced metabolic acidosis. Urine output gradually dwindles. They may develop acute respiratory distress syndrome, that is, pul- monary oedema after fluid volume repletion of more than 40 ml/​kg. Circulatory collapse dominates the clinical picture during the first 48–​96 h. Fifty per cent of the non​survivors die within 12 h of hospital admission. Few patients die after 48 h. Later, acute respira- tory distress syndrome, renal failure, and the consequences of the diffuse thrombosis of the extremities and the skin dominate the pic- ture. The case fatality rates ranged from 16 to 53% in four European studies. Rapidly evolving symptoms with fever, circulatory shock, and extensive skin haemorrhages in a person without a history of splenectomy makes the diagnosis of fulminant meningococcal septicaemia likely. The same clinical picture is, however, observed in cases of overwhelming infections caused by Streptococcus pneu- moniae, Haemophilus influenzae, Streptococcus pyogenes, and Capnocytophaga canimorsus (after animal bite) and with viral haem- orrhagic fevers (Fig. 8.6.5.11). Pathophysiological background The pathophysiological changes are explained by the very rapid pro- liferation of N. meningitidis in the circulation. On admission 5 × 105 to 5 × 108 meningococci/​ml plasma are detectable by quantitative PCR (Fig. 8.6.5.10). This massive bacterial growth generates very high levels of endotoxin and other bacterial molecules in the blood, lungs, heart, liver, spleen, and kidneys. Few meningococci have yet penetrated into the subarachnoid space, which is explained by the short duration of symptoms. The levels of lipopolysaccharides in the plasma are closely associated with the copy number of meningococcal DNA and predict the devel- opment of persistent septic shock, multiple organ failure, and death. Plasma levels of lipopolysaccharides below 10 endotoxin units/​ml were associated with 1% mortality due to circulatory impairment whereas levels above 250 endotoxin units/​ml (i.e. 1.4 log higher), were associated with 100% mortality among 150 Norwegian patients (Fig. 8.6.5.12). Coagulopathy Coagulation is activated primarily via the extrinsic (tissue factor, FVIIa) pathway. In patients with fulminant meningococcal septicaemia there are increased levels of bioactive tissue factor in monocytes and on microparticles released from monocytes. The platelets disappear rapidly and remain at a low level for many days due to extensive consumption at the altered endothelial surface. Thrombopoietin increases in plasma without detectable increase of circulating platelets. The activation of the coagulation system, as measured by formation of fibrin, gradually reduces after antibiotic and fluid therapy is initiated (Table 8.6.5.2). Inhibited fibrinolysis Concurrently with activation of coagulation, fibrinolysis is inhibited by high levels of plasminogen activator inhibitor 1 (PAI-​1) released from activated endothelial cells and platelets. High levels of PAI-​1 are associated with development of persistent septic shock and a fatal outcome. Allelic variations in the promoter region of the PAI-​1 gene enhance production and are associated with an increased risk of dying. Thrombus formation Thrombosis occurs particularly in the vessels of the skin, adrenals, kidneys, muscles, choroid plexus, peripheral extremities, and to some extent in the lungs. The thrombomodulin–​thrombin complex on the endothelial cells converting protein C to activated protein Fig. 8.6.5.11  The ‘tumbler test’ used to differentiate haemorrhagic skin lesions from viral or drug rash in an infant with meningococcal meningitis caused by Neisseria meningitidis group B. There was complete recovery after 5 days treatment with benzylpenicillin. n = 68 100 80 60 40 20 0 <0.5 Case fatality rate (%) Endotoxin in plasma (endotoxin units/ml) 0.5–10 10–50 50–250

250 n = 31 n = 24 n = 20 n = 7 Fig. 8.6.5.12  Relationship between the levels of endotoxin (lipopolysaccharides) in plasma and case fatality rate related to the development of septic shock and multiple organ failure in 150 Norwegian patients with systemic meningococcal disease. Table 8.6.5.2  Factors contributing to the coagulopathy in fulminant meningococcal septicaemia Procoagulant factor Tissue factor in monocytes and microvesicles ↑ Anticoagulant factors Antithrombin ↓ Protein C ↓↓ Tissue factor pathway inhibitor ↑ Profibrinolytic factor Tissue plasminogen activator ↑ ↓ Antifibrinolytic factor Plasminogen activator inhibitor 1 ↑↑

section 8  Infectious diseases 1020 C, and the protein C endothelial cell receptor enhancing this ac- tivity, are down-​regulated. Glycosaminoglycans including heparan sulphate, molecules with an antithrombotic effect, are released from the endothelial surface. Numerous meningococci are pre- sent in and around small vessels. These processes facilitate forma- tion of thrombi. Concomitantly natural coagulation inhibitors are consumed. Protein C is reduced to 20% and antithrombin to me- dian 50% of normal functional plasma levels (>70%). Tissue factor pathway inhibitor increases. Proinflammatory and anti-​inflammatory mediators A multitude of bioactive proinflammatory and anti-​inflammatory mediators are released into the plasma and tissues of the lungs, heart, liver, spleen, and kidneys. The complement and the kallikrein–​kinin systems generate anaphylatoxins (C3a, C4a, C5a) and bradykinin, which are potent vasodilators. Proinflammatory cytokines, not- ably TNF, IL-​1β, IL-​6, and various chemokines are massively up-​ regulated. Concomitantly, high levels of soluble receptors of the same cytokines are released. The anti-​inflammatory cytokines IL-​10 and IL-​1 RA are present at high levels partly suppressing the cell-​ activating effect of the bacterial lipopolysaccharides (endotoxin) and the many proinflammatory cytokines. Nitric oxide production is increased in meningococcal septic shock and is thought to con- tribute to the vasodilation. The subarachnoid space The number of meningococci is very low owing to short onset ad- mission time, if present at all. However, they can be cultured from cerebrospinal fluid in up to 50% of untreated cases. The inflamma- tory response is very limited with a leucocyte count usually in the range of 10 to 100 × 106/​litre and normal contents of protein and glucose. Laboratory findings The erythrocyte sedimentation rate and C-​reactive protein are only moderately elevated on admission, rising to high levels within 48 h. The leucocyte count is usually low owing to upregulation of adhe- sion molecules with a marked shift to young band forms of neutro- phils. There is evidence of a partly compensated metabolic acidosis with decreased levels of pH and bicarbonate. Hyperventilation reduces Pco2. Creatinine and urea are elevated, serum glucose is variable (high, normal, or low), and potassium, calcium, and magnesium are low. Potassium rises with the renal failure. Serum aspartate aminotransferase and alanine aminotransferase are slightly elevated, whereas γ-​glutamyl transferase remains normal. Creatine kinase rises within 1 to 3 days, indicating rhabdomyolysis. Prothrombin, activated partial thromboplastin, and thrombin times are prolonged. The levels of platelets, fibrinogen, coagulation factors VII, X, and V, and prothrombin are low. Antithrombin and protein C are low, whereas tissue factor pathway inhibitor is ele- vated. Fibrin(ogen) degradation products, thrombin–​antithrombin complexes, PAI-​1, and plasmin-​α2-​antiplasmin complexes are ele- vated. Lumbar puncture should be avoided since the procedure may deteriorate the general condition of the patient, particularly the unstable circulation. For unknown reasons this clinical picture is uncommon in Africa but occurs among Africans migrating to industrialized countries. The case fatality rate was 14–​52% in four European studies. The mortality was highest during the outbreak of a serogroup B epidemic. Distinct meningitis and persistent shock There are meningeal and circulatory symptoms. Usually the symp- toms from the inflamed meninges dominate the picture. On ad- mission there are classic signs and symptoms of meningitis such as headache and nausea, nuchal and back rigidity, and a positive Kernig’s sign. The blood pressure remains low despite fluid volume repletion. Circulating levels of endotoxin and inflammatory mediators are lower than in patients with fulminant septicaemia, and case fatality is lower (Fig. 8.6.5.12). However, it is higher than in patients with meningitis without compromised circulation. The case fatality rate is 7–​14% in Europe. Meningococcaemia without distinct meningitis
and persistent shock Twenty to thirty per cent (20–​30%) of patients with invasive meningococcal disease are hospitalized because of fever and pe- techial or uncharacteristic rash. They lack distinct signs of men- ingitis although slight cerebrospinal fluid pleocytosis (less than 100 × 106 leucocytes/​l) may be present. The circulation is not se- verely compromised. They represent a composite group of pa- tients. Many are admitted to hospital early, 12 to 24 h after their first symptoms. Left untreated they might have developed symp- toms of meningitis or fulminant shock. The endotoxin level in plasma is less than 7 endotoxin units/​ml and the number of N. meningitidis DNA copies less than 104/​ml (Fig. 8.6.5.10). The case fatality rate was 0–​5% in Europe. Transient benign meningococcaemia These patients develop fever and often an uncharacteristic rash, but no meningism. They are diagnosed as most likely having a viral in- fection and receive no antibiotic. When the blood culture results are known, the symptoms have disappeared spontaneously, usually within 1–​3 days. This syndrome may occur in all age groups. Subacute meningococcaemia A few patients develop fever, an uncharacteristic maculopapular rash, general malaise, and arthralgia but no signs of meningitis or shock. They feel uncomfortable but are not severely ill. Meningococci are isolated from blood cultures. Untreated the symptoms may last for days to several weeks but disappear within 1–​2 days after peni- cillin therapy is initiated. Chronic meningococcaemia The patient develops undulating fever, arthralgia, and maculopapular rash (Fig. 8.6.5.13). The symptoms may last for months, but at times they may disappear completely. Blood cultures are sometimes re- peatedly negative. Patients are often treated with corticosteroids because an underlying autoimmune disease is suspected. The fever disappears temporarily before reappearing and at this stage menin- gococci may well be isolated from blood cultures. Antibiotic treat- ment clears the symptoms within a few days. Meningococci with an abnormal lipopolysaccharide (endotoxin) containing five and not

8.6.5  Meningococcal infections 1021 six fatty acids in the lipid A moiety have been isolated in many of such patients. Other organ manifestations Pericarditis The pericardium is seeded during a transient meningococcaemia. Subsequent inflammation and exudate may lead to cardiac tam- ponade if left untreated. The patient is febrile, nauseated, and may complain of epigastric pain. The condition is often misdiagnosed as an acute abdominal condition. Blood cultures may be negative. N. meningitidis can be cultured, detected by PCR, and seen in aspir- ated pus by direct microscopy. Treatment consists of evacuating the pus and administering antibiotics. The condition should be followed daily by ultrasound examination. Serogroup C organisms have been particularly implicated in these cases. Arthritis Acute meningococcal arthritis is an uncommon clinical mani- festation of a preceding, often low-​grade, meningococcaemia. It is usually located to one, or more rarely, several large joints. If the characteristic petechial rash is absent, detection of meningo- cocci in blood or joint fluid is necessary for a correct diagnosis. Arthritis caused by Neisseria gonorrhoeae is considerably more common than primary meningococcal arthritis. The symptoms disappear rapidly after penicillin treatment and there are no long-​ term complications. Arthritis induced by immune complexes This is more common than the meningococcal arthritis. One or several large joints become swollen and painful. The symptoms usually develop at the end of the first week of treatment. Blood and joint cultures are negative. The temperature and inflammatory markers may rise after an initial decline. The symptoms disappear gradually after some days of treatment with non​steroidal anti-​ inflammatory drugs. Extended antibiotic therapy is not necessary. Cutaneous vasculitis and episcleritis This appears simultaneously with the immune complex arthritis and is commonly observed in sub-​Saharan Africa (Figs. 8.6.5.14, 8.6.5.15). The vasculitis causes multiple blisters that readily rupture leading to multiple superficial skin ulcers. Ocular infections Conjunctivitis or panophthalmitis may precede other symptoms of invasive meningococcal infection. They are primarily observed in infants and children. The patient develops a red eye which in the case of panophthalmitis becomes painful with impaired vision. Formation of microthrombi and haemorrhage in retina and corpus vitreum, leading to blindness, may complicate the infection. Pneumonia Strains belonging to serogroups Y and W or more rarely other sero- groups may cause pneumonia in adults and children. The diagnosis depends on detecting meningococci in a representative specimen from the low respiratory tract or blood culture. It cannot be differ- entiated from pneumonia caused by other agents on the clinical symptoms alone. Treatment Prehospital antibiotic treatment Early antibiotic treatment to stop further bacterial growth is re- garded as vital because as many as 30% of the patients in industri- alized countries infected with N. meningitidis develop septic shock Fig. 8.6.5.14  Vasculitic lesion in an African child with meningococcal group A meningitis. Copyright D. A. Warrell. Fig. 8.6.5.15  Episcleritis in an African child with meningococcal group A meningitis. Copyright D. A. Warrell. Fig. 8.6.5.13  Maculopapular rash and peri-​articular swellings in an adult patient with chronic meningococcaemia. Copyright D. A. Warrell.

section 8  Infectious diseases 1022 characterized by rapidly increasing levels of meningococci and lipo- polysaccharides (endotoxin) in the blood. Consequently, health authorities in many countries advise general practitioners to start prehospital antibiotic treatment with ceftriaxone 100 mg/​kg, (max- imum dose is 4 g) or benzylpenicillin in suspected cases of men- ingococcal infection. The doses in Table 8.6.5.3 rapidly lead to bactericidal concentrations in plasma. The ceftriaxone or penicillin is injected intravenously or intramus- cularly (into one or both thighs). The patients most likely to benefit from this strategy, if applied early enough, are those who are distant from the hospital and have rapidly evolving symptoms leading to a compromised circulation and extensive haemorrhagic skin lesions. Initial evaluation in hospital The patients should be regarded as emergency cases. The main clin- ical presentation and severity should be evaluated immediately. A variety of prognostic scores have been developed. The Glasgow Meningococcal Septicaemia Prognostic Score is the one most com- monly used. Scores can be used to select patients for intensive care treatment. They should never be used to justify withholding treat- ment as they may overestimate case fatality. Antibiotic treatment Adequate doses of benzylpenicillin, cefotaxime, ceftriaxone, or chloramphenicol effectively stop further proliferation of N. menin- gitidis in the circulation, cerebrospinal fluid, and other extravascular sites. Induction of an explosive release of bacterial lipopolysac- charides leading to a Jarisch–​Herxheimer reaction has never been documented in patients receiving antibiotics for meningococcal infection. Plasma levels of lipopolysaccharides and the levels of important inflammatory mediators decline immediately after treat- ment with antibiotics is initiated in these patients (Table 8.6.5.4). Benzylpenicillin, chloramphenicol, cefotaxime, ceftriaxone, and meropenem are bactericidal to N.  meningitidis. Benzylpenicillin remains the drug of choice in most countries. It is effective, cheap, and non​toxic in high doses as long as renal function is normal. High doses are necessary since it penetrates the cerebrospinal fluid rela- tively poorly. In patients with fulminant septicaemia and severe renal dysfunction the doses should be reduced after 24 to 48 h. Strains whose sensitivity to penicillin is reduced because of altered penicillin-​binding protein 2 are an increasing problem. In most in- dustrialized countries they account for less than 5% of all menin- gococcal isolates, but the frequency is higher in Mediterranean countries, particularly Spain. Patients infected with these strains have been adequately treated with benzylpenicillin as long as dosage is adequate. A recent study from the United Kingdom indicated the same outcome among patients infected with fully sensitive strains as compared with strains with reduced sensitivity to penicillin. Penicillinase-​producing meningococci remain extremely rare. Chloramphenicol is a good alternative in patients hypersensitive to β-​lactam antibiotics. In developing countries, it is the best and cheapest alternative to benzylpenicillin. Meningococcal strains re- sistant to chloramphenicol occur in certain areas. In many industrial- ized countries cefotaxime or ceftriaxone is combined with vancomycin as empirical treatment of bacterial meningitis until the aetiological agent has been identified. Cefotaxime and ceftriaxone are highly ef- fective antibiotics that penetrate the blood–​brain barrier better than benzylpenicillin. Meropenem is a carbapenem highly active against N. meningitidis, H. influenzae, and S. pneumoniae. It does not induce seizures as observed with the imipenem–​cilastatin combination. In each country the health authorities and microbiological labora- tories should recommend the optimal and affordable drug regimen. Antibiotic treatment should be initiated promptly. Therapy should start immediately after the first clinical evaluation and collection of the necessary samples for microbiological diagnosis. If there are contra- indications to lumbar puncture or if it is delayed until after brain imaging, antibiotic treatment should be started immediately. Three to 4 days of treatment is adequate to eradicate sensitive meningococci. Supportive treatment Patients with persistent shock should be given extensive volume re- placement, whereas patients with meningitis should receive a mod- erate amount of fluid. All patients should be monitored closely to detect early signs of a deteriorating circulation, renal and pulmonary failure, or increasing intracranial pressure. Volume treatment Patients in industrialized countries with persistent hypotension and signs of inadequate peripheral circulation have routinely been treated with massive fluid volume repletion. The extensive capillary leak syndrome increases the volume required. Children and adults may require an infused volume that is one to several times their circu- lating blood volume in the first 24 h. Sodium chloride 0.9% solution is recommended as basic treatment, later supplemented with Ringer’s solution. However, in a recently conducted clinical controlled trial in several African countries evaluating children with severe febrile illness and impaired circulation, the results showed unexpectedly that an initial bolus infusion with 20–​40 ml/​kg of 5% albumin or NaCl 0.9% increased the mortality significantly. Whether these re- sults also apply to meningococcal shock in Africa is unknown since the pathophysiology with capillary leakage, cardiac dysfunction, and disseminated intravascular coagulation differs from malaria. In many countries the use of fresh frozen plasma is no longer recommended because of the risk of transmitting pathogens, especially HIV. Table 8.6.5.3  Doses of prehospital antibiotic to be administered in suspected cases of meningococcal infection Age (years) Dose All 100 mg/​kg ceftriaxone intramuscularly, maximum single dose is 2 g <2 300 mg (0.5 × 106 IU) benzylpenicillin intramuscularly 2–​7 600 mg (1 × 106 IU) benzylpenicillin intramuscularly

7 1.2 g (2 × 106 IU) benzylpenicillin intravenously or intramuscularly Table 8.6.5.4  Antibiotics in meningococcal meningitis or sepsis Antibiotic Dose/​24 h Dose interval (h) Adult (g) Child (mg/​kg) Benzylpenicillin 14.4 (24 × 106 IU) 200 (300 000 IU/​kg) 4–​6 Cefotaxime 9 200 6–​8 Ceftriaxone 4 100 12–​24 Chloramphenicol 3 100 6

8.6.5  Meningococcal infections 1023 Patients presenting with distinct signs of meningitis without shock should receive the basic daily requirement of fluid supple- mented with extra volume for dehydration and loss due to vomiting and fever to ensure a normal diuresis (≥1 ml/​kg per hour for chil- dren). Excessive hydration should be avoided since it may precipitate irreversible brain oedema and cerebellar herniation. In patients with persistent shock and meningitis, treatment of shock is the priority. Inotropic support If initial volume repletion fails to improve the circulation, inotropic support should be added. Dopamine, dobutamine, noradrenaline, and adrenaline are used. Most physicians start with dopamine at 3–​10 µg/​kg per min which at an early stage is combined with noradrenalin at 0.03–​3.0 µg/​kg per min or dobutamine at 1–​10 µg/​ kg per min. Ideally, infusions should be via infused a central line. Corticosteroid therapy for shock In adults with septic shock and reduced adrenal function, low doses of cortisol increased survival in one study but was not con- firmed in a larger follow up study. Similar studies do not exist for children. Adrenal haemorrhage is common in patients with ful- minant meningococcal septicaemia. Serum cortisol is lower and adrenocorticotropic hormone higher in non​surviving than sur- viving children with meningococcal shock; a relative adrenal in- sufficiency may therefore exist. Recently many clinicians have treated meningococcal shock with low doses of cortisol in an at- tempt to reduce inotropic support. Corticosteroid therapy for meningitis The United Kingdom National Institute of Health and Clinical Excellence (NICE) guidelines advocate the use of dexamethasone 0.15 mg/​kg × 4/​24 h (maximum dose 10 mg × 4/​24 h) for 4 days for bacterial meningitis. Dexamethasone should ideally be given 15 min before or at least within 4 h after antibiotic treatment is initiated. However, the benefit of dexamethasone in meningo- coccal meningitis is controversial and has not been documented in randomized clinical controlled trials in Europe and North America. Dexamethasone did not improve the outcome in any type of bacterial meningitis in two large double-​blind random- ized clinical controlled trials (children and adults) in Malawi, one (adults) in Vietnam and one (children) in Chile. Corticosteroids reduce the penetration of antibiotics over the blood–​brain bar- rier. In developing countries corticosteroids are presently not re- commended as adjunct treatment for meningitis. The American Academy of Pediatrics does not advise any adjunct therapy, given the lack of evidence. Ventilatory support Patients receiving volume treatment for profound shock are in danger of developing acute respiratory distress syndrome. Hyperventilation, increasing oxygen demand, decreased pulmonary compliance, and the appearance of diffuse infiltrates on chest radiograph indicate the development of acute respiratory distress syndrome. At a par- tial oxygen pressure in arterial blood (Pao2) of less than 8 kPa with the fraction of inspired oxygen (Fio2) above 0.6 (60% O2 in the in- spiration air), the patient usually requires intubation and artificial ventilation. Infants and children often require mechanical ventila- tion if the resuscitation fluid volume exceeds 40 ml/​kg per 24 h to combat the septic shock, even if the oxygenation is normal. Renal support Patients with persistent septic shock and coagulopathy develop renal dysfunction from acute proximal tubular necrosis. Thrombosis in the small peritubular vessels and in glomeruli, and myoglobinaemia, may contribute to renal dysfunction. Serum creatinine and urea are ele- vated on admission and continue to increase for many days without ad- equate treatment. Hyperkalaemia, which may develop during the first 24 to 48 h, is an immediate threat. Haemodialysis or peritoneal dialysis and continuous haemofiltration are used to treat the renal failure and remove oedema. The renal failure is usually reversible but may last for weeks. Complete kidney failure is uncommon in survivors. Treatment of disseminated intravascular coagulation The first priority is to stop further bacterial proliferation with anti- biotics. This reduces the thrombin activity by 50% within 2 to 6 h. In the 1970s heparin was extensively used. Two small controlled trials did not document any survival benefit in patients receiving heparin. Infusion of a continuous low-​dose unfractionated heparin (10–​15 IU/​kg per h) has been advocated as supplement to treatment with concentrated protein C. The antithrombin levels should be kept above 35 to 40 IU/​ml. Antithrombin does not reduce the fatality rate in other types of severe sepsis. Infusion of the natural anticoagulant protein C (loading dose 100 IU/​ kg, followed by 15 IU/​kg per h for 4 days to keep the plasma concen- tration between 0.8 and 1.2 IU/​ml) may possibly limit thrombus for- mation, skin necrosis, and the need for amputation. If used it should be started early. In the few uncontrolled studies that have been pub- lished, several patients treated with protein C concentrate still needed amputation. Randomized controlled trials are lacking. Recombinant human activated protein C (Xigris) did not improve the outcome in patients with septic shock in a randomized con- trolled trial. The drug was withdrawn from the market (November 2011). Routine transfusion of platelets is controversial. In patients with life-​threatening bleeding and thrombocytopenia, massive platelet transfusion can be lifesaving; however, it may also aggravate thrombus formation. Fibrinolysis To overcome inhibition by PAI-​1, recombinant human tissue plas- minogen activator (0.25–​0.5 mg/​kg in 1.5–​4 h) has been infused to enhance fibrinolysis. Retrospective studies suggest that it increases the rate of cerebral haemorrhage. It is not recommended for routine use in severe meningococcaemia. Plasmapheresis and blood exchange Plasmapheresis and exchange blood transfusion have been tried to remove pathologically activated plasma and leucocytes; 50 ml plasma/​kg body weight has been exchanged with fresh plasma. These techniques do not increase the clearance of bacterial lipo- polysaccharide (endotoxin) substantially. Results suggest improved survival, but adequate control groups are lacking. Even desperately ill patients have tolerated the procedures.

section 8  Infectious diseases 1024 Extracorporeal membrane oxygenation A limited number of children have been treated with extracorporeal membrane oxygenation in a few centres with apparently good re- sults. However, equally good results have been achieved in another paediatric intensive care unit without using the procedure, sug- gesting that the experience of the intensive care unit is more im- portant than the procedure per se. Neutralization of bacterial lipopolysaccharides Three different antiendotoxin principles, the anti-​J5 serum, the human monoclonal IgM (HA-​1A) antibody, and the recombinant bactericidal/​permeability increasing protein (BPI21) have been evalu- ated in randomized double-​blind controlled clinical trials. None in- creased survival significantly; however, fewer patients treated with BPI21 required multiple severe amputations and more patients had a functional outcome similar to that before illness 60 days after treat- ment. None of the principals are presently commercially available. Antimediator therapy Strategies to neutralize TNF, IL-​1β, bradykinin, platelet-​activating factor, and prostaglandins in patients with septic shock have not increased the 28-​day survival rate. They have not been specifically evaluated in meningococcal septic shock. Sequelae Meningitis Sensorineural hearing loss or impaired vestibular function occurs in 4–​19% of patients. It develops at an early stage, is usually irreversible, and is more common in adults than children. Epilepsy, hydroceph- alus, and diffuse brain damage are at present rare complications in industrialized countries. Persistent headache, altered sleep pattern, concentration difficul- ties, irritability, and neurasthenia may persist in 5–​8% of all patients. Shock and coagulopathy Most long-​term complications are related to development of gan- grene of the extremities requiring amputation and necrotic skin lesions requiring extensive grafting. The renal failure is usually re- versible although reduced function may persist. Permanent adrenal insufficiency (i.e. Addison’s disease), develops very rarely in sur- vivors. Acute respiratory distress syndrome may lead to permanent pulmonary fibrosis and reduced function. Prevention Vaccination Conjugate protein capsule polysaccharide vaccines
(A, C, Y, and W) Serogroup A, C, W, Y conjugate vaccines are immunogenic from 2 months of age. They are very effective, reduce transmission, and induce immunological memory. Booster doses are required for those vaccinated in the first year of life. In United Kingdom babies are offered the C conjugate vaccine at 3 and 12 months. Combined conjugate vaccines containing serogroups A, C, Y, and W have been licensed in many countries for children and adults. Menveo® has been licensed in the United Kingdom for use in infants ≥2 months of age. In babies 2–​12 months old, two doses given 1 month apart are required. In United Kingdom students are presently offered A, C, W, and Y conjugate vaccine. Menactra® is licensed for use for ages ≥9 months to 55 years in the United States and other coun- tries. Booster doses are recommended. Mass vaccination of 1–​29-​ year-​olds in sub-​Saharan Africa with serogroup A conjugate vaccine MenAfriVac® has proven to be very effective. It induces bacteri- cidal anticapsule antibodies and reduces carriage profoundly in the first two years after mass vaccination, resulting in heard immunity. Serogroup A, combined A–​C, and various other combinations of conjugate vaccines are marketed or under development, and will be licensed in the near future for use in infants. Capsule polysaccharide vaccine (A, C, Y, and W) The protective effect of these vaccines in infants below 2 years of age is uncertain. When vaccination is required to prevent serogroup A infection, infants of less than 24 months should receive two doses with at least a 1-​month interval, whereas those above 2 years should receive one dose. For serogroup C infection, one dose should be given from 18 months. Revaccination with serogroup C polysac- charide may reduce the antibody level. Malaria reduces the immune response. They do not induce immunological memory. An antibody level of 1–​2 µg/​ml appears to be necessary for protection which lasts for 3–​5 years. The vaccines are cheap and have for many years been used successfully to contain outbreaks for those above 2 years of age. Outer membrane vesicle vaccine (B) Since the capsule polysaccharide of serogroup B strains induces a short-​lived IgM but no lasting IgG response, several countries (Cuba, Norway, New Zealand) have developed and used an outer membrane vesicle vaccine protecting against outbreaks of one viru- lent clone. The protection rate in adolescents after two doses is lower (57–​80%) than for the non-​B polysaccharide and conjugate vaccines and is relatively strain specific. The immunodominant epitope is the outer membrane protein PorA. Three doses given 6 weeks apart and a fourth dose 8 months later induce a significantly better immune re- sponse than two doses. Studies in New Zealand with a strain-​specific vaccine resulted in 73% protection. The duration of the protection is not known. The Norwegian and New Zealand vaccines are presently not on the market. Serogroup B vaccines Two new serogroup B vaccines are available. The method known as ‘reverse vaccinology’ has identified genes in the N. meningitidis DNA coding for previously unknown surface-​exposed outer mem- brane proteins present in most of the invasive strains. Bexsero® was granted marketing authorization by the European Medicines Agency in November 2012. It contains three cloned proteins: neisserial ad- hesion A  (NadA), factor H binding protein (fHbp) and neisserial heparin binding antigen. These are produced in E. coli, purified, and, combined with outer membrane vesicles from the New Zealand epi- demic strain (exposing PorA P1.4). The vaccine induces bactericidal antibodies in all age groups from 2 months. The United Kingdom is the first country to include Bexsero® in its immunization programme starting in September 2015 and vaccinating at 2, 4, and 12 months. If starting after 6 months of age 2 doses are given 2 months apart with a

8.6.6 Neisseria gonorrhoeae 1025

8.6.6 Neisseria gonorrhoeae 1025

8.6.6  Neisseria gonorrhoeae 1025 third dose in the second year of life. The vaccine reduces but does not eliminate carriage. A vaccine combining the components of Bexsero® with the conjugate polysaccharide A, C, W, Y is presently being tested. Such a vaccine could protect against all major serogroups except serogroup X. In October 2014 a new vaccine Trumenba® containing factor H binding protein (fHbp) subfamily A and B was licensed in the United States and Europe in 2017 for the age group 10–​25 years. It is given as three doses at 0, 2, and 6 months. The use of this vaccine in other age groups and the influence on carriage has yet to be defined. Indications for vaccination Routine immunization with the A, C, Y, and W vaccine is advocated for people with documented deficiencies in the alternative pathway and late complement components. Non-​outbreak situation Indications for vaccination are close contacts of an index case (in addition to antibiotic prophylaxis), travellers to high-​risk areas, mili- tary recruits, persons with asplenia, HIV infection, and alcoholics. Outbreak situation Vaccination has been recommended if two or more persons are attacked by the same strain in a school class or day care centre, the at- tack rate exceeds 10 cases/​100 000 population per 3 months, or the at- tack exceeds 1/​1000 with three or more cases in a closed group setting. Epidemic situation An advocated threshold for mass vaccination is 15 cases/​100 000 population per week for two consecutive weeks caused by the same strain. A steadily increasing number of cases and an increase in the median age of the patients indicate an epidemic. Secondary prophylaxis Antibiotic prophylaxis Household contacts of an index case have 100 to 1000 times in- creased relative risk for developing meningococcal infections. Usually the second case occurs within 2 weeks of the index case if no eradication treatment is given. However, there is doubt about the effectiveness of eradication treatment when the causative strain belongs to serogroup B. Health authorities in most countries advise that close con- tacts have eradication treatment. Presently, adults receive 500 mg ciprofloxacin or 400 mg ofloxacin as a single dose. Pregnant women and children of less than 12  years should receive 250 mg and 125 mg ceftriaxone, respectively, as one intramuscular injection. Alternatively, children are treated with rifampicin 10 mg/​kg, max- imum dose 600 mg, every 12 h for 48 h. FURTHER READING American Academy of Pediatrics (2012). Meningococcal infections. In: Red book, pp. 500–​9. Elk Grove Village, IL. https://​redbook.solu- tions.aap.org/​chapter.aspx?sectionid=88187187&bookid=1484 Brower MC, Read RC, van de Beek (2010). Host genetics and outcome in meningococcal disease:  a systematic review and metaanalysis. Lancet Infect Dis, 10, 262–​74. Christodoulides M (ed) (2012). Neisseria meningitidis, advanced meth- ods and protocols. Humana Press, New York, NY. Davila S, et al. (2010). Genome-​wide association study identifies vari- ants in the CFH region associated with host susceptibility to menin- gococcal disease. Nat Genet, 42, 772–​6. Dretler AW, Rouphael NG, Stephens DS (2018). Progress toward the global control of Neisseria meningitidis: 21st century vaccines,
current guidelines, and challenges for future vaccine development. Hum Vaccin Immunother, 14, 1146–60. Frosch M, Maiden M (eds) (2006). Handbook of meningococcal disease. Wiley, Weinheim. Hellerud BC, et  al. (2015). Massive organ inflammation in experi- mental and clinical meningococcal septic shock. Shock, 44, 458–69. Immunisation against infectious disease: The Green Book, Public Health England, 2017, Chap 22, page 1–24. Kristiansen PA, et al. (2015). Serogroup A meningococcal conjugate vaccines in Africa. Expert Rev Vaccines, 14, 1441–58. Lewis LA, Ram S (2014). Meningococcal disease and the complement system. Virulencei, 5, 98–​126. Molyneux E, et al. (2011). 5 versus 10 days of treatment with ceftriaxone for bacterial meningitis in children:  a double-​blind randomised equivalence study. N Engl J Med, 377, 1837–​45. Nathan N, et al. (2005). Ceftriaxone as effective as long-​acting chloram- phenicol in short-​course treatment of meningococcal meningitis during epidemics: a randomised non-​inferiority study. Lancet, 366, 308–​13. National Institute for Health and Care Excellence (NICE) (2010). Guidelines for bacterial meningitis and meningococcal septicaemia. https://​www.nice.org.uk/​guidance/​CG102 Pace D (2013). Glycoconjugate vaccines. Expert Opin Biol Ther, 13, 11–​33. Rollier CS, et al. (2015). The capsular group B meningococcal vaccine, 4CMenB: clinical experience and potential efficacy. Expert Opin Biol Ther, 15, 131–42. Rosenstein NE, et al. (2001). Meningococcal disease. N Engl J Med, 344, 1378–​88. Sadarangani M, Feavers I, Pollard AJ (eds) (2016). Handbook of me- ningococcal disease management. Springer, Gewerbestrasse, Cham. Stephens DS, Greenwood B, Brandtzaeg P (2007). Epidemic meningitis, meningococcemia and Neisseria meningitidis. Lancet, 369, 2196–​3210. Stoof SP, et al. (2015). Disease burden of invasive meningococcal disease in the Netherlands between June 1999 and June 2011: a subjective role for serogroup and clonal complex. Clin Infect Dis, 61, 1281–​92. Van Deuren M, Brandtzaeg P, van der Meer JWM (2000). Update on meningococcal disease, with special emphasis on pathogenesis and clinical management. Clin Microbiol Rev, 13, 144–​66. Welch SB, Nadel S (2003). Treatment of meningococcal infection. Arch Dis Child, 88, 608–​14. 8.6.6  Neisseria gonorrhoeae Jackie Sherrard and Magnus Unemo ESSENTIALS Neisseria gonorrhoeae is a Gram-​negative, intracellular diplococcus that is transmitted by direct inoculation of infected secretion from one mucosa to another. It primarily colonizes the columnar epithelium of lower genital tract, only occasionally spreading to the upper genital tract or causing systemic disease. Oropharyngeal and rectal infections

section 8  Infectious diseases 1026 are common in men who have sex with men but also occur in women. N. gonorrhoeae is almost exclusively transmitted by sexual activity. Clinical features Men—​dysuria (50%) and urethral discharge (80%); complications (e.g. epididymitis, orchitis), are rare. Women—​there are no specific symptoms in the absence of com- plications (e.g. salpingitis, bartholinitis). Oropharyngeal and rectal infections usually produce no symp- toms. Disseminated gonococcal infection is a comparatively benign bacteraemia affecting joints (particularly shoulder and knee) and skin; traditionally more common in women than men. Diagnosis—​appropriate laboratory diagnostics are essential for the diagnosis of gonorrhoea. Microscopy of a Gram-​stained or methy- lene blue smear from a genital site (male urethral or endo-cervical) will give a presumptive diagnosis of gonorrhoea. Nucleic acid amp- lification tests are now the most sensitive tests for a confirmed diag- nosis of gonorrhoea but a single result from an extragenital site or from samples from low-​prevalence populations should be verified using an additional nucleic acid amplification test with a different nucleic acid target. Culture is the most specific test and provides a viable organism for antimicrobial susceptibility testing but can lack sensitivity, particularly for oropharyngeal and rectal samples. Treatment—​the gonococcus has adapted rapidly to prevalent anti- microbial usage, leading to resistance to all antibiotics used for treat- ment, notably penicillins, fluoroquinolones, macrolides, tetracycline, and cephalosporins. This development has resulted in major concerns internationally and the introduction of international and national ac- tion/​response plans as well as dual antimicrobial therapy. In the United Kingdom as well as in Europe, Australia, United States, and Canada, re- commended first-​line treatment of uncomplicated infection in adults is now ceftriaxone intramuscularly as a single dose plus azithromycin or- ally as a single dose. Spectinomycin intramuscularly plus azithromycin orally as a single dose is suitable for those with penicillin allergy. Introduction Gonorrhoea is an ancient disease. Galen coined its name in the 2nd century ad (from Greek words meaning ‘semen’ and ‘flow’), but there are older references including Chapter 15 of Leviticus in the Old Testament. The name of the causative obligate pathogenic bacterium, Neisseria gonorrhoeae, credits Albert Neisser with its discovery in 1879, although Hallier had described its characteristic microscopic appearance 7 years earlier. In the era of HIV/​AIDS, gonococcal infections additionally play an important role as an indicator of risky sexual activity. Epidemiology The World Health Organization (WHO) estimated there were 78 million global cases of gonorrhoea among adults in 2012. In European Union/​European Economic Area (EU/​EEA), gonor- rhoea is the second most commonly reported sexually transmitted infection (STI) after chlamydial infections. However, the incidence in several EU/​EEA countries is underestimated due to suboptimal diagnostics, testing, case reporting, and epidemiological surveil- lance. In 2016, 75 349 gonorrhoea cases (18.8 per 100 000 popu- lation) were reported in 27 EU/​EEA countries (data unavailable from Austria, Germany, Greece, and Liechtenstein). The highest incidence was reported in the United Kingdom (61.4 per 100 000 population), which also reflects effective diagnostics, high levels of testing and a well-​functioning epidemiological surveillance system. Young people (15–​24 years of age) accounted for 36% of all EU/​EEA cases. Forty-​six per cent (46%) of the cases were reported in men who have sex with men. Since 2008, the incidence in United Kingdom has significantly increased and the reported incidence in EU/​EEA has increased by more than 80%, which is mainly due to an increased number of cases in men, especially among men who Year 70 000 60 000 50 000 40 000 30 000 20 000 10 000 0 1918 1920 1922 1924 1926 1928 1930 1932 1934 1936 1938 1940 1942 1944 1946 1948 1950 1952 1954 1956 1958 1960 1962 1964 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Total number of cases Benzyl- penicillin Sulphonamides Ampicillin Tetracyclines Erythromycin 3rd generation cephalosporins Fluoro- quinolones NHS Cefixime or ceftriaxone Ceftriaxone PLUS azithromycin Fig. 8.6.6.1  Reported cases of gonorrhoea and introduction of different treatments for gonorrhoea in England between 1918 and 2017 (Public Health England, formerly Health Protection Agency). NHS, National Health Service.

8.6.6  Neisseria gonorrhoeae 1027 have sex with men. In the United Kingdom since the Second World War, the peak incidence of reported gonorrhoea cases in 1946 re- sulted from a combination of returning infected soldiers and ascer- tainment bias (Fig. 8.6.6.1). Changing incidence thereafter seemed independent of the availability of effective antibiotics. The rising numbers of infections since the late 1950s, peaking in 1974, coin- cided with the introduction of the oral contraceptive pill, greater sexual promiscuity, effective diagnostics (culture) in women, and an increasing availability of different classes of effective antibiotics and was mainly unrelated to antimicrobial resistance. The rapid fall in incidence in the late 1980s coincided with a self-​imposed regime of safer sex in the general population and particularly in the men who have sex with men community, which was primarily due to the awareness of spread of HIV. The rates of complications of gonorrhoea have declined in parts of the world where diagnosis and treatment of STIs is readily avail- able. However, the availability of effective antimicrobial agents for gonorrhoea is declining worldwide as resistance rapidly emerges to current agents. Changes in N. gonorrhoeae have been driven, until now, mainly by antibiotic pressures. An intriguing and worrying problem, with the increasing use of nucleic acid amplification tests (NAATs) for diagnosis, is the evolution of strains that become ‘invisible’ to the NAAT as a result of altered or loss of the target sequence; this has al- ready happened with NAATs for Chlamydia trachomatis in Sweden in 2006 as well as with porA pseudogene NAATs for N. gonorrhoeae in several countries. Pathogenesis N. gonorrhoeae has evolved mechanisms for evading host defences and causing repeated infections. Major outer membrane antigens exposed to the immune response are pili, lipo-​oligosaccharide, and major outer membrane proteins, for example, PorB, Opa, and Rmp. N. gonorrhoeae primarily colonizes the columnar epithelium of the lower genital tract, only occasionally spreading to the upper genital tract or causing systemic disease. To colonize successfully, the organism must attach to and invade the epithelial layer to avoid being swept away by cervical secretions in women or urine in men. Iron is essential to replication; N. gonorrhoeae expresses transferrin or lactoferrin receptors on its surface. In vivo, gonococci resist the bactericidal activity of serum by sialylation of lipo-​oligosaccharide. In vitro, most strains become serum sensitive. Pili, Opa, and lipo-​ oligosaccharide antigens can alter the part of the molecule reacting with the immune response. This antigenic variation occurs at a fre- quency higher than the normal mutation rate. On each encounter between the organism and the host, the gonococcus presents a range of immunologically distinct proteins that are not recognized by the host. Host cell receptors are complex carbohydrates, glycosamines, lipoproteins, and glycoproteins. Signs and symptoms of gonorrhoea Classically, urethral gonorrhoea in men causes discharge (>80%) and dysuria (>50%) although the severity and frequency of the dys- uria has diminished in recent years. The diagnostic thick, profuse, purulent, white or off-​white exudate is less often seen and men more commonly complain of a mucopurulent or scanty mucoid discharge. Even if untreated, the discharge might, after some weeks, diminish to a simple clear mucus or resolve completely; asymptomatic patients (<10%) thus include presymptomatic, postsymptomatic, and unob- servant men. Rectal and urethral gonorrhoea acquired by fellatio is increasingly seen in men who have sex with men practising ‘safe’ sex. Uncomplicated gonorrhoea in women affects the cervix (90%), ur- ethra (75%), rectum (40%), or oropharynx (5–​15%). Initially, there are no specific symptoms. Gonococcal cervicitis might result in an increased purulent or mucopurulent exudate from the os, which can present as an increased vaginal discharge (50%). This vaginal discharge has no specific characteristic. Dysuria (12%), without fre- quency, is not found consistently enough to make it a diagnostically helpful symptom. The occasional urethral discharge is not profuse enough to cause symptoms. Rectal infection can occur without anal intercourse and rarely produces slight dampness or discharge. Abdominal pain signifies spread to the pelvic organs. Rectal and oropharyngeal infections are asymptomatic in most cases. Development of a sore throat after oral sex does not indicate any particular STI. Complications Complications in men which include tysonitis (infection of the Tyson’s or preputial glands, Fig. 8.6.6.2), epididymitis, prostatitis, periurethral abscess, and infection of the median raphe are rare in the United Kingdom and other developed countries. If the urogenital infection is undetected or not appropriately treated, spread to the endometrium, fallopian tubes, and pelvic adnexae is the most common complication (5%) in women. It usually occurs at, or soon after, the menstrual period, probably resulting from retrograde flow of menses. Pelvic pain might be unilateral causing confusion with acute appendicitis. Pelvic inflammatory disease can result in severe sequelae such as infertility or ectopic pregnancy. Coincidental infection with C. trachomatis is sufficiently common to justify treatment of both organisms. Infection of Bartholin’s, Skene’s, or periurethral glands is now rare in the United Kingdom and other developed countries. Vertical transmission can occur at the time of delivery, resulting in purulent conjunctivitis in the neonate which characteristically develops in the first week of life. Fig. 8.6.6.2  Gonococcal urethritis and tysonitis. Courtesy of Peter Greenhouse.

section 8  Infectious diseases 1028 Perihepatitis (Fitz-​Hugh–​Curtis syndrome) occurs more fre- quently with C.  trachomatis than with N.  gonorrhoeae. Right hypochondrial pain, referred to the shoulder, occasionally with pleural effusion and rub, might lead to referral to a surgical or gen- eral medical clinician rather than a genitourinary physician. Disseminated gonococcal infection is rare but traditionally more common in women than men, reflecting the lack of genital symp- toms in women. It has traditionally been caused mainly by penicillin-​ sensitive organisms and is a comparatively benign bacteraemia affecting joints and skin. The shoulder and knee are most commonly affected, followed by wrist, elbow, and small joints of the hands and feet, often with an associated tenosynovitis. The pathognomonic painless, usually 4 to 10, skin lesions evolve through vesicular, pus- tular, and haemorrhagic stages before healing (Figs. 8.6.6.3, 8.6.6.4). Erythema nodosum-​like lesions have been described. Systemic symp- toms are minimal. White cell count and erythrocyte sedimentation rate are usually not greatly raised. The response to appropriate anti- biotic treatment is rapid, but joints may need to be aspirated. Blood or joint fluid culture can yield gonococci, but the quickest diagnosis usually comes from anogenital and throat NAAT. In both sexes, gonococcal infection is associated with increased risk of acquisition and transmission of HIV due to genital tract inflammation. Diagnosis Microscopy Microscopy (×1000) of an appropriately methylene blue-​ or Gram-​ stained smear is the first line in diagnosis. For adequate diagnosis, Gram-​negative intracellular (within the cytoplasm of a polymorpho- nuclear leucocyte) diplococci should be identified (Fig. 8.6.6.5). In samples from the male urethra (Fig. 8.6.6.6), microscopy is sensitive (identifying up to 98% of culture positives in symptomatic men and ra- ther fewer in those without symptoms) and highly specific (<1% will be found on culture to be Neisseria meningitidis or other bacterial species). Microscopy of stained samples from the cervix is much less sensi- tive (≤55%) and comprises a suboptimal specificity, but when posi- tive, immediate treatment is enabled. Because of the preponderance of other Neisseria species in the oropharynx, microscopy of stained samples from this site is not helpful. Microscopy of stained smears from blind anorectal swabs is not recommended because of the large number of other Gram-​ negative cocci present in faecal flora. However, microscopy of stained smear of a rectal discharge taken through a proctoscope can be helpful. Laboratory detection of N. gonorrhoeae Isolation (culture) of N. gonorrhoeae was the previous diagnostic gold standard because of its high sensitivity and 100% specificity (if adequate species confirmation is performed), but can be ham- pered by inadequacies in sample taking, storage, transport to the laboratory, and culture methodology, including culture media used. NAATs have been shown to be more sensitive than culture for gono- coccal infection. NAATs are significantly more sensitive than cul- ture in samples from the oropharynx and rectum, although they are not currently licensed for use at these sites, but cross-​reacting other Neisseria species can reduce specificity and require confirmation using a second NAAT with a different nucleic acid target. Because NAATs are the standard test methodology for C. trachomatis in- fection, commercial kits offering testing for both organisms have become the norm. Care is needed in their use in low-​prevalence populations; false-​positives, resulting in a low positive predictive value, may be unacceptably common. Culture is recommended fol- lowing a positive NAAT test, to allow susceptibility testing of the Fig. 8.6.6.3  Disseminated gonococcal infection, haemorrhagic vesiculopustule. Fig. 8.6.6.4  Disseminated gonococcal infection: healing lesions with desquamation and deposition of haemosiderin. Fig. 8.6.6.5  Gram-​stained urethral discharge showing Gram-​negative intracellular diplococci.

8.6.6  Neisseria gonorrhoeae 1029 organism. In general, only validated and quality-​assured laboratory methods should be used for diagnosis of gonorrhoea. Isolation and identification of N. gonorrhoeae N. gonorrhoeae requires an enriched culture medium, such as (modi- fied) Thayer–​Martin or modified New York City which consist of gonococcal (GC) agar base supplemented with a source of iron (lysed horse blood) and essential amino acids and glucose, and incubation in moist atmospheric conditions enriched with 5–​7% carbon di- oxide at 37°C. Appropriate specimen collection, storage, and efficient transport to the laboratory are crucial for successful isolation. Specimens are taken from appropriate sites using disposable loops or swabs for inoculation in the clinic or transfer to the labora- tory in transport medium. Isolation is enhanced by adding anti- biotics to the medium to suppress other organisms that colonize the anogenital tract. Vancomycin or lincomycin inhibit Gram-​positive organisms, colistin and trimethoprim inhibit other Gram-​negative organisms, and amphotericin B or nystatin inhibit yeasts. Gram-​negative cocci on primary isolation that are oxidase positive (produce cytochrome c oxidase) are considered to be Neisseria species. In the developed world, confirmation of species identity as N. gonor- rhoeae is considered normal practice. This is frequently achieved using carbohydrate utilization tests, either alone or in combination with detection of the enzyme prolyliminopeptidase in commercial kits; N. gonorrhoeae differs from other species in that it utilizes glucose only during growth. An alternative approach is to use immunological re- agents such as coagglutination tests (e.g. Phadebact Monoclonal GC test) that utilize antibodies against antigenic epitopes on the N. gonor- rhoeae major outer membrane protein PorB (Por or PI). These sensitive and specific reagents can identify colonies directly from the primary isolation medium and a result can be obtained on the same day as the organism is isolated. Correct identification of N. gonorrhoeae is always desirable but is most important in cases of sexual or child abuse when more than one identification test should be used to confirm an iso- late as N. gonorrhoeae. In recent years, NAATs have become commonly used for species verification of N. gonorrhoeae. Molecular detection of N. gonorrhoeae NAATs have historically not been used as extensively for N. gonor- rhoeae as for C. trachomatis, with which it commonly coexists. This is because the NAATs initially failed to offer much advantage over Gram staining and culture for urogenital specimens, most particu- larly earlier generations of NAATs had a clearly suboptimal speci- ficity for N. gonorrhoeae, and NAATs do not provide an organism for antimicrobial susceptibility testing. However, the increasing pressure to screen more patients attending for sexual healthcare, or asymptomatic individuals in other healthcare settings, together with the evolution of improved commercial NAATs, has signifi- cantly escalated their use. The sensitivity of NAATs is high, they are less affected by suboptimal handling or transport of specimens and can be used with specimens such as urine (recommended for males) or self-​taken swabs (recommended for females). NAATs are also rapid, allow automation, and enable simultaneous detection of several STIs. However, despite the fact that the specificity of newer generations of N. gonorrhoeae NAATs has improved, confirmation using a second NAAT with a different nucleic acid target remains recommended in low-​prevalence populations and for extragenital specimens. Furthermore, the performance characteristics of the various commercially available or in-​house N. gonorrhoeae NAATs differ significantly. No molecular tests are available for complete determination of antibiotic susceptibility and, accordingly, a rep- resentative sample of viable organisms will be required for surveil- lance purposes to guide antimicrobial therapy. Substantial research is ongoing internationally to develop molecular tests for prediction of antimicrobial resistance or susceptibility for surveillance and, ideally, to guide individually tailored treatment. Epidemiological typing This has been used to study evolution and bacterial population gen- etics, identify specific strains transmitted globally in specific popu- lations and/​or in core groups, identify temporal and geographic changes in strain types and the emergence and transmission of in- dividual strains (e.g. antimicrobial resistant strains), establish strain identity/​difference in contact tracing, reinfection, or test of cure, confirm/​disprove treatment failures, resolve forensic issues, and confirm presumed epidemiological connections or discriminate iso- lates of suspected sexual networks clusters and outbreaks. Molecular typing has largely replaced phenotypic typing methods (auxotyping (based on nutritional requirement) and serovar determination (based on reactivity with monoclonal antibodies)) because it is more robust, reproducible, objective, and discriminating. Sequence-​based methods, such as the N. gonorrhoeae multiantigen sequence typing (NG-​MAST), which examines diversity in two more variable genes (porB and tbpB), have proved useful in many countries. However, N.  gonorrhoeae is highly competent for genetic exchange during its entire life and particularly genes with products exposed to the immune response can have high mutational and recombinational rates. Accordingly, a highly discriminatory typing method, such as NG-​MAST, is more appropriate for short-​term studies such as ana- lysis of sexual networks or outbreaks, rather than long-​term tem- poral studies or comparisons at different geographical locations, nationally and particularly internationally. The combination of an appropriate typing method and detailed epidemiological, anti- microbial resistance and behavioural data can provide information that can be used for public health purposes, and has been proven on a national level in United Kingdom as well as on a European level. Ideally, it would be possible to apply such information when designing public health preventive measures and interventions. In Fig. 8.6.6.6  Urethral swab for gonorrhoea. Courtesy of Peter Greenhouse.

section 8  Infectious diseases 1030 recent years, whole genome sequencing has become more access- ible, user-​friendly, and cost-​effective. Whole genome sequencing provides ideal discriminatory and more accurate data that can be ap- plied to microepidemiological (short-​term), macroepidemiological (long-​term), and evolutionary studies, and allows molecular predic- tion of antimicrobial susceptibility or resistance. Antimicrobial resistance N. gonorrhoeae was originally inherently susceptible to most antimicro- bial agents but with increased usage both chromosomally mediated and plasmid-​mediated resistance has developed to all antimicrobials available for treatment of gonorrhoea. Penicillin was used as first-​line therapy for many years, until in 1989 when WHO issued new guide- lines for the treatment of gonorrhoea following increasing levels of plasmid-​mediated and chromosomally mediated resistance worldwide. Alternative treatments were recommended: ciprofloxacin (a fluoro- quinolone), ceftriaxone (a third-​generation cephalosporin), or spec- tinomycin (an aminocyclitol), with penicillin recommended only if the gonococcal population was known to be susceptible. Ciprofloxacin was the treatment of choice in the United Kingdom because it is ad- ministered orally and was highly effective and inexpensive, whereas in the United States of America ceftriaxone was more widely used. In 2002 resistance to ciprofloxacin reached levels over 5% in England and Wales resulting in a change in guidelines for first-​line therapy to a third-​generation cephalosporin (i.e. ceftriaxone or cefixime). In 2011, reports of treatment failure to the oral third-​generation cephalosporins such as cefixime and in vitro surveillance data indicating increasing prevalence of resistance to cefixime were worrying and national guide- lines in the United Kingdom were revised to recommend the inject- able agent ceftriaxone 500 mg intramuscularly plus azithromycin 1 g oral single dose as first line. The lack of new alternative treatments after ceftriaxone is a major concern internationally and raises the pos- sibility of gonorrhoea as a potentially untreatable infection. In recent years, the drug pipeline has fortunately opened slightly again and a few new antimicrobials for future treatment of gonorrhoea have been developed. Particularly the novel spiropyrimidinetrione, zoliflodacin, (ETX0914 or AZD0914), is promising and currently in randomized clinical trials for treatment of gonorrhoea. Chromosomally mediated resistance Decreased susceptibility to penicillin was detected as early as 1958 but this could be overcome by increasing the dose of penicillin and by adding probenecid. It was not until the 1970s that strains began to appear with minimum inhibitory concentrations (MIC) to peni- cillin of more than 1.0 mg/​litre and posed a therapeutic problem. Chromosomal resistance to penicillin in N. gonorrhoeae is the re- sult of the cumulative effects of mutations at multiple loci: penA (decreasing the drug affinity to the main lethal target penicillin-​ binding protein 2 (PBP2)), mtrR (increasing the drug efflux through an overexpressed MtrCDE efflux pump), penB (decreasing the drug influx through the porin PorB), ponA (decreasing the drug affinity to the second target PBP1), and penC (or pilQ; decreasing the drug influx through the secretin PilQ). Resistance to ciprofloxacin emerged initially in N. gonorrhoeae strains primarily originating from the Western Pacific region, par- ticularly Japan, with single nucleotide polymorphisms in the DNA gyrase gene gyrA and the topoisomerase IV gene parC. These are the main resistance-​determining loci, however, mutations in the gyrB gene and overexpressed efflux pumps, such as NorM, might also in- crease the MIC further. Fluoroquinolone-​resistant gonorrhoea is now endemic and highly prevalent in most countries globally. In vitro and clinical resistance to third-​generation cephalosporins appear also to have initially emerged in the Western Pacific region, particularly in Japan. Therapeutic failure of the oral cephalosporin, cefixime, has now been verified in many countries. Rare failures to treat pharyngeal gonorrhoea with ceftriaxone have also been veri- fied in several countries. The main mechanism of resistance to third-​ generation cephalosporins is specific mosaic penA alleles, encoding mosaic PBP2 with a remodelled target with less affinity for the drug. The mtrR and penB resistance determinants (see earlier) further in- crease the MIC of the third-​generation cephalosporins. The emer- gence of resistance to ceftriaxone, which is the last remaining option for empiric first-​line monotherapy is a major concern globally. Azithromycin, primarily used for chlamydial infection, is being used also for gonorrhoea, particularly in the currently recommended, dual antimicrobial therapy. However, resistance has emerged, pos- sibly under selective pressure of the lower 1-​g dose recommended for chlamydial infection compared to the 2-​g dose for gonorrhoea. The azithromycin resistance is mostly due to specific single nucleo- tide polymorphisms in the 23S rRNA (decreasing the drug affinity to the target 23S rRNA), but also the mtrR resistance determinant, uptake of erm genes encoding 23S rRNA methylases and additional efflux pumps (MacAB efflux pump and Mef effux pump) increase the MIC of azithromycin. Since 2015, an outbreak of N. gonorrhoeae with high-​level resistance (MIC ≥256 mg/​litre) has been recorded in England. It is of grave concern that the first gonococcal strain with ceftriaxone resistance combined with high-level resistance to azithromycin globally was identified in the United Kingdom in 2018. Spectinomycin can be an alternative for treatment of gonorrhoea as resistance is only reported very sporadically worldwide. However, if spectinomycin is used frequently, resistance might quickly be selected. Spectinomycin also has poor efficacy (just above 52%) against pharyngeal gonorrhoea and can be difficult to access in many countries, including the United Kingdom. High-​level resistance to spectinomycin (MIC >1024 mg/​litre) is due to a single nucleo- tide polymorphism in the 16S rRNA gene or, more rarely, specific muta­tions in the rpsE gene (encoding the 30S ribosomal protein S5), which decrease the drug affinity for the ribosomal target. Plasmid-​mediated resistance N. gonorrhoeae exhibiting plasmid-​mediated high-​level resistance to penicillin was first described in 1976. Simultaneous reports ap- peared of two strains, one from Africa carrying a plasmid of 3.2 MDa (5599 base pair) and the second from the Far East carrying a plasmid of 4.4 MDa (7426 base pair). Both plasmids encoded the TEM-​1 type β-​lactamase (penicillinase). Penicillinase-​producing N.  gon- orrhoeae carrying the African (3.2-​MDa) and Asian (4.4-​MDa) plasmids have now disseminated worldwide although their preva- lence is greatest in countries of the developing world. Penicillinase-​ producing N. gonorrhoeae carrying plasmids of differing size have been described, such as the Rio/​Toronto, Nimes, New Zealand, and Johannesburg plasmids, but these have had more limited spread. The Asian plasmid appears to be the ancestral plasmid from which the other plasmids evolved, through deletions and/​or insertions.

8.6.6  Neisseria gonorrhoeae 1031 In 1985, plasmid-​mediated resistance to tetracycline was first de- tected. This high-​level resistance (MIC ≥16 mg/​litre) is due to the acquisition of the tetM determinant by the conjugative plasmid of N. gonorrhoeae resulting in a plasmid of 25.2 MDa. Tetracycline is not the treatment of choice for gonorrhoea but was commonly used, par- ticularly in African countries, until the emergence of high-​level resist- ance to tetracycline, because it was inexpensive and readily available. Antimicrobial susceptibility testing The primary aim of antimicrobial susceptibility testing of N. gonor- rhoeae is to predict therapeutic failure. However, it is also important to monitor temporal drifts in susceptibility and to detect the emer- gence of resistant strains to therapies recommended, which should inform revisions of the guidelines for empiric treatment of gonor- rhoea. The quantitative agar dilution method determines the MICs of antimicrobials and is the gold standard method. However, particularly for testing a low number of isolates, agar dilution method is laborious and not appropriate for routine antimicrobial susceptibility testing. Instead, the quantitative Etest for MIC determination is commonly used internationally. Etests, which are strips that contain a gradient of concentrations of antibiotics and give a MIC, are very useful for laboratories testing small numbers of strains, albeit still an expensive choice. A qualitative susceptibility testing can also be performed using a disc diffusion method. This determination of zone sizes of inhibition around antibiotic-​containing discs has been the method of choice by most clinical laboratories in the United Kingdom. However, N. gonor- rhoeae is a fastidious organism and different strains significantly vary in their growth patterns and therefore this method can be difficult to quality assure, control, and interpret. Accordingly, for adequate repro- ducibility and interpretation to appropriately reflect the MIC values of given antibiotics, these methods require considerable quality as- surance and appropriate quality controls. Determination of the full MIC might not be necessary for most clinical laboratories. However, for susceptibility testing of cefixime and ceftriaxone, Etests are par- ticularly useful, reliable, and are preferable to disc testing. When disc diffusion methods are used, any new, emerging, or rare antimicrobial resistance should be confirmed by MIC determination. Treatment of gonococcal infection Since the spectrum of antibiotic resistance is continually chan- ging, up-​to-​date advice regarding resistance and treatment should be sought, for example from the websites of the Public Health England (PHE; formerly Health Protection Agency (HPA)), the British Association of Sexual Health and HIV (BASHH), and the International Union against STIs (IUSTI) European STI guidelines. In the United Kingdom, first-​line treatment of uncomplicated uro- genital, rectal, or oropharyngeal infection in adults is ceftriaxone intramuscularly with azithromycin orally. Spectinomycin intramus- cularly, also with azithromycin orally, is an alternative and is suitable for those with penicillin allergy. Both regimens can be used to treat infection in pregnant or breastfeeding women. When an infection is confirmed by laboratory testing before treatment to be susceptible, ciprofloxacin orally as a single dose, ofloxacin orally as a single dose or cefixime orally as a single dose can also be used. Azithromycin should not be used on its own for treatment of gonorrhoea because of the ease of selection of resistance and presence of high-​level resistance in the United Kingdom as well as in many other countries globally. Pelvic infection and epididymo-​orchitis might be due to N. gonor- rhoeae, C. trachomatis, mixed anaerobes or any combination of organ- isms, and treatment regimens reflect this. British guidelines suggest that gonococcal pelvic infection and perihepatitis should be treated with parenteral antibiotics. Ceftriaxone intramuscularly or intraven- ously as a single dose with doxycycline and metronidazole, both twice daily intravenously or orally for 14 days, are recommended. For gono- coccal epididymo-​orchitis, the treatment is a single dose of ceftriaxone intramuscularly plus doxycycline orally twice daily for 2 weeks. For disseminated gonococcal infection, British guidelines suggest ceftriaxone intramuscularly or intravenously daily; or cefotaxime intravenously every 8 h; or spectinomycin intramuscularly every 12 h continuing for 7 days. This can be switched 24–​48 h after symp- toms improve to an oral regimen such as cefixime twice daily or, if quinolone resistance is excluded, ciprofloxacin or ofloxacin twice daily. Laboratory testing should provide full antibiotic susceptibil- ities of the causative organism. Individuals infected with gonorrhoea should be given a detailed explanation of their infection with emphasis on the long-​term im- plications for the health of themselves and their partner(s). Patients should also be advised to abstain from sexual intercourse until they and their partner(s) have completed treatment. Finally, patients should be screened for other STIs and their partners identified and, ideally, tested for infection before treatment. Both the United Kingdom national guideline for management of gonorrhoea in adults, and the European guideline on the diagnosis and treatment of gonorrhoea in adults recommend a test of cure in all cases of gonorrhoea. Where resource or practical considerations require test of cure to be selective, priority should be given to pa- tients with persisting symptoms or signs, pharyngeal infection (sig- nificantly harder to eradicate with any treatment), and treatment with a regimen other than the first-​line recommendation. FURTHER READING Barlow D, Phillips I (1978). Gonorrhoea in women: diagnostic, clin- ical and laboratory aspects. Lancet, i, 761–​4. Bignell CJ, Fitzgerald M (2011). UK National guideline for the manage- ment of gonorrhoea in adults, 2011. https://​www.bashh.org/​guidelines Bignell C, Unemo M (2013). 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int J STD AIDS, 24, 85–​92. Eyre DW, et  al. (2018). Gonorrhoea treatment failure caused by a Neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance, England, February 2018. Euro Surveill. Fifer H, et al. (2016). Failure of dual antimicrobial therapy in treatment of gonorrhea. N Engl J Med, 374, 2504–6. Harris SR, et  al. (2018). Public health surveillance of multidrug- resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey. Lancet Infect Dis, pii, S1473-3099 (18)30225-1. doi: 10.1016/ S1473-3099(18)30225-1. Hook III EW, Handsfield HH (2008). Gonococcal infections in the adult. In: Holmes KK, et al. (eds) Sexually transmitted diseases, 4th edition, Chapter 35. McGraw Hill, New York, NY. Ison CA, et al. (2013). Current and future treatment options for gonor- rhoea. Sex Transm Infect, 89 Suppl 4, iv52–​6.

8.6.7 Enterobacteria and bacterial food poisoning

8.6.7 Enterobacteria and bacterial food poisoning 1032

section 8  Infectious diseases 1032 Nassif X, et al. (1999). Interactions of pathogenic neisseria with host cells. Is it possible to assemble the puzzle? Mol Biol, 32, 1124–​32. Newman L, et al. (2015). Global estimates of the prevalence and inci- dence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One, 10, e0143304. Ohnishi M, et al. (2011). Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea? Detailed characterization of the first strain with high-​level resistance to ceftriaxone. Antimicrob Agents Chemother, 55, 3538–​45. Public Health England (2014). Guidance for the detection of gon- orrhoea in England. 2014. https://​www.bashh.org/​documents/​ Guidance%20for%20the%20detection%20of%20gonorrhoea%20 in%20England%20Aug%202014.pdf Sherrard J, Barlow D (1996). Gonorrhoea in men: clinical and diag- nostic aspects. Genitourin Med, 72, 422–​6. Tabrizi SN, et al. (2011). Evaluation of six commercial nucleic acid amplification tests for the detection of Neisseria gonorrhoeae and other Neisseria species. J Clin Microbiol, 49, 3610–​16. Taylor SN, et al. (2018). Single-dose zoliflodacin (ETX0914) for treat- ment of urogenital gonorrhea. N Engl J Med, 379, 1835–45. Unemo M (2015). Current and future antimicrobial treatment of gonorrhoea—​the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infect Dis, 15, 364. Unemo M, Ison C (2013). Gonorrhoea. In:  Unemo M, et  al. (eds) Laboratory diagnosis of sexually transmitted infections, in- cluding human immunodeficiency virus, pp. 21–​54. World Health Organization (WHO), Geneva, Switzerland. Unemo M, Shafer WM (2014). Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev, 27, 587–​613. Whiley DM, et al. (2008). Exploring ‘best practice’ for nucleic acid de- tection of Neisseria gonorrhoeae. Sex Health, 5, 17–​23. 8.6.7  Enterobacteria and bacterial food poisoning Hugh Pennington ESSENTIALS The worldwide impact of food poisoning is very great. Such in- fections kill many children in the developing world, where diar- rhoeal diseases stunt their physical and cognitive development. The number of illnesses is also large elsewhere: in the United Kingdom the most common cause of food poisoning, Campylobacter, accounts for about 500 000 cases every year. The most common bacterial pathogens are Campylobacter and various members of the Enterobacteriaceae, a large family of Gram-​negative organisms, of which Escherichia coli, shigella, and salmonella are considered in this chapter. Escherichia coli Pathogenic E. coli include the following: Enteropathogenic—​virulence-​positive enteropathogenic E.  coli are now rare in industrialized countries; food-​ and water-​borne and person-​to-​person spread occur, resulting in diarrhoeal illness; fewer than 500 cases are recorded annually in the United Kingdom. Enteroaggregative—​first isolated from malnourished children in Chile suffering from chronic diarrhoea; not routinely tested for in industrialized countries but probably common. Enterotoxigenic—​an important cause of mortality in children under 5 years of age in developing countries, and causes travellers’ diar- rhoea; adheres to the mucosal surface of epithelial cells of the prox- imal small bowel, a process mediated by at least 12 different kinds of pili encoded by transferable plasmids, and produces enterotoxins. Enteroinvasive—​like shigella, for all practical purposes. Enterohaemorrhagic—​the most important enterohaemorrhagic E. coli is E. coli O157:H7, which produces a toxin virtually identical to that of Shigella dysenteriae. E. coli O157 is a normal non​pathogenic inhabitant of the gastrointestinal tract of cattle and sheep; most human infections are contracted either by the consumption of foods contaminated with animal manure or by its direct ingestion, prob- ably from hands that have touched contaminated surfaces. Clinical presentation is with diarrhoea (becoming bloody in 90% of cases) and abdominal pain, with a few cases (15% of children <10) going on to develop haemolytic uraemic syndrome. Diagnosis is by culture. Management is supportive. An enterohaemorrhagic E. coli /​Eagg E. Coli hybrid caused the ser- ious E. coli O104:H4 outbreak in Germany in May to July 2011; attrib- uted to an organic farm producing fenugreek seed sprouts. Shigella Infections are exclusively human, spread by the faecal–​oral route from person-​to-​person, and with a very low infectious dose. Shigellosis is endemic in developing countries in tropical areas, and it probably kills about 600 000 annually, mostly young children. Presentation is with watery diarrhoea, fever, and malaise, with se- vere infections (most often caused by S. dysenteriae) progressing to diarrhoea comprising mucus, blood, and pus, along with severe ab- dominal cramps and tenesmus. Management of mild cases is sup- portive; severe cases are given antibiotics (ampicillin, co-​trimoxazole, tetracycline, ciprofloxacin, others) as guided by local antimicrobial susceptibility data. Salmonella There are over 2000 salmonella serotypes, all belonging to the single species Salmonella enterica. Those that cause food poisoning infect both animals and humans, and most infections are food-​borne, most often by poultry, with S. enteritidis (strictly a serotype rather than a species) the paradigmatic organism. Clinical presentation is typic- ally with headache, vomiting (not usually a prominent feature), diar- rhoea, abdominal pain, and fever. Metastatic infection sometimes occurs, particularly osteomyelitis and in atherosclerotic vessels, ab- normal heart valves, and joint prostheses. Management of mild cases is supportive; severe cases are given antibiotics, usually a quinolone or macrolide. Campylobacter This is by far the most common cause of bacterial gastroenteritis in the industrialized world, with an annual incidence of infection perhaps as high as 1 per 100 in the United Kingdom. The organ- isms are very common in the intestines of wild birds, poultry, cattle, and sheep, but the source of infection in most human cases is unknown. A  prodrome of fever and general aching sometimes

8.6.7  Enterobacteria and bacterial food poisoning 1033 precedes abdominal pain (sometimes severe) and diarrhoea (fre- quently bloody). Complications include reactive arthritis (1% of cases) and Guillain–​Barré syndrome. Most infections are self-​limiting, but aside from supportive care, antibiotics (often erythromycin or ciprofloxacin) are given to severe cases. Prevention Prevention of food poisoning depends on Hazard Analysis and Critical Control Points, which identifies hazards, identifies the points in a pro- cess where they may occur, and decides which points are critical to control to ensure consumer safety (e.g. in milk pasteurization the crit- ical control points are the temperatures reached during heating, its dur- ation, and the measures taken to prevent subsequent contamination). Introduction Food poisoning denotes gastrointestinal diseases caused by microbes transmitted in food or by microbial toxins preformed there. Food spoilage by microbes also has important consequences for human health because of its impact on food supply. Each year 10–​20% of the world’s annual cereal crop of approximately 2 × 109 tonnes is lost through spoilage by moulds. Much of this loss occurs in the humid tropics and contributes there to the nutritional deficiencies caused by other factors. The terms food poisoning and food-​borne disease overlap but are not synonymous. Thus, variant Creutzfeldt–​Jacob disease (vCJD), contracted by eating meat products from cows with bovine spongi- form encephalopathy, only fits under the food-​borne rubric because of its very long incubation period despite the absence of gastrointes- tinal symptomatology. It is the same for bovine tuberculosis trans- mitted by milk. The worldwide impact of food poisoning is very great, as rec- ognized by the World Health Organization Global Strategy for Food Safety (2002), and is the cause of death of many children in the developing world. Diarrhoeal diseases stunt the growth of children and impair their physical and cognitive development. Mortality rates are much lower in developed countries, but the number of illnesses is still large. Quantitation is difficult because of underreporting. A large national study of the number and causes of cases of infectious intestinal disease in the United Kingdom es- timated that in 2009 there were 16.9 million cases and over 1 mil- lion GP consultations due to infectious intestinal disease every year. Routes of transmission were not established in this study, but aetiologies indicate that food-​borne transmission occurred in a sig- nificant minority; for example, campylobacter was responsible for 500 000 cases. In the United States of America, it has been estimated that each year 3.645 million people contract bacterial food-​borne disease with 35 796 being hospitalized and 865 deaths. The human intestine is home to 1013 to 1014 microorganisms. Their collective genome contains at least 100 times as many genes as the human one. They metabolize glycans and amino acids, detoxify xenobiotics, and synthesize isoprenoids and vitamins. A prominent member of the distal gut and faecal flora is the methane synthe- sizer, Methanobrevibacter smithii. However, the taxonomic identity and precise properties of most gut microbes is unknown because they cannot be grown in the laboratory. Cultivable ones that cause disease comprise a small minority, even when those opportunistic pathogens which occur primarily as commensals are included, such as members of the Enterobacteriaceae. This family contains several important causes of disease. Salmonella Typhi and S. Paratyphi, the causes of typhoid and paratyphoid fevers, are members of the Enterobacteriaceae. They cause systemic disease and are described in detail in Chapter 8.6.9. Gastroenteritis caused by non-​Enterobacteriaceae (i.e. the Gram-​negative organisms Aeromonas, Plesiomonas, Vibrio para- haemolyticus and other non-​cholera vibrios), and, most important quantitatively by incidence, Campylobacter, are included here, to- gether with accounts of food poisoning caused by Bacillus spp. For descriptions of diseases caused by Clostridium botulinum and C. perfringens, see Chapter 8.6.25, C. difficile see Chapter 8.6.24, and Staphylococcus aureus see Chapter 8.6.4. An overview of infections of the intestinal tract is given in Chapter 15.18. Enterobacteriaceae The Enterobacteriaceae is a large family of Gram-​negative bacteria. Many species are free-​living, some are associated with plants and can be plant pathogens, and others live in the intestines of animals and humans. The pathogens considered in detail in this chapter be- long to the genera Escherichia, Shigella, and Salmonella. The formal bacteriological definition of the family is that its mem- bers are non-​sporing Gram-​negative rods that are often motile, usu- ally by peritrichous flagella. They are easily cultivable on ordinary laboratory media. They might or might not have capsules. All are aerobes, although many grow anaerobically as well. All ferment glu- cose with the formation of acid and sometimes gas, and most reduce nitrate to nitrite. They are oxidase negative and, with the exception of one type of Shigella dysenteriae, are catalase positive. For a century, species in the family have been identified by carbo- hydrate fermentation patterns and by testing the reactivity of the bacteria to antisera prepared against their surface structures. Salmonella and Shigella do not ferment lactose. With the exception of proteus, providencia, and morganella, all other Enterobacteriaceae ferment this sugar freely with acid production. After cultivation on agar medium containing lactose and a pH indicator, non-​lactose-​ fermenting colonies stand out because of their colour differ- ence, making the initial detection of salmonella or shigella a fairly straightforward task. A similar approach is used to detect the most frequently occurring enterohaemorrhagic Escherichia coli sero- type, E. coli O157:H7, most isolates of which do not ferment sorb- itol. Antigenic epitopes key in identification schemes reside in the thick outer bacterial layer (the O antigens) and in flagella (the H antigens). The outer layer is a complex of lipopolysaccharide protein and lipid. The lipopolysaccharide has a hydrophobic lipid A com- ponent (responsible for the pathological effects of endotoxin) and a hydrophilic polysaccharide made up of an O-​specific polysaccharide and a core oligosaccharide. K-​antigen epitopes reside on a capsular polysaccharide which when present covers the O antigens. It can be removed by boiling. Enterobacteriaceae have a clonal population structure. Each in- dividual pathogen has a common ancestor and the incidence of the disease it causes correlates with the population size of the clone that has grown from it. The task of the diagnostic laboratory is to detect

section 8  Infectious diseases 1034 and identify these clones as quickly and as cheaply as possible. In general, the traditional tests described here satisfy these require- ments. Enterobacteriaceae clones evolve in real time, however, so markers can often be found to distinguish strains, even those with a recent common clonal origin. Tests that determine the suscepti- bility of isolates to a range of bacterial viruses, bacteriophage typing, have been widely used for this purpose, particularly in the United Kingdom. Molecular methods that detect DNA sequence differences are widely used internationally and have universal applicability and high discriminatory power. The Enterobacteriaceae considered here live in the intestines of animals and humans. This environment facilitates gene exchange between individual bacteria. It has been known for many years that plasmids, bacteriophages, and transposons are mobile gen- etic elements. Studies on E. coli virulence factors in the 1990s led to the discovery of pathogenicity islands, large genomic regions that are present in pathogenic strains but not in related non​pathogens. They carry genes associated with virulence, are often associated with tRNA genes, and are frequently flanked by repeat sequences. Their G + C content is different from the rest of the bacterial chromosome. DNA sequencing studies have shown more recently that similar islands also occur in non​pathogenic strains. The functions they en- code contribute to increased adaptability, fitness, and competitive- ness. Genomic islands have been acquired from other bacteria, not necessarily closely related ones. With the other mobile genetic elem- ents they form part of a flexible gene pool which confers beneficial traits supplementing the essential functions encoded by the con- served core genome. Promiscuous plasmid exchange coupled with the spread of clones has been responsible for the emergence and spread of Enterobacteriaceae that produce New Delhi metallo-​β-​lactamase-​1 (NDM-​1). This inactivates all β-​lactam antibiotics except aztreonam. The blaNDM-​1 gene responsible usually occurs in isolates already resistant to most antibiotics; they only remain sensitive to colistin, fosfomycin, and, less consistently, to tigecycline. The first NDM-​1 infection was identified in 2008. Since then they have been iden- tified worldwide, mostly in E. coli and Klebsiella pneumoniae and usually in patients who have been treated in hospitals in the Indian subcontinent. In New Delhi the blaNDM-​1 gene has been found in Enterobacteriaceae (including shigella), aeromonads, and Vibrio cholerae, and in tap water and environmental water. Molecular genetics has shown that shigella and E. coli are so closely related that formally they belong to a single genus. Escherichia has priority; however, Shigella is a useful name and is likely to continue in use for the foreseeable future. Likewise, the enthusiasm of those who gave hundreds of specific names to salmonella strains distin- guished by serotyping was misplaced. The strains are so closely related they are now referred to as serovars of the single species, Salmonella enterica. Escherichia coli Theodor Escherich was the first to grow E. coli in pure culture. He employed the ‘Plattenmethode’ described by Robert Koch in 1881 in his investigation in Munich in 1885 of the intestinal bacterial flora of newborns. It was the first detailed study of human commensal bacteria; appropriately so, because an overwhelming majority of the hundred billion billion E. coli bacteria that live in the world at any time are normal inhabitants of the intestines of healthy humans and animals. The perception in the 1940s by molecular biologists of its harmless nature coupled with its non​fastidious cultural require- ments and rapid growth (2–​3 generations/​h in the laboratory) led to its choice as a model organism. The strain most often studied is K12, isolated from the faeces of an American convalescent diphtheria pa- tient in 1922. More Nobel prizes have been won by researchers on E. coli than any other species (with the exception of Homo sapiens). The identification of E.  coli using traditional bacteriological methods is straightforward. It is a non-​sporing Gram-​negative rod, usually motile with peritrichous flagella, facultatively anaerobic, and a gas producer from fermentable carbohydrates. The methyl red re- action is positive and the Voges–​Proskauer reaction negative. Many strains have a polysaccharide capsule or microcapsule and most rapidly ferment lactose. Finding such an organism in a normally sterile site such as cerebrospinal fluid or in larger numbers in urine than can be accounted for by contamination is sufficient to indicate an aetiological role. Different approaches have to be used to detect enterovirulent E. coli in stools. Selective indicator media have been developed for E. coli O157. Other kinds are not looked for routinely; the best methods for detection use DNA probes or polymerase chain reaction amplification procedures that are only available in refer- ence laboratories. Few studies have been done on the carriage of commensal E. coli by healthy individuals but it is known that some carry a single clone for long periods, whereas others carry several simultaneously, acquiring and losing different clones rapidly. Some clones have a worldwide distribution; others seem to be only local. The genome sequence of strain K12 was published in 1997 and since then the genomes of representative pathogenic clones have been sequenced. A general principle has emerged that within the spe- cies there is an enormous amount of genetic diversity. Comparison of K12, a uropathogenic isolate, and an E. coli O157 showed that only 39.2% of the combined set of proteins was common to all. The genomes of the pathogens were as different from each other as each pathogen was from the commensal strain. Another E. coli charac- teristic is that different clones share a common genomic backbone of vertically evolved genes which is punctuated by many islands that have been acquired by different horizontal transfer events in each strain. All pathogenic E. coli are sticky, in that they produce structures on their surfaces that act as organelles of attachment. The pro- teins that make them sticky are adhesins, which recognize host cell structures—​receptors—​with stereochemical specificity. This fit is an important determinant of host specificity and tissue tropism. Adhesins are often assembled into hair-​like fibres, pili. Some take the form of a fuzzy mass on the bacterial surface, curli. Others form no particular oligomeric structures. The genomes of uropathogenic strains are also rich in genes coding for autotransporters, phase-​switch recombinases, and iron-​ sequestration systems. Enteropathogenic E. coli (EPEC) The isolation of antigenically identical E. coli strains during the in- vestigation of outbreaks of diarrhoea in young babies in the 1940s in London, Aberdeen, and Liverpool provided the first clear evidence that E. coli could be an intestinal pathogen. Subsequent serotyping showed the isolates to be O111 and O55. The disease they caused had a mortality of about 50% and mostly occurred in babies aged 6 months or less. Although volunteer studies in Liverpool showed

8.6.7  Enterobacteria and bacterial food poisoning 1035 that isolates from babies caused gastroenteritis in adults, a dose of 2 × 109 organisms only led to a mild short illness. Typical EPEC cause illness in infants and children under 2 years old; the hospital outbreaks that occurred in the 1940s are no longer seen. Intestinal colonization by typical EPEC involves virulence plasmid-​encoded type IV bundle-​forming pili which mediate bacterium to bacterium adherence and the formation of compact microcolonies on the surface of host cells, a pattern called localized adherence. EPEC fall into two related groups. Each contains several clones, some of which have been circulating for many years and have been found on several continents. O type does not always correlate with clonal type; thus type O142 marked two clones, one respon- sible for a high mortality outbreak in a Mexico City hospital in 1965 and the other for much less lethal infections of infants in Indonesia in 1960, hospital outbreaks in England, Scotland, and Ireland from 1969 to 1972, and sporadic cases in Canada in 1972 and Arizona in 1975. Virulence-​positive EPEC are now rare in industrialized coun- tries. Surveys in Brazil showed that they were common there in the 1980s and 1990s. In Europe and North America, EPEC lacking the virulence plasmid are now much more frequent causes of diarrhoea. These atypical EPEC are now becoming proportionally commoner in Brazil as well. A mechanism central to EPEC pathogenesis is the attaching and effacing (A/​E) lesion. At the sites of adhesion in the colon, intes- tinal cell microvilli disappear. Actin accumulates beneath the bac- teria, which become seated on pedestal-​like structures. The bacterial genes for the production of attaching and effacing lesions are located on the locus of enterocyte effacement pathogenicity island. It codes for intimin, an outer membrane protein responsible for adherence of the bacteria to enterocytes, Esp molecules, which are involved in the machinery that translocates bacterial proteins into enterocytes, and tir, which is translocated and inserts into the enterocyte cell mem- brane to act as the receptor for intimin. The incubation period of the diarrhoeal illness caused by EPEC ranges from 12 to 72 h, and the illness can last for several days. Food-​ borne, water-​borne, and person-​to-​person spread occur. Fewer than 500 cases are recorded annually in the United Kingdom. Enteroaggregative E. coli (EAggEC) These adhere to cell cultures in a ‘stacked brick’ pattern, a property often encoded on a 60-​MDa plasmid. Enteroaggregative E. coli were first isolated from malnourished children in Chile who had chronic diarrhoea and have been found since in Brazil, Mexico, India, and Zaire. They are not routinely tested for industrialized countries but are probably common. They have diverse O and H types. Little is known about their virulence factors or their precise pathogenic potential. Enterotoxigenic E. coli (ETEC) ETEC are an important cause of mortality in children under 5 years old in developing countries, and a significant cause of travellers’ diar- rhoea; 31 to 75% of Peace Corps volunteers in Africa with diarrhoea have been found to have ETEC in their stools. An incubation period of 12 to 72 h is followed by diarrhoea and vomiting lasting 3 to 5 days. ETEC adhere to the mucosal surface of epithelial cells of the prox- imal small bowel, a process mediated in different strains by at least 12 different kinds of pili encoded by transferable plasmids. There they produce enterotoxins, either a heat-​labile (LT) or a heat-​stable (ST) one, or both. LTs resembles cholera toxin in structure, mode of entry into cells, and toxic effects therein (see Chapter 8.6.12). There are different forms but are all made up of one A subunit and five B subunits. There are two kinds of the low molecular weight ST; ST-​1 increases intestinal secretion through a route that involves the acti- vation of cyclic guanosine monophosphate. ETEC enterotoxins are often plasmid encoded. Many E. coli O serotypes have ETEC virulence factors; different clones vary in pilus type and in the enterotoxins they express. Enteroinvasive E. coli (EIEC) For all practical purposes EIEC are like shigella (see next); they have the same virulence factors and cause watery diarrhoea. Enterohaemorrhagic E. coli (EHEC) The most important EHEC is E. coli O157:H7. Because it produces a toxin which is lethal to cultured African green monkey (Vero) cells and is virtually identical to that of Shigella dysenteriae serotype 1 it is often called VTEC or STEC. Epidemiology E.  coli O157 is a new pathogen. It came to notice abruptly and dramatically in the United States of America in 1982 where it in- fected consumers of beef burgers at a well-​known chain of fast food restaurants. The first outbreak in England was in 1983. There is a rough correlation between closeness to the north and south poles and the national incidence of infection, which is higher in Scotland than England, in Canada than the United States of America, and in Argentina than Brazil. Accurate figures on its incidence in tropical countries are not available; it is probably uncommon. E. coli O157 is a normal non​pathogenic inhabitant of the gastrointestinal tract of cattle and sheep. A significant minority of animals, up to 9%, carry it at any one time. Most tissue-​associated E. coli O157 adhere to mucosal epi- thelium in a region extending up to 5 cm proximal to the rectoanal junction characterized by a high density of lymphoid follicles. Transmission of infection in humans is by the faecal–​oral route. Person-​to-​person spread between young children occurs, and most infections are contracted either by the consumption of foods con- taminated with animal manure or by its direct ingestion, probably from hands that have touched contaminated surfaces. Prevention of the contamination of carcasses in slaughter houses is difficult, which explains why transmission by meat occurs. Transmission by burgers has been significant in the United States of America be- cause they are often consumed rare; maintaining 60° C for 2 min in their centre makes them safe. Many ready-​to-​eat foods have been vectors (e.g. lettuce). Poorly pasteurized milk, unpasteurized apple juice, and untreated drinking water have been important vehicles of transmission. Contamination of meats after cooking was important in the big Scottish outbreak in 1996, in which about 500 people were infected and 17 died. About 80% of infections are sporadic. In North America and Europe, they are more common in people who live in or who have visited rural areas; in a majority of infections a food vehicle cannot be identified and direct transmission probably occurs.

section 8  Infectious diseases 1036 Pathogenesis E. coli O157 has the locus of enterocyte effacement pathogenicity island and adheres to enterocytes with the production of attaching and effacing lesions. In this respect it resembles EPEC; it may be that the latter was its progenitor. It also produces Shiga toxins (Stx1, Stx2). They are made of a single A subunit and a B pentamer. Stx1 is almost identical to the toxin produced by Shigella dysenteriae type 1; there are several allelic variants of Stx2 which are 50% homolo- gous to Stx1 in amino acid sequence. The B subunit binds to the glycosphingolipid globotriaosylceramide on the surface of host cells; the A subunit enters and turns off protein synthesis by disrupting the large ribosomal subunit in a ricin-​like fashion. Shiga toxins induce apoptosis in human renal cells as well. Most pathogenic E. coli O157 are Stx2 gene positive; about two-​thirds are positive for Stx1. Clinical features After an incubation period ranging from 2 to 12 days, most com- monly 3 days, diarrhoea starts. In up to 90% of cases it becomes bloody after another 1 to 3 days. Asymptomatic infections are not rare. Most symptomatic cases are afebrile; abdominal pain is more severe than in other forms of bacterial gastroenteritis and abdom- inal tenderness is common. After between 5 and 13 days of diar- rhoeal onset, a minority of cases develop haemolytic uraemic syndrome (HUS). The risk is much greater at the extremes of age; about 15% of children under 10 years develop HUS. Other risk fac- tors are antibiotic administration and the use of antimotility agents. Thrombocytopenia is the first abnormality to develop. There is in- creased activity of plasminogen activator inhibitor 1 and the concen- tration of fibrin D-​dimers and thrombin fragments 1 and 2 becomes high. In full HUS (some cases never progress beyond thrombocyto- penia) the kidneys fail. Neurological complications—​thrombotic or haemorrhagic strokes, seizures, and coma—​occur in 10% of HUS cases and cardiac dysfunctions occur in about the same propor- tion; they are important determinants of mortality. No treatment has been shown to prevent the development of haemolytic uraemic syndrome or specifically affect its course; the vascular damage that causes it is almost certainly well underway when patients present with diarrhoea. There is no bacteraemia and at this time the Shiga toxin has probably already reached its target organs via the blood stream. Management is supportive rather than specific. Antibiotics, antimotility agents, and non​steroidal anti-​inflammatory drugs should not be given. Fluid balance should be monitored and treated carefully to avoid cardiac overload. Platelet monitoring will indicate whether the haemolytic uraemic syndrome risk period has passed. Anaemia sometimes requires transfusion. Renal failure requires specialist management, and renal function returns in the majority. The sequelae of E. coli O157 haemolytic uraemic syndrome mostly relate to renal function; risk factors for long-​term problems are the severity of the HUS itself and the need for dialysis. In most cases of haemolytic uraemic syndrome, long-​term problems have not been described. Laboratory diagnosis The diagnosis of E. coli O157 infection is by culture. Growth on se- lective media containing sorbitol leads to the formation of colourless colonies that are provisionally identified using O157 antiserum. For the detection of small numbers or organisms (e.g. in food suspected to be a vehicle of transmission), a procedure using enrichment cultures followed by a specific concentration step using magnetic beads covered with O157 antiserum is carried out. Direct tests for Shiga toxin have been developed. Subtyping by phage typing and by DNA sequence-​based profiling is an essential tool in outbreak investigation. E. coli O157 is the most common EHEC and cause of haemolytic uraemic syndrome. Other serotypes fall into these categories, and O26:H11, O103:H2, O111:H−, and O113:H21 have caused outbreaks in Australia and in continental Europe. Some pathogenic strains of E. coli O157 ferment sorbitol. Routinely used selective media detect none of these. Control The inability to influence the outcome of EHEC infections once es- tablished means that prevention is paramount. The development and implementation of preventive policies has been driven by the impact of big dramatic outbreaks, particularly those associated with burger chains in the United States of America in the 1990s and a butcher’s shop in Scotland in 1996. In the United States of America, the Food and Drug Administration classifies E. coli O157 as a food adulterant; in consequence its detection has very bad commercial effects. In the United Kingdom and in Europe as a whole, the implementa- tion of Hazard Analysis and Critical Control Points (HACCP)—​the evidence-​based food safety system—​has probably been driven more rapidly than it otherwise would have been. With occasional excep- tions these measures have worked well. For example, in Scotland rural/​environmental risk factors for infection now far outweigh food ones. Further reductions in the number of cases will be difficult to achieve. No effective measures for reducing E. coli O157 in rumin- ants have been devised, and ruminants shed large numbers into the environment. In north-​east Scotland (human population 5 × 105) it has been estimated that cattle and sheep drop about 3 × 1013 live E. coli O157 on the ground every day; the infectious dose of E. coli O157 for humans is very small, less than 100. Fortunately, the chain of events that leads to transmission from manure to mouth only oc- curs infrequently. In most years since the mid-​1990s the annual in- cidence of infection in Scotland by E. coli O157 has been the highest in the world, but it is usually about 4 per 100 000, so infections are uncommon. EHEC/​EAggEC hybrid Between May and July 2011 more than 3500 cases of gastroenteritis caused by E. coli O104:H4 occurred in Germany. A small outbreak in France at the same time caused by the same organism assisted epi- demiological investigations which showed a very clear association with the consumption of fenugreek seed sprouts. Big differences with E. coli O157 were that many (c.25% of those with gastroenter- itis) went on to develop haemolytic uraemic syndrome and that most of these were adults. The incubation period (median 8 days) was longer but the interval between the onset of diarrhoea (median 5 days) was shorter. The causative organism had EHEC character- istics (the Stx2 gene, a high pathogenicity island encoding an iron uptake system, and adhesin genes) as well as those characteristic of EAggEC (the virulence plasmid pAA, the aggA (coding for the pilin subunit of aggregative adherence fimbriae), aggR (a transcriptional regulator), plc (coding for an intestinal colonization protein) and

8.6.7  Enterobacteria and bacterial food poisoning 1037 set1 (coding for Shigella enterotoxin) genes. The fenugreek was con- taminated with faeces during growth, or harvesting, or processing. The source—​animal or human—​is not known. Shigella Bacteriologists working in Japan, Germany, and the Philippines in the early 20th century demonstrated the bacterial aetiology of many cases of dysentery, and that the causative organisms belonged to a group of related but different non​motile, non​capsulate Gram-​ negative bacilli closely resembling E. coli but differentiated from it by their inability to ferment lactose on overnight incubation. The names of the genus, Shigella, and three of the four species, S. flex- neri, S. boydii, and S. sonnei, commemorate them. The pioneer was Kiyoshi Shiga, who discovered S. dysenteriae in Tokyo in 1898. Epidemiology As countries become more affluent there is a fall in the number of shigella types circulating as common causes of disease. There is also a relative shift towards types that cause milder disease. S. dysente- riae type 1 causes the most severe disease. In the United Kingdom it had disappeared by the mid-​1920s, when several S. flexneri types and S. sonnei were endemic. In England and Wales after 1950, 95 to 98% of infections were caused by S. sonnei, although S. flexneri was still more common in Scotland. In the United States of America S. flexneri became less common than S. sonnei in 1968; currently in Thailand S. sonnei is becoming more common than S. flexneri. However, the propensity of shigella to cause epidemics has meant that the change in incidence of infection has not been one of unre- mitting reduction. Thus in England and Wales after a postwar peak of 49 000 notifications of S. sonnei dysentery in 1956, the incidence declined steadily to an annual average of 3000 notifications between 1970 and 1990. However, they rose sharply in 1991 and again in 1992, peaking at 17 000 cases and then falling again. S. dysenteriae type 1 became more common in Mexico and Central America in 1968, in the Indian subcontinent in 1975, and Central Africa during 1985. Shigella infections are exclusively human (monkeys are suscep- tible but very probably catch their infections from humans) and are spread by the faecal–​oral route. Volunteer studies and information from outbreaks caused by the faecal contamination of water and food on cruise liners have shown that the infectious dose is very low; dys- entery can follow the ingestion of 10 viable organisms. Most spread is person-​to-​person and infection is greatly facilitated by bringing people close together in institutions and circumstances where un- sanitary defaecation and inadequate hand washing is common; well-​ described examples are prisons in England in the early 19th century, mental hospitals in the United Kingdom, Germany, Denmark, and the United States of America later in the 19th century and in the early 20th century, British soldiers in Greece and Mesopotamia (now Iraq) in the First World War, and children in nursery and primary (elementary) schools in the United Kingdom in the early 1990s. It is considered that those with diarrhoea are by far the most effective transmitters of infection. After recovery many individuals continue to excrete organisms for a few weeks; temporary carriers of this kind are not thought to be important sources of infection, even if they are food handlers. Large and dramatic water-​borne outbreaks have occurred occasionally in industrialized countries; milk and ice cream have also been vectors. Vegetables contaminated with human faeces during growth, harvesting, or preparation have also caused outbreaks. Molecular typing has revealed the international nature of some; for example, more than 100 cases in the United Kingdom, Denmark, Norway, and Sweden in 1994 were shown in this way to be due to lettuce contaminated with an identical strain of S. sonnei. Shigellosis is endemic in developing countries in tropical areas; a long-​standing estimate is that it kills about 600 000 people—​mostly young children—​annually. More recently, there have been outbreaks of S. sonnei and S. flexneri in men who have sex with men, with global spread of antibiotic-​resistant strains. Pathogenesis Central to the pathogenesis of shigellosis (including that of enteroinvasive E. coli, which can be regarded as a variant of S. son- nei) is invasion of the colonic mucosa. Organisms gain access to the basolateral pole of enterocytes through M cells, components of in- testinal lymphoid follicles (Peyer’s patches). Bacteria infect macro- phages in these structures and kill them by apoptosis. Their release allows direct invasion and is associated with a cytokine-​induced in- flammatory response that facilitates bacterial invasion by disrupting the epithelial architecture. The entry of shigella into intestinal cells is actin-​microfilament dependent. Shortly after entry the bac- terium lyses its phagocytic vacuole and grows in the cytoplasm at a rate of about 40 min/​generation; most of the bacterial proteins responsible are plasmid encoded. Bacteria then spread from cell to cell. Infected cells die, and bacterial spread continues deep into the lamina propria. There is an acute inflammatory response dominated by polymorphonuclear leucocytes. Proctocolitis with epithelial des- quamation and purulent necrosis with ulcers leads to the produc- tion of bloody mucus. Spread of bacteria from the intestines to other parts of the body is rare. Shiga toxin is only produced by S. dysenteriae type 1 (see earlier). As with EHEC, infections with S. dysenteriae type 1 lead, in a mi- nority of cases, to haemolytic uraemic syndrome. The increased severity of S. dysenteriae type 1 proctocolitis compared with that caused by other Shigella spp. is probably due to the local effects of Shiga toxin on the colonic vasculature. Clinical features After an incubation period ranging from 12 h to 7 days, but most commonly 2 to 3 days, symptoms usually start suddenly, often with abdominal colic. Watery diarrhoea follows, usually with fever and malaise. The symptomatology of most S. sonnei infections progresses no further and, usually, the number of watery stools is small. The most severe infections are caused by S. dysenteriae. After 1 to 3 days the diarrhoea becomes bloody and very frequent, being composed of mucus, blood, and pus. Abdominal cramps and tenesmus are se- vere. Serious complications, sometimes lethal, are hyponatraemia, hypoglycaemia, septic shock, and haemolytic uraemic syndrome. Recovery in complicated cases is slow. More straightforward but se- vere illnesses, such as those not infrequently caused by S. flexneri and S. boydii, usually last about 4 days but might continue for 10 days or more. Laboratory diagnosis Diagnosis is by culture and traditional bacteriological methods work well; faeces are the best samples. Shigella dies rapidly when swabs dry and such samples should be transported to the labora- tory quickly. Inoculation of enrichment cultures from broths or

section 8  Infectious diseases 1038 direct inoculation onto special media gives colonies recognizable as shigella by morphology. Further identification is by biochemical tests and type-​specific antisera. DNA probes for plasmids are avail- able and Shiga toxin can be looked for. Treatment S. sonnei infections in healthy individuals other than those at the extremes of age do not benefit from antibiotic treatment. Agents re- ducing gut motility should be avoided. Antibiotic treatment of se- vere infections must be guided by antimicrobial susceptibility data; antibiotic-​resistant strains are common in areas where these infec- tions have a high incidence. Ampicillin, co-​trimoxazole, tetracycline, or ciprofloxacin have worked well; ceftriaxone and pivmecillinam have been successfully used to treat infections in children caused by antibiotic-​resistant strains. Control The occurrence of urban epidemic shigellosis in countries like the United Kingdom long after the universal provision of treated town water shows that, while the provision of safe water in parts of the world where serious shigella infections are common is a necessary general public health measure, it will not be sufficient. Interrupting faecal–​oral spread needs the provision of toilets and wash hand ba- sins in homes—​more a concomitant of economic development than of public health programmes. Salmonella The number of different salmonella clones is very large, but they all belong to the single species Salmonella enterica. Traditional bacterio- logical methods—​serotyping using O and H antigens and simple biochemical tests—​have been used to identify different kinds of sal- monella since the 1930s and they are good markers of clonal identity. The custom of referring to the entities they define as though they were species, for example, Salmonella enteritidis is taxonomically in- correct (they are serotypes) but operationally useful. A minority of salmonella serotypes has a host range limited to a single species, for example, for humans Salmonella typhi (see Chapter 8.6.9), and these serotypes not considered further here. The serotypes that cause food poisoning infect both animals and humans, and well over 2000 have been described. Epidemiology Person-​to-​person spread is uncommon and the infected/​carrier food handler is not an important source; faecal–​oral spread after contact with carrier animals such as terrapins and other reptiles occurs from time to time, but most infections are food-​borne. In the United Kingdom a big increase in microbiologically confirmed salmonella infection rates and the number of serotypes causing in- fections occurred in the late 1940s and early 1950s. A common pat- tern, which continues, is that a serotype appears, persists, and then declines. Their source for humans is food animals. Cattle, sheep, and pigs are far less important than poultry, although S. Typhimurium of bovine origin has remained quite common for many years. However, poultry dominate and the paradigmatic organism is S. enteritidis. It caused a panzootic in broiler and layer chicken flocks in Europe and the United States of America starting in the 1980s and concomi- tantly a human pandemic. In England and Wales it peaked in 1993; more than 525 000 fell ill during its course. In chickens, S. enteritidis not only grows in the intestines but also invades the reproductive tract leading to egg contamination. Since the early 1990s, control in flocks by slaughter, vaccination, and heightened biosecurity in hen houses has markedly reduced carriage levels in poultry, accounting for the decline in the number of human cases; in England and Wales in 2004 there were 2201 infections. The propensity of certain Salmonella serotypes to expand their population size has been enor- mously facilitated by the scale and nature of the poultry industry. The increase in the number of human infections since the 1940s has followed the expansion of broiler production. In 1950, United States broiler production was 631 million heads and per capita consump- tion was 8.7 pounds ready to cook; in 1990, it was 5864 million and 61.0 pounds, respectively. Cross-​contamination, where organisms from chicken car- casses have been transferred to ready-​to-​eat foods in the kitchen, has caused large outbreaks. Undercooked egg products are im- portant vectors of S. Enteritidis. Many other foods have been ve- hicles of transmission: unpasteurized milk, dried milk, desiccated coconut, alfalfa sprouts, mung bean sprouts, lettuce, and chocolate. Multicontinental outbreaks occur because of international trade (e.g. 4000 cases of S. Agona infection were caused by a contamin- ated kosher snack in the United Kingdom, Israel, the United States of America, and Canada in 1996). Pathogenesis Volunteer studies give an infectious dose ranging from 125 000 to 50 million organisms. For some foods, particularly those with much fat (e.g. cheese, potato chips, peanut butter, and chocolate), it is much less and ranges from fewer than 10 to 100. Organisms attack the distal small intestine and large intestine. At points of contact there is a transient denaturation of brush border microvilli, bacteria are internalized, and they remain in membrane-​bound compart- ments. Replication is necessary for virulence; their presence triggers a transepithelial migration of neutrophils. These processes need the action of many bacterial genes, some of which are in pathogenicity islands. Clinical features The incubation period ranges from 4 to 48 h but most commonly it is between 8 and 24 h. Onset is often sudden, with headache, vomiting (not usually a prominent feature), diarrhoea, and abdom- inal pain; fever is common. The clinical course is usually short, up to 2 to 3 days; in a minority it is severe and prostrating with dehy- dration. Mortality rates are low but are higher in infants (meningitis sometimes occurs) and elderly people with preexisting pathologies. Bloodstream infections are common in Africa in association with malaria and malnutrition in children and HIV infections in adults. Some serotypes (e.g. S. Dublin) are more virulent; bacteraemia with any serotype is usually transient but sometimes leads to metastatic infection, particularly in atherosclerotic vessels, abnormal heart valves, and joint prostheses. Osteomyelitis most frequently occurs in long bones, costochondral junctions, and the spine. Sickle cell anaemia is an important predisposing condition. Arthritis can be septic or reactive; the latter follows more than 1% of infections. It is most commonly seen in those with the HLA-​B27 haplotype. Faecal excretion of organisms continues for 4 to 8 weeks and is longer for infants; the number of organisms excreted is usually low. Carriage for longer than 6 months is rare.

8.6.7  Enterobacteria and bacterial food poisoning 1039 Laboratory diagnosis Diagnosis is by culture. Direct plating of faeces onto selective media and testing of suspicious colonies by slide agglutination for O antigens can give a presumptive diagnosis in 24 h. Enrichment broth cultures increase test sensitivity and are used to search for small numbers of bacteria in faeces or food. Phage typing schemes, available for S. enteritidis, S. typhimurium, and S. virchow, and DNA sequence-​based profiling methods have high resolution and are used to type isolates from patients and other sources in outbreaks. Treatment Fluid and electrolyte replacement is the management mainstay. Drugs that reduce gut motility are contraindicated. In uncom- plicated cases antibiotics have no place and they may prolong the excretion of organisms. In patients with a high risk of bac- teraemia and invasive disease (infants under 3 months, im- munosuppressed patients, patients with cancer, and those with haemoglobinopathies) antibiotics should be considered. Ciprofloxacin is usually the agent of choice but antibiotic-​ resistant strains have emerged and therapy must be guided by susceptibility testing. Cefotaxime and ceftriaxone have been of value in treating meningitis in infants. Prevention Preventing the infection of food animals is central and it has been successful in poultry in Northern Europe and North America. HACCP has been adopted worldwide; refrigeration and adequate cooking are very important critical control points. Campylobacter Campylobacter was discovered as a pathogen of sheep at the begin- ning of the 20th century, but 70 years elapsed before it was recog- nized as a common cause of human gastroenteritis. Its high optimum growth temperature (42°C), need for a microaerobic atmosphere, and requirement for help from selective medium to inhibit other competing gut bacteria hindered its detection. Campylobacter shares about 50% of its genes with helicobacter; both have a spiral shape and flagella. Most human infections are caused by Campylobacter jejuni and some by C. coli. Occasional infections are caused by C. fetus, an important pathogen of cattle and sheep, and sometimes in patients with immune deficiency. Epidemiology Campylobacter is by far the most common cause of bacterial gastro- enteritis in the industrialized world. In England and Wales in 2016, 52 381 laboratory isolates were recorded, 7.3 times more than for non​typhoidal salmonellae. In the United Kingdom it is estimated that for every case reported to national surveillance there are 9.3 cases in the community. Campylobacteriosis is a zoonosis. The organisms are very common inhabitants of the intestines of wild birds, poultry, cattle, and sheep. Mechanized processes in chicken abattoirs mean that most carcasses leave with surface contamination. However, the source of infection in most human cases is unknown; outbreaks, an invaluable epidemiological investigative tool, are rare, and the very great genotypic and phenotypic diversity of the C. jejuni genome caused by frequent horizontal gene exchange seriously impedes the development of epidemiologically useful typing systems. Multilocus sequence typing allows the identification of genetically related clonal complexes. The most common, ST-​21 has been iso- lated from human cases and healthy cattle, broiler chickens, wild birds, and sheep. Unlike Salmonella, the organisms do not grow on contamin- ated food, so outbreaks are uncommon. They have been associ- ated with failures in milk pasteurization and water chlorination. The incidence of sporadic human cases in the United Kingdom rises sharply in weeks 21 to 24 (May and June); the reason for this is unknown. Pathogenesis The infectious dose is low, fewer than 1000 viable organisms. The jejunum and ileum are colonized first, with extension distally, often to the colon and rectum. Infection is invasive; the mesenteric lymph glands enlarge and become inflamed and neutrophil polymorpho- nuclear leucocytes accumulate in the intestinal mucosa. A cytolethal distending toxin, phospholipase A, and flagellar structural proteins as well as other bacterial proteins with unknown functions are pro- duced by all pathogenic isolates. Clinical features The incubation period ranges from 1 to 7 days and averages 3 days. A prodrome of fever and general aching sometimes precedes ab- dominal pain and diarrhoea; vomiting is not a prominent feature. Abdominal pain can be severe and acute appendicitis is a frequent differential diagnosis. The diarrhoea contains leucocytes, is fre- quently bloody, and seldom lasts more than 2 to 3 days. Most pa- tients have culture-​negative stools after 5 weeks. Ten to 15% of patients have a recurrence of symptoms. About 1% of patients develop reactive arthritis 1 to 3 weeks after the onset of illness. It is indistinguishable from that which follows Salmonella infections. Campylobacter gastroenteritis is the most frequent event that leads to the development of Guillain–​Barré syndrome (Chapter 24.16); 26–​41% of cases have a history of its occurring 1 to 3 weeks after the onset of diarrhoea. Laboratory diagnosis Laboratory diagnosis is by culture. Stools are plated onto selective media and incubated for 48 h at 42–​43°C in 5–​15% oxygen and 1–​ 10% CO2. Infectivity is labile; if delays in transport to the laboratory are expected, faeces should be refrigerated or placed in transport medium. Diagnosis of recent infections is by serology. Treatment Most Campylobacter infections are self-​limiting. Fluid and elec- trolyte replacement may be needed. Most strains are sensitive to erythromycin; ciprofloxacin and other fluoroquinolones are also ef- fective in more severe infections, but resistant strains are becoming more common. Miscellaneous food poisoning bacteria Listeria monocytogenes See Chapter 8.6.38.

section 8  Infectious diseases 1040 Vibrio parahaemolyticus Vibrio parahaemolyticus is the most common bacterial cause of diar- rhoea (usually watery, sometimes explosive) in Japan. Infection fol- lows the consumption of seafoods, particularly those prepared raw in the Japanese style. The incubation period is usually 10 to 20 h (range 4–​9 h) and the illness lasts 1 to 2 days. Pathogenic strains produce a heat-​stable toxin and are Kanagawa positive (produce haemolysis on Wagatsuma’s agar). Other vibrios that cause seafood-​ associated gastroenteritis are V. fluvialis, V. hollisae, V. mimicus, and V. vulnificus. Aeromonas hydrophila This Gram-​negative rod is frequently isolated from diarrhoea. Virulence factors remain unidentified. Exotoxin producers See Chapters 8.6.24 and 8.6.25 for Clostridium difficile, C. botu- linum, and C. perfringens, and Chapter 8.6.4 for Staphylococcus aureus. Bacillus cereus This Gram-​positive saprophyte produces heat-​resistant spores. It is common in raw foods, especially rice, and causes two kinds of food poisoning, emetic and diarrhoeic. Vomiting occurs 6 h or less after eating food containing preformed toxin, usually lightly cooked rice that has then been stored at room temperature and reheated, condi- tions which stimulate the bacterium to produce the low molecular weight heat-​, acid-​, and protein-​resistant peptide toxin. Diarrhoea occurs 8–​24 h after eating contaminated food. A heat-​labile entero- toxin is produced in the intestine. Both kinds of illness are short lived. Other Bacillus spp., B. licheniformis, B. pumilis, and B. subtilis, have caused B. cereus-​like illnesses. Prevention of food poisoning The production of safe food rests on evidence-​based practical tech- nologies and management systems; HACCP is central to their de- livery. The system was developed by the National Aeronautics and Space Administration (NASA) and others in the 1960s to prevent food poisoning in space; the notion of diarrhoea and vomiting in zero gravity was too awful to contemplate. HACCP is now used worldwide and in many countries for some food businesses it is a legal requirement. It identifies hazards, identifies the points in a pro- cess where they may occur, and decides which points are critical to control to ensure consumer safety. A good example is milk pasteur- ization; critical control points are the temperatures reached during heating, its duration, and the measures taken to prevent subsequent contamination. As a written scheme testable by food law enforcers, HACCP stops at the farm gate and the dwelling door. However, its prin- ciples apply on the farm and in the home, and their promulgation there currently exercises all promoters of food safety. Ignorance of them is not restricted to these environments; large food poisoning outbreaks have followed failures of food processors to follow them. Milk pasteurization is again a good example. Political resistance to its implementation in England meant that 65 000 died there from milk-​borne bovine tuberculosis between 1912 and 1937. Thirty-​ nine milk-​borne salmonella outbreaks with deaths in Scotland between 1970 and 1981 drove legislation preventing the sale of un- pasteurized milk there, and now nearly all United Kingdom milk is pasteurized. However, pasteurization failures or postpasteurization contamination still lead to campylobacter and E.  coli O157 outbreaks. FURTHER READING Advisory Committee on the Microbial Safety of Food (2005). Second report on campylobacter. Food Standards Agency, London. Carlin F, Nguyen-​The C (2013). Pathogen update:  bacillus species. In: Sofos J, (ed). Advances in microbial food safety, Vol 1. Woodhead Publishing, Cambridge. Cheasty T, Smith HR (2010). Escherichia. In: Borriello SP, Murray PR, Funke G (eds) Topley and Wilson’s microbiology and microbial infec- tions (bacteriology). Wiley. DOI:10.1002/​9780470688618. taw 0052. Maskell D, Mastroeni P (eds) (2006). Salmonella infections:  clin- ical, immunological and molecular aspects. Advances in molec- ular and cellular microbiology. Cambridge University Press, Cambridge, UK. Nair GB, et al. (2007). Global dissemination of Vibrio parahaemo- lyticus serotype 03; K6 and its serovariants. Clin Microbiol Rev, 20, 39–​48. Nordmann P, Naas T, Poirel L (2011). Global spread of carbapenemase-​ producing Enterobacteriaceae. Emerg Infect Dis, 17, 1791–​8. Pennington TH (2010). Review. Escherichia coli O157. Lancet, 376, 1428–​35. Ricke SC, Dawoud TM, Kwon YM (2015). Application of molecular methods for traceability of foodborne pathogens in food safety systems. In:  Ricke SC, Donaldson JR, Phillips CA (eds) Food Safety Emerging Issues, Technologies, and Systems. Academic Press, London. Schroeder GN, Hilbi H (2008). Molecular pathogenesis of shigella spe- cies: controlling host cell signaling, invasion and death by type III secretion. Clin Microbiol Rev, 21, 134–​56. Tarr PI, Gordon CA, Chandler WI (2005). Shiga-​toxin-​producing Escherichia coli and haemolytic uraemic syndrome. Lancet, 365, 1073–​86. The Pennington Group (1997). Report on the circumstances leading to the 1996 outbreak of infection with E. coli O157 in Central Scotland, the implications for food safety and the lessons to be learned. The Stationery Office, Edinburgh. The Public Inquiry into the September 2005 outbreak of E. coli O157 in South Wales 2009. http://​gov.wales/​docs/​dhss/​publications/​ 150618ecoli-​reporten.pdf Threlfall EJ (2010). Salmonella. In: Borriello SP, Murray PR, Funke G (eds) Topley and Wilson’s microbiology and microbial infections (bac- teriology). Wiley. DOI:10.1002/​9780470688618. taw0054. Toro C, et al. (2015). Shigellosis in subjects with traveler’s diarrhea versus domestically acquired diarrhea: implications for antimicro- bial therapy and human immunodeficiency virus surveillance. Am J Trop Med Hyg, 93, 491–​6. Young KT, Davis LM, Dirita VJ (2007). Campylobacter jejuni:  mo- lecular biology and pathogenesis. Nat Rev Microbiol, 5, 665–​79.

8.6.8 Pseudomonas aeruginosa 1041

8.6.8 Pseudomonas aeruginosa 1041

8.6.8  Pseudomonas aeruginosa 1041 8.6.8  Pseudomonas aeruginosa G.C.K.W. Koh and Sharon J. Peacock ESSENTIALS Pseudomonas aeruginosa is a highly versatile environmental Gram-​ negative bacterium that can be isolated from a wide range of habi- tats, including soil, marshes, and the ocean, as well as from plant and animal tissues. It is resistant to many disinfectants and antibiotics, giving it a selective advantage in hospitals. It rarely causes infection in the healthy host but is a major opportunistic pathogen. Clinical features—​(1) Hospitals—​causes a range of infections, including bacteraemia (often in association with neutropenia), ventilator-​associated pneumonia, urinary tract infection, skin and soft tissue infections, and bacteraemia associated with burns. (2) Community—​the largest group of people affected by P. aeruginosa are those with cystic fibrosis, who develop long-​term colonization of the airways, punctuated by episodes of clinical infection. Diagnosis—​Diagnosis is usually straightforward when the or- ganism is cultured from samples collected from normally sterile sites, but is often challenging when infection is suspected in non-​ sterile sites such as a catheterized urinary tract, burns, or skin ulcers, because P. aeruginosa may be either a pathogen or an innocent bystander. Treatment—​P. aeruginosa is intrinsically resistant to a broad range of antimicrobials. Appropriate and effective prescribing requires (1) awareness of risk factors for P. aeruginosa, combined with know- ledge of the spectrum of diseases it causes; (2) carefully considered empirical regimens based on local antimicrobial susceptibility data—​ typically a β-​lactam (e.g. ceftazidime, meropenem, or piperacillin); and (3) attention to susceptibility profiles once the causative strain has been isolated and tested. Genetics and pathogenesis The Pseudomonas aeruginosa genome is composed of a single chromosome of 6.3 Mbp containing around 5700 predicted open reading frames. This is markedly larger than most other sequenced bacterial genomes (for comparison, the genome of the simple eu- karyote Saccharomyces cerevisiae encodes around 6200 proteins). The P. aeruginosa genome contains a high proportion of regulatory genes and many genes involved in catabolism, transport, and efflux of organic chemicals. The size and complexity of the genome under- pins its ability to thrive in diverse environments. P. aeruginosa produces a single polar flagellum (which makes it motile) and type IV pili (which allow it to adhere to the respiratory epithelium). More than half of all clinical isolates produce pyocyanin (a blue pigment) and pyoverdin (a green pigment), which are re- sponsible for the characteristic blue-​green colour of P. aeruginosa colonies growing on solid media. Pyocyanin is an exotoxin that has immunomodulatory effects on respiratory epithelial cells, is toxic to neutrophils, and is involved in iron acquisition. P. aeruginosa is able to produce an alginate-​containing biofilm that increases its resistance to antimicrobials and protects it from the host immune response. P. aeruginosa in the environment P. aeruginosa is ubiquitous in the environment. In homes, it is often found in the aerators and traps of sinks, shower heads, water coolers, contact lens solutions, and cosmetics, as well as in swimming pools, whirlpool baths, and jacuzzis. It may also be cultured from a wide variety of raw fruit and vegetables. It is difficult to eradicate from the hospital environment, where it has been found in soap dishes, dia- lysis fluid, irrigation fluids, eye drops, disinfectants, ointments, and mechanical ventilators. P. aeruginosa is resistant to several commonly used disinfectants:  ammonium acetate-​buffered benzalkonium chloride solution will support the growth and division of P. aerugi- nosa, and the organism readily develops resistance to chlorhexidine. P. aeruginosa is killed by povidone-​iodine, glutaraldehyde, bleach, and alcohol, but may be relatively resistant to these when present in a biofilm or embedded within proteinaceous material. Human colonization and disease Colonization P. aeruginosa is probably consumed regularly and is capable of col- onizing the human gastrointestinal tract. It is rarely present on the intact skin or mucous membranes of healthy individuals but often colonizes severely ill patients, particularly those on broad-​spectrum antibiotics. P. aeruginosa often colonizes broken skin (e.g. ulcers) and medical devices in contact with the environment, such as long-​ term urinary catheters. The organism may cause a broad range of infections, most commonly in patients with one or more risk factors. Bacteraemia Bacteraemia occurs primarily in immunocompromised patients, particularly those with haematological malignancies, neutropenia, or severe burns. P.  aeruginosa accounts for approximately one-​ quarter of all hospital-​acquired bacteraemias, and has a mortality of c.20%. In 2017, the incidence of Pseudomonas aeruginosa bacter- aemia in England, Wales and Northen Ireland was 8.1 per 100 000 population. The highest rates were in children less than 1 year of age (8.8 per 100 000) and those aged more than 85 years (73.5 per 100 000). In the elderly, males were three-times more commonly affected than females. The clinical features of sepsis associated with P.  aeruginosa infection do not differ from those associated with other bacterial infections, and empirical antimicrobial prescribing for high-​risk patients should include cover for P. aeruginosa. A pri- mary source of infection (e.g. a chronic ulcer in a diabetic patient, a urinary catheter, and so on) should be sought and removed wher- ever possible. In rare cases of P. aeruginosa infection, patients may develop a skin lesion called ecthyma gangrenosum (Fig. 8.6.8.1) which, although not pathognomonic for P.  aeruginosa, is rarely a feature of infection by any other organism. This presents as a painful, well-​circumscribed, erythematous lesion anywhere on the body that progresses to necrosis within hours or days. Ecthyma rarely appears in a non​neutropenic host, and its appearance marks the failure of the host immune response to control the infection. In these patients, P. aeruginosa may often be cultured both from blood and from the lesion, but not every patient with ecthyma is detect- ably bacteraemic.

section 8  Infectious diseases 1042 Pulmonary infection P. aeruginosa consistently ranks as the most common cause of ven- tilator-​associated pneumonia in the USA and Europe (National Healthcare Safety Network). Diagnosis is complicated by the fact that severely ill patients commonly become colonized by P. aeruginosa, and appropriate sampling of patients with suspected ventilator-​associated pneumonia requires the use of bronchoalveolar lavage or protected-​ specimen brush sampling of the distal airways. Tracheal aspirates are easier to obtain but less helpful (positive cultures are suggestive but not diagnostic). The diagnosis and treatment of ventilator-​associated pneumonia is described in Section 18 and Chapter 18.4.3. P. aeruginosa commonly colonizes the respiratory tract of people with cystic fibrosis and is the leading cause of respiratory infection in this group. Asymptomatic P. aeruginosa colonization is associ- ated with a more rapid decline in lung function and increased mor- tality from respiratory failure in this patient group. Bronchoscopy is sometimes the only available diagnostic technique in children: some clinicians have attempted to avoid invasive sampling by using sero- logical tests, but the results are unreliable. Early treatment with nebulized tobramycin, inhaled colistin or oral ciprofloxacin is cap- able of eradicating P. aeruginosa from cystic fibrosis patients, but the impact of P. aeruginosa eradication on mortality and morbidity is unclear. Cystic fibrosis is discussed in Chapter 18.10. P. aeruginosa may cause a fulminant necrotizing pneumonia in neutropenic patients as part of a syndrome of disseminated infection. Skin and soft tissue infection P. aeruginosa rarely invades healthy skin and a breach of the in- tegument (e.g. skin maceration from chronic immersion in water, a burn, a cut, or nick from a razor blade or rose thorn, a surgical wound, and so on) is usually required for infection to become established. ‘Hot tub’ dermatitis is a self-​limiting skin infection in healthy people caused by exposure to water contaminated with P.  aeruginosa and manifests as folliculitis or vesicular lesions. Outbreaks have been associated with jacuzzis, spas, and swimming pools. P. aeruginosa can cause surgical wound infections but is far less common than Staphylococcus aureus or Escherichia coli. P. aeruginosa colonization of chronic leg ulcers is common, but it is rarely the only organism found from superficial swabs taken from this type of lesion and is usually a colonizer rather than an invader. Superficial swabs of ulcers are best avoided in the absence of clinical signs of active infection, since the results are difficult to interpret. When infection is present (e.g. cellulitis, associated osteomyelitis, bacteraemia), cultures from deep tissue that does not communi- cate with the ulcer or wound surface should be obtained. Ecthyma gangrenosum is described under the section on bacteraemia (see earlier). P. aeruginosa is an important cause of infection in patients with burns, the other important pathogen being S. aureus. Urinary tract The initiating event in P. aeruginosa urinary tract infection is usually urinary catheterization or instrumentation of the urinary tract, al- though infection may occasionally occur by haematogenous spread to the kidneys. Patients with long-​term indwelling urinary catheters are at particular risk (a combined effect of the presence of prosthetic material that provides a nidus for infection and because frequent antimicrobial therapy for recurrent urinary infection selects for re- sistant organisms such as P. aeruginosa). No specific clinical features distinguish P. aeruginosa urinary infections from infection caused by other pathogens. The diagnosis is made on urine culture in the presence of appropriate clinical features, predominant of which is fever. P. aeruginosa infection in this patient group is rarely cured without removal/​replacement of the urinary catheter on which or- ganisms persist within a biofilm. Catheter change should be per- formed towards the end of therapy once the burden of planktonic bacteria (bacteria free in urine) is much reduced. Routine urine cul- ture of patients with long-​term urinary catheters provides no useful information in the absence of clinical features of active infection. Renal imaging may be useful to exclude renal abscesses or calculi if the reason for the infection is not obvious. The most commonly identified source of P. aeruginosa blood stream infections is compli- cated urinary tract infections (30%). Ear infection P. aeruginosa is a leading cause of otitis externa, an infection of the external auditory canal that causes inflammation, pain (exacerbated by traction on the pinna), and, if severe, a purulent discharge. It is common to find lymphadenopathy just anterior to the tragus. The disease is usually seen in children and the source of infection in- cludes underchlorinated swimming pools or fresh water lakes and rivers. The diagnosis is based on signs and symptoms, and empiric treatment with eardrops is usually effective. Malignant otitis externa is rare but much more serious. It is not a neoplastic process, but is so called because of the risk of localized destructive spread to the central nervous system. It most commonly occurs in elderly patients with diabetes and people with HIV infection, and is essentially an osteomyelitis of the mastoid and petrous temporal bone. Affected patients present with an erythematous, oedematous, and inflamed external auditory canal, and the tympanic membrane is often hidden by oedema. Otoscopy is necessary to make the diagnosis, but is often poorly tolerated because of pain. Lymphadenopathy of the ipsilat- eral cervical lymph nodes may be present; facial nerve involvement produces an ipsilateral lower motor neuron seventh nerve palsy. Spread to the temporomandibular joint causes pain on mastica- tion, and spread to the apex of the petrous temporal nerve produces Gradenigo’s syndrome (trigeminal and trochlear nerve palsies). Features of malignant otitis externa should prompt immediate re- ferral to an ear, nose, and throat surgeon for assessment and de- bridement of the ear canal and adjacent bone. The diagnosis is made by demonstrating osteomyelitis of the skull base on a technetium-​99 bone scintigram or on MRI, along with P. aeruginosa cultured from the discharge or from a bone biopsy. Fig. 8.6.8.1  Ecthyma gangrenosum lesion in a patient with Pseudomonas aeruginosa septicaemia. Courtesy of the late Dr BE Juel-​Jensen.

8.6.8  Pseudomonas aeruginosa 1043 Eye infection The most common manifestation of P. aeruginosa eye infection is keratitis, which occurs following direct inoculation from trauma (e.g. contact sports, industrial accidents) or minor abrasions (e.g. contact lens use). Contact lens keratitis has been associated with contamin- ated contact lens disinfectant solutions. P. aeruginosa keratitis requires prompt ophthalmological referral and treatment since infection may be rapidly progressive and can result in corneal opacification and even perforation within 48 h. Pseudomonal endophthalmitis most com- monly occurs as a consequence of penetrating injury or surgery, but there is also a rare syndrome of neonatal endophthalmitis that may be bilateral, the main risk factor for which is prematurity. Clinical fea- tures include severe pain, chemosis, loss of the red reflex, hypopyon, and corneal clouding. Neonatal pseudomonal endophthalmitis most commonly arises from haematogenous spread, frequently in associ- ation with a syndrome of disseminated disease that includes men- ingitis and pneumonia, and is commonly fatal. Endophthalmitis is diagnosed by culture of vitreous humour. Endocarditis P.  aeruginosa endocarditis is a disease confined almost exclu- sively to injecting drug users, in whom it is usually right-​sided. Extended intravenous combination therapy with a β-​lactam and an aminoglycoside is required, and valve replacement is often neces- sary. In the case of left-​sided endocarditis, antibiotic therapy alone is rarely sufficient and valve replacement is mandatory. Bone and joint infection Patients with diabetes may develop osteomyelitis of the foot fol- lowing penetrating injury or local extension of an untreated chronic ulcer. Results from superficial swabs are of minimal clinical rele- vance, and diagnosis should be based on the results of bone bi- opsy which should be processed for culture and histopathology. Parenteral antimicrobials are not always successful and radical de- bridement or amputation may be necessary to clear the infection. Intravenous drug users are susceptible to P. aeruginosa septic arth- ritis and osteomyelitis of the axial skeleton. HIV infection Patients with HIV infection are more susceptible to P. aeruginosa infection when the CD4 count is below 100 cells/​µl. The incidence has fallen since the advent of highly active antiretroviral therapy (HAART). The presentation of P. aeruginosa infection in HIV pa- tients is more indolent than that in neutropenic patients, but mor- tality is 22–​34%. The fever is frequently low grade and ecthyma gangrenosum is rare. It is most commonly intravenous device re- lated. Pneumonia is the most common community-​acquired pres- entation, followed by sinusitis, and infections of the urinary tract, all of which may be associated with bacteraemia. Antimicrobial therapy P.  aeruginosa elaborates a range of β-​lactamases (penicillinases and cephalosporinases) and has a relatively impermeable outer membrane which makes it intrinsically resistant to a wide variety of antimicrobials, including all first-​generation and second-​gen- eration cephalosporins, most penicillins, and all macrolides. The antipseudomonal cephalosporins ceftazidime and cefepime are effective; of the carbapenems imipenem and meropenem are ef- fective. The antipseudomonal penicillins are piperacillin and ticarcillin (commonly available in combined preparations with tazobactam or clavulanate). The β-​lactams are bactericidal and there is good clinical evidence for their efficacy and safety. However, cefepime monotherapy is associated with a high all-cause mortality and cannot be recommended. There is evidence from animal studies that continuous infusions of β-​lactams are superior to intermittent dosing. Aztreonam, has not found widespread use, because isolates that are resistant to ceftazidime or piperacillin are generally also resistant to aztreonam. There are rare metallo-​β-​lactamase-​produ- cing strains of P. aeruginosa that can be resistant to carbapenems but sensitive to aztreonam. Therapeutic combinations which con- tain beta-lactamase inhibitors, such as ceftazidime-avibactam, ceftolozone-tazobactam and meropenem-vaborbactam have re- cently become available. The aminoglycosides (gentamicin, amikacin, kanamycin, tobra- mycin, and so on) are effective in vitro, but their use in combin- ation with β-​lactam drugs in patients with febrile neutropenia has been called into question by a Cochrane review (2013). This con- cluded that β-​lactam monotherapy was advantageous compared with β-​lactam-​aminoglycoside combination therapy with regard to survival, adverse events, and fungal super-​infections. Some clinicians may choose instead to use β-​lactam-​fluoroquinolone combinations, although there is currently little evidence to support this. β-​lactams and aminoglycosides are commonly used in com- bination when treating serious infections such as P.  aeruginosa ventilator-​associated pneumonia. The toxicity of systemic aminoglycosides means inhaled or top- ical aminoglycosides may be preferred, depending on the site of infection (e.g. inhaled tobramycin for cystic fibrosis patients, or topical gentamicin for otitis externa and superficial eye infections). Ciprofloxacin is active when administered orally, an attribute that makes it almost unique among the therapeutic options available for P. aeruginosa treatment. Acquired drug resistance is a problem in patients who are anti- biotic experienced (an important example being patients with cystic fibrosis), but resistance to commonly used antibiotics is a problem even outside this patient group. The Public Health England re- ported that of the P. aeruginosa strains isolated from blood in 2016, 8% were not susceptible to ciprofloxacin, 3% to gentamicin, 2% to amikacin, 7% to piperacillin-tazobactam, 6% to ceftazidime, and 5% to meropenem. It is not uncommon for resistance to develop during the course of treatment, an event that is associated with excess mor- tality. Gentamicin-​resistant strains are usually also resistant to tobra- mycin, but may remain susceptible to amikacin. Plazomicin is a new aminoglycoside that is resistant to inactivation by aminoglycoside- modifying enzymes, and may be useful in gentamicin-or amikacin- resistant strains. Strains that colonize patients with cystic fibrosis frequently become multiply resistant:  older antimicrobial agents such as colistin and polymyxin B may then be required. The antimicrobial treatment and management of P. aeruginosa infection is complex because the infections are often system-​ or patient-​group specific:  a single guideline is not appropriate. For patients with serious suspected P. aeruginosa infection, increasing resistance rates mean first line therapy should include a β-​lactam (e.g. piperacillin–​tazobactam or meropenem) in combination with a second agent in order to achieve adequate coverage. Therapy should be reviewed when culture and susceptibility results are known.

8.6.9 Typhoid and paratyphoid fevers 1044

8.6.9 Typhoid and paratyphoid fevers 1044

section 8  Infectious diseases 1044 There is good in vitro evidence that monotherapy is associated with a slower rate of bacterial killing and the emergence of resistance; however, for uncomplicated infections, therapy with a single agent is probably adequate. Decisions on empirical antimicrobial therapy should be taken in the light of local information on patterns of re- sistance. The reader is encouraged to study this section in conjunc- tion with other relevant chapters on the management of conditions including neutropenic sepsis, ventilator-​associated pneumonia, cystic fibrosis, and urinary tract, ear, and eye infections. Prevention Groups of patients (e.g. neutropenic patients, or patients with severe burns) who are particularly susceptible to invasive pseudomonal in- fection may be housed in clean units. Such units are equipped with filtered air supplies, and incoming water is chlorinated and continu- ously heated to 60°C. Attention is paid to the regular maintenance of air conditioning, hydrotherapy units, and water coolers. Visitors and staff are required to wear protective gowns and gloves, and to re- move their shoes to avoid contaminating the hospital environment with bacteria brought in from outside the hospital. Fresh flowers and fruit are prohibited for the same reasons, and a rigorous regimen of hand washing is instituted for all visitors and staff. The emergence over the last decade of highly transmissible strains of multidrug-​ resistant P. aeruginosa in people with cystic fibrosis has necessitated the institution of measures to segregate affected patients. Several vaccine candidates have been evaluated for using in clinical trials, but none are currently recommended for clinical use. FURTHER READING Chenoweth CE, Gould CV, Saint S (2014). Diagnosis, management, and prevention of catheter-​associated urinary tract infections. Infect Dis Clin North Am, 28, 105–​19. Torres A, et al. (2017). International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquiredpneumonia and ventilator-associated pneumonia: Guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneu- monia (VAP) of the EuropeanRespiratory Society (ERS), European Society of Intensive Care Medicine (ESICM), EuropeanSociety of Clinical Microbiology and Infectious Diseases (ESCMID)
and Asociación Latinoamericana del Tórax (ALAT). Eur Respir J,
50, 1700582. Fujitani S, Sun HY, Yu VL, Weingarten JA (2011). Pneumonia due to Pseudomonas aeruginosa: part I: epidemiology, clinical diagnosis, and source. Chest, 139, 909–​19. Jain K, Wainwright C, Smyth AR (2013). Bronchoscopy-​guided anti- microbial therapy for cystic fibrosis. Cochrane Database Syst Rev, 12, CD009530. Kaushik V, Malik T, Saeed SR (2010). Interventions for acute otitis externa. Cochrane Database Syst Rev, 1, CD004740. Langton Hewer SC, Smyth AR (2014). Antibiotic strategies for eradicating Pseudomonas aeruginosa in people with cystic fibrosis. Cochrane Database Syst Rev, 11, CD004197. Paul M, et al. (2013). Beta-​lactam versus beta-​lactam-​aminoglycoside combination therapy in cancer patients with neutropenia. Cochrane Database Syst Rev, 6, CD003038. 8.6.9  Typhoid and paratyphoid fevers Christopher M. Parry and Buddha Basnyat ESSENTIALS Typhoid and paratyphoid fever (the enteric fevers) are caused by specific serovars of the Gram-​negative bacillus, Salmonella enterica. Sources of typhoid transmission are excreting chronic or convales- cent carriers and the acutely infected, with transmission occurring through contamination by carriers of food or water by effluents containing infected faeces or urine. Global estimates have varied be- tween 12 and 27 million cases of enteric fever in the world each year, almost all in low-​ and middle-​income countries, with about 200 000 deaths. Clinical features—​the main symptom is fever (39–​40°C); head- ache and malaise are common; constipation is a frequent early symptom, but most patients will experience diarrhoea; abdominal pain is usually diffuse and poorly localized. Physical examination is often unremarkable, apart from fever, but rose spots and relative bradycardia may be observed. In developing countries, patients may progress in the second to fourth week, with life-​threatening manifestations including gastrointestinal bleeding, intestinal per- foration, and the syndrome of mental confusion. Diagnosis—​the principal method for confirming the diagnosis is by isolating Salmonella Typhi or Salmonella Paratyphi from blood or bone marrow. The organisms may also be isolated from stool, urine, and bile aspirates, but such demonstration should be interpreted with caution in areas with many chronic carriers as the acute illness may be due to another cause. Treatment—​aside from supportive care, antibiotic therapy reduces mortality and complications and shortens the illness. Antibiotic re- sistance is a common and increasing problem, hence the choice of antibiotic should be informed by knowledge of likely local suscep- tibility. Fluoroquinolones are often given as first-​line treatment, al- though low and high level resistance to these agents is becoming widespread in Asia, with extended-​spectrum cephalosporins and azithromycin as alternatives. Prevention—​typhoid has been eliminated from industrialized countries by (1) the provision of safe drinking water and safe disposal of sewage; (2) legal enforcement of high standards of food hygiene, and programmes to detect, monitor, and treat chronic carriers; and (3) prompt investigation and intervention when these safeguards are breached. Measures for individual protection are to (1)  kill the organism in water by heating to 57°C, iodination, or chlorination; (2) take care with uncooked or reheated food; and (3) immunization—three typhoid vaccines are available and widely used in travellers, but their role as a public health tool in endemic areas is undefined; there is no paratyphoid vaccine. Acknowledgement: The authors acknowledge the contribution of Dr John Richens to previous editions of this chapter.

8.6.9  Typhoid and paratyphoid fevers 1045 Introduction The organisms classically responsible for enteric fever are Salmonella Typhi and Salmonella Paratyphi A, with occasional infections due to Salmonella Paratyphi B and C. They commonly present as a pro- longed febrile illness with a paucity of physical signs. The spectrum of disease varies from a mild self-​limiting febrile illness to severe dis- ease associated with gastrointestinal bleeding, intestinal perforation, or mental confusion with shock. In the 19th century, typhoid fever was a leading cause of death in Europe and America. The disease today is predominantly found in low-​ and middle-​income countries. Aetiology The Gram-​negative bacilli Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and S. Paratyphi A are the principal causa- tive agents of enteric fever. Three antigens are important for identi- fication: in Typhi the somatic oligosaccharide O antigen (9 and 12), the protein flagellar H-​d antigen, and the polysaccharide envelope Vi antigen; in Paratyphi A the relevant O antigens are 1212 and H antigens a:[1,5]. Antibiotic resistance is conferred by R plasmids, usually of the incompatibility group IncH-​1 (chloramphenicol, amoxicillin, co-​trimoxazole), and by mutations in the chromosomal gyrA gene (fluoroquinolones). Whole genome sequencing of iso- lates of S. Typhi and S. Paratyphi A has shed light on the epidemi- ology and pathogenicity of these organisms. Evidence of a degraded genome compared with other Salmonella enterica serovars (deleted and inactivated genes) are consistent with its high level of adap- tion causing an invasive disease restricted to humans. The multiply antibiotic-​resistant H58 lineage has emerged and spread throughout Asia and Africa. Transmission Sources of typhoid transmission are excreting chronic or convales- cent carriers and the acutely infected. Transmission occurs through contamination by carriers of food or water by effluents containing infected faeces or urine. ‘Typhoid Mary’ was a faecal carrier and cook who infected 53 people early last century, while the Aberdeen outbreak in 1964 was traced to a leaking corned beef tin which had been cooled with faecally contaminated river water. Transmission of typhoid has also been attributed to flies, laboratory mishaps, un- sterile instruments, and anal intercourse. Recent human challenge experiments have demonstrated that 103 organisms of Quailes strain of S. Typhi given orally in sodium bicarbonate solution infected 55% of experimental subjects. Susceptibility is increased by medi- cines which decrease the gastric acidic environment or by vagotomy. Infection may lead to acute disease, transient symptoms, or a symp- tomless carrier state. Multiplication and dissemination Bacteria are thought to pass from the gut through the cytoplasm of enterocytes and M cells overlying lymphoid tissue (Peyer’s patches) of the small intestine to reach the lamina propria from which they are conveyed to the mesenteric nodes before reaching the blood stream via the thoracic duct. During a transient primary bacter- aemia the organism is seeded to reticuloendothelial sites where intracellular multiplication occurs during a 7-​ to 14-​day incuba- tion period. A second bacteraemia follows, accompanied by symp- toms as the infection spreads throughout liver, gallbladder, spleen, Peyer’s patches, and bone marrow. Multiplication occurs mainly in macrophages. Concentrated sites of infection in reticuloendothelial tissues, known as typhoid nodules, are characterized by infiltrates of lymphocytes and macrophages. At post-​mortem examination, hypertrophy of lymphoid tissue is often visible within liver, spleen, mesenteric nodes, and Peyer’s patches. Ulceration of Peyer’s patches is seen where the inflammatory process has resulted in ischaemia and necrosis. Cytokines, such as tumour necrosis factor and interleukin-​6, are elevated in the blood of patients with enteric fever but to a lesser de- gree than other Gram-​negative septicaemias and the correlation be- tween cytokine levels and clinical outcome is less clear. The capacity of whole blood to produce proinflammatory cytokines following stimulation is reduced in patients with severe typhoid. HLA-​DRB1 has been shown to be a major contributor to resistance against en- teric fever, presumably through an effect on antigen presentation Immune response There is a cell-​mediated immune response lasting about 16 weeks, a mucosal immune response lasting for up to 48 weeks, and per- sistent circulating anti-​O and anti-​H agglutinins for up to 2 years. The predominance of clinical typhoid among children and young adults in endemic areas suggests a degree of acquired immunity. Only 25% of volunteers given a standard inoculum of S. enterica ser. Typhi 20 months after an initial infection developed clinical illness. Prolonged elevation of Vi antibody occurs in typhoid carriers. Immunodeficiency reduces the ability to clear salmonella infections. Epidemiology Worldwide, there are estimated to be between 12 and 27 million cases of enteric fever occur each year with about 200 000 deaths. In affluent countries, enteric fever is seen in returned travellers vis- iting friends and relatives abroad in areas of endemicity or when food or water safety measures fail. With appropriate antibiotic treat- ment, death is rare. In parts of the Indian subcontinent, Central and Southeast Asia, Indonesia, and sub-​Saharan Africa, high rates of transmission are seen and annual incidence rates of 100 to 1600 cases per 100 000 population have been recorded. In these countries, transmission has been exacerbated by antibiotic resistance. Peaks of transmission occur in dry weather or at the onset of rains. Case fatality rates have exceeded 10% in some reports of hospitalized patients in Indonesia and Papua New Guinea. S. Paratyphi A was previously thought to cause less severe disease that S. Typhi; a study of 609 cases of bacteraemic enteric fever in Nepal (409 with S. Typhi and 200 with S. Paratyphi) found that the clinical syndromes were indistinguishable and of similar severity. Prevention The elimination of typhoid from industrialized countries can be attributed to the provision of safe drinking water, safe disposal of sewage, legal enforcement of high standards of food hygiene,

section 8  Infectious diseases 1046 programmes to detect, monitor, and treat chronic carriers, and prompt investigation and intervention when these safeguards are breached. Outbreaks were previously investigated using phage typing of isolates and pulsed-​field gel electrophoresis but whole genome sequencing methods are now increasingly used. Registers are kept of known carriers. Bacterial cultures from sewer swabs have previously been used to trace isolates back to their source. This may be replaced by molecular detection of isolates in environmental samples in the future. Measures for individual protection are to kill the organism in water by heating to 57°C, iodination, or chlorination, care with un- cooked or reheated food, and immunization. Patients and conva- lescents with typhoid should be advised to wash their hands after using the toilet and before preparing food and to use separate towels. Western travellers visiting friends and relatives in areas of endemicity are vulnerable to acquiring enteric fever and counselling needs to be targeted on this group. The approach of travel medi- cine, which has evolved around the tourist industry, might miss this susceptible group. Clinical features Enteric fever is predominantly an infection of infants, children, and young adults, affecting both sexes equally. The incubation period ranges from 3 to 60 days, but most infections occur 7–​14 days after exposure. The main focus of typhoid is in the small bowel, but sys- temic symptoms often overshadow abdominal symptoms. The predominant symptom is the fever which rises gradually to a high plateau of 39–​40°C, and then shows little diurnal variation unless antipyretics are given. Rigors are uncommon, except in late or com- plicated typhoid or in patients treated with antipyretics. Patients usu- ally complain of headache and malaise, and constipation is a frequent early symptom. Most patients will experience diarrhoea, and typhoid can present as an acute gastroenteritis and occasionally bloody diar- rhoea. Severe diarrhoea or colitis have been reported in HIV-​infected patients. The abdominal pain is usually diffuse and poorly localized but occasionally sufficiently intense in the right iliac fossa to sug- gest appendicitis. Nausea and vomiting are infrequent in uncompli- cated typhoid but are seen with abdominal distension in severe cases. Other early symptoms include cough, sore throat, and epistaxes. In developing countries, patients with typhoid in its second to fourth week present with accelerating weight loss, weakness, altered mental state, intestinal haemorrhage and perforation, refractory hypoten- sion, pneumonia, nephritis, and acute psychosis. Those infected with antibiotic-​resistant infections may have more severe disease. Physical examination is often unremarkable apart from fever. A coated tongue might be observed. Rose spots appear at the end of the first week and form a sparse collection of maculopapular lesions on the abdominal skin, which blanch with pressure and fade after 2 or 3 days (Fig. 8.6.9.1). Osler found them in 90% of white-​skinned patients and 20% of patients with black skin. The rash can extend on to the trunk and arms. Melanesian typhoid patients develop purpuric macules that do not blanch (Fig. 8.6.9.2). Petechiae are sometimes visible on the conjunctivae (Fig. 8.6.9.3) Tachycardia is common although temperature–​pulse dissociation (relative brady- cardia) is considered characteristic. Hypotension has important im- plications (see ‘Severe typhoid’, later in this chapter). Adventitious Fig. 8.6.9.1  Rose spots on the abdomen in typhoid fever. Copyright CM Parry. Fig. 8.6.9.2  Typhoid rash in a Melanesian child: sparse purpuric (non-​ blanching) macules. Copyright D. A. Warrell. Fig. 8.6.9.3  Conjunctival petechial haemorrhage in an African child with typhoid. Copyright D. A. Warrell.

8.6.9  Typhoid and paratyphoid fevers 1047 lung sounds, especially scattered wheezes, are common and might suggest pneumonia. These findings, with a normal chest radiograph and high fever, should prompt consideration of typhoid. Abdominal examination might reveal the typhoid rash, distension, or a diffuse tenderness, occasionally localized to the area of the terminal ileum. Intra-​abdominal inflammation sometimes provokes retention of urine. A moderate soft tender hepatosplenomegaly eventually de- velops in most patients but it less likely to be found early. Patients with advanced illness may display the ‘typhoid’ facies (Fig. 8.6.9.4), a thin flushed face with a staring apathetic expression. Mental apathy may progress to an agitated delirium, frequently ac- companied by tremor of the hands, tremulous speech, and ataxic gait. If the patient’s condition deteriorates further, the features described in the writings of Louis and Osler make their appearance—​muttering delirium, twitching of the fingers and wrists, agitated plucking at the bedclothes, and a staring unrousable stupor (coma vigil). Typhoid in children Community-​based studies in highly endemic areas have shown that enteric fever can be common in children less than 5 years old. The main differences, compared to adults, are a greater frequency of diarrhoea and vomiting, jaundice, febrile convulsions, and nephritis or typhoid meningitis may occur. Relative bradycardia is of greater diagnostic significance for typhoid in febrile children. In some re- ports case fatality rates are high in the under-​fives. The disease can also take a milder course in very young children, behaving like a non​specific febrile illness or mild respiratory illness that is not clin- ically recognized as enteric fever. Typhoid may also occasionally de- velop in neonates born to infected mothers. Differential diagnosis Many viral, bacterial, and protozoal infections as well as non​infectious conditions characterized by fever, including lymphoproliferative disorders and vasculitides, resemble enteric fever. Typhoid should al- ways be considered when suspected malaria has not been confirmed or has not responded to antimalarial therapy. In areas of endemicity, typhus, leptospirosis, and dengue should be considered in the differ- ential diagnosis. Diagnosis Culture The definitive diagnosis of enteric fever rests on the isolation of S. Typhi or S. Paratyphi from blood, bone marrow, cerebrospinal fluid, or rose spots. In mild typhoid, the number of bacteria in blood may be less than 1 colony-​forming unit/​ml. The median number of bacteria in the blood of children is higher than adults and declines with increasing duration of illness. Successful culture from blood can be achieved in up to 80% of patients but depends on taking a generous volume of blood and using the correct volume of blood to broth (1:10). Although bone marrow generally gives a higher yield than blood, including those exposed to antibiotics, it is rarely per- formed. Rose spots, when present, can give a positive culture in 70% of patients. The organisms may also be isolated from stool, urine, and bile as- pirates. The number of organisms recoverable from faeces increases through the illness. The results should be interpreted with caution in areas with many carriers, as the acute illness may be due to another cause in chronic carriers. Isolation from urine is more common in areas endemic for schistosomiasis. Culture of bile obtained from an overnight duodenal string capsule gives a similar yield to blood and offers additional means to isolate S. Typhi and S. Paratyphi from children or from carriers. Serology The use of a tube or slide agglutination test (the Widal test) to diag- nose typhoid is cheaper and simpler than culture but fraught with pitfalls. The demonstration of a fourfold rise in titre of antibodies to S. Typhi or S. Paratyphi antigens suggests enteric fever but is too delayed to help clinical decision-​making and is not observed in all patients. Single measurements of antibody titres have been found useful in populations where accurate up-​to-​date informa- tion about the predictive value of the test at specific cut-​off points is available. False-​positive serological tests are obtained from per- sons with previous infection, infection with cross-​reacting organ- isms, or following vaccination. Several commercially available enzyme-​linked immunosorbent assay (ELISA) and point-​of-​care rapid diagnostic tests perform somewhat better than the Widal test, but with sensitivities and specificities that are still not ad- equate for routine diagnostic use. An ELISA for antibodies to the Vi antigen can be useful for detecting carriers in the context of an outbreak. Other tests for typhoid Many other tests for the detection of antibodies, antigens, and sal- monella DNA in body fluids have been described. Few have so far been adopted for routine use. The detection of IgA antibodies in the supernatant from blood lymphocytes and real-​time polymerase chain reaction after short-​term blood culture are two potentially promising new approaches. Fig. 8.6.9.4  Typhoid facies: a man with the apathetic expression seen in severe typhoid. Copyright B Basnyat.

section 8  Infectious diseases 1048 Other laboratory findings in typhoid A mild normochromic anaemia, mild thrombocytopenia, and an increased erythrocyte sedimentation rate are common. Most pa- tients have a total white cell count within, or just below, the normal range. Leucocytosis suggests either perforation or another diag- nosis. Laboratory evidence of mild disseminated intravascular co- agulation is common but rarely of clinical significance. Common biochemical findings include hyponatraemia, hypokalaemia, and elevation of liver enzymes, which may mimic acute viral hepatitis. The urine often contains some protein and white cells. Examination of the cerebrospinal fluid may be normal or show a mild pleocytosis (<35 cells/​mm3) in patients with central nervous system symptoms. Treatment The aims of management are to eliminate the infection swiftly with antibiotics, to restore fluid and nutritional deficits, and to monitor the patient for dangerous complications. In many parts of the world antibiotic treatment for typhoid fever is started empirically based on the syndrome of fever of 3 or 4 days and constitutional symptoms with no apparent source of infection and a negative malaria smear. Because there are no reliable clinical predictors, in areas of endem- icity concurrent treatment with doxycycline to cover for typhus and leptospirosis must be considered. Supportive care Cooling is preferred to antipyretics for relief of fever, and simple an- algesics may be used to relieve headache. Most patients can eat and drink normally; special diets do not protect the bowel from perfor- ation. Daily assessment of the patient’s mental and circulatory status is required plus examination of the abdomen for signs of impending perforation. Severely ill patients require intensive care with paren- teral fluids, intravenous steroids (see next), inotropic support, and sedation. Antibiotics Effective antibiotic therapy in typhoid reduces mortality and min- imizes complications and shortens the illness (see Table 8.6.9.1 for doses). Chloramphenicol was the first antibiotic found to be effective and the standard against which subsequent antibiotics have been measured. Symptom resolution occurs over a period of 3 to 6 days although it is generally suggested that the antimicrobial should be given for at least 2 weeks to prevent relapse. Ampicillin, amoxicillin, and co-​trimoxazole have been shown to have comparable efficacy Table 8.6.9.1  Guidelines for the antibiotic treatment of enteric fever Antibiotic Daily dose Routea Doses/​day Duration in non​severe enteric fever (days) Duration in severe enteric feverb Acute infection Chloramphenicolc 50–​100 mg/​kg O/​IM/​IVd 4 14 14–​21 Co-​trimoxazolee Trimethoprim 6.5–​10 mg/​kg O/​IM/​IV 2–​3 14 14 Sulfamethoxazole 40 mg/​kg Amoxicillin 75–​100 mg/​kg O/​IM/​IV 3 14 14 Ceftriaxone 50–​60 mg/​kg IM/​IV 1–​2 7–​14 14 Cefixime 20 mg/​kg O 2 7–​14 Ciprofloxacinf 20–​25 mg/​kg O/​IV 2 7–​14 14 Ofloxacinf 15–​20 mg/​kg O/​IV 2 7–​14 Pefloxacinf 800 mg O/​IV 2 7–​14 Fleroxacinf 400 mg O/​IV 1 7–​14 Gatifloxacin 10 mg/​kg O 1 7 Azithromycin 10–​20 mg/​kg O 1 7 Treatment of carriers Ampicillin or amoxicillin with probenecid 100 mg/​kg O 3–​4 90g 30 mg/​kg Co-​trimoxazole 6.5–​10 mg trimethoprim O 2 90 Ciprofloxacin 1500 mg O 2 28 O, oral; IM, intramuscular; IV, intravenous. a Oral therapy is satisfactory for most patients. Parenteral therapy is generally reserved for severely ill patients. b In intestinal perforation, the antibiotic therapy should also cover other aerobic and anaerobic gastrointestinal bacteria contaminating the peritoneum. In severe typhoid (characterized by delirium, obtundation, coma, or shock) dexamethasone is beneficial (see text). c May cause bone marrow suppression. d The oral route is preferred; there are reports of lower blood levels of chloramphenicol in patients given parenteral therapy. e May cause allergic reactions and nephrotoxicity. Not suitable for children younger than 2 years or during pregnancy. f Infection with isolates that have low-​level fluoroquinolone resistance (nalidixic acid resistance) may not respond. g The duration of treatment can be shortened if parenteral therapy is given (e.g. 8-​hourly intravenous ampicillin for 2 weeks).

8.6.9  Typhoid and paratyphoid fevers 1049 to chloramphenicol while having less toxicity; they are also recom- mended to be given for at least 2 weeks. In many areas these drugs are no longer used because of the spread of multidrug-​resistant strains of S. Typhi and S. Paratyphi A. Alternative antibiotics active against multidrug-​resistant infec- tions include the fluoroquinolones, although resistance has, in turn, emerged to these agents, the extended-​spectrum cephalosporins (e.g. parenteral ceftriaxone), and azithromycin. In recent years many physicians have given a fluoroquinolone, ciprofloxacin, or ofloxacin as first-​line therapy. Treatment can be completed in a week or less with minimal toxicity. In controlled trials in endemic areas, infections with fully susceptible isolates have resulted in a rapid resolution of symptoms with high cure rates and low levels of relapse and faecal carriage. Response rates in endemic areas may be better than those of non​immune travellers. There have been questions about the safety of fluoroquinolones in children and during pregnancy. Careful follow-​up studies of children in Asia fol- lowing fluoroquinolone therapy have not shown toxicity and there has been a growing consensus that where alternatives are limited the advantages of therapy outweigh the potential dangers. Strains of S. Typhi and S. Paratyphi A with low-​level resistance (or intermediate susceptibility) to the commonly used fluoroquinolones (ciprofloxacin and ofloxacin) are common in Asia and sporadic- ally reported in sub-​Saharan Africa. These strains are usually nali- dixic acid, or pefloxacin, resistant and this can be a useful, although not a completely sensitive, laboratory marker. Where possible ciprofloxacin and ofloxacin should be avoided in patients infected with these strains. The newer fluoroquinolone gatifloxacin is effective but safety concerns mean it is unavailable in some countries. These infections can be treated with extended-​spectrum cephalosporins (ceftriaxone) or, in non​severe cases, with azithromycin. Another alternative is cefixime, an oral third-​generation cephalosporin, al- though there have been concerns about its efficacy in some studies. Many areas in the Indian subcontinent now report isolates that are fully resistant to all the fluoroquinolones including gatifloxacin. A recent outbreak in Pakistan of MDR typhoid that is also resistant to ceftriaxone and fluorquinolones is giving cause for concern. In some places there has been an increase in isolates that have regained sus- ceptibility to the old first-​line drugs, chloramphenicol, ampicillin, and co-​trimoxazole. In such circumstances these older drugs may be appropriate. Some antibiotics such as gentamicin appear sensi- tive in vitro but are ineffective in vivo and should not be used in enteric fever. Ampicillin, amoxicillin, or ceftriaxone are considered safe in pregnancy with enteric fever. There are limited data on the manage- ment of immunocompromised patients with enteric fever, but data from patients with non​typhoidal salmonella infections suggest that they may need extended treatment to prevent relapse. Complications Box 8.6.9.1 lists the complications of typhoid. Most are rare and only likely to be encountered in patients who present with untreated dis- ease lasting 2 weeks or more. Occasionally, a complication domin- ates the clinical picture and deflects attention from the underlying diagnosis of typhoid. Box 8.6.9.1  Complications of typhoid Abdominal • Intestinal perforation • Intestinal haemorrhage • Hepatitis • Cholecystitis (usually subclinical) • Spontaneous splenic rupture • Rupture and haemorrhage from mesenteric nodes • Pancreatitis Genitourinary • Retention of urine • Glomerulonephritis • Pyelonephritis • Cystitis • Orchitis Cardiovascular • Asymptomatic electrocardiogram changes • Myocarditis • Pericarditis • Endocarditis • Phlebitis and arteritis • Deep venous thrombosis • Gangrene • Haemodynamic shock • Sudden death Respiratory • Bronchitis • Laryngeal ulceration • Glottal oedema • Pneumonia (S. enterica ser. Typhi, Streptococcus pneumoniae) Neuropsychiatric • Delirium • Psychotic states • Depression • Deafness • Meningitis • Encephalomyelitis • Transverse myelitis • Signs of upper motor neuron lesions • Signs of extrapyramidal disorder • Impairment of coordination • Optic neuritis • Peripheral and cranial neuropathy • Guillain–​Barré syndrome • Pseudotumour cerebri Haematological • Disseminated intravascular coagulation (usually subclinical) • Anaemia • Haemolysis • Haemolytic uraemic syndrome Focal infections • Abscesses of brain, liver, spleen, breast, thyroid, muscles, lymph nodes • Parotitis • Pharyngitis • Osteitis, especially tibia, ribs, spine • Arthritis (continued)

section 8  Infectious diseases 1050 Severe typhoid Studies from Indonesia and Papua New Guinea have revealed an im- portant subgroup of patients with mental confusion or shock (de- fined as a systolic blood pressure of less than 90 mm Hg in adults or less than 80 mm  Hg in children), with evidence of decreased skin, cerebral, or renal perfusion, who have a 50% fatality rate and account for many typhoid deaths. In a small study in Jakarta, high doses of dexamethasone substantially reduced the mortality of such severe cases. The criteria for severe typhoid were marked mental confusion or shock. In adults treated with chloramphenicol, 3 mg/​ kg dexamethasone infused intravenously over 30 min, followed by eight doses of 1 mg/​kg every 6 h, resulted in a 10% case fatality rate compared to 55.6% in controls. This study has not been repeated. Intestinal haemorrhage and perforation Perforation of ileal ulcers occurs in less than 5% of typhoid patients (Fig. 8.6.9.5). The development of acute abdominal signs is often gradual, making diagnosis difficult. Severely ill patients display only restlessness, hypotension, and tachycardia. A chest radiograph may show free gas under the diaphragm. Ultrasonography is useful for demonstrating and aspirating faeculent fluid in the peritoneal cavity. Management includes nasogastric suction, administration of fluids to correct hypotension, and prompt surgery. Although simple closure of perforations can be adequate, experienced surgeons use procedures to bypass the worst-​affected sections of the ileum in order to reduce postoperative morbidity. Closure of perforations should be accom- panied by vigorous peritoneal toilet. Metronidazole or clindamycin should be added to the therapy of ceftriaxone or fluoroquinolone-​ treated patients. Metronidazole and aminoglycosides are recom- mended for patients receiving chloramphenicol, ampicillin, or co-​trimoxazole. In a recent systematic review, the mean case fatality rate of patients with intestinal perforation was 15.4%, reaching less than 5% in the best series, but postoperative complications were common. This compares with historical case fatality rates of around 70% in patients managed without surgery. Evidence for silent gastrointestinal bleeding may be sudden col- lapse of a patient or a steadily falling haematocrit. Most bleeding episodes are self-​limiting. Severe bleeding is sometimes seen in ad- vanced typhoid but is rarely fatal. A few require transfusion. In ex- ceptional circumstances surgery or intra-​arterial vasopressin have been used to halt haemorrhage. Relapse Relapse in typhoid is a second episode of fever, usually milder than the first, occurring a week or two after recovery from the first epi- sode. Isolates from relapsing patients usually have identical anti- biotic susceptibility to those identified during the first episode. Relapse rates of 10% have been described in untreated typhoid and chloramphenicol-​treated patients. Relapse is managed with a similar or abbreviated course of the same therapy used in the initial episode. Reinfection may also occur but can only be distinguished by differ- ences in the sensitivity pattern or molecular typing of isolates. Carriers Many patients will continue to excrete S. Typhi or S. Paratyphi in faeces, and occasionally urine, for several days or weeks after starting antibiotic treatment. For most patients this eventually stops but if they are still excreting at 3 months, they are unlikely to cease and at 1 year meet the formal definition of ‘chronic carrier’. Among carriers detected by screening, 25% give no history of acute typhoid. Faecal carriage is more frequent in individuals with gallbladder disease and is most common in women over 40; in the Far East there is an as- sociation with opisthorchiasis. Urinary carriage is associated with schistosomiasis and nephrolithiasis. Chronic carriage is occasion- ally complicated by acute typhoid and there is a long-​term increased risk of carcinoma of the gallbladder. Patients discharged after treatment for typhoid with six negative faeces and three negative urine specimens and negative Vi serology are considered free of infection. Many public health authorities aim for the pragmatic requirement of three negative faeces samples. Most patients with positive faeces at the completion of treatment excrete temporarily and can be safely followed up. Antibiotic eradication of carriage is advised in those still excreting at 3 months, or earlier in those at particular risk of communicating infection to others. The pa- tient with a persistently elevated or rising Vi antibody titre is likely to be a carrier. Repeated checks of urine and faeces should be made and consideration given to obtaining bile cultures if these are negative. Eradication of carriage requires prolonged, high-​dose anti- biotics (Table 8.6.9.1). Ampicillin, amoxicillin, co-​trimoxazole, and fluoroquinolones have been used with some reported success. The choice depends on the antibiotic susceptibility of the organism. Cholecystectomy and nephrectomy, once used to eliminate carriage (and not without operative mortality), are hard to justify on public health grounds alone, but can be considered if antibiotic methods fail and there are additional indications for operation. The success rates of surgery are increased by giving antibiotics as well. Box 8.6.9.1  Continued Other • Myopathy • Hypercalcaemia • Decubitus ulceration • Abortion • Relapse Fig. 8.6.9.5  Typhoid perforation of the distal ileum at operation.

8.7 Fungi (mycoses) 1338

8.7 Fungi (mycoses) 1338

8.7.1 Fungal infections 1338

8.7.1 Fungal infections 1338

8.7 Fungi (mycoses) CONTENTS 8.7.1 Fungal infections  1338 Roderick J. Hay 8.7.2 Cryptococcosis  1359 William G. Powderly, J. William Campbell, and Larry J. Shapiro 8.7.3 Coccidioidomycosis  1361 Gregory M. Anstead 8.7.4 Paracoccidioidomycosis  1364 M.A. Shikanai-​Yasuda 8.7.5 Pneumocystis jirovecii  1371 Robert F. Miller and Christopher P. Eades 8.7.6 Talaromyces (Penicillium) marneffei infection  1375 Romanee Chaiwarith, Khuanchai Supparatpinyo,
and Thira Sirisanthana 8.7.7 Microsporidiosis  1378 Louis M. Weiss 8.7.1  Fungal infections Roderick J. Hay ESSENTIALS The mycoses are disorders caused by fungi, which are saprophytic or parasitic organisms found in every continent and environment. Many are common commensals in nature, but others cause agricul- tural disease. The mycoses that are human infections include diseases ranging from those that are worldwide and common, such as derm- atophytosis and candida infections, to those that are rare and often potentially life-​threatening (e.g. histoplasmosis). In humans, fungi usually adopt one of two morphologies: (1) the yeast form—​where individual cells produce daughter cells by a process of budding and subsequently separate; or (2) the hyphal form—​where cells do not separate but multiply to produce chains of cells joined end to end. Diagnosis Mycological diagnosis is often complex because many fungi are also commensals or transiently carried in humans, hence it is necessary to show both that the organisms are present and that they are causing disease, which is particularly difficult in the context of opportunistic fungal infection. The main laboratory diagnostic tests involve (1) visu- alization of fungi in tissue—​by direct microscopy or histopathology; (2) culture—​often using a glucose peptone agar (Sabouraud’s agar); (3) detection of antibody, fungal antigens, or DNA—​assimilation of genetic tests such as polymerase chain reaction-​based methods into routine diagnosis has been slow, and they are offered by few laboratories. Superficial infections Superficial fungal infections may reach prevalence rates of 15–​25% in some communities, with the common infections being dermato- phytosis or ringworm, pityriasis versicolor, and superficial candidiasis. Dermatophytoses—​otherwise known as tinea infections—​ commonly affect the feet (tinea pedis), the body (tinea corporis), the scalp (tinea capitis) and the finger and toe nails (onychomycosis). They occur in all climates and usually present in primary care as scaly rashes. Diagnosis is made by direct microscopy of skin scales mounted in potassium hydroxide (20%) to demonstrate hyphae, and by culture. Pityriasis versicolor—​caused by a skin surface commensal, Malassezia globosa, and often triggered by sun exposure. Presentation is with hypo–​ or hyperpigmented scaling on the trunk. Laboratory diagnosis (if required) is by demonstration of the yeasts and hyphae in skin scales removed by scraping. Superficial candidiasis—​these infections affect the mouth, vagina, and body folds, often in the context of some form of predisposition (e.g. recent antibiotic therapy or, in the case of severe oral infec- tion, immunosuppression including that associated with HIV/​AIDS). Infections are diagnosed by microscopy and culture, the latter being particularly important where non-​albicans Candida species may be involved. Treatment—​the main treatments for superficial mycoses are top- ical agents that include imidazole preparations (e.g. ketoconazole, clotrimazole), but for widespread infections or those involving hair or nails, oral imidazoles (e.g. itraconazole, fluconazole) or the allylamine, terbinafine, are employed. Subcutaneous mycoses Subcutaneous fungal infections, for example, mycetoma (Madura foot), chromoblastomycosis and sporotrichosis, are not common and usually restricted to the tropics and subtropics. They might present in immigrants from tropical areas, sometimes years after the person has left the tropics, and hence cause diagnostic confusion. Diagnosis is

8.7.1  Fungal infections 1339 by histological examination of affected tissues or culture. Treatment is often difficult, with only partial responses being achieved, but oral imidazole drugs or terbinafine are helpful in some cases. Systemic mycoses Systemic mycoses are deep and often disseminated infections that involve many different sites, including the blood and bone marrow. They can be caused by organisms which invade normal hosts (en- demic mycoses) and those which only cause disease in comprom- ised patients (opportunistic mycoses). Endemic mycoses—​these include histoplasmosis, coccidioido- mycosis (see Chapter  8.7.3) and infections due to Talaromyces (Penicillium marneffei) (see Chapter 8.7.6), all of which can occur in healthy people, although many are also common complications of HIV/​AIDS. Initial manifestations are as respiratory infections, but they can spread haematogenously to other sites (e.g. skin, liver, and brain). Diagnosis is made on culture or biopsy of affected areas. Opportunistic mycoses—​these occur in those who are immuno- compromised (e.g. patients with neutropenia secondary to cancer). The routes of fungal entry into the body are very variable (e.g. skin, gastrointestinal tract, lung). Infections include systemic candidiasis, aspergillosis, and mucormycosis, but in severely compromised pa- tients (e.g. those with profound neutropenia, many organisms not usually associated with human disease can cause invasive infections, e.g. Fusarium species). Cryptococcus neoformans, a yeast that can in- vade the lungs, often presents with meningitis or other signs of intra- cranial infection. Prognosis and treatment—​the endemic mycoses are often fatal if untreated, and even with treatment the mortality of opportunistic fungal infection can be high (e.g. over 40% for the severely neutro- penic patient with aspergillosis). Aside from supportive care, oral or parenteral agents such as amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin are the treatments of choice, but detecting the organisms and successfully treating the in- fections remains a challenge. Introduction Fungi are saprophytic or parasitic organisms that are normally as- signed to a distinct kingdom. As eukaryotes, they have the complex subcellular organization and highly organized genetic material seen in both animal and plant cells. The cell wall is a distinctive feature of fungi and has a complex cytoskeleton based on mannan, glucan, or chitin subunits. The arrangement and reproduction of indi- vidual cells is also characteristic. Most fungi form new cells termin- ally, which remain connected to form long, branching filaments or hyphae (the mould fungi). Some reproduce in a similar manner but each new cell separates from the parent by a process of budding (the yeast fungi). It is a feature of certain fungi to be yeast-​like during one phase of their life history but hyphal at another, a phenom- enon known as dimorphism. In culture, mould fungi usually form a cottony growth on laboratory media while yeasts normally have a smooth, shiny appearance. Fungi adversely affect humans in several ways. They cause disease indirectly by spoilage and destruction of food crops, with subsequent malnutrition and starvation. Many of the common moulds produce and release spores, which can act as airborne allergens to produce asthma or hypersensitivity pneumonitis. Fungi elaborate complex metabolic byproducts, some of which are useful to humans, such as the penicillins. However, others are toxic. Disease caused by the ingestion of fungal toxins includes both poisoning by eating certain mushrooms (mycetism) and damage caused by the ingestion of mi- nute quantities of toxin (mycotoxicosis), for example, in contam- inated grain. The contribution of the latter mechanism to human disease remains largely unexplored, as does the question of whether inhalation of toxic fungal spores may cause pathology. Finally, fungi might invade human tissue. Medical mycology is largely concerned with this last group. Invasive fungal diseases are normally divided into three groups: the superficial, subcutaneous, and deep mycoses. In superficial infections, such as ringworm or thrush, fungi are con- fined to the skin and mucous membranes. Extension deeper than the surface epithelium is rare. Subcutaneous infections are usually tropical: the main site of involvement is within subcutaneous tissue, although secondary invasion of adjacent structures such as bone or skin can occur. In deep or systemic infections, deep organs such as the lung, spleen, or brain are invaded. This classification of mycoses is based on the main ‘sphere of involvement’ by the causal organ- isms, but there are exceptions. For instance, brain involvement has been recorded in patients with chromoblastomycosis, which is nor- mally a subcutaneous infection. The fungi causing systemic mycoses are often classified in two groups: the opportunists (which cause disease in immunocomprom- ised individuals) and the endemic pathogens. These contrast with the true pathogens, which cause infection in all subjects inhaling airborne spores. Superficial fungal infections The main superficial mycoses are the dermatophyte infections, superficial candidiasis, and tinea versicolor (see Section 23). These are both common and widespread. Rare superficial infections in- clude tinea nigra, and black or white piedra. Dermatophyte infections (dermatophytoses) Aetiology The dermatophyte or ringworm infections are caused by a group of organisms capable of existing in keratinized tissue such as stratum corneum, nails, or hair. The mechanism of invasion is thought to be linked to production of extracellular enzymes; at least three distinct metalloproteinase genes are found in Microsporum canis. Epidemiology Some dermatophyte fungi have a worldwide distribution; others are more restricted. The most common and most widely distributed is Trichophyton rubrum, which causes different types of infection in different parts of the world. It is commonly associated with athlete’s foot (tinea pedis) in temperate areas as well as tinea corporis or tinea cruris in the tropics. This distinction is not based solely on climatic factors, as immigrants from tropical countries, particularly eastern Asia, might still have tinea corporis caused by T. rubrum when living in northern Europe. Certain dermatophytes are limited to defined

section 8  Infectious diseases 1340 areas. For instance, tinea imbricata caused by T. concentricum, is found in hot, humid areas of the eastern Asia, Polynesia, and South America. Scalp ringworm tends to occur in well-​defined endemic areas in Africa and elsewhere. In different regions, different spe- cies of dermatophytes might predominate. Thus, in North Africa, the most common cause of tinea capitis is T. violaceum; in southern parts of the continent, the major agents might be Microsporum aud­ ouinii, M. ferrugineum, and T. soudanense. Not all dermatophyte infections are endemic and dominant species can disappear to be replaced by others. M. audouinii, once endemic and common in the United Kingdom, is now infrequent. By contrast, T tonsurans is now established as a major cause of tinea capitis in urban areas in the United Kingdom, parts of Europe, and the United States of America. Dermatophytes can be passed from person-​to-​person (anthropophilic infections), from animal to person (zoophilic), or from soil to person (geophilic). Sources of zoophilic organisms in Europe include cats and dogs, cattle, hedgehogs, and small rodents. Rarer sources include horses, monkeys, and chickens. Lesions pro- duced by zoophilic species can be highly inflammatory. Factors governing the invasion of stratum corneum are largely un- known, but heat, humidity, and occlusion have all been implicated. Susceptibility to certain infection, such as tinea imbricata, might be genetically determined. Clinical features The clinical features of dermatophyte infections are best considered in relation to the site involved. Often the term tinea, followed by the Latin name of the appropriate part (such as corporis, meaning ‘body’) is used to describe the clinical site of infection. Tinea pedis Scaling or maceration between the toes, particularly in the fourth interspace, is the most common form of dermatophytosis seen in temperate countries. Itching is variable, but can be severe. Sometimes blisters might form both between the toes and on the soles of the feet. The causative organisms are commonly T. rubrum and T. inter­ digitale, the latter being responsible for the vesicular forms. Similar appearances can be caused by Candida albicans and in the bacterial infection, erythrasma. Gram-​negative bacterial infection causes erosive interdigital disease associated with discomfort. ‘Dry type’ infections of the soles and palms These are normally caused by T. rubrum. Palms (Fig. 8.7.1.1) or soles have a dry, scaly appearance, which in the soles may encroach on to the lateral or dorsal surfaces of the foot. The palmar involve- ment is often unilateral, an important diagnostic feature. Nail inva- sion is often seen (see next). Itching is not prominent, and infections are usually chronic. Tinea cruris Infections of the groin, most often caused by T.  rubrum or Epidermophyton floccosum, are relatively common. They occur in both tropical and temperate climates, although in the former the infection can spread to involve the whole waist area in both males and females. Tinea cruris in females is uncommon in Europe. An erythematous and scaly rash with a distinct margin extends from the groin to the upper thighs or scrotum. Itching can be severe. Coincident tinea pedis is common, and patients should be examined for this. The rash of crural erythrasma shows uniform scaling without a margin, whereas in candidiasis, satellite pustules occur distal to the rim. Onychomycosis (caused by dermatophytes) Invasion of the nail plate is most often seen with T. rubrum infec- tions. The plate is invaded distally and becomes thickened and fri- able with terminal loss of the nail plate. Onycholysis might be seen. More rarely, and most often with T. interdigitale, the dorsal surface of the plate is invaded, causing superficial white onychomycosis. Tinea corporis (body ringworm) Dermatophyte or ringworm infection on the trunk or limbs might produce the characteristic annular plaque with a raised edge and central clearing (Fig. 8.7.1.2). Scaling and itching is variable. Lesions caused by zoophilic organisms can be highly inflammatory and in certain cases, particularly those caused by T. verrucosum, intense itching, oedema, and pustule formation (kerion) can de- velop. This reaction is seldom secondarily infected by bacteria but is a response to the fungus on hairy skin. Infections of the beard, tinea barbae, are often highly refractory to treatment. Facial derm- atophyte infections can mimic a variety of non​fungal skin dis- eases, including acne, rosacea, and discoid lupus erythematosus. However, the underlying annular configuration can usually be distinguished. The term tinea incognito is used to describe such atypical lesions. Fig. 8.7.1.1  Palmar scaling due to Trichophyton rubrum. Fig. 8.7.1.2  Tinea corporis due to Microsporum gypseum.

8.7.1  Fungal infections 1341 Tinea capitis (scalp ringworm) In the United Kingdom as in the United States of America, the most common cause of scalp ringworm is T. tonsurans, an anthropophilic fungus which mainly occurs in inner cities, particularly in black Caribbean or African children. This has now replaced Microsporum canis, originating from an infected cat or dog, although this derm- atophyte is dominant elsewhere in the United Kingdom and Europe. Scalp ringworm is mainly a disease of childhood, but infections can occur in adult women. Spontaneous clearance at puberty is the rule. M. canis causes an ‘ectothrix’ infection where spores form on the out- side of the hair shaft and the scalp hair breaks above the skin surface. Scaling, itching, and loss of hair occur. Other causes of ectothrix in- fection include M. audouinii, which is still seen in West Africa. This infection can be spread from child to child and causes serious social handicap. The infection can occur in epidemic form, particularly in schools. By contrast, infections with M. canis are acquired from a pri- mary animal source rather than by spread from human lesions. In endothrix infections where sporulation is within the hair shaft, scaling is less pronounced, and hairs break at scalp level (black dot ringworm). Examples include T. tonsurans and T. violaceum, the latter being most prevalent in the Middle East, parts of Africa, and India, although it also is being recognized with increasing frequency in Europe. Favus, now most often seen in isolated foci in the tropics, is a par- ticularly chronic form of ringworm caused by T. schoenleinii where hair shafts become surrounded by a necrotic crust or scutulum (Fig. 8.7.1.3). Individual crusts coalesce to form a pale, unpleasant-​ smelling mat over parts of the scalp. Such infections can cause exten- sive and permanent hair loss. Tinea imbricata (tokelau) This infection is endemic in parts of eastern Asia, West Pacific, and Central and South America, and is caused by T. concentricum. In many cases the trunk is covered with scales laid down in concen- tric rings producing a ripple effect (Fig. 8.7.1.4). Alternatively, large, loose scales can form (hence the name; imbricata is the Latin word for ‘tiled’). The infection is often chronic, and can constitute a ser- ious social handicap. There is some evidence that susceptibility of this disease in Papua New Guinea might be inherited as an auto- somal recessive trait. Infection in HIV and immunocompromised patients While dermatophyte infections are no more common in the im- munocompromised patient, they might differ clinically. In patients with untreated HIV infection there can be (1)  more tinea facei, (2) more widespread and atypical skin lesions, and (3) a distinct pat- tern of nail infection characterized by white discoloration spreading rapidly through the nail plate from the proximal nail fold. Laboratory diagnosis The mainstays of diagnosis are direct microscopy of skin scales mounted in potassium hydroxide (20%) to demonstrate hyphae, and culture. Scalp hairs can also be examined in a similar way, and the site of arthrospore formation, inside or outside the shaft, deter- mined. Fluorescent whitening agents (Calcofluor) or chlorazol black stain have been used to highlight fungi in scales. Further tests, such as the ability to penetrate hair, can be used to separate similar cul- tures. Identification of organisms is important, as it will indicate the source of infection in scalp ringworm, for example. When large numbers of children are involved, screening of scalp infections with a filtered ultraviolet lamp (Wood’s light) is useful. Certain species, including M. canis and M. audouinii, cause infected hair to fluoresce with a vivid greenish light. Scalps can also be screened for infection by passing a sterile brush or scalp massager through the hair and plating this directly on to an agar plate. Fig. 8.7.1.3  Advanced favus of scalp in a Nigerian child caused by Trichophyton schoenleinii. Copyright D. A. Warrell. Fig. 8.7.1.4  Tinea imbricata, Papua New Guinea. Courtesy of Dr B. Hudson, Sydney.

section 8  Infectious diseases 1342 Treatment The treatment of dermatophyte infections depends, to an extent, on the nature and severity of infection. Topical therapy is reserved for circumscribed infections such as athlete’s foot and tinea corporis, not involving hair or nail keratin. Scalp and nail infections, severe or widespread ringworm, and failures of topical therapy are usually treated orally with griseofulvin, itraconazole, or terbinafine. Specific antifungal drugs in topical form are effective and well tolerated. The important compounds in this group are miconazole, clotrimazole, ketoconazole, and econazole, which are imidazole derivatives, undecenoic acid, and tolnaftate and the allylamine, terbinafine. Generally treatment is given for 7–​30 days. They are all very similar in their clinical efficacy, but topical terbinafine is particularly rapid in foot infection (≤7 days). Adverse reactions are rare. For oral therapy the main alternatives are terbinafine, itraconazole, or fluconazole. Terbinafine (250 mg/​day) is rapidly effective in most forms of dermatophytosis that require oral therapy and also produces rapid responses in toenail (12 weeks) and sole infections (2 to 4 weeks), without a high rate of relapse. Side effects include headache and nausea, but loss of taste might also occur. Itraconazole is somewhat similar in its profile, but is given intermittently (200 mg twice daily for 7 days). This course is given once for sole infections but repeated three times at monthly intervals for toenail infections, as pulsed therapy. Side effects include nausea and abdominal dis- comfort. Fluconazole is also active and is given in a dose of 150 mg weekly; 300 mg might be necessary for toenail infections. This side effect profile is similar to itraconazole. All three drugs are ex- tremely rare causes of hepatic toxicity. Griseofulvin is still used for tinea capitis in a dose of 10 to 20 mg/​kg daily. Treatment should be continued for at least 6 weeks in tinea capitis. Side effects are not common, but include headache, nausea, and urticaria. The drug can also precipitate acute intermittent porphyria and systemic lupus erythematosus in predisposed subjects. Neoscytalidium infections The organisms Neoscytalidium dimidiatum (Hendersonula toru­ loidea) and N hyalinum, can cause a superficial scaly condition that resembles the ‘dry type’ of dermatophyte infection on the palms or soles. Nail plate destruction can also occur, the lateral border of the nail being the initial site of invasion. The disease has been seen in Europe, almost invariably in immigrants from the tropics, particu- larly the Caribbean, West Africa, India, or Pakistan. Its prevalence in the tropics is unknown, although in some surveys it has been shown to be relatively common. In skin scrapings the tortuous hyphae might resemble those of a dermatophyte, but the organisms do not grow on media containing cycloheximide, which is often incorpor- ated into agar for routine dermatophyte isolation. Treatment is difficult, but some improvement might follow the use of keratolytic compounds such as salicylic acid. Nail infections seldom respond to terbinafine, griseofulvin, or azoles. Miscellaneous nail infections Occasionally, fungi other than dermatophytes or Neoscytalidium spe- cies are isolated from dystrophic nails. These include Scopulariopsis brevicaulis, Onychocola canadensis, acremonium, and fusarium species, and certain types of aspergillus. These infections are usu- ally seen in elderly or immunosuppressed individuals. It is often difficult, particularly with aspergillus, to establish that the organism is playing a pathogenic role. Pityriasis versicolor (tinea versicolor) Aetiology Pityriasis versicolor is a superficial infection caused by Malassezia species, usually M.  globosa. Although most common in tropical countries, it has a worldwide distribution. Dermal penetration does not occur. There are six species of malassezia that can be found on normal skin, the commonest of which are M. sympodialis and M. globosa. In pityriasis versicolor there is transformation of yeast cells to pro- duce hyphae. It is likely that the state of host immunity plays some part in pathogenesis and depression; for instance, endogenous or exogenous corticosteroids potentiate the disease in some individ- uals. However, it is also commonly seen in normal individuals, and climatic factors or sun exposure are believed to trigger the infection in many cases. There is no effective animal model for studies of this disease. Epidemiology Pityriasis versicolor is very common in the tropics, where it might be widespread on the body. Its incidence in temperate climates has increased over the last 20–​30 years. It is not more common in HIV-​ infected individuals. Clinical features The rash of pityriasis versicolor is asymptomatic or mildly pruritic. Its presents with scaling, confluent macules on the trunk, upper arms, or neck. These can be hypopigmented or hyperpigmented. In some people and in the tropics, other areas including face, forearms, and thighs might be involved. The diagnosis is rarely confused with other complaints, although eczema or ringworm infections are sometimes considered. Patients are often anxious to exclude leprosy, but the two are unlikely to be mistaken. In vitiligo, depigmentation is complete and there is no scaling. Laboratory diagnosis The diagnosis is made by demonstration of the yeasts and hyphae of malassezia in skin scales removed by scraping. Culture is difficult and unnecessary. Treatment Topical ketoconazole, miconazole, clotrimazole, or econazole is ef- fective. Oral itraconazole can be used in recalcitrant cases. Whatever the treatment, relapse is common. Other malassezia-​associated conditions Malassezia yeasts have been implicated in the pathogenesis of several other skin diseases such as seborrhoeic dermatitis and a form of itchy folliculitis, malassezia folliculitis. The evidence con- necting seborrhoeic dermatitis, one of the most common of skin diseases, and Malassezia is largely concerned with the response of antifungal drugs and the observation that improvements in the rash mirror disappearance of organisms from the skin as well as the production of fungal specific inflammatory mediators such as indolocarbazoles.

8.7.1  Fungal infections 1343 Superficial candidiasis Aetiology Superficial candidiasis is a term used to describe a group of in- fections of skin or mucous membranes caused by species of the genus Candida. They range in severity from oral thrush to chronic mucocutaneous candidiasis, a chronic infection refractory to con- ventional antifungal treatment. Candida albicans is the species most frequently involved. It is a saprophytic yeast often found as a commensal in the mouth and gastrointestinal tract, and is commonly present in the vagina. Several factors influence the incidence of carriage. For instance, oral colonization is more common in hospital staff than in equiva- lent non​hospital employees. Vaginal carriage is more common in pregnancy. Other factors (Box 8.7.1.1) are known that predispose to conversion from a commensal to a parasitic role with the causation of disease—​candidosis. The list includes factors that influence host immunological response, such as carcinoma, AIDS, or cytotoxic therapy; those that disturb the population of other microorganisms, such as antibiotics; and those that affect the character of the epithe- lium, such as dentures. Other species of candida can also cause superficial infections, but are less common. They include C. glabrata, C. dubliniensis, and C. parapsilosis. There is evidence that the first two species are more common in oral infection in patients with HIV and C. glabrata in vaginal candidiasis. Epidemiology Superficial candida infections are seen in all countries. Clinical features There are several clinically distinct types of superficial infection caused by candida species, as follows. Oral candidiasis (thrush) Oral infection by candida is fairly common, particularly in infancy and old age, or in association with antibiotic or cytotoxic therapy, or in diseases where the neutrophil or T-​lymphocyte responses may be impaired. In older people, the wearing of dentures is a predisposing factor. The lesions present with discomfort both in the mouth and at the corners of the lips. The mouth and buccal mucosa show patchy or confluent, white adherent plaques; less commonly the mucosa and tongue are sore and glazed—​erythematous candidosis. Angular cheilitis usually accompanies the oral lesions. In long-​standing cases, the plaque might become hypertrophic, with oedema of the mucosal surfaces, or the mucosa can appear glazed and raw. There is a significant correlation between leucoplakia and oral candidiasis, and it has been suggested that the infection might lead to epithelial dysplasia. The diagnosis is made by the demonstration of yeasts and hyphae of candida in smears, and by culture. Vaginal candidiasis (thrush) See Chapter 9.4 for further detail. Paronychia Infection around the nail fold is seen in people whose occupations involve frequent wetting of the hands (such as cooks) or in those with eczema or psoriasis. The aetiology is complicated and there might be a mixture of bacterial infection and irritant or allergic con- tact dermatitis as well as candida infection. The condition presents with painful, red swelling of the nail fold. Pus might be discharged. Secondary invasion of the lateral border of the nail plate by candida can occur from this site. Candida intertrigo Infection of the moist folds of the skin in the groin or under the breasts causes itching and discomfort. The area becomes macerated and erythematous. Candida might contribute to this condition, but is certainly not the only factor. It might also superinfect the napkin area in infants. The presence of satellite pustules (see earlier) is a useful indicator of involvement by candida in the disease process. Direct invasion of toe-​web folds by candida closely resembles ‘ath- lete’s foot’ caused by dermatophytes. A similar erosive infection can occur in the finger webs—​interdigital candidiasis—​and is seen most commonly in the tropics. Chronic superficial candidiasis Chronic candida infections of the mouth, vagina, and nail pre- sent problems in management. Chronic oral candidiasis, for in- stance, is associated with leucoplakia. Predisposing causes should be searched for. The most serious of this group of infections is chronic mucocutaneous candidiasis, a rare condition in which chronic skin, nail, and mucosal infection coexist (Fig. 8.7.1.5). A  series of underlying genetic, endocrine (hypoparathyroidism, hypoadrenalism, or hypothyroidism), and immunological abnor- malities has been found; in some cases, it has been associated with mutations in the autoimmune regulator (AIRE) or STAT1 genes. Extensive human papillomavirus (wart) or dermatophyte infections might also be present in these patients, whose condition is normally diagnosed in childhood. Oral candidiasis is one of the earliest signs of untreated AIDS, occurring in a high proportion of patients. The appearances are similar to those seen with other groups, although plaque forma- tion might be very extensive. Oesophageal infection is common in this group. Box 8.7.1.1  Predisposing factors in superficial candidiasis • Local epithelial defects, occlusion, constant immersion in water (e.g. damaged nail folds, beneath dentures) • Defects of immunity (primarily T cell or phagocytosis) —​ Primary immunological disease (e.g. chronic granulomatous disease) —​ Immunodefects secondary to intercurrent illness (e.g. leukaemia) —​ Immunodefects secondary to therapy (e.g. cytotoxic therapy in organ transplantation) • Drug therapy (e.g. antibiotics) • Carcinoma or leukaemia • Endocrine disease —​ Diabetes mellitus —​ Hypothyroidism, hypoparathyroidism, hypoadrenalism (all in chronic mucocutaneous candidiasis) • Physiological changes (e.g. infancy, pregnancy, old age) • Miscellaneous disorders, for example: —​ Iron deficiency —​ Zinc deficiency —​ Malabsorption

section 8  Infectious diseases 1344 Laboratory diagnosis All these infections are diagnosed by microscopy and culture. When associated with the condition, candida cells are always evident on microscopy. Culture establishes the specific identity and is important particularly where species other than C. albicans might be involved. Treatment Two groups of drugs are effective in superficial candidiasis. The polyenes such as nystatin and amphotericin B are topically active in many forms of candidiasis. They are often less effective in oral can- didiasis in immunodeficient patients, including those with AIDS. Likewise, topical azole drugs such as miconazole and clotrimazole are usually effective in superficial candidiasis. For unresponsive cases, oral therapy with fluconazole and itraconazole might be ne- cessary. Fluconazole resistance can occur and C. glabrata is seldom responsive to this drug. For vaginal infections, topical creams or vaginal preparations should be used—​many requiring only a single treatment. Single-​ dose oral fluconazole is an alternative. In recalcitrant cases it might be necessary to use longer courses of fluconazole or itraconazole. Miscellaneous superficial mycoses There are several relatively rare, superficial fungal infections such as tinea nigra, and black or white piedra. They never cause invasive disease, and are mainly confined to the tropics. Tinea nigra Tinea nigra is a superficial infection confined to the epidermis of the palms or soles, and more rarely elsewhere. The initial lesion is a dark macule without scaling, which resembles a brown stain on the skin and spreads slowly over the palmar or plantar surface. The disease is normally asymptomatic. On scraping the skin, brown pigmented hyphae can be seen by direct microscopy, and the causative organism, Phaeoanellomyces werneckii, isolated. The lesion responds to Whitfield’s ointment. Black piedra Black piedra is a disease of the tropics in which small, dark nodules form on hair shafts in the scalp or, less commonly, elsewhere. There are no symptoms. Each nodule consists of a dense mat of hyphae containing the sexual spores (ascospores) of the fungus. The diagnosis is made by direct microscopy of infected hair, and the isolation of Piedraia hortae. Treatment using a 1% azole solution or amphotericin B lotion is usually effective. White piedra White piedra occurs in both temperate and tropical climates, and is rare. It produces pale nodules on the hair of the beard, groin, or scalp. The hair shaft may fracture. The nodule consists of hyphae, arthrospores (spores formed by fragmentation of hyphae), and blastospores (budding yeast cells). The organism Trichosporon spe- cies can be readily cultured. The treatment is similar to that for black piedra. Subcutaneous mycoses Subcutaneous infections caused by fungi are rare, and are mainly seen in the tropics. The organisms gain entry via the skin; in my- cetoma, organisms may be implanted subcutaneously via a thorn. Most of the causative organisms in this group of infections can be isolated from vegetation or soil. Involvement of deep viscera is rare. Attempts to establish experimental infections that resemble the human diseases have been largely unsuccessful. A clearer under- standing of the pathogenesis therefore awaits such a model system. These infections tend to be chronic, chemotherapy might be lengthy, and in the case of mycetoma, often unsuccessful. Mycetoma (Madura foot) Aetiology Mycetoma is a chronic infection involving subcutaneous tissue, bone, and skin, in which colonies of infecting fungi or actinomy- cetes (grains) are found within a network of burrowing abscesses and sinuses (Fig. 8.7.1.6). The more common organisms that cause mycetoma are listed in Box 8.7.1.2. The organisms are divided into two groups, the actinomycetomas and the eumycetomas, caused by actinomycetes and fungi, respectively. The size and colour of the grains (red, pale, Fig. 8.7.1.5  Oral candidiasis in a patient with chronic mucocutaneous candidiasis. Fig. 8.7.1.6  Grains in abscess in actinomycetoma (Nocardia brasilensis) (haematoxylin and eosin stain).

8.7.1  Fungal infections 1345 or dark) are important clues to their identification. The organisms can be found in the natural environment such as soil, and some have even been identified in association with acacia thorns in an endemic area. The infection is initiated when an infected thorn is implanted in deep tissue. However, many years might elapse before the forma- tion of a clinically apparent mycetoma. Epidemiology The disease is seen primarily in the tropics, although rare cases, apart from imported ones, can occur in temperate areas. Countries with the most reported cases include India, Mexico, Senegal, Sudan, and Venezuela. However, the disease is widely distributed in the tropics, particularly in Africa to the south and east of the Sahara Desert. The pattern of prevalence of infections caused by certain or- ganisms differs strikingly in different parts of the world. For in- stance, Streptomyces somaliensis is most common in the Sudan and Middle East, but Madurella grisea is mainly found in the New World. Altogether about 60% of reported infections are caused by actinomycetes, of which Nocardia brasiliensis is the most common (Chapter 8.6.31). Clinical features Early mycetomas might present with a circumscribed area of hard painless subcutaneous swelling (Fig. 8.7.1.7). Later, sinus tracts open on to the skin surface and visible grains might be discharged, along with serosanguinous fluid (Fig. 8.7.1.8). Bone erosion and de- struction, leading to deformity, can occur. However, severe pain is rarely a problem. Local lymph node invasion can occur, but more widespread involvement is very rare. Feet and lower legs are the areas most commonly involved, but the arms, buttocks, chest, and head can all be sites of infection. Mycetoma caused by N. brasiliensis can occur in any site, but one favoured area is the chest wall. The radiological features of mycetoma are cortical erosion, fol- lowed by the development of lytic deposits in bone. Periosteal pro- liferation and destruction, leading to deformity, may follow. MRI provides a clearer picture of bone involvement and might be positive earlier than radiography. Laboratory diagnosis The diagnosis is made by the demonstration and identification of grains obtained from the sinus openings by gentle pressure or curettage. If these measures are not successful, tissue should be obtained by deep surgical biopsy. Grains can be mounted in potas- sium hydroxide and examined microscopically. Those containing filaments 3–​4 µm or more in diameter are caused by true fungi (eumycetomas), and those with filaments of less than 1 µm by ac- tinomycetes (actinomycetomas). These features can usually be dis- tinguished by direct microscopy. The morphology of grains fixed, sectioned, and stained with haematoxylin and eosin is typical. Special stains are less helpful. Grains can be used for culture, although several attempts at isolation may have to be made. Serology (such as immunodiffusion) can also be helpful, although the tests are not widely available. Treatment Actinomycetomas might respond to sulphones such as dapsone (50–​ 100 mg daily) or sulphonamides such as sulphadiazine. The treatment of choice for many is long-​term co-​trimoxazole (2–​3 tablets twice daily) with an initial 2 to 3 months of streptomycin or rifampicin. Treatment might have to be continued for many months or years. Dapsone is an effective and cheaper alternative to co-​trimoxazole. Extensive actinomycetomas might respond poorly and additional treatment with amikacin, moxifloxacin, or linezolid might be ne- cessary. The eumycetomas seldom respond to antifungal therapy. But in some infections griseofulvin, amphotericin B, voriconazole, ketoconazole, and itraconazole have rarely produced remission or Box 8.7.1.2  Causes of mycetoma • Fungi, for example: —​ Madurella mycetomatis, M fahalii, M. pseudomycetomatis —​ Madurella grisea —​ Scedosporium apiospermum —​ Exophiala jeanselmei —​ Plenodomus senegalensis —​ Species of Acremonium, Aspergillus, Fusarium • Actinomycetes, for example: —​ Nocardia brasiliensis —​ Actinomadura madurae —​ Actinomadura pelletieri —​ Streptomyces somaliensis Fig. 8.7.1.7  A mycetoma caused by Madurella grisea. Fig. 8.7.1.8  Nocardia brasiliensis actinomycetoma draining sinus.

section 8  Infectious diseases 1346 cure. A trial of therapy can be attempted, where the patient can be monitored closely in outpatient departments. Otherwise, radical surgery or amputation is usually necessary. Small, local excisions are rarely successful. Mycetoma is slowly progressive and increasingly disabling. However, wider dissemination is very rare, and therefore cases are seldom fatal, except where the skull is involved. However, the de- formity caused by the disease can be severely disabling. Chromoblastomycosis (chromomycosis) Aetiology Chromoblastomycosis, one of the intermediate subcutaneous my- coses, is a chronic granulomatous fungal infection characterized histologically by the presence of brown, spherical fungal cells known as sclerotic cells or fumagoid bodies. In most cases, the lesions are confined to the skin and subcutaneous tissues. In the past there has been great confusion over nomenclature of the aetiological agents of chromoblastomycosis. At present, five agents assigned to four genera are recognized as causing chromoblastomycosis—​most are due to the first two. They are: • Fonsecaea pedrosoi, which occurs in high-​rainfall areas and is found worldwide • Cladophialophora carrionii, the sole cause of chromoblastomycosis in arid areas • Phialophora verrucosa, the first agent to be described • Fonsecaea compactum, an uncommon cause and isolated only a few times • Rhinocladiella aquaspersa, a rare cause Sporadic cases caused by other dematiaceous fungi such as Cladosporium trichoides and Taeniolella boppii have been reported from Uganda and Brazil. Epidemiology The principal endemic areas for chromoblastomycosis are tropical and subtropical countries including Central and South America, Costa Rica, Africa, Japan, Australia, Madagascar, and Indonesia. Curiously, sporadic cases have been reported from Finland and Russia. Although soil itself does not seem to be a particularly good sub- strate, the various agents of chromoblastomycosis occur as saprobic fungi in the environment and have been isolated from soil, decaying vegetation, and rotting wood. Strains of F. pedrosoi and P. verrucosa have been isolated from the atmosphere but proved less virulent than those isolated from human lesions or organic material. Infection occurs as a result of trauma, however minor, the fungi gaining entrance through a cut, abrasion, or thorn prick. Farmers and labourers in agricultural areas are most likely to be exposed to contaminated material. Although lesions on exposed areas might be accounted for in this way, it was suggested by Wilson in 1958 that lesions on non​exposed areas might result from a previously unrec- ognized pulmonary focus. Bacquero later demonstrated the pres- ence of F. pedrosoi in bronchial washings and subsequently proved their pathogenicity by inoculating those strains into normal skin of human volunteers and recovering the fungus from the ensuing skin lesions. Other methods of transmission have included metal particles from automobiles, and acupuncture. Person-​to-​person and animal-​to-​human transmission have not so far been reported. Chromoblastomycosis has rarely been reported in children, and it might be that factors other than trauma and exposure to contamin- ated material are necessary for its development. Pathogenesis Host resistance and virulence of the organism are the two main factors associated with the pathogenesis of this disease. Chromoblastomycosis occurs mainly in healthy individuals. However, it has been found in immunosuppressed patients. Although the mechanism of granuloma formation is not well under- stood, it appears that lipids extracted from these fungi and cell-​wall constituents might be responsible for this reaction. Clinical features The initial lesion of chromoblastomycosis is a small papule at the site of trauma, which gradually enlarges (Fig. 8.7.1.9). Nodules and tumours develop, producing a malodorous discharge; eventu- ally, over a period of years, a wide variety of morphological patterns may emerge including dry, hyperkeratotic plaques, verrucose le- sions, and large, cauliflower-​like masses (Fig. 8.7.1.10). Extensive cicatricial plaques, surrounded by peripherally spreading vegetative lesions, can also be present. Evolution is slow, and lesions usually in- volve the lower limb. However, any part of the body can be involved and the sites can be multiple. Dissemination occurs by (1) surface spread; (2) the lymphatics, the most common method; (iii) autoinoculation from scratching; and (iv) haematogenously, resulting in subcutaneous lesions at sites distant from the primary. Visceral metastases are known to occur and involvement of the central nervous system, respiratory system, larynx, and vocal folds has been recorded. Therapeutically, there- fore, early diagnosis is important. Fig. 8.7.1.9  Chromoblastomycosis. Early lesion in a Brazilian patient. Copyright D. A. Warrell.

8.7.1  Fungal infections 1347 Complications of long-​standing chromoblastomycosis include lymphoedema, flexion deformity of joints, and development of squamous carcinoma. Diagnosis Although the history and clinical presentation might suggest the diagnosis, the varied clinical presentation of chromoblastomycosis necessitates consideration of other granulomatous diseases such as sporotrichosis, cutaneous tuberculosis, Hansen’s disease, blasto- mycosis, candidiasis, leishmaniasis, paracoccidioidomycosis, rhinosporidiosis, tertiary syphilis, squamous carcinoma, and even psoriasis, sarcoidosis, and discoid lupus erythematosus. Therefore, to establish a definitive diagnosis, histological and myco- logical investigations are essential. Diagnosis is confirmed by the presence of the characteristic brown, sclerotic bodies in histological sections. From both epidemiological and therapeutic points of view, culture is necessary as F. pedrosoi is the most difficult of the causative fungi to eradicate whereas C. carrionii responds rapidly to treatment. Treatment Small, single, localized lesions are satisfactorily eradicated by cryo- surgery, but long-​term follow-​up is needed to assess accurately the success of this treatment. Thermotherapy has been found effective by some, again principally in the management of small, single le- sions, but here the possibility of a burn must be borne in mind. Rapid spread of the disease has been associated with inadequate sur- gery, curettage, and electrodesiccation. Itraconazole and terbinafine have both been reported as effective agents. A combination of 5-​flucytosine with either thiabendazole or itraconazole can also be efficacious, particularly in long-​standing disease. Whatever method of treatment is used, chromomycosis although clinically healed, should be followed-​up for at least 2 years before its total eradication can be assumed. Sporotrichosis Aetiology The most common clinical form of sporotrichosis is a subcutaneous infection, which can spread proximally from its initial site in a series of nodules along the course of a lymphatic (Fig. 8.7.1.11a, b). More rarely, systemic involvement is seen, for example, in the lung (see ‘Systemic mycoses’, next). The causative organism, Sporothrix schenckii, which is a complex of closely related species such as S. braziliensis, S. mexicana can be found in soil, in vegetation, or in association with plants or bark. People who develop the subcutaneous infection might have had contact with material that harbours the organism, such as moss or flowers (e.g. florists). It is assumed that the pathogen gains entry via an abrasion and in some endemic areas there is often a preceding history of a cat scratch or insect bite. Epidemiology Although sporotrichosis was once prevalent in Europe, particularly France, non​imported cases are now very rare in this area. However, the disease is seen in the United States of America, Mexico, Central and South America, and Africa. In the late 1930s, there was a remark- able epidemic of sporotrichosis in workers in the Witwatersrand gold mines (South Africa). The source of infection was a large number of wooden pit props contaminated with the organism. Other, smaller Fig. 8.7.1.10  Chromoblastomycosis: late lesion. Courtesy of João LC Cardoso, São Paulo, Brazil. (a) (b) Fig. 8.7.1.11  (a) Sporotrichosis. (b) Histopathological appearances. (a) Courtesy of João LC Cardoso, São Paulo, Brazil; (b) copyright Professor R. Hay.

section 8  Infectious diseases 1348 ‘epidemics’ have been described in certain groups, such as Mexican pottery workers packing ceramics in straw. Normally, however, cases are sporadic in incidence. There are also ‘hyperendemic’ areas where there is an unexpectedly high incidence of this infection (e.g. Rio de Janeiro State, Brazil). Systemic sporotrichosis is much rarer, and cases have mainly been described from the United States. Clinical features There are two main clinical types of subcutaneous sporotrichosis. The first, the fixed type, presents with a solitary cutaneous ulcer or nodule. In this form of the disease, infection does not spread along lymphatics. It has been suggested that it is most common in children, and it has been described most frequently in Central and South America. In the lymphangitic form, an initial nodule forms on a limb or extremity, such as a finger. This may break down and ulcerate. Subsequently, one or more secondary nodules develop along the draining lymphatic channel, which may ulcerate through the skin (Fig. 8.7.1.11a). Other variants include the psoriasiform or verru- cous types or a superficial granuloma that resembles lupus vulgaris. These usually represent chronic infection. Rarer forms include secondary spread via scratching, which might present with multiple widespread ulcers or multiple cutaneous le- sions secondary to systemic disease. In HIV-​positive individuals, widespread cutaneous lesions can develop. Fixed-​type sporotrichosis can resemble many other forms of cuta- neous ulceration. However, in endemic areas a major source of con- fusion is cutaneous leishmaniasis. The lymphangitic variety can also resemble other infections, notably atypical mycobacterial infections, particularly fish-​tank granuloma, or ‘sporotrichoid’ leishmaniasis. Treatment Some cases of sporotrichosis heal spontaneously. However, treat- ment is usually advised to prevent scar formation. The cheapest treatment is potassium iodide, which is administered in a satur- ated aqueous solution. The starting dose is 0.5–​1 ml, given three times daily, and this is increased drop by drop per dose to 3–​6 ml, three times daily. The mixture is more palatable if given with milk. Treatment should be given for a month after clinical resolution. However, both itraconazole and terbinafine are also effective; min- imal durations of treatment for these agents have not been defined. Subcutaneous mucoromycosis due to Basidiobolus Subcutaneous mucoromycosis is an infection primarily seen in chil- dren in Africa or eastern Asia (Indonesia). It is characterized by the development of localized woody swellings on the limbs or trunk. The swelling is rarely inflammatory, but has a well-​defined leading edge, and is hard. Progression is slow. The causative organism, Basidiobolus haptosporus, can be cultured or demonstrated histologically in biopsy material. Although resolution has been recorded without treatment, therapy is normally given. Potassium iodide solution is the treatment of choice, and is given in as high a dose as possible (see ‘Sporotrichosis’, earlier). Itraconazole might also be effective. Subcutaneous mucoromycosis due to Conidiobolus (conidiobolomycosis or rhinoentomophthoromycosis) Conidiobolomycosis is a similar infection confined to subcutaneous tissue and presenting with painless swelling. The infection is mainly seen in West Africa, but a case has been seen in the Caribbean. There are important differences from the subcutaneous mucoromycosis caused by basidiobolus. The disease is most common in young adults, and is confined to facial tissues around the nose, the forehead, and the upper lip (Fig. 8.7.1.12). The initial site of infection is in the region of the inferior turbinate in the nose. The diagnosis is estab- lished by biopsy or culture. The causative organism is Conidiobolus coronatus. Treatment with itraconazole or ketoconazole is effective, but an alternative is high-​dose potassium iodide. Relapse after treat- ment is common, and residual fibrosis can be severely disfiguring. Lobo’s disease (lobomycosis) Lobo’s disease is a subcutaneous infection. The organism, Lacazia loboi, in tissue, appears to be a yeast. It has a tendency to form chains of four to six yeast cells with prominent nucleoli, joined by a narrow intercellular bridge. However, the organism has never been cultured from human cases and can only be identified by biopsy and hist- ology. The disease is seen in countries of South America around and to the north of the Amazon basin, and cases are also seen in Central America. Apart from humans, the only other species affected are freshwater dolphins. Often, exposed sites (such as earlobes) are in- vaded and small nodules containing the organisms develop. These may resemble keloids (Fig. 8.7.1.13). More diffuse plaques may also be seen. Deep invasion has not been documented. The treatment is excision, and there is no effective chemotherapy, although there have been recent reports that posaconazole might be effective. Systemic mycoses The systemic or deep visceral mycoses include some of the rare and more serious fungal infections. There are two main types of infection Fig. 8.7.1.12  Subcutaneous mucoromycosis (Conidiobolus coronatus). Copyright Professor R. Hay.

8.7.1  Fungal infections 1349 in this group: (1) the endemic mycoses, caused by organisms that in- vade normal hosts, and (2) the opportunistic mycoses, which cause disease only in compromised patients. The fungi associated with these two types of infection differ in their innate levels of pathogen- icity, but an element of opportunism, depending on host suscepti- bility, is usually recognizable in all cases of systemic mycoses. The endemic pathogens cause infections such as histoplasmosis or coccidioidomycosis. These diseases have well-​defined endemic zones and most of those exposed remain symptomless but usually develop positive skin tests. However, in certain patients, chronic local or disseminated disease can occur. In the systemic infections caused by opportunistic fungi, there is usually a serious underlying abnormality in the patient affecting T lymphocytes (such as HIV) or neutrophils (such as cancer chemotherapy). Such infections are worldwide in occurrence: where tissue invasion occurs, the mor- tality is high. Cryptococcosis, a systemic yeast infection, has features of both types of systemic disease and occurs in both normal and immunosuppressed subjects (Chapter 8.7.2). The systemic endemic infections are histoplasmosis, coccidioido- mycosis (Chapter  8.7.3), blastomycosis, paracoccidioidomycosis (Chapter  8.7.4), and infections due to Talaromyces marneffei (Chapter 8.7.6). The significance of various laboratory tests in these infections is shown in Table 8.7.1.1. Histoplasmosis There are two forms of histoplasmosis. In both types, the organism is present in tissue in its yeast phase. In small-​form or classic histo- plasmosis, the diameter of the yeast cells is between 3 and 4 µm. Infections are most common in the United States of America, but sporadic cases are reported widely from the New World, Africa, and eastern Asia. By contrast, large-​form or African histoplasmosis is most common in Central Africa, south of the Sahara, and north of the Zambezi River. Yeast forms in infected tissue are much larger, 10–​15 µm in diameter. Both infections are clinically distinct (see next), but cultural isolates are indistinguishable. Histoplasmosis (classic or small-​form histoplasmosis) Aetiology Histoplasmosis is a systemic infection caused by Histoplasma capsu­ latum. The main route of infection is pulmonary. Most of those ex- posed are sensitized without overt signs of infection, but more rarely chronic pulmonary or disseminated forms of the disease are seen. Table 8.7.1.1  Laboratory tests in systemic mycosesa Direct microscopy Significance of positive cultures Serology Histopathology Histoplasmosis   Classic (small form) Sometimes positive Significant ID, CIE, CFT Urine antigen detection Yeasts (3–​4 µm)   African histoplasmosis Positive in pus (valuable) Significant ID, CFT Yeasts (10–​15 µm) Coccidioidomycosis Positive in pus, sputum, etc. (valuable) Significant, NB Handle with caution ID, CFT, TP, CIE Spherules (50–​150 µm) Blastomycosis Positive in pus, sputum, etc. (valuable) Significant ID, CFT, CIE (unreliable) Yeasts (4–​10 µm) Broad-​ based buds Paracoccidioidomycosis Positive in pus, sputum etc. (valuable) Significant ID, CFT, TP Antigen detection Yeasts (5–​15 µm) Multiple buds Cryptococcosis Often positive in CSF (rare in urine, pus), NB Indian ink Significant Latex agglutination or ELISA
for antigen (ID, CFT, WCA, IF) Encapsulated yeasts (5–​10 µm) Mucicarmine positive Systemic candidiasis Positive in oral smzears, sputum, etc. (interpret with caution) Significance depends on site
and presence of positive microscopy ID, CFT, WCA, CIE Antigen detection Yeasts (5–​10 µm) and hyphae Invasive aspergillosis Rarely positive, depends on site Positive sputum cultures not always significant ID, CIE, rarely positive Antigen detection, e.g. Pasteurex Hyphae—​dichotomous branching Mucormycosis Rarely positive Depends on site Rarely positive Hyphae—​broad and aseptate CFT, complement fixation test; CIE, counterimmunoelectrophoresis; CSF, cerebrospinal fluid; ID, immunodiffusion; IF, immunofluorescence; RIA, radioimmunoassay; TP, tube precipitation; WCA, whole-​cell agglutination. a Molecular diagnostic techniques are increasingly used but are not standardized. Fig. 8.7.1.13  Lobo’s disease in a Brazilian man. Copyright D. A. Warrell.

section 8  Infectious diseases 1350 The organism, H. capsulatum, can be found in soil in endemic areas. Its growth is facilitated by the presence of bird excreta (e.g. in old chicken houses, bird roosts, and barns). In tropical and some temperate areas, bat guano plays a similar role. Exposure to a suitable source, such as a cave containing bats, is often recorded in acute epidemic histoplasmosis (see next). It is rarely identified in more slowly evolving cases. The condition of the host is important in determining the clin- ical course and manifestations of histoplasmosis. Slowly evolving (chronic), disseminated disease can occur in normal individuals. However, infants, elderly people, or those with untreated AIDS ap- pear to be more likely to develop the more rapidly progressive forms of disseminated infection. Epidemiology The major endemic area, as shown by skin testing, is in the central region of the United States around the Ohio and Mississippi valley basins. Prevalence is highest in the states of Tennessee, Kentucky, and Ohio. Up to 95% of those skin-​tested in certain parts of these areas have positive delayed reactions to intradermal histoplasmin. Scattered cases of active disease, healed calcified foci in chest radiographs, and foci found at autopsy representing inactive histoplasmosis also pro- vide evidence of spread within this area. However, the disease also oc- curs in other parts of the United States, Mexico, Central and South America, Africa, eastern Asia, and Australia. Outside the major en- demic areas in the United States, human cases are less frequent, and much of the evidence of the endemicity comes from positive skin tests or the presence of the organism in selected sites, such as caves. Although there has been considerable discussion on the nature of soil factors responsible for the growth of H. capsulatum, the conditions limiting its occurrence to certain areas are largely unknown. Clinical features The clinical forms of histoplasmosis can be placed in several groups: • asymptomatic • acute symptomatic pulmonary: ■ acute epidemic ■ acute reinfection • chronic pulmonary • disseminated (acute, subacute, and chronic) • primary cutaneous (by inoculation) Asymptomatic infection Over 99% of patients becoming infected in endemic areas record no overt symptoms but develop a positive skin test. The incidence of positive skin tests declines in individuals above the age of 60 years. Acute (symptomatic) pulmonary histoplasmosis Acute epidemic histoplasmosis Groups of people exposed to a source of infection (e.g. during cave exploration, or those who might have inhaled a large infecting dose, often develop a symptomatic illness 12–​21 days after exposure). The main features are pyrexia, cough, chest pain, and malaise. Flitting arthralgia and, less commonly, erythema nodosum or multiforme may occur. The radiological appearances might be much more se- vere than would be supposed from the symptoms, and enlargement of hilar lymph nodes and diffuse or patchy consolidation suggesting pneumonitis can occur (Fig. 8.7.1.14). These patients develop precipitating or complement-​fixing anti- body, but this often follows the peak of illness. About 50% of those with symptoms do not develop positive antibody responses. Likewise, skin-​test conversion is often too late to be of diagnostic value, and its use is normally contraindicated, as a single histoplasmin test might cause the development of false-​positive serological results. Cultures are often negative. The symptoms and history of exposure to a suit- able source, combined with a rising antibody titre, are often the best evidence of infection. Most cases require no specific therapy apart from rest. Those with severe or prolonged symptoms or impaired gas exchange require intravenous amphotericin B or itraconazole. The lung lesions often heal to leave multiple scattered pulmonary calcifications. Acute reinfection histoplasmosis Massive acute exposure to H. capsulatum in sensitized individ- uals is believed by some physicians to cause a less severe infection associated with bilateral pulmonary infiltrates. The incubation period is shorter than with acute epidemic histoplasmosis, namely 5–​10 days. Chronic pulmonary histoplasmosis Chronic pulmonary disease caused by H. capsulatum is mainly seen in the United States. It is more common in men and smokers, and there is often underlying pulmonary disease such as emphysema. Early cases might present with pyrexia and cough, but malaise and weight loss occur later. Lesions might heal initially, but relapse is common, leading to established consolidation and cavitation. The most common radiological appearance of early lesions is of uni- lateral, wedge-​shaped, segmental shadows in the apical zones. Subsequently, the disease can become bilateral, with fibrosis and cavitation. In some cases, extensive and progressive destruction of lung tissue may occur. Fig. 8.7.1.14  Acute pulmonary histoplasmosis. Copyright Professor R. Hay.

8.7.1  Fungal infections 1351 Culture and serology are both helpful methods of diagnosis in this form of histoplasmosis, but repeated attempts might be required be- fore positive results are obtained. In early cases, resolution might occur with rest alone. However, relapse occurs in at least 25% of cases, and these patients might re- quire amphotericin B therapy or itraconazole. Although chemo- therapy might virtually sterilize lesions, fibrosis persists, and relapse can occur. Surgical excision or lobectomy is sometimes effective. Solid lung tumours might persist after the primary infection. These can be single (coin lesions) or multiple, and have to be distin- guished from carcinomas. The diagnosis is normally made at sur- gery, although the presence of calcification might give a clue to the nature of the lesion (histoplasmoma). The organisms can be demon- strated by histopathology, but they are seldom viable. Disseminated histoplasmosis There is considerable variation in the rate of progression of histo- plasmosis that has spread beyond the initial focus in the lung. In rapid or acutely disseminated cases, widespread infiltration of re- ticuloendothelial cells of bone marrow, spleen, and liver might occur. Gastrointestinal lesions, endocarditis, and meningitis are less common, and meningitis is more usually associated with a slower course of disseminated disease. Infants, elderly people, or immuno- suppressed patients are more susceptible to acute dissemination. The most prominent symptoms are fever and weight loss, with ac- companying hepatosplenomegaly. Extensive purpura and bruising secondary to thrombocytopenia can occur. The blood picture can reflect marrow infiltration with organisms, leading to pancytopenia. Disseminated histoplasmosis is also seen in patients with AIDS. The clinical manifestations are not significantly different, although skin papules and ulcers have been reported in many (Fig. 8.7.1.15); isolation of histoplasma from blood has also been reported more frequently in these patients. Cultures, including sputum or bone marrow, should be taken. Serology is often positive, with high titres of complement-​fixing antibodies occurring in some patients. However, new antigen detection systems in serum or urine provide a better means of confirming the diagnosis and monitoring treatment. A much more slowly progressive form of disseminated histoplas- mosis can present with fewer localized lesions, such as persistent oral ulcers, chronic laryngitis, or adrenal insufficiency. Granulomas, few of which contain organisms, can be found in the liver in some patients. Such cases present up to 30 years after the patient has left an endemic area. Outside endemic areas this form is the most widely recognized presentation of histoplasmosis, occurring in Europeans, for instance, who have worked in Africa or eastern Asia. The diagnosis of disseminated histoplasmosis is made on culture or biopsy of affected areas. Antibodies might only be positive in low titres and in all cases adrenal involvement should be looked for. Treatment is required in all forms of disseminated histoplasmosis. Itraconazole is preferred by most physicians, although amphotericin B might be necessary in some patients. Posaconazole is an alterna- tive. In patients with AIDS who are acutely ill, the disease is often controlled by a short (2-​week) course of amphotericin B and there- after patients receive continuous itraconazole indefinitely or until their immune system improves with antiretrovirals. Primary cutaneous histoplasmosis Primary infection sometimes follows accidental inoculation of vi- able organisms in a laboratory or autopsy room. This type of infec- tion is normally associated with a chancre at the site of inoculation and regional lymphadenopathy. The condition is self-​limiting. African histoplasmosis Overt pulmonary involvement is rare in this form of histoplasmosis, and the normal portal of entry of the pathogen is not known. The most common presenting features are skin lesions (papules, nodules, abscesses, or ulcers) (Fig. 8.7.1.16) or lytic bone deposits. Solitary or Fig. 8.7.1.16  Nodular subcutaneous lesions of African histoplasmosis in a Nigerian man. Copyright D. A. Warrell. Fig. 8.7.1.15  Histoplasmosis. Molluscum-​like skin lesions in an HIV- positive Peruvian patient. Copyright D. A. Warrell.

section 8  Infectious diseases 1352 multiple foci may be present, and in the latter instances rapid pro- gression and death may occur. In such cases, gastrointestinal and lung lesions may develop. The diagnosis is normally made by culture, smear, or biopsy. The organism H.  capsulatum var. duboisii is identical to that causing classic histoplasmosis in culture, but in lesions the yeast forms are considerably larger (10–​15 µm). Although local excision of skin nodules has been reported to be curative, treatment with itraconazole, ketoconazole, or amphotericin B is usual. Some patients will respond to co-​trimoxazole. A skeletal scan should be made to detect occult foci of infection. Blastomycosis (See also Section 23.) Blastomycosis (North American blastomycosis) caused by Blastomyces dermatitidis is a systemic fungal infection in which skin and lung involvement are common features. The infective organism, B.  dermatitidis, has only been isolated from the environment on rare occasions. Positive sites have included soil and rotten timbers. The organism infects humans and domestic animals, particularly dogs. Epidemiology Blastomycosis was originally thought to be confined to North America, where it occurs sporadically throughout the south and east-​central area, and in areas of central Canada. ‘Epidemics’ of acute disease are rare, and where these occur a source of infection is rarely demonstrated. There is evidence that sources might include areas exposed to flooding. More recently, cases have been found in Africa. Again, these are widely scattered from the north coast to the southern parts of the continent, and are rare in all areas. Patients with the disease have also been reported from the Middle East and central Europe. Clinical features The clinical forms of blastomycosis differ from histoplasmosis in sev- eral important aspects. The existence of an asymptomatic form has not been proved conclusively, because there is no reliable skin test. Acute infections or infections in groups are rare, and the features are often similar to histoplasmosis (acute pulmonary). However, specific serological tests might be negative in 30–​50% of cases. The demon- stration of the organisms in sputum and positive cultures are more reliable diagnostic criteria. Although some cases undoubtedly re- solve without sequelae, some physicians advise chemotherapy, with a short course of amphotericin B in acute cases of blastomycosis. Chronic pulmonary blastomycosis Chronic consolidation or cavitation of the upper or mid zones occur with chronic pulmonary infections. Fever, malaise, and cough with sputum are seen. Weight loss might be prominent. Culture is again the most reliable method of diagnosis. The mainstays of treatment are itraconazole or amphotericin B. Disseminated blastomycosis Although generalized infiltration in skin, lungs, and liver can occur over a short period, leading to rapid death, signs of chronic extrapulmonary dissemination are more usual. The skin is an area that is frequently involved (chronic cutaneous blastomycosis). The face or forearms and hands are common sites for skin lesions. These are slow, spreading, verrucose plaques with central scarring. The initial lesion is often a dermal nodule. Many such cases have underlying pulmonary consolidation, or cavities. The diagnosis is established by biopsy and culture. Bone deposits in the form of lytic lesions, and involvement of the genitourinary tract, particularly the epididymis, are also seen in chronic disseminated blastomycosis. Unlike tuberculosis, the kidneys are usually spared. In slowly progressive forms of blastomycosis, itraconazole (200–​400 mg daily) has proved to be very effective. Alternatively, amphotericin B can be given intravenously and is indicated where there is rapidly progressive disease. Coccidioidomycosis See Chapter 8.7.3. Paracoccidioidomycosis See Chapter 8.7.4. Systemic sporotrichosis In addition to causing cutaneous disease, Sporothrix schenckii may be responsible for a systemic mycosis. The infection is rare and has been mainly reported from the United States of America. Involvement might be confined to a single site such as a lung or a joint, or it might be multifocal. Cavitation in the lung associated with weight loss and pyrexia is probably the most common variety of systemic sporotrichosis. Unlike cutaneous forms of the disease, systemic sporotrichosis responds poorly to potassium iodide, and amphotericin B is the treatment of choice. Rare systemic infections These include pulmonary invasion by Geotrichum candidum (geotrichosis) and adiaspiromycosis, a respiratory infection caused by Emmonsia crescens or E. parva. Isolated examples of human disease caused by fungi are consistently reported and almost always occur in the immunosuppressed host. In these patients many fungi that are nor- mally saprophytes in the environment invade and cause disease. Systemic mycoses caused by opportunistic fungi The opportunistic mycoses are a worldwide problem, although fortunately rare in most countries. In recent years they have been recognized more frequently with the increase in transplant- ations of organs such as heart or bone marrow and in the more effective but immunocompromising regimes of cancer chemo- therapy. Opportunistic invasion by organisms such as candida or mucoromycetes (rhizopus, absidia) may also occur in cases of mal- nutrition. One of the recent trends in the management of the pa- tients with neutropenia has been the emergence of new pathogens such as non-​albicans species of Candida or other organisms such as fusarium, trichosporon, or scedosporium species. The opportunists present particular problems in diagnosis and management. Because many of the organisms are normally sapro- phytic, it has to be positively established that they have assumed an invasive role. Mere isolation might not provide sufficient evidence and in some instances low titres of antibody can be present even in normal hosts. The significance of various laboratory tests in these

8.7.1  Fungal infections 1353 infections is shown in Table 8.7.1.1. Treatment is also difficult and it is important in most cases to attempt to reverse the process that led to the establishment of the infection. Systemic candidiasis Aetiology In addition to their role in superficial infections, candida yeasts can also cause invasive systemic disease. The clinical forms described range from bloodstream isolation or candidaemia to disseminated invasive disease, sometimes with involvement of a single organ, site, or body cavity (deep focal candidiasis) as can occur in peritonitis or meningitis. Urinary tract infections can also be caused by candida species. The factors underlying systemic candida infections are shown in Box 8.7.1.3. All these factors are important in disrupting the balance by which candida is maintained as a saprophyte. Intravenous or central venous pressure lines can serve as a portal of entry or as a nidus for circulating yeasts in a candidaemia. Antibiotic therapy might upset the balance by inhibiting a potentially competitive bacterial flora. Candida albicans is the most common species involved but other species might be isolated, particularly in cases of endocarditis (e.g. C. parapsilosis). C. tropicalis has been implicated in infections of pa- tients with neutropenia. These non-​albicans Candida species are now more frequent causes of systemic infection and are important to recognize as their antifungal susceptibility can differ from that of C.  albicans. Portals of entry include the gastrointestinal tract (common), skin, and urinary tract (rare). However, superficial candidosis or saprophytic colonization of mouth, skin, or airways can also occur in compromised patients and does not necessarily indicate systemic invasion. Epidemiology Systemic infections caused by candida species are worldwide in distribution. However, they are particularly associated with sev- eral predisposing factors such as neutropenia, antibiotic usage, indwelling lines, and abdominal surgery. Clinical features Candidaemia The isolation of candida in blood culture may be linked to any of the factors listed in Box 8.7.1.3. Common predisposing features are the presence of intravenous lines, previous surgery (mainly gastrointes- tinal), antibiotic therapy, hepatic failure, or neutropenia. Patients develop a swinging fever and feel generally unwell. Clinical shock might occur. Some such cases resolve following removal of predisposing fac- tors, particularly the intravenous lines. Generally, however, all such patients receive treatment and a careful investigation should be made to identify the presence of established invasive disease. Other sites should be searched for evidence of infection (e.g. urine by culture or the presence of white cells). Signs of muscle invasion (tenderness) or metastatic skin nodules should be excluded (Fig. 8.7.1.17). Other signs of invasion include the development of new cardiac murmurs or of soft, white, retinal plaques caused by candida. Persistently posi- tive blood cultures or serum candida antigen levels or high antibody titres might also indicate possible deep invasion. Disseminated candidiasis Although multiorgan invasive candidiasis can follow candidaemia, at least 50% of disseminated infections develop in patients without initially positive blood cultures. The features of some forms of inva- sive candidiasis are listed earlier (under ‘Candidaemia’). Although candida might be isolated from the sputum in these patients, there is rarely objective evidence of lung invasion. Moreover, there is no radiological appearance that is diagnostic of pulmonary candidiasis and, indeed, chest radiographs might even appear normal. General localizing signs are a late feature of disseminated candidiasis. Laboratory diagnosis of disseminated candidiasis The diagnosis can be made by culture or PCR, and repeated attempts to isolate should be made where cultures are initially negative. Numerous techniques have been used to detect antibody or antigen in disseminated candidiasis. However, in many patients, particularly those with neutropenia, it might not be possible to confirm the diag- nosis using laboratory tests and treatment is often initiated on the basis of clinical suspicion (empirical therapy) as the risk of delaying antifungal therapy is great. By themselves, positive cultures, particularly from sputum, or the presence of antibodies do not necessarily prove the existence of deep-​seated candidiasis. A positive isolation might simply indicate the presence of colonization and normal individuals can have low Fig. 8.7.1.17  Candidiasis disseminated to skin (methenamine silver, × 516). Box 8.7.1.3  Predisposing factors in deep candida infections • Local defects, foreign bodies (e.g. prosthetic heart valves, intravenous lines) • Defects of immunity (primarily T cell or phagocytosis) (e.g. cytotoxic therapy or systemic lupus erythematosus) • Drug therapy (e.g. antibiotics) • Carcinoma or leukaemia • Endocrine disease (e.g. diabetes mellitus in urinary tract candidiasis) • Physiological changes (e.g. infancy, old age, and pregnancy) (urinary tract) • Miscellaneous disorders, for example: —​ Malnutrition —​ Surgery such as gastrointestinal resections —​ Drug addiction

section 8  Infectious diseases 1354 titres of antibody to candida. If there is a readily accessible lesion from which to take a biopsy, such as a skin nodule or even a pul- monary infiltrate, this might provide the best evidence of invasion, although such procedures carry their own risk (Fig. 8.7.1.16). Treatment of disseminated candidiasis Untreated disseminated candidiasis is normally progressive and fatal. The signs must be separated from, for instance, bacterial septicaemia, which might coexist with the candida infection. The treatment of invasive candidiasis is intravenous amphotericin B or caspofungin or intravenous or oral fluconazole given until there is a clinical and mycological response. This might take between 2 and 20 weeks depending on the site of infection and the underlying state of the patient. Fluconazole is usually used in infections where the pa- tient is not neutropenic. Lipid-​associated forms of amphotericin B are also useful and carry a lower risk of renal impairment. An alternative approach is to add flucytosine in doses of 150–​200 mg/​kg body weight daily to amphotericin B in serious infections or where cure might be hampered by poor penetration of amphotericin B, such as in the eye. A biologic, Mycograb, which is an antibody against candida heat shock protein 70 has been shown to improve treatment responses in candidaemia when used in combination with amphotericin B. Deep focal candidiasis Candida infections in the peritoneum or meninges most often follow direct implantation after dialysis or surgery. Alternatively, secondary invasion from the middle ear or a perforated bowel is also possible. The signs and symptoms are similar to bacterial men- ingitis or peritonitis, but candida is isolated. Sometimes these in- fections clear spontaneously, but normally treatment is instituted with fluconazole, which penetrates areas such as peritoneum, or amphotericin B. Candida endocarditis Invasion of heart valves, mainly the mitral or aortic valves, most commonly follows homograft replacement, but it can occur also in patients with neutropenia or drug addicts. The symptoms are similar to bacterial endocarditis. However, candida vegetations might reach considerable size. Embolic phenomena may involve obstruction of large vessels including the femoral artery or large cerebral vessels. The detection of large vegetations using an echocardiography, par- ticularly in cases with negative blood cultures, should raise the pos- sibility of fungal endocarditis. Blood cultures are usually positive at some stage in the illness but repeated sampling might be necessary. High antibody titres are usually seen in such cases and serological tests are therefore of considerable value. Untreated candida endocarditis is uniformly fatal. There is also a high mortality associated with cases in which early surgical interven- tion is precipitated by impending heart failure. Normally, treatment consists of amphotericin B given intravenously and, where possible, valve replacement. There is no evidence to suggest that the addition of flucytosine to the regimen increases the effectiveness of treatment. However, the relapse rate is high and combination therapy might, therefore, be a reasonable approach on theoretical grounds. Urinary tract candidiasis Candida species might be isolated from the urine, particularly in conditions associated with urinary stasis such as neurogenic bladder or where there is an indwelling catheter. Type 2 diabetes is another predisposing factor. There is no value in using the presence of pyuria or quantitative yeast-​colony counts to assess the significance of in- fection. Treatment is normally given where there are symptoms such as dysuria or frequency or where there is a potential risk of invasion such as in immunosuppressed patients. Fluconazole is very useful in these patients as urinary levels are above inhibitory concentrations. Aspergillosis (See also Chapter 8.2.4 and Section 18.) Aspergillosis is the name given to diseases associated with spe- cies of mould fungi of the genus Aspergillus. As such, it comprises a series of clinically distinct infections:  aggressive pulmonary infec- tions with angio-​invasion and the potential for widespread systemic haematogenous spread (invasive pulmonary aspergillosis); slow but progressive paranasal sinus infection mainly seen in the tropics (paranasal aspergillus granuloma); and colonization of a pre-​existing space or cavity (aspergilloma) which can give rise to medical problems including severe haemorrhage. They are also associated with both superficial and subcutaneous fungal infections. Aspergillus species cause several different allergic disorders including asthma and allergic bronchopulmonary aspergillosis (Chapter 18.14.2). Box 8.7.1.4 indi- cates the range of diseases associated with aspergillus. Aspergillus species are ubiquitous and have established them- selves in every conceivable terrain and environment. As they propagate through the production of large number of airborne spores, exposure is difficult to avoid. Production of spores is also determined by local and environmental conditions. For example, construction or destruction of buildings and turnover of soil have been associated with focal outbreaks of infection in predisposed and immunosuppressed individuals. Susceptibility to aspergillus in- fections is dependent, to a large extent, on defective immunity or structural abnormalities, and therefore the major diseases caused Box 8.7.1.4  Diseases caused by aspergillus species Superficial infections • Onychomycosis • Otitis externa • Keratomycosis Subcutaneous infections • Mycetoma • Systemic infections • Localized invasive aspergillosis: —​ Aspergilloma, chronic aspergillosis of the paranasal sinuses, chronic pulmonary aspergillosis, paranasal aspergillus granuloma • Invasive aspergillosis with potential for systemic spread: —​ Invasive (pulmonary) aspergillosis (common sites for dissemination are brain, liver, skin) —​ Aspergillus endocarditis Allergic disease • Asthma, allergic rhinitis, • Extrinsic hypersensitivity pneumonitis (A. clavatus) • Allergic bronchopulmonary aspergillosis • Allergic aspergillus sinusitis Toxicosis • Mycotoxin-​producing aspergilli (e.g. A. flavus—​aflatoxins)

8.7.1  Fungal infections 1355 by these organisms are usually seen in immunosuppressed individ- uals, including, in particular, neutropenic patients or people with anatomocal abnormalities such as lung cavities. The incidence of in- fection can reach high levels in certain populations such as patients following bone marrow transplantation (Chapter 8.2.4). Aspergillus can produce several potent metabolic byproducts or myxotoxins, such as the aflatoxins produced by A. flavus which, if present in contaminated food, can induce liver necrosis. The most common human pathogen among the aspergillus spe- cies is A. fumigatus, followed by A. flavus which causes infections more commonly in warmer climates. A. niger causes aspergilloma rather than invasive disease but A. nidulans rarely causes mycetoma. A. terreus is sometimes found as a cause of onychomycosis. Hence aspergillus infections might present to a wide range of different specialities and, in the severely immunocompromised patient, dis- semination of aspergillus through the blood stream can result in in- fection of almost any organ. Cryptococcosis See Chapter 8.7.2. Invasive mucuromycosis (mucormycosis, zygomycosis, phycomycosis) Aetiology Invasive disease caused by mucor-​like (mucoromycete) fungi is rare. In the compromised host it may lead to paranasal destruction, nec- rotic lung or skin lesions, and disseminated disease. The causative organisms commonly belong to three genera: Absidia, Rhizopus, and Rhizomucor. More rarely other or- ganisms such as Cunninghamella or Saksenaea have been implicated. Most of the agents are associated with decaying vegetable matter and are common airborne moulds. The route of infection is highly vari- able: they might invade via the lungs, paranasal sinuses, gastrointes- tinal tract, or damaged skin. The predisposing illness might, in some way, determine the site of clinical invasion. Underlying factors in- clude diabetic ketoacidosis (rhinocerebral involvement), leukaemia and immunosuppressive therapy (lung and disseminated infection), malnutrition (gastrointestinal infection), and burns and trauma or wounds (cutaneous invasion). These patterns are not always strictly followed. Epidemiology Mucoromycosis is rare but has a worldwide distribution. Its inva- sive nature, particularly the tendency to involve blood vessels and its selection of compromised hosts, distinguishes this form of in- fection from subcutaneous mucuromycosis, which is also caused by mucuromycete species. Clinical features The most characteristic features of this type of infection are the ex- tensive necrosis and infarction that follow blood vessel invasion leading to thrombosis. A similar type of invasion can occur with in- vasive aspergillosis, but is usually less prominent. Mucuromycosis follows several different patterns. The infection might initially localize in one of several sites. The most common is in the paranasal sinuses and this is most often seen in diabetic patients with ketoacidosis. The patient presents with fever and unilateral facial pain. Subsequently, there might be facial swelling with nasal obstruction and proptosis. There can be invasion into the orbit leading to blindness, into the brain, and into the palate. Palatal ulceration should be searched for. Widespread dissemin- ation with infarction of major organs or limbs might occur subse- quently. A similar pattern of invasion of surgical wounds or burns might occur and has on occasions been associated with contam- ination of dressing packs. Infections are initially localized causing extensive necrosis around the original wound. Gastrointestinal in- vasion might be heralded by perforation of viscera, and diarrhoea or haemorrhage. Alternatively, a patient can present with established pulmonary or widespread dissemination. Such patients are usually leukaemic, or are severely immunosuppressed. Neutropenia is often seen. Once infection has spread beyond the original site, mucuromycosis is almost invariably fatal with or without treatment. Laboratory diagnosis The diagnosis is suggested by the combination of infection and ex- tensive infarction, particularly if it occurs in any of the sites men- tioned. The organisms can be difficult to culture, even from biopsy, and histology is often the quickest way of establishing the diagnosis. Serology is frequently negative. Treatment Treatment should be initiated as soon as possible and extensive sur- gical debridement combined with intravenous amphotericin B in maximum daily dosage offers the best chance of success. Local instil- lations of amphotericin B might also be used where appropriate (such as nasal sinuses). Some physicians also recommend anticoagulation with heparin to forestall thrombosis. Despite therapy, the mortality remains high. Liposomal amphotericin B also has been used with some success is cases of mucormycosis. Rhinosporidiosis Rhinosporidiosis is an infection found in India, Sri Lanka, parts of East Africa, and South America. It is characterized by polypoid growth from the nose or conjunctiva. The causative organism can be demonstrated in tissue and consists of aggregates of large sporangia containing spores in various phases of development. However, they have never been successfully cultured and they appear to be related genetically most closely to aquatic protista, members of the Mezomycetozoa, and not fungi. The treatment is surgical excision. Otomycosis and oculomycosis External otitis is often multifactorial, but in some cases dense fungal colonization can contribute to the picture. In severe cases, the ex- ternal ear might be plugged by a dense mat of mycelium. Aspergillus species are the most common organisms cultured, particularly A. niger, but candida, penicillium, and mucor might all contribute. Intensive ear toilet can eradicate the infection without recourse to antifungal agents. Infections of the eye, particularly the cornea, caused by fungi (oculomycosis) are rare. They often follow penetrating injuries to the globe or contamination of lacerations. An opacity develops within the cornea with associated pain and chemosis. An exudate is usually present in the aqueous humour. Prompt treatment with intensive topical instillation of drugs containing an antifungal drug such as

section 8  Infectious diseases 1356 miconazole or econazole is necessary every 2 to 4 h. Perforation of the eye can occur in advanced cases. Approaches to management of fungal infections Antifungal agents can be considered in four main groups: the poly- enes, azoles, morpholines, and allylamines, and an assortment of unrelated drugs with specific activity. Polyenes The polyene antifungals are macrolide substances derived origin- ally from species of Streptomyces. They include amphotericin B, natamycin, and nystatin. More recent additions to this group are partricin and mepartricin. Amphotericin B is the only one widely used as a parenterally administered drug. Nystatin and natamycin are purely topical. Amphotericin B is metabolized in the liver with low penetration of body cavities, cerebrospinal fluid, and urine. The polyenes have broad activity against a wide range of fungi. The mode of action of the polyenes appears to involve inhibition of sterol syn- thesis in the fungal cell membrane. The combination of an amphotericin B with a lipid, for in- stance a liposome, has been proposed as a means of reducing the nephrotoxicity of this drug. Three commercial lipid amphotericins are available:  AmBisome (a true liposome), amphotericin B lipid complex—​ABLC or Abelcet (a ribbon-​like lipid binding amphotericin B), and amphotericin B colloidal dispersion (ABCD) (a dispersion of lipid discs). Azoles The imidazoles are synthetic antifungal agents. They include miconazole, clotrimazole, econazole, isoconazole, ketoconazole, tioconazole, and bifonazole. The triazole series contains two po- tent oral agents, fluconazole and itraconazole. Voriconazole, posaconazole, and isavuconazole are newer additions. Most are used topically except for ketoconazole (oral), itraconazole (oral), voriconazole (oral and intravenous), posaconazole (oral and intravenous) and isavuconazole (oral and intravenous. These are metabolized in the liver and, like amphotericin B, affect fungal cell-​ membrane synthesis and penetrate cerebrospinal fluid and urine in low concentrations. The imidazoles have a broad spectrum of activity against many fungi, particularly those causing superficial infectiond. Fluconazole is less active against moulds and there are instances of both primary (Candida krusei, C. glabrata) and sec- ondary resistance to this compound. New triazoles, voriconazole, posaconazole and isavuconazole, are now available; voriconazole is an effective treatment for invasive aspergillosis. The allylamines such as terbinafine are primarily active against superficial fungi, but in vitro appear to have fungicidal activity at low concentrations. Other antifungals in this category include flucytosine, which is a synthetic pyrimidine analogue. Given either intravenously or orally it is mainly useful for chromomycosis and certain yeast infections. Drug resistance is a major problem with flucytosine, particularly with cryptococcus. The drug shows several modes of action including disruption of RNA transcription following up- take by the cell. Caspofungin, an echinocandin, is an effective treatment for deep candida, including fluconazole-​resistant, in- fections. Newer echinocandins are anidulafungin and micafungin. Griseofulvin is derived from a species of penicillium. It can be given orally and is only useful against dermatophytes. It is best absorbed when given with a meal and selectively accumulates in stratum corneum in concentrations approximately 10 times greater than serum levels. Griseofulvin acts by inhibiting intracel- lular microtubule formation. Management of superficial infections Specific details of therapy are included under the separate diseases. Benzoic acid compound (Whitfield’s ointment), which contains 2% salicylic acid and 2% benzoic acid, acts as a keratolytic agent by causing exfoliation of the superficial layers of the stratum corneum. Other topical agents with only weak antifungal activity include gentian violet (candidiasis or dermatophytosis); Castellani’s paint, which contains magenta and resorcinol (candidiasis or dermato- phytosis); and brilliant green (dermatophytosis). Selenium sul- phide (2%) remains a highly effective method of treating pityriasis versicolor by application once daily for 2 weeks. The more specific antifungals such as the polyenes, amphotericin B, nystatin, and natamycin (candidiasis) or the imidazoles (can- didiasis, dermatophytosis, and pityriasis versicolor) are highly effective and probably quicker than the keratolytics or dyes, al- though more expensive. Local irritation can be a problem, par- ticularly with Whitfield’s ointment, which is usually given as a half-​strength preparation. Allergic contact dermatitis is rare but has been recorded from some imidazoles (miconazole, clotrimazole, tioconazole) and tolnaftate. Topical terbinafine is highly active in tinea pedis with cures being effected with less than 1 week of therapy. Terbinafine or itraconazole are more effective in many forms of dermatophytosis requiring oral therapy than griseofulvin. In onychomycosis they are preferred. Terbinafine has occasional side effects, mainly related to gastrointestinal intolerance, although it might also cause transient loss of taste. It is given in daily doses of 250 mg. Itraconazole is usually given in ‘pulses’ (e.g. 200 mg twice daily for 1 week monthly). Itraconazole likewise can cause gastro- intestinal discomfort and nausea. Both drugs rarely cause hepatic injury, with a frequency of less than 1 in 70 000 to 1 in 120 000. Fluconazole is also effective in dermatophytosis and is given in weekly doses of 150–​300 mg. Griseofulvin is still the principal treat- ment for tinea capitis (10–​20 mg/​kg per day). In onychomycosis caused by dermatophytes both terbinafine and itraconazole lead to remission of toenail infections in only 3 months. Terbinafine is used on a daily basis, whereas itraconazole is given in a pulsed regimen, 200 mg twice daily for 1 week every month for 3–​4 months. There is one study which shows better responses with terbinafine for toenail disease. Amorolfine, a morpholine drug, is used in the topical treatment of nail disease where there is less than complete involvement of the nails. It can be given together with other drugs, such as terbinafine. Management of deep mycoses Very few drugs are effective in systemic fungal infections, and those that are used should always be accompanied by supportive measures and, if possible, an attempt to eliminate any predisposing conditions. For instance, if their condition permits, patients who have devel- oped a candidaemia while a central venous line is in place should be managed by removal of the line. However, fluconazole is also usually

8.7.1  Fungal infections 1357 given as well. In the patient with neutropenia, a positive blood cul- ture would be regarded as evidence of dissemination and antifungal therapy would be required. Amphotericin B is given intravenously in a 5% dextrose infusion not containing additional drugs, if possible. A test dose of 1–​5 mg is given over 2 h and this is followed by gradually increasing doses over the next 3–​9 days to the normal maximum of 0.6–​1.0 mg/​kg body weight daily depending on the infection. In some cases, this slow approach might help the patient to tolerate the drug better, or may define the dose at which side effects such as pyrexia start. In severely ill patients, half of the full dose can be given 4 h after a test dose of 5 mg, usually under hydrocortisone cover. The full dose is given 24 h later. Side effects include thrombophlebitis, nausea, hypotension, and pyrexia. Renal clearance might fall in the initial period but this usually returns to normal after a tem- porary halt in therapy. More permanent renal tubular damage can follow a total dose of 4 g or more. Amphotericin B does not pene- trate urine, cerebrospinal fluid, or peritoneal fluid in significant concentrations. Local instillations (such as the peritoneum) can be used, but can be highly irritant. Amphotericin B is normally given until clinical or mycological cure is induced. This is often difficult to judge accurately and in many of the mycoses caused by the systemic pathogens a course of at least 2 g is often used on an empirical basis. In the opportunistic infections, lower total doses are probably effective, and the length of treatment should depend on the clinician’s judgement. This approach is not necessary with the lipid-​associated amphotericin B formulations, which can be given without the slow build-​up. The initial dose is usually 1 mg/​kg, but standard daily doses of 3 mg/​kg are common. Patients are less likely to develop renal impairment although it can occur. There have been a few clinical trials comparing these formulations with amphotericin B and these show equal efficacy with less toxicity; however, these for- mulations are expensive. The main lipid-​associated formulations are given earlier. The azole drugs are also used in systemic mycoses. Fluconazole is given in systemic candidiasis, urinary tract infections, and as a long-​ term suppressive, in addition to primary therapy, in cryptococcosis in patients with AIDS. Side effects are uncommon, although it can cause nausea and vomiting. Fluconazole can be given orally or intra- venously. It penetrates urine in effective concentrations. Its daily dosage varies from 100 to 200 mg for oropharyngeal infections to 600 to 800 mg for disseminated candidiasis. It is highly active in can- dida infections. It can also be used in some endemic mycoses such as histoplasmosis. Resistance to fluconazole has mainly been re- corded with oropharyngeal candidiasis, principally in HIV-​positive patients, although it can occur with other candida infections; for example, C. krusei and C. glabrata are often primarily resistant to this drug. Itraconazole has been evaluated in a variety of systemic mycoses from aspergillosis to cryptococcosis. Its active range includes histo- plasmosis, sporotrichosis, chromoblastomycosis, blastomycosis, coccidioidomycosis, and paracoccidioidomycosis. Itraconazole is used as an oral preparation, but an intravenous formulation is now available. Oral absorption is often defective in individuals with AIDS and patients after bone marrow transplantation and in these groups the mean daily dosage is doubled (200 mg). An itraconazole suspension is also available for treatment of oral infections. Voriconazole is now the treatment of choice for many cases of invasive aspergillosis and for some other systemic mycosis. Long-​term administration might lead to photosensitivity and increased incidence of skin cancers. The indications for posaconazole include fluconazole unresponsive infections but it also appears be effective in some mould infections including some cases of fusarium infection as well as prophylaxis in neu- tropenic patients. Flucytosine (5-​fluorocytosine) is an effective oral and intra- venous antifungal agent that is primarily active against yeasts such as candida and cryptococcus. It enters urine, cerebrospinal fluid, and peritoneal fluid. Its excretion is reduced in renal failure and the daily dose should be reduced accordingly and blood levels moni- tored. The main disadvantage of flucytosine is the development of either primary or secondary drug resistance in a significant number of isolates, and when given in toxic doses it may cause bone marrow depression. The serum level should not be allowed to rise above 100–​120 µg/​ml. Combination amphotericin B and flucytosine therapy might offer an alternative but effective method of treatment. Theoretically, as the drugs synergize, the dose of amphotericin B may be re- duced. In cryptococcal meningitis, combination therapy using a dose of 0.3–​0.6 mg/​kg body weight of amphotericin B with the normal dose of flucytosine is more effective at sterilizing the cere- brospinal fluid and preventing relapse. In other forms of systemic infection such as candidiasis there is little evidence that it is more effective than amphotericin B alone, although this might be the case. Combinations of other drugs have not been critically evalu- ated in vivo. Caspofungin is used in fluconazole-​resistant deep candidiasis. FURTHER READING General Dismukes WE, Pappas PG, Sobel J (2006). Clinical mycology. Oxford University Press, New York, NY. Kibbler CC, MacKenzie DWR, Odds FC (1996). Principles and prac­ tice of clinical mycology. John Wiley & Sons, Chichester. Merz W, Hay RJ (eds) (2005). Mycology. Topley and Wilson’s micro­ biology and microbial infections, 10th edition, Vol. 4. Arnold, London. Midgley G, Clayton YM, Hay RJ (1997). Diagnosis in colour: medical mycology. Mosby-​Wolfe, London. Dermatophytosis Aly R (1994). Ecology and epidemiology of dermatophyte infec- tions. J Am Acad Dermatol, 31, S21–​5. Hay RJ (2005). Fungal infections. In: Bos JD (ed) Skin immune system (SIS), pp. 593–​604. CRC Press, Boca Raton, FL. Hay RJ, et  al. (1996). Tinea capitis in south-​east London—​a new pattern of infection with public health implications. Br J Dermatol, 135, 955–​8. Munoz-​Perez MA, et al. (1998). Dermatological findings correlated with CD4 lymphocyte counts in a prospective 3-​year study of 1161 patients with human immunodeficiency virus disease predom- inantly acquired through intravenous drug abuse. Br J Dermatol, 139, 33–​9.

section 8  Infectious diseases 1358 Scytalidium Hay RJ, Moore MK (1984). Clinical features of superficial fungal infec- tions caused by Hendersonula toruloidea and Scytalidium hyalinum. Br J Dermatol, 110, 677–​83. Malassezia Ashbee, HR (2006). Recent developments in the immunology and biology of Malassezia species. FEMS Immunol Med Microbiol, 47, 14–​23. Candidiasis Bodey GP (ed) (1993). Candidiasis: pathogenesis, diagnosis and treat­ ment. Raven Press, New York, NY. Greenspan D, et al. (2000). Oral mucosal lesions and HIV viral load in the Womens Interagency HIV study (WIHS). J Acquir Immune Defic Syndr, 25, 89–​104. Pappas PG, et al. (2009). Clinical practice guidelines for the manage- ment of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis, 48, 503–​35. Sobel JD (1992). Pathogenesis and treatment of recurrent vulvovaginal candidiasis. Clin Infect Dis, 14, S148–​53. Mycetoma Fahal AH (2004). Mycetoma: a thorn in the flesh. Trans Roy Soc Trop Med Hyg, 98, 3–​11. Hay RJ (2005). Eumycetomas. In: Merz WG, Hay RJ (eds) Mycology. Topley and Wilson’s microbiology and microbial infections, 10th edi- tion, Vol. 5, pp. 385–​95. Arnold, London. Chromoblastomycosis Banks IS, et al. (1985). Chromomycosis in Zaire. Int J Dermatol, 24, 302–​7. Bayles MAH (1989). Chromomycosis. In: Hay RJ (ed) Tropical fungal infections, Baillière’s clinical tropical medicine and communicable diseases, Vol. 4, pp. 45–​70. Baillière Tindall, London. Esterre P, et al. (1997). Natural history of chromo-​blastomycosis in Madagascar and the Indian Ocean. Bull Soc Pathol Exot, 90, 312–​17. Minotto R, et al. (2001). Chromoblastomycosis: a review of 100 cases in the state of Rio Grande do Sul, Brazil. J Am Acad Dermatol, 44, 585–​92. Silva JP, et al. (1999). Chromoblastomycosis: a retrospective study of 325 cases on Amazonic Region (Brazil). Mycopathologia, 143, 171–​5. Sporotrichosis Carvalho MT, et al. (2002). Disseminated cutaneous sporotrichosis in a patient with AIDS: report of a case. Rev Soc Bras Med Trop, 35, 655–​9. da Rosa AC, et al. (2005). Epidemiology of sporotrichosis: a study of 304 cases in Brazil. J Am Acad Dermatol, 52, 451–​9. Kauffman CA (1999). Sporotrichosis. Clin Infect Dis, 29, 231–​236. Systemic mycoses de Pauw BE, Meunier F (1999). The challenge of invasive fungal infec- tion. Chemotherapy, 45, Suppl 1, 1–​14. Histoplasmosis Ashford DA, et al. (1999). Outbreak of histoplasmosis among cavers attending the National Speleological Society Annual Convention, Texas, 1994. Am J Trop Med Hyg, 60, 899–​903. Barton EN, et al. (1988). Cutaneous histoplasmosis in the acquired immunodeficiency syndrome: a report of three cases from Trinidad. Trop Geogr Med, 40, 153–​7. Khalil MA, Hassan AW, Gugnani HC (1998). African histoplasmosis:
report of four cases from north-​eastern Nigeria. Mycoses, 41, 293–​5. Wheat LJ, Kaufman CA (2003). Histoplasmosis. Infect Dis Clin North Am, 17, 1–​19. Wheat J, et al. (2000). Practice guidelines for the management of pa- tients with histoplasmosis. Infectious Diseases Society of America. Clin Infect Dis, 30, 688–​95. Blastomycosis Chapman SW, et al. (2000). Practice guidelines for the management of patients with blastomycosis. Infectious Diseases Society of America. Clin Infect Dis, 30, 679–​83. Emerson PA, Higgins E, Branfoot A (1984). North American blasto- mycosis in Africans. Br J Dis Chest, 78, 286–​91. Lemos LB, et al. (2000). Blastomycosis: organ involvement and etio- logic diagnosis. A  review of 123 patients from Mississippi. Ann Diagn Pathol, 4, 391–​406. Opportunistic systemic mycoses De Pauw BE (2006). Increasing fungal infections in the intensive care unit. Surg Infect, 7 Suppl 2, S93–​6. Magill SS, et al. (2006). The association between anatomic site of can- dida colonization, invasive candidiasis, and mortality in critically ill surgical patients. Diagn Microbiol Infect Dis, 55, 293–​301. Wingard JR (1999). Fungal infections after bone marrow transplant. Biol Blood Marrow Transplant, 5, 55–​68. Aspergillosis Marr KA (2008). Fungal infections in hematopoietic stem cell trans- plant recipients. Med Mycol, 46, 293–​302. Pini G, et al. (2008). Invasive pulmonary aspergillosis in neutropenic patients and the influence of hospital renovation. Mycoses, 51, 117–​22. Walsh TJ, et  al. (2008). Treatment of aspergillosis:  clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis, 46, 327–​60. Mucormycosis Nenoff P, et  al. (1998). Rhinocerebral zygomycosis following bone marrow transplantation in chronic myelogenous leukaemia: report of a case and review of the literature. Mycoses, 41, 365–​72. Rhinosporidiosis Fredericks DN, et  al. (2000). Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites. Emerg Infect Dis, 6, 272–​6. Therapy Bassetti M, et  al. (2006). Candida infections in the intensive care unit: epidemiology, risk factors and therapeutic strategies. Expert Rev Anti-​Infect Ther, 4, 875–​85. Bennett JE (2006). Echinocandins for candidemia in adults without neutropenia. N Engl J Med, 355, 1154–​9. Elweski B (ed) (1996). Cutaneous fungal infections. Marcel Dekker, New York, NY. Gupta AK, et al. (2006). Onychomycosis therapies: strategies to im- prove efficacy. Dermatol Clin, 24, 381–​6. Maertens J, et al. (2009). Antifungal prophylaxis in leukemia patients; 2009 update of the ECIL-​1 and 2 guidelines. https://​www.leukemia-​ net.org/​content/​treat_​research/​supportive_​care/​standards_​sop_​ and_​recommendations/​e4702/​infoboxContent5849/​ECIL3Antifun galprophylaxisupdate2009.pdf Vanden Bossche H, et al. (1998). Antifungal drug resistance in patho- genic fungi. Med Mycol, 36 Suppl 1, 119–​28.

8.7.2 Cryptococcosis 1359

8.7.2 Cryptococcosis 1359

8.7.2  Cryptococcosis 1359 8.7.2  Cryptococcosis William G. Powderly, J. William Campbell,
and Larry J. Shapiro ESSENTIALS Cryptococcus neoformans, which is found worldwide as a soil organism and thought to be transmitted by inhalation, most often causes disease in patients with abnormal cell-​mediated immunity, notably patients with HIV infection and solid-​organ transplant recipients, but the in- fection also occurs rarely in apparently immunocompetent people in restricted geographical areas, especially involving C. neoformans var. gattii. The most common presentation is with subacute meningo- encephalitis, but other manifestations (e.g. isolated pulmonary disease or disseminated infection, are well described). Diagnosis is usually by culture or serology. Untreated cryptococcal meningitis is fatal: aside from supportive care (including monitoring for raised intracranial pres- sure), the therapy of choice is an initial period (at least two weeks) of amphotericin B (ideally with flucytosine), followed by at least 3 months of fluconazole. Most immunocompromised patients subsequently re- quire maintenance suppressive therapy, usually with fluconazole. Aetiology and epidemiology Infection with the fungus Cryptococcus neoformans occurs mainly in patients with impaired cell-​mediated immunity. It is the most common systemic fungal infection in patients infected with HIV and is also seen as a complication of solid-​organ transplantation, lymphoma, and corticosteroid therapy. C.  neoformans is found worldwide as a soil organism; it is an encapsulated yeast measuring from 4 to 6 μm with a surrounding polysaccharide capsule ranging in size from 1 to over 30 μm. Two varieties exist, distinguishable by serology: C. neoformans var. neoformans (serotypes A and D) and C.  neoformans var. gattii (serotypes B and C). Virtually all HIV-​ associated infection is caused by C.  neoformans var. neoformans. About 5% of HIV-​infected patients in the Western world develop disseminated cryptococcosis; the disease is much more prevalent in sub-​Saharan Africa and South-​East Asia, with estimates of 750 000–​ 1 million cases annually. C. neoformans var. gattii infection is more common in tropical and subtropical areas (Australia, New Guinea, and the Philippines) in apparently immunocompetent people. Cases of Cryptococcus gattii were described on Vancouver Island, British Columbia, Canada, in 1999. The Pacific Northwest of North America (British Columbia in Canada, and Washington and Oregon in the United States) now has one of the highest incidences of this infection worldwide, and cases of C. neoformans var. gattii infection have occurred throughout North America. It has only rarely been reported in HIV-​immunosuppressed patients. The exact mechanism of infection is unknown. It is assumed that transmission occurs via inhalation of the organism leading to colonization of the airways and subsequent respiratory infection. Throughout the world, the excreta of birds such as pigeons are the richest environmental source of C. neoformans var. neoformans. The ecological association of C. neoformans var. gattii is with river red and forest river gum trees (Eucalyptus camaldulensis and E. tereti­ cornis) and with mammals such as koalas. It has been suggested that infective basidiospores are released at flowering. In the case of C. neoformans var. neoformans, the absence of an intact cell-​mediated response results in ineffective clearance with subsequent dissemination. The polysaccharide capsule, composed mainly of glucuronoxylomannan, is thought to be its primary viru- lence factor. It is not clear whether cryptococcal infection in im- munocompromised patients represents acute primary infection or reactivation of previously dormant disease. Clinical features The most common presentation of cryptococcosis is a subacute meningitis or meningoencephalitis with fever, malaise, headache, and altered behaviour and level of consciousness. Symptoms are usually present for 2 to 4 weeks before diagnosis. Classic menin- geal symptoms and signs (such as neck stiffness or photophobia) (Fig. 8.7.2.1) occur in only about a quarter to a third of patients. Papilloedema and cranial nerve palsies (especially VI and VII) are common (Fig. 8.7.2.2). Patients might present with encephalopathic symptoms such as lethargy, altered mentation, personality changes, and memory loss. Analysis of the cerebrospinal fluid usually shows a mildly elevated serum protein, normal or slightly low glucose, and a lymphocytic pleocytosis. India ink staining of the cerebrospinal fluid will usually reveal the yeast. Cryptococcal antigen is almost in- variably detectable in the cerebrospinal fluid. The opening pressure in the cerebrospinal fluid is elevated in most patients. Infection with C. neoformans can involve sites other than the men- inges. Isolated pulmonary disease has been well described and usu- ally presents as a solitary nodule in the absence of other symptoms. Cryptococcal pneumonia also occurs. In immunocompromised Fig. 8.7.2.1  Neck stiffness in a Papua New Guinean patient with Cryptococcus neoformans var. gattii meningitis. Copyright D. A. Warrell.

section 8  Infectious diseases 1360 patients, especially those with AIDS, subsequent dissemination is common but presentations such as cough or dyspnoea, and abnormal chest radiographs can be the initial findings. Many patients have posi- tive blood cultures. Skin involvement is common; several types of skin lesion have been described (Fig. 8.7.2.3) but the most common form is that resembling molluscum contagiosum. Osteolytic bone le- sions and prostatic involvement have also been described. Infection with C. neoformans var. gattii is more likely to occur in older patients with other comorbid conditions. Infection with C. neoformans var. gattii appears to cause cryptococcomas in the lung and brain (often large, multifocal lesions) more commonly than C. neoformans var. neoformans, and patients with C. neofor­ mans var. gattii appear to have more aggressive retinal involvement with papilloedema and haemorrhagic papillitis in more than a half of patients, leading to blindness in one-​third of survivors. Diagnosis Cryptococcal disease can be diagnosed through culture, by visual- ization of the organism in microscopy or tissue, or by detection of cryptococcal polysaccharide antigen (CrAg) in blood or cerebro- spinal fluid. CrAg testing is highly sensitive and specific in the diag- nosis of infection with C. neoformans. Three methods currently exist for antigen detection:  latex agglutination, enzyme immunoassays, and lateral flow assay (a newly developed dipstick test that can be used as a point-​of-​care test). Cerebrospinal fluid CrAg is usually posi- tive in patients with cryptococcal meningoencephalitis. Serum CrAg is usually positive in both meningeal and non​meningeal infection and might be detectable before symptoms are evident. Such patients should be evaluated for possible meningeal involvement. Culture of C. neoformans from any site should also be regarded as significant and is an indication for further evaluation and initiation of therapy. Treatment Management of patients with cryptococcal infection depends on the extent of the disease and the immune status of the patient. The finding of a solitary pulmonary nodule in a normal host might not need treatment, provided patients have careful follow up. Fluconazole (200–​400 mg/​day) can be given for 3 to 6 months in most patients with localized pulmonary disease. Extrapulmonary disease is gener- ally managed in the same way as meningitis. In patients who are not known to be immunosuppressed, a search for underlying problems should be initiated. An HIV antibody test should be performed, as cryptococcal meningitis can be the initial AIDS-​defining event. Additionally, a CD4+ lymphocyte count should be considered, as cryptococcal infection has been described as one of the manifest- ations of so-​called ‘isolated CD4 T lymphocytopenia’. Untreated, cryptococcal meningitis is fatal. In patients with AIDS, the combination of amphotericin B and flucytosine (100 mg/​kg per day in three or four divided doses) given for 2 weeks followed by fluconazole (400 mg orally) for a further 8 weeks is associated with the best outcome to date in prospective trials, with a mortality of 10–​15% and a mycological response of approximately 70%. This regimen is also reasonable for treatment of meningitis in other circumstances. In the Western world, the liposomal formulation of amphotericin B is pre- ferred and dosed at 5 mg/​kg/​day. In resource-​poor settings, the more nephrotoxic deoxycholate formulation of amphotericin is usually the only available formulation; its dosage for cryptococcal meningitis in 0.7–​1.0 mg/​kg/​day. A recent study in Africa found that one week of amphotericin B with flucytosine was adequate as induction therapy. Clinical deterioration in patients with meningitis might be due to cerebral oedema, which can be diagnosed by a raised opening pres- sure of the cerebrospinal fluid. All patients with cryptococcal men- ingitis should have the opening pressure measured when a lumbar puncture is performed; if the opening pressure is high (>25 cmH2O), pressure should be reduced by repeated lumbar punctures, a lumbar drain, or a shunt. The use of adjunctive corticosteroids has been shown to be deleterious in the setting of HIV infection. Provided HIV infection and isolated CD4 lymphopenia have been excluded, immunocompetent patients with cryptococcal meningitis can be generally managed with a shorter (3–​4  months) course of Fig. 8.7.2.2  Right cranial VI (abducens) nerve paralysis in an African HIV-​seropositive patient with Cryptococcus neoformans var. neoformans meningitis. Copyright D. A. Warrell. Fig. 8.7.2.3  Cryptococcal cutaneous ulcer. Courtesy of Professor R. Hay.

8.7.3 Coccidioidomycosis 1361

8.7.3 Coccidioidomycosis 1361

8.7.3  Coccidioidomycosis 1361 treatment. An approach similar to that recommended for immuno- suppressed patients is still recommended, such as an initial (2–​4 weeks) of amphotericin B-​based induction therapy followed by 8–​10 weeks of fluconazole. More prolonged use (4–​6 weeks) of amphotericin B and flucytosine might be more rapidly curative but is also more toxic. Cryptococcal meningitis in AIDS requires lifelong suppressive therapy unless the immunosuppression is reversed with effective treatment of HIV infection. In that circumstance, treatment can be discontinued if the CD4+ lymphocyte count increases to over 200 cells/​mm3. In other immunocompromised patients, suppressive treatment for 6 to 12 months can be given. Effective antiretroviral therapy might also sufficiently improve the immune system such that there is an immunological response to the fungal infection. This might be associated with clinical deterioration and apparent relapse of symptoms; this immune reconstitution inflammatory syndrome (IRIS) should not prompt change in antifungal therapy and patients should receive anti-​inflammatory therapy, as needed. It has also been described in transplant patients whose immunosuppressive therapy is decreased during management of the cryptococcal infection. Timing of initiation of antiretroviral therapy in HIV-​infected pa- tients is a very important consideration. Recent randomized clinical trials suggest that early (within two weeks of diagnosis) initiation of antiretroviral therapy is associated with an increased mortality in patients with cryptococcal meningitis, possibly because of the devel- opment of immune reconstitution syndrome. This means that there is often a delay in starting antiretrovirals for a few weeks to avoid this complication. However, recent studies suggest that screening for CrAg, with preemptive fluconazole for those infected, before starting antiretrovirals reduces IRIS and mortality. Fluconazole, 200 mg daily, is the suppressive treatment of choice. Fluconazole, in dosages ranging from 400 mg weekly to 200 mg daily, and itraconazole, 100 mg twice daily, are very effective in preventing invasive cryptococcal infections, especially in HIV-​positive patients with CD4 counts less than 50–​100 cells/​mm3. However, because of the relative infrequency of invasive fungal infections, antifungal prophylaxis does not prolong life and is not routinely recommended where antiretroviral therapy is readily available. An approach using CrAg screening and pre-​emptive antifungal therapy in ART-​naive individuals with a CD4 count of less than 100 cells/​mm3 is currently being evaluated in high incidence settings. FURTHER READING Beardsley J, et al. (2016). Adjunctive dexamethasone in HIV-​associated cryptococcal meningitis. N Engl J Med, 374, 542–​54. Bicanic T, Harrison TS (2005). Cryptococcal meningitis. Br Med Bull, 72, 99–​118. Boulware D, et al. (2014). ART initiation within the first 2 weeks of cryptococcal meningitis is associated with higher mortality:  a multisite randomized trial. N Engl J Med, 370, 2487–​98. Datta K, et al. (2009). Spread of Cryptococcus gattii into Pacific north- west region of the United States. Emerg Infect Dis, 15, 1185–​91. Day JN, et  al. (2013). Combination antifungal therapy for
HIV-​associated cryptococcal meningitis. N Engl J Med, 368, 1291–​302. Ellis DH, Pfeiffer TJ (1990). Ecology, lifecycle, and infections propa- gule of Cryptococcus neoformans. Lancet, 36, 923–​5. Graybill JR, et  al. (2000). Diagnosis and management of increased intracranial pressure in patients with AIDS and cryptococcal men- ingitis. Clin Infect Dis, 30, 47–​54. Molloy SF, et al. (2018). Antifungal combinations for treatment of cryptococcal meningitis in Africa. N Engl J Med, 378, 1004–17. Perfect JR, et al. (2010). Clinical practice guidelines for the manage- ment of cryptococcal disease: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis, 50, 291–​322. Rajasingham R, et al. (2017). Global burden of disease of HIV- associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis, 17, 873–81. Shelbourne S, et al. (2005). The role of immune reconstitution inflamma- tory syndrome in AIDS-​related Cryptococcus neoformans disease in the era of highly active antiretroviral therapy. Clin Infect Dis, 40, 1049–​52. Speed B, Dunt D (1995). Clinical and host differences between infec- tion of the two varieties of Cryptococcus neoformans. Clin Infect Dis, 21, 28–​34. Temfack E, et al. (2018). Impact of routine cryptococcal antigen screening and targeted preemptive fluconazole therapy in antiretroviral-naive human immunodeficiency virus-infected adults with CD4 cell counts < 100/ul: A systematic review and meta- analysis. Clin Infect Dis, ciy567, https://doi.org/10.1093/cid/ciy567 8.7.3  Coccidioidomycosis Gregory M. Anstead ESSENTIALS Coccidioidomycosis results from inhalation of arthroconidia of Coccidioides spp., which are soil fungi endemic to the south-​western United States of America and parts of Latin America. Most infections are asymptomatic, but primary infection may resemble community-​ acquired pneumonia, sometimes with hypersensitivity manifestations such as erythema nodosum, erythema multiforme, and arthritis. Acute pulmonary infection usually resolves spontaneously, but—​ especially in immunocompromised patients, African Americans, and Filipinos—​it may progress to persistent pulmonary disease or dissem- inate to skin, soft tissues, the osteoarticular system, and the central nervous system. Diagnosis is by culture, histopathology, or serology. Fluconazole and itraconazole are usually the initial drugs of choice, with amphotericin B reserved for severe pulmonary and dissemin- ated disease, and in pregnancy. In refractory cases, posaconazole, voriconazole, and isavuconazole are alternative antifungal agents. Introduction Coccidioidomycosis results from inhalation of arthroconidia of di- morphic fungi of the genus Coccidioides, of which the two species are C. immitis (Californian isolates) and C. posadasii (non-​Californian

section 8  Infectious diseases 1362 isolates). Both species produce similar clinical effects. These soil fungi inhabit semiarid to arid areas in the south-​western United States of America and parts of Latin America. Hyperendemic areas include the San Joaquin Valley (California) and Pima, Pinal, and Maricopa counties in Arizona. There are approximately 150 000 infections per year in the United States of America, and about one-​third of those infected become symptomatic. Persons at risk Residence in or travel to endemic areas is the key risk factor for ac- quiring coccidioidomycosis. Arizona accounts for about 60% of re- ported American cases. At increased risk of more serious disease are people of Filipino or African American descent, those with blood group B, those exposed to soil, and the immunocompromised (organ transplant recipients; HIV infection, cancer, and diabetes; pregnancy; recipients of tumour necrosis factor α antagonists). Outbreaks may follow dust storms, earthquakes, droughts, and ac- tivities causing soil disruption, such as archaeological digs. Recent data from Arizona have defined a primary exposure season with peaks in May and September, which correlates with seasonal rainfall. Pathogenesis Inhaled coccidioides arthroconidia are ingested by pulmonary macrophages and, over 3 days or more, convert to thick-​walled round spherules containing hundreds of endospores. When spher- ules rupture, the endospores may disseminate to meninges, bones, skin, or other soft tissues. Resolution of coccidioidomycosis de- pends on intact cell-​mediated immunity. Diagnosis This is based on clinical findings supported by microbiological, histopathological, and/​or serological evidence. Coccidioides mycelia grow readily on many culture media. They are formed by barrel-​ shaped arthroconidia, with intercalated ‘ghost’ cells. The mycelia are extremely fragile, and the minimum infective dose approaches one arthroconidium, so these fungi must be handled with great caution by laboratory personnel. Coccidioides is considered a potential agent for bioterrorism, and there are strict rules for its handling in the United States. Histopathological findings can vary, from abscesses with many spherules, large endospores, and neutrophils (in uncon- trolled disease) to well-​formed granulomas with few organisms (in patients with competent cell-​mediated immunity). These findings are readily seen with haematoxylin and eosin staining. Serological methods are often used for the diagnosis of coccidi- oidomycosis. IgM antibodies, detected by the tube precipitin (TP) test or immunodiffusion TP, appear within the first few weeks of in- fection and clear within 1 or 2 months. IgG is detectable by comple- ment fixation (CF) or immunodiffusion CF after several months and persists for years. Serum CF titres of 1:16 or higher suggest deterior- ation or dissemination. In coccidioidal meningitis, any positive titre confirms the diagnosis; the cerebrospinal fluid IgG titre is positive more than 75% of the time, whereas cerebrospinal fluid cultures are positive in less than 50% of patients. More recently, enzyme-​linked immunosorbent assay (ELISA) has been used for coccidioidal IgG and IgM antibodies. ELISA optical density correlates roughly with immunodiffusion CF titre. Negative ELISA results do not require confirmation by other tests. However, positive tests may not be entirely specific, and should be confirmed by immunodiffusion or complement fixation tests. A diagnostic test based on the detection of coccidioidal antigens in the serum and urine has been commercialized by Miravista Laboratories. However, problems with this test include low sensitivity and cross-​reaction with histoplasma and blastomyces antigens. Clinical presentation Primary infection About 60% of subjects are asymptomatic. Symptomatic primary infec- tion presents from 1 to 3 weeks after exposure, with fever, cough, and pulmonary infiltrates, and may be accompanied by hypersensitivity manifestations, such as erythema nodosum, erythema multiforme, and arthritis. Eosinophilia or eosinophilic pleocytosis (in meningitis) may be present. Usually, however, the clinical syndrome of primary coccidioidal pneumonia is similar to other forms of community-​acquired pneu- monia, and this contributes to the difficulty of making a specific diag- nosis. In high-​incidence areas, such as Pima or Maricopa Counties in Arizona, coccidioides is the cause of up to 29% of community-​acquired pneumonia. It is now recommended that patients presenting with community-​acquired pneumonia in highly endemic areas should be tested for coccidioidomycosis. Although antifungal therapy is not re- quired for the treatment of primary infection, it is now understood that primary coccidioidal pneumonia can be an infection with significant morbidity, resulting in prolonged respiratory symptoms and delays in return to normal activity levels. Treatment of primary disease should be undertaken with immunocompromised patients. Recent appreciation of the clinical significance of primary coccidioidomycosis makes up a substantial percentage of community-​acquired pneumonias in Arizona again raises the question whether fluconazole should be used more rou- tinely for primary disease. In addition to uneventful resolution, there are various outcomes of primary coccidioidomycosis, which include those given below. Coccidioma formation Pulmonary infiltrates may contract into an asymptomatic mass (coccidioma), which can persist for years. In an immunocompetent person, antifungal therapy is unnecessary. Progressive/​persistent pneumonia Heavily exposed immunosuppressed patients may develop acute respiratory failure. Amphotericin B treatment is recommended. Pneumonia persists more than 2 months, with extensive infiltrates and, often, cavitation. Initial treatment with amphotericin B is re- commended if the patient is severely ill. The Infectious Disease Society of America guidelines suggest between 3 and 6 months for the duration of therapy, but we would favour treatment for more

8.7.3  Coccidioidomycosis 1363 than 6 months after resolution of symptoms, and for more than a year with diffuse miliary disease. Conversion to an oral azole is ap- propriate when the patient is improving. Chronic pulmonary coccidioidomycosis This occurs in about 5% of patients with symptomatic primary coccidioidomycosis and may have a fluctuating course over years. Nodular lesions may cavitate, with surrounding infiltrates and fi- brosis. Cavitary disease might be asymptomatic or be associated with rupture and pneumothorax, haemorrhage, or secondary infec- tion. Cavities smaller than 2.5 cm in diameter tend to resolve, while cavities larger than 5 cm persist. Cavities may remain stable for years or become infected with Aspergillus, or fluctuate with inter- mittent infiltrates and fibrocavitary disease. Chronic pulmonary coccidioidomycosis can progressively destroy the lungs and re- quires medical therapy with either fluconazole or itraconazole. The appropriate duration of therapy is uncertain. If large asymptomatic cavities persist for several years, resection should be considered. Coccidioidal mycetoma can occur in pre-​existing cavities and is treated by resection. Disseminated coccidioidomycosis Pleura and pericardium may be invaded during pulmonary coc- cidioidomycosis. Haematogenous dissemination occurs within a few months after infection and may involve skin, soft tissue, osteoarticular tissue, and meninges (Figs. 8.7.3.1–​8.7.3.3). Papules, nodules, abscesses, verrucous plaques, or ulcers are seen. Medical therapy is often combined with surgical therapy to debulk lesions. In chronic coccidioidomycosis, fluconazole at 400 or 800 mg/​day or itraconazole at 200 mg twice daily are used but death may ensue despite intensive medical and surgical intervention. Osteoarticular disease Any bone or joint may be targeted, but those that are weight bearing are more vulnerable (Fig. 8.7.3.4). Infection can destroy the verte- bral body, with collapse and joint instability. Paraspinous abscesses should be drained and, if necessary, the joint(s) stabilized. Fig. 8.7.3.2  Ulcerative ankle lesion with underlying osteomyelitis in a patient with coccidioidomycosis. Fig. 8.7.3.4  Coccidioidal arthropathy. Copyright R. Hay. Fig. 8.7.3.3  Abscesses on the chest in a patient with coccidioidomycosis. Fig. 8.7.3.1  CT of paraspinous abscess in a patient with coccidioidomycosis.

8.7.4 Paracoccidioidomycosis 1364

8.7.4 Paracoccidioidomycosis 1364

section 8  Infectious diseases 1364 Central nervous system involvement Coccidioidal meningitis may be accompanied by coccidioma, vas- culitis, infarction, and hydrocephalus. Most clinicians initiate treat- ment of meningitis with high-​dose fluconazole (800–​2000 mg/​day), which can be reduced as the patient improves. Lifelong treatment is necessary. Obstructive hydrocephalus requires ventriculoperitoneal shunting. Selection of antifungal agents Antifungal therapy of primary coccidioidomycosis remains contro- versial, with no randomized trials comparing different treatments. One observational study indicated that clinicians were more likely to treat patients with more severe disease or culture positivity. Disseminated disease developed in 10% of the treated patients, indicating that treatment of primary disease does not guarantee a benign future course. Azoles are preferred for treating most forms of coccidioidomycosis. Fluconazole and itraconazole appear simi- larly effective, but fluconazole is usually chosen because of fewer adverse reactions. For either drug, 400 mg/​day is usually given for a year or more after clinical cure. In HIV-​infected patients with non​meningeal disease, antifungal therapy may be stopped if their fungal disease is quiescent and their CD4 count has increased above 250 cells/​μl with antiretroviral therapy. In meningitis, fluconazole has replaced intrathecal amphotericin B. Intrathecal amphotericin B is difficult to administer, and can cause arachnoiditis and vasculitis. A case-​controlled study of coccidioidal meningitis comparing treat- ment with amphotericin B (primarily intrathecal) to fluconazole indicated that the neurologic complication rate (strokes, hydroceph- alus, and so on) and the overall mortality (39–​40%) were similar in both groups, with survivors commonly having persistent neuro- logical deficits. Thus, fluconazole therapy for coccidioidal menin- gitis, although better tolerated and easier to administer, has not been associated with an improved prognosis. Posaconazole has also been used for primary therapy in a series of 20 patients with chronic pulmonary or non​meningeal dissemin- ated disease. Of these 20 patients treated for up to 6 months, 17 had a satisfactory clinical response. Posaconazole is licensed in Europe for salvage therapy of coccidioidomycosis, based on limited clin- ical experience. Posaconazole may succeed in cases of dissemin- ated non​meningeal coccidioidomycosis in which other azoles and amphotericin B have failed. The dose is 200 mg four times daily, given orally with a high-​fat meal. Voriconazole has been used successfully in cases of refractory coccidioidomycosis, including meningitis, and there are now some data showing efficacy of isavuconazole. Amphotericin B should be used largely as salvage therapy and in pregnancy, since azoles are teratogenic. If amphotericin B is toxic or not successful, in the last two trimesters of pregnancy fluconazole may be used with less risk of teratogenicity. After therapy has been stopped, the patient should be observed for years as coccidioido- mycosis has an unpleasant propensity to relapse. In vitro testing of antifungals in coccidioidomycosis typically shows susceptibility, and such testing is not helpful. The echinocandins have not been shown to be of value for the treatment of coccidioidomycosis. Recently, antifungal prophylaxis has been recommended for trans- plant recipients living in highly endemic areas. Nevertheless, the mor- tality rate remains high (29%) for transplant recipients who develop coccidioidomycosis while receiving prophylaxis. Patients with a his- tory of coccidioidomycosis may receive solid organ transplants when the disease is inactive and if they maintain lifelong azole therapy (e.g. fluconazole 400 mg/​day). Antifungal prophylaxis for transplant re- cipients visiting the endemic zone is not recommended. Patients re- ceiving biological modifiers, such as tumour necrosis factor (TNF) antagonists, are probably at increased risk in endemic areas but there are no data on prophylaxis. It is recommended that these patients are screened for coccidioides by serology prior to starting therapy. FURTHER READING Ampel NM (2010). New perspectives on coccidioidomycosis. Proc Am Thorac Soc, 7, 181–​5. Crum NF, et al. (2004). Coccidioidomycosis: a descriptive survey of a reemerging disease. Clinical characteristics and emerging contro- versies. Medicine (Baltimore), 83, 149–​75. Fitterer C, et al. (2016). Coccidioidomycosis in patients with selected solid organ cancers: a case series and review of medical literature. Mycopathologica, 181, 787–​98. Galgiani JN, et al. (2016). 2016 Infectious Diseases Society of America (IDSA) clinical practice guideline for the treatment of coccidioido- mycosis. Clin Infect Dis, 63, e112–​46. Lewis ERG, Bowers JR, Barker BM (2015). Dust devil: the life and times of the fungus that causes valley fever. PLoS Pathog, 11, e1004762. Limper AH, et  al. (2011). An official American Thoracic Society statement: treatment of fungal infections in adult pulmonary and critical care patients. Resp Crit Care Med, 183, 96–​128. Mathisen G, et al. (2010). Coccidioidal meningitis: clinical presenta- tion and management in the fluconazole era. Medicine (Baltimore), 89, 251–​84. 8.7.4  Paracoccidioidomycosis M.A. Shikanai-​Yasuda ESSENTIALS Paracoccidioidomycosis is a systemic mycosis caused by dimorphic fungus Paracoccidioides spp., which is found in soil and in a variety of animals, and transmitted to humans by inhalation. This fungus is re- stricted geographically to Central and South America, where it is the most common endemic chronic human mycosis, acquired in rural and periurban areas, equally distributed among prepubescent boys and girls, but more frequent in men than women (10:1). Clinical features—​manifestations range from an asymptomatic course to severe and potentially fatal disseminated disease. (1) Acute form (juvenile type)—​1 to 20% of cases; presentation is with progres- sive lymphadenopathy; fever and weight loss are common; liver and spleen are usually moderately enlarged; other manifestations include mucocutaneous lesions and bone and small bowel involvement. (2) Chronic form—​usually occurs in men aged 30–​50 years who have worked in agricultural areas; frequently involves the lung, skin, and

8.7.4  Paracoccidioidomycosis 1365 mucous membranes (mainly pharynx, larynx, and trachea); may involve lymph nodes and adrenals, also (less frequently) intestine, spleen, bones, central nervous system (brain, cerebellum, meninges) and genitourinary system. Complications include microstomia, la- ryngeal/​tracheal/​bronchial stenosis, pulmonary emphysema/​fi- brosis, respiratory and adrenal insufficiencies, and cor pulmonale. Diagnosis and treatment—​diagnosis is made by (1) direct microscopy or culture from sputum, pus, or other lesions; (2) histopathology—​ silver or periodic acid–​Schiff staining reveals fungal cell granulomas containing fungal cells with either proliferative and/​or exudative reactions; or (3) serological testing. Treatment of mild cases is with itraconazole or sulfamethoxazole-trimethoprim; severe cases of acute or chronic disease require intravenous amphotericin B or other amphotericin formulations, followed by oral drugs. Long courses of treatment (6–​36 months) are required until stabilization or dis- appearance of antibodies detected by immunodifusion or counter immunoelectrophoresis tests. Definition Paracoccidioidomycosis is a systemic granulomatous disease caused by a dimorphic fungus of the complex Paracoccidioides brasiliensis and Paracoccidioides lutzii, that mainly involves the lungs, phago- cytic mononuclear system, mucous membranes, skin, and adrenals. History The disease was first described in 1908 by Lutz, a Brazilian scientist. In 1912, Splendore classified the organism as a yeast of the genus Zymonema and, in 1928, Almeida and Lacaz suggested the name Paracoccidioides. In 1930, Almeida named the fungus Paracoccidioides brasiliensis. Formerly, the disease was known as South American blastomycosis or Lutz–​Splendore–​Almeida disease. In 1977, it was renamed paracoccidioidomycosis. In 2005, molecular biology ana- lyses revealed that isolates from Brazil, Colombia and Venezuela were not similar and constituted the complex Paracoccidioides brasilien­ sis, and in 2009 a new species Paracoccidioides lutzii was reported in North and Central area of Brazil. Epidemiology Paracoccidioidomycosis is the most common endemic human my- cosis in Latin America but is restricted geographically to Central and South America, ranging from Mexico to Argentina. The disease is prevalent in Brazil, Colombia, Venezuela, Argentina, Uruguay, Paraguay, Guatemala, Ecuador, Peru, and Mexico. Imported cases have been recorded in the United States of America, Europe, and Asia. Paracoccidioidomycosis is the eighth most important cause of mortality from chronic infectious diseases in Brazil, the highest among systemic mycoses. Recent registered data suggested epi- demic levels of disease in the North Region of Brazil. Prevalence, inferred from the result of intradermal paracoccidioidin testing, ranges from 6 to 60.6% among rural and urban populations of endemic and non​endemic areas; lower rates were observed in the same region when a more specific antigen, 43 kDa glycoprotein, was employed in comparison with paracoccidioidin. The disease is equally distributed among prepubescent boys and girls but among adults the sex ratio of clinical cases is 10 or more men to each woman. This may be explained by the ability of oestrogens to inhibit the transformation of mycelium or conidia to yeast. The disease is most common among 20-​ to 50-​year-​ old agricultural workers or those who have lived in rural endemic areas. Spouses of patients are rarely affected by the disease, which suggests that hormonal and genetic factors play a part in the distribution of this my- cosis. Transmission from one person to another has not been shown. Ecology The geographical regions where paracoccidioidomycosis is most prevalent are humid with more acidic soils and a temperature range from 15 to 30°C. P. brasiliensis has been isolated from soil, animals such as armadillos, and rarely in dog food, penguin faeces, and in the intestinal contents of bats. Efforts to maintain the fungus in bat in- testines have been unsuccessful. The saprophytic habitat of P. brasil­ iensis has yet to be discovered. Aetiology Phylogenetic studies of eight regions in five nuclear loci of 65 P.  brasiliensis isolates indicated initially that this fungus consisted of at least three distinct, previously unrecognized species: S1 (spe- cies 1 with 38 isolates from Brazil, Argentina, Paraguay, Peru, and Venezuela isolates), PS2 (phylogenetic species 2 with five Brazilian and one Venezuelan isolates), and PS3 (phylogenetic species 3 with 21 Colombian isolates). Another phylogenetic species of P. brasiliensis, PS4, is further registered from analysis of samples of soil in Venezuela. Additionally, other Brazilian isolate ‘Pb01-​like’ species exhibits great sequence and morphological divergence from the S1/​PS2/​PS3 spe- cies clade and was named as Paracoccidioides lutzii. Recently, re- naming of species of the Paracoccidoides brasiliensis complex was suggested based on divergences on nuclear gene genealogies but not found on mitocondrial genes (Turrissini et al., 2017): Paracoccidioides americana sp., formely known as “PS2”; Paracoccidioides venezuelensis sp., formely known as “PS4”. Paracoccidioides restrepiensis sp., formely known as “PS3”; Paracoccidioides brasiliensis stricto sensu, previously known as “S1’. Mycology P. brasiliensis is a dimorphic fungus that can be cultivated either as a mould or a yeast. When cultured at 25°C, it appears after 15–​30 days as white colonies. When Sabouraud’s dextrose agar is used, the my- celium shows hyaline septate hyphae with branches. P.  brasiliensis grows as a yeast in human and animal tissues (Fig. 8.7.4.1) and in cultures maintained at 37°C. Colonies can be observed after 7 to 20 days. Under direct microscopy, yeast forms are seen as oval or spherical cells with doubly refractile walls; the cells vary in size from buds of 2–​10 μm in diameter to mature cells of 20–​30 μm. Mother cells may produce 10–​12 uniform or variably sized buds (Fig. 8.7.4.2), forming the characteristic ‘pilot wheel’ shape observed in biological samples or in infected tissues.

section 8  Infectious diseases 1366 Conidia produced by mycelium represent the infectious form and are inhaled through the respiratory tract. Analysis of 6022 assembled groups from mycelium and yeast phase expressed sequence tags of about 80% of the estimated genome of P. brasiliensis. The transcriptome analysis reported information about sequences related to the cell cycle, stress response, drug resist- ance, and signal transduction pathways of the pathogen. Virulence Virulence, defined as the ability to produce disseminated infection in experimental animals, varies between different fungal isolates but little is understood of the biochemical basis for these differences. The pres- ence of higher levels of α-​1,3-​glucan in virulent strains of P. brasilien­ sis compared with avirulent strains was initially related to virulence, but no correlation has been shown between glucans and virulence in experimentally induced infections. Binding of laminin to yeast cells (possibly through binding to gp43) enhanced their pathogenicity in the hamster testicle model. However, in a murine model, previous treatment of laminin with high and low virulent isolates of P. brasilien­ sis showed lower inflammatory responses with the virulent isolate and decreased pulmonary fungal burden with the low-​virulence isolate, suggesting an inhibitory effect of laminin treatment on P. brasiliensis infectivity. More recently the importance of a 32 kDA hydrolase from P. brasiliensis during initial adherence to host cells has been reported. Pathogenesis Experimental and clinicopathological observations indicate that the respiratory route is the main portal of entry and the lung is the pri- mary site of infection. The first fungus–​host contact occurs through inhalation of airborne conidia. When mice are experimentally infected through the respira- tory route, conidia have been observed in the alveoli soon after inocu- lation. Some 12–​18 h after the exposure, yeast forms can be observed in the alveoli. There is an initial inflammatory response, which is me- diated by polymorphonuclear cells, followed by granuloma formation. The primary infective complex develops at the inoculation site and in- volves the surrounding lymphatic vessels and regional lymph nodes. The fungus spreads to other parts of the lung through peribronchial lymph- atic vessels and drains into regional lymph nodes. Haematogenous dis- semination to a variety of organs and tissues can occur at this time. The lesions usually undergo involution and the fungi remain dormant if the host’s immune response can control their proliferation. A balanced host–​fungus relationship is associated with the absence of symptoms, although, in some children or young adults, acute disease might arise, primarily affecting the phagocytic mononuclear system. In adult life, previously quiescent lesions can become reactivated, especially in the lungs, leading to the adult or chronic form of the disease. Pathology The characteristic lesion is a granuloma containing P. brasiliensis cells. The infected tissue might exhibit a predominantly prolifera- tive, granulomatous inflammatory response and/​or an exudative re- action, sometimes resulting in necrosis, with variable numbers of neutrophils and large numbers of extracellular yeast cells, leading to a chronic epithelioid granuloma. Autopsy studies, mainly of adult patients, indicate that the or- gans most frequently involved are the lungs (42–​96%), adrenals (44–​80%), lymph nodes (28–​72%), pharynx/​larynx (18–​60%), and skin/​other mucosal surfaces (2.7–​64%). Host–​fungal interaction Non​specific immune response The influence of genetic factors on the individual susceptibility to this mycosis is suggested by the observation of higher rates of HLA phenotypes A9, B13, B40, and Cw3 among patients than in con- trols and higher rates of HLA DRB1*11 in patients with unifocal disease than with other forms of the disease. Analysing cytokine polymorphisms, the AA genotype of IL12RB1 was more frequent in the disseminated chronic disease in comparison to patients with unifocal chronic disease. In isogenic mice, resistance to P. brasilien­ sis is controlled by a single autosomal gene. The ability of circulating human neutrophils, obtained by bronchoalveolar washing, to digest the yeast forms of fungi was im- paired in severe cases, while this defect was absent in uninfected family members of patients. Interaction between a glycoprotein of 43 kDA of the fungus and monocytes was shown to modulate the expression of TLR2 (by inducing IL10 production), TLR4 (inducing TNF​α secretion) and mannose receptors. Fig. 8.7.4.1  Small and large yeast forms of Paracoccidioides brasiliensis in the lung of a transplant recipient (methenamine silver stain). Courtesy of C. S. Lacaz. Fig. 8.7.4.2  Scanning electron micrograph of a multiple budding yeast cell of Paracoccidioides brasiliensis. Courtesy of C. S. Lacaz.

8.7.4  Paracoccidioidomycosis 1367 Specific immune response Host–​fungal interaction in infection and disease was analysed through in vivo intradermal tests for ubiquitous fungal antigens, in vitro lymphoblastic transformation tests, and intra-​ and extracel- lular cytokine secretion, chemokines, and regulatory T-​cell activity after stimulation with mitogens or P. brasiliensis antigens (PbAg). Infected people (asymptomatic individuals without disease) showed a positive skin test to PbAg, absence of specific antibodies, a vigorous lymphoproliferative response to PbAg, and a typical T-​helper (Th) type 1 pattern of cytokines (see Table 8.7.4.1). They had a higher ex- pression of CD80 monocytes and lower expression of CD86 mono- cytes compared to patients with chronic or acute disease. Patients with acute disease showed impairment of proliferative response to PbAg and a mixed Th2/​Th9 cytokine pattern. This pattern is asso- ciated with poor granuloma formation, spreading of the fungus and high levels of antibody production (immunoglobulins IgG 1, IgG 4, and IgE). Patients with chronic disease present a predominant Th17/​ Th22 response. The specific lymphoproliferative response was lower than in asymptomatic paracoccidioidomycosis-​infected patients but higher than in patients with acute disease (see Table 8.7.4.1). More recent research indicates that regulatory T cells exhibiting suppressive activity in patients’ cells seem to play a role in control- ling local and systemic immune responses. In biopsies of mucosa and skin lesions, Foxp3+ cells were shown in compact granulomas sug- gesting their role in the modulation of the local immune response. The presence of IL17 in mucosa and skin lesions also suggests that this cytokine plays a role in cutaneous and mucosal lesions, perhaps in the clearance of the fungal antigens. In mice, treatment of dendritic cells with gp43 plus lipopolysac- charide was followed by an increase of fungal colony forming units in the lungs in comparison with controls, suggesting that gp43 might reduce effectiveness of the immune response in the primary infec- tion. In pulmonary murine paracoccidioidomycosis, a dual role of interleukin 4 (IL-​4, a Th2 cytokine) was observed in IL-​4-​depleted mice depending on the host genetic pattern: isogenic resistant mice showed better control of the disease. Conversely, susceptible mice showed enhanced pulmonary infection, suggesting a role for IL-​4 in the modulation of immune response, not only as a Th2 cytokine. Antibodies may enhance phagocytosis through opsonization of the fungus, but their role in resistance is not established. The im- portance of late hypersensitivity in protection has been observed recently in patients receiving cytotoxic therapy for associated neo- plasms and in those with AIDS presenting severe disease. Clinical features The range is from an asymptomatic course to severe and potentially fatal disseminated disease. The incubation period is unknown except in a laboratory worker, who developed a skin lesion some days after an accidental inoculation. The disease has been reported in children 3 years of age or older who had lived for some years in the endemic area. A proposed classification of clinical forms of paracoccidioidomycosis is shown in Box 8.7.4.1. Localization in a particular tissue or organ and the degree of se- verity of the disease according to established criteria make this clas- sification easily and uniformly applicable. General and nutritional debility and organ dysfunction (lung, brain, adrenals, bone marrow) indicate the severity of the disease. In immunosuppressed patients, signs of chronic and acute disease are observed simultaneously, with dissemination of fungi through phagocytic mononuclear cells. Acute form (juvenile type) Children, adolescents, and young adults (under 30 years of age) are affected, men and women equally. Only 1–​20% of patients fall into this group. There is progression for 2 or 3 months or longer, char- acterized by involvement of the phagocytic mononuclear system. Cervical, axillary, and inguinal nodes are the most commonly en- larged (Fig. 8.7.4.3). Nodes are initially hard but are sometimes fluc- tuant and drain pus rich in fungi. Less frequently, deep-​seated lymph nodes can also be affected. When the hepatic perihilar lymph nodes are enlarged, they sometimes produce symptoms of obstructive jaundice. The liver and spleen are usually moderately enlarged. Bones (clav- icle, scapulae, ribs, skull, long, and flat bones) and, rarely, the bone marrow may be involved. Radiographs show lytic lesions without periosteal reaction. Involvement of the small bowel may be asymp- tomatic or produce abdominal pain, diarrhoea, constipation, and even intestinal obstruction. Radiological studies of the digestive tract reveal intestinal tract involvement in about 50% of clinical cases. Fever and weight loss are common. Multiple mucocutaneous lesions are more frequent in some geographical areas (Fig. 8.7.4.4). High transient blood eosinophilia (up to 30 000/​mm3) has some- times been described. Clinical lung involvement is rarely described in this form of paracoccidioidomycosis. In some case reports, either bronchopneu- monia or primary complex-​like disease have been observed. Table 8.7.4.1  Host–​fungal interaction in paracoccidioidomycosis: cytokine secretion and P. brasiliensis antigenaemia/​antigenuria in infection and disease Groups Cytokine secretion and antigenaemia/​antigenuria Intracellular cytokines Infectiona IFγ, Ab undetectable IFγ↑, TNFα↑, IL-​2↑ Acute disease IFγ↓, IL-​4↑↑, IL-​5↑↑, IL9↑,
IL21↑, IL-​10↑, Ab↑ (IgG 4), antigenaemia/​antigenuria↑ IFγ↓, TNFα↓, IL-​2↓ Chronic disease IFγ↓, IL-​4↑, IL-​5↑, IL-​10↑, Ab↑
(IgG 2), IL17↑,IL22↑ IL17 in mucosa and skin biopsies, Foxp3 in compact granulomas IFγ↓, TNFα↓, IL-​2↓ Immunosuppressed patients ? IFγ↓, IL-​10↑,? IL-​4,? IL-​5↓, Ab increased or lower levels, antigenaemia/​antigenuria ↑ ?b Ab, antibodies; IF, interferon; IL, interleukin; TNFα, tumour necrosis factor α; ↓, decrease; ↑, increase. a Asymptomatic individuals sensitized by P. brasiliensis antigens without signs and symptoms. b Decrease in lymphoproliferation in response to P. brasiliensis antigens: intracellular cytokines unknown. Box 8.7.4.1  Paracoccidioidomycosis: proposed classification of clinical forms • Paracoccidioidomycosis infection • Regressive (self-​healing) paracoccidioidomycosis • Paracoccidioidomycosis disease —​ Acute form (juvenile type)—​moderate or severe —​ Chronic form (adult type)—​mild, moderate, or severe • Sequelae

section 8  Infectious diseases 1368 Chronic form This form of the disease usually occurs in 30-​ to 50-​year-​old men who have worked in agricultural areas. The male to female ratio varies from 10:1 to 25:1. The evolution is insidious and, in many cases, clinically mild. The organ most frequently involved is the lung, followed by skin and mucous membranes, mainly pharynx, larynx, and trachea. Lymph nodes and adrenals might be compromised. More than one organ or tissue is usually involved. Less frequently, intestine, spleen, bones, cen- tral nervous system (brain, cerebellum, meninges), eyes, genitourinary system, myocardium, pericardium, and arteries are involved. The patients might be asymptomatic or complain of dyspnoea, cough, sometimes purulent sputum, and, rarely, haemoptysis. Fever is unusual. Physical examination is frequently normal or there can be scattered rales. In contrast, chest radiography commonly reveals bilateral, asymmetrical, reticulonodular infiltrates in the middle and lower parts of the lungs (Fig. 8.7.4.5). Apical cavities and pleural ef- fusions are less frequently observed. Fig. 8.7.4.3  Lymph node and skin involvement in a patient with the acute form of paracoccidioidomycosis. Courtesy of C. S. Lacaz. Fig. 8.7.4.4  Multiple molluscum-​like lesions in a young Peruvian patient. Copyright Francisco Bravo, Lima. Fig. 8.7.4.5  Alveolar and interstitial infiltrates in both lungs in a patient with chronic paracoccidioidomycosis. Department of Infectious and Parasitic Diseases, School of Medicine, University of São Paulo. Fig. 8.7.4.6  Mucocutaneous lesions in a patient with chronic paracoccidioidomycosis. Courtesy of C. S. Lacaz. Fig. 8.7.4.7  Disseminated skin lesions. Courtesy of Universidad Peruviana Cayetano Heredia.

8.7.4  Paracoccidioidomycosis 1369 Cutaneous lesions include papules, pustules, ulcers, crusted ul- cers, vegetations, tuberculoids, verrucoids, or acneiform lesions mainly on the face (Fig. 8.7.4.6) or limbs. Multiple, scattered le- sions result from haematogenous dissemination (Fig. 8.7.4.7). Subcutaneous cold abscesses, more commonly associated with bone lesions, can occur. Mucosal lesions are usually in the mouth and/​or oropharynx, including the palate (Fig. 8.7.4.8), uvula, and tonsils, or in the re- spiratory tract, involving mainly the larynx (vocal cords, glottis, and epiglottis) and trachea. Pain is usually intense and might hamper mastication and swallowing. Hoarseness and dysphonia result from laryngeal lesions and can lead to obstruction of the upper respira- tory tract. Examination shows ulcerative, verrucous, vegetant, and infiltrative ‘moriform’ stomatitis, resembling a raspberry, with pap- ules, vesicles, and haemorrhagic spots. The last is characteristic of this mycosis and appears as shallow ulcers, with a granular surface showing multiple, fine, haemorrhagic points. Few lymph nodes are involved, in contrast to the acute form of the disease. Uni-​ or bilateral lesions in the adrenal glands have been found in about half of patients coming to autopsy. Partial adrenal insuffi- ciency has been documented in about 40% of the cases but only 7.4% were symptomatic. Concomitant tuberculosis is observed in about 10–​15% of cases of pulmonary paracoccidioidomycosis and has also been described in cases of lymph node involvement by P. brasiliensis. Carcinomas may arise in pulmonary or mucosal mycotic lesions. Sequelae Nowadays, sequelae constitute one of the most important problems in the management of paracoccidioidomycosis. Although fungal multiplication can be controlled by chemotherapy, impairment of vital functions might prove fatal. Acute form Inflammmatory lesions in the small intestine and mesenteric lymph nodes can result in fibrosis, causing lymphatic obstruction, intestinal malabsorption, or protein-​losing enteropathy. A clinical picture of severe malnutrition and immunodeficiency has been re- ported (Fig. 8.7.4.9). Chronic form As the lesions usually tend to heal by fibrosis, sequelae such as microstomia and laryngeal, tracheal, or even bronchial stenosis might be observed. Corrective surgery is indicated. Pulmonary emphysema, fibrosis, respiratory insufficiency, and, finally, cor pulmonale are frequent sequelae. Obstructive and re- strictive patterns of ventilatory defect have been found in about 36 and 16% of patients, respectively. As many as 30% of these pa- tients might die as a result of respiratory or cardiorespiratory failure. Adrenal reserve is decreased in 15–​50% of patients and there is cen- tral nervous system dysfunction in about 6–​25% of patients. Diagnosis Microbiological identification Isolated or budding (single or multiple) mother cells are observed under direct microscopy in sputum, pus from lymph nodes, and ma- terial from the skin or mucous membrane lesions. Specimens are cultured at 37°C on blood, chocolate, or yeast ex- tract agar. The colonies are produced after 7 days, usually in 10 to 20 days. Cultures can be maintained at 25°C on Sabouraud’s dextrose agar, where the colonies might be noticed after 15 to 30 days. Histopathology Silver or periodic acid–​Schiff staining is required to detect the fungus in sputum. Diagnostic features are the variable size (1–​30 μm) of the yeast cells, and their multiple budding. Proliferative or exudative re- actions, as described in the section on pathology, may be observed. Immunological tests Serological reactions Immunodiffusion (Ouchterlony) and counterimmunoelectrophoresis are the best techniques initially. Sensitivities and specificities are as Fig. 8.7.4.8  Palatal lesion. Copyright D. A. Warrell. Fig. 8.7.4.9  Ascites, cachexia, and immunodeficiency due to malabsorption and protein-​losing enteropathy as sequelae of acute paracoccidioidomycosis. Courtesy of M. Shiroma.

section 8  Infectious diseases 1370 high as 95%. Cross reactions are mainly with other systemic mycoses such as histoplasmosis, aspergillosis, cryptococcosis, and candidiasis. Complement fixation and indirect immunofluorescence are less reliable tests for diagnosis and have not been employed even for treatment follow-​up. Recently, enzyme immunoassays employing PbAgs, including a 43 kDa glycoprotein, have shown high sensitivity and specificity. Antibody titres tend to decrease about 3 to 6 months after starting specific therapy and to disappear after 9 months to 5 years or more. For diagnosis of paracoccidioidomycosis in Central and North regions of Brazil, it has been shown that the sensitivity of immuno- diffusion or counterimmunoelectrophoresis is only appropriate when antigens from local isolates were employed. This sensitivity is lower than 40% when antigens classically employed for other re- gions were used, indicating differences in antigenic composition of Paracoccidioidodes species from isolates of these regions. However, antigenic preparations without cells derived from P. lutzii have been able to diagnose by immunodiffusion test 100% of sera from patients with paracoccidioidomycosis due to P. lutzii. Antigenaemia and antigenuria have been considered useful in- dications in patients presenting low levels of antibodies in the sera, both for diagnosis and follow-​up after treatment, particularly in an immunocompromised host. Circulating gp43 and gp70 antigens were detected in 100% of cerebrospinal fluid and almost all serum samples of patients with neuroparacoccidioidomycosis. The correlation between immunological and histopathological findings and clinical forms is outlined in Table 8.7.4.1. Treatment Clinically active disease is usually treated for 6–​36 months, according to the severity of the disease, until disappearance of antibodies detected by immunodiffusion or counterimmunoelectrophoresis tests or their stabilization at low levels (1:2 and 1:4, respectively) in test performed 6 months apart. In mild or moderately severe cases, co-​trimoxazole (160 mg of trimethoprim and 800 mg of sulfamethoxazole, twice or three times a day) or imidazoles (itraconazole 100–​400 mg/​day) have been shown to be effective. In a randomized trial with 42 cases, sulphadiazine (150 mg/​kg per day), itraconazole (50–​100 mg/​day), and ketoconazole (200–​400 mg/​day) were equally effective in pa- tients with moderately severe disease. More recently, a study of 177 cases found no difference in the efficacy and effectiveness of initial treatment of 47 individuals given itraconazole compared with 130 given sulfamethoxazole–​trimethoprim, but the time to clinical cure was shorter in those given itraconazole (105 vs. 159 days), specifically in patients with the chronic form of the disease. Voriconazole has been used in a randomized study in comparison with itraconazole with similar results and, since it achieves high levels in cerebrospinal fluid, it could be useful in neuroparacoccidioidomycosis. Severe cases of acute or chronic disease should be treated with intravenous infusion of amphotericin B. Both amphotericin B and liposomal amphotericin preparations are employed during a short induction phase, aiming to control the evolution and dissemination of the lesions, and should be replaced by maintenance therapy as soon as signs of clinical improvement are achieved. The daily dose of amphotericin begins at 0.1–​0.2 mg/​kg, increasing up to 1.0 mg/​kg, with the total dose ranging from 1 to 3 g or more. Toxic reactions to amphotericin B include fever, chills, headache, anaemia, and nephro- toxicity characterized by tubular acidosis and potassium urinary excretion and resultant hypokalaemia and azotaemia. In most cases, these reactions can be controlled until the end of the course of therapy. Co-​trimoxazole (80 mg of trimethoprim and 400 mg of sulphamethoxazole), twice a day, or itraconazole (100–​200 mg/​day) are recommended as maintenace therapy. Liposomal amphotericin has been used in severe cases of paracoccidioidomycosis, but a short period of treatment was followed by relapses. In a Brazilian study, relapses were seen in 5.2% of 400 registered cases, most of them in the chronic form (71.4%). Prognosis Even though the disease is easily controlled in most cases, the course of treatment is long and abandonment of treatment is the most important cause of therapeutic failure, e.g. in Brazil. Normalization of cellular specific responses, particularly of the skin test (paracoccidioidin) indicates a good prognosis. Death may occur in severe acute or chronic cases and severe cases with sequelae. FURTHER READING André DC, et al. (2004). Binding of laminin to Paracoccidioides brasilien­ sis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-​virulence isolates. Microbes Infect, 6, 549–​58. Bocca AL, et al. (2013). Paracoccidioidomycosis: eco-​epidemiology, taxonomy and clinical and therapeutic issues. Future Microbiol, 28, 1177–​91. Borges-​Walmsley MI, et al. (2002). The pathobiology of Paracoccidioides brasiliensis. Trends Microbiol, 10, 80–​7. Calich VLG, et al. (1985). Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis. Br J Exp Pathol, 66, 585–​94. Cavalcante RS, et al. (2014). Comparison between Itraconazole and Cotrimoxazole in the Treatment of Paracoccidiodomycosis. PloS Neglected Trop Dis, 8, e2793. de Castro LF, et al. (2013). Characterization of the immune response in human paracoccidioidomycosis. J Infect, 67, 470–​85. Hollanda FMC, et  al. (2016). Polymorphisms on IFNG, IL12 and IL12RB1 genes and paracoccidioidomycosis in the Brazilian popu- lation. Infect Genet Evol, 43, 245–51. Matute DR, et al. (2006). Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol, 23, 65–​73. Pagliari C, et  al. (2011). Paracoccidioidomycosis:  cells expressing IL17 and Foxp3 in cutaneous and mucosal lesions. Microb Pathog, 50, 263–​7. Shikanai-​Yasuda MA (2005). Pharmacological management of paracoccidioidomycosis. Expert Opin Pharmacother, 6, 385–​97. Shikanai Yasuda MA (2017). Brazilian guidelines for the clinical management of paracoccidioidomycosis. Rev Soc Bras Med Trop, 50, 715–40. Teixeira MM, et al. (2009). Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus. Mol Phylogenet Evol, 52, 273–​83. Turissini DA, et al. (2017). Species boundaries in the human pathogen Paracoccidioides. Fungal Genet Biol, 106, 9–25.

8.7.5 Pneumocystis jirovecii 1371

8.7.5 Pneumocystis jirovecii 1371

8.7.5  Pneumocystis jirovecii 1371 8.7.5  Pneumocystis jirovecii Robert F. Miller and Christopher P. Eades ESSENTIALS The ascomycete fungus Pneumocystis jirovecii (previously called Pneumocystis carinii) is the cause of pneumocystis pneumonia in humans, which occurs largely among people with impaired CD4+ T-​lymphocyte function or numbers (e.g. those infected with HIV, re- cipients of solid organ or haematopoietic stem cell transplants, and those taking therapeutic immunosuppressive agents). The organism is restricted to humans, and disease is now thought to arise from
de novo infection by inhalation from an exogenous source. Clinical features and diagnosis—​presentation of pneumocystis pneu- monia is non​specific, with progressive dyspnoea and non​productive cough. Examination of the chest is typically normal, but fine bibasal end-​inspiratory crackles may be heard. Diagnosis is usually by demon- stration of organisms on microscopy (preferably with immunofluor- escence staining) of induced sputum or bronchoalveolar lavage fluid. Detection of P. jirovecii-​specific DNA by polymerase chain reaction is increasingly used for diagnosis. Treatment and prognosis—​aside from supportive care, first-​line therapy of pneumocystis pneumonia is sulphamethoxazole–​ trimethoprim (co-​trimoxazole, which has a high rate of treatment-​ limiting adverse drug reactions), with adjunctive corticosteroids indicated for those with severe disease (i.e. hypoxaemia). In pa- tients whose disease is failing to respond, or those intolerant of co-​ trimoxazole, the main alternatives are intravenous pentamidine or clindamycin with primaquine. Among HIV-​infected patients, early initiation of antiretroviral therapy (i.e. within 14  days of starting antipneumocystis pneumonia therapy) is beneficial. Prevention—​primary prophylaxis is recommended for (1)  HIV-​ infected patients—​when the CD4+ count falls below 200 cells/​µl or they have HIV-​constitutional features or other AIDS-​defining diagnoses; and (2) other at-​risk groups—​for example, recipients of solid organ or haem- atopoietic stem cell transplants, and those taking therapeutic immuno- suppressive agents for underlying rheumatological diseases. Secondary prophylaxis is given after an episode of pneumocystis pneumonia. The first-​choice prophylactic agent is co-​trimoxazole; alternative options include dapsone with pyrimethamine, and nebulized pentamidine. Introduction What is Pneumocystis jirovecii? Pneumocystis species are ascosmycetous fungi which infect a wide variety of mammalian hosts asymptomatically but sometimes cause pneumonia, which is known as pneumocystis pneumonia (PCP). Pneumocystis jirovecii (previously called Pneumocystis carinii) is the cause of PCP in humans. Who gets PCP? Most patients have abnormalities of T-​lymphocyte function or numbers but, rarely, PCP develops in patients with isolated B-​cell defects and in people without evidence of immunosuppression. In non-​HIV-​infected people, glucocorticoid administration is an inde- pendent risk factor for development of PCP irrespective of the type or intensity of immunosuppression or the nature of the underlying disease process. In HIV-​infected people, those at greatest risk of PCP have CD4+ T-​lymphocyte counts less than 200 cells/​μl. In the early years of the AIDS epidemic, PCP was the AIDS-​defining diagnosis for almost two-​thirds of patients. Since the introduction of antiretro- viral therapy (ART), although there has been a marked decline in incidence of PCP, it remains the most common serious opportun- istic infection in HIV-​infected people in Europe, the United States of America, and Australasia. Patients living in areas without access to PCP prophylaxis or ART remain at high risk of PCP. Aetiology Pneumocystis cannot reliably be cultured in vitro. Pneumocystis or- ganisms from different mammalian host species show antigenic, karyotypic, and genetic heterogeneity. Cross-​infection between host species has not been successful, suggesting host specificity and that pneumocystis infection in humans is not a zoonosis. The demonstration of antibodies against pneumocystis in most healthy children and adults has been regarded previously as supportive of the hypothesis that PCP arises in an immunocompromised indi- vidual by reactivation of a childhood-​acquired latent infection. However, this hypothesis is challenged by the failure to demonstrate pneumocystis in bronchoscopic alveolar lavage fluid or necropsy lung tissue of immune competent people and the observation that Pneumocystis DNA is detectable only at low levels in less than 25% of HIV-​infected people with low CD4+ T-​lymphocyte counts pre- senting with respiratory episodes and with diagnoses other than PCP. Human Pneumocystis infection is currently thought to arise from de novo infection from an exogenous source. Finding different genotypes in each episode in patients with recurrent PCP supports the reinfection model. Pathogenesis After inhalation, the organism reaches the alveoli where the trophic form attaches to type 1 pneumocytes. In an immune competent person, the organism is eliminated; in the immunodeficient, PCP will develop. The major surface glycoprotein of Pneumocystis binds to macro- phages and induces T-​lymphocyte proliferation and increased se- cretion of L1 (L1CAM, CD171), L2 and tumour necrosis factor-​α (TNF​α). Monocytes respond to major surface glycoprotein by re- leasing interleukin-​8 and TNF​α. Pneumocystis induces changes in the quantity and quality of pulmonary surfactant; total cholesterol, glycerol, and phospholipase A2 are increased while phospholipid is reduced. Clinical presentation Patients typically present with progressive exertional dyspnoea, a non​productive cough, and fever of several days or weeks duration.

section 8  Infectious diseases 1372 They often report an inability to take in a deep breath that is not due to pleural pain. Purulent sputum, haemoptysis, and pleural pain are atypical for PCP and suggest a bacterial or mycobac- terial pathogen. In HIV-​infected patients, the presentation is usu- ally more indolent than in patients immunosuppressed for other reasons. However, in a small proportion of HIV-​infected patients, the disease course of PCP is fulminant with an interval of 7 days or fewer between onset of symptoms and progression to respira- tory failure. Rarely, PCP might present as pyrexia of undetermined origin. Examination of the chest is usually normal; occasionally, fine bibasal end-​inspiratory crackles are heard. Signs of focal consolida- tion or pleural effusion suggest an alternative diagnosis. Pathology Within the lung, Pneumocystis infection is characterized by an eo- sinophilic, foamy intra-​alveolar exudate, associated with a mild plasma-​cell interstitial pneumonitis. Morphologically, two forms of Pneumocystis can be identified: thick-​walled cystic forms (6–​7 µm diameter) that lie freely within the alveolar exudate are demon- strated by Grocott-​Gömöri methenamine silver, toluidine blue O, or cresyl violet stains (Fig. 8.7.5.1). The exudate consists largely of thin-​walled, irregularly shaped, single-​nucleated trophic forms (2–​5 µm diameter) that are shown by Giemsa stain but lack dis- tinctive features. Uncommonly, interstitial fibrosis, diffuse alveolar damage, granulomatous inflammation, nodular and cavitary lesions, and pneumatocele formation may occur. Before the availability of ART Pneumocystis infection extending beyond the airspaces; extrapulmonary pneumocystosis involving liver, spleen, gut, or eye was reported and was strongly associated with use of nebulized pentamidine for prophylaxis. Investigations Chest radiograph The chest radiograph can be normal in early or mild PCP. With more severe disease or later presentation, bilateral perihilar inter- stitial or reticular infiltrates are seen (Fig. 8.7.5.2). These might progress to diffuse bilateral alveolar (air space) consolidation that mimics pulmonary oedema. In the late stages, the lungs can be massively consolidated and almost airless. Radiographic de- terioration from near normal at presentation to being markedly abnormal might occur over 48 h or less. Up to 20% of chest radio- graphs are atypical, showing intrapulmonary nodules, cavitary le- sions, lobar consolidation, pneumatoceles (Fig. 8.7.5.3), or hilar/​ mediastinal lymphadenopathy. All of these typical and atypical radiographic appearances can also be seen in bacterial, mycobac- terial, and fungal infections and in non​specific pneumonitis and pulmonary Kaposi sarcoma. Despite treatment and clinical recovery, in some individuals the chest radiograph might remain abnormal for many months in the absence of symptoms. In others, postinfectious bronchiectasis or fi- brosis develops. Arterial blood gases/​oximetry Less than 10% of patients with PCP have a normal partial pressure of oxygen (PaO2) and a normal Alveolar-​arterial gradient (P(A–​a)O2). These measures are sensitive, though not specific, for PCP as they Fig. 8.7.5.1  Cystic form of Pneumocystis jirovecii in bronchoalveolar lavage fluid. The walls of the cysts are stained black (Grocott-​Gömöri methanamine silver stain). Fig. 8.7.5.2  Chest radiograph showing bilateral interstitial infiltrates typical of Pneumocystis pneumonia.

8.7.5  Pneumocystis jirovecii 1373 can also occur in bacterial pneumonia, pulmonary Kaposi sarcoma, and tuberculosis. CT High-​resolution CT of the chest might be useful in the symptom- atic patient with a normal or equivocal chest radiograph. Areas of ‘ground-​glass’ shadowing indicate active pulmonary disease (Fig. 8.7.5.4). Subpleural sparing is a common radiological feature. Such appearances can be caused by other fungal infections and by cytomegalovirus, as well as by PCP. Alveolar haemorrhage might also be a relevant differential in haematopoietic stem cell transplant recipients. Induced sputum Spontaneously expectorated sputum is inadequate for diagnosis of PCP. Sputum induction by inhalation of ultrasonically nebu- lized hypertonic (3–​5%; 513–​856 mmol/​litre) saline might provoke a suitable sample. Pneumocystis is usually found in clear saliva-​ like samples. Purulent samples suggest an alternative diagnosis. The sensitivity varies between 55 and 90% and a negative result for Pneumocystis should prompt further diagnostic tests. Induced sputum might have a higher yield for PCP diagnosis in HIV-​infected patients, in whom sputum organism-​load is higher than in those with immunosuppression of alternate aetiology. Bronchoscopy Fibre-​optic bronchoscopy with bronchoalveolar lavage has a sensitivity exceeding 90% for detection of Pneumocystis. Immunofluorescence (IF) staining increases the diagnostic yield compared to conventional histochemical staining. Transbronchial biopsies add very little to the diagnostic yield and are associated with a relatively high complication rate (c.8%). As Pneumocystis persists in the lung for many days (and even weeks) after the start of antimicrobial therapy, bronchoscopy can be performed up to 1 week after commencing antimicrobial therapy without a reduc- tion in diagnostic yield. Molecular detection tests Detection of Pneumocystis-​specific DNA by the polymerase chain reaction (PCR) on bronchoalveolar lavage fluid and induced sputum is superior to conventional histochemical methods and offers sen- sitivity approaching 100%. However, specificity is reduced, as Pneumocystis-​specific DNA can also be detected in immunosup- pressed patients who are colonized with P. jirovecii, and who have alternative explanations for their presentation (e.g. bacterial pneu- monia). PCR might, therefore, be of most diagnostic benefit for the patient with a high pretest probability of PCP and negative results from histochemical staining and IF of bronchoalveolar lavage fluid or induced sputum. Detection of Pneumocystis DNA by PCR might also be achieved on oropharyngeal samples obtained by gargling with 10 ml normal saline. Empirical therapy Many centres in the United Kingdom and North America seek to confirm a diagnosis in every suspected case of PCP. Others treat HIV-​infected patients empirically when they present with fea- tures typical of PCP: symptoms and signs, chest radiographic ab- normalities, and hypoxaemia. Bronchoscopy is reserved for those who fail to respond to empirical therapy by day 5 or who have atypical presentations. Both strategies are equally effective in clin- ical practice. Treatment It is important to stratify PCP as mild (PaO2 on air >11.0 kPa, SaO2

96%), moderate (PaO2 8.0–​11.0 kPa, SaO2 91–​96%), or severe Fig. 8.7.5.3  Chest radiograph showing atypical appearances for Pneumocystis pneumonia, including bilateral apical shadowing and a right mid-​zone thin-​walled pneumatocele. Fig. 8.7.5.4  CT of thorax showing diffuse bilateral ‘ground-​glass’ shadowing typical of Pneumocystis pneumonia.

section 8  Infectious diseases 1374 (PaO2 <8.0 kPa, SaO2 <91%) as some drugs are unproven or inef- fective in severe disease. First-​choice treatment is high-​dose co-​trimoxazole (sulphameth- oxazole 100 mg/​kg per day and trimethoprim 20 mg/​kg per day, in two to four divided doses orally or intravenously). In HIV-​infected patients with PCP, 21 days are recommended; in those with other causes of immunosuppression, between 14 and 21 days are frequently given. In mild disease, oral medication may be given throughout; in moderate or severe disease, intravenous therapy is usually given for the first 7 to 10 days, then orally. Another treatment in patients with severe disease is clindamycin (450–​600 mg three to four times daily orally or intravenously) with primaquine (15–​30 mg once daily orally). Despite its toxicity, pentamidine (4 mg/​kg per day intravenously) can be used if other treatments have failed. In patients with mild or moderate disease, alternatives to co-​trimoxazole include clindamycin with prima- quine (doses as described here), dapsone (100 mg once daily or- ally) with trimethoprim (20 mg/​kg per day orally), or atovaquone (750 mg twice daily orally). Nebulized pentamidine has no role in treatment of PCP; treatment response is delayed, early relapse is common, and extrapulmonary dissemination of pneumocystosis is not suppressed. Current US and UK Guidelines recommend that HIV-​infected patients presenting with PCP should commence ART within 14 days after starting anti-​PCP therapy. There is a risk of immune reconsti- tution inflammatory syndrome; patients experiencing paradoxical worsening of symptoms, oxygenation, and radiographic appearances. Adjuvant steroids HIV-​infected patients with moderate or severe PCP and PaO2 less than 9.3 kPa (on air) benefit from adjuvant corticosteroids, which might reduce the need for mechanical ventilation and risk of death. Many non-​HIV-​infected patients with PCP are already receiving glucocorticoids as part of their regimen of immuno- suppression and the benefits of dose increases have not clearly been demonstrated. Adjunctive glucocorticoid regimens include prednisolone (40 mg twice daily orally for 5 days, then 40 mg once daily on days 6 to 10, then 20 mg once daily on days 11 to 21) or methylprednisolone (intravenously at 75% of these doses). The benefit of adjuvant corticosteroids has only been documented when they are started within 72 h of starting specific anti-​PCP therapy. Adverse reactions Adverse reactions to co-​trimoxazole, which usually occur between days 6 and 14 of treatment, are more common in HIV-​infected pa- tients than in patients with other causes of immunosuppression. Anaemia and neutropenia (≤40% of patients), rash and fever (≤30% each), and biochemical hepatitis (≤15%) are the most frequent ad- verse reactions. Coadministration of folic or folinic acid does not at- tenuate haematological toxicity. Diarrhoea and rash (≤30% each) are the most frequent adverse reactions to clindamycin. Stool should be examined for Clostridium difficile in patients developing diarrhoea on clindamycin. Glucose-​6-​phosphate dehydrogenase deficiency Glucose-​6-​phosphate dehydrogenase levels should be checked before (or as soon as possible after) starting treatment with co-​trimoxazole, dapsone, or primaquine, but treatment initiation should not be de- layed pending the result. If haemolysis occurs, or enzyme deficiency is identified, alternative treatment can be started (e.g. intravenous pentamidine or atovaquone). Prophylaxis HIV-​infected patients are at increased risk of PCP as the CD4+ T-​ lymphocyte count decreases. Primary prophylaxis (to prevent a first episode of Pneumocystis pneumonia) is given when the CD4 count falls below 200 cells/​µl or the CD4:total lymphocyte ratio is less than 1:5, to patients with HIV-​constitutional features (unexplained fever of three or more week’s duration or oral candida irrespective of CD4 count), and to patients with other AIDS-​defining diagnoses, for ex- ample, Kaposi sarcoma. Secondary prophylaxis is given after an epi- sode of PCP. The first-​choice agent for primary and secondary prophylaxis is co-​ trimoxazole (960 mg daily: 800 mg sulphamethoxazole and 160 mg trimethoprim). A  lower dose (i.e. 960 mg three times weekly or 480 mg daily) might be equally effective and have fewer side effects. Co-​trimoxazole may also protect against bacterial infections and re- activation of cerebral toxoplasmosis. Alternative, less effective options include nebulized pentamidine (300 mg once monthly, or once per fortnight if the CD4 count is 50 µl or less), dapsone (100 mg daily) with pyrimethamine (25 mg once weekly (and folinic acid)), atovaquone (750 mg twice daily), and azithromycin (1.25 g once weekly). Non-​HIV-​infected patients with high attack rates of PCP should receive prophylaxis (drug choice and doses as described here). At-​ risk groups include those with acute leukaemias, severe combined immunodeficiency syndrome, Hodgkin lymphoma, rhabdomyo- sarcoma, primary and secondary central nervous system tumours, granulomatosis with polyangitis, and organ transplantation including allogenic haematopoietic stem cell, renal, heart, heart/​lung, and liver. Infection control considerations The mode of transmission of human Pneumocystis infection is un- clear but recent molecular data suggest that transmission might occur from infected patients to susceptible immunocompromised individuals. Patients with minor immune suppression, including those with moderate to severe chronic obstructive lung disease, and those receiving long-​term corticosteroids (prednisolone 20 mg/​day or more), irrespective of the cause of underlying immune suppres- sion, might be colonized by Pneumocystis, thus acting as a poten- tial infectious reservoir. Nosocomial outbreaks have been described in cohorts of both HIV-​infected and non-​HIV-​infected patients. Pneumocystis DNA can be isolated from air in the environs of pa- tients with PCP. Where available, respiratory isolation might be prudent, particularly on inpatient units responsible for care of im- munocompromised patients. Areas of uncertainty/​future research The drug target for sulphamethoxazole and dapsone is dihydropteroate synthase (DHPS). Mutations in the DHPS gene of Pneumocystis occur

8.7.6 Talaromyces (Penicillium) marneffei infectio

8.7.6 Talaromyces (Penicillium) marneffei infection 1375

1375 8.7.6  Talaromyces (Penicillium) marneffei infection more commonly in individuals who have prior exposure. There is con- flicting evidence as to whether DHPS mutations are associated with poor outcomes (failure to respond to co-​trimoxazole, or death) from PCP. Drug toxicity remains the biggest issue in the treatment of PCP, particularly with co-​trimoxazole therapy. Novel treatment regimens with reduced toxicity would be clinically useful. Caspofungin (and the other echinocandins) have been shown in animal models to be fungistatic. In a mouse model of PCP a combination of caspofungin together with low dose co-​trimoxazole (at a dose normally used for prophylaxis) was more effective than high-​dose co-​trimoxzole alone. Preliminary data in humans suggests a combination of caspofungin with low dose co-​trimoxazole is clinically effective and is associated with a low incidence of co-​trimoxazole-​associated adverse events. Rtt109 is a fungus-​specific histone acetyltransferase, recently charac- terized in Pneumocystis jirovecii. This protein plays a significant role in the virulence of pathogenic fungi by reducing genomic instability; Rtt109-​knockout strains have significantly attenuated pathogenesis in animal models. This provides an attractive molecular target for future drug development. FURTHER READING Carmona EM, Limper AH (2011). Update on the diagnosis and treatment of Pneumocystis pneumonia. Ther Adv Respir Dis, 5, 41–​59. Miller RF, Huang L, Walzer PD (2013). Pneumocystis pneumonia as- sociated with human immunodeficiency virus. Clin Chest Med, 34, 229–​41. Morris A, et al. (2004). Current epidemiology of Pneumocystis pneu- monia. Emerg Infect Dis, 10, 1713–​20. National Institutes of Health, AIDSinfo (2013). Guidelines for preven­ tion and treatment of opportunistic infections in HIV-​infected adults and adolescents, 2013. http://​www.aidsinfo.nih.gov Redhead SA, et al. (2006). Pneumocystis and Trypanosoma cruzi: no- menclature and typifications. J Eukaryot Microbiol, 53, 2–​11. Thomas CF, Limper AH (2004). Pneumocystis pneumonia. N Engl J Med, 350, 2487–​98. Walzer PD, Cushion MT (eds) (2004). Pneumocystis carinii pneu- monia, 3rd edition. Marcel Dekker, New York, NY. 8.7.6  Talaromyces (Penicillium) marneffei infection Romanee Chaiwarith, Khuanchai Supparatpinyo, and Thira Sirisanthana ESSENTIALS Talaromyces (formerly Penicillium) marneffei infection is very rare in the immunocompetent but one of the most common opportunistic infections in HIV-​infected people in Southeast Asia, north-​eastern India, southern China, Hong Kong, and Taiwan. Presentation is usually with fever, chills, lymphadenopathy, hepatomegaly, and splenomegaly, with skin lesions—​most commonly papules with central necrotic umbilication—​in two-​thirds of cases. Diagnosis is made by microscopy of bone marrow aspirate or biopsy specimens. Standard treatment, which is usually effective, is with amphotericin B followed by itraconazole. Introduction Talaromyces (formerly Penicillium) marneffei was first isolated from bamboo rats Rhizomys sinensis in Vietnam in 1956. The fungus is endemic in Southeast Asia, north-​east India, south China, Hong Kong, and Taiwan. Fewer than 40 cases of infec- tion with T. marneffei were reported before the HIV epidemic. Since then, the incidence of disseminated T.  marneffei infec- tion has increased markedly. This increase is mainly due to in- fection in patients immunocompromised by HIV. Most patients have been reported from Thailand, Vietnam, Hong Kong, and Taiwan. Cases have also been reported in HIV-​infected indi- viduals from the United States of America, Europe, Japan, and Australia following visits to the endemic region. T.  marneffei infection has also been reported in HIV-​negative immunocom- promised patients (e.g. solid organ and bone marrow transplant recipients). Recently, the increased number of patients with adult-​onset immunodeficiency in Southeast Asia, especially in Thailand and Taiwan, means that these patients are at risk for T. marneffei infection. Aetiology T. marneffei is the only dimorphic fungus of the genus Talaromyces (formerly Penicillium). The fungus grows in a mycelial phase at 25°C on Sabouraud dextrose agar. Mould-​to-​yeast conversion is achieved by subculturing the fungus onto brain-​heart-​infusion agar and incubating at 37°C. Microscopic examination of the mycelial form shows typical structures of the genus Talaromyces; examination of the yeast form reveals unicellular, pleomorphic, ellipsoidal-​to-​rectangular cells (2 μm × 6 μm in dimension) that divide by fission and not by budding. Natural history Many features of the natural reservoir, mode of transmission, and natural history of T.  marneffei infection remain unknown. The fungus was isolated from several species of bamboo rats in the en- demic area. Since the bamboo rats usually live near the forest and have limited contact with people, it is believed that both humans and bamboo rats are infected with T. marneffei from a common source, rather than by direct exposure to bamboo rats. By analogy with other endemic systemic mycosis, such as histoplasmosis, it is likely that T. marneffei conidia are inhaled from a contaminated reservoir in the environment and subsequently disseminate from the lungs if and when the host becomes immunosuppressed. The

section 8  Infectious diseases 1376 disease is significantly more likely to occur in the rainy season, suggesting that there may be an expansion of the environment reservoirs with favourable conditions for growth during these rainy months. In endemic areas, it is likely that a certain proportion of the population is infected, but remains asymptomatic. Patients have been reported with long periods of asymptomatic infection before presentation with clinical T. marneffei infection. In other cases, the clinical manifestation of T.  marneffei infection occurred within weeks of exposure to the fungus. A study from Vietnam using a mathematical model estimated that the incubation period of this fungal infection was approximately 1 week. Clinical features Most patients with T. marneffei infection have already been in- fected with HIV and usually present late in the course of the HIV disease. The patient’s CD4+ cell count at presentation is typically 100 cells/​μl or less. Commonly, they present with symptoms and signs of infection of the reticuloendothelial system. These include fever, chills, lymphadenopathy, hepatomegaly, and splenomegaly. Cough, dyspnoea, and lung crepitations may be present. Other manifestations are secondary to dissemination of the fungus via the bloodstream. Cutaneous and subcutaneous lesions are ob- served in up to 80% of the patients. As in other systemic mycoses, such as histoplasmosis or paracoccidioidomycosis, skin lesions resemble molluscum contagiosum. (Fig. 8.7.6.1). They may break down and bleed (Fig. 8.7.6.2) while some larger lesions become in- durated and appear infarcted. Mucosal and palatal lesions are also seen (Fig. 8.7.6.3). Arthritis and osteomyelitis are not uncommon. Cases with mesenteric lymphangitis, colitis, genital or oropharyn- geal ulcer, retropharyngeal abscess, brain abscess, or pericarditis have been reported. HIV-​negative immunocompromised patients with T.  marneffei infection are less likely to have fever, splenomegaly, unbilicated skin lesions, and fungaemia, but more likely to have bone and joint infec- tions, with a longer duration of illness before the diagnosis can be made. Subcutaneous nodules, with or without subsequent abscess formation, are more commonly seen. Biochemical and haematological laboratory findings are non-​ specific and include elevation of liver enzymes, anaemia, and leucocytosis. The chest radiograph may show diffuse interstitial, localized alveolar, or diffuse alveolar infiltrates. Cases with chest radiographs showing cavitary lesions or lung masses have been re- ported (Fig. 8.7.6.4). Fig. 8.7.6.1  T. marneffei in an HIV-​infected Thai patient: typical molluscum-​like lesions. Copyright G. Watt, Bangkok, Thailand. Fig. 8.7.6.2  Bleeding into T. marneffei skin lesions. Copyright D. Walsh. Fig. 8.7.6.3  T. marneffei palatal lesions. Copyright D. Walsh.

1377 Diagnosis Diagnosis depends on familiarity with the clinical syndrome and a high index of suspicion. Presumptive diagnosis can be simply made by microscopic examination of Wright-​stained samples of bone marrow aspirate, touch smears of the skin biopsy specimen, and/​ or the lymph-​node biopsy specimen. Many intracellular and extra- cellular basophilic, spherical, oval, and elliptical yeast cells can be seen with this technique, some of which have clear central septation, a characteristic feature of T. marneffei (Fig. 8.7.6.5). The diagnosis is confirmed by histopathological sections and/​or by culturing the fungus from the blood, skin biopsy specimens, bone marrow, or lymph nodes. Cases of T. marneffei infection can clinically resemble tuberculosis, histoplasmosis, and cryptococcosis. Tests to detect the antibody or antigen of T. marneffei as well as tests based on the polymerase chain reaction have been developed. Clinical trials are needed to show their usefulness in the diagnosis of active T. marnef­ fei infection and in predicting relapses. They might also be used to identify HIV-​infected individuals who are infected with T. marnef­ fei but are still asymptomatic. These individuals might then benefit from pre-​emptive treatment with an antifungal agent. Treatment T. marneffei infection is a potentially fatal disease. The mortality rate is high in patients with delayed diagnoses. The fungus is sensitive to azoles, flucytosine, and amphotericin B. Among azoles, the fungus is sensitive to ketoconazole, miconazole, and itraconazole, but less sensitive to fluconazole. In HIV-​infected patients, the recommended Fig. 8.7.6.4  Pulmonary lesion in an HIV-​infected patient from Hong Kong. Copyright D. A. Warrell. (a) (b) Fig. 8.7.6.5  Microscopic appearance of T. marneffei yeasts in (a) skin biopsy and (b) bone marrow aspirate, showing characteristic septation. Copyright Thira Sirisanthana. 8.7.6  Talaromyces (Penicillium) marneffei infection

8.7.7 Microsporidiosis 1378

8.7.7 Microsporidiosis 1378

section 8  Infectious diseases 1378 treatment is amphotericin B 0.6 mg/​kg per day intravenously for 2 weeks, followed by itraconazole 400 mg/​day orally for 10 weeks. Patients with less severe disease can be initially treated with oral itraconazole for 12 weeks. In the latter case, itraconazole 600 mg/​ day in 2–​3 divided doses may be considered in the first 3 days be- fore reducing to 400 mg/​day. Most patients respond well, with reso- lution of fever and other signs of infection, and clearance of fungus from bloodstream within the first 2 weeks. In patients with severe immunodeficiency (i.e. CD4+ cell count <50 cells/​μl), antiretroviral therapy should be initiated as soon as possible. After successful pri- mary treatment, HIV-​infected patients should be given 200 mg/​day of itraconazole orally as secondary prophylaxis for life or until im- mune recovery after antiretroviral therapy (i.e. CD4+ cell counts of 200 cells/​μl or above for at least 6 months). For HIV-​uninfected indi- viduals, the optimal duration of treatment and the role of secondary prophylaxis remain unknown. FURTHER READING Chaiwarith R, et al. (2007). Discontinuation of secondary prophylaxis against penicilliosis marneffei in AIDS patients after HAART. AIDS, 21, 365–​7. Deng Z, et al. (1998). Infection caused by Penicillium marneffei in China and Souhtheast Asia: review of eighteen published cases and report of four more Chinese cases. Rev Infect Dis, 10, 640, 52. Kawila R, et  al. (2013). Clinical and laboratory characteristics of penicilliosis marneffei among patients with and without HIV infec- tion in Northern Thailand: a retrospective study. BMC Infect Dis, 13, 464. Le T, et al. (2011). Epidemiology, seasonality, and predictors of out- come of AIDS-​associated Penicillium marneffei in Ho Chi Minh City, Vietnam. Clin Infect Dis, 52, 945–​52. Sirisanthana T, Supparatpinyo K (1998). Epidemiology and manage- ment of penicilliosis in human immunodeficiency virus-​infected patients. Int J Infect Dis, 3, 48–​53. Supparatpinyo K, et al. (1994). Disseminated Penicillium marneffei infection in Southeast Asia. Lancet, 344, 110–​13. Supparatpinyo K, et al. (1998). A controlled trial of itraconazole to prevent relapse of Penicillium marneffei infection in patients in- fected with the human immunodeficiency virus. N Engl J Med, 339, 1739–​43. 8.7.7  Microsporidiosis Louis M. Weiss ESSENTIALS Microsporidia are obligate intracellular eukaryotic pathogens re- lated to the Fungi that can infect both vertebrates and inverte- brates. They were first identified about 150 years ago as the cause of pebrine, a disease of silkworms, with the description of Nosema bombycis in these economically important insects. They were first described in mammalian tissue samples about 75 years ago and starting 30 years ago they were recognized as pathogenic organ- isms responsible for a diarrhoeal syndrome in patients with AIDS. In addition to the gastrointestinal tract, it is now appreciated that microsporidia can infect virtually any organ system. The phylum Microsporidia contains about 1400 species, distributed in 200 genera, and 15 microsporidian species have been reported in human infections. Microsporidia are found in surface water and it appears that at least some cases are due to ingestion of spores of the causative organism in water or food. In addition, many of the species seen in humans are also seen in various animals, sug- gesting that zoonotic transmission of this infection occurs. Clinical manifestations are most frequently reported in patients with ad- vanced immune suppression, such as patients with HIV infection, and include diarrhoea and wasting syndrome. This is usually due to infection of the small intestinal mucosa by either Enterocytozoon bieneusi or Encephalitozoon intestinalis. Microsporidia also cause
keratoconjunctivitis in both immune competent and immune compromised hosts. These corneal infections have been most commonly caused by either Encephalitozoon hellem or Vittaforma corneae. Other reported manifestations of infection include acalculous cholecystitis, sinusitis, cough/​dyspnoea, urethritis, myositis, and encephalitis. Gastrointestinal microsporidiosis is diagnosed by microscopic examination of faecal specimens, after appropriate staining for microsporidian spores, or by detection of microsporidian DNA in faecal specimens. Aside from sup- portive care, albendazole is an effective drug for treating infec- tion due to Encephalitozoonidae. Ent. bieneusi does not respond to albendazole and fumagillin is an effective drug for diarrhoeal syndromes due to this pathogen, although it is not commercially available for humans. Topical fumagillin solutions have been used successfully to treat microsporidian keratoconjunctivitis. In HIV-​ infected patients, remission of microsporidiosis can be achieved by immune restoration due to antiretroviral drug treatment. As transmission can occur via food or water, prevention can be achieved by safe food and water practices, such as boiling water. As microsporidian spores are minuscule, water filtration methods need to be able to remove organisms that are 1 to 3 µm in size to be an effective preventative measure. Introduction The class or order Microsporidia was elevated to the phylum Microspora by Sprague in 1977 and in 1998 Sprague and Becnel suggested that the term Microsporidia instead be used for the phylum name. While microsporidia were historically considered ‘primitive’ protozoa, molecular phylogenetic analysis has led to the recognition that these organisms are not primitive but degenerate, and that they are related to the Fungi, either as a basal branch of the Fungi or as a sister group. Microsporidia infect almost all animal phyla, including other protists. They are important agricultural parasites in insects, fish, laboratory rodents, and mammals. Several species of microsporidia are used as biological control agents for destructive species of insects. In their hosts, most microsporidia in- fect the digestive tract, but infections of almost all organ systems have been documented. Microsporidiosis occurs in both immune

8.7.7  Microsporidiosis 1379 compromised and immune competent hosts. These pathogens can be transmitted by food or water and are likely zoonotic. Several different genera and species of microsporidia cause disease in hu- mans. The genera infecting humans include Nosema, Vittaforma Pleistophora, Encephalitozoon, Enterocytozoon, Trachipleistophora, Anncaliia, Tubulonosema, Endoreticulatus, and Microsporidium. Diagnosis can be made by finding characteristic spores in body fluids (e.g. stool, urine, conjunctival scraping, and so on) using stains, such as chromotrope 2R or Uvitex 2B. Definitive identifica- tion of the microsporidia causing an infection can be done using ultrastructural examination or molecular techniques. Historical perspective Before the HIV/​AIDS pandemic, most of the literature on microsporidian infections dealt with such infections in non​human hosts (e.g. silk moths, honeybees, fish, and rabbits). Microsporidia were first described in 1959 as being human pathogens when they were found in a child with encephalitis. In the immune suppressed host, for example, those treated with immune suppressive drugs or infected with human immunodeficiency virus (particularly those at advanced stages of the disease), microsporidia can produce a wide range of clinical diseases. Reports of diarrhoeal syndromes associ- ated with microsporidiosis and HIV infection were first reported in 1985, and the number of articles describing human disease increased rapidly after 1990. Since the advent of combination antiretroviral therapy, the incidence of microsporidiosis has declined in the HIV infected population. In addition to gastrointestinal tract involvement, immune compromised patients with encephalitis, ocular infection, sinusitis, myositis, and disseminated infection are well described in the literature. The burgeoning literature on human microsporidiosis in HIV-​infected individuals has been recently complemented by an increasing awareness of microsporidia infections in immune compe- tent people, most notably infections resulting in keratoconjunctivitis. Although initially regarded as rare, microsporidia are now believed to be common enteric pathogens that cause self-​limited or asymp- tomatic infections in most immune competent hosts. Aetiology, pathogenesis, and pathology Microsporidia are obligate intracellular parasites, whose lifecycle comprises an extracellular stage (spore) and reproductive stages occurring in host cells. They are eukaryotes containing a nucleus with a nuclear envelope, an intracytoplasmic membrane system, chromosome separation on mitotic spindles, vesicular Golgi, and a mitochondrial remnant organelle called a mitosome. Microsporidia are ubiquitous in the environment and infect almost all inverte- brates and vertebrates. They form characteristic unicellular spores (Fig. 8.7.7.1) which are environmentally resistant. Microsporidia are currently classified based on their ultrastructural features, including the size and morphology of the spores, number of coils of the polar tube, developmental life cycle, and host-​parasite relation- ship. The microsporidia infecting humans have spores that range from 1.0 to 4.0 μm in size and are usually ovoid. Spore structure is characteristic of the phylum; consisting of an electron-​dense, pro- teinaceous exospore, an electronlucent endospore composed of chitin and protein, and an extrusion apparatus that consists of a polar tube attached to the inside of the anterior end of the spore by an anchoring disc and, depending on the species, the polar tube has 4 to approximately 30 coils within the spore. Spores induce in- fection by a high velocity extrusion of their polar tube bringing the sporoplasm into intimate contact with the host cell thereby forming a channel for delivering sporoplasm (spore contents) into its target host cell. The overall process of germination, formation of the polar tube and delivery of the sporoplasm into the host cell functions es- sentially like a hypodermic needle. Replication of the parasite and subsequent production of spores occurs in host cells. When spores are phagocytosed they can also germinate infecting adjacent cells. Microsporidia can invade and survive in macrophages and den- dritic cells allowing their dissemination within the host. Microsporidia were historically considered ‘primitive’ protozoa. Molecular phylogenetic data, however, indicate that microsporidia are related to the Fungi (either as a basal branch of the Fungi or as a sister group within the Cryptomycota) and are not primitive but degenerate eukaryotes. Molecular phylogeny has also led to the rec- ognition that traditional phylogeny based on structural observations may not reflect the relationships among the various microsporidian species and genera. The genome size of the microsporidia varies from 2.3 to 19.5 Mbp, with the genomic size of the Encephalitizoonidae being under 3.0Mbp, making them among the smallest eukaryotic nuclear genomes so far identified. There are almost no introns in the compact genome of Enc. cuniculi, the gene density is high, and proteins are shorter than the corresponding genes in Saccharomyces cerevisiae. Genome data for the Microsporidia are available on MicrosporidiadB (http://​microsporidiadb.org/​micro/​), which is part of the EuPathdB (http://​eupathdb.org/​ eupathdb/​) website. In HIV-​infected patients, diarrhoea is the clinical feature that has been most frequently associated with microsporidiosis. In par- ticular, this symptom has been historically associated with infections by either Ent. bieneusi or Enc. intestinalis. The diarrhoea reported in these infections is a consequence of infection of the mucosa of the small intestine and, in advanced HIV disease, is often associated with a wasting syndrome and historically with an elevated mortality rate. Parasitization of the intestinal mucosa can be seen on micro- scopic examination of biopsy specimens. Microsporidia that infect human subjects are listed in Table 8.7.7.1. Human infections with microsporidia other than with Ent. bieneusi, Encephalitozoon spe- cies, or Vittaforma corneae have been reported in only a few case reports for each organism. Polaroplast membranes Nucleus Endospore Exospore Anchoring disc (polar sac) Straight part of polar tube Polar tube coil Plasmalemma Fig. 8.7.7.1  Diagram of a microsporidian spore, showing internal structure. Courtesy of Professor Elizabeth U. Canning. Modified from Canning EU, Hollister WS (1992). Human infections with microsporidia. Rev Med Microbiol, 3, 35–​42, with permission.

section 8  Infectious diseases 1380 The immune suppressive states (e.g. AIDS and transplantation) associated with microsporidiosis in humans are those that inhibit cell-​mediated immunity. In mice, interferon-​γ and interleukin-​12 (IL-​12) contribute to protective immunity against Enc. intestinalis and Enc. cuniculi infections. Adoptive transfer of sensitized syn- geneic T-​enriched spleen cells protects athymic or severe com- bined immunodeficiency (SCID) mice against lethal Enc. cuniculi infection. The major killing mechanism exhibited by CD8+ T cells in murine microsporidosis models is due to the perforin pathway (e.g. mice lacking perforin die when infected with Enc. cuniculi). Infection with Enc. cuniculi in many mammals re- sults in chronic infection with persistently high antibody titres and ongoing inflammation (e.g. persistent encephalitis in rabbits and chronic renal disease and congenital transmission in foxes). Transmission of infection to transplant recipients by kidneys used in renal transplantation suggests that chronic infection is also seen in humans. Epidemiology Most infections due to microsporidia are transmitted by oral inges- tion of spores, with the site of initial infection being the gastrointes- tinal tract. Microsporidian spores are commonly found in surface water, and human pathogenic species have been found in muni- cipal water supplies, tertiary sewage effluent, and ground water. Water contact has been found to be an independent risk factor for microsporidiosis (e.g. an outbreak of V. corneae keratoconjunctivitis was associated with hot spring exposure). Risk factors for Ent. bieneusi infection, in a population of HIV-​infected patients sur- veyed in France, included swimming in a pool in the 12 months before the survey. In rural Mexican households, faecal excretion of Encephalitozoon spores was associated with the use of unboiled water for drinking and for preparing food. Spores are viable for a long time in water, but can be killed by boiling. Microsporidia are also foodborne pathogens, for example, an outbreak of foodborne Ent. bieneusi infection (characterized by abdominal pain, nausea, and diarrhoea) occurred in Sweden in 2009. Viable infective spores of Microsporidia are present in several body fluids (e.g. stool, urine, respiratory secretions) during infection, suggesting that person-​to-​person transmission can occur. Heavy parasitization of respiratory tract epithelial cells with Enc. hellem, in at least one HIV-​infected patient examined at autopsy, raises the possibility that some microsporidian infections can be acquired by inhaling spores. Congenital transmission has been seen in many mammals, but has not been reported in humans. Many microsporidia are also probably zoonotic infections in hu- mans. Spores and/​or DNA of potentially human-​infective strains of Ent. bieneusi and of Enc. hellem have been found in faecal samples and intestinal contents from pigeons. In addition, spores and/​or DNA of Encephalitozoon spp. have been identified in faecal samples of aquatic animals. Spores of Enc. intestinalis have been identified in faecal specimens from dogs, pigs, goats, cows, and donkeys. Ent. bie­ neusi has been identiied in faecal samples from dogs, cats, pigs, goats, cows, horses, and rhesus monkeys. House mice in central Europe have been found to be infected with Ent. bieneusi, Enc. hellem, and Enc. cuniculi. Genotyping of microsporidia is beginning to shed light on possible interspecies transmissibility of these organisms. For example, Ent. bieneusi organisms excreted by rhesus monkeys in a public park in China included genotypes known to infect human subjects. Contact with pigs is a risk factor for infection by a porcine-​ associated genotype of Ent. bieneusi, among human inhabitants of a rural area of China. Several human pathogenic microsporidia (e.g. Anncaliia algerae and Trachipleistophora hominis), can also infect mosquitoes, suggesting that vector borne transmission might also occur for some of the human pathogenic microsporidia. Although initially regarded as rare, microsporidia are now be- lieved to be common enteric pathogens that cause self-​limited or asymptomatic infections in normal hosts. Cases of microsporidiosis have been identified from all continents except Antarctica. Surveys of pathogens seen in stool samples in Africa, Asia, South America, and Central America have demonstrated that microsporidia are often found during careful stool examinations. In immune deficient hosts, most reported cases have manifested as diarrhoea with wasting syndrome and disseminated infection. Microsporidiosis prevalence rates varied between 2% and 70%, in 25 studies conducted on pa- tients with HIV infection from 1989 to 1998, before the widespread use of combination highly active antiretroviral therapy. The rates varied depending on the symptoms of the population studied and the diagnostic techniques used. Overall, these studies identified 375 E. bieneusi infections among 2400 patients with chronic diar- rhoea, for a prevalence of 15% in this population. The aggregate data Table 8.7.7.1  Species of microsporidia that infect humans Species Reported sites of infection Enterocytozoon bieneusi Small intestinal epithelium, gallbladder epithelium, rarely in respiratory tract and maxillary sinus Encephalitozoon (formerly Septata) intestinalis Intestinal epithelium, gallbladder epithelium, paranasal sinuses, respiratory tract, liver, kidney, pituitary, conjunctiva. Encephalitozoon hellem Corneal epithelium, respiratory tract, kidney, paranasal sinuses Encephalitozoon cuniculi Kidney, urinary bladder, duodenal mucosa, conjunctiva, respiratory tract, adrenal glands, brain, heart, spleen, lymph nodes, cerebrospinal fluid Vittaforma corneae (formerly Nosema corneum) Corneal stroma, urinary tract Trachipleistophora hominis Skeletal muscle, conjunctiva, corneal stroma, nasopharynx Trachipleistophora anthropophthera Brain, kidney, heart, pancreas, thyroid, parathyroid glands, liver, spleen, lymph nodes, bone marrow, cornea Pleistophora ronneafiei Skeletal muscle Anncaliia algeraea Skeletal muscle, skin, corneal stroma Anncaliia vesicularuma Skeletal muscle Anncaliia connoria Generalized Tubulinosema sp. Skeletal muscle Tubulinosema acridophagus Generalized Nosema ocularum Corneal stroma ‘Microsporidium ceylonensis’ Corneal stroma ‘Microsporidium africanum’ Corneal stroma a Organisms in the genus Anncaliia were formerly designated by the generic names Brachiola and Nosema.

8.7.7  Microsporidiosis 1381 available have demonstrated microsporidia demonstrate strength of association, coherence, and reproducibility with respect to being causative for a diarrhoeal syndrome. Studies have demonstrated that asymptomatic carriage can occur in immune compromised patients. There are numerous reports of microsporidiosis in patients who have undergone kidney, liver, heart-​lung, pancreas, or bone marrow transplantation. There has been speculation that microsporidiosis in immune deficient subjects might sometimes reflect activation of microsporidian infection acquired before the onset of immunodefi- ciency. Three recipients of transplanted organs (lung and kidneys) from one donor developed Enc. cuniculi infection caused by an iden- tical genotype of this organism; an archival serum sample from the organ donor had a high titre of antibody to Enc. cuniculi, suggesting that the donated organs were the source of the infection seen in the recipients. The phylum Microsporidia (Microspora) contains at least 1400 species distributed into over 200 genera, of which the following have been demonstrated in human disease (Table 8.7.7.1): Nosema (N.  corneum renamed Vittaforma corneae; N.  algerae reclassi- fied initially as Brachiola algerae and now as Anncaliia algerae), Pleistophora, Encephalitozoon, Enterocytozoon, Septata (re- classified as Encephalitozoon1), Trachipleistophora, Brachiola Anncaliia, Tubulonosema, Endoreticulatus, and Microsporidium. Microsporidium is a non​taxonomic genus created for microsporidia of unclear identity. Encephalitozoon hellem (Enc. hellem) has been associated with superficial keratoconjunctivitis, sinusitis, respira- tory disease, prostatic abscesses, and disseminated infection. Encephalitozoon cuniculi (Enc. cuniculi) has been associated with hepatitis, encephalitis, and disseminated disease. Encephalitozoon (Septata) intestinalis is associated with diarrhoea, disseminated in- fection, and superficial keratoconjunctivitis. Nosema, Vittaforma, and Microsporidium have been associated with stromal keratitis associated with trauma. Pleistophora, Anncaliia, Tubulonosema, Endoreticulatus, and Trachipleistophora have been associated with myositis sometimes with associated disseminated disease. Trachipleistophora has been associated with encephalitis, keratitis, and disseminated disease. Enterocytozoon bieneusi (Ent. bieneusi), originally described in humans, is associated with malabsorption, diarrhoea, and cholangitis. Prevention There are limited data on effective preventative strategies for microsporidiosis, however, the most effective prophylaxis for im- mune compromised patient is restoration of immune function. There are no data that demonstrate an effective prophylactic medi- cation that prevents infection in humans. It is probable that the usual sanitary measures that prevent contamination of food and water will decrease the chance of infection; these measures for water include boiling water, the use of bottled water, ultraviolet treatment, chlor- ination, or the use of filters that remove particles of at least 1 µm in size. Both immune capture and molecular methods have been de- veloped to evaluate the presence of microsporidia in water samples. Hand washing and general hygienic habits reduce the chance of con- tamination of conjunctiva and cornea with microsporidian spores. Spores can be rendered non​infectious by a 30-​minute exposure to most common disinfectants, so the procedures used to clean most hospital rooms should be sufficient to limit infection. Spores are also killed by the methods commonly used for sterilization. Given the presence of microsporidia in the respiratory secretions of pa- tients with disseminated microsporidiosis, it is reasonable to isolate infected patients from contact with immune suppressed patients to prevent transmission. It is reasonable to screen close contacts of pa- tients with microsporidiosis for the presence of these organisms. Clinical features Clinical features of microsporidian infections reflect the anatomical site colonized by the microsporidia (Table 8.7.7.1). Watery diar- rhoea, weight loss, and fat malabsorption have been reported in HIV-​ infected patients with intestinal microsporidiosis. Microsporidian infection of the gallbladder and billiary system can result in acalculous cholecystitis, which might necessitate cholecystectomy, or sclerosing cholangitis or AIDS cholangiopathy. Infection of the paranasal sinuses and respiratory tract can result in symptoms of si- nusitis, cough, and dyspnoea. Symptomatic urethritis and prostatitis have been ascribed to Encephalitozoon spp. infection. Encephalitis with mass lesions has been seen with both Encephalitozoon and Trachipleistophora infections associated with headache, cognitive impairment, nausea, vomiting, focal neurological deficit, and epi- leptic seizures. Both Trachipleistophora and Anncaliia have been re- ported in cases of myositis with muscle pain, tenderness, weakness, and wasting. Disseminated microsporidiosis can present as a fever of unknown origin with negative blood cultures. Hepatitis with liver function abnormalities can be seen in disseminated infection. There have been case reports of microsporidian infections causing nodular skin lesions. Microsporidian infection of the conjunctiva and corneal epi- thelium causes symptoms of keratoconjunctivitis (e.g. foreign body sensation in the eye, ocular discomfort and redness, photo- phobia, blurred vision, and sometimes reduced visual acuity). Microsporidian infections of the corneal stroma lead to reduced visual acuity, with or without corneal ulceration. Microsporidian keratoconjunctivitis, in individuals without HIV infection, has been recognized increasingly in Asia. Exposure to mud after recent rain- fall appears to be a risk factor for this microsporidian eye infection. In India, Vittaforma corneae DNA has been demonstrated (by poly- merase chain reaction testing) in corneal scrapings from patients with keratitis. This organism has been described as a cause of kera- titis resulting from bathing in hot springs in Taiwan, and has also caused keratoconjunctivitis in rugby football players in Singapore. Laboratory diagnosis Intestinal microsporidiosis can be diagnosed by microscopic exam- ination of faecal samples. The spores (which are ovoid) can be de- tected by microscopy after staining with chromotrope 2R or with optical brighteners such as Uvitex 2B or Calcofluor White M2R (which bind to chitin in the spores, resulting in fluorescence), or with fluorescent antibodies directed against the spores. In a study that examined 50 electron microscopy-​proven microsporidia-​positive stool specimens, both the chromotrope 2R and optical brighteners identified 100% of specimens if at least fifty 100× objective fields

section 8  Infectious diseases 1382 were examined. The limit of detecting microsporidia by these tech- niques appears to be 50 000 organisms/​ml. Spores of Ent bieneusi are 1.5 μm × 0.9 μm whereas those of Encephalitozoonidae are larger, ca. 2.5 μm × 1.5 μm. Definitive identification of a microsporidium causing an infection can be done by ultrastructural examination (Fig. 8.7.7.2) or molecular techniques. Patients with diarrhoea or keratoconjunctivitis should have urine examined to look for dis- seminated infection. This has therapeutic implications because microsporidia that disseminate, such as Encephalitozoon spp, are sensitive to albendazole, whereas those that do not dissem- inate, such as Ent. bieneus), are resistant to albenazole. Species specific diagnosis can be obtained by electron microscopy or by molecular tests. Generally, it is easier to identify microsporidian spores in body fluids other than in stool because of the absence of bacteria and debris, which can be confused with microsporidian spores. Because these infections usually involve mucosa or epithe- lium, cytologic preparations that have been useful for diagnosing infection include intestinal and biliary epithelium, epithelium of the cornea and conjunctivae, epithelium of the sinonasal and tracheobronchial regions, renal tubular epithelium, and urothelium. Histologically, microsporidia can be seen in sections prepared from tissue fixed using routine procedures by employing a modified tissue chromotrope 2R (Fig. 8.7.7.3), tissue Gram stain (Brown–​Hopp or Brown–​Brenn) or Luna stain. Serology has not proven useful for the diagnosis of active infection. Several molecular diagnostic tests have been developed for pathogenic microsporidia and these tests are available from some reference laboratories. Treatment and prognosis Among the compounds tested in vitro and in vivo for treatment of microsporidiosis, fumagillin and albendazole have demonstrated the most consistent activity and have been demonstrated to have clinical efficacy in human infections with various microsporidia. Encephalitozoonidae infections can be treated with albendazole. In contrast, albendazole is not an effective treatment for Ent. bie­ neusi infection. In a small controlled trial, HIV-​infected patients with Enc. intestinalis infection were treated with albendazole (400 mg orally twice daily) or with placebo. Albendazole treatment led to clearance of gastrointestinal Enc. intestinalis infection in this study. Uncontrolled trials and anecdotal case reports describe par- tial or complete resolution of symptoms (diarrhoea, sinusitis, and keratoconjunctivitis) in patients with Enc. intestinalis, Enc. hellem, or Enc. cuniculi infection following albendazole treatment. Pregnancy is a contraindication to albendazole treatment. Microsporidiosis is seen most commonly in immune compromised hosts, particularly in those with HIV infection and CD4+ cell counts lower than 50/​μl. Clinical studies have demonstrated that improved immune function can result in the clinical response of patients with gastrointestinal microsporidiosis, with elimination of the organism and normalization of the intestinal architecture. In HIV-​infected pa- tients with Ent. bieneusi infection, remission of this microsporidian in- fection can be achieved by treatment of the HIV disease with effective antiretroviral therapy that leads to restoration of immune competence, as evidenced by a raised CD4+ count and reduced HIV load. Clinical trials and case reports in patients with AIDS or organ transplantation Fig. 8.7.7.2  Transmission electron micrograph of jejunal biopsy from a patient with AIDS and Encephalitozoon intestinalis infection. The microvillus border (epithelial surface) is at the top of the photograph. Epithelial cells and lamina propria leukocytes are heavily infected with Enceph. intestinalis (arrows). Courtesy of the Electron Microscopy and Histopathology Unit, London School of Hygiene and Tropical Medicine. From Croft SL, Williams J, McGowan I (1997). Intestinal microsporidiosis. Semin Gastrointest Dis, 8, 45–​55, with permission. Fig. 8.7.7.3  Light micrograph of intestinal villus biopsy (plastic section) from a patient with a gastrointestinal infection with Encephalitozoon intestinalis. Note the presence of spores (arrows) on the apical and basal side of the epithelial cells as well as in the lamina propria. From Wittner M and Weiss LM (eds.) The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, DC, 1999; with permission.

8.7.7  Microsporidiosis 1383 have demonstrated that fumagillin (20 mg orally thrice daily) is ef- fective for the treatment gastrointestinal infection with Ent. biene­ usi. Fumagillin is also active against other microsporidia including Encephalitozoonidae. The major limiting toxicity of systemic therapy with fumagillin has been thrombocytopenia which has been reversible on stopping treatment. Transient clinical remission has been reported in Ent. bieneusi with furazolidone or nitazoxanide (1000 mg twice daily) treatment. Individual patients infected with Trachipleistophora hominis, Anncaliia vesicularum, or Anncaliia algerae have demon- strated clinical improvement after treatment with albendazole com- bined with azoles (e.g. itraconazole or voriconazole). A solutions of the soluble salt fumagillin bicylohexylammonium (3 mg/​ml which is equivalent to fumgaillin, 70 µg/​ml) applied topic- ally has been demonstrated to be non​toxic to the cornea and effective for the treatment of ocular microsporidiosis. It should be appreci- ated that ocular infection can be associated with systemic infection and if microsporidia are demonstrated in urine or nasal smears then oral albendazole should also be administered. Successful treat- ment of microsporidian keratoconjunctivitis has also been reported with voriconazole eye drops. In a study of the natural history of microsporidian keratoconjunctivitis in India in immune competent hosts it was observed that this infection resolves spontaneously in many patients. HIV-​negative patients with microsporidian infection of the cor- neal stroma have been treated by corneal transplantation, with results that have ranged from failure (opacification of the transplant) to apparent success, as judged by transparency of the graft 6 months after transplantation. FURTHER READING Didier ES, Weiss LM (2011). Microsporidiosis: not just in AIDS pa- tients. Curr Opin Infect Dis, 24, 490–​5. Didier ES, et al. (2005). Therapeutic strategies for human microsporidia infections. Expert Rev Anti Infect Ther, 3, 419–​34. Field AS (2002). Light microscopic and electron microscopic diag- nosis of gastrointestinal opportunistic infections in HIV-​positive patients. Pathology, 34, 21–​35. Franzen C (2008). Microsporidia: a review of 150 years of research. The Open Parasitology Journal, 2, 1–​34. Ghosh K, Weiss LM (2009). Molecular diagnostic tests for micro­ sporidia. Interdiscip Perspect Infect Dis, 2009, 926521. Ghosh K, Weiss LM (2012). T cell response and persistence of the microsporidia. FEMS Microbiol Rev, 36, 748–​60. Molina JM, et al. (2002). Fumagillin treatment of intestinal micro­ sporidiosis. N Engl J Med, 346, 1963–​69. Sharma S, et al. (2011). Microsporidial keratitis: need for increased awareness. Surv Ophthalmol, 56, 1–​22. Weiss LM, Becnel JJ (eds) (2014). Microsporidia: pathogens of oppor­ tunity. Wiley-​Blackwell, Chichester.

8.8 Protozoa 1384

8.8 Protozoa 1384

8.8.1 Amoebic infections 1384

8.8.1 Amoebic infections 1384

8.8 Protozoa CONTENTS 8.8.1 Amoebic infections  1384 Richard Knight 8.8.2 Malaria  1395 Nicholas J. White and Arjen M. Dondorp 8.8.3 Babesiosis  1414  Philippe Brasseur 8.8.4 Toxoplasmosis  1416 Oliver Liesenfeld and Eskild Petersen 8.8.5 Cryptosporidium and cryptosporidiosis  1424 Simone M. Cacciò 8.8.6 Cyclospora and cyclosporiasis  1432 Paul Kelly and Ralph Lainson 8.8.7 Cystoisosporiasis  1436 Louis M. Weiss 8.8.8 Sarcocystosis (sarcosporidiosis)  1438 John E. Cooper 8.8.9 Giardiasis and balantidiasis  1440 Lars Eckmann and Martin F. Heyworth 8.8.10 Blastocystis infection  1449  Richard Knight 8.8.11 Human African trypanosomiasis  1451  Reto Brun and Johannes Blum 8.8.12 Chagas disease  1459 Michael A. Miles 8.8.13 Leishmaniasis  1467  Antony D.M. Bryceson and Diana N.J. Lockwood 8.8.14 Trichomoniasis  1475 Jane Schwebke 8.8.1  Amoebic infections Richard Knight ESSENTIALS Two very different groups of amoebic species infect humans. (1) Obligate anaerobic gut parasites, including the major pathogen Entamoeba histolytica, Dientamoeba fragilis (which causes relatively mild colonic involvement with diarrhoea), and eight non​pathogenic species including Entamoeba dispar. (2) Aerobic free-​living, water and soil amoebae—​these can become facultative tissue parasites in hu- mans after cysts or trophozoites are inhaled, ingested, or enter dam- aged skin or mucosae. Entamoeba histolytica infection The term amoebiasis (when unqualified) generally refers to E. histolytica infection, which is especially common in Mexico, South America, Natal, the west coast of Africa, and Southeast Asia; nearly all amoebic disease seen in temperate countries is acquired elsewhere. Transmission is faeco-​oral; following ingestion of infective cysts, a population of trophozoites becomes established in the caecum and proximal colon. Clinical features—​these range from minimal changes in bowel habit to severe dysentery or liver abscess. Onset of bowel disease is usu- ally gradual or intermittent, with initially mild constitutional upset, colicky abdominal pain, and foul-​smelling stools that always contain visible or occult blood. Less typical presentations of amoebic colitis include (1)  fulminant colitis; (2)  amoebic colitis without dysentery; (3) amoeboma—​presenting as an abdominal mass, most frequently in the right iliac fossa; (4) localized perforation and amoebic appendicitis; (5) rectal bleeding. An important complication is hepatic amoebiasis. Diagnosis, treatment, and prognosis—​examination of dysenteric stool, bowel-​wall scrapings, liver abscess aspirate, or other sam- ples in temporary wet mounts is critical, with identification of live erythrocytophagous trophozoites confirming the diagnosis of inva- sive amoebic disease. Other diagnostic methods include (1) dem- onstration of amoebal DNA in faeces/​tissues by polymerase chain reaction; (2) serology—​but seropositivity does not distinguish current and past tissue invasion. Aside from supportive care, metronidazole for 5 days is usually the first-​choice treatment, with the addition of diloxanide to eliminate infection from the bowel and so prevent re- currence of tissue invasion or transmission to others. Uncomplicated invasive intestinal disease (and uncomplicated hepatic amoebiasis) should have mortality less than 1%, but this may reach 40% for amoebic peritonitis with multiple gut perforation. Hepatic amoebiasis—​less than 50% of patients give any convincing history of dysentery and few have concurrent dysentery. Presentation is typically with fever, sweating, liver or diaphragmatic pain, weight loss, and tender hepatomegaly. Diagnosis is usually achieved by demon- stration of a (most often solitary) liver abscess on ultrasonography or CT and positive serological testing, with a therapeutic amoebicide trial generally being preferable to diagnostic needling of the liver.

8.8.1  Amoebic infections 1385 Prevention—​simple hygienic measures and health education provide considerable protection: boiling water for 5 min kills cysts. Travellers to endemic areas may need a medical check on their re- turn; but chemoprophylaxis is not appropriate. Free-​living amoebae Three genera of free-​living amoebae, Naegleria, Acanthamoeba, and Balamuthia, cause human disease. Naegleria causes a primary men- ingoencephalitis after bathing or diving or other nasal exposure to fresh water; amphotericin B is an effective drug, but most cases are fatal, partly because of diagnostic delays. Acanthamoeba causes a painful keratitis, mainly in contact lens users, which usually responds to intensive local amoebicides, although corneal grafting may be needed; it also causes a highly fatal granulomatous encephalitis in immunocompromised patients. Balamuthia causes an encephal- itis similar to that of Acanthamoeba in both immunocompromised and immunocompetent individuals; primary skin or facial lesions are common; both genera are potential donor risks in transplantation. Introduction The amoebic species infecting humans belong to two very different groups. The obligate anaerobic gut parasites include the major pathogen Entamoeba histolytica, which ranks second to malaria as the most dangerous parasite in humans; Dientamoeba fragilis, a minor pathogen; and eight non​pathogenic species including the common and important Entamoeba dispar. The second group in- cludes certain aerobic free-​living, water and soil amoebae which produce cytopathic changes in cultured cell monolayers and cerebral invasion after intranasal inoculation into mice. They can become facultative tissue parasites in humans after cysts or trophozoites are inhaled, ingested, or enter damaged skin, cornea or mucosae. All motile feeding amoebae are called ‘trophozoites’; they move with pseudopodia and divide by binary fission. The hyaline external cytoplasm, the ‘ectoplasm’, is a contractile gel that surrounds the sol endoplasm containing numerous phagocytic and pinocytic vacu- oles. Noninvasive trophozoites feed on bacteria. All species can form environmentally resistant transmissive cysts by rounding up and se- creting a chitinous cyst wall. The definitive taxonomic separation of E. dispar as a non​pathogenic species separate from E.  histolytica was made in 1993. This was based upon genomic and biochemical differences. This distinction is of fundamental importance because their cysts and non​invasive trophozoites are morphologically indistinguishable, but they are now separated by specific antigen and polymerase chain reaction (PCR) assays. All strains of E. histolytica are now regarded as pathogenic, whereas the commoner E. dispar is never pathogenic. Entamoeba histolytica infection Biology and pathogenicity Following ingestion of infective cysts, a population of tropho- zoites becomes established in the caecum and proximal colon. Some degree of tissue invasion occurs in all subjects with at least low-​titre seroconversion. Tissue invasion is frequently mild, self-​ limiting, and with minimal symptoms, but at the other end of the clinical spectrum it can lead to extensive destruction of the colonic mucosa. Parasite genotype may partly determine clinical outcome. Invasive trophozoites have a characteristic morphology; they may reach 30–​40 μm in diameter and are very active with apparently pur- poseful, unidirectional movements during which they become con- siderably elongated. Their most important diagnostic characteristic is the presence of host erythrocytes within the endoplasm, which otherwise appears clear and contains no bacteria. Trophozoites containing red blood cells are described as erythrocytophagous. Progression through tissues is by active movement, facilitated by secreted collagenase; leucocytes are drawn chemotactically towards the amoebae but most are rapidly destroyed on contact. The transmissive cystic form of the parasite is derived entirely from a commensal population within the colonic lumen. Live com- mensal amoebae measure from 10 to 20 μm in diameter, the endo- plasm is granular and contains bacteria, and the pseudopodia are blunt and movement is sluggish. Intestinal hurry from any cause, including the use of laxatives, can lead to the appearance of com- mensal trophozoites in the faeces. Cysts are spherical and measure from 11 to 14 μm in diameter; when mature, they contain four nu- clei, several chromatoid bodies that are ribosome aggregates, and a glycogen vacuole. Host factors may increase susceptibility to overt disease. Steroid therapy given systemically or locally into the rectum carries great risk, as may cytotoxic therapy. Severe amoebic bowel disease is par- ticularly common in late pregnancy and the puerperium. Before pu- berty, both sexes are equally susceptible to hepatic amoebiasis, but in adults this condition is much more common in males. Local dis- ease can also favour tissue invasion; thus, amoebic ulceration may be superimposed upon colonic and rectal cancers, or those of the uterine cervix. Colonic disease is favoured by concurrent Trichuris infection or intestinal schistosomiasis. Infection with HIV ap- pears to have little effect on colonic disease but may facilitate liver involvement. Epidemiology The incidence of disease is particularly high in Mexico, South America, Natal (South Africa), the west coast of Africa, and Southeast Asia. In most temperate countries, E. histolytica is now rare and nearly all amoebic disease seen in such countries will have been acquired elsewhere. Symptomless or convalescent carriers are the main source of infection; patients with dysentery normally pass only trophozoites in their stool and are therefore non​infectious. Cysts remain viable in the environment for up to 2 months. The in- fection is eventually self-​limiting and rarely exceeds 4 years. Tissue invasion can occur at any time during an infection but is much more common during the first 4 months; the incubation period may be as short as 7 days. E. histolytica-​associated diarrhoea can retard growth in preschool children. The incidence of invasive amoebiasis in a population is best esti- mated from seropositivity surveys. Prevalence of infection is esti- mated by specific faecal antigen or DNA; microscopic surveys for cysts are of no value as their differentiation from E. dispar is impos- sible. Zoonotic sources are uncommon but transient infections do occur in dogs, and molecular methods have confirmed infections in

section 8  Infectious diseases 1386 Malaysian rats. All modes of faeco-​oral transmission occur in amoeb- iasis. Of special importance are the food handler and contaminated vegetables; transmission by flies and drinking-​water is less common. Drinking-​water can be contaminated in the home or at surface-​water sources. Direct spread can produce outbreaks; it occurs within insti- tutions for children, people with learning difficulties, and with con- taminated colonic irrigation equipment. Household clustering is common; hand-​fed infants are frequently infected from the fingers of their mother. Contamination of piped water supplies can lead to ser- ious disease outbreaks, as happened in the Chicago hotels epidemic in 1933. Interruption of piped water supplies probably caused the re- cent outbreak in Georgia. Entamoeba infections are common among male homosexuals, but most are due to E. dispar; oro-​anal contact is probably responsible. However, invasive amoebiasis in HIV-​positive homosexual men is an emerging problem in Japan. Pathology The basic lesion is cell lysis and tissue necrosis, which, by creating lo- cally anoxic and acidic conditions, favours further penetration of the parasite. Most amoebae are seen at the advancing edge of the lesion with little inflammatory cell response. In tissue sections, amoebae stain indistinctly with haematoxylin and eosin but appear bright red with periodic acid–​Schiff stain; iron haematoxylin is necessary to show nuclear detail. Cysts of E. histolytica are never seen in tissue. Amoebic lesions of the gut are most common in the rectosigmoid and caecum but can occur anywhere in the large bowel; involve- ment may be patchy or continuous. Less commonly, the appendix or terminal ileum are affected. The initial lesions are either small, discrete erosions of the mucosa or minute crypt lesions Fig. 8.8.1.1). Unrestrained, the lesions extend through the mucosa, across the muscularis mucosa and into the submucosa, where they expand laterally to produce lesions that are typically flask shaped in cross-​ section Fig. 8.8.1.2). Further lateral spread of the submucosal lesions leads to their coalescence and, later, to denudation of over- lying mucosa. The bowel wall may become appreciably thickened. Blood vessels involved in the disease may thrombose, bleed into the gut lumen, or, in the case of portal-​vein radicles, enable dissemin- ation of amoebae to the liver. In very severe lesions, and usually in association with toxic megacolon, there is an irreversible coagulative necrosis of the bowel wall. Amoebomas are tumour-​like lesions of the colonic wall measuring up to several centimetres in length; they are most common in the caecum and may be multiple. Histologically there is tissue oedema, with a mixed picture of healing and new areas of epithelial loss and tissue destruction; round-​cell infiltration is patchy. Lesions may be annular and rarely an amoeboma initiates an intussusception; narrow, stricture-​like amoebomas may occur in the anorectal region. Amoebae reach the liver in the portal vein. Once initiated, the amoebic lesion extends progressively in all directions to produce the liver-​cell necrosis and liquefaction that constitute an amoebic liver abscess. The lesions are well demarcated from surrounding liver tissue; untreated nearly all will eventually extend into adjacent structures. Secondary bacterial infection is rare and usually follows rupture or aspiration. Clinical manifestations Invasive intestinal amoebiasis The clinical features show a wide spectrum from minimal changes in bowel habit to severe dysentery. Lesions may be limited to a small part of the large bowel or extend throughout its length. A relapsing course is common. Amoebic colitis with dysentery Dysentery, the passage of loose or diarrhoeal stools containing fresh blood, occurs when there is generalized colonic ulceration or when more localized lesions occur in the rectum or rectosigmoid. Onset may be gradual, intermittent, or, much less commonly, acute. Typically, constitutional upset is initially mild and the patient remains ambulant; mild or moderate abdominal pain is common, often col- icky and maximal over affected parts of the gut. Tenesmus can occur but is rarely severe. Stools vary in consistency from semiformed to watery. They are foul smelling and always contain visible or occult blood; even when they are watery, faecal matter is nearly always pre- sent. Symptoms frequently wax and wane over a period of weeks or even months and such patients can become debilitated and wasted. Fig. 8.8.1.1  Amoebic colitis. Crypt abscess. Periodic acid–​Schiff stains amoebae red. Copyright Viqar Zaman. Fig. 8.8.1.2  Amoebic colitis. Superficial ulcer breaching the muscularis mucosae. Copyright Viqar Zaman.

8.8.1  Amoebic infections 1387 In a few patients the disease runs a fulminating course. The most frequent physical sign is abdominal tenderness in one or both iliac fossae, but tenderness may be generalized. The affected gut may be palpably thickened. A low fever is common, but dehydration is un- common. Abdominal distension occurs in the more severely ill pa- tients, who sometimes pass relatively small amounts of stool. A careful proctoscopy or sigmoidoscopy should be done. The endoscopic appearances may be non​specific in early, acute, or very severe colitis; the findings are hyperaemia, contact bleeding, or con- fluent ulceration. In more chronic cases, the presence of normal-​ looking intervening mucosa is highly suggestive of amoebiasis. Early lesions are often elevated, with a pouting opening only 1 to 2 mm in diameter; later, ulcers may reach 1 cm or more in diameter, with an irregular outline, and often a loosely adherent, yellowish, or grey ex- udate. Mucosal scrapings or superficial biopsies taken at endoscopy should be examined immediately by wet-​preparation microscopy. Special forms of amoebic colitis Fulminant colitis—​This may arise de novo, for example, in preg- nant women or during steroid therapy, or it may evolve during a dysenteric illness. Patients show progressive abdominal distension, vomiting, and watery diarrhoea. Bowel sounds are absent and there may be little or no abdominal tenderness, guarding, or rigidity. Plain radiographs may reveal free peritoneal gas, together with acute gas- eous dilatation of the colon; affected segments of bowel may ap- pear relatively narrow and show visible mucosal pathology. Barium enema and full sigmoidoscopy are contraindicated. Stools contain erythrocytophagous trophozoites. Amoebic colitis without dysentery—​When ulceration is limited to the caecum or ascending colon, or when early, mild, or localized le- sions occur elsewhere in the colon, there may be no dysenteric symp- toms. Patients complain of change in bowel habit, blood-​staining of the stool, flatulence, and colicky pain. Often the only physical sign is tenderness in the right iliac fossa or elsewhere along the course of the colon. Some patients eventually go into complete remission; others progress to a dysenteric illness. The most important diagnostic measure is repeated stool exam- ination for erythrocytophagous amoebae; the finding of cysts or commensal trophozoites is of little diagnostic value, especially in endemic areas, unless E. histolytica specific methods are used. Sigmoidoscopy is often normal when the distal bowel is not involved but colonoscopy may reveal typical lesions. Amoeboma  This presents as an abdominal mass, most frequently in the right iliac fossa. The lesion may be painful, tender, and associated with fever. Bowel habit is altered and some patients have intermittent dys- entery, especially if lesions are multiple or distal. Evidence of partial or intermittent bowel obstruction may be present, particularly when lesions are distal and annular. Localized perforation and amoebic appendicitis Sudden perforation with peritonitis can occur from any deep amoebic ulcer; alternatively, leakage may lead to a pericolic abscess or retroperitoneal cellulitis. Amoebic appendicitis is an uncommon but important condition that occurs when amoebic lesions are con- fined to the appendix and caecum. The clinical presentation can re- semble that of simple appendicitis, often with some clinical evidence of dysentery. If it is unrecognized at appendicectomy the outcome can be disastrous, with gut perforation; fresh smears should be made from the resected appendix and examined immediately. Rectal bleeding Some patients with amoebiasis present with rectal bleeding, with or without tenesmus; this occurs particularly in children. Massive bleeding into the gut lumen can occur in any form of amoebic colitis but is rare. Differential diagnosis Amoebic colitis must be differentiated from other causes of in- fective colitis. High-​volume diarrhoea, copious mucus, and severe tenesmus are all uncommon in amoebiasis. In temperate countries, non​specific ulcerative colitis, Clostridium difficile colitis, and colo- rectal carcinoma create the greatest diagnostic problems. Parasitic conditions to be considered are intestinal schistosomiasis, heavy Trichuris infection, and balantidiasis. More chronic amoebic path- ology may clinically resemble Crohn’s disease, ileocaecal tubercu- losis, diverticulitis, or anorectal lymphogranuloma venereum. Hepatic amoebiasis Less than half of all patients give any convincing history of dysen- tery and few have concurrent dysentery. In those with no dysenteric history, the interval between presumed infection and presentation may be as short as 3 weeks or as long as 22 years; for most, it is be- tween 8 weeks and 1 year. The dominant symptoms are fever and sweating, liver or dia- phragmatic pain, and weight loss. Onset of constitutional symp- toms is often insidious, but pain may begin abruptly. Most patients seek medical help between 1 and 4 weeks. Fever is typically remit- tent, with a prominent evening rise, brief rigors, and very profuse sweating. Liver pain may be poorly localized initially and later be- come pleuritic, referred to the right shoulder tip or localized to the abdominal wall. Within a few weeks, patients lose much weight and often become anaemic; a painful dry cough is common. The most important clinical finding is liver enlargement Fig. 8.8.1.3) with localized tenderness, which should be searched for in the right hypochondrium, the epigastrium, and along all the intercostal spaces overlying the liver. Liver pain, on compression or heavy digital percussion, is a less useful sign. Left-​lobe lesions can present as an epigastric mass. Hepatomegaly may be difficult to de- tect by abdominal palpation when enlargement is mainly upwards, but bulging of the right chest wall may be noted, together with a raised upper level of liver dullness on percussion. Reduced breath sounds or crepitations may be heard at the right lung base. Important radiological findings are a raised or locally upward-​ bulging right diaphragm Fig. 8.8.1.4) with immobility on screening, areas of lung collapse or consolidation, and sometimes a pleural ef- fusion. A neutrophil leukocytosis is almost invariable, the erythro- cyte sedimentation rate is raised, and normochromic normocytic anaemia is common. Liver function tests are frequently completely normal or there may be a raised alkaline phosphatase; less commonly the serum transaminase or bilirubin is elevated. Liver scanning to demonstrate a filling defect is of great value; about 70% of lesions are solitary, but multiple lesions are common in children and those with concurrent dysentery. Ultrasonographic and CT scans are the most useful. Lesions appear round or oval and are usually between 4 and 10 cm in diameter at the time of presentation. On ultrasonography

section 8  Infectious diseases 1388 most are hypoechoic with well-​defined walls without enhanced echoes. Even when concurrent dysentery is absent, the stools are frequently, but not always, positive for E. histolytica. Colonoscopy may reveal unsuspected lesions. Complications Most complications involve extension of hepatic lesions into adja- cent structures: usually the right chest, the peritoneum, and the peri- cardium. Upward extension usually produces adhesions between the liver, the diaphragm, and the lung; in consequence, subphrenic rupture and amoebic empyema are rare, although a right serous pleural effusion is not uncommon. Untreated, the disease process advances upwards through lung tissue leading to hepatobronchial fistula and expectoration of brownish, necrotic liver tissue, the so-​ called ‘anchovy sauce’ sputum. Rupture into the peritoneum can occur at any time; it is sometimes the mode of presentation of an amoebic liver abscess, the cause of peritonitis being discovered only at laparotomy. Amoebic pericarditis usually results from upward ex- tension of a left-​lobe liver lesion. Initially patients have retrosternal pain and a pericardial friction rub; later rupture or large serous ef- fusion produces cardiac tamponade. The diagnosis is most difficult when an underlying liver abscess was not suspected. Less commonly the lesion extends through the skin, producing a sinus and cutaneous lesion. The gut, stomach, vena cava, spleen, and kidney are occasionally involved by direct spread. Blood-​borne spread to the lung produces a lesion resembling an isolated pyo- genic lung abscess. Amoebic brain abscesses due to E. histolytica are rare; most are discovered postmortem Fig. 8.8.1.5). Jaundice occurs when a large lesion compresses the common bile duct or when multiple lesions compress several intrahepatic bile ducts. Rupture into a major bile duct can cause haemobilia. Portal-​vein compression occasionally produces portal hypertension and con- gestive splenomegaly. Differential diagnosis Amoebic serology and scanning have now greatly simplified diag- nosis. However, a few patients (generally less than 5%) are initially seronegative; scanning patterns may be atypical before lesions have liquefied. Pyogenic abscess, especially when cryptogenic, may be clinically indistinguishable and this condition is quite common in some Asian countries. Other conditions to be distinguished are pri- mary and secondary carcinoma of the liver, lesions of the right lung base and right pleura, subphrenic abscess, cholecystitis, septic chol- angitis including that resulting from aberrant Ascaris worms, and liver hydatid cysts. Fig. 8.8.1.3  Amoebic liver abscess. Hepatic enlargement with focal tenderness in a Thai woman. Courtesy of the late Professor Sornchai Looareesuwan. (c) (b) (a) Fig. 8.8.1.4  Amoebic liver abscess. Radiographic changes showing (a) elevated right diaphragm; (b) enormous abscess in the right lobe of the liver outlined with air (fluid level) and contrast medium introduced during the aspiration of more than 1 litre of pus; and (c) same patient as (b), lateral view. Courtesy of the late Professor Sornchai Looareesuwan.

8.8.1  Amoebic infections 1389 Needle aspiration of the liver Fig. 8.8.1.6) may be necessary for diagnostic or therapeutic purposes (see next). Suspected pyo- genic abscess is the main indication for the former; blood cultures should also be taken. Typically, the aspirate in hepatic amoebiasis is pinkish-​brown, odourless, and bacteriologically sterile Fig. 8.8.1.7); a thinner, malodorous, or frothy aspirate suggests bacterial infec- tion. A therapeutic amoebicide trial is generally preferable to diag- nostic needling of the liver. Cutaneous and genital amoebiasis Skin ulceration due to E. histolytica produces deep, painful, and foul-​smelling lesions that spread rapidly. Secondary bacterial in- fection is common and may mask the amoebic pathology. Lesions are most frequent in the perianal area, but also occur at colostomy stomas, laparotomy scars, and at the site of skin rupture by a hep- atic lesion. Female genital involvement results from faecal contamination, the extension of perianal lesions, or by the formation of internal fis- tulae from the gut, which can involve the bladder. Lesions of the vulva and uterine cervix may resemble carcinoma. Male genital le- sions follow rectal coitus, the lesion beginning as a balanoposthitis and progressing rapidly. Laboratory diagnosis Microscopy and culture The identification of live erythrocytophagous trophozoites in tem- porary wet mounts is of prime importance because it confirms the diagnosis of invasive amoebic disease. Amoebae should be sought in dysenteric bowel-​wall scrapings, the last portion of aspirate from a liver abscess Fig. 8.8.1.8), sputum, and tissue scrapings from skin lesions. In non​dysenteric stools, flecks of pus, blood, or mucus should be looked for and examined. The amoebae re- main active for about 30 min at room temperature. Other micro- scopical features of faeces in amoebic colitis are scanty or absent leucocytes, clumped or degenerating red cells, and, sometimes, Charcot–​Leyden crystals. If wet preparations are not made or are negative, a portion of the specimen should be preserved in poly- vinyl alcohol or sodium acetate–​acetic acid–​formalin fixative for later smear preparation; alternatively, drying faecal smears should be fixed in Schaudinn’s solution. In either case, fixed smears Fig. 8.8.1.5  Metastatic brain abscess in a patient with an amoebic liver abscess. Courtesy of the late Professor Sornchai Looareesuwan. Fig. 8.8.1.6  Diagnostic/​therapeutic aspiration of ‘anchovy sauce’ pus from a patient with amoebic liver abscess. Contrast medium is being injected after aspiration of the abscess. Copyright D. A. Warrell. Fig. 8.8.1.7  ‘Anchovy sauce’ pus drained from and amoebic liver abscess. Copyright Viqar Zaman. Fig. 8.8.1.8  Aspirate from amoebic liver abscess showing margin of hepatocytes and erythrocytophagous trophozoites of E. histolytica. Copyright Viqar Zaman.

section 8  Infectious diseases 1390 should be stained with Gomori trichrome or Heidenhain’s iron haematoxylin. Cysts and commensal trophozoites of E.  histolytica found in wet faecal mounts are indistinguishable from those of E. dispar. The cysts of both species are four-​nucleated and can be differenti- ated from the smaller E. hartmanni using an eyepiece micrometer. Direct mounts are made by emulsifying a small portion of stool in 1% eosin and in Lugol’s iodine; however, the diagnostic sensitivity, per specimen, is only about 30%. Concentration methods for cysts such as formol-​ether sedimentation give a 70% sensitivity per spe- cimen. Cultivation of intestinal amoebae from faeces in Robinson’s medium is relatively easy. Species identification requires immuno- fluorescent staining. Amoebae are often difficult to find microscop- ically in liver aspirates. Positive cultures from extraintestinal sites do confirm invasive E. histolytica. DNA and immunological tests PCR methods can now be used for both E. histolytica and E. dis­ par using either faecal or tissue material. E.  histolytica antigen can be detected in faecal specimens, and assays for antigen in serum have also been used in extraintestinal disease. These new methodologies have excellent sensitivity and specificity. Where they are available, they greatly simplify diagnosis in both amoebic disease and in carriers. They are already revolutionizing our ideas on epidemiology. E. histolytica DNA can now be detected in the blood, urine, and saliva of patients with invasive disease using real-​time PCR assay. Many serodiagnostic methods have been applied to amoeb- iasis. The most detectable antibody is IgG, with some IgM in ac- tive disease. However, seropositivity does not distinguish current and past tissue invasion. The more sensitive methods are indirect haemagglutination, enzyme immunoassay, and indirect immuno- fluorescence. Latex agglutination and gel-​diffusion precipitation are also used, the former being commercially available as a slide test, taking only minutes to perform. Using sensitive tests, over 95% of patients with liver abscess are seropositive, as are about 60% of those with invasive bowel disease; patients with amoeboma are nearly all seropositive. All patients with tissue invasion eventually become seropositive. Titres decline after therapy but may remain positive for 2 years or more with the most sensitive tests. Patient management Chemotherapy Metronidazole for 5 days will be the first choice in most patients. The usual adult dose of metronidazole is 800 mg thrice daily for 5 or 8 days; the daily paediatric dose is 35 to 50 mg/​kg in three div- ided doses. The alternative is tinidazole, which has the advantage of a single daily dose, 2 g in adults and 50 to 60 mg/​kg in children. A 5-​ or even a 3-​day course may be sufficient for tissue amoebae but rates of parasite elimination from the intestine are low. When nitroimidazoles are contraindicated, or not available, erythromycin is useful in non-​severe colitis. The synthetic derivative dehydroemetine is a potent tissue amoebicide. It has less cumulative cardiotoxicity than the alkaloid emetine and is more rapidly excreted in the urine. Where appro- priate nitroimidazoles are unavailable, as continues to be the case in some tropical contexts, this drug will continue to be lifesaving, especially when a parenteral drug is needed. A daily intramuscular dose of dehydroemetine of 1.25 mg/​kg (maximum 90 mg) is given for 5 days. Cutaneous and genital amoebiasis responds well to metronida- zole, partly perhaps because the lesions often contain anaerobic bacteria. Amoebiasis at other sites is nearly always secondary to hepatic lesions and the chemotherapy will be the same. Metronidazole crosses the blood–​brain barrier and should be used in the desperate situation of amoebic brain abscess due to E. histolytica. All patients with E.  histolytica infection treated with a tissue amoebicide should also be given diloxanide to eliminate infection from the bowel and so prevent recurrence of tissue invasion or transmission to others. The dosage of diloxanide for adults is 500 mg thrice daily for 10 days; the daily dose in children is 20 mg/​kg daily in three divided doses. Alternatives to diloxanide when it is not avail- able are paromomycin 30 mg/​kg daily for 5 to 10 days or iodoquinol 650 mg thrice daily for 20 days, but iodoquinol may cause optic or peripheral neuropathy if the dose is exceeded. Early re-​infection with E. histolyica after diloxanide is reported to be a problem among male homosexuals in Japan. Convalescent carriers, and also infected family contacts, should always be treated. Persons entering temperate countries from the tropics or new residents from such countries should be screened if there is a significant risk of infection; those with E. his­ tolytica faecal antigen, or who are seropositive and have four-​nu- cleated Entamoeba cysts in their stools, should be treated. In these contexts, diloxanide is the drug of choice. Metronidazole is less effective even using an 8-​day course and side-​effects are trouble- some. Unfortunately cure rates with tinidazole are very low when followed up at 1 month. Supportive and surgical management Intestinal amoebiasis Supportive management plays a major role in patients with com- plicated amoebic colitis, with emphasis on fluid and electrolyte re- placement, gastric suction, and blood transfusion as necessary. Gut perforation complicating extensive colitis carries a very poor prog- nosis; management may have to be medical. Parenteral metronida- zole is invaluable in these situations because of its activity against anaerobic bacteria in the peritoneum and blood stream. A cephalo- sporin plus gentamicin will normally be given as well. Amoebomas respond well to metronidazole; a slow response should arouse suspicion that the amoebic lesion is superimposed upon other pathology, particularly a carcinoma. Surgical manage- ment is important in several situations. Acute colonic perforation in the absence of diffuse colitis or ruptured amoebic appendicitis may be amenable to local repair. In the case of diffuse colitis, local repair, or end-​to-​end anastomosis, may not be possible because of the poor condition of the gut wall: temporary exteriorization with an ileos- tomy may be necessary. In fulminant colitis with multiple perfor- ation the viability of the gut wall is uncertain and the only definitive option is total colectomy. Hepatic amoebiasis A favourable response to medical treatment alone can be expected in about 85% of patients. Liver abscesses may rupture before, during, or after oral chemotherapy; this requires parenteral metronidazole or dehydroemetine. Intra-​abdominal rupture will always require

8.8.1  Amoebic infections 1391 laparotomy. Extension into the pleural or pericardial cavities neces- sitates drainage of these structures, together with aspiration of the liver lesion; pericardial drainage is most urgent when tamponade is present. Hepatopulmonary lesions generally require drainage of the liver lesion but medical treatment alone has been successful in some cases. Antibiotics will always be needed when the abscess ruptures into the peritoneum or lung. The most common management problem is slow response to the amoebicide. Patients whose pain and fever do not subside by 72 h are at significantly greater risk of rupture or therapeutic failure, and aspiration is generally to be recommended. A  likely explanation of poor initial response is a tense lesion that restricts drug entry. Regular ultrasonographic monitoring is of great value as it will in- dicate the risk of rupture and guide the aspiration procedure. No change in lesion size on ultrasound can be expected during the first 2 weeks, although its outline may become clearer. Percutaneous as- piration with a wide-​bore needle will be possible in most patients; if unsuccessful or anatomically contraindicated, then surgical help should be sought. Catheter drainage is a better alternative to re- peated needle aspiration with large abscesses. Resolution times for small or moderate lesions are unaffected by aspiration. All patients with hepatic amoebiasis should be given diloxanide to eliminate bowel infection. Prognosis Uncomplicated invasive intestinal disease and uncomplicated hepatic amoebiasis should normally have a mortality rate of less than 1%. In complicated disease, the mortality is much greater and may reach 40% for amoebic peritonitis with multiple gut perfor- ation. Prognosis is usually better in centres where the disease is common and more likely to be recognized early. Late diagnosis increases the probability of complicated disease and mortality rises accordingly. Unless parasitological cure is achieved and the gut completely freed of E. histolytica, clinical relapse is quite common, although probably limited by immunological responses. There is, so far, no evidence of naturally occurring strains of E. histolytica being re- sistant to normally used drugs. Hepatic scans show that nearly all liver abscesses completely disappear within 2 years; the median reso- lution time is 8 months. In secondarily infected lesions, bizarre hep- atic calcification may be seen years afterwards. Healing of the bowel is remarkably rapid and complete; occasionally fibrous strictures persist after severe dysentery. Prevention Chlorination of water supplies does not destroy amoebic cysts, but adequate filtration will remove them. Regular stool screening by microscopy of food handlers and domestic staff is of no value, but health education is important with encouragement to have a med- ical check if diarrhoea occurs. Visitors to the tropics should not attempt chemoprophylaxis; in particular, long-​term unsupervised use of hydroxyquinoline drugs must be strongly deprecated. Simple hygienic measures provide considerable protection. Boiling water for 5 min kills cysts. Routine examinations in temperate countries for returning visitors from the tropics or for new residents coming from such countries is of no value unless E. histolytica can be differentiated from E. dispar. Amoebic serology is useful in those with gut symptoms or a history of dysentery. Other parasitic gut amoebae including Dientamoeba fragilis The nuclei of Entamoeba species have a fine ring of peripheral chro- matin and a small central endosome. Six cyst forming Entamoeba species are non​pathogenic colonic commensals. Entamoeba coli has eight-​nucleated cysts and is the commonest species in most surveys. E. dispar and E. hartmanni both have cysts with four nu- clei; the former was previously known as ‘non​pathogenic E. his­ tolytica’ and the latter as ‘small race E. histolytica’. Size is the only microscopic diagnostic criterion for E. hartmanni; its cysts are less than 10 μm in diameter. The relative prevalence of E. dispar and E. histolytica varies greatly, but the former is usually much more common, especially where sanitation and water supplies are better. E. chattoni is primarily a pig and primate parasite; the cyst has one nucleus and an ‘inclusion body’. Human infections are common in highland Papua New Guinea where humans and pigs may share a peridomestic environment; elsewhere it is rare. Recently an- other species E. bangladeshi has been found to be quite common in Bangladesh infants. Lastly there is E. moshkovskii, which nor- mally lives in soil and sewage; it infects and can be transmitted between humans. It was previously incorrectly referred to a low-​ temperature variant of E. histolytica. It should be mentioned that the complete non​pathogenicity of E. dispar, at least in experimental animals, has been challenged. E. gingivalis has no cystic stage and lives in the mouth within gin- gival pockets and tonsillar crypts. It is spread by kissing or more indirect oral contact. Its possible role in periodontal disease was for- merly dismissed but there is now renewed interest following recog- nition of its high prevalence in individual lesions in people with this condition; it may act as a bacterial vector within the lesions. It has been found on intrauterine devices, sometimes causing symptoms. Both in the uterus and in the mouth, this amoeba occurs in associ- ation with the bacterium Actinomyces israelii. Endolimax nana and Iodamoeba bütchlii both have nuclei with large endosomes and no visible peripheral chromatin. Cysts of the former are oval in shape with four nuclei; those of the latter are somewhat irregular in shape with a single nucleus and a large glycogen vacuole that stains prominently with iodine. Neither spe- cies is pathogenic. Dientamoeba fragilis is overlooked in most parasitological labora- tories and most reports are from developed countries. There is good evidence that it can cause colonic inflammation; however, this is not severe and there is no ulceration or systemic spread. No cystic stage is found using standard methods and, unless this organism is specif- ically looked for, it will be missed. In fixed stained smears, about 60% of trophozoites have two nuclei; the endosome is large and lobulated and there is no peripheral chromatin. Using special techniques, however, both cysts and precysts have recently been demonstrated in stools. Infected patients may shed the parasite intermittently. Alternatively, D. fragilis may be identified in faeces or cultures using immunofluorescence with specific antibody or of parasite DNA by PCR; some patients are seropositive. Transmission is direct but

section 8  Infectious diseases 1392 possibly within eggs of the threadworm Enterobius. It causes a rela- tively mild diarrhoeal illness that may persist for several weeks and sometimes there is a superficial eosinophilic colitis. Irritable bowel syndrome may be suspected. Protein-​losing enteropathy is reported and blood eosinophilia is quite common. This infection is frequent in some institutional contexts. It is found within some resected ap- pendices but a causal role is unlikely. Electron micrographs and gen- etic studies indicate that D. fragilis is a trichomonad rather than a true amoeba. The infection responds to metronidazole, but a single dose of ornidazole is also effective. Series of symptomatic patients who improve after treatment continue to be reported, but in a recent placebo-​controlled study in Danish children metronidazole treat- ment conferred no benefit. Free-​living amoebae A shared feature of these species is the very large central nuclear endosome, quite different from that of E. histolytica, from which dif- ferentiation may be necessary in tissue sections. Under dry condi- tions, trophozoites form resistant cysts that permit survival and also airborne dispersal; cysts can resist chlorination. Many species are thermophilic and they are one of the causes of ‘humidifier fever’, a form of extrinsic allergic alveolitis presenting with fever, cough, and dyspnoea. Some bacteria including Legionella and Parachlamydia acanthamoebae may live symbiotically within these amoebae per- sisting within the phagosome, being resistant to lysosomal enzymes. Surprisingly, Legionella can survive encystment: the amoebae pro- vide a refuge for these bacteria when chlorination or other anti- bacterial measures are applied. Four groups of free-​living amoebae cause human infections: 1 Naegleria fowleri is an amoeboflagellate with two trophozoite forms. The amoeba moves rapidly with a single pseudopodium, it can transform into a non​feeding flagellate in hypotonic media, and these free-​swimming forms facilitate dispersal. Cysts are thin walled and spherical. 2 Acanthamoeba has no flagellate form. The small pseudopodia are multiple, thin, and spike-​like; they are called acanthopodia Fig. 8.8.1.9). Cysts are thick walled, angulated, and buoyant Fig. 8.8.1.10); their dispersal may be wind borne. Several species are pathogenic but morphological classification is unsatisfac- tory; rRNA sequences differentiate 15 genotypes. Acanthamoeba is sometimes isolated from throat or nasal swabs or from stool specimens. 3 Balamuthia is closely related to Acanthamoeba and not a leptomyxid amoeba; it shows little directional movement and has an irregular or branched shape. Cysts are thick walled and spherical. Human infections formerly attributed to Hartmanella are now all thought to be due to Balamuthia mandrillaris, a species described in 1993 from a mandrill baboon that died of meningoencephalitis in San Diego zoo. Balamuthia can only be cultured on tissue culture monolayers. About 200 cases have been reported worldwide, with many from Latin America. 4 Sappinia pedata has caused at least one case of granulomatous amoebic encephalitis. Its trophozoites contain a double nucleus and when living the ectoplasm has a rippled appearance. Primary amoebic meningoencephalitis due to
Naegleria fowleri Epidemiology and pathology In temperate countries most patients give a history of swimming or diving in warm fresh water or spa water between 2 and 14 days before the illness began, common-​source outbreaks occur during warm summer months. Amoebic trophozoites cross the cribriform plate from the nasal mucosa to the olfactory bulbs and subarachnoid space. At autopsy the brain shows cerebral softening and damage to the olfactory bulbs; cysts are never formed in the tissues. The first human case was reported in 1965, at least 450 cases have now been reported from temperate countries, some retrospectively, and many Fig. 8.8.1.9  Acanthamoeba trophozoite showing spike-​like acanthopodia. Courtesy of the late Professor Sornchai Looareesuwan. Fig. 8.8.1.10  Acanthamoeba cysts. Copyright Viqar Zaman.

8.8.1  Amoebic infections 1393 from the United States of America. Some are undoubtedly missed clinically and are discovered at autopsy or in preserved pathological material. Specific antisera enable amoebae to be recognized by im- munofluorescence staining. In the tropics this is an emerging problem that is being increas- ingly recognized, for instance in Pakistan. High temperatures and lack of clean water for washing encourage bathing in waters con- taining N.  fowleri. In addition, religious practices and ablutions add to the risk. A particular local hazard is nasal irrigation from a spouted pot called a ‘neti’. Clinical features and diagnosis Patients are immunocompetant; most are young adults and chil- dren. Initial nasal symptoms and headache are soon followed by fever, neck rigidity, coma, and, later, convulsions; most die within a few days. Cerebrospinal fluid is often turbid and bloodstained with high protein, low glucose, and neutrophils. Amoebae must be urgently looked for in wet specimens using phase-​contrast mi- croscopy. Unless amoebae are seen, bacterial meningitis will be sus- pected; on Gram staining amoebae appear as indistinct smudges. Fixed preparations stained with iron haematoxylin will show full details of nuclear structure. Confirmation is by culture at 37°C using a bacterial lawn on non​nutrient agar. Amphotericin B can be an effective drug, it should be given by daily intravenous infusion, and intrathecally; other additional drugs that have been used are intra- venous fluconazole and rifampicin; in mouse models, azithromycin is effective. So far, very few patients have survived but this may partly be due to diagnostic delays. Cadaver organ donation from such pa- tients carries potential risk. Amoebic keratitis due to Acanthamoeba Most patients, but not all, are contact lens users; some are using disposable lenses. Among contact lens users, annual incidence rates of 1.49 and 0.33 per 10 000 are reported from Scotland and Hong Kong, respectively, but most figures are lower. Risk factors include poor hygiene when handling lenses and their cases, use of chlorine-​based disinfectants, swimming or washing eyes while wearing lenses, handling lenses after gardening, and too pro- longed use of plastic or unwashed lenses. The most appropriate disinfectants are chlorhexidine and hydrogen peroxide. Corneal lesions are painful with photophobia, and present as indolent and progressive ulcers leading eventually to perforation. Recognition may be in the context of lesions unresponsive to anti- biotics or corticosteroids. Differentiation must be made from commoner causes of microbial keratitis, including Pseudomonas, Staphylococcus, and herpes simplex. Inflammatory cells are mainly neutrophils. Infection may be by wind-​borne cysts upon a damaged epithelium or from contact lenses. Solutions used to store or wash lenses can be contaminated by these amoebae, many of which are resistant to some antiseptics, especially as cysts. Amoebae are found in corneal scrapings or histologically in corneal tissue, but can be missed unless stained with special stains or by immunofluorescence. PCR and DNA methods are now available. Cysts may be seen in tissue. Cultures from fresh material, using a bacterial lawn on non-​ nutrient agar, should be at 30°C. The majority (90%) of cases are due to genotype T4. Early aggressive topical treatment is with chlorhexidine 0.02% with a aromatic diamidine such as 0.1% propamidine or 0.1% hexamidine, plus neomycin. Both trophozoites and cysts must be destroyed. Hourly application is needed for three days and then 3-​hourly for 3–​4 weeks. Regular surgical debridement may be needed and sometimes corneal grafting. Topical steroids are not recommended. Granulomatous amoebic encephalitis due to Acanthamoeba, Balamuthia, and Sappinia The main route of infection is the lower respiratory tract followed by haematogenous spread to the brain. Other routes of entry are the skin (Fig.  8.8.1.11a), the nasopharynx (Fig.  8.8.1.11b), the lungs and the stomach. Primary lesions have been described at all these sites. Soil contamination of skin and craniofacial wounds is an important risk factor. Almost all patients infected by Acanthamoeba are immunocom- promised; this is associated with malignancy, collagen disorder, alcoholism, diabetes mellitus, AIDS, and steroid or immunosup- pressant therapy, including that used in transplant patients. More patients with B mandrillaris are now being reported, many immuno- compromised, but in Peru most of the patients are not. These infections are now important in transplantation medi- cine. Acanthamoeba encephalitis has been reported in immunosup- pressed transplant recipients of liver or haematopoietic stem cells. Transplant donors can also be the source of infection. In 2009 two patients with B.  mandrillaris were reported who received kidney graft from the same donor. In 2010 four patients received organs from a presumed stroke patient. Two recipients developed B. man­ drillaris encephalitis and died but two others who received heart and kidney transplants remained asymptomatic; the donor had had a large chronic skin lesion on his back and this was the presumed source of the infections. Pathologically lesions resemble chronic bacterial brain ab- scesses or localized subacute haemorrhagic necrosis; involvement of the meninges is common. Some patients present with head- ache and meningism, others with evidence of a focal brain lesion (Fig. 8.8.1.11c, Fig. 8.8.1.12). Unless these amoebae are found in wet tissue preparations or cerebrospinal fluid, the diagnosis will be usually based on histology, often at autopsy. Cysts may be seen in tissue but trophozoites may be missed unless stained with iron haematoxylin or immunofluores- cence using specific antisera. Cultural diagnosis at 37°C from fresh biopsies or cerebrospinal fluid is sometimes possible. PCR and DNA methods are becoming available. Survival of patients with this condition is still only rarely re- ported. Intracranial pressure can be relieved by mannitol and cerebrospinal fluid drainage, and total excision of cerebral le- sions is occasionally possible. Drug treatment with combinations of miltefosine, fluconazole, pentamidine, and cotrimoxazole, may be successful. Acanthamoeba encephalitis has been success- fully treated with cotrimoxazole plus rifampicin in a liver trans- plant recipient. Amoeba-​infected skin lesions, especially following cranio-​facial trauma, may precede the encephalitis by several weeks and should be recognized early and treated.

section 8  Infectious diseases 1394 FURTHER READING Gut amoebae Barwick RS, et al. (2002). Outbreak of amebiasis in Tbilisi, Republic of Georgia, 1998. Am J Trop Med Hyg, 67, 623–​31. Diamond LS, Clark CG (1993). A redescription of Entamoeba histo­ lytica Schaudinn, 1903 (emended Walker 1911) separating it from Entamoeba dispar Brumpt, 1925. J Eukaryot Microbiol, 40, 340–​4. Gilchrist CA (2014). Entamoeba bangladeshi: an insight. Trop Parasitol, 4, 96–​8. Jha AK, et al. (2015). Clinicopathological study and management of liver abscess in a tertiary care center. J Nat Sci Biol Med, 6, 71–​5. Karin IMA, et al. (2003). Entamoeba moshkovskii infection in children in Bangladesh. Emerg Infect Dis, 9, 580–​4. Lau YL, et al. (2014). Molecular detection of Entamoeba histolytica and Entamoeba dispar infections among wild rats in Kuala Lumpur, Malaysia. Tropical Biomedicine, 31, 721–​7. Marie C, Petri WA Jnr. (2014). Regulation of virulence of Entamoeba histolytica. Ann Rev Microbiol, 68, 493–​520. Nagata N, et al. (2012). Risk factors for intestinal invasive amoebiasis in Japan, 2003–​2009. Emerg Infect Dis. 18, 717–​24. Nespola B, et al. (2015). First case of amoebic liver abscess 22 years after the first occurrence. Parasite, 22, 20. Oliveira FMS, et al. (2015). Entamoeba dispar: could it be pathogenic. Trop Parasitol, 5, 9–​14. Panja SK, et  al. (2014). Laboratory methods of identification of Entamoeba histolytica and its differentiation from look-​alike Entamoeba spp. Trop Parasitol, 4, 90–​5. Singh O, et  al. (2009). Comparative study of catheter drainage and needle aspiration in management of large liver abscess. Ind J Gastroenterol, 28, 88–​92. (a) (b) (c) Fig. 8.8.1.11  Balamuthia mandrillaris infection. Cases at Instituto de Medicina Tropical ‘Alexander von Humboldt’ Universidad Peruana Cayetano Heredia, Lima, Peru: (a) cutaneous lesion in a 26-​year-​old man from Ica, (b) perforating lesion of palate in 16-​year-​old boy from Piura, and (c) encephalitis in a 57-​year-​old man from Piura showing the skin lesion that was the likely portal of entry. Copyright D. A. Warrell. Fig. 8.8.1.12  Balamuthia mandrillaris infection. MRI scan in same patient as in Fig. 8.8.1.11c. Copyright D. A. Warrell.

8.8.10 Blastocystis infection 1449

8.8.10 Blastocystis infection 1449

1449 concurrent malignant disease (including chronic lymphocytic leu- kaemia and anal cancer). Furthermore, rare case reports have also suggested that B. coli can be associated with osteomyelitis of the cer- vical spine, and urogenital tract infections and kidney failure. Laboratory diagnosis Balantidiasis is most commonly diagnosed by microscopic exam- ination of freshly obtained diarrhoeal stools or colonic mucus obtained at sigmoidoscopy. Examination of wet mount slide pre- parations within less than 6 h after faecal collection shows cysts or motile trophozoites displaying characteristic spiralling move- ments. A preponderance of trophozoites is often found in diarrheal stool, while a greater proportion of cysts is seen in formed stool. Identification is greatly aided by the large size of the parasite, with ovoid-​shaped trophozoites of 30–​150 μm in length and 40–​55 μm in width, or ovoid-​shaped cysts of 40–​65 μm in size. The large cyst size allows differentiation from the smaller cysts (10–​20 μm) of Entamoeba histolytica, which can cause dysenteric symptoms similar to those of severe B.  coli infection. Fixation and staining (with Lugol’s iodine) can be done, but may obscure cellular details visible by phase-​contrast microscopy of fresh specimens, and could lead to misdiagnosis as helminthic ova. Stools in suspected cases should be examined repeatedly over several days because parasite excretion may be intermittent. Histological examination of rectal biopsies may reveal B.  coli trophozoites on sections stained with haematoxylin and eosin. Pulmonary balantidiasis can be diagnosed by bronchoalveolar lavage and finding the parasite in the lavage fluid. B. coli has also been detected by PCR in faecal samples, but the technology is not fully developed and not presently available for routine clinical diagnostics. Treatment and prevention Balantidiasis has been treated empirically with various antimicro- bial drugs (Table 8.8.9.2), although available reports are mostly an- ecdotal in regard to the effectiveness of such treatments. One trial in the 1970s showed that metronidazole was effective in eradicating the parasites in all of 20 patients over a range of doses (2.5 g over 5 days or 7.5 g over 10 days in children, or 5 g over 5 days or 12.5 g over 10 days in adults). Several case studies further support the general efficacy of metronidazole against balantidiasis, although at least one report exists in which the drug was apparently inef- fective. As an alternative, tetracycline has been shown to be effi- cacious against B. coli infection. Iodoquinol and nitazoxanide may also have therapeutic benefit, although these drugs have not been employed as widely as metronidazole and tetracycline. The mech- anisms of action of any of these drugs have not been specifically in- vestigated in B. coli, but in the absence of such information it would be reasonable to assume that they resemble those described in other target microorganisms of the respective drugs. Furthermore, resistance of B. coli to any of the commonly used drugs has not been reported. However, this possibility has not been adequately exam- ined, so it cannot be excluded that reported treatment failures are related to resistance. In rare situations, surgical intervention might be necessary in patients with extracolonic spread, such as liver ab- scess or appendicitis, or with colonic perforation. Prevention of balantidiasis involves avoidance of B. coli cyst ingestion, via filtra- tion or boiling of drinking water, hand washing before handling food, and careful cleaning and adequate cooking of food. A vaccine has not been developed. FURTHER READING Giardiasis Ankarklev J, et  al. (2010). Behind the smile:  cell biology and
disease mechanisms of Giardia species. Nat Rev Microbiol, 8, 413–​22. Feng Y, Xiao L (2011). Zoonotic potential and molecular epidemi- ology of Giardia species and giardiasis. Clin Microbiol Rev, 24, 110–​40. Fink MY, Singer SM (2017). The intersection of immune responses, microbiota, and pathogenesis in giardiasis. Trends Parasitol, 33, 901–13. Miyamoto Y, Eckmann L (2015). Drug development against the major diarrhea-​causing parasites of the small intestine, Cryptosporidium and Giardia. Front Microbiol, 6, 1208. Soares R, Tasca T (2016). Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis. J Microbiol Methods, 129, 98–102. Upcroft P, Upcroft JA (2001). Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin Microbiol Rev, 14, 150–​64. Balantidiasis Arean VM, Koppisch E (1956). Balantidiasis. A review and report of cases. Am J Pathol, 32, 1089–​115. Garcia-​Laverde A, de Bonilla L (1975). Clinical trials with metro- nidazole in human balantidiasis. Am J Trop Med Hyg, 24, 781–​3. Schuster FL, Ramirez-​Avila L (2008). Current world status of Balantidium coli. Clin Microbiol Rev, 21, 626–​38. 8.8.10  Blastocystis infection Richard Knight ESSENTIALS Blastocystis is an anaerobic unicellular non​invasive colonic parasite of animals and humans. It is transmitted faeco-​orally, with human infection associated with travel, institutions, animal handlers, and immunodeficiency. Case reports strongly suggest that it causes a self-​ limited diarrhoeal illness. Diagnosis is by microscopic examination of Table 8.8.9.2  Oral drug regimens for treating balantidiasis in adults Drug Dose Treatment duration Metronidazole 250–​400 mg/​dose,
3 × doses/​day 5–​10 days Tetracycline 500 mg/​dose, 4 × doses/​day 10 days 8.8.10  Blastocystis infection

section 8  Infectious diseases 1450 faecal smears or concentrates. A trial of treatment with metronida- zole is justified in patients who are immunocompromised, also when symptoms are prolonged. Aetiology and biology of the parasite Molecular and ribosomal RNA studies now indicate that Blastocystis is a Stramenopile (a synonym for kingdom Chromista), currently only one species is recognized. Blastocystis has no flagellae, unlike other stramenopiles, which include slime nets, water moulds, and brown algae. The form commonly described in faeces and also in cultures is spherical, from 4 to 15 μm in diam- eter, with one prominent central vacuole, surrounded by periph- eral cytoplasm (Fig. 8.8.10.1) that electron microscopy shows to contain a nucleus, a Golgi complex, and mitochondrion-​like or- ganelles (Fig. 8.8.10.2). It grows readily in cultures with mixed bacteria but axenic cultures can also be established; division is by binary fission. Transmission is by small, resistant, faecal cysts, from 3 to 8 μm in diameter. The basic life cycle alternates between the univacuolar and cystic stages, but electron microscopy of faeces and cultures may also show granular, and amoeboid forms of uncertain significance. Bizarre environmentally induced forms with huge vacuoles may develop in cultures (Fig. 8.8.10.3). The common ‘univacuolar’ form was named Blastocystis by Brumpt in 1912 as a yeast, although it was first described by Alexieff in 1911 as a protozoan cyst. Epidemiology Prevalence may exceed 35% in some human populations associ- ated with high faeco–​oral transmission. This infection is associated with travel, institutions, animal handlers, and immunodeficiency. Blastocystis is genetically diverse and occurs in a wide range of domesticated and wild animals. Currently only one species is rec- ognized, but at least 17subtypes are described, with subtype ST3 the most common in humans. Important zoonotic sources are pigs, cattle, non​human primates, and birds, including chickens and ducks. The resistant cysts can occur in both sewage influents and effluents. Diagnosis Blastocystis is usually recognized as univacuolar forms in direct wet faecal smears or formol ether concentrates. Wet mounts can be stained with iodine, giving a brownish central body, or with tolui- dine blue. The organism is often numerous in symptomatic subjects. Permanent mounts stain well with trichrome. Blastocystis can re- semble amoebic cysts but lack their characteristic nuclei. In fixed smears stained specifically for Cryptosporidium, there is no oocyst wall. Special techniques are used to concentrate and identify cysts in environmental samples. Inflammatory cells in faecal exudates or inflammation seen at endoscopy should promote search for an additional invasive pathogen. Fig. 8.8.10.1  Blastocystis from culture showing binary fission; the cytoplasm is at the periphery. v, vacuole. Phase contrast, ×400. Fig. 8.8.10.3  Blastocystis from culture showing the great variation in size. v, vacuole. Dark field, ×400. Fig. 8.8.10.2  Blastocystis. Electron micrograph showing the peripheral cytoplasm (c) and the central vacuole (v); the inclusions in the cytoplasm are mitochondria. ×5000.

8.8.11 Human African trypanosomiasis 1451

8.8.11 Human African trypanosomiasis 1451

8.8.11  Human African trypanosomiasis 1451 Clinical features and treatment A non​invasive diarrhoeal illness lasting from 3 to 10 days is attrib- uted to this organism, sometimes symptoms continue for weeks or months. Associated features are abdominal bloating, flatu- lence, and anorexia. Symptoms are more prolonged in immuno- compromised subjects. Some studies of patients with AIDS report higher prevalences or more numerous parasites in the stool. There is no definite association with irritable bowel syndrome or urticaria. Illnesses are self-​limiting in most people, but infection and symp- toms can usually be eliminated with metronidazole or tinidazole. The organism is also sensitive to cotrimoxazole, furazolidine, and hydroxyquinoline. Blastocystis has been reported once in a liver abscess aspirate but the patient was later shown to have amoebiasis. Another pa- tient with faecal Blastocystis and rectocolitis responded rapidly to metronidazole but it appears that amoebiasis was not properly excluded. Evidence for pathogenicity Definite histopathology in humans is still lacking, although serum antibody has been reported in symptomatic subjects. A good labora- tory animal model remains elusive; pigs are a natural host in whom the parasite is located in the caecal and colonic lumen with no mu- cosal attachment or pathology, rats are not a normal host and when experimentally infected some showed lamina propria inflammatory cells. A convincing in vitro cytopathic model awaits discovery al- though cultured colonic epithelial cells release cytokines in the presence of Blastocystis. The parasite may damage actin filaments in epithelial cells. Cysteine proteinase has been postulated as a viru- lence factor. The genetic heterogeneity of Blastocystis isolates correlates weakly with host species. In some human studies, subtype determined by polymerase chain reaction correlated with symptoms. Clinical re- sponse to metronidazole is hardly compelling evidence for patho- genicity since concurrent infection with other enteropathogens is common and this drug has a wide spectrum of activity includ­ ing an effect upon small bowel bacterial overgrowth. More well-​ documented outbreaks and cytopathic evidence are needed. FURTHER READING Andersen LO, Stensvold CR (2016). Blastocystis in health and disease: are we moving from a clinical to a public health perspective? J Clin Microbiol, 54, 524–8. Janarthanan S, Khoury N, Antaki F (2011). An unusual case of invasive Blastocystis hominis infection. Endoscopy, 43 Suppl 2 UCTN, E185–​6. Li J, et al. (2013). A rat model to study Blastocystis subtype 1 infections. Parasitol Res, 112, 3537–​41. Roberts T, et al. (2014). Update on the pathogenic potential and treat- ment options for Blastocystis sp. Gut Pathogens, 6, 1–​9. Tan KS, et  al. (2010). Current views on the clinical relevance of Blastocystis spp. Curr Infect Dis Rep, 12, 28–​35. Wawrzyniak I, et al. (2013). Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. Ther Adv Infect Dis, 1, 167–​78. 8.8.11  Human African trypanosomiasis Reto Brun and Johannes Blum ESSENTIALS Human African trypanosomiasis (sleeping sickness) is caused by subspecies of the protozoan parasite Trypanosoma brucei. The dis- ease is restricted to tropical Africa where it is transmitted by the bite of infected tsetse flies (Glossina spp.). Control programmes in the 1960s were very effective, but subsequent relaxation of control measures led to recurrence of epidemic proportions in the 1980s and 1990s. Control is now being regained and elimination is being envisaged. Clinical features Trypanosomal chancre—​a papule at the site of the bite is sometimes seen, and may be associated with regional lymphadenopathy. Haemolymphatic stage (human African trypanosomiasis stage 1)—​manifests with fever, chills, rigors, headache, and joint pains; hepatosplenomegaly and generalized lymphadenopathy are common. Meningoencephalitic stage (human African trypanosomiasis stage 2)—​insidious onset of headache, sometimes with change in behav- iour and personality; convulsions are common; sleep pattern be- comes fragmented, eventually leading to somnolence and coma. Progress tends to be fast in rhodesiense human African tryp- anosomiasis and slow—​sometimes lasting years—​in gambiense human African trypanosomiasis. Diagnosis, staging, prognosis, and treatment Diagnosis—​by detection of trypanosomes (usually by direct mi- croscopy) in chancre aspirate, blood, lymph, or cerebrospinal fluid. Serology and polymerase chain reaction-​based tests can be useful for mass screening. Staging—​the cerebrospinal fluid must be examined in every pa- tient found positive for trypanosomes in blood or lymph aspirate. Treatment—​untreated human African trypanosomiasis is almost invariably fatal. Specific treatment depends on the trypanosome subspecies and the stage of the disease. Drugs used for stage 1 in- clude pentamidine and suramin, and for stage 2 include melarsoprol, eflornithine, and nifurtimox, but regimens are not standardized and treatment is difficult and dangerous; all of the drugs used have many side effects, some potentially lethal. However, a new oral drug should soon be available. Prevention Control can be achieved by a combination of mass screening pro- grammes, treatment of patients, and vector control, which together can lead to a complete break of the transmission cycle. There is no vaccine. Acknowledgement: The authors and editors gratefully acknowledge the inclu- sion in this chapter of material contributed to previous editions of the Oxford Textbook of Medicine by Professor August Stich.

section 8  Infectious diseases 1452 Introduction Human African trypanosomiasis (HAT) or sleeping sickness is caused by subspecies of the protozoan flagellate Trypanosoma bru­ cei and transmitted to humans and animals by tsetse flies (Glossina spp.). The distribution of the vector restricts sleeping sickness to the African continent between 14° north and 29° south. Human dis- ease occurs in two clinically and epidemiologically distinct forms, gambiense or Central/​West African and rhodesiense or East African sleeping sickness (Table 8.8.11.1). A third subspecies of the parasite, T. b. brucei, causes disease in animals but is non​pathogenic for hu- mans. Uganda is the only country where gambiense and rhodesiense sleeping sickness both occur (Fig. 8.8.11.1). The first case reports of the disease go back to the 14th century. In the past, its impact on health in Africa was enormous. Many areas were long rendered uninhabitable for people and livestock. During the early decades of the 20th century, millions may have died in Central Africa around Lake Victoria and in the Congo basin (Fig. 8.8.11.2). The success of control programmes in the 1960s promised the disappearance of sleeping sickness as a public health problem. After all African countries had gained independence, the rigorous control measures introduced by the colonial powers were not maintained. As a consequence, the numbers of cases increased again mainly in the Democratic Republic of Congo, northern Angola, southern Sudan, the Central African Republic, and Uganda. According to estimates by the World Health Organization (WHO) at the end of the 20th century, the achievements in sleeping sickness control during colonial times had been nearly completely reversed. However, strong efforts of control programmes run by national institutions and various non​governmental organizations reduced transmission and prevalence to fewer than 4000 cases in 2014. The goal is now to eliminate sleeping sickness; that is, to bring down the number of cases below 2000 by the year 2020 (see Box 8.8.11.1). Today, HAT is a focal disease with probably fewer than 15 000 infected people in about 10 countries. However, millions of Africans in about 20 countries are exposed to the potential risk of HAT. Almost all patients are infected with T.  b.  gambiense, while only about 100 cases of T. b. rhodesiense were reported from Uganda, Tanzania, Zambia, and Malawi in 2013. For tourists and expatriates, sleeping sickness has always been a rare disease; occasional cases have been reported in tourists visiting National Parks in Tanzania, Zambia, and Malawi. Aetiology In 1895, Sir David Bruce (1855–​1931) suggested an association between trypanosomes and ‘cattle fly fever’, a major problem for livestock in southern Africa. In 1902, Robert M Forde and Everett Dutton from the Liverpool School of Tropical Medicine identified trypanosomes in the blood of a patient during a research exped- ition in the Gambia (see Fig. 8.8.11.3a and b), and in 1903, Aldo Castellani isolated trypanosomes from the cerebrospinal fluid of a patient. In the same year, tsetse flies were identified as vectors. Trypanosoma brucei (phylum Sarcomastigophora, order Kinetoplastida) is an extracellular protozoan parasite. Like leish- mania, it possesses a centrally placed nucleus and a kinetoplast, a distinct organelle containing mitochondrial DNA. The kinetoplast is the starting point of the flagellum which extends on the surface of the cell body forming an undulating membrane and ending as a free flagellum. Table 8.8.11.1  The principal features of Gambiense and Rhodesiense sleeping sickness Disease Gambiense sleeping sickness Rhodesiense sleeping sickness Parasite Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense Vector Transmitted by riverine tsetse flies (Palpalis group) Transmitted by savannah tsetse flies (Morsitans group) Clinical course Insidious onset, slow progression, death in stage II after many months or years Acute onset, chancre frequent, rapid course, death frequently in stage I (cardiac failure) Diagnosis Parasitaemia scanty, Winterbottom’s sign, serology Parasitaemia usually higher and easily detectable, serological tests unreliable Treatment See Table 8.8.11.3 Epidemiology Tendency for endemicity, humans as main reservoir, animal reservoir unlikely, latent public health problem in many
Central and West African countries Wild (antelopes e.g. bushbuck) and occasionally domestic animals as reservoir and source of case clusters and epidemic outbreaks Fig. 8.8.11.1  Geographical distribution of human African trypanosomiasis cases in endemic populations and in travellers. Courtesy of Dr. Bernhard Beck, Swiss Tropical and Public Health Institute.

8.8.11  Human African trypanosomiasis 1453 The three subspecies of T. brucei are indistinguishable morpho- logically. However, they differ in their interaction with their mam- malian host and the epidemiological pattern of the diseases they cause. Formerly, T. b. gambiense and T. b. rhodesiense isolates were characterized either by isoenzyme analysis or by animal inocula- tion. The advent of molecular techniques created expectations of more reliable tools for their differentiation. However, genomic characterization has revealed several more subdivisions than the three that were expected. Whereas West African isolates proved relatively homogeneous, East African isolates from humans and animals did not simply conform to what is still called T. b. rhod­ esiense and T. b. brucei but showed a complex relationship with evidence of genetic exchange in the vector. T. b. rhodesiense and T. b. brucei are almost identical, the only difference being the pres- ence of the SRA (human serum resistance associated) gene present in T. b. rhodesiense. Transmission The main mode of transmission is through the bite of an infected tsetse fly (Glossina spp., order Diptera; Fig. 8.8.11.4). Congenital transmission may play an occasional role; other modes of transmis- sion are highly unlikely. Tsetse flies are biologically unique insects, which occur only in Africa, with 31 distinct species and subspecies, of which less than half are potential vectors of HAT. Their distinctive behaviour, ecology, and chosen habitat explain many epidemio- logical features of sleeping sickness. Tsetse flies can live for many months in the wild, are viviparous, and give birth to maximal eight larvae per lifetime. Both sexes feed on blood. They are fastidious in requiring warm temperatures, shade, and humidity for resting and larviposition and so their distribution is highly focal. Mapping and monitoring of possible HAT transmission foci has become possible with the use of satellite imaging techniques. During the blood meal on an infected mammalian host, the tsetse fly takes up trypanosomes (‘short-​stumpy form’) into its mid-​ gut, where they differentiate to procyclic forms and multiply. After 2–​3 weeks, they migrate to the salivary glands as epimastigote forms where they attach to the gland epithelium and finally develop into infective metacyclic forms. At the next blood meal, some of them are injected with the saliva into a new vertebrate host where they develop to ‘long-​slender’ trypomastigotes and multiply by binary Fig. 8.8.11.2  Sleeping sickness patients on an island in Lake Victoria; historical photograph taken during Robert Koch’s research expedition to East Africa. Box 8.8.11.1  Control of human African trypanosomiasis • Diagnosis and treatment of patients • Active case finding • Vector control • Implementation and continuation of a surveillance system • Training, health education, and community participation (a) (b) Fig. 8.8.11.3  (a) Trypanosomes in thin human blood film (Giemsa stain, x1000). (b) Everett Dutton’s painting of trypanosomes. Fig. 8.8.11.4  Adult tsetse fly Glossina morsitans.

section 8  Infectious diseases 1454 fission. The mature (salivary glands) infection rate in the field is extremely low with less than one fly in a thousand. In contrast to Leishmania species and T. cruzi, T. brucei is an exclusively extracel- lular parasite. Antigenic variation The bloodstream forms of T. brucei are covered with a dense coat of identical glycoproteins. Being highly immunogenic, they stimulate the production of specific antibodies, mainly of the IgM subclass. Once the surface glycoproteins have been recognized by host anti- bodies, the parasite will be attacked and destroyed through com- plement activation and cytokine release, giving rise to local and systemic inflammatory reactions. However, about 2% of T. brucei in each new generation change the expression of their specific surface glycoprotein. The ‘coat’ will then be different in the new clone (thus called variant surface glycopro- tein, VSG). This phenotypic switch is done mainly by programmed DNA rearrangements, moving a silent VSG gene into an active, telomeric expression site. Each T. brucei parasite has hundreds of different VSG genes, and within all trypanosome populations, the potential repertoire for such different VSG types seems to be al- most infinite. Antigenic variation is the major hindrance for vaccine development. Every new VSG copy is antigenically different, thus stimulating the production of a new IgM population. This antigenic variation is the major immune evasion strategy of the parasite, enabling the trypanosome to persist in its vertebrate host. It also reduces parasite load and prolongs the infection. But the inevitable outcome is im- mune exhaustion of the host (supported by additional immunosup- pressive metabolites of the parasites), penetration of trypanosomes into immune-​privileged sites such as the central nervous system, and finally death of the host. Clinical features The clinical presentation of HAT depends on the parasite species, the stage of the disease and on the host. T.b. rhodesiense HAT is usually an acute disease progressing to stage II within a few weeks and death within 6 months (Fig. 8.8.11.5). T.b. gambiense HAT is characterized by a chronic progressive course that is mostly fatal if untreated. The disease occurs in two stages, the first, or haemolymphatic stage, and the second, or meningoencephalitic stage, with invasion of the central nervous system by the trypanosomes. Neurological signs and symptoms, including sleep disturbances, are characteristic of stage II. However, most of the symptoms of both stages overlap, making the distinction between the stages based on clinical features not clear. Therefore, this distinction relies on the analysis of the cere- brospinal fluid (see diagnosis). Clinical signs and symptoms are non​specific, and their frequency varies between individuals and between disease foci. T.b.gambiense HAT T.b.gambiense HAT is characterized by a chronic progressive course leading mostly to death if untreated. Fever, headache, pruritus (Fig. 8.8.11.6), lymphadenopathy and, to a lesser extent, hepatosplenomegaly are the leading signs and symptoms of stage I, but are also present to a lesser extent in stage II. Fever is intermit- tent, with attacks lasting from a day to a week, separated by inter- vals of a few days to a month or longer, and is rarely seen in stage II. Lymphadenopathy with enlarged firm, mobile, non​suppurate, and painless lymph nodes and enlargement of the posterior cervical lymph nodes is the so-​called Winterbottom’s sign. Oedema mainly affects the face (puffy face). HAT causes a meningoencephalitis involving different parts of the brain. Non​specific neurological or psychiatric symptoms, such as headaches and mood or behavioural changes, are commonly found in both stage I and II, but their intensity and persistence increase as the illness evolves. The neurological symptoms include tremor, fasciculation, general motor weakness, paralysis of an extremity, Fig. 8.8.11.5  Patient with stage II sleeping sickness. Fig. 8.8.11.6  Scratch marks on the back of a stage I sleeping sickness patient. Photo by Dr. Johannes Blum, Swiss Tropical and Public Health Institute.

8.8.11  Human African trypanosomiasis 1455 epilepsy, akinesia, and abnormal movements such as dyskinesia or choreoathetosis, Parkinson-​like movements, unspecific movement disorders, speech disorders, and abnormal archaic reflexes. Sensory involvement is often described as hyperaesthesia, paraesthesia, an- aesthesia, or pruritus. In addition, psychiatric symptoms, such as irritability, psychotic reactions, aggressive behaviour, or inactivity with apathy, might dominate the clinical picture. Sleep disorder is a leading symptom, hence the name ‘sleeping sickness’. Somnographic studies have revealed a fragmentation with frequent sleep episodes of short durations at day and night caused by a dysregulation of the circadian rhythm of the sleep/​wake cycle: The patient has somnolence during the day, with uncontrollable urges to sleep, and nocturnal insomnia. Cardiac involvement includes QTc prolongation with risk of fatal arrhythmias, repolarization changes, and low voltage (observed in 50–​70% of patients) but rarely leads to relevant clinical heart failure. Endocrine disorders of the thyroid, adrenocortical, and sexual func- tion comprise hypo-​ and hyperfunction, but rarely demand specific treatment. T.b. rhodesiense HAT T.b. rhodesiense HAT is an acute febrile disease rapidly progressing to stage II and leading to death within 6 months. Recent descriptions of the clinical presentation show a high variability in different foci, possibly due to different parasite strains. The clinical presentation is similar to T.b. gambiense HAT, but more acute and trypanosomal chancres as a primary lesion at the site of the infection bite are more frequently seen. It appears a few days following the bite of an in- fected tsetse fly as an erythematous and tender swelling, which later becomes indurated and eventually may ulcerate. It is often accom- panied by a satellite lymphadenopathy. The localization of enlarged lymph nodes is usually submandibular, axillary, and inguinal rather than nuchal, and oedema is more frequently observed. Compared to T.b. gambiense HAT, thyroid dysfunction, adrenal insufficiency, and hypogonadism are more frequent and myopericarditis can be more severe. Liver involvement with hepatomegaly is usually moderate. HAT in travellers The symptomatology in Caucasians differs markedly from the textbook descriptions of African HAT patients. The disease onset is almost invariably acute and of the febrile type, regardless of the involved trypanosome species. The incubation time of T.b. gambi­ ense HAT in travellers is often shorter than 1 month, but might be as long as 7 years in immigrants. T.b. rhodesiense HAT has an incu- bation time of less than 3 weeks. It is an acute, life-​threatening dis- ease with the cardinal symptoms being high fever, headache, and a trypanosomal chancre (Fig. 8.8.11.7). The classical sleep disorders and neurological findings of HAT are not a hallmark in travellers, irrespective of the trypanosome species. Since most of the travellers are in the first stage and have a short duration of the disease, sleep disorders and neuropsychi- atric findings might not be present at the time of the first clinical assessment. Headache, lymphadenopathy, hepatosplenomegaly, and even icterus are non​specific findings seen in about a quarter to half of the patients. Gastrointestinal symptoms such as nausea, vomiting, and diarrhoea may dominate the clinical presentation. Electrocardiographic abnormalities due to myopericarditis and conduction abnormalities such as transient second and third degree atrioventricular block, supraventricular tachycardia, and ventricular premature captures have been reported. In a few travellers HAT has been complicated by renal failure requiring haemodialysis, multiorgan failure, disseminated intravascular coagulopathy, and coma with a fatal outcome. Differential diagnosis The differential diagnosis depends on the stage of the disease. In stage I the differential diagnosis is broad and includes any febrile dis- ease such as malaria, typhoid fever, viral diseases (dengue, chikun- gunya), rickettsiosis (also causes chancre), meningitis, leptospirosis, brucellosis, and gastrointestinal infection. In travellers the history of visit to a game park or hunting, the bite of a tsetse fly and the pres- ence of a chancre should alert the clinician. In stage II HAT other causes of chronic meningoencephalitis in- clude HIV-​related diseases such as cryptococcal meningitis, toxo- plasmosis, and tuberculosis. Clinical investigations Diagnosis The diagnosis is based on the visualization of the parasite in lymph node aspirate, peripheral blood, or cerebrospinal fluid, polymerase chain reaction (PCR) technology, and serologic testing. Parasite numbers in the peripheral blood of patients with T.b. gambiense HAT vary between more than 10 000 trypanosomes/​ ml to fewer than 100 trypanosomes/​ml, which is below the de- tection limit of microscopic examination of wet blood films, Giemsa-​stained thin blood films or thick blood films (5000–​10 000 trypanosomes/​ml). The sensitivity can be improved by using con- centration methods such as the microhaematocrit centrifugation technique or quantitative buffy coat (detection limit:  450–​500 Fig. 8.8.11.7  Trypanosomal chancre on the calf of a missionary returning from the Congo.

section 8  Infectious diseases 1456 trypanosomes/​ml) or the mini-​anion-​exchange centrifugation technique (50–​100 trypanosomes/​ml), or a combination of both techniques (10 trypanosomes/​ml). In contrast, the parasitaemia is more constant and higher in T.b. rhodesiense patients and the visu- alization of the parasite in the blood smear poses fewer problems. A simple LED-​based microscope for both bright-​field and fluor- escence microscopy has recently been developed by FIND, the Foundation for Innovative New Diagnostics, and Carl Zeiss. The use of fluorescence microscopy increases the sensitivity and ease of demonstration of trypanosomes. Lymph node aspiration is widely used, especially for the diagnosis of T.b. gambiense HAT. Fluid in enlarged lymph nodes, preferably of the posterior triangle of the neck (Winterbottom’s sign), is as- pirated and examined immediately at ×400 magnification without additional staining. Mobile trypanosomes can be detected for a few minutes between the numerous lymphocytes. The sensitivity of parasitological examination of lymph node aspirate varies between 40% and 80%. The Card Agglutination Test for Trypanosomiasis (CATT) is a cost-​efficient antibody detection test for mass screening of T.b. gam­ biense HAT. In most endemic regions its sensitivity varies from 87% to 98%. However, the CATT test is not suitable for T.b. rhodesiense. A first rapid diagnostic test has recently been developed by FIND and Standard Diagnostics. It is a lateral flow test that requires a drop of finger prick blood with the result available in 15 minutes. Molecular diagnosis with PCR has a good sensitivity, but is la- borious and mostly not available in the field. A real alternative is loop-​mediated isothermal amplification, which amplifies target DNA at a constant temperature. FIND and partners developed this technology which is feasible for field laboratories. Some practical issues are crucial for the correct diagnosis. A delay between sampling and examination can lead to a false negative re- sult since trypanosomes do not survive a long time after the blood sample is taken. Additionally, the sample should be sent to the la- boratory at a temperature of 2–​8°C (not frozen), be protected from sunlight, and tested within 12 hours. As treatment differs markedly between stage I and stage II HAT, staging of the disease by examination of the cerebrospinal fluid is essential. Stage II HAT is defined by an elevated white blood cell count (WBC >5 /​mm3) or the presence of trypanosomes in the cere- brospinal fluid. However, this has limited sensitivity and may lead to incorrect staging. Laboratory findings Among T.b. gambiense HAT patients in endemic regions anaemia and impaired renal function are frequent, but liver enzymes, lac- tate dehydrogenase, creatinine kinase, and blood sugar are usually normal. In tourists with T.b. rhodesiense HAT, elevated creatinine (81%), liver enzymes (82%), low platelets (92%), and elevated levels of C reactive protein are frequent. Severe haematological disorders and abnormal kidney function tests have been reported. Magnetic resonance imaging Since the knowledge on imaging changes in HAT is scarce, this method cannot be used for the diagnosis of HAT. The findings are multifarious and include symmetrical focal lesions, diffuse hyperintensity, brain oedema with demyelination, brain atrophy, and multiple abnormal signals. Treatment General considerations HAT is curable, especially if the diagnosis is made at an early stage of the disease. In the stark reality of the African setting, however, there are many obstacles to successful patient management: Sleeping sickness is a disease of rural places. The active foci of sleeping sickness are usually in remote and insecure places, which are difficult to reach. Many treatment centres work under emer- gency conditions with extremely restricted resources. Numerous affected patients, without proper access to healthcare, are left unattended. • Diagnosis is difficult. Initial diagnosis and exact staging of sleeping sickness require sophisticated methods that are often dangerous to the patient and justified only in the hands of experienced per- sonnel. Repetitive training programmes, constant supervision, and continuous quality control are necessary but in reality, rarely available. • Treatment of trypanosomiasis is extremely costly, although the drugs themselves are now covered by a donation programme. Invariably, demand exceeds the locally available resources. External funding and sustainable donor commitments for rural Africa are generally decreasing. • Treatment is complicated. Treatment of HAT is dangerous, pro- longed, and usually requires hospitalization. Most patients with stage II trypanosomiasis are severely ill and malnourished. Adverse drug reactions during treatment are difficult to assess because of concomitant pathologies. Their management requires considerable medical skill and good nursing care. Hospitals in rural Africa are often inadequately equipped and staffed to accom- plish good patient care. • HAT treatment is not standardized. Trypanosomiasis treatment regimens vary considerably between countries and treatment centres. Results from different centres are comparable to only a very limited extent. Few properly conducted and sufficiently powered clinical trials are available to evaluate duration, dosage, and possible combinations of drugs. Sufficient infrastructure for carrying out clinical research exists in only a handful of places. Stage I drugs The treatment of HAT depends on the trypanosome subspecies (T. b. gambiense or T. b. rhodesiense) and the stage of the disease (stage I or stage II) (Table 8.8.11.2). Pentamidine Since its introduction in 1940, pentamidine has become the drug of choice for gambiense HAT stage I, achieving cure rates as high as 98%. However, there are frequent failures in rhodesiense HAT. Table 8.8.11.2  The choice of drugs in the treatment of sleeping sickness Gambiense sleeping sickness Rhodesiense sleeping sickness Stage I Pentamidine Stage I Suramin Stage II NECT (nifurtimox + eflornithine combination therapy) Stage II Melarsoprol

8.8.11  Human African trypanosomiasis 1457 Some cures of stage II infections have also been reported, but cere- brospinal fluid drug levels are usually not sufficiently high to guar- antee a reliable trypanocidal effect in the central nervous system. Pentamidine is usually given by deep intramuscular injec- tion, often to outpatients. If hospital care and reasonable moni- toring conditions are available, an intravenous infusion, given in normal saline over 2 h, might be used instead. The main ad- vantage of pentamidine over other drugs is the short treatment course and ease of administration. Adverse effects are related to the route of administration or its dose and are usually reversible (Table 8.8.11.3). Pentamidine is also used as second-​line therapy for visceral leishmaniasis and in the prophylaxis and treatment of opportun- istic Pneumocystis jiroveci pneumonia in AIDS patients. Since the start of the HIV pandemic, the cost of pentamidine has been in- creased more than 10-​fold by producers, making it unaffordable for health institutions in low-​income countries. After an intervention by WHO, pentamidine is now made available for use in HAT as part of a donation programme. Suramin In the early 20th century, the development of suramin, resulting from German research on the trypanocidal activity of various dyes (‘Bayer 205’), was a major breakthrough in the field of tropical medi- cine. For the first time, human African trypanosomiasis, at least in its early stages, became treatable without causing major harm. Even today, suramin is still used to treat stage I HAT, especially rhodesiense. Like pentamidine, it does not reach therapeutic levels in cerebrospinal fluid. Suramin is injected intravenously after dilu- tion in sterile water. Adverse effects depend on nutritional status, concomitant illnesses (especially onchocerciasis), and the patient’s clinical condi- tion. Although life-​threatening reactions have been described, ser- ious adverse effects are rare (Table 8.8.11.3). Table 8.8.11.3  Dosage and principal adverse reactions of antitrypanosomal agents Drug Dosage regimen Adverse drug reactions Pentamidine 4 mg/​kg body weight intramuscular daily or on alternate days for 7 to 10 injections (3 dose regimen currently under investigation) Hypotensive reaction with tachycardia, dizziness, even collapse and shock, especially after intravenous administration, close monitoring of pulse rate and blood pressure after injection is mandatory Inflammatory reactions at the site of injection (sterile abscesses, necrosis) Renal, hepatic, and pancreatic dysfunction Neurotoxicity: peripheral polyneuropathy Bone marrow depression Suramin Day 1: Test dose of 4–​5 mg/​kg body weight Pyrexia (very common) Day 3, 10, 17, 24, and 31: 20 mg/​kg body weight, maximum dose per injection 1 g Early hypersensitivity reactions such as nausea, circulatory collapse, urticaria Late hypersensitivity reactions: skin reactions (exfoliative dermatitis), haemolytic anaemia Renal impairment: albuminuria, cylinduria, haematuria (high renal tissue concentrations); regular urine checks during treatment are mandatory Neurotoxicity: peripheral neuropathy Bone marrow toxicity: agranulocytosis, thrombocytopenia Melarsoprol New regimen: Treatment-​induced encephalopathy Day 1–​10: 2.2 mg/​kg body weight Pyrexia Neurotoxicity: peripheral motor or sensory polyneuropathy Dermatological reactions: pruritus, urticaria, exfoliative dermatitis Cardiotoxicity Renal and hepatic dysfunction Eflornithine Most commonly used dosage regimen: Gastrointestinal symptoms such as nausea, vomiting, and diarrhoea 100 mg/​kg body weight at 6-​hourly intervals for 14 days Bone marrow toxicity: anaemia, leucopenia, thrombocytopenia Alopecia, usually towards the end of the treatment cycle Neurological symptoms such as convulsions Nifurtimox 5 mg/​kg body weight 3 times daily for 30 days Abdominal discomfort such as nausea, pains, and vomiting in half of the treated patients, often leading to a disruption of the treatment course Neurological complications: convulsions Impairment of cerebellar function, polyneuropathy Skin reactions NECT Eflornithine: 200 mg/​kg body weight at 12-​hourly intervals for 7 days; Nifurtimox: 5 mg/​kg body weight 3 times daily for 10 days Neurological symptoms and gastrointestinal disorders are the leading adverse reactions, however, much less pronounced compared to the eflornithine monotherapy

section 8  Infectious diseases 1458 Stage II drugs Melarsoprol Until the systematic introduction of the arsenical compound melarsoprol in 1949, advanced trypanosomiasis was virtually un- treatable. Since then, it has remained the most widely used stage II antitrypanosomal drug both for gambiense and rhodesiense infec- tions. It has saved thousands of lives, but has a high rate of dangerous adverse effects. Increasing frequency of relapses and resistance have been reported in some parts of the Democratic Republic of Congo, Angola, Sudan, and Uganda (Fig. 8.8.11.8). Melarsoprol clears trypanosomes rapidly from the blood, lymph, and cerebrospinal fluid. Its toxicity usually restricts its use to stage II disease. It is given by slow intravenous injection; extravascular leakage must be avoided. A new, simpler regimen is based on pharmacokinetic investiga- tions and modelling (Table 8.8.11.3). The most important adverse effect is an acute encephalopathy, provoked around day 5 to 8 of the treatment course in 5 to 14% of all patients. Other adverse re- actions are severe headache, convulsions, rapid neurological de- terioration, or deepening of coma. Characteristically, the comatose patient’s eyes remain open. The overall case fatality under treatment ranges between 2 and 6%, depending on the stage of disease and the quality of medical and nursing care. Simultaneous administra- tion of glucocorticosteroids (prednisolone 1 mg/​kg body weight; maximum 40 mg daily) reduces mortality. However, in areas where tuberculosis, amoebiasis, and strongyloidiasis are highly prevalent, their use is problematic. Eflornithine (DFMO) Initially developed as antitumour agent, eflornithine (α-​ dif­luoro­methylornithine) was introduced in 1980 as an antitry­ panosomal drug, in the hope that it might replace melarsoprol for treatment of stage II trypanosomiasis. However, exorbi- tant costs and limited availability have restricted its use mostly to melarsoprol-​refractory cases of gambiense sleeping sickness. T. b. rhodesiense is much less sensitive, because of a much higher turnover rate of the target enzyme ornithine decarboxylase, and therefore cannot be treated with eflornithine. Eflornithine should be administered slowly over a period of at least 30 min. Continuous 24-​h administration is preferable if facilities allow. The range of adverse reactions to eflornithine is wide, their occurrence and intensity increase with the duration of treatment and the severity of the patient’s general condition (Table 8.8.11.3). In 2000 WHO established a public-​private partnership with Sanofi which resulted in supply of eflornithine free of charge. The partnership was renewed in 2006 and in 2011. NECT (Nifurtimox eflornithine combination therapy) NECT, a combination treatment of nifurtimox and eflornithine was introduced in 2009. It reduces the duration of treatment of eflornithine to 7 days and the number of IV infusions to 14, com- bined with oral nifurtimox at 5 mg/​kg 3-​times a day for 10 days. But, unfortunately, it has not been studied in T.b. rhodesiense infection. Nifurtimox is registered for Chagas’ disease (T. cruzi) but not for HAT. As a monotherapy nifurtimox is not very effective for HAT; however, in combination with eflornithine it acts synergistically producing oxidative stress to the parasite. NECT is superior to the eflornithine monotherapy regarding ef- ficacy and safety. The relapse rate after 18 months was 1.4% com- pared to 5.7% for the monotherapy. Adverse events were lower in the NECT group than in the eflornithine group. Both drugs are provided free of charge by WHO to endemic countries with a kit containing all the material needed for its administration. 50 000 40 000 30 000 20 000 10 000 1940 1943 1946 1949 1952 1955 1958 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012 0 Cases Year Fig. 8.8.11.8  Number of annually reported cases of human African trypanosomiasis. Reprinted from Franco JR et al. (2014). Epidemiology of human African trypanosomiasis. Clin Epidemiol, 6, 257–​275 (based on data from the WHO).

8.8.12 Chagas disease 1459

8.8.12 Chagas disease 1459

8.8.12  Chagas disease 1459 New drug candidates Fexinidazole, a 5-​nitroimidazole, was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after having reviewed over 700 nitroheterocyclic compounds. The molecule was able to cure mouse models of infection after oral dosing and to cross the blood-​brain barrier. Fexinidazole is rapidly metabolized to a sulf- oxide and a sulfone, both metabolites are trypanocidal, and reach significant plasma levels. Phase I clinical studies were completed successfully demonstrating the safety of a 10-​day oral treatment regime. Phase II/​III clinical trials are almost completed. Several hundred patients with stage II disease were treated and all cleared parasitaemia after treatment, the 2-​year follow-​up is still ongoing. There is hope that the new drug will be on the market and freely available by 2019. A second clinical candidate is the benzoxaborole SCYX-​7158 which is also in the portfolio of DNDi. This novel class of boron-​ containing molecules is orally bioavailable and able to pass the blood-​brain barrier. SCYX-​7158 completed phase I clinical studies successfully. The long terminal half-​life gives hope for a short treat- ment course, maybe even single dose treatment. Phase II/​III trials are in progress. Prevention/​Individual protection A vaccine for sleeping sickness is not available and chemo- prophylaxis is not recommended because of the toxicity of the available drugs and the low risk of infection. Travellers should take notice of the risk of sleeping sickness, be informed on the transmission and clinical presentation of the disease and preven- tion. The only preventive measure is the protection from tsetse fly bites. The flies are attracted to bright or contrasting colours, particularly blue, as well as to the dust and motion of vehicles. As routine preventive measures, travellers should avoid known areas of tsetse flies, travel in endemic foci in cars with screened or closed windows, use insect repellent, and wear wrist-​ and ankle-​ length clothes. Efforts to eliminate HAT The first milestone in the effort to eliminate HAT was the London Declaration on Neglected Tropical Diseases launched in 2012. It set the goal to eliminate HAT and four other neglected dis- eases by the year 2020 (i.e. reduce the number of reported cases <2000/​year and the number of active foci). In 2014 WHO estab- lished a Coordination Network for HAT to strengthen efforts to eliminate the disease. Stakeholders are national sleeping sickness control programmes, organizations developing drugs and diagnos- tics, governmental organizations, NGOs, and philanthropic organ- izations. New diagnostics and drugs are prerequisites for elimination especially an oral drug for both disease stages, rendering staging unnecessary. Another important element in the elimination effort is vector control which is based on new types of targets, attractants, sterile male flies, or on the treatment of domestic animals which the flies feed on. Elimination is certainly a realistic goal provided the new tools become available. FURTHER READING Balmer O, et al. (2011). Phylogeography and taxonomy of Trypanosoma brucei. PLoS Negl Trop Dis, 5, e961. Blum JA, et al. (2008). Cardiac involvement in African and American trypanosomiasis. Lancet Infect Dis, 8, 631–​41. Brun R, et al. (2010). Human African trypanosomiasis. Lancet, 375, 148–​59. Eperon G, et al. (2014). Treatment options for second-​stage gambiense human African trypanosomiasis. Expert Rev Anti Infect Ther, 12, 1407–​17. Franco JR, et al. (2014). The journey towards elimination of gambiense human African trypanosomiasis: not far, nor easy. Parasitology, 141, 748–​60. Kennedy PGE (2013). Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol, 12, 186–​94. Kuepfer I, et al. (2011). Clinical presentation of T.b. rhodesiense sleeping sickness in second stage patients from Tanzania and Uganda. PLoS Negl Trop Dis, 5, e968. Mäser P, et al. (2012). Antiparasitic agents: new drugs on the horizon. Curr Opin Pharmacol, 12, 562–​6. Mesu VKBK, et  al. (2018). Oral fexinidazole for late-stage African Trypanosoma brucei gambiense trypanosomiasis: a pivotal multi­ centre, randomised, non-inferiority trial. Lancet, 391, 144–54. Schmid C, et  al. (2012). In-​hospital safety in field conditions of nifurtimox eflornithine combination therapy (NECT) for T.b. gam­ biense sleeping sickness. PLoS Negl Trop Dis, 6, e1920. Simarro PP, et al. (2015). Monitoring the progress towards the elim- ination of Gambiense human African trypanosomiasis. PLoS Negl Trop Dis, 9, e3785. Steinmann P, et al. (2015). Contemporary and emerging strategies for eliminating human African trypanosomiasis due to Trypanosoma brucei gambiense: review. Trop Med Int Health, 20, 707–​18. Vale GA, Torr S (2004). Development of bait technology to control tsetse. In: Maudlin I, Holmes PH, Miles MA (eds). The trypanoso­ miases, pp. 509–​24. CABI, Wallingford. Welburn SC, et al. (2016). Beyond Tsetse—Implications for Research and Control of Human African Trypanosomiasis Epidemics. Trends Parasitol, 32, 230–41. World Health Organization (2013). Control and surveillance of African trypanosomiasis. WHO Technical Report Series 984. WHO, Geneva. 8.8.12  Chagas disease Michael A. Miles A poeira de Curvelo não az mal para ninguém não Do pulmão lá ninguém morre O que mata é o coração The dust of Curvelo does not harm anybody No one dies there of lung disease What kills is the heart (From the poem O galo cantou na serra by Luiz Claudio and Guimarães Rosa)

section 8  Infectious diseases 1460 ESSENTIALS Trypanosoma cruzi, the protozoan parasite that causes Chagas dis- ease, is a zoonotic infection with many mammal host and vector species. It is transmitted to humans by contamination of mucous membranes or abraded skin with infected faeces of bloodsucking triatomine bugs, also by blood transfusion, organ transplantation, transplacentally, and orally by food contaminated with infective forms. It multiplies intracellularly (pseudocysts) as amastigotes in a wide range of mammalian cells, particularly heart and smooth muscle, from which flagellated trypomastigotes emerge to reinvade cells or circulate in blood. Around 7 million people are infected in Latin America; imported cases and congenital cases occur elsewhere. Clinical features There are classically two principal phases. (1) Acute—​asymptomatic or with manifestations that can include fever, myalgia, headache, vomiting, diarrhoea, anorexia, facial or generalized oedema, rash, generalized lymphadenopathy, and hepatosplenomegaly; there may be a lesion at the portal of entry; the acute phase is fatal in less than 10% of cases. (2) Chronic—​indeterminate (asymptomatic) or, in up to 30% of those recovering from the acute phase, with cardiac in- volvement (typically cardiomyopathy leading to congestive cardiac failure, arrhythmias and ECG abnormalities due to focal inflamma- tory lesions of the conducting system), also megaoesophagus and megacolon. Infection is opportunistic, relapsing in the immunocom- promised. Meningoencephalitic involvement may occur in the im- munocompromised (most typically with HIV/​AIDS), and is also seen in congenital cases. Diagnosis (1) Acute phase—​parasitaemia is scanty, but circulating trypomastigotes may be detectable in the acute phase by microscopy of blood, en- hanced by concentration methods. (2) Chronic phase—​multiple cultures of centrifuged (plasma de- pleted) blood or feeding and subsequent dissection of laboratory-​ reared triatomines (xenodiagnosis) may reveal infection. (3) Serological testing—​can demonstrate evidence of infection, but needs to be standardized with reference sera and by external quality control. Treatment (1) Acute phase—​proven cases should be treated promptly with benznidazole or nifurtimox, but there is no guarantee that a full course of treatment will eliminate the infection. (2) Chronic phase—​the value of drug treatment for adults in the chronic phase is still debated; supportive care may include the following (a)  for heart disease—​conventional drug treatment for cardiac failure and arrhythmias; cardiac pacemaker; (b)  for megaoesophagus—​dilatation; segmentary removal of stomach muscle; replacement of the distal oesophagus; (c)  megacolon—​ resection and anastomosis with the rectal stump. Prevention Proven methods of controlling domestic triatomine bugs include insecticide spraying (with pyrethroids), health education, commu- nity support, and house improvement. Serological surveillance of children detects residual endemic foci or congenital transmission and is vital for monitoring the success of control programmes.
The Southern Cone programme against Triatoma infestans is con- sidered a model for international cooperation in disease control. There is no vaccine. Introduction and aetiology In 1907, in the space of a few months, the Brazilian scientist Carlos Chagas discovered the disease that bears his name and described the entire life cycle of the causative organism. Chagas first found the protozoan agent Trypanosoma cruzi in the gut of the large blood- sucking insect vector, the triatomine bug (order Hemiptera, family Reduviidae, subfamily Triatominae) (Fig. 8.8.12.1). Later he re- turned to bug-​infested houses and detected T. cruzi in the blood of sick children. T. cruzi is a kinetoplastid protozoan. In addition to the nucleus, it has a second, microscopically visible DNA-​containing organelle, the kinetoplast. The main life cycle stages (trypomastigote, amastigote, epimastigote) are distinguished by the position of the kinetoplast relative to the nucleus and by the presence or absence of a free flagellum. Vector-​borne transmission of T. cruzi is by contamination of the mammal host with infected faeces of triatomine bugs, not by their bite. During or shortly after feeding, bugs release blackish liquid faeces and urine on to the skin of the host. Infective forms (metacyclic trypomastigotes) penetrate mucous membranes or abraded skin. Inside the mammal, T.  cruzi is primarily an intracellular para- site. Trypomastigotes enter non​phagocytic or phagocytic cells, in which they transform to ovoid or round aflagellate amastigotes that Fig. 8.8.12.1  Adult female triatomine bug (Panstrongylus megistus), with a single egg shown adjacent to the tip of the abdomen. Courtesy of Dr T. V. Barrett.

8.8.12  Chagas disease 1461 multiply inside the cytoplasm of the cell by binary fission to produce a pseudocyst (Fig. 8.8.12.2). After 5 days or more, the pseudocyst ruptures to release numerous new trypomastigotes, which reinvade cells or circulate in the blood. Multiplication may occur at the site of infection. Pseudocysts may occur in a wide range of cell types but subsequently predominate in muscle, especially heart and smooth muscle. In the blood, trypomastigotes are small, often C-​shaped, with a large terminal kinetoplast (Fig. 8.8.12.3). In fulminating or experimental infections, slender highly motile trypomastigotes may also sometimes be seen. Trypomastigotes do not multiply in the blood. Triatomine bugs become infected by taking a blood meal from an infected mammal; birds and reptiles are not susceptible to infection. Infection in the bug is confined to the alimentary tract, where T. cruzi multiplies by binary fission as epimastigotes (kineto- plast adjacent to the nucleus). Metacyclic trypomastigotes are pro- duced in the hindgut and rectum of the bug. All stages of the T. cruzi life cycle can be cultured in vitro. T. cruzi can also be transmitted by blood transfusion and organ transplantation, across the placenta, via breast milk (rarely), and orally through food contaminated by triatomine faeces and the raw meat of infected mammals. Sexual transmission has not been documented. Epidemiology Vector-​borne T. cruzi infection is confined to the Americas. Closely related organisms of the same subgenus (Schizotrypanum) are cosmopolitan in bats. The vast majority of more than 140 recognized triatomine bug species are restricted to the Americas. Their natural habitats are the refuges of mammals, birds, and reptiles, in trees, in burrows, and among rocks. All mammals are thought to be suscep- tible to T. cruzi, which has been reported from at least 150 mammal species. The opossum (Didelphis spp.) is the most commonly re- ported sylvatic host. A few triatomine species thrive as domestic colonies. More than 10 000 bugs have been found in a single house. Before the recent Southern Cone initiative to control Triatoma infestans, this vector species was widespread in rural housing of the Southern Cone countries of South America (Argentina, Bolivia, Brazil, Chile, Paraguay, Uruguay, and southern Peru). Rhodnius pro­ lixus is the common vector in northern South America and has also been an important vector in Central America, with Triatoma dim­ idiata as secondary vector in the same regions. Panstrongylus meg­ istus (Fig. 8.8.12.1) infests central and eastern Brazil, and Triatoma brasiliensis north-​eastern Brazil. Animals that share human dwell- ings, such as guinea pigs, dogs, cats, rats, and mice are domestic res- ervoirs of T. cruzi infection. Chickens, although not susceptible to T. cruzi, encourage triatomine infestation and can sustain large bug colonies. Serological surveys suggest that 6–​7 million people may still be infected with T. cruzi in South and Central America, a level that has been reduced from up to 20 million around four decades ago. In some communities, seropositivity rates may still exceed 50%. As expected from the precarious contaminative route of trans- mission, prevalence rises with age. Based on prevalence, before recent control initiatives, it was estimated that up to 300 000 new infections might occur in Latin America each year; this is now reduced to less than 60 000/​year. Only approximately 1500 cases are known from the Amazon basin, about half of these due to oral transmission by drinking plant juices contaminated by live triatomine bugs during juice extraction or storage (e.g. juice from berries of açaí or bacaba palms or sugar cane). Oral outbreaks also occur elsewhere; one among schoolchildren in Caracas, Venezuela, due to guava juice, involved 103 cases. There are rela- tively few Amazonian cases because the local forest vectors do not colonize houses. For the same reason, autochthonous vector-​ borne infection is very rare in the United States of America. However, there is an autochthonous cycle of T. cruzi infection among dogs in Texas. Abundant local vectors (Triatoma species) and established sylvatic transmission cycles, involving mammals such as opossums, racoons and woodrats, are widespread in the United States. Not surprisingly, sporadic T.  cruzi infections can be found among migrants from Latin America. Serological studies indicate that there may be up to 300 000 carriers of T. cruzi infection in the United States among migrants, and that universal surveillance of blood donors should, therefore, be introduced. It is estimated (a) (b) Fig. 8.8.12.2  Pseudocyst of Trypanosoma cruzi. Pseudocyst in (a) heart muscle and (b) umbilical cord, from a congenital case of Chagas disease. (a) Courtesy of J E Williams; (b) courtesy of Dr Hipolito de Almeida. Fig. 8.8.12.3  Trypanosoma cruzi C-​shaped trypomastigote in blood. Note the large posterior kinetoplast.

section 8  Infectious diseases 1462 that there are more than 100 000 carriers of infection in Europe. Cases of transmission by blood or organ donors and rare con- genital cases can thus occur worldwide. In 2007, the World Health Organization (WHO) launched a ‘Global Network for Chagas Elimination’ to raise global awareness and coordinate prevention of transmission. Initial acute infections are frequently asymptomatic or over- looked. It is thought that less than 10% of acute infections in chil- dren or young adults are fatal. Morbidity due to Chagas disease arises primarily from the chronic infection. Once acquired, infec- tion is usually carried for life. Around 30% of those infected will subsequently display ECG abnormalities and chagasic cardiomy- opathy, and a proportion of those have associated megaoesophagus or megacolon. There are regional differences in the epidemiology of Chagas disease. Research in molecular genetics has shown that the spe- cies T. cruzi is remarkably diverse genetically. Six distinct genetic lineages are currently described, TcI-​TcVI. The six lineages have complex disparate but partially overlapping geographical and ecological distributions and are circumstantially associated with the different epidemiological features. TcI is the principal agent north of the Amazon, in association with chagasic heart disease but where gastrointestinal involvement, megaoesophagus, and megacolon are considered to be rare. TcII is one of three principal agents of Chagas disease in the Southern Cone region of South America, where chagasic cardiomyopathy, megaoesophagus, and megacolon are found. TcIII is seldom isolated from humans but is widely distributed with the natural armadillo host Dasypus nove­ mcinctus. TcIV is a sporadic secondary agent of Chagas disease in Venezuela. TcV and TcVI, like TcII, are also agents of Chagas dis- ease in the Southern Cone region, and are known to be relatively recent hybrids of TcII and TcIII. Pathogenesis and pathology At the portal of entry, local multiplication of T. cruzi may lead to uni- lateral conjunctivitis or to a skin lesion (Fig. 8.8.12.4). Unruptured pseudocysts in muscle apparently generate no inflammatory re- sponse. Pseudocyst rupture is followed by infiltration of lympho- cytes, monocytes, and/​or polymorphonuclear cells. Antigens released from pseudocysts may spread and be adsorbed on to ad- jacent uninfected cells. Such uninfected cells may be attacked by the immune response of the host and be destroyed. In this way, expanded focal lesions may be produced. Post-​mortem histology of human hearts and experimental studies in dogs have demon- strated a clear association between ECG abnormalities and focal lesions in the conducting system of the heart. Much damage may occur in the acute phase of infection, particularly if pseudocysts are numerous. Post-​mortem histology has demonstrated that neuron loss is a feature of chagasic cardiopathy and of megasyndromes, which may be exacerbated by further disease or age-​related loss. Thus, a threshold may be reached, often many years after the acute infection, at which organ function is perturbed. Further ECG ab- normalities, aperistalsis, and organ enlargement may ensue. This ‘neurogenic’ pathogenesis has been linked to sudden death. It is proposed that pathological exposure of normal host-​ sequestered antigens, or sharing of antigens between T. cruzi and its host, may precipitate autoimmune pathogenesis. Some chronic chagasic cardiomyopathy is said to display a renewed intense in- flammatory response and a progressive diffuse myocarditis, and a slow decline in cardiac function. The contribution of the lifelong infection to the pathogenesis of chronic Chagas disease continues to be controversial. Although some studies have suggested that elimination of residual infection improves prognosis a recent randomized study (BENEFIT) treat- ment of 2854 patients already with some chagasic cardiomyop- athy showed no reduction in cardiac clinical deterioration during five years of follow-​up, despite evidence of reduced parasitaemias; asymptomatic trials are desirable. After the initial acute phase, trypomastigotes are detectable in the blood only by sensitive in- direct methods such as polymerase chain reaction (PCR). Similarly, pseudocysts in the tissues are infrequent, but are detectable im- munologically and by amplification of T. cruzi DNA. In vivo imaging of mice with prolonged chronic infections of bioluminescent trans- genic T. cruzi has revealed the presence of cryptic, dynamic, wide- spread, and unpredictably distributed foci of parasite replication. Severity of T. cruzi infection is considered to be influenced by the balance between the Th1 (cell mediated) and Th2 (antibody) arms of the immune response and the extent of the inflammatory response, the indeterminate form being associated with an anti-​inflamma- tory cytokine profile. Patients immunocompromised by AIDS have impaired Th1 responses. Thus, HIV-​positive patients chronically infected with T. cruzi may suffer reactivation of the acute phase of Chagas disease, with microscopically patent parasitaemia, poor prognosis, and risk of meningoencephalitic invasive lesions. At the level of gross pathology, substantial megacardia may be seen. Thinning of the myocardium may be present, with focal an- eurysms visible upon transillumination, especially at the apex of the left ventricle (Fig. 8.8.12.5) and thrombus in the right atrial appendage (Fig. 8.8.12.6). Apical aneurysm is considered to Fig. 8.8.12.4  Romaña’s sign in acute Chagas disease.

8.8.12  Chagas disease 1463 be a pathognomonic sign of chronic chagasic cardiomyopathy. Megaoesophagus (Fig. 8.8.12.7) and megacolon (Fig. 8.8.12.8) may show enormous dilatation and thinning of the wall. Chagasic megaoesophagus is more frequent than chagasic megacolon, but both may occur in the same patient and are often accompanied by chagasic heart disease. Chagasic megaoesophagus may be a prelude to carcinoma. Occasionally megasyndromes may arise in infants, following congenital infection. Clinical features Classically, there are two principal clinical phases of Chagas dis- ease. In the acute phase, symptoms can include fever, myalgia, head- ache, hepatosplenomegaly, lymphadenopathy, facial or generalized oedema, rash, vomiting, diarrhoea, and anorexia. If T. cruzi invasion has been via the conjunctiva, Romaña’s sign might be present: uni- lateral conjunctivitis, chemosis, and periophthalmic oedema (Fig. 8.8.12.4). If the portal of entry is the skin, an indurated oedema- tous cutaneous lesion (chagoma) may be seen. Regional lymph- adenopathy may be present. Multiple chagomas occasionally occur in acute-​phase infections in infants or in reactivated immunocom- promised cases. ECG abnormalities include sinus tachycardia, in- creased PR interval, T-​wave changes, and low QRS voltage. The incubation period can be as short as 2 weeks or as long as several months if infection is due to transfusion of contaminated blood. General lymphadenopathy and splenomegaly are frequent in blood transfusion-​acquired infections. Fig. 8.8.12.5  Apical aneurysm of the left ventricle in chronic Chagas disease. Courtesy of Dr J. S. de Oliveira. Fig. 8.8.12.6  Mural thrombus filling the right atrial appendage. Copyright D. A. Warrell. Fig. 8.8.12.7  Megaoesophagus seen by radiography in chronic Chagas disease. Courtesy of Dr J. S. de Oliveira. Fig. 8.8.12.8  Megacolon post-​mortem in chronic Chagas disease. Courtesy of Dr J. S. de Oliveira.

section 8  Infectious diseases 1464 Congenital acute infection may cause fever, oedema, metastatic chagomas, neurological signs such as convulsions, tremors, and weak reflexes, and apnoea. Hepatosplenomegaly is frequent. The ECG is usually normal but low-​voltage complexes, reduced T-​wave height, and longer atrioventricular (AV) conduction time may be present. Meningoencephalitis is rare in adults, more frequent in infants, and common in immunocompromised patients. It carries a poor prognosis. The clinical picture of AIDS-​associated chagasic meningoenceph- alitis may be similar to toxoplasmosis. Haemorrhagic necrotic en- cephalitis is described in the nests of trypanosomes in microglia. Congenital infection may resemble toxoplasmosis, cytomegalovirus infection, or syphilis, with an increased likelihood of abortion and premature birth. Congenital infection is well known in Bolivia but less frequently reported from Venezuela and Brazil. The symptomatic or asymptomatic acute phase infection is followed by the chronic phase, which may be symptom-​free (indeterminate) for life. However, symptoms emerge in up to 30% of patients recovering from the acute phase. Cardiac symptoms include arrhythmias, pal- pitations, chest pain, oedema, dizziness, syncope, and dyspnoea. The cardiac enlargement may be massive with chronic congestive cardiac failure, apical aneurysm (Fig. 8.8.12.5), and thrombus in the right atrial appendage (Fig. 8.8.12.6). The cardiac conducting system is in- volved, especially the sinus node, bundle of His and AV node, in which there is mononuclear and mast-​cell infiltration, inflammation, and fibrosis. Characteristic ECG abnormalities are right bundle branch block (RBBB) and left anterior hemiblock (LAH). AV conduction ab- normalities, including AV block, may be present. Arrhythmias may include sinus bradycardia, sinoatrial block, ventricular tachycardia, primary T-​wave changes, and abnormal Q-​waves. The severity of heart disease is graded by the degree of disturbance; there are several alter- native published scales of severity. Sudden death is attributable, not to ruptured aneurysm, but to arrhythmias often precipitated by exercise (e.g. on the football field). Radiography may reveal megacardia (Fig. 8.8.12.9). Signs of oesophageal involvement include loss of peristalsis, regurgitation, and dysphagia (Fig. 8.8.12.7). Parotid enlargement may be associated. In megacolon, there may be failure of defaecation, constipa- tion, and faecaloma (Fig. 8.8.12.8). Progressive dilatation of either the oesophagus or colon can be graded clinically according to se- verity and may be detectable by radiography. Megaduodenum and megaureter are also described. The lymph nodes between the pul- monary trunk and the aorta are frequently enlarged. The differential diagnosis includes other types of heart disease and causes of ECG abnormalities. RBBB and LAH are indicative, but a history of exposure to T. cruzi infection and laboratory diagnostic evidence must be considered (see next). Laboratory diagnosis A history of exposure to triatomine bugs, to transfused blood that is potentially contaminated or a prolonged stay in endemic regions must be considered. Rarely short-​term visitors to endemic regions may acquire infection. In the acute phase motile trypomastigotes might be seen in unstained, wet blood preparations examined by microscopy (Fig. 8.8.12.3). Nevertheless, parasitaemia is often scanty or undetectable by this method. The sensitivity of parasitological diagnosis may be enhanced by microscopy of samples prepared with concentration methods, such as the centrifugation pellet from separated serum (Strout’s method), the haematocrit buffy coat layer, Giemsa-​stained thick films, or the centrifugation sediment after lysis of red blood cells with 0.87% am- monium chloride. All these tests may be negative if parasitaemia is low. Potentially infected blood must be handled with care, especially during haematocrit centrifugation, as a single trypomastigote can cause infec- tion. Multiple blood cultures may also be performed, with a sensitive blood agar-​based medium and physiological saline overlay. However, before culture the blood cells and potential trypanosomes must be pel- leted by centrifugation and the plasma removed, because antibodies in plasma can lyse the culture form (epimastigote) stage. Even more sensitive than blood culture is xenodiagnosis, in which hungry fourth or fifth instar bugs from a clean triatomine colony, raised from bug eggs and fed only on birds, are allowed to feed on the patient. Bugs are applied in a plastic pot contained discretely in a black bag, which is tied beneath the patient’s forearm. The bugs are dissected 20–​25 days later. The hindgut and rectum are drawn out into a drop of sterile physio- logical saline, mixed with a blunt instrument (microspatula), and ob- served microscopically for motile epimastigotes and trypomastigotes. Dissection should be performed behind a small, perspex safety screen or in a microbiological safety cabinet. R. prolixus is the most avid feeder for xenodiagnosis but may cause delayed hypersensitivity reactions in sensitized patients. Anaphylaxis is rare but two cases are known. The local vector should be used as the susceptibility of triatomine species varies with the strain of T. cruzi. If pericardial effusion is a feature of acute infection and the fluid is drained it should be examined by microscopy for the presence of trypanosomes, which might be detectable even if blood parasit- aemia is subpatent. After the acute-​phase infection, all the aforementioned methods of parasitological diagnosis will fail except xenodiagnosis and, possibly, multiple blood cultures. Up to 50% of patients in the chronic phase may yield a positive xenodiagnosis, providing at least 10 triatomine bugs are used. Polymerase chain reaction or loop-mediated iso- thermal amplification (LAMP) of multicopy T. cruzi DNA targets may be sensitive and specific but are not yet available as routine diag- nostic tests. Serum antibody is produced within a few days of T. cruzi Fig. 8.8.12.9  Chest radiograph showing gross cardiac enlargement in a Brazilian woman with chronic Chagas disease. Copyright D. A. Warrell.

8.8.12  Chagas disease 1465 infection and persists for life in untreated patients. There is an early IgM response, but it is not sustained at the high levels seen in African trypanosomiasis. Persistent IgG may be detected by the enzyme-​ linked immunosorbent assay, the indirect fluorescent-​antibody test or the indirect haemagglutination test. Cross reactions may occur with visceral and mucocutaneous leishmaniasis, with treponematoses, and possibly with other hyperimmune responses or autoimmune diseases. Recombinant antigens have been used to improve species specificity and several commercial kits and rapid tests are available, with differing sensitivities, specificities, ease of use, and costs. Most diagnostic kits are prepared from TcII or TcVI antigen preparations; sensitivity of serological diagnosis has been reported to be lower in TcI endemic regions. Quality of commercial tests should not be presumed without reference to authoritative comparative studies. Serological assays must be standardized with negative and positive control sera and by reference to experienced external reference centres to check reprodu- cibility. Transplacentally acquired IgG may persist for up to 9 months in infants born of seropositive mothers. IgM-​specific seropositivity in such infants might be an indicator of congenital infection but is not definitive. IgM may decline rapidly in filter paper blood spots if they are used as the source of serum. Serology may be performed post-​ mortem using pericardial fluid. After early successful treatment of acute cases seropositivity may revert to negative within months; however, treated chronic cases may remain seropositive for decades. There is as yet no definitive biomarker for cure of infection, although PCR with blood samples is considered to be currently the most informative method, parasites, and different T. cruzi genetic lineages, may be sequestered in the tis- sues and undetectable. Treatment Proven acute cases must be treated promptly as it may be life-​saving, and in an effort to minimize tissue damage and neuron loss. The syn- thetic oral nitrofuran, nifurtimox was the first successful drug for the treatment of Chagas disease. However, the oral nitroimidazole, benznidazole is now considered to be the first line drug of choice due to fewer side effects. The adult dosage of benznidazole is 5–​7 mg/​kg in two divided doses for 60 days; for children, 7–​10 mg/​kg also in two divided doses for 60 days. Adverse effects may demand inter- ruption of treatment. These include rashes, fever, nausea, peripheral polyneuritis, leukopenia, and, rarely, agranulocytosis. Rare cases of Steven’s Johnson syndrome have been reported. Double or even higher doses have been used for immunocompromised patients, es- pecially if meningoencephalitis is present. Nifurtimox for adults is given in three divided daily doses at 8–​10 mg/​kg for 90 days, up to double doses for infected children. Adverse effects, which may lead to interruption of treatment, can include anorexia, loss of weight, psychological disturbances, excitability, nausea, and vomiting. Up to 40% of adult patients may fail to complete treatment with ei- ther drug, particularly if severe dermatological side effects arise within the first two weeks of treatment. There is no guarantee that a full course of treatment will eliminate the infection. Although the value of drug treatment for chronic infections is still debated, it is favoured for infants, for children under 12 years or by some author- ities for young adults under 15 or 18 years, because children tolerate treatment much better than adults. If not already available locally, favourable access to these drugs may be obtained via WHO; pedi- atric doses are available. As mentioned, there is as yet no definitive diagnostic biomarker of cure of infection after drug treatment. It has been argued that the chronic phase should treated more ag- gressively to eliminate T. cruzi and so prevent newly emergent in- flammatory foci, further cardiomyopathy and megasyndromes and reduce cardiac block and arrhythmia, and that not only children and recently infected cases but all cases of the indeterminate chronic form of Chagas disease, including chronic chagasic cardiomyopathy grade II of the New York Heart Association classification should be treated unless this is contraindicated by concomitant diseases or pregnancy. Nevertheless, as mentioned, the BENEFIT treatment trial, for patients who already display some chagasic cardiomyop- athy, showed no reduction in the progression of cardiac disease. Posoconazole and ravuconazole, inhibitors of ergosterol me- tabolism, were proposed as promising new drugs for treatment of T. cruzi infection. However, both drugs have failed in clinical trials and in highly sensitive mouse models using bioluminescent trans- genic T. cruzi and in vivo imaging. In acute-​phase heart failure, sodium intake is restricted, and di- uretics and digitalis may be indicated. Meningoencephalitis may re- quire anticonvulsants, sedatives, and intravenous mannitol. Heart failure due to Chagas disease may require vasodilatation (angio- tensin-​converting enzyme inhibitors) and maintenance of normal serum potassium levels; digitalis is a last resort because it may ag- gravate arrhythmias. A pacemaker may be fitted to improve brady- cardia not responding to atropine, or for atrial fibrillation with a slow ventricular response that is not responsive to vagolytic drugs, or for complete AV block. Amiodarone has been suggested as the most useful drug to treat arrhythmias, but it may still be aggravating. For ventricular extrasystoles lidocaine, mexiletine, propafenone, flecainide, and β-​adrenoreceptor antagonists may be effective. Lidocaine may be used intravenously in emergencies. It is essential to consult detailed WHO expert reports and physicians with sub- stantial experience in the management of chagasic heart disease. Surgery is a vital part of case management for Chagas dis- ease. Resection of ventricular aneurysms has been suggested. Specialized surgery has been developed in Brazil for the treatment of megaoesophagus and megacolon. Early megaoesophagus may respond to balloon dilatation. The Heller–​Vasconcelos operation, in which a portion of muscle at the junction of the oesophagus and stomach is removed, may alleviate megaoesophagus. Severe megaoesophagus requires replacement of the distal oesophagus (e.g. with a portion of jejenum). The modified Duhamel–​Haddad oper- ation has been considered the most successful surgery for correc- tion of a megacolon: after resection, the colon is lowered through the retrorectal stump as a perineal colostomy. Subsequent suturing, under peridural anaesthesia, gives a wide junction between the colon and the rectal stump. Prognosis, even in treated patients who show serological rever- sion, is unpredictable as the sequelae of damage due to the acute phase of Chagas disease cannot be foreseen. Prevention and control There is no vaccine against Chagas disease and no immunotherapy. With the aim of activating a Th1 immune profile with stimulation of

section 8  Infectious diseases 1466 CD8+ T cells, several experimental vaccines, including recombinant proteins, DNA and viral vectors, and heterologous prime-​boost com- binations have proved immunogenic and protective against mor- tality in mice. However, there have been no clinical trials. Chagas disease flourishes where there is poverty and poor housing conditions. There are proven methods of controlling domestic triatomine bugs. These depend on insecticide spraying, health edu- cation, community support, and house improvement. Synthetic pyrethroids are the insecticides of choice and several commercial sources are available. Vector control programmes consist of prepara- tory, attack, and vigilance phases. In the preparatory phase, the dis- tribution of all dwellings must be mapped, the presence of infested houses assessed, and the attack and vigilance phases costed and planned. The attack phase involves high quality spraying all houses and peridomestic buildings, irrespective of whether bugs have been found. During the vigilance phase, the community plays an essential role in reporting residual bug infestations, which elicit a rapid re- spraying response for the affected and neighbouring houses. Serology is vital for monitoring the success of control programmes. Children born after control programmes begin should be serologically nega- tive beyond 9 months of age (to exclude transplacental transfer of IgG) except for infrequent cases of congenital transmission. Blood donors in or from endemic areas should be screened sero- logically. If emergency conditions demand the use of seropositive blood, it can be decontaminated with crystal violet (250 mg/​litre) and storage at 4°C for at least 24 h. Organ donors or recipients who are potentially infected should be screened serologically. Seropositive immunosuppressed recipients are likely to suffer reactivated acute-​ phase infection. Prophylactic chemotherapy with benznidazole may be effective. The Southern Cone programme launched a massive effort to elim- inate T. infestans from Argentina, Bolivia, Brazil, Chile, Paraguay, Uruguay, and southern Peru. Domestic infestation in Brazil has been reduced by 85%. Uruguay and Chile are essentially free of vector-​ borne and blood transfusion transmission. Substantial progress has also been made in the other participating countries. However, triatomine infestation and high prevalence of T. cruzi infection re- main persistent and urgent public health problems in parts of the Gran Chaco region of South America, particularly among indi- genous communities. Similar international collaborations have been initiated in Central America and the Andean Pact countries. Reinvasion of sylvatic bugs into domestic habitats may compli- cate vector control in some regions. One example is T. brasilien­ sis in north-​eastern Brazil, which reinvades houses from adjacent rock piles. A second example is R. prolixus, which, in some regions of Venezuela and Colombia, has the capacity to reinvade houses from adjacent infested palm trees, as demonstrated by compara- tive triatomine population genetics. A surveillance programme and rapid responses to new domestic triatomine populations has been planned to protect the Amazon against domiciliation of vectors. Unanswered questions and future research T. cruzi is of immense research interest. It is not yet entirely clear how the organism evades the host immune response; the patho- genesis of Chagas disease is not fully understood. Further research is required, especially to produce a non​toxic, low-​cost oral drug, which would eliminate the reservoir of infection in humans. New drugs are under development via the Drugs for Neglected Diseases initiative (DNDi). Drug discovery will be aided by new mouse models with transgenic bioluminescent T.  cruzi strains, which have suggested prolonged sequestration of chronic infection in the alimentary tract. An improved, standardized universal, point of care, rapid diagnostic test is required, and post-​chemotherapeutic biomarkers of cure. Peptide-​based T.  cruzi lineage-​specific ser- ology may contribute to understanding the unclear association be- tween sequestered T. cruzi genetic lineages, clinical presentations, and and the complexity of transmission cycles. Molecular methods have already radically changed our understanding of T. cruzi and they will continue to clarify the epidemiological significance of di- verse and hybrid strains, and genetic exchange in natural popu- lations. High resolution comparative genomics of T.  cruzi may provide incisive new insight. The origins and evolution of the or- ganism and its vectors are also of considerable academic interest. Trypanosoma rangeli The second human trypanosomiasis in the New World is due to T.  rangeli infection. T.  rangeli is also transmitted by triatomine bugs, in particular the genus Rhodnius. In Rhodnius species, how- ever, T. rangeli traverses the wall of the alimentary tract, infects the haemocoel, and reaches the salivary glands, in which the metacyclic infective trypomastigotes are produced. T. rangeli is thus transmitted by the bite of the triatomine bug and not by contamination with bug faeces. Although enzootic T. rangeli infection is widespread in Latin America, transmission to humans is virtually confined to areas in which R. prolixus is the domestic vector of T. cruzi. Coinfections of T. cruzi and T. rangeli may occur. The organism appears to be non​pathogenic in humans. T. rangeli can be pathogenic to Rhodnius species. The importance of T. rangeli lies in the fact that it may con- fuse xenodiagnoses or blood culture to detect T. cruzi. With care and experience, T. rangeli can be distinguished from T. cruzi either by its long slender epimastigotes (up to 80 μm in length) and its smaller kinetoplast or by its presence in the haemolymph or salivary glands of some xenodiagnosis bugs with established infections. The life cycle in the mammalian host is uncertain, but T. rangeli is thought Fig. 8.8.12.10  Trypanosoma rangeli in a blood smear from an infected mouse. Courtesy of J. Williams.

8.8.13 Leishmaniasis 1467

8.8.13 Leishmaniasis 1467

8.8.13  Leishmaniasis 1467 to divide in the blood, possibly in sequestered sites. Trypomastigotes are rarely seen in human blood: they are much larger than T. cruzi, with a small subterminal kinetoplast (Fig. 8.8.12.10). Antibodies to T. cruzi certainly crossreact strongly with T. rangeli. Based on ex- perimental work in mice, T. rangeli infections are thought to induce very low crossreactive antibody titres to T. cruzi. As with T. cruzi, there is subspecies genetic heterogeneity, with up to four distinct T. rangeli lineages, thought to be linked to two species groups within the triatomine genus Rhodnius. FURTHER READING Bern C (2015). Chagas disease N Eng J Med, 373, 456–​66. Bhattacharyya T, et al. (2018). Severity of chagasic cardiomyopathy is associated with response to a novel rapid diagnostic test for Trypanosoma cruzi TcII/V/VI. Clin Infect Dis, 67, 519–24. Carter YL (2012). Acute Chagas disease in a returning traveller. Am J Trop Med Hyg, 87, 1038–​40. Dutra WO, et  al. (2014). Immunoregulatory networks in human Chagas disease. Parasit Immunol, 36, 377–​87. Garcia MN, et al. (2015). Historical perspectives on the epidemiology of human Chagas disease in Texas and recommendations for en- hanced understanding of clinical Chagas disease in the southern United States. PLoS Negl Trop Dis, 9, e00003981. Lewis MD, et al. (2011). Recent, independent and anthropogenic ori- gins of Trypanosoma cruzi hybrids. PLoS Negl Trop Dis, 5, e1363. Lewis MD, et  al. (2014). Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-​specific parasite dy- namics and heart disease in the absence of locally persistent infec- tion. Cell Microbiol, 16, 1285–​300. Maudlin I, Holmes P, Miles MA (eds) (2004). The trypanosomiases. CABI Publishing, Wallingford. Messenger LA, Bern C. (2018). Congenital Chagas disease: current diagnostics, limitations and future perspectives. Curr Opin Infect Dis, 31, 415–21. Messenger LA, Miles MA, Bern C (2015). Between a bug and a hard place: Trypanosoma cruzi genetic diversity and the clinical outcomes of Chagas disease. Expert Rev Anti Infect Ther, 13, 995–​1029. Miles MA (2004). The discovery of Chagas disease:  progress and prejudice. Infect Dis Clin North Am, 18, 247–​60. Molina I, et al. (2014). Randomized trial of posaconazole and ben­ znidazole for chronic Chagas disease. N Eng J Med, 370, 1899–​908. Morillo CA, et al. (2015). Randomized trial of benznidazole for chronic Chagas cardiomyopathy. N Eng J Med, 373, 1295–​306. Pinazo MJ, et al. (2017). A strategy for scaling up access to compre- hensive care in adults with Chagas disease in endemic countries: The Bolivian Chagas Platform. PLoS Negl Trop Dis, 11, e0005770. Raia AA (1983). Manifestações digestivas da moléstia de Chagas. Sarvier, São Paulo, Brazil. Riera C, et al. (2006). Congenital transmission of Trypanosoma cruzi in Europe (Spain): a case report. Am J Trop Med Hyg, 75, 1078–​81. Sanchez-​Camargo CL, et al. (2014). Comparative analysis of 11 com- mercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity. J Clin Microbiol, 52, 2506–​12. World Health Organization (WHO) (2002). Control of Chagas disease, Technical Report Series 905. WHO, Geneva. Bhattacharyya T, et al. (2018) Severity of chagasic cardiomyopathy is as- sociated with response to a novel rapid diagnostic test for Trypanosoma cruzi TcII/V/VI. Clin Infect Dis, 67, 519–524. 8.8.13  Leishmaniasis Antony D.M. Bryceson and Diana N.J. Lockwood ESSENTIALS Leishmaniasis is caused by parasites of the genus Leishmania, which are transmitted to humans from human or animal reservoirs by the bites of phlebotomine sandflies. In places the disease is common and important, with perhaps 500 000 cases of visceral leishmaniasis and 1.5–​2 million cases of cutaneous leishmaniasis worldwide each year. As an imported disease, cutaneous leishmaniasis is common in travellers, military per- sonnel, and immigrants coming from endemic areas, while the diagnosis of the less common visceral leishmaniasis is frequently overlooked. Cutaneous leishmaniasis Clinical features—​at the site of the infected sandfly bite, an erythema- tous nodule typically develops into a sore which fails to heal spon- taneously and might progress to mucosal leishmaniasis (espundia)—​a condition in which mucosal lesions develop in 4 to 40% of patients with untreated cutaneous ulcers due to L. brasiliensis. Diagnosis and treatment—​diagnosis is by demonstration of leish- mania organisms in tissue smears or biopsy material by microscopy, cul- ture, or detecting leishmaniai DNA by polymerase chain reaction. Many leishmanial sores will heal naturally, but treatment is indicated for those that are severe, or failing to heal spontaneously, or due to particular spe- cies (e.g. L. brasiliensis). Treatment may be (1) local (e.g. surgery/​curettage); infiltration with a pentavalent antimonial; or (2) systemic—​most cutaneous species of leishmania are sensitive to pentavalent antimonials. Visceral leishmaniasis Zoonotic disease is common around the Mediterranean littoral, across the Middle East and central Asia, in northern and eastern China, and in South and Central America. Anthroponotic disease causes large outbreaks in Northeast India and the Sudan. Clinical features—​most infections are subclinical, but clinical pres- entation is with gradual onset of fever, discomfort from an enlarged spleen, abdominal swelling, weight loss, cough, or diarrhoea. The illness can be associated with HIV infection. Diagnosis and treatment—​diagnosis is by isolation of leishmania from spleen, bone marrow, liver, lymph node, or buffy coat. Serology is useful for diagnosis, and might replace direct demonstration of parasites in remote areas. The best treatment is intravenous liposomal amphotericin B, but (much cheaper) pentavalent antimonials are most often used in countries where visceral leishmaniasis is endemic. An oral agent, miltefosine is now being used in the Indian subcon- tinent and this has simplified treatment Prevention Prevention is by controlling reservoir hosts and sandfly vectors, or by avoiding bites by vectors. There is no vaccine. Introduction Leishmaniasis is caused by parasites of the genus Leishmania, which are transmitted by sandflies of the genus Phlebotomus in the Old

section 8  Infectious diseases 1468 World and Lutzomyia in the New World. The infection may be anthroponotic or zoonotic, having human or animal reservoirs, respectively. In humans, the disease is usually either cutaneous or visceral. The most important variant is mucosal leishmaniasis of South and Central America. In places the disease is common and important, but there are few accurate statistics. The World Health Organization (WHO) estimates 500 000 cases of visceral leishman- iasis and 1.5 to 2 million cases of cutaneous leishmaniasis occur annually, with 200 million people at risk of each disease, but these figures may underestimate the problem. As an imported disease, cu- taneous leishmaniasis is common in travellers, military personnel, and immigrants coming from endemic areas, while the diagnosis of the less common visceral leishmaniasis is frequently overlooked. Aetiological agent and lifecycle In its vertebrate host, the oval amastigote form of the parasite (2–​3 μm in diameter) is found in cells of the reticuloendothelial system (Fig. 8.8.13.1). In the sandfly or in culture medium, it is in the elongated, motile, promastigote form with an anterior flagellum. The most important species of Leishmania that cause disease in humans and their own reservoir hosts are shown in Table 8.8.13.1. Isoenzyme patterns and DNA hybridization are used to distinguish species. Sandflies require a precise microclimate that is provided in cer- tain places in each endemic focus at particular seasons of the year. Transmission is often seasonal. Amastigotes are ingested from blood or tissues of the mammalian host by the female fly and transform into promastigotes in the gut, rendering the fly infective after about 10 days. Cutaneous leishmaniasis Epidemiology The vectors of Leishmania major live in rodent burrows. Visiting hunters, travellers, soldiers, and tourists, and dwellers at oases or in new settlements, are affected. The disease may be sporadic or epidemic, as seen recently among Afghan refugees in camps in Fig. 8.8.13.1  Amastigotes of L. donovani in a reticuloendothelial cell. From the splenic aspirate of a child with visceral leishmaniasis in Kenya. Copyright A. D. M. Bryceson. Table 8.8.13.1  Epidemiology of leishmaniasis Organism Geographical location Reservoir Major vectors Old World L. donovani North-​east India, Bangladesh, Nepal Humans Phlebotomus argentipes L. infantum Mediterranean basin, Sudan, West Africa, Middle East, China, central Asia Dogs, foxes, jackals P. perniciosus, P. perfiliewi, P. chinensis, etc. L. donovani (Africa) Sudan, Kenya, Horn of Africa Humans P. orientalis, P. martini L. major Semideserts in North Africa and Middle East, north India, Pakistan, central Asia Gerbils (especially Rhombomys, Meriones) P. papatasi L. major Sub-​Saharan savannah, Sudan Rodents (especially Arvicanthis, Tatera) P. duboscqi L. tropica Towns in Middle East, Mediterranean basin, central Asia Humans,?dogs P. sergenti L. aethiopica Highlands of Kenya, Ethiopia, Uganda Hyraxes (Procavia, Heterohyrax) P. longipes, P. pedifer New World L. chagasi ( = L. infantum) Most of Central and South America, especially Brazil Dogs, foxes, opossums (Didelphis) Lutzomyia longipalpis, Lu. evansi L. mexicana Central and northern South America Forest rodents (especially Ototylomys) Lu. olmeca L. amazonensis Tropical forests of South America Forest rodents (especially Proechimys, Oryzomys) Lu. flaviscutellata L. brasiliensis Tropical forests and cultivated land throughout South
and Central America Rodents, opossums, dogs, and equines Lu. wellcomei, Lu. whitmani, etc. L. guyanensis Northern South America Sloths (Choleopus), arboreal anteaters (Tamandua) Lu. umbratilis L. panamensis Central America, Ecuador, Colombia Sloths (Choleopus) Lu. trapidoi, etc. L. peruviana West Andes of Peru Dogs, rodents, opossums Lu. verrucarum, Lu. peruensis

8.8.13  Leishmaniasis 1469 Pakistan. The vectors of L.  tropica live in crevices in buildings and walls. The disease may be endemic or epidemic. The vector of L. aethiopica bites people sleeping in their huts. The disease is en- demic, and most people are affected by early adulthood. L. infantum causes simple, self-​healing skin lesions in some parts of southern Europe and North Africa. L. donovani causes post-​kala-​azar dermal leishmaniasis (PKDL) in India. In the New World, transmission is usually in the forest. L. brasil­ iensis, the major cause of American cutaneous and mucosal leish- maniasis, is the most widely distributed of the New World species. Its vectors are highly anthropophilic and human infection is common. Periurban and urban foci of infection are increasing. Malnutrition is a risk factors for mucosal leishmaniasis. Infection with L. peruviana occurs in high Andean valleys, where it may be locally common. Pathogenesis and pathology Leishmania, when inoculated by the sandfly, invade and multiply in macrophages in the skin. The parasitized macrophage granuloma is in- filtrated by lymphocytes and plasma cells. Piecemeal or focal necrosis destroys parasitized cells. The overlying epidermis shows hyperkera- tosis and ulcerates. In chronic lesions, epithelioid cells and Langhans giant cells produce a picture similar to that of non​caseous tubercu- losis. Rarely, the cellular immune response is suppressed, and histology shows heavily parasitized macrophages with little or no lymphocytic infiltrate, characteristic of diffuse cutaneous leishmaniasis. L. aethiopica, L. mexicana, and L. brasiliensis may invade cartilage. Cartilaginous lesions are extremely chronic. L. brasiliensis, and oc- casionally L. panamensis or L. guyanensis, may metastasize through the bloodstream to sites deep in the mucosa of the upper respiratory tract, where they can lie dormant. After months or years, a lesion de- velops, characterized by necrosis, vasculitis, and tissue destruction. Immunity to a given species of leishmania is usually lifelong. Second infections occur occasionally, especially in older people or immunosuppressed. Clinical features After an incubation period of a few days to several months, an ery- thematous nodule develops at the site of the infected sandfly bite. A golden crust forms (Fig. 8.8.13.2). The sore reaches its final size, usually 1 to 5 cm in diameter, over weeks or months. The crust may fall away, leaving an ulcer with a raised edge (Fig. 8.8.13.3). Satellite papules are common. After months or years, the lesion starts to heal leaving a depressed, mottled scar. Any secondary bacterial infection is superficial and unimportant. The lesion is not normally painful, but can disfigure or disable if scarring is severe or over a joint. Draining lymphatic vessels might be thick- ened or nodular. There are many variations on this classical pattern. Sores due to L. major form and heal rapidly (mean 3–​5 months) and might be inflamed and exudative: the so-​called wet or rural sore. Sores due to L. tropica tend to be less inflamed and to heal more slowly (mean 10–​14 months):  the so-​called dry or urban sore (Fig. 8.8.13.4). Fig. 8.8.13.2  Nodular lesion of cutaneous leishmaniasis. Showing crusting and small satellite papules, typical of early lesions of all species; in this case L. brasiliensis. Copyright A. D. M. Bryceson. Fig. 8.8.13.3  Cutaneous leishmaniasis due to L. brasiliensis. Shallow ulcer with raised edge. Copyright A. D. M. Bryceson. Fig. 8.8.13.4  Cutaneous leishmaniasis due to L. tropica in a young man in Kabul. Crusty nodular lesions are spreading on the face. There is a typical depressed scar of a previous lesion on the right cheek. Copyright Dr Mark Bailey.

section 8  Infectious diseases 1470 Lesions due to L.  infantum have an incubation period of many months and can persist over several years. In L. aethiopica infec- tions, lesions are usually central on the face. Satellite papules accu- mulate to produce a slowly growing, shiny tumour or plaque that might not crust or ulcerate, taking between 2 and 5 years to heal (Fig. 8.8.13.5); mucocutaneous leishmaniasis may develop, produ- cing swelling of the lips and expansion and elongation of the nose. Leishmanial lymphangitis might accompany sores of any species but is more common in the New World than the Old World (Fig. 8.8.13.6). On occasion, hard thickened lymphatics might accom- pany an insignificant cutaneous lesion. L. brasiliensis often causes deep, spreading ulcers, which heal over 6 to 24 months. Up to 15% of patients will relapse after spontaneous or therapeutic cure. L. mexicana lesions are commonly on the limbs or side of the face and heal in 6 to 8 months. Sores on the pinna of the ear can invade the cartilage, persist for many years, and destroy the pinna. Three forms of cutaneous leishmaniasis do not heal spontan- eously:  diffuse cutaneous leishmaniasis, leishmaniasis recidivans, and American mucosal leishmaniasis. Diffuse cutaneous leishmaniasis This occurs with L. aethiopica and L. amazonensis infections but is rare. The primary nodule spreads locally without ulceration and secondary blood-​borne lesions appear at other sites in the skin, af- fecting especially the face and the cooler extensor surfaces of the limbs (Fig. 8.8.13.7). The eye, mucosae, viscera, and peripheral nerves are spared, which differentiates it clinically from lepromatous leprosy. The infection proceeds gradually over many years. Leishmaniasis recidivans (lupoid leishmaniasis) This is a rare complication of L. tropica infection. The initial sore heals, but papules recrudesce in the edge of the scar and the lesion spreads slowly over many years (Fig. 8.8.13.8). American mucosal leishmaniasis (espundia) Depending on the geographical location, between 4 and 40% of patients with untreated cutaneous ulcers due to L.  brasiliensis Fig. 8.8.13.5  Spreading nodular lesion typical of L. aethiopica, Kenya. Fig. 8.8.13.6  Leishmanial lymphangitis in a man with cutaneous leishmaniasis from Belize. On occasion, hard thickened lymphatics may accompany an insignificant cutaneous lesion. Copyright A. D. M. Bryceson. Fig. 8.8.13.7  Diffuse cutaneous leishmaniasis caused by L. aethiopica, Ethiopia. Fig. 8.8.13.8  Lupoid or recidivans leishmaniasis in a citizen of Baghdad. Courtesy of Dr Ahmed.

8.8.13  Leishmaniasis 1471 develop mucosal lesions, half of them within 2 years of the appear- ance of the original lesion and 90% within 10 years. About one in six patients gives no history of a previous skin lesion. In most cases the nasal mucosa is affected, and in one-​third another site is also involved: in order of frequency, the pharynx, palate, larynx, or upper lip. The initial lesion is a nodule and the initial symptom is of nasal obstruction. It commonly presents as protuberant new growth of the nose or lips (Figs. 8.8.13.9 and 8.8.13.10), or cicatrization, which causes an elongated ‘tapir’ nose. Mucosal leishmaniasis is slowly destructive, the septum perforates, and eventually the whole nose and mouth might be destroyed. Death can result from secondary sepsis, starvation, or laryngeal obstruc- tion. Mucosal leishmaniasis is occasionally seen in travellers re- turning from South America. Mucosal lesions are occasionally caused by Old World species, usually in the mouth or larynx, and these patients might have risk factors such as old age, corticosteroid medication, or other immuno- suppression (Fig. 8.8.13.11). Laboratory findings Parasitological diagnosis Leishmania organisms can be isolated from 80% of sores during the first half of their natural course. An incision a few millimetres long is made into the dermis with the point of a scalpel, which is used to scrape dermal tissue and juice. Material obtained can be used to prepare smears for staining with Giemsa, Wright’s, or Leishman’s stains (Fig. 8.8.13.1). Biopsy material can be used to make impres- sion smears, for culture and for histology for differential diagnosis. Leishmanai DNA can be detected by polymerase chain reaction (PCR) using kinetoplast DNA primers and is nearly 99% sensitive and 93% specific. Diagnosis of mucosal leishmaniasis requires a deep punch biopsy specimen. Species identification by PCR is de- sirable in patients with American leishmaniasis to assess the risk of mucosal leishmaniasis. Treatment Old World sores, or those due to L. mexicana, L. amazonensis, and L. peruviana that are not troublesome, can be left to heal naturally. But those that are disfiguring, potentially disabling, inconvenient, or around the ankle where they heal slowly, should be treated ei- ther locally or systemically. Systemic treatment is required when there is risk that the sore might be due to L. brasiliensis, L. pana­ mensis, or L. guyanensis, when the sore is too large or badly sited for local treatment, when there is lymphatic spread, and for mucosal leishmaniasis, diffuse cutaneous leishmaniasis, and recidivans leishmaniasis. Fig. 8.8.13.9  Espundia. Swollen upper lip and ‘tapir’ nose due to mucosal leishmaniasis, at Instituto de Medicina Tropical ‘Alexander von Humboldt’ Universidad Peruana Cayetano Heredia, Lima, Peru. Copyright D. A. Warrell. Fig. 8.8.13.10  Infiltration of lip and palate due to mucosal leishmaniasis in Peru. Fig. 8.8.13.11  Mucosal leishmaniasis due to L. infantum. Showing erythematous infiltration of the hard palate in an elderly British expatriate living in southern Spain and taking steroids for asthma. Copyright A. D. M. Bryceson.

section 8  Infectious diseases 1472 Local treatment Surgery, curettage, CO2 laser, cryotherapy, and thermotherapy are effective methods of removing small sores. Infiltration into the le- sion with a pentavalent antimonial, weekly for 2 or 3 weeks or longer, can be successful. The technique needs practice and the infiltration is transiently painful (Fig. 8.8.13.12). An ointment containing 12% paromomycin and 15% methylbenzethonium chloride cures 70% lesions due to L. major in 20 days and might be suitable for lesions caused by other species, except L. brasiliensis, but is not always well tolerated. Systemic treatment All cutaneous species of leishmania are sensitive to pentavalent antimonials in conventional dosage except L.  aethiopica, where paromomycin can be used. Pentamidine is effective but seldom warranted because of toxicity. Miltefosine is effective for L. major, L. mexicana, L. guyanensis and L. panamensis infections. Patients with diffuse cutaneous leishmaniasis should be treated with two drugs for at least 2 months longer than it takes to clear parasites from the skin, Miltefosine is also effective for mucoal leishmaniasis. In addition, they might require antibiotics for secondary sepsis, atten- tion to nutrition, and, later, plastic surgery. Visceral leishmaniasis Epidemiology Visceral leishmaniasis is found in four main zoogeographical zones:  the Ganges Brahmaputra plains, the Mediterranean basin extending into West and Central Asia, Sudan and East Africa, and Brazil (see Table 8.8.13.1). Around the Mediterranean littoral, across the Middle East and central Asia, and in northern and eastern China, zoonotic visceral leishmaniasis is endemic in many places, where as many as 50% of domestic and stray dogs are infected. Children under 5 years of age are especially affected. It is the second most common infectious cause of fever of unknown origin in children in the Balkan countries. HIV infection is a risk factor for adults. In other places, the disease is sporadic. Non​immune adults such as tourists, hunters, and soldiers are susceptible. The Ganges and Brahamputra river valleys of India and Bangladesh are the home of epidemic anthroponotic visceral leishmaniasis, or kala-​azar, which returns approximately every 15–​20 years. Most cases are in young people under 15 years of age and are found in clusters. The annual incidence is about 250 per 100 000. About 50% of household contacts of cases in Bihar India are seropositive, one in four of whom will develop disease. Malnutrition predisposes to clinical disease. In the interepidemic period, the parasite survives in patients with post-​kala-​azar dermal leishmaniasis. Visceral leishmaniasis is endemic in parts of Sudan, where it can be both anthroponotic and zoonotic, and in adjacent parts of Ethiopia and Kenya. Older children and teenagers are most com- monly affected. Sporadic cases also occur in nomads and visitors. In remote areas, half the cases do not reach a medical facility and 90% of deaths go unreported. In South America, the disease is most common in north-​eastern Brazil, where older children are affected. Previously a rural disease, it is becoming increasingly important in towns. Visceral leishmaniasis might appear unexpectedly in immuno- suppressed patients (e.g. after renal transplantation), with haem- atological malignancies, while receiving immunosuppressive drugs, and in pregnant women. In endemic areas, it is an opportunistic in- fection in patients with HIV infection. Visceral leishmaniasis can be transmitted by blood transfusion from subclinical cases; parasites were cultured from 2 to 4% of donor blood samples in endemic areas of France and Spain. Pathogenesis and pathology For every case of classical visceral leishmaniasis, there are about 30 subclinical infections that cause leishmanin positivity and lifelong immunity to the infecting species. Established visceral infections are Fig. 8.8.13.12  Infiltrating a lesion of cutaneous leishmaniasis with sodium stibogluconate. The edge of the lesion is demarcated using a ballpoint pen and infiltrated radially from several points on its perimeter using an intradermal syringe and needle. Copyright A. D. M. Bryceson. Table 8.8.13.2  Dosage regimens for the treatment of leishmaniasis Drug Dose Sodium stibogluconate or meglumine antimoniate By body surface area, so that: 10–​20 mg Sb/​kg body weight is given once daily for 21 days (visceral or cutaneous disease) or 28 days (visceral or mucosal disease)—​PKDL may need treatment for 2–​4 months. By body weight: 20 mg/​kg per dose as described, but less well tolerated by adults Amphotericin B desoxycholate 1 mg/​kg body weight on alternate days for 2 weeks (visceral disease) or 4–​6 weeks (mucosal disease) Liposomal amphotericin B Ampoules of 50 mg, 3–​5 mg/​kg body weight per daily dose over a 3–​6 day period, to a total of 21 mg/​kg. In India, a total dose of 15 mg/​ kg is sufficient. A single dose of 10 mg/​kg has a comparable cure rate. A 20-​day regimen of 2.5 mg/​ kg cures PKDL in Sudan Miltefosine Adult dose 100–​150 mg daily for 28 days; paediatric dose 2.5 mg/​kg body weight daily for 28 days Paromomycin 15 mg (11 mg base)/​kg body weight daily for 21 days Ketoconazole 60 mg/​day (adult) for 4–​6 weeks Coadministered combinations Two of the three drugs liposomal amphotericin B, miltefosine, and paromomycin, given in the doses described for 7–​10 days See text for choice of drug regimen.

8.8.13  Leishmaniasis 1473 characterized by the failure of specific cell-​mediated immunity. The leishmanin test is negative. The parasite multiplies freely in macro- phages in the spleen, bone marrow, lymphoid tissues, jejunal sub- mucosa, and Kupffer cells of the liver. Histology shows a variable degree of granuloma formation and interstitial inflammation in the liver that might lead to fibrosis. In the spleen especially, there is mas- sive reticuloendothelial hyperplasia and infiltration with plasma cells. Small splenic infarcts might develop. Antibodies, polyclonal IgG, and immune complexes circulate at high concentration but rarely cause complications. About half of the patients have mild malabsorption but seldom diarrhoea. When present, jaundice usually has another cause such as viral hepatitis. Spontaneous bleeding is unusual and is associated with hypoprothrombinaemia. Visceral leishmaniasis is characterized by anaemia, leukopenia, thrombocytopenia, and hypoalbuminaemia. The anaemia results mainly from shortened red-​cell survival with destruction of cells in the spleen, together with splenic pooling and sequestration (hypersplenism). In young children, profound an- aemia might develop rapidly as a result of severe haemolysis. Death is usually due to secondary infection. Clinical features The male/​female ratio is between 3:1 and 4:1. The incubation period is usually 2 to 8 months. In endemic areas, the onset is usually ill defined. The patient develops fever, discomfort from an enlarged spleen, abdominal swelling, weight loss, cough, or diarrhoea. Classically, the fever spikes twice daily, usually without rigors, but daily, irregular, or undulant fevers are common. During an epidemic or in visitors to an epidemic area, symptoms can start abruptly with high fever and rapid progression of illness with toxaemia, weakness, dyspnoea, and acute anaemia. Physical examination of early cases might show only symptom- less splenomegaly. Patients with advanced disease are wasted, with hair changes and pedal oedema typical of hypoalbuminaemia. Hyperpigmentation is characteristic of visceral leishmaniasis in India (kala-​azar means ‘black disease’). The spleen is huge, smooth, and non​tender unless there has been a recent infarct. The liver is moderately enlarged in one-​third of cases. In African patients, a generalized lymphadenopathy is common. Over months or years, the patient becomes emaciated, with a distended abdomen (Fig. 8.8.13.13). Intercurrent infections are common, especially pneumococcal otitis media, pneumonia, septicaemia, tuberculosis, measles, dysentery, other locally im- portant infections, and rarely, cancrum oris. Untreated, between 80 and 90% of patients die. Post-​kala-​azar dermal leishmaniasis (PKDL) Up to 10% of Indian patients and up to 50% of Sudanese and East African patients develop a rash on the face, extensor surfaces of the arms and legs, and trunk after recovery from visceral leishman- iasis. In India, the rash begins after an interval of 1 or 2 years and progresses over many years:  pale macules become erythematous plaques, papules, or nodules resembling lepromatous leprosy, and almost the entire body surface may be involved, including buccal and genital mucosa, and conjunctiva (Fig. 8.8.13.14). In Kenya, the rash usually appears while the patient is still recovering, as dis- crete nodules which show a granulomatous histology with scanty parasites. It heals spontaneously within 6 months (Fig. 8.8.13.15). Sudanese patients show a mixture of these two forms. PKDL is rarely seen after L. infantum infections. Visceral leishmaniasis immunosuppression
and HIV infection Visceral leishmaniasis can be associated with HIV infection. The presentation might be atypical and with unusual skin lesions. Antiretroviral treatment has greatly reduced the clinical impact of coinfection, but in some patients leishmaniasis now presents as an immune reconstitution inflammatory syndrome. The parasite might be found by chance, for example, in a rectal or skin biopsy taken for other purposes, or in bronchoscopic lavage. The bone marrow has abundant parasites, but two-​thirds of cases have no detectable antileishmanial antibodies. In 90% of cases, the CD4 count is less than 200 cells/μl. Response to treatment is poor and relapse usual (see ‘Treatment’, next). Patients are now regularly seen who have Fig. 8.8.13.13  Visceral leishmaniasis in a Kenyan child. Note the wasting, massive enlargement of liver and spleen, and increased pigmentation. Fig. 8.8.13.14  Post-​kala-​azar dermal leishmaniasis in an Indian child. Showing the typical hypopigmented macular rash. Note also the nodules on the lower lip.

section 8  Infectious diseases 1474 acquired their leishmaniasis in the Mediterranean and have a risk factor such as autoimmune disease, treatment with biological agents of diabetes. Laboratory diagnosis Parasitological diagnosis Leishmania organisms can be isolated from reticuloendothelial tissue. Yields are of the order of: spleen, over 95% cases; bone marrow or liver, 85%; lymph node in Sudan, 65%; and buffy coat, 70%. Bone marrow aspiration is most commonly used, but splenic aspiration is simple, painless, and safe if the prothrombin time is normal and the platelet count above 40 × 109/​litre. PCR for leishmanial DNA in bone marrow is even more sensitive. PCR for leishmanial DNA in blood is useful for follow up HIV coinfected patients. Serological diagnosis Except in HIV coinfections, antibodies are present in high titre, useful for diagnosis, and may replace parasite diagnosis in remote areas. Indirect immunofluorescence is the gold standard but, for fieldwork, it has been replaced by enzyme-​linked immunosorbent assay, direct agglutination, and the rK39 antigen dipstick. All give comparable results with sensitivities of about 90% and specificities above 95% (positive predictive value c.99% and negative predictive value c.70%). The leishmanin skin test is negative. Other findings There is a normochromic, normocytic anaemia without reticulocytosis, and neutropenia, eosinopenia, and thrombocytopenia. Serum albumin is low (c.20 g/​litre) and globulin high (c.70 g/​litre), IgG and IgM are ap- proximately thrice and twice the normal population values. Hepatic en- zymes and prothrombin and partial thromboplastin times are usually normal. Treatment Chemotherapy Liposomal amphotericin B by intravenous infusion is the best drug for visceral leishmaniasis in adults and children. It is concentrated and retained in reticuloendothelial cells and is not toxic. Over 99% patients respond promptly, but HIV coinfected patients might re- lapse. The drug is also effective against PKDL in India and Sudan and it is the drug of choice in pregnancy. The drug is becoming afford- able in endemic countries where the World Health Organization has negotiated a 90% reduction in price. Otherwise, a pentavalent an- timonial remains the drug of choice in most situations, except in Bihar, India. See Table 8.8.13.2 for dosage regimens. Conventional amphotericin B deoxycholate is cheaper than liposomal amphotericin B and just as effective, though more toxic, and is useful for patients unresponsive to antimonials. Sodium stibogluconate containing 100 mg Sb/​ml and meglumine antimoniate containing 85 mg Sb/​ml are of equal efficacy and tox- icity. The drug is administered by intramuscular injection, which can be painful, or by intravenous injection through a fine-​gauge needle, slowly or by infusion in 50–​100 ml of 5% dextrose over 20 min to reduce the risk of venous thrombosis. Treatment is given daily for 28 days. Usually the drug is well tolerated but towards the end of treatment there may be malaise, anorexia, nausea, vomiting, and muscle pains. Should toxic effects develop, rest for 1 day and re- duce each dose by 2 mg Sb/​kg. Hepatic and pancreatic enzyme levels might rise, and haemoglobin levels fall, but they return to normal when treatment is stopped. The electrocardiogram develops unim- portant T-​wave changes. At higher doses, the corrected QT interval might be prolonged, heralding the development of a serious ar- rhythmia. Cure rates exceed 95%, except in Bihar, north of the river Ganges, where primary antimony resistance is spreading and up to 60% patients do not respond to antimonials. The aminoglycoside antibiotic paromomycin, is equally effective and well tolerated, but cure rates vary between countries and en- demic foci. It is given by intramuscular injection or intravenous infusion over 90 min. Renal function and hearing should be moni- tored. Paromomycin is not readily available outside countries with control programmes. The sole oral drug, miltefosine, cures from 90 to 94% of HIV-​ negative adults and children with visceral leishmaniasis in Sudan and India, even in areas of parasite resistance to antimonials. Trials in Bihar have shown that 7–​10 days courses of combined treatment with any two of liposomal amphotericin B, paromomycin and miltefosine are highly effective. It has now been recommended for use in treating patients with visceral leishmaniasis in the Indian subcontinent. Miltefosine is associated with significant nausea and vomiting. It is also potentially teratogenic, so patients should avoid pregnancy. Patients who are immunosuppressed as a result of HIV coinfec­ tion or immunosuppressive drugs respond slowly, require longer treatment, and are more liable to relapse than immunocompe- tent patients. Ideally, treatment of such patients should be moni- tored by splenic aspirate counts of parasites and continued for 2 or 3 weeks beyond parasitological cure. Antimonials cause adverse ef- fects in two-​thirds of HIV coinfected patients and can precipitate clinical pancreatitis. Liposomal amphotericin B and paromomycin are effective and well tolerated. Relapse might be prevented by secondary prophylaxis with pentamidine given every 2 weeks. Antiretroviral therapy (ART) reduces the number of relapses and delays their onset. PKDL can be treated in India with miltefosine for 12 weeks or amphotericin B deoxycholate for 3–​4 months; and in Sudan and East Fig. 8.8.13.15  Post-​kala-​azar dermal leishmaniasis in a Kenyan child. Showing the typical collection of small discrete nodules on the face. Copyright A. D. M. Bryceson.

8.8.14 Trichomoniasis 1475

8.8.14 Trichomoniasis 1475

8.8.14  Trichomoniasis 1475 Africa with pentavalent antimonials for 30–​60 days or liposomal amphotericin B for 20 days. Supportive treatment Intercurrent infection must be sought and treated, and nutritional deficiencies corrected. Blood transfusion is rarely needed. Response to treatment Fever, splenic size, haemoglobin, serum albumin, and body weight are useful monitors of progress. Proof of parasitological cure is not usually necessary. Reassessment at 6 weeks and 6 months will detect over 90% of relapses. Serology is unhelpful in monitoring progress. Relapse rates should be under 4%. Relapsed patients are slower to respond and run a 40% chance of further relapses and of becoming unresponsive to antimony. Economic impact Visceral leishmaniasis is a major economic burden on affected fam- ilies. The direct costs of an episode of visceral leishmaniasis in rural India or Bangladesh, where the drug is, in principle, provided free, are equivalent to the household’s annual income. Prevention and control of cutaneous and visceral leishmaniasis Prevention is a matter of controlling reservoir hosts and sandfly vec- tors or of avoiding bites by vectors. Successful control requires an accurate knowledge of transmission in each ecological focus. In the Old World, urban cutaneous leishmaniasis is controlled by case-​finding and treatment, better housing, and domestic spraying with residual insecticides, while rural leishmaniasis is controlled in the Middle East and North Africa by poisoning or destruction of gerbil colonies. Mediterranean and urban visceral leishmaniasis in South America can be controlled by the destruction or treatment of dogs, but dogs are infectious to flies before they become symptom- atic and screening of dogs is problematic. Dog collars impregnated with permethrin reduce the numbers of flies that become infected. In India, mass campaigns to spray houses and cattle sheds are needed in addition to case-​finding and treatment. In the interepidemic period, cases of PKDL should be sought and treated. Currently no nation has an effective control programme in place. In endemic populations, infection can be prevented during the season of transmission by the use of insect repellent creams and of fine mesh bed nets, top sheets or chadors (women’s outer garments or cloaks) impregnated with permethrin during the hours of biting, usually around dusk and dawn. In endemic foci, a higher level of education in households is associated with lower rates of disease. Vaccines have proved disappointing. FURTHER READING Blum J, et  al. (2014). LeishMan recommendations for treatment of cutaneous and mucosal leishmaniasis in travellers. J Trav Med, 21, 116–​29. den Boer M, Davidson RN (2006). Treatment options for visceral leishmaniasis. Expert Rev Anti Infect Ther, 4, 187–​97. Desjeux P (2001). The increase in risk factors for leishmaniasis world- wide. Trans R Soc Trop Med Hyg, 95, 239–​43. Lockwood DNJ, Sundar S (2006). Serological tests for visceral leish- maniasis. Br Med J, 333, 711–​12. Murray HW, et  al. (2005). Advances in leishmaniasis. Lancet, 366, 1561–​77. Sundar S, et al. (2011) Comparison of short-​course multidrug treat- ment with standard therapy for visceral leishmaniasis in India:
an open-​label, non-​inferiority, randomised controlled trial. Lancet, 377, 477–​86. World Health Organization (WHO) (2010). Control of the leishman­ iasis:  report of a meeting of the WHO Expert Committee on the Control of Leishmaniases. Geneva, 22–​26 March 2010. WHO tech- nical report series; no. 949. World Health Organization, Geneva. Websites Centres for Disease Control. http://​www.cdc.gov/​parasites/​leishman- iasis/​index.html World Health Organization (WHO). Leishmaniasis. http://​www.who. int/​leishmaniasis/​en/​ 8.8.14  Trichomoniasis Jane Schwebke ESSENTIALS Trichomonas vaginalis is a sexually transmitted protozoan pathogen that may cause more than one-​half of all curable sexually trans- mitted genital infections worldwide. Women with trichomoniasis are often asymptomatic, but they may develop vaginal malodour, discharge, erythema, or itching, and their male or female sexual part- ners may also be infected, although urethritis in men is less likely to cause symptoms. Women with trichomoniasis have an increased risk of HIV acquisition, HIV shedding, pelvic inflammatory disease, and preterm birth. For diagnosis, rapid antigen detection, culture, and polymerase chain reaction methods have advantages over con- ventional microscopy, but are more expensive. Oral metronidazole is usually an effective treatment, with both sexual partners needing to be treated to prevent reinfection. Introduction Trichomoniasis is an infection of the human urogenital tract caused by the flagellated protozoan Trichomonas vaginalis. There are about 170 million new cases each year, making it the world’s most common Acknowledgement: The editors acknowledge the inclusion of material from Dr Sharon Hillier’s chapter in the previous edition of the Oxford Textbook of Medicine.

section 8  Infectious diseases 1476 non​viral sexually transmitted infection and, according to the World Health Organization (WHO), it accounts for more than one-​half of all curable sexually transmitted infections worldwide. Pregnant women who have trichomoniasis are at increased risk of preterm delivery. Trichomoniasis is a risk factor for HIV acquisition and shedding. Aetiology Although there are more than 100 species of this protozoan, only T.  vaginalis parasitizes the human genital tract and has specific tropism for this environment. Trichomonas tenax can be found in the human oral mucosa and Pentatrichomonas homininis might be found in the human gastrointestinal tract. In women, T. vaginalis can be found in the vagina and the exterior cervix in over 95% of infections, but is recovered from the endocervix in 13%. The urethra and Skene’s glands are also commonly infected. In men, the urethra is the most common site of infection but the organism has also been recovered from epididymal aspirates and semen. Epidemiology Epidemiological evidence suggests that T. vaginalis is transmitted almost exclusively by sexual intercourse, both during heterosexual intercourse and in sexual activity between female sexual part- ners. Although the organism can survive for many hours at room temperature if kept damp, there is only limited evidence that this pathogen is transmitted among household members in the absence of sexual exposure. No cyst form of the organism is known to exist. A very small proportion of female babies of infected mothers will become infected during birth, but this infection is transient and trichomoniasis discovered in a child should immediately raise the suspicion of sexual abuse. The recent availability of nucleic acid amplification tests (NAATS) for women and men has provided newer and more accurate data on the prevalence of infection. The US National Health and Nutrition Examination Survey conducted 2001–​2004 projected that 3.1% of US women are infected. Rates were higher among minority popu- lations. HIV infection and illicit drug use are risk factors for infec- tion. T. vaginalis has been reported in 18–​24% of women attending sexual health clinics in the United States of America and in 3–​34% of women in four African cities. The epidemiology of this pathogen is less well understood among men, but has been reported in 3–​20% of men attending sexual health clinics. In several developed countries, there has been a steady decline in the incidence of trichomoniasis in the past few decades, but this has not occurred in less-​developed countries nor in deprived inner-​city areas in industrialized nations. Human trichomoniasis is becoming a disease of the underprivileged. Pathogenesis In vitro, T. vaginalis has a well-​defined, contact-​mediated, cytotoxic effect, but its relationship to pathogenesis in vivo is unknown. It activates complement and attracts neutrophils, which may kill the parasite but, in large numbers, might also contribute to the pathology. The organism produces several proteolytic enzymes which degrade genital tract mucins. Several potential T. vaginalis adhesions have been identified but, apart from its surface lipophosphoglycan, there is little evidence supporting a role in adhesion. Availability of the T. vaginalis genome sequence has allowed wider search for surface, soluble, and secreted proteins involved in host–​parasite interactions. Clinical features In women, T.  vaginalis can infect the vagina, urethra, and the Bartholin’s and Skene’s glands. From 10 to 50% of cases are asymp- tomatic but acute inflammatory diseases can occur, with copious and malodorous vaginal discharge, vulvovaginal soreness and ir- ritation, dysuria, and dyspareunia. Trichomoniasis is significantly associated with purulent yellow vaginal discharge, vulvar itching, and colpitis macularis (strawberry cervix) detectable by colposcopy, with vulvar and vaginal erythema. The discharge fluctuates with time and, if untreated, might disappear spontaneously or persist for months or even years. Most men with trichomoniasis are asymptomatic, but the parasite is responsible for a significant number of cases of non​gonococcal urethritis. Differential diagnosis In women, vaginal discharge syndromes including bacterial vagin- osis, yeast vulvovaginitis, and trichomoniasis should be considered (see Chapter  9.4). Women who present with vaginal discharge, vulvar itching, and/​or vaginal malodour might have no infection, or could have any combination of these common vaginal infections. In men, other causes of urethritis should be ruled out. An accurate diagnosis cannot be made based upon signs or symp- toms elicited during the clinical evaluation. Trichomoniasis in women The most commonly used method for diagnosis is identification of the pathogen in vaginal (not endocervical) secretions examined under the microscope at ×400 magnification. In clinical specimens or culture, T. vaginalis is a motile and round or oval flagellate, 10–​ 13 μm long and 8–​10 μm wide. Fixed and stained, it is about 25% smaller (Fig. 8.8.14.1). Diagnostic features include the jerky motility, undulating membrane, and microtubular rod (axostyle), which runs through the body and projects as a thin spine from the posterior end. Vaginal pH is usually elevated (>4.5) but can be normal. Microscopy is inexpensive and can be used as a bedside diagnostic test, allowing immediate treatment of infected people. However, its sensitivity is only 65–​80% and it requires a microscope. Broth culture methods for detection of T. vaginalis have the advantage of greater sensitivity, but require up to 5 days’ incubation (Diamond’s TYM and the InPouch system). Rapid antigen tests can be per- formed within the clinic in a few minutes. Their sensitivity is 80% compared to culture, with the advantage of providing results during the clinic visit. The current gold standard for diagnosis is NAATS testing which can be performed on the same sample as collected for gonorrhoea

8.8.14  Trichomoniasis 1477 and chlamydia. These specimens include vaginal, endocervical, urine, or liquid-​based Pap smears. Vaginal specimens can be self-​collected. The sensitivity and specificity of NAATS testing exceeds 95%. Trichomoniasis in men Although currently male testing with NAATS has not been pre- sented to the US Food and Drug Administration (FDA) for poten- tial approval, NAATS is the diagnostic test of choice for men and has excellent sensitivity and specificity. The test can be run as an analyte specific reagent test in laboratories which have internally validated the assay in men. Urethral swab specimens or urine can be tested. Treatment 5-​nitroimidazoles are the first line therapy. A single 2 g dose of oral metronidazole is most widely used. The alternative is 500 mg twice daily for 7 days. The latter is the recommended dose for women in- fected with HIV. Metronidazole is not contraindicated in pregnancy. Recurrence occurs in 8 to 20% of women in the first month after therapy. About half the occurrences are attributed to reinfection by the same or a new sexual partner. Sexual partners must also be treated. Only the 7-​day regimen has been extensively evaluated in men, in whom it appears as effective as in women. Treatment failures with metronidazole due to resistance occur 5–​ 10% of the time. If reinfection can be ruled out, metronidazole for a longer treatment course can be tried. However, tinidazole should be considered due to its more favourable pharmacokinetic profile against T. vaginalis. There is no standardized dose for treating metro- nidazole resistant infections. One approach is 2 gm orally per day for 5–​7 days. In cases of continued treatment failure, the combination of intravaginal paromomycin cream with high dose oral tinidazole has been reported to be successful. Because up to half of infected individuals are asymptomatic, the only way to reduce the population prevalence of this pathogen is through screening of individuals and providing treatment to in- dividuals and their sexual partners. There is no effective vaccine against T. vaginalis. Prognosis In most women who receive appropriate treatment, symptoms will resolve but they are at increased risk of becoming infected with Trichomonas in the future. About 50% of men will spontaneously clear their infections, but unless both sexual partners are treated, reinfection is common. Complications Trichomoniasis in women was previously regarded as unpleasant but harmless; however, epidemiological studies have now linked it with a modest increase in the risk of heterosexual HIV transmission, and with complications in pregnancy. Areas of uncertainty, controversy, and future developments Trichomonisis has been linked with preterm birth, pelvic inflamma- tory disease (Chapter 9.8), and an increased risk of HIV. However, metronidazole treatment failed to reduce the risk of preterm delivery or acquisition of HIV in a limited number of previously conducted clinical trials which used culture for diagnosis as opposed to the currently available, more sensitive, NAATS. No study has yet docu- mented that accurate diagnosis and treatment of trichomoniasis provides a long-​term health benefit for men and women. In preg- nant women, single-​dose metronidazole treatment achieves para- sitological cure, but one trial suggested an increased risk of preterm birth. Broader implementation of specific and sensitive screening tests and additional prospective studies of treatment should reveal whether routine screening and treatment of T.  vaginalis reduces morbidity. FURTHER READING Kissinger P, et al. (2013). Trichomoniasis and HIV interactions: a re- view. Sex Trans Infect, 89, 426–​33. Meites E, et al. (2015). A review of evidence-​based care of symptomatic trichomoniasis and asymptomatic Trichomonas vaginalis infections. Clin Infect Dis, 61 (Suppl 8), S837–​48. Nye MB, et al. (2009). Comparison of APTIMA Trichomonas vagina­ lis transcription-​mediated amplification to wet mount microscopy, culture, and polymerase chain reaction for diagnosis of trichomon- iasis in men and women. Am J Obstet Gynecol, 200, 188.e181–​7. Sena AC, et al. (2007). Trichomonas vaginalis infection in male sexual partners: implications for diagnosis, treatment, and prevention. Clin Infect Dis, 44, 13–​22. Fig. 8.8.14.1  Trichomonads in vaginal secretions (Giemsa stain). Copyright J. P. Ackers.

8.8.2 Malaria 1395

8.8.2 Malaria 1395

8.8.2  Malaria 1395 Shirley DA, Moonah S (2016). Fulminant amoebic colitis after cortico- steroid therapy: a review. PLos Negl Trop Dis, 10, e0004879. Entamoeba gingivalis and Dientamoeba fragilis Bonner M, et al. (2014). Detection of the amoeba Entamoeba gingiva­ lis in periodontal pockets. Parasite, 21, 30. Foda AA, et  al. (2012). Prevalence of genital tract infection with Entamoeba gingivalis among copper plate T380A intrauterine device users in Egypt. Contraception, 85, 108–​12. Roser D, et al. (2014). Metronidazole therapy for treating dientamoebiasis in children is not associated with better clinical outcomes: a random- ized, double-​blinded and placebo-​controlled clinical trial. Clin Infect Dis. 58, 1692–​9. Stark D, et al. (2014). Description of Dientamoeba fragilis cyst and precyst forms in human samples. J Clin Microbiol, 52, 2680–​3. Free-​living amoebae Bravo SG, Alvarez PJ, Gotuzzo E (2011). Balamuthia mandrillaris infection of the skin and central nervous system: an emerging disease of concern to many specialities in medicine. Curr Opin Infect Dis, 24, 112–​7. Carter RF (1972). Primary amoebic meningo-​encephalitis. Trans R Soc Trop Med Hyg, 66, 193–​208. Fung KT-​T, et al. (2008). Cure of Acanthamoeba cerebral abscess in a liver transplant patient. Liver Transplant, 14, 308–​12. Garg P, Kaira P, Joseph J (2017). Non-contact lens related Acanthamoeba keratitis. Indian J Ophthalmol, 65, 1079–86. LaFleur M, et al. (2013). Balamuthia mandrillaris meningoencephalitis associated with solid organ transplantation—​a review. J Radiol Case Rep, 7, 9–​18. Lorenzo-​Morales J, Khan NA, Walochnik J (2015). An update on Acanthamoeba keratitis:  diagnosis, pathogenesis and treatment. Parasite, 22, 10. Ong TYY, Khan NA, Siddiqui R (2017). Brain-eating amoebae: predilec- tion sites in the brain and disease outcome. J Clin Microbiol, 55, 1989–97. Panja SC, Dinoop K, Venugopal H. (2015). Management granuloma- tous amebic encephalitis: laboratory diagnosis and treatment. Trop Parasitol, 5, 23–​8. Qvarnstrom Y, et el. (2009). Molecular confirmation of Sappinia pedata as a causative agent of amoebic encephalitis. J Infect Dis, 199, 1139–​42. Roy SL, et al. (2014). Risk of transmission of Naegleria fowleri from solid organ transplantation. Am J Transplant, 14, 163–​71. Siddiqui R, Khan NA. (2014). Primary amoebic meningoencephal- itis caused by Naegleria fowleri: an old enemy presenting new chal- lenges. PLoS Neg Trop Dis, 8, e3017. Sood A, et al. (2013). Prompt diagnosis and extraordinary survival from Naegleria fowleri meningitis:  a rare case report. Ind J Med Microbiol, 32, 193–​6. Visvesvara GS (2013). Infections with free-​living amoebae. Handb Clin Neurol, 113, 153–​68. 8.8.2  Malaria Nicholas J. White and Arjen M. Dondorp ESSENTIALS In 2015, 3.4 billion people were at risk for malaria, of which 1.1 bil- lion were at high risk. According to World Health Organization’s World Malaria Report for 2016, there were an estimated 212 million cases of malaria worldwide in 2015 with 429 000 deaths. Africa accounted for 90% of the cases and 92% of deaths; 70% of the deaths were in children aged less than 5 years. Malaria remains endemic in 91 countries. Human malaria parasites, mosquitoes, and transmission
of malaria Malaria parasites and their impact on the human genome—​six species of Plasmodium commonly cause malaria in humans: P. falciparum, P. vivax, P. ovale (two species), P. malariae, and P. knowlesi. P. falciparum, the most pathogenic species accounts for 99% of malaria deaths and has exercised immense selection pressure on the human genome. Biology of the parasite and mosquito vector—​sporozoites are ijected into humans during the female Anopheles mosquito’s blood meal. They invade hepatocytes. Hepatic schizogony releases merozoites into the blood stream where they invade red blood corpuscles and undergo further asexual multiplications before gametocytes form. If these are ingested by mosquitoes, male and female gametes fuse, resulting in ookinetes that penetrate the mosquito’s midgut and de- velop into oocysts. Daughter sporozoites are released. They invade the mosquito’s salivary glands, ready to infect a new human host. Persistent latent forms (hypnozoites) of P. vivax and P. ovale remain in the liver to give rise to later relapses of parasitaemia and symptoms. Other mechanisms of transmission—​malaria can also be trans- mitted by transfusion of blood products, marrow transplants, and contaminated needles. Innate resistance and immunity In most stably endemic areas, acquisition of immunity, although never complete, ensures that death due to malaria is rare after the age of 5 years and hardly ever occurs in normally immune compe- tent adults. Immunity allows tolerance of levels of parasitization that would cause illness in a naive individual. Malnutrition and advanced HIV infection increase the risk of severe falciparum malaria in children. Molecular pathology, organ pathology,
and pathophysiology Pathophysiology—​intravascular, asexual forms are responsible for all the pathological effects of malaria in humans. The main pathophysio- logical hallmark of severe P. falciparum infection is the cytoadherence and sequestration of parasitized red blood cells to capillary and postcapillary venular endothelium of vital organs, especially in the brain, intestines, lungs, and kidneys, resulting in reduced perfusion and tissue damage. Anaemia results from destruction/​phagocytosis of both uninfected and parasitized red blood cells, as well as from dyserythropoiesis. Thrombocytopenia is attributable to splenic se- questration, dysthrombopoiesis, and perhaps endothelial mediated binding and lysis. Pulmonary oedema may result from fluid overload, but more often there is increased pulmonary capillary permeability. Clinical features Malaria causes periodic febrile paroxysms with afebrile asymp- tomatic intervals: every other day in falciparum and vivax malaria (‘tertian fever’), every second day in P. malariae (‘quartian fever’). Severe falciparum malaria—​defined by (1)  clinical features—​ prostration, impaired consciousness, respiratory distress/​acidotic breathing, multiple convulsions, circulatory collapse, pulmonary oe- dema, and acute respiratory distress syndrome, abnormal bleeding, jaundice, and haemoglobinuria; and (2)  laboratory tests—​severe

section 8  Infectious diseases 1396 anaemia, hypoglycaemia, metabolic acidosis, hyperlactataemia, and renal impairment, which are of proven prognostic significance. Cerebral malaria—​defined by coma in patients with acute P. fal- ciparum infection in whom other causes of coma, including hypo- glycaemia and transient postictal coma, have been excluded. Convulsions, dysconjugate gaze, malaria specific retinal changes, and abnormal posturing are common. So-​called benign malarias, P. ovale, P. malariae, and particularly P. vivax, can cause even more severe feverish symptoms than falcip- arum malaria. P. knowlesi, one of the monkey malarias, is increasingly recognized as an important and potentially fatal zoonosis in humans in several Southeast Asian countries. Malaria in pregnancy—​malaria is an important cause of maternal anaemia and death, abortion, stillbirth, premature delivery, low birth weight, and neonatal death. Chronic immunological complications of malaria—​these include
quartan malarial nephrosis, tropical splenomegaly syndrome (hyper­ reactive malarial splenomegaly) and endemic Burkitt’s lymphoma. Diagnosis The diagnosis is made by microscopy of a peripheral blood thin or thick smear or using a rapid diagnostic antigen test. Differential diag- noses include other acute febrile illness: falciparum malaria has been misdiagnosed as influenza, viral hepatitis, epilepsy, viral encephalitis, or bacterial meningitis, sometimes with fatal consequences. Treatment Uncomplicated P. falciparum malaria in malaria endemic areas—​ treatment is with artemisinin combination therapies. Currently used artemisinin combination therapies include artemether–​ lumefantrine, dihydroartemisinin-​piperaquine, artesunate-​mefloquine, artesunate-​amodiaquine (mainly Africa), artesunate-​sulphadoxin-​ pyrimethamine (only selected countries in Africa), and artesunate-​ pyronaridine (still limited use). Resistance to artemisinins, and increasingly also to the artemisinin combination therapy partner drugs, has emerged in the Greater Mekong Subregion (Cambodia, Laos, Vietnam, Thailand, and Myanmar). Further spread westward could occur over the coming years. For presumed non​immune travellers returning to non​endemic areas with uncomplicated falciparum malaria, artemether–​lumefantrine, other artemisinin combination therapies, or atovaquone–​proguanil are recommended. During first trimester pregnancy, quinine combined with clindamycin is recommended. P. vivax, P. ovale, P. malariae, P. knowlesi malarias—​these are treated with chloroquine. Resistant P.  vivax (New Guinea, Indonesia) is treated with artemisinin combination therapies. Radical treatment in P. vivax and P. ovale malaria, eliminating the liver hypnozoites to pre- vent relapse infections, is with a 14-​day course of primaquine. Severe falciparum, vivax, and knowlesi malaria—​urgent parenteral antimalarial treatment with artesunate is essential. Intramuscular artemether, or quinine by intermittent or continuous intravenous infusion or intramuscular injection are second and third choice. Quinine therapy requires a loading dose. Rectal artesunate has shown benefit as a prereferral therapy in African villages. Supportive care—​patients with severe malaria should be transferred to the highest possible level of care for treatment of convulsions, hypoglycaemia, severe anaemia (by blood transfusion) and organ failure. Prevention Modern malaria control and prevention aims to limit human–​vector contact by indoor residual spraying and insecticide (pyrethroid) treated nets. Repellents such as diethyltoluamide are used for per- sonal protection. Intermittent preventive treatment in pregnant women with sulphadoxine–​pyrimethamine in sub-​Saharan Africa improves birth weight and maternal anaemia, and such treatment of infants has been implemented in several high-​transmission countries in Africa. Seasonal malaria chemoprevention in children is currently rolled out in the Sahel subregion. Malarial vaccines—​the RTS,S/​ASO1 P. falciparum malaria vaccine has been registered recently by the European Medicines Agency. It provides short-​term protection of approximately 30–​50% for one year, but declines thereafter. RTS,S is now field tested on a larger scale in several African countries (2018). Travellers—​prevention of malaria in people from non​malarious areas who are visiting endemic regions has become more difficult because of resistance to antimalarial drugs. Travellers are advised to (1) be aware of the risk; (2) prevent exposure to anopheline mosqui- toes; (3) take chemoprophylaxis where appropriate—​malarone, mef- loquine, or doxycycline; in Southeast Asia mefloquine resistance in prevalent (4) seek immediate medical advice in case of any feverish illness developing while abroad, or within 3 months of returning, and to mention malaria as a possibility—​regardless of the precautions taken—​to any doctor who sees them. Pregnant women are best ad- vised to avoid malarious areas. Introduction Malaria is a protozoan disease transmitted by the bite of infected Anopheles mosquitoes. Malaria is the most important of the para- sitic diseases of humans. It is transmitted in 106 countries containing 3 billion people and still causes approximately 2000 deaths each day. Malaria has been eliminated from the United States, Canada, Europe, and Russia. The global mortality has decreased over the last decade as a result of substantial increases in funding for con- trol efforts, but this progress had stalled in 2017. This follows a resurgence in malaria between the 1970s and early 2000s as a re- sult of insufficient investment and support for control activities in endemic countries, increased human population movement and worsening resistance of the malaria parasites to antimalarial drugs, and of the mosquito vectors to insecticides. Occasional local transmission after importation of malaria has occurred in Europe (notably Greece) and several southern and eastern areas of the United States, indicating the continued danger to non​malarious countries. Malaria remains today, as it has been for centuries, a heavy burden on tropical communities, a threat to non​endemic countries, and a danger to travellers. Epidemiology Malaria occurs throughout most of the tropical regions of the world. P. falciparum predominates in Africa, New Guinea, and Hispaniola (i.e. the Dominican Republic and Haiti); P. vivax is more common

8.8.2  Malaria 1397 in Central America. The prevalence of these two species is approxi- mately equal in South America, the Indian subcontinent, eastern Asia, and Oceania. P. malariae is found in most endemic areas, es- pecially throughout sub-​Saharan Africa, but is much less common. P. ovale (which comprises two species) is relatively unusual outside of Africa and, where it is found, comprises less than 1% of infections. P. knowlesi malaria occurs on the island of Borneo and to a lesser extent elsewhere in Southeast Asia. Unlike the human malarias, its main hosts are the long-​tailed and pig-​tailed macaques (Fig. 8.8.2.1). The epidemiology of malaria is complex and can vary consid- erably even within relatively small geographic areas (Fig. 8.8.2.2). Endemicity traditionally has been defined by the prevalence of parasitaemia or palpable spleens in children 2–​9  years of age (hypoendemic: <10%, mesoendemic: 11–​50%, hyperendemic: 51–​ 75%, and holoendemic: >75%). Many countries conduct national surveys using these indices to assess control programme progress. In holo-​ and hyperendemic areas (e.g. parts of tropical Africa or coastal New Guinea) where there is intense P. falciparum transmis- sion, people might receive more than one infectious mosquito bite each day. They are infected repeatedly throughout their lives. As a consequence, the morbidity and mortality due to malaria are con- siderable during early childhood. But if the child survives, immunity against disease is gradually acquired and by adulthood most malaria infections are asymptomatic. Constant, frequent, year-​round infec- tion is termed stable transmission. In areas where transmission is low, erratic, or focal, full protective immunity is not acquired, and symptomatic disease may occur at all ages. This is the usual situ- ation in hypoendemic areas. It is termed unstable transmission. Even in stable transmission areas, the number of malaria cases often in- crease during the rainy season, coinciding with increased mosquito breeding. In areas with unstable malaria, such as the Punjab region of northern India, the horn of Africa, Rwanda, Burundi, southern Africa, and Madagascar, sudden environmental, social, or eco- nomic changes can cause malaria epidemics. Examples are heavy P. falciparum P. vivax P. ovale P. malariae P. knowlesi Fig. 8.8.2.1  Asexual stage parasites of the different Plasmodium species infecting humans. Courtesy Dr Kesinee Chotivanich. Fig. 8.8.2.2  (a) Global epidemiology of falciparum malaria. (b) Global epidemiology of vivax malaria. © 2010 Malaria Atlas Project, available under the Creative Commons Attribution 3.0 Unported License.

section 8  Infectious diseases 1398 rains following drought, or migrations of refugees or workers from a non​malarious region to an area of high transmission together with failure to invest in malaria control activities. Breakdowns in malaria control and prevention services caused by war or civil disorder can also cause epidemics. This usually results in considerable mortality among all age groups if the population is non​immune. The epidemiology of malaria is determined largely by the number (density), the human-​biting habits, and the longevity of the anoph- eline mosquito vectors. The c.40 species that can transmit malaria vary considerably in their efficiency as malaria vectors. Mosquito longevity is particularly important because malaria parasite devel- opment within the mosquito—​from gametocyte ingestion to sub- sequent inoculation (sporogony)—​takes 8–​30 days, depending on ambient temperature. Thus, to transmit malaria, the mosquito must survive for at least 7 days. Sporogony is not completed at cooler tem- peratures (i.e. <16°C for P. vivax and <21°C for P. falciparum) and so transmission does not occur below these temperatures. Malaria does not occur at high altitudes either, although malaria outbreaks and transmission have occurred in the highlands (>1500 m) of east Africa, which were previously free of vectors, possibly as a result of global warming. The most effective mosquito vectors of malaria are those, which are long-​lived, occur in high densities in tropical cli- mates, breed readily, and bite humans in preference to animals. The main vector in Africa, Anopheles gambiae, is a prime example. The entomologic inoculation rate (the number of sporozoite-​positive mosquito bites per person per year) is the most common measure of malaria transmission and varies from less than one in some parts of Latin America and Southeast Asia to more than 300 in parts of tropical Africa. Aetiology and pathogenesis Six species of the sporozoan (apicomplexan) genus Plasmodium cause nearly all malarial infections in humans. These are P. falcip­ arum, P. vivax, P. ovale, P. malariae, and—​in Southeast Asia—​the monkey malaria parasite P.  knowlesi (Table 8.8.2.1). Recent evi- dence shows that P. ovale comprises two morphologically identical sympatric species, P. ovale curtisi and P. ovale wallikeri. P. falciparum causes most malaria deaths but P. knowlesi and occasionally P. vivax can also cause severe illness. Life cycle Human infection begins when a female anopheline mosquito in- oculates plasmodial sporozoites from its salivary gland while sucking blood (Fig. 8.8.2.3). These microscopic motile malaria parasites are carried rapidly via the bloodstream to the liver, where they invade hepatic parenchymal cells and there begin asexual reproduction. By this amplification process (known as preerythrocytic schizogony or merogony), a single sporozoite eventually produces from 10 000 to Fig. 8.8.2.2  Continued

Table 8.8.2.1  Characteristics of Plasmodium species infecting humans Speciesa P. falciparum P. vivax P. ovale P. malariae P. knowlesi Duration of intrahepatic phase (days) 5.5 8 9 15 5 Number of merozoites released per infected hepatocyte 30 000 10 000 15 000 15 000 Duration of erythrocytic cycle (hours) 48 45 50 72 24 Average number of merozoites per schizont 16 16 8 8 10 Red cell preference Younger cells (but can invade cells of all ages) Reticulocytes and cells up to 2 weeks old Reticulocytes Older red cells cells Red cells of all ages Morphology Usually only ring formsb; banana-​shaped gametocytes Irregularly shaped large rings and trophozoites; enlarged erythrocytes; Schüffner’s dots Infected erythrocytes, enlarged and oval with tufted ends; Schüffner’s dots Band or rectangular forms of trophozoites Young rings resemble P. falciparum, mature trophozoites resemble P. malariae Pigment colour Black Yellow-​brown Dark brown Brown-​black Yellow-​black Parasitaemias may exceed 2% Ability to cause relapses Yes No No Yes No Yes No No Yes No a Reliable identification, particularly with low-​density ring form parasitaemia, requires molecular genotyping. b P. ovale comprises two sympatric species P. ovale wallikeri and P. ovale curtesi. Salivary gland sporozoites Midgut sporozoites Oocyst Ookinete Meiosis Zygote Gametes 2 Transmission to mosquito D Sexual stages Gametocytes Asexual cycle Ring Blood stage Liver Merozoites 105 108–1012 C Liver stage Hepatocyte invasion Sporozoites B A Infection 10 Inoculation Mosquito stage 5 x 104 0–6 H 6–16 H 16–26 H 26–30 H 30–34 H 34–38 H 38–44 H 44–48 H (a) (b) Fig. 8.8.2.3  Lifecycle of Plasmodium falciparum. Female anopheline mosquitoes inoculate around 10 motile sporozoites into the dermis (a), which invade hepatocytes within one hour (b). Within the hepatocyte, each sporozoite produces a liver schizont with 10 000 to 30 000 nuclei. After about a week, the liver schizonts ruptures, together releasing around 100 000 merozoites into the bloodstream that invade red blood cells and begin the asexual cycle (c). During the asexual cycle (48-​hours in P. falciparum; inset), the parasite develops from a small ring, to a trophozoite (when malaria pigment becomes visible), to a schizont stage (when the nucleus starts to divide). At schizont rupture around 10 new erythrocytes are being infected, resulting in a multiplication factor of around 10 every 48 hours. Illness starts when total asexual parasite numbers in the circulation reach roughly 100 million. After a few cycles, some parasites develop into sexual forms (gametocytes), which are taken up by a feeding anopheline mosquito (d) and reproduce sexually, forming an ookinete, and then an oocyst in the mosquito gut. The oocyst bursts and liberates sporozoites, which migrate to the salivary glands to await inoculation at the next blood feed. The entire cycle can take roughly 1 month. Estimated numbers of parasites are shown in boxes—​a total body parasite burden of 10¹² corresponds to roughly 2% parasitaemia in an adult. Reprinted from The Lancet, Vol. 383, White NJ et al., Malaria, pages 723–​735, Copyright © 2014, with permission from Elsevier.

section 8  Infectious diseases 1400 more than 30 000 daughter merozoites. The swollen infected liver cell eventually bursts, discharging the motile merozoites into the bloodstream where they invade red blood cells. They progressively consume the red cell contents and so develop from tiny ring forms into large malaria pigment-​containing trophozoites, and then start nuclear division, thereby becoming schizonts. This erythrocytic life cycle takes 48 h for P. falciparum, P, vivax, and P. ovale, but 24 h for P. knowlesi, and 72 h for P. malariae. After erythrocyte schizont rupture, the released merozoites rapidly invade new erythrocytes, resulting in a multiplication rate of around 10 per cycle in non-​ immune hosts. When the logarithmically expanding parasite num- bers reach densities of c.50/​µl of blood (c.100 million parasites in the blood of an adult), the symptomatic stage of the malaria infec- tion begins. In P. vivax and P. ovale infections, a proportion of the intrahepatic forms do not divide immediately but remain inert for a period ranging from 3 weeks to a year or longer before reproduction begins. These dormant forms, or hypnozoites, are the cause of the re- lapses that characterize infection with these two species. Merozoite invasion requires attachment to specific erythrocyte surface receptors (the glycophorins are particularly important). For P. falciparum erythrocyte invasion is dependent on the reticulocyte-​ binding protein homologue 5 (PfRh5), for which basigin (CD147, EMMPRIN) is the erythrocyte receptor. In P. vivax, this receptor ap- pears to be CD71 with an important supporting role from the Duffy blood-​group antigen Fya or Fyb. Most West Africans and people with origins in that region have the Duffy-​negative FyFy phenotype and are therefore largely resistant to P. vivax malaria. During the early stage of intraerythrocytic development, the small ‘ring forms’ of the different parasite species appear very similar under light micros- copy. But as the trophozoites grow, species-​specific characteristics become evident, malaria pigment (the waste product of digested haemoglobin) becomes visible, and the parasite assumes an irregular or amoeboid shape. Then multiple nuclear divisions take place (schi­ zogony or merogony) before the schizont ruptures releasing 6–​30 daughter merozoites, each potentially capable of invading a new red blood cell and repeating the asexual cycle. The disease malaria in human beings is caused by the direct effects of red blood cell invasion and destruction by the asexual parasite and the host’s reaction. Only in P. falciparum malaria, which causes most severe disease, erythrocytes containing the more mature asexual stage parasites sequester in the microcirculation impairing tissue flow. This process is central to pathogenesis. After release from the liver some of the blood stage parasites develop into mor- phologically distinct, longer-​lived sexual forms (gametocytes) that can transmit malaria. In falciparum malaria there is a delay of sev- eral asexual cycles before this switch to gametocytogenesis, and the developing gametocytes (stages 1 to 4) are sequestered for about one week–​ particularly in the bone marrow. Only the stage 5 P. fal­ ciparum gametocytes circulate. There are usually 3 to 5 times more female gametocytes than males in the blood. After ingestion by a feeding mosquito each male gametocyte will undergo three rounds of rapid mitosis and produce eight flagellated microgametes each capable of fertilizing a female macrogamete. In the female anopheline mosquito the male and female gametes fuse to form a zygote in the insect’s midgut. This zygote matures into an ookinete, which penetrates and encysts in the mosquito’s gut wall. The resulting oocyst expands by asexual division until it bursts to liberate myriad motile sporozoites, which migrate in the mosquito hemolymph to the salivary glands to await inoculation into another human at the next feeding. Pathogenesis After red cell invasion, the growing malarial parasite progressively consumes and degrades the erythrocyte proteins. By the end of the intraerythrocytic life cycle, the parasite has consumed two-​thirds of the cell’s haemoglobin. The potentially toxic haem is detoxified by lipid-​mediated crystallization to biologically inert haemozoin (malaria pigment). The parasite also modifies the red cell mem- brane by changing its transport properties, revealing cryptic sur- face antigens, and inserting new parasite-​derived proteins. The erythrocyte becomes more irregular in shape, more antigenic, and in P.  falciparum infections it becomes less deformable, as most of the erythrocyte volume becomes occupied by the rigid schizont. In contrast, P. vivax enlarges the infected erythrocyte making it more deformable. In P. falciparum infections, membrane protuberances appear on the erythrocyte’s surface 12–​15 h after the cell’s invasion. These ‘knobs’ extrude a high-​molecular-​weight, strain-​specific, antigenically variant, erythrocyte membrane adhesive protein (PfEMP1) that adheres to receptors on venular and capillary endothelium—​a pro- cess termed cytoadherence (Fig. 8.8.2.4). Several vascular receptors have been identified, of which endothelial protein C receptor and intercellular adhesion molecule 1 (ICAM-​1) are important recep- tors on brain endothelium, chondroitin sulfate B on the placenta syncytiotrophoblast, and CD36 on the vascular endothelium of most other organs. Cytoadherence compromises blood flow in capillaries and venules and causes endothelial activation. At the same stage of parasite development, these P. falciparum-​infected red cells can also adhere to uninfected red blood cells (to form rosettes) and might agglutinate with other parasitized erythrocytes. These processes of cytoadherence, rosetting, and agglutination are central to the patho- genesis of falciparum malaria. They result in the sequestration of Fig. 8.8.2.4  Electron microscopic photograph of a post-​mortem brain biopsy, showing a P. falciparum trophozoite stage infected erythrocyte cytoadhered to the endothelium, causing partial obstruction of the capillary. Courtesy Dr Emsri Pongponratn.

8.8.2  Malaria 1401 erythrocytes containing mature forms of the parasite in vital or- gans (particularly the brain), interfering with microcirculatory flow, tissue oxygenation, and metabolism. Sequestered parasites develop out of reach of the principal host defence mechanism: splenic pro- cessing and filtration. As a consequence, only the younger ring forms of the asexual parasites are seen circulating in the peripheral blood in falciparum malaria. This means that the level of periph- eral parasitaemia can underestimate considerably the true number of parasites within the body. Severe malaria is also associated with reduced deformability of the uninfected erythrocytes, which com- promises their passage through the partially obstructed capillaries and venules and shortens their survival. In the other human malarias, significant sequestration does not occur, and so all stages of the parasites’ development are evident on peripheral blood smears. However, all the malarias cause rosetting. Whereas P. vivax, P. ovale, and P. malariae show a marked predilec- tion for either young red blood cells (P. vivax, P. ovale) or old cells (P.  malariae) and produce parasitaemias that seldom exceed 2%, P. falciparum and P. knowlesi can invade erythrocytes of all ages and can cause very high—​and often lethal parasite densities. Host responses Initially, the host responds to malaria infection by activating non-​ specific defence mechanisms. Splenic immunologic and filtrative clearance functions are augmented in malaria, and the removal of both parasitized and uninfected erythrocytes is accelerated. The spleen is able to remove damaged ring form parasites (‘pitting’) and return the once infected erythrocytes to the circulation, where they have shortened survival. The parasitized cells escaping splenic removal are destroyed when the schizont ruptures. The material released by the bursting schizonts induces the activation of leuko- cytes and complement factors and the release of proinflammatory cytokines, which cause fever, and exert other pathologic effects. Temperatures of ≥40°C damage mature parasites; in untreated in- fections these temperatures further synchronize the asexual parasite cycle, with eventual production of the regular fever spikes and rigors that originally characterized the different malarias. These regular fever patterns (quotidian, daily; tertian, every 2 days; quartan, every 3 days) are seldom seen today in patients who receive prompt and effective antimalarial treatment. The global distributions of the haemoglobinopathies (thalassaemias, sickle cell disease, haemoglobins C and E, hereditary ovalocytosis) and glucose-​6-​phosphate dehydrogenase (G6PD) deficiency closely resemble the world map of malaria over a century ago (before large control initiatives). These genetic disorders evolved to confer protec- tion against death from malaria. For example, HbA/​S heterozygotes (sickle cell trait) have a sixfold reduction in the risk of dying from se- vere falciparum malaria. HbA/​S impairs parasite growth at low oxygen tensions and P. falciparum-​infected red cells containing haemoglobins S and C have reduced cytoadherence because of reduced surface pres- entation and disturbed organization of the cytoadhesion molecule PfEMP1. Parasite multiplication in HbA/​E heterozygotes is reduced at high parasite densities. In Melanesia, children with α-​thalassemia have more frequent malaria (both vivax and falciparum) in the early years of life, and this pattern of infection appears to protect them against severe disease. In Melanesian ovalocytosis, rigid erythrocytes resist merozoite invasion, and provide a hostile intraerythrocytic milieu for the parasite. Malaria has evolved to avoid the immune response. As a result, immunity to malaria is slowly acquired and imperfect. Both hu- moral immunity and cellular immunity are necessary for protection, but the mechanisms of each are incompletely understood. Initially non​specific host defence mechanisms stop the exponential expan- sion of malaria parasite numbers, and the subsequent strain-​specific immune response later controls the infection. Eventually, with re- peated infections, exposure to sufficient numbers of strains confers protection from high-​level parasitaemia and disease, but not from infection. As a result of this infection without illness (premunition), asymptomatic parasitaemia is common among adults and older children living in malaria endemic regions. The more intense the transmission, the earlier in life is this ‘illness protecting’ immunity acquired (Fig. 8.8.2.5). Gradually species and then strain-​specific immunity is acquired against local parasites. This protects against infection. Some immunity is also gradually acquired against the preerythrocytic liver stage and the sexual stage of the infection. Immune individuals have a polyclonal elevation in serum IgM, IgG, and IgA, although much of this antibody is unrelated to protection. Antibodies to a variety of parasitic antigens limit in vivo replica- tion of the parasite. In the case of falciparum malaria, one of the most important antigens is the variant surface cytoadhesion protein PfEMP1. Passive transfer of maternal antibody contributes to the relative protection of infants from severe malaria in the first months of life. This complex immunity to disease declines when a person lives outside an endemic area for several months or longer. Several factors slow the development of cellular immunity to mal- aria; these include the absence of major histocompatibility antigens on red cells precluding direct T cell recognition; malaria antigen-​ specific immune unresponsiveness; reduced formation of long-​lived plasma cells and memory B-​cells, the enormous strain diversity of malarial parasites, and redundancy of surface protein functions, all of which is compounded by the ability of the parasites to express Severe Mild Parasitaemia 100 80 60 40 20 0 0 5 10 15 20 25 30 35 40 45 50 Age Maximum response (%) Fig. 8.8.2.5  Clinical manifestations of P. falciparum infection in relation to age in an area of moderate to high-​transmission intensity. With repeated exposure protection is acquired, first against severe malaria, then against illness with malaria, and, much more slowly, against microscopy-​detectable parasitaemia. Modified with permission from Marsh K and Kinyanjui S (2009). Immune effector mechanisms in malaria. Parasite Immunology 28, pages 51–​60, Copyright © 2005, John Wiley and Sons.

section 8  Infectious diseases 1402 variant immunodominant antigens on the erythrocyte surface that change during the infection. Parasites may persist in the blood for months or sometimes years (or, in the case of P. malariae, for life) if treatment is not given. These factors have all contributed to the slow progress toward an effective vaccine. Clinical features Malaria is a very common cause of fever in tropical countries. The first symptoms of malaria are non​specific; the lack of a sense of well-​being, fatigue, headache, abdominal discomfort, and muscle aches followed by fever are all similar to the symptoms of a minor viral illness. Sometimes prominent headache, chest pain, abdom- inal pain, cough, arthralgia, myalgia, or diarrhoea may suggest a different diagnosis. Although headache can be severe in malaria, there is no neck stiffness or photophobia as in meningitis. Myalgia might be prominent, but it usually milder than in dengue fever, and the muscles are not tender as in leptospirosis or typhus. Nausea, vomiting, and orthostatic hypotension are common. The classic malarial paroxysms, in which fever spikes, chills, and rigors occur at regular intervals, are relatively unusual, and suggest relapse infection with P. vivax or P. ovale. The fever is initially irregular (that of falcip- arum malaria may never become regular); in non​immune individ- uals pyrexia often rises above 40°C with tachycardia and, sometimes, delirium. Although childhood febrile convulsions might occur with any malaria, generalized seizures are specifically associated with falciparum malaria and may herald the development of encephal- opathy (cerebral malaria). Many clinical abnormalities have been described in acute malaria, but most patients have few abnormal physical findings initially other than fever, malaise, mild anaemia, and (in some cases) a palpable spleen. This is sometimes called ‘un- differentiated fever’. Anaemia is common among young children living in areas with stable malaria transmission, particularly where antimalarial drug resistance results in recurrent infections. In acute malaria, the spleen enlarges but usually takes several days to become palpable. In malaria endemic areas splenic enlargement is found in a high proportion of otherwise healthy individuals as a result of re- peated infections. Hepatomegaly is also common, particularly in young children. Mild jaundice is common among adults; it may de- velop in patients with otherwise uncomplicated malaria and usu- ally resolves over 1–​3 weeks. Malaria is not associated with a rash, differentiating it from meningococcal septicaemia, rickettsial infec- tions, enteric fever, viral exanthems, and drug reactions. Petechial haemorrhages in the skin or mucous membranes—​features of viral haemorrhagic fevers and leptospirosis—​develop only very rarely in severe falciparum malaria. Diagnosis The diagnosis of malaria requires demonstration of asexual forms of the parasite in suitably stained peripheral blood smears, or de- tection of blood stage antigens by rapid diagnostic tests (RDTs) (Fig. 8.8.2.6; Table 8.8.2.2). After a negative blood smear, repeat smears should be made if there is a high degree of suspicion. Both thin and thick blood smears should be examined. The thin blood smear should be air-​dried rapidly, fixed in anhydrous methanol, and stained; the red cells in the tail of the film should then be examined under oil immersion (×1000 magnification). The parasite density is recorded as the number of parasitized erythrocytes per 1000 red cells. The thick blood film has the advantage of concentrating the parasites (by 40-​ to 100-​fold compared with a thin blood film) and thus increasing diagnostic sensitivity. The thick film should be of uneven thickness. It should be dried thoroughly and stained without fixing. Both parasites and white blood cells are counted, and the number of parasites per unit volume is calculated from the total leukocyte count (or assuming a white blood cell count of 8000/​µl). A minimum of 200 white blood cells should be counted under oil immersion. Interpretation of blood films requires some experience because artefacts are common. Before a thick smear is called nega- tive, 100–​200 fields should be examined under oil immersion mi- croscopy. In high-​transmission areas, parasite densities up to 10 000 parasites/​µl of blood may be tolerated without symptoms or signs in partially immune individuals. In these areas the detection of malaria parasites is sensitive but has low specificity in identifying malaria as the cause of illness because incidental low-​density parasitaemia is commonly found in other conditions causing fever. Rapid, simple, sensitive, and specific antibody-​based RDTs that detect P.  falciparum–​specific, histidine-​rich protein 2 (PfHRP2), lactate dehydrogenase or aldolase antigens in finger-​prick blood samples are now being used widely in malaria control programmes (Fig. 8.8.2.6). Some of these RDTs carry a second antibody, which allows falciparum malaria to be distinguished from the less dan- gerous malarias. PfHRP2-​based tests may remain positive for several weeks after acute infection. This is a disadvantage in high-​transmission areas where infections are frequent, but is useful in the diagnosis of Thin film Thick film Rapid diagnostic test Result window Well for blood sample Well for buffer solution Contol line (C) Test line (T) Fig. 8.8.2.6  Giemsa stained peripheral blood slide, showing with 400x magnification in the thin film large ring stage P. falciparum parasites inside red blood cells (some with multiple invasion), and in the (haemolysed) thick film more concentrated ring stage parasites amid two white blood cells. In the lower panel, an example of a rapid diagnostic test with a positive test result.

8.8.2  Malaria 1403 severe malaria in patients who have taken antimalarial drugs and cleared peripheral parasitaemia (but in whom the PfHRP2 test re- mains strongly positive). RDTs are replacing microscopy in many areas because of their simplicity and speed, and similar sensitivity. The disadvantage is that they do not quantify peripheral blood para- sitaemia PfHRP2-based tests can be falsely negative in P. falcip­ arum infections with PfHRP2 gene deletions, reported mainly from South-America as well as increasingly from Africa. Prognosis In falciparum malaria patients with more than 105 parasites/​µl (c.2% parasitaemia) are at increased risk of dying, but as the peripheral blood parasitaemia variably underestimates the total body parasite burden because of the sequestration which is responsible for organ dysfunc- tion, non​immune patients may die with much lower counts. Conversely partially immune persons may tolerate relatively high parasitaemias with only minor symptoms. In severe falciparum malaria, a poorer prognosis is indicated at any parasite density by a predominance of more mature P. falciparum parasites (i.e. >20% of parasites with visible pigment) in the peripheral blood film and by the presence of phagocyt- osed malarial pigment in more than 5% of neutrophils. In P. falciparum infections, gametocytaemia peaks one week after the peak of asexual parasites. The mature gametocytes of P. falcip­ arum are not affected by most antimalarial drugs (whereas those of the other malarias are), so their persistence does not indicate drug resistance. Phagocytosed malarial pigment in peripheral blood monocytes can provide a clue to recent infection if malaria parasites are not detectable. Molecular diagnosis by polymerase chain reaction (PCR) ampli- fication of parasite nucleic acid is more sensitive than microscopy or RDTs for detecting low-​density malaria parasitaemia and is more ac- curate in speciation. In parts of Southeast Asia, PCR is important in the identification of P. knowlesi, which mimics P. malariae morphologic- ally. In malaria endemic areas PCR is mostly used in reference centres, although loop-​mediated isothermal amplification is a low technology PCR variant adapted to the resource-​poor setting. Sensitive PCR Table 8.8.2.2  Methods for the diagnosis of malariaa Method Procedure Advantages Disadvantages Thick blood filmb Round blood spot (1–​2 cm2) should be of uneven thickness but sufficiently thin to read the hands of a watch through part of the spot. Stain well dried, unfixed blood spot with Giemsa, Field’s, or another Romanowsky stain. Count the number of asexual parasites per 200 WBCs (or per 500 at low densities) at ×1000 magnification. Count the gametocytes separately.c Sensitive (0.001% parasitaemia); species specific; inexpensive Requires experience (artefacts may be misinterpreted as low-​level parasitaemia); underestimates true count Thin blood filmd Stain fixed smear with Giemsa, Field’s, or another Romanowsky stain. Count the number of RBCs containing asexual parasites per 1000 RBCs at ×1000 magnification. In severe malaria, assess stage of parasite development and count neutrophils containing malaria pigment.e Count gametocytes separately.c Rapid; species specific; inexpensive; in severe malaria, provides prognostic informatione Insensitive (<0.05% parasitaemia); uneven distribution of P. vivax, as enlarged infected red cells concentrate at leading edge Plasmodium LDH dipstick or card test A drop of blood is placed on the stick or card, which is then immersed in washing solutions. Monoclonal antibodies capture the parasite antigens and read out as coloured bands. One band is genus specific (all malarias) or P. vivax specific, and the other is specific for P. falciparum. Rapid; sensitivity similar to or slightly lower than that of thick films for P. falciparum (c.0.001% parasitaemia) Slightly more difficult preparation than PfHRP2 tests; may miss low-​level parasitaemia with P. vivax, P. ovale, and P. malariae and may not speciate these organisms; does not quantitate P. falciparum parasitaemia. PfHRP2 dipstick or
card test A drop of blood is placed on the stick or card, which is then immersed in washing solutions. Monoclonal antibody captures the parasite antigen and reads out as a coloured band. Robust and relatively inexpensive; rapid; sensitivity similar to or slightly lower than that of thick films (c.0.001% parasitaemia) Detects only Plasmodium falciparum; remains positive for weeks after infectionf; does not quantitate P. falciparum parasitaemia. Increasing reports of PfHRP2 gene deltions in
P. falciparum, causing a false negative test result Microtube concentration methods with acridine orange staining Blood is collected in a specialized tube containing acridine orange, anticoagulant, and a float. After centrifugation, which concentrates the parasitized cells around the float, fluorescence microscopy is performed. Sensitivity similar or superior to that of thick films (c.0.001% parasitaemia); ideal for processing large numbers of samples rapidly Does not speciate or quantitate; requires fluorescence microscopy LDH, lactate dehydrogenase; PfHRP2, P. falciparum histidine-​rich protein 2; RBCs, red blood cells; WBCs, white blood cells. a Malaria cannot be diagnosed clinically with accuracy, but treatment should be started on clinical grounds if laboratory confirmation is likely to be delayed. In areas where malaria transmission is high, low-​level asymptomatic parasitaemia is common in otherwise healthy people. Thus finding a positive test for malaria does not necessarily mean it is the cause of a fever, although in this context the presence of >10 000 parasites/​µl (c.0.2% parasitaemia) does indicate that malaria is the likely cause of illness. Antibody and polymerase chain reaction tests have no role in the diagnosis of malaria except that PCR is increasingly used for genotyping and speciation in mixed infections, and for detection of low-​density parasitaemias in asymptomatic residents of endemic areas. b. Clean blood slides and filtered fresh stains should be used. Asexual parasites/​200 WBCs × 40 = parasite count/​µl (assumes a WBC count of 8000/​µl). c P. falciparum gametocytaemia may persist for days or weeks after clearance of asexual parasites. Gametocytaemia without asexual parasitaemia does not indicate active infection. d Clean blood slides and filtered fresh stains should be used. Parasitized RBCs (%) × haematocrit × 1256 = parasite count/​µl. e Parasite densities of >100 000 parasites/​µl (c.2% parasitaemia) are associated with an increased risk of severe malaria, but some patients have severe malaria with lower counts. At any level of parasitaemia, if >50% of parasites are tiny rings (cytoplasm width less than half of nucleus width) carries a relatively good prognosis whereas if there is visible pigment in >20% of parasites or phagocytosed pigment in >5% of polymorphonuclear leukocytes (indicating massive recent schizogony) the prognosis is worse. f Slow clearance of PfHRP2 may result in false positive results in patients who have recently recovered from malaria but fall ill again (common in high-​transmission settings), but can be used to diagnostic advantage in low-​transmission settings when a sick patient has already received antimalarial drugs treatment. A positive PfHRP2 test indicates that the illness is falciparum malaria, even if the blood smear is negative.

section 8  Infectious diseases 1404 detection can be used in epidemiological surveys to identify asymp- tomatic infections to guide elimination initiatives. Malaria antibody measurement using either IFA or ELISA assays may have a role in future epidemiological studies as measures of transmission intensity, but serology has no place in the diagnosis of acute malaria illness. Severe falciparum malaria Appropriately and promptly treated, uncomplicated falciparum mal- aria (i.e. the patient can swallow medicines and food) carries a mor- tality rate of less than 0.1%. However, once vital organ dysfunction occurs or the total proportion of erythrocytes infected increases to more than 2% (a level corresponding to >1012 parasites in an adult), mortality risk rises steeply. The major manifestations of severe falcip- arum malaria are shown in Table 8.8.2.3, and features indicating a poor prognosis are listed in Table 8.8.2.4. Severe malaria is a multiorgan dis- ease; and the organ systems involved vary by age group (Table 8.8.2.5, Fig. 8.8.2.7). Severe anaemia and hypoglycaemia are common mani- festations in small children whereas deep jaundice, renal failure, and pulmonary oedema are more common in adult patients. Coma and metabolic (lactic) acidosis are common in both children and adults, and have the strongest prognostic significance for death. Cerebral malaria Coma is a characteristic and ominous feature of falciparum mal- aria and, despite treatment, is associated with death rates of 15–​20% among adults and 10–​15% among children. Any obtundation, de- lirium, or abnormal behaviour should be taken very seriously. The onset may be gradual or sudden following a convulsion. Cerebral malaria is a diffuse symmetric encephalopathy; focal neurologic signs are unusual (Fig. 8.8.2.8). Some passive resistance to head flexion may be detected but signs of meningeal irritation are absent. The eyes may be divergent. The corneal reflexes are pre- served, except in deep coma. A pout reflex is common, but other primitive reflexes are usually absent. Muscle tone can be increased or decreased. The tendon reflexes are variable, and the plantar re- flexes can be flexor or extensor; the abdominal and cremasteric reflexes are absent. Flexor or extensor posturing might occur. On funduscopy, c.15% of patients have retinal haemorrhages, but with pupillary dilatation and indirect ophthalmoscopy the preva- lence is much higher (30–​40%). Other funduscopic abnormalities (Fig. 8.8.2.9) include retinal whitening due to capillary obstruction, discrete spots of retinal opacification (30–​60%), papilloedema (8% among children, rare among adults), cotton wool spots (<5%), and decolourization of part or all of a retinal vessel of vessel (occasional cases in paediatric cases). Generalized, and often repeated, convulsions occur in c.10% of adults and up to 50% of children with cerebral malaria. More covert seizure activity is common, particularly among children, and may manifest as repetitive tonic-​clonic eye movements or hypersalivation. Adults rarely suffer neurologic sequelae (in <3% of cases), but c.10% of children surviving cerebral malaria—​ especially those with hypoglycaemia, severe anaemia, repeated Table 8.8.2.3  Manifestations of severe falciparum malaria Signs Manifestations Major Unarousable coma/​cerebral malaria Failure to localize or respond appropriately to noxious stimuli; coma persisting for >30 min after generalized convulsion. Adults: Glasgow Coma scale <11, young children: Blantyre coma scale <3 Acidaemia/​acidosis Arterial pH of <7.25, plasma bicarbonate of <15 mmol/​litre or venous lactate >5 mmol/​litre; manifests clinically as laboured deep breathing, often termed ‘respiratory distress’ Severe normochromic, normocytic anaemia Haematocrit of <15% or haemoglobin <50 g/​litre (<5 g/​dl) with parasitaemia >10 000/​μl Renal failure Serum or plasma creatinine level of >265 μmol/​litre (>3 mg/​dl) in the adult patienta. Urine output (24 h) of <400 ml in adults or <12 ml/​kg in children; nonoliguric renal failure is also common;
no improvement with rehydration Pulmonary oedema/​adult respiratory distress syndrome Non​cardiogenic pulmonary oedema, often aggravated by overhydration Hypoglycaemia Plasma glucose <2.2 mmol/​litre (<40 mg/​dl) Hypotension/​shock Systolic blood pressure of <50 mmHg in children 1–​5 years or <80 mmHg in adults; core/​skin temperature difference of >10°C; capillary refill >2 s Bleeding/​disseminated intravascular coagulation Significant bleeding and haemorrhage from the gums, nose, and gastrointestinal tract and/​or evidence of disseminated intravascular coagulation Other manifestations Convulsions More than two generalized seizures in 24 h; signs of continued seizure activity sometimes subtle (e.g. tonic-​clonic eye movements without limb or face movement) Extreme weakness Prostration; inability to sit unaidedb Hyperparasitaemia Parasitaemia level of >5% in non​immune patients (>10% in any patient) Jaundice Serum bilirubin level of >50 mmol/​litre (>3 mg/​dl) with a parasite density of 100 000/​ul or other evidence of vital organ dysfunction a Normal range is lower in children so the threshold for diagnosing kidney injury in paediatric patients should be correspondingly lower. b In a child who is normally able to sit.

8.8.2  Malaria 1405 seizures, and deep coma—​have an evident residual neurologic deficit when they regain consciousness; hemiplegia, cerebral palsy, cortical blindness, deafness, and impaired cognition may all occur. Most of these neurological deficits improve significantly or resolve completely within 6 months. Meanwhile the prevalence of other deficits increases; approximately 10% of children surviving cere- bral malaria have a persistent language deficit. There may also be deficits in learning, planning, and executive functions, attention, memory, and non​verbal functioning. The incidence of epilepsy is increased, and the life expectancy of paediatric cerebral malaria survivors is decreased. Hypoglycaemia Hypoglycaemia is an important and common complication of se- vere malaria. It is associated with a poor prognosis and is particu- larly problematic in children and pregnant women. Hypoglycaemia in malaria results from a failure of hepatic gluconeogenesis and an increase in the consumption of glucose mainly by the host. To compound the situation, quinine, which is still widely used for the treatment of both severe and uncomplicated falciparum malaria, is a powerful stimulant of pancreatic insulin secretion. Hyperinsulinaemic hypoglycaemia is especially troublesome in pregnant women receiving quinine treatment. In severe disease, the clinical diagnosis of hypoglycaemia is difficult: the usual physical signs (sweating, gooseflesh, tachycardia) can be absent, and the neurologic impairment caused by hypoglycaemia cannot be distin- guished from that caused by cerebral malaria. Acidosis Acidotic breathing, often described as respiratory distress, is a sign of poor prognosis in severe malaria. It is often followed by circu- latory failure unresponsive to volume expansion or inotropic drug treatment, and ultimately by respiratory arrest. Acidosis results from accumulation of organic acids, in particular lactic acid. Lactic acid- osis is caused by the combination of anaerobic glycolysis in tissues where sequestered parasites interfere with microcirculatory flow, lactate production by the parasites, and a failure of hepatic and renal lactate clearance. Hypovolaemia is not a major contributor. Severe hyperlacataemia commonly coexists with hypoglycaemia. In adults acidosis is often compounded by coexisting renal dysfunction; in children, ketoacidosis may also contribute. Other still-​unidentified organic acids are important contributors to acidosis. The plasma concentrations of bicarbonate or lactate or the ‘base deficit’ are the Table 8.8.2.4  Clinical and laboratory features indicating a poor prognosis in severe falciparum malaria Clinical Haematology Biochemistry Parasitology Deep coma Severe anaemia (PCV <15%) Hyperlactataemia (>5 mmol/​litre) Increased mortality is associated with: parasitaemia >100 000/​µl Marked agitation Leukocytosis (>12 000/​μl) Acidosis (arterial pH <7.3, serum HCO3 <15 mmol/​litre) High mortality at >500 000/​µl Repeated convulsions Severe thrombocytopenia (<50 000/​ul) Hypoglycaemia (<2.2 mmol/​litre)

20% of parasites identified as pigment-​containing trophozoites and schizonts Laboured hyperventilation (respiratory distress) Prolonged prothrombin
time (>3 s) Elevated serum creatinine (>265 μmol/​litre) 5% of neutrophils with visible pigment Hypothermia (<36.5°C) Prolonged partial thromboplastin time Elevated urate (>600 μmol/​litre) High plasma PfHRP2 Shock Decreased fibrinogen (<200 mg/​dl) Elevated transaminases (AST/​ALT 3 times upper limit of normal) Bleeding Elevated total bilirubin (>50 μmol/​litre) Anuria Elevated muscle enzymes (CPK ↑, myoglobin ↑) PCV, packed cell volume; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CPK, creatine phosphokinase. Table 8.8.2.5  Relative incidence of severe complications of falciparum malaria Complication Non​pregnant adults Pregnant women Children Anaemia

++ +++ Convulsions + + +++ Hypoglycaemia + +++ +++ Jaundice +++ +++ + Renal failure +++ +++ − Pulmonary oedema ++ +++ + Note: −, rare; +, infrequent; ++, frequent; +++, very frequent. 60% 40% 20% 0% 0 5 10 20 30 40 60 50 Age (years) Proportion of patients Jaundice Renal failure Anaemia Shock Acidosis Convulsions Hypoglycaemia Coma Pulmonary oedema ? Fig. 8.8.2.7  The different manifestations of severe falciparum malaria, according to patient age. Reprinted from The Lancet, Vol. 383, White NJ et al., Malaria, pages 723–​735, Copyright © 2014, with permission from Elsevier.

section 8  Infectious diseases 1406 best biochemical prognosticators in severe malaria. The prognosis of severe acidosis is poor. Non​cardiogenic pulmonary oedema Adults with severe falciparum malaria might develop non​cardiogenic pulmonary oedema even after several days of antimalarial therapy. The pathogenesis of this form of the acute respiratory distress syn- drome is unclear. Although it is well recognized in adult patients, pulmonary oedema might be underrecognized in paediatric severe malaria. The mortality rate is more than 80% and higher in the absence of positive pressure mechanical ventilation. Malaria non-​ cardiogenic pulmonary oedema can be aggravated by overly vig- orous administration of IV fluid. Pulmonary oedema can also develop in otherwise uncomplicated vivax malaria, where the prog- nosis is substantially better. Renal impairment Acute kidney injury is common in severe falciparum malaria, but oliguric renal failure is rare among children. The patho- genesis of malaria renal failure is unclear but may be related to erythrocyte sequestration and agglutination interfering with renal microcirculatory flow and oxidative damage by free haem. Clinically and pathologically, this syndrome manifests as acute tubular ne- crosis. Renal cortical necrosis never develops. Acute renal failure might occur simultaneously with other vital organ dysfunction (in which case the mortality is high) or may progress as other disease manifestations resolve. In survivors, urine flow resumes in a median of 4 days, and serum creatinine levels return to normal in a mean of 17 days. Early dialysis or hemofiltration considerably enhances the likelihood of survival. Haematologic abnormalities Anaemia results from accelerated red cell removal by the spleen, ob- ligatory erythrocyte destruction at parasite schizogony, and in re- peated infections from ineffective erythropoiesis. In severe malaria, both infected and uninfected red cells have reduced deformability, which correlates with prognosis and the development of anaemia. The survival of all red cells is shortened in severe malaria. In pa- tients with little or no pre-​existing immunity, anaemia can develop rapidly, and transfusion is often required. Some patients have such severe haemolysis that haemoglobinuria results (blackwater fever). In many areas of Africa and on the island of New Guinea children have repeated malarial infections and commonly develop severe an- aemia as a result of both shortened survival of uninfected red cells and marked dyserythropoiesis. Anaemia is a common consequence of antimalarial drug resistance, which results in repeated or con- tinued infection. Moderate coagulation abnormalities are common in falciparum malaria, and mild thrombocytopenia is usual (a normal platelet count should question the diagnosis of malaria). Profound thrombo- cytopenia can occur in severe malaria (<20 000/​µl). In adult patients with severe malaria, less than 5% have significant bleeding with evidence of disseminated intravascular coagulation. Hematemesis from stress ulceration or acute gastric erosions might also occur. Liver dysfunction Mild haemolytic jaundice from unconjugated bilirubin is common in malaria. Severe jaundice is associated with P. falciparum infections and is more common in adults than among children. It results from haemolysis, hepatocyte injury, and cholestasis. When accompanied by other vital organ dysfunction (often renal impairment), liver dys- function carries a poor prognosis. Hepatic dysfunction contributes to hypoglycaemia, lactic acidosis, and impaired drug metabolism. Fig. 8.8.2.8  Examples of patients with cerebral malaria. Left: adult Asian patient with unrousable coma, without signs of lateralization. Middle: African child with cerebral malaria and decerebrate posturing. Right: African child with cerebral malaria and decorticate posturing. (a) (b) Fig. 8.8.2.9  Malaria retinopathy. Left panel: indirect ophthalmoscopy picture showing small haemorrages, and peripheral retinal whitening. These areas correspond to the filling defects in the fluorescent angiogram shown in the right panel. Reprinted from The Lancet Infectious Diseases, Vol. 10, Glover SJ et al., Malarial retinopathy and fluorescein angiography findings in a Malawian child with cerebral malaria, page 440, Copyright © 2010, with permission from Elsevier.

8.8.2  Malaria 1407 Occasional patients with falciparum malaria may develop deep jaundice (with haemolytic, hepatic, and cholestatic components) without evidence of other vital organ dysfunction, in which case the prognosis is good. Other complications Septicaemia may complicate severe malaria, particularly in chil- dren. Up to 20% have concomitant bacteraemia. Differentiating severe malaria from sepsis with incidental parasitaemia in child- hood is very difficult. In endemic areas, non​typhoid Salmonella and Strep. pneumoniae bacteraemia, have been associated specifically with P. falciparum infections. Chest infections and catheter-​induced urinary tract infections are common among hospitalized patients who are unconscious for more than 3 days. Aspiration pneumonia may follow generalized convulsions. HIV/​AIDS and malnutrition predispose to more severe malaria. Malaria anaemia is worsened by concurrent infections with intestinal helminths (hookworm in particular). Some residents of malaria endemic areas in tropical Africa and Asia exhibit an abnormal immunologic response to repeated infec- tions that is characterized by massive splenomegaly (tropical spleno- megaly or hyperreactive malarial splenomegaly), hepatomegaly, pancytopenia with anaemia, marked elevations in serum IgM and malarial antibody, hepatic sinusoidal lymphocytosis, and (in Africa) peripheral B cell lymphocytosis. Malarial parasites can be absent in the peripheral blood smear. Patients should receive antimalarial chemoprophylaxis, which usually results in reversal of splenomegaly and the haematological abnormalities. Chronic or repeated infections with P.  malariae can cause an immune-​complex membranoproliferative glomerulonephritis, re- sulting in nephrotic syndrome. This quartan nephropathy usually responds poorly to treatment with either antimalarial agents or cor- ticosteroids and cytotoxic drugs. Malaria-​related immune dysregulation may provoke infection with lymphoma viruses. Burkitt’s lymphoma is strongly associated with the Epstein–​Barr virus. The prevalence of this childhood tu- mour is high in malarious areas of Africa. Laboratory findings in severe malaria The laboratory abnormalities in severe malaria reflect multiple vital organ dysfunction. Metabolic acidosis, with low plasma concentra- tions of glucose, sodium, bicarbonate, calcium, phosphate, and al- bumin together with elevations in plasma lactate, urea, creatinine, urate, muscle, and liver enzymes, and conjugated and unconjugated bilirubin may all occur. Normochromic, normocytic anaemia is usual. Typically, the leukocyte count is normal, although it may be raised in very severe infections. There is slight monocytosis, lymphopenia, and eosinopenia, followed by reactive lymphocytosis and eosinophilia in the weeks after the acute infection. The erythro- cyte sedimentation rate, plasma viscosity, and levels of C-​reactive protein and other acute-​phase proteins are all high. The platelet count is usually reduced to c.105/​µl, but can be lower. Severe in- fections may be accompanied by prolonged prothrombin and par- tial thromboplastin times and by more severe thrombocytopenia. Antithrombin III is reduced even in mild infections. In uncompli- cated malaria, plasma concentrations of electrolytes, urea, and cre- atinine are usually normal. Hypergammaglobulinaemia is usual in immune and semi-​immune subjects. Urinalysis is generally normal. In both adults and children with cerebral malaria, the mean opening pressure at lumbar puncture is c.160 mm of cerebrospinal fluid. The cerebrospinal fluid is usually normal or has a slightly elevated total protein level (<100 mg/​dl) and cell count (<20/​µl). Malaria in children Most of the 438 000 malaria deaths each year (2015 estimate) are in young African children. Convulsions, coma, hypoglycaemia, meta- bolic acidosis, and severe anaemia are relatively common among children with severe malaria, whereas deep jaundice, oliguric acute renal failure, and acute pulmonary oedema are unusual. In areas of high malaria transmission severe anaemia is the usual presentation of life-​threatening falciparum or vivax malaria. Severely anaemic children may present with deep laboured breathing, previously at- tributed incorrectly to ‘anaemic congestive cardiac failure’ but in fact is usually a manifestation of metabolic acidosis. In general, children tolerate antimalarial drugs well and respond more rapidly to treat- ment than adults. Malaria in pregnancy Malaria in early pregnancy causes abortion. In areas of high mal- aria transmission, falciparum malaria in primi-​ and secundigravid women is associated with low birth weight (average reduction, c.170 g) and consequently increased infant mortality. In these areas infected mothers remain asymptomatic despite intense accumula- tion of parasitized erythrocytes in the placental microcirculation. Maternal HIV infection predisposes pregnant women to more fre- quent and higher density malaria infections, exacerbates the reduc- tion in birth weight associated with malaria, and predisposes their newborns to congenital malarial infection. In areas with low transmission of malaria, pregnant women are prone to severe falciparum malaria. They are particularly vulnerable to high parasitaemias with anaemia, hypoglycaemia, and acute pul- monary oedema. Fetal distress, fetal death, premature labour, and stillbirth or low birth weight are common consequences. Congenital falciparum malaria occurs in less than 5% of newborns whose mothers are infected particularly if they have high parasite densities. Congenital malaria often self-​terminates. P. vivax malaria in preg- nancy is also associated with a reduction in birth weight (average, 110 g), but, in contrast to falciparum malaria, low birthweight is more pronounced in multigravid than in primigravid women. About 350 000 women die in childbirth each year. Most deaths occur in low-​income countries with malaria-​induced anaemia, a major risk factor for maternal death from haemorrhage at childbirth. Transfusion malaria Malaria can be transmitted by blood transfusion, needle-​stick in- jury, sharing of needles by infected injection drug users, or organ transplantation. The incubation period in these settings is often short because there is no pre-​erythrocytic stage of development. The clinical features and management of these cases are the same as for naturally acquired infections. Radical chemotherapy with

section 8  Infectious diseases 1408 primaquine is unnecessary for transfusion-​transmitted P. vivax and P. ovale infections. Treatment of malaria When a patient in or from a malaria endemic area presents with fever or a history of fever, thick and thin blood smears should be prepared and examined immediately, or a rapid test performed. Repeated blood smears should be performed if the first smears are negative and malaria is strongly suspected. Patients with severe mal- aria or those unable to take oral medicines reliably should receive prompt parenteral antimalarial therapy. If there is any doubt about antimalarial drug resistance, the infection should be considered re- sistant. In all endemic areas, the World Health Organization (WHO) now recommends artemisinin-​based combinations (ACTs) as first-​ line treatment for uncomplicated falciparum malaria. They are well tolerated and are rapidly and reliably effective. ACTs are also highly effective for the other malaria species and can therefore be used as first-​line treatment for all malarias. ACTs are sometimes unavailable in temperate countries, where treatment recommenda- tions are limited by the registered available drugs. Chloroquine re- mains effective for the non-​falciparum malarias (P. vivax, P. ovale, P. malariae, P. knowlesi) except in Indonesia and Papua New Guinea, although resistance is increasing. Fake or substandard antimal- arials are commonly sold in many Asian and African countries. Thus, careful attention is required at the time of purchase and later, especially if the patient fails to respond as expected. Treatment of severe malaria Severe falciparum malaria is a medical emergency requiring inten- sive nursing care and careful management. The patient should be weighed if possible and, if comatose, placed on his or her side in the recovery position with frequent turning to avoid aspiration pneu- monia and decubitus pressure sores. Antimalarial treatment should be started immediately. Antimalarial treatment (Table 8.8.2.6). In the largest randomized controlled trials conducted in severe malaria, parenteral artesunate, a water-​soluble artemisinin derivative, was compared with quinine—​ the previous treatment of choice. Artesunate reduced mortality rates in adults by 35% and by 22.5% in African children. Artesunate has, therefore, become the drug of first choice for all patients with se- vere malaria everywhere. Artesunate is usually given by IV injection but it can also be given by IM injection. Artemether and the closely related drug artemotil (β-​arteether) are oil-​based formulations of artemisinin derivatives given by IM injection. They are erratically absorbed and do not confer the same survival benefit as artesunate, but they are probably more effective than quinine. If artemisinin de- rivatives are not immediately available, then treatment should start with quinine until they can be obtained. If none of these are avail- able, then the antiarrhythmic quinidine gluconate is as effective as quinine, but it is significantly more cardiotoxic and so requires close monitoring for dysrhythmias and hypotension. A rectal formulation of artesunate has been developed as a community-​based prereferral treatment for patients in the rural tropics who cannot take oral medications. Prereferral administration of rectal artesunate has been shown to decrease the mortality of severely ill children without access to immediate parenteral treatment. Treatment of complications (Table 8.8.2.7). Frequent evaluation of the patient’s condition is essential. Many adjunctive treat- ments including high-​dose corticosteroids, urea, heparin, dextran, desferrioxamine, antibody to tumour necrosis factor, high-​dose phenobarbital, mannitol, or large volume fluid or albumin bo- luses have proved either ineffective or harmful in clinical trials and should not be used. In acute renal failure or severe metabolic acid- osis, haemofiltration, or haemodialysis should be started as early as possible. Exchange transfusion might be considered for severely ill patients, although recent retrospective studies have shown no additional benefit. The rapid clearance of peripheral blood parasit- aemia achieved by exchange transfusion is also achieved by the rapid administration of parenteral artesunate. Convulsions should be treated promptly with intravenous (or rectal) benzodiazepines. The role of prophylactic anticonvulsants in children is uncertain. If re- spiratory support is not available, then a full loading dose of pheno- barbital (20 mg/​kg) to prevent convulsions should not be given as it has shown to increase mortality, presumable by causing respira- tory arrest. When the patient is unconscious, the blood glucose level should be measured every 4–​6 h, or at any time if the level of con- sciousness falls. All patients should receive a continuous infusion of dextrose, and blood concentrations ideally should be maintained above 4 mmol/​litre. Hypoglycaemia (<2.2 mmol/​litre or 40 mg/​dl) should be treated immediately with bolus IV glucose. The para- site count and haematocrit level should be measured every 6–​12 h. Anaemia develops rapidly in severe malaria; if the haematocrit falls to less than 20%, then whole blood (preferably fresh) or packed cells should be transfused slowly, with careful attention to circula- tory status. In endemic areas where there is limited availability of safe blood the transfusion threshold is often a haematocrit of 15%. Renal function should be checked daily. Children presenting with severe anaemia and acidotic breathing require immediate blood transfusion. Accurate assessment is vital. Management of fluid balance is difficult in severe malaria, because of the thin dividing line between overhydration (leading to pulmonary oedema) and underhydration (contributing to renal impairment). Except in hypotensive shock, fluid therapy should be restricted (e.g. 2 to 4 ml/​ kg/​hr). Fluid bolus therapy is contraindicated. As soon as the pa- tient can take fluids, a full three-​day course of ACT should be started (except that mefloquine should not be given following cerebral malaria). In areas of high transmission where severe malaria and bacterial septicaemia commonly coexist all patients should also re- ceive broad-​spectrum antibiotics. Treatment of uncomplicated malaria (See Tables 8.8.2.6 and 8.8.2.8). Malaria caused by chloroquine-​sensitive P.  vivax, P.  knowlesi, P. malariae, and P. ovale can be treated by oral chloroquine (total dose, 25 mg base/​kg divided over three days), or a three day course of an ACT (except for artesunate-​sulfadoxine-​pyrimethamine, P.  vivax is often resistant to antifolates). Uncomplicated falcip- arum malaria should also be treated with a three-​day course of an ACT. The ACTs are safe and effective in adults, children, and pregnant women. There is increasing evidence that they are also safe in the first trimester of pregnancy. The rapidly eliminated artemisinin component of ACT is usually an artemisinin deriva- tive (artesunate, artemether, or dihydroartemisinin) given for 3 days, and the partner drug is usually a more slowly eliminated

8.8.2  Malaria 1409 Table 8.8.2.6  Treatment of malaria Indication Regimen(s) Uncomplicated Malaria Known chloroquine-​sensitive strains of Plasmodium vivax, P. malariae, P. ovale, P. knowlesi, P. falciparuma Chloroquine (10 mg of base/​kg stat followed by 5 mg/​kg at 12, 24, and 36 h or by 10 mg/​kg at 24 h and 5 mg/​kg at 48 h) or Any ACT (except artesunate-​sulfadoxine-​pyrimethamine where there is resistance) Radical treatment for P. vivax or P. ovale infection (to prevent relapse) In addition to chloroquine or an ACT give primaquine once daily for 14 days; in SE Asia and Oceania, 0.5 mg of base/​kg/​day elsewhere 0.25 mg/​kg/​day In mild G6PD deficiency, 0.75 mg of base/​kg should be given once weekly for 8 weeks Primaquine should not be given in severe G6PD deficiency Sensitive P. falciparum malariab Artesunatec (4 mg/​kg once daily for 3 days) plus sulfadoxine (25 mg/​kg)/​pyrimethamine (1.25 mg/​kg) as a single dose or Artesunatec (4 mg/​kg once daily for 3 days) plus amodiaquine (10 mg of base/​kg once daily for 3 days)d Multidrug-​resistant P. falciparum malaria Either artemether-​lumefantrinec (1.5/​9 mg/​kg bid for 3 days with food) or Artesunatec (4 mg/​kg once daily for 3 days) plus Mefloquine (24–​25 mg of base/​kg—​either 8 mg/​kg once daily for 3 days or 15 mg/​kg on day 2 and then 10 mg/​kg on day 3)d or Dihydroartemisinin-​piperaquinec (2.5/​20 mg/​kg once daily for 3 days) Second-​line treatment/​ treatment of imported malaria Quinine (10 mg of salt/​kg tid for 7 days) plus 1 of the following 3:

  1. Tetracyclinee (4 mg/​kg qid for 7 days)
  2. Doxycyclinee (3 mg/​kg once daily for 7 days)
  3. Clindamycin (10 mg/​kg bid for 7 days) or Atovaquone–​proguanil (20/​8 mg/​kg once daily for 3 days with food) In of low malaria transmission, a single dose of primaquine 0.25 mg base/​kg should be added to all falciparum malaria treatments to prevent transmission, except in pregnant women and infants. This is considered safe even in G6PD deficiency. Severe falciparum malariaf Artesunatec (2.4 mg/​kg stat IV followed by 2.4 mg/​kg at 12 and 24 h and then daily if necessary)g or, if unavailable, Artemetherc (3.2 mg/​kg stat IM followed by 1.6 mg/​kg once daily) or, if unavailable Quinine dihydrochloride (20 mg of salt/​kgh infused over 4 h, followed by 10 mg of salt/​kg infused over 2–​8 h q8hi) or, if unavailable Quinidine (10 mg of base/​kgh infused over 1–​2 h, followed by 1.2 mg of base/​kg per houri with electrocardiographic monitoring) ACT, artemisinin combination therapy; G6PD, glucose-​6-​phosphate dehydrogenase. See WHO guidelines for the treatment of malaria for full details: http://​apps.who.int/​iris/​bitstream/​10665/​162441/​1/​9789241549127_​eng.pdf a Chloroquine-​sensitive P. falciparum malaria is now found only in Central America and Haiti. b In areas where the longer acting partner drug to artesunate is known to be effective. c Artemisinin derivatives are not readily available in some temperate countries. d Fixed-​dose coformulated combinations are available. The World Health Organization now recommends artemisinin combination regimens as first-​line therapy for falciparum malaria in all tropical countries and advocates use of fixed-​dose combinations. e Tetracycline and doxycycline should not be given to pregnant women or to children <8 years of age. f Oral treatment should be substituted as soon as the patient recovers sufficiently to take fluids by mouth. A full course of ACT should be given, except that mefloquine should not be given following cerebral malaria. g Artesunate is the drug of choice when available. The data from large studies in Southeast Asia showed a 35% lower mortality rate than with quinine, and very large studies in Africa showed a 22.5% reduction in mortality rate compared with quinine. Children weighting <20 kg should receive a higher dose of artesunate of 3mg/kg per dose, to ensure equivalent exposure to the drug. h A loading dose should not be given if therapeutic doses of quinine or quinidine have definitely been administered in the previous 24 h. Some authorities recommend a lower dose of quinidine. i Infusions can be given in 0.9% saline and 5–​10% dextrose in water. Infusion rates for quinine and quinidine should be carefully controlled.

section 8  Infectious diseases 1410 Table 8.8.2.7  Management of complications of severe malaria Hypoglycaemia. An initial slow intravenous injection of 20% dextrose (0.5 g/​kg) should be followed by an infusion of 10% dextrose (0.10 g/​kg per hour). The blood glucose level should be checked regularly as recurrent hypoglycaemia is common, particularly among patients receiving quinine or quinidine. In severely ill patients, hypoglycaemia commonly occurs together with metabolic (lactic) acidosis and carries a poor prognosis. Acute renal failure. If the plasma concentrations of urea or creatinine rise despite adequate rehydration, fluid administration should be restricted to prevent volume overload. Renal replacement therapy is best performed early. Haemofiltration and haemodialysis are more effective than peritoneal dialysis and are associated with lower mortality. Some patients with renal impairment do pass small volumes of urine sufficient to allow control of fluid balance; these cases can be managed conservatively if other indications for dialysis do not arise. Renal function usually improves within days, but full recovery may take weeks. Acute pulmonary oedema. Patients should be positioned with the head of the bed at a 45° elevation and given oxygen and IV diuretics. Positive pressure ventilation should be started early if the immediate measures fail. Other complications. Patients who develop spontaneous bleeding should be given fresh blood and IV vitamin K. Convulsions should be treated with IV or rectal benzodiazepines and, if necessary, respiratory support. Aspiration pneumonia should be suspected in any unconscious patient with convulsions, particularly with persistent hyperventilation; IV antimicrobial agents and oxygen should be administered, and the airway secured. Hypoglycaemia or Gram-​negative septicaemia should be suspected when the condition of any patient suddenly deteriorates during antimalarial treatment. In malaria endemic areas where a high proportion of children are parasitaemic, it is impossible to distinguish severe malaria from bacterial sepsis with confidence. In addition, severe malaria is often (ca 20%) accompanied by bacteraemia. Therefore, all children with severe malaria should be treated with both antimalarials and broad-​spectrum antibiotics from the outset. Because non​typhoidal Salmonella infections and Streptococcus pneumonia are particularly common, empirical antibiotics should be selected to cover these organisms. Antibiotics should be considered for severely ill patients of any age who are not responding to antimalarial treatment. Table 8.8.2.8  Properties of antimalarial drugs Drug(s) Pharmacokinetic properties Antimalarial activity Minor toxicity Major toxicity Quinine (and quinidine) Good oral and IM absorption (quinine); Cl and Vd reduced, but plasma protein binding (principally to ∝1 acid glycoprotein) increased (90%) in malaria; quinine mean t1/​2: 18h in severe malaria 16 h in uncomplicated malaria, 11 h in healthy persons; quinidine t1/​2:
13 h in malaria, 8 h in healthy persons Acts mainly on trophozoite blood stage; kills gametocytes of P. vivax, P. ovale, and P. malariae (but not P. falciparum); no action on liver stage Common: ‘Cinchonism’: tinnitus, high-​tone hearing loss, nausea, vomiting, dysphoria, postural hypotension; ECG QTc interval prolongation (quinine usually by <10% but quinidine by up to 25%). Rare: Diarrhoea, visual disturbance, rashes Note: Very bitter taste Common: Hypoglycaemia Rare: Hypotension, blindness, deafness, cardiac arrhythmias, thrombocytopenia, haemolysis, haemolytic-​uremic syndrome, vasculitis, cholestatic hepatitis, neuromuscular paralysis Note: Quinidine substantially more cardiotoxic Chloroquine Good oral absorption, very rapid IM and SC absorption; complex pharmacokinetics; enormous Cl and Vd (unaffected by malaria); blood concentration profile determined by distribution processes in malaria; t1/​2: 1–​2 months As for quinine but acts slightly earlier in asexual cycle Common: Nausea, dysphoria, pruritus in dark-​skinned patients, postural hypotension Rare: Accommodation difficulties, keratopathy, rash Note: Bitter taste, well tolerated Rare: Hypotensive shock (parenteral), cardiac arrhythmias, neuropsychiatric reactions Chronic: Retinopathy (cumulative dose, >100 g), skeletal and cardiac myopathy Piperaquine Adequate oral absorption, may be enhanced by fats; similar pharmacokinetics to chloroquine; t1/​2: 21–​28 days As for chloroquine Epigastric pain, diarrhoea, ECG QTC prolongation None identified Amodiaquine Good oral absorption; largely converted to active metabolite desethylamodiaquine As for chloroquine Nausea (tastes better than chloroquine) Agranulocytosis (rare); hepatitis, mainly with prophylactic use; should not be used with efavirenz Primaquine Complete oral absorption; active metabolite not known; t1/​2: 5–​7 h Radical cure; eradicates hepatic forms of P. vivax and P. ovale; kills all stages of gametocyte development of P. falciparum Nausea, vomiting, diarrhoea, abdominal pain, hemolysis, methaemoglobinemia Massive hemolysis in subjects with severe G6PD deficiency Mefloquine Adequate oral absorption; no parenteral preparation; t1/​2:
14–​20 days (shorter in malaria) As for quinine Nausea, giddiness, dysphoria, fuzzy thinking, sleeplessness, nightmares, sense of dissociation Neuropsychiatric reactions, convulsions, encephalopathy Lumefantrine Highly variable absorption related to fat intake; t1/​2: 3–​4 days As for quinine Nausea, giddiness, dysphoria, fuzzy thinking, sleeplessness, nightmares, sense of dissociation Neuropsychiatric reactions, convulsions, encephalopathy Pyronaridine t1/​2: 10 days children, 13 days adults As for quinine Nausea, vomiting, abdominal pain, diarrhoea, headache Hepatotoxicity (continued)

8.8.2  Malaria 1411 antimalarial to which P.  falciparum is sensitive. Five ACT re- gimens are currently recommended by the WHO. In areas with multidrug-​resistant falciparum malaria (parts of Asia and South America), artemether–​lumefantrine, artesunate-​mefloquine, or dihydroartemisinin-​piperaquine should be used; these regimens provide cure rates of more than 90% except in Thailand and Eastern Myanmar where there is increasing resistance to mefloquine, and in Cambodia and adjacent Vietnam where there is resist- ance to piperaquine. In areas with sensitive parasites, these ACTs and also artesunate-​sulfadoxine-​pyrimethamine, or artesunate-​ amodiaquine may also be used. Artesunate-​pyronaridine has been registered in a limited number of countries and appears a safe and effective alternative. Atovaquone–​proguanil is also effective everywhere, although it is seldom used in endemic areas because of its high cost and the propensity for high-​level atovaquone re- sistance. Of great concern is the emergence of artemisinin re- sistance in P.  falciparum in the Greater Mekong subregion. Artemisinin resistant P. falciparum is now found from the coast of Vietnam to the Myanmar–​India border. Infections with arte- misinin resistant parasites are cleared slowly from the blood, with parasite clearance times which typically exceed 3 days, and cure rates with ACTs are reduced. High ACT failure rates with DHA-​ piperaquine have been reported in Cambodia and with artesunate-​ mefloquine on the Thai-​Myanmar border. Elsewhere these ACTs, and artemether–​lumefantrine can be relied upon. Amodiaquine and sulfadoxine-​pyrimethamine-​resistance compromises the use of ACTs containing these antimalarials in several endemic areas. In low-​transmission settings for the treatment of falcip- arum malaria a single dose of primaquine (0.25 mg/​kg) should be added to the ACT as a gametocytocide to reduce transmission. Primaquine should not be given to young infants (<6 months) or to pregnant women. The 3-​day ACT regimens are all generally well tolerated. Mefloquine is associated with increased rates of vomiting and minor central nervous system reactions (nausea, dizziness, dysphoria, sleep dis- turbances) are common. The incidence of serious adverse neuro- psychiatric reactions to mefloquine treatment is c.1 in 1000 in Asia but may be as high as 1 in 200 among Africans and Caucasians. All the antimalarial quinolines (chloroquine, amodiaquine, mef- loquine, and quinine) exacerbate the orthostatic hypotension associated with malaria, and all are tolerated better by children than by adults. Several antimalarials, notably quinidine, quinine, chloroquine, amodiaquine and piperaquine prolong ventricular repolarization (QT prolongation on the electrocardiogram), but they have not been linked with dysrhythmias in the treatment of malaria. If falciparum malaria recrudesces following first-​line ACT therapy, second-​line treatment with a different ACT regimen may be given. An alternative is a 7-​day course of either artesunate or quinine plus tetracycline, doxycycline, or clindamycin. Tetracycline and doxycycline cannot be given to pregnant women or to children less than 8 years of age. Oral quinine is extremely bitter and regu- larly produces cinchonism comprising tinnitus, high-​tone deafness, nausea, vomiting, and dysphoria. Adherence is poor with the re- quired 7-​day regimens of quinine. Patients should be monitored for vomiting for 1 h after the admin- istration of any oral antimalarial drug. If there is vomiting within the first half hour, the full dose should be repeated. Symptom-​based Drug(s) Pharmacokinetic properties Antimalarial activity Minor toxicity Major toxicity Artemisinin and derivatives (artemether, artesunate) Good oral absorption, slow and variable absorption of IM artemether; artesunate and artemether biotransformed to active metabolite dihydroartemisinin; all drugs eliminated very rapidly; t1/​2: <1 h Broader stage specificity and more rapid than other drugs; no action on liver stages; kills all but fully mature gametocytes of P. falciparum Reduction in reticulocyte count (but not anaemia); neutropenia at high doses. Following treatmen t of severe malaria with hyperparasitaemia, delayed anaemia may occur. Rare: Anaphylaxis, urticaria, fever Pyrimethamine Good oral absorption, variable IM absorption; t1/​2: 4 days For blood stages, acts mainly on mature forms; causal prophylactic Well tolerated Megaloblastic anaemia, pancytopenia, pulmonary infiltration Proguanil Good oral absorption; biotransformed to active metabolite cycloguanil; t1/​2: 16 h; biotransformation reduced by oral contraceptive use and in pregnancy Causal prophylactic; not used alone for treatment Well tolerated; mouth ulcers and rare alopecia Megaloblastic anaemia in renal failure Atovaquone Highly variable absorption related to fat intake; t1/​2: 30–​70 h Acts mainly on trophozoite blood stage None identified None identified Tetracycline, doxycyclinea Excellent absorption; t1/​2: 8 h for tetracycline, 18 h for doxycycline Weak antimalarial activity; should not be used alone for treatment Gastrointestinal intolerance, deposition in growing bones and teeth, photosensitivity, candidiasis, benign intracranial hypertension Renal failure in patients with impaired renal function (tetracycline) Cl, systemic clearance; Vd, total apparent volume of distribution. ECG, electrocardiogram; G6PD, glucose-​6-​phosphate dehydrogenase; a Tetracycline and doxycycline should not be given to pregnant women or to children <8 years of age. Table 8.8.2.8  Continued

section 8  Infectious diseases 1412 treatment, with tepid sponging and paracetamol (acetaminophen) administration, lowers fever and may help to prevent vomiting. Pregnant women, young children, patients unable to tolerate oral therapy, and non​immune individuals (e.g. travellers) with sus- pected malaria should be evaluated carefully and hospitalization considered. If there is any doubt as to the identity of the infecting malarial species, treatment for falciparum malaria should be given. Non​immune patients receiving treatment for malaria should have daily parasite counts performed until the thick films are negative. If the parasite density does not fall below 25% of the admission value in 48 h or if parasitaemia has not cleared by 7 days (and adherence is assured), drug resistance is likely, and the regimen should be changed. Radical cure In infections with P. vivax or P. ovale infections primaquine (0.5 mg of base/​kg, adult dose in Southeast Asia and Oceania, 0.25 mg/​kg else- where) should be added to treatment of the blood stage treatment to eradicate persistent liver stages and prevent relapse (radical treat- ment). Primaquine should be given daily for 14 days after labora- tory tests for G6PD deficiency have proved negative. If the patient has a mild variant of G6PD deficiency, primaquine can be given in a dose of 0.75 mg base/​kg (45 mg maximum) once weekly for 8 weeks. Pregnant women or infants less than 6 months with vivax or ovale malaria should not be given primaquine. Pregnant women should receive suppressive prophylaxis with chloroquine (5 mg of base/​kg per week) until delivery, after which radical treatment can be given. Prevention of malaria Malaria may be contained and controlled by: insecticides to kill the mosquito vector; rapid diagnosis, and treatment of symptomatic malaria and in endemic areas, where effective and feasible; admin- istration of intermittent preventive treatments; seasonal malaria chemoprevention: or chemoprophylaxis to high-​risk groups such as pregnant women, young children, and travellers from non​endemic regions. Insecticides are the cornerstone of mosquito control. The most important group are the pyrethroid insecticides which are used to impregnate mosquito nets. Insecticide treated bed-​nets pro- vide protection against malaria for those sleeping under or near the nets in areas where the anopheline vectors bite at night, although increasing pyrethroid resistance threatens their future. Insecticide treated bed-​nets have been shown to reduce mortality in African children by 17%. Their widespread distribution is one of the main reasons for recent reductions in global malaria mortality. Indoor re- sidual spraying (IRS) with insecticides can be highly effective against indoor resting (endophylic) Anopheles species, but sustaining high coverage has proven a challenge. It has proved very difficult to develop an effective malaria vaccine. The RTS,S/​ASO1 P. falciparum malaria vaccine has been registered recently by the European Medicines Agency. This vaccine provides short-​term protection of approxi- mately 30–​50% for one year but declines thereafter. Protection from RTS,S in infants dropped to 16% four years after vaccination. While there is great promise for one or more malaria vaccines on the more distant horizon, prevention and control measures continue to rely on vector control and antimalarial drugs. Worryingly the recent gains in malaria control are threatened by increasing insecticide resistance and behaviour change (to avoid contact with insecticide treated bed-​nets) in anopheline mosquito vectors, and spreading ar- temisinin and ACT partner drug resistance in P. falciparum. Personal protection against malaria Simple measures to reduce the frequency of infected-​mosquito bites in malarious areas are very important. These include the use of insecticide treated bed-​nets, avoidance of exposure to mosqui- toes at their peak feeding times (usually dusk to dawn), suitable (long-​sleeve) clothing, and the use of insect repellents containing 10–​35% diethyltoluamide (DEET) (or, if DEET is unacceptable, 7% picaridin). Chemoprophylaxis (See Table 8.8.2.9.) Recommendations for prophylaxis depend on knowledge of the risks of acquiring malaria and local patterns of antimalarial drug Table 8.8.2.9  Drugs used in the prophylaxis of malaria Drug Usage Adult dose Paediatric dose Comments Atovaquone/​ proguanil Prophylaxis in areas with chloroquine-​ or mefloquine-​resistant Plasmodium falciparum 1 adult tablet POa 5–​8 kg: ½ paediatric tabletb once daily Begin 1–​2 days before travel to malarious areas. Take once daily at the same time each day while in the malarious areas and for 7 days after leaving such areas. Atovaquone–​proguanil is contraindicated in severe renal impairment (creatinine clearance rate <30 ml/​min). In the absence of data, it is not recommended for children weighing <5 kg, pregnant women, or women breastfeeding infants weighing <5 kg. Atovaquone/​ proguanil should be taken with food or a milky drink. ≥8–​10 kg: ¾ paediatric tablet once daily ≥10–​20 kg: 1 paediatric tablet once daily ≥20–​30 kg: 2 paediatric tablets once daily ≥30–​40 kg: 3 paediatric tablets once daily ≥40 kg: 1 adult tablet once daily Chloroquine Prophylaxis only in areas with P. vivax only 300 mg of base (500 mg of phosphate salt) PO once weekly 5 mg/​kg of base (8.3 mg of salt/​kg) PO once weekly, up to maximum adult dose of 300 mg of base Begin 1–​2 weeks before travel to malarious areas. Take weekly on the same day of the week while in the malarious areas and for 4 weeks after leaving such areas. Chloroquine may exacerbate psoriasis. (continued)

8.8.2  Malaria 1413 Drug Usage Adult dose Paediatric dose Comments Doxycycline Prophylaxis in areas with chloroquine-​ or mefloquine-​resistant P. falciparumc 100 mg PO once daily ≥8 years of age: 2 mg/​kg, up to adult dose Begin 1–​2 days before travel to malarious areas.
Take once daily at the same time each day while in the malarious areas and for 4 weeks after leaving such areas. Doxycycline is contraindicated in children <8 years of age and in pregnant women. Mefloquine Prophylaxis in areas with chloroquine-​resistant P. falciparumc 250 mg of base PO once weekly ≤9 kg: 4.6 mg of base/​kg (5 mg of salt/​kg) PO once weekly Begin 1–​2 weeks before travel to malarious areas. Take weekly on the same day of the week while in the malarious areas and for 4 weeks after leaving such areas. Mefloquine is contraindicated in persons allergic to this drug or related compounds (e.g. quinine and quinidine) and in persons with active or recent depression, generalized anxiety disorder, psychosis, schizophrenia, other major psychiatric disorders, or seizures. Use with caution in persons with psychiatric disturbances or a history of depression. 10–​19 kg: ¼ tablet once weekly 20–​30 kg: ½ tablet once weekly 31–​45 kg: ¾ tablet once weekly ≥46 kg: 1 tablet once weekly Primaquine For prevention of malaria in areas with mainly P. vivax 30 mg of base (52.6 mg of salt) PO once daily 0.5 mg of base/​kg (0.8 mg of
salt/​kg) PO once daily, up to adult dose; should be taken with food Begin 1–​2 days before travel to malarious areas. Take once daily at the same time each day while in the malarious areas and for 7 days after leaving such areas. Primaquine prophylaxis is contraindicated in persons with G6PD deficiency. It is also contraindicated during pregnancy and in lactation unless the infant being breast-​fed has a documented normal G6PD level. Primaquine Used for presumptive antirelapse therapy (terminal prophylaxis) to decrease risk of relapses of P. vivax and P. ovale 30 mg of base PO once daily for 14 days after departure from the malarious area 0.5 mg of base/​kg (0.8 mg of
salt/​kg), up to adult dose, PO once daily for 14 days after departure from the malarious area This is indicated for persons who have had prolonged exposure to P. vivax and/​or P. ovale. It is contraindicated in persons with G6PD deficiency as well as during pregnancy and in lactation unless the infant being breast-​fed has a documented normal G6PD level. a An adult tablet contains 250 mg of atovaquone and 100 mg of proguanil hydrochloride. b A paediatric tablet contains 62.5 mg of atovaquone and 25 mg of proguanil hydrochloride. c Very few areas now have chloroquine-​sensitive malaria. Atovaquone–​proguanil (Malarone; 3.75/​1.5 mg/​kg or 250/​100 mg, once daily adult dose) is a fixed-​combination, once daily prophylactic agent that is very well tolerated by adults and children, with fewer adverse gastrointestinal effects than chloroquine-​proguanil and fewer adverse central nervous system effects than mefloquine. It is proguanil itself, rather than the antifolate metabolite cycloguanil, that acts synergistically with atovaquone. This combination is effective against all types of malaria, including multidrug-​resistant falciparum malaria. Atovaquone–​proguanil is best taken with food or a milky drink to optimize absorption. There are insufficient data on the safety of this regimen in pregnancy. Mefloquine (250 mg of salt weekly, adult dose) has been widely used for malarial prophylaxis because it is usually effective against multidrug-​resistant falciparum malaria and is reasonably well tolerated. The drug has been associated with rare episodes of psychosis and seizures at prophylactic doses; these reactions are more frequent at the higher doses used for treatment. More common side effects with prophylactic doses of mefloquine include mild nausea, dizziness, fuzzy thinking, disturbed sleep patterns, vivid dreams, and malaise. The drug is contraindicated for use by travellers with known hypersensitivity to mefloquine or related compounds (e.g. quinine, quinidine) and by persons with active or recent depression, anxiety disorder, psychosis, schizophrenia, another major psychiatric disorder, or seizures; mefloquine is not recommended for persons with cardiac conduction abnormalities although the evidence that it is cardiotoxic is very weak. There increasing confidence in the safety of mefloquine prophylaxis during pregnancy; in studies in Africa, mefloquine prophylaxis was found to be effective and safe during pregnancy. However, in one study from Thailand, treatment of malaria with mefloquine was associated with an increased risk of stillbirth, but this effect was not seen subsequently. Once daily administration of doxycycline (100 mg daily, adult dose) is an effective alternative to atovaquone–​proguanil or mefloquine. Doxycycline is generally well tolerated but may cause vulvovaginal thrush, diarrhoea, and photosensitivity and cannot be used by children <8 years old or by pregnant women. Chloroquine can no longer be relied upon to prevent P. falciparum infections in most areas but is used to prevent and treat malaria due to the other human Plasmodium species and for P. falciparum malaria in Central American countries west and north of the Panama Canal, Caribbean countries, and some countries in the Middle East. Chloroquine-​resistant P. vivax has been reported from parts of eastern Asia, Oceania, and Central and South America. This drug is generally well tolerated, although some patients cannot take it because of malaise, headache, visual symptoms (due to reversible keratopathy), gastrointestinal intolerance, or pruritus. Chloroquine is considered safe in pregnancy. With chronic administration for >5 years, a characteristic dose-​related retinopathy may develop, but this condition is rare at the doses used for antimalarial prophylaxis. Idiosyncratic or allergic reactions are also rare. Skeletal and/​or cardiac myopathy is a potential problem with protracted prophylactic use; they are more likely to occur at the high doses used in the treatment of rheumatoid arthritis. Neuropsychiatric reactions and skin rashes are unusual. When used continuously, amodiaquine, a related aminoquinoline, is associated with a high risk of agranulocytosis
(c.1 person in 2000) and hepatotoxicity (c.1 person in 16 000); thus, this agent should not be used for prophylaxis. Primaquine (once daily adult dose, 0.5 mg of base/​kg or 30 mg taken with food), an 8-​aminoquinoline compound, has proved safe and effective in the prevention of drug-​resistant falciparum and vivax malaria in adults. This drug can be considered for persons who are travelling to areas with or without drug-​resistant P. falciparum and who are intolerant to other recommended drugs. Abdominal pain and oxidant haemolysis—​the principal adverse effects—​are not common as long as the drug is taken with food and is not given to G6PD-​ deficient persons, in whom it can cause haemolysis that is sometimes fatal. Travellers must be tested for G6PD deficiency and be shown to have a level in the normal range before receiving primaquine. Primaquine should not be given to pregnant women or neonates. The 8-​aminoquinolines (primaquine, tafenoquine) given in a single dose with ACT are being considered for widespread use in treatment regimens in malaria elimination programmes because of their gametocytocidal effect on P. falciparum. In the past, the dihydrofolate reductase inhibitors pyrimethamine and proguanil (chloroguanide) were administered widely, but the rapid selection of resistance in both P. falciparum and P. vivax has limited their use. Whereas antimalarial quinolines such as chloroquine (a 4-​aminoquinoline) act on the erythrocyte stage of parasitic development, the dihydrofolate reductase inhibitors also inhibit preerythrocytic growth in the liver (causal prophylaxis) and development in the mosquito (sporontocidal activity). Proguanil is safe and well tolerated, although mouth ulceration occurs in c.8% of persons using this drug; it is considered safe for antimalarial prophylaxis in pregnancy. The prophylactic use of the combination of pyrimethamine and sulfadoxine is not recommended because of an unacceptable incidence of severe toxicity, principally exfoliative dermatitis and other skin rashes, agranulocytosis, hepatitis, and pulmonary eosinophilia (incidence, 1:7000; fatal reactions, 1:18 000). The combination of pyrimethamine with dapsone (0.2/​1.5 mg/​kg weekly; 12.5/​100 mg, adult dose) has been used in some countries. Dapsone may cause methaemoglobinemia and allergic reactions and (at higher doses) may pose a significant risk of agranulocytosis. Proguanil and the pyrimethamine-​dapsone combination are not available in the United States. There is an increasingly appreciated problem of falsified (fake, counterfeit) and substandard antimalarial drugs (and other medicines) on the shelves of pharmacies in Southeast Asia and sub-​Saharan Africa; hence, travellers should purchase their preventive drugs from a reputable source before going to a malarious country. Table 8.8.2.9  Continued

8.8.3 Babesiosis 1414

8.8.3 Babesiosis 1414

section 8  Infectious diseases 1414 sensitivity. When there is uncertainty, drugs effective against re- sistant P.  falciparum should be used (atovaquone–​proguanil, or doxycycline). Chemoprophylaxis is never entirely reliable, and mal- aria should always be considered in the differential diagnosis of fever in patients who have travelled to endemic areas, even if they have been taking prophylactic antimalarial drugs. Pregnant women travelling to malarious areas should be warned about the potential risks. All pregnant women at risk in endemic areas should be encouraged to attend regular antenatal clinics. Mefloquine is the only drug advised for pregnant women travel- ling to areas with drug-​resistant malaria, except for areas with mefloquine-​resistant falciparum malaria in Southeast Asia; this drug is generally considered safe in the second and third trimesters of pregnancy, and the data on first-​trimester exposure, although limited, are reassuring. Chloroquine and proguanil are regarded as safe, but are no longer effective in the prevention of falciparum malaria. The safety of other prophylactic antimalarial agents in pregnancy has not been established. Children born to non​immune mothers in endemic areas (usually expatriates moving to malaria endemic areas) should receive prophylaxis from birth. Travellers should start taking antimalarial drugs 2 days to 2 weeks before departure so that any untoward reactions can be detected and so that therapeutic antimalarial blood concentrations will be present when needed. Antimalarial prophylaxis should continue for 4 weeks after the traveller has left the endemic area, except if atovaquone–​proguanil or primaquine has been taken; these drugs have significant activities against the liver stage of the infection (causal prophylaxis) and can be discontinued 1 week after departure from the endemic area. If suspected malaria develops while a trav- eller is abroad, obtaining a reliable diagnosis and antimalarial treat- ment locally is a top priority. Presumptive self-​treatment for malaria with atovaquone–​proguanil (for three consecutive days) or another drug can be considered under special circumstances; medical advice on self-​treatment should be sought before departure for malarious areas and as soon as possible after illness begins. Every effort should be made to confirm the diagnosis by parasitologic studies. FURTHER READING Dondorp A, et al. (2010). Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): An open-​label randomized trial. Lancet, 376, 1647 (erratum Lancet (2011), 377, 126). Dondorp AM, et al. (2009). Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med, 361, 5. Gomes MS, et al. (2009). Pre-​referral rectal artesunate to prevent death and disability in severe malaria: a placebo-​controlled trial. Lancet, 373, 557. Moore KA, et al. (2016). Safety of artemisinins in first trimester of prospectively followed pregnancies: an observational study. Lancet Infect Dis, 16, 576–​83. Nayyar GML, et  al. (2012). Poor-​quality antimalarial drugs in Southeast Asia and sub-​Saharan Africa. Lancet, 12, 6. White NJ (2013). Primaquine to prevent transmission of falciparum malaria. Lancet Infect Dis, 13, 175–​81. White NJ, et al. (2014). Malaria. Lancet, 383, 723. World Health Organization (WHO) (2013). Policy recommendation: seasonal malaria chemoprevention (SMC) for plasmodium falciparum malaria control in highly seasonal transmission areas of the Sahel sub-​region in Africa. http://​www.who.int/​malaria/​publications/​atoz/​ who_​smc_​policy_​recommendation/​en/​ World Health Organization (WHO) (2014). Severe malaria. Trop Med Int Health, 19 (Suppl 1), 7–​131. World Health Organization (WHO) (2015). Guidelines for the treat- ment of malaria, 3rd edition. http://​apps.who.int/​iris/​bitstream/​ handle/​10665/​162441/​9789241549127_​eng.pdf;jsessionid=E2DC3 90064A9EA2C038A2E509DC6D863?sequence=1 8.8.3  Babesiosis Philippe Brasseur ESSENTIALS Babesia are intraerythrocytic, tick-​transmitted, protozoan para- sites that infect a broad range of wild and domesticated mammals including cattle, horses, dogs, and rodents. Human babesial infec- tion is uncommon, mainly caused by B. microti in North America and B. divergens in Europe, with most infections occurring in asplenic people. Presentation is typically with non​specific ‘viral-​type’ symp- toms. Haemolytic anaemia is a characteristic feature and can be severe, particularly with B.  divergens. Diagnosis is by discovering babesia organisms in Giemsa-​stained blood smears, or detection of its DNA in blood by polymerase chain reaction. Aside from sup- portive care, treatment is usually with combinations of clindamycin and quinine or atovaquone and azithromycin. Mortality ranges from 5 to 40%. Prevention is by use of repellents, removing ticks from the skin, and avoidance of exposure in asplenic and immuno- compromised individuals: there is no vaccine. Epidemiology Although several species of babesia can infect humans, two species, Babesia microti and B. divergens, are responsible for most cases of human babesiosis. In the United States of America, thousands of cases of B. microti infections have been reported since 1988, mostly from the north-​east coast including Nantucket, Martha’s Vineyard, and Block Island. B.  microti is transmitted by Ixodes scapularis
(previously I. dammini) and its reservoir host is the common white-​ footed mouse Peromyscus leucopus. B. duncani, a new species, has been identified in nine patients in Washington State. B.  equi cases have been identified in California and a single fatal case of B. divergens infection in Missouri. The zoonotic Borrelia burgdor­ feri, causing Lyme disease, is also transmitted by I. scapularis and coinfections are documented. The risk of both babesiosis and Lyme disease is highest in June when nymphal I.  scapularis are most abundant. Babesiosis may also be acquired through infected blood transfusion or rarely by transplacental transmission. More than 170 cases of transfusion-​transmitted babesiosis have been reported in

8.8.3  Babesiosis 1415 the United States of America since 1980. Several cases have been reported of infection exported to other countries in visitors from the United States. Since the first description of human babesiosis in Europe in 1957, more than 50 cases have been reported. Most of them were due to B. divergens, a common cattle pathogen transmitted by I. ricinus. France, the United Kingdom, and Ireland account for more than 50% of the cases reported in Europe. Farmers, foresters, campers, and hikers are affected, usually between May and October, the season of activity of I.  ricinus. Most infections occur in asplenic people. Only one autochthonus case of B. microti transfusion-​trans- mitted has been reported in Europe in a German patient with a mye- loid leukaemia, but the risk exists for B. divergens which may survive in packed red blood cells for at least 4 weeks at 4°C. B. venatorum, closely related to but distinct from B. odocoilei that infects white-​tail deer in United States of America has been isolated in three asplenic patients in Europe (Italy, Austria and Germany) and recently in China. Few human babesiosis cases have been observed in Africa, Asia, Australia and South America. Pathogenesis Ticks infected with babesia inoculate parasites while feeding on a vertebrate. Babesia enter red blood cells directly and multiply by budding to form two or four parasites, rarely more, in 8 to 10 h. They are released and invade other erythrocytes. The spleen plays a major role in resistance to babesial infections, especially in the case of B. divergens babesiosis. Clinical features B. microti infection In humans, B.  microti babesiosis is characterized by gradually developing malaise, anorexia, and fatigue with subsequent develop- ment of fever, sweats, and generalized myalgia, starting from 1 to 4 weeks after a tick bite; 95% of those infected have intact spleens. Headache, shaking chills, nausea, depression, and hyperaesthesia are less frequent. Mild hepatomegaly and splenomegaly might be detected and spontaneous splenic rupture can occur. A mild to se- vere haemolytic anaemia, sometimes complicated by acute kidney injury, thrombocytopenia, and normal white blood cell count, is generally present. Lactate dehydrogenase, liver enzymes, and unconjugated bilirubin levels are sometimes increased. Parasites are found in peripheral blood of 1–​20% of patients with intact spleens, but in up to 80% of those who are asplenic. The illness is usually more severe in asplenic and older patients. Complications are more likely in the immunocompromised. Acute illness lasts from 1 to 4 weeks, but weakness and malaise often persist for several months. A low, asymptomatic parasitaemia can persist for several weeks after recovery. Case fatality is about 5%. B. divergens infection In Europe, B. divergens infections are usually more severe than those caused by B. microti, with a case fatality up to 42%. After an incu- bation period of 1 to 3 weeks, there is sudden severe intravascular haemolysis resulting in haemoglobinuria, severe anaemia, and jaundice, associated with non​periodic high fever (40–​41°C), hypo- tension, shaking chills, intense sweats, headache, myalgia, lumbar pain, vomiting, and diarrhoea. Peripheral blood B. divergens para- sitaemia varies from 5 to 80%. Patients rapidly develop renal failure, which can be associated with pulmonary oedema, coma, and death. Infection with B. venatorum is usually mild or moderate, even in asplenic patients. Diagnosis Babesiosis should be suspected in any patient from any area who presents with fever and a history of tick bite. Initially, Plasmodium falciparum malaria might be suspected, but lack of recent travel in malaria-​endemic areas or recent blood transfusion and lack of a spleen should lead to suspicion of babesiosis. Diagnosis is based on discovering babesia in Giemsa-​stained blood smears (Fig. 8.8.3.1). Babesia can be distinguished from plasmodia by the absence of gametocytes and pigment in erythrocytes. B. microti is characterized by multiple basket-​shaped parasites. In some cases, parasitaemia is sparse and detection of antibodies, using an indirect fluorescent antibody assay, can be useful for diagnosis. Antibody titres rise during the first weeks and fall after 5 months, but correlation between antibody titre and severity of the disease is poor. A real-​time polymerase chain reaction (RT-​PCR) assay targeting the 18S rRNA gene of B. microti has been developed. B. divergens is characterized in Giemsa-​stained blood smears by double piriform intraerythrocytic parasites or tetrads, but annular, punctiform, and filamentous forms might also be encountered. Serology cannot be used for a rapid diagnosis of B. divergens infec- tion. Amplification of babesial DNA by polymerase chain reaction, using species-​specific primers can establish the diagnosis of both B. microti and B. divergens within 24 h. These assays are more sensi- tive than, but equally specific as, smear detection. Clearance of DNA seems to be related to disappearance of parasites. Treatment and prevention Chloroquine, sulphadiazine, co-​trimoxazole, pentamidine, or diminazene aceturate appear ineffective in completely eliminating babesia parasites. For B. microti infection, the standard treatment is a combination of atovaquone (750 mg every 12 h) and azithromycin (500–​1000 mg orally on day 1, and 250–​1000 mg thereafter) for 7 days. Alternatively, a combination of clindamycin (600 mg intra- venously or orally) with quinine (650 mg orally) every 6 to 8 h for at least 7 days in adults; treatment for children is atovaquone (20 mg/​ kg every 12 h, maximum 750 mg/​dose) and azithromycin (10 mg/​kg per day on day 1 and 5 mg/​kg per day thereafter) or alternatively a combination of clindamycin (7–​10 mg/​kg) and quinine (8 mg/​kg) every 6 to 8 h for at least 7 days. For immunocompromised patients, a treatment for 6 weeks and two additional weeks after blood para- site clearance is recommended. For patients with high parasitaemias (≥10%), haemolysis, or renal failure or those that are immunocom- promised, these therapies might not be sufficient and exchange transfusion should be considered. In Europe, babesiosis should be treated as a medical emergency. Immediate chemotherapy with either a combination of clindamycin

8.8.4 Toxoplasmosis 1416

8.8.4 Toxoplasmosis 1416

section 8  Infectious diseases 1416 and quinine or clindamycin alone reduces parasitaemia and pre- vents extensive haemolysis and renal failure. Exchange transfu- sion should be used in fulminating B. divergens cases. Imidocarb dipropionate, which has been used for treatment of cattle babesiosis, has been successfully used in two patients in Ireland, although this drug is not approved for human treatment. Preventive measures consist of use of repellents containing N,N-​diethyl-​3-​methylbenzamide for clothing or skin, removing ticks from the skin, and avoiding exposure for asplenic and im- munocompromised individuals. To date, no vaccine against human babesiosis is available. FURTHER READING Hildebrandt A, Gray JS, Hunfeld KP (2013). Human babesiosis in Europe: what clinicians need to know. Infection, 41, 1057–​72. Lobo CA, et al. (2013). Babesia: an emerging infectious treat in trans- fusion medicine. Plos Pathog, 9, e1003387. Vannier E, Krause PJ, (2012). Human babesiosis. N Eng J Med, 366, 2397–​407. 8.8.4  Toxoplasmosis Oliver Liesenfeld and Eskild Petersen ESSENTIALS Toxoplasma gondii is a protozoan parasite with worldwide distribu- tion that infects up to one-​third of the world’s population. Human infection is acquired through ingestion in water or food of oocysts shed by cats, or by ingestion of bradyzoites released from cysts contained in uncooked or undercooked meat (e.g. sheep, swine, cattle). Following invasion in the intestine, tachyzoites rapidly dis- seminate throughout the host. Immune mechanisms mediate the formation of cysts, primarily in the brain, eye, and skeletal and heart muscles, where they persist for the life of the host. Presence of in- fection can be established by direct detection of the parasite in clinical samples (often by polymerase chain reaction) or by sero- logical techniques. Clinical features and treatment Immunocompetent adults and children—​primary infection is usually subclinical, but some patients develop cervical lymphadenopathy; specific treatment is not usually required. Ocular disease—​choroidoretinitis; treatment with pyrimethamine and sulphadiazine is usually recommended if there are severe in- flammatory responses and/​or proximity of retinal lesions to the fovea or optic disc. Immunocompromised patients—​the central nervous system is the most commonly affected site. Reactivation of latent infection can cause life-​threatening encephalitis. Empirical anti-​T. gondii therapy is given to patients with single or multiple ring-​enhancing brain lesions on imaging, positive serology, and advanced immunodefi- ciency, most commonly with the combination of pyrimethamine/​ sulphadiazine and folinic acid. Patients with suspected cerebral T. gondii infection should be tested for HIV. (a) (b) (c) Fig. 8.8.3.1  (a) Babesia divergens infection in a 29-​year-​old Frenchman infected in Normandy. He had a splenectomy 4 months previously for idiopathic thrombocytopenia. Parasitaemia reached 30%. He was successfully treated with exchange transfusion, clindamycin, and quinine. (b) Babesia microti in a male patient, Missouri, United States of America (×100). (c) Babesia microti in a 72-​year-​old female patient, Massachusetts, United States of America (×150). (a) Copyright P. Brasseur; (b, c) courtesy of Centers for Disease Control, Atlanta, GA.

8.8.4  Toxoplasmosis 1417 Congenital toxoplasmosis—​infection acquired in early pregnancy may cause severe damage to the fetus or intrauterine death; infection in the second and third trimesters goes unnoticed in the newborn in most cases, but signs of disease (e.g. chorioretinitis), may occur later in life. Suspected or established maternal infection acquired during pregnancy must be confirmed by prenatal diagnosis of fetal infection using polymerase chain reaction on amniotic fluid: if this is positive it is highly probable that the fetus is infected and pyrimeth- amine/​sulphadiazine and folinic acid should be given and continued throughout the pregnancy. Prevention Prevention of infection by avoiding ingestion is the strategy of choice in seronegative people. Trimethoprim-​sulfamethoxazole can be used for primary and secondary prophylaxis of seropositive immunocompromised patients or seronegative recipients of organ transplants from seropositive donors. Spiramycin can be used for secondary prevention of transmission from the acutely infected mother to her fetus. Historical perspective The first human case ascribed to infection with Toxoplasma gondii was a child with hydrocephalus reported by Janku in 1923. Sabin reported the first case of encephalitis due to T.  gondii in 1941. Lymphadenopathy was recognized as a key symptom by Siim, Gard, and Magnusson (1951). Encephalitis due to T. gondii in im- munocompromised patients was first reported from patients with Hodgkin’s disease during immunosuppressive treatment in 1967. Aetiology, genetics, pathogenesis, and pathology Aetiology T.  gondii is an obligate intracellular protozoan of the phylum Apicomplexa, subclass  Coccidiasina. The parasite exists in three life-​stages of medical importance: the oocyst (10 × 12 μm in size), which is the product of the parasite’s sexual cycle in the intestine of all members of the cat family; the tachyzoite (2–​4 μm wide and 4–​8 μm long), which is the asexual invasive form; and the tissue cyst, which contains hundreds or thousands of bradyzoites in tissues (Fig. 8.8.4.1). Tissue cysts (the latent stage) remain viable through­ out the life of the host. Ingestion of T. gondii cysts or oocysts (the natural route of infec- tion) results in cyst (or oocyst) rupture and release of bradyzoites (or sporozoites) into the intestinal lumen, followed by rapid entry into intestinal cells and multiplication as tachyzoites. Tachyzoites are spread by disruption of infected cells, invasion of neighbouring cells, and via the bloodstream. In intermediate hosts and extraintestinal tissues of the cat, cysts containing bradyzoites are formed and persist for the life of the host. Immunodeficiency may result in reactivation of latent infection and severe disease, whereas reinfection does not appear to cause clinically apparent disease. A single case of symp- tomatic infection with an exotic strain despite previous infection with a type II strain has been published. T. gondii consists of three clonal lineages designated types I, II, III, and archetypes, which differ in virulence and geographical distribu- tion. Archtypes not belonging to type I, II or III, are more common in South America compared to Europe and the United States of America, and clinical toxoplasmosis is more severe in South America compared to Europe (Gilbert et al., 2008). The recent description of strain-​specific peptides has allowed typing of strains using serum. The generation of specific gene-​deficient strains of T. gondii and the sequencing of the Toxoplasma genome (http://​toxodb.org) will pro- vide further insight into parasite virulence factors and specific host immune responses. Pathogenesis The inoculum size and virulence of the organism, and the genetic background and immunological status of the individual, appear to influence the course of the infection in humans. Following ac- tive invasion, T. gondii induces the formation of a parasitophorous (a) (b) (c) Fig. 8.8.4.1  Toxoplasma gondii: (a) rosette-​forming tachyzoites inside
a macrophage, (b) bradyzoites inside a tissue cyst, and (c) oocyst in
cat faeces.

section 8  Infectious diseases 1418 vacuole containing secreted parasite proteins but excluding host proteins that would normally promote phagosome maturation, thereby preventing lysosome fusion. The molecular characterization and function of several proteins from organelles including rhoptries (specialized secretory organelles), micronemes (also a secretory or- ganelle), and dense granules have been reported. These molecules and the immunodominant tachyzoite surface antigen SAG1 are among the most promising vaccine candidates. Following intracel- lular replication and host cell disruption, parasites are disseminated via the blood stream and infect multiple organs including the cen- tral nervous system, eye, skeletal and heart muscle, and placenta. The developing immune response causes the formation of cysts in the central nervous system and skeletal muscle during the first week of infection. These persist lifelong. In immunocompromised hosts, cysts may disrupt and cause recrudescence of the infection, which then presents as life-​threatening toxoplasmic encephalitis. Infection with T. gondii results in a strong and persistent Th1 re- sponse characterized by the production of interleukin 12 (IL-​12), interferon-​γ, and tumour necrosis factor α (TNF​α). Strain-​specific differences in the modulation of host cell transcription are mediated by protein kinases: ROP16 and ROP18 are released from rhoptries and injected into the host, resulting in the activation of signalling pathways and IL-​12 production. The combined action of these cyto- kines and specific antibodies protects the host against rapid replica- tion of tachyzoites and subsequent pathological changes. Dendritic cells and their capacity to produce IL-​12 were identified as the main activators of Th1 immune reactions. Granulocytes might also con- tribute to the early production of IL-​12. The activated macrophage inhibits or kills intracellular T. gondii, which counteract these ac- tions by down-​regulating surface molecules and interfering with apoptosis pathways in antigen-​presenting cells, suggesting a role for these cells as ‘Trojan horses’ in early stages of infection. Sensitized CD4+ and CD8+ T lymphocytes are cytotoxic for T.  gondii-​ infected cells. Both proinflammatory (e.g. interferon-​γ and TNF​α) and down-​regulatory cytokines (e.g. IL-​10 and transforming growth factor β) are involved in balancing this response. Within 2 weeks after infection, IgG, IgM, IgA, and IgE antibodies against multiple T. gondii proteins can be detected. Reinfection may occur, and rare cases of congenital infection have been reported in subjects infected with a type II strain and reinfected with a virulent atypical geno- type, demonstrating that cross-​immunity between genotypes is not absolute. Pathology Histopathological changes in toxoplasma lymphadenitis in im- munocompetent people are frequently distinctive and often diag- nostic. They consist of reactive follicular hyperplasia, irregular clusters of epithelioid histiocytes encroaching on and blurring the margins of the germinal centres, and focal distension of sinuses with monocytic cells. Eye infection in immunocompetent patients pro- duces acute choroidoretinitis characterized by severe inflammation and necrosis. The pathogenesis of recurrent choroidoretinitis is con- troversial. Rupture of cysts may release viable organisms that induce necrosis and inflammation; alternatively, choroidoretinitis may re- sult from a hypersensitivity reaction of unknown cause. Damage to the central nervous system by T. gondii, toxoplasmic encephalitis, is characterized by multiple foci of enlarging necrosis and microglia nodules. In infants, periaqueductal and periventricular vasculitis and necrosis are distinctive of congenital toxoplasmosis. The nec- rotic areas can calcify and lead to radiographic findings suggestive but not pathognomonic of toxoplasmosis. Hydrocephalus can result from obstruction of the aqueduct of Sylvius or foramen of Monro. Tachyzoites and cysts are seen in and adjacent to necrotic foci. The presence of multiple brain abscesses is the most characteristic fea- ture of toxoplasmic encephalitis in severely immunodeficient pa- tients and is especially characteristic in AIDS. At autopsy in AIDS patients with toxoplasmic encephalitis, there is almost universal involvement of the cerebral hemispheres and a remarkable predi- lection for the basal ganglia. In cases of congenital toxoplasmosis, necrosis of the brain is most intense in the cortex and basal ganglia. Epidemiology Infection with T. gondii in humans is naturally acquired through in- gestion of cysts or oocysts. Humans can be infected by ingestion of undercooked or raw meat (e.g. sheep, swine, cattle) containing tissue cysts, or of water or food contaminated by faeces containing oocysts from infected cats. The differences in seroprevalence of T. gondii depend on eating habits and customs that support the ingestion of cysts as the major source of infection. Epidemics of toxoplasmosis in humans and sheep attributed to exposure to infected cats indi- cate the importance of oocyst excretion by cats. Several outbreaks of toxoplasmosis through contamination of drinking water by oocysts have been reported. This is a major route of transmission under poor socioeconomic conditions, where untreated surface water is drunk. Transmission of T.  gondii in organs transplanted from seroposi- tive donors to seronegative recipients remains an important cause of infection in immunocompromised patients. T. gondii can also be transmitted by blood or leucocytes from immunocompetent or im- munocompromised donors. In congenital transmission, the parasite gains access to the fetal circulation by infection of the placenta following maternal parasit- aemia. The reported birth prevalence of congenital toxoplasmosis ranges from 1 to 10 per 10 000 live births in Europe and North America. The frequency of congenital transmission depends on the time during gestation when the mother acquired her infection (Fig. 8.8.4.2). Maternal infection acquired weeks or a few months before gestation poses very little or no risk to the fetus. Infection acquired around the time of conception and within the first 2 weeks of gestation in most cases does not result in transmission, whereas rates of transmission are above 60% in the last trimester. There is an inverse relationship between frequency of transmission and severity of disease. Infection in the first and second trimester, al- though less frequent than infection in the third trimester, results in severe congenital toxoplasmosis more often (Fig. 8.8.4.3). In con- trast, maternal infection during the third trimester, although more frequent than infection in the first or second trimester, usually re- sults in subclinical infection of the newborn. It is important to be aware that the overall frequency of subclinical infection in new- borns with congenital toxoplasmosis is as high as 85%. The vast ma- jority of these neonatal infections are initially unnoticed, of which a fraction later develop choroidoretinitis. Treatment of the mother during pregnancy aims to reduce the frequency and severity of fetal infection. However, the efficacy of such treatment is debatable (see next). Treatment aimed at preventing mother-​to-​child transmission

8.8.4  Toxoplasmosis 1419 should be given within 3 weeks of infection. In practice, this is very difficult because most infections are asymptomatic. Seroprevalence increases with age. It does not vary signifi- cantly between sexes and tends to be less in cold, hot, and arid areas, and at high altitudes. Incidence of infection varies with the population group and geographical location. In El Salvador and France, seropositivity is as high as 40–​50% by the fourth decade of life, compared with an overall seroprevalence of 15% in the United States of America. In various countries, seroprevalence of T. gondii has decreased by approximately one-​third over the past decades. Prevention Since the infection is naturally acquired through ingestion of under- cooked cyst-​containing meat or food contaminated with oocysts, infection is preventable in almost all cases. Primary prophylaxis (prevention of infection) by avoiding ingestion is the strategy of choice in seronegative people, whereas in seropositive immunocom- promised patients (e.g. people with AIDS) or seronegative recipients of organ transplants (e.g. heart, bone marrow) from seropositive donors, primary prophylaxis using trimethoprim/​sulfamethoxazole has proved effective. Secondary prevention is employed to prevent transmission from the acutely infected mother to her fetus using spiramycin, in immunocompromised patients following treatment of reactivated toxoplasmosis (maintenance therapy) using pyri- methamine/​sulphadiazine. Systematic serological screening of all pregnant women is performed only in some countries. Uncertainty about the incidence of congenital infection, problems with the sensitivity and specificity of serological tests especially in the first trimester, and doubts of the benefit of treating newborns with asymptomatic congenital toxoplasmosis has hampered attempts to implement screening programmes in several countries. Neonatal screening programmes have allowed the identification of as many as 80% of infected newborns. Clinical features Infection with T. gondii may be subclinical or it may cause clin- ical signs and symptoms that vary according to the immune status of the patient and their clinical situation (‘toxoplasmosis’). Four clinical situations can be distinguished:  the immunocompetent patient, patients with ocular disease, the immunocompromised patient, and the patient with congenital toxoplasmosis. Immunocompetent adults and children Primary T.  gondii infection in children and adults is generally asymptomatic. In approximately 10% of the patients, it causes a self-​limited and non​specific illness that very seldom requires treat- ment. The most frequently observed clinical manifestation is iso- lated cervical or occipital lymphadenopathy. Lymph nodes are not tender, do not suppurate, are usually discrete, and stay enlarged for less than 4 to 6 weeks. Very infrequently, chronic lymphadenitis, myocarditis, polymyositis, pneumonitis, hepatitis, or encephal- itis can occur in otherwise healthy individuals. Acute toxoplasma infection during pregnancy is asymptomatic in the vast majority of women. Ocular toxoplasmosis Toxoplasma choroidoretinitis can be observed in congenital or postnatally acquired disease where it results from acute infection or reactivation. Choroidoretinitis can present in infancy or early childhood or might reactivate later. It is uncommon after the age of 40. Bilateral disease, old retinal scars, and involvement of the macula are hallmarks of retinal disease in these cases. In contrast, in patients who present with toxoplasma choroidoretinitis in acute Probability of congenital infection 0 5 10 15 20 Gestational age at seroconversion (weeks) 25 30 35 40 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.8 0.9 1.0 Fig. 8.8.4.2  Risk of mother-​to-​child transmission of T. gondii by gestational age at maternal seroconversion. Reprinted from The Lancet, Vol. 369, Thiebaut R et al. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-​analysis of individual patients’ data, pages 115–​22, copyright © 2007, with permission from Elsevier.

section 8  Infectious diseases 1420 toxoplasmosis typically only one eye is involved, the macula is spared, and there is no old scarring. AIDS and non-​AIDS immunocompromised patients In contrast to the relatively favourable course of toxoplasmosis in most immunocompetent people, it is life-​threatening in the im- munosuppressed. Toxoplasmosis almost always occurs as a result of reactivation of chronic infection. It can occur when a heart, kidney, or liver from a seropositive donor is transplanted into a seronega- tive recipient; patients with HIV/​AIDS and patients receiving sec- ondary immunosuppression, including biologic therapies, are also at risk. The central nervous system is the most commonly af- fected site. Toxoplasmic encephalitis may present subacutely, grad- ually evolving over weeks, or as an acute confusional state with or without focal neurological deficits, evolving over days. Clinical features include changes in level of consciousness, seizures, focal motor deficits, cranial nerve disturbances, sensory abnormalities, cerebellar signs, movement disorders, and neuropsychiatric dis- turbances. The differential diagnosis of toxoplasmic encephalitis lesions includes central nervous system lymphoma, progressive multifocal leukoencephalopathy, infection with cytomegalovirus, cryptococcosis aspergillosis, bacterial abscess, and tuberculosis. In 0.5 Probability of intracranial lesions 0.4 0.3 0.2 0.1 0 0 5 10 15 20 Gestational age at seroconversion (weeks) (b) Risk of eye lesions (n-526) (a) Risk of intracranial lesions (n-473) 25 30 35 40 0.6 0.7 0.8 0.9 1.0 0.5 Probability of eye lesions 0.4 0.3 0.2 0.1 0 0 5 10 15 20 Gestational age at seroconversion (weeks) 25 30 35 40 0.6 0.7 0.8 0.9 1.0 Fig. 8.8.4.3  Risk of intracranial and eye lesions in children infected with T. gondii by gestational age at maternal seroconversion. Reprinted from The Lancet, Vol. 369, Thiebaut R et al. Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-​analysis of individual patients’ data, pages 115–​22, copyright © 2007, with permission from Elsevier.

8.8.4  Toxoplasmosis 1421 immunocompromised patients, toxoplasmosis can also present as choroidoretinitis, pneumonitis, or multiorgan disease, presenting with acute respiratory failure and haemodynamic abnormalities re- sembling septic shock. Congenital toxoplasmosis Prenatal ultrasound examination often fails to detect a fetus with congenital toxoplasmosis. Abnormalities include intracra- nial calcification, ventricular dilatation, hepatic enlargement, ascites, and increased placental thickness. Approximately 85% of newborns with congenital infection appear normal at birth. However, if untreated, congenital toxoplasmosis may result later in loss of vision, and children born with symptoms of congenital infection can later develop psychomotor retardation, intellectual disability, and hearing loss. Fetal and neonatal disease is more se- vere the earlier in gestation the acute infection was acquired. The classic triad of chorioretinitis, hydrocephalus, and cerebral calci- fication is rather rare. None of the signs described in newborns with congenital disease are pathognomonic for toxoplasmosis and may be mimicked by other congenital infection such as cyto- megalovirus, herpes simplex virus, rubella, and syphilis. Early maternal infection can result in death of the fetus in utero and spontaneous abortion. Clinical investigation and criteria for diagnosis Infection in the immunocompetent host Immunocompetent adults and children with toxoplasma lymph- adenitis are usually not treated unless symptoms are severe or per- sistent. Characteristic histological criteria and a panel of serological tests (IgG, IgM, IgG avidity index) consistent with recently acquired infection establish the diagnosis of toxoplasma lymphadenitis in older children and adults. If required, treatment is usually admin- istered for 2 to 4 weeks, followed by reassessment of the patient’s condition. The combination of pyrimethamine, sulphadiazine, and folinic acid for 4 to 6 weeks is the most common drug combination used (Table 8.8.4.1). Table 8.8.4.1  Suggested regimens for the treatment of infection with T. gondii Therapy/​drug Dosage Duration Acute acquired infection Symptomatica Acute toxoplasmosis in pregnant womenb Spiramycin 3 g once a day in three divided doses without food Until termc or until fetal infection is documented Documented fetal infection (after 18 weeks of gestation)d Pyrimethamine Loading dose: 100 mg once a day in two divided doses for 2 days, then 50 mg once a day Until term plus Sulphadiazine Loading dose 75 mg/​kg once a day in two divided doses (max. 4 g once a day) for 2 days, then 100 mg/​kg once a day in two divided doses (max. 4 g once a day) Until term plus Leucovorin (folinic acid) 5–​20 mg once a day During and for 1 week after pyrimethamine therapy Congenital toxoplasma infection in the infante Pyrimethamine Loading dose 2 mg/​kg once a day for 2 days, then 1 mg/​kg once a day for 2–​6 months, then this dose every Monday, Wednesday, Friday 1 year plus Sulphadiazine 100 mg/​kg once a day in two divided doses 1 year plus Leucovorin 10 mg three times weekly During and for 1 week after pyrimethamine therapy Corticosteroids (prednisone)f 1 mg/​kg once a day in two divided doses Until resolution of signs and symptoms Choroidoretinitis in adults Pyrimethamine Loading dose 200 mg once a day, then 50–​75 mg once a day Usually 1–​2 weeks after resolution of symptoms plus Sulphadizine Oral, 1–​1.5 g once a day Usually 1–​2 weeks after resolution of symptoms plus Leucovorin 5–​20 mg three times weekly During and for 1 week after pyrimethamine therapy Corticosteroidsf 1 mg/​kg once a day in two divided doses Until resolution of signs and symptoms Acute/​primary therapy of toxoplasmic encephalitis in AIDS patients Standard regimens: Pyrimethamine Oral, 200 mg loading dose, then 50–​75 mg once a day At least 4–​6 weeks after resolution of signs and symptoms (continued)

section 8  Infectious diseases 1422 Management of maternal and fetal infection The IgG and IgM antibody status of a pregnant woman should be obtained before or early in pregnancy. The absence of IgG antibodies before or early in pregnancy allows identification of those women at risk of acquiring the infection. The presence of IgG and IgM anti- bodies indicates recent infection in approximately 40% of patients. The presence of high-​avidity IgG antibodies essentially rules out an infection acquired in the previous 3 or 4 months, whereas low avidity antibodies can persist for more than 3 months after infection, especially in pregnant women. Detection of IgG and IgM antibodies establishes that the patient has been infected, whereas seronegative women should be provided with necessary information to prevent primary infection (see earlier). Absence of IgM antibodies during the first two trimesters virtually rules out recently acquired infec- tion unless the sera were obtained too early for the IgM antibody response to be detectable or too late after IgM antibodies had be- come non​detectable. The definitive diagnosis of acute toxoplasma infection or toxoplasmosis requires demonstration of a rise in titres in serial specimens (either conversion from a negative to a positive titre or a significant rise from a low to a higher titre). Treatment of women with acute acquired infection using spiramycin was thought to reduce the incidence and severity of fetal infection by approxi- mately 60%, but a recent meta-​analysis of data from children diag- nosed by prenatal screening showed an effect only on intracranial lesions and not on choroidoretinitis at birth. Therapy should be started as soon as possible after diagnosis of recently acquired ma- ternal infection (Table 8.8.4.1). Since maternal infection does not necessarily result in fetal infection, suspected or established ma- ternal infection acquired during pregnancy (based on ultrasonog- raphy or serology) must be confirmed by prenatal diagnosis of fetal infection using polymerase chain reaction (PCR) on amniotic fluid. PCR has an overall reported sensitivity of between 64 and 98.8%. When the PCR is positive or it is highly probable that the fetus is infected, pyrimethamine/​sulphadiazine is given in combination with folinic acid after gestational week 20 and continued throughout the pregnancy. Spiramycin is used before gestational week 18. If the initial ultrasound reveals no abnormalities, it should be repeated at least monthly until term. Hydrocephalus is an indication for thera- peutic abortion. Since fetal infection is undetected in 85% of new- borns, serology is commonly performed for neonatal diagnosis. The presence of IgG antibodies in the neonate’s serum may reflect maternal and/​or its own antibodies. Testing for IgM and IgA anti- bodies will identify up to 75% of infected newborns. Maternally transferred IgG antibodies usually decline and disappear within 6 to 12 months. Immunoblots can, in most but not all cases, distin- guish maternal and fetal T. gondii specific IgG and IgM antibodies. Treatment of the fetus is followed by treatment of the symptomatic newborn throughout the first year of life, but the benefit of treating Therapy/​drug Dosage Duration Leucovorin Oral, IV, or IM, 10–​20 mg once a day (up to 50 mg once a day) During and for 1 week after pyrimethamine therapy plus Sulphadiazine Oral, 1–​1.5 g four timses daily g or Clindamycin Oral or IV, 600 mg four times daily (up to IV 1200 mg four times daily) g Possible alternative regimens: (1) Co-​trimoxazole Oral or IV, 10 mg (trimethoprim component)/​kg four times daily g (2) Pyrimethamine plus leucovorin As in standard regimens g plus one of the following: Atovaquone Oral, 750 mg four times daily g Clarithromycin Oral, 1 g two times daily g Azithromycin Oral, 1200–​1500 mg once a day g Dapsone Oral, 100 mg once a day g IM, intramuscular; IV, intravenous; q6 h, every 6 h; q12 h, every 12 h. a Acute acquired infection in immunocompetent patients does not require specific treatment unless there are severe or persistent symptoms or evidence of damage to vital organs.
If such signs or symptoms occur, treatment with pyrimethamine/​sulphadiazine, and leucovorin should be initiated (for dosages, see ‘Toxoplasmic choroidoretinitis in adults’). b Practices vary widely between centres. c German and Austrian guidelines recommend using spiramycin prophylaxis until 17 weeks of pregnancy followed by a 4-​week course of pyrimethamine plus sulphadiazine plus leucovorin). d Practices vary widely between centres (pyrimethamine plus sulphadoxine is used in some centres, monthly alternating cycles of pyrimethamine plus sulphadiazine and spiramycin). e Practices vary widely between centres (monthly alternating cycles of pyrimethamine plus sulphadiazine and spiramycin). f When cerebrospinal protein is more than 1 g/​dl and when active choroidoretinitis threatens vision. g Duration of treatment as for pyrimethamine in patient with toxoplasmic encephalitis. Table 8.8.4.1  Continued

8.8.4  Toxoplasmosis 1423 asymptomatic newborns with congenital toxoplasmosis after birth is debatable (Table 8.8.4.1). Retinochoroiditis The decision to treat active toxoplasma choroidoretinitis should be based on examination by an experienced ophthalmologist. Low titres of IgG antibody are usual in patients with active choroidoretinitis due to reactivation of congenital T. gondii infection. IgM antibodies are usually not detected. Patients with retinochoroiditis due to postnatally acquired disease usually have serological tests results consistent with an infection acquired in the recent past. PCR per- formed on aqueous humour has shown sensitivities of up to 55% that increased to 85% when used in combination with serological tests. Most ophthalmologists recommend treatment if there are se- vere inflammatory responses and/​or proximity of retinal lesions to the fovea or optic disc (Table 8.8.4.1). The combination of pyri- methamine and sulphadiazine is the most commonly used regimen. Prednisolone is added if the lesion threatens the macula. The inci- dence of recurrent toxoplasma retinochoroiditis has been signifi- cantly reduced by using long-​term intermittent co-​trimoxazole (trimethoprim/​sulfamethoxazole). Infection in the immunocompromised host In immunocompromised patients with suspected reactivation, PCR rather than serological methods are strongly recommended. Pre-​emptive antiparasitic therapy should be considered in all symptomatic seropositive immunosuppressed patients suspected to have toxoplasmosis. If the clinical features suggest central ner- vous system and/​or spinal cord involvement, CT or MRI is man- datory. In most studies PCR performed on cerebrospinal fluid showed sensitivities between 60% and 75% while PCR on blood samples did not achieve sensitivities greater than 30% in most studies. Empirical anti-​T. gondii therapy is accepted practice for patients with multiple ring-​enhancing brain lesions (usually es- tablished by MRI), positive IgG antibody titres against T. gondii, and advanced immunodeficiency. Clinical and radiological re- sponse to specific anti-​T. gondii therapy supports the diagnosis of central nervous system toxoplasmosis. The most commonly used and successful regimen continues to be the combination of pyri- methamine and sulphadiazine with folinic acid (Table 8.8.4.1). Clindamycin can be used instead of sulphadiazine in patients in- tolerant of sulphonamides. Duration of treatment is recommended for 4 to 6 weeks after resolution of all signs and symptoms (often for several months or longer). After treatment of the acute phase (primary or induction treatment) in immunosuppressed patients, maintenance treatment (secondary prophylaxis) should be insti- tuted using the same regimen as for the acute phase but at half the dose. In patients with AIDS, secondary prophylaxis is usually discontinued when the patient’s CD4 count has returned to above 200 cells/μl and HIV viral load has been controlled by antiretro- virals for at least 6 months. Areas of uncertainty and future developments • Epidemiology: ■​ Sources of infection, relative importance (e.g. water, meat, cats) • Pathogenesis/​pathology: ■​ Susceptibility of the host to infection (e.g. human leukocyte antigen types) ■​ Strain differences and clinical presentation ■​ Virulence factors • Diagnosis: ■​ Improved avidity testing using recombinant antigens ■​ Increased sensitivity of PCR on amniotic fluid • Treatment/​prophylaxis: ■​ Clinical treatment trials in different clinical situations, for ex- ample, eye disease and congenital toxoplasmosis using new drugs (e.g. atovaquone) • Prevention strategies/​screening: ■​ Co-​trimoxazole for prevention of multiple episodes of recur- rent episodes of chorioretinitis ■​ Atovaquone for prophylaxis of toxoplasmic encephalitis ■​ Prophylaxis and treatment in bone marrow transplant recipients ■​ Effectiveness of prevention strategies in pregnancy ■​ Cost-​effectiveness of routine screening programmes ■​ Vaccination: proteins, DNA, adjuvants, and mucosal strategies FURTHER READING Cook AJ, et  al. (2000). Sources of toxoplasma infection in preg- nant women: European multicentre case–​control study. European Research Network on Congenital Toxoplasmosis. BMJ, 321, 142–​47. Elbez-​Rubinstein A, et  al. (2009). Congenital toxoplasmosis and reinfection during pregnancy:  case report, strain characteriza- tion, experimental model of reinfection, and review. J Infect Dis, 199, 280–​5. Gilbert RE, et  al. (2008). The European Multicentre Study on Congenital Toxoplasmosis (EMSCOT). Ocular sequelae of con- genital toxoplasmosis in brazil compared with Europe. PLoS Negl Trop Di, 2, e277. Gras L, et al. (2005). Association between prenatal treatment and clin- ical manifestations of congenital toxoplasmosis in infancy: a cohort study in 13 European centres. Acta Paediatr, 94, 1721–​31. Hernadez AV, et al. (2017). A systematic review and meta-​analysis of the relative efficacy and safety of treatment regimens for HIV-​associated cerebral toxoplasmosis:  is trimethoprim-​sulfamethoxazole a real option? HIV Med 18, 115–​124. Holland GN (2003). Ocular toxoplasmosis:  a global reassessment. Part I: epidemiology and course of disease. Am J Ophthalmol, 136, 973–​88. Holland GN (2004). Ocular toxoplasmosis:  a global reassessment. Part II: disease manifestations and management. Am J Ophthalmol, 137, 1–​17. Luft BJ, et al. (1984). Toxoplasmic encephalitis in patients with ac- quired immune deficiency syndrome. JAMA, 252, 913–​17. McLeod R, et al. (2006). Outcome of treatment for congenital toxo- plasmosis, 1981–​2004: the National Collaborative Chicago-​Based, Congenital Toxoplasmosis Study. Clin Infect Dis, 42, 1383–​94. Montoya JG, Liesenfeld O (2004). Toxoplasmosis. Lancet, 363, 1965–​76. Pomares C, Montoya JG (2016). Laboratory diagnosis of congenital toxoplasmosis. Clin Microbiol, 54, 2448–54.

8.8.5 Cryptosporidium and cryptosporidiosis 1424

8.8.5 Cryptosporidium and cryptosporidiosis 1424

section 8  Infectious diseases 1424 Robert-​Gagneux S, Balas F (2016). Molecular diagnosis of toxo- plasmosis in immunocompromised hosts. Curr Opin Infect Dis, 29, 330–​9. Saeij JP, et al. (2006). Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science, 314, 1780–​3. Schmidt DR, et al. (2006). Treatment of infants with congenital toxo- plasmosis:  tolerability and plasma concentrations of sulfadiazine and pyrimethamine. Eur J Pediatr, 165, 19–​25. Syrocot (2007). Effectiveness of prenatal treatment for congenital toxoplasmosis: a meta-​analysis of individual patients’ data. Lancet, 369, 115–​22. Thalib L, et  al. (2005). Prediction of congenital toxoplasmosis by polymerase chain reaction analysis of amniotic fluid. BJOG, 112, 567–​74. Thiebaut R, et al. (2007). Effectiveness of prenatal treatment for con- genital toxoplasmosis: a meta-​analysis of individual patients’ data. Lancet, 369, 115–​22. 8.8.5  Cryptosporidium and cryptosporidiosis Simone M. Cacciò ESSENTIALS Parasites within the genus Cryptosporidium infect the mucosal epi- thelia of a variety of vertebrate hosts, including humans, affecting the health, survival, and economic development of millions of people and animals worldwide. Human infection is mainly caused by two species, Cryptosporidium parvum and C. hominis. The former species is also prevalent in young livestock and has a demonstrated zoonotic potential, whereas the latter species is essentially a human parasite. Direct and indirect (through contaminated water and food) transmis- sion routes exist for both species. Clinical features—​infection involves either children or adults, but is a major cause of diarrhoea in children under 5 years old in both de- veloped and developing countries. A recent study demonstrated that Cryptosporidium is a significant cause of moderate to severe diarrhoea and associated mortality among very young children in Sub-​Saharan and South Asia. Patients may be asymptomatic or experience acute or chronic diarrhoea, depending on their age and immune status. In the immunocompetent, infection usually results in acute self-​limiting diarrhoea, whereas in immunocompromised patients (e.g. AIDS) and those with concurrent infections such as measles or chickenpox, clinical symptoms are more severe and persistent and may become chronic, leading to electrolyte imbalance, wasting, and even death. Since 2004, Cryptosporidium has been included in the World Health Organization’s ‘Neglected Diseases Initiative’, in recognition of the importance of this infection in developing countries. Diagnosis and treatment—​diagnosis is usually based on detection of oocysts in stool, often by use of direct fluorescent-​antibody tests. Detection of sol- uble Cryptosporidium antigens in faecal samples by enzyme-​linked immunosorbent assay or by an immuno-​chromatographic lateral flow assay is useful for the screening of large numbers of specimens. Molecular methods allow reliable identification of species and geno- types, and are therefore of paramount importance for environmental or epidemiological research purposes. Treatment of immunocompe- tent patients, when necessary, is based on nitazoxanide, a thiazolide drug with broad antiparasitic activities. Nitazoxanide is the only US Food and Drug Administration-​approved drug for the treatment of cryptosporidiosis, but it is not licensed in Europe. Management of patients who are immunocompromised is difficult: aside from sup- portive care, highly active antiretroviral therapy is effective, both by immune reconstitution (in patients with HIV/​AIDS) and by direct in- hibition of parasite proteases. There is little evidence for efficacy of nitazoxanide in immunocompromised individuals. Prevention—​primary control is by limiting the opportunity for faecal–​oral transmission, both direct and indirect, with maintenance of drinking-​water quality and general hygiene (especially in hos- pitals, wards, and so on) essential for the prevention of the infection. Secondary control, when water supplies are contaminated, can be achieved by boiling or filtering water before drinking. Introduction The cryptosporidia are obligate intracellular parasites of many species from all vertebrate classes. In humans, infection is caused mainly by two species, Cryptosporidium parvum, which is also prevalent in young livestock and can be transmitted zoonotically, and C.  hominis, which is essentially a human parasite. First de- scribed in laboratory mice by Tyzzer in 1912, Cryptosporidium was recognized as a cause of human infection in 1976. In the 1980s it emerged worldwide as a common cause of severe or life-​threatening infection in severely immunocompromised patients, especially those with AIDS, and of acute, self-​limiting gastroenteritis in other- wise healthy subjects, especially children. Biology Cryptosporidium species have been traditionally considered as members of the coccidia (phylum Apicomplexa), but recent in- vestigations have revealed a closer phylogenetic affinity with the Gregarinae, which are parasites of invertebrates. The oocyst, con- taining four sporozoites, is an environmentally robust transmissible stage and is fully sporulated and infective upon excretion with the host faeces. Cryptosporidia are monoxenous; this is, they complete their lifecycle in a single host (Fig. 8.8.5.1). C. parvum is not tissue specific but shows a predilection for the lower ileum during the primary stages of infection. Following ingestion of oocysts, the motile sporozoites are released, through a suture in the oocyst wall, in the lumen of the small bowel. Acknowledgement: The author and editors acknowledge the inclusion of material from the chapter by Dr D. P. Casemore in the 4th edition of this textbook. Plates for this chapter were kindly provided from photographs by A. Curry and D. P. Casemore.

8.8.5  Cryptosporidium and cryptosporidiosis 1425 They quickly attach superficially to cells, rounding up to form fixed trophozoites (meronts). The initial site of infection is the brush border of enterocytes in the small bowel, but the parasite is able to infect other epithelial and parenchymal cells. The complex life cycle includes both asexual and sexual stages of replication (Figs. 8.8.5.1 and 8.8.5.2). The endogenous (tissue) stages develop within a parasitophorous vacuole, the outer layer of which is derived from the host cell’s outer mem- branes, in a unique intracellular but extracytoplasmic location. Molecular biology The sequences of the genome of both C. parvum and C. hominis have been described and have revealed many peculiar characteristics. The two genomes are remarkably similar, displaying 95–​97% DNA sequence identity and c.30% GC content, and are organized in eight chromosomes that are apparently completely collinear. The compacted nature of the genome, which is comprised of only c.9.2 Mbp, is reflected in its high coding capacity (about 4000 genes that account for two-​thirds of the genome). The highly streamlined metabolic pathways imply that Cryptosporidium heavily relies on scavenging nutrients from the host, salvage rather than de novo biosynthesis, and glycolysis or substrate-​level phosphorylation for energy production. The increasing availability of whole Cryptosporidium genome sequences and functional genomics and metabolomics data will assist in the identification of new drug targets. Moreover, a recent study showed the possibility of genetically engineering the parasite, Sporozoite Ingested Exit host Thick-walled oocyst (sporulated) Thin-walled (sporulated) Microgamont Type II meront Merozoite Macrogamont Zygote Auto infection Merozoite Type I meront Trophozoite Fig. 8.8.5.1  Diagrammatic representation of the lifecycle of C. parvum. Following ingestion of oocysts, the motile sporozoites are released, attach to cells, and develop into fixed trophozoites (uninucleate meronts) in an intracellular but extracytoplasmic location. These undergo schizogony (asexual multiple budding), the first-​stage meronts producing eight merozoites, some of which recycle to form further type I meronts. Type II meronts produce four merozoites, which form gamonts (sexual stages) that mature as either macrogametes or as microgamonts containing 16 motile microgametes. Most of the zygotes formed after fertilization develop into thick-​walled, environmentally resistant, transmissible oocysts, which then sporulate, usually by the time they are excreted. Some have only a thin unit membrane, which ruptures to release the sporozoites in situ to produce an autoinfective cycle. Adapted from a drawing by Kip Carter, University of Georgia, and shown by courtesy of W I Current and CRC Press, Inc., Boca Raton, FL.

section 8  Infectious diseases 1426 opening avenues for investigating the basis of drug susceptibility by gene knockout experiments. Protocols based on nucleic acid amplification of specific markers are available to differentiate Cryptosporidium species and genotypes in both clinical and environmental samples. Epidemiology C. parvum occurs worldwide and is common in humans and in young livestock animals, especially lambs and calves, and has been reported in goats, horses, pigs, and farmed deer as well as in mammalian wild- life. Prevalence in humans varies both geographically and temporally. Because of the diversity of host species that can infect humans, the epidemiology of the infection is complex and involves both direct and indirect routes of transmission from animals to man (zoonotic trans- mission) and from person-​to-​person (urban cycle). A recent study has demonstrated that respiratory involvement commonly occurs in HIV-​ seronegative children with intestinal cryptosporidiosis and cough, sug- gesting the potential for respiratory transmission of the infection. Zoonotic transmission Transmission from livestock is common, particularly in children, including those from urban homes and schools visiting educational farms and rural activity centres. Companion animals have long been considered potential sources for human cryptosporidiosis. However, they appear to be most commonly infected with host-​specific and non​zoonotic Cryptosporidium species; they are, therefore, not con- sidered important reservoirs of infection. Cryptosporidiosis is rarely seen in adults in rural areas, presumably as a result of frequent ex- posure and the development of immunity. Zoonotic transmission of other species, such as C. meleagridis, a parasite of turkeys and other birds, has been recently demonstrated. Human-​to-​human transmission Cases of human-​to-​human transmission have been reported be- tween family members, sexual partners, children in daycare centres, and hospital patients and staff. Outbreaks in daycare centres have been reported in the United Kingdom and the United States of America, mainly as a result of direct (person-​to-​person) faecal–​oral transmission, although the infection may be introduced in the first instance through zoonotic contact. Affected adults may acquire in- fection from young children in the home or occupationally. Infection may be transmitted sexually where this involves faecal exposure. In developing countries, the high prevalence of C. hominis and of anthroponotic subtypes of C. parvum, particularly in children, has been interpreted as an indication of the importance of person-​to-​ person transmission. Cryptosporidium is a cause of traveller’s diar- rhoea, although apparently not as frequently as Giardia. A  new species, Cryptosporidium viatorum, has been identified as a cause of infection among travellers, and appears to be restricted to humans. Waterborne transmission In the United Kingdom, the United States of America, and elsewhere, there have been numerous well-​documented outbreaks resulting from contamination of public drinking-​water supplies. Outbreaks, which can be massive, have been associated with C. hominis, which indicates contamination of the supply by human sewage, or with C.  parvum, which suggests an animal source of contamination. Recently, another species, C. cuniculus, which infects rabbits, has been associated with a waterborne outbreak in the United Kingdom. Isolates from endemic (sporadic) cases, some of which will be water- borne, fall into both categories. Oocysts have been demonstrated widely in both raw and treated water and legislation has been intro- duced in the United Kingdom and the United States of America in an attempt to limit the latter. Cryptosporidium is also one of the most commonly recognized causes of recreational waterborne disease. Most outbreaks are the re- sult of faecal accident or cross-​connection in swimming pools. Faecal contamination coupled with oocyst resistance to chlorine, low in- fectious dose, and high bather densities facilitate transmission. The potential for intentional contamination of water supplies has led to in- clusion of Cryptosporidium as a Category B pathogen for biodefence. Food-​borne transmission Food-​borne transmission is probably underestimated, because the long incubation period (3–​7 days or more) makes the relationship between cryptosporidiosis and a possibly contaminated food item difficult to establish. Cryptosporidiosis has been attributed to in- gestion of contaminated apple juice, chicken salad, milk, and food items prepared by sick food handlers. A large outbreak occurred Fig. 8.8.5.2  Electron micrograph of a transverse section of small bowel of a mouse infected with C. parvum. The section shows numerous developmental stages: uninucleate meronts (trophozoites); type I meronts (schizonts) containing merozoites in which may be seen
the darker granules of the apical complex organelles; the degenerate remains of a schizont and a free-​swimming merozoite within the
lumen; and macrogamonts showing dark wall-​forming granules and electron-​lucent amylopectin (polysaccharide) food-​storage granules.
The parasitophorous vacuole can be clearly seen surrounding the parasite stages. Some of the intracellular stages appear to be free within the lumen because of the plane of sectioning.

8.8.5  Cryptosporidium and cryptosporidiosis 1427 in 2012 across England and Scotland, and was strongly associ- ated with the consumption of pre-​cut mixed salad leaves sold by a single retailer. Typing revealed the outbreak strain to be C. parvum. Methods for the detection of oocysts in fruits and vegetables have been developed and validated. Nosocomial transmission Transmission has been reported between healthcare staff and pa- tients and between patients, particularly the immunocomprom- ised. Large numbers of oocysts may be present in patients’ stools and in vomit; transmission via fomites occurs, although this route is limited by the susceptibility of oocysts to desiccation. Poor hand-​ washing practice has been identified as an important risk factor. In an outbreak with high mortality in a ward of immunocompromised patients in Denmark, transmission was probably by patients’ hands via a ward ice-​making machine. Demography Age and sex distribution In the United Kingdom, approximately two-​thirds of Cryptosporidium-​ positive samples are from children between 1 and 10 years of age, with a secondary peak in adults under 45 years; the infection is uncommon in infants less than 1 year old and in older people. Distribution appears to be the same in both sexes. Other EU countries reported a similar age and sex distribution of cryptosporidiosis cases. A relative increase in adult cases is often seen in waterborne outbreaks. In developing coun- tries, infection is common in infants less than 1 year old and asymp- tomatic infection is common in older subjects. Temporal distribution Data from the European Centre for Disease Control, and especially the United Kingdom, indicate that cryptosporidiosis exhibits a strong sea- sonality in Europe, with low endemic levels followed by pronounced seasonal increases, particularly during late spring and late summer to early autumn. Springtime cases are more often due to C. parvum, and are likely the result of an increased exposure to oocysts shed by young animals, as this coincides with the calving/​lambing season. In recent years, the spring peak has reduced, largely due to improved drinking-​ water supplies. On the other hand, the late summer-​early autumn peak is mainly due to C. hominis, and has not reduced in recent years; it is likely linked to increased travel and exposure to recreational water at this time of year. In the United States of America, the peak onset of cryptosporidiosis occurs annually from early summer to early au- tumn. This might reflect the increased use of communal swimming venues, particularly by susceptible hosts like young children. Frequency of occurrence Laboratory rates of detection in immunocompetent subjects average about 2% in developed countries (range <1–​5%) and about 8% in developing countries (range 2–​30%), and Cryptosporidium is about fourth in the list of pathogens detected in stools submitted to the laboratory. In the United Kingdom, about 5000 to 6000 confirmed cases are reported annually; it is generally somewhat less frequent than giardiasis. Among young children in the United Kingdom, cryptosporidiosis is more common than salmonellosis and detec- tion rates may exceed 20% during peak periods. Cryptosporidiosis is one of the most common causes of diarrhoea in patients with AIDS and in some studies prevalence has exceeded 50%. The infection rate in patients with AIDS in industrialized countries has been falling in recent years, due to infection control advice and the use of antiretroviral therapy. Infection rates are not generally increased for most other immunocompromised groups. Clinical aspects Pathology Histopathology There is mucosal involvement of the small bowel, other parts of the gastrointestinal tract, and sometimes beyond. Moderate to severe abnormalities of villous architecture occur, with stunting and fusion of villi and lengthening of crypts. There may be evidence of mild in- flammation, with some cellular infiltration into the lamina propria. The endogenous stages of the parasite in the luminal surface are generally inconspicuous and appear as small (2–​8 μm) bodies, apparently superficially attached to the brush border, unevenly distributed over the apical cells, and within the crypts of the villi (Figs. 8.8.5.1 and 8.8.5.2). Peaking and apoptosis of infected cells have been reported. There is usually little intracellular change at the ultrastructural level beyond the attachment zone of the parasite. Rectal biopsy might reveal mild non​specific proctitis. Extensive and chronic involvement of the bile duct and gallbladder can occur in some patients with AIDS. Immunological response T cells play a crucial role in the elimination of cryptosporidial in- fections. In humans, T-​cell immunosuppression caused by other infection or chemotherapy increases susceptibility to infection. Moreover, severe cryptosporidiosis has been reported in individ- uals with mutations affecting the costimulatory CD40 or CD40L re- quired for T-​cell activation. In particular, CD4+ T cells are necessary to control infection and achieve sterile immunity in adults, whereas the role of CD8+ T cells is not fully established. In agreement with these findings, low levels of CD4+ T cells counts (<100 cells/μl) in- dicate a poor prognosis if infection occurs. The most important cytokine in resistance to Cryptosporidium is interferon-​γ and the principal sources are CD4+ T cells. Therefore, it appears that a Th1 immune response is involved in the clearance of the infection. IL12, produced by dendritic cells and macrophages upon exposure to antigens, plays an important role in the activation of interferon-​γ production by T cells. During infection, antigen-​specific antibodies can be detected in serum, including IgG, IgA, and IgM. If the in- fection is brought under control, the IgM titre declines very soon, whereas IgG may persist for several months. Experimental studies in the murine model and data from studies involving AIDS patients have shown that, although antibodies may contribute to the pro- tective immune response against the parasite, they are not normally essential for establishing host resistance. Possible pathogenic mechanisms The watery diarrhoea is characteristic of non​inflammatory infection of the small bowel, especially that associated with toxin-​producing

section 8  Infectious diseases 1428 organisms and enteric viruses. Several mechanisms have been sug- gested to explain the symptoms: reduction in absorptive capacity, particularly for water and electrolytes; increase in secretory capacity from crypt hypertrophy; osmotic effects from loss of brush-​border enzymes (e.g. disaccharidases) resulting in malabsorption of sugars, increased osmolality of chyme, and subsequent microbial fermenta- tion of sugars in the colon (which may account for the characteristic offensive smell); and toxic activity. Clinical presentation in otherwise healthy (immunocompetent) people Cryptosporidiosis in the immunocompetent person is a self-​ limiting, acute gastroenteritis with a variety of presenting symptoms. In cases where the time of exposure has been known, the incuba- tion period was about 5 to 7 days (range probably 2–​14 days; wider limits have been suggested but are unlikely). There may be a pro- drome of 1 day to a few days, with malaise, abdominal pain, nausea, and loss of appetite. Gastrointestinal symptoms start suddenly, the stools being described as watery, greenish with mucus in some cases, without blood or pus, and very offensive. Patients may open their bowels more than 20 times a day but more usually 3 to 6 times. Other symptoms include colicky, abdominal pain, especially after meals, anorexia, nausea, and vomiting, abdominal distension, and marked weight loss. Influenza-​like systemic effects, including malaise, head- ache, myalgias, and fever, commonly occur. Gastrointestinal symp- toms usually last about 7 to 14 days (average 12 days), but weakness, lethargy, mild abdominal pain, and intermittent loose bowels some- times persist for up to a further month. There is no evidence of transplacental transmission but infec- tion during late pregnancy may cause metabolic disturbances in the mother, leading to the infant’s failure to thrive. Failure to thrive has also been observed in older infants and children and may be associated with persistent infection and enteropathy, especially in developing countries. Reported sequelae include pancreatitis (associated with se- vere abdominal pain), toxic megacolon, and reactive arthritis. In immunocompetent patients, deaths are rarely attributable to cryptosporidiosis. Recent studied in the United Kingdom and Sweden have fur- ther demonstrated that the impact of cryptosporidiosis on public health extends beyond that of acute diarrhoeal illness. Notably, an increased risk of extra-​intestinal sequelae (joint pain, eye pains, recurrent headache, and fatigue) is associated with infection with C. hominis but not with C. parvum. A follow-​up study after two large waterborne outbreaks in Sweden found that outbreak cases were more likely to report diarrhoea, watery diarrhoea, abdominal pain, joint pain, fatigue, and nausea compared to non​cases. Therefore, these studies showed a significant burden of illness even after out- breaks are over. Clinical presentation in immunocompromised patients Susceptibility to cryptosporidiosis and the severity of the disease is increased in patients who are immunocompromised because of AIDS, hypo-​ or agammaglobulinaemia, severe combined immuno- deficiency, leukaemia, malignant disease, and bullous pemphigoid. Disease susceptibility and severity are also increased during im- munosuppressive treatment with cyclophosphamide and cortico- steroids, as in patients undergoing bone marrow transplantation, and in children immunosuppressed by measles and chickenpox, especially where there is associated malnutrition. Infection in pa- tients with leukaemia may be unusually severe and has sometimes proved fatal, particularly when associated with aplastic crisis, and may then require modification of chemotherapy to control the infection. Symptoms of cryptosporidiosis are generally similar to those in immunocompetent patients but often develop insidiously. In those with late-​stage AIDS and very low CD4 cell counts, or in some other profound deficiency states, diarrhoea may be frequent, profuse, and watery, like cholera. Patients may open their bowels frequently, passing up to 20 litres of infected fluid stool per day; persistent nausea and vomiting is usually associated with severe diarrhoea and suggest a poor prognosis. Associated symptoms include colicky, abdom- inal pain often associated with meals, severe weight loss, weakness, malaise, anorexia, and low-​grade fever. Infection in immunocom- promised patients can spread to the pharynx, oesophagus, stomach, duodenum, jejunum, ileum, appendix, colon, rectum, gallbladder, bile duct, pancreatic duct, and the bronchial tree. Cryptosporidial cholecystitis (presenting with severe right upper quadrant ab- dominal pain), sclerosing cholangitis, pancreatitis, hepatitis, and respiratory-​tract symptoms may occur, with or without diarrhoea. The clinical picture might include other features of HIV infection and there is often coinfection with other pathogens such as cyto- megalovirus, Pneumocystis jiroveci, and Toxoplasma gondii. Patients with less severe impairment of immunity can experience resolution or a more chronic course, with less profuse diarrhoea, sometimes with remission and then recurrence, possibly associ- ated with biliary tract involvement. Except in those patients whose immune suppression can be relieved by stopping immunosuppres- sant drugs, or, in the case of HIV, intensifying antiretroviral therapy, severe symptoms may persist until the patient dies. This is either because of dehydration, acid–​base or electrolyte disturbances and cachexia, from some other opportunistic infection or malignant dis- ease, or a combination of these. Recently, a role of C.  parvum infection in the development of cancer in the digestive tract has been suggested. A zoonotic C. par­ vum isolate, that caused a fulminant cryptosporidiosis in a trans- planted patient, was able to induce invasive gastrointestinal and biliary adenocarcinoma in severe combined immune deficiency (SCID) mice. The hypothesis that cryptosporidiosis increases the risk for some types of gastrointestinal cancer is of interest, even though experimental evidence for this correlation is limited to animal models with immunodeficiency. Laboratory investigations In early acute cases, the stools are usually watery, greenish, some- times with mucus but without blood or pus. Peripheral leucocytosis and eosinophilia are found rarely. Serum electrolyte abnormalities will develop in patients who become severely dehydrated. In im- munocompromised patients with cryptosporidial cholecystitis, serum alkaline phosphatase and γ-​glutamyl transpeptidase levels increase, while aminotransferase and bilirubin levels may remain normal. In patients with AIDS, cytomegalovirus and Cystoisospora belli are commonly associated with Cryptosporidium; in immunocompetent

8.8.5  Cryptosporidium and cryptosporidiosis 1429 patients, mixed infection with Campylobacter, Giardia, and Cyclospora species may be found. In the bowel mucosa, there is histological evidence of entero- cyte damage, villous blunting, and inflammatory-​cell infiltration of the lamina propria; cell peaking and apoptosis have been reported. Histopathological appearances of the affected biliary tract resemble primary sclerosing cholangitis. Radiographic abnormalities in- clude dilatation of the small bowel, mucosal thickening, prominent mucosal folds, and abnormal motility. In the biliary system, ab- normalities include dilated distal biliary ducts, stenosis with an ir- regular lumen, and other changes reminiscent of primary sclerosing cholangitis. Differential diagnosis The absence of blood, pus cells, or Charcot–​Leyden crystals may distinguish cryptosporidiosis from some acute bacterial diarrhoeas, and that associated with amoebiasis and cystoisosporiasis. In im- munocompetent patients, the symptoms of cryptosporidiosis re- semble those of giardiasis or cyclosporiasis. Intense abdominal pain and cramps are generally more common in cryptosporidiosis, but bloating and weakness less common. In immunocompromised pa- tients, especially in those with AIDS, cystoisosporiasis is clinically indistinguishable, but can be diagnosed by finding the organisms in the stool, where Charcot–​Leyden crystals may also be found. This infection responds to treatment with co-​trimoxazole, as does cyclosporiasis. Treatment of cryptosporidiosis Several groups may benefit from an effective treatment, particularly patients with HIV/​AIDS, transplant recipients, patients undergoing cancer chemotherapy, those with severe malnutrition, and older people. However, existing therapeutics for other apicomplexan dis- eases are largely ineffective against Cryptosporidium infection, prob- ably because of the unique intracellular, extracytoplasmic location of cryptosporidia, and limited understanding of the host–​parasite interaction. Hundreds of drugs have been tested in the laboratory, but results have suggested that only paromomycin, azithromycin, spiramycin, and albendazole are partially effective. The failure to develop effective therapy for cryptosporidiosis is also related to the limited attempts undertaken by health agencies and the pri- vate sector, mostly because of a perceived limited market for such drugs in developed countries. Recent developments, which include the sequencing of the genomes of C. parvum and C. hominis and the possibility of genetic engineering of the parasite, will help the identification of new molecular targets for drug development. The availability of a substantial number of chemical libraries for drug discovery should also facilitate screening for effective drugs. Today, the therapy of choice is nitazoxanide (2-​acetyloloxy-​N-​ (5-​nitro-​2-​thiazolyl) benzamide), a synthetic agent that has a dem- onstrated activity against a broad range of parasites as well as some bacteria. In vitro studies showed inhibition of growth at concen- trations of less than 10 μg/​ml, and studies in adults have shown that single doses of up to 4 g are well tolerated without important adverse effects. Nitazoxanide has been approved by the US Food and Drug Administration (FDA) for the treatment of cryptospor- idiosis in immunocompetent patients in the United States. There is little evidence for efficacy of nitazoxanide in immunocompromised individuals. Immunocompromised patients with persistent severe diarrhoea, malabsorption, and other complications may require prolonged palliative treatment. They should avoid excess milk, as lactose in- tolerance may develop. Parenteral feeding and fluid, electrolyte, and nutrient replacement may be needed. Antiperistaltic agents such as loperamide, diphenoxylate, or opiates may increase abdominal pain and bloating. Antiemetics may be needed for symptomatic re- lief. Temporary relief of biliary obstruction has been achieved by endoscopic papillotomy and of cholecystitis by cholecystectomy. Diarrhoea and vomiting, however, may prove intractable. Antiretroviral therapy (ART) is the treatment of choice for cryptosporidiosis in immunocompromised patients with HIV ART is effective against cryptosporidiosis and acts both by immune re- constitution and direct inhibition of parasite proteases. Laboratory detection and diagnosis The characteristic endogenous stages (Figs. 8.8.5.1 and 8.8.5.2) may be found in histological sections, using light and electron mi- croscopy, but diagnosis is usually by detection of oocysts in stools. Oocysts have also been found in vomit and sputum in some cases, especially those associated with AIDS. The oocysts of C. parvum and C. hominis are spherical or slightly ovoid, about 4–​6 μm, and appear refractile in wet faecal preparations with a highly refractile inner body, the cytoplasmic residuum; the four sporozoites within the oocyst may be distinguished with difficulty using special optical systems (Figs. 8.8.5.3–​8.8.5.12). Several conventional stains have been adapted for diagnostic purposes, such as the modified Ziehl–​Neelsen method and phenol-​ auramine fluorescent stain. Fig. 8.8.5.3  Modified Giemsa-​stained faecal smear showing oocysts of C. parvum, examined with × 100 oil-​immersion objective lens. The uniformity of size (4.5–​5 µm) but variability of staining of oocysts can be seen. The eosinophilic nuclei and basophilic bodies of the sporozoites can be clearly seen within the oocysts that have taken up the stain.

section 8  Infectious diseases 1430 Fig. 8.8.5.4  Modified Ziehl-​Neelsenstained faecal smear showing oocysts of C. parvum examined with × 100 oil-​immersion objective lens. The uniformity of size (4.5–​5 µm) but variability of staining of oocysts can be seen. Fig. 8.8.5.5  Modified Ziehl-​Neelsen-​stained faecal smear showing oocysts of C. parvum. The uniformity of size (4.5–​5 µm) is apparent but the oocysts in this preparation show a definite increase in refractility and marked failure to take up the stain (identity confirmed by immunofluorescence and electron microscopy). Fig. 8.8.5.6  Modified Ziehl-​Neelsen-​stained faecal smear showing oocyst-​like bodies (mushroom spores) examined with × 100 oil-​immersion objective lens (from specimen submitted to Reference Unit for identification). Fig. 8.8.5.7  Modified Ziehl-​Neelsen-​stained faecal smear showing oocyst-​like bodies (mould spores) examined with × 100 oil-​immersion objective lens. The spores are uniform in size but a little smaller (4.0 µm) than oocysts of C. parvum. They are generally more uniform in their acid-​fast staining (identity confirmed by mycological culture and electron microscopy). Fig. 8.8.5.8  Phenol-​auramine/​carbol fuchsin-​stained faecal smear showing oocysts of C. parvum, examined with × 720 dry objective lens (screening magnification) on a fluorescence microscope. Fig. 8.8.5.9  Phenol-​auramine/​carbol fuchsin-​stained faecal smear showing oocysts of C. parvum, examined with × 100 oil-​immersion objective lens on a fluorescence microscope.

8.8.5  Cryptosporidium and cryptosporidiosis 1431 Direct fluorescent-​antibody tests, which detect intact organisms using monoclonal antibodies that label the oocyst wall, are widely used due to their excellent sensitivity and specificity. Detection of Cryptosporidium soluble antigens in faecal samples by an enzyme-​linked immunosorbent assay (ELISA) or by an immuno-​ chromatographic lateral flow assays is very easy to perform and particularly useful for the screening of large numbers of speci- mens, albeit its specificity is limited by cross-​reactions with other antigens of parasitic and non​parasitic origin that can generate false positives. None of the abovementioned methods can differen- tiate Cryptosporidium species and genotypes. Therefore, molecular methods, including conventional and real-​time polymerase chain reaction, are increasingly used for environmental or epidemiological research purposes. The high specificity and sensitivity of PCR-​based methods and the possibility of detecting multiple gastrointestinal pathogens in a single reaction, suggest that these methods may find application in routine diagnostics in the close future. Standardization of approach to screening and to reporting is essential for epidemiological purposes. Ideally, all stool samples from cases of diarrhoea should be screened; restriction, where un- avoidable, should be based on age group (see demography) and not on factors such as stool consistency. Concentration of stool speci- mens is not usually required for diagnosis in acute cases. Fungal spores, yeasts, cysts of Balantidium, sporocysts of Cystoisospora, and oocysts of Cyclospora might be mistaken for oocysts of Cryptosporidium. Infectivity, resistance, and control Infectivity In studies using monkeys and lambs, the infective dose for C. parvum was fewer than 10 oocysts. In human volunteer studies in the United States of America, the minimum infective dose for C. parvum and C. hominis appeared to be similar (ID50 was 132 and 83, respectively). In contrast to C. parvum, however, C. hominis elicted a serum IgG response in most infected persons. A recent study has demonstrated the infectivity of Cryptosporidium melea­ gridis in healthy adult volunteers, albeit the minimum infective dose was not determined. Resistance and disinfection Oocysts can survive for several months in a cool, moist environ- ment but are highly susceptible to desiccation, prolonged freezing, and moderate heat (pasteurization temperatures). They are re- markably resistant to most disinfectants and antiseptics, including chlorine at concentrations far greater than those used in water treatment and even to glutaraldehyde under normal use conditions. Some disinfectants may be more effective if used at elevated tem- perature (37°C or higher). Oocysts are sensitive to 10 volume (3%) hydrogen peroxide, to appropriate levels of ozone, and to medium or high-​pressure ultraviolet. In hospitals, adequate disinfection of faecal contamination or of endoscopes is difficult. If such instruments have been used for patients with cryptosporidiosis, prolonged immersion in glutaraldehyde at a temperature higher than 37°C, or in hydrogen peroxide, after careful cleaning, may be required to ensure safety. Fig. 8.8.5.10  Fluorescent dye-​tagged monoclonal antibody-​stained faecal smear showing oocysts of C. parvum, examined with × 50 oil-​ immersion objective lens (screening magnification) on a fluorescence microscope. The suture or associated surface cleft or fold, through which the sporozoites are released, can be seen. Fig. 8.8.5.11  Modified Ziehl-​Neelsen-​stained sputum smear from an AIDS patient with respiratory involvement (examined with × 100 oil-​ immersion objective lens). The C. parvum bodies present may include endogenous (tissue) stages attached to exfoliated cells. For this reason, oocyst wall-​specific indirect immunofluorescence may show a poor reaction. There may also be less uniformity of size and differences in the staining appearance of the internal structures. Fig. 8.8.5.12  Toluidine blue-​stained semithin section of human rectal biopsy tissue of an AIDS patient with cryptosporidiosis. The apparent pseudo-​external location of the parasite can be seen, the true location being intracellular but extracytoplasmic. Plates for this chapter were kindly provided from photographs by A. Curry and
D. P. Casemore.

8.8.6 Cyclospora and cyclosporiasis 1432

8.8.6 Cyclospora and cyclosporiasis 1432

section 8  Infectious diseases 1432 Control of transmission Primary control is by limiting the opportunity for faecal–​oral trans- mission, both direct and indirect. Symptom-​free subjects not in con- tact with immunocompromised patients can normally be permitted to work if their hygiene is scrupulous. Spread via fomites is possible but this route is limited by the susceptibility of oocysts to desiccation. Patients with AIDS are more susceptible to infection with uncommon species or genotypes and advice may be needed to limit exposure. Contamination of water supplies is inevitable, even in developed countries, and may be the source of some sporadic cases as well as outbreaks. When a public advisory notice is issued to boil water, raising the water just to boiling point is sufficient. In general, bot- tled water and water from point-​of-​use filters are unlikely to con- tain parasites but may carry an increased bacterial load, the health significance of which is uncertain for the immunocompromised. Patients with AIDS and others who are profoundly compromised should be advised never to drink water that has not been boiled or filtered through a suitable device. Users of filters should remember that these devices may concentrate potential pathogens and care is needed in replacing and disposing of filter elements. Hospitals involved in the care of profoundly immunocomprom- ised patients should be particularly vigilant in the management of patients with cryptosporidiosis. Long-​term arrangements should be made for the provision of safe water for the immunocompromised to avoid difficulties when a notice to boil water is issued. FURTHER READING Cacciò SM, Putignani L (2014). Epidemiology of human cryptospor- idiosis. In: Cacciò SM, Widmer G, editors. Cryptosporidium: para­ site and disease, pp. 43–​79. Springer, Vienna. Chalmers RM, Katzer F (2013). Looking for Cryptosporidium: the ap- plication of advances in detection and diagnosis. Trends Parasitol, 29, 237–​51. Checkley W, et al. (2015). A review of the global burden, novel diag- nostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis, 15, 85–​94. Hunter PR, Nichols G (2002). Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin Microbiol Rev, 15, 145–​54. Sparks H, et al. (2015). Treatment of Cryptosporidium: what we know, gaps, and the way forward. Curr Trop Med Rep, 2, 181–​7. Xiao L (2010). Molecular epidemiology of cryptosporidiosis: an up- date. Exp Parasitol, 24, 80–​9. 8.8.6  Cyclospora and cyclosporiasis Paul Kelly and Ralph Lainson† ESSENTIALS Most species of Cyclospora (Protozoa:  Apicomplexa:  Eimeriidae)
are parasites of various reptiles and mammals. C.  cayetanensis, which probably infects only humans, is transmitted by way of re- sistant oocysts voided in the faeces and contaminating food or water. Distribution is worldwide, particularly in regions with a low level of hygiene. Clinical presentation is with explosive outbreaks of acute diarrhoea, with this infection now regarded as an important causa- tive agent of traveller’s diarrhoea. Diagnosis is dependent on detec- tion of oocysts in faeces by direct examination or in stained faecal smears. Aside from supportive care, treatment with trimethoprim–​ sulfamethoxazole has proved effective in eliminating the parasite in immunocompetent patients, but relapses are common in those with AIDS. Prevention is by ensuring good general hygiene, and in areas of high endemicity water should be boiled before drinking or use in preparation of fruits/​vegetables that are to be eaten raw. Introduction Species of the coccidian genus Cyclospora (Protozoa:  Apicomple xa: Eimeriidae) have been recorded in invertebrates (millipedes), reptiles (principally snakes), insectivores (moles), rodents, and pri- mates (monkeys and humans). The genome of this parasite has been partially sequenced. Endogenous development of most species is within the epithe- lial cells of the small intestine, culminating in the production of oocysts, which are voided in the faeces and serve as the means of transmission. Small coccidial oocysts detected in the faeces of patients with diarrhoea in Papua New Guinea almost certainly represented the first discovery of Cyclospora in humans in 1979, but the parasite was not identified to generic level. Oocysts of the same parasite, seen by other authors in patients with diarrhoea, were for many years referred to as ‘cryptosporidium-​like oocysts’, ‘cyanobacterium-​like bodies’ (bodies resembling blue-​green algae), or even ‘fungal spores’, and it was not until 1992 that the parasite was named as Cyclospora cayetanensis. Life cycle Cyclospora species have been most extensively studied in non-​ human hosts. Asexual reproduction (merogony) (Fig. 8.8.6.1a) is followed by the production of female gametocytes (macrogamonts) (Figs. 8.8.6.1b–​f) and male gametocytes (microgamonts) that pro- duce many flagellated gametes (Figs. 8.8.6.1g–​j). Following fer- tilization of the female parasites, the zygotes develop a resistant membrane (Fig. 8.8.6.1k). The resulting oocysts (Fig 8.8.6.2) are voided, unsporulated, in the host’s faeces. During periods varying from a few days to 1 or 2 weeks, depending on the species of Cyclospora and ambient temperature, the zygote within the oocyst (Fig. 8.8.6.3a) undergoes division to produce two sporoblasts (Fig. 8.8.6.3b), each of which develops a resistant membrane, the sporocyst (Fig. 8.8.6.3c). Division of each sporoblast then gives rise to two elongate sporozoites, leaving a conspicuous residual body (Figs. 8.8.6.2b and 8.8.6.3c). The sporo- zoites are the stages that infect the next host when oocysts are in- gested with contaminated food or water. † It is with great regret that we report that Ralph Lainson died on 5 May, 2015.

1433 8.8.6  Cyclospora and cyclosporiasis Epidemiology Failure to infect a variety of animals experimentally or to detect C. cayetanensis in those living in or near houses with human infection has led to the conclusion that humans are the specific host of this coccidian and the sole source of its oocysts. The parasite is globally distributed, although risk of infection is greatest in developing countries with low standards of hygiene where prevalence rates up to 40% (Peru) have been reported. It is particularly prevalent in Central America and southern Asia, and uncommon in southern Africa. Serious outbreaks of acute diarrhoea have been re- ported across the world, related to consumption of contaminated foods such as raspberries and salads. Oocysts of C. cayetanensis have been detected on green leafy vegetables, in sewage, and even in tap water. Airborne and zoonotic transmission is suspected but not confirmed. Clinical features An acute, watery, and non​bloody diarrhoea is variously accom- panied by abdominal pain, steatorrhoea, headache, fever, nausea, and general malaise. The diarrhoea can be persistent and last for several weeks. Asymptomatic infections are known to occur, notably in the indigenous population of developing countries. Infection is more pro- longed in immunocompromised hosts, such as in AIDS or in trans- plant recipients. (a) (f) (g) (i) (j) (k) (h) (b) (c) (d) (e) 10 μm Fig. 8.8.6.1  Intracellular development in epithelial cells of the ileum (haematoxylin and eosin stained sections) in a typical life cycle of a Cyclospora species: (a) segmented meronts; (b–​e) developing macrogamonts; (f) mature macrogamont with small wall-​forming bodies (arrow) and large wall-​forming bodies (arrowhead); (g, h) developing microgamonts; (i, j) mature microgamonts shedding microgametes; (k) intracellular zygote, with developing oocyst wall (OW). From Lainson R (2004). The genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil—​a review. Mem Inst Oswaldo Cruz, 100, 103–​110, with permission.

section 8  Infectious diseases 1434 Diagnosis Diagnosis is dependent on the demonstration of oocysts of C. cayetanensis in the faeces by direct microscopic examination. Flotation methods, using saturated sugar or aqueous zinc sulphate solutions, are useful in concentrating the oocysts, which measure from 8.0 to 10.0 μm in diameter (average 8.6 μm). The living oocysts are autofluorescent using ultraviolet illu- mination, which is useful for rapid diagnosis. In addition, most diagnostic laboratories use a variety of staining methods to colour the oocysts in faecal smears fixed in 10% formalin: notably, modi- fied Ziehl–​Neelsen acid-​fast staining and safranin stain. The polymerase chain reaction with primers specific for C. cayetan­ ensis also affords a highly sensitive, but more costly, diagnostic technique. There are four other intestinal coccidia that infect humans and may produce similar symptoms, but they are morphologic- ally readily differentiated from C.  cayetanensis (Fig. 8.8.6.4). Cryptosporidium oocysts are only 4.5–​5.0 μm in diameter and they contain four naked sporozoites. Cystoisospora belli oocysts are elongated, measure from 25 to 33 μm in length and from 12 to 16 μm in width, and have two sporocysts, each of which contains four sporozoites. Sarcocystis hominis and S. suihominis oocysts are easily differentiated from C. cayetanensis oocysts by their larger size (average 16 μm × 10.5 μm) and ellipsoidal shape. Pathology Histology of jejunal biopsies from patients with cyclosporiasis has shown blunting and widening of infected villi and an in- tense lymphocytic infiltration in the lamina propria and overlying epithelium (Fig. 8.8.6.5), features which are common to all coc- cidian infections of the small intestine. Treatment Co-​trimoxazole (960 mg two times daily for 1 week) has proved ef- fective in eliminating the parasite in immunocompetent patients and has been shown successfully to control relapses in those with AIDS by the administration of 960 mg three times a week, indefin- itely. Ciprofloxacin (500 mg two times daily for 1 week) is recom- mended for patients who react badly to sulphonamides. (a) (b) 10 μm Fig. 8.8.6.2  Extracellular stages in a typical life cycle of a Cyclospora:
ss(a) unsporulated oocyst in the intestinal lumen; (b) sporulated and ruptured oocyst in faeces. L, lumen; Sp, sporozoite; Sr, sporocystic residuum. Bar, 10 μm (all figures). From Lainson R (2004). The genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil—​a review. Mem Inst Oswaldo Cruz, 100, 103–​110, with permission. (a) (b) (c) Fig. 8.8.6.3  Developing oocysts of C. cayetanensis as seen by Nomarski interference-​contrast microscopy: (a) unsporulated oocyst; (b) formation of the two sporoblasts; (c) formation of the two sporocysts. Bar, 5 μm. From Ortega YR, Gilman RH, Sterling CR (1994). A new coccidian parasite (Apicomplexa: Eimeriidae) from humans. J Parasitol, 80, 625–​9, with permission.

1435 (a) (b) 10 μm (c) (d) Fig. 8.8.6.4  Faecal stages of the five intestinal coccidia that infect humans: (a) oocyst of Cryptosporidium parvum, (b) oocyst of Cyclospora cayetanensis, (c) sporocyst of Sarcocystis hominis or S. suihominis (the two are morphologically indistinguishable), and (d) oocyst of Cystoisospora belli. Bar, 10 μm. (c) (a) (d) (b) Fig. 8.8.6.5  Comparison of normal human jejunal villi (a, c) and villi infected with C. cayetanensis (b, d):
low-​power appearance (a, b) and high-​power appearance (c, d). Note the blunting and widening of the villi, with inflammatory lymphocytic infiltrate in the lamina propria and infiltration of overlying epithelium. From Ortega YR, et al. (1997). Pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-​cycle stages. J Infect Dis, 176, 1584–​9, with permission. 8.8.6  Cyclospora and cyclosporiasis

8.8.7 Cystoisosporiasis 1436

8.8.7 Cystoisosporiasis 1436

section 8  Infectious diseases 1436 Prevention As with all other organisms dependent on faecal–​oral transmission, simple precautions will help prevent infection with C. cayetanensis. Water should be boiled before drinking or when used to wash fruits (although these are best peeled) or green leafy vegetables that are to be eaten raw. These measures are important in endemic areas and also when consuming fruit or vegetables that are imported from such regions, as seen with the serious outbreaks of cyclosporiasis in the United States of America due to unwashed raspberries imported from Guatemala. FURTHER READING Ashford RW (1979). Occurrence of an undescribed coccidian in man in Papua New Guinea. Ann Trop Med Parasitol, 73, 497–​500. Chacín-​Bonilla L (2010). Epidemiology of Cyclospora cayetanensis:
a review focusing in endemic areas. Acta Trop, 115, 181–​93. Eberhard ML, Pieniazak NJ, Arrowood MJ (1997). Laboratory diag- nosis of Cyclospora infections. Arch Pathol Lab Med, 121, 792–​7. Lainson R (2005). The genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil—​a review. Mem Inst Oswaldo Cruz, 100, 103–​110. McDonald V, Kelly MP (2005). Intestinal coccidia: cryptosporidi- osis, isosporiasis, cyclosporiasis. In: Cox FEG, et al. (eds) Topley & Wilson’s microbiology & microbial infections: parasitology, 10th edition, pp. 399–​421. Hodder Arnold ASM Press, London. Ortega YR, Sanchez R (2010). Update on Cyclospora cayetanensis, a food-​borne and waterborne parasite. Clin Microbiol Rev, 23, 218–​34. Ortega YR, et  al. (1992). Cyclospora cayetanensis:  a new protozoan pathogen of humans. Abstract 289 in proceedings of the 41st annual meeting of the American Society of Tropical Medicine and Hygiene. Am J Trop Med Hyg, (Suppl), p. 210. Ortega YR, et al. (1997). Pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-​ cycle stages. J Infect Dis, 176, 1584–​9. Qvarnstrom Y, et al. (2015). Draft genome sequences from Cyclospora cayetanensis oocysts purified from a human stool sample. Genome Announcements, 3, e01324–​15. 8.8.7  Cystoisosporiasis Louis M. Weiss ESSENTIALS Cystoisospora belli is a coccidian protozoan that colonizes epithelial cells of the small intestine. Infection occurs by ingestion of para- site oocysts in water or food. Clinical features of infection include watery diarrhoea, dehydration, fever, and weight loss. Cystoisospora belli forms tissue cysts that allow for relapses of this infection. Cystoisosporiasis presenting as chronic diarrhoea is an opportunistic infection associated with HIV infection. Diagnosis is by microscopic examination of faecal specimens for C. belli oocysts, which show blue autofluorescence under ultraviolet light. Cystoisospora belli infection, even in patients with AIDS, responds rapidly to treatment with co-​trimoxazole. Introduction Cystoisospora belli causes diarrhoea in both immune competent and immune deficient patients. This diarrhoeal syndrome, however, is both more chronic and more frequently seen in patients with AIDS. Infection with this pathogen can be acquired in any country, but it is more common in tropical environments. Unlike most causes of chronic diarrhoea in the setting of AIDs, infection with this pathogen responds rapidly to drug therapy. Historical perspective While previously referred to as Isospora belli, and occasionally as I. hominis (some of these reported cases may have been due to mis- identified Sacrcocystis species), both the life cycle of this human pathogen and molecular phylogeny indicate that this organism should be a member of genus Cystoisospora, which contains over 200 species, and not Isospora. Organisms formerly included in the genus Isospora that are parasitic to mammals have now been as- signed to the genus Cystoisospora and the generic name Isospora has been retained for avian parasites. Aetiology, pathogenesis, and pathology Cystoisospora belli is a parasite of the human small intestine that causes diarrhoea. There is limited evidence that C.  belli infects non​human hosts (e.g. oocysts of C. belli have been isolated from dog faeces in India and this parasite has been transmitted experi- mentally to gibbons). Cystoisospora belli is the only member of this genus that has been clearly documented to cause human infection. There are a few reports from South Africa, in the literature prior to 1955, of a second species, C. natalensis, causing infections; however, these reports have not been subsequently confirmed. The mechan- isms responsible for the watery diarrhoea seen in infection by this pathogen are not known. The life cycle of C. belli generally occurs in a single host, with the formation of oocysts occurring 9–​17 days following infection which are passed in faeces. C. belli oocysts are ellipsoidal structures that are excreted in the faeces of infected individuals (Fig. 8.8.7.1). Studies of Cystoisospora species that parasitize non​human hosts indicate that infection occurs by ingestion of oocysts that contain sporozoites that then penetrate small intestinal epithelial cells. Evidence exists that this organism is facultatively heteroxenous (i.e. utilizes two-​hosts) and forms tissue cysts. These tissue cysts are probably the source of relapsing infections seen in immune com- promised hosts. Oocysts are generally non​infectious when passed in the faeces and complete sporulation within 1 to 5 days depending

8.8.7  Cystoisosporiasis 1437 upon the environmental conditions. Shedding of oocysts usually lasts for 30 to 50 days; however, in immune suppressed patients shedding might be prolonged, lasting over 6 months, and in such patients recrudescence of oocyst shedding occurs. This prolonged shedding is presumably due to activation of the dormant monozoic tissue cysts that can be found in a variety of tissues (e.g. lamina propria, mesenteric lymph nodes, liver, and spleen). The presence of these tissue cysts suggests that a paratenic host could be involved in the life cycle of C. belli, as has been shown to occur in Cystoisospora spp. which infect cats and dogs. Epidemiology C.  belli infection has been documented in immune competent hosts as an occasional cause of travellers’ diarrhoea and in immune suppressed individuals where it can cause a chronic diarrhoeal syndrome. In a study of 397 HIV-​infected patients in Venezuela, 56 (14%) were found to have C. belli infection (as judged by the presence of oocysts in faecal specimens). Of these 56 patients with C. belli infection, 98% had diarrhoea. C. belli infections ac- counted for 2–​3% of AIDS-​defining conditions in the 1980s, but with the widespread use of co-​trimoxazole (trimethoprim-​ sulfamethoxazole; TMP-​SMX) prophylaxis for pneumocystis pneumonia, this decreased to 0.1% by the late 1990s. Depending on the geographic region, C. belli infection was aetiologic in up to 25% of cases of chronic diarrhoea prior to the wide spread use of combination antiretroviral therapy, but has declined dramatically since its widespread use. Vehicles for transmission of C. belli oocysts to human subjects have not been identified, but presumably include water and food. Oocysts of this parasite are among the human pathogens that were found on cockroaches in a study in Nigeria, suggesting that in- sects could act as transport hosts for this pathogen. Infection with C.  belli is more commonly found in tropical, subtropical, and warm temperate region; however, indigenous infection has been reported from temperate regions. Transmission is most likely due ingestion of sporulated oocysts, and food-​ and water-​borne outbreaks have occurred. Sexual practices that permit faecal oral spread are also associated with infection. As with most faecal oral pathogens, improved sanitation and water quality decreases the risk of transmission. Clinical features In immune competent hosts a self-​limiting diarrheal illness results that lasts 2 to 3 weeks and is associated with malaise, weight loss, cramps, watery diarrhea, steatorrhea, headache, low grade fever, ab- dominal pain, vomiting, dehydration, and weight loss is seen about a week after ingestion of oocysts. In general, clinical disease is more severe in infants, young children, and in immune compromised hosts. Infection can result in a very severe illness and fatalities have occurred. Blood is not usually present in faeces. Eosinophilia is ob- served in many patients. Oocysts can be present in the faeces or bi- opsies for several months to years. Recurrences occur and have been reported as long as 10 years after initial infection. In patients infected with HIV, C.  belli infection is associated with chronic watery diarrhoea, abdominal cramps, nausea, fever, and weight loss. Severe dehydration and profound hypokalaemia can result from diarrhoea attributable to C. belli infection in HIV-​ infected patients. This can be associated with fever and weight loss. Infection has been described in patients on systemic corticosteroids being treated for eosinophilic gastroenteritis, in patients with renal or liver transplants, and in patients with haematological malignan- cies such as Hodgkin’s disease, non-​Hodgkin’s lymphoma, and adult T-​cell leukaemia. Persistent diarrhoea ascribed to isosporiasis has been described in a few HIV-​infected patients on antiretroviral therapy whose circulating CD4+ T-​cell counts were within a range that suggested immune competence. Rarely, extraintestinal C. belli infection has been described in patients with AIDS; in the relevant patients, tissues parasitized by C. belli have included gallbladder epi- thelium, liver, spleen, and mesenteric lymph nodes. In general, im- mune suppressed hosts respond to TMP-​SMX and other anti-​C. belli treatments. Laboratory diagnosis Cystisosporiasis can be diagnosed by microscopic examination of faecal specimens for C. belli oocysts. Although these structures are relatively large (20–​30 μm in length), they are translucent and can be difficult to see in unstained samples. Concentration techniques like formalin-​ethyl acetate sedimentation or sucrose centrifugal float- ation are helpful when few oocysts are present. Oocyst detection is increased by staining with carbol fuchsin, which stains oocyst in- ternal structures red, or by incubation with lactophenol cotton blue. Staining, however, can be variable and some oocysts do not stain. An alternative approach is to examine faecal smears under ultraviolet light; with this type of illumination C.  belli oocysts demonstrate blue autofluorescence. There is neither a commercial immunofluor- escence stain nor copro-​antigen assay available for the detection of this pathogen in stool. Molecular assays (e.g. polymerase chain re- action), using primers based on the small subunit RNA gene have been described and three genotypes of C. belli can be identified using restriction length polymorphisms of the small subunit RNA locus. Fig. 8.8.7.1  Light micrograph of a Cystoisospora belli oocyst (×2500). Courtesy of Dr William L. Current. From Garcia LS (2001). Diagnostic medical parasitology, 4th edition. ASM Press, Washington DC, with permission.

8.8.8 Sarcocystosis (sarcosporidiosis) 1438

8.8.8 Sarcocystosis (sarcosporidiosis) 1438

section 8  Infectious diseases 1438 Polymerase chain reaction assays have been able to identify infec- tions that were negative by traditional stool microscopy. Treatment and prognosis The drug of choice for the treatment of C. belli-​induced diarrhoea C.  belli is oral co-​trimoxazole (TMP-​SMX). There are no docu- mented reports of drug resistant infections. A dose of TMP 160 mg-​ SMX 800 mg 2–​4 times a day for 10–​14 days results in rapid (within 2–​3 days) clearance of parasites and diarrhoea. In patients with HIV-​ 1 infection and CD4+ cell counts less than 200 cells/μl secondary prophylaxis with TMX 320 mg/​SMX 1600 mg once daily or three times a week prevents relapses. In general, secondary prophylaxis can be stopped if the CD4+ count exceeds 200/μl; however, there are case reports of chronic infection with relapse still occurring despite patients have CD4+ counts above 200/μl and having re- ceived primary therapy with TMP/​SMX. In patients unable to tol- erate sulfonamides due to allergy or intolerance there is no standard treatment. Pyrimethamine has been used for both treatment and secondary prophylaxis in patients with AIDS and sulfa allergy with success. When pyrimethamine is administered it should be given with folinic acid (5–​10 mg/​day) to minimize bone marrow suppres- sion. Ciprofloxacin can be used as an alternative treatment, although it is less effective than either TMP/​SMX or pyrimethamine. In a ran- domized study of 22 patients with cystoisosporiasis and HIV infec- tion, 10/​10 patients on TMP/​SMX had cessation of diarrhoea within 2 days and 10/​12 on ciprofloxacin (500 mg BID) had a cessation of diarrhoea within 4.5 days. All three patients who had persistent C. belli oocysts in their stools responded to treatment with TMP/​ SMX with clearance of the parasite. In patients who responded to ciprofloxacin continued prophylaxis with ciprofloxacin prevented recurrence of disease. Nitazoxanide has also been used to treat C. belli infections. Two patients on 500 mg nitazoxanide twice daily for 3 days were oocyst negative after treatment; a patient treated with 500 mg nitazoxanide twice daily for 7 days became oocyst negative by day 14 after treatment. FURTHER READING Boyles TH, et al. (2012). Failure to eradicate Isospora belli diarrhoea despite immune reconstitution in adults with HIV—​a case series. PLoS One, 7, e42844. Field AS (2002). Light microscopic and electron microscopic diagnosis of gastrointestinal opportunistic infections in HIV-​positive patients. Pathology, 34, 21–​35. Fox LM, Saravolatz LD (2005). Nitazoxanide:  a new thiazolide antiparasitic agent. Clin Infect Dis, 40, 1173–​80. Lindsay DS, et al. (1997). Examination of extraintestinal tissue cysts of Isospora belli. J Parasitol, 83, 620–​5. Pape JW, Verdier RI, Johnson WD Jr (1989). Treatment and prophy- laxis of Isospora belli infections in patients with the acquired im- munodeficiency syndrome. New Eng J Med, 320, 1044–​7. Weiss LM, et al. (1988). Isospora belli infection: treatment with pyri- methamine. Ann Int Med, 109, 474–​5. Verdier RI, et al. (2000). Trimethoprim-​sulfamethoxazole compared with ciprofloxacin for treatment and prophylaxis of Isospora belli and Cyclospora cayetanensis infection in HIV-​infected patients—​a randomized, controlled trial. Ann Int Med, 132, 885–​8. 8.8.8  Sarcocystosis (sarcosporidiosis) John E. Cooper ESSENTIALS Sarcocystosis is characterized by the invasion of various tissues by protozoa of the genus Sarcocystis. S. hominis (intermediate host domestic cattle) and S. suihominis (domestic pig) are the most significant to humans, to whom they are transmitted by ingestion of uncooked beef or pork. Camel meat can be a significant source of S. cameli in Arabia. Humans and other primates serve as either intermediate or final host: (1) inter- mediate host—​presence of cysts in muscle is usually asymptomatic, but might cause myositis or myopathy; detected on clinical examination or muscle biopsy; (2) final host—​can be asymptomatic or cause fever and gastrointestinal upset; oocysts or sporocysts can be detected in faeces. There is no specific treatment. Prevention is by not eating uncooked meat from any animal and by improving food hygiene in poorer countries. Introduction Although often described as uncommon in humans, sarcocystosis ap- pears to be widespread but undetected. The causal organism is a proto- zoon that was first described in a deer-mouse in Switzerland in 1843 but the life cycle of which was not determined until 1972. Over the succeeding four decades there has been growing interest in the para- site. In 2015 the first Sarocystis genome was elucidated. Sarcocystis is considered part of the Apicomplexa phylum, along with Eimeria species such as Toxoplasma gondii. It has been reported from most continents, but the exact distribution of the different species remains uncertain, largely on account of the absence of definitive clinical signs in many cases. Sarcocystosis is one of the most prevalent infections of endothermic and ectothermic animals throughout the world. In 2011, 9 travellers who had stayed on an island in Malaysia presented in Germany 4–​41  days later with fever, pruritus, myalgia, fatigue, nausea, and headache. Laboratory abnormalities included eosino- philia (15–​20%) and mildly elevated serum creatine kinase concen- trations. Muscle biopsies demonstrated sarcocystis-​like bradyzoites. Over the past decade, veterinary studies, especially serological sur- veys, have indicated that Sarcocystis species are present in a wide range of domesticated and wild mammals and other animals, often at a high prevalence (Fig. 8.8.8.1). Snakes and their rodent prey are definitive and intermediate hosts for many species of Sarcocystis; there is evidence of coevolution of the parasites with their vertebrate hosts. Equine protozoal myeloencephalitis, a highly fatal disease of domestic horses due to S. neurona—​and characterized by the presence of schiz- onts in neural cells—​has prompted a considerable body of research on Sarcocystis in recent years because of its economic importance. Sarcocystosis presents both actual and perceived public health problems. Some species, such as S. hominis and S. suihominis, can be transferred from animals to humans but others, while often causing alarm among those who encounter them, do not appear to be Acknowledgement: I am most grateful to Dr Sarah Cooper for reading and com- menting on an early version of this chapter.

8.8.8  Sarcocystosis (sarcosporidiosis) 1439 transmissible. For example, S. rileyi, which commonly affects ducks and geese in North America, presents with readily visible cream-​ coloured cysts generally running in parallel lines in the muscles of affected birds. This condition, often termed ‘rice breast disease’, is fa- miliar to hunters and to those who skin waterfowl before they are cooked. Many affected carcasses are discarded, but meat containing the cysts presents no known hazard to people who eat it. More re- cently S. nesbitti has been identified as infecting humans, as inter- mediate hosts, probably after consuming contaminated water or food. This species usually has reptiles, such as snakes, as the final host. The role of host resistance in sarcocystosis has not, however, been extensively studied and it is possible that reduced host resistance might render humans susceptible to species of Sarcocystis that are pri- marily parasites of wild birds, reptiles, or mammals. In non​human primates, immunodeficiency does appear to be a predisposing factor. Clinical features Humans as the final host Depending on the species of parasite and the previous health of the host, infection might cause symptoms. Humans who have ingested raw or undercooked pork or beef containing mature sarcocysts of Sarcocystis can be considered final hosts. Infection can have effects that range from gastrointestinal symptoms, pyrexia, and hypersensi- tivity-​like clinical signs to an asymptomatic state. Humans as the intermediate host Sometimes humans ingest sarcocysts from other species and become ab- errant intermediate hosts. In these circumstances the presence of cysts in human skeletal, visceral, or cardiac muscle is usually not associated with symptoms or clinical signs. However, it is likely that large numbers of cysts might, as in the definitive host animals, cause myositis or myop- athy, especially if calcification occurs, sometimes with vasculitis. Diagnosis Humans as the final host Oocysts or sporocysts can be detected in faeces in smears (especially using Heine’s method), in wet saline preparations, or, better, using a so- dium chloride or sucrose flotation method (Fig. 8.8.8.2). The oocysts/​ sporocysts, measuring about 10 by 15um, are usually readily recognized by an experienced parasitologist but can easily escape the attention of those who are less familiar with the organism. Sarcocystis must be dis- tinguished from other sporozoal organisms that are either being pro- duced in the intestine or are in transit in the lumen following ingestion. Humans as the intermediate host Occasionally, tissue cysts are detected during routine clinical examin- ation, especially if calcification has occurred. They might also be seen in muscle biopsies, either as an incidental finding or because samples have been taken specifically for diagnostic purposes. Calcified cysts found in biopsies or located at autopsy have a gritty texture when cut. Sarcocystosis of muscle (Figs. 8.8.8.1 and 8.8.8.3) must be differ- entiated from toxoplasmosis, in which tissue cysts can also be found. The morphology of the two protozoa differs. In particular, cysts of Sarcocystis have a distinct wall, which is thick and striated in some species, and do not stain with periodic acid–​Schiff stain, which usu- ally gives Toxoplasma cysts a magenta colour. (a) (b) Fig. 8.8.8.1  Sarcocystis in skeletal muscle of a little penguin Eudyptula minor (haematoxylin and eosin). Courtesy of Dr Richard Norman. Fig. 8.8.8.2  Sporocyst containing sporozoites in faeces of a fox Vulpes vulpes. There are two within the oocyst when freshly passed but single sporocysts are often seen. Courtesy of Dr John McGarry.

8.8.9 Giardiasis and balantidiasis 1440

8.8.9 Giardiasis and balantidiasis 1440

section 8  Infectious diseases 1440 Treatment There is no specific therapy for sarcocystosis in humans or ani- mals, although various compounds, including albendazole, metronidazole, cotrimoxazole, and corticosteroids, have been used empirically in both humans and animals. Experimentally infected calves and lambs appeared to respond to amprolium and salinomycin. Ponazuril prevented infection of the central ner- vous system of mice experimentally given sporocysts of S. neu­ rona and is currently used in the New World for the treatment of equine protozoal myeloencephalitis in horses. When humans are the final host, symptomatic and supportive treatment is indicated. Prevention Sarcocystosis can be best prevented by not eating raw or under- cooked meat from any animal and by improvements to food hygiene, especially in poorer countries. Experimental vaccines have been shown to produce cellular immunity to Sarcocystis species in horses and studies on experimentally inoculated pigs demonstrated a persistent immunity to further infection. FURTHER READING AbuBakar S, et al. (2013). Outbreak of human infection with Sarcocystis nesbitti, Malaysia 2012. Emerg Infect Dis, 19, 1989–91. Arness MK, et al. (1999). An outbreak of acute eosinophilic myositis attrib- uted to human Sarcocystis parasitism. Am J Trop Med Hyg, 61, 548–​53. Bunyaratvej S, Bunyawongwiroj P, Nitiyanant P (1982). Human intestinal sarcosporidiosis: report of six cases. Am J Trop Med Hyg, 31, 36–​41. Dubey JP, et al. (2015). Sarcocystosis of animals and humans, 2nd edi- tion. CRC Press, Boca Raton, FL. Fayer R (2004). Sarcocystis spp. in human infections. Clin Microbiol Rev, 17, 894–​902. Hilali M, et al. (1995). Isolation of tissue cysts of Toxoplasma, Isospora, Hammomdia and Sarcocystis from camel (Camelus dromedarius) meat in Saudi Arabia. Veterinary Parasitology, 58, 353–​6. Hofmann P, et al. (1999). Sarcocystosis and Malassezia infection in an im- munodeficient rhesus macaque—​a case report. Primate Report, 55, 19–​24. Marsh AE, et al. (2004). Evaluation of immune responses in horses im- munized using a killed Sarcocystis neurona vaccine. Vet Ther, 5, 34–​42. Mehrotra R, et al. (1996). Diagnosis of human Sarcocystis infection from biopsies of the skeletal muscle. Pathology, 28, 281–​2. Mohammed OB, et  al. (2000). Sarcocystis infections in gazelles at the King Khalid Wildlife Research Centre, Saudi Arabia. Veterinary Record, 146, 218–21. Palmer SR, et  al. (2011). Oxford textbook of zoonoses, 2nd edition. Oxford University Press, Oxford. Slapeta JR, et  al. (2003). Evolutionary relationships among cyst-​ forming coccidia Sarcocystis spp. (Alveolata: Apicomplexa: Coccid ea) in endemic African tree vipers and perspective for evolution of heteroxenous life cycle. Mol Phylogenet Evol, 27, 464–​75. Velásquez JN, et al. (2008). Systemic sarcocystosis in a patient with acquired immune deficiency syndrome. Hum Pathol, 39, 1263–​7. Wong KT, Pathmanathan R (1992). High prevalence of human skeletal muscle sarcocystosis in south-​east Asia. Trans R Soc Trop Med Hyg, 86, 631–​2. Zaman V, Colley FC (1975). Light and electron microscopic observa- tions of the life cycle of Sarcocystis orientalis sp. n. in the rat (Rattus norvegicus) and the Malaysian reticulated python (Python reticula­ tus). Z Parasitenkd, 47, 169–​85. Websites https://www.sciencedirect.com/topics/medicine-and-dentistry/sarcocystis https://www.cdc.gov/parasites/sarcocystosis/index.html https://www.britannica.com/science/Sarcocystis https://www.msdvetmanual.com/musculoskeletal-system/sarcocystosis/ overview-of-sarcocystosis 8.8.9  Giardiasis and balantidiasis Lars Eckmann and Martin F. Heyworth ESSENTIALS Giardiasis Infection with Giardia intestinalis, a common flagellate protozoan that colonizes the lumen of the small intestine, is acquired by ingesting environmentally resistant cysts of the parasite, typically in water or Fig. 8.8.8.3  Cysts of bovine origin, containing crescent-​shaped bradyzoites that are infective in the definitive host. Courtesy of Dr John McGarry.

8.8.9  Giardiasis and balantidiasis 1441 food, or after contact with faecal material from infected individuals. Strains of the parasite that can potentially infect humans are also har- boured by various mammals, including dogs and cattle. Clinical features—​manifestations include watery diarrhoea, abdom- inal discomfort, distension and pain, nausea and vomiting, weight loss, and malabsorption, with the infection typically being persistent and severe in individuals with certain immunodeficiencies. Chronic G. intestinalis infection can lead to micronutrient deficiencies, and im- pairment of growth and cognitive development in children. Diagnosis and treatment—​diagnosis is by faecal examination for evi- dence of G. intestinalis infection, including (1) by light microscopy of fresh or stained smears to detect cysts (ova and parasites test; a simple historic and still common approach that lacks sensitivity); (2) by immunofluores- cence microscopy of samples stained with direct fluorescent antibodies; or (3) by nucleic acid amplification techniques, such as the polymerase chain reaction, to detect parasite DNA or RNA. Treatment is primarily with metronidazole or tinidazole, and less commonly with nitazoxanide, albendazole, paromomycin, or quinacrine. Resistance to all common drugs has been reported, but can presently be overcome by switching antimicrobial classes or with combination therapies. Prevention—​cysts of G. intestinalis are relatively resistant to several common disinfectants, including chlorine-​ and iodine-​based chem- icals, but can be killed by boiling or exposure to ultraviolet light, or removed by filtration. A vaccine is available and moderately effective in animals, but a human vaccine has not been developed. Balantidiasis Balantidium coli is a relatively rare ciliate protozoan that invades the colonic mucosa. Infection—​which may or may not be acquired from pigs or other animals—​might be asymptomatic or cause diarrhoea that can be watery or contain blood and mucus. Perforation of the colon can occur, leading to peritonitis, and the parasite can also spread to the liver, lungs, and spine. Diagnosis is by recognition of the parasite on microscopic examination of freshly obtained diarrhoeal stools, co- lonic mucus, or rectal biopsies. Aside from supportive care, treatment with metronidazole or tetracycline has reportedly eradicated infection. Prevention is by filtration or boiling of drinking water, hand washing before handling food, and careful cleaning and cooking of food. Introduction The two organisms covered in this chapter, Giardia and Balantidium, are protozoa that can cause diarrheal disease. Transmission of both occurs through ingestion of environmentally resistant life cycle stages of the parasites. Giardia is one of the two most common protozoan causes (along with Cryptosporidium) of diarrheal disease world- wide, while infections with Balantidium are less common and occur primarily in tropical and subtropical countries. Other important protozoan causes of diarrheal disease, including Cryptosporidium, Entamoeba, and Cyclospora, are discussed in separate chapters. Giardiasis Aetiology Giardia, the causative agent of giardiasis, was first discovered in 1681, when van Leeuwenhoek undertook a microscopic examination of his own diarrhoeal stool. Historically, the literature on human giardiasis had emphasized a relationship between drinking unfiltered water in wilderness areas and acquiring this infection, as well as occurrence of Giardia species in muskrats and beavers (which led to the now out- dated term ‘beaver fever’ for giardiasis). Although it is possible that faeces from these animals were sources of Giardia cysts that could infect humans, it is also possible that cysts of human origin could have infected the amphibious mammals. The host range of organ- isms morphologically classifiable as Giardia is wide and include birds and various terrestrial and marine mammals. Giardia organisms have been divided into eight genetic assemblages (designated A-​H), on the basis of their DNA sequences. Assemblages A and B cause human in- fection; the other assemblages infect non​human hosts. Besides human subjects, hosts for assemblages A and B include domestic pets (dogs, cats, rabbits, chinchillas, and ferrets), and livestock (cattle, sheep, and pigs). Proving that an assemblage A or B organism from a non​human host can cause human infection is difficult; although giardiasis is clas- sified as a zoonosis, evidence of animal-​to-​human (and vice versa) transmission is largely circumstantial. In this chapter, the terms ‘as- semblage’ and ‘genotype’, as applied to Giardia, are used interchange- ably. Because they lack mitochondria and some other features of ‘higher’ eukaryotic (nucleated) cells, Giardia was formerly considered to be primitive. Following the discovery of gene sequences homolo- gous with mitochondrial DNA and of organelles (mitosomes) that ap- pear to be derived from mitochondria, the organism is now regarded as highly specialized rather than primitive. The apparently primitive features are almost certainly adaptations to the parasitic lifestyle, re- flecting the colonization of a microaerophilic or anaerobic niche (the vertebrate intestinal lumen) by Giardia species. Giardia intestinalis (synonyms G. lamblia and G. duodenalis) is a flagellated parasite that belongs to the order of diplomonads in the phylum of metamonads, a large group of flagellate amitochondriate protozoa. The parasite has a two-​stage life cycle (Fig. 8.8.9.1), which generally occurs in a single host (monoxenous) and involves an in- fectious thick-​walled cyst and a motile trophozoite. Hosts become infected by ingesting G. intestinalis cysts, from which trophozoites emerge (excystation) in the small intestine upon prior exposure to gastric acid in the stomach. Trophozoites are the vegetative, replicating forms of the parasite. They are pear-​shaped, dorsoven- trally flattened organisms with dimensions of 12–​15 μm in length and 5–​9 μm in width (Fig. 8.8.9.2). Four pairs of flagella located at the anterior, posterior, caudal and ventral sides of the organism confer motility, and together with a ventral adhesive disc enable attachment to the luminal surface of intestinal epithelial cells. The parasite resides in the intestinal lumen and in close proximity to the epithelium, but does not invade the mucosa or spread systemically. It replicates in its luminal niche and ultimately forms cysts that are shed in the faeces for transmission to other hosts. The genome sequences of multiple G.  intestinalis isolates have been determined and analysed. The compact haploid genome of G. intestinalis comprises c.12 Mb of DNA encoding c.5000 genes. Trophozoites have two morphologically similar nuclei, which each carry diploid genomes (making the trophozoite genome overall tetraploid) and are both transcriptionally active. Although the polyploid genome sequences have generally been considered to be identical, some evidence exists for allelic sequence heterozygosity of specific genes within single parasites. Genome comparison of isolates from the human-​pathogenic assemblages A and B shows only 77% nucleotide and 78% amino acid identity in protein-​coding

section 8  Infectious diseases 1442 regions, indicating that the two assemblages truly represent different Giardia species, despite the historic convention of a single species name. The metabolism of G. intestinalis is fermentative, and electron transport proceeds in the absence of mitochondrial oxidative phos- phorylation. However, the parasite is microaerotolerant and can re- duce molecular oxygen and thus protect the highly oxygen-​sensitive, central metabolic enzyme, pyruvate:ferredoxin oxidoreductase (PFOR), and iron-​containing ferredoxins. PFOR decarboxylates pyruvate and donates electrons to ferredoxin, which in turn reduces other components in the electron transport chain and leads to ad- enosine triphosphate generation. Epidemiology G. intestinalis is a major cause of diarrheal disease with c.180 mil- lion annual cases worldwide and prevalence rates ranging from 1 to 20% in different countries and regions. Infections are more frequent and severe in young children, particularly in day-​care centres, and persons changing their diapers, as well as among individuals with a history of foreign travel, use of swimming pools, hiking, and keeping a dog. A slight predominance of males (60%) over females (40%) has been reported in giardiasis cases in Austria, but the underlying reasons are unclear. The infection is acquired by ingestion of water or, less commonly, food contaminated with cysts, or by direct faecal–​oral transmission of cysts, particularly among young children. Waterborne giardiasis occurs as a result of drinking contaminated water from streams and lakes containing G. intestinalis cysts. Swimming in (and inad- vertently drinking) water in lakes and rivers containing the cysts is also a risk factor for giardiasis. Outbreaks of this infection have re- sulted from the unintended presence of G. intestinalis cysts in public Fig. 8.8.9.1  Life cycle of Giardia intestinalis in humans. Transmission occurs by ingestion of cysts from contaminated water or food (step 2). Once ingested, trophozoites emerge from the cysts in the small intestine, where they reside and replicate (steps 3 and 4) in the lumen or in close proximity to the epithelial surface. Trophozoites differentiate into infective cysts in the small intestine (step 5), and are passed in the stool (step 1). Reprinted from the Public Health Image Library of the Centers for Disease Control and Prevention, Atlanta, Georgia, USA; image generated by Dr Alexander J. da Silva and Melanie Moser.

8.8.9  Giardiasis and balantidiasis 1443 drinking water supplies and in swimming pools due to inadequate disinfection. Giardiasis is one of several parasitic and bacterial diseases that are potentially or actually transmitted by eating raw vegetables grown on fields irrigated or contaminated with untreated human sewage or animal manure. From the 1990s onwards, numerous comparisons have been made of genome sequences of G. intestinalis isolated from human and non​human hosts. Close similarity between DNA sequences of G. intestinalis from different host species suggests, rather than proves, interspecies transmissibility of the parasite. Genotyping has revealed genetic similarity between Giardia isolates from people and from dogs occupying the same households in India, a finding that suggests transmission of G. intestinalis between dogs and people. Approximately 10% of Giardia isolates from cattle belong to geno- types that can potentially cause human infection. Flies that feed on garbage and sewage are able to carry Giardia cysts on their exoskel- etons and in their alimentary tracts and may therefore contaminate human food with viable cysts. Immunodeficiency predisposes to the occurrence of severe and persistent giardiasis. Human immunodeficiency states that are as- sociated with giardiasis include conditions that impair host anti- body responses, notably ‘common variable’ immunodeficiency and X-​linked immunoglobulin deficiency (XID). Impairment of intes- tinal IgA production is a feature of these particular immunodefi- ciencies that may contribute to increased susceptibility to giardiasis. Furthermore, HIV-​induced acquired immunodeficiency syndrome (AIDS) is associated with an increased prevalence of giardiasis (and other enteric protozoan infections), particularly in patients with low CD4+ T cell numbers and untreated for HIV. An association be- tween selective IgA deficiency and increased prevalence of giardiasis has been mentioned in some reports, although other studies have not seen such an association, suggesting that effective host defence against Giardia can occur in humans in the absence of normal intes- tinal IgA production. Pathogenesis and pathology The mechanisms responsible for diarrhoea and malabsorption in giardiasis are incompletely understood. Giardia is not known to pro- duce classical enterotoxins, making it likely that the host response to infection is mostly responsible for the diarrhoea. A large endoscopic and histological study of 567 patients with confirmed giardiasis in Austria revealed that trophozoites colonized predominantly the duo- denum (83% of cases), but were also found in jejunum (2%), ileum (12%), colon (0.4%), and stomach antrum (9%), suggesting that the relevant host-​parasite interactions that lead to diarrhoea most likely occur in the proximal small intestine. The study also demonstrated that the mucosal response to infection was morphologically unre- markable, as 96% of cases displayed normal light-​microscopic ap- pearance of the duodenal mucosa. In only a small percentage (4%) of acutely infected patients, moderate villous shortening and mild in- flammation of the lamina propria were observed in the duodenum. These findings indicate that acute giardiasis is not a classical inflam- matory infection as seen, for example, in amoebiasis or shigellosis. Consequently, increased production of inflammatory mediators, such as prostaglandins or interleukin 1, which can mediate diar- rhoeal responses in other enteric infections, is not likely to explain the diarrhoea in giardiasis. Prolonged Giardia infection can be asso- ciated with more pronounced villus shortening and significant in- creases in intraepithelial lymphocytes, as well mixed inflammatory cell infiltrates in the lamina propria of the small intestine, which may contribute to diarrhoeal symptoms, although these tend to become less prominent in protracted infections. Shortening of microvilli on the luminal surface of intestinal epithelial cells has been observed in small intestinal biopsies from patients and animals with giardiasis. In parallel, reduced activity of intestinal disaccharidases occurs in Giardia-​infected human subjects and rodents. This functional en- zyme deficiency, which depends on intact immune responses to the parasite, might lead to osmotic diarrhoea (via the presence of undigested disaccharides in the intestinal lumen). Furthermore, intestinal hypermotility occurs in infected patients and animals, which may further contribute to an osmotic form of diarrhoea by reducing the contact time available for complete absorption of nu- trients from the lumen. Other potential mechanisms of diarrhoea may be operational in giardiasis. Electrophysiologic investigations of duodenal biopsy specimens from patients with chronic infection revealed epithelial barrier dysfunction, possibly related to down regulation of tight junction proteins and increased epithelial cell death. Concomitantly, sodium-​dependent glucose absorption was impaired and active electrogenic anion secretion was found to be ac- tivated, suggesting that diarrhoea in human chronic giardiasis may be, in part, caused by a combination of leak flux, and malabsorptive and secretory processes. Giardia-​induced barrier disruption has also been observed in several in vitro and animal models, giving further support to the notion that paracellular mechanisms may contribute to the infection-​associated diarrhoea. Giardiasis is self-​limiting in more than 85% of cases in non-​ endemic areas, strongly suggesting that effective immune defences exist. Consistent with this, experimentally infected rodents eradi- cate the infection over weeks to months. Furthermore, symptoms of giardiasis are much less severe in endemic than non​endemic Fig. 8.8.9.2  Scanning electron micrograph of three Giardia intestinalis trophozoites on a jejunal biopsy specimen from a patient with giardiasis. The dorsal surfaces of two trophozoites are visible (D), and the ventral adhesive disc of the other trophozoite is shown (V). Courtesy of Dr Robert L. Owen; modified from Carlson JR, Heyworth MF, Owen RL (1984). Giardiasis: Immunology, diagnosis and treatment. Survey of Digestive Diseases, 2, 210–​23, with permission.

section 8  Infectious diseases 1444 regions, suggesting gradual build-​up of immunity. IgA produced in the intestinal mucosa and secreted into the intestinal lumen has long been considered a primary mechanism of adaptive host defence against the parasite. In human volunteers who were deliberately in- fected with G. intestinalis, an intestinal IgA response to the parasite occurred, as it does in experimentally infected animals. IgA directed against trophozoites can inhibit their attachment to the intestinal epithelium (mostly by randomly cross-​linking trophozoites with each other, which presumably interferes with their proper attach- ment orientation and dynamics; specific parasite ligands or recep- tors for attachment that could be blocked by antibodies have not been identified), such that they become susceptible to peristaltic expulsion from the host. Trophozoite antigens recognized by IgA include structural proteins that show little or no variability in amino acid sequence between different Giardia isolates, and surface pro- teins whose amino acid sequences show considerable variability. Giardia-​infected human subjects and mice also generate a serum IgG response against trophozoites, although it is not clear whether this contributes to parasite clearance. Despite the compelling nature of secretory IgA as a key antigiardial defence, several clinical and experimental studies have cast doubts on the general importance of IgA, and have explored alternative defence mechanisms against the parasite. Infection in humans and in mice leads to T lymphocyte-​ dependent increases in small intestinal motility, which can promote clearance of the parasite in the luminal bulk flow. Furthermore, anti- microbial peptides and nitric oxide can directly kill or inhibit the parasite, although their relative contributions to clearance of Giardia remain to be firmly established. Beyond direct effectors, several immune cells and regulators are known to be involved in antigiardial immune defence in animal models. Mast cells and CD4+ T cells, but not CD8+ T cells, are re- quired for clearing Giardia infection in mice. CD4+ T cells might act in part by controlling antigiardial IgA responses. Their functions are probably not related to classical T helper 1 cells or T helper 2 cell subsets, because their signature cytokines, IFN-​γ or IL-​4, play no role in immune defence. In contrast, IL-​6 and IL-​17 are important in Giardia clearance. IL-​6 appears to act by promoting dendritic cell functions during infection, although it has many other activities, including activation of monocytes, enhancement of follicular helper T cell responses, and stimulation of B cell proliferation and antibody production. Clinical features Giardia infection can be asymptomatic (as shown by cyst excretion in the absence of symptoms) or cause clinical symptoms, ranging from abdominal pain, discomfort and distension, and watery diar- rhoea, to nausea and vomiting, and anorexia and weight loss. Other clinical features are a sensation of fullness, heartburn, flatulence, and steatorrhoea. In one study of adult patients in Austria, the fol- lowing frequencies of symptoms were observed: pain on abdominal palpation (52% of cases), abdominal discomfort (43%), sensation of fullness (41%), abdominal distension (38%), epigastric pain (38%), nausea and/​or vomiting (36%), heartburn (27%), and diarrhoea (26%). In contrast, foul-​smelling stools, anecdotally considered typ- ical for giardiasis, were observed in fewer than 6% of cases in that study. Micronutrient deficiencies can occur in giardiasis. Impaired absorption of iron and zinc has been observed in Giardia-​infected patients, which can be associated with mild to modest anaemia. Occasional case reports have also found defects in vitamin B12 ab- sorption and megaloblastic anaemia, although several larger surveys reported normal serum vitamin B12 levels in Giardia-​infected indi- viduals. Together, these findings suggest that no particular symp- toms allow reliable clinical recognition of giardiasis, demanding a high index of suspicion if one or several of the possible symptoms are encountered in patients with gastrointestinal complaints. In immunologically competent persons, untreated giardiasis typ- ically lasts for several weeks to months, with symptoms that fluc- tuate in severity. Most patients clear infection spontaneously over extended periods even without treatment, although asymptomatic carriers exist, and re-​infections are probably common in endemic regions. An outbreak of G. intestinalis infection in Norway in 2004 predisposed individuals who had been infected with the parasite to irritable bowel syndrome and chronic fatigue for years after the out- break, despite successful eradication of the parasite by treatment. G.  intestinalis infection can be associated with other important long-​term sequelae in children, including stunting, low weight, and impairment of cognitive functions, particularly in less developed countries and regions. Laboratory diagnosis The traditional approach to diagnosing giardiasis is detection of G. intestinalis cysts in stool samples by light microscopy, which is often referred to as the ova, cysts, and parasites (OCP) test. Samples can be examined directly as fresh smears, or preserved with a suit- able fixative (e.g. formalin-​ethyl acetate) and stained with various dyes, such as trichrome stain. Cysts are recognizable by their oval shape and size of approximately 8  ×  12  μm. Internal structures including four nuclei, axonemes, and median bodies might be vis- ible to varying degrees in the cysts. Trophozoites are not commonly observed in faecal samples, but are found in duodenal specimens collected during endoscopy or by a string test. Negative results in the faecal OCP test can be related to variable faecal parasite shed- ding, so repeated stool tests on 2–​3 different days might increase the diagnostic yield, although it is generally not necessary to confirm a positive result. The standard OCP test can be performed under conditions of limited resources and is widely used, but the test has a relatively low sensitivity, is labour-​intensive and time-​consuming, and is highly dependent on operator skill and experience, so many clinical microbiology laboratories no longer offer the test routinely. Assay sensitivity can be increased by staining faecal samples with direct fluorescent antibodies (DFA) and immunofluorescence microscopy. Highly specific antibodies against abundant cyst wall antigens allow ready recognition of brightly stained cysts against a dark background of unstained faecal materials. DFA tests are moderately labour-​intensive and have some degree of subjectivity, although less than standard OCP tests, and depend on access to a fluorescence microscope. Nonetheless, compared to the standard OCP test, the method has markedly improved sensitivity and spe- cificity, approaching 95–​100% on both measures, and can be con- sidered the current standard in Giardia diagnostics. Enzyme-​linked immunosorbent assay (ELISA) tests for detection of faecal G. intestinalis antigen(s) are available, and can be useful as an objective diagnostic technique that can obviate the need for faecal microscopy if there is a high initial suspicion of giardiasis. However, microscopy might be preferable over testing exclu- sively for G. intestinalis antigen(s) in cases of unspecified intestinal

8.8.9  Giardiasis and balantidiasis 1445 parasitic infections, particularly because combination DFA tests can be used to detect more than one parasitic species in the same test. While the specificity of ELISA tests is similar to DFA tests (>95%), reported sensitivities of 85–​95% are slightly inferior to DFA tests, so stool antigen ELISA tests are less common in routine diagnos- tics. Serum antibodies are elicited by Giardia infection, but cannot distinguish between past and current infection, and are not used in clinical diagnostics. Detection of parasite-​specific DNA or RNA in stool by nucleic acid amplification techniques is a relatively new diagnostic strategy with excellent sensitivity and specificity. Numerous protocols and technical approaches have been reported, but they all involve initial sample lysis and nucleic acid extraction, followed by a combination of hybridization-​based gene enrichment, reverse transcription (for RNA targets), and one or two rounds of polymerase chain reaction (PCR) amplification of pathogen-​specific genes. PCR products are then de- tected by DNA-​binding dyes, or with fluorescent microspheres coated with matching capture probes for hybridization. Specific methods differ in the lysis conditions, amplification parameters, and target genes, whose sequences must be sufficiently conserved to allow de- tection of different strains and isolates of the respective pathogen, but sufficiently different from other microbes to avoid false-​positive results. A major advantage of PCR-​based diagnostics is the ability to detect multiple (>20) relevant pathogens in one sample, increasing diagnostic yield and shortening the time to diagnosis. This is particu- larly useful for identifying species of pathogens that might not have been suspected as the cause of a patient’s gastroenteritis symptoms. PCR-​based technologies have outstanding sensitivity and specificity, although they can exhibit an increased risk of false-​positive results due to their exquisite sensitivity (<10 organisms can be routinely de- tected). Even minor contaminations during initial sample collection or subsequent laboratory processing can pose problems, particularly in endemic regions where environmental contamination might be high. Furthermore, the need for highly specialized equipment and technical expertise limits the use to well-​equipped clinical laboratories with stringent work practices. Nonetheless, PCR-​based diagnostics are increasingly supplanting microscopic methods in the routine la- boratory diagnostics of giardiasis and other enteric infections. As an alternative to PCR-​based nucleic acid detection, loop-​ mediated isothermal amplification protocols have more recently been developed. The initial assay steps, including meticulous sample collection, lysis, and nucleic acid extraction, are similar to PCR methods, and the technical development and validation of new assays is at least as challenging as it is for PCR-​based methods. However, unlike PCR, loop-​mediated isothermal amplification as- says do not require a thermal cycler for the amplification steps and can be performed with a water-​bath or heating block at a constant temperature, a feature that can simplify use in resource-​limited con- ditions. This technology has the promise to promote point-​of-​care diagnostics over a wide range of clinical settings, although it is not currently employed in routine laboratory testing. Treatment Table 8.8.9.1 summarizes the major drug regimens used in the treat- ment of giardiasis. The most commonly utilized drugs worldwide are members of the 5-​nitroimidazole class, including metronidazole and tinidazole. Metronidazole, which was developed and approved for clinical use in the 1950s and 1960s, is typically given in three divided daily 250 mg oral doses for 5–​10 days, and has a reported efficacy of 80–​95%. More recently, tinidazole, first approved in 2004, has become a good alternative for giardiasis because of its efficacy (85–​90%), tol- erability, and convenience (a single oral dose is recommended). All 5-​ nitroimidazole drugs are prodrugs whose microbial specificity is due to the requirement for reduction to toxic free radical intermediates by low redox potential reductive reactions present only in the anaer- obic target microbes including Giardia. The radicals that result from nitro drug reduction form covalently bonded adducts on microbial target molecules, leading to their inactivation. The specific molecular targets of 5-​nitroimidazole drugs have not been defined in G. intesti­ nalis. In spite of the general efficacy of 5-​nitroimidazole drugs, treat- ment failures in giardiasis are not uncommon (up to 20%), clinical resistance is proven, and in vitro resistance can be induced so that parasites grow in clinically relevant levels of metronidazole. Nitazoxanide is a nitrothiazole with broad-​spectrum activity against intestinal parasites. Like 5-​nitroimidazole drugs, it is a pro- drug that must be reduced to form toxic radicals, which inactivate various microbial target molecules. It is usually given in two daily 500 mg doses for three days, which is more convenient dosing than metronidazole, but has slightly lower efficacy (70–​80%) than 5-​ nitroimidazole drugs and can also be impacted by metronidazole resistance. In a clinical trial involving children with diarrheal illness, nitazoxanide reduced symptom duration in those afflicted with giar- diasis as well as in those without a microbiological diagnosis, further underlining its utility as a broad-​spectrum antimicrobial agent. Benzimidazoles, such as albendazole and mebendazole, are gen- erally used to treat intestinal helminth infections. Albendazole is also effective in giardiasis, although its efficacy varies markedly (25–​90%) depending on the dosing regimen. The drug binds to β-​tubulin in G. intestinalis and blocks tubulin polymerization and hence microtubules assembly and normal parasite motility and at- tachment. Albendazole can be taken once daily for five days, making it more convenient than three-​times-​a-​day metronidazole, and its antihelminth activity makes it an attractive agent for dual use Table 8.8.9.1  Major oral drug regimens for treating giardiasis Drug Dose Treatment duration Metronidazole 250 mg/​dose, 3 x doses/​day (adult) 5–​10 days 5 mg/​kg/​dose, 3 x doses/​day (paediatric) 5 days Tinidazole 2 g/​dose (adult) Single dose 50 mg/​kg/​dose (paediatric) Single dose
(2 g maximum) Nitazoxanide 500 mg/​dose, 2 × doses/​day (adult) 3 days 100 mg/​dose, 2 × doses/​day
(age 1–​3 years) 3 days 200 mg/​dose, 2 × doses/​day
(age 4–​11 years) 3 days Albendazole 400 mg/​dose, 1 × dose/​day
(adult and paediatric >2 years) 5 days Paromomycin 500 mg/​dose, 3 × doses/​day (adult) 7–​10 days 10 mg/​kg/​dose, 3 × doses/​day (paediatric) 7–​10 days Quinacrine 100 mg/​dose, 3 × doses/​day (adult) 5–​7 days

section 8  Infectious diseases 1446 purposes. However, results from a Bolivian cohort study (a region with endemic Giardia and helminth infections) found that treat- ment with mebendazole reduced hookworm infections but para- doxically led to an increase in giardiasis, suggesting an antagonistic relationship between the two parasites and complicating the pro- spect of multitarget therapies. Quinacrine, an old malaria drug, is an acridine derivative that binds to DNA and interferes with transcription and DNA replica- tion in many target microorganisms, although different mechan- isms of action, including membrane breakdown and inhibition of critical enzymes, appear to be operating in G. intestinalis. The drug has excellent efficacy against giardiasis (c.90%). In a randomized trial in children with giardiasis, chloroquine was equally effective as tinidazole and superior to albendazole. Anecdotal reports also sug- gest that quinacrine can be effective when other drug regimen failed. Despite its efficacy, quinacrine has potentially severe adverse effects, including major psychiatric and dermatologic manifestations, and is no longer commercially available in the United States or Canada. Finally, paromomycin, an aminoglycoside antibiotic that inhibits bacterial protein synthesis by targeting ribosomal RNA, is active against G. intestinalis in vitro and in vivo. Although Giardia is an eu- karyote, its ribosomal RNAs display features that resemble those in susceptible bacteria (but not human cells), thus providing a poten- tial explanation for the antigiardial activity of paromomycin. Oral formulations of this antibiotic are not absorbed from the gastro- intestinal tract, which presumably accounts for the rarity of systemic adverse effects (such as nephrotoxicity and ototoxicity) compared to other aminoglycosides. Paromomycin is moderately effective against giardiasis, with reported response rates of 55–​90%. It has also been used in cases of metronidazole-​refractory giardiasis. Treatment failure and resistance of G. intestinalis to all common antigiardial drugs have been reported. For the most commonly used 5-​nitroimidazoles, resistance has been observed in 1–​20% of cases. Therapeutic strategies for treatment-​refractory giardiasis include longer duration and/​or higher doses of the original agent, switching to a different class of drug, or combination therapy. In a case series of ten patients who failed 5-​nitroimidazole therapy, all were cured with one of the following combinations: metronidazole or tinidazole

8.8.9  Giardiasis and balantidiasis 1447 vaccine may be feasible. Several vaccine antigen candidates have been identified. The veterinary vaccine has also been used as an immunotherapeutic agent in dogs with chronic giardiasis that had failed standard drug treatment, raising the possibility that a future human vaccine may also be effective postexposure. Balantidiasis Aetiology Balantidium was first described by Malmsten in 1857 in the stool of two cases of dysentery in Sweden, and was given its current designation as Balantidium coli in 1863. It has long been con- sidered one of the rarest pathogenic organisms, with only 600 cases reported in the literature over a 100-​year period until the 1950s. However, the parasite is now recognized as being more prevalent than earlier appreciated, partly due to more compre- hensive epidemiologic surveys, as well as the realization that many infected individuals are colonized but exhibit no overt dis- ease symptoms. B. coli, the cause of balantidiasis, is the largest protozoan and only ciliated parasite of man. The parasite has a two-​stage life cycle comprised of non​motile cysts and motile, replicating tropho- zoites (Fig. 8.8.9.3). Spread of the infection to new hosts occurs Fig. 8.8.9.3  Life cycle of Balantidium coli in humans. Transmission occurs by ingestion of cysts from contaminated food or water (step 2). Excystation occurs in the small intestine, and trophozoites colonize the large intestine (step 3), where they replicate (step 4) and potentially invade the mucosa. Trophozoites undergo encystation to produce infective cysts (step 5), which are passed with faeces (step 1). Reprinted from the Public Health Image Library of the Centers for Disease Control and Prevention, Atlanta, Georgia, USA; image generated by Dr Alexander J. da Silva and Melanie Moser.

section 8  Infectious diseases 1448 by the faecal-​oral route via ingestion of the cyst form, which is presumably resistant to gastric acid during stomach passage, and release of the vegetative trophozoite form in the small intestine or colon. Trophozoites are large (30–​150 μm) and ovoid in shape with a funnel-​like depression (peristome, mouth) at the tapering anterior end, and a cytopyge (anus) at the rounded posterior end. The entire trophozoite surface is covered by numerous short (4–​6 μm) hair-​like cilia (Fig. 8.8.9.4), whose beating leads to a straight swimming motion combined with rotation around the longitudinal axis. Cilia in the peristomal region are elongated and have feeding functions. Trophozoites carry two nuclei, a large macronucleus and a small micronucleus, whose relative roles in metabolism, growth, and reproduction are poorly defined in B. coli. In other ciliates, macronuclei are typically polyploid and primarily re- sponsible for the metabolic cell functions in the vegetative stage, while micronuclei are diploid and contribute little during vege- tative metabolism, but play a key role in reproduction. Although the morphology of the parasites has been well characterized, not much is presently known about its biochemical and molecular biologic features. Selected DNA regions have been sequenced for taxonomic purposes, but the entire genome sequence has not been reported to date. Epidemiology Humans are not primary hosts for B. coli (or any other intestinal ciliates), and the pathogen is maintained in populations of mam- malian reservoir hosts, yet the exact relationship between animal and human infections has not been established. Substantial cir- cumstantial evidence suggests that humans can acquire B. coli from animals, making it likely that the infection is zoonotic in origin. The parasite is frequently identified in pigs and several species of non​human primates, and more rarely in cows, donkeys, horses, and ostriches (but not cats, dogs, or rodents). Parasites from the faeces of infected humans can infect piglets, and cause severe diar- rhoea and mucosal destruction from terminal ileum to rectum, strongly implying that no species barrier exists between humans and swine in regard to B. coli infectivity and pathogenicity. A high prevalence of the infection has been seen in communities that live in close proximity to B. coli-​infected pigs (e.g. in New Guinea and subtropical China). Consequently, it is assumed that pigs are an important reservoir for the spread of B. coli to humans. However, balantidiasis has also occurred in human subjects who had no known contact with pigs or other animals. It is possible that veget- ables and fruits grown on fields fertilized with contaminated animal manure carry the parasite, as suggested by identification of B. coli on field-​grown strawberries in Brazil. In India, B. coli cysts have been found in water for drinking and cooking, and the organism has been identified on cockroaches in Nigeria and Ethiopia, and house flies in Egypt. Overall, the prevalence of B.  coli infection varies considerably across the world, with the highest levels gener- ally in tropical and subtropical regions. Prevalences as high as 0.4% in the general population in rural Thailand, 1–​5% in asymptomatic children in Bolivia, and 2.4% in children with diarrhoea in India have been reported. Infections are rarely seen in most developed nations, although clusters of B. coli infection have been described in institutional facilities such as psychiatric hospitals and prisons in the United States and Italy, and sporadic cases have been reported worldwide. Pathogenesis and pathology Once trophozoites are present in the intestinal lumen, they pri- marily interact with the host in the colon, where they can invade the mucosa and cause ulcerations that are accompanied by inflam- matory cell infiltrates (Fig. 8.8.9.4). The parasite is not known to produce toxins. It can probably break down extracellular matrix, and may be able to lyse host cells during invasion, but the detailed mechanisms responsible for tissue invasion and destruction are not known. It is also not clear why some infections cause marked tissue destruction and dysenteric symptoms, while most are asymptom- atic and presumably not accompanied by significant tissue damage. Differences in virulence genes of the parasite or host susceptibility to the actions of such genes are predictably important but remain to be investigated. Clinical features Human subjects infected with B. coli are commonly asymptomatic, but a subset of individuals can develop diarrhoea with stools that are either watery or consist of blood and mucus, resembling amoebic dysentery. In severe cases, patients can develop colonic perforation, peritonitis, gangrene of the appendix. Spread of the parasite to the liver or lungs can occur, although balantidiasis is only an exceed- ingly rare cause of liver abscess and of pulmonary haemorrhage. Severe colonic balantidiasis may be clinically indistinguishable from amoebiasis, bacillary dysentery, ulcerative colitis, and Crohn’s disease, and can be fatal if undiagnosed and untreated. B. coli in- fection in the lungs has been described in occasional patients with Fig. 8.8.9.4  Light micrograph of Balantidium coli trophozoite (arrow) in colonic tissue (×705). Cilia are visible on the surface of the organism (arrow). Arrowheads indicate tissue plasma cells. Modified from Neafie RC (1976). Balantidiasis. In: Binford CH, Connor DH (eds) Pathology of tropical and extraordinary diseases, vol. 1, pp. 325–​7. Armed Forces Institute of Pathology, Washington DC, with permission.

8.9 Nematodes (roundworms) 1478

8.9 Nematodes (roundworms) 1478

8.9.1 Cutaneous filariasis 1478

8.9.1 Cutaneous filariasis 1478

8.9 Nematodes (roundworms) CONTENTS 8.9.1 Cutaneous filariasis  1478 Gilbert Burnham 8.9.2 Lymphatic filariasis  1487 Richard Knight 8.9.3 Guinea worm disease (dracunculiasis)  1495 Richard Knight 8.9.4 Strongyloidiasis, hookworm, and other gut strongyloid nematodes  1500 Michael Brown 8.9.5 Gut and tissue nematode infections acquired
by ingestion  1506 Peter L. Chiodini 8.9.6 Angiostrongyliasis  1516 Richard Knight 8.9.1  Cutaneous filariasis Gilbert Burnham ESSENTIALS The cutaneous filariae are transmitted by biting insects. Some, such as Onchocerca volvulus, are transmitted by Simulium flies and can cause debilitating conditions such as visual impairment and disfiguring skin conditions. The Mansonella infections are transmitted either by Simulium flies or biting midges (genus Culicoides), but consequences of infections are general mild. Loa loa is transmitted by the bite of the Chrysops fly. Loaisis is manifest by adult worms periodically passing beneath the sclera and by subcutaneous swellings, usually of the forearm. Onchocerciasis Onchocerciasis (river blindness), caused by O. volvulus, infects per- haps 18 million people, once in six countries in the Americas, but now almost entirely in Africa. Clinical features—​the larvae are introduced into the body when the Simulium vector takes a blood meal, they then develop into male or female adult worms within palpable nodules, commonly located over bony prominences. Adults give rise to microfilariae which are responsible for clinical manifestations. Most important are: (1) Eye damage—​microfilariae enter the cornea from the skin and con- junctiva; manifestations include sclerosing keratitis, iridocyclitis and (sometimes) choroidoretinal lesions; without treatment, permanent visual impairment or blindness are common. (2) Skin disease—​which ranges from itching with a localized maculopapular rash, to in- tense itching with a chronic generalized papular rash, or lichenified hyperkeratotic lesions. Diagnosis, treatment, and prevention—​diagnosis is usually made by finding microfilariae in skin snips. Treatment is with ivermectin (Mectizan®), often given as a single or twice annual dose. Ivermectin has dramatically reduced the eye and skin lesions that ravaged many communities in Africa and Latin America. Methods of disease pre- vention include adding insecticides to rivers to interrupt Simulium breeding and the regular mass distribution of ivermectin. Loa loa Loiasis, for which humans are the only host, is transmitted by the Chrysops fly in West and Central Africa. Clinical manifestations in- clude transient localized inflammatory oedema (Calabar swellings), the appearance of a migrating worm under the skin or (most dra- matically) crossing the conjunctiva, and (rarely) meningoencephal- itis. Diagnosis is based on typical clinical findings, or traditionally by finding microfilariae in a daytime blood sample. Treatment is usu- ally with diethylcarbamazine, although ivermectin is effective, and albendazole less so. All treatments risk serious adverse reactions in the heavily affected persons. The best prevention is avoidance of Chrysops fly bites. Mansonellas The Mansonellas are filarial infections is transmitted by Culicoides midges and is common to many countries, but of negligible clinical importance under most circumstances. Only Mansonella streptocerca produces clear-​cut manifestations, most typically a mild chronic papular skin lesions. Diagnosis is by finding characteristic micro- filariae in the blood or skin. People who are asymptomatic do not require treatment, but M. streptocerca responds well to ivermectin. M. perstans and M. ozzardi are widespread in tropical countries, with few specific symptoms.

8.9.1  Cutaneous filariasis 1479 Onchocerciasis Onchocerciasis, or river blindness, historically occurred in 34 coun- tries in Africa, Yemen, and Latin America (Fig. 8.9.1.1). It is esti- mated that 18 million people are infected, and 87 million at risk of infection. Most are in Africa. In 1995, it was estimated that in- fection with Onchocerca volvulus had caused blindness in 270 000 people, and left another 500 000 with severe visual impairment. Mass treatment with ivermectin has now greatly lessened the ocular burden of infection. Besides eye changes, onchocerciasis has chronic systemic effects, causing extensive and disfiguring skin changes, musculoskeletal complaints, weight loss, changes to the immune system, and perhaps also epilepsy and growth arrest. Skin lesions are the most common manifestation of onchocerciasis. Changes in- clude acute and chronic itchy papular disease, and intensely prur- itic lichenification. Lesions can be localized or widespread. In the later stages, severe degenerative skin disease develops, with a loss of elastic tissue, and extensive pigmentary changes. The disease, endemic to some of the world’s poorest areas, has a great impact on the economic and social fabric of communities. A complex (a) (b) Fig. 8.9.1.1  Distribution of onchocerciasis in Africa, Yemen, and Latin America, where transmission is now largely interrupted.

section 8  Infectious diseases 1480 human–​parasite tolerance allows people who host millions of para- sites to continue daily existence. Mass treatment with ivermectin (Mectizan®) has interrupted or eliminated transmission in almost all foci in the Americas and has controlled the public health consequences of this disease in Africa. A goal of control of onchocerciasis by 2020 was set by the London Declaration on Neglected Tropical Diseases in 2012. The elimination of transmission in Africa, long thought not feasible, is now accepted as possible in many locations. Epidemiology The microfilariae of O. volvulus were first observed by O’Neill in Ghana in 1875 in an intensely pruritic chronic skin condition called ‘craw-​craw’. Leuckart described the adult worm 20 years later, and in 1923 Blacklock in Sierra Leone showed the blackfly Simulium damnosum to be the vector. Hissette in the Congo and Robles in Guatemala linked blindness with onchocerciasis. Long before, Ghanaians along the Red Volta River had associated the biting flies with skin lesions and blindness. The Onchocerciasis Control Programme suppressed vector breeding in West Africa’s Volta basin between 1974 and 2002, and is thought to have prevented 600 000 cases of blindness. Today, the largest numbers of infected people live in Nigeria, Cameroon, Chad, Ethiopia, Uganda, Angola, and the Democratic Republic of the Congo. In the Americas, aggressive treatment with ivermectin has eliminated or interrupted onchocerciasis transmission in Guatemala, Mexico, Colombia, and Ecuador. A small focus remains among the Yanomami Indians on the Brazil-​Venezuela border. In Africa, blindness had been noted to be more common in sa- vannah and woodland than rainforest areas, but people in forest areas had more depigmented skin disease. Different strains or forms of the parasite were shown to be present in savannah and woodland areas, particularly in West Africa. Environmental changes and mi- grations have now lessened these distinctions. Pathology The larvae of O. volvulus enter the human during a blood meal taken by an infected female Simulium fly. Within 1 to 3  months, larvae develop into male or female adult worms within palpable nodules commonly located over the bony prominences of the thorax, pelvic girdle, or knees (Fig. 8.9.1.2). Nodules can also be found on the head, particularly among chil- dren. These average 3 cm in diameter and are easily palpable, but some are deep, particularly around the pelvis. A female worm might release 1300–​1900 microfilariae per day for 9 to 11 years. From the nodules, these microfilariae find their way mainly to the skin and eyes. In the skin they are found predominantly in the subepidermal lymphatics. In the eye, most microfilariae are in the anterior chamber, but are also found in the retina and optic nerve. When an infected human is bitten, anticoagulants from the Simulium fly create a pool of blood from which blood and microfilariae are in- gested. Within the fly, those microfilariae that survive moult twice over the following 6 to 12 days to become infective larvae. Microfilariae are about 250–​300 µm in length and may live for up to 2 years. They move easily through the skin and connective tissue, ordinarily remaining within lymphatic vessels and provoking little reaction while alive. They have been found in blood, urine, cerebro- spinal fluid, and internal organs. Millions of microfilariae can be present in a heavily infected person. Although live microfilariae are tolerated by their human hosts, dead and dying microfilariae can evoke intense inflammatory reactions, which are mainly responsible for the eye and skin damage. Important Simulium spp. are complexes made up of sibling spe- cies, identifiable through the banding patterns of their larval chromo- somes. In Africa, the main vectors are members of the S. damnosum complex or sensu lato (s.l.), which can fly long distances. The vectors in areas of Uganda, Tanzania, Ethiopia, and the Congo are members of the S. neavei complex. In the Americas, complexes of S. ochraceum, S. metallicum, and S. exiguum are the principal vectors; these cover shorter distances. Some Simulium flies will bite humans almost exclu- sively, whereas other species are to varying degrees zoophilic. Simulium breed in water courses varying in size from broad rivers to small streams, depending on the individual sibling species. Rapidly flowing water provides the oxygenation needed for the development of the immature stages. Most larvae and pupae develop on rocks or vegetation just below the water surface, but those of S. neavei develop on amphibious Potamonautes crabs. During this developmental period the larvae are susceptible to insecticides. These breeding pat- terns have made the larviciding of water sources an effective con- trol approach. Unique relationships have developed between the Simulium fly and local parasites, so that flies from one geographical area do not efficiently transmit parasites from other areas. Clinical features The manifestations of onchocerciasis are almost entirely caused by localized host inflammatory responses to dead or dying microfil- ariae. In a heavily infected person, 100 000 or more microfilariae die every day. The predominant immune response in onchocerciasis is antibody mediated, but with an important cellular component. Inflammatory responses might vary considerably between groups of people, depending on the length of exposure to antigens and the down-​regulating activities of the host’s immune system. Eosinophils play an important role in the inflammatory re- sponse. Cellular proteins derived from eosinophils are deposited in connective tissues throughout the dermis, and bind to elastic fibres causing their destruction and, thereby, skin damage (see ‘Skin disease’, next). Fig. 8.9.1.2  A 3-​cm subcutaneous nodule.

8.9.1  Cutaneous filariasis 1481 An important discovery was that filarial parasites host endosymbi- otic Wolbachia bacteria. The inflammatory response to onchocerciasis seems associated with the Wolbachia rather than to the parasite itself. When the parasites were depleted of their Wolbachia by doxycycline they did not induce corneal lesions. Further studies showed that in- flammatory changes in the cornea in response to Wolbachia were de- pendent on the expression of myeloid differentiation factor 88. Exposure of the fetus to antigens associated with the parasite in utero and later through breast milk might induce immune tolerance in residents of endemic areas. This could explain the difference in the disease patterns seen in people from non​endemic areas who become infected. Eye damage The risk of visual impairment increases as the prevalence and in- tensity of infection rises in a community. Microfilariae enter the cornea from the skin and conjunctiva. Punctate keratitis develops around dead microfilariae, and clears when inflammation settles. In those exposed to years of heavy infection, sclerosing keratitis and iridocyclitis are likely to develop, causing permanent visual impair- ment or blindness. The first sign of sclerosing keratitis (Fig. 8.9.1.3a) is haziness at the medial and lateral margins of the cornea. This is followed by the migration of pigment onto the cornea, accompanied by a progres- sive ingrowth of vessels. Gradually the cornea becomes opacified. The central and superior areas are the last involved. Although eye lesions can be found wherever onchocerciasis occurs, blindness is most common in the West African savannah. Before control efforts began in Burkina Faso, 46% of men and 35% of women would even- tually become blind. Posterior segment lesions, which can coexist with anterior eye lesions, might be caused by inflammation around microfilariae entering the retina along the posterior ciliary vessels (Fig. 8.9.1.3b). Chorioretinal lesions are commonly seen at the outer side of the macula, or encircling the optic disc. Posterior segment changes have been an important cause of loss of vision in some countries. Loss of peripheral vision is well recognized in onchocerciasis. Skin disease Of all the consequences of onchocerciasis, skin lesions are the most pervasive. Surveys of seven endemic sites in five African countries found that between 40 and 50% of adults had troublesome itching, which was so intense in some cases that those affected slept on their elbows and knees to minimize the symptom. In its mildest form, onchocerciasis presents as itching with a lo- calized maculopapular rash (Fig. 8.9.1.4). These reactive lesions and itching may be evanescent, clearing completely without treat- ment in a few months. In other instances, the papular lesions may become chronic, generalized, and accompanied by severe itching (Fig. 8.9.1.5). Oedema and excoriations can be associated, and le- sions may heal with hyperpigmentation. Particularly distressing are lichenified hyperkeratotic lesions, which can be widespread and intensely itchy (Fig. 8.9.1.6). A localized form of chronic papular dermatitis, often confined to one extremity, is known as ‘sowda’, Arabic for dark. In this condition, first described from Yemen, there is an exceptionally strong IgG antibody response. Light-​skinned expatriates infected while visiting an endemic area might present a year or more later with intensely itchy and red macular, or maculopapular, lesions. These might be confined to one (a) (b) Fig. 8.9.1.3  (a) Bilateral sclerosing keratitis in a man blinded by onchocerciasis in Nigeria and (b) onchocerciasis producing a Hissette–​ Ridley fundus and optic atrophy in a person with central keyhole vision remaining. (a) Courtesy of Professor A. D. M. Bryceson; (b) courtesy of the Royal Tropical Institute, Amsterdam. Fig. 8.9.1.4  Maculopapular rash. Courtesy of Mauricio Sauerbrey.

section 8  Infectious diseases 1482 area of the body or be more generalized, and can be associated with fever, muscle and joint pain, and sometimes oedema. The rash can sometimes persist for several months following ivermectin treatment. In endemic areas, degenerative skin changes might develop in some people with long-​standing infection. Elastic fibres are des- troyed, leaving the skin thinned with a wrinkled cigarette-​paper ap- pearance. The atrophied skin begins to sag, the most extreme state being ‘hanging groin’ with its apron-​like skin folds (Fig. 8.9.1.7). Depigmentation of the pretibial areas, or ‘leopard skin’, is a charac- teristic finding in older people living in endemic areas (Fig. 8.9.1.8). Onchocercal skin disease reduces marital prospects (and dowry size), disrupts social relationships, and decreases the productivity of agricultural workers. As mass treatment with ivermectin has controlled onchocerciasis in many areas, appearance of these condi- tions is becoming increasingly rare. Other conditions associated with onchocerciasis Both men and women with onchocerciasis weigh less than unin- fected people and report more musculoskeletal pains. Evidence, first from Uganda and more recently from other African countries, has suggested an association between epilepsy and onchocerciasis. There is also evidence for an association between an increasing microfilarial load and excess mortality. Fig. 8.9.1.5  Excoriated papular lesions of onchocerciasis with hyperpigmentation. Fig. 8.9.1.6  Lichenified skin lesions with atrophy. Fig. 8.9.1.7  ‘Hanging groin’. Courtesy of the late Dr B. O. L. Duke. Fig. 8.9.1.8  Depigmented ‘leopard skin’.

8.9.1  Cutaneous filariasis 1483 A peculiar pattern of growth arrest beginning around the age of 6–​10 years was reported from a Ugandan onchocerciasis focus near Jinja in 1951. This Nakalanga syndrome now seems to have disap- peared from the area following the elimination of onchocerciasis, but has been noted in western Uganda, and might be present in Burundi. A condition of children in South Sudan, known as ‘nodding disease’, occurs in areas of onchocerciasis endemicity. ‘Nodding disease’ has also affected small areas of Uganda and Tanzania. Clinical features include head nodding, mental retardation, stunted growth, blind- ness, body stiffness, endless running nose and saliva, and faecal and urinary incontinence. This condition is still poorly understood, though infection with onchocerciasis might play some role. In nor- thern Uganda there have been no new cases in areas receiving twice yearly ivermectin distribution, augmented by insecticide application to rivers. Diagnosis Finding visible microfilariae in skin snips has been the time-​honoured method of diagnosis. Microfilariae lie close to the surface, and are most plentiful in the iliac crest area, except in Latin America, where they were previously more common in the shoulder and scapular areas. This remains a common method for detection in both indi- vidual diagnosis and to assess interruption of transmission in a popu- lation. Using polymerase chain reaction (PCR) analysis of skin snips for microfilaria greatly improves diagnosis where parasite counts are low. The examination of excised onchocercal nodules shows sections of adult worms. Immunoassay detecting the IgG4 antibody response to parasite Ov.16 antigen is an available method of diagnosing a cur- rent or previous infection and has a high degree of sensitivity and spe- cificity. Eosinophilia is common in onchocerciasis. The Mazzotti test, in which people with onchocerciasis react with itching and a skin rash to 50 mg of oral diethylcarbamazine (DEC), is seldom needed for diagnosis, and can be dangerous in heavy infec- tions. However, DEC patch tests, evoking a skin response at 24/​30hr are accurate and tolerated. Treatment The introduction of ivermectin for onchocerciasis in 1987 was one of the milestones of tropical disease treatment. Its discovery and de- velopment earned the 2016 Noble Prize in medicine. The symptoms of onchocerciasis can be effectively controlled by the treatment of individuals attending clinics, or through the mass treatment of en- demic communities. Ivermectin is derived from Streptomyces avermitilis. A  single dose of 150–​200 µg/​kg clears microfilariae from the skin for sev- eral months through the killing of microfilariae as well as blocking embryogenesis among adult female worms. Annual treatment con- trols microfilarial counts, and prevents the progression of clinical findings, although increasingly it is given twice yearly, with the in- tention of interrupting transmission. Treatment can be repeated if itching returns before the next dose is due. In the absence of reinfec- tion, individual treatment should probably be continued annually for 10 years or more, or until microfilariae are no longer detectable. In Nigeria, after 8 years of treatment, gross visual impairment de- creased from 16% to 1%, nodule prevalence fell from 59% to 18%, and papular skin dermatitis reduced from 15% to 2%. Treatment during pregnancy and under the age of 5 years is not recommended, although there has been no clear evidence of harm (increased risk of malformations or abortions) where treatment has been given inad- vertently in mass treatment programmes. Limiting the numbers of microfilariae through annual ivermectin treatment improves early and advanced anterior segment eye lesions, halts the development of optic nerve disease, and improves severe onchocercal skin lesions. Adverse reactions to ivermectin commonly consist of increased itching, swelling of the face or extremities, and headache and body pains. Hypotension has been reported rarely after treatment in heavily infected people. Bullae have been seen occasion- ally. The most pronounced adverse reactions occur after the first ivermectin treatment, decreasing after subsequent treatment cycles. Ivermectin has no adverse effects in uninfected people. Although ivermectin temporarily reduces the release of microfilariae by adult worms, it does not destroy the adults. Those coinfected with Loa loa, are at risk of developing potentially fatal central nervous system events after treatment with ivermectin. Although most severe reac- tions occur with L. loa counts more than 30 000 microfilariae/​ml, great caution should be observed when treating anyone with counts greater than 8000 microfilariae/​ml. Pretreatment with six weeks of Abendazole reduces levels of L. loa but levels might still put patients at risk of severe reactions after ivermectin. Ivermectin appears to have several separate actions against the parasite. In microfilariae it acts primarily on parasite neurotrans- mitters, producing paralysis. This action appears to be mediated by the potentiation or direct opening of glutamate-​gated chloride channels. The prolonged disappearance of microfilariae after a single treatment is the result of the drug’s effect on embryogenesis in the adult female worm. Treatment with ivermectin does not prevent the development of new infections by additional larvae introduced by bites of infected flies. Resistance to ivermectin has been reported where animal para- sites have been exposed to high and prolonged drug selection pres- sures. In 2007, a suboptimal response to ivermectin was noted among some persons in an area in Ghana who received annual mass treatment for many years. In several areas of Africa, twice annual treatment has now been implemented as standard. Through its ac- tion against Wolbachia bacteria, doxycycline given for six weeks has been shown to be effective in clearing microfilariae and killing some adult worms. A new medicine, moxidectin, has recently shown an excellent pattern of clearing microfilariae after a single treatment. Its role in mass treatment for onchocerciasis is not yet clear. Prevention and control Treatment and prevention methods have included insecticides added to rivers to interrupt Simulium breeding, mass distribution of ivermectin, and nodulectomy in an attempt to prevent blindness. Vector control Killing Simulium larvae by adding the insecticide dichlorodiphenyltrichloroethane (DDT) to rivers eliminated on- chocerciasis in Kenya and the Mabari forest of Uganda. In 1974, the Onchocerciasis Control Programme was formed to control Simulium by larviciding rivers in the Volta basin of West Africa using ecologic- ally suitable compounds. This highly successful vector control pro- gramme, later supplemented with ivermectin distribution, helped eliminate onchocerciasis as a public health problem from most foci in West Africa. Vector control continues to be appropriate in some locations, especially where transmission is with S. naevi.

section 8  Infectious diseases 1484 Ivermectin mass distribution After the effectiveness of ivermectin had been shown, its manufac- turer, Merck & Co., established the Mectizan Donation Program to provide the drug free ‘for as long as necessary to as many as ne- cessary’. Between 1988 and 2016, 2 billion ivermectin treatments had been provided for endemic countries through the Mectizan Donation Program which oversees drug approvals. This has in- volved the treatment of some 146 000 communities. The goal of a control programme in Latin America has been the elimination of disease transmission through twice yearly treatment. In all locations but two, there are no new infections or no evidence of disease transmission. The remaining ongoing transmission is among a small number of the Yanomami Indians who move back and forth across the Brazil-​Venezuela border in difficult-​to-​reach circumstances. In Africa, the 20-​year African Programme for Onchocerciasis Control oversaw mass distribution of annual ivermectin treatment which has eliminated the public health consequences of onchocer- ciasis in 20 countries. This has been accomplished using community selected distributors whose goal is to treat 85% of the eligible popu- lation each year. In several foci in several African countries the evi- dence suggests transmission has already been interrupted, but the final evidence is still awaited on elimination of transmission. The movement from control to elimination of transmission requires new public health approaches involving post-​treatment methods to verify the end of vector transmission and postelimination surveillance to verify the absence of new human infections. Some countries such as Uganda, Ethiopia, and Nigeria are embarking on these, of which twice annual treatment might play an important part. Treatment has so far been largely confined to meso-​ and hyperendemic areas. An elimination strategy would need to encompass the larger area where infection is hypoendemic (so previously excluded from mass treat- ment) and where transmission can still occur. Modelling suggests that elimination, with an accelerated strategy, is achievable in many countries by 2025, though some in countries this might be protracted, especially those with extensive Loa infections, and those with weak infrastructure. Countries such as the Democratic Republic of the Congo, Cameroon, and Angola have presented particular challenges. Conflict has retarded mass distribution in several areas. Population migration has changed the distribution of the disease in some loca- tions. Treatment of onchocerciasis in urban areas, such as in Congo (Brazzaville), present unique challenges. With the closure of the African Programme for Onchocercias Control in 2015, onchocerciasis mass treatment is now combined with mass drug distribution for the control or elimination of four other diseases in most African countries. Onchocerciasis control in Africa is now coordinated by the World Health Organiation’s Expanded Special Project for Elimination of Neglected Tropical Diseases (ESPEN). Nodulectomy A third form of onchocerciasis control has been the nodulectomy programmes of Mexico and Guatemala. For many years, health workers have moved from village to village removing nodules, es- pecially around the head. Evidence for this approach in preventing blindness is not strong. Eliminating infection Although ivermectin brings great relief to the individual, and has a clear impact on the disease in mass distribution programmes for affected populations, it does not kill adult worms. While symptoms and risks are controlled through annual ivermectin treatment, the disease itself is not eliminated, and the potential for the development of drug resistance remains. Several macrofilaricidal drugs capable of eliminating the disease through the killing of adult worms have been tested, but none has so far proved suitable for either individual or mass treatment. Treatment with doxycycline combined with ivermectin does show promise as do early trials with flubendazole. The filarial endosymbiont Wolbachia is a target for further drug development. Loiasis Loa loa is a filaria transmitted by Chrysops spp. flies in West and Central Africa. The adult worm migrates beneath the skin, and some- times across the eye, moving at about 1 cm per minute. Periodically, the infection causes sudden but transient localized inflammatory oedema known as Calabar swellings. Parasitology The larvae of L. loa burrow into human skin during feeding of the Chrysops or mangrove fly (C. silacea or C. dimidiata). In hu- mans, the parasites mature and live in the fascial layers. After 1  year or more, microfilariae are produced. Microfilariae are most heavily present in the blood in the daytime, between 10.00 and 15.00, when the Chrysops fly bites. Once taken up by the fly, microfilariae go through developmental stages in the fly’s thoracic muscles. After 10 days the fly is able to infect a human, and can do so for another 5 days. Epidemiology Infection is most commonly reported from around the Gulf of Guinea, particularly in Cameroon, Gabon, Congo (Brazzaville), Equatorial Guinea, Central African Republic, and Democratic Republic of the Congo. With continuing deforestation in some areas, infections are less common than in the past (Fig. 8.9.1.9). Humans are the only host, although a similar parasite is found in monkeys in the same areas. The fly lives in the rainforest canopy, and descends to bite humans, attracted perhaps by movement. Transmission may be most intense during the rainy season, when flies are breeding on the muddy banks of forest streams. Clinical features The first clinical symptoms of loiasis can appear as soon as 5 months after infection, or as late as 13 years. Calabar swellings appear sud- denly, most commonly on the forearms or wrists, and sometimes following heavy exercise or exposure to heat. These oedematous le- sions are red and itchy, and might be associated with fever and irrit- ability, but are generally non​tender. After several days the affected part returns to normal. However, recurrence is common at irregular intervals. Swellings are not confined to the arms, but can be present in the face, breasts, or legs. Calabar swellings are a hypersensitivity reaction to worm antigens which might be released in the process of migration or perhaps during the maturation of the worm. A high proportion of eosinophils are seen in peripheral blood smears, often exceeding 70%.

8.9.1  Cutaneous filariasis 1485 A second common feature is the appearance of a migrating worm (Fig. 8.9.1.10). This can be under the skin in any location, but is most dramatic when it crosses the eye (‘eye worm’; Fig. 8.9.1.11). Other than local irritation of the conjunctiva while the worm is passing, and the obvious concern of the host, there are no serious consequences. The time of passage can last from 30 min to more than 1 day. Rare but potentially serious consequences of L. loa are meningo- encephalitis, renal disease, and endomyocardial fibrosis. Arthralgias have also been noted. The meningoencephalitis can occur spontan- eously, although usually after treatment with diethylcarbamazine or Fig. 8.9.1.9  Map of the reported instances of Loa loa eyeworms using RAPLOA observations. From Zouré HGM et al. (2011). The geographic distribution of Loa loa in Africa: results of large-​scale implementation of the rapid assessment procedure for loiasis (RAPLOA). PLoS Negl Trop Dis 5(6), e1210, © 2011 Zouré et al. Fig. 8.9.1.10  Migrating adult Loa loa. Fig. 8.9.1.11  Loa loa crossing the bulbar conjunctiva.

section 8  Infectious diseases 1486 ivermectin. Fatalities have been reported following treatment. The renal and endocardial complications of loiasis are likely due to the deposition of immune complexes. Laboratory diagnosis Diagnosis has traditionally been by the finding of microfilariae in a daytime blood sample, or by a history of typical clinical findings. The use of more sensitive PCR methods has shown that many people, even perhaps most, of those infected do not have microfilariae in their peripheral blood. Treatment The standard treatment has been diethylcarbamazine, which kills microfilariae and many adult worms. The treatment is commonly given in doses of 8–​10 mg/​kg orally in three divided doses daily for 21 days. Fever, arthralgia, and itching can occur during treat- ment. Ivermectin at 200 µg/​kg dramatically decreases the number of microfilariae and some of the loiasis symptoms, but has little macrofilaricidal effect. Two courses of treatment may be required. As with diethylcarbamazine, there is a high risk of potentially fatal meningoencephalitis in persons with microfilarial counts over 30 000 microfilariae/​ml. Caution should be observed in the treat- ment of all persons with over 8000 microfilariae/​ml. Albendazole given in six doses substantially reduced high Loa microfilarial counts in some patients but not all. Treatment with small doses of iver- mectin does not offer any advantages. As L. loa does not harbour Wolbachia, treatment with doxycycline is ineffective. Since many people with loiasis also have onchocerciasis, careful monitoring for severe eye and skin inflammation is important when giving diethylcarbamazine. Blood films for microfilariae or PCR tests should be followed to indicate the need for retreatment. Prevention The best prevention is avoiding Chrysops fly bites. Having window screens on dwellings, wearing clothing to protect the legs and fore- arms, and avoiding areas where biting is frequent can reduce the risk. Chemoprophylaxis with diethylcarbamazine has been sug- gested, using either 5 mg/​kg on three consecutive days in a month, or a weekly dose of 300 mg while living in an area of transmission. Mansonellosis Mansonella spp. are a group of filarial species common to many countries, but are of negligible clinical importance under most circumstances. Infection is transmitted by Culicoides spp. biting midges. Epidemiology Mansonella (formerly Dipetalonema) perstans is found widely in sub-​Saharan Africa, as well as Trinidad and several parts of South America. The adult worms live free in the abdominal cavity, and microfilariae are found in the blood and skin. Mansonella ozzardi is found in the West Indies and Central and South America. In addition to Culicoides, Simulium flies have been reported to transmit M. ozzardi in the Amazon basin. Mansonella streptocerca is a common infection in West and Central Africa, extending into western Uganda. Both microfilariae and adult worms are found in the skin, but without the nodules seen in onchocerciasis. Unless M.  streptocerca microfilariae are differentiated parasitologically from those of O. volvulus, inappropriate mass onchocerciasis treat- ment programmes could inadvertently be implemented. Clinical manifestations Of the Mansonellas, only M. streptocerca produces clear-​cut symp- toms, although even these can be confused with those of O. volvulus, and which might be a coinfection. Chronic papular lesions are com- monly present, often associated with postinflammatory hyperpig- mentation. Lichenification occurs less commonly. Hypopigmentation has been noted in areas of skin overlying the location of adult worms in the skin. In general, these findings are not easily distinguishable from those of onchocerciasis. Eosinophilia is common. M. perstans has been reported to produce Calabar-​like swellings, pruritus, fever, and headache. M. ozzardi infections are generally asymptomatic, although vague complaints of fever, arthralgia, head- ache, and itching have been associated with infection. There have been reports of ocular lesions in Brazil. Diagnosis Diagnosis is by finding characteristic microfilariae in the blood or skin. The tails of the microfilariae have a distinctive walking-​stick shape, and contain four prominent nuclei, distinguishing them from microfilariae of O. volvulus. A PCR assssay has been described for M. streptocerca, and both quantitative buffy coat fluorescent staining and enzyme immunoassay methods for M. perstans. Eosinophilia is a characteristic finding. Treatment In asymptomatic people no treatment is required. M. streptocerca re- sponds well to ivermectin, producing prolonged suppression of circu- lating microfilariae. Mild reactions similar to those in onchocerciasis may be seen. The treatment of M. perstans with doxycycline is ef- fective, consistent with the effect of the drug on Wolbachia endosym- bionts. A combination of both diethylcarbamazine and mebendazole is highly effective against M. perstans, while ivermectin has little effect. For M. ozzardi infections, a single ivermectin dose of 0.14-0.2 mg/kg has been found to be highly effective, though the risks of Mazzotti-​like reactions are well described in persons with high parasite loads. FURTHER READING Onchocerciasis African Programme for Onchocerciasis Control (2015). African Pro­ gramme for Onchocerciasis Control: progress report, 2014–​2015. Wkly Epidemiol Rec, 90, 661–​80. Coffeng LE, et  al. (2013). African Programme for Onchocerciasis Control 1995–​2015: model-​estimated health impact and cost. PLoS Negl Trop Dis, 13, e2032. Colebunders R, et al. (2015). Nodding syndrome since 2012: recent progress, challenges and recommendations for future research. Trop Medi Int Health, 22, 194–​200. Debrah, AY, et al. (2015). Doxycycline leads to sterility and enhanced killing of female onchocerca volvulus worms in an area with per- sistent microfilaridermia after repeated ivermectin treatment: a ran- domized, placebo-​controlled, double-​blind trial. Clin Infect Dis, 61, 517–​26.

8.9.2 Lymphatic filariasis 1487

8.9.2 Lymphatic filariasis 1487

8.9.2  Lymphatic filariasis 1487 Gillette-​Ferguson I, et al. (2006). Wolbachia-​ and Onchocerca volvulus-​ induced keratitis (river blindness) is dependent on myeloid differen- tiation factor 88. Infect Immun, 74, 2442–​5. Hopkins AD (2015). From ‘control to elimination’: a strategic change to win the end game. Int Health, 7, 304–​5. Kaiser C, et al. (1996). The prevalence of epilepsy follows the distribu- tion of onchocerciasis in a west Ugandan focus. Bull World Health Organ, 74, 361–​7. Katabarwa MN, et al. (2016). Community-​directed interventions are practical and effective in low-​resource communities: experience of ivermectin treatment for onchocerciasis control in Cameroon and Uganda, 2004–​2010. Int Health, 8, 116–​23. Lawrence J, et  al. (2015). Growth, challenges, and solutions over 25 years of mectizan and the impact on onchocerciasis control. PLoS Negl Trop Dis, 9, e0003507. Ottesen EA (1995). Immune responsiveness and the pathogenesis of human onchocerciasis. J Infect Dis, 171, 659–​71. Stanimira P, et  al. (2015). African Programme for Onchocerciasis Control 1995–​2010: impact of annual ivermectin mass treatment on off-​target infectious diseases. PLoS Negl Trop Dis, 9, e0004051. Uniting to Combat NTDs (2012). London declaration on neglected tropical diseases. http://​unitingtocombatntds.org/​resource/​london-​declaration Vlaminck J, et al. (2015). Diagnostic tools for onchocerciasis elimin- ation programs. Trends Parasitol, 31, 571–​82. Zouré HG, et  al. (2014). The geographic distribution of onchocer- ciasis in the 20 participating countries of the African Programme for Onchocerciasis Control: (2) pre-​control endemicity levels and estimated number infected. Parasit Vectors, 22, 326. Loa loa Kelly-​Hope LA, et al. (2014). Innovative tools for assessing risks for severe adverse events in areas of overlapping Loa loa and other fil- arial distributions: the application of micro-​stratification mapping. Parasit Vectors, 7, 307. Metzger WG, Mordmüller B (2014). Loa loa—​does it deserve to be neglected? Lancet Infect Dis, 14, 353–​57. Zouré H (2011). The geographic distribution of Loa loa in Africa: re- sults of large-​scale implementation of the Rapid Assessment Procedure for Loiasis (RAPLOA). PLoS Negl Trop Dis, 6, e1210. Mansonella Gehringer C (2014). Molecular evidence of wolbachia endosymbiosis in Mansonella perstans in Gabon, Central Africa. J Infect Dis, 210, 1633–​8. Stensgaard A-​S (2016). Ecological drivers of mansonella perstans infection in Uganda and patterns of coendemicity, with lymphatic filariasis and malaria. PLoS Negl Trop Dis 10, e0004319. 8.9.2  Lymphatic filariasis Richard Knight ESSENTIALS Wuchereria bancrofti, Brugia malayi, and B. timori are mosquito-​borne lymphatic-​dwelling nematode parasites that are important causes of morbidity, disability, and social stigma in tropical and subtropical countries. Bancroftian filariasis due to W.  bancrofti, which has no animal reservoir, accounts for 90% of human infections worldwide. Clinical features Acute lymphatic filariasis—​(1) lymphadenitis and lymphangitis—​most common in the inguinal and femoral nodes; (2)  acute genital—​ usually tender fusiform or cylindrical swelling of the spermatic cord; (3) abscess and fever—​affected nodes may break down to produce an open ulcer. Chronic lymphatic filariasis—​(1) lymphoedema and elephantiasis—​ initially transient pitting oedema occurs during acute inflammatory episodes in proximal nodes; eventually brawny, non​pitting oedema becomes permanent; (2) chronic genital—​most commonly hydro- cele; (3) chronic lymphadenitis and lymphangitis; (4) chyluria and lymphuria; (5)  non​lymphatic pathology—​including tropical pul- monary eosinophilia, filarial arthritis, and filarial glomerulonephritis. Diagnosis, treatment, and control Diagnosis—​microfilariae are typically found in Giemsa-​stained blood films; the sample is best taken at night (22.00–​02.00), except in Oceania and parts of Southeast Asia. Microfilariae are also some- times found in aspirates from lymph varix, hydrocele, lymphocele of the cord, or in urine. A rapid antigen detection test allows the mapping of prevalence and assessment of the impact of mass drug distribution. Individual treatment—​diethylcarbamazine, which may provoke both local and systemic reactions, or doxycycline are needed in some situations, including infected visitors, people leaving infected areas, and those with tropical pulmonary eosinophilia or other clinical features where elimination of adult worms is a priority. Concurrent bacterial infection requires prompt treatment with antibiotics, and supportive bandaging can reduce chronic oedema. Community wide treatment and control—​The Global Programme for the Elimination of Lymphatic Filariasis usually involves four or six annual rounds using two-​drug combinations of ivermectin, albendazole, or diethylcarbamazine to all eligible persons to interrupt transmission by reducing the numbers of circulating micro- filariae, together with (in appropriate circumstances) vector control. This regimen will eliminate acute disease episodes and significantly reduce chronic morbidity. Introduction Wuchereria bancrofti, Brugia malayi, and B.  timori are mosquito-​ borne nematode parasites. They are important causes of morbidity, disability, and social stigma in tropical and subtropical countries (Fig. 8.9.2.1). The total population at risk is estimated to be 856 mil- lion in some 52 countries where these infections are endemic. In 2000 bancroftian filariasis due to W. bancrofti infected 120 million people of whom about 40 million had clinical disease and some 80 million had hidden lymphatic damage; it was introduced into the Americas from Africa by the Atlantic slave trade. The two Brugia species in- fected about 13 million people in South and Southeast Asia. B. timori has a localized distribution in a few islands in eastern Indonesia. Lymphatic filariasis ranks second as a cause of disability worldwide.

section 8  Infectious diseases 1488 Fig. 8.9.2.1  Lymphatic filariasis endemic countries and territories by mass drug administration (MDA), 2013. Reproduced from WHO Weekly Epidemiological Record No. 38, 2014, 89, 409–​420, with kind permission of the World Health Organization.

8.9.2  Lymphatic filariasis 1489 Aetiology: The biology of the parasite The adult worms live in the larger lymphatic vessels and lymph nodes; many live as ‘worm nests’ within dilated lymphatics of the limbs and male genitals. The worms are smooth, creamy-​white, and threadlike; females measure 8 to 10 cm in length and males 4 cm. Their lifespan is estimated to be 4 to 6 years, but may be longer—​a critical issue for planning, implementation, and duration of elimin- ation programmes. Mated females produce numerous microfilariae throughout their life; these actively motile embryonic worms are sheathed by the remnants of the egg shell. They are 180–​290 µm in length and 7–​10 µm in diameter, with diagnostic species morpholo- gies in stained blood films. Microfilariae migrate via the lymphatic system to the blood, where they have an estimated lifespan of up to 12 months. Their numbers in the peripheral blood vary during the day and night, a phenomenon known as periodicity, and when not circulating they are sequestered in lung and reticuloendothelial capillaries. Maximum counts in the blood coincide with the biting cycle of the vector. The species and strain of parasite determine the periodicity. Most common is the nocturnally periodic form, with maximum microfilarial counts found between 22.00 and 02.00, and virtual absence during the day. Alternatively, microfilariae may be pre- sent throughout the 24-​h cycle, with prominent peaks during the day or the night; referred to as diurnal or nocturnal subperiodicity, respectively. After ingestion by the mosquito, microfilariae penetrate the midgut and migrate to the thoracic muscles, where they mature over 9 to 15 days to infective third-​stage larvae. These then migrate to the head of the mosquito and escape through the arthrodial membranes around the proboscis during a blood meal. Larval worms enter the puncture wound made by the vector, enter the peripheral lymphatic system, and most eventually reach the lymph vessels of the prox- imal limb and male genitalia. Sexual maturity and the appearance of microfilariae in the blood usually take 8 to 18 months, but some- times only 3 months. Both adult worms and microfilariae harbour Wolbachia bacterial endosymbionts that are essential for the repro- duction and survival of the parasite. Epidemiology and transmission In endemic areas microfilaria prevalence rates increase steadily from early childhood and often reach a maximum in early adult life, when a prevalence of 10–​30% is not unusual in highly endemic areas. However, a recent meta-​analysis suggests that prevalence, in the absence of a decline in transmission, may often continue to rise with age. Prevalence in males is generally higher, perhaps as a result of greater vector exposure. The cord blood of some infants shows microfilariae. Recent studies using an immunochromatographic card test to detect adult worm antigen showed that in a population of Haitian children prevalence reached 25% by the age of 4 years. The test is used to map the distribution of Wuchereria bancrofti and gives a prevalence of approximately double that detected by night blood films. Detailed mapping, using districts, assists in defining pro- gramme implementation units for the purposes of planning mass drug distribution. W.  bancrofti has no animal reservoir. Brugia malayi, however, is a zoonosis in some areas of its distribution (southern Thailand, Indonesia, and Malaysia), with a reservoir in cats and leaf monkeys, although their importance in terms of maintaining the cycle in hu- mans is not known; elsewhere it is an anthroponosis with only a human source of infection. Mosquito vectors and geographical distribution W. bancrofti infection Culex spp. transmission This vector, mainly C. quinquefasciatus, breeds mostly in organic- ally polluted water, usually in urban and suburban areas, but also villages where there are suitable latrine and cesspit habitats. Culex is the most widely distributed vector and is increasing with urban- ization; it occurs in India, Sri Lanka, Central and South America, some Caribbean islands, urban and coastal villages in East Africa and Egypt, and formerly in parts of China, where transmission has been eliminated. Culex bites at night; the microfilariae are noctur- nally periodic. Culex is the most efficient vector and can maintain transmission at low microfilarial densities, making control difficult. Anopheles spp. transmission The same species of Anopheles, notably An. gambiae sensu lato and An. funestus, commonly transmit both filariasis and malaria in East and West Africa. In Papua New Guinea and Vanuatu, the vectors are Anopheles of the punctulatus complex. Anopheles bites at night, mainly on the legs; microfilariae are nocturnally periodic. Aedes spp. transmission This is limited to southern Oceania, especially Fiji, Samoa, Tonga, the Cook Islands, and New Caledonia; but also patchily in Thailand, the Philippines, Vietnam, and the Nicobar islands. Aedes feeds throughout the 24-​h cycle, but predominantly with a daytime biting peak, and bites all over the body; the microfilariae are diurnally subperiodic. Ochlerotatus spp. transmission This genus of mosquito transmits subperiodic and aperiodic strains of W.  bancrofti in Asian forest habitats and in the Philippines, Samoa, and New Caledonia. Until recently this genus was classified in the genus Aedes. B. malayi infection Zoonotic Mansonia spp. transmission in swamp forests This occurs in Malaysia, Indonesia, and southern Thailand. Mansonia bites mainly by night, but also during the day, usually on the legs below the knee; the microfilariae are nocturnally subperiodic. Transmission in agricultural areas In parts of Malaysia, Buru in Indonesia, and southern Thailand a mixed anthroponosis and zoonosis occurs in transitional zones, with monkeys and cats as reservoirs, and both Anopheles and Mansonia as vectors. Microfilariae have periodicities intermediate between nocturnally periodic and nocturnally subperiodic.

section 8  Infectious diseases 1490 In India (mainly Kerala), Malaysia, Sulawesi, southern Thailand, and Vietnam infection involves humans only, with Anopheles as the main vector and Mansonia as an accessory vector; the microfilariae are nocturnally periodic. B. timori infection This is confined to Timor Leste and islands in the Lesser Sundas group in eastern Indonesia. Anopheles barbirostris is the vector, and the microfilariae are nocturnally periodic. Pathogenesis Local immunological reactions to worm antigens provoke acute and subacute responses, with dilatation of lymphatics and infiltra- tion of tissues with eosinophils and monocytes. The antigens derive from the moulting fluids of developing worms, excretory products, microfilariae trapped within the lymphatic system, and also dying worms, including those killed by chemotherapy. Wolbachia lipopro- teins from living and dying worms also provoke inflammation; thus, living worms can create lymph vessel dilatation and stasis. Several immunological mechanisms and cytokines are involved including vascular endothelial growth factors. Living worms also induce sup- pressive immunomodulatory responses that facilitate worm survival so that subjects with high blood microfilaraemias may have no evi- dent clinical disease. Dead and disintegrating worms become surrounded by granula- tion tissue with giant cells and epithelioid cells. Stasis and blockage of lymph vessels leads to distal dilatation, with varicosities and valve incompetence. Prolonged or recurrent lymph stasis leads to the ac- cumulation of protein-​rich interstitial tissue fluid, fibroblast prolif- eration, dilated dermal lymphatics, and epithelial acanthosis and hyperkeratosis. Determinants of pathology include the duration of exposure, in- tensity of transmission, anatomical sites of infective mosquito bites, human genetic factors, and the species and strain of parasite. Prenatal exposure to filarial antigen is of great importance and induces im- munological tolerance. Residents in high-​transmission areas often show patent microfilaraemia, but little immunopathology. However, in some adults a later decline in microfilarial prevalence parallels in- creased host immunological reactivity and pathology. New residents and visitors show marked local reactivity to worms, and often no blood microfilariae; the latter situation was well documented among American troops in the Pacific in the Second World War, and French troops in former Indochina. Clinical manifestations Acute lymphatic filariasis In endemic areas acute episodes are recurrent from the age of 10 years, and most frequent 4 to 8 months after the peak of seasonal transmission. Episodes last several days or weeks; fever and malaise are common, but blood eosinophilia is not marked. Persons leaving endemic areas cease to have acute episodes after 1 year, although they may experience recurrent pain in previously affected tissues, especially after unusual exercise. Filarial lymphadenitis and lymphangitis Tender lymphadenopathy is most common in the inguinal and fem- oral nodes, but axillary and epitrochlear nodes are also affected. Tender retrograde lymphangitis typically spreads peripherally below the node. Acute genital filariasis This is uncommon in boys before puberty, but common thereafter. The typical lesion is funiculitis, with a tender fusiform or cylindrical swelling of the spermatic cord; epididymitis and orchitis are less common. Filarial abscess and filarial fever Affected nodes in the groin or elsewhere may break down producing an open ulcer that heals slowly leaving characteristic scars. Pelvic and retroperitoneal lymphadenitis can produce a febrile illness that is difficult to diagnose. Chronic lymphatic filariasis Lymphoedema and elephantiasis Initially, transient pitting oedema occurs during acute inflamma- tory episodes in proximal nodes. Bacterial infection, often caused by Streptococcus pyogenes, is common in those with compromised lymphatics, especially when the skin is fissured, breached in an inter- digital cleft, or when there is minor injury, an ulcer, or insect bite; this presents as cellulitis and ascending lymphangitis. Later, oedema persists between episodes, becoming distally non​pitting. Eventually, brawny non​pitting oedema becomes permanent (Fig. 8.9.2.2). In patients with leg involvement, epidermal thickening, papillomatosis, and fissuring are common (Fig. 8.9.2.3). Fig. 8.9.2.2  Chronic elephantiasis in a man in Belém, northern Brazil. Note the scars of unsuccessful surgery. Copyright Pedro Pardal.

8.9.2  Lymphatic filariasis 1491 Chronic genital filariasis Hydrocele (Fig. 8.9.2.4) is the most common lesion, and preva- lence rates may reach 30% in men over 35 years in highly en- demic areas; many patients give a history of preceding episodes of funiculitis or epididymitis. Hydrocele fluid is usually a transudate, but lymph or blood may be present. The tunica vaginalis is often thickened. Nodular lesions of the spermatic cord and epididymis are common, and the testis itself may become enlarged and indur- ated. Lymphoceles occur on the cord. Dilated dermal lymphatics in the scrotal wall associated with atrophic epidermis produce ‘lymph scrotum’, the skin having a velvety appearance. Rupture of these lymphatics leads to weeping skin lesions and often secondary in- fection, occasionally complicated by Fournier’s gangrene caused by anaerobic bacterial sepsis. Lymphoedema of the scrotum is a late sequel; often the testes are unaffected, and penile lesions are rare. Vulval lymphoedema is underrecognized; it is associated with dilated retroperitoneal lymph- atics, and must be distinguished from lymphogranuloma venereum. Chronic lymphadenitis and lymphangitis Recurrent episodes of acute inflammation lead to persisting and sometimes massive lymph node enlargement. Thickened lymph- atic cords may be palpable connecting the axillary and epitrochlear, or the femoral and popliteal nodes. Varicose lymph vessels may be visible in these areas. ‘Lymph varices’ are fluctuant sacs of lymph- atic tissue derived usually from the capsule of a node, hence the alternative term ‘lymphadenocele’. They partially empty when the part is raised, and aspiration reveals lymph or occasionally chyle. They occur in the medial thigh, groin, axilla, and sometimes even the neck. Chyluria and lymphuria Dilated pelvic and retroperitoneal lymphatics may rupture into the urinary tract in the renal pelvis, ureter, or bladder. When there is lymph stasis above the cisterna chyli then small-​bowel chyle may re- flux into the urine postprandially. Chyluria is often intermittent and blood stained (Fig. 8.9.2.5). Continued loss of protein and lipids in the urine may lead to weight loss and cachexia. Chyluria may even- tually be self-​limiting. Non​lymphatic pathology Tropical pulmonary eosinophilia This presents as a subacute or chronic illness with cough, wheezing, and reticular or miliary pulmonary shadowing. Microfilariae are absent from the blood, but eosinophilia is marked, and titres of filarial antibody are very high. Some patients have features of lymphadenopathic or genital filariasis, but many do not. The defect in of lung function is restrictive. The response to antifilarial treat- ment is good, but untreated the condition can lead to pulmonary fibrosis and pulmonary hypertension. The syndrome is the result of a heightened immunological response to dead microfilariae, which may be found in biopsies of lung and other tissue, surrounded by eosinophilic microabscesses. It occurs in most endemic areas, but is rare in Africa. It is more common in men, and rare in children, and many patients are not long-​term residents. Filarial arthritis Joint involvement is subacute, and often recurrent with effusion; it usually affects the knee. Fig. 8.9.2.4  Gross hydrocele in a patient with chronic filariasis. Courtesy of the late P. E. C. Manson-​Bahr. Fig. 8.9.2.3  Chronic elephantiasis with epidermal thickening, fissuring, and papillomatosis in a man in north-​east Nigeria. Copyright D. A. Warrell.

section 8  Infectious diseases 1492 Filarial glomerulonephritis This results from filarial and streptococcal immune-​complex deposition on the glomerular basement membrane, but there is also tubular damage. Clinical findings include proteinuria and haematuria, which usually respond to chemotherapy. The incidence of clinically significant disease and its prognosis are uncertain. Diagnosis Clinical Many patients will have several clinical features that, together with a history of preceding acute episodes, will be strongly suggestive diagnostically—​manifestations such as varicose lymphatics, lymphadenocele, retrograde lymphangitis, and lymph scrotum are highly specific to filariasis. Genital lesions are rare in Brugia infec- tions, which usually present with lymphoedema below the knee. In B. timori infection lymph node pathology in the legs is often severe, sometimes with skin ulceration. Upper limb and breast le- sions are common in diurnally subperiodic W. bancrofti infections in the Pacific, but they do occur elsewhere with other strains of this parasite. Parasitological Microfilariae (Fig. 8.9.2.6) are typically found in Giemsa-​stained blood films, but also in aspirates from a lymph varix, hydrocele, or lymphocele of the cord, or in urine in chyluria patients. Blood should be taken to coincide with the expected microfilarial peri- odicity. For quantitative studies, measured 10-​ or 20-​µl volumes are used to prepare thick blood films. For measuring changes in intensity, larger measured quantities of blood (60 microlitres) should be used to increase the sensitivity and accuracy of a key parameter. Counting chambers taking 100 microlitres of lysed blood can be used, or larger volumes may be lysed and the spun de- posit examined. A sensitive method which allows quantitation of parasite density is filtration of 1–​5 ml of heparinized venous blood through a nucleopore filter of pore size 5 microns; microfilariae on the filters can then be stained. Nocturnally periodic W. ban­ crofti microfilariae appear transiently in the blood 30 to 60 min after a 100-​mg dose of diethylcarbamazine, which forms the basis of the provocation test. Species diagnosis of stained microfilariae is made by their sheath characteristics and the arrangement of caudal nuclei. The microfilariae of Loa loa, the tropical eye worm (found only in West and Central Africa) are diurnally periodic and also have a sheath; they must be distinguished from those of lymphatic filariasis. Immunodiagnosis Positive filarial antibody and skin tests are common in those ex- posed to infection, and may be of value in visitors to an endemic area. Several tests are now available for W.  bancrofti antigen in serum, including a card test for field use. A positive test indicates persisting adult worms; antigen may be present in the absence of microfilaraemia. For Brugia infections, techniques for DNA detec- tion by polymerase chain reaction are available, and also specific IgG4 antibody tests. Imaging of lymphatic vessels Lymphangiography will delineate the anatomical details of ab- normal lymphatic tissues, such as lymph varices and lymphatic connections to the urinary tract in chyluria. They are not usually diagnostic for filariasis. Scrotal ultrasonography can show nests of live worms—​the ‘filarial dance’ sign; this can be used to assess the impact of chemotherapy on adult worms. Lymphoscintigraphy using technetium-​labelled dextran or al- bumin is a less invasive technique for demonstrating lymphatic pathology. Abnormal dermal lymphatics occur in many asymptom- atic infected persons in endemic areas. Fig. 8.9.2.5  Chyluria and haematuria in a patient with chronic filariasis. Courtesy of the late P. E. C. Manson-​Bahr. Fig. 8.9.2.6  Microfilaria of W. bancrofti on a Giemsa-​stained blood film showing sheath and row of terminal nuclei (right).

8.9.2  Lymphatic filariasis 1493 Drugs for the control and treatment
of lymphatic filariasis Filaricides may act against adult worms (macrofilaricides), against microfilariae (microfilaricides), or both. They often also act against other filarial infections such as Onchocercaa volvulus and L. loa; this may cause severe reactions in patients coinfected with these para- sites. Side effects of filaricides are mainly due to immunological re- actions to dying worms and the release of Wolbachia lipoproteins. Diethylcarbamazine Diethylcarbamazine was first introduced for treatment of lymphatic filariasis in 1948 and until 25 years ago it was the only drug available. It acts against both adult worms and microfilariae, but predominantly the latter; its mode of action is poorly understood but involves the arachi- donic pathway. The resulting damage to the worm surface leads to vig- orous immunological reactions that may be dangerous. A single dose of 6 mg/​kg body weight greatly reduces the level of blood microfilariae for a year and kills some adult worms. In communities with low and declining endemicity many of the remaining adult worms are near the end of their lifespan and more easily killed with diethylcarbamazine. Ivermectin Ivermectin has a broad spectrum of antiparasitic activity. In lymph- atic filariasis it acts only against microfilariae, and a dose of 100–​ 200 µg/​kg will clear microfilariae as well as diethylcarbamazine. Its action is rapid and side effects correlate with microfilaria load. Ivermectin immobilizes microfilariae by hyperpolarization of glutamate-​sensitive channels. Albendazole Albendazole is a broad spectrum anthelminthic and is widely used to treat intestinal nematodes. It acts by inhibiting polymerization of β-​tubulin and microtubules. A 400 mg dose will clear most intestinal nematode infections and will reduce microfilaria levels in lymphatic fil- ariasis progressively over 6–​12 months; greater reductions occur when it is given with either diethylcarbamazine or ivermectin, with which it acts synergistically against microfilariae. It does not kill adult worms. Doxycycline Doxycycline is a broad spectrum antibiotic that kills the Wolbachia endosymbionts. A 100 mg or 200 mg dose given daily for 3 to 8 weeks will kill both adult worms and microfilarariae; its action is slow over 12 months and this greatly reduces the severity of side effects that occur in response to the rapidly dying worms and Wolbachia with other filaricides. Treated patients show a significant improvement of their hydrocele, lymphoedema, and other clinical manifestations of lymphatic filariasis. Unfortunately, the drug is contraindicated in children under 9 years of age and in pregnant women. Filariasis at the community level The Global Programme to Eliminate
Lymphatic Filariasis A World Health Assembly Resolution in 1997 launched a pro- gramme to eliminate lymphatic filariasis as a public health problem by 2020. This resolution was based on new evidence of the impact of two-​drug combinations on microfilaraemia, and the availability of a rapid antigen test, the immunochromatographic card test, that allows mapping of the prevalence of disease and assessment of the impact of mass drug administration (MDA). Clinical studies had demonstrated that the annual distribution of diethylcarbamazine and albendazole, or albendazole and ivermectin, for 4 or 6 years re- duced microfilaraemia levels by about 95%. Mathematical models suggested that this would interrupt transmission, provided that an- nual coverage exceeded 65%. The programme was backed by generous commitments by the manufacturers to donate albendazole and ivermectin. A  global public–​private partnership was formed in 2000, the Global Alliance to Eliminate Lymphatic Filariasis (GAELF). The extensive geo- graphic distribution of lymphatic filariasis required a regional ap- proach to programme management and planning with detailed mapping, baseline data collection, and the establishment of evalu- ation and monitoring based on sentinel-​site selection. Training drug distributors; selecting appropriate drug distribution systems; infor- mation, education, and communication; social mobilization needs; and reporting systems were recognized as being of great importance. By 2015, having reviewed the global situation, the World Health Organization (WHO) reported that 73 countries were endemic and 63 of these had implemented mass drug distribution to stop trans- mission; 18 countries that were previously included in the list of endemic countries were deemed to be free of endemic disease. In 2015, a total of 698 million people were targeted for MDA, of whom 556 million received the recommended WHO two-​drug combin- ation of albendazole plus either ivermectin or diethylcarbamazine, an average coverage of 80%. The estimated numbers of cumulative treatments since the programme began in 2000 is around 6.7 billion treatments. An important advantage in using albendazole is the re- duction of the burden of soil-​transmitted helminths such as hook- worm, Trichuris, and Ascaris. To assess the impact of MDA WHO has provided guidelines for Transmission Assessment Surveys which are based on using immunochromatographic tests in cohorts of children aged 6 to 10 years who were born shortly after the annual drug distributions began. Additional assessment of transmission or the existence of microfilarial parasites in the population can be achieved by a technique known as xenomonitoring when mosqui- toes are caught and examined for the presence of filarial DNA. Drug coverage in an MDA programme may be misleading as compliance, actual ingestion of the tablets, may be lower; in an extensive review of the programme in India it was 22% lower. Compliance varies be- tween households and relate to absences, lack of perceived need, drug contraindications, or fears of toxicity. The programmes are not yet including all those who need it; In the African Region 395 mil- lion were estimated to need MDA but only 44% were covered, the respective figures in the Southeast Asian Region were 501 million and 72%. The needs of patients already afflicted with lymphoedema and hydrocele must also be addressed. To this end WHO has issued guidelines for home-​based care, whereby lymphoedema patients and their families are taught how to treat lymphatic filariasis-​related lymphoedema and prevent acute attacks. WHO also aims to increase access to hydrocele surgery that uses new reconstructive techniques. Despite resource constraints, particularly in sub-​Saharan Africa, there are encouraging signs that the programme is reducing the

section 8  Infectious diseases 1494 prevalence of the disease. Egypt has reported the elimination of transmission in formerly endemic areas of the Nile Delta. China has been certified by WHO as free of transmission in a population of some 350 million who were previously at risk. The Republic of Korea has also eliminated a focus of Brugia malayi and certified free of transmission. Several smaller countries previously demonstrated disease elimination following a range of different interventions. These are Suriname, Costa Rica, Trinidad and Tobago, and also the Solomon Islands, where vector control for malaria, using indoor re- sidual spraying with dichlorodiphenyltrichloroethane (DDT) in the 1970s, appears to have been effective. Population-​based chemotherapy In the past, different dosage regimens of diethylcarbamazine alone were used in many endemic areas. Drugs were given annually or 6 monthly, either to the whole population or to those found to be infected; medicated salt being the alternative. The main aim was to eliminate microfilaraemia and hence transmission, but with re- peated doses many adult worms are eventually killed. The availability of ivermectin offered an effective alternative for reducing microfilaraemia, but does not kill adult worms. A single dose of 200 µg/​kg of ivermectin is as effective as a 6 mg/​kg dose of diethylcarbamazine. Both will virtually eliminate microfilaraemia for 6 or 12 months, adverse reactions are probably equally common with both drugs. Albendazole is also effective as a microfilaricide, and probably has some activity against adult worms. A 400 mg dose given annually can replace either diethylcarbamazine or ivermectin in a two-​drug annual regimen. Annual dosage with either of these two-​drug combinations, continued for 4 to 6 years (the lifespan of nearly all adult worms), will interrupt transmission. It is not recommended that diethylcarbamazine be given in areas where onchocerciasis or loiasis are endemic to avoid dangerous reactions. Loa-​associated encephalopathy occurs especially in people with Loa microfilarial loads of more than 8000/​ml of blood. Similarly, extreme caution needs to be exercised when community treatment with ivermectin and albendazole is implemented in areas where Loa is endemic; in such areas twice yearly albendazole 400 mg should be given alone. As there are few areas in sub-​Saharan Africa where Loa and Onchocerca do not have potential overlap with W. bancrofti, the use of diethylcarbamazine has been discouraged or abandoned there in recent years. As there are many areas of Africa where W. bancrofti and Onchocerca are coendemic there is urgent need to integrate the two previously separate MDA programmes for these infections, which both employ ivermectin, more closely. In areas where population-​based annual chemotherapy is in pro- gress there is a reduced incidence of worm-​related acute manifest- ations, and often reductions in hydrocele size. In Brugia areas recent studies have shown that that mass drug administration programmes can reverse the subclinical lymphatic damage in children. In most areas the MDA also provides what is termed ‘beyond LF benefits’ because the drugs have a wide spectrum of antihelminthic benefits as well as the impact of ivermectin on scabies. In addition, there is stabilization or regression in lymphoedema when this is managed by health education, skin hygiene, and antibiotics. Vector control The vector control method used depends on the habits of the local vector to be targeted: Aedes breeding sites, such discarded tins, tyres, or coconut shells, can be removed; Culex numbers can be reduced by improved sanitation, larvicides, and polystyrene beads applied to the water surface of latrines and cesspits. Bed nets and repellents are universally applicable. However, vector control as part of the GAELF must be planned ac- cording to the cost of MDA, the collateral benefits from annual inter- vention with broad spectrum drugs, and the costs of vector control itself. Thus, vector control targeted specifically at the transmission of lymphatic filariasis itself has not been a major part of the global elimination programme. This is because a lymphatic filariasis spe- cific vector control activity, while it may reduce the number of rounds of MDA, is not likely to be a sustainable or cost-​effective exercise. WHO promotes the principles of integrated vector management (IVM); hence in any country the vector control capacity must be as- sessed and opportunities for synergy and optimization of resources taken into account in initiating vector control. There is no doubt, however, that where there is vector control in Anopheles transmis- sion areas to control malaria (i.e. bed nets, particularly long-​lasting impregnated nets (LLINs) and indoor residual spraying), there will be an impact on the transmission of W. bancrofti which will likely re- duce the number of rounds of MDA required. Evidence is emerging that LLINs alone at full coverage can arrest transmission, this has now happened in The Gambia. This strategy could be applied where L. loa is coendemic with W. bancrofti to reduce the risks of severe adverse reactions if ivermectin is used. There is also a case for imple- mentation of vector control in settings where MDA has not achieved the required reduction in prevalence (<1%) and intensity to reduce transmission below the threshold for parasite elimination. The risk of parasite re-​emergence after MDA depends on the vectorial capacity or ‘force of infection through the mosquito population’. The vectorial capacity is the average number of new infections generated by the vector population during the lifetime of one mated female worm, if this remains below unity the infection will be eradicated. Management of patients in clinics and hospitals Chemotherapy Individual chemotherapy with diethylcarbamazine or doxycycline is needed in some situations, including infected visitors, people leaving infected areas, and those with tropical pulmonary eosino- philia or other clinical features where elimination of adult worms is a priority. Doxycycline has the advantage that side effects are much fewer because of its much slower mode of action; it also has a signifi- cant morbidity reducing effect on both hydrocele and lymphoedema. Treatment with diethylcarbamazine may provoke both local and sys- temic reactions, and thus requires care and supervision in the initial stages, especially in Brugia infections. Coinfection with L. loa must be treated with great care as both ivermectin and diethylcarbamazine can cause encephalopathy when Loa microfilaria counts are high; patients coinfected with Onchocera volvulus should not be given diethylcarbamazine as serious ocular damage or systemic reactions may develop (see Chapter 8.9.1). Diethylcarbamazine treatment should be started at 1 mg/​kg on the first day, increasing over 3 days or more to 6 mg/​kg daily. In the standard regimen 6 mg/​kg is continued for 12  days; alterna- tively, this dose is given weekly for 12 weeks. These regimens are poorly evidence based. Ultrasonography reveals the variable killing

8.9.3 Guinea worm disease (dracunculiasis) 1495

8.9.3 Guinea worm disease (dracunculiasis) 1495

8.9.3  Guinea worm disease (dracunculiasis) 1495 of adult worms, both within and between individuals. For tropical pulmonary eosinophilia a full 21 days of treatment is indicated, and may need to be repeated. Doxycycline is given at a dose of 200 mg/​kg daily for 4 weeks. Unless the patient is coinfected with L. loa a single dose of iver- mectin 200 µg/​kg is then given to clear microfilariae. For patients with hydrocele, and perhaps those with other morbidities, the doxy- cycline is given for 6 weeks. For Brugia infections, doxycycline 100 mg/​kg for 6 weeks has been recommended. Surgical and supportive management The acute manifestations of filariasis can mimic strangulated hernia and testicular torsion. The surgical treatment of filarial hydrocele is the same as that for non​filarial disease. Scrotal lymphoedema can be treated surgically, usually with preservation of the testes. Lymphosaphenous anastomosis is being used for leg elephant- iasis; many other procedures have been used in the past, often with disappointing results (Fig. 8.9.2.2). Bacterial infection is common in those with lymphoedema, espe- cially when skin integrity is breached. Early use of antibiotics, anti- bacterial soaks, together with resting the affected limb, lessens the risk of increasing lymphoedema; supportive bandaging re-​applied each morning reduces chronic oedema. FURTHER READING Alexander NDE (2015). Are we nearly there yet? Coverage and com- pliance of mass drug administration for lymphatic filariasis elimin- ation. Trans R Soc Trop Med Hyg. 109, 173–​4. Babu BV, Babu GR (2014). Coverage of, and compliance with, mass drug administration under the programme to eliminate lymphatic filariasis in India: a systematic review. Trans R Soc Trop Med Hyg, 108, 538–​49. Beaver PC (1970). Filariasis without microfilaremia. Am J Trop Med Hyg, 19, 181–​9. Dreyer G, et al. (1999). Acute attacks in the extremities of persons living in an area endemic for bancroftian filariasis: differentiation of two syndromes. Trans R Soc Trop Med Hyg, 93, 413–​1. Eigege A et al. (2013). Long-​lasting insecticidal nets are synergistic with mass drug administration for interruption of lymphatic filar- iasis transmission in Nigeria. PLoS Negl Trop Dis, 7, e2508. Evans DS, Unnasch TR, Richards RO (2015). Onchocerciasis and lymphatic filariasis elimination in Africa:  it’s about time. Correspondence. Lancet, 385, 2151–​2. Gyapong JO, Chinbuah MA, Gyapong M (2003). Inadvertent ex- posure of pregnant women to ivermectin and albendazole during mass drug treatment for lymphatic filariasis. Trop Med Int Health, 8, 1093–​101. Ichimori K, et al. (2014). Global programme to eliminate lymphatic fil- ariasis: the processes underlying programme success. PLoS Negl Trop Dis, 8, e3328. Kelly-​Hope LA, et al. (2014). Innovative tools for assessing risks for severe adverse events in areas of overlapping Loa loa and other fil- arial distributions: the application of micro-​stratification mapping. Parasites & Vectors, 7, 307. Kisoka WJ, et al. (2014). Factors influencing drug uptake during mass drug administration for control of lymphatic filariasis in rural and urban Tanzania. PLoS One, 9, e109316. Langhammer J, Birk HW, Zahner H (1997). Renal disease in lymphatic filariasis: evidence for tubular and glomerular disorders at various stages of the infection. Trop Med Int Health, 2, 875–​84. Mand S, et  al. (2009). Macrofilaricidal activity and amelioration of lymphatic pathology in bancroftian filariasis after 3 weeks of doxycyc- line followed by a single dose of diethylcarbamazine. Am J Trop Med Hyg, 81, 702–​11. Mathieu E, et al. (2013). It is possible: availability of lymphedema case management in each health facility in Togo. Program description, evaluation, and lessons learned. Am J Trop Med Hyg, 89, 16–​22. Molyneux DH (2014). Multidimensional complexities of filariasis con- trol in an era of large-​scale mass drug administration programmes: a can of worms. Parasit Vectors, 7, 363. Mullerpattan JB, Udwadia ZF, Udwadia FE (2013). Tropical pulmonary eosinophilia—​a review. Indian J Med Res, 138, 295–​302. Njomo DW, et al. (2014). Increasing coverage in mass drug admin- istration for lymphatic filariasis elimination in an urban setting: a study of Malindi Town, Kenya. PLoS One, 9, e83413. Oliveira P, et al. (2014). Evaluation of diagnostic tests for Wuchereria bancrofti infection in Brazilian schoolchildren. Rev Soc Bras Med Trop, 47, 359–​66. Ramaiah KD, Ottesen EA (2014). Progress and impact of 13 years of the global programme to eliminate lymphatic filariasis on reducing the burden of filarial disease. PLoS Negl Trop Dis, 8, e3319. Ray S, et al. (2012). Tropical pulmonary eosinophilia misdiagnosed as miliary tuberculosis: a case report and literature review. Parasitol Int, 61, 381–​4. Rebollo MP et al. (2015). Elimination of lymphatic filariasis in the Gambia. PLoS Negl Trop Dis, 9, e0003642. Taylor MG, et al. (2005). Macrofilaricidal activity after doxycycline treatment for Wuchereria bancrofti: double-​blind randomised pla- cebo-​controlled trial. Lancet, 365, 2116–​21. World Health Organization (WHO) (2014). Global programme to eliminate lymphatic filariasis:  progress report, 2013. Weekly Epidemiological Record, 89, 409–​18. World Health Organization (WHO). Global programme to eliminate lymphatic filariasis; progress report, 2015. Weekly Epidemiological Record, 91, 441–60. 8.9.3  Guinea worm disease (dracunculiasis) Richard Knight ESSENTIALS Guinea worm disease (dracunculiasis)—​now limited to sub-​Saharan Africa—​is caused by the nematode Dracunculus medinensis, whose life cycle involves aquatic copepod crustaceans. Humans are infected when they drink water containing infective larvae. Adult worms enter subcutaneous tissue and can reach a metre in length. Clinical presenta- tion is usually with a skin blister, most often on the leg, sometimes pre- ceded by allergic prodromal symptoms. Bacterial infection and local scarring with disability are common complications. Most patients in endemic areas recognize their condition, but irrigation of ulcers can re- veal larvae. Treatment is by physical removal of the worm; anthelmin- tics have no role in management. Provision of safe water for drinking is the key to prevention. The disease is now nearing eradication.

section 8  Infectious diseases 1496 Introduction The clinical manifestations of Guinea worm and its surgical removal were known in antiquity. Attention was drawn to the seasonal occur- rence of painful limb blisters that broke down to reveal a ‘worm’ in the floor of an ulcer. Dracunculus medinensis is the longest nematode infecting humans; in the Bible it is described as the ‘fiery serpent’. It was the first human parasite to be shown to have an arthropod inter- mediate host: in 1870 the Russian naturalist Fedtschenko described the worm’s early development in Cyclops, a ‘water flea’. Eradication programmes based on public health measures alone have been very successful. In 1986 3.2 million cases were reported from a total of 20 countries, but by 2016 this had been reduced to 25 in 4 African countries: Mali, Chad, South Sudan, and Ethiopia. Aetiology: The biology of the parasite The life cycle of the Guinea worm is shown in Fig. 8.9.3.1. Mature female worms, 70–​120 cm in length, migrate along fascial planes and subcutaneous tissue to reach the skin, usually below the knee. Tissue damage caused by worm products produces a blister that soon ulcerates (Fig. 8.9.3.2). Immersion of the affected part in water causes the worm to contract and expel numerous rhabditiform first-​stage larvae from the uterus at the ruptured anterior end of the worm (Fig. 8.9.3.3). The larvae swim vigorously in water for up to 7 days, and some are ingested by predatory copepod crustaceans of the genus Cyclops. They penetrate the gut of the intermediate host, and develop with two moults in the haematocele over a period of 14 days to become infective third-​stage larvae. When water containing infected Cyclops is swallowed, the re- leased infective larvae burrow though the wall of the duodenum to reach retroperitoneal tissue. After about 60 to 90 days the worms mate, and the females begin their migration towards the limbs; the male worms die and may later calcify. Ten months after infection most female worms, containing fully formed larvae, have reached their destination; within the next month they will rupture through the skin to begin the cycle anew. Epidemiology Guinea worm transmission is predominantly rural, with an annual cycle that often coincides with the planting or harvesting season. Usually, young adults and farmers are most at risk, and there is no immunity. The seasonal morbidity causes great economic hard- ship. Water sources containing Cyclops are easily contaminated by infected persons, including those seeking relief by immersing their painful lesion. In semiarid areas transmission occurs in temporary ponds during the rainy season; in wetter areas flooding and water turbidity limits transmission during the rains, and infection oc- curs in shallow wells during the dry season. There is normally no (a) (b) (c) (d) Copepod Released larva Third- stage larva First-stage larva Emerging worm Fig. 8.9.3.1  Life cycle of Guinea worm in humans: (a) copepods infected with third-​stage larvae are ingested in drinking water; larvae are released in the intestine, migrate to the body cavity, mature, and mate; (b) gravid female worms migrate to the limbs, cause a blister to form, and release first-​stage larvae into water; (c) first-​stage larvae are ingested by copepods; and (d) larvae undergo two moults in the copepod and are infective after 2 weeks. Fig. 8.9.3.2  Blister at site of imminent emergence of the female worm. Courtesy of the late P. E. C. Manson-​Bahr. Fig. 8.9.3.3  Emergent female worm being wound out on a stick. Copyright D. A. Warrell.

8.9.3  Guinea worm disease (dracunculiasis) 1497 (a) Fig. 8.9.3.4  (a) Localities/​villages reporting dracunculiasis cases, 2014. (b) Annual number of new dracunculiasis cases reported worldwide, 1989–​2014. Reproduced with permission from WHO Weekly Epidemiological Record No. 19, 2015, 90, 201–​216, © WHO 2015.

section 8  Infectious diseases 1498 (b) Fig. 8.9.3.4  Continued

8.9.3  Guinea worm disease (dracunculiasis) 1499 zoonotic reservoir, although infected dogs have recently been found in a few endemic areas, and primates can be experimentally infected. Related Dracunculus spp. are found in mink, raccoons, and otters in North America. Geographical distribution This infection was previously endemic over wide areas of the Middle East and the Indian subcontinent. Largely as a result of improved and protected water sources the infection disappeared from the central Asian republics between 1926 and 1933, from Iran in the 1970s, and from Saudi Arabia in the 1980s. It was eradicated from Pakistan in 1996 and India in 2000. It is now limited to sub-​Saharan Africa within the Sahel and Guinea savannah, between latitudes 2° north and 18° north (Fig. 8.9.3.4). Previously it occurred in the Americas, having been introduced with the slave trade, but by the 1880s it had disappeared. Clinical features The blister (Fig. 8.9.3.2) is the first sign of infection in most patients. In others, pre-​emergent worms may be seen or felt under the dermis; some are actively motile (Fig. 8.9.3.5). Allergic prodromal symp- toms, with urticaria, facial oedema, dyspnoea, and gastrointestinal manifestations, may precede the blister by a few days and disappear when the blister ruptures. Most patients have one or two worms each season, but up to 50 have been recorded. Most gravid worms emerge from the lower limb, but other sites include the buttocks, trunk, arms, scrotum, and vulva. Uncomplicated cases resolve within 4 weeks. Local complications derive from sensitization to worm products, inappropriate self-​ treatment, and bacterial infection; these can cause severe pain and prolonged disability. Gravid worms failing to reach the skin release larvae within the host’s body, inducing vigorous tissue reactions and abscesses, sometimes presenting as buboes, epididymo-​orchitis, or acute arthritis. Joint involvement, often with secondary bacterial in- fection, is also common near the site of emergence; this leads to an- kylosis and tendon contractures, with deformities and permanent disability. Immature female worms may die before reaching the skin and become encapsulated by host tissue; some calcify. They may also enter ectopic sites, including the orbit, pericardium, and central ner- vous system. Mortality is usually less than 1% and results from sys- temic or local bacterial infection. Tetanus is a significant risk when spores contaminate open lesions. Diagnosis Most patients in endemic areas recognize their condition. Worms release larvae on contact with water, and these can be seen as a milky cloud. When the worm is not visible, ulcers may be irrigated with sa- line and the centrifuged deposit examined for larvae. Onchocerciasis may also be endemic in Guinea worm infected countries and a worm found in the floor of an ulcer could be O. volvulus; another source of confusion is spargana tapeworm larvae. Patient management Local treatment can be very painful and must often be repeated. Warm moist packs should be applied for several hours, followed by gentle massage along the tract of the worm towards the ulcer. Light traction is then applied to the worm; breakage must be avoided as this greatly aggravates the situation. Analgesics and antibacterial soaks are useful, and oral antibiotics are often necessary. Between local treatments the lesion must be bandaged to reduce the risk of bacterial infection and contamination of water sources. Pre-​emergent worms can be surgically removed, a practice originating in India. A small incision is made adjacent to the worm near its midpoint, and a loop of worm is lifted out with a blunt curved probe. Massage is applied along the length of the worm to- wards the incision, and by gentle traction the whole worm can usu- ally be removed. In the event of breakage, the worm ends should be ligated to minimize contact between host tissue and worm antigens. Deep abscesses require surgical treatment. Anthelmintics have no role in the treatment of Guinea worm. Control and eradication Several factors facilitate control: Guinea worm is recognized by local communities as a major health problem, there are no carriers beyond the annual cycle, and there is usually no animal reservoir. The provision of safe water for drinking is the key to control; it is unrealistic to expect piped water supplies in most endemic areas, but covered tube wells or hand-​dug wells provided with parapets are appropriate. Additional measures are filtration of household water with finely woven cloth, and the application of temephos to ponds to kill copepods. National programmes have played a major role in many endemic areas. Case-​detection surveys and health education can be inte- grated into existing primary healthcare systems. Rumours of cases are common especially when cash rewards are offered. Unhygienic local treatments such as mud or leaf poultices and crude methods of worm extraction must be discouraged. Several international health agencies took up the challenge of Guinea worm eradication in the mid-​1980s, with an initial target eradication date of 1995. Much has been achieved, but the target was Fig. 8.9.3.5  Guinea worm in the scrotum. Copyright D. A. Warrell.

section 8  Infectious diseases 1500 missed. The initial expensive hydrological programmes were later replaced by the training of local cadres who could recruit patients within 24 hours of worm emergence to ‘containment centres’ for treatment and education to prevent water source contamination. In some areas, private-​sector initiatives have been able to gain commer- cially from the publicity achieved by adopting control in a defined area. Transmission from a patient is reported as ‘contained’ when it is detected less than 24 h after emergence, the patient has not entered a water source since emergence, and has been properly managed by the volunteer worker and seen by the supervisor with 7 days of worm emergence. Countries reach the precertification stage of eradication 1 year after reporting their last indigenous case. Ghana was certified free of Guinea worm in 2014 and there were no cases in Sudan. Cases were reported from 54 villages in 2014 compared with 103 in 2013 and 92 of the 126 cases reported were indigenous to the re- porting village, the others were infected elsewhere. The infection remains endemic in Mali, Chad, South Sudan and Ethiopia. There were 22 cases in 2015, 25 in 2016 and 30 in 2017. Dogs infected with Dracunculus are present in Chad and a few in Ethiopia, the worms are genetically identical to the human parasite and the epidemio- logic role of dogs is uncertain, but the cycle may include river fish guts or perhaps frogs; infected dogs must be kept away from water sources. Chad had reported no cases for a decade until 2010, there are now many infected dogs along the Chari river. The last stages of eradication will be the most difficult, as vertical programmes then become inefficient. Unfortunately, some of the major residual foci are in situations of civil disorder where there are mobile refugees; in others, a lack of resources, infected dogs or an absence of democratic institutions may slow progress. FURTHER READING Biswas G, et al. (2013). Dracunculiasis (guinea worm disease): eradi- cation without a drug or a vaccine Philos Trans R Soc Lond B Biol Sci, 368, 20120146. Callahan K, et  al. (2013). Contributions of the Guinea worm dis- ease eradication campaign toward achievement of the Millennium Development Goals. PLoS Negl Trop Dis, 7, e2160. Eberhard ML, et al. (2014). The peculiar epidemiology of dracunculiasis in Chad. Am J Trop Med Hyg, 90, 61–​70. Enserink M (2014). Guinea worm eradication at risk in south Sudanese war news and analysis. Science, 343, 236. Galan-Puchades MT (2016). Dogs and Guinea worm eradication. Lancet Infect. Dis. 16, 770. Glenshaw MT, et al. (2009). Guinea worm disease outcomes in Ghana: determinants of broken worms. Am J Trop Med Hyg, 81, 305–​12. Hopkins DR, et al. (2013). Dracunculiasis eradication: and now, south Sudan. Am J Trop Med Hyg. 89, 5–​10. Mbong EN, et al. (2015). Not every worm wrapped around a stick is a guinea worm: a case of Onchocerca volvulus mimicking Dracunculus medinensis. Parasit Vectors, 8, 374. Molyneux D, Sankara DP (2017). Guinea worm eradication: Progress and challenges-should we beware of the dog? Plos Negl Trop Dis. 11, e0005495. Muller R (1971). Dracunculus and dracunculiasis. Adv Parasitol, 9, 73–​151. Rhode JE, et al. (1993). Surgical extraction of Guinea worm: disability reduc- tion and contribution to disease control. Am J Trop Med Hyg, 48, 71–​6. World Health Organization (WHO) (2015). Dracunculiasis eradication: global surveillance summary, 2014. Weekly Epidemiological Record, 90, 201–​16. 8.9.4  Strongyloidiasis, hookworm, and other gut strongyloid nematodes Michael Brown ESSENTIALS Strongyloides stercoralis and hookworms are common soil-​transmitted nematodes in tropical and subtropical regions. After the organisms penetrate exposed skin, most infections are asymptomatic, but heavy infections can result in significant morbidity. Strongyloidiasis The roundworm S. stercoralis infects an estimated 30 million to 100 million people. Clinical manifestations include: (1) skin—​often the only clinical manifestation, commonly in the form of larva currens, a serpiginous, pruritic, erythematous eruption at the site of migrating larvae; (2) lungs—​ cough and tracheal irritation; less commonly wheeze; patchy infiltrates on chest radiography with eosinophilia; (3) intestinal—​epigastric pain and diarrhoea; (4) Strongyloides hyperinfection—​occurs in patients who are immunosuppressed; severe diarrhoea is a common feature; mor- tality is high. Infection is persistent and may present decades after ex- posure. Diagnosis is usually by microscopy or culture of stool; serology is useful as a screening test. Treatment is typically with ivermectin or albendazole. Improved sanitation and appropriate footwear can reduce the acquisition of infection. Hookworms Hookworm infections, mainly caused by Ancylostoma duodenale and Necator americanus, affect more than 500 million people, predomin- antly in sub-​Saharan Africa and Asia. Clinical manifestations include: (1) migratory/​larval—​ground itch (a pruritic, papular, and erythematous rash on the feet or hands); occasionally pneumonitis with eosinophilia; (2) intestinal—​occasionally profuse watery diarrhoea, but most people are asymptomatic excepting for iron-​deficiency anaemia (sometimes with haemoglobin <2 g/​dl) in those with heavy infections, which are a particular problem in infants and pregnant women, in whom it affects pregnancy adversely. Diagnosis of acute infection is clinical and of chronic infection by discovering eggs in the stool by microscopy. A single dose of albendazole will reduce the worm load to levels below those likely to cause disease; complete eradication can be achieved with repeated doses. Population-​based control programmes, using single-​dose antihelmintic therapies, aim to reduce anaemia, and improve child- hood growth and cognitive development in countries with high prevalence of soil-​transmitted helminths. Integration with other hel- minth control programmes is most effective. Increasing attention is being paid to the effect of coinfection with hookworm on other dis- eases such as malaria, tuberculosis, HIV, and asthma. Non​human hookworms These cannot complete their life cycle in humans but are cap- able of causing significant morbidity, including:  (1) cutaneous larva migrans—​usually due to dog hookworms; presents as in- tensely pruritic lesions on exposed areas of the skin; diagnosis is clinical, although the worm may be visualized in skin biopsies; albendazole is effective; (2) Ancylostoma caninum-​associated enter- itis; (3) oesophagostomiasis; (4) trichostrongyliasis.

8.9.4  Strongyloidiasis, hookworm, and other gut strongyloid nematodes 1501 Strongyloides stercoralis Aetiology Human strongyloidiasis is due to infection with Strongyloides ster­ coralis, a roundworm (nematode). The organism is one of the few helminths that can complete its life cycle in humans. Infection is by percutaneous penetration of exposed skin, with subsequent migra- tion and maturation of the worms in the human host. Epidemiology S. stercoralis infects an estimated 30–​100 million people, with a dis- tribution throughout tropical and subtropical areas. Prevalence in rural areas of sub-​Saharan Africa, Southeast Asia, and Central and South America is between 10 and 40%, with Brazil and Thailand having particularly high prevalence; a lower level of active trans- mission persists in temperate regions such as southern Europe and the southern United States of America. In highly endemic areas, in- fection intensities peak in childhood and then plateau or decline. Because of the chronicity of infection, prevalence remains high in immigrants from endemic areas, reaching 30–​80% in Southeast Asian immigrants screened in North America. A prevalence greater than 30% has been observed among former British servicemen who were prisoners of war in the Far East in 1941–​1945, screened 30 years or more after exposure. Case–​control studies have identified HIV, HTLV-​1, alcoholism, and some malignancies as risk factors for infection, some of these also being risk factors for hyperinfection (see next). Pathogenesis and life cycle Filariform larvae in moist soil penetrate exposed skin and pass, via the bloodstream, to the lungs, and into the alveolar spaces. From there, the larvae ascend the trachea and are swallowed, reaching their final habitat in the crypts of Lieberkühn in the duodenum and upper jejunum, where they mature. Once the worms have reached maturity in the small intestine, the adult males are rapidly elimin- ated, leaving parthenogenetic adult parasitic females, 2.5 mm in length, attached to the mucosa, where they deposit eggs. One month after infection, the resulting rhabditiform larvae bore through the epithelium into the gut lumen. At this stage, most larvae follow an indirect developmental route—​they are excreted in the faeces and develop into free-​living adults, which produce eggs from which in- fectious filariform larvae develop, with potential to penetrate ex- posed skin and re-​infect humans percutaneously, thus completing the life cycle. Some larvae by contrast develop directly via three moults into filariform larvae. These are usually passed in the faeces and can survive in the soil for many weeks. However, some can re- invade the host in the lower gastrointestinal tract or perianal skin before evacuation. It is this process of autoinfection that explains the persistence of S. stercoralis infections for decades after exposure, and allows for the multiplication of worms that might lead to the phe- nomenon of hyperinfection seen in immunosuppressed patients. Strongyloidiasis, like most helminth infections, is associated with a type 2 immune response, with raised IgE levels and increased cir- culating eosinophil numbers. In immunosuppressed patients, some elements of this immune response are lacking. The pathogenesis of hyperinfection is not well understood. A possible explanation is that immunosuppression facilitates the direct route of strongyloides development, leading to multiplicative autoinfection, increasing larval intensities, and ultimately, dissemination of larvae beyond the gut mucosa into other organs. Pathogenic Gram-​negative bacteria, carried from the intestine into the bloodstream and other organs, contribute to the severe morbidity and mortality associated with dis- seminated strongyloidasis. Clinical features Most infected individuals are asymptomatic. In such patients, diag- nosis might only be considered as part of investigation for peripheral eosinophilia. Cutaneous Skin symptoms are often the only manifestation of infection, com- monly in the form of larva currens, a serpiginous pruritic erythema- tous eruption on the legs, buttocks, and back, at the site of migrating larvae, that can advance as quickly as 15 cm/​h (Fig. 8.9.4.1). The rash is more diffuse and migrates more rapidly than the cutaneous larva migrans associated with hookworm infections. It can occur with the initial infection, but is also sometimes seen in people with chronic strongyloidiasis. Pulmonary The migratory phase might be associated with cough and tracheal ir- ritation and, less commonly, with wheeze, which may be persistent. Patchy infiltrates might be seen on chest radiographs. When larvae become trapped in the lung during migration, eosinophilic pneu- monia occasionally occurs. Pulmonary manifestations, including Fig. 8.9.4.1  Characteristic serpiginous rash of larva currens on the shoulder of a traveller with Strongyloides stercoralis infection acquired in India. The rash was transient, but recurrent and widespread. Courtesy of R. H. Behrens, Hospital for Tropical Diseases, London.

section 8  Infectious diseases 1502 pneumonia, bacterial lung abscesses, and acute respiratory distress syndrome are more prominent in hyperinfected patients (see next). Intestinal Intestinal symptoms are generally mild in people with light infec- tion. Epigastric pain mimicking peptic ulcer disease may occur within 3 weeks of infection and persist. Diarrhoea is usually chronic and mild, but can occur early and be associated with bloody stools. In more severe cases, usually associated with hyperinfection, intes- tinal oedema with malabsorption, mesenteric lymphadenopathy, and ascites may occur. An eosinophilic granulomatous enterocolitis resembling Crohn’s disease is well described in older patients on cor- ticosteroids. Subacute intestinal obstruction, biliary stenosis, and necrotizing enteritis are occasionally seen. Special circumstances/​Complications Strongyloides hyperinfection Patients on long-​term corticosteroids, and those undergoing chemotherapy or organ transplantation, are at risk from severe manifestations of strongyloidiasis. This also occurs in patients with lymphoma or leukaemia without chemotherapy, most commonly in those with T-​cell leukaemia caused by human T-​cell leukaemia virus (HTLV)-​1 infection. It is also well described in HTLV-​1-​infected patients without overt malignancy, and very rarely in patients with AIDS. Disseminated disease has a mortality rate of 60–​70%. Severe diarrhoea is a common feature. Gram-​negative pneumonia, bacter- aemia, or meningitis caused by enteric pathogens that have breached the mucosal barrier along with the strongyloides larvae are frequent manifestations. Petechial haemorrhages in the skin, especially around the umbilicus, and hepatitis may occur. Peripheral eosino- philia is usually absent in disseminated disease. Clinical investigations Microscopy of stool might reveal rhabditiform larvae, but the sen- sitivity of direct smears is low. Formol-​ether concentration tech- niques are more useful, but multiple stool samples may be required to detect light infections. Culture techniques have been developed to enhance the diagnostic yield. Agar plate cultures have the highest yield; tracks made by larvae migrating across the plate can be seen. Charcoal culture and filter-​paper methods make use of the indirect life cycle: stool is incubated for several days to allow the develop- ment of adults and second-​generation filariform larvae. Larvae might also be isolated by duodenal aspiration, or by the string test, although these are of limited sensitivity. Molecular techniques, such as real-​time and nested polymerase chain reaction (PCR) on stool, perform well. In disseminated infection, larvae are found in the sputum and in biopsies from tissues such as the gastrointestinal tract and lung. Enzyme-​linked immunosorbent assays (ELISA) for strongyloides-​ specific IgG have high sensitivity for strongyloides infection. It is useful as a screening test, particularly before embarking on im- munosuppressive therapy. There is cross-​reaction with filarial anti- bodies, and levels may take over a year to become negative after treatment, so specificity may be limited. Treatment and prevention Albendazole 400 mg once or twice daily for 3 days is moderately effective in chronic infections, and is better tolerated than thiaben- dazole 25 mg/​kg twice daily for 3 days, which is now rarely used. The treatment of choice is ivermectin 200 µg/​kg as a single dose or for two doses 1–​14 days apart. Cure rates are more than 90% with a single dose, compared with less than 75% with albendazole. Prolonged courses of treatment are necessary in patients with severe or disseminated disease. Subcutaneous veterinary pre- parations have been used when parenteral treatment is required, although encephalopathy can develop, in association with high serum levels. Improved sanitation and appropriate footwear can reduce the acquisition of infection. Once established, strongyloides should be eradicated in any patient being considered for immunosup- pressive therapy, because of the potentially lethal consequences of hyperinfection. Areas of uncertainty, controversy, and future developments Global prevalence and disease burden are not well understood, due to the limited number of high quality community-​based preva- lence studies, or indeed hospital-​based prevalence studies among patients at risk of hyperinfection. The limited diagnostic sensitivity of stool microscopy, and limited specificity of serology, are also factors. There has been much interest in use of PCR techniques to provide better diagnostic precision and thus better analyses of risk factors for infection; and in newer serological methods to assess cure. Several specialist guidelines have advocated screening for strongyloides before immunosuppression (e.g. for haematological malignancies, inflammatory bowel disease, transplantation). It is likely that, given the high prevalence of chronic infection in these populations, most patients do not progress to severe disease; further evidence on the pathogenesis of immunosuppression-​associated hyperinfection, and on the cost-​benefits of screening, are needed to improve practice in these settings. The same is true for screening of immunocompetent immigrants, in whom the health burden of chronic infection is not well established. Another area of interest is the impact of strongyloides coinfection on other infections, for example, tuberculosis. Studies suggest a sup- pressive effect of coinfection on anti-​TB immune responses, which suggests there could be an impact of strongyloides infection and of antihelminthic treatment on risk of progression to and of severity of, active tuberculosis. Strongyloides fuelleborni Strongyloides fuelleborni fuelleborni is a parasite of primates in tropical Africa and Asia, which can also infect human populations sharing similar habitats. Prevalence rates up to 20% have been re- ported among forest-​dwelling communities. Infections are gen- erally asymptomatic. Unlike S. stercoralis, eggs are passed in the stool, and might be confused with hookworm ova. Benzimidazole therapy is effective. A phylogenetically distinct nematode, Strongyloides fuelleborni kellyei, has been found in rural communities in Papua New Guinea. Infection intensities are highest among young children. It is associ- ated with ‘swollen belly sickness’ in 2-​month-​old infants, which is characterized by abdominal distension, respiratory distress, gener- alized oedema, and gastrointestinal disturbance.

8.9.4  Strongyloidiasis, hookworm, and other gut strongyloid nematodes 1503 Hookworm Aetiology Human hookworm disease is principally caused by the two species that can complete their life cycles in humans: Ancylostoma duode­ nale and Necator americanus. Infection is by percutaneous penetra- tion of exposed skin, with subsequent migration and maturation of the worms in the human host. Epidemiology Estimates suggest that more than 1 billion people are infected with hookworm, predominantly sub-​Saharan Africa and Asia. Although significant overlap occurs, the distribution of A. duo­ denale is more restricted geographically than that of N. ameri­ canus. Necator is more widespread in sub-​Saharan Africa, the Americas, Southeast Asia, and India; Ancylostoma is also widely distributed in Southeast Asia, but is more common in temperate regions, North Africa, and the Middle East. As with other intes- tinal helminths, most infected people harbour a few adult worms, but a minority are heavily infected. Social, behavioural, and gen- etic factors determine which individuals within a community are most heavily infected. Unlike most other intestinal helminth infections, the prevalence and intensity of hookworm infection increases with age. Pathogenesis and life cycle The life cycles of the two hookworm species are similar. Larvae in moist soil penetrate exposed skin, usually on the feet or buttocks. They enter the circulation after 10 days, are carried to the lungs, and cross into the alveolae, from where they are transported to the pharynx and swallowed. The adults, approximately 10 mm in length, attach themselves to the small intestinal mucosa with their buccal cavities, which contain hooked teeth (ancylostoma) or cut- ting plates (necator). After 3 to 6 weeks the females produce up to 30 000 eggs per day, which are passed in the faeces. The eggs hatch within 48 h, but the larvae can remain viable for up to 6 weeks in appropriate soil conditions. Adult hookworms live for 1 to 9 years. Infection can also be acquired by the ingestion of contaminated soil, and the transmission of infective larvae via breast milk is well recognized. Infection is associated with tissue and peripheral eosinophilia, and specific IgG and non​specific IgE responses. Regulatory cyto- kine responses are probably crucial in limiting immunopathology in established infection. Equally important are a range of worm-​ derived immunomodulatory molecules that interfere with neutro- phil migration and adhesion, inhibit complement, induce T-​cell apoptosis, and prevent blood coagulation. Secreted anticoagulants, including serine protease inhibitors of factor Xa, are responsible for anaemia, the main consequence of infection. Radioisotope studies have demonstrated that hookworm infections produce a daily blood loss of up to 0.3 ml per worm per day. This translates into a loss of up to 100 ml per day in heavily infected people. The degree of anaemia is partly a function of worm burden, but also of iron stores. Variations in dietary iron intake, as well as coinfection with other parasites such as schistosomes and malaria, account for some of the geographical differences in the incidence of hook- worm anaemia. Clinical features Migratory/​larval Repeated exposure to penetrating hookworm larvae results in ground itch, a pruritic papular erythematous rash on the feet or hands. Larval pulmonary migration is generally asymptomatic, but can cause a pneumonitis characterized by fever, cough, wheeze, haemoptysis, and peripheral eosinophilia. Symptoms can last sev- eral weeks, but are rarely severe. Oral ingestion of A.  duodenale larvae can result in Wakana disease, which presents with nausea, vomiting, cough, pharyngeal irritation, and dyspnoea. Intestinal Recently acquired human hookworm infection occasionally causes profuse watery diarrhoea. Most people with established infection are asymptomatic. The major morbidity associated with hookworm infection is iron-​deficiency anaemia in those with heavy infections. Haemoglobin concentrations of less than 2 g/​dl are not uncommon. Hookworms were first identified in the investigation of anaemic miners in 19th century Europe, notably in Cornish tin mines, where an extreme form of anaemia (chlorosis or ‘green disease’) was preva- lent. Now eradicated in developed countries, hookworm remains a major cause of anaemia in the developing world. It is particularly common among women of reproductive age, and a major con- tributor towards adverse outcomes in pregnancy, being responsible for over 20% of pregnancy-​associated anaemia. Studies have also highlighted an even higher burden of anaemia due to hookworm among elderly residents in high prevalence settings. Fatigue and list- lessness are the principal symptoms, and probably have a significant economic impact in areas of high prevalence. High-​output cardiac failure is a major cause of death in patients with severe anaemia. Malabsorption is not a frequent consequence of hookworm infec- tion, but protein loss does occur and might contribute to the oedema seen in severe infections. Dyspepsia, nausea, and a range of non​specific symptoms are common in those with heavy worm burdens. Pica, a craving for eating soil, is well described in patients with hookworm anaemia. Growth and cognitive development There has been debate about the effect of chronic hookworm infec- tion on growth and cognitive development in childhood. Seminal work in an impoverished community in the southern United States of America in the 1920s demonstrated an inverse association be- tween IQ and hookworm intensity. The results of subsequent studies have been inconclusive. Intervention trials in East Africa have sug- gested a modest effect of heavy hookworm infection on growth, and the balance of evidence suggests that heavily infected subjects do have impairments of memory and other specific cognitive functions. It is not known to what extent these effects are mediated by, or are independent of, anaemia. Treatment results in improved cognitive performance and school attendance among those with the heaviest infections. Clinical investigations The diagnosis of acute hookworm infection is clinical. Characteristic symptoms are usually associated with peripheral eosinophilia. The diagnosis of chronic infection is made by discovering eggs in the stool by microscopy. Symptomatic infection is readily diagnosed by

section 8  Infectious diseases 1504 direct microscopy of a single stool sample, as the worm burden is high in these patients. Where diagnosis of lighter infections is required (e.g. in the investigation of eosinophilia), the diagnostic yield is in- creased by examining multiple stool samples, using concentration methods, or by culture techniques. The latter allow for the develop- ment of third-​stage larvae, which can be used to identify the infecting hookworm species and differentiate from related nematode species. Semiquantitative techniques, such as the modified Kato smear, or more sensitive faecal flotation methods, can be used to estimate in- fection intensity. Hookworm eggs degenerate rapidly after excretion, and laboratory processing should be performed as soon as possible. Treatment, prevention, and control A single dose of albendazole 400 mg, will kill more than 80% of adult hookworms and thus reduce the worm load to a level below that likely to cause disease. Complete eradication can be achieved with repeated doses. Treatment is well tolerated, and can safely be given to children and in pregnancy (although not recommended in the first trimester). Single-​dose mebendazole is much less effective. For patients with anaemia, anthelmintic treatment should be combined with iron replacement. Patients with heart failure or severe anaemia during pregnancy frequently require transfusion of packed red cells. In developing countries where the prevalence of hookworm and other intestinal helminths is high, the increasing availability of safe, affordable, single-​dose anthelmintics makes mass deworming pro- grammes feasible (Fig. 8.9.4.2). The impact of empirical population-​ based treatment (e.g. in schools), can be sustained in the face of ongoing transmission by repeated treatment at 3-​ to 12-​month inter- vals. These strategies can be integrated with control programmes for other helminthiases. The World Health Organization set a target date of 2020 for routine anthelmintic treatment to be provided to 75% of school-​age children at risk of infection. Current global coverage is close to this target, which has already been achieved by many African and southeast Asian countries. The outcomes of national control programmes on anaemia have been mixed. School-​based programmes miss the most vulnerable populations (preschool chil- dren and pregnant women), and new targets for expanded coverage to include these groups have been introduced. Furthermore, pro- grammes combining albendazole with antischistosomal treatment have had more impressive outcomes, suggesting that in coinfected populations both helminths contribute to anaemia. The age–​intensity distribution of hookworm infection is likely to limit any benefit of mass treatment programmes on hookworm transmission. The provision of better footwear (although clearly protective against infection) and improved sanitation for infected communities probably has only a marginal role, at least in the me- dium term, in reducing transmission. Vaccines offer potential for better hookworm control, but development has been hampered by challenges in generating relevant animal models and in eliciting safe immune responses in early clinical studies. Non​human hookworms Non​human hookworms cannot complete their life cycle in the human host, but are capable of causing significant morbidity in the skin and gastrointestinal tract. Cutaneous larva migrans Cutaneous larva migrans presents as intensely pruritic lesions on exposed areas of the skin. The rash is commonly seen on the feet or buttocks, but can occur elsewhere (Fig. 8.9.4.3). Dog hookworms, Fig. 8.9.4.2  School-​based treatment as part of a mass anthelmintic treatment programme in Burkina Faso. Courtesy of A. Gabrielli, Schistosomiasis Control Initiative. (a) (b) Fig. 8.9.4.3  Cutaneous larva migrans: (a) caused by probable Ancylostoma braziliense infection acquired on a beach in the Caribbean, and (b) heavy infection with Ancylostoma braziliense acquired in Brazil. (a) Courtesy of D. Webster; (b) copyright D. A. Warrell.

8.9.4  Strongyloidiasis, hookworm, and other gut strongyloid nematodes 1505 such as Ancylostoma braziliense, are usually implicated. Like human hookworms, these species thrive in sandy soil and frequently in- fect travellers visiting beaches in the Caribbean, Southeast Asia, and Africa. Untreated, the rash can persist for months, with gradual spread through the epidermis leaving serpiginous tracks. Secondary bacterial infection can sometimes occur. Diagnosis is clinical, al- though the worm can be visualized in biopsies taken from the leading edge of the lesion. Topical albendazole or thiabendazole are usually effective, although short courses of oral albendazole or iver- mectin are more effective. Ancylostoma caninum-​associated gastroenteritis First described in Australia in the 1980s, a distinctive eosinophilic enteritis has been linked to infection with A. caninum, a dog hook- worm. The global distribution of this disease is unknown. Oral in- gestion might be the predominant route of infection. Manifestations range from a limited aphthous ileitis with tissue and peripheral eosinophilia, which may be asymptomatic, to a severe painful eo- sinophilic gastroenteritis with gut oedema, ascites, and regional lymphadenopathy. Immature hookworm larvae can be identified in the lesions, although the diagnosis might only be made at lapar- otomy. Benzimidazoles are effective. Oesophagostomum spp. Human oesophagostomiasis, usually caused by Oesophagostomum bifurcum, is common in forested areas of West Africa, but rare else- where. Prevalence in some areas of Togo and northern Ghana has reached 75%. The nematode can complete its life cycle in humans, and is distinct from related species in primates. Humans probably ac- quire the disease by ingesting infective third-​stage larvae, although a percutaneous route of infection has not been excluded. The larvae migrate to, and develop within, the colonic wall before returning to the intestinal lumen where they reach adulthood and excrete eggs. Intense tissue reactions occur around the larvae, forming nodules along the wall of the (usually ascending) colon. Although these in- fections are generally asymptomatic, heavy infections can result in the development of multiple pea-​sized nodules, with gross mucosal and serosal oedema, and microabscess formation (Fig. 8.9.4.4). Children are most commonly affected and present with abdominal pain, diarrhoea, and weight loss. Solitary palpable painful inflam- matory masses, known as Dapaong tumours, also occur within the bowel wall and in extraintestinal sites, such as the mesentery or ab- dominal wall. Nodules can be detected ultrasonographically. Ova are morphologically indistinguishable from those of hookworm, but can be differentiated by culturing third-​stage larvae. Treatment with short courses of albendazole is effective and may obviate the need for surgery. Mass treatment campaigns have reduced the burden of disease. Trichostrongylus spp. Trichostrongylus spp. are ubiquitous nematode parasites of herbi- vores, particularly domesticated animals such as sheep, goats, cattle, and donkeys. Human infection occurs most commonly among herders, with a prevalence of more than 80% reported among Iranian nomads. Sporadic human infections occur in urban en- vironments through contact with the faeces of domestic animals. Infective larvae hatch in the soil, and are ingested with contaminated vegetables. There is no migratory phase; the adults develop in the duodenal mucosa, and produce eggs after a long prepatent period. Most infected people are asymptomatic, although eosinophilia is common. Epigastric pain, diarrhoea, and rectal bleeding may occur. Diagnosis is made by finding eggs (which might be mistaken for hookworm ova) by stool microscopy. The adults are occasionally seen at endoscopy. Benzimidazole anthelmintics may be effective, although resistance is increasing; a single dose of ivermectin 200 µg/​ kg is usually sufficient. Areas of uncertainty, controversy, and future developments Recent molecular studies have demonstrated that a proportion of patent hookworm infections is, in fact, due to infection with Ancylostoma ceylanicum, a hookworm species whose natural hosts are cats and dogs. Surveys in Asia have demonstrated that this is the second most common hookworm infection among humans, comprising nearly 25% total patent infections in some surveys. Experimental infections demonstrate significant morbidity, with ‘ground itch’, abdominal pain, eosinophilia, and establishment of chronic infection with anaemia. There are limited data, however, on the health burden associated with naturally occurring infection. One particular area of interest is the effect of hookworm infection on the immunes system. Helminth infections have a similar global distribution to other pathogens. Our understanding of the immune response to helminth infection has stimulated interest in the effect of helminths on subjects coinfected with other microorganisms, particularly malaria, tuberculosis, and HIV. Hookworm infection in pregnancy or early childhood is associated with a slight increase in incidence and severity of malaria. There are demonstrable effects of hookworm infection on immune responses to mycobacterial antigens, although hookworm does not appear to increase the inci- dence of tuberculosis or accelerate the progression of HIV infection. There is evidence that childhood or maternal infection with some helminths, especially hookworm, may protect against the develop- ment of infantile eczema and/​or atopy. These findings lend support to the ‘hygiene hypothesis’ that the increasing prevalence of allergy Fig. 8.9.4.4  Excised colon from a young Ghanaian adult with Oesophagostomum bifurcum-​associated disease. Multiple nodules and serosal oedema are present. The diagnosis was made at laparotomy after the patient presented with abdominal pain and peritonism. Courtesy of A. M. Polderman, Leiden University Medical Center, Netherlands.

8.9.4 Strongyloidiasis, hookworm, and other gut st

8.9.4 Strongyloidiasis, hookworm, and other gut strongyloid nematodes 1500

section 8  Infectious diseases 1550 and oesophageal varices. Previous episodes of haematemesis indi- cate a 70% risk of rebleeding. Urogenital schistosomiasis caused by S. haematobium may have an impact on the reproductive health of people and genital schis- tosomiasis has been associated with infertility. Eggs are deposited during active infections in childhood but when the infection levels decrease in adults many egg calcify in the tissue, so called sandy patches. Sandy patches can cause contact bleeding and disruption of the mucosal surface which may increase the risk of HIV transmis- sion. Calcified eggs are dead and treatment with praziquantel has no impact on these lesions. Prevention and control Despite the substantial risk of reinfection, chemotherapy is usu- ally highly beneficial at both the individual and population levels, as those suffering high intensities of infection are at greatest risk of the more severe forms of schistosomiasis. Furthermore, even low-​ intensity infections may lead to anaemia and have a negative im- pact on the well-​being of the infected individual. This is important especially among vulnerable groups such as children and pregnant women. Various chemotherapy-​based control strategies can be em- ployed depending on intensity of transmission and the available re- sources. In the Nile Delta region of Egypt, injections of tartar emetic were used for mass treatment from the 1960s to the 1980s. Tragically, the needles were not adequately sterilized and, as a result, hepatitis C virus was widely spread in this population to reach its highest re- corded prevalence. In areas of high transmission, population-​based mass drug ad- ministration can avoid the time and expense required for diagnosis and reduce the prevalence and severity of morbidity. Alternatively, schoolchildren can be targeted for treatment, as they invariably have the heaviest worm burdens and contribute most to ongoing transmission. In areas of less intense transmission, treatment can be restricted to diagnosed cases. While the mass drug treatment of school-​age children in endemic areas is a very important and promising development, recent studies indicate that many infants and preschool-​aged children have schistosomiasis and they are not presently targeted to receive praziquantel within current mass drug administration. The provision of safe water supplies and sanitation, where it can be achieved, will make an important additional contribution. Mortality can be prevented and morbidity best controlled by a combination of health education, chemotherapy, provision of safe water sup- plies and sanitation, and, where appropriate, snail control. Health education should be aimed at improving practices of water use and preventing indiscriminate urination and defecation. The role of molluscicides in control programmes depends on the local epidemiological and ecological circumstances and the re- sources available. Within the context of a larger concerted interven- tion, focal mollusciciding of major transmission sites can be useful. Eradication of host snail species is not usually feasible, although modification of the environment to eliminate snails has been suc- cessful in parts of China. In general, it has only been through sus- tained effort with integrated control strategies that disease control has been achieved. In May 2001 the World Health Assembly passed Resolution 54.19, which called for efforts to reduce morbidity caused by schistosom- iasis and soil-​transmitted helminths in school-​age children. As a re- sponse to this call, the Schistosomiasis Control Initiative, supported by the Bill and Melinda Gates Foundation, with the objective of encouraging the development of sustainable schistosomiasis con- trol programmes throughout sub-​Saharan Africa, was launched in Uganda in March 2003. In the World Health Assembly Resolution 65.21 from May 2012 countries were urged to intensify interventions to control schistosomiasis and to strengthen surveillance of schistosom- iasis transmission and it is recommended that endemic countries embark on elimination programmes where appropriate with the aim of eliminating schistosomiasis as a public health problem by 2025. A long-​term solution for schistosomiasis control could be pro- vided by a protective vaccine. Although more than 100 schisto- some vaccine candidates have been identified only three vaccine antigens, S.  mansoni fatty acid binding protein (Sm14), S.  man- soni tetraspanin (Sm-​TSP-​2) and S.  haematobium glutathione S-​transferase (Sh28GST; Bilhvax), have entered human clinical trials. Both Sm14 and Sm-​TSP-​2 have been tested in phase I clinical trials and further safety and immunogenicity phase II clinical trials in Brazil and Africa are scheduled for Sm14. Bilhvax (Sh28GST) was tested in phase III clinical trials in Senegal in 2012 but the results from the trial have not yet been released. FURTHER READING Andrade G, et al. (2017). Decline in infection-​related morbidities fol- lowing drug-​mediated reductions in the intensity of Schistosoma in- fection: a systematic review and meta-​analysis. PLoS Negl Trop Dis, 11, e0005372. Clerinx J, Van Gompel A (2011). Schistosomiasis in travellers and mi- grants. Travel Med Infect Dis, 9, 6–​24. Danso-​Appiah A, et al. (2013). Drugs for treating Schistosoma mansoni infection. Cochrane Database Syst Rev, 2, CD000528. Fairley J (1991). Bilharzia:  a history of imperial tropical medicine. Cambridge University Press, Cambridge. Ferrari TC, Moreira PR (2011). Neuroschistosomiasis: clinical symp- toms and pathogenesis. Lancet Neurol, 10, 853–​64. Gavilanes F, Fernandes CJ, Souza R (2016). Pulmonary arterial hyper- tension in schistosomiasis. Curr Opin Pulm Med, 5, 408–​14. Gryseels B, et  al. (2006). Human schistosomiasis. Lancet, 368, 1106–​18. Jordan P, Webbe G, Sturrock RF (eds) (1993). Human schistosomiasis. CAB International, Wallingford. King CH, Dickman K, Tisch DJ (2005). Reassessment of the cost of chronic helmintic infection:  a meta-​analysis of disability-​related outcomes in endemic schistosomiasis. Lancet, 365, 1561–​9. Kramer CV, et al. (2014). Drugs for treating urinary schistosomiasis. Cochrane Database Syst Rev, 8, CD000053. Magnussen P, Vennervald BJ, Aagaard-​Hansen J (2011). Schistosomiasis. In: Selendy JMH (ed) Water and sanitation-​related diseases and the environment:  challenges, interventions, and preventive measures, Chapter 13: pp. 167–​74. Wiley-​Blackwell, Chichester. Olds GR (2003). Administration of praziquantel to pregnant and lactating women. Acta Tropica, 86, 185–​95.

8.9.5 Gut and tissue nematode infections acquired

8.9.5 Gut and tissue nematode infections acquired by ingestion 1506

section 8  Infectious diseases 1506 worldwide may partly be a result of reduced exposure to the im- munosuppressive effects of helminths, and lead to concerns that mass deworming may have detrimental as well as beneficial ef- fects. A trial of experimental hookworm infection as a treatment for asthma did not demonstrate a significant benefit, however hookworm-​derived molecules have been demonstrated to have po- tent immunomodulatory effects in vitro and could form the basis for future drug development. FURTHER READING Bottazzi ME (2015). The human hookworm vaccine: recent updates and prospects for success. J Helminthol, 89, 540–​4. Buonfrate D, et  al. (2015). Novel approaches to the diagnosis of Strongyloides stercoralis infection. Clin Microbiol Infect, 21, 543–​52. George PJ, et al. (2015). Modulation of pro-​ and anti-​inflammatory cytokines in active and latent tuberculosis by coexistent Strongyloides stercoralis infection. Tuberculosis (Edinb), 95, 822–​8. Karagiannis-​Voules DA, et al. (2015). Spatial and temporal distribu- tion of soil-​transmitted helminth infection in sub-​Saharan Africa:
a systematic review and geostatistical meta-​analysis. Lancet Infect Dis, 15, 74–​84. Schär F, et al. (2013). Strongyloides stercoralis: global distribution and risk factors. PLoS Negl Trop Dis, 7, e2288. World Health Organization (WHO) (2017). Chistosomiasis and soil-​ transmitted helminthiases:  number of people treated in 2016. Wkly Epidemiol Rec, 92, 49–60. 8.9.5  Gut and tissue nematode
infections acquired by ingestion Peter L. Chiodini ESSENTIALS Ascariasis Ascaris lumbricoides (the human roundworm) is widespread in the tropics and subtropics where sanitation is poor and the soil is con- taminated with its eggs. Ascaris suum (the pig roundworm) is also capable of infecting humans. Some authorities consider them to be the same species, making A. suum a synonym of A. lumbricoides. Ingested eggs hatch in the small bowel, the larvae released mi- grate via the bloodstream and lungs, then return to the small bowel and develop into adult worms 15 to 30 cm long. Most infections are asymptomatic, but there may be pulmonary infiltrates with eosinophilia, abdominal discomfort and—​in children with heavy infections—​intestinal obstruction. Infection is diagnosed by finding eggs in the faeces. Treatment is with mebendazole, albendazole, or pyrantel pamoate. Anisakidosis This is caused by larvae of roundworms in the family Anisakidae, which are parasites of marine mammals. The larvae cannot mature to adult form in humans who thus represent dead-​end hosts for these parasites. After ingestion in uncooked fish or squid, immature larvae burrow into the gastric or intestinal mucosa and may cause abdom- inal pain. Diagnosis is usually made at endoscopy, with treatment by endoscopic removal (if possible) of the larvae, although symptoms resolve spontaneously in most cases. Capillariasis Intestinal capillariasis, caused by Paracapillaria philippinensis, is ac- quired by ingestion of larvae in undercooked freshwater fish and may cause a severe diarrhoeal disease. Diagnosis is by finding eggs in the stool. Treatment is with albendazole or mebendazole. Prevention is by properly cooking fish. Hepatic capillariasis—​caused by Capillaria hepatica, a parasite of wild rodents and other mammals. Ingested eggs hatch and larvae pass to the liver and cause a syndrome similar to visceral larva migrans (see next). Serology is rarely available and definitive diagnosis is made by identifying the parasite or eggs in a liver biopsy. Treatment is usu- ally with albendazole. Adjunctive corticosteroids have been used. Enterobiasis Enterobius vermicularis (threadworm) is cosmopolitan. Ingested eggs develop directly into adult worms in the intestine; fertilized female worms emerge from the rectum at night and deposit eggs on the perianal skin. Most infections are asymptomatic, but pruritus ani may be troublesome at night. Diagnosis is made by finding eggs on clear adhesive tape applied to the perianal skin or identification of worms found on the perianal skin or surface of the stool. Mebendazole or albendazole or pyrantel pamoate are effective in combination with sanitary measures. Recurrent threadworm due to reinfection can be troublesome in a minority of patients. Gnathostomiasis Gnathostomiasis is due to ingestion of the third stage larva, usually from Gnathostoma spinigerum, a parasite of domestic and wild fe- lines and canines, or G. hispidum, a parasite of pigs, via raw or under- cooked freshwater fish. It is endemic in Southeast Asia, the Indian subcontinent, Southern Africa, Mexico, and Peru, and is increasingly seen in ever-​more adventurous travellers. It causes both migratory cutaneous swellings and visceral disease. Toxocariasis This is due to invasion by larvae of Toxocara canis or T. cati, acquired by ingestion of eggs from dog or cat faeces contaminating the en- vironment. It occurs in three clinical forms—​visceral larva migrans, ocular larva migrans, and covert toxocariasis. Visceral larva migrans usually afflicts children; larvae migrate to the viscera and may be asymptomatic or cause protean manifest- ations including muscular pain, lassitude, anorexia, cough, urticarial rashes, hepatomegaly, and occasionally splenomegaly, lymphaden- opathy and skin lesions, and (rarely) central nervous system involve- ment resulting in convulsions. Eosinophilia is prominent. While definitive diagnosis is by finding larvae in a liver biopsy this should not be necessary to diagnose visceral larva migrans due to toxocar- iasis. A negative serological test for toxocara antibody rules out the diagnosis. Most patients recover spontaneously. Severe cases can be treated with albendazole plus adjunctive corticosteroid therapy. Ocular larva migrans is more commonly seen in older chil- dren and due to granuloma formation around a larva in the retina.

8.9.5  Gut and tissue nematode infections acquired by ingestion 1507 Diagnosis depends upon positive serology together with consistent fundoscopic features. Anthelmintic therapy is not routinely given. Covert toxocariasis results in relatively mild, non​specific symptoms. Trichinosis This is acquired by ingestion of larvae of Trichinella spiralis in under- cooked meat, usually pork. Adult worms in the small bowel produce larvae which seed the muscles and other tissues, where they develop. Most infections are asymptomatic, but heavy infections typically cause diarrhoea, followed by fever and myositis. Definitive diagnosis depends upon finding larvae in muscle biopsies, although this is usu- ally unnecessary; serological tests become positive several weeks after infection. Treatment is mainly symptomatic. Anthelminthic agents are used to kill adults and thus minimize the production of new larvae. Thorough cooking of pork is the best safeguard against infection. Trichuriasis Trichuris trichiura (whipworm) is most prevalent in the tropics and subtropics where sanitation is poor. Ingested eggs hatch in the small bowel and then develop within the gut into adult worms, the an- terior end of which becomes embedded in the large bowel mucosa. Very heavy infections may cause dysentery or rectal prolapse. Infection is diagnosed by finding eggs in the faeces. Treatment is with mebendazole or albendazole. Ascariasis (giant roundworm infection) Life cycle Ascariasis is an infection caused by the giant roundworm, Ascaris lumbricoides. Infection is acquired when an egg is ingested (Fig. 8.9.5.1). The infective larva hatches out in the small intestine (Fig. 8.9.5.2) and penetrates the intestinal wall to enter the portal cir- culation. From here it enters the systemic circulation and reaches the lungs, where it breaks out of the capillaries into the alveoli and undergoes another moult to become a fourth-​stage larva. From the lungs the larva moves up the bronchial tree to the mouth and is then swallowed. In the intestine it moults again to become a sexually ma- ture worm about 6 to 8 weeks after ingestion of the egg. The mature worm is cylindrical with tapering ends and creamy white to light-​ brown in colour (Fig. 8.9.5.3). The female measures 20 to 35 cm in length and 3 to 6 mm in width, whereas the male is 12 to 31 cm long and 2 to 4 mm wide and has a curved tail. Normally, the adult worms live in the lumen of the small intes- tine, primarily the jejunum. The worm is able to maintain its pos- ition in the small intestine by the activity of its somatic muscles; if these are paralysed by anthelmintics it is expelled by peristalsis. The lifespan of an adult worm is usually 1 to 2 years, after which it is ex- pelled spontaneously. The worms mate and eggs are passed in the faeces, with gravid females each producing 200 000 to 250 000 eggs daily. When freshly passed, these eggs are not infective and contain a single cell. This develops in the soil over the next 2–​6 weeks (faster in warmer temperatures) into an infective larva. The ova are resistant to chemicals and low temperatures, and may remain viable for up to 10 years in moist soil. Epidemiology and control Ascariasis is cosmopolitan, and is arguably the most common hel- minth infection. It is prevalent in areas where there is poor sanitation, with contamination of the soil with eggs. Infection is more common in tropical climates, especially where human faeces are used to fertilize vegetable gardens. Infection is usually acquired by eating contamin- ated food, or from soil ingested by children when playing and does not induce resistance to reinfection. It is relatively more common in children and intensity of infection peaks in the first decade of life in high transmission areas. In hyperendemic areas with constant warm temperatures and high humidity, children are continuously being in- fected, so that as some worms are being expelled, others are maturing to take their place. Transmission might only be associated with the Trachea Lungs Pharynx Swallowed female male Circulation Ingested HUMANS EXTERNAL ENVIRONMENT Fertilized Unfertilized Two cell stage Embryonated egg (infective stage) Eggs in faeces (diagnostic stage) Larva hatches in intestine Advanced cleavage Adults in small intestine Fig. 8.9.5.1  Life cycle of Ascaris lumbricoides and Ascaris suum. Fig. 8.9.5.2  Decorticated eggs of Ascaris lumbricoides, showing emergence of larvae. Copyright Viqar Zaman.

section 8  Infectious diseases 1508 rainy season in areas that are generally hot and arid. The pig ascarid A. suum can infect humans and mature into adult worms and is re- garded by some as a synonym of A. lumbricoides. Environmental sani- tation is the best control measure, but when this is not possible, mass chemotherapy given at intervals of 6 to 12 months to preschool age and school age children reduces the severity and intensity of infection. Clinical features The first passage of larvae through the lungs usually causes no symp- toms or pathological changes, but subsequent infections can be associ- ated with hypersensitivity reactions, causing Ascaris pneumonitis (Fig. 8.9.5.4). When this causes fever, cough, dyspnoea, bronchospasm, per- ipheral eosinophilia, and infiltrates on a chest radiograph that are often migratory, it is known as Löffler’s syndrome, but the syndrome is not spe- cific to ascariasis. The condition usually subsides after 7 to 10 days, unless reinfection occurs. In areas where pig farming is common, the larvae of A. suum may also produce severe pneumonitis and bronchospasm. Most people with established infection with A. lumbricoides are asymptomatic, especially if the worm burden is small. Some might complain of anorexia, nausea, and abdominal discomfort or dis- tension. Heavy infections in children can cause malnutrition and hinder normal development in terms of both stature and cogni- tive performance. Mechanical complications probably occur in less than 1% of infected individuals. Occasionally, usually in chil- dren, large numbers of worms may become entangled to form a bolus that blocks the intestinal lumen, usually near the ileocaecal valve, producing signs and symptoms of acute intestinal obstruc- tion. This might be complicated by perforation, intussusception, volvulus, and death. In some circumstances, such as fever, irrita- tion caused by drugs, anaesthesia, or bowel manipulation during surgery, the worms may migrate to ectopic sites. Migration into the common bile duct can be complicated with cholangitis and liver abscesses, whereas entry into the pancreatic duct might precipi- tate acute pancreatitis. Worms may migrate into the appendix, oc- casionally come out through the mouth and nose, and are rarely found in other ectopic locations. Diagnosis Ascariasis is usually diagnosed by finding plentiful numbers of the characteristically oval fertilized eggs measuring 60 × 30 to 70 × 50 μm in the faeces (Fig. 8.9.5.5). Sometimes the patient brings developing or adult worms that have been passed in the faeces or have emerged from the anus or nose of a sick child. Occasionally, adult worms are outlined in the intestines during barium-​meal examination, or are seen at upper gastrointestinal endoscopy. Treatment It is desirable to treat all infected individuals, even when the worm load is small. There are several effective drugs (listed next). None of them is recommended by its manufacturer for use in pregnancy, especially in the first trimester, or children under 1 to 2 years of age, although this has been disputed by some authorities. For preventive chemotherapy, the World Health Organization manual Preventive chemotherapy in human helminthiasis recommends that albendazole or mebendazole be offered in the second and third trimesters of pregnancy in targeted interventions where the prevalence of infec- tion with any soil-​transmitted helminth is greater than 20%. • Mebendazole is given is given as 100 mg twice daily for 3 days in adults and children over 2 years of age. It should not be given in the first trimester of pregnancy. Fig. 8.9.5.3  Adult Ascaris lumbricoides (scale in mm). Copyright Viqar Zaman. Fig. 8.9.5.4  Ascaris lumbricoides in the lungs, surrounded by an inflammatory reaction. Copyright Viqar Zaman. Fig. 8.9.5.5  Egg of Ascaris lumbricoides.

8.9.5  Gut and tissue nematode infections acquired by ingestion 1509 • Albendazole is given as a single dose of 400 mg in adults and chil- dren over 2 years of age. It should not be given in the first trimester of pregnancy. • Pyrantel pamoate given in a single dose of 11 mg/​kg body weight (maximum 1 g) is effective in curing more than 90% of cases of as- cariasis. Side effects are usually mild and the drug is well tolerated. It should not be given in the first trimester of pregnancy. In cases of intestinal obstruction caused by an Ascaris bolus, a pi- perazine salt given at a dose of 75 mg/​kg (maximum 3.5 g) daily for two consecutive days has been recommended as it induces flaccid paralysis of the worms, which may relieve the obstruction. This should be supplemented with decompression of the bowel through an intestinal tube with constant suction; rehydration and restoration of electrolyte balance with intravenous fluids. In most cases this con- servative therapy will relieve the obstruction and the child will rap- idly recover. If, however, the signs of obstruction persist, laparotomy is required. Obstruction of the bilary tract by Ascaris is diagnosed by ultrasound. Anthelminthics are given and worms not passed spon- taneously may be removed endoscopically, reducing the need for op- eration. Surgery is required for gangrenous cholecystitis. Anisakidosis Life cycle Anisakidosis (previously known as Anisakiasis) is an infection caused by the larvae of various species of nematode belonging to the family Anisakidae, most commonly Anisakis simplex and Peudoterranova decipiens. Adults live in the lumen of the intestine of cetaceans (whales, dolphins, and porpoises). Eggs are passed in water, embryonate, and release second-​stage larvae which are in- gested by crustaceans in which they develop to third-​stage larvae. When crustaceans are ingested by fish or squid they enter the muscles of these hosts. Cetaceans and humans become infected by eating infected saltwater fish or squid. Epidemiology and control The adult worms are commonly found in marine mammals in many parts of the world. Humans are infected when they eat raw or im- properly cooked fish or squid. The incidence is highest in Japan, countries on the northern European seaboard and the Pacific coast of the Americas, especially in the South. Infection is prevented by not eating raw, pickled, smoked, or undercooked fish and squid. Larvae are killed by cooking fully to at least 63°C; or freezing solid and storing at minus 20°C for 7 days; or freezing solid at minus 35°C and storing at minus 35°C or below for 15 hours. Clinical features The larvae do not develop to maturity in humans, but attach them- selves to, and then burrow into, the mucosa of the stomach (especially Pseudoterranova species) or small intestine (especially Anisakis spe- cies), and rarely the large bowel (Fig. 8.9.5.6). Symptoms commonly develop 4 to 24 h after eating infected fish. Gastric invasion produces severe epigastric pain, nausea, vomiting, and sometimes haematem- esis during the acute stage of the disease. Involvement of the intestine may cause severe lower abdominal pain, which might be misdiag- nosed as appendicitis. If symptoms are mild and the patient is left untreated the infection can become chronic, with pseudotumour formation around the parasite. Sometimes the larva precipitates an allergic reaction, with urticaria, angio-​oedema, or anaphylaxis. Diagnosis A definitive diagnosis is made by upper gastrointestinal endoscopy which reveals the lesion and the presence of white or yellow larvae up to 3 cm in length attached to the mucous membrane. Intestinal anisakiasis is more difficult to diagnose, but imaging studies might show thickening of the intestinal wall, and narrowing of the jejunum or ileum. Serology for anti-​Anisakis IgE or IgG is deployed in some specialist centres. Treatment In acute infection, an attempt should be made to remove the larva through an endoscope, although in most patients symptoms resolve spontaneously within 2 weeks. In chronic cases, surgical removal of the ulcerated areas or the tumour may be required. No chemo- therapy has been proven to be effective in a clinical trial, though albendazole has been reported to be beneficial in a case report. Capillariasis There are two forms of capillariasis: intestinal capillariasis caused by Paracapillaria philippinensis, and hepatic capillariasis caused by Capillaria hepatica. Intestinal capillariasis This infection is caused by a worm still generally referred to as Capillaria philippinensis, although it has been renamed Paracapillaria philippinensis. Fish-​eating birds are the definitive reservoir. Adult C. philippinensis measure 2.5 to 4.3 mm in length and produce eggs that are deposited in water and ingested by fish, in which they de- velop into infective larvae. Humans are infected by eating under- cooked freshwater or brackish-​water fish. In humans, the parasite has the capacity to autoinfect; female worms produce eggs that hatch into larvae that reinvade the intestinal mucosa, resulting in prolonged infection, so that the original source and time of infection Fig. 8.9.5.6  Third-​stage larva of Anisakis simplex, showing the tip of the boring tooth (arrow) (×400).

section 8  Infectious diseases 1510 may be forgotten. Autoinfection can result in extremely heavy worm loads, especially in immunocompromised patients. The parasite is endemic in parts of South-​East Asia, especially the Philippines and Thailand, and has more recently been found in Egypt. Infection is prevented by properly cooking fish. Intestinal capillariasis can be a severe and even fatal disease. Patients often present with abdominal pain, diarrhoea, and bor- borygmi. As the worm load increases, diarrhoea becomes more severe, with anorexia, nausea, and vomiting. Prolonged diarrhoea leads to cachexia. There might also be signs of hypotension and cardiac failure. The mortality rate in untreated cases approaches 20%. The diagnosis is made by finding eggs in the faeces, 36–​ 45 µm in length by 19–​21 µm in breadth (Fig. 8.9.5.7), which can superficially resemble those of Trichuris trichiura. Larvae or adult worms may also be present and repeated stool examination may be required in some cases. The parasite might also be found in je- junal aspirate or biopsy. The treatment of choice is albendazole for 10 days. If it is not available, the alternative is mebendazole for 21 days. Stools should be re-​examined to ensure the eradication of infection; if not, the course of treatment should be repeated. Supportive measures to overcome malnutrition and diarrhoea are required in severely ill patients. Hepatic capillariasis The adults of C. hepatica measure 52–​104 mm in length and live in the liver of various mammals, especially rats. Eggs are produced that are retained in the liver parenchyma; they measure 28 × 48 to 36 × 66 µm, and have bipolar plugs. The ova eventually reach the soil, ei- ther by decomposition of a carcass, or when the host is eaten by an- other animal and the eggs pass through the gut of that animal and are deposited in the faeces. Eggs embryonate in the soil and can sur- vive there for more than a year. Infective eggs are ingested by an- other definitive host, then hatch and the larvae reach the liver via the portal system. Human infection with C. hepatica occurs when eggs in the soil are accidentally swallowed, but human infection is rare and is usually a dead end for the parasite. Clinical features may resemble those of visceral larva migrans, with tender hepatomegaly, fever, and eosinophilia. The diagnosis is made by identifying the parasite or eggs in a liver biopsy (Fig. 8.9.5.8). The most effective treatment is unclear, although cases have been reported to respond to albendazole which is given as described for intestinal capillariasis. Enterobiasis (threadworm infection) Life cycle Enterobiasis is an infection caused by the threadworm or pinworm, Enterobius vermicularis. Infection is acquired by the ingestion of eggs (Fig. 8.9.5.9). Larvae hatch in the upper intestine and mi- grate to the region of the caecum, where they mature and copulate. Worms do not invade the tissues. About 1 month after infection, the white thread-​like gravid female worms, about 10–​13 mm long by 0.3–​0.5 mm in diameter, move down the bowel and pass out of the anus at night (Fig. 8.9.5.10). Each worm deposits approximately 10 000 eggs on the perianal skin. The worms then usually die, but sometimes migrate elsewhere, for example, into the vagina. The eggs are infective within a few hours of deposition. Epidemiology and control This worm is found worldwide and is extremely common, being found most frequently in children. Infections are commonly Fig. 8.9.5.7  Capillaria philippinensis egg (×1400). Fig. 8.9.5.8  Capillaria hepatica eggs in the liver (×250). Ingested Embryonated egg (infective stage) HUMANS Penetrates and develops in mucosa Larva hatches in intestine Gravid migrates to perianal region Egg on perianal skin (diagnostic stage) EXTERNAL ENVIRONMENT Fig. 8.9.5.9  Life cycle of Enterobius vermicularis.

8.9.5  Gut and tissue nematode infections acquired by ingestion 1511 clustered in families and institutions. Humans are the only res- ervoir of infection. Eggs can remain viable for up to 3 weeks, and can be transmitted via contaminated clothing, bedding, and dust. Resistance to reinfection does not develop and autoinfection may occur by contamination of the fingers. Clinical features Most infected people are asymptomatic. The most common pre- senting symptom is pruritus ani. This can be very troublesome and occurs more often during the night, causing restless sleep, especially in children. Persistent itching may lead to inflammation and sec- ondary bacterial infection of the perianal region. Occasionally, adult worms migrate to aberrant sites such as the urethra. In females they may enter the genital tract, causing vulvovaginitis or rarely salpin- gitis. E. vermicularis is sometimes found lodged in the lumen of the appendix (Fig. 8.9.5.11), but whether or not this causes appendicitis is controversial. Diagnosis The eggs are not usually found in the faeces. They are most easily found around the anus first thing in the morning by using cellulose adhesive tape applied with the sticky side against the perianal skin, which is then examined under the microscope; they are 33 × 55 μm in size and flattened on one side (Fig. 8.9.5.12). Sometimes intact worms are passed in the faeces and can easily be recognized by their size and shape. Treatment All the children and adults in a household should be treated at the same time. Several drugs are available, including mebendazole 100 mg in a single dose, repeated after 2 weeks; albendazole 400 mg in a single dose, repeated after 2 weeks; and pyrantel pamoate 11 mg/​kg to a max- imum dose of 1 g in a single dose, repeated after 2 weeks. Adult thread- worm live for no longer than about 6 weeks, so recurrent threadworm infestation is the result of reinfection. Therefore, attention to personal hygiene is an important part of treatment and prevention. The patient should be advised to keep fingernails short; wash hands with soap and water after defaecating and before preparing food; wear close fit- ting underwear in bed and change it every morning; shower or bath in the morning. The bed linen, mattress cover, soft toys, and towels should be washed at first diagnosis and at least weekly for six weeks thereafter. Over the same time period the bedroom, including the mattress, should be vacuum cleaned and the bathroom and kitchen damp-​dusted. Some individuals, often only one member of a household, suffer regular recurrences which can prove very distressing. There is no in vivo or in vitro assay for anthelminthic drug resistance to this para- site, so it is not possible to assess the potential contribution of drug resistance to this condition. Such cases require management in a parasitology clinic as they might need more intensive treatment re- gimens under expert supervision. Gnathostomiasis Life cycle There are 12 species of Gnathostoma, but only four are known to infect humans:  G.  spinigerum, G.  hispidum, G.  doloresi and G.  nipponicum. Most human cases are due to G.  spinigerum or Fig. 8.9.5.10  Adult Enterobius vermicularis (scale in mm). Copyright Viqar Zaman. Fig. 8.9.5.11  Histological section of Enterobius vermicularis in the lumen of the appendix (×250). Fig. 8.9.5.12  Enterobius vermicularis egg.

section 8  Infectious diseases 1512 G. hispidum (Fig. 8.9.5.13). Gnathostoma does not mature to the adult form in human infections. Its larvae are up to 12.5 mm long and 1.2 mm wide and have a cephalic bulb armed with rows of hooks (see Fig. 8.9.5.14). These enable the larva to move readily through human tissue and inflict substantial damage, especially if nervous tissue is involved. Epidemiology and control Humans are accidental hosts and become infected with the third stage larva after eating raw or undercooked freshwater fish or other intermediate hosts (e.g. snakes, frogs, and chickens, and possibly via ingestion of infected copepods). Given the large variety and wide distribution of its intermediate hosts, it is not possible to eradicate Gnathostoma from the environment, so individual protection is re- quired via thorough cooking plus avoidance of consuming raw or undercooked freshwater fish. Freezing to minus 20°C for 2 to 5 days is also effective. The common practice of marinating fresh fish in lime juice is not effective. Clinical features Cutaneous disease—​Gnathostomiasis can present as migratory cu- taneous swellings (nodular migratory panniculitis). Visceral disease—​Gnathostoma can produce a wide range of pres- entations, including pleuritic chest pain, pleural effusion, pneumo- thorax; ocular disease with uveitis, intraocular haemorrhage, or retinal detachment; central nervous system involvement with radiculomyeloencephalitis, eosinophilic meningitis, or subarach- noid haemorrhage. = Infective Stage = Diagnostic Stage L3 Gnathostomiasis Cutaneous Ocular Visceral Neurological Gnathostoma spinigenum Domestic and wild felines and canines are the definitive hosts Gnathostoma hidpidum Domestic and wild pigs are the definitive host. Unembryonated egg Embryonated egg in water. Infected second intermediate host ingested by definitive host. Second intermediate host Paratenic host L2 develops into L3. Copepod first intermediate host L1 develops into L2. Egg hatches and releases L1 larva. 2 1 5 6 4 3 7 L2 L3 L1 L3 develop into adult worms. L3 i i i i d d Fig. 8.9.5.13  Life cycle of Gnathostoma spinigerum and Gnathostoma hispidum. Courtesy of CDC. Fig. 8.9.5.14  Cephalic bulb of a Gnathostoma larva showing characteristic rows of hooks. Courtesy of P. L. Chiodini.

8.9.5  Gut and tissue nematode infections acquired by ingestion 1513 Diagnosis Clinical suspicion in cases of eosinophilia, migratory lesions, and ex- posure history. Occasionally a larva is extruded from the skin spon- taneously or removed intact from the eye by an ophthalmologist and its very characteristic morphology then makes diagnosis straightfor- ward. Serology by Western blot is available in specialist centres. Treatment Albendazole 400  mg twice daily for 21  days or ivermectin 200 micrograms per kilogram daily for two days. Repeat courses may be required. Surgical removal is required for ocular lesions. Toxocariasis Toxocariasis in humans occurs in two clinical forms, visceral larva migrans and ocular larva migrans, and is caused mainly by the migrating larvae of Toxocara canis, to a lesser extent T. cati and rarely other nematodes. T. canis and T. cati are parasites that live in the intestines of dogs and cats, which pass eggs in the faeces. Humans, usually young children, become infected by inadvertently ingesting embryonated eggs in the soil. The larvae hatch in the small intestine and migrate to various organs of the body, including the liver, lungs, eyes, and brain. The larvae, which are about 15–​20 μm in length, do not mature in humans, but granulomas eventually develop around them (Fig. 8.9.5.15). In a fully formed granuloma the larvae are surrounded by layers of fibrous tissue and inflammation subsides (Fig. 8.9.5.16). Eggs are never seen in human faeces. Toxocara in- fection occurs wherever there are large domestic dog and cat popu- lations in close association with humans and is more common in children. Deworming dogs and stopping children from eating dirt (pica) especially when playing in areas frequented by dogs, are im- portant control measures. Visceral larva migrans This disease is most often seen in young children, because of pica. Most people remain asymptomatic. In a minority, symptoms consist of muscular pain, lassitude, anorexia, cough, itching, and urticarial rashes. Physical signs might include wheezing and hepatomegaly. Occasionally there is splenomegaly, lymphadenopathy, and skin lesions. Central nervous system involvement can result in convul- sions. The acute phase generally lasts for 2–​3 weeks, followed by recovery. Sometimes the resolution of all the signs can take up to 18 months. Rarely, the infection ends fatally if a massive number of eggs has been ingested. The hallmark of visceral larva migrans is marked eosinophilia, which can reach a level of 75%. Serological tests for toxocara anti- body can be helpful; a negative test can rule out the diagnosis, but positive titres are often found in normal individuals. The defini- tive diagnosis is by finding larvae on biopsy, usually of the liver, but is seldom indicated. Most patients recover spontaneously; the larvae cannot multiply and eventually die. There is no proven therapy. Anthelmintics, including mebendazole, albendazole, and diethylcarbamazine have been tried, but may be ineffective or precipitate an inflammatory reaction. Corticosteroids and non-​ steroidal anti-​inflammatory agents have been suggested in order to suppress inflammation. Ocular larva migrans This condition is caused by granuloma formation around a larva in the eye and is most commonly seen in older children. If it is near the macula, impairment of vision or even blindness may re- sult. A rounded swelling, often near the optic disc, might be de- tected on fundoscopy. The features of visceral larva migrans are usually lacking in ocular larva migrans cases. There is usually no marked peripheral eosinophilia. Diagnosis depends upon posi- tive serology with consistent fundoscopic features; the major dif- ferential diagnosis is retinoblastoma. Antibody tests on vitreous or aqueous fluid can be positive when serum antibody tests are negative. Western blot is the assay of choice for use on aqueous or vitreous fluid. Anthelmintic therapy is not routinely given. Visible larvae can be photocoagulated by laser. Vitrectomy has been used in some cases and local and intraocular steroids are used to sup- press inflammation. Fig. 8.9.5.16  Histological section. Granuloma formation in the same animal as Fig. 8.9.5.15 at a later stage when the larva is completely surrounded by fibroblasts (×400). Fig. 8.9.5.15  Histological section. Granuloma formation in a monkey experimentally infected with Toxocara canis, showing many giant cells and some fibroblastic reaction. The arrow marks the larva (×400).

section 8  Infectious diseases 1514 Trichinosis (trichinellosis) Life cycle Trichinosis is an infection usually caused by Trichinella spiralis and related species. Humans become infected by eating undercooked meat, usually pork or pork products from domestic and wild pigs (boars) (Fig. 8.9.5.17). After ingestion the larvae are liberated in the stomach, then pass into the small bowel, where they invade the columnar epithelium and develop into adult worms living in the cytoplasm of a row of enterocytes. Male trichinellae are about 1.5 × 0.05 mm in size and female worms measure 3.5 × 0.06 mm. Over 2 to 3 weeks or so before they are expelled, female worms release about 500 newborn larvae (Fig. 8.9.5.18), which enter the bloodstream and seed the skeletal muscles. Over the next few weeks the larvae in the muscles increase in size, moult, coil, usually develop a cyst wall and become capable of infecting a new host (Fig. 8.9.5.19); they may remain viable in muscle for several years. Epidemiology and control Trichinella species are widely distributed in many geographical areas among many carnivorous hosts found in three classes of ver- tebrates: mammals, birds and reptiles (Table 8.9.5.1). Domestic pigs become infected by eating infected scrap from abattoirs or farms. Humans are incidental hosts and are usually infected with T. spiralis, but occasionally other species, depending upon the animal eaten. Infection is best prevented by properly cooking meat. Clinical features Most people with light infections are asymptomatic. In heavy in- fections, diarrhoea develops in the first week and is associated with abdominal discomfort and vomiting. Fulminating enteritis may develop in patients with extremely heavy infections. Symptoms of larval invasion develop during the second week, and include fever, myositis with pain, swelling, and weakness, usually first involving the extraocular muscles, then the masseters, neck muscles, limb flexors, and lumbar muscles. Some patients may develop one or more HUMANS Larva in mucosa Circulation SWINE, OTHER CARNIVORES Adults in small intestine Larva released in small intestine Encysted larva (infective stage) Encysted larva (diagnostic stage) Larva deposited in mucosa Encysted larva in muscle (diagnostic stage) Larva released in small intestine MEAT (Pork, wild boar, bear) Adults in small intestine Fig. 8.9.5.17  Life cycle of Trichinella spiralis. Fig. 8.9.5.18  In vitro preparation of infected mouse small bowel, showing adult worms of Trichinella spiralis surrounded by newborn larvae. Courtesy of D. I. Grove. Fig. 8.9.5.19  Trichinella spiralis third-​stage larvae in human muscle (×100). Table 8.9.5.1  Major Trichinella spp. and their epidemiology Species Code Distribution Most common hosts Cyst wall T. spiralis T1 Worldwide except Australasia Pig, horse, bear, rodent, fox Yes T. nativa T2 Arctic, subarctic Bear, fox, dog Yes T. britovi T3 Temperate, subarctic Dog, bear, cat, boar Yes T. pseudospiralis T4 Cosmopolitan Bird No T. murrelli T5 North America Bear, coyote, dog Yes Uncertain T6 Subarctic Bear Yes T. nelsoni T7 Sub-​Saharan Africa Hyena, cat Yes Uncertain T8 Southern Africa Lion, panther Yes Uncertain T9 Japan Bear Yes T. papuae T10 Papua New Guinea Pig, crocodile No T. zimbabwensis T11 Central Africa Crocodile, mammals No Uncertain T12 Argentina Cougars No

8.9.5  Gut and tissue nematode infections acquired by ingestion 1515 of cough, dyspnoea, headache, periorbital oedema, subconjunctival haemorrhages, and a petechial rash. These symptoms slowly subside over several weeks, although symptoms persist longer in a minority of patients. In fulminant infections a potentially fatal myocarditis or meningoencephalitis might develop. Diagnosis The diagnosis is suggested by a combination of fever, periorbital oe- dema, myositis, and eosinophilia in a patient who gives a history of eating undercooked meat, often in the context of an outbreak. Elevated creatine kinase and lactate dehydrogenase levels indicate considerable muscle involvement. Serological tests become posi- tive several weeks after infection. A definitive diagnosis depends upon finding larvae in muscle biopsies, although this is usually unnecessary. Treatment and prevention If the diagnosis is made early in the illness, treatment with albendazole or mebendazole for 14 days is given to kill adult worms in the bowel and thus reduce the load of larvae able to invade the muscles. In established infections these benzimidazole agents can be also be used, but usually have little influence on the course of the dis- ease. In established symptomatic infections the mainstays of treat- ment are rest and the administration of corticosteroids, especially in severe cases. Mortality rates as high as 5% have been reported with very heavy parasite loads. Trichinosis in the pig population can be greatly reduced or eliminated by hygienic rearing methods. Larvae in pork may be killed by freezing at –​18 °C for 1 week. Whether or not that is done, thorough cooking of pork is the best safeguard against infection in all endemic areas. Trichuriasis (whipworm infection) Life cycle Trichuriasis is an infection caused by Trichuris trichiura. Infection is acquired when an egg is ingested (Fig. 8.9.5.20). The infective larva hatches in the small intestine and enters the mucosal crypts of the caecum, where it moults several times to become an adult worm 30–​50 mm long. The anterior three-​fifths of the worm are thin and elongated, and the posterior two-​fifths bulbous and fleshy. The thin end is embedded in a syncytial tunnel in the large bowel epithelium (Fig. 8.9.5.21). Nearly 3 months after infection the fertilized female worms begin to produce about 10 000 eggs per day. Adult worms live for 1 to 3 years. After passage in the faeces, eggs embryonate in the soil and become infective after several weeks. Epidemiology and control Trichuriasis has a worldwide distribution, particularly in the warmer parts and is most common in areas where sanitation is poor, espe- cially where human faeces are used as fertilizer in vegetable gardens. Environmental sanitation is the best control measure. Ground-​ growing fruits and vegetables should be carefully washed. Clinical features Most infections are light and asymptomatic. In heavy infections there is colitis and/​or proctitis, with the passage of blood and mucus in the faeces. In some cases, prolapse of the oedematous parasitized rectum occurs. Chronic heavy infection can be associated with iron-​ deficiency anaemia and growth retardation. Diagnosis This is based on finding characteristically barrel-​shaped eggs 50 × 20 mm in size (Fig. 8.9.5.22) in the faeces. Sigmoidoscopy, proctoscopy, or colonoscopy may show worms attached to the mu- cous membrane and sometimes intact worms may be passed in the faeces. Treatment Benzimidazole anthelminthics (mebendazole or albendazole) are effective when given for between 3 and 5 days, depending upon the severity of infection. Repeat courses may be required. Ivermectin is an alternative if benzimidazoles cannot be used. EXTERNAL ENVIRONMENT HUMANS Embryonated egg (infective stage) Eggs in faeces (diagnostic stage) Penetrate and develops in mucosa Adults in large intestine Two cell stage Advanced cleavage Larva hatches in intestine Fig. 8.9.5.20  Life cycle of Trichuris trichiura. Fig. 8.9.5.21  Histological section. Anterior end of an adult Trichuris trichiura embedded superficially in the large bowel mucosa (×250).

8.9.6 Angiostrongyliasis 1516

8.9.6 Angiostrongyliasis 1516

section 8  Infectious diseases 1516 Uncommon intestinal or tissue nematode infections From time to time a patient may be encountered who harbours an un- usual nematode. Some of these organisms are free-​living parasites and the patient has a spurious infection, usually as the result of ingesting the worm, or following the in vitro contamination of a clinical spe- cimen such as faeces or urine. Other individuals may have true infec- tions, with worms being found in either the gastrointestinal tract or the tissues. Many of these infections are with parasites that are poorly adapted to the human host and are unable to complete their devel- opment in humans. Thus, worms in varying stages of development, including larvae, adults, and eggs, may be found in clinical specimens. If there is uncertainty in identifying the worm, help may often be obtained from a veterinary parasitologist, who might be more used to dealing with the species concerned. Sometimes parasites are seen only in histological sections and should be reviewed by a tropical histo- pathologist. The texts by Connor et al.; Gutierrez; and Orihel and Ash provide helpful aids to histopathological identification. FURTHER READING Connor DH, et al. (eds) (1997). Pathology of infectious diseases, Vol. 2, pp. 1305–​588. Appleton and Lange, Stamford, CT. Cooper AJ, Hollingsworth TD (2018). The impact of seasonality on the dynamics and control of Ascaris lumbricoides. J Theor Biol, 453, 96–107. Cross JH (1992). Intestinal capillariasis. Clin Microbiol Rev, 5, 120–​9. Fuehrer HP, et al. (2011). Capillaria hepatica in man—​an overview of hepatic capillariosis and spurious infections. Parasitol Res, 109, 969–​79. Gottstein B, et al. (2009). Epidemiology, diagnosis, treatment, and con- trol of trichinellosis. Clin Microbiol Rev, 22, 127–​45. Gutierrez Y (2000). Diagnostic pathology of parasitic infections with clinical correlations, 2nd edition. Oxford University Press, Oxford. Herman JS, Chiodini PL (2009). Gnathostomiasis, another emerging imported disease. Clin Microbiol Rev, 22, 484–​92. Knopp S, et  al. (2012) Nematode infections:  soil-​transmitted hel- minths and trichinella. Infect Dis Clin North Am, 26, 341–​58. Kocięcki J, Kocięcka W, Dmitriew A (2016). Toxocarosis of the organ of sight—​the complex pathological and diagnostic problem. Acta Parasitol, 61, 1–​9. Leles D, et al. (2012). Are Ascaris lumbricoides and Ascaris suum a single species? Parasit Vectors, 5, 42. Lu LH, et al. (2006). Human intestinal capillariasis (Capillaria philip­ pinensis) in Taiwan. Am J Trop Med Hyg, 74, 810–​13. Luna J, et al. (2018). Updated evidence of the association between toxocariasis and epilepsy: Systematic review and meta-analysis. PLoS Negl Trop Dis, 12, e0006665. Ojha SC, et  al. (2014). Geohelminths:  public health significance.
J Infect Dev Ctries, 8, 5–​16. Orihel TC, Ash LR (1995). Parasites in human tissues. American Society of Clinical Pathologists, Chicago, IL. Shimamura Y, et al. (2016). Common symptoms from an uncommon infection: gastrointestinal anisakiasis. Can J Gastroenterol Hepatol, 2016, 5176502. Wani I, et al. (2010). Intestinal ascariasis in children. World J Surg, 34, 963–​8. Woodhall DM, Fiore AE (2014). Toxocariasis: a review for pediatri- cians. J Pediatric Infect Dis Soc, 3, 154–​9. World Health Organization (WHO) (2006). Preventive chemotherapy in human helminthiasis. World Health Organization, Geneva. Websites Photographs of the various stages of these parasites and diagrams of their life cycles, may be found at several useful websites: Centers for Disease Control and Prevention. Laboratory investigation of parasites of public health concern. http://​www.dpd.cdc.gov/​DPDx/​ Korean Society for Parasitology. Web atlas of medical parasitology. http://​www.atlas.or.kr UK NEQAS Parasitology. http://​www.ukneqasmicro.org.uk/​parasitology/​ 8.9.6  Angiostrongyliasis Richard Knight ESSENTIALS Angiostrongylus cantonensis The rat lungworm causes outbreaks of eosinophilic meningitis pre- dominantly in Southeast Asia, East Asia, Oceania, and the Caribbean. Elsewhere the condition is usually seen in travellers. Human infec- tions follow ingestion of raw snails (the primary intermediate hosts), food contaminated by snail mucus, or one of several paratenic hosts. Clinical manifestations include headache, meningism, vomiting, cranial nerve lesions, and (less commonly) other neurological fea- tures such as seizures. Ocular lesions are quite common. Diagnosis is made by lumbar puncture revealing eosinophilic meningitis, with larval or immature adult worms sometimes seen. Treatment is usu- ally with prednisolone alone, or with albendazole and prednisolone. Mortality is usually below 2%. Prevention is by avoidance of raw high-​risk dietary items and unwashed salads. Fig. 8.9.5.22  Egg of Trichuris trichiura. Courtesy of A R Butcher.

8.9.6  Angiostrongyliasis 1517 Angiostrongylus costaricensis The cotton rat is the principal reservoir host. Unwitting ingestion of slugs, the intermediate hosts, in salads or fruit leads to human infections in tropical American countries, especially in Costa Rica, Nicaragua, Guatemala, and Honduras. The organism causes granulomatous le- sions of the right colon and sometimes the liver: most patients present with right-​sided or right iliac fossa pain, with tenderness and some- times a palpable mass. Diagnosis is usually made histologically on resected material. Surgery may be necessary, but the value of anthel- minthics is uncertain. Preventive measures include washing and careful inspection of vegetables, and hand washing before meals by children and those preparing salads. Introduction Human disease is caused by two nematode species of the genus Angiostrongylus. Both parasites normally infect rodents, and mol- luscs are the primary intermediate hosts. Angiostrongylid worms with rodent hosts were placed in the genus Parastrongylus in 1986 but this name is no longer used. Infection follows accidental or de- liberate ingestion of molluscs or paratenic hosts. The epidemiology is complex because of multiple potential routes of transmission. Angiostrongylus cantonensis This is the rat lungworm and was first described in 1935 from China. The first known human case, reported in 1944, was a 15-​year-​old Taiwanese boy with meningoencephalitis, in whose cerebrospinal fluid an immature adult worm was found. Detailed clinicopathological studies were made in 1962 during epidemics of eosinophilic menin- gitis in Tahiti. Aetiology: The biology of the parasite Adult worms live in the pulmonary arteries of rats; larvae from hatched eggs ascend the airways, are swallowed, and so reach the faeces. Molluscs ingest these larvae, and after two moults they are infective when eaten by a rodent. In the rat, infective larval worms migrate to the cerebral grey matter, where they start to mature. They then move to the meninges and enter the venous sinuses, thereby reaching the pulmonary arteries, where maturation is completed. Infective larvae from a mollusc can also enter, by ingestion, a second or third inter- mediate host, in which they undergo no further development until they enter a mammalian host. Such supernumerary hosts are termed paratenic hosts, and are important sources of infection in humans. Development in humans reaches the immature adult stage, measuring 11 to 15 mm in length. Nearly all will die in the superfi- cial cortex, brainstem, meninges, and occasionally the eye, causing vigorous tissue reactions; very few reach the lungs. Epidemiology The parasite is endemic and causes human outbreaks in Southeast Asia, East Asia, Oceania, and the Caribbean. Sporadic cases are reported in many other countries, usually in travellers. Most recent outbreaks have been in mainland China, Taiwan, Thailand, and Japan. In the Pacific epidemics have occurred in Hawaii, Samoa, and the Solomon Islands. In the Caribbean most cases are reported from Cuba, Costa Rica, and Jamaica; it has also been reported from Ecuador, Brazil, Egypt, and South Africa. It is now present, at least in rats, in several African and tropical American countries including southern USA. All ages can be affected, and outbreaks have occurred after weddings and feasts; infections are often seasonal. The modes of transmission differ geographically, by age and social group, and with time. The principal rodent hosts are Rattus rattus, R. norvegicus, R. alex­ andrinus, and R. exulans. The prevalence in rats in endemic areas may be 40% or more. The geographical spread and population increase of these peridomestic rodents has increased the zoonotic reservoir; wildlife is now infected in the southern United States of America. Another factor leading to the increase in human infection has been the dispersal by human agency of the edible giant African land snail Lissachatina fulica, from Madagascar in 1800, eastwards across the Indian Ocean and the Pacific, to reach Hawaii in 1936. The freshwater golden apple snail Pomacea canaliculata, which is highly susceptible to the parasite, was recently introduced into Asia, where it has colon- ized paddy fields and caused disease when served raw in restaurants in China, Taiwan, and Japan. The popularity of heliculture, the cultiva- tion of exotic snails for food, and keeping them as pets, facilitates the spread of the parasite. Raw snails are eaten as a delicacy and for me- dicinal purposes; salads may contain small undetected molluscs, their slime trails, or planarians. During food preparation infected mucus from snails may reach the mouth directly or contaminate other foods or water. An outbreak in Taiwan followed the drinking of raw vege- table juice. In Thailand, Pila spp. snails are a seasonal delicacy eaten by all the family, but only young men take them raw with alcohol. Paratenic hosts include freshwater prawns, land and coconut crabs, frogs, and land planarians, which cause infection if eaten raw; drinking-​water may contain tiny immature prawns, especially after heavy rains. In Thailand, the yellow tree monitor lizard is an important paratenic host. In the Ryukyu Islands of Japan, patients are usually in- fected by eating raw snails or toad liver for medicinal purposes. Pathology Inflammatory granulomatous lesions, sometimes track-​like, occur predominantly in the cortical grey matter and the meninges, but also in the brain stem and cerebellum; nerve roots and the spinal cord may also be affected. Live worms are occasionally found at autopsy, and dead worms are found in many lesions. The number of worms found varies greatly, and may reach several hundred; worm tracks in the tissue and meninges are surrounded by a cuff of eosinophils; Charcot–​ Leyden crystals derived from eosinophils are numerous. Rarely, adult worms have been found in human lung at autopsy. Ocular infection derives from worms that have migrated across the cribriform plate. Clinical features Illness severity depends mainly on the number of larvae ingested. After an incubation period of 1 to 4 weeks the onset is acute, with headache (intermittent at first), together with nausea and vomiting. There is constitutional upset and frequently menigism; fever is un- usual. The illness is often self-​limiting over a period of 4 weeks. Cranial nerve lesions are seen in the optic, abducens, and facial nerves. Less common are seizures, confusion, and radiculopathy (with paraesthesia, root pains, or weakness). Long-​tract signs and impaired consciousness are uncommon, except in severe cases, but spinal cord damage can cause sphincter disturbance.

section 8  Infectious diseases 1518 Fig. 8.9.6.1  Angiostrongylus under the conjunctiva in a Thai girl with a left facial nerve palsy. Copyright D. A. Warrell. Ocular complications include retinitis, retinal haemorrhages, optic neuritis, and larval worms in the vitreous, anterior chamber, or beneath the conjunctiva Fig. 8.9.6.1). Rarely, migration to the lungs produces clinical evidence of pneumonitis. Numerous eosinophils occur in the cerebrospinal fluid, and there is blood eosinophilia. Diagnosis Lumbar puncture reveals high opening pressure, with a clear or lightly turbid cerebrospinal fluid containing 500–​2000 cells/​mm3 (of which 10–​>90% are eosinophils). Protein levels are usually elevated with normal or, less commonly, reduced glucose. Detailed examination at low power reveals larval or immature adult worms in up to 25% of cases, measuring 5–​15 mm in length. Cerebrospinal fluid changes may persist for up to 3 months. CT or MRI may reveal focal cortical abnormalities. Serology using antigens from fourth-​stage larvae is useful, but cross-​reactions with other nematodes can cause difficulty. Commercial serological tests are not yet available. Techniques to de- tect worm antigens in cerebrospinal fluid and serum have also been developed. Parasite DNA can also be detected by PCR. Differential diagnosis is from other helminth infections affecting the nervous system, as eosinophils are otherwise rare in cerebro- spinal fluid. A detailed geographical and dietary history is essential. Conditions to be considered include paragonimiasis, schistosom- iasis, and neurocysticercosis. A particular problem in Thailand is confusion with Gnathostoma spinigerum, which more commonly causes long-​tract signs, bloody or xanthochromic cerebrospinal fluid, neck stiffness, and clouding of consciousness. Treatment, prognosis, and control In a recent prospective trial prednisolone alone was as effective as prednisolone plus albendazole. Prednisolone alone for 14 days is now often the recommended treatment, although prednisolone plus albendazole is still used in many centres using albendazole 15 mg/​kg daily in divided doses. Treatment hastens recovery and relieves headache and it probably improves the prognosis in severe cases. Repeated lumber puncture has also been used to relieve head- ache. Ocular disease may require laser therapy and larvae in the eye chambers should be removed surgically. Anthelminthics should be avoided in eye involvement. Mortality rates are generally low in uncomplicated cases, but some patients develop encephalitis and pass into coma after about 2 weeks, and their prognosis is then very poor. Most patients improve in 2 to 4 weeks, but focal neurological deficits can persist for longer. Partial relapse after 2 months of illness may represent a reaction to dying worms. Some cases are relatively mild and can be discharged within a few days; during epidemics, mild cases may need only outpatient care. Control measures include health education to limit the inges- tion of raw high-​risk dietary items and unwashed salads. Warnings may be necessary regarding raw molluscs, amphibians, and reptiles used for medicinal purposes. Rodents in vegetable gardens and the peridomestic environment should be controlled. Angiostrongylus costaricensis This was first recognized in Costa Rica in 1950 in surgical specimens simulating bowel malignancy. The parasite was described from such specimens in 1967, and the complete life cycle in rodents was eluci- dated over the next 3 years. Aetiology: the biology of the parasite In both the rodent and human hosts, the worms are located in the ileocaecal mesenteric arteries. The cotton rat Sigmodon hispidus is the principal reservoir host, but other species of rodent (including the coatimundi) are also involved, and even dogs and marmosets. In the rodent hosts worm eggs embolize to gut-​wall capillaries, and the hatched larvae pass into the gut lumen. Veronicellid slugs, es- pecially Vaginulus plebeius, eat rodent faeces containing larvae, and these develop into infective larvae in the fibromuscular tissue of the mollusc after two moults over a period of 18 days. Infective larvae can persist in the slug for several months or be shed in slime trails. The prepatent period in rats eating infected slugs is 24 days. In human infections the worms reach maturity, but the embryon- ated eggs do not hatch. Epidemiology Infections occur especially in Costa Rica, Nicaragua, Guatemala, and Honduras, but also sporadically elsewhere in the Americas from the United States of America to Argentina, and some Caribbean islands. Recently, infections have been increasingly recognized from southern Brazil. Small veronicellid slugs are the source of infection in man; infection rates in intermediate hosts can reach 85%. Small or chopped slugs may be unnoticed on fallen fruits or in salads; the mucus of their slime trails also contains infective larvae. Many cases are in schoolchildren, but infants and older persons are also affected. Seropositivity in endemic areas suggests that there are unrecognized infections. Pathology and clinical features Lesions primarily affect the small arteries, producing subacute or chronic granulomatous inflammatory masses in the wall of the

8.9.6  Angiostrongyliasis 1519 caecum, right colon, and less often the small intestine or elsewhere in the colon. Rarely, the predominant feature is ischaemic infarction. The finding of an adult nematode measuring 18 to 42 mm in length within a gut arterial vessel is diagnostic of infection; eggs may be seen in vessels or in tissue, where they are surrounded by eosinophil granulomas. Lesions also occur in regional abdominal lymph nodes or the omentum. Some larvae enter the hepatic artery and cause granulomatous or necrotic lesions in the liver; others enter testicular arteries causing similar lesions of the testis. In a recently reported case an adult worm was shown histologically within a hepatic arteriole. Clinically, most patients present with right-​sided or right iliac fossa pain, with tenderness and sometimes a palpable mass in this region. Other features are eosinophilia, fever, diarrhoea, or rectal bleeding. Tender hepatomegaly with high blood eosinophilia occurs in some patients. Serious complications are bowel obstruction and perforation, and rarely testicular infarction. Diagnosis and treatment The confirmation of diagnosis is usually made histologically on re- sected material, PCR can be used to detect parasite antigen. The condition can mimic appendicitis, bowel neoplasm, Meckel’s di- verticulitis, testicular torsion, or other surgical problems. Parasite eggs are not found in faeces, but serology using enzyme-​linked im- munosorbent assay (ELISA) or latex agglutination is useful. More recently indirect immunofluorescence using an egg antigen has given good specificity. Serology suggests that subclinical infections may be common. Contrast radiology reveals filling defects and al- tered motility of the terminal ileum, caecum, or ascending colon. Laparoscopy can reveal the bowel and hepatic lesions; biopsy may then be diagnostic. The value of anthelmintic treatment remains unproven; thiaben- dazole or high doses of mebendazole have been used. Surgery is often necessary, but can sometimes be deferred in uncomplicated cases when the diagnosis is strongly suspected, as spontaneous re- mission is common. Preventive measures include washing and careful inspection of vegetables, and hand washing before meals by children and those preparing salads. Rinsing salads in 1.5% bleach kills larvae. FURTHER READING Angiostrongylus cantonensis Cowie RH (2013). Biology, systematics, life cycle, and distribution of Angiostrongylus cantonensis, the cause of rat lungworm disease. Hawaii J Med Public Health, 72 (6 Suppl 2), 6–​9. Cowie RH (2013). Pathways for transmission of angiostrongyliasis and the risk of disease associated with them. Hawai J Med Public Health, 72, (6 Suppl 2), 70–​4. Cowie RH, et al. (2012). Workshop on research priorities for manage- ment and treatment of angiostrongyliasis. Emerg Infect Dis, 18, e3. Damien KY Ming, et  al. (2017). Angiostrongylus cantonensis DNA in cerebrospinal fluid in persons with eosinophil meningitis. Laos Emerg Inf Dis, 23, 2112–13. Eamsobhana P (2014). Eosinophilic meningitis caused by Angiostrongylus cantonensis – a neglected disease with escalating importance. Trop Pubmed, 31, 569–78. Evans-​Gilbert T, et al. (2014). Severe eosinophilic meningitis owing to Angiostrongylus cantonensis in young Jamaican children: case report and literature review. Paediatr Int Child Health, 34, 148–​52. Flerlage T, et al. (2017). Angiostrongylus cantonensis eosinophilic men- ingitis in an infant, Tennessee, USA. Emerg Inf Dis, 25, 1756–58. Jitpimolmard S, et  al. (2007). Albendazole therapy for eosinophilic meningitis caused by Angiostrongylus cantonensis. Parasitol Res, 100, 1293–​6. Sawanyawisuth K, Sawanyawisuth K (2008). Treatment of angio­ strongyliasis. Trans R Soc Trop Med Hyg, 102, 990–​6. Sawanyawisuth K, et al. (2013). Clinical manifestations of eosinophilic meningitis due to infection with Angiostrongylus cantonensis in chil- dren. Korean J Parasitol, 51, 735–​8. Spratt DM (2015). Species of Angiostrongylus (nematoda: meta­ strongyloidea) in wildlife: A review. Int J Parasitol Parasites Wildl, 4, 178–​89. Thanaviratananich S, Thanaviratananich S, Ngamjarus C (2015). Corticosteroids for parasitic eosinophilic meningitis. Cochrane Database Syst Rev, 2, CD009088. Wang QP, et al. (2012). Human Angiostrongylus cantonensis: an update. Eur J Clin Microbiol Infect Dis, 31, 389–​95. Ying Feng Y, et  al. (2013). Comprehensive review of ocular angio­ strongyliasis with special reference to optic neuritis. Korean J Parasitol, 51, 613–​19. Angiostrongylus costricensis Abrahams-​Sandi E, et al. (2011). An indirect immunofluorescence anti- body test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis. Mem Inst Oswaldo Cruz, 106, 390–3. Dard C, et  al. (2018). Angiostrongylus costaricensis infection in Martinique, Lesser Antilles, from 2000–2017. Parasite, 25, 22. Incani RN, et al. (2007). Human infection by Angiostrongylus costar­ icensis in Venezuela: first report of a confirmed case. Rev Inst Med Trop Sao Paulo, 49, 197–​200. Rodriguez R, et al. (2008). Abdominal angiostrongyliasis: report of two cases with different clinical presentations. Rev Inst Med Trop Sao Paulo, 50, 339–​41. Rodriguez R, et  al. (2014). PCR for the diagnosis of abdominal angiostrongyliasis in formalin-​fixed paraffin-​embedded human tissue. PLoS One, 9, e93658. Valente R (2018). Angiostrongylus spp. in the Americas: geographical and chronological distribution of definitive hosts versus disease. Mem Inst Oswaldo Cruz, 113, 143–52.