unit 5. application of biomarkers to

chapter 23. Infectious Betsy Foxman

Summary Historical perspectives

Molecular tools have enhanced our rapidly detect infectious agents and The development of understanding of the epidemiology predict disease course. Integration as a discipline was roughly of infectious diseases by describing of molecular tools into etiologic simultaneous with the development the system, including studies has identified infectious of microbiology. As the presence identifying novel transmission causes of chronic diseases, and of infectious agents was linked to modes and reservoirs, identifying characteristics of the agent and disease, laboratory methods were characteristics of the infectious that modify disease risk. The incorporated into epidemiologic agent that lead to transmission and combination of molecular tools with studies. One epidemiologic hero is pathogenesis, identifying potential epidemiologic methods provides , who identified a strong vaccine candidates and targets for essential information to guide epidemiologic association between therapeutics, and recognizing new clinical treatment, and to design and sewage-contaminated water and infectious agents. Applications of implement programmes to prevent cholera. Despite extremely well- molecular fingerprinting to public and control infectious diseases. documented evidence supporting health practice have enhanced However, incorporating molecular thoroughly researched and outbreak investigation by objectively tools into epidemiologic studies of reasoned arguments, his findings confirming epidemiologic evidence, infectious diseases impacts study remained in doubt for some time. and distinguishing between time- design, conduct, and analysis. Max Von Pettenkofer, 1818–1901, space clusters and sporadic cases. a contemporary of Snow and

Clinically, molecular tools are used to also an early epidemiologist, is Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 421 related (in a perhaps apocryphal whooping cough and polio, and novel transmission modes and story) to have drunk a glass of the the identification of , reservoirs, identifying characteristics stool of someone with cholera to led to a rather simplistic view of of the infectious agent that lead to test the hypothesis; Pettenkofer infectious disease, and ultimately transmission and pathogenesis, remained disease-free. Snow’s to the incorrect impression that we revealing potential targets for conclusions were not generally might “close the book” on infectious vaccines and therapeutics, and accepted until 25 years after his disease during the 20th century. This recognizing new infectious agents. death, when the cholera vibrio was assertion was quickly undermined The combination of molecular tools discovered by Joseph Koch, who in the last quarter of the 20th with epidemiologic methods thus definitively demonstrated the causal century by the emergence of new provides essential information to relationship between the vibrio and infectious agents such as human design and implement programmes cholera (1). The strategy of isolating immunodeficiency virus (HIV), Ebola to prevent and control infectious an organism from an ill individual, and Hantavirus, the re-emergence diseases. showing it can cause disease in a of tuberculosis and malaria, and disease-naïve individual, and then the transcontinental transmission of Outbreak investigations be re-isolated as described in the agents such as West Nile Virus and landmark postulates of Henle and Dengue. Molecular tools have substantially Koch reflects how incorporating Infectious disease improved inferences from outbreak laboratory methods enhances our epidemiologists were early adapters investigations. We can more rapidly ability to make causal inferences of modern molecular biologic provide laboratory confirmation about disease transmission and techniques to epidemiology, such of disease diagnosis, and detect pathogenesis from even the most as those used in genomics. Indeed, the presence of difficult-to-culture carefully researched epidemiologic the term molecular epidemiology or uncultivable agents, enhancing evidence. came from infectious disease work the sensitivity and specificity of Early epidemiologists made (5). Modern molecular techniques case definitions. Molecular tools tremendous strides with what are have fundamentally changed our make it possible to determine if now relatively simple molecular understanding of the epidemiology the epidemiologically identified tools: using microscopy for of infectious agents. Characterizing outbreak source, such as a identification, which showed that the genetics of human pathogens food item, contains the infecting agents not visible by microscope has revealed the tremendous organism, and if the identified caused disease (“filterable”); and heterogeneity of various infectious organism has the same genotype detection of protective antibodies agents, and the rapidity with which causing the outbreak, enhancing with haemagglutination assays. they evolve. This heterogeneity causal inference. Molecular typing For example, Charles Nicolle and and rapid evolution helps can also be used to determine order Alphonse Laveran showed that a explain our difficulties in creating of transmission (Table 23.1). It is not protozoan caused malaria (2,3), successful vaccines for the more surprising that molecular typing has and Charles Nicolle demonstrated, heterogeneous organisms, such become a standard tool in outbreak by injecting a monkey with small as Neisseria gonorrhoeae. With investigation. amounts of infected louse, that the increased ability to detect host Case definition is an essential lice transmitted typhus. He also immunologic response to infectious component of successful outbreak observed that some animals carry agents, we increase our ability to investigation. The detection of the asymptomatically (3). test and refine our theories about infectious agent from all cases Wade Hampton Frost used the the extent and duration of immunity, more accurately classifies cases presence of protective antibodies a key parameter in disease spread. than clinical diagnosis alone; in the serum of polio patients to Molecular analysis has also revealed molecular typing further minimizes explain the emergence of polio the role of infectious agents in the misclassification. For point-source (4). These early, initiation and promotion of previously outbreaks, all individuals are dramatic successes combined with classified chronic diseases. Further, expected to be infected with the the successful development and molecular tools have enhanced our same strain of a particular genus and implementation of vaccines against understanding of the epidemiology species; for propagated outbreaks major childhood diseases, including of infectious diseases by describing similar molecular fingerprints are smallpox, measles, diphtheria, the transmission systems, identifying expected that vary only in the

422 Table 23.1. Applications of molecular tools in outbreak investigations cases occurred in three other states among individuals suspected to • Enhance case definitions have consumed frozen strawberries from the same processor. The • Determine whether cases occurring in the same time frame are part of the same outbreak genetic sequences of the virus

• Confirm or refute epidemiologic inferences regarding etiologic pathways from all tested individuals were the same, confirming a common • Determine the order of transmission source of infection for all cases. Without molecular fingerprinting, it mutations accrued over repeated the same PFGE pattern as the S. would have been very difficult, if not transmission events. The resulting agona causing the outbreak (7). impossible, to link these apparently reduction of misclassification of This was the first time a commercial disparate cases to one common disease status increases the study cereal product was implicated in source using solely epidemiologic power and the validity of inferences. a Salmonella outbreak, so the methods. This example also The first and most typical PFGE evidence was particularly highlights the importance of using application of molecular tools in an compelling. molecular tools with surveillance, outbreak situation is to confirm or A second application in an discussed in detail in the next refute epidemiologic information. For outbreak investigation is determining section. example, in a foodborne outbreak, whether cases occurring in the A third application is to isolates might be collected from same time frame are part of the determine the order of transmission. all those with disease, the person same outbreak. With disseminated This application has been suspected to have introduced the outbreaks, there can be apparent particularly useful in forensic cases. infected agent into a food item, and sporadic cases that are actually Understanding the evolution of an the suspected food item. Figure 23.1 linked. For example, in 1997 a large organism and the ability to trace shows the molecular fingerprints, (n = 126) foodborne outbreak of the order of that evolution has determined using pulsed-field hepatitis A occurred in Michigan (8). made it possible to detect cases gel eletrophoresis (PFGE), of a Epidemiologic evidence implicated where an individual deliberately foodborne Staphylococcus aureus frozen strawberries from a single infected others. For example, a (S. aureus) outbreak isolated from processor. During the same time gastroenterologist was convicted of the suspected food that had the period, a much smaller outbreak (n = infecting a former girlfriend with the same molecular type as S. aureus 19) occurred in Maine, and sporadic blood of an HIV patient. A variety found in the food handler, and in those with disease. Figure 23.1. Example of molecular typing using pulsed-field gel electrophoresis In most foodborne outbreaks, (PFGE). In this foodborne outbreak of methicillin sensitive Staphylococcus aureus, neither specimens from all isolates from food handler (lane 6), cases of food poisoning (lanes 7-9, 11) and infected food (lane 12) had the same PFGE type (6). individuals that meet the case definition, nor the putative food is available for testing by the time it is identified. With clear epidemiologic evidence, the demonstration of genetic relatedness between cases and the putative item is solely confirmatory, and adds little unless the food vehicle has not been previously identified. For example, epidemiologic evidence linked consumption of toasted oats cereal with a multistate outbreak of Salmonella in the USA. A culture of the cereal from one opened and two unopened boxes found

Salmonella agona (S. agona) with Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 423 of molecular analyses, including Table 23.2. Applications of molecular tools in surveillance phylogenetic analyses of HIV-1 reverse transcriptase and env DNA • Distinguish between time-space clusters and sporadic cases of the same infection sequences isolated from the victim, the patient, and a local population • Identify clusters requiring further investigation sample of HIV-1-positive individuals, • Detect the emergence of strains with new resistance profiles strongly supported not only that there was transmission between • Estimate of infection and observe trends over time the two individuals, but who had infected whom (9). causing infection among patients, a E. coli O157:H7 with the putative distinction can be made between a pulsed-field type was isolated from Surveillance strain from the community and one an unopened package of spinach circulating endemically or causing from an individual’s home. Molecular Surveillance is an essential an outbreak within the hospital. The typing enabled rapid linkage of cases component of a successful public prevention and control strategies occurring across several states, the health infrastructure. Laboratories are different in each case, and thus identification of the disease source, are key components of many it is important to make a distinction and facilitated quick surveillance systems; hospital between them. intervention. laboratories may be part of regional A second application of A third application is the surveillance networks, as well as molecular tools in surveillance is detection of infectious agents part of a local surveillance system. to identify clusters requiring further resistant or insensitive to prevailing Monitoring of infectious disease investigation. By monitoring isolates therapies. A cluster of drug-resistant isolates identifies time-space from time-space clusters for the agents is often the first indication of clusters of infection; molecular typing presence of a common molecular an outbreak, particularly in a hospital distinguishes between infectious type, one can distinguish between setting. Mobile genetic elements that agents of the same species, allowing common-source outbreaks that confer resistance can be exchanged differentiation among clusters of are local and those that are widely between bacteria, even across disease occurring by chance and disseminated. Processed foods species, complicating outbreak true outbreaks. True outbreaks and may be distributed widely, as investigation. Molecular tools can clusters of the same strain can be demonstrated in the earlier example distinguish between a common traced back to a common source of the Michigan hepatitis A outbreak strain of a single bacterial species or and presumably are amenable caused by frozen strawberries a mobile genetic element, conferring to public health intervention. (8), so adding molecular typing to resistance across strains Spurious clusters cannot, and laboratory monitoring of specimens of the same or even different their investigation wastes time and is essential. The US Centers for species. Outbreak control must resources. Applying molecular tools Disease Control and Prevention’s take into account whether a mobile to surveillance isolates can also PulseNet, a molecular subtyping genetic element is being exchanged identify new strains with increased surveillance system for foodborne between species or if there is clonal virulence or changing patterns of bacterial disease, monitors spread of a single organism. resistance (Table 23.2). Escherichia coli (E. coli) O157:H7, In the United States there is Hospitals have high Salmonella, Shigella, and Listeria selective culture of organisms. rates of bacterial infection, but monocytogenes, and other bacterial In outpatient settings the most the are often due to a pathogens (11) causing disease common bacterial infections, urinary bacterial strain that was colonising throughout the United States. In tract infection and pneumonia, are an individual before entering the 2006, clusters of a common E. coli generally treated empirically. Only hospital, for example, S. aureus. O157:H7 pulsed-field type were if treatment fails is a culture taken. The prevalence of S. aureus observed at several monitoring sites. Thus surveillance for colonization among the general An investigation revealed the source resistance reflects a biased sample, population is 32% in the nares (10), of the outbreak to be washed, pre- suggesting the subset most likely but much higher in patients and packaged, fresh spinach. Once the to have a resistant infection. The personnel in hospitals and long- epidemiologic investigation identified inherent selection biases should term care facilities. By typing strains spinach, the public was notified and be taken into consideration when

424 suggesting policy changes in infectious agents are circulated as the duration of infectivity and the therapies based on surveillance within a population, and includes rate of effective contact. Molecular data. the transmission mode, interactions tools can usefully be applied to Molecular tools have also between the infectious agent and estimate each of these parameters. been applied to screen biological the host, the natural history of the Prior to the availability of modern specimens collected as part of infection, and interactions between molecular tools, our ability to ongoing national databases for hosts that lead to infection. The empirically estimate transmission the presence of known and newly emergence and re-emergence probabilities was limited. For discovered infectious agents. For of a variety of infectious agents example, the transmission of a example, blood samples are collected highlights the utility of understanding sexually transmitted infection can as part of the National Health and the various transmission systems, be estimated by following couples Nutrition Examination Survey, a as this understanding is central where one is infected and the other multistage probability sample of to identifying effective prevention is susceptible; however, without the United States conducted every and control strategies. Combining molecular tools it is difficult to 10 years. This has enabled the molecular typing methods with ensure that the transmission event is estimation of the prevalence of questionnaire data can confirm not attributable to a person outside various infectious agents, including self-reported behaviours, especially the partnership. For respiratory hepatitis B and C viruses, human important when the validity of infections, such as pulmonary herpes virus 8 (which causes Kaposi self-report may be in doubt, such tuberculosis, our estimates of sarcoma) and herpes simplex as contact tracing of sexually the transmission probability and viruses 1 and 2. These studies transmitted diseases. As described natural history have been based on provide insight into the frequency in detail below, molecular tools careful documentation of outbreaks. of new agents, and the distributions facilitate estimating parameters key However, as we have been able to of agents by spatial-temporal and to understanding the transmission type individual strains, it has been host characteristics. Such studies system, including the , determined that tuberculosis cases are extremely useful for generating prevalence, transmission probability, that previously were considered hypotheses about transmission duration of carriage, effective dose, sporadic, and not part of apparent systems, potential prevention and and probability of effective contact. time-space clusters (because the control strategies, evaluating the exposure to the was effectiveness of ongoing prevention Estimation of key parameters very limited), were indeed part of the and control programmes, and same outbreak (12). observing time trends. When using simple transmission Key transmission system

models to estimate R0, the average parameters are incidence, Describe the transmission number of new cases generated prevalence, duration of infection, system from each infectious case in and transmission probabilities. The a fully susceptible population, estimation of these parameters The transmission system of an the transmission probability per assumes the accurate measure infectious agent determines how effective contact is needed, as well of identical strains or subtypes of an infectious agent. As we Table 23.3. Components of the transmission system and associated parameters have increased our ability to type infectious agents, we have been Component Parameter forced to re-evaluate many of our previous assumptions. One key Incidence Occurrence in a population Prevalence assumption is that pathogens are clonal; that is, during active infection Transmission mode Probability of transmission given contact all infecting organisms are the same. A second, parallel assumption is Natural history of infection Duration of infection that during an infectious process the

Interactions between agent and host Effective dose pathogenic organism will be the one most frequently isolated from the Interactions between hosts leading to Probability of contacting an infected indivi- infected site. For many diseases, transmission dual we now know these assumptions Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 425 are false. For example, individuals The duration of carriage can Increase understanding can be infected with different strains be estimated from the prevalence of the epidemiology of human papillomavirus (HPV), and incidence, presuming that the of infectious diseases gonorrhea and even tuberculosis. average duration across strain During a diarrheal episode, the type is of interest. However, if While the contributions of molecular predominant organism isolated duration is short but incidence is tools to outbreak investigation and from the stool may not be the one high, an individual might become surveillance have been substantial, causing the symptoms: a toxin- re-infected with a different strain there have also been significant secreting organism occurring at type, suggesting a longer duration if contributions to our understanding low frequency may be the culprit. strain types are not determined. By of the epidemiology of infectious For infectious agents that also contrast, if a strain mutates rapidly diseases. Molecular tools enable are human commensals, such as within the human host, duration of us to trace the dissemination of a Streptococcus agalactiae, strains carriage might be underestimated. particular subtype across time and causing disease may be different Thus, strain-specific estimates space, and thus develop theories from normal inhabitants and of prevalence and incidence are of transmission and dissemination; different strains may have different essential to our understanding determine the origin of an , transmission systems. of disease etiology, especially if and therefore test theories These observations have different strains have different about reservoirs and evolution profound impacts on the conduct propensities to cause diseases. of a particular agent; follow the of future studies. If the population emergence of new infections as genetic structure of pathogens is not Using molecular tools they cross species, testing our clonal and the pathogen is not readily to estimate contact patterns hypotheses about the apparent isolated, this must be reflected in transmissibility and rate of evolution; the sampling of isolates for study. Molecular tools can also assist in and follow mobile genetic elements Multiple isolates must be sampled the estimation of contact patterns conferring and tested from an individual. by identifying asymptomatic and low or virulence between strains within For example, if there is a second levels of infections. Asymptomatic a species or between species, strain only 5% of the time and the infection is often a key component and so develop theories about pathogen is uniformly distributed in maintaining disease transmission. evolution and transmission within in the sample, 28 different isolates For example, in a study of intra- the populations of infectious agents. must be sampled from an individual family transmission of Shigella, to reliably detect the second strain. asymptomatic carriage increased Tracing the dissemination Further, if an infectious agent risk of a symptomatic episode of infectious agents across mutates rapidly within a host, such within 10 days by nine-fold (14). time and space as HIV, determining the mutation Molecular typing can also be used rate will be essential for accurately to enrich and validate contact Infectious agents are constantly estimating transmission probabilities tracing information. The addition of emerging and re-emerging. Some and following transmission chains. molecular typing to epidemiologic agents, like influenza, have a well- As molecular tools can detect information on gonorrhea cases in understood pattern, where new the presence of the organism or host Amsterdam identified large clusters strains generally emerge from response to a specific organism, of individuals with related strains, southeastern Asia. This allows not in some cases, for a particular individuals infected with different only set up of sentinel surveillance strain (13), studies can detect strains at different anatomical sites, points, but prediction, with some both incidence and prevalence of and persons with high rates of re- accuracy, of which influenza asymptomatic infection and clinical infection (15). The results suggested strain type(s) are most likely to disease. Understanding the full that the transmission networks for cause the next epidemic. As the extent of the circulation of a particular men who have sex with men and genetics of influenza is fairly well- infectious agent is essential for for heterosexuals were essentially understood, appropriate vaccines making accurate predictions and separate—a key public health for known variants can be prepared. determining appropriate prevention insight for planning interventions. The difficultly is when the virus and control strategies. undergoes an antigenic shift. At this writing, an influenza A strain

426 Table 23.4. Ways molecular tools increase understanding of the epidemiology of resistant to fluoroquinolones infectious diseases has been due to a diverse set of genetic mutations (17), suggesting • Tracing the dissemination of infectious agents across time and space spontaneous emergence following treatment. As S. pneumonia resistant • Determine the origin of an epidemic to fluoroquinolones rapidly followed the introduction of fluoroquinolones, • Follow emergence of new infections alternative antibiotics will be needed • Follow mobile genetic elements conferring virulence of antimicrobial resistance in relatively short order to treat S. pneumonia infections.

Table 23.5. Applications of evolutionary theory to infectious disease epidemiology Determine the origin of an epidemic

• Identify genetic lineages Molecular tools enable us to trace

• Estimate rate of evolution an outbreak or epidemic back in time to its origin, and back in space • Generate theories about the emergence and maintenance of specific lineages of infectious to its reservoir. Knowing the origin agents in time is essential for predicting future spread and identifying the (H5N1, also known as avian or bird has been single or multiple points reservoir for infection is central for influenza) has repeatedly caused of entry, and if emerging resistance controlling disease spread. The use human infection with very high case was from multiple spontaneous of molecular techniques has solved fatality rates (~50%), although the mutations or from dissemination of long-standing mysteries, such as chains of transmission have been a single clone. For example, until cholera’s reservoir betweencholera relatively short and the total number 2004, only occasional isolates of epidemics. The same strains of of cases is relatively small. However, gonorrhea found in Sweden were cholera that infect humans also there is an ongoing widespread resistant to azithromycin, and these thrive in aquatic environments epidemic among wild birds, and cases were attributed to acquisition (19). While the importance of pigs there have been several outbreaks elsewhere (16). However, in 2004, and fowl as the origin of antigenic among domestic birds, resulting epidemiologic evidence suggested shifts in the genetics of influenza is in large-scale culling of birds and that domestic transmission might understood, molecular tools have considerable adverse economic have occurred; this was confirmed clarified that avian influenza need impact. by molecular typing. The ongoing not first pass through the pig before Other infectious agents have hit transmission of the azithromycin- jumping to humans, and that direct by surprise, such as the emergence resistant strain in Sweden has short- transmission from birds to humans of HIV, and the migration of West term implications for surveillance is often more virulent (20). Molecular Nile virus to the United States. and long-term implications for tools can also provide insight into the Further, some infectious agents treatment recommendations. origins of infection in highly endemic have mutated in unpredicted Streptococcus pneumoniae populations, such as hospitals. The ways, such as the emergence of (S. pneumonia) is a major cause prevalence of methicillin resistant multidrug-resistant tuberculosis, of pneumonia, but also causes Staphylococcus aureus (MRSA) penicillin-resistant Streptococcus meningitis and otitis media. A has been steadily increasing in pneumoniae, and community- major human pathogen, it is one hospitals in the United States; acquired methicillin resistant S. of the most common indications in 2004 the prevalence among aureus. In addition to understanding for antibiotic use. Resistance to some intensive care units was as the transmission system, the origin penicillin emerged relatively slowly, high as 68% (21). However, in the and source of entry of infectious but once it emerged it was widely early 2000s, new strains of MRSA agents into the population must be disseminated in relatively few emerged among individuals in the traceable to prevent and control the clones as defined by multilocus community that could not be traced spread of infection. By comparing sequence typing. By contrast, the back to hospitals. Genetic typing of strains, it can be determined if there recent emergence of S. pneumonia the strains confirmed that strains Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 427 isolated from those who had no improves our ability to predict outbreak was the ability to detect epidemiologic linkage with hospitals transmission routes, and identify mild cases and confirm that widely had genotypically different strains potential therapies and prevention disseminated cases were caused (Figure 23.2) (18). More recently, strategies by analogy to similar by the same agent, which required community-acquired MRSA has organisms. a validated antibody test (24). Early joined hospital-acquired strains in Severe acute respiratory in the epidemic there were many causing infection in hospital settings syndrome (SARS) was the first possible candidates identified as (22). emerging disease identified this the causative agent, but these century. The story of the rapid agents were not found in all SARS Emergence of new infectious isolation, identification, and patients. A variety of state-of-the-art agents sequencing of the coronavirus and standard molecular techniques causing SARS, is illustrative were used to identify the viral agent, Surveillance, outbreak investigation, of the synergistic effects of the a new coronavirus. Molecular sentinel networks, and the astute marriage of molecular methods techniques established that the healthcare worker are keystones with epidemiology. SARS was genetic sequences were the same for identifying the presence of new first reported in southern China in throughout the world, and a rapidly disease syndromes. While a clearly 2002 and rapidly spread worldwide developed test demonstrated that defined clinical syndrome facilitates (Figure 23.3). Basic epidemiologic SARS patients had antibodies to the epidemiologic investigation, the methods were essential for tracking new coronavirus. Further, healthy potential for misclassification and the outbreak; a carefully collected controls not having SARS had no associated bias can be high for non- epidemiologic case definition was evidence of either past or present specific syndromes. Molecular tools, sufficient for case ascertainment, infection (25). such as non-culture techniques, clinical management, infection have dramatically improved our control, and identifying chains of Trace mobile genetic elements ability to rapidly identify the etiologic transmission (23). However, key agent and develop diagnostic tools. to characterizing and ultimately Mobile genetic elements are In addition, detection of the agent preventing and controlling the sequences of genetic material that can change places on a Figure 23.2. Pulsed-field gel electrophoresis pattern relatedness of community- chromosome, and be exchanged associated and health care-associated methicillin resistant Staphylococcus aureus between chromosomes, between (MRSA) isolates to a reference strain. The reference strain was MR14, which was bacteria, and even between species. the most commonly identified pattern among Minnesota MRSA isolates with a community-associated case definition (18). A type of mobile genetic element, known as a plasmid, can integrate directly into the chromosome or extra-chromosomal in the cytoplasm of bacteria and still code for proteins. The recognition of mobile genetic elements, and the ability to trace these genetic elements as they move within and between species, has caused a re-thinking of the rate of, and potential for, evolution of infectious agents. For example, Figure not available Shiga toxin-producing E. coli probably emerged from the transfer of genes coding for Shiga toxin from Shigella into E. coli. Antibiotic resistance is often spread via a mobile genetic element. These elements tend to code for genes providing resistance against multiple antibiotics. This explains

428 Figure 23.3. The rapid dissemination of severe acute respiratory syndrome (SARS) (http://yaleglobal.yale.edu/reports/images/SARS_MAP1.jpg, permission given by Yale Center for the Study of Globalization and YaleGlobal Online).

several apparent mysteries, such as elements, or conserved elements demonstrate that two subtypes the spread across several bacterial on the chromosome. introduced into drug networks in species within a hospital of the same Human immunodeficiency the 1990s still contributed to the antibiotic resistance profile, and virus (HIV), which causes acquired epidemic in 2001 and 2002, and why treating an individual with one immunodeficiency syndrome that one subtype spread widely antibiotic can result in resistance to (AIDS), evolves quite rapidly even throughout the Ukraine and into the multiple unrelated antibiotics. with a single host. Thus, the strain Russian Federation, the Republic that infects an individual is not of Moldova, Georgia, Uzbekistan Determine phylogenetic genetically identical to the strains and Kyrgyzstan. Further studies to relationships that the individual might transmit determine the biologic and social to others. This property of HIV has contributions to the success of Genetic sequence and other made it possible to confirm the the one subtype over another will molecular typing methods enable deliberate infection of one individual provide important insights into how the construction of phylogenetic by another using a single blood to control HIV. trees. Phylogenetics enables sample from an individual (9) and A second application is to the use of evolutionary theory to to gain insight into the origin and determine the rate of evolution. explain epidemiologic phenomena, spread of HIV worldwide. This is a standard application in particularly emergence and There are three primary biology, but understanding how transmission of more (or less) applications of phylogenic analyses fast infectious agents evolve has virulent strains, strains resistant to in an epidemiologic context. First, profound implications for choosing , simply to trace the phylogenic analysis enables us to a molecular typing technique and transmission of a rapidly evolving determine genetic lineages. Using interpreting epidemiologic data. For species, or, in an outbreak situation, this type of analysis, researchers example, some agents change very determine order of transmission. traced the introduction and spread rapidly, so that the agent infecting an Separate phylogenies can be of HIV in the Ukraine (Figure individual is different from the agent constructed for mobile genetic 23.4) (26). They were able to that is transmitted to another, for Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 429 Figure 23.4. Phylogenetic analysis of strains from different cities in Ukraine. Test hypotheses about Phylogenetic trees of strains from Kiev (a), Crimea (b), Nikolayev (c), Odessa (d), transmission systems Donetsk (e), and Poltava (f). A, B, C, D (capital letters) shown on tree branches are the HIV-1 subtypes. A scale bar of 0.01 substitutions per site is shown under each tree (26). The publisher for this copyrighted material is Mary Ann Liebert, Inc. Applying molecular typing to ongoing publishers. or endemic disease transmission increases our understanding of how contact patterns produce observed patterns of disease, revealing novel prevention and control strategies. In addition to characterizing ongoing chains of transmission, molecular typing can clarify who had contact with whom, and who was the source of infection, and thus identify a transmission network. Identifying transmission networks provides essential information for targeting intervention programmes, particularly when designing and implementing vaccine programmes. Using polymerase chain reaction (PCR)-restriction length polymorphism typing of the porin and opacity genes of Neisseria gonorrhoreae and questionnaire data, a study of successive gonorrhea cases in Amsterdam identified several ongoing transmission chains. The epidemiologic characteristics, including number of sexual partners and choice of same or opposite partners of patients with different molecular types differed, suggesting example HIV. Other agents change maintenance of specific genetic that the transmission chains very slowly, such as tuberculosis. lineages. This application is a by- represented different transmission Thus, the appropriate typing product of studies of genetic lineages networks (15). Molecular typing has technique must be chosen, so that and the rate of evolution. A key insight also improved our understanding phylogeny can be used to determine from the study in the Ukraine was of tuberculosis transmission. Until if rapidly changing agents evolved the differential spread of different confirmed by molecular typing, from a common ancestor, and to be HIV subtypes (26). A study of the tuberculosis was not believed to be able to distinguish between slowly molecular epidemiology of norovirus transmitted by short-term casual evolving isolates. A typing technique outbreaks in Norway demonstrated contact. Several investigations have for a rapidly evolving agent might the emergence of a new variant demonstrated that this assumption is focus on a region of the genome that that accounted for a change in incorrect, such as clusters associated evolves relatively slowly; a typing both the seasonal distribution with use of services at day shelters technique for a slowly evolving agent and common transmission mode (28), and even linked to only a few might focus on a genetic region that (27). A next step for furthering our brief visits to an infected individual’s evolves fairly quickly, so that the understanding of the epidemiology worksite (12). Molecular typing has investigator can distinguish between of HIV and norovirus would be to also demonstrated linkage between outbreak and non-outbreak strains. generate theories to explain these apparently sporadic tuberculosis A final application is to generate phenomena. cases, and determined that at theories about the emergence and least some recurrent tuberculosis

430 is attributable to exogenous re- Table 23.6. Molecular epidemiologic strategy for gene discovery infection (reviewed by (29)). • Identify candidate genes by combining bioinformatics information with molecular data Identify agent characteristics that lead to transmission and • Screen well-characterized representative samples of isolates causing different pathologies and asymptomatic infection pathogenesis • Analyse to determine relative frequency of selected characteristics in various populations The Microbial Genome Program of the US Department of Energy has different in genetic content from a novel microarray platform that sequenced more than 500 microbial the well-studied E. coli K12 strain, enables the screening of thousands genomes (http://microbialgenomics. which was originally isolated from of bacterial isolates for the presence energy.gov/brochure.pdf); the human feces in 1922 (http://www. of a putative virulence gene in a genetic sequence of numerous sgm.ac.uk/pubs/micro_today/ single experiment. The genomes human pathogens have already pdf/080402.pdf). Epidemiologic of up to 5000 bacterial isolates can been published, and many more are studies can take advantage of be arrayed, in duplicate, on a single ongoing, as well as experiments to this variation in genetic content to array, and screened for the presence compare the sequences of other compare the frequency of a putative or absence of a single gene using dot strains to a sequenced strain. A virulence factor present among blot hybridization (Figure 23.5) (31). great deal can be learned from strains isolated from individuals Library on a Slide has been created sequence data; of particular interest with a specific pathology with the for Mycobacterium tuberculosis, here is the identification of new frequency among commensal Haemaphilis influenza, E. coli, S. open reading frames (ORF) which isolates. For infectious agents with pneumoniae, Group B Streptococcus correspond to gene sequences. less diverse genomes, studies can and Streptococcus mutans. Although inferences can be made determine differences in genetic The molecular epidemiologic about a particular ORF based on alleles or in gene expression. strategy has been productively the genetic sequence by comparing Studies using a molecular applied to many bacteria species. it to other gene sequences of epidemiologic strategy can be done For example, a study screening known function, we cannot be using high-throughput methods, such collections of middle ear and certain of the gene’s function or its as multiplex PCR or microarrays. throat non-typeable Haemaphilis importance to disease transmission For example, Library on a Slide is influenzae isolates, a major cause of or pathogenesis. However, Figure 23.5. Library on a slide microarray platform compared with a United States conducting epidemiologic studies on quarter. Each spot on the slide contains the total genomic DNA of a strain of E. coli. appropriately collected samples can Photograph and slide by Dr. Lixin Zhang. Reprinted from (32), Copyright (2007), with be done to increase understanding permission from Elsevier. of the potential function of the genes and their relative prevalence using a molecular epidemiologic strategy (Table 23.6) (30). Many bacterial species are found in both diseased and healthy individuals and have highly diverse genomes. For example, E. coli, the most common cause of urinary tract infection and diarrhoea, is found in the normal bowel flora of virtually all humans and animals. When disease and commensal isolates are compared, the genome of E. coli is quite diverse, even when limited to human isolates. E. coli O157:H7, which causes diarrhoea and haemolytic urea syndrome, is substantially Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 431 otitis media, identified a gene found more effective and specific therapies 8 as the infectious cause of Kaposi significantly more frequently among and prevention strategies may be sarcoma, and the rapid identification middle ear isolates, lic2B. lic2B was more successful than attempts at of the coronavirus as the cause of found 3.7 times more frequently changing human behaviour. SARS. among middle ear isolates than Infectious agents may also The development of a vaccine in throat isolates from children contribute to the evolution of against cervical cancer was a attending day care (33,34). humans. A well-documented case is direct result of our ability to use the impact of malaria on the human molecular tools in an epidemiologic Identify infectious agents host: there are several different context. Cervical cancer has an that are adapted to particular genetic variations that protect epidemiology that strongly suggests human hosts against malaria. These variations a sexually transmitted infection. The are found in countries that either disease is associated with a greater Humans and infectious agents are currently or in the past had endemic lifetime number of sex partners, extremely well adapted to each malaria. The effectiveness of the early age at first intercourse, and other. Some infectious agents are human genetic variant at reducing history of a sexually transmitted essential to human development, malarial disease varies with infection. The precursors of such as for digestion of foods and Plasmodium species. The most cervical cancer (cervical dysplasia production of nutrients such as well-known variant is the sickle detectable via PAP smear), were vitamins (35). Thus, it is not surprising cell trait, but there are others, such studied extensively, but widespread that recent evidence suggests that as the Duffy blood group. When misclassification obscured the normal microbiota co-evolved with the Duffy blood group is absent, results. Cervical infection with their human hosts (36). Further, Plasmodium vivax is unable to different HPV types have different several studies suggest certain enter the red blood cells (39). Using propensities to progress to cervical pathogenic species are adapted different molecular techniques, cancer, but the clinical presentation at to certain human populations, and other human adaptations can be the initial stages of infection, cervical that infectious agents may select for identified. These may provide dysplasia, is indistinguishable mutations in human lineages. insight into potential therapeutics, among types. It was not until the Evidence that a particular such as understanding the role of tools were available to identify HPV genetic lineage of a pathogen may CCR5 in blocking HIV, or generate and to determine the different HPV be better adapted to certain human theories to explain observed human types that the epidemiology was populations, has been gathered variation. truly understood and an effective by comparing the epidemiology vaccine developed (40). of specific lineages in human Identify new infectious agents The Kaposi sarcoma (KS) populations of mixed genetic origin. causing disease story demonstrates the potential of For example, HPV variants of African combining an exquisitely sensitive origin persist longer among African Epidemiologic studies have often molecular detection technique American women than among white suggested a possible infectious with epidemiologic study design. women, and European variants origin for a clinical syndrome. Using representational difference persist longer in white women than However, our ability to detect the analysis to identify DNA sequences among African American women etiologic agent has been hampered present in KS lesions but absent or (37). A similar association between by our inability to culture most present in low copy number in non- the infected host’s region of origin infectious agents. The development diseased tissue obtained from the and the infecting strain has been of the polymerase chain reaction same patient, researchers identified observed for tuberculosis (38). (PCR) has dramatically increased non-human DNA sequences in Social and behavioural factors have our ability to detect infectious KS lesions of HIV patients (41). been associated with acquisition agents, both those cultivable The sequences were determined and persistence of each of these and uncultivable. PCR has been to be herpes-like, subsequently infectious agents, but tangible essential to such public health designated human herpes virus evidence of host susceptibility tied triumphs as the development of a 8. A randomized, blind, evaluation to specific agent characteristics vaccine against HPV (the primary of tissue from patients with KS of implies that strategies based on the cause of cervical cancer), the different origin was then performed: identification and development of identification of human herpes virus AIDS-associated, classic, and

432 among homosexual men who Table 23.7. Selected infectious causes of chronic diseases were HIV-seronegative (42). This confirmed that the DNA sequences Chronic disease Infectious agent were present in all types of KS, Arthritis Borrelia burgdorferi, Epstein-Barr Virus, suggesting that the sequences Salmonella spp, Campylobacter spp, were not found only among AIDS Yersinia spp, Chlamydia spp patients. Seroepidemiology studies Bladder cancer Schistosoma spp. confirmed that seroprevalence was correlated with risk of KS, and that Cervical, anal, penile, head and neck cancers Human papillomavirus seroconversion and seropositivity predicted development of KS (43). Chronic liver diseases, hepatocellular Hepatitis B, Hepatitis C The ability to detect non- carcinoma culturable infectious agents provides Creutzfeldt-Jakob disease Variant Creutzfeldt-Jakob disease new strategies to more rapidly identify emerging infections. A surveillance Gastric cancer Helicobacter pylori system has been established in the Heart disease Chlamydia pneumoniae United States to identify the infectious components of unexplained deaths Kaposi sarcoma Human herpes virus 8 and critical illnesses possibly due to Leukemia Human T-lymphotropic virus type 1 infectious causes (http://www.cdc. gov/ncidod/eid/vol8no2/01-0165. Lymphoma Epstein-Barr virus, htm). This system enabled the Human T-lymphotropic virus type 1 rapid detection of West Nile Virus Peptic ulcer disease, chronic gastritis Helicobacter pylori encephalitis when it first appeared in the United States (44). Whipple disease Tropheryma whipplei

Identify infectious agents Modified and expanded from (45). involved in the initiation and promotion of chronic disease Figure 23.6. Schematic showing how multiple factors interact leading to chronic sequelae of infectious diseases (45). Infectious agents are popularly associated with acute disease processes, but many well-studied infectious processes lead to chronic diseases, such as tuberculosis and AIDS. However, other diseases that were previously attributed to genetics, behavioural, or lifestyle factors are now known to have an infectious component, including stomach ulcers, chronic liver disease, and arthritis (Table 23.7). Pathogenesis occurs at the infectious-chronic axis complex (Figure 23.6), and includes interactions between the agent, host and environment. Some of these diseases result from molecular mimicry, that is, the infectious agent has epitopes so similar to the host that the host response attacks itself, such as in reactive arthritis or rheumatic fever following infection. Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 433 Alternatively, there may be disease human endogenous retroviruses These methods have profound that results from a host primed for are the remnants of ancient germ implications for public health practice, infection that does not happen. cell infections (48). However, as we clinical diagnosis and epidemiologic Molecular tools have definitively increase our ability to detect specific studies. Rapid detection methods demonstrated that there are host response to infectious agents are based on either PCR, the infectious causes of cancer: hepatitis and detect traces of infection within amplification of genetic sequences of B and C can cause liver cancer; the host, we will likely increasingly the infectious agent that can identify serotypes of human papillomavirus detect infectious causes of many the agent present, or an antigen- cause cervical, anal, penile, and diseases of unknown etiology. antibody reaction that detects the head and neck cancers; and herpes host response to the infectious virus 8 causes KS (45). Infectious Guide clinical treatment agent or a metabolite of the agent. diseases, such as Chlamydophila and intervention strategies Rapid detection means faster pneumoniae (C. pneumoniae), are and more accurate diagnosis. For hypothesized to lead to the promotion Knowledge of the molecular example, intrapartum prophylaxis of atherosclerotic plaques and thus genetics of infectious agents and with antibiotics has reduced by 50% coronary artery disease. Numerous the interaction of the infectious the incidence of neonatal Group studies have demonstrated C. agent with the host gained from B streptococcal (GBS) disease. pneumoniae in atherosclerotic molecular epidemiologic studies However, GBS colonization is tissue, and severity of disease has can be used to more rapidly detect often transient, so many women been positively associated with infectious agents and thus improve may be treated unnecessarily. antibodies to C. pneumoniae after patient diagnosis, predict disease Detection of GBS colonization at adjustment for other known risk course and identify potential vaccine time of labour and delivery would factors. However, results of antibiotic candidates. Molecular techniques minimize inappropriate antibiotic therapy in preventing progression can also be applied to characterize use, decreasing unnecessary of cardiovascular disease among the ecology of normal human flora, pressure for the development of those who already have disease detect disruptions in the flora that antibiotic resistance. Further, GBS is have been disappointing. The lead to disease, and to detect the increasingly resistant to the second- totality of the evidence suggests presence of biofilms (microbial line antibiotics used for women that C. pneumoniae is neither a structures which often contain sensitive to the first-choice antibiotic. necessary nor sufficient cause of multiple species that can initiate or Rapid detection of antibiotic coronary artery disease, but is likely promote disease or protect the host resistance, based on the detection a modifiable risk factor (46). from disease) (Table 23.8). of resistance genes and the ability to Determining an infectious cause discriminate between more virulent of a chronic disease is difficult: Rapid detection of infectious GBS strains, will potentially improve active infection often has ceased agents medical care. by the time the chronic disease is Rapid techniques are often manifest. Disease clusters may be Increasingly, there are rapid methods extremely sensitive, so when quite informative: Lyme disease for the detection of infectious agents. applied in an epidemiologic context was identified as a cause of juvenile arthritis because of careful Table 23.8. Using molecular tools in a clinical epidemiologic context epidemiologic investigation of a disease cluster (47). As the disease process is not solely a function • Rapidly detect infectious agents of the presence of the infectious • Distinguish between pathogens and commensals agent, but an interaction of the agent with the host, it is as likely • Distinguish between relapse and re-infection to detect the presence of specific genes associated with the disease • Predict disease course as the infectious agent. Moreover, there is evidence that infectious • Evaluate potential vaccine candidates agents can incorporate their genetic • Guide intervention material into the human genome:

434 may be cost saving. For example, determine the causal agent and the that have dramatically improved epidemiologic studies of colonization physician to prescribe appropriate public health worldwide: smallpox with MRSA, which has emerged as therapy. has been eradicated, and measles a community-acquired pathogen of and polio are largely under control some significance, can be screened Distinguish between relapse in developed countries. Tetanus, for using rapid techniques; only and re-infection influenza, diphtheria, mumps, specimens screening positive by rubella, chickenpox, hepatitis A and rapid methods might be cultured. Some infections have a chronic, B, and yellow fever can be prevented, Culture is not only more time- recurring nature. In this situation, and the Bacille Calmette–Guérin consuming, but costly in terms of it is extremely useful to distinguish vaccine for tuberculosis minimizes reagents and personnel. However, between a relapse, which implies the most adverse manifestations the ability to propagate an infectious treatment failure, and a new of tuberculosis in children. Most agent is highly desirable, as it infection. Strain typing can be recently, a vaccine against the HPV facilitates more detailed studies at extremely useful in this situation, serotypes 16 and 18, which are most the molecular level. as typing allows us to distinguish likely to cause cancer, was licensed. between strains of the same Nonetheless, many other infectious Distinguish between species. For example, molecular diseases that cause significant commensals and pathogens typing demonstrated that individuals morbidity and mortality have can be infected with more than one remained intractable to prevention Increasingly, it is recognized that strain of tuberculosis (50) and of HIV via vaccine using conventional many species of infectious agents (51). strategies, but not from lack of previously thought to be harmless trying. Some of these infectious commensals can, under certain Predict disease course agents, such as the bacteria that circumstances, cause disease (49). cause gonorrhea and bacterial For example, fungal infections are Disease course is a function of meningitis, Neisseria gonorrhoeae only a problem among immune- both host and agent factors. While and Neisseria menigitidis, can suppressed patients. Acquired an individual who receives a larger rapidly vary their surface antigens immunosuppression can result infectious dose of an infectious (53,54) making it difficult to identify either from medical therapy, such agent is, on average, more likely an appropriate target. as chemotherapy for cancer to become ill and to manifest The human pathogen or immunosuppressive drugs symptoms more rapidly, this may not sequencing project has resulted in prescribed to transplant patients, always be the case. The virulence of a new strategy for the identification or as a result of infection, such as the infectious agent, whether the of vaccine candidates based on HIV. It has been discovered that all host has had previous exposure to the predicted protein products strains within a species do not have the same or a similar strain of the based on the genetic sequence equal disease potential; indeed, an infectious agent, or if the host has (Figure 23.7) (55). In silico analyses may arise an underlying genetic predisposition using bioinformatics enable the because of special characteristics or presence of predisposing factors, detection of potential epitopes. of the infectious agent itself. This such as co-morbidities, all influence Many species are very diverse at makes the identification of the causal the infectious course. For example, the genetic level, thus not only must agent in the laboratory difficult, initial viral load in HIV patients appropriate epitopes be identified, as there are cases where basic has been demonstrated as a good but epitopes that are found across clues such as quantity of the agent predictor of disease prognosis, as the range of potentially diverse or agent type may be insufficient well as potential to transmit to others members of a particular species. to identify the cause. Molecular (52). Similar predictors for other Thus, epidemiologic screening of tools can be used to identify and infections are sure to follow. population-based samples of the characterize the specific virulence species of interest for the presence potential, as well as identify humans Identify potential vaccine of candidate epitopes can assist particularly susceptible. This ability candidates in selecting between potential should eventually translate into candidates by ruling out those with improved laboratory tests, making it Early microbiology led to the limited geographic distribution (56). much easier for the laboratorian to development of several vaccines Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 435 Figure 23.7. Reverse vaccinology for identification of novel vaccine antigens (55). of microbial growth can create local Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology, variations in pH, and in the presence copyright (2006). of oxygen result in the growth of biofilms that enable colonization by other infectious agents. Biofilms are complex, often polymicrobial structures formed on a variety of surfaces in the environment and human body. The scum that forms on the insides of water pipes is a biofilm, but so is plaque on teeth, and the slimy surface on the tongue, inside the nose and throat, in the vaginal cavity and on other surfaces. There are also natural synergies or anergies between organisms of the same or different species; for example, the anergy between S. pneumoniae and S. aureus colonization in the nasal cavity (59). There are also synergies, such Guide intervention strategies in their contacts (58). A better as is observed in the vaginal flora understanding of the transmission with lactobacillus modifying the pH The ability of molecular tools to of resistance genes between and enabling the growth of other detect asymptomatic infection not bacteria, and of resistant bacteria species. only increases our understanding between individuals, will aid in PCR and high-throughput of the transmission system, but also designing effective policies. New sequencing have enabled the has implications for clinical practice. therapies or combination therapies description of the complex For example, using culture, the may be introduced or steps taken to microbiota found on and in the protozoan parasite Trichomonas minimize the spread of resistance. human body. These studies use the vaginalis (T. vaginalis) is detected fact that all cells have a ribosome, in only 8–20% of male partners of Polymicrobial infections and that the sequence of genes women infected with T. vaginalis; and interactions that code for the ribosome can testing urine with PCR detects T. be used for taxonomy (61). These vaginalis in up to 70% of partners Many diseases result from infection techniques have a great advantage (57). This has profound implications not with a single infectious agent, over detection by culture, as culture for preventing the spread of this but as a result of a change in the requires at least a rough idea of common infection, suggesting that microbial community that results what organisms might be present routine PCR testing of sex partners from microbial activities (35). and their growth requirements. For is in order. Microbes have certain nutrient and example, applications of non-culture Detecting the emergence and other requirements, but as they grow techniques to microorganisms in the spread of resistance to therapy and die, they themselves serve as human gut suggest that as many should lead to changes in clinical a source of nutrients for additional as 93% of the rRNA sequences practice. Resistance genes are microorganisms. For example, identified are from uncultured often carried on the same mobile an upper respiratory infection organisms (62). Most non-culture genetic elements, so that resistance caused by a virus can enhance the techniques are based on some type to one drug often implies resistance environment for bacterial growth. of PCR and detect highly-conserved to others. Further, not only does Thus, upper respiratory infections genes, such as those coding for treating an individual with antibiotics are often a precursor to otitis media 16sRNA, which vary at the species select for resistant organisms within or bacterial pneumonia; influenza level and are semiquantitative. that host, it also increases risk of children reduces rates Others involve in situ hybridization of acquiring resistant organisms of otitis media. Further, by-products techniques, enabling both detection

436 Figure 23.8. Vaginal epithelium from a healthy premenopausal woman hybridized with a universal probe (x400) and lactobacillus probe (inset x1000). Only a small number of bacteria are scattered over the surface of intact epithelium (A). Long rods can be seen with high magnification (inset). Bacteria are found in similar concentrations on the subepithelial surface of the biopsy that was exposed by mechanical trauma of the tissue (B) (60).

of organism presence and Table 23.9. Impact of using molecular tools on the design, conduct and interpretation visualization of structure, such as of epidemiologic studies vaginal epithelium shown in Figure

23.8 (60). There is much to learn • Study design: design choice, sampling about what constitutes normal flora; the dynamics of colonization; how • Conduct: specimen collection and handling colonization varies with normal biovariations such as the menstrual • Analysis: translating laboratory measures to interpretable variables cycle, pregnancy and aging; and in • Interpretation: limitations of measures the face of antibiotic therapy and disease. conduct, analysis, and interpretation technique, while others require a Implications of using of the study results (Table 23.9). prospective design. molecular tools for the Some molecular measures are The requirements of the design, conduct and analysis relatively invariate with time, such as molecular tool also affect sampling. of epidemiologic studies human genes. Studies associating For example, if a test must be genetic susceptibility to an infectious conducted on fresh samples, the Modern molecular techniques agent might be conducted using sampling of cases and controls combined with epidemiologic genetic material collected long in a case–control study should methods allow us to identify novel before or after the disease occurred. be done so that the groups are methods of disease prevention By contrast, studies of host response sampled and tested in similar time and control, markers of disease to infection must be collected within periods to minimize potential biases diagnosis and prognosis, and a fairly tight time frame. Antibodies to resulting from assay drift, which is fertile research areas for potential an infectious agent may not appear where a method gives increasingly new therapeutics and/or vaccines. until a defined period after infection, higher or lower results with time. However, the success of these such as for HIV, or the infectious For nested case–control studies, studies depends not only on the agent may be present only for a short how specimens are collected molecular measure chosen, but duration, such as for Streptococcus may determine whether controls also on whether the strengths and agalactiae. Thus, some studies might can be sampled from the base limitations of the chosen measure be nested in large cohort studies or population (case-based, also called are considered in the design, conducted using a case–control case–cohort, sampling) or at time Unit 5 Chapter 23 Chapter Unit 5 • Chapter 23. Infectious diseases 437 of incidence disease (incidence Conclusions to form, such as pedestals on which density sampling) (63). and future challenges E. coli O157:H7 sit. The conduct of the study must Another area to explore is the take into account the requirements The applications of molecular interaction between the host and of molecular testing. Some tests tools to the study of infectious the agent. With molecular tools it are sensitive to freezing and disease are varied, including was possible to identify why some thawing and must be tested applications to public health practice, individuals are repeatedly exposed immediately, while others degrade diagnostics, and understanding to HIV but do not develop disease: over time, even if stored properly. of the transmission, evolution and these individuals have a variant in Multiple different strains of a single pathogenesis. To date, the major their CCR5 receptor that makes it infectious agent might be isolated potentials have been explored using difficult for HIV to invade the cell from one individual, such as from genomics, but applying the power (65). It is also possible to identify different body sites. Labelling of proteomics and transcriptomics human genes that explain why some should make it possible to identify to the understanding of disease individuals infected with HIV do not the appropriate strain and link it transmission and pathogenesis and progress to AIDS. For example, a back to the appropriate individual host-agent interactions will open new genome-wide association study and isolation site. Further, infectious avenues to understanding. (GWAS) identified the HCP5 gene agents may be grown in different Much remains to be learned of the HLA region in chromosome media, passed in multiple cultures about infectious agents. Some 6 (66). GWAS have been applied to that might change phenotypic future challenges are listed in Table hepatitis C virus to identify why the characteristics, or a plasmid might 23.10. One area that is particularly infection spontaneously resolves be lost. Thus, noting the number of amenable to study using molecular in some individuals and treatment times an isolate is cultured and on techniques is the normal human of chronic disease only eradicates what media is important. flora or microbiota. Extremely little infection in 40% of cases (67). Epidemiologic analyses often is known about normal human Other human genetic variants likely use simple cut-offs: e.g. diseased microbiota, its response to invasion modify risk of infection and response versus not diseased. Laboratory by pathogens, and its response to to infection, both positively and measures often are continuous, but therapeutic treatment. Disruptions of negatively, for many other infectious the scales may be ordinal, that is, normal microbiota are associated with agents. the differences between values are a variety of pathogenic syndromes, We have only begun to explore not consistent. The interpretation of such as bacterial vaginosis, that put these interactions, and many the measures might vary with the the affected host at increased risk of challenges, both technological and study population or the presence acquiring other, often more serious, methodological, remain (68). By of other ancillary information. For infectious agents. Interactions combining modern molecular tools example, >100 000 of a single between disrupted normal with epidemiologic methods, we have bacterial species in the urine of microbiota and the host may also a powerful means to understand host an asymptomatic, healthy, non- be important in explaining chronic agent interactions—an understanding pregnant individual has no clinical recurring infections. Relatively little essential for us to learn to live significance. If the individual is understood about the structures peacefully with microbes within our has symptoms referable to the formed by microbes within the human bodies, which, after all, outnumber the urinary tract, such as urgency and body; there are also structures that human cells that comprise us. frequency, the individual probably microbes stimulate the human host has a urinary tract infection (64). By contrast, >100 000 of a single Table 23.10. Future challenges in the study of infectious disease bacterial species in the urine of an asymptomatic, healthy, pregnant woman, is a treatable condition, • Normal flora because of the increased risk of • Biofilm formation pyelonephritis due to physiologic changes that occur during • Host agent interactions pregnancy. • Successive infection

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