4

Clinical and Forensic Approaches to Microbial Identification

TECHNOLOGIES FOR MICROBIAL FORENSICS

In this chapter we review both the biological and physical science techniques and methods that are commonly used or have potential for further development for use in microbial forensics. The law enforcement and legal context of microbial forensics and the challenges imposed by this context are emphasized and contrasted with clinical diagnostics in medicine.

Dr. Dana Kadavy, Senior Microbiologist at Signature Science LLC, reviewed the microbial forensic process from systems and technical perspectives, described challenges in microbial forensics compared with the needs of clinical diagnostics and public health, and reviewed technologies applicable to identification of organisms for microbial forensics. These technologies can be roughly grouped into four classes depending on their targets for detection: protein (antibody and toxin) signatures, nucleic acid signatures other than sequencing (e.g., PCR), gene sequencing, and mass spectrometry (IOM/NRC, 2014).

As previously noted, microbial forensic analyses are conducted in the context of a criminal investigation, which shapes the differences between its technologies and/or best practices and those used for clinical diagnostics and public health. The drivers for the analytical differences between microbial forensics and other sectors are legal requirements it must meet regarding (1) sample type, (2) level of characterization, and (3) interpretation and reporting. Sampling and sample types are discussed elsewhere in this report.



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4 Clinical and Forensic Approaches to Microbial Identification TECHNOLOGIES FOR MICROBIAL FORENSICS In this chapter we review both the biological and physical science techniques and methods that are commonly used or have potential for further development for use in microbial forensics. The law enforcement and legal context of microbial forensics and the challenges imposed by this context are emphasized and contrasted with clinical diagnostics in medicine. Dr. Dana Kadavy, Senior Microbiologist at Signature Science LLC, reviewed the microbial forensic process from systems and technical per- spectives, described challenges in microbial forensics compared with the needs of clinical diagnostics and public health, and reviewed technologies applicable to identification of organisms for microbial forensics. These technologies can be roughly grouped into four classes depending on their targets for detection: protein (antibody and toxin) signatures, nucleic acid signatures other than sequencing (e.g., PCR), gene sequencing, and mass spectrometry (IOM/NRC, 2014). As previously noted, microbial forensic analyses are conducted in the context of a criminal investigation, which shapes the differences between its technologies and/or best practices and those used for clinical diagnos- tics and public health. The drivers for the analytical differences between microbial forensics and other sectors are legal requirements it must meet regarding (1) sample type, (2) level of characterization, and (3) interpreta- tion and reporting. Sampling and sample types are discussed elsewhere in this report. 79

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80 SCIENCE NEEDS FOR MICROBIAL FORENSICS In addition, the level of characterization and reporting requirements are specific for the microbial forensics “space.” Some are shared with clinical diagnostic practices and food analysis systems, and Dr. Kadavy suggested that the two sectors should leverage one another. At the same time, microbial forensics must drill very deep for pathogen identification. Analysis extends to strain, subtype, or isolate; type and/or abundance of organism present in simple to complex samples; the presence of antibiotic resistance and virulence genes; evidence of genetic engineering and/or isolate evolution (is it endemic wild type or a cultured strain repeatedly passed around labs?); and in-depth, sample-to-sample comparison that may be informed by SNPs. When reporting to a court,1 investigators must have confidence in their results and understand the power and limitations of the procedures they have used, which can only be gained through accumulated experi- ence in testing and validation. Investigators also must consider reason- able alternative explanations. Moreover, they must provide known error rates and detection limits when possible for a judicial audience that is unlikely to include scientists. These factors inform the choice of analyti- cal technologies to be used. Traditional microbiology and culture are the current gold standard in clinical diagnostics, but it is not always possible or practical to culture microbes in a forensic investigation. A capability needed in microbial forensics is sample processing to achieve desired sensitivity/accuracy in complex sample matrices, such as soil samples from forensic exhumations of graves linked to the Bosnian– Serbian war. Sensitive nucleic acid–based and antibody-based detection technologies also are needed. Although these diagnostic assays may not provide in-depth information, they potentially offer rapid, inexpensive, high-throughput screening to rule in or rule out various samples. Inves- tigators and responders need to know quickly what kind of threat they face; additional in-depth analysis can be performed later. Traditional microbiology methods and features do have forensic value. These methods include culture, phage sensitivity, staining, and microscopy (all used in the U.S. anthrax letters case), as well as fatty acid analysis and serotyping. It is not always possible or practical, however, to use these on complex, degraded, and/or inactivated samples, or for isolates or mixtures that cannot be cultured. During the 1980s and 1990s, antibody-based techniques dominated biological agent detection. The “backbone” of antibody-based identifica- tion relies on the ability of the immune system to recognize “non-self” components of pathogens, especially antigenic proteins and polysac- 1  Before trial, experts must prepare reports summarizing their analysis and conclusions, and share the reports with all other parties.

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CLINICAL AND FORENSIC APPROACHES 81 charides (Schaudies, 2014). Most antibody-based identification methods (such as enzyme-linked immunosorbent assay, or ELISA) involve immobi- lizing antibodies for particular antigenic targets on solid substrates. These “capture” antibodies then bind to the antigens produced by the micro- organismal cells. This binding is then detected with a second antibody to the same antigen that contains a “reporter molecule” that produces a detectable signal based on optical absorbance or fluorescence. Examples of antibody-based platforms include Luminex, PathSensors, Inc., and TacBioHawk. (See Schaudies, 2014, for a more in-depth review.) The most notable advantage of antibody- or protein-based detection systems is their speed—many of them can provide answers in 3 to 10 minutes. Some of the systems can reach sensitivities approaching that of nucleic acid amplifica- tion techniques, such as PCR-based assays. However, for microbial foren- sic purposes, they often lack sufficient specificity to discriminate beyond the level of species. Platforms that perform protein and antibody/antigen detection, such as electrochemiluminescence (ECL), are also needed to detect protein toxins, such as ricin and botulinum. Schaudies (2014:175) states that antibody-based systems are “more effective and consistent than nucleic-acid-based systems for the detection and identification of toxins.” This statement is based not on efficacy of an assay, but more on the degree of toxin purification; in some toxin preparations, the DNA that codes for production of the toxin may not be present or nucleic acid concentra- tions may be reduced below limits of detection. Toxin treatment also may degrade DNA from the sample. As with PCR, these platforms are only as good as the assays developed for the intended target. Knowledge of the target being pursued is necessary as are thoughtfully designed and validated assays to use ECL effectively. Nucleic acid–based detection and identification technologies pro- vide the ability to examine the genetic as well as structural information associated with a pathogen. Among the early tools developed to access an organism’s genetic information were nucleic acid amplification tech- niques, such as PCR. PCR allows specific fragments of genomic DNA to be isolated and their copy number amplified. Over time, variations of the basic PCR technique have developed, such as “multiplex PCR,” which allows several DNA genomic regions to be amplified in the same reac- tion set, and quantitative or “real-time” PCR (qPCR), which can measure the quantity of a target sequence in real time. Multiplex PCR technolo- gies do have limitations. Most allow up to five different fluorophores to be detected simultaneously. Other platforms have attempted to increase this to as many as 20 fluorophores simultaneously, which would be a big advance in multiplex capability. Real-time qPCR is a rapid and sensi- tive nucleic acid signature detection technology that has proved effec- tive in both clinical and microbial forensic applications. Some platforms

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82 SCIENCE NEEDS FOR MICROBIAL FORENSICS have a closed system, which may be fine for some situations. However, a closed system may not be optimal if an open-architecture system is needed to port novel assays to determine genetic elements more fully or to screen quickly for elements that are most likely of diagnostic value in isolate characterization. The power of qPCR lies with assay selection, some multiplex capability, optimization, and validation of the PCR assays themselves. There is a wealth of available PCR assays that are validated to certain standards, although they must be validated again in each laboratory that implements the assay(s). Many are Food and Drug Administration (FDA) approved and enable qualitative detection of hundreds of pathogens and hospital-acquired infections to the level of species and sometimes strain. Qualitative tests can provide solid evidence for determining the next step of an analysis as well as generating a quick answer. Some detect antimicrobial resistance genes. There are also assays in the food safety arena to draw upon. Matrices in the food industry are complex, and food is a common target of intentional and accidental contamination. There are many food industry investigations about such events, and microbial forensics should learn from these, understand the assays, and have access to the assays should a food-related microbial forensic investigation arise. There are fewer assays for animal and plant pathogens, yet they are important, and the microbial forensics community can communicate their importance. The limitation of PCR is that the list of what can be detected is con- tingent on the validated assays in one’s toolbox. Even if one has many assays, it is still limiting and restricts one’s multiplexed capability. The MassTag PCR technology marries PCR with traditional chemistry tech- niques (mass spectrometry) to provide the resolution quality of mass spectrometry coupled with the sensitivity and specificity of PCR. It has, for example, revolutionized the ability to identify respiratory pathogens and hemorrhagic fever viruses. Mass spectrometry also is useful in identifying many non–nucleic acid chemical species that may provide clues to microbial identity, ori- gins, and production processes. Proteins, peptides, lipids, carbohydrates, inorganic metals, and organic metabolites may provide information about an organism’s source environment, how it was produced, and the level of sophistication of the preparation (Wahl et al., 2011). For example, micro- organisms respond to the conditions in their environment by altering which of their genes are transcribed and then translated into proteins. For pathogens isolated from samples other than patients, the profile of proteins an organism produces can sometimes provide valuable informa- tion about the environment in which the organism originated. A mass spectrometer generally consists of an inlet for introducing the sample,

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CLINICAL AND FORENSIC APPROACHES 83 an ionization source to turn the analyte into charged gas-phase particles (ions), and an analyzer that detects and separates the charged particles based on their mass-to-charge ratio. Two ionization methods are used for biological mass spectrometry: electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). Wahl and colleagues (2011:456-457) point out that although nucleic acid sequencing is invaluable in micro- bial forensics, proteins “may offer improved stability over DNA markers . . . and have fewer or different inhibitors to analysis methods.” It is also useful to have additional detection modalities. More in-depth reviews of mass spectrometry and its role in microbial forensics are available in Wahl et al. (2011) and Snyder and Jabbour (2014). Dr. Jongsik Chun of Seoul National University pointed out that in Korea, use of MALDI—specifically the MALDI–time of flight (MALDI-TOF)2—is becoming very popular in routine clinical laboratory analyses. The poten- tial of this technique exceeds most techniques discussed by Kadavy except for sequencing. Kadavy agreed that the MALDI-TOF technology is a very good option although it does not have capacity equal to sequencing. The CDC is using MALDI-TOF to identify the protein toxins of B. anthracis and Clostridium botulinum (Boyer et al., 2011; Kalb et al., 2011). The CDC’s tests are used both for assays for the presence of the toxins and for identifying the correct botulinum neurotoxin subtype for administration of the most effective therapeutic immunoglobulins to affected patients. Microarray platforms provide similar capabilities but in a more mul- tiplexed format. They have the ability to detect both nucleic acid and pro- tein signatures. Many panels have 1 million assays on a single array chip; some have up to 5 million assays. There are custom and standard micro- array panels. Microarray panels are available for microbial detection, SNP detection, and genome-wide association studies, gene expression, and protein presence and abundance. Laboratories that use a microarray reader for other purposes, such as oncology or infectious disease screen- ing, could be leveraged by the development of custom chips that are directed to answering microbial forensic questions. Most assays are avail- able; it is a matter of customizing a single chip to combine them. Micro- arrays will not achieve the level of information provided by sequencing, but they are quite impressive. They may be a rapid and cost-effective alternative or supplement to sequencing. For microbial forensics purposes, NGS is a powerful set of technolo- 2  Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spec- trometry can establish the identity, purity, or other aspects of protein samples, genetic materials, and microorganisms. MALDI-TOF uses the time for a target particle to traverse a specific distance after being dislodged from a surface by a precise amount of energy. This allows a determination of the molecular weight of the target. http://www.ndif.org/public/ terms/11543-MALDITOF_mass_spectrometry.

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84 SCIENCE NEEDS FOR MICROBIAL FORENSICS gies that offers a state-of-the-art approach. However, NGS also poses a new set of issues for implementation. For example, the same data run on two different systems—the benchtop MPS (massively parallel sequencing) instruments, Personal Genome Machine™ or PGM (or Ion Torrent, Life Technologies) and the MiSeq (Illumina)—can produce somewhat different answers. These issues will have to be resolved or at least understood for the microbial forensics context. Detter and Resnick (2014) provide a brief review of NGS. In general, sequencing consists of four steps: preparation of the sample by nucleic acid (either DNA or RNA) extraction, library preparation, sequencing, and data analysis. Following extraction of the material from the sample as described above and in Chapter 5, the DNA molecules to be sequenced must be converted into sequencing “libraries.” This preparation entails fragmenting the DNA and then adding adapter molecules to the ends of the fragments to facilitate sequencing. Each DNA fragment in a library is then clonally amplified before sequencing. The precise process and how automated it is depends on the NGS platform being used (Detter and Resnick, 2014). NGS produces huge amounts of data as “reads,” which are strings of DNA that correspond to the sequence of the original DNA or RNA being investigated. Reads have three important features: length, number (or “coverage”), and quality. All of these differ according to which platform and sequencing kit are used as well as the quality of the sample and library preparation. Kadavy discussed three platforms that were described in a 2012 article that compared the three, head-to-head, using real data (Quail et al., 2012). However, new improved instruments come out frequently and displace those already on the market. The new high-throughput sequencing methods have greatly improved the overall process. Some novel approaches beyond these platforms incorporate single-molecule real time, or “smart cell,” and also preparation adaption-ligation strate- gies; alternative chemistries, and so, less RNA/DNA input is required; and longer read lengths. These decrease the time to results. The approxi- mate sequencing capacity of thousands of bases per day of 1995 has been increased to billions per instrument per day in 2013. All sequencing-platform workflows share general themes— fragmenting DNA, various adaptor-ligation strategies, and novel chemistries—and efforts are under way to reduce preparation time and sample input requirements and improve the quality of results. The CDC, the College of American Pathologists, and others have developed guide- lines for its use with human diagnostics (Ellard et al., 2012; Gargis et al., 2012; Pont-Kingdon et al., 2012; Rehm et al., 2013). But these guidelines do not address the range of possibilities encountered in microbial forensics, that is, from the homogeneous sample to the very complex metagenomic

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CLINICAL AND FORENSIC APPROACHES 85 or microbiome samples. Sequencing remains a huge investment in time, materials, and money and also demands a huge investment in data analy- sis because none of the data are meaningful without bioinformatics, and bioinformatics and software issues must be factored in (see Chapter 7). Bioinformatics pipelines that can handle a continuum of samples from pure isolates to complicated metagenomic samples that may contain only trace amounts of the threat agent are needed. Kadavy stated that it is necessary to be prepared for all possibilities, so building on the current technologies to meet future needs must continue. In terms of bioinformatics, Kadavy stressed that accurate and updated genomic databases are critical. The dialogue has only just begun on how to share such data. Bioinformatics tools must be usable and practical. They must be applicable to the questions posed and validated for forensic application. A challenge is that in a field that is evolving so quickly, how can methods that are themselves continually evolving be validated in a timely fashion and in an economically feasible manner? Kadavy suggested that investments in technology should be pri- oritized according to what is best for the common good. It is possible to leverage advancement that helps microbial forensics and public health simultaneously. These fields should not compete for resources, but should share them to meet their respective goals. The issue of proprietary restrictions that make some technologies inaccessible was raised by Dr. Raymond Lin. For example, there are good publications on MassTag spectrometry, but many of the technologies are proprietary. Consequently investigators have had to develop their own panels, and results have not been good enough for application. Published microarrays are often proprietary, so other groups have had to develop their own chips. One group in Singapore has licensed arrays for about 70,000 pathogens, but they tend to be undersensitive, and do not perform as well on respiratory pathogens. Lin believes that the future may be in using one of the NGS instruments that is less biased as far as target analy- sis and target enriched with certain non-eukaryotic sequences. Kadavy agreed that the proprietary issues are an important consid- eration. It is also important to know what technologies people are using. How does the MALDI-TOF operate in one’s own hands? To what do other countries have access? What technologies should one be considering? She suggested the workshop consider how to link up experts—in microbiology, clinical medicine, public health, research and diagnostics, and food science—in order to leverage and build on knowledge and for reach-back purposes. She noted that small laboratories and research groups can leverage existing infrastructure and expertise in public health systems. How can information, including sequencing data, be shared in

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86 SCIENCE NEEDS FOR MICROBIAL FORENSICS the international microbial forensics community? This will require policy decisions. Dr. Munirul Alam raised the problem of sharing data in areas where limited resources restrict technologies. Bangladesh is part of PulseNet Asia Pacific (part of PulseNet International3), which enables access to a database for threat pathogens. He asserted that labs in developing coun- tries cannot afford everything, so it is important to collaborate as much as possible. Dr. Ruifu Yang agreed that sharing is key for developing microbial forensics. He has collaborated with others to standardize methods, using the same data but analyzing them in different laboratories, and then pub- lishing the results. If scientists work together, they can share more than just published data. Piers Millet of the U.N. Office for Disarmament Affairs pointed out the potential of the technology and international collaborative arrange- ments that enable sharing sequences and related data via the Internet. He cited the example of the rapid response to the posting of the viral sequences of the H7N9 avian influenza by the Global Initiative on Shar- ing Avian Influenza Data (GISAID) by the Chinese CDC in March 2013.4 This process enabled Novartis, in partnership with a unit of the J. Craig Venter Institute and support from the U.S. CDC, to synthesize the genes of the new virus and begin initial work on developing a vaccine using its cell-culture production process, which is itself much faster than tradi- tional egg-based flu vaccine development (Brennan, 2013). The synthesis was completed and shared with CDC before physical samples of the virus arrived by mail. Dr. Gilles Vergnaud, Institut de Génétique et Microbiologie, France, works for the French Ministry of Defense, Defense Procurement (DGA). In 2001, he headed the department of dangerous biological agents in the DGA. Following the U.S. anthrax event, many countries experienced thousands of threat hoaxes. In France alone, there have been 3,000; the defense establishment dealt with most, and his division undertook about 1,500. They needed an easy way to determine which incidents were hoaxes and which, if any, were real threats. A hoax is massively disruptive; many involved hospitals, post offices, and other public spaces. In every case, evidence was processed as quickly as possible to differentiate hoaxes 3  PulseNet International is a network of national and regional laboratories that track foodborne infections worldwide. Each laboratory uses standardized genotyping methods and shares information in real time More information available at http://www.pulsenet international.org/. See also Global Food Safety, http://www.slideshare.net/Adrienna/ global-food-safety2013. 4  Further information about GISAID can be found at http://platform.gisaid.org/epi3/ frontend#283e41; accessed March 31, 2014.

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CLINICAL AND FORENSIC APPROACHES 87 from real threat agents, although even this simple question requires a confident answer. He went on to say that he has long had an interest in database issues, but there are many political problems. He believes it will never be possible to share all forensic-quality data, but some level of slightly degraded information may be shared, and he believes that even that would be a great achievement. Dr. Rocco Casagrande of Gryphon Scientific added a cautionary note by suggesting that a guiding principle when developing new tech- nology should be to consider the consequences of its day-to-day use. Better surveillance capabilities and better technologies for identifying unusual microorganisms can result in “events” that otherwise would not be interpreted as such. Had it been possible, for example, to detect Aum Shinrikyo’s release of anthrax spores in Japan in 1993, there might have been an “event” even though there were no public health consequences. Similarly, in the United States, when grapes imported from Chile were allegedly contaminated with cyanide (Rushing, 1994), the cyanide was insufficient to cause human health concerns, but the detection caused sig- nificant economic harm to the exporter. John Clements further noted that it is important to distinguish between meaningful events and background “noise.” For instance, there are approximately seven cases of plague due to Y. pestis diagnosed in the United States every year. Although a sharp increase in number or distribution may be important, the mere existence of the disease is “background.” Also, in the Aum Shinrikyo anthrax case, cult members failed to spread the disease because they used the nonviru- lent vaccine strain of the organism. Use of an insufficiently discriminatory assay would have picked up B. anthracis and may have started a full-scale panic even though the public was at no great risk. There are consequences for public safety, the economy, and individuals for making declarative statements about things that are merely suggestive. INVESTIGATING INFECTIOUS DISEASES IN CLINICAL MEDICINE Professor Alemka Markotić shared her perspective as a scientist and a clinician who daily faces a variety of infectious diseases in patients and has used elements of microbial forensics to deal with public health outbreaks. She offered lessons learned through experience with zoonotic diseases—diseases transmitted between animal species and from animals to humans—which she believes could be useful to public health experts, clinicians, and microbial forensic investigators. She pointed out natural vulnerabilities that terrorists could exploit, and proposed areas where research is needed. Zoonotics can cause severe disease both in animals and humans and

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88 SCIENCE NEEDS FOR MICROBIAL FORENSICS therefore constitute a serious public health problem. Animals and their vectors play an essential role in maintaining zoonotic infections in nature. Zoonoses may be due to bacterial, viral, or parasitic organisms or may involve unconventional agents. In addition to being a public health prob- lem, many of the major zoonotic diseases (e.g., brucellosis, salmonellosis, listeriosis, trichinellosis, campylobacteriosis, and hepatitis A or E) prevent the efficient production of food of animal origin and create obstacles to the international trade of animal products (World Health Organization, 2014). Markotić noted that many of the agents regarded as potential bio- weapons are zoonotic. Preparing for a biological threat event requires knowledge of patho- genic agents; a clinical picture of disease incubation, transmission mode, and treatment; knowledge of which molecular diagnostics and technolo- gies to apply to which pathogen; and characterization of a usual versus an unusual outbreak (see Box 3-1). But Markotić stressed that in real life, the picture is not always clear. Hantaviruses are transmitted to humans through contact with hanta- virus-infected rodents or their urine and droppings. Infection with Old World hantaviruses can progress to hemorrhagic fever with renal syn- drome (HFRS), and infection with New World viruses to hantavirus pul- monary syndrome (HPS). The clinical picture for hantavirus ranges from mild illness to a severe form with fulminant hemorrhagic fever. Mortality can be as high as 20 percent with Old World hantaviruses and 60 percent with New World hantaviruses. Croatia is a natural center for many rodentborne zoonoses (e.g., lep- tospirosis, babesiosis, and HFRS) because of to its diverse forest ecology and abundance of small rodents (Markotić et al., 2002a; Tadin et al., 2012). In January through April 2012, 33 patients were hospitalized in Zagreb, experiencing something that resembled flu but did not exhibit its typical symptoms (Tadin et al., 2014). This outbreak occurred against the back- drop of a normal flu season, including cases of influenza A subtype H1N1. However, it was subsequently determined that all patients had either attended the “Snow Queen Trophy” skiing competition at Medvednica Mountain, a popular ski resort, or lived near or had links to the mountain. Acting on their suspicions, Markotić and her colleagues performed point- of-care tests and found that all patients were serologically positive for specific antibodies to Puumala virus, a hantavirus (Markotić et al., 2002b). There are three lessons to be learned from this event. First, a simple clinical test can quickly distinguish between influenza and HFRS. Results were received within hours of patient admission. Such tests can be a pow- erful tool for providing orientation in the field at the onset of an unusual outbreak. This outbreak comprised an extremely high number of HFRS cases

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CLINICAL AND FORENSIC APPROACHES 89 within a small geographical area. Moreover, the timing was unusual because HFRS cases typically peak during the summer months. The patients’ clinical symptoms were first compared with those of previously published cases and there were no differences. To confirm the origin of the infection, both human and rodent samples were analyzed. Through collaboration with the forestry and veterinary medicine sectors, rodent samples from different altitudes of the mountain were collected. Markotić and colleagues discovered that almost 80 percent of rodents at the altitudes of the mountain’s ski track were infected with hantaviruses—a rate never reported previously in Croatia or in the litera- ture. Molecular and phylogenetic analyses of viral nucleic acid sequences obtained from human and rodent samples confirmed an almost 100 per- cent similarity between the samples, which helped rule out the virus having been imported or deliberately created. The second major lesson learned was that molecular analysis enabled linking the human outbreak with a pathogen in a natural reservoir, rodents, in a specific geographic location. A major contributor to the dispersal of hantaviruses in the Medvednica Mountain outbreak was that, because of warm winter temperatures, the ski resort had distributed aerosolized artificial snow to prepare the snow track for the competition. Moreover, high winds blew the snow off the track. A third major lesson came from an unusual outbreak of HFRS cases that began in a community of former drug users whose rehabilitation program included gardening near a forest (Medved et al., 2002). Many community members became infected with hantaviruses and developed HFRS, and one died. Fifty-two percent of all community members were infected with hepatitis B or C, or both. Of those with HFRS, 82 percent were co-infected with one or both types of hepatitis virus (Medved et al., 2002). It appeared that the multiple infections were associated with altered immune responses. Markotić suggested that there is a need to consider whether multiple infections pose an investigative problem, both in terms of public health and microbial forensics. Markotić posed the questions, “What if mother nature can do it better? And what if terrorists copy it?” She and her colleagues, using an existing bank of rodent samples, investigated what pathogens rodents in northern Croatia carry. Their research revealed that the rodents typically carry one to four zoonotic infections (Tadin et al., 2012). In light of this fact, one must consider the public health implications of multiple infections. When physicians diagnose a patient with one microorganism, they tend to be satisfied with that diagnosis, and treat the patient accordingly. Even if we record unusual immunopathogenic responses (e.g., strong inflammatory response or immune suppression) in patients, the possibility of multiple infections is not typically considered. Co-infection and multiple infections

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90 SCIENCE NEEDS FOR MICROBIAL FORENSICS should be the target of public health research. The literature shows that more severe disease occurs in the presence of co-infection (e.g., influenza with pneumonia), as well as higher mortality (Chertow and Memoli, 2013). Bacterial co-infection has occurred in influenza pandemics through- out the world, including those with H1N1 influenza (Wang et al., 2011). In the event of co-infection or multiple infections, there exists a different immunopathogenic basis for disease (Guidi et al., 2011). Immune response is altered in the presence of more than one infec- tious disease. Markotić’s research shows the level of proinflammatory cytokines to be several times higher in patients infected with both hanta- viruses and leptospira than in patients with a single infection (Markotić et al., 2002a). A study examining the interacting roles of cytokines and viral load in Crimean-Congo hemorrhagic fever showed that viral load and immune response parameters can be useful as biomarkers to predict disease severity and outcomes (Saksida et al., 2010). Therefore, in addition to using multiplex technology to detect co-infection or multiple infections, the capability to measure immune response in unusual outbreaks or dis- ease presentations should be developed. An important “omics” research field that holds great promise for both clinical medicine and microbial forensics is immunogenomics, an emerg- ing field that focuses on the intersection between genomics and immu- nology. It takes into consideration the host and pathogen proteomes, and combines bioinformatics technology, genomics, proteomics, immunology, and clinical medicine (Gupta et al., 2009). Professor Indrani Karunasagar of the Karnataka Veterinary, Animal & Fisheries Sciences University of India, commented that a syndrome may be precipitated by a certain fac- tor, but the condition is exacerbated by the presence of bacteria. Perhaps addressing changes in climate or other environmental factors might be used for prevention. She agreed that immunogenomic bioinformatics is very important. It might answer, for example, why when two people are exposed to the same situation, one becomes infected and the other does not. The genetic makeup of an individual could aid in addressing possible contributing environmental factors. Markotić emphasized that there are very powerful simple techniques. Flow cytometry, a well-known cell sorting tool, can sometimes quickly orient one to whether a pathogen is a virus or a bacterium when the clini- cal picture and diagnostic results are not clearly distinctive. At the Zagreb hospital where Markotić practices, they have used this technology to dif- ferentiate HFRS from leptospirosis (Markotić et al., 2011). To improve clinical and forensic approaches to microbial identifica- tion, Markotić believes interdisciplinary approaches and advanced tech- nology are needed, specifically:

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CLINICAL AND FORENSIC APPROACHES 91 1. Molecular epidemiology data and field research. 2. More extensive clinical databases, for example, descriptions of “usual” and “unusual” cases and outbreaks. 3. A translational medicine approach to link basic and applied research to clinical data and diagnostic tool development and a One Health medicine approach to zoonotic diseases. 4. Development of robust, powerful, multiplex molecular diagnostic tools and bioinformatic analysis tools. 5. Development of rapid and inexpensive diagnostic tests. 6. Powerful immune-response measurement and bioinformatics analysis tools. 7. The ability to understand and synthesize a huge amount of data, and react promptly and properly. 8. The ability to quickly communicate (e.g., via telemedicine) with world experts knowledgeable about the clinical manifestation and detection of dangerous pathogens. In a final note on zoonotics, it was pointed out that the National Cen- ter for Foreign Animal and Zoonotic Disease Defense (FAZD, http://fazd. tamu.edu/) at Texas A&M University in the United States has a program in which they are trying to integrate data and information from clinical and field observations provided by any stakeholders—from pet owners to production/processing facilities. Participants can electronically enter observations (e.g., presentation, diagnostics) into the system. Normally, there is a problem with unwillingness of people to share such information because any indication of a serious zoonotic situation can have significant financial ramifications. FAZD provides a shield for reporting this informa- tion and they are assimilating the data at a high level, looking for multiple loci or anything that will give them information about natural outbreaks, deliberate attacks, or other sources. CULTURE-INDEPENDENT TESTS: CLINICAL AND PUBLIC HEALTH PERSPECTIVES Dr. Stephen Morse of the U.S. CDC spoke about how rapid develop- ments in technology and clinical laboratory test methodologies are lead- ing to culture-independent testing. He discussed the advantages and limi- tations of culture-independent tests from the clinical and public health perspectives, noting that his comments should not be assumed to reflect CDC policies. In the case of a large outbreak of an infectious disease, par- ticularly an enteric infection, careful epidemiological investigation will likely be needed to differentiate between an intentional and a naturally occurring event.

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92 SCIENCE NEEDS FOR MICROBIAL FORENSICS Currently public health surveillance is isolate-based, but this approach is changing. For example, PulseNet, which was founded in 1996, is the molecular subtyping laboratory network for conducting foodborne dis- ease surveillance. It relies on the availability of isolates for testing using pulse field gel electrophoresis. This culture-based diagnostic and typing method is, however, likely to be replaced by more rapid PCR-based tests (CDC, 2011). A move toward rapid nonculture testing is expected to improve the speed and cost-effectiveness of diagnostics. However, it also likely will result in a decrease in the availability of isolates for PulseNet subtyping and susceptibility testing. The issue of how to adapt to rap- idly changing technology is a major one for the U.S. CDC. CDC and its partners in the State Public Health Laboratory network are working to develop higher resolution diagnostics, new rapid and standardized data collection methods, new analytical software, and improved environmen- tal assessments (CDC, 2011). President Obama’s FY 2014 budget proposed the establishment of the advanced molecular detection (AMD) program to begin to replace CDC’s decades-old methods of detecting microbes, which will soon be obsolete, as well as increasing understanding of their lethality to humans.5 In the United States, public health surveillance has a number of func- tions (Cronquist et al., 2012). Surveillance assists in individual case man- agement and investigation at the local level. It is used to assess disease burden and trends in order to prioritize and assess the impact of popu- lation-based control methods. It is relied upon for outbreak detection to protect the population, as well as to identify gaps in control measures. Isolates collected for public health purposes undergo characterization to improve understanding of pathogens and their virulence mechanisms and to assess infection epidemiology. The needs for testing in patient management differ from those in pub- lic health. For patients, an ideal test is fast, accurate and, when necessary, can establish antimicrobial susceptibility. In the case of most infections, a patient will be treated with an antibiotic before the test results arrive, and subtyping or virulence testing is seldom used. As we understand more about the genetics of virulence factors, however, such testing may play a greater role. In contrast, population-based testing values specificity and sensitiv- ity over speed; the goal is to ensure that outbreak-associated cases are identified unambiguously. The public health sector requires more detailed information on isolates, and subtyping becomes important because it enables detection of outbreaks due to particular strains. In this context, 5  More information on AMD can be found at http://www.cdc.gov/amd/; accessed April 7, 2014.

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CLINICAL AND FORENSIC APPROACHES 93 monitoring antimicrobial susceptibility is particularly important for two reasons. First, antibiotic-resistant organisms are associated with more severe disease. Second, livestock and poultry are commonly treated with antibiotics to enhance their growth, which selects for antibiotic-resistant organisms. Monitoring can aid in attribution, particularly in foodborne outbreaks in which antibiotic-resistant organisms are associated with meat and poultry. From a clinical perspective, the advantages of culture-independent testing to patients include more rapid diagnoses, the potential for easier specimen collection and decreased costs, and the detection of a wider range of pathogens (e.g., non-O157 Shiga toxin–producing E. coli) as well as better sensitivity for some pathogens, such as those that are difficult to culture. Challenges include false-positive findings that result in unneces- sary treatment or incorrect diagnosis, and an inability to test for antimi- crobial susceptibility (Cronquist et al., 2012). Specifically, the presence of a genetic sequence coding for a factor associated with antimicrobial resistance (e.g., an enzyme) does not mean that the gene is expressed or that the microbe is resistant, only that the associated genetic sequences are present. From a public health/population-level perspective, the advantages of culture-independent testing include rapid case detection; a potential for increased testing, leading to greater case capture; detection of a wider range of pathogens; and again, better sensitivity for some pathogens (Cronquist et al., 2012). Limitations can include resources and time wasted following up noncases, incorrect or unstable estimates of the number of illnesses, and disruption of trend monitoring. Other challenges are the loss of subtyping for outbreak detection, the possible investigation of pseudo-outbreaks, and a decreased ability to monitor trends in subtypes,6 such as Salmonella enteritidis (Cronquist et al., 2012). PERSPECTIVE OF A CLINICAL MICROBIOLOGIST IN THE PUBLIC HEALTH SECTOR Dr. Raymond Lin of the National University of Singapore offered a perspective on the challenges and issues clinical microbiologists who work in the public health sector face in terms of technology, policy, and legal issues. 6  Infectious disease outbreaks are currently monitored by culturing organisms from pa- tients or contacts, identifying those organisms by serology, biochemistry, fermentation pat- terns, and antimicrobial susceptibility. Changing to a nonculture system would not provide sufficient continuity to allow us to continue monitoring infectious disease trends (down to the subtype level of the microorganisms) that have been established over the last 100 years using the aforementioned technologies.

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94 SCIENCE NEEDS FOR MICROBIAL FORENSICS New technologies, such as the MALDI-TOF mass spectrometry tech- nique, have brought great change into clinical microbiology and public health labs because they enable earlier pathogen identification. However, as with any technology, limitations exist. Currently MALDI-TOF cannot always provide needed differentiation because some important public health pathogens are genomically very similar to nonpathogenic species. For example, B. anthracis may not be easily differentiated from B. cereus or B. thuringiensis, so extra testing may be required. Dr. Lin pointed out that public communications about the meaning of data generated by the new technologies can also present a major prob- lem. The media and the public typically do not understand the science underpinning a report of “a new pathogen,” “a new chimera,” or “a new mutation.” They assume the worst and fear there is a serious danger to public health, whether there is or not. Better communication could pre- vent unnecessary disruptions and delays. In public health, the needs for gene typing are relatively simple. Most investigations focus on outbreaks, and seek clustering over a short period of time to provide leads for further epidemiological investigation. Most commonly, action needs to be taken to address the most likely cause of an outbreak based on the epidemiology, with or without laboratory results. In his experience with foodborne outbreaks, one or two people with diar- rhea meeting the case definition may prove to have an unrelated pathogen or unrelated strain. Usually it is not difficult to piece the story together, but a capacity that is very much needed is a database of the baseline types of the worst pathogens—even something as common as Salmonella. Certain species are so common that adequate baseline databases are not available to public health authorities to help identify how different a cer- tain isolate may be from what is known to be in the community. Lin also believes that a problem with typing/sequencing capability is that it may encourage litigation. He asks if, in foodborne outbreaks, whether action should be taken against the food vendor? He tries to avoid being drawn into cases alleging, for example, that one person trans- mitted HIV to another person. The courts ask many questions, and Lin believes that public health lacks the manpower to spend time in court for such cases. Public health investigators are obligated, however, to be involved when commercial products are suspect in pathogen transmis- sion. In Singapore, there are many imported disease cases, and these must be resolved. Were a “local” transmission of cholera to rise above a certain threshold, for example, the country would lose its cholera-free status. Similarly, people who come from malaria-endemic countries may be infected by more than one strain of Plasmodium, be fairly asymptom- atic, but still transmit the infection(s), possibly with antigenic variations. Lin would like to see studies using benchtop NGS instruments. Much

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CLINICAL AND FORENSIC APPROACHES 95 of the literature has focused on the large expensive machines. The PGM (Ion Torrent, Life Technologies) and MiSeq (Illumina), both benchtop machines, are not used much in public health in Singapore. He agrees that there is a need to build databases and to standardize how to report the data internationally. How does one communicate in plain language so that the implications of an analysis with MLST+, for example, are understandable? Lin agreed with Morse in his concern that viral isolation has become almost a thing of the past in diagnostic labs. He believes that public health labs must make an effort to collect speci- mens of bacteria and viruses. Even something as common as investigating foodborne outbreaks is still done by culture, but Lin thinks that with an increasing move toward multiplexed PCR, public health will have to take over the responsibility of collecting isolates so that long-term bacterial and viral archives can be created and maintained.

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