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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics 8 FINDINGS AND CONCLUSIONS: INITIAL PRIORITIZED SCIENCE NEEDS FOR MICROBIAL FORENSICS The speakers and participants at the Zagreb workshop represent a collection of expertise from many disciplines, such as microbial forensics, epidemiology, public health, clinical medicine, genetics and genomics, phylogenetic analysis, and bioinformatics. Despite the great diversity of backgrounds, there were clear themes that emerged from the presentations and discussions. Most of these themes are consistent with the current literature in the fields represented and with the views of the committee members. In this chapter, the committee synthesizes its views on the needs that emerged and the questions that require answers. BASIC SCIENCE As discussed in Chapter 2, although the world of living things is dominated by microorganisms, very little is known about the vast majority of microbes. Until recently, much of what is known was based on the very few microorganisms that are culturable and that were studied in laboratories using growth characteristics, Gram stains, serology, and other traditional techniques. Today, however, the ability to sequence the genomes of microbes has provided a great deal of knowledge about nutrient cycling, gene regulation, and reproduction for some bacteria and viruses. These processes, however, remain unknown for most microorganisms and “knowledge of the evolution and ecology of microbial communities lags far behind cellular microbiology” (NRC, 2007). In addition, until recently there have been few systematic efforts to collect and describe the microbes living in soil, seawater, freshwater lakes and streams, on plants, and even commensally in the guts or other surfaces of humans and other animals. The new techniques of metagenomics circumvent the inability to culture the majority of microbes by directly assaying microbial genes, sequences, and genomes from entire communities, such as those available from environmental samples and the human microbiome. The use of metagenomic techniques, however, is still an area of active development. Meanwhile, knowledge of the natural microbial communities residing throughout the world is still highly incomplete. Although the biology of the small fraction of bacteria and viruses that are pathogenic to human beings, livestock and companion animals, and crop or forestry plants is somewhat better known and understood, there is still much to be learned about their phylogeny and evolution, how many strains of pathogenic species exist in nature, what their distribution is throughout the world, and how this distribution interacts with ecological conditions. Finding: For microbial forensics purposes, the dearth of information about microbial diversity, ecology, population genetics, evolution and phylogeny, and worldwide distribution represents a major scientific   Prepublication Copy 110 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics knowledge gap. Determining an organism’s source in the event of a biothreat will be greatly hampered by the absence of baseline information on the natural abundance and distribution of pathogens. If the normal distribution of a pathogen in background settings is unknown, it would be difficult, for example, to discern whether there is an unusually high abundance of that pathogen in an area experiencing an infectious disease outbreak. Background knowledge is also important in determining whether the presence of that pathogen is natural or the result of a deliberate or inadvertent release. Understanding the endemic microbial background is necessary to provide proper context for microbial forensics analyses, interpretations, communication, and resulting decision making. Currently there is insufficient understanding of microbial diversity and endemism to facilitate determining whether a pathogen has been intentionally released, where and how a biothreat agent was developed, and whether the presence of naturally occurring or endemic organisms could be exploited. Conclusion 1: Although efforts to characterize the diversity of bacteria and viruses existing in nature are under way, there is no comprehensive effort ongoing to describe microorganisms. An international collaboration engaging the worldwide scientific community in a systematic effort to identify, monitor, and characterize a far higher proportion of global microbial species to increase knowledge about endemism and background is needed. The effort should begin with known pathogens and then expand to their close relatives as well as emerging pathogens. To mount such an effort, formal international scientific collaborations will need to be created to ensure that technological resources are accessible to all nations, including developing countries that currently lack such resources, and that funding can be leveraged better. Other questions will need to be addressed: for example, what kinds of sampling and forensic characterization programs would be required to provide the most useful information? In addition, should there be an international committee to establish standards for typing and characterization of strains? Finding: Skowronski and Lipkin (2011:173) state that Perhaps the most significant challenge in microbial forensics is the ability to differentiate the natural from the intentional biological event. Mother Nature continues her relentless assault on human, animal, and plant populations with an impressive array and diversity of microbes, and situational awareness of unnatural malicious intent can be hard to come by. Although there are criteria (see Box 3.1) for considering whether a disease outbreak is unusual, determining whether such an event is due to natural, accidental, or deliberate causes, and collecting the information to work through these criteria at the time of an event is likely to be time-consuming with slow response time. As noted in the Biological Response and Recovery Science and Technology Roadmap recently issued by the National Science and Technology Council (NSTC, 2013), a biological event demands “a quick and effective response in order to minimize loss of life and other adverse consequences and, in the case of suspected criminal activity or terrorism, to thwart ongoing activity and prevent follow- on attacks.” Time lines for making this crucial determination are currently unacceptable from both public health and law enforcement standpoints, which require development and management of appropriate and rapid responses, recovery measures, and resolution. Conclusion 2: There is a strong need for the development of high-confidence methodologies to distinguish among natural, accidental, and deliberate outbreaks of infectious disease. Although the research and development agendas of the U.S. federal departments and agencies are now being coordinated under the NSTC Roadmap mentioned above (NSTC, 2013), such coordination on an international basis is justified by the potential for rapid spread of disease through global transportation networks. This is a high-priority need for the research and funding agendas both inside and outside the United States that requires a coordinated effort on an international scale.   Prepublication Copy 111 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics Metagenomic techniques offer the possibility of sequencing the genomes of unculturable microbes, which represent the vast majority of microorganisms, particularly from environmental samples (e.g., soil and water). In epidemiology and public health, the need to move from culture-dependent to culture-independent methods to identify pathogens is also being recognized as an important diagnostic capability. Metagenomics would avoid culture bias, but it is difficult to study rare taxa, which comprise many of the organisms in such samples. The probative value of the “small signal” of the rare microbe of interest in the “big noise” of a cluttered metagenomic sample is undetermined, and validation of the processes to detect rare organisms is necessary but problematic, particularly because of artifacts, stochastic effects, abundance effects, and contamination. Questions that arise include: What is required to demonstrate that this is a viable approach and what are the limits to what could be stated from such analyses? Can these problems be overcome to meet forensic standards? How should the scientific and legal significance be determined and supported when the agent of interest is a minority constituent in a “probative sample”? How much of the threat agent of interest must be in a sample to be significant or meaningful in context? Finding: Metagenomics has great promise for microbial forensics if it can be adapted to improve and speed up the ability to characterize microbial species and communities derived from environmental samples. But it is as yet unclear whether the “clutter” in metagenomics samples can be exploited for forensic value. Finding: The other “omics” (e.g., proteomics, metabolomics, transcriptomics, and glycomics) may also provide applications of value to microbial forensics. Wahl et al. (2011) state that “Molecular variations in DNA sequence are widely used for organism and strain identification, but many other molecules and chemical species may be useful in determining microbial identity and origins; these include proteins, peptides, lipids, carbohydrates, inorganic ions, and organic metabolites.” For example, proteomics may be of forensic value because protein expression profiles can potentially provide information that can be traced back to the environment in which the organism was cultured. Given that microorganisms respond to environmental conditions by changing patterns of gene expression, the types of proteins expressed by an organism can reveal information for forensics about aspects of growth conditions. The chemical species produced by microbes must, of course, be correctly detected and identified and the results appropriately interpreted using computational algorithms and rigorous statistics (Wahl et al, 2011). Conclusion 3: Priority research is needed to realize the promise of metagenomics and its application to microbial forensics and the development of the forensic value of the other “omics”: proteomics, metabolomics, transcriptomics, glycomics, immunogenomics, etc. NEEDS COMMON TO MEDICINE, PUBLIC HEALTH, AND MICROBIAL FORENSICS Human and veterinary medicine, public health, agriculture, and microbial forensics clearly share many techniques and approaches and all are similarly motivated by the need to protect the health of humans, animals, and crops. With any infectious disease outbreak, the starting point is determining what the agent is, whether for epidemiological or microbial forensic purposes. The use of molecular techniques to identify pathogen species and strains has, over the last 10 years, become fundamental to microbial forensics and is rapidly being adopted in clinical medicine. Although hospitals do not yet routinely use sequencing strategies for diagnostics, recent examples (e.g., Snitkin et al., 2012) have demonstrated that hospitals can deal more quickly and effectively with nosocomial infections if they have the ability to integrate genomic and epidemiological data. The importance of being able to access rapid molecular   Prepublication Copy 112 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics diagnostic capabilities in developing countries was described in the report Biosecurity Challenges of the Global Expansion of High-Containment Biological Laboratories (NRC, 2012). Finding: Access to rapid and accurate molecular diagnostic equipment and techniques is important for controlling and responding to disease outbreaks as well as for microbial forensics. Conclusion 4: Improved worldwide access to molecular diagnostics (polymerase chain reaction [PCR], whole-genome sequencing [WGS], etc.), including refinement and distribution of bench-top next-generation sequencing (NGS) instruments that are fast and affordable and have simple workflow procedures, is a critical need. At a minimum, PCR technology should be universally available. WGS also could be more broadly available with the advent of small benchtop instruments that offer speed and accuracy of analysis and have become less costly. These tools should be in place as soon as is feasible, to allow more rapid detection, higher sample throughput, and swift response for both public health and microbial forensics. Finding: In addition to increasing basic knowledge of the diversity, ecology, phylogeny, and other aspects of basic microbes, there is a need to improve our knowledge of mechanisms of pathogenicity, virulence factors, antibiotic resistance traits, mutation rates, prevalence and impacts of lateral gene transfer (AAM, 2009b), and host immune responses to pathogens. Some participants at the Zagreb meeting expressed the need for the development of databases on clinical infectious disease agents, which is not currently common practice in medicine and would be a daunting task. Skowronski and Lipkin (2011) pointed out that between 1998 and 2006 there were 40 million hospital admissions for infectious diseases in the United States alone. They stated that it is literally impossible for the etiological agent to be identified, much less to a standard that would be considered a validated fact generated by a tertiary testing facility (CDC/Laboratory Response network/State Public Health Laboratories) and that would withstand legal scrutiny. . . . In many cases, presumptive diagnoses and minimal screening tests are often used when clinical signs are sufficient to treat. In practice, this has been sufficient for medical care but is not adequate to address the needs of microbial forensics. (Skowronski and Lipkin, 2011:174) Nonetheless, insights into pathogenesis and the development of drugs and vaccines require live organisms and clinical samples from individuals who have been exposed to live organisms. Skowronski and Lipkin also noted that sequencing, microarrays, high-throughput NGS, and bioinformatics represent new sensitive and exacting techniques for identifying and characterizing pathogens that will eventually have broad applicability to public health, biosurveillance, and forensics and attribution. It also is worth noting that in November 2013, the U.S. Food and Drug Administration (FDA) approved Illumina’s MiSeq Dx DNA sequencing system for diagnostics, which until then had only been used for research (Dubay, 2013). Conclusion 5: An increased emphasis on research to determine mechanisms of pathogenicity, including virulence factors and host immune responses, is needed. Finding: A number of the participants in the Zagreb workshop called for better global disease surveillance. Although there are a number of programs already in existence for this purpose (see discussion of biosurveillance in Chapter 2), there is considerable evidence that these programs have yet to fully bear fruit. For example, as noted previously, only 16 percent of countries were able to meet the 2012 deadline for implementing the International Health Regulations, and the U.S. government has recently announced a new Global Health Security Agenda to intensify worldwide efforts to achieve global health security.   Prepublication Copy 113 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics Conclusion 6: Greatly improved global disease monitoring and surveillance in humans, animals, and plants is needed to facilitate rapid response and better disease control. It also would increase our understanding of background levels for aiding in the detection of unusual outbreaks. Although a daunting task, clinical databases of infectious disease cases in hospitals need to be developed to improve our understanding of pathogen distribution and characterization. An early-warning geographic information system could also be valuable for aiding these purposes. METHODS AND TECHNOLOGIES USED IN MICROBIAL FORENSICS Methods of great value in microbial forensics for identification of microbial agents and analysis of their source include both molecular genetic and nongenetic technologies. PCR, sequencing of 16S ribosomal RNA, multilocus sequence typing, and WGS are molecular genetic technologies used to characterize microbial agents. Other physical science techniques, such as mass spectrometry and electron beam–based methods, can be used to analyze the physical properties of microbial forensic evidence, for example, the presence of additives for stabilization and/or dispersability and physical signatures from the locale where the material was produced (Michael et al., 2011). Mass spectrometry is also useful for analyzing biological toxins, such as ricin and botulinum toxin, which may not contain co-purifying nucleic acid signatures (Johnson et al., 2011). In addition, proteins and other biochemical products may provide clues about an organism’s place or conditions of origin. Nucleic Acid–Based Technologies There are a number of approaches for interrogating nucleic acids for attribution. As described in Chapter 4, real-time PCR (qPCR) is a rapid and sensitive detection technology that has proven effective in both clinical and microbial forensic assay applications. The power of qPCR lies in simplicity, assay selection, multiplex capability, optimization, and already-validated assays. There are many available assays that are validated to certain standards, although they must be validated internally in each laboratory that uses the assay. Many methods enable qualitative detection of hundreds of pathogens and hospital- acquired infections to the species and sometimes the strain level and a number of assays are FDA approved. There also are assays in the food safety arena on which microbial forensics can draw. Matrices in the food industry are complex, and food is a potential target of intentional and accidental contamination. There are many food industry microbial and epidemiological investigations, and microbial forensics can learn from these events and have access to the assays. Mass spectrometry exploits the different masses of atoms or molecules in a sample material. MassTag PCR combines PCR with mass spectrometry to provide high resolution, sensitivity, and specificity. This technology already has revolutionized the ability to identify respiratory pathogens and hemorrhagic fever viruses. Microarray platforms accomplish a similar goal of organism identification via detecting nucleic acid or protein signatures in a higher-multiplexed format. Many panels have 1 million hybridization or binding assays on a single array chip, and some have up to 5 million assays. Microarray panels are available for microbial detection, single-nucleotide polymorphism (SNP) detection, genome-wide association studies, gene expression analyses, and protein presence and abundance. Microarray readers for other purposes, such as oncology or infectious disease screening, could be leveraged by the development of custom chips that are directed to addressing microbial forensic issues. Many assays are available, but will need to be customized on a single chip to be more comprehensive for microbial forensic needs. Typically an array is dedicated to a specific organism or small group of organisms, although arrays suited to larger numbers of organisms do exist. Microarrays do not provide as much information as sequencing, but they approach   Prepublication Copy 114 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics the discriminatory power afforded by sequencing. These platforms may be a rapid and cost-effective alternative or a supplement to sequencing. Until recently, it was laborious and expensive to sequence an entire microbial genome, and researchers and funding agencies were obliged to choose carefully, organism by organism, what would be sequenced (AAM, 2009b). The Sanger chain termination method dominated for about 30 years before a new generation of technologies significantly changed how sequencing is performed. Today, NGS technologies are allowing rapid sequencing of thousands of microbial isolates as well as complex microbial communities at far less cost and effort (Fricke et al., 2011). A review of various early high- throughput machines coming onto the scene can be found in Parla et al. (2011). There have been numerous comparisons of current NGS devices, among them Quail et al. (2012), Loman, et al. (2012), and Jünemann et al. (2013), that evaluate the advantages and disadvantages of the many devices already on the market or expected soon. NGS technology will continue to improve rapidly. Any reference in this report to a related methodology or specific technology for a case study is used for illustration and not to represent the state of the art. Nevertheless, as a consequence of these recent advances, sequencing and genomics are revolutionizing our understanding of bacterial and viral evolution and function. It is now possible to sequence the genomes of an unprecedented variety of microorganisms. However, there has not been a strong open effort to prioritize which organisms to sequence for microbial forensics purposes. In 2009, the White House’s NSTC (2009) recommended that a survey of all full genome sequences available at the time be conducted to assess whether the number and diversity of sequences are adequate for forensic purposes. On the basis of results of the survey, additional strains for WGS were to be recommended. However, this effort appears to be restricted to the United States and it is unclear what the outcome of the effort has been. The NSTC strategy document in which this recommendation appeared did not specifically address whether the effort was to be international in scope and the role of other countries in the task, if any, is not clear. In addition, the enormous increase in sequencing has brought with it other needs that must be addressed. For example, the competing technology platforms for sequencing lack standardization, and there are no standards for how sequencing data should be reported. One consequence of this is that it is unclear whether differences in sequence data generated using different devices are a consequence of error or have some other significance. Sequencing error may inflate the similarity or dissimilarity between reference and evidence samples, increasing uncertainty. Defining and quantifying error rates associated with each sequencing platform are critically important, but it is unclear how to go about this task. “Deep” sequencing routinely produces vast (terabyte) quantities of data that demand novel methods for analysis, handling, storage, etc. These data require expert annotation and curation, but there are no standardized annotation software systems. (This topic is covered in more detail under Bioinformatics and Data.) Finding: The development, validation, and standardization of rapid new analytical methods, including sequencing and non–nucleic acid assay technologies, will continue to be important. It is not likely that prolonged research, development, testing, evaluation, validation, and technology transfer programs such as those developed for the anthrax letter attacks case, which took 8 years, will be acceptable each time a biothreat event occurs, particularly if a novel agent is used or if exigent circumstances arise. To be as prepared as possible for the next outbreak event, this work needs to be carried out now, whether it is in response to a natural, accidental, or deliberate event involving a biological agent. Conclusion 7: Development of more advanced, faster, and cheaper assay and sequencing technologies that can be standardized and made more accessible to benefit both microbial forensics and public health is necessary. Although industry is likely to pursue technology improvements on its own, increased accessibility of small and affordable benchtop NGS machines, such as those used in the German Escherichia coli O104:H4 outbreak, would “democratize” use and enable many countries that cannot afford sequencing to be able to implement such capabilities for routine diagnostic purposes.   Prepublication Copy 115 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics Further development of NGS would also enable detection and identification of organisms without a specific a priori assay design. Standards for reporting sequence data also need to be developed. Indeed, small handheld devices to digitize DNA results and send sequences over smart phones for real-time detection in the field might be a reasonable technology hope in the future. Of course, if handheld devices come to fruition, they will have to be applicable to, and meet the requirements of, microbial forensic analyses. Applications of Physical Science Finding: As noted, a variety of nonsequencing techniques are available for identifying organisms and their biochemical properties. Although nucleic acid–based technologies are very sensitive, they will not identify all threats that may be encountered, such as toxins. Toxins can be purified to a degree that it is impossible to identify even trace levels of DNA or treatment may degrade DNA in the sample. Platforms that perform protein and antibody/antigen detection are being exploited to address this problem. One system is electrochemiluminescence (ECL), which enables antibody-based assays, as well as nucleic acid–based assays. Knowledge of the target being pursued is necessary, as are thoughtfully designed and validated assays, to use ECL effectively. The textbook by Budowle et al. (2011) has several chapters devoted to non–nucleic acid chemical and physical analysis technologies, such as mass spectrometry and electron beam–based methods. These technologies also appear to be useful, particularly for helping to determine the manufacturing method for a biothreat agent by analyzing the materials associated with the sample, such as agar and silicon in scenarios where residual material, a dispersal device, or other materials are discovered. Conclusion 8: High priority needs to be placed on continued research and development to improve physical science applications to microbial forensics. SAMPLING Finding: Chapter 5 contains a great deal of detailed information on sampling and preservation of microbial forensic evidence. Ideally, sampling should be conducted according to a plan or design appropriate for the context and must include provisions for clear quality assurance and quality control (QA/QC) measures, documentation, handling, storage, packaging, and transportation. It was stressed that results are more reliable if specialists in microbial forensics can advise those who collect samples (e.g., first responders) before they begin work on the crime scene. For microbial forensics, guidelines are needed that will support the criminal justice system and intelligence community and comply with standards for crime scene examiners. Type of release influences the type of sampling that is appropriate. A covert release may result in a delayed response, whereas an overt release allows a more rapid, and possibly more focused, response. It also needs to be recognized that evidence collection for microbial forensics is sometimes incompatible with other forensic techniques. This concern must be taken into account, and triage strategies are needed for sample collection so that critical evidence is not destroyed. Investigative protocols that optimize the choice and priority of methods are needed and should consider sample availability, preservation and conservation of evidence, trade-offs between speed and accuracy and/or precision, as well as other factors. First and foremost, consideration of the health and safety of the people collecting and handling samples is required. Collection protocols also should be based on analytical capability, preservation and integrity of evidence, low cost and high efficiency, documentation, training, quality and transparency, and flexibility, because matrices in which evidence might be collected could be highly diverse and may be difficult to handle or preserve.   Prepublication Copy 116 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics Conclusion 9: The development (and validation) of processes (sample collection, preservation, handling, storage, packaging, and transportation) and analytical methods for microbial forensics, including establishing standards for most components, require a much higher priority. Existing processes need to be standardized, compiled, and shared worldwide, while new, more efficient ones need to be sought. VALIDATION AND STANDARDS Finding: All components of a microbial forensics investigation, from methods for collection and sampling, preservation and handling to identification of the agent, etc., need to be validated. Although validation is recognized as necessary and essential, it is ill defined. There is a challenge in translating the requirements because there is no standard way to validate an assay or method. This includes developmental validation of one’s own method as well as internal validation by other laboratories implementing the method. Minimum validation criteria include: sensitivity, specificity, reproducibility, precision, accuracy, robustness, analyses consistent with the samples and the intended application of an assay, for example, reference panels and mock or nonprobative materials. Other criteria that tend to apply to many assays but may or may not apply in every situation are resolution, purity, critical equipment calibration, critical reagents, and databases. Validation presents many other challenges. For example, validating sampling methods is very difficult because there are many types of surfaces and environmental matrices. Another variable is the individual who performs sample collection. Quality management needs for performing microbial forensic work include scientific rigor and validity; methods that will support attribution for criminal investigations; and establishing national and international working guidelines for quality assurance and quality control as applied to microbial forensics. There is a serious need for methods for characterizing uncertainty and resolving conflicting results. All analyses are subject to bias and error, so the limits of a method need to be understood. Interpretations may need to be made with incomplete and mutable data due to the fact that the diversity and variability of microorganisms are unknown and organisms undergo genetic change in response to environmental and other factors. Consequently, methods to accommodate this functional knowledge gap must be devised. Although many analysts develop new tests, there is also a need to look at old, less-used, or abandoned techniques, since nonstate actors may not have access to the latest sophisticated technology. As noted above, although the precise identification of an analyte (DNA, etc.) can now be accomplished to the level of a distinct strain using, for example, canonical SNPs at few- or even the single-molecule level, the validation of the processes to detect rare and low-level signatures is problematic. The limits of interpretation also need to be clearly understood, and statistical approaches should be rigorously applied in determining whether two microbial samples “match” or are identical, their degree of similarity, their association, and/or their most recent common ancestor. Given what current science and informatics can provide, what is the best that can be expected, accepted, and communicated regarding the probative characteristics of a microorganism and determining its source with acceptable confidence? It is important to understand what a negative result means, particularly for metagenomic analyses. Finally, analysis is not equal to interpretation, and there can be alternative explanations, so there is a need to test hypotheses and try to disprove them, not just attempt to prove them. Velsko (2011a; see also Conclusion 10) emphasized that we must begin an interactive and committed process to address these issues now. Scrambling to prepare as an event occurs is an undesirable scenario. Conclusion 10: There is a great need for establishing criteria and requirements for validation. In addition, rapid and accurate development of additional validated analytical methods for microbial forensics must continue. These efforts should include establishment of standards for most components of   Prepublication Copy 117 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics microbial forensic analyses. A compilation of all protocols in use (e.g., for sampling, DNA extraction and isolation, and sequencing) and whether and how they have been validated is essential. BIOINFORMATICS AND DATA Bioinformatics is an interdisciplinary field that develops and improves on methods for storing, retrieving, organizing, and analyzing biological data. A major activity in bioinformatics is to develop software tools to generate useful biological knowledge. To do so, bioinformatics uses many areas of computer science, mathematics and engineering to process biological data. Complex machines are used to generate biological data at a much faster rate than before. Databases and information systems are used to store and organize biological data. Analyzing biological data may involve algorithms in artificial intelligence, soft computing,74 data mining, image processing, and simulation. The algorithms in turn depend on theoretical foundations such as control theory, system theory, information theory, and statistics. The quality of sequence data and the results of bioinformatics analyses must be as high as possible. Factors to consider that impact data interpretation and quality include the quality metrics of sequence data; sequence errors and uncertainties; reliable standards for genomic data representation; uncertainty with databases used; inferences based on available data, including meta-data; formulation of well-defined hypotheses; and testing methods for assessing the weight of microbial forensics evidence. Data quality and interpretation both require reliable standards for genomic data representation. First, there must be an understanding of sequence quality, sequence errors, and uncertainties about the output data. Second, for interpretation with respect to attribution, criteria are needed for comparisons: match, similar, different, inconclusive; rigor of reasoning by the expert; and well-defined hypotheses and testing methods for assessing the weight of microbial forensic evidence. Understanding what various bioinformatics results mean may be difficult because one laboratory cannot precisely replicate all essential details of any particular bioinformatics analysis pipeline used by another laboratory. Analyses are complex, different versions of programs exist, and software and hardware change rapidly. Software libraries are updated frequently, and investigators often find that the software they were using is obsolete before their project is completed or validated. No bioinformatics software comes as a stand-alone. All programs depend on multiple libraries, each of which has its own versions, “bug” fixes, and potential parameter settings. It is sometimes uncertain how changes in any of those library modules might affect answers coming from any particular program. For example, changes in a random number generator seed or in a floating-point math library can cause many programs to exhibit different behavior if they incorporate Monte Carlo calculations. At an even more fundamental level, the version and patch level of the underlying operating system and the CPU chip architecture and release level could affect outputs. Although some differences may be insignificant, others may be quite important and these need to be understood. In addition, there are important issues regarding how much documentation is required. The baseline assumptions that are needed to ensure assessments and effective comparisons of technologies need to be defined. Validating bioinformatics pipelines requires data acquisition at all phases of the process, from extraction to data analysis to interpretation. Finding: The new “deep-sequencing” methods generate data in the range of tens to hundreds of gigabases. Although the capability to detect polymorphisms for attribution at a level that was not possible                                                              74 Use of inexact or approximate solutions for computationally difficult tasks, often when the computing power needs exceed the task.   Prepublication Copy 118 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics only a few years ago is a great advance, it also presents new challenges. It requires novel ways to handle and analyze terabytes of data. Given this new demand, bioinformatics is both a necessity and a challenge. Conclusion 11: Refinement of bioinformatics and statistical methods for evaluating evidence in microbial forensics is needed, including new algorithms that scale to very large and complex datasets. Bioinformatics also needs to be made understandable and user-friendly to laboratory users, first responders, the public, and the policy makers. Clear requirements are critical in order to attract industry to develop needed hardware and software for bioinformatics. Data Sharing Finding: One highly consistent theme heard at the workshop was that data sharing is critical and desperately needed, encompassing biological information on gene sequences, software, and protocols, and standard operating procedures for all microbial forensic methods. Sharing of such data has the potential to promote international collaboration and cooperation among scientists and, more importantly, to inspire innovation. It might improve security efforts in the sense of promoting more rapid responses to infectious disease outbreaks, which could be crucial in this age of rapid global transportation. Although most participants agreed on the importance of data and information sharing, many expressed concerns about its feasibility and acceptability to governments and policy makers. While cloud computing offers solutions to data sharing as well as storage issues, these possibilities generate security concerns for microbial forensics. In addition, the quality of what is shared is crucial, so standardization of characterization assays was deemed to be key for data sharing. Conclusion 12: Discussions are needed under the auspices of an international body that has the respect of the international political and scientific communities about how to share microbial forensic data, and for developing and presenting cogent arguments that can be persuasive to political leaders and scientists worldwide. Databases and Reference Collections Finding: The importance of having a comprehensive archive, or set of archives, of reference materials was emphasized by numerous participants at the workshop, although it was not clear to what extent an archive should contain organisms, nucleic acids only, or just sequence information, the latter of which is technically not a reference collection but a database. An institution could facilitate the development of standardized nomenclature, typing techniques, and characterization systems; uniform QA/QC requirements; standardization of new techniques and analytical evaluations; and reference samples for high-resolution genomic comparisons. It is important that any centralized reference entity maintain a reasonably comprehensive collection; have sufficient long-term support; maintain the highest possible QA/QC standards; use accepted and standardized methods; strengthen standards for curation and material preparation; be responsive to the needs of research and development and support R&D that updates our understanding of diversity; be governed as a community resource; and maintain procedures that balance access and security concerns. Ideally, a reference archive would meet the needs of both criminal investigators and researchers. However, there is skepticism about the feasibility of capturing the full diversity of the microbial world and all the permutations necessary for the multitude of analytical methods in a single archive. However, if highest priority were given to archiving the pathogens and near neighbors that may be used in biocrimes or bioterrorism, this would have considerable value for microbial forensics. Even if less comprehensive collections are likely to be inadequate for most microbial forensic investigations, they would still be suitable for basic research and provide at least a nominal degree of   Prepublication Copy 119 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics centralization, which is required to move forward on an effective system for dynamic response and accountability. Conclusion 13: An international effort to design and establish more systematic and comprehensive reference collections and databases for pathogens and other microorganisms could take advantage of existing models, such as the World Data Centre for Microorganisms and the American Type Culture Collection. A model system for a consortium of reference collections and data storage centers could be created and later scaled up to become more inclusive. TRAINING AND EDUCATION Velsko (2011a:522) provides an informative concluding paragraph: If there is any lesson to be drawn from past experience, it is that microbial forensic collection and analysis are not very effective when they are conducted as ad hoc activities, by non-specialists, using improvised methods and on-the-fly attempts at validation, without prior review by a knowledgeable community. The utility of microbial forensic analysis rises in proportion to the extent that it is anticipatory, well planned, driven by a cadre of qualified experts, and resourced adequately. Finding: The committee agrees with this statement. Microbial forensics is still a relatively new discipline. Training is needed for a number of purposes, including  Increasing the availability of trained microbial forensics practitioners. Given that microbial forensics encompasses a number of complex technical areas, any such training would necessarily require multidisciplinary, or even transdisciplinary, approaches. A core microbial forensics training program that is available worldwide is needed, and a determination of who would be responsible for developing and implementing such a program is required as soon as possible. Training, and perhaps even certifications, in microbial forensics disciplines would help make microbial forensics a more widely accepted basis for law enforcement actions for use in both domestic and international settings.  Increasing the awareness and preparedness of first responders, which is essential for both safety purposes (to prevent accidental exposures of responder personnel to hazardous pathogens) and for law enforcement needs to ensure that evidence samples are not compromised, crime scenes are not contaminated, etc.  Improving policy-maker and public understanding of what microbial forensics is and what it can accomplish. Currently, there is scant literature available for either the general public or policy makers that explains either the nature or significance of microbial forensics or the fact that moving microbial forensics to a fully mature and accepted set of law enforcement tools requires substantial long-term scientific effort as well as new policies to facilitate that effort and its implementation. Conclusion 14: An expansion of technically based training is needed to “professionalize” microbial forensics and increase the number of qualified practitioners worldwide by engaging international professional organizations or other entities that have experience providing training in related fields.   Prepublication Copy 120 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics SUMMING UP In its efforts to identify what is required to develop microbial forensics further, the committee used a generous definition of “science,” including research to improve fundamental scientific understanding of microbes, specialized research intended for particular applications in public health, law enforcement, or elsewhere, and an array of technologies and methods that support both basic and applied research. The committee chose breadth over depth and gave particular attention to those science and technology areas that would benefit from international cooperation and collaboration. The result was an extensive list of needs, as outlined in the preceding section. The committee also identified a variety of procedural and policy needs, such as common understandings and protocols for taking and managing samples within and between nations. These additional needs must be addressed if the scientific and technical advances the committee calls for are to make microbial forensics a more effective tool for responding to natural, accidental, or deliberate disease outbreaks. This report is not meant to, nor can it, provide a detailed roadmap for the international development of microbial forensics, but rather elucidates the major issues that the committee believes need to be addressed for the global development of the science of microbial forensics. SETTING PRIORITIES The list of needs identified by the committee is long, but the successful development of microbial forensics will require addressing all of them. The committee emphasizes, however, that there are considerable differences in how difficult it will be to address the needs and in whether there are already existing national or international efforts, for example, in basic research, public health, or industry, that can be drawn upon to help achieve the desired results. There are also a number of high-priority needs that are particularly challenging tasks with long lead times to achieving real progress so that efforts should begin or expand soon. The latter needs will also require substantial and sustained support from governments as major funders of the research, development, and implementation that will be essential for achieving success. Table 8.1 presents the needs identified in this chapter organized according to the key features discussed above:  One set of needs represents tasks, for example, the need to identify and characterize a significantly increased number of microbial species that are particularly challenging and/or require a long lead time to achieve the desired results. Such efforts will require the involvement of governments to provide the research resources to carry them out over many years and should be given priority by participating institutions.  The second set represents needs that could take advantage of ongoing efforts to advance the development of microbial forensics, but will require deliberate communication efforts and in some cases funding to ensure that microbial forensics applications are actually included and implemented.  The third set of needs has the advantage of either a relatively short lead time to make substantial progress or the existence of significant markets that will provide incentives for industry to produce what is required. For example, the production of faster and cheaper instruments for diagnostics for medicine and genomic analyses for microbial forensics will probably be conducted by industry, which is always seeking to put improved devices on the market. The committee recognizes that there is overlap among the categories and that some of the needs would fit within more than one of them. It nevertheless believes that exercises like this can be helpful in   Prepublication Copy 121 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics thinking about implementation issues and for the eventual development of a more detailed roadmap to guide future efforts. TABLE 8.1 Priority Categories for Microbial Forensics Needs Challenging Tasks and/or Long Lead Times  An international collaboration engaging the worldwide scientific community in a systematic effort to identify, monitor, and characterize a far higher proportion of global microbial species to increase knowledge about endemism and background. The effort should begin with known pathogens and then expand to their close relatives as well as emerging pathogens. (Conclusion 1, Basic Science)  Development of high-confidence methods to distinguish among natural, accidental, and deliberate outbreaks of infectious disease. (Conclusion 2, Basic Science)  Increased emphasis on development and validation of processes (sample collection, preservation, handling, storage, packaging, and transportation) and analytical methods for microbial forensics, including establishing standards for most components. (Conclusion 9, Validation and Standards)  Discussions under the auspices of an international body that has the respect of the international political and scientific communities about how to share microbial forensic data, and for developing and presenting cogent arguments that can be persuasive to political leaders and scientists worldwide. (Conclusion 12, Bioinformatics and Data/Data Sharing)  An international effort to design and establish more systematic and comprehensive reference collections and databases for pathogens and other microorganisms. This effort could take advantage of existing models, such as the World Data Centre for Microorganisms and the American Type Culture Collection. A model system for a consortium of reference collections and data storage centers could be created and later scaled up to become more inclusive. (Conclusion 13, Bioinformatics and Data/databases and Reference Collections) Ongoing Efforts on Which to Build  Increased emphasis on research to determine mechanisms of pathogenicity, including virulence factors and host immune responses. (Conclusion 5, Needs Common to Medicine, Public Health, and Microbial Forensics)  Priority research is needed to realize the promise of metagenomics and its application to microbial forensics and the development of the forensic value of the other “omics”: proteomics, metabolomics, transcriptomics, glycomics, immunogenomics, etc. (Conclusion 3, Basic Science)  Greatly improved global disease monitoring and surveillance in humans, animals, and plants to facilitate rapid response and better disease control. (Conclusion 6, Needs Common to Medicine, Public Health, and Microbial Forensics)  Improved worldwide access to molecular diagnostics (PCR, WGS, etc.), including refinement and distribution of benchtop NGS instruments that are fast and affordable and have simple workflow procedures. (Conclusion 4, Needs Common to Medicine, Public Health, and Microbial Forensics)  High priority placed on continued research and development to improve physical science applications to microbial forensics. (Conclusion 8, Methods and Technologies)  Refinement of bioinformatics and statistical methods for evaluating evidence in microbial forensics, including new algorithms that scale to very large or complex datasets. (Conclusion 11, Bioinformatics and Data)   Prepublication Copy 122 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics Shorter Lead Times or Industry Incentives  Development of more advanced, faster, and cheaper assay and sequencing technologies that can be standardized and made more accessible to benefit both microbial forensics and public health. (Conclusion 7, Methods and Technologies)  A compilation of all protocols in use (e.g., for sampling, DNA extraction and isolation, and sequencing) and whether and how they have been validated. (Conclusion 10, Validation and Standards)  Expansion of technically based training to “professionalize” microbial forensics and increase the number of qualified practitioners worldwide by engaging international professional organizations or other entities that have experience providing training in related fields. (Conclusion 14, Training and Education) Microbial forensics has specialized needs because of the demand for “evidence” and “proof” in the context of law enforcement or international policy. Many of these relate as much to the quality of the process by which material is collected and analyzed as to the science and technology employed. The strong presence of public health needs in this report reflects the global reality that in most countries the only capabilities and activities relevant to microbial forensics occur in the context of public health. The United States is largely the exception in having a microbial forensics science community that is differentiated from public health, reflecting its experiences in the early 2000s with the anthrax letters. Some militaries in developed countries are creating microbial forensic capabilities, and nations such as the United Kingdom, Sweden, Germany, China, and France have basic microbial forensics infrastructures. But for most of the rest of the world, microbial forensics is a side activity of public health officials. Since most disease outbreaks will first be recognized through the public health infrastructure, strengthening detection and diagnostic capacities there serves both public health and microbial forensics. In this regard, the new Global Health Security Agenda as an alliance among over 25 countries, the European Union, and three international organizations (World Health Organization [WHO], World Organization for Animal Health (OIE), and Food and Agriculture Organization (FAO) illustrates the potential for initiatives to address global disease threats of any origin to contribute to building fundamental microbial forensic capabilities. As one of the documents announcing the new collaboration framed the challenge: An interconnected world is increasing the opportunities for human, animal and zoonotic diseases to emerge and spread globally. Today’s health security threats arise from at least 5 sources: the emergence and spread of new microbes; the globalization of travel and food supply; the rise of drug-resistant pathogens; the acceleration of biological science capabilities and the risk that these capabilities may cause the inadvertent or intentional release of pathogens; and continued concerns about terrorist acquisition, development, and use of biological agents. The recent emergence of the H7N9 influenza virus and Middle East Respiratory Syndrome Coronavirus underscore infectious disease as a serious global threat. Since the emergence of Severe Acute Respiratory Syndrome in 2003, the world has made great progress in strengthening local, regional, and international capacity to prevent, detect and respond to emerging infectious disease threats. Yet, despite important accomplishments, much remains to be done to achieve our shared global health security vision. Only 16% of countries reported reaching full compliance with the core IHR [International Health Regulations] competencies by the June 2012 deadline set by the WHO. Vulnerabilities include geographic areas with limited disease surveillance systems, reluctance to share outbreak information or biological samples, emergence of new pathogens and development of drug-resistance,   Prepublication Copy 123 

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Findings and Conclusions: Initial Prioritized Science Needs for Microbial Forensics and the specter of intentional or accidental release of biological agents. Multi-sectoral collaboration and the combined resources and expertise of the health and security sectors will be required to efficiently match resources to needs, avoid redundant efforts, and identify gaps. In 2013, for the first time, the G20 called upon countries to strengthen compliance with the WHO IHR—the standard by which the world measures its preparedness for emerging disease threats, as well as bioterrorist events. (Global Health Security Agenda, 2014) The committee commends the Global Health Security Agenda and hopes that its implementation will include a conscious effort to develop new capacities and connections among institutions, including those in the scientific community, that have a role in responding to this range of threats. Although the new agenda promotes a welcome multisectoral collaboration between the health and security sectors, it will be important to consider how the needs of law enforcement and legal standards for “evidence” and “proof” can be related to the new Agenda. It is hoped that the Agenda will eventually accommodate clear considerations of the needs of microbial forensics.   Prepublication Copy 124