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Introduction: What Is Microbial Forensics and Why Is It Important?

Many people have some notion about forensics from their exposure to the many “police procedural” TV shows being broadcast in countries around the world. Popular entertainment has made many familiar with the concept of using human DNA to identify criminals, although the TV version of this practice gives what is probably a less than realistic impression of the complexity of analyses and degree of certainty surrounding DNA evidence. Nevertheless, human DNA typing is a widely accepted means of identifying and convicting perpetrators, exonerating the innocent, and identifying missing persons from such tragedies as mass disasters.

“Microbial forensics” has been defined as “a scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime, or inadvertent microorganism/toxin release for attribution purposes” (Budowle et al., 2003). This emerging discipline is still in the early stages of development and faces substantial scientific challenges to provide a robust suite of technologies for identifying the source of a biological threat agent and attributing a biothreat act to a particular person or group. The unlawful use of biological agents poses substantial dangers to individuals, public health, the environment, the economies of nations, and global peace. It also is likely that scientific, political, and media-based controversy will surround any investigation of the alleged use of a biological agent, and can be expected to affect significantly the role that scientific information or evidence can play. For these reasons, building awareness of and capacity in microbial forensics can assist in our understanding of what may



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1 Introduction: What Is Microbial Forensics and Why Is It Important? Many people have some notion about forensics from their exposure to the many “police procedural” TV shows being broadcast in countries around the world. Popular entertainment has made many familiar with the concept of using human DNA to identify criminals, although the TV version of this practice gives what is probably a less than realistic impression of the complexity of analyses and degree of certainty sur- rounding DNA evidence. Nevertheless, human DNA typing is a widely accepted means of identifying and convicting perpetrators, exonerating the innocent, and identifying missing persons from such tragedies as mass disasters. “Microbial forensics” has been defined as “a scientific discipline dedi- cated to analyzing evidence from a bioterrorism act, biocrime, or inadver- tent microorganism/toxin release for attribution purposes” (Budowle et al., 2003). This emerging discipline is still in the early stages of develop- ment and faces substantial scientific challenges to provide a robust suite of technologies for identifying the source of a biological threat agent and attributing a biothreat act to a particular person or group. The unlawful use of biological agents poses substantial dangers to individuals, public health, the environment, the economies of nations, and global peace. It also is likely that scientific, political, and media-based controversy will surround any investigation of the alleged use of a biological agent, and can be expected to affect significantly the role that scientific information or evidence can play. For these reasons, building awareness of and capac- ity in microbial forensics can assist in our understanding of what may 7

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8 SCIENCE NEEDS FOR MICROBIAL FORENSICS have occurred during a biothreat event, and international collaborations that engage the broader scientific and policy-making communities are likely to strengthen our microbial forensics capabilities. One goal would be to create a shared technical understanding of the possibilities—and limitations—of the scientific bases for microbial forensics analysis. Toward this end, a group of national and international organizations held a workshop from October 14 to October 16, 2013, in Zagreb, Croatia. The workshop was organized by the U.S. National Academy of Sciences (NAS), the Croatian Academy of Sciences and Arts, the International Union of Microbiological Societies, and the U.K.’s Royal Society. The Croatian Academy of Sciences and Arts hosted the workshop at their headquarters in Zagreb. A planning meeting between NAS and the Royal Society in June 2011 began the process of designing the project with the intent of the Zagreb workshop to (1) identify the scientific challenges that should be met to improve the capability of microbial forensics to differentiate among natural outbreaks, unintentional release, biocrimes, or bioterror attacks; and (2) provide evidence of sufficient quality to sup- port legal proceedings and the development of government policies. The workshop also was designed to increase awareness of microbial forensics among the members of the larger international scientific communities and to engage these communities in the development of a plan on how to address scientific challenges. An international ad hoc committee was appointed by the NAS’s National Research Council to organize the meet- ing; brief committee member biographies can be found in Appendix A. The committee’s statement of task is in Box 1-1. Fifty-nine expert participants from 21 countries took part in the work- shop. Participants included researchers and clinicians from numerous scientific and technological disciplines related or applicable to microbial BOX 1-1 Statement of Task An ad hoc committee with substantial international membership will plan an inter- national symposium and prepare a consensus report of findings and conclusions to address the science needs underlying the development of microbial forensics. The results of the symposium, supported by additional information and data gathering by the committee, are intended to •  oster collaboration within the international scientific community to support F technical understanding and enhanced research on microbial forensics, and •  evelop the beginnings of an international roadmap for how to do the neces- D sary science, including priorities among potential topics.

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INTRODUCTION 9 forensics as well as technical experts and policy makers with a strong interest in the contribution of science and technology (S&T) to security. The two-and-a-half-day meeting included plenary sessions featuring talks by scientific experts and policy makers who reviewed current and devel- oping practices and technologies in, or applicable to, microbial forensics, with an emphasis on defining the challenges confronting the field. Smaller breakout groups enabled more targeted discussion of perceived gaps in microbial forensic capacity, as well as identification and prioritization of possible ways to develop and strengthen the field and its applications. Information about the convening organizations may be found in Appendix B. Support for the workshop and report was provided by the U.S. Navy through the Naval Postgraduate School, the U.S. Depart- ment of State, and the NAS itself. The workshop agenda may be found in Appendix C and a list of participants in Appendix D. A list of all the presentations and biographies for the speakers may be found in Appen- dixes E and F, respectively. John Clements, the chair of the organizing committee, asked workshop participants to address what tools are needed to enable us to successfully confront a bioterrorism or biocrime event, an unintentional release of a harmful agent, or a public health crisis caused by microorganisms or toxins. In all cases, the goal is the same—to protect the health and well-being of the public. He posed the following questions: What will help move microbial forensics forward in supporting such pro- tection and how should these needs be prioritized? The committee prepared this final consensus report, which draws on the workshop presentations and discussions as well as on additional information obtained from other experts and the literature to reach con- clusions for moving forward. The report is not meant to, nor can it, provide a detailed roadmap for the international development of micro- bial forensics, but rather elucidates the major issues highlighted at the workshop that the committee believes need to be addressed for the global development of the science of microbial forensics. For the purposes of this project, the committee chose breadth over depth.1 The committee also gave particular attention to those areas, such as increased scientific knowl- edge about microbial communities and common standards and protocols for analysis, which would benefit from international cooperation and collaboration. The number and variety of issues meant that not all issues could be examined in sufficient detail to develop a true roadmap. 1  Basic information about microbial forensics is available in a number of textbooks (see, e.g., Budowle et al., 2011, and Primorac and Schanfield, 2014). Various national and inter- national advisory bodies have also addressed many of the issues covered in the report (see, e.g., the reports of the U.S. National Biosurveillance Advisory Subcommittee, 2009, 2011).

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10 SCIENCE NEEDS FOR MICROBIAL FORENSICS WHAT IS BIOTERRORISM? Seth Carus (2001) published a now classic compilation of cases from 1900 to 2000 of the illicit use of biological agents by criminals and ter- rorists. His definition of “bioterrorism” is that it is “assumed to involve the threat or use of biological agents by individuals or groups moti- vated by political, religious, ecological, or other ideological objectives.” He clearly noted, however, that “most individuals and groups who have used biological agents had traditional criminal motives.” He believes that it is, therefore, essential to separate the clearly criminal perpetrators from those with political agendas, whether the motive is sectarian, religious, or ecological. The available evidence, in fact, suggests that the vast major- ity of cases involve criminal motives. Such motives include extortion, revenge, a desire to terrorize particular victims to make them worry about their health, and murder. Both biocrimes and bioterrorism exist on a continuum of risk associ- ated with biological agents (Figure 1-1). The other end of the spectrum deals with natural outbreaks or accidental releases of infectious disease agents. In all cases on the spectrum, public health protection requires that the microorganism first be identified and its source located to stop further cases of exposure. To this degree, medicine, public health, and law Spectrum of Biorisks Natural Accidental Biocrime Bioterrorism Biowarfare Outbreak Release FIGURE 1-1  Spectrum of risks due to biological agents.

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INTRODUCTION 11 enforcement initially have common aims and methods. Microbial foren- sics, however, has requirements and needs that, in many ways, go beyond those of medicine and public health. Though often applicable to medicine and public health, the methods used in microbial forensics delve deeper into identification of organisms, require standardization and validation, and must meet legal standards for evidence. Hence these topics are the subjects of much of the detailed discussion in this report, while keeping in mind the commonalities with public health that provide opportunities to leverage methods and information across fields. THE EMERGING FIELD OF MICROBIAL FORENSICS Forensic science involves the application of science to the investiga- tion of legal and policy matters. Science may not offer definitive answers in all cases, but often plays a special investigative role. “Science and tech- nology are used to serve as independent ‘witnesses’ in criminal or civil matters, intelligence, and policy” (Murch presentation, 2013). The law enforcement goal is “attribution”—that is, determining who committed the offense. Based on the analysis of biological and other evidence, law enforcement builds a case for attribution to a specific source or sources. The evidence supporting attribution must be robust and suitable for use in legal proceedings and to inform decision making at the highest levels. Microbial forensics seeks to produce reliable conclusions quickly to protect public health and with sufficient validity and quality to serve law enforcement and policy purposes. In microbial forensics, law enforce- ment may partner with scientists from microbiology, genetics, public health, agriculture, and many other disciplines to identify and character- ize pathogens, or their toxins, implicated in biological events. Dr. Randall Murch proffered five important questions to frame the background on microbial forensics: 1. Why is there an important need for microbial forensics? 2. What is the current state of the art? 3. How do the forensics used for criminal investigations differ from epidemiological investigations for public health? 4. What are the major research challenges for the field? 5. How can basic science be used to solve the current challenges for microbial forensics and how might this help in other areas, such as public health? Murch explained that microbial forensics in the United States began in the 1990s with the formation of the Federal Bureau of Investigation’s (FBI’s) Hazardous Materials Response Unit (HMRU). The Unit was cre-

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12 SCIENCE NEEDS FOR MICROBIAL FORENSICS ated to support suspected or known bioterrorism investigations by pro- viding investigative leads and supporting prosecutions or exonerations with scientific evidence. The FBI unit initially drew upon legacy science being developed or performed by scientists at national laboratories and universities across the United States and employed forensic science prin- ciples and practices to try to produce evidence that would be admissible in court according to U.S. legal requirements and standards. The FBI also recognized the importance of collaborating with the public health com- munity. At the time of preparations for the 1996 Summer Olympic Games in Atlanta, a collaboration with the U.S. Centers for Disease Control and Prevention (CDC) was established that is still ongoing. The CDC pro- vides, for example, training materials on forensic epidemiology. 2 Although microbial forensics incorporates basic research to develop techniques or methodologies, the questions asked, processes engaged in, expectations for, and outcomes sought may be different or more demand- ing than for basic research. The science in microbial forensics, like all sci- ence, must (1) be properly validated and accepted by peers and stakehold- ers (scientific, legal, policy making) before being used, but must also (2) demonstrate that the information generated can answer key investigative and legal questions. Microbial forensics borrows, transitions, and devel- ops science from related disciplines for its own purposes. Meeting these challenges will increase the value of microbial forensics by providing “leaps” in value, confidence, and timeliness for critical decision making and will advance other related disciplines. In the event of a suspected biological attack, leaders would have ques- tions about the identity and source of the biological threat that intelligence, public health, law enforcement, and forensics must try to answer (see Box 1-2A). Policy leaders want correct answers—and quickly—so they can act appropriately. Forensic science can help answer these questions, and it is essential that the answers be reliable. At the same time, policy leaders have to assess not just whether information is objectively credible and defensible, but also whether it will be accepted by key intended audi- ences. This can be a major issue at the national level, but becomes a much larger consideration when dealing with international relations, security, and terrorism issues (see next section). Both “elites” and “publics” may reject information because of distrust of the source and other emotive fac- tors. When using the results of science, policy makers (and international negotiators) must take these factors into consideration, too, not just the objective science. It is beyond the scope of this report to examine these political factors in any detail, but they have to be taken into account in developing and applying microbial forensic capabilities. 2  http://www.cdc.gov/phlp/publications/forensicepidemiology/index.html.

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INTRODUCTION 13 BOX 1-2 Questions Forensics Can Help Answer A.  Leader’s Perspective: A B.  Investigative, Legal, Key and Time-Sensitive Questions  Intelligence and Policy Perspective Questions • What is or was it? •  id a crime (event of interest) D • Who did it? occur? • Did it come from a program or • What happened? lab we know? • How, where, when, and why did • Will there be more attacks? it occur? • What are we—and nations • Who was involved and involved­ doing about it? responsible? — • What can we know, by when, • What evidence exists? What and with what confidence? does it tell us? How strong are he links? t • How reliable and credible is the evidence? • What alternative explanations are there for the evidence? • Can we defend our conclusions and actions? Consequence Crisis Mgmt Mgmt Min Hours Days Weeks Months SOURCE: Adapted from Murch (2010). However, a somewhat varied set of questions could arise from the investigative/legal and intelligence/policy perspectives, as listed in Box 1-2B. These parties will share the same questions as national leaders, but they also can have many additional questions, particularly regarding the accuracy, reliability, validity, credibility, and defensibility of scientific evidence. They may also have information about the likelihood of evi- dence being accepted and of ways in which different actors might attempt to manipulate information for political reasons. Forensics can contribute to answers, but the evidence is rarely of a clear-cut “smoking gun” type. While legal frameworks exist for evidence, policy frameworks are being developed. In the United States, evidence in a criminal case must align with the standards of proof of the criminal justice system in order to be admis- sible in legal proceedings, and standards must be met at every step of the investigation. Investigative leads, for example, must be based on validated, verifiable evidence, in order to enable authorities to gather fur-

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14 SCIENCE NEEDS FOR MICROBIAL FORENSICS ther evidence and/or compel suspected parties to cooperate in a variety of ways. In the United States and many other countries, the “standard of proof in a criminal trial is ‘beyond a reasonable doubt,’ which means the overall evidence must be so strong that there is no reasonable doubt that the defendant committed the crime.”3 Reasonable doubt, of course, depends on the jurors’ (or the policy makers’) assessment of all the evi- dence presented during a trial. Murch defined “scientific attribution” as the assignment of a sample of questioned origin to a source, or sources, of known origin, to the high- est possible degree of scientific certainty—while excluding origination from other sources (Murch, 2010). Ideal examples are fingerprint and DNA analyses, which can provide a high degree of scientific certainty that evidence came from one source to the exclusion of all others. However, according to Murch, microbial forensics cannot yet claim that degree of certainty and in many cases may never reach such specificity. In addition, the standard for attribution required in court may be different from the standard required to drive a policy decision. The process of forensic investigation begins with gathering intel- ligence and information to lay a foundation for and justify an investi- gation. This initial information gathering is followed by a time-driven, multidisciplinary, multisource investigation to pursue a rule-in and rule- out process. Crime scene investigation includes evidence identification, collection, preservation, and transport, as well as presumptive testing, which seeks to prove that either (a) the sample probably is a certain sub- stance or (b) the sample is definitely not a certain substance. Laboratory analyses provide deeper characterization and comparison of questioned source and known source samples. Throughout the process, investigators must place the interpretations of analyses and the conclusions drawn into a context, transitioning them to meet the needs of both real-time and end users. Murch, a former FBI official, stated that investigators also should provide alternative interpretations of analyses. Throughout the investiga- tion, forensics is assisting in building and shaping decisions and actions. The process is iterative, aids meeting the burden of proof, is part of the build toward prosecution, and supports exoneration. Finally, all the evi- dence and information generated are channeled to communication and decision making. Unbiased results, conclusions, and explanations about an event, as well as alternative explanations, must be provided to the legal and policy decision systems and to other stakeholders. Science plays a role in every phase of a forensic investigation, not only in the establishment of guilt or innocence. For example, science helps to 3  See, for example, the United States Courts website, http://www.uscourts.gov/Federal Courts/UnderstandingtheFederalCourts/HowCourtsWork/CriminalCases.aspx.

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INTRODUCTION 15 generate leads, and did so repeatedly during the investigation of the 2001 anthrax letters case. Ultimately investigators will weigh the combined forensic evidence to exclude or attribute to a source (Figure 1-2) to sup- port a finding of guilt or innocence. Because the information generated by forensic methods is to be used by law enforcement in litigation, the goals of these methods differ from those of traditional research. In addition to the accuracy, reliability, and repeatability demanded in traditional research, forensic methodolo- gies are subject to other rigorous requirements. There are admissibility requirements for “new science” in the U.S. legal process, and similar requirements are being developed for the policy process. The ideal forensic science methodology would incorporate the goals listed in Box 1-3A. Achieving these goals would help to ensure that sam- ples are collected and handled appropriately to preserve the target evi- dence as well as possible; analyses and comparisons of known-source “The Forensic Continuum” Individual Sample Types and Compilation Exclusion Attribution Consistent With Having Originated From Could Not Have Did Absolutely Come From Come From Power of and Confidence in Analysis, Interpretation, Meaning “Not Integrate with Other Evidence & Intelligence “Guilty” Guilty” FIGURE 1-2  The forensic continuum represents the evaluation and analysis of a microbial sample to determine its exclusion or attribution value. These results will be integrated with other evidence and information to determine innocence or guilt. SOURCE: Budowle et al. (2013).

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16 SCIENCE NEEDS FOR MICROBIAL FORENSICS BOX 1-3 Forensic Science Methods and System Elements A. Forensic Methods B. Forensic Science System • Robust collection and preservation • Capabilities are matched to of evidence submitter and stakeholder needs • Relevant exploitation of samples nd requests a • High discrimination • Appropriate deployable assets, • Enables comparison of “K” and and facilities with sufficient “Q” samples resources for operation and • Utility across known, encountered maintenance sample types • Field, transport, and laboratory • Accuracy evidence integrity and security • Reliability • Properly credentialed, trained and • Defined and acceptable error rate certified personnel • Speed and responsiveness • Full suite of equipment, fully • Repeatability validated methods, matched to • Transferabilitya questions samples received and • Validity can be independently asked established • Comprehensive quality assurance • Probative results are interpretable, and control program explainable, defensible • Appropriate repositories and database •  ppropriate resources: research, A development, test and evaluation, validation and technology transfer a Transferability means that the method can be performed in other laboratories. It is similar to the concept of reproducibility and important for reanalysis or some form of verification. SOURCE: Murch (2008, 2010). (K) and questioned-source (Q) samples are performed with applicable discrimination and resolution; analyses are reliable and repeatable, with clearly defined error rates or defined limitations; and the process yields interpretable, probative results that can be communicated and sup- ported. The ideal forensic science system would comprise the elements in Box 1-3B and enable the marshaling of appropriate scientific methods, tools, equipment, infrastructure, and personnel to meet the needs of the submitter and stakeholders. Such a system would help ensure that qual- ity control and quality assurance can be maintained through the stages of field assessment and analysis, evidence collection and preservation, lab analysis to characterize an unknown or questioned sample and/or

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INTRODUCTION 17 compare it with a sample from a known source, interpretation and conclu- sions, and reporting and communication. The founders of the HMRU recognized that building a microbial forensics discipline would require aggregating a broad range of disci- plines (e.g., epidemiology, genomics,4 metagenomics,5 and other “omics” disciplines, biostatistics and population genetics,6 analytical chemistry and biochemistry, microscopy, bacteriology, mycology, virology, clinical medicine for infectious diseases, veterinary medicine, plant pathology, food science, ecology, materials science, process engineering, physical sci- ences, and bioinformatics and computational science). Murch emphasized that many of these disciplines also are fundamental to public health and medical science. As noted above, the need for a strong union and dynamic collaboration between law enforcement and public health to investigate possible bioattacks from event outset to post-event was recognized early in the development of microbial forensics. Public health, infectious dis- ease medicine, and law enforcement investigations all need to establish whether an event is deliberate, accidental, or natural. Importantly, each can leverage the other’s resources to achieve the same initial objectives. The major difference between the two approaches is that the public health investigation’s goal is to manage the public health response and protect the public’s health and safety, whereas law enforcement’s is to provide safety and security by apprehending and convicting those who commit- ted the attack. Microbial forensics seeks to answer these questions: 1. What is the threat agent? Usually establishing this has not been difficult, although it may not occur in an optimal time frame. 2. Is it probative or relevant? Establishing certainty here is more diffi- cult. Scientists may be working with trace quantities, for example, or analysis may require an understanding of the sample back- ground to understand the source. 3. Can it be linked to a source? Establishing this demands understand- ing the power of methods used to discriminate and characterize with acceptable confidence limits. 4. What are the meaning and weight of the conclusion? The goal of the microbial forensics process is to use microbial analy- 4  The science of studying DNA sequences and properties of entire genomes (Alberts et al., 2002). 5  Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms (http://ghr.nlm.nih.gov/glossary=metagenomics). 6  The study of genetic composition in populations and how natural selection and other factors produce changes in genetic composition.

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24 SCIENCE NEEDS FOR MICROBIAL FORENSICS grams and actions, in 2004 the UN Security Council adopted Resolution 1540, which puts binding obligations on all UN members to address the risks posed by nonstate actors.16 None of these agreements has its own investigative or enforcement capacities. Under the BWC, an allegation of use of biological weapons would be handled through the UN Security Council. The treaty also calls on States Parties to provide assistance to states that have been determined by the Security Council to have suffered an attack (see Box 1-7 for the relevant articles). In the event of an allegation, the U.N. Secretary General has the authority to launch an investigation; the formal title for the process is the Secretary-General’s Mechanism for Investigation of Alleged Use of Chemical and Biological Weapons. This is the process that was used to investigate allegations that chemical weapons had been used in the con- flict in Syria in 2013 with support from the Organization for the Prohibi- tion of Chemical Weapons (OPCW) and the World Health Organization (WHO). Once Syria joined the Chemical Weapons Convention in October 2013, the subsequent elimination of its chemical weapons program has been verified by the OPCW, which implements the treaty. Depending on the scenario, efforts to investigate a case of alleged use would face substantial practical challenges. There could be issues of access to sites, availability and conditions of samples, analytical capac- ity on the ground, concealment of evidence or deliberate efforts to mis- lead investigators, and concerns for their safety (Casagrande presenta- tion, 2013). Depending on the mandate for an investigation, there would almost certainly be questions related to the sensitivity of information and the willingness and capacity of various actors to share, and at present no international agreement or standard governs what will or will not be shared in a given set of circumstances. The successful use of micro- bial forensics in such cases involves difficult policy as well as technical issues. Although the BWC does not have a formal investigative capacity, Dr. Piers Millet from the BWC Implementation Support Unit argued that the treaty is a key international platform for addressing challenges in microbial forensics, on both the technical and policy levels. His presenta- tion focused on drivers for enabling and directing advances in microbial forensic capabilities. Chemical Weapons Convention (Convention on the Prohibition of the Development, Produc- tion, Stockpiling and Use of Chemical Weapons and on their Destruction) also covers toxins. 16  U.N. Security Council Resolution 1540 addresses all weapons of mass destruction and obliges U.N. member states “to refrain from supporting by any means non-State actors from developing, acquiring, manufacturing, possessing, transporting, transferring or using nuclear, chemical or biological weapons and their delivery systems” (UN 1540 Committee website, http://www.un.org/en/sc/1540/#&panel1-16; accessed December 14, 2013).

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INTRODUCTION 25 BOX 1-7 Relevant Articles of the Biological Weapons Convention Article V The States Parties to this Convention undertake to consult one another and to cooperate in solving any problems which may arise in relation to the objective of, or in the application of the provisions of, the Convention. Consultation and Cooperation pursuant to this article may also be undertaken through appropriate international procedures within the framework of the United Nations and in ac- cordance with its Charter. Article VI (1) Any State Party to this convention which finds that any other State Party is acting in breach of obligations deriving from the provisions of the Convention may lodge a complaint with the Security Council of the United Nations. Such a complaint should include all possible evidence confirming its validity, as well as a request for its consideration by the Security Council. (2) Each State Party to this Convention undertakes to cooperate in carrying out any investigation which the Security Council may initiate, in accordance with the provisions of the Charter of the United Nations, on the basis of the complaint re- ceived by the Council. The Security Council shall inform the States Parties to the Convention of the results of the investigation. Article VII Each State Party to this Convention undertakes to provide or support assistance, in accordance with the United Nations Charter, to any Party to the Convention which so requests, if the Security Council decides that such Party has been exposed to danger as a result of violation of the Convention. SOURCE: Text of the Biological Weapons Convention, signed April 10, 1972. With 170 participating states, the consensus achieved in the BWC is meaningful. Since 2002 the BWC has held a series of annual intersessional meetings between the treaty review conferences held every 5 years. The two annual meetings of experts and states parties provide both an expert and a diplomatic component, with different issues addressed each year along with some standing topics. The BWC 2004 annual meetings, for example, examined mechanisms in place in the event a biological/toxic weapon was used or an allegation of use was made. In the final report of the meetings, states parties agreed on the value of (1) continuing to develop national capacities to respond to, investigate, and mitigate poten- tial use of bioweapons; and (2) of doing so in cooperation with relevant regional and international organizations (Biological Weapons Conven-

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26 SCIENCE NEEDS FOR MICROBIAL FORENSICS tion, 2004). Although not explicitly defined, relevant organizations could include regional and international scientific bodies. Moreover, the BWC agreed to encourage and assist other states to pursue these activities. Consensus exists that bioweapon defense should be a global enterprise. The BWC revisited this topic in 2010. Additions to previous agree- ments were made that stressed the importance of 1. Effective efforts, regardless of whether the outbreak is natural or deliberate. Diseases and toxins that harm humans, animals, plants, and the environment fall within the purview. 2. Recognition that capabilities to quickly and effectively detect, respond to, and recover from an alleged use of a biological/toxic weapon must be in place before they are used (United Nations, 2011). 3. Investigating and mitigating the potential impact of an alleged use of biological/toxic weapons in accordance with national laws and regulations (e.g., for data handling). 4. Coordinating a state-government approach to emergency man- agement since multiple arms of a government would be involved. 5. Addressing the full range of possible implications of meeting these expectations (Biological Weapons Convention, 2010). Reviewing advances in S&T was a main focus of the 7th BWC review conference in 2011. The work program for 2012 to 2015 that resulted from the meeting comprises three pillars that are addressed each year—one of which is the examination of relevant advances in S&T (Biological Weap- ons Convention, 2011). Included within its purview are the potential for misuse of S&T (e.g., to make biological weapons) and beneficial applica- tions (e.g., investigating and mitigating bioweapon attacks), as well as interaction between the security and scientific communities. International interest in ensuring that the S&T reviews focus heavily on potential benefits—including advances in microbial forensics—con- tinues, and the BWC now has an international platform to address such issues. Moreover, mechanisms to respond to the use of bioweapons will be a topic at the next two biannual meetings.17 Dr. Dana Perkins from the U.N. 1540 Committee added that microbial forensics is an essential element of a national and international biosecurity infrastructure, both as a deterrent and as a support tool. Similar to nuclear forensics, microbial forensics may be used to detect, prevent, and deter 17  The language in the report is “How to strengthen implementation of Article VII, includ- ing consideration of detailed procedures and mechanisms for the provision of assistance and cooperation by States Parties” (Biological Weapons Convention, 2011:21).

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INTRODUCTION 27 acts of bioterrorism and illicit trafficking or use of biological materials. The potential applications of microbial forensics thus may contribute to strengthening biosecurity in the context of Resolution 1540 and to achieving cooperation and synergy among various international security frameworks. Perkins stated that, in addition to its role in supporting investigations of alleged use, developing and improving microbial forensics methods to detect illicit trafficking and biological materials outside regulatory con- trol, and to prevent and respond to biosecurity events, will strengthen the implementation and enforcement of Resolution 1540. She pointed out for nuclear forensics, the International Atomic Energy Agency (IAEA) has a leading role in facilitating the exchange of information and international collaboration and providing assistance to support law enforcement and assessment of nuclear security vulnerabilities. In contrast, microbial foren- sics lacks that level of international leadership. She suggested, however, that there is potential for much more widespread and effective coop- eration, not only among countries but also among organizations such as WHO, the World Organization for Animal Health (OIE), the Food and Agriculture Organization (FAO) of the United Nations, the Chemi- cal Weapons Convention/Organization for the Prohibition of Chemical Weapons, and the BWC/BWC Implementation Support Unit. (The contri- butions of WHO, OIE, and FAO are discussed further in the “Biosurveil- lance” section of Chapter 2.) Another interesting point can be made about the International Atomic Energy Agency, which has a major role in the development and oversight of the peaceful applications of nuclear energy, as well as nuclear weapons security. This suggests that policy mechanisms for mitigating rare events, such as bioterrorism, should be coupled when possible to more routine-use drivers so that the applicable technologies are tried, tested, and familiar. A sustained effort will be required to (1) build communities of micro- bial forensics specialists, (2) formulate projects to develop microbial foren- sics S&T foundations, and (3) raise awareness of possible synergies among its different applications. Perkins believes that these plans should not be confined to investigating the use and alleged use of weapons but should also focus on contributing to prevention and deterrence of weapon pro- liferation and terrorist threats. Identifying major technical and policy challenges that these plans could support is the subject of the next section and the remainder of this report. AN OVERVIEW OF KEY QUESTIONS AND UNMET NEEDS FOR MICROBIAL FORENSICS Significant scientific challenges or needs have long been recognized in microbial forensics, and have persisted with little or no progress toward

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28 SCIENCE NEEDS FOR MICROBIAL FORENSICS resolution. Murch pointed out that raising the bar in science improves our capabilities of innovation and use and also raises the bar against adver- saries. In addition, many challenges in microbial forensics are shared by other disciplines,18 and so, bridging these gaps could provide leaps in “operationally useful” capabilities and knowledge for more than just microbial forensics. One way to meet the needs would be to enable inte- gration of fundamental science, advancing technologies, and forensic applications with those of related fields for multivariate benefits. The ben- efit would be to push the value of and expectations for microbial forensic investigation/attribution capabilities to the limits of science, technology, and knowledge. This will require recognizing pragmatic priorities—for example, iterative evolutions of technologies, platforms, and methods— that must be designed and implemented to resolve chokepoints and bar- riers that impede progress and success. In addition, we should identify what the “big leaps” are and invest in them. Advancing national and global biosecurity also requires fostering international, cross-disciplinary collaboration in ways not yet recognized. The benefits would apply to both microbial forensics and other basic and applied science fields. The needs of microbial forensics share many aspects with medicine and public health, which are discussed elsewhere in this report. Many of the capabilities required for detecting and responding to the whole spectrum of natural, intentional, and man-made events are essentially the same. The advantages of the microbial forensics and public health communities working together are that systems created for rare events— for example, bioterrorism—may suffer because of lack of use, but those created for addressing natural and accidental outbreaks of infectious disease are likely to be used frequently. Availability of tools and systems compatible with both rare and common occurrences suggest that when a rare event does occur, these tools and systems will be ready and detection and response will not be delayed by lack of familiarity with them. Drs. Randall Murch, Bruce Budowle, and Paul Keim, three of the pioneers of the emerging field of microbial forensics, collaborated on a list of key unmet needs—and questions—in microbial forensics, address- ing methodologies, technologies, applications, and practices. This list was presented in the opening plenary session of the Zagreb workshop to encourage the other participants to define their own ideas about needs and questions throughout the workshop. As Murch, Keim, and Budowle see it, the challenges include 18  This is exemplified by a colloquium convened by the American Academy of Micro- biology in Washington, DC, on September 27–28, 2006, to discuss problems in microbial taxonomy.

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INTRODUCTION 29 • Being able to discriminate with a high level of confidence among similarly presenting natural, accidental, and deliberate out- breaks, within a matter of hours, anywhere in the world. This capability would aid decision-tree design and inform actions of decision makers at all levels to manage response, recovery, and resolution. • Establishing the limits of current and near-term microbial foren- sic characterization methods for identification of priority threat agents at levels more specific than the strain/isolate level. What is the probative value for different methods (e.g., method-specific, agent-specific, single approach, and combined approaches)? • Being able to rapidly develop and validate19 new and agile foren- sic analytical methods as a response to a “surprise” event. Inves- tigators need on-the-shelf capabilities that can be adapted for a wide variety of circumstances. • Sampling and forensic characterization of any relevant microbial background to provide key context for microbial forensic analy- ses, interpretation, communication, and resulting decision mak- ing. There is insufficient understanding of microbial diversity and endemism to inform assessment of where an attack effort may have been developed or perpetrated, or how perpetrators may have exploited the microbial background. And as technologies become more sensitive, it is more likely that organisms that may not have been known to be endemic may be discovered to reside in a particular region. False positives may increase and methods for assessing the significance of false positives and false negatives are necessary. With higher throughput systems, it is conceivable that background sampling could be performed when an event occurs to attempt to define what may be endemic. Of course such an approach requires access to the geographic location of an event, which may not always be feasible. • Determining the probative value of a “small signal” (microbe of interest) in a “big noise” (highly cluttered with other material or microorganisms, or “dirty”) sample, with defined confidence. Environmental samples are very dirty samples. What if we find one cell of interest, but do not understand the background—what does that cell really mean? What can the clutter tell us? How 19  “Validation” describes a number of activities in forensics. Scientific validity depends on two major factors: reliability, which is the ability of a technique to produce consistent and objective results with known precision and accuracy; and relevance, which is necessary to make evidence admissible in court. Both data quality and the interpretation of the data must be validated (Velsko, 2011b). See Chapter 6 for a more detailed discussion of all the elements of validation.

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30 SCIENCE NEEDS FOR MICROBIAL FORENSICS should scientific and legal significance be determined and sup- ported when the agent of interest is a minority constituent in a “probative sample”? How much of the threat agent of interest must be contained in a sample to be considered significant? • Exploiting the “clutter” (microbiota20 other than the threat agent of interest) in metagenomic samples for forensic value, including potential use in the comparison of samples from known and ques- tioned sources. In metagenomic samples (including highly com- plex samples), can the “clutter” provide more power to micro- bial forensic analyses? Can it meet “forensic standards”? What is required to demonstrate viability of this approach, including the limits on analysis conclusions? • Determining other-than-genomic approaches that can be devel- oped, validated, and integrated for deeper forensic characteriza- tion, including other omics (e.g., proteomics), and approaches such as multitarget analysis of culture media. • Determining the maximal characterization for forensic value that can be achieved for biological toxins. In addition to identification, can analyses be developed to aid rule-in/rule-out determinations? • Maximally reducing the “discovery-to-decision” timeline, across all threat agents, with maximal probative value and confidence. The answer could be integrated with the decision process. • Establishing how to best validate low-level analytics (very small quantities of a target analyte) in an operational setting. Precise identification of an analyte (e.g., DNA) might be made to the level of a distinct strain (e.g., canonical single-nucleotide polymor- phism [canSNP])21 or other molecule (e.g., isotope signature)22 at the few- or even single-molecule(s) level. 20  The microorganisms of a particular site, region, or habitat. 21  “A single nucleotide polymorphism (SNP) is a DNA sequence variation that occurs when a single nucleotide (A, T, C, or G) in a gene sequence is altered (Orho-Melander, 2006). SNPs are useful for both diagnostic identification and phylogenetic population analysis. There is a model for identifying nucleic acid “signatures” (a set of DNA-related molecular markers) that is based on evolutionary rules and comparative genomic analysis. This model is the foundation for identifying key diagnostic features called “canonical characters,” one of which is a canonical SNP (canSNP). A canonical diagnostic character marks a pivotal evolutionary point in the development of an organism and, therefore, represents multiple evolutionary differences. These “signatures” can be used to discriminate between target and nontarget species. A single canSNP can identify a particular species, subpopulation, and/or isolate (Engelthaler and Balajee, 2011; Keim et al., 2004). 22  “An isotopic signature (or isotopic fingerprint) is a ratio of stable or unstable isotopes of particular elements found in an investigated material. The atomic mass of different iso- topes affects their chemical kinetic behavior, leading to natural isotopic separation process.” (http://www.springerreference.com/docs/html/chapterdbid/330809.html).

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INTRODUCTION 31 • International data-sharing forums and quality and nomencla- ture standards. These are essential. Governmental restrictions on sharing material have limited development of global databases needed to provide confidence in microbial forensic analysis. The political obstacles are likely greater than the S&T obstacles. • Deep sequencing23 offers methodological advantages over the Sanger method24 (throughput, speed, cost) but generates a lot of data. Should databases be established in advance or be generated as events occur? • Determining how to measure with certainty and report whole- genome–sequencing25 comparisons performed during forensic analysis (e.g., comparing an evidence sample “profile” with a reference sample that may be considered a direct link or have a common ancestor). Sequencing errors and other factors will likely inflate dissimilarity between samples, creating a degree of uncer- tainty. Defining and quantifying the error rates associated with each platform and chemistry are critical. How do we accomplish this in a communal way? • Ensuring that the quality of sequence data and the results of bioinformatics analyses is as high as possible. Factors to consider that affect data interpretation and quality include reliability of standards for genomic data representation; uncertainty about databases (e.g., inferences based on available data, including metadata26); sequence errors and uncertainties; criteria for com- parisons (match, similar, different, inconclusive); and the rigor of expert reasoning, which should include formulating well-defined hypotheses, and testing methods for assessing the weight of microbial forensics evidence. • Replicating all the essential details of any particular bioinfor- matics analysis pipeline by different labs is as much art as it is mathematics and science. Investigators must understand it to use it. It is not easy to transfer; there are analytical complexi- ties. There are also multiple technologies, and different versions 23  “Techniques of nucleotide sequence analysis that increase the range, complexity, sen- sitivity, and accuracy of results by greatly increasing the scale of operations and thus the number of nucleotides, and the number of copies of each nucleotide sequenced” (http:// www.urmc.rochester.edu/profiles/display/123618). 24  A widely used method of determining the order of bases. A base is one of the molecules (adenine, thymine, guanine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA) that forms part of a DNA and RNA nucleotide. 25  Determining the complete nucleotide sequence of an organism’s DNA. 26  Data about other data.

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32 SCIENCE NEEDS FOR MICROBIAL FORENSICS of programs and data assessments, and software and hardware change rapidly. • No bioinformatics software comes as a stand-alone. How much documentation is needed? What baseline truths are needed to ensure that assessments and comparisons of technologies can be made effectively? • Integrating disparate data to provide a single value (e.g., can genomics analysis be added to physical/chemical analysis to yield a single value or answer; can traditional forensics, intelli- gence, prior odds, networks, be incorporated?) Is simplicity desir- able for conveying information “up the chain” to decision mak- ers? Should disparate data be maintained as separate entities? • Avoiding the filtering of data on the basis of individual prefer- ences and bias. • Instituting processes to inform decision makers in a way that ensures that the science is properly understood. Many nonscien- tists who make decisions based on forensic science make those decisions based in large part on media, such as television. The overall goal for microbial forensics is to move as far to the left as possible on the time-risk continuum portrayed in Figure 1-3. Microbial forensics results that are very informative, have high confidence, and are rapidly obtained—and perhaps better leveraged with other capabilities— could enable investigators to manage risks so that energy is dedicated to anticipating and preparing for an event rather than reacting to a surprise, scrambling to mitigate consequences, and seeking attribution. Currently, microbial forensics directs much of its science to operations on the wrong end of this time-risk timeline (Murch presentation, 2013). Dynamic cross-disciplinary international collaboration, however, could serve to improve and develop better microbial forensics capacities, shift- ing the focus of microbial forensics to the left on the continuum, and at the same time contribute to the strength of other sciences. ORGANIZATION OF THE REPORT As discussed at the beginning of the chapter, the report prepared by the committee draws upon the material presented in the symposium as well as other evidence. This chapter has provided an introduction to the field and to major technical and policy issues. Among its key messages: • The law enforcement goal of microbial forensics is “attribution”— that is, determining who committed the offense. Based on the

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INTRODUCTION 33 Time-Risk Continuum Overall Goal is to Move to the Left as Far As Possible (Very Informative, High Confidence and Rapid Microbial Forensics Better Leveraged with Other Capabilities Could Help) We should be working smarter to plan and operate We plan and operate mostly more here over here Nothing Should Get Past Here Very Good Time-Risk Very Bad Anticipation Attribution, Prosecution Preemption Mitigation Disruption & Defeat Surprise Recovery, Cleanup Finger Pointing Reaction Lesson Learned Response Risk Reduction FIGURE 1-3  Time-risk continuum. The goal is to collapse time—to reduce risk by planning and preparing for an event rather than targeting efforts to reacting to an event. SOURCE: Murch presentation, 2013. analysis of biological and other evidence, law enforcement builds a case for attribution to a specific source or sources. The evidence supporting attribution must be robust and suitable for use in legal proceedings and to inform decision making at the highest levels. • Public health, infectious disease medicine, and law enforcement investigations all need to establish whether an event is deliber- ate, accidental, or natural. Importantly, each can leverage the other’s resources to achieve the same initial objectives. The major difference between the two approaches is that the public health investigation’s goal is to manage the public health response and protect the public’s health and safety, whereas law enforcement’s is to provide safety and security by apprehending and convicting those who committed the attack. • The needs of microbial forensics share many aspects with med- icine and public health. Many of the capabilities required for detecting and responding to the whole spectrum of natural, intentional, and man-made events are essentially the same. The advantages of the microbial forensics and public health commu-

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34 SCIENCE NEEDS FOR MICROBIAL FORENSICS nities working together are that systems created for rare events (e.g., bioterrorism) may suffer through lack of use, whereas those created for addressing natural and accidental outbreaks of infec- tious disease are likely to be used frequently. Developing tools and systems compatible with both rare and common occurrences means that when a rare event does occur, these tools and systems will be ready and detection and response will not be delayed by lack of familiarity with them. These messages are illustrated and developed in the remainder of the report, with a focus on the major scientific, technological, and policy and process issues that need to be addressed to develop the field of microbial forensics. Chapters 2 through 7 follow the general order of the sympo- sium, while Chapter 8 presents the committee’s findings and conclusions, which follow from these chapters and respond to the major messages as well as to the “Key Questions and Unmet Needs” presented in the sec- tion above.