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Summary Summary One of the fundamental components of any investigation of a suspected accidental release of a dangerous pathogen or the alleged hostile use of biological agents, whether by states or nonstate actors, will be scientific analysis to support efforts for attribution. Science will not offer definitive solutions in such scenarios, but it often plays a special role in supporting other aspects of an investigation. The United States and a number of other governments and international and regional organizations are actively working to identify and support the research that is needed to build those capabilities, which are known generally as “microbial forensics.” Microbial forensics deals with the genetic and other materials associated with microorganisms. Contrary to the images from popular media, 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 threat agents poses substantial dangers to individuals, public health, the environment, the economies of nations, and global peace. It is likely that scientific, political, and media-based controversy will surround any investigation of the alleged use of a biological agent. For these reasons, building awareness of and capacity in microbial forensics can assist in our understanding of what may have occurred during a biothreat event and facilitate international collaborations that engage the broader scientific and policy-making communities that are likely to strengthen our microbial forensics capabilities. One goal is to create a shared technical understanding of the possibilities—and limitations— of the scientific bases for microbial forensics analyses. Another is to identify the range of scientific needs to continue the field’s development. Microbial forensics has additional 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 public health needs highlighted in this report reflect the global reality that in most countries the capabilities and activities relevant to microbial forensics occur only in the context of public health. Particularly the United States has a microbial forensics science community that is truly differentiated from public health, and a limited number of other countries have some basic microbial forensics infrastructure. 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 law enforcement. With these needs and realities in mind, a group of national and international scientific organizations undertook a collaboration whose centerpiece was a workshop held in fall 2013 in Zagreb, Croatia. As mentioned above, the specific goals of the workshop (and the larger collaboration) were to:  Foster collaboration within the international scientific community to support technical understanding and enhanced research on microbial forensics, and  Develop the beginnings of an international roadmap for how to do the necessary science, including priorities among potential topics. The partner organizations were 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 hosted the workshop at its headquarters in Zagreb. Participants included 59 experts from 21 countries and several international organizations, spanning a range of researchers and clinicians from numerous scientific and technological disciplines related or applicable to Prepublication Copy 1

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Summary microbial forensics as well as technical experts and policy makers with a strong interest in the contributions of science and technology to security. The two-and-a-half-day meeting was the primary evidence-gathering mechanism for an ad hoc committee with substantial international membership appointed by the NAS’s National Research Council (NRC). The NRC committee supplemented the information obtained during the workshop with other evidence to prepare a final consensus report, which offers a series of conclusions for advancing the field. Funding for the project was provided by the U.S. Navy, the U.S. Department of State, and the NAS. 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 are enabling dramatic advances in both basic and applied research. The committee also identified important procedural and policy needs, such as common understandings and protocols for taking and managing samples within and across nations. Meeting these additional needs is essential to ensure that the scientific and technical developments the committee calls for are to make microbial forensics a more effective tool for investigating suspicious disease outbreaks. 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 conclusions for moving forward. The report is not meant to provide a detailed roadmap for the international development of microbial 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 knowledge 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. The statement of task for this project calls for the committee to “develop the beginnings of an international roadmap for how to do the necessary science, including priorities among potential topics.” The list of needs identified in Table S.1 is long, but the successful development of microbial forensics will require addressing all of them. There are considerable differences in how difficult it will be to achieve the priorities and in whether there are already existing national or international efforts, for example, in basic research or public health or by industry, which can be drawn upon to help achieve them. Many scientific and technical communities will be able to make contributions. These communities should also be able to take advantage of other initiatives, for example, to advance disease surveillance and diagnostics for public health purposes, particularly if there is a conscious effort to foster communication across the many relevant disciplines and technical fields. There are 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 require substantial and sustained support from governments as major funders of the research, development, and implementation that will be essential for achieving success. Box S.1 presents the needs identified in this report 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                                                              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 international 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). Prepublication Copy 2

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Summary 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 thinking about implementation issues and for the development of a more detailed roadmap to guide future efforts. Box S-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 diseases. (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) Prepublication Copy 3

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Summary  Priority research 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 (polymerase chain reaction, whole-genome sequencing, etc.), including refinement and distribution of benchtop next generation gene sequencing 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 ) 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, sequencing, etc.) 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)                   Prepublication Copy 4