- Email: [email protected]
Microbial forensics
C H A P T E R
33 Microbial forensics: what next? Stephen A. Morse1, Bruce Budowle2, Steven E. Schutzer3 1
IHRC, Inc., Atlanta, GA, United States; 2Center for Human Identification, Department of Microbiology, Immunology and Genetics, University of North Texas Health Science Center, Ft Worth, TX, United States; 3Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ, United States
Microbial forensics combines genomic, microbiologic, and epidemiologic methods to identify, characterize, and ascribe the cause of an incident or the processes used to produce a weaponizable agent resulting from the intentional or unintentional release of a harmful pathogen or toxin. Before October 2001, several forensic investigations of events involving microbial agents had been carried out to address public health questions as well as attribution. Microbial forensic approaches, although not termed as such at the time, were used to attribute the “reemergence” of H1N1 human influenza in 1977 to the escape from a laboratory in China or Russia of a virus sample that had been originally isolated around 1950 (Zimmer and Burke, 2009). Similarly, microbial forensic techniques were used to retrospectively show that inadequately inactivated vaccines were likely responsible for most VEE outbreaks that occurred in Latin America between 1938 and 1972 (Powers et al., 1997). Perhaps, more notable were investigations on intentional releases such as Salmonella contamination of salad bars in the Dalles, OR (Torok et al., 1997), contamination of muffins with
Microbial Forensics, Third Edition https://doi.org/10.1016/B978-0-12-815379-6.00033-7
Shigella dysenteriae in Dallas, TX (Kolavic et al., 1997), the Aum Shinrikyo Kameido “anthrax” attack (Keim et al., 2001), and the retrospective analysis of the unintentional Sverdlovsk anthrax incident (Meselson et al., 1994). In all these examples, the subsequent investigations involved infectious disease epidemiology and the use of laboratory methods, including molecular genetic typing, which played an important role. However, the technical analyses employed were primitive by today’s standards, and the involvement of law enforcement forensic laboratories was limited or nonexistent. Microbial forensics as a discipline was affected dramatically by the dissemination of spores of Bacillus anthracis through the US postal system and the intense investigatory effort associated with the Amerithrax investigation that followed. The Amerithrax case involved highly sophisticated technologies of the time, the development of novel scientific analytical approaches, and was driven by federal law enforcement efforts (National Research Council, 2011). In addition to the massive scale of the Amerithrax investigation, the engagement of the law enforcement
497
© 2020 Elsevier Inc. All rights reserved.
498
33. Microbial forensics: what next?
community led to new standards for microbial analyses that have had a positive effect on how epidemiologists and public health officials approach normal disease outbreaks. Doubtlessly, future criminal investigations will quickly result in law enforcement-driven forensic analysis and will capitalize on the very latest in technological and bioinformatics innovations. Consequently, analysis standards will be set high to support the prosecution of the perpetrators within the judicial system and to exculpate individuals not involved in an attack. Microbial forensics is no longer just a “side activity” for epidemiologists but rather a discipline all its own that will rely on specialists trained in multiple disciplines. Since the publication of the second edition of this book (Budowle et al., 2011), several excellent books have been published (Cano and Toranzos, 2018; Iftikhar, 2019; Carter et al., 2017; Cliff et al., 2012) or already existed (Riley, 2004; Emanuel et al., 2008) on various aspects of forensics, pathogen tracking, and molecular epidemiology. However, this book is unique in trying to combine the essential components and timely topics of microbial forensics into a coherent body of work. The third edition of Microbial Forensics covers a broad range of topics relevant to microbial forensics and in a manner focused on this topic. As such, we believe that it remains the definitive guide on the topic, to date, and will be useful to a broad range of readers interested in microbial forensics. While molecular forensic methods can enhance an epidemiological investigation of a naturally occurring disease outbreak, an important difference between an epidemiological investigation and a microbial forensics epidemiological investigation is that results of the latter need to withstand the scrutiny of the legal system, especially an adversarial one. Controlling the crime scene, maintaining chain of custody, validating methods, proficiency testing, defendable interpretation of results, and associated degrees of uncertainty will need to be performed to very high standards.
Likewise, the actual methodologies for microbial forensics are evolving rapidly and new methods are being devised and applied to investigations of disease outbreaks as well as biocrimes. The explosion in genomics, phylogeography, and population dynamics is evident to all; however, tremendous improvements are also being made in the physical and material sciences for understanding the exact evidentiary composition from isotopes to elements. Coupling highresolution microscopy to elemental analysis was a key to understanding the anthrax letter spores (National Research Council, 2011). A complementary suite of methodologies will ultimately provide scientists with better investigative strategies. Preparing to investigate future criminal events has largely been focused on a small list of pathogens and toxins, primarily developed from knowledge of state biological weapons programs (Khan et al., 2000). The biology of each pathogen and toxin will necessitate agent-specific expertise and analysis. Inevitably, an unanticipated pathogen or toxin will be used in a future attack, requiring microbial forensics to adapt previous approaches to a novel event. Practitioners will gain considerable expertise by using these approaches for natural outbreaks of novel, emerging, or reemerging infectious diseases. A better understanding of these agents will lead to a quicker response when one is used in a criminal event. Finally, microbial forensic investigations must be approached with an eye toward the final legal stage. Investigators must be using methods that will meet legal standards for scientific evidence (e.g., the Daubert admissibility standard) and validated with a level of rigor that can be defended successfully against inevitable critical objections. This required rigor and special demands lead to the question of whether microbial forensics will become a stand-alone science or whether it will remain a subspecialty of others. It is currently organized as either a subspecialty of infectious disease epidemiology or of traditional
V. Miscellaneous
33. Microbial forensics: what next?
forensics, and its growth into its own discipline likely will proceed for many years. The necessity to be a separate discipline will be dependent on the number of biocrime events and the resources available for such investigations. The specialized need to conduct forensic examinations in a highcontainment environment for biosafety purposes does not separate it from molecular epidemiology and traditional forensics. The National Bioforensic Analysis Center located at Ft. Detrick, Frederick, MD, is the US government’s response to this need for an infrastructure with both capabilities. However, if microbial forensics is to be practiced outside this single-focused environment, it will likely be closely associated with either infectious disease epidemiology or forensics. Indeed, other national assets currently are relied on for their unique expertise to address the novel events that occur. Biocrimes may remain rare events; thus, while local disease control and law enforcement efforts will need to be prepared continuously (Treadwell et al., 2003), microbial forensics will need to be practiced less frequently than many traditional forensic applications. Maintaining widespread capacity focused solely on microbial forensics would appear to be unneeded and expensive. However, preparedness is essential. We would envision some capacity, training, and preparedness in all regions and locations and across both public health and law enforcement agencies. As technological advances occur, such as next-generation sequencing, the democratization of the capability to perform genomic analyses and chemical analyses will allow for ready access by investigating agencies of the tools to meet the challenges and carry out attribution of a biocrime or bioterrorist act. Challenges still exist for efficient and effective microbial forensic investigations, including the development and maintenance of large strain repositories as references to evidentiary material. While genetic methods have become less expensive, faster, and with better discrimination power, strain repositories remain problematic.
499
Federal regulations, agency-specific restrictions, and international paranoia have placed additional barriers for strain acquisition and sharing. Complicating the situation is the need for comprehensive strain (or source) collections for each pathogen and toxin investigated, which multiplies the size of the challenge, effective coordination, and the expense. In addition, genomic technologies are changing so fast that the standardization of methods and even data formats remains a hurdle. Error rates and differential quality across data sets must be estimated, captured, and understood to minimize false investigative leads, which would expend resources unnecessarily, degrade public confidence in microbial forensic analysis, and, ultimately, impact the ability to prosecute perpetrators successfully. Evolutionary models, coupled with repository databases, need to be applied to evidence to generate confidence estimates associated with any results. Regardless of analysis results, their interpretation and subsequent communication to peers, lawyers, judges, juries, and other stakeholders will have to be effective. The microbial forensics community must strive for a common language to describe investigative outcomes effectively and accurately. Prospects for this are good if the science is rigorous and the community is open to critical review. Crimes of all types cost societies valuable resources in term of money, productivity, safety, and peace of mind. Indeed, in extreme cases, the cost is measured in lives lost. Ineffective and inaccurate forensics compounds these societal problems with injustice while failing to curtail crime. Scientific rigor needs to be applied to microbial forensics to maximize its effectiveness and benefits. Hopefully, this will result in fewer biocrimes and bioterrorist events by removing perpetrators as well as providing a deterrent to future perpetrators. Performing microbial forensics accurately with valid interpretation will not always result in the identification of the perpetrator, but inappropriate use of microbial
V. Miscellaneous
500
33. Microbial forensics: what next?
forensics that attributes a crime to an individual, group, or state erroneously will come with a much greater cost. The work and developments from the nascence of microbial forensics to date suggest that we are on the right track. Foundations have been laid and we must remain vigil to ensure that the standards and high quality that we advocate continue to be a major part of the growth of the very important discipline microbial forensics.
References Cano, R.J., Toranzos, G.A. (Eds.), 2018. Environmental Microbial Forensics. ASM Press, Washington, DC. Budowle, B., Schutzer, S.E., Breeze, R.G., Keim, P.S., Morse, S.A. (Eds.), 2011. Microbial Forensics, second ed. Academic Press, San Diego. Carter, D.O., Tomberlin, J.K., Benbow, M.E., Metcalf, J.L. (Eds.), 2017. Forensic Microbiology, first ed. John Wiley & Sons, Hoboken, NJ. Cliff, J.B., Kreuzer, H.W., Ehrhardt, C.J., Wunschel, D.S. (Eds.), 2012. Chemical and Physical Signatures for Microbial Forensics. Humana Press, NY. Emanuel, P., Roos, J.W., Niyogi, K., 2008. Sampling for Biological Agents in the Environment. ASM Press, Washington, DC. Iftikhar, S. (Ed.), 2019. Trends of Environmental Forensics in Pakistan. Academic Press, London. Keim, P., Smith, K.L., Keys, C., Takahashi, H., Kurata, T., Kaufmann, A., 2001. Molecular investigation of the
Aum Shinrikyo anthrax release in Kameido, Japan. J. Clin. Microbiol. 39, 4566e4567. Khan, A.S., Morse, S., Lillibridge, S., 2000. Public-health preparedness for biological terrorism in the USA. Lancet 356, 1179e1182. Kolavic, S.A., Kimura, A., Simmons, S.L., Slutsker, S.L., Barth, S., Haley, C.E., 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA. 278, 396e398. Meselson, M., Guillemin, J., Hugh-Jones, M., et al., 1994. The Sverdlovsk anthrax outbreak of 1979. Science 266, 1202e1208. National Research Council, 2011. Review of the Scientific Approaches Used during the FBI's Investigation of the 2001 Anthrax Letters. The National Academies Press, Washington, DC. Powers, A.M., Oberste, M.S., Brault, A.C., Rico-Hesse, R., Schmura, S.M., Smith, J.F., et al., 1997. Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis from a single genotype of enzootic subtype ID virus. J. Virol. 71, 6697e6705. Riley, L.W., 2004. Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. Torok, T.J., Tauxe, R.V., Wise, R.P., Livengood, J.R., Sokolow, R., Mauvais, S., et al., 1997. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA. 278, 389e395. Treadwell, T.A., Koo, D., Kuker, K., Khan, A.S., 2003. Epidemiologic clues to bioterrorism. Public Health Rep. 118, 92e98. Zimmer, S.M., Burke, D.S., 2009. Historical perspective e emergence of influenza A (H1N1) viruses. N. Engl. J. Med. 361, 279e285.
V. Miscellaneous
33 Microbial forensics: what next? Stephen A. Morse1, Bruce Budowle2, Steven E. Schutzer3 1
IHRC, Inc., Atlanta, GA, United States; 2Center for Human Identification, Department of Microbiology, Immunology and Genetics, University of North Texas Health Science Center, Ft Worth, TX, United States; 3Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ, United States
Microbial forensics combines genomic, microbiologic, and epidemiologic methods to identify, characterize, and ascribe the cause of an incident or the processes used to produce a weaponizable agent resulting from the intentional or unintentional release of a harmful pathogen or toxin. Before October 2001, several forensic investigations of events involving microbial agents had been carried out to address public health questions as well as attribution. Microbial forensic approaches, although not termed as such at the time, were used to attribute the “reemergence” of H1N1 human influenza in 1977 to the escape from a laboratory in China or Russia of a virus sample that had been originally isolated around 1950 (Zimmer and Burke, 2009). Similarly, microbial forensic techniques were used to retrospectively show that inadequately inactivated vaccines were likely responsible for most VEE outbreaks that occurred in Latin America between 1938 and 1972 (Powers et al., 1997). Perhaps, more notable were investigations on intentional releases such as Salmonella contamination of salad bars in the Dalles, OR (Torok et al., 1997), contamination of muffins with
Microbial Forensics, Third Edition https://doi.org/10.1016/B978-0-12-815379-6.00033-7
Shigella dysenteriae in Dallas, TX (Kolavic et al., 1997), the Aum Shinrikyo Kameido “anthrax” attack (Keim et al., 2001), and the retrospective analysis of the unintentional Sverdlovsk anthrax incident (Meselson et al., 1994). In all these examples, the subsequent investigations involved infectious disease epidemiology and the use of laboratory methods, including molecular genetic typing, which played an important role. However, the technical analyses employed were primitive by today’s standards, and the involvement of law enforcement forensic laboratories was limited or nonexistent. Microbial forensics as a discipline was affected dramatically by the dissemination of spores of Bacillus anthracis through the US postal system and the intense investigatory effort associated with the Amerithrax investigation that followed. The Amerithrax case involved highly sophisticated technologies of the time, the development of novel scientific analytical approaches, and was driven by federal law enforcement efforts (National Research Council, 2011). In addition to the massive scale of the Amerithrax investigation, the engagement of the law enforcement
497
© 2020 Elsevier Inc. All rights reserved.
498
33. Microbial forensics: what next?
community led to new standards for microbial analyses that have had a positive effect on how epidemiologists and public health officials approach normal disease outbreaks. Doubtlessly, future criminal investigations will quickly result in law enforcement-driven forensic analysis and will capitalize on the very latest in technological and bioinformatics innovations. Consequently, analysis standards will be set high to support the prosecution of the perpetrators within the judicial system and to exculpate individuals not involved in an attack. Microbial forensics is no longer just a “side activity” for epidemiologists but rather a discipline all its own that will rely on specialists trained in multiple disciplines. Since the publication of the second edition of this book (Budowle et al., 2011), several excellent books have been published (Cano and Toranzos, 2018; Iftikhar, 2019; Carter et al., 2017; Cliff et al., 2012) or already existed (Riley, 2004; Emanuel et al., 2008) on various aspects of forensics, pathogen tracking, and molecular epidemiology. However, this book is unique in trying to combine the essential components and timely topics of microbial forensics into a coherent body of work. The third edition of Microbial Forensics covers a broad range of topics relevant to microbial forensics and in a manner focused on this topic. As such, we believe that it remains the definitive guide on the topic, to date, and will be useful to a broad range of readers interested in microbial forensics. While molecular forensic methods can enhance an epidemiological investigation of a naturally occurring disease outbreak, an important difference between an epidemiological investigation and a microbial forensics epidemiological investigation is that results of the latter need to withstand the scrutiny of the legal system, especially an adversarial one. Controlling the crime scene, maintaining chain of custody, validating methods, proficiency testing, defendable interpretation of results, and associated degrees of uncertainty will need to be performed to very high standards.
Likewise, the actual methodologies for microbial forensics are evolving rapidly and new methods are being devised and applied to investigations of disease outbreaks as well as biocrimes. The explosion in genomics, phylogeography, and population dynamics is evident to all; however, tremendous improvements are also being made in the physical and material sciences for understanding the exact evidentiary composition from isotopes to elements. Coupling highresolution microscopy to elemental analysis was a key to understanding the anthrax letter spores (National Research Council, 2011). A complementary suite of methodologies will ultimately provide scientists with better investigative strategies. Preparing to investigate future criminal events has largely been focused on a small list of pathogens and toxins, primarily developed from knowledge of state biological weapons programs (Khan et al., 2000). The biology of each pathogen and toxin will necessitate agent-specific expertise and analysis. Inevitably, an unanticipated pathogen or toxin will be used in a future attack, requiring microbial forensics to adapt previous approaches to a novel event. Practitioners will gain considerable expertise by using these approaches for natural outbreaks of novel, emerging, or reemerging infectious diseases. A better understanding of these agents will lead to a quicker response when one is used in a criminal event. Finally, microbial forensic investigations must be approached with an eye toward the final legal stage. Investigators must be using methods that will meet legal standards for scientific evidence (e.g., the Daubert admissibility standard) and validated with a level of rigor that can be defended successfully against inevitable critical objections. This required rigor and special demands lead to the question of whether microbial forensics will become a stand-alone science or whether it will remain a subspecialty of others. It is currently organized as either a subspecialty of infectious disease epidemiology or of traditional
V. Miscellaneous
33. Microbial forensics: what next?
forensics, and its growth into its own discipline likely will proceed for many years. The necessity to be a separate discipline will be dependent on the number of biocrime events and the resources available for such investigations. The specialized need to conduct forensic examinations in a highcontainment environment for biosafety purposes does not separate it from molecular epidemiology and traditional forensics. The National Bioforensic Analysis Center located at Ft. Detrick, Frederick, MD, is the US government’s response to this need for an infrastructure with both capabilities. However, if microbial forensics is to be practiced outside this single-focused environment, it will likely be closely associated with either infectious disease epidemiology or forensics. Indeed, other national assets currently are relied on for their unique expertise to address the novel events that occur. Biocrimes may remain rare events; thus, while local disease control and law enforcement efforts will need to be prepared continuously (Treadwell et al., 2003), microbial forensics will need to be practiced less frequently than many traditional forensic applications. Maintaining widespread capacity focused solely on microbial forensics would appear to be unneeded and expensive. However, preparedness is essential. We would envision some capacity, training, and preparedness in all regions and locations and across both public health and law enforcement agencies. As technological advances occur, such as next-generation sequencing, the democratization of the capability to perform genomic analyses and chemical analyses will allow for ready access by investigating agencies of the tools to meet the challenges and carry out attribution of a biocrime or bioterrorist act. Challenges still exist for efficient and effective microbial forensic investigations, including the development and maintenance of large strain repositories as references to evidentiary material. While genetic methods have become less expensive, faster, and with better discrimination power, strain repositories remain problematic.
499
Federal regulations, agency-specific restrictions, and international paranoia have placed additional barriers for strain acquisition and sharing. Complicating the situation is the need for comprehensive strain (or source) collections for each pathogen and toxin investigated, which multiplies the size of the challenge, effective coordination, and the expense. In addition, genomic technologies are changing so fast that the standardization of methods and even data formats remains a hurdle. Error rates and differential quality across data sets must be estimated, captured, and understood to minimize false investigative leads, which would expend resources unnecessarily, degrade public confidence in microbial forensic analysis, and, ultimately, impact the ability to prosecute perpetrators successfully. Evolutionary models, coupled with repository databases, need to be applied to evidence to generate confidence estimates associated with any results. Regardless of analysis results, their interpretation and subsequent communication to peers, lawyers, judges, juries, and other stakeholders will have to be effective. The microbial forensics community must strive for a common language to describe investigative outcomes effectively and accurately. Prospects for this are good if the science is rigorous and the community is open to critical review. Crimes of all types cost societies valuable resources in term of money, productivity, safety, and peace of mind. Indeed, in extreme cases, the cost is measured in lives lost. Ineffective and inaccurate forensics compounds these societal problems with injustice while failing to curtail crime. Scientific rigor needs to be applied to microbial forensics to maximize its effectiveness and benefits. Hopefully, this will result in fewer biocrimes and bioterrorist events by removing perpetrators as well as providing a deterrent to future perpetrators. Performing microbial forensics accurately with valid interpretation will not always result in the identification of the perpetrator, but inappropriate use of microbial
V. Miscellaneous
500
33. Microbial forensics: what next?
forensics that attributes a crime to an individual, group, or state erroneously will come with a much greater cost. The work and developments from the nascence of microbial forensics to date suggest that we are on the right track. Foundations have been laid and we must remain vigil to ensure that the standards and high quality that we advocate continue to be a major part of the growth of the very important discipline microbial forensics.
References Cano, R.J., Toranzos, G.A. (Eds.), 2018. Environmental Microbial Forensics. ASM Press, Washington, DC. Budowle, B., Schutzer, S.E., Breeze, R.G., Keim, P.S., Morse, S.A. (Eds.), 2011. Microbial Forensics, second ed. Academic Press, San Diego. Carter, D.O., Tomberlin, J.K., Benbow, M.E., Metcalf, J.L. (Eds.), 2017. Forensic Microbiology, first ed. John Wiley & Sons, Hoboken, NJ. Cliff, J.B., Kreuzer, H.W., Ehrhardt, C.J., Wunschel, D.S. (Eds.), 2012. Chemical and Physical Signatures for Microbial Forensics. Humana Press, NY. Emanuel, P., Roos, J.W., Niyogi, K., 2008. Sampling for Biological Agents in the Environment. ASM Press, Washington, DC. Iftikhar, S. (Ed.), 2019. Trends of Environmental Forensics in Pakistan. Academic Press, London. Keim, P., Smith, K.L., Keys, C., Takahashi, H., Kurata, T., Kaufmann, A., 2001. Molecular investigation of the
Aum Shinrikyo anthrax release in Kameido, Japan. J. Clin. Microbiol. 39, 4566e4567. Khan, A.S., Morse, S., Lillibridge, S., 2000. Public-health preparedness for biological terrorism in the USA. Lancet 356, 1179e1182. Kolavic, S.A., Kimura, A., Simmons, S.L., Slutsker, S.L., Barth, S., Haley, C.E., 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA. 278, 396e398. Meselson, M., Guillemin, J., Hugh-Jones, M., et al., 1994. The Sverdlovsk anthrax outbreak of 1979. Science 266, 1202e1208. National Research Council, 2011. Review of the Scientific Approaches Used during the FBI's Investigation of the 2001 Anthrax Letters. The National Academies Press, Washington, DC. Powers, A.M., Oberste, M.S., Brault, A.C., Rico-Hesse, R., Schmura, S.M., Smith, J.F., et al., 1997. Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis from a single genotype of enzootic subtype ID virus. J. Virol. 71, 6697e6705. Riley, L.W., 2004. Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. Torok, T.J., Tauxe, R.V., Wise, R.P., Livengood, J.R., Sokolow, R., Mauvais, S., et al., 1997. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA. 278, 389e395. Treadwell, T.A., Koo, D., Kuker, K., Khan, A.S., 2003. Epidemiologic clues to bioterrorism. Public Health Rep. 118, 92e98. Zimmer, S.M., Burke, D.S., 2009. Historical perspective e emergence of influenza A (H1N1) viruses. N. Engl. J. Med. 361, 279e285.
V. Miscellaneous