Table Of Content

TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. L ABORATORY A SPECTS OF B IOLOGICAL W ARFARE A GENTS Edited Mark J. Wolcott, PhD, MT(ASCP) UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Department of Defense. The mention of specific commercial entities does not imply endorsement by the US Army or the Department of Defense. Updated: Jan 2016 Cleared for all audiences: PR-12-206 1 Oct 12 (MRMC) UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Table of Contents Foreword ........................................................................................................................................................................ 9 Bacillus anthracis ......................................................................................................................................................... 11 OVERVIEW ............................................................................................................................................................. 11 ORGANISM INFORMATION ................................................................................................................................... 11 Taxonomy Information. ....................................................................................................................................... 11 Virulence Factors. ............................................................................................................................................... 12 Epidemiology and Endemic Areas. ..................................................................................................................... 13 LABORATORY DIAGNOSTICS .............................................................................................................................. 14 Biosafety Information. ......................................................................................................................................... 14 Diagnostic Information. ....................................................................................................................................... 14 Immunoassay Tests. .......................................................................................................................................... 19 Nucleic Acid Detection Tests. ............................................................................................................................. 21 Other Diagnostic Methods. ................................................................................................................................. 22 Characterization. ................................................................................................................................................ 22 Confirmation. ...................................................................................................................................................... 22 REFERENCES ........................................................................................................................................................ 25 Yersinia pestis .............................................................................................................................................................. 30 OVERVIEW ............................................................................................................................................................. 30 ORGANISM INFORMATION ................................................................................................................................... 30 Taxonomy Information. ....................................................................................................................................... 30 Virulence Factors. ............................................................................................................................................... 31 Epidemiology and Endemic Areas. ..................................................................................................................... 34 LABORATORY DIAGNOSTICS .............................................................................................................................. 36 Biosafety Information. ......................................................................................................................................... 36 Diagnostic Information. ....................................................................................................................................... 36 Immunoassay Tests. .......................................................................................................................................... 41 Nucleic Acid Detection Tests. ............................................................................................................................. 42 Other Diagnostic Methods. ................................................................................................................................. 43 Characterization. ................................................................................................................................................ 43 Confirmation. ...................................................................................................................................................... 44 REFERENCES ........................................................................................................................................................ 45 Francisella tularensis ................................................................................................................................................... 50 OVERVIEW ............................................................................................................................................................. 50 ORGANISM INFORMATION ................................................................................................................................... 50 Taxonomy Information. ....................................................................................................................................... 50 Virulence Factors. ............................................................................................................................................... 51 Epidemiology and Endemic Areas ...................................................................................................................... 52 LABORATORY DIAGNOSTICS .............................................................................................................................. 53 Biosafety Information. ......................................................................................................................................... 53 Diagnostic Information ........................................................................................................................................ 54 Metabolic Information ......................................................................................................................................... 54 Immunoassay Tests. .......................................................................................................................................... 58 Nucleic Acid Detection Tests. ............................................................................................................................. 59 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Other Diagnostic Methods. ................................................................................................................................. 60 Characterization. ................................................................................................................................................ 60 Confirmation. ...................................................................................................................................................... 61 REFERENCES ........................................................................................................................................................ 63 Brucella ........................................................................................................................................................................ 69 OVERVIEW ............................................................................................................................................................. 69 ORGANISM INFORMATION ................................................................................................................................... 69 Taxonomy Information. ....................................................................................................................................... 69 Virulence Factors. ............................................................................................................................................... 70 Epidemiology and Endemic Areas. ..................................................................................................................... 70 LABORATORY DIAGNOSTICS .............................................................................................................................. 72 Biosafety Information. ......................................................................................................................................... 72 Diagnostic Information. ....................................................................................................................................... 72 Immunoassay Tests: .......................................................................................................................................... 77 Nucleic Acid Detection Tests. ............................................................................................................................. 79 Other Diagnostic Methods. ................................................................................................................................. 80 Characterization. ................................................................................................................................................ 80 Confirmation. ...................................................................................................................................................... 80 REFERENCES ........................................................................................................................................................ 82 Burkholderia ................................................................................................................................................................. 87 OVERVIEW ............................................................................................................................................................. 87 ORGANISM INFORMATION ................................................................................................................................... 87 Taxonomy Information. ....................................................................................................................................... 87 Virulence Factors. ............................................................................................................................................... 88 Epidemiology and Endemic Areas. ..................................................................................................................... 89 LABORATORY DIAGNOSTICS .............................................................................................................................. 90 Biosafety Information. ......................................................................................................................................... 90 Diagnostic Information. ....................................................................................................................................... 91 Immunoassay Tests. .......................................................................................................................................... 95 Nucleic Acid Detection Tests. ............................................................................................................................. 97 Other Diagnostic Methods. ................................................................................................................................. 97 Characterization. ................................................................................................................................................ 97 Confirmation. ...................................................................................................................................................... 98 REFERENCES ...................................................................................................................................................... 101 Coxiella burnetii.......................................................................................................................................................... 108 OVERVIEW ........................................................................................................................................................... 108 ORGANISM INFORMATION ................................................................................................................................. 108 Taxonomy Information. ..................................................................................................................................... 108 Virulence Factors. ............................................................................................................................................. 109 Epidemiology and Endemic Areas. ................................................................................................................... 109 LABORATORY DIAGNOSTICS ............................................................................................................................ 110 Biosafety Information. ....................................................................................................................................... 110 Diagnostic Information. ..................................................................................................................................... 111 Immunoassay Tests. ........................................................................................................................................ 112 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Nucleic Acid Detection Tests. ........................................................................................................................... 113 Other Diagnostic Methods. ............................................................................................................................... 114 Characterization ............................................................................................................................................... 114 Confirmation ..................................................................................................................................................... 114 REFERENCES ...................................................................................................................................................... 116 VIRAL AGENTS ......................................................................................................................................................... 120 OVERVIEW ........................................................................................................................................................... 120 GENERAL INFORMATION ................................................................................................................................... 120 Taxonomy Information. ..................................................................................................................................... 120 LABORATORY DIAGNOSTICS ............................................................................................................................ 122 General Diagnostic Information. ....................................................................................................................... 122 Immunoassay Tests. ........................................................................................................................................ 125 Nucleic Acid Detection Tests. ........................................................................................................................... 125 Other Diagnostic Methods. ............................................................................................................................... 125 Characterization ............................................................................................................................................... 126 Confirmation ..................................................................................................................................................... 126 REFERENCES ...................................................................................................................................................... 126 Poxviridae family ........................................................................................................................................................ 127 OVERVIEW ........................................................................................................................................................... 127 ORGANISM INFORMATION ................................................................................................................................. 127 Taxonomy. ........................................................................................................................................................ 127 Epidemiology and Endemic Area ...................................................................................................................... 129 LABORATORY DIAGNOSTICS ............................................................................................................................ 129 Biosafety Information. ....................................................................................................................................... 129 Diagnostic Information. ..................................................................................................................................... 129 Immunoassay Tests. ........................................................................................................................................ 131 Nucleic Acid Detection Tests. ........................................................................................................................... 132 Other Diagnostic Methods. ............................................................................................................................... 133 REFERENCES ...................................................................................................................................................... 134 Alphaviruses; Family Togaviridae .............................................................................................................................. 138 OVERVIEW ........................................................................................................................................................... 138 ORGANISM INFORMATION ................................................................................................................................. 138 Taxonomy. ........................................................................................................................................................ 138 Epidemiology and Endemic Area ...................................................................................................................... 139 LABORATORY DIAGNOSTICS ............................................................................................................................ 139 Biosafety Information. ....................................................................................................................................... 139 Diagnostic Information. ..................................................................................................................................... 140 Immunoassay Tests. ........................................................................................................................................ 142 Nucleic Acid Detection Tests. ........................................................................................................................... 143 Other Diagnostic Methods. ............................................................................................................................... 143 REFERENCES ...................................................................................................................................................... 144 Arenaviruses: Family Arenaviridae ............................................................................................................................. 148 OVERVIEW ........................................................................................................................................................... 148 Taxonomy. ........................................................................................................................................................ 148 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Epidemiology and Endemic Area ...................................................................................................................... 148 LABORATORY DIAGNOSTICS ............................................................................................................................ 149 Biosafety Information. ....................................................................................................................................... 149 Diagnostic Information. ..................................................................................................................................... 150 Immunoassay Tests. ........................................................................................................................................ 151 Nucleic Acid Detection Tests. ........................................................................................................................... 152 Other Diagnostic Methods. ............................................................................................................................... 152 REFERENCES ...................................................................................................................................................... 153 Bunyaviridae Family ................................................................................................................................................... 155 OVERVIEW ........................................................................................................................................................... 155 ORGANISM INFORMATION ................................................................................................................................. 155 Taxonomy. ........................................................................................................................................................ 155 Epidemiology and Endemic Area ...................................................................................................................... 155 LABORATORY DIAGNOSTICS ............................................................................................................................ 157 Biosafety Information. ....................................................................................................................................... 157 Diagnostic Information. ..................................................................................................................................... 157 Immunoassay Tests. ........................................................................................................................................ 158 Nucleic Acid Detection Tests. ........................................................................................................................... 159 Other Diagnostic Methods. ............................................................................................................................... 159 REFERENCES ...................................................................................................................................................... 160 Filoviridae Family ....................................................................................................................................................... 164 OVERVIEW ........................................................................................................................................................... 164 ORGANISM INFORMATION ................................................................................................................................. 164 Taxonomy. ........................................................................................................................................................ 164 Epidemiology and Endemic Area ...................................................................................................................... 164 LABORATORY DIAGNOSTICS ............................................................................................................................ 165 Biosafety Information. ....................................................................................................................................... 165 Diagnostic Information. ..................................................................................................................................... 165 Immunoassay Tests. ........................................................................................................................................ 166 Nucleic Acid Detection Tests. ........................................................................................................................... 166 Other Diagnostic Methods. ............................................................................................................................... 166 REFERENCES ...................................................................................................................................................... 167 Flaviviridae Family ..................................................................................................................................................... 169 OVERVIEW ........................................................................................................................................................... 169 ORGANISM INFORMATION ................................................................................................................................. 169 Taxonomy. ........................................................................................................................................................ 169 Epidemiology and Endemic Area ...................................................................................................................... 169 LABORATORY DIAGNOSTICS ............................................................................................................................ 171 Biosafety Information. ....................................................................................................................................... 171 Diagnostic Information. ..................................................................................................................................... 171 Immunoassay Tests. ........................................................................................................................................ 172 Nucleic Acid Detection Tests. ........................................................................................................................... 173 Other Diagnostic Methods. ............................................................................................................................... 173 REFERENCES ...................................................................................................................................................... 174 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Biological Toxins ........................................................................................................................................................ 177 Botulinum ................................................................................................................................................................... 178 OVERVIEW ........................................................................................................................................................... 178 TOXIN CHARACTERISTICS ............................................................................................................................ 178 ORGANISM INFORMATION ................................................................................................................................. 178 Taxonomy Information. ..................................................................................................................................... 178 Virulence Factors. ............................................................................................................................................. 179 Epidemiology and Endemic Areas .................................................................................................................... 179 LABORATORY DIAGNOSTICS ............................................................................................................................ 179 Biosafety Information ........................................................................................................................................ 179 Diagnostic Information. ..................................................................................................................................... 180 Immunoassay Tests ......................................................................................................................................... 181 Nucleic Acid Detection Tests ............................................................................................................................ 182 Other Diagnostic Methods. ............................................................................................................................... 182 Characterization. .............................................................................................................................................. 183 Confirmation ..................................................................................................................................................... 184 REFERENCES ...................................................................................................................................................... 185 Ricin ........................................................................................................................................................................... 189 OVERVIEW ........................................................................................................................................................... 189 TOXIN CHARACTERISTICS ............................................................................................................................ 189 LABORATORY DIAGNOSTICS ............................................................................................................................ 191 Biosafety Information. ....................................................................................................................................... 191 Diagnostic Information. ..................................................................................................................................... 191 Immunoassay Tests ......................................................................................................................................... 191 Nucleic Acid Detection Tests: ........................................................................................................................... 192 Other diagnostic methods: ................................................................................................................................ 192 Characterization ............................................................................................................................................... 192 Confirmation ..................................................................................................................................................... 192 REFERENCES ...................................................................................................................................................... 194 Staphylococcal Enterotoxin B .................................................................................................................................... 195 OVERVIEW ........................................................................................................................................................... 195 TOXIN CHARACTERISTICS ............................................................................................................................ 195 ORGANISM INFORMATION ................................................................................................................................. 195 Taxonomy Information ...................................................................................................................................... 195 Virulence Factors. ............................................................................................................................................. 195 LABORATORY DIAGNOSTICS ............................................................................................................................ 195 Biosafety Information ........................................................................................................................................ 195 Diagnostic Information ...................................................................................................................................... 196 Immunoassay Tests ......................................................................................................................................... 198 Nucleic Acid Detection Tests ............................................................................................................................ 199 Other diagnostic methods ................................................................................................................................. 199 Characterization ............................................................................................................................................... 199 Confirmation ..................................................................................................................................................... 199 REFERENCES ...................................................................................................................................................... 200 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Appendix A: Glossary of Terms.................................................................................................................................. 203 Appendix B: Biological Agent Disease Characteristics ............................................................................................... 217 Appendix B: Biological Agent Disease Characteristics ............................................................................................... 217 Appendix C. Diagnostic Technologies ........................................................................................................................ 218 Appendix D. Confirmation Levels ............................................................................................................................... 222 Appendix E. Commercial Identification Systems ........................................................................................................ 223 UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. Foreword The three primary methods typical laboratories use for detection/diagnostics of infectious agents is: (1) cultivation of a viable agent, (2) use of the infected host to obtain evidence of infection (typically a serological response or other host response such as encephalitis), and (3) direct detection of the agent using laboratory methods such as amplification of the infectious agent’s nucleic acids or an immunoassay targeted toward the infectious agent’s proteins. Direct cultivation of the agent is always the most discriminant evidence that an infectious agent is present. Direct cultivation, however, has several logistical and practical constraints. First is that not all agents are easily cultivated. Many of bacterial agents are fastidious, taking special media or conditions to support cultivation. Similarly, viral agents take special media and systems, and are not deployable outside a fixed facility. In addition, the time for culture takes anywhere from days to sometimes several weeks before results are available to enable a health care provider to take action based on the data. The use of host responses, while sometimes the only evidence available, also has significant constraints on routine application, including the availability of the highly specific reagents necessary. Figure 1. Cartoon of a typical infection-host response Serological responses are rarely useful in time course demonstrating potential diagnostic time points. direct patient treatment and only provide a look at what ‘was.” Direct detection, principally through nucleic acid amplification, has become the most significant method for patient treatment since the constraints to obtain meaningful results for patient treatment are the most minimal. While useful when a patient is displaying acute symtoms, direct detection is contrained by the availability of specific reagents for purported the disease. All three systems have constrains, and acknowledgement of the constraints and mechanisms to understand and supplement the constraint with knowledge is the purpose of this manual. UNCLASSIFIED TR-16-088 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. One of the best strategies to minimize the laboratorian from reporting a false-positive or a false-negative report is having sufficient information on which to base the final detection/diagnostic decision. Having multiple supportive answers improves the probability of the the final answer being correct. Obtaining multiple supportive answers is often referred to as orthogonal testing. Knowing the pros and cons of the individual assays Figure 2. Depiction of the concept of orthogonal testing. Each of the five pieces of information will helps understand the probability of help determine the correct answer with the intent to obtaining a false-positive or a false-negative minimize the potential for false-negatives or false- assay result. Orthogonal testing can not only positives. be multiple assays, but also should include information from a physician’s clinical assessment of the disease presentation or medical intelligence about infectious disease prevellance in the area of operations. May this manual provide you a reference to understand and provide good quality laboratory results for the protection of our war fighter’s from biothreat agents, and infectious diseases in genera. Supporting the latter, information on the geographic dispersion of these agents is included in this manual to help provide a basis of information on the agent’s occurance naturally to help put in context any medical intelligence that is being developed. UNCLASSIFIED

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resistant isolates of the biowarfare agent Yersinia pestis. Ganapathy R, Padmanabhan S, Eric YP, Moochhala S, Lionel LK, Ponnampalam.
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resistant isolates of the biowarfare agent Yersinia pestis. Ganapathy R, Padmanabhan S, Eric YP, Moochhala S, Lionel LK, Ponnampalam.

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