Natural pandemics and deliberate covert introduction of threat agents share in common the need for a significant response by the health care community. It is critical in both natural outbreaks and outbreaks triggered by a deliberate covert introduction of a threat agent, to provide a rapid definitive identification of the causative agent in order to reduce its "footprint." The footprint in the context of morbity and potential mortality, from a natural or terrorist release of a given agent may be the same; but the footprint from a terrorist release has potentially far more profound psychological, economical, and national security aspects.

Signs and symptoms produced by infectious agents overlap; particularly in the most critical time of presentation; i.e. early on when our chances for halting their effects are greatest. The clinician can only suspect. It is the role of the laboratory to provide the confirmation. The confirmation of the diagnosis is critical to optimal medical management. One key difference between a natural event and a deliberate event is that there is a law enforcement aspect to the later. Here too the laboratory confirmation is critical to appropriate legal management of bioterrorist event, and this introduces the need for handling specimens with proper chain of custody.

The Centers for Disease Control and Prevention (CDC; Atlanta, GA) considers an infectious disease diagnose to be either suspected, probable, or confirmed based on the level of laboratory involvement. Thus, a suspected diagnoses is one where there exists a clinically compatible case without presumptive or confirmatory laboratory results. The diagnosis is probable when there is a clinically compatible case with presumptive laboratory results, and a confirmed diagnosis as one in which there is a clinically compatible case with confirmatory laboratory results.

Clinical and anatomic pathologists have vital roles in the national response to bioterrorism. As it is a daunting task to consider the vast number of organisms of medical importance, it is fortunate that organisms with the virulence and stability characteristics that make them likely nefarious agents are much more limited. Unfortunately, despite great efforts in recent months, many biological threat agents with high impact potential (if used by terrorists or rogue states) continue to be unfamiliar to pathologists, microbiologists, cytologists, and medical technologists. It behooves the pathologist, and microbiologist to be aware and skilled in techniques and mechanisms that enable us to establish a rapid definitive diagnosis.

Definitive identification of pathogenic agents integrates laboratory results. Laboratory tools available include microscopy, cytology, classical culture methods, anatomic examination of tissue samples, immunodiagnostic assays, and nucleic acid analysis. The ideal combination varies depending on the agent, thus it is incumbent on the pathologist to be knowledgeable about these agents so as to provide a rapid definitive identification. The purpose of this presentation is to review the current threat agents and to describe current and future diagnostic approaches that are compatible with our maturing response to bioterrorism.

As anatomic pathologists we must increase our knowledge of the clinical and epidemiologic characteristics of threat agents and develop skills that enable us to recognize in tissue or cytologic samples:

1. unique features of the host response to various infectious agents,
2. the general features of the various categories of infectious agents, and
3. characteristics that help distinguish the various infectious agents from one another in tissue section and from artifacts that can mimic infectious agents. These characteristics can be structural, immunological, genetic, and/or biochemical.

As clinical pantologists we must familiarize ourselves with the tiered network of civilian laboratories developed by the CDC and the Association of Public Health Laboratories (APHL) to provide the expertise required to rapidly identify the biological threat agents and with the similar network of military laboratories to support global counterproliferation and counterbioterrorism initiatives in the U.S. Department of Defense (DoD).

There are multiple national and international lists of threat agents. Thus, in addition to the CDC's list there are lists by the World Health Organization (WHO), North Atlantic Treaty Organization (NATO), National Institute of Allergy and Infectious Disease (NIAID), the United States Department of Agriculture (USDA), and the Australia Group (AG), and others. These lists offer a Ven Diagram of overlapping possible agents. And some of the agents we know have been weaponized and or studied for weaponization do not appear on any of these lists!

I cannot cover all of these agents in all of these lists in this small presentation. Consider for example AG's list. The AG is a consortium of countries organized to slow the proliferation of chemical and biological warfare programs, their list of human pathogens with nefarious potential includes 37 bacteria and viruses, 10 toxins and associated genetically modified organisms. The AG also has a list of animal and plant pathogens.

Military biological defense programs focus on biological agents that can be dispersed in stable aerosols of particle size 1 to 10 um. Other routes of infection are considered less important in the context of strategic weapons. With regard to bioterrorism, the biological agents of greatest concern to the NIAID and the CDC, are also agents that can be dispersed as aerosols and have been on military lists. These include Bacillus anthracis (anthrax), Yersinia pestis (plague), Francisella tularensis (tularemia), variola major (smallpox), botulinum toxin (botulism) and viral hemorrhagic fevers (Filoviruses and Arenaviruses). Federal law (42CFR72.6; 1997) restricts the transfer of these and other select agents between registered facilities and laboratories. Terrorists and those who perform biocrimes often prefer enteric agents such as Salmonella and Shigella species. More recently, there have been intercepts of terrorist interest in E. coli 0157.

If there is an overt bioterrorist it is likely that there will be many patients who present before the onset of clinical symptoms as well as many non-exposed individuals with fear that they might have been exposed. In a covert bioterrorist event while some may present before the onset of clinical symptoms, it is more likely that there will be patients who present as acutely ill patients and critically ill patients.

The earlier an infection can be detected, preferably before symptoms are apparent, the greater is the impact on both individual patient care and on public health and safety. Unfortunately, many of the biological threats present with a nonspecific flu-like febrile illness and most classical microbiological approaches for identifying biological agents are designed to work best when high concentrations of the agent are present and the patient is already critically ill. Thus it behooves us to develop diagnostic methods that allow for a definitive diagnosis when clinical symptoms are still non-specific.

The military has developed an integrated approach that combines clinical diagnosis or medical intelligence with the collection of specific specimens, classical culture methods, sensitive immunodiagnostic assays, and rapid nucleic acid analysis. In particular, electrochemiluminescence assay and rapid nucleic acid analysis may provide confirmed identity (detection of at least two independently derived markers) in 1 to 6 hours depending upon the physical characteristics of the specimen and the concentration of agent. This approach can be used with clinical or environmental samples. Selected diagnostic systems have been incorporated into emerging domestic and military laboratory networks for bioterrorism response. In addition to these methods anatomic pathology can offer similar tests for cytologic and tissue samples.

References

1. Marty AM, Conran RM, Kortepeter MG. Recent challenges in infectious diseases. Biological pathogens as weapons and emerging endemic threats. Clin Lab Med. 2001 Sep;21(3):411-20, vii. Review.
2. Marty AM. History of the development and use of biological weapons. Clin Lab Med 2001 Sep;21(3):421-34
3. Schoneboom BA, Catlin KM, Marty AM, Grieder FB. Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice. J Neuroimmunol 2000 Sep 22;109(2):132-46
4. Gibb TR, Bray M, Geisbert TW, Steele KE, Kell WM, Davis KJ, Jaax NK. Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice. J. Comp Pathol 2001 Nov;125(4):233-42
5. Burgess TH, Steele KE, Schoneboom BA, Grieder FB. Clinicopathologic features of viral agents of potential use by bioterrorists. Clin Lab Med 2001 Sep;21(3):475-93, viii