The twentieth century saw a logarithmic rise in scientific knowledge and technologic
capability which has continued exponentially in the twenty-first century. Science and technology are
inherently of "dual use," in that the knowledge can be used to cause great good or great harm. Among the
sciences none is more ambiguous and with more complex in terms of dual use issues than biology. Thus,
the global proliferation of biological warfare and terrorism technologies has become among the greatest
threats to public health.
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
- unique features of the host response to various infectious agents,
- the general features of the various categories of infectious agents, and
- 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
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.
- 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.
- Marty AM. History of the development and use of biological weapons. Clin Lab Med 2001
- 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
- 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
- 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