Wun-Ju Shieh, M.D., M.P.H., PH.D., Infectious Disease Pathology Activity, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

The tragic event of the terrorist attacks on September 11 and the juxtaposed anthrax attack have impacted profoundly on the society in the United States and worldwide. The prospect of anthrax as a weapon of biologic warfare is not new, but this is the first time that anthrax was deliberately used in this manner and probably will not be the last. These events have transformed a theoretical threat to harsh reality, and clearly illustrated the United States' homeland is vulnerable to hostile attack and will not be sufficiently protected by our military superiority. These events also demarcated the beginning of a novel form of warfare in the 21st century. Before World War I, the United States had little knowledge about the potential of chemical and biological warfare. By the end of World War I, the situation had drastically changed. Biological and chemical warfare had already been used covertly on several fronts and the U.S. army realized that it had failed to recognize and prepare for these already existing types of warfare. During the 1950s, the biological warfare program was one of the most highly classified programs; because of its nature and the ongoing Cold War, many of the details of the program have never been declassified. The use of chemical and biological weapons for terrorism became a major concern of the U.S. Army in the 1990s. In 1996, the U.S. Congress passed a new antiterrorism training bill to prepare the United States for future chemical and biological terrorism incidents. In addition to using military experts to equip and train local chemical and biological response teams, the bill also provided funding for former Soviet republics to destroy their own chemical and biological weapons to keep them out of the hands of terrorists. However, such effort did not stop the hostile countries and terrorists from developing and using these weapons. In an era of expanding global communications and increasing trade and travel, countries or small groups of terrorists can directly attack the United States' homeland without using conventional military weapon. Chemical, biological, radiological, or nuclear (CBRN) weapons can be exploited by accessing information through Internet or modern telecommunication systems. The potential use of biological agents – anthrax, plague, mycotoxins, for example – is particularly disconcerting. Such agents are more attractive options for a hostile country or terrorists with intention of mass destruction. The potential lethality of these agents is many times that of chemical agents or nuclear weapons and the manufacture cost of them is much lower. In the future, terrorists using biological weapons are likely to have a much greater capacity for mass destruction. These terrorists may overcome technological obstacles through the support of hostile countries and increase the efficient use of any kind of CBRN weapons. Future advances in molecular genetics may also make it easier to create more potent and more easily useable biological agents. To increase the capacity of handling a large-scale CBRN terrorist attack, two major forms need to be taken: 1) intelligence and law enforcement activities to prevent attacks, and 2) medical and public health activities to prepare for, respond to, and lessen the impact of attacks. With regard to the latter, our medical and public health systems must be an integral part of a multifaceted and comprehensive preparation. Classic public health preparedness and activities at the federal and local levels have to be implemented. These include revitalizing the capacity of local public health facilities, education and training of teams of first responders, developing and updating plans and guidelines for immediate responses, and providing for the availability of vaccines, antibiotics and other medical supplies for emergency deployment.

To cope with the unprecedented demand of bioterrorism preparedness, Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and the Federal Bureau of Investigation (FBI) collaboratively developed a system called The Laboratory Response Network (LRN). LRN is a multi-level arrangement of public and private laboratories that links basic state and local public health laboratories with laboratories with more advanced capabilities. The main components of the APHL system are the state public health laboratories representing each of the 50 states.  In addition, there are laboratories in some counties and larger cities, at the CDC and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Each laboratory has been assigned a designation, predicated on the scope of their diagnostic capability, ranging from Level A (fewest resources) through Level D (most resources).  Local hospital laboratories are generally designated as Level A; they play an upfront role with "rapid rule out and forward" mission when dealing with presumptive clinical cases.  County, city, and state public health laboratories are designated Level B (core) or Level C (advanced), depending on their degree of containment capacity and technical proficiency. The Level D designation is currently reserved for the CDC and the USAMRIID laboratories. There are no "regional" laboratories; the network functions by channeling the specimens through the designated levels to a pathogen-specific conclusion.

Medical examiners are essential partners in bioterrorism preparedness. They play an important role in the response to a known biological attack as well as in the surveillance for a covert attack because of their state statutory authority to investigate deaths that are sudden, suspicious, violent, unattended, and unexplained. The principal investigative tool of the medical examiners is the autopsy that enables them to identify the dead, observe the condition of the body, and deduce the cause and manner of death. Autopsies are valuable in diagnosing unrecognized infections, evaluating therapy, understanding the pathogenesis and route of infection for uncommon or emerging infections, and in developing evidence for subsequent legal proceedings. The same algorithm and principle apply to the investigation and surveillance of an event of biological warfare or biological terrorism.

"Biological warfare" was defined as "warfare involving the use of biological agents against men, animals, or plants. A working definition of a biological agent is "a microorganism (or a toxin derived from it) which causes disease in man, plants or animals or causes deterioration of material. Biological terrorism was defined as "the use or threatened use of biologic agents against a person, group, or larger population to create fear or illnesses for purposes of intimidation, gaining an advantage, interruption of normal activities, or ideologic activities."  The consequence depends upon the nature of the actual event and the population involved; it can vary from a minimal effect to disruption of ongoing activities, emotional reaction, illness, or death. The list of potential biological warfare and bioterrorism agents is long. However, these agents have been assigned priority based on the risk to national security.  Agents are classified as high-risk or "Category A" (Fig. 1) because they can be easily disseminated or transmitted person-to-person; cause high mortality, with potential for major public health impact; might cause public panic and social disruption; and require special action for public health preparedness. The second highest priority or "Category B" agents (Fig. 2) include those that are moderately easy to disseminate; cause moderate morbidity and low mortality; and require enhanced disease surveillance. The third highest priority or "Category C" agents (Fig. 3) include emerging pathogens that could be engineered for mass dissemination in the future because of availability; ease of production and dissemination; and potential for high morbidity and mortality and major health impact. The importance of recognizing the pathological features of the different bioterrorism agents was illustrated by the inhalational and cutaneous anthrax cases that occurred in the United States during October 2001. The autopsy on the index inhalational anthrax case was performed to determine the exact route of infection (cutaneous, gastrointestinal, or inhalational). Once inhalational anthrax was diagnosed, public health officials were able to better define potential sources of airborne B. anthracis spores.

Autopsy diagnostic procedures for the Category A agents should include routine microscopic examination and combine with the collection of specimens for additional tests that will aid in determining a definitive organism-specific diagnosis. Blood, cerebrospinal fluid, and tissue samples or swabs should be placed in transport media that will allow bacterial and viral isolation. Serum should be collected for serological and biological assays. Tissue samples need to be frozen for polymerase chain reaction (PCR). Tissue samples should also be placed in electron microscopy fixative (glutaraldehyde) if possible. Microscopic examination of formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin (H&E) is indispensable because it will characterize the patterns of tissue damage defining a syndrome, and it will establish a list of possible microorganisms in the differential diagnosis (see Tables). Special stains, such as tissue Gram and silver impregnation stains (Steiner's or Warthin-Starry), can be helpful in identifying bacterial agents. Additionally, specific immunohistochemical (IHC) and direct fluorescent assays (DFA) for the Category A bioterrorism agents have been developed at the CDC. These tests can be performed on formalin-fixed tissues and paraffin-embedded blocks. Specimens from suspected bioterrorism-related cases should be submitted to the state public health laboratory through the LRN channel.

Biosafety is of paramount importance for autopsy prosectors who might handle human remains contaminated with potential bioterrorism agents. Infections can be transmitted during performing autopsies via percutaneous inoculation (injury), splashes to unprotected mucosal surfaces, and inhalation of infectious aerosols. All of the Category A pathogens are potentially transmissible to autopsy personnel, though the degree of risk varies considerably among these organisms. Existing guidelines for biosafety and infection control for patient care are designed to prevent transmission of infections between living patients and care providers, or between laboratory specimens and laboratory technicians.

Figure 1. Category A Biological Agents

• Variola major (smallpox)
• Bacillus anthracis (anthrax)
• Yersinia pestis (plague)
• Clostridium botulinum toxin (botulism)
• Francisella tularensis (tularemia)
• Hemorrhagic Fever Viruses: Including:
• Filoviruses, including Ebola and Marburg hemorrhagic fever
• Arenaviruses, including Lassa (Lassa fever), Junin (Argentine hemorrhagic fever), Machupo (Bolivian hemorrhagic fever), and related viruses

Figure 2. Category B Biological Agents

• Coxiella burnetii (Q fever)
• Brucella species (brucellosis)
• Burkholderia mallei (glanders)
• Alphaviruses, including Venezuelan equine encephalomyelitis, eastern and western equine encephalomyelitis
• Ricin toxin from Ricinus communis (castor beans)
• Epsilon toxin of Clostridium perfringens
• Staphylococcus enterotoxin B
• A subset of List B agents includes food-or waterborne pathogens.
• Salmonella species
• Shigella dysenteriae
• Escherichia coli O157:H7
• Vibrio cholerae
• Cryptosporidium parvum

Figure 3. Category C Biological Agents

• Nipah virus
• Hantaviruses
• Tickborne hemorrhagic fever viruses
• Tickborne encephalitis viruses
• Yellow fever virus
• Multidrug-resistant Mycobacterium tuberculosis

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