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Descriptions of the use of microbial pathogens as potential weapons of war or terrorism date from ancient times. Among the most frequently cited of such episodes are the poisoning of water supplies in the sixth century B.C. with the fungus Claviceps purpurea (rye ergot) by the Assyrians, the hurling of the dead bodies of plague victims over the walls of the city of Kaffa by the Tartar army in 1346, and the efforts by the British to spread smallpox via contaminated blankets to the native American population loyal to the French in 1767. Although the use of chemical weapons in wartime took place in the not-too-distant past, the tragic events of September 11, 2001, followed closely by the anthrax attacks through the U.S. Postal System, dramatically changed the mindset of the American public regarding both our vulnerability to microbial bioterrorist attacks and the seriousness and intent of the Federal government to protect its citizens against future attacks. Modern science has revealed methods of deliberately spreading or enhancing disease in ways not appreciated by our ancestors.The combination of basic research, good medical practice, and constant vigilance will be needed to defend against such attacks.


Although the potential impact of a bioterrorist attack could be enormous, leading to thousands of deaths and extensive morbidity, acts of bioterrorism would be expected to produce their greatest impact through the fear and terror they generate. In contrast to biowarfare, where the primary goal is destruction of the enemy through mass casualties, an important goal of bioterrorism is to destroy the morale of a society through fear and uncertainty. Although the actual biologic impact of a single act may be small, the degree of disruption created by the realization that such an attack is possible may be enormous. This was readily apparent with the impact on the U.S. Postal System and the functional interruption of the activities of the legislative branch of government after the anthrax attacks noted above. Thus the key to the defense against these attacks is a highly functioning system of public health surveillance and education so that attacks can be quickly recognized and effectively contained.This is complemented by the availability of appropriate countermeasures in the form of diagnostics, therapeutics, and vaccines, both in response to and in anticipation of bioterrorist attacks.


The Working Group for Civilian Biodefense has put together a list of key features that characterize the elements of biologic agents that make them particularly effective as weapons (Table 6-1). Included among these are the ease of spread and transmission of the agent as well as the presence of an adequate database to allow newcomers to the field to quickly apply the good science of others to bad intentions of their own. Agents of bioterrorism may be used in their naturally occurring forms, or they can be deliberately modified to provide maximal impact. Among the approaches to maximizing the deleterious effects of biologic agents are the genetic modification of microbes for the purposes of antimicrobial resistance or evasion by the immune system, creation of fine-particle aerosols, chemical treatment to stabilize and prolong infectivity, and alteration of host range through changes in surface proteins. Certain of these approaches fall under the category of weaponization, which is a term generally used to describe the processing of microbes or toxins in a manner that would ensure a devastating effect of a release. For example, weaponization of anthrax by the Soviets comprised the production of vast amounts of spores in a form that maintained aerosolization for prolonged periods of time; the spores were of appropriate size to reach the lower respiratory tract easily and could be delivered in a massive release, such as via widely dispersed bomblets.


The U.S. Centers for Disease Control and Prevention (CDC) classifies potential biologic threats into three categories: A, B, and C (Table 6-2). Category A agents are the highest-priority pathogens. They pose the greatest risk to national security because they (1) can be easily disseminated or transmitted from person to person, (2) result in high mortality rates and have the potential for major public health impact, (3) might cause public panic and social disruption, and (4) require special action for public health preparedness. Category B agents are the second highest-priority pathogens and include those that are moderately easy to disseminate, result in moderate morbidity rates and low mortality rates, and require specifically enhanced diagnostic capacity. Category C agents are the third highest priority. These include certain emerging pathogens, to which the general population lacks immunity, that could be engineered for mass dissemination in the future because of availability, ease of production, ease of dissemination, potential for high morbidity and mortality, and major public health impact. A potential pandemic strain of influenza, such as avian influenza, is one such example. It should be pointed out, however, that these designations are empirical, and, depending on evolving circumstances such as intelligencebased threat assessments, the priority rating of any given microbe or toxin could change. The CDC classification system also largely reflects the severity of illness produced by a given agent, rather than its accessibility to potential terrorists.


CATEGORY A AGENTS

ANTHRAX

Bacillus Anthracis as a Bioweapon Anthrax may be the prototypic disease of bioterrorism. Although rarely, if ever, spread from person to person, the illness embodies the other major features of a disease introduced through terrorism, as outlined in Table 6-1. U.S. and British government scientists studied anthrax as a potential biologic weapon beginning approximately at the time of World War II (WWII). Offensive bioweapons activity including bioweapons research on microbes and toxins in the United States ceased in 1969 as a result of two executive orders by President Richard M. Nixon. The 1972 Biological and Toxin Weapons Convention Treaty outlawed research of this type worldwide. Clearly, the Soviet Union was in direct violation of this treaty until at least the Union dissolved in the late 1980s. It is well documented that during this post-treaty period, the Soviets produced and stored tons of anthrax spores for potential use as a bioweapon. At present there is suspicion that research on anthrax as an agent of bioterrorism is ongoing by several nations and extremist groups. One example of this is the release of anthrax spores by the Aum Shrinrikyo cult in Tokyo in 1993. Fortunately, there were no casualties associated with this episode because of the inadvertent use of a nonpathogenic strain of anthrax by the terrorists. The potential impact of anthrax spores as a bioweapon was clearly demonstrated in 1979 after the accidental release of spores into the atmosphere from a Soviet Union bioweapons facility in Sverdlosk, Russia. Although actual figures are not known, at least 77 cases of anthrax were diagnosed with certainty, of which 66 were fatal. These victims were exposed in an area within 4 km downwind of the facility, and deaths due to anthrax were also noted in livestock up to 50 km further downwind. Based on recorded wind patterns, the interval between the time of exposure and development of clinical illness ranged from 2–43 days.The majority of cases were within the first 2 weeks. Death typically occurred within 1–4 days after the onset of symptoms. It is likely that the widespread use of postexposure penicillin prophylaxis limited the total number of cases. The extended period of time between exposure and disease in some individuals supports the data from nonhuman primate studies suggesting the anthrax spores can lie dormant in the respiratory tract for at least 4–6 weeks without evoking an immune response. This extended period of microbiologic latency after exposure poses a significant challenge for management of victims in the postexposure period. In September 2001, the American public was exposed to anthrax spores as a bioweapon delivered through the U.S. Postal System.


The CDC identified 22 confirmed or suspected cases of anthrax as a consequence of this attack. These included 11 patients with inhalational anthrax, of whom 5 died, and 11 patients with cutaneous anthrax (7 confirmed), all of whom survived (Fig. 6-1). Cases occurred in individuals who opened contaminated letters as well as in postal workers involved in the processing of mail.A minimum of five letters mailed from Trenton, NJ, served as the vehicles for these attacks. One of these letters was reported to contain 2 g of material, equivalent to 100 billion to 1 trillion weapon-grade spores. Since studies performed in the 1950s using monkeys exposed to aerosolized anthrax suggested that ∼10,000 spores were 69 required to produce lethal disease in 50% of animals exposed to this dose (the LD50), the contents of one letter had the theoretical potential, under optimal conditions, of causing illness or death in up to 50 million individuals when one considers an LD50 of 10,000 spores. The strain used in this attack was the Ames strain. Although it was noted to have an inducible beta-lactamase and to constitutively express a cephalosporinase, it was susceptible to all antibiotics standard for B. anthracis.


Microbiology and Clinical Features Anthrax is caused by B. anthracis, a gram-positive, nonmotile, spore-forming rod that is found in soil and predominantly causes disease in herbivores such as cattle, goats, and sheep. Anthrax spores can remain viable for decades.The remarkable stability of these spores makes them an ideal bioweapon, and their destruction in decontamination activities can be a challenge. Naturally occurring human infection is generally the result of contact with anthrax-infected animals or animal products such as goat hair. Although an LD50 of 10,000 spores is a generally accepted number, it has also been suggested that as few as one to three spores may be adequate to cause disease in some settings. Advanced technology is likely to be necessary to generate spores of the optimal size (1–5 ìm) to travel to the alveolar spaces as a bioweapon. The three major clinical forms of anthrax are gastrointestinal, cutaneous, and inhalational. Gastrointestinal anthrax typically results from the ingestion of contaminated meat; the condition is rarely seen and is unlikely to be the result of a bioterrorism event.The lesion of cutaneous anthrax typically begins as a papule after the introduction of spores through an opening in the skin. This papule then evolves to a painless vesicle followed by the development of a coal-black, necrotic eschar (Fig. 6-2).


It is the Greek word for coal (anthrax) that gives the organism and the disease its name. Cutaneous anthrax was ∼20% fatal before the availability of antibiotics. Inhalational anthrax is the form most likely to be responsible for death in the setting of a bioterrorist attack. It occurs after the inhalation of spores that become deposited in the alveolar spaces.These spores are phagocytosed by macrophages and transported to the mediastinal and peribronchial lymph nodes where they germinate, leading to active bacterial growth and elaboration of the bacterial products edema toxin and lethal toxin. Subsequent hematogenous spread of bacteria is accompanied by cardiovascular collapse and death. The earliest symptoms are typically a viral-like prodrome with fever, malaise, and abdominal and/or chest symptoms that progress over the course of a few days to a moribund state. A characteristic finding is mediastinal widening and pleural effusions on chest x-ray (Fig. 6-3). Although initially thought to be 100% fatal, the experiences at Sverdlosk in 1979 and in the United States in 2001 (see below) indicate that, with prompt initiation of antibiotic therapy, survival is possible. The characteristics of the 11 cases of inhalational anthrax diagnosed in the United States in 2001 after exposure to contaminated letters postmarked September 18 or October 9, 2001, followed the classic pattern established for this illness, with patients presenting with a rapidly progressive course characterized by fever, fatigue or malaise, nausea or vomiting, cough, and shortness of breath. At presentation, the total white blood cell counts were ∼10,000 cells/ìL; transaminases tended to be elevated, and all 11 had abnormal findings on chest x-ray and CT. Radiologic findings included infiltrates, mediastinal widening, and hemorrhagic pleural effusions. For cases in which the dates of exposure were known, symptoms appeared within 4–6 days. Death occurred within 7 days of diagnosis in the 5 fatal cases (overall mortality rate 55%). Rapid diagnosis and prompt initiation of antibiotic therapy were key to survival.


Treatment:

ANTHRAX

Anthrax can be successfully treated if the disease is promptly recognized and appropriate therapy is initiated early. Although penicillin, ciprofloxacin, and doxycycline are the currently licensed antibiotics for this indication, clindamycin and rifampin also have in vitro activity against the organism and have been used as part of treatment regimens. Until sensitivity results are known, suspected cases are best managed with a combination of broadly active agents (Table 6-3). Patients with inhalational anthrax are not contagious and do not require special isolation procedures. vaccine licensed for human use is a product produced from the cell-free culture supernatant of an attenuated, nonencapsulated strain of B. anthracis (Stern’s strain), referred to as anthrax vaccine adsorbed (AVA). Clinical trials for safety in humans and efficacy in animals are currently underway to evaluate the role of recombinant protective antigen (one of the major components, along with lethal factor and edema factor, of B. anthracis toxins) as an alternative to AVA. In a postexposure setting in nonhuman primates, a 2-week course of AVA plus ciprofloxacin was found to be superior to ciprofloxacin alone in preventing the development of clinical disease and death. Although the current recommendation for postexposure prophylaxis is 60 days of antibiotics, it would seem prudent to include immunization with anthrax vaccine if available. Given the potential for B. anthracis to be engineered to express penicillin resistance, the empirical regimen of choice in this setting is either ciprofloxacin or doxycycline.


PLAGUE

Yersinia Pestis as a Bioweapon Although it lacks the environmental stability of anthrax, the highly contagious nature and high mortality of plague make it a close to ideal agent of bioterrorism, particularly if delivered in a weaponized form. Occupying a unique place in history, plague has been alleged to have been used as a biologic weapon for centuries. The catapulting of plague-infected corpses into besieged fortresses is a practice that was first noted in 1346 during the assault of the city of Kaffa by the Tartars. Although unlikely to have resulted in disease transmission, some believe that this event may have played a role in the start of the Black Death pandemic of the fourteenth and fifteenth centuries in Europe. Given that plague was already moving across Asia toward Europe at this time, it is unclear whether such an allegation is accurate. During WWII, the infamous Unit 731 of the Japanese army was reported to have repeatedly dropped plague-infested fleas over parts of China, including Manchuria. These drops were associated with subsequent outbreaks of plague in the targeted areas.


After WWII, the United States and the Soviet Union conducted programs of research on how to create aerosolized Y. pestis that could be used as a bioweapon to cause primary pneumonic plague. As mentioned above, plague was thought to be an excellent bioweapon due to the fact that, in addition to causing infection in those inhaling the aerosol, significant numbers of secondary cases of primary pneumonic plague would likely occur due to the contagious nature of the disease and person-to-person transmission via respiratory aerosol. Secondary reports of research conducted during that time suggest that organisms remain viable for up to 1 h and can be dispersed for distances up to 10 km. Although the offensive bioweapons program in the United States was terminated before production of sufficient quantities of plague organisms for use as a weapon, it is believed that Soviet scientists did manufacture quantities sufficient for such a purpose. It has also been reported that more than 10 Soviet Institutes and >1000 scientists were working with plague as a biologic weapon.


Of concern is the fact that, in 1995, a microbiologist in Ohio was arrested for having obtained Y. pestis in the mail from the American Type Culture Collection, using a credit card and a false letterhead. In the wake of this incident, the U.S. Congress passed a law in 1997 requiring that anyone intending to send or receive any of 42 different agents that could potentially be used as bioweapons first register with the CDC. Microbiology and Clinical Features Plague is caused by Y. pestis, a nonmotile, gram-negative bacillus that exhibits bipolar, or “safety pin,” staining with Wright’s, Giemsa’s, or Wayson’s stains. It has had a major impact on the course of history, thus adding to the element of fear evoked by its mention. The earliest reported plague epidemic was in 224 B.C. in China.The most infamous pandemic began in Europe in the fourteenth century, during which time one-third to one-half of the entire population of Europe was killed. During a plague outbreak in India in 1994, even though the number of confirmed cases was relatively small, it is estimated that 500,000 individuals fled their homes in fear of this disease. The clinical syndromes of plague generally reflect the mode of infection. Bubonic plague is the consequence of an insect bite; primary pneumonic plague arises through the inhalation of bacteria.


Most of the plague seen in the world today is bubonic plague and is the result of a bite by a plague-infected flea. In part as a consequence of past pandemics, plague infection of rodents exists widely in nature, including in the southwestern United States, and each year thousands of cases of plague occur worldwide through contact with infected animals or fleas. After inoculation of regurgitated bacteria into the skin by a flea bite, organisms travel through the lymphatics to regional lymph nodes, where they are phagocytized but not destroyed. Inside the cell, they multiply rapidly, leading to inflammation, painful lymphadenopathy with necrosis, fever, bacteremia, septicemia, and death. The characteristic enlarged, inflamed lymph nodes, or buboes, give this form of plague its name. In some instances, patients may develop bacteremia without lymphadenopathy after infection, a condition referred to as primary septicemic plague. Extensive ecchymoses may develop due to disseminated intravascular coagulation, and gangrene of the digits and/or nose may develop in patients with advanced septicemic plague. It is thought that this appearance of some patients gave rise to the term Black Death in reference to the plague epidemic of the fourteenth and fifteenth centuries. Some patients may develop pneumonia (secondary pneumonic plague) as a complication of bubonic or septicemic plague. These patients may then transmit the agent to others via the respiratory route, causing cases of primary pneumonic plague.


Primary pneumonic plague is the manifestation most likely to occur as the result of a bioterrorist attack, with an aerosol of bacteria spread over a wide area or a particular environment that is densely populated. In this setting, patients would be expected to develop fever, cough with hemoptysis, dyspnea, and gastrointestinal symptoms 1–6 days after exposure. Clinical features of pneumonia would be accompanied by pulmonary infiltrates and consolidation on chest x-ray. In the absence of antibiotics, the mortality of this disease is on the order of 85%, and death usually occurs within 2–6 days. Treatment: PLAGUE Streptomycin, tetracycline, and doxycycline are licensed by the U.S. Food and Drug Administration (FDA) for the treatment of plague. Multiple additional antibiotics licensed for other infections are commonly used and are likely effective. Among these are aminoglycosides such as gentamicin, cephalosporins, trimethoprim/sulfamethoxazole, chloramphenicol, and ciprofloxacin (Table 6-3). A multidrug- resistant strain of Y. pestis was identified in 1995 from a patient with bubonic plague in Madagascar. Although this organism was resistant to streptomycin, ampicillin, chloramphenicol, sulfonamides, and tetracycline, it retained its susceptibility to other aminoglycosides and cephalosporins. Given the subsequent identification of a similar organism in 1997 coupled with the fact that this resistance is plasmid-mediated, it seems likely that genetically modifying Y. pestis to a multidrug-resistant form is possible. Unlike patients with inhalational anthrax (see above), patients with pulmonary plague should be cared for under conditions of strict respiratory isolation comparable to that used for multidrugresistant tuberculosis. Vaccination and Prevention A formalin-fixed, whole-organism vaccine was licensed by the FDA for the prevention of plague.


That vaccine is no longer being manufactured, but its potential value as a current countermeasure against bioterrorism would likely have been modest at best, as it was ineffective against animal models of primary pneumonic plague. Efforts are underway to develop a second generation of vaccines that will protect against aerosol challenge. Among the candidates being tested are recombinant forms of the F1 and V antigens of Y. pestis. It is likely that doxycycline or ciprofloxacin would provide coverage in a chemoprophylaxis setting. Unlike the case with anthrax, in which one has to be concerned about the persistence of ungerminated spores in the respiratory tract, the duration of prophylaxis against plague need only extend to 7 days after exposure.


SMALLPOX

Variola Virus as a Bioweapon

Given that most of the world’s population was vaccinated against smallpox, variola virus would not have been considered a good candidate as a bioweapon 30 years ago. However, with the cessation of immunization programs in the United States in 1972 and throughout the world in 1980 due to the successful global eradication of smallpox, close to 50% of the U.S. population is fully susceptible to smallpox today. Given its infectious nature and the 10–30% mortality in unimmunized individuals, the deliberate spread of this virus could have a devastating effect on our society and unleash a previously conquered deadly disease. It is estimated that an initial infection of 50–100 persons in a first-generation of cases could expand by a factor of 10–20 with each succeeding generation in the absence of any effective containment measures. Although the likely implementation of an effective public health response makes this scenario unlikely, it does illustrate the potential damage and disruption that can result from a smallpox outbreak. In 1980, the World Health Organization (WHO) recommended that all immunization programs be terminated; that representative samples of variola virus be transferred to two locations: one at the CDC in Atlanta, GA, in the United States and the other at the Institute of Virus Preparations in the Soviet Union; and that all other stocks of smallpox be destroyed. Several years later, it was recommended that these two authorized collections be destroyed. However, these latter recommendations were placed on hold in the wake of increased concerns of the use of variola virus as a biologic weapon and thus the need to maintain an active program of defensive research. Many of these concerns were based on allegations made by former Soviet officials that extensive programs had been in place in that country for the production and weaponization of large quantities of smallpox virus.The dismantling of these programs with the fall of the Soviet Union and the subsequent weakening of security measures led to fears that stocks of V. major may have made their way to other countries or terrorist organizations. In addition, accounts that efforts had been taken to produce recombinant strains of Variola that would be more virulent and more contagious than the wild-type virus have led to an increase in the need to be vigilant for the reemergence of this often fatal-infectious disease. Microbiology and Clinical Features Smallpox is caused by one of two variants of variola virus, V. major and V. minor.Variola is a double-strand DNA virus and member of the Orthopoxvirus genus of the Poxviridae family. Infections with V. minor are generally less severe than those of V. major, with milder constitutional symptoms and lower mortality rates; thus V. major is the only one considered to be a viable bioweapon.


Infection with V. major typically occurs after contact with an infected person from the time that a maculopapular rash appears on the skin and oropharynx, through the resolution and scabbing of the pustular lesions. Infection occurs principally during close contact, through the inhalation of saliva droplets containing virus from the oropharyngeal exanthem. Aerosolized material from contaminated clothing or linen can also spread infection. Several days after exposure, a primary viremia is believed to occur that results in dissemination of virus to lymphoid tissues. A secondary viremia occurs ∼4 days later that leads to localization of infection in the dermis. Approximately 12–14 days after the initial exposure, the patient develops high fever, malaise, vomiting, headache, backache, and a maculopapular rash that begins on the face and extremities and spreads to the trunk (centripetal), with lesions in the same developmental stage in any given location. This is in contrast to the rash of varicella (chickenpox) that begins on the trunk and face and spreads to the extremities (centrifugal), with lesions at all stages of development. The lesions are initially maculopapular and evolve to vesicles that eventually become pustules and then scabs. The oral mucosa also develops maculopapular lesions that evolve to ulcers.


The lesions appear over a period of 1–2 days and evolve at the same rate. Although virus can be isolated from the scabs on the skin, the conventional thinking is that once the scabs have formed, the patient is no longer contagious. Smallpox is associated with a 10–30% mortality, with patients typically dying of severe systemic illness during the second week of symptoms.His- 75 torically, ∼5–10% of naturally occurring smallpox cases take either of two highly virulent atypical forms, classified as hemorrhagic and malignant.These are difficult to recognize because of their atypical presentations. The hemorrhagic form is uniformly fatal and begins with the relatively abrupt onset of a severely prostrating illness characterized by high fevers and severe headache and back and abdominal pain.This form of the illness resembles a severe systemic inflammatory syndrome, in which patients have a high viremia, but die without developing the characteristic rash. Cutaneous erythema develops accompanied by petechiae and hemorrhages into the skin and mucous membranes.


Death usually occurs within 5–6 days. The malignant, or “flat,” form of smallpox is frequently fatal and has an onset similar to the hemorrhagic form, but with confluent skin lesions developing more slowly and never progressing to the pustular stage. Vaccination and Prevention In 1796 Edward Jenner demonstrated that deliberate infection with cowpox virus could prevent illness on subsequent exposure to smallpox. Today, smallpox is a preventable disease after immunization with vaccinia. The current dilemma facing our society regarding assessment of the risk and/or benefit of smallpox vaccination is that the degree of risk that someone will deliberately and effectively release smallpox into our society is unknown. As a prudent first step in preparedness for a smallpox attack, virtually all members of the U.S. armed services have received primary or booster immunizations with vaccinia. In addition, tens of thousands of civilian health care workers who comprise smallpox-response teams at the state and local public health level have been vaccinated. Initial fears regarding the immunization of a segment of the American population with vaccinia when there are more individuals receiving immunosuppressive drugs and other immunocompromised patients than ever before have largely been dispelled as data are generated from the current military and civilian immunization campaigns. Adverse event rates for the first 450,000 immunizations are similar to and, in certain categories of adverse events, even lower than those from historic data, in which most severe sequelae of vaccination occurred in young infants (Table 6-4). In addition, 11 patients care. Although several antiviral agents, including cidofovir, that are licensed for other diseases have in vitro activity against V. major, they have never been tested in the setting of human disease. For this reason, it is difficult to predict whether or not they would be effective in cases of smallpox and, if effective, whether or not they would be of value in patients with advanced disease. Research programs studying the efficacy of new antiviral compounds against V. major are currently underway. Treatment:


SMALLPOX

Given the infectious nature of smallpox and the extreme vulnerability of contemporary society, patients who are suspected cases should be handled with strict isolation procedures. Although laboratory confirmation of a suspected case by culture and electron microscopy is essential, it is equally important that appropriate precautions be employed when obtaining samples for culture and laboratory testing. All health care and laboratory workers caring for patients should have been recently immunized with vaccinia, and all samples should be transported in doubly sealed containers. Patients should be cared for in negativepressure rooms with strict isolation precautions. There is no licensed specific therapy for smallpox, and historic treatments have focused solely on supportive with early-stage HIV infection have been inadvertently immunized without problem. One significant concern during the recent immunization campaign, however, has been the description of a syndrome of myopericarditis, which was not appreciated during prior immunization campaigns with vaccinia.


TULAREMIA

Francisella tularensis as a Bioweapon

Tularemia has been studied as an agent of bioterrorism since the mid-twentieth century. It has been speculated by some that the outbreak of tularemia among German and Soviet soldiers during fighting on the Eastern Front during WWII was the consequence of a deliberate release. Unit 731 of the Japanese Army studied the use of tularemia as a bioweapon during WWII. Large preparations were made for mass productive of F. tularensis by the United States, but no stockpiling of any agent took place. Stocks of F. tularensis were reportedly generated by the Soviet Union in the mid-1950s. It has also been suggested that the Soviet program extended into the era of molecular biology and that some strains were engineered to be resistant to common antibiotics. F. tularensis is an extremely infectious organism, and human infections have occurred from merely examining an uncovered petri dish streaked with colonies. Given these facts, it is reasonable to conclude that this organism might be utilized as a bioweapon through either an aerosol or contamination of food or drinking water.


Microbiology and Clinical Features Although similar in many ways to anthrax and plague, tularemia, also referred to as rabbit fever or deer fly fever, is neither as lethal nor as fulminant as either of these other two category A bacterial infections. It is, however, extremely infectious, and as few as 10 organisms can lead to establishment of infection. Despite this fact, it is not spread from person to person. Tularemia is caused by F. tularensis, a small, nonmotile, gram-negative coccobacillus. Although it is not a spore-forming organism, it is a hardy bacterium that can survive for weeks in the environment. Infection typically comes from insect bites or contact with organisms in the environment. Large waterborne outbreaks have been recorded. It is most likely that the outbreak among German and Russian soldiers and Russian civilians noted above during WWII represented a large waterborne tularemia outbreak in a Tularensis-enzootic area devastated by warfare. Humans can become infected through a variety of environmental sources. Infection is most common in rural areas where a variety of small mammals may serve as reservoirs. Human infections in the summer are often the result of insect bites from ticks, flies, or mosquitoes that have bitten infected animals. In colder months infections are most likely the result of direct contact with infected mammals and are most common in hunters. In these settings infection typically presents as a systemic illness with an area of inflammation and necrosis at the site of tissue entry.


Drinking of contaminated water may lead to an oropharyngeal form of tularemia characterized by pharyngitis with cervical and/or retropharyngeal lymphadenopathy (Chap. 59). The most likely mode of dissemination of tularemia as a biologic weapon would be as an aerosol, as has occurred in a number of natural outbreaks in rural areas, including Martha’s Vineyard in the United States. Approximately 1–14 days after exposure by this route, one would expect to see inflammation of the airways with pharyngitis, pleuritis, and bronchopneumonia. Typical symptoms would include the abrupt onset of fever, fatigue, chills, headache, and malaise (Table 6-3). Some patients might experience conjunctivitis with ulceration, pharyngitis, and/or cutaneous exanthems. A pulse-temperature dissociation might be present. Approximately 50% of patients would show a pulmonary infiltrate on chest x-ray. Hilar adenopathy might also be present, and a small percentage of patients could have adenopathy without infiltrates. The highly variable presentation makes acute recognition of aerosol-disseminated tularemia very difficult. The diagnosis would likely be made by immunohistochemistry or culture of infected tissues or blood. Untreated, mortality rates range from 5–15% for cutaneous routes of infection and 30–60% for infection by inhalation. Since the advent of antibiotic therapy, these rates have dropped to <2%.


Treatment: TULAREMIA

Both streptomycin and doxycycline are licensed for treatment of tularemia. Other agents likely to be effective include gentamicin, chloramphenicol, and ciprofloxacin (Table 6-3). Given the potential for genetic modification of this organism to yield antibiotic-resistant strains, broad-spectrum coverage should be the rule until sensitivities have been determined. As mentioned above, special isolation procedures are not required.  Vaccination and Prevention There are no vaccines currently licensed for the prevention of tularemia. Although a live, attenuated strain of the organism has been used in the past with some reported success, there are inadequate data to support its widespread use at this time. Development of a vaccine for this agent is an important part of the current biodefense research agenda. In the absence of an effective vaccine, postexposure chemoprophylaxis with either doxycycline or ciprofloxacin appears to be a reasonable approach Hemorrhagic Fever Viruses as Bioweapons 77 Several of the hemorrhagic fever viruses have been reported to have been weaponized by the Soviet Union and the United States. Nonhuman primate studies indicate that infection can be established with very few virions and that infectious aerosol preparations can be produced. Under the guise of wanting to aid victims of an Ebola outbreak, members of the Aum Shrinrikyo cult in Japan were reported to have traveled to central Africa in 1992 in an attempt to obtain Ebola virus for use in a bioterrorist attack. Thus, although there has been no evidence that these agents have ever been used in a biologic attack, there is clear interest in their potential for this purpose.


Microbiology and Clinical Features The viral hemorrhagic fevers are a group of illnesses caused by any one of a number of similar viruses (Table 6-2). These viruses are all enveloped, single-strand RNA viruses that are thought to depend upon a rodent or insect host reservoir for long-term survival. They tend to be geographically restricted according to the migration patterns of their hosts. Great apes are not a natural reservoir for Ebola virus, but large numbers of these animals in Sub-Saharan Africa have died from Ebola infection over the past decade. Humans can become infected with hemorrhagic fever viruses if they come into contact with an infected host or other infected animals. Personto- person transmission, largely through direct contact with virus-containing body fluids, has been documented for Ebola, Marburg, and Lassa virus and rarely for the New World arenaviruses. Although there is no clear evidence of respiratory spread among humans, these viruses have been shown in animal models to be highly infectious by the aerosol route. This, coupled with mortality rates as high as 90%, makes them excellent candidate agents of bioterrorism. The clinical features of the viral hemorrhagic fevers vary depending upon the particular agent (Table 6-3). Initial signs and symptoms typically include fever,myalgia, prostration, and disseminated intravascular coagulation with thrombocytopenia and capillary hemorrhage.These findings are consistent with a cytokine-mediated systemic inflammatory syndrome. A variety of different maculopapular or erythematous rashes may be seen.


Leukopenia, temperature-pulse dissociation, renal failure, and seizures may also be part of the clinical presentation. Outbreaks of most of these diseases are sporadic and unpredictable. As a consequence, most studies of pathogenesis have been performed using laboratory animals. The diagnosis should be suspected in anyone with temperature >38.3°C for <3 weeks who also exhibits at least 2 of the following: hemorrhagic or purpuric rash, epistaxis, hematemesis, hemoptysis, or hematochezia in the absence of any other identifiable cause. In this setting, samples of blood should be sent after consultation to the CDC or the U.S. Army Medical Research Institute of Infectious Diseases for serologic testing for antigen and antibody as well as reverse transcriptase polymerase chain reaction (RT-PCR) testing for hemorrhagic fever viruses. All samples should be handled with double-bagging. Given how little is known regarding the human-tohuman transmission of these viruses, appropriate isolation measures would include full-barrier precautions with negative-pressure rooms and use of N95 masks or powered air-purifying respirators (PAPRs). Unprotected skin contact with cadavers has been implicated in the transmission of certain hemorrhagic fever viruses such as Ebola, so it is recommended that autopsies be performed using the strictest measures for protection and that burial or cremation be performed promptly without embalming.


Treatment: VIRAL HEMORRHAGIC FEVERS

There are no approved and effective antiviral therapies for this class of viruses (Table 6-3). Although there are anecdotal reports of the efficacy of ribavirin, interferon-á, or hyperimmune immunoglobulin, definitive data are lacking.The best data for ribavirin are in arenavirus (Lassa and New World) infections. In some in vitro systems, specific immunoglobulin has been reported to enhance infectivity, and thus these potential treatments must be approached with caution. Vaccination and Prevention A live attenuated virus vaccine is available in limited quantities for prevention of yellow fever. There are no other licensed and effective vaccines for these agents. Studies are currently underway examining the potential role of DNA, recombinant viruses, and attenuated viruses as vaccines for several of these infections. Among the most promising at present are vaccines for Argentine, Ebola, Rift Valley, and Kyasanur Forest viruses.


BOTULISM TOXIN (CLOSTRIDIUM BOTULINUM) 

Botulinum Toxin as a Bioweapon

In a bioterrorist attack, botulinum toxin would likely be dispersed as an aerosol or as contamination of a food supply. Although contamination of a water supply is possible, it is likely that any toxin would be rapidly inactivated by the chlorine used to purify drinking water. Similarly, toxin can be inactivated by heating any food to >85_C for >5 min. Without external facilitation, the environmental decay rate is estimated at 1% per minute, and thus the time interval between weapon release and ingestion or inhalation needs to be rather short. The Japanese biologic warfare group, Unit 731, is reported to have conducted experiments on botulism poisoning in prisoners in the 1930s.The United States and the Soviet Union both acknowledged producing botulinum toxin, and there is some evidence that the Soviet Union attempted to create recombinant bacteria containing the gene for botulinum toxin. In records submitted to the United Nations, Iraq admitted to having produced 19,000 L of concentrated toxin—enough toxin to kill the entire population of the world three times over. By many accounts, botulinum toxin was the primary focus of the pre-1991 Iraqi bioweapons program. In addition to these examples of state-supported research into the use of botulinum toxin as a bioweapon, the Aum Shrinrikyo cult unsuccessfully attempted on a least three occasions to disperse botulism toxin into the civilian population of Tokyo.


Microbiology and Clinical Features Unique among the category A agents for not being a live microorganism, botulinum toxin is one of the most potent toxins ever described and is thought by some to be the most poisonous substance in existence. It is estimated that 1 g of botulinum toxin would be sufficient to kill 1 million individuals if adequately dispersed. Botulinum toxin is produced by the gram-positive, spore-forming anaerobe C. botulinum (Chap. 42). Its natural habitat is soil. There are seven antigenically distinct forms of botulinum toxin, designated A–G. The majority of naturally occurring human cases are of types A, B, and E. Antitoxin directed toward one of these will have little to no activity against the others.The toxin is a 150-kDa zinc-containing protease that prevents the intracellular fusion of acetylcholine vesicles with the motor neuron membrane, thus preventing the release of acetylcholine. In the absence of acetylcholine-dependent triggering of muscle fibers, a flaccid paralysis develops. Although botulism does not spread from person to person, the ease of production of the toxin coupled with its high morbidity and 60–100% mortality make it a close to ideal bioweapon. Botulism can result from the presence of C. botulinum infection in a wound or the intestine, the ingestion of contaminated food, or the inhalation of aerosolized toxin.The latter two forms are the most likely modes of transmission for bioterrorism.


Once toxin is absorbed into the bloodstream it binds to the neuronal cell membrane, enters the cell, and cleaves one of the proteins required for the intracellular binding of the synaptic vesicle to the cell membrane, thus preventing release of the neurotransmitter to the membrane of the adjacent muscle cell. Patients initially develop multiple cranial nerve palsies that are followed by a descending flaccid paralysis. The extent of the neuromuscular compromise is dependent upon the level of toxemia.The majority of patients experience diplopia, dysphagia, dysarthria, dry mouth, ptosis, dilated pupils, fatigue, and extremity weakness. There are minimal true central nervous system effects, and patients rarely show significant alterations in mental status. Severe cases can involve complete muscular collapse, loss of the gag reflex, and respiratory failure, requiring weeks or months of ventilator support. Recovery requires the regeneration of new motor neuron synapses with the muscle cell, a process that can take weeks to months. In the absence of secondary infections, which may be common during the protracted recovery phase of this illness, patients remain afebrile. The diagnosis is suspected on clinical grounds and confirmed by a mouse bioassay or toxin immunoassay.


Treatment: BOTULISM

Treatment for botulism is mainly supportive and may require intubation, mechanical ventilation, and parenteral nutrition (Table 6-3). If diagnosed early enough, administration of equine antitoxin may reduce the extent of nerve injury and decrease the severity of disease. At present, antitoxins are available on a limited basis as a licensed bivalent product with activity against toxin types A and B and as an experimental product with activity against toxin type E. In the event of attack with another toxin type, an investigational antitoxin with activity against all seven toxin types is also available through the U.S. Army. A single dose of antitoxin is usually adequate to neutralize any circulating toxin. Given that these preparations are all derived from horse serum, one needs to be vigilant for hypersensitivity reactions, including serum sickness and anaphylaxis after their administration. Once the damage to the nerve axon has been done, however, there is little possible in the way of specific therapy. At this point, vigilance for secondary complications such as infections during the protracted recovery phase is of the utmost importance. Due to their ability to worsen neuromuscular blockade, aminoglycosides and clindamycin should be avoided in the treatment of these infections. Vaccination and Prevention A botulinum toxoid preparation has been used as a vaccine for laboratory workers at high risk of exposure and in certain military situations; however, it is not currently available in quantities that could be used for the general population. At present, early recognition of the clinical syndrome and use of appropriate equine antitoxin is the mainstay of prevention of full-blown disease in exposed individuals. The development of human monoclonal antibodies as a replacement for equine antitoxin antibodies is an area of active research interest.


CATEGORY B AND C AGENTS

The category B agents include those that are easy or moderately easy to disseminate and result in moderate morbidity and low mortality rates. A listing of the current category B agents is provided in Table 6-2. As can be seen, it includes a wide array of microorganisms and products of microorganisms. Several of these agents have been used in bioterrorist attacks, although never with the impact of the agents described above. Among the more notorious of these was the contamination of salad 79 bars in Oregon in 1984 with Salmonella typhimurium by the religious cult Rajneeshee. In this outbreak, which many consider to be the first bioterrorist attack against U.S. citizens, >750 individuals were poisoned and 40 were hospitalized in an effort to influence a local election. The intentional nature of this outbreak went unrecognized for more than a decade. Category C agents are the third highest-priority agents in the biodefense agenda.These agents include emerging pathogens to which little or no immunity exists in the general population, such as the severe acute respiratory syndrome (SARS) coronavirus or pandemic-potential strains of influenza that could be obtained from nature and deliberately disseminated.These agents are characterized as being relatively easy to produce and disseminate and as having high morbidity and mortality rates as well as a significant public health impact.There is no running list of category C agents at the present time.


PREVENTION AND PREPAREDNESS

As noted above, a large and diverse array of agents has the potential to be used in a bioterrorist attack. In contrast to the military situation with biowarfare, where the primary objective is to inflict mass casualties on a healthy and prepared militia, the objectives of bioterrorism are to harm civilians, as well as to create fear and disruption among the civilian population. Although the military needs only to prepare their troops to deal with the limited number of agents that pose a legitimate threat of biowarfare, the public health system needs to prepare the entire civilian population to deal with the multitude of agents and settings that could be utilized in a bioterrorism attack.This includes anticipating issues specific to the very young and the very old, the pregnant patient, and the immunocompromised individual. The challenges in this regard are enormous and immediate. Although military preparedness emphasizes vaccines toward a limited number of agents, civilian preparedness needs to rely upon rapid diagnosis and treatment of a wide array of conditions. The medical profession must maintain a high index of suspicion that unusual clinical presentations or the clustering of cases of a rare disease may not be a chance occurrence but rather the first sign of a bioterrorist event. This is particularly true when such diseases occur in traditionally healthy populations, when surprisingly large numbers of rare conditions occur, and when diseases commonly seen in rural settings appear in urban populations. Given the importance of rapid diagnosis and early treatment for many of these conditions, it is essential that the medical care team report any suspected cases of bioterrorism immediately to local and state health authorities and/or to the CDC (888-246-2675).


Recent enhancements have been made to the public health surveillance network to facilitate the rapid sharing of information among public health agencies. At present, a series of efforts are taking place to ensure the biomedical security of the civilian population of the United States. The Public Health Service is moving toward a larger, more highly trained, fully deployable force. A Strategic National Stockpile (SNS) has been created by the CDC to provide rapid access to quantities of pharmaceuticals, antidotes, vaccines, and other medical supplies that may be of value in the event of biologic or chemical terrorism.The SNS has two basic components.The first of these consists of “push packages” that can be deployed anywhere in the United States within 12 h. These push packages are a preassembled set of supplies, pharmaceuticals, and medical equipment ready for immediate delivery to the field.They provide treatment for a variety of conditions given the fact that an actual threat may not have been precisely identified at the time of stockpile deployment. The contents of the push packages are constantly updated to ensure that they reflect current needs as determined by national security threat assessments; they include antibiotics for treatment of anthrax, plague, and tularemia as well as a cache of vaccine to deal with a smallpox threat.The second component of the SNS comprises inventories managed by specific vendors and consists of the provision of additional pharmaceuticals, supplies, and/or products tailored to the specific attack.


The number of FDA-approved and -licensed drugs and vaccines for category A and B agents is currently limited and not reflective of the pharmacy of today. In an effort to speed the licensure of additional drugs and vaccines for these diseases, the FDA has proposed a new rule for the licensure of such countermeasures against agents of bioterrorism when adequate and well-controlled clinical efficacy studies cannot be ethically conducted in humans. Thus, for indications in which field trials of prophylaxis or therapy for naturally occurring disease are not feasible, the FDA is proposing to rely on evidence solely from laboratory animal studies.

For this rule to apply, it must be shown that

(1) there are reasonably well-understood pathophysiologic mechanisms for the condition and its treatment;

(2) the effect of the intervention is independently substantiated in at least two animal species, including species expected to react with a response predictive for humans;

(3) the animal study endpoint is clearly related to the desired benefit in humans; and

(4) the data in animals allow selection of an effective dose in humans. Finally, an initiative referred to as Project BioShield has been established to facilitate biodefense research within the federal government, create a stable source of funding for the purchase of countermeasures against agents of bioterrorism, and create a category of “emergency use authorization” to allow the FDA to approve the use of unlicensed treatments during times of extraordinary unmet needs, as might be present in the context of a bioterrorist attack.


Although the prospect of a deliberate attack on civilians with disease-producing agents may seem to be an act of incomprehensible evil, history shows us that it is something that has been done in the past and will likely be done again in the future. It is the responsibility of health care providers to be aware of this possibility, to be able to recognize early signs of a potential bioterrorist attack and alert the public health system, and to respond quickly to provide care to the individual patient.Among the websites with current information on microbial bioterrorism are www.bt.cdc.gov, www.niaid.nih.gov, www. jhsph.edu/preparedness, and www.cns.miis.edu/research/cbw/index.htm.