EDMG541Wk4

Koenig_and_Schultz_s_Disaster_Medicine_Ch_29.pdf

Biological Events

Robert G. Darling, Jon B. Woods, and Theodore J. Cieslak

The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as necessarily reflecting the views of the Department of Defense, the U.S. Department of Health and Human Services, or any of their component institutions.

OVER V IEW

A Brief History of Biological Warfare

Biological warfare is ancient in its origins, dating back at least as far as 1346. In that year, Tatar invaders laid siege to the city of Kaffa, in present-day Ukraine. When an outbreak of bubonic plague afflicted the Tatar invaders, they catapulted bodies of their own victims over the city walls in an attempt to inten- tionally spread plague within the city. The plan appeared to succeed. Genoese and Venetian merchants stranded within the city contracted plague, fled the city, sailed home, and took the disease with them, thus firmly establishing the “Black Death” in continental Europe.1 Current understanding suggests that the catapults fail to explain the extension of plague into Kaffa. Bubonic plague is now known to be transmitted by fleas, which rapidly abandon the cooling cadaver, making it unlikely that these corpses would remain infectious. Similarly, the American experience with biological warfare dates back at least as far as 1763. During the French and Indian Wars, the British Colonial Commander, Sir Jeffrey Amherst, allegedly ordered the use of smallpox-laden blankets presented as gifts to his Native Ameri- can adversaries,2 apparently resulting in outbreaks of smallpox among these natives.

The advent, in the 19th century, of modern microbiology, germ theory, and Koch’s postulates opened the way for a stock- piling of infectious pathogens and more robust efforts at state- sponsored biological weapons programs. During the First World War, Imperial Germany experimented with anthrax and glan- ders, intending them as antianimal weapons directed at adver- saries’ livestock food sources and beasts of burden.3 In the1930s, Japan used dozens of different biological agents in conducting an extensive series of macabre human biological warfare experi- ments on civilians and prisoners of war in occupied Manchuria.4

Largely in response to these efforts, the United States, in 1943, established its own biological warfare research center at Camp Detrick in Maryland. In 1953, a defensive medical countermea- sures program (which continues to exist today) was added to Camp Detrick’s previously offensively oriented efforts. A more thorough review of the history of biological warfare is available elsewhere.5

In 1925, in response to the horrors produced by chemical weapons in the trenches of WW I, the “Geneva Gas Protocol” was promulgated, forbidding, among other things, the “practice of bacteriological warfare.” Cynics noted that it said nothing about toxin warfare, and it did not prohibit production and storage of agents. Moreover, the United States, for various reasons, was not a signatory to that protocol at the time. In 1969, however, Pres- ident Nixon, speaking for the nation, unilaterally renounced the use of biological weapons. Moreover, he ordered that the United States offensive program be halted and existing weapons stock- piles be destroyed. This destruction occurred during the period from 1969–1972 and culminated in the signing of the Biological Weapons Convention (BWC) by the Soviet Union, the United Kingdom, and the United States. This treaty has since been ratified by more than 140 nations, and prohibits the possession, stockpiling, and use of biological weapons. In 1975, the United States ratified both the Geneva Gas Protocol and the BWC.

Developing a Threat List

Biological warfare, according to language used during crafting of the BWC, is “the use, for hostile purposes, of living organisms, whatever their nature, or infective material derived from them, which are intended to cause disease or death in man, animals, or plants.”6 Little about the topic of biowarfare and bioterrorism is, in reality, as straightforward as this definition might make it appear. In fact, the wording of the BWC has complicated attempts to develop a universally accepted list of biological agents of con- cern. For example, the Soviet Union accused the U.S. government of violating the terms of the BWC by using antiplant agents such as “Agent Orange” during the Vietnam War. The U.S. govern- ment, for its part, considered this to be a nonprohibited use of a chemical agent. Similarly, during that same war, the United

454 Koenig and Schultz's Disaster Medicine : Comprehensive Principles and Practices, edited by Kristi L. Koenig, and Carl H. Schultz, Cambridge University Press, 2009. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/apus/detail.action?docID=564432. Created from apus on 2018-03-08 08:57:10.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 455

Table 29.1: Former Components of the (now-Destroyed) U.S. Biological Arsenal

Lethal Agents Incapacitating Agents Anticrop Weapons

Bacillus anthracis Venezuelan Equine Encephalitis

Wheat-Stem Rust

Botulinum Toxin Staphylococcal Enterotoxin B

Rye-Stem Rust

Francisella tularensis Brucella suis Rice-Blast Spore

Coxiella burnetii

States accused the Soviets of waging biological warfare by using trichothecene mycotoxins (“Yellow Rain”), a charge the Sovi- ets denied. Moreover, they apparently considered toxins to be chemical, rather than biological, weapons. As science advances, consideration of novel substances such as biomodulators (kinins, leukotrienes, substance P, δ-sleep-inducing peptide) as potential agents of warfare will likely only serve to heighten this semantic controversy.

To gain insight into prospective biological weapons candi- dates, it is useful to consider those agents contained within the U.S. arsenal during the 1943–1969 period of offensive weapons research and development. Ten agents were weaponized in the 1950s and 1960s and are listed in Table 29.1. These can be divided into three anticrop and seven antipersonnel agents; the antiper- sonnel agents can be further subdivided into lethal agents and incapacitants.7 Russian sources have provided different perspec- tives.8 A list, in some order of priority, of agents considered for weaponization by Russian (and presumably, Soviet) scientists is provided in Table 29.2.

Biological Warfare versus Bioterrorism

Although the weapons caches of the Cold War Superpowers pro- vide an interesting starting point, it is useful to examine the bio- logical threat on multiple levels. Strategically speaking, biologi- cal agents might be used to strike fear into a nation’s inhabitants and to diminish the resolve of the citizenry. As such, strategic weapons, in this context at least, need only be effective enough to create such fear. Perception of a weapon’s abilities might thus be more important than its actual ability to cause disease. Strategic weapons might also be used against domestic targets important to the conduct of a foreign war, such as command and control nodes, staging bases, and ports of embarkation. Thus, even agents capable of infecting a few key personnel might be strategically beneficial.

Conversely, contagion might be a useful property in a strategic weapon, permitting its propagation among the pop- ulation. Such contagion, however, might limit a commander’s enthusiasm for using the same weapon on the battlefield, where friendly forces might inadvertently contract it. This property of contagion likely explains the inclusion of smallpox, plague, and Marburg virus in the Soviet arsenal (the Soviets favored the strategic use of biological agents)9 and, perhaps, their exclusion from the U.S. arsenal (as the United States saw these primarily as operational agents).

A number of biological agents might be adapted for oper- ational use. That is, they possess characteristics (such as atmo- spheric stability) that could enable their use over large geograph- ical areas. The list of such agents is short, enabling public health

Table 29.2: “Rating System (Russian) of Bioagent Distribution According to Probability of Use as Bioweapons”

Smallpox Virus

Yersinia pestis

Bacillus anthracis

Botulinum Toxin

Venezuelan Equine Encephalitis Virus

Francisella tularensis

Coxiella burnetii

Marburg Virus

Influenza Virus

Burkholderia mallei

Rickettsia typhi

See text and reference 8 for details.

authorities to concentrate countermeasure efforts on a finite number of threats. Anthrax, plague, tularemia, and perhaps a few other agents might pose viable operational threats. In one regard, the threat posed by tactical use of biological agents is more problematic than the operational threat in that certain viruses and toxins, which lack the stability necessary for effective operational use, might nonetheless be used effectively against smaller, more concentrated targets (such as individual buildings or smaller areas of terrain). Conversely, some strategists would argue that biological agents are a poor choice for such tactical use. This contention stems from a unique characteristic not typically shared by conventional, chemical, and nuclear weapons, namely, the incubation periods inherent with infectious agents. These incubation periods might vary from hours in the case of staphy- lococcal enterotoxin B to weeks in the case of brucellosis or Q- fever but are more typically several days in duration. Comman- ders assigned tactical objectives (such as seizing an important terrain feature) would likely balk at the thought of pausing for several days while waiting for biological agents to slowly produce their effects. Even if one could conceive of a tactical use for bio- logical weapons, the list of viable candidates remains short, with only a handful of agents possessing the desirable characteristics.

In addressing the terrorist threat, one, in a sense, comes full circle. Here, public perception is of paramount importance, and any attack capable of generating headlines (and publicity for the cause of a given extremist group) might be considered “viable.” Moreover, the delay induced by the incubation periods of biological agents would not likely dissuade the terrorist from their use. The list of potential agents of terrorism thus becomes quite lengthy. Designing effective countermeasures and defen- sive strategies against such a large list of possible threats becomes a daunting task. Agents and diseases such as human immun- odeficiency virus, Escherichia coli, “mad cow,” rabies, and Ebola, which might otherwise lack characteristics desirable in a weapon, possess the “brand-name” recognition sought by certain terror- ists. Similarly, terrorists might select “weapons of opportunity,” that is, weapons that might be widely available and/or readily procured by a member of the group. This further lengthens the list of potential agents. The intentional contamination of restau- rant salad bars in The Dalles, Oregon, with Salmonellae in 1984 highlights this problem as does the 1996 use of Shigella-laden

Koenig and Schultz's Disaster Medicine : Comprehensive Principles and Practices, edited by Kristi L. Koenig, and Carl H. Schultz, Cambridge University Press, 2009. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/apus/detail.action?docID=564432. Created from apus on 2018-03-08 08:57:10.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

456 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

pastries in a notable biocrime.10,11 Although not considered credible military threats by most planners, these two common gastrointestinal pathogens were nonetheless somewhat effective in the hands of criminals. Presumably, dozens of similar organ- isms and many toxins might be used in an analogous manner. Taking these factors into consideration, many intelligence and law enforcement professionals contend that the magnitude of the terrorist threat surpasses, in some ways, that of the military threat. Moreover, this threat remains nebulous and difficult to monitor. Finally, although response to the military use of such weapons might occupy defense planners, it is the civilian medical and public health community that must shoulder the responsi- bility of reacting to a terrorist release of a biological agent. Con- sequently, the remainder of this chapter focuses on this terrorist threat.

CURRENT STATE OF THE AR T

The Terrorist Threat

Examples of the terrorist use of conventional weapons abound. Mumbai, Beirut, Oklahoma City, the Khobar Towers, the first World Trade Center attacks, and the bombing of American embassies in Nairobi and Dar es Salaam all highlight the con- ventional threat. The use of airplanes in the second attack on the World Trade Center and on the Pentagon on September 11, 2001 gives new meaning to unconventional weaponry without requir- ing access to nuclear or biological arms. The release of sarin in the Tokyo subway system by members of the Aum Shinrykyo cult is a reminder that terrorists have the ability to procure and deploy chemical weapons. By comparison, the intentional mailing of anthrax-laced letters in October 2001 in the U.S. sickened 22 and resulted in a relatively modest (by comparison) death toll of five people.12 Why, then, might biological weapons interest terrorist factions? There are multiple characteristics that make biological weapons potentially attractive to such groups.

First, biological weapons are relatively easy to procure. Clostridium botulinum is ubiquitous in soil and easily cultured by anyone with modest training in microbiology. Bacillus anthracis is similarly readily cultivatable from soil in many parts of the world. Ricin extraction from castor beans, readily available throughout the world, is easily accomplished using recipes widely published on the Internet. Many putative biological weapons, such as Coxiella burnetii, the encephalitic alpha viruses, the Bru- cellae, Francisella tularensis, and even Yersinia pestis and Bacillus anthracis, continue to cause endemic disease in many parts of the world. Clinical laboratories in those locales handle cultures of such organisms and constitute a potential source for their acquisition. Culture repositories organized for legitimate scien- tific purposes may be accessed by those with sinister motives. Although the U.S. Department of Health and Human Services (HHS), Centers for Disease Control and Prevention (CDC) man- aged Select Agent Program seeks to limit access to particularly hazardous pathogens and toxins in the United States (regulated agents are listed in Table 29.3), hundreds of such repositories exist in dozens of foreign nations; many of these sell and ship these hazardous agents.13,14 Devices able to disseminate biolog- ical agents are also widely available. Crop-dusting assemblies may be adapted by terrorists for sinister purposes and can, in some cases, generate aerosolized particles of 2–6 µm in diame- ter, the ideal size for impinging on the human lower respiratory tract.

Second, biological weapons are potentially inexpensive to produce. In 1969, a United Nations study considered the cost to a belligerent of producing mass casualties (defined as 50%) and found crude biological weapons to be far less costly than chemical, nuclear, or even conventional arms on a “casualties per square kilometer” basis.15

Third, unless the terrorists announced the release of a bio- logical agent, detection of an attack would be challenging. Aerosolized biological agents would likely be odorless, colorless, tasteless, and otherwise invisible (in contrast to chemical agents, many of which have characteristic odors and generally cause immediate symptoms in all victims within a confined space). At the time of this writing, standoff detection systems (such as the Joint Biological Point Detection System) are under devel- opment, but are expensive, often cumbersome to use, and not yet readily available.16 Although standoff detection systems are used during certain high-profile public events, their widespread real-time deployment remains limited. In fact, initial detection of a bioterrorist attack will probably not hinge on the finding of explosive devices, missiles, or even crop-dusting equipment. It would not be likely to involve environmental detection or meteorological perturbations. Rather, it more likely will involve the presentation (perhaps widely dispersed geographically) of patients with nonspecific symptoms, to various practitioners, clinics, and emergency departments. This is especially prob- lematic to the clinician because treatment of diseases such as anthrax, plague, and botulism is most effective when begun as early as possible (when such nonspecific symptoms are likely to predominate), ideally during the incubation period. By the time hallmark findings (mediastinitis in the case of anthrax, hemoptysis in plague victims, and neuromuscular symptoms for botulism) appear, treatment is of dubious benefit and prognosis is often poor. Moreover, with easy access to jet travel, incubation periods ensure that perpetrators can safely depart for any foreign land before nonspecific effects are noticed. Additionally, conta- gious biological agents (e.g., smallpox and pneumonic plague) can continue to propagate among successive generations of vic- tims prior to discovery and diagnosis.

Finally, victims of a biological attack, more so than conven- tional or chemical casualties, can potentially overwhelm medical response capabilities. Botulism provides an instructive example. In isolated cases with access to modern medical interventions (such as lengthy courses of mechanical ventilation and other intensive care modalities), it is a survivable disease; however, the sudden requirement to provide critical care and ventilation to hundreds or thousands of casualties in a given city would render a large botulism outbreak unmanageable.

With these considerations in mind, it is clear that the specter of biological terrorism warrants a level of planning and pre- paredness at least as great as that devoted to conventional and chemical terrorism. In fact, it is precisely a lack of prepared- ness that might amplify the allure of biological weapons in the minds of some terrorists. Capitalizing on this lack of prepared- ness, a terrorist need not even possess a weapon. The simple threat of release might be enough to influence policymaking and engender a massive commitment of resources. For exam- ple, many hundreds of anthrax threats have come to the atten- tion of law enforcement agencies over the past decade. With the notable exception of the anthrax letter attacks of October 2001 in the United States, however, virtually all of these threats have proven to be unfounded.17 Yet, even the most amateur hoax (or a concerned person calling about a benign substance) has often

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 457

Table 29.3: HHS and U.S. Department of Agriculture (USDA) Select Agents and Toxins (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73)

HHS SELECT AGENTS AND TOXINS Coxiella burnetii

Alirin Eastern Equine Encephalitis virus

Cercopithecine herpesvirus 1 (Herpes B virus) Francisella tularensis

Coccidioides posatfasii Hendra virus

Conotoxins Nipah virus

Crimean-Congo hemorrhagic fever virus Rinn Valley fever virus

Diacetoxyscirpenol Shigatoxin

Ebola virus Staphylococcal enterotoxins

Lassa fever virus T-2 toxin

Marburg virus Venezuelan Equine Encephalitis virus

Monkeypox virus USDA SELECT AGENTS AND TOXINS

Reconstructed replication-competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all 8 gene segments (reconstructed 1918 influenza virus)

African horse sickness virus

Ricin

African swine fever virus

Rickettsia prowazekii, Rickettsia rickettsii, Saxitoxin

Akabane virus

Shigalike ribosome-inactivating proteins

Avian influenza virus (highly pathogenic)

South American Hemorrhagic Fever viruses

Bluetongue virus (exotic)

Flexall

Bovine spongiform encephalopathy agent

Guanarito

Camel pox virus

Junin

Classic swine fever virus

Machupo

Cowdria ruminantium (Heartwater)

Sabia

Foot-and-mouth disease virus

Tetrodotoxin tick-borne encephalitis complex (flavi) viruses

Goat pox virus

Central European tick-borne encephalitis-Far Eastern tick-borne

Japanese encephalitis virus

encephalitis

Kyasanur Forest disease

Lumpy skin disease virus

Omsk Hemorrhagic Fever

Malignant catarrhal fever virus

Russian Spring and Summer encephalitis

(Alcelaphine rierpesvims type 1) Menangle virus Mycoplasma capricola/ M.F38/M. mycoides Capri (contagious caprine pleuropneurnonia) Mycoplasma inycoides mycoides

Variola major virus (Smallpox virus) and (Contagious bovine pleuropneumonia) Newcastle disease virus (velogenic) Peste des petits ruminants virus, Rinderpest virus, Sheep pox virus, Swine vesicular disease virus, Vesicular stomatitis virus (exotic)

Variola minor virus (Alastrim) Yersinia pestis USDA PLANT PROTECTION AND QUARANTINE (PPC) SELECT

AGENTS AND TOXINS OVERLAP SELECT AGENTS AND TOXINS

Candidatus Liberobacter africanus Bacillus anthracis

Candidatus Liberobacter asiaticus Botulinum neurotoxins

Perorws clerospora phi/ippinensis Botulinum neurotoxin producing species of Clostridium

Ralstonia solanacearum race 3. biovar 2 Brucella abortus

Ciliophora rayssiae var zeae Brucella melitensis

Synclinorium endobioticum Brucella suis

Xanthomonas oryzae pv. oryzicola Burkholderia mallei (formerly Pseudomonas mallei)

Xylella fastidiosa (citrus variegated chlorosis strain) Burkholderia pseudomallei (formerly Pseudomonas pseudomallei)

Clostridium perfringens epsilon toxin

Coccidioides iritis

resulted in a response that forced the expenditure of hundreds or thousands of dollars.

For bioterrorism defense planning and preparedness pur- poses, it is necessary to be familiar with the specific agents that might be used by terrorists. The agents developed and studied by the Cold War Superpowers (catalogued in Tables 29.1 and 29.2) provide a starting point for consideration. An examination

of a terrorist’s potential motives permits further refinement of these lists. A study conducted by the World Health Organization (WHO) showed that anthrax was somewhat unique in its ability to produce widespread mortality.18 Table 29.4 compares the mor- bidity and mortality figures derived from this study, which con- sidered the release of 50 kg of agent along a 2-km line upwind of a city of 500,000 inhabitants. For a terrorist group interested

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

458 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

Table 29.4: Results of the Hypothetical Aerosol Dissemination of Various Infectious Agents

Downwind Total Agent Carriage Deaths Casualties

Venezuelan Equine Encephalitis 1 km 400 35,000

Tick-borne Encephalitis 1 km 9,500 35,000

Epidemic Typhus 5 km 19,000 85,000

Brucellosis 10 km 500 100,000

Plague 10 km 55,000 100,000

Q-Fever >20 km 150 125,000

Tularemia >20 km 30,000 125,000

Anthrax >>20 km 95,000 125,000

Casualty figures assume 50 kg of dried agent, disseminated along a 2-km line upwind of a population center of 500,000. Adapted from reference 18 with permission.

in producing widespread lethality, anthrax would seem an ideal choice (assuming it could be procured, weaponized, and deliv- ered optimally). Weaponization refers to the process of modify- ing a biological agent in the laboratory to optimize its dispersal in the environment or its pathogenicity for use as a weapon. Smallpox, a disease not considered in the WHO study, might be expected to pose problems of a similar or greater magnitude. Smallpox and pneumonic plague, and to a lesser degree certain viral hemorrhagic fevers, are noteworthy in that they, in contrast to other agents mentioned in this chapter, are contagious. Terror- ists might thus leverage the results of an attack by weaponizing smallpox or plague, infecting a modest number of persons in a first wave, and depend on contagion to assist the agent in prop- agating through a population, thereby overcoming some of the technical challenges of widespread aerosol delivery.

Although certain assumptions and generalizations can be made in attempting to define and combat the terrorist threat, it is clear that the motives and rationale of terrorists cannot always be

elucidated. Shigellae, Giardia, and even roundworms have been used as weapons by terrorists, criminals, or other disgruntled per- sons.19 Envisioning and preparing for each of these scenarios in advance of their occurrence would have been impossible. Given this factor and the constraint of limited resources, it is not useful to consider those agents most likely to be used (because this can- not be ascertained), but rather those that, if used, would produce the most devastating consequences and require the most critical medical responses. In June of 1999, U.S. public health experts met at CDC headquarters and used this rationale to develop a list of “critical biological agents for health preparedness” (Table 29.5).17,20 Agents in “category A” are those that, if released effectively, would be expected to have a high overall public health impact. Consequently, significant medical intervention would be required and intensive public health preparedness is ongoing. Preparedness measures include medication and supply stockpil- ing and improvements to surveillance and response capabilities of local, state, and federal health authorities. Category B agents present a somewhat lesser requirement for preparedness, whereas category C agents require vigilance to guard against their future development as threat agents, but can otherwise likely be ade- quately managed within the framework of the existing public health infrastructure.

CATEGOR Y A AGENTS

Anthrax

The causative agent of anthrax, Bacillus anthracis, is a Gram- positive, sporulating rod-shaped bacterium. Anthrax is primarily an endemic and epidemic disease of livestock. Ungulates such as sheep, goats, and cattle are exposed while grazing through the ingestion of soil-borne spores. These spores germinate within the animal, multiplying rapidly in the bloodstream and leading to death within days. At the time of death, the animal’s blood may contain as many as 108 bacteria per cubic centimeter; these bacteria, once exposed to oxygen as the animal decomposes, sporulate, enter the soil, and thus continue the cycle.

Table 29.5: Critical Agents for Public Health Preparedness

Category A Category B Category C

Variola virus Coxiella burnetii Emerging threat agents (e.g., Nipah virus, hantaviruses, pandemic influenza viruses)

Bacillus anthracis Brucellae

Yersinia pestis Burkholderia mallei

Botulinum toxin Burkholderia pseudomallei

Francisella tularensis Alphaviruses

Filoviruses and arenaviruses Rickettsia prowazekii

Certain toxins (e.g., Ricin, SEB)

Chlamydia psittaci

Food safety threat agents (e.g., Salmonellae, E. coli O157:H7)

Water safety threat agents (e.g., Vibrio cholera)

Category A agents have high public health impact requiring intensive public health preparedness and intervention; Category B agents have a somewhat lesser need for public health preparedness. Category C agents are emerging infections that may pose a threat in the future.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 459

Figure 29.1. Cutaneous anthrax. Note the painless black eschar and moderate surrounding erythema. Photograph: Courtesy of the Centers for Disease Control and Prevention, Atlanta, Georgia. www.bt.cdc.gov/ agent/anthrax/anthrax-images/cutaneous.asp. See color plate.

B. anthracis has several characteristics that make it a useful biological weapon: 1) it is easy to obtain – the organism can be found virtually anywhere in the world where livestock are kept but are not routinely immunized against anthrax; 2) it grows readily in easily prepared media; 3) it can be easily be induced to form spores, which are not only highly infective via the aerosol route, but can also be stored for an extended time with minimal degradation; and 4) spore size and durability facilitate highly efficient aerosol delivery as a biological weapon.

Human anthrax takes three primary forms – cutaneous, gas- trointestinal, and inhalational. Cutaneous anthrax is the most common naturally occurring form of human disease. Approx- imately 7 days (range 1–12 d) following exposure to infected hides or meat, a painless or mildly pruritic papule forms at the site of exposure. The lesion rapidly enlarges and ulcerates, often developing vesicles or bullae at the margins, and is often accompanied by significant surrounding edema and regional lymphadenopathy. As the ulcer dries, it forms a coal-black scab (hence the name, “anthrax,” from the Greek anthracis meaning coal), which resolves over 1–2 weeks (Figure 29.1). Up to 20% of untreated cutaneous anthrax cases progress to systemic disease and are fatal. Of note, 11 of the 22 suspected or documented cases of anthrax from the 2001 “Amerithrax” mailings were cutaneous in nature.12

Oropharyngeal anthrax is a variation of cutaneous anthrax in which ingestion of contaminated meat leads to an oropharyngeal lesion and associated neck edema and adenopathy; the mortality rate in oropharyngeal anthrax can be much higher than that for cutaneous anthrax, likely due to the increased incidence of systemic spread as well as airway compromise resulting from oropharyngeal edema.21

Gastrointestinal anthrax results from consumption of the insufficiently cooked meat of infected animals. One to 6 days fol- lowing ingestion, fever, nausea, vomiting, and focal abdominal pain develop. Victims then typically develop massive gastroin- testinal bleeding and sepsis, with a fatal outcome occurring in more than 50% of cases.

Historically, inhalational anthrax has been an extraordinar- ily rare disease found only in wool or hide mill workers after exposure to high concentrations of anthrax spores that are aerosolized by manipulation of contaminated animal products. Disease is the result of inhalation of these aerosolized spores, which are then ingested by alveolar macrophages and car- ried to mediastinal lymph nodes, where they multiply and release toxins. Typically 1–6 days after exposure (but perhaps up to several months later) disease onset is heralded by a non- specific febrile illness, often accompanied by malaise, fatigue, and drenching sweats. Pneumonia is rare, and an auscultatory examination of the lungs is often normal at this phase of the illness, although radiological studies may demonstrate pleural effusions and the classic widened mediastinum of hemorrhagic mediastinitis (Figure 29.2). Upper respiratory symptoms such as rhinorrhea or nasal congestion are rare in inhalational anthrax. Cough, if present, is nonproductive. If untreated, disease that has progressed this far will typically, within 2–5 days, lead to severe respiratory distress, shock, and death. Historically, mor- tality for inhalational anthrax was greater than 85%, although only five of the 11 victims (45%) of inhalational anthrax suc- cumbed in the Amerithrax mailings of 2001. This improve- ment in outcome likely reflects the aggressive modern-day management of these patients. Patients with all forms of anthrax disease should be managed using standard infection control pre- cautions. Person-to-person spread of anthrax is extremely rare, even in inhalational cases. Invasive procedures that can generate infectious aerosols should, however be avoided in patients who may be bacteremic. In addition it is at least theoretically possi- ble for person-to-person transmission of cutaneous anthrax via nonintact skin; therefore, standard precautions should always be followed in individuals with open skin or mucosal lesions.

Effective diagnosis of anthrax relies on a strong clinical sus- picion to drive appropriate confirmatory laboratory studies. For systemic febrile disease resulting from any form of anthrax, blood cultures may be diagnostic if performed prior to receipt of antibi- otics. For mild cutaneous disease, culture of the lesion (ideally vesicle fluid) may be positive; Gram stain of vesicle fluid may show large, Gram-positive bacilli, and immunohistochemical stains can identify anthrax in culture-negative lesions. In patients with gastrointestinal anthrax, stool cultures can sometimes be positive, and hemorrhagic peritoneal fluid can also be cultured or immunostained for B. anthracis. A widened mediastinum with or without pleural effusions on chest x-ray or computed tomog- raphy suggests inhalational anthrax. Gram stain of pleural fluid or cerebrospinal fluid (in the presence of meningitis, seen in up to 50% of cases and hemorrhagic in character) can be positive, and specific immunostaining or polymerase chain reaction (PCR) of these fluids can be diagnostic.21 The first patient diagnosed dur- ing the 2001 U.S. attacks presented with fever, confusion, and respiratory distress with hemorrhagic meningitis secondary to B. anthracis (Figure 29.3).

Uncomplicated cutaneous anthrax known to have been con- tracted via infected animal contact can be treated with a num- ber of oral antibiotics. Because naturally occurring strains of B. anthracis are nearly always susceptible to penicillin, 500 mg orally every 6 hours (40–80 mg/kg/d every 6 h in children) or amoxicillin 500 mg orally every 8 hours (40–80 mg/kg/d every 8 h in children) would be effective. Ciprofloxacin 500 mg twice per day (20–30 mg/kg divided twice per day for children) or doxycycline 100 mg twice per day (5 mg/kg divided twice per day for children) would be acceptable alternatives.22

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

460 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

C Figure 29.2. (A) Chest x-ray demonstrates mediastinal and hilar widening, and bilateral pleural effusions. (B) Chest computed tomog- raphy scan demonstrates enlarged, hyperdense subcarinal (arrow) and left hilar (arrowhead) lymph nodes probably secondary to intranodal hemorrhage. (C) Note the peribronchial consolidation which reflects lymphatic spread of anthrax infection. Radiologic Images: Courtesy of JR Galvin, MD and AA Frazier, MD, Department of Radiologic Pathology, Armed Forces Institute of Pathology, Washington, DC.

Figure 29.3. Meningitis with subarachnoid hemorrhage in a man from Thailand who died 5 days after eating undercooked carabao (water buffalo). Reproduced from: Binford CH, Connor DH, eds. Pathology of Tropical and Extraordinary Diseases. Vol 1. Washington, DC: Armed Forces Institute of Pathology; 1976: 121. AFIP Negative 75-12374-3. See color plate.

Although fluoroquinolone and tetracycline antibiotics are generally not recommended for use in children and pregnant women, a consensus group, as well as the American Academy of Pediatrics, has suggested that ciprofloxacin or doxycycline should nonetheless be used as first-line therapy in life-threatening anthrax disease or in disease suspected to be of sinister ori- gin (because penicillin-resistant strains can readily be selected for in the laboratory) until strain susceptibilities are known. The U.S. Food and Drug Administration (FDA) has approved ciprofloxacin for prophylaxis and treatment of anthrax in children, and as the first choice for antibiotics in pregnant women.23 If the infecting strain later proves to be penicillin sus- ceptible, a switch to oral penicillin VK or amoxicillin can be made in cases of mild cutaneous anthrax.

In the event that the exposure route is unknown or that expo- sure is potentially related to a biological terrorism event in which anthrax may have been aerosolized, antibiotics should be contin- ued for at least 60 days (as postexposure prophylaxis). Moreover, for all forms of symptomatic anthrax aside from mild cuta- neous disease (to include inhalational, gastrointestinal, oropha- ryngeal, and severe cutaneous forms), combination intravenous antibiotics are strongly advised. Initial empirical therapy should include ciprofloxacin or doxycycline plus one or two additional antibiotics effective against anthrax. Some additional antibiotics to which naturally occurring strains of B. anthracis are suscepti- ble include imipenem, meropenem, daptomycin, quinupristin- dalfopristin, linezolid, vancomycin, rifampin, the macrolides, clindamycin, chloramphenicol, and the aminoglycosides. It is at least theoretically possible to induce antimicrobial resistance to any of these antibiotics; however, there are no reports of this in the literature. A switch from intravenous to oral therapy may be made as the patient’s clinical course dictates, although in most situations, the optimal duration of therapy and antibiotic combination is not known. At the time of this writing, human anthrax immune globulin, collected from recipients of Anthrax Vaccine Adsorbed (AVA), is in clinical trials and may be available as adjunctive therapy under an investigational new drug (IND) application from the FDA.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 461

AVA (BioThraxTM, Bioport, Lansing, MI) is licensed for the prevention of anthrax and has been administered to certain U.S. laboratory workers, selected first responders, and military service members. AVA is a protein vaccine produced from the supernatant of a culture of an attenuated strain of B. anthracis. It is administered subcutaneously in an initially six-dose series over 18 months (0, 2, and 4 weeks and then 6, 12, and 18 months) followed by annual boosters. Although AVA is licensed only for preexposure prophylaxis of anthrax in adults aged 18–65 years, it is available under IND protocol for preexposure use in children, and postexposure prophylaxis in adults and children, although its role in such postexposure prophylaxis has not yet been deter- mined.24 Patients who have had hypersensitivity reactions to previous doses should not receive AVA, and the vaccine should be deferred in those who are: pregnant, currently suffering from a febrile infectious disease, or taking immunosuppressant drugs such as corticosteroids. The U.S. FDA has determined that the AVA is both “safe and effective” in preventing all forms of anthrax disease.25

Following an aerosolized attack with B. anthracis, exposed persons should receive antibiotic prophylaxis to prevent devel- opment of disease. Even previously immunized victims should immediately receive oral ciprofloxacin, levofloxacin, or doxycy- cline, all of which have been licensed for this application. Should the offending strain later be determined to be penicillin sensitive, penicillin VK or amoxicillin can be substituted in those who can- not tolerate first-line antibiotics. Antibiotics should be continued for at least 60 days because spores can remain dormant within a victim’s lungs for extended periods only to germinate later, after the victim may have completed weeks of prophylaxis. Some patients may be noncompliant due to side effects such as diarrhea. Because of this potential spore dormancy, the U.S. Department of Defense (DoD) recommends that, following exposure, previ- ously unimmunized persons receive at least three doses of AVA (under IND, as this is not an FDA-approved use) over 4 weeks prior to discontinuing prophylactic antibiotics. After complet- ing antibiotics, patients should receive close follow-up for return of fever or other signs and symptoms of anthrax infection.26 A July 2009 New England Journal of Medicine study reported that raxibacumab, a human monoclonal antibody, is effective in the treatment and prevention of inhalational anthrax in two animal models, however it is not yet clear whether this newer modality can be routinely recommended.27

Smallpox

Smallpox is a disease limited to humans and caused by the Orthopox virus Variola major. Variola minor causes a milder form of the disease termed alastrim. Historically, smallpox was a significant cause of human suffering and death worldwide, responsible for as many as 50 million cases per year in the mid- dle of the last century. The World Health Organization declared smallpox to be eradicated in 1980 after a monumental worldwide vaccination campaign. Significant concern remains that existing laboratory-based variola virus isolates could be reintroduced as a weapon into an increasingly susceptible population. Variola is easily grown in cell culture or chicken eggs and is readily dried into a stabile form, which can survive prolonged stor- age and is suitable for aerosolization. Smallpox is a contagious disease and can spread through a susceptible population, with secondary attack rates as high as 50% in nonimmune household contacts.28

People typically acquired smallpox via mucous membrane contact with infectious respiratory droplets from an infected, coughing person. Less commonly, disease was transmitted via direct contact with lesions or secretions, fomites, or via infec- tious aerosols. Approximately 12 days (range 7–19 d) after inoc- ulation, the patient experienced the sudden onset of high fever (38.8◦C–40.0◦C), malaise, headache, and shaking chills. The patient would often be bedridden with severe backache, abdom- inal pain, and vomiting. Two to 3 days after the onset of symp- toms the patient would experience a mild improvement in symp- toms, with decreased fever, and the onset of an enanthem in the form of small, painful ulcerations of the tongue and oropharynx. Oral secretions at this phase of illness are teeming with variola virus, and the patient is a significant infection risk. Within a day of enanthem onset, 2–3 mm erythematous macules appear on the face and distal extremities (Figure 29.4). The macules progress to papules, and then to clear vesicles over the next 3–5 days, and finally to tense, painful, centrally umbilicated pustules shortly thereafter – often accompanied by a second fever spike. As the lesions evolve, they spread centrally, although they typ- ically remain more pronounced and abundant on the face and distal extremities. Death, if it occurs, typically does so within this second week of infection. Among survivors, pustules further progress to scabs, which separate to leave depressed, hypopig- mented, permanent scars that are often quite disfiguring.29 Scabs contain viable virus; the patient is thus considered contagious and requires appropriate isolation until scabs are entirely shed.

The severity of smallpox disease in the past was quite variable, with several forms of disease described. Severity and ultimate mortality were directly related to the number and concentration of skin lesions; confluent lesions portended a particularly bad outcome. Disease was generally more severe in women (especially if pregnant), children, the elderly, certain ethnic groups (e.g., Native Americans and Pacific Islanders), and immunocompro- mised individuals. Partially immune individuals (i.e., vaccinated) tended to have mild disease, with few lesions and lower mortal- ity – a syndrome that closely resembled that seen in variola minor. Flat-type smallpox (Figure 29.5) probably represented an extreme form of confluent smallpox in which the skin took on a uniform “crepe rubber” appearance instead of forming classic lesions. This was most commonly seen in children. Hemorrhagic smallpox (Figure 29.6) was a rare form of fulminant disease with associated bleeding diatheses seen predominantly during preg- nancy and in immunocompromised individuals. Mortality from classic smallpox varied from 10% to 30% in nonimmune indi- viduals, to approximately 3% in the immunized. Among preg- nant women the mortality rate was up to 65%, and flat and hemorrhagic forms of disease were fatal in 95% of cases. Long- term complications of smallpox included blindness from corneal scarring (1%–4% of cases),30 growth abnormalities in children secondary to variola osteomyelitis (2%–5% of child cases),31 and disfiguring or even physically debilitating dermal scarring from the pox lesions themselves.

With an increasing immunologically naive population (e.g., routine immunization of U.S. civilians ceased in 1972) and the ease of global travel, there is concern that smallpox would spread faster than it has historically. The degree of person-to-person spread of smallpox in the past varied according to many factors. Such spread was associated with exposure to cases with con- fluent rash or severe enanthem and to cases with severe bron- chiolitis and cough. Typically, close person-to-person contact was required for reliable transmission; however, variola’s spread

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

462 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

B2 C1 C2

A2 B1

Figure 29.4. Series of photographs illustrating the evolution of skin lesions in an unvaccinated infant with the classic form of Variola major. (A1, A2) The third day of rash shows synchronous eruption of skin lesions; some are becoming vesiculated. (B1, B2) On the fifth day of rash, almost all papules are vesicular or pustular. (C1, C2) On the seventh day of rash, many lesions demonstrate central umbilication, and all lesions are in the same general stage of development. Reproduced with permission from reference 27. See color plate.

via aerosol is well documented in hospital outbreaks.32 Vari- ola could also spread by contact with contaminated bedding, especially in the hospital setting, although such factors typically played a small role in overall transmission through a population. In past outbreaks, environmental conditions are thought to have factored prominently in disease propagation. Smallpox spread more quickly in conditions of low humidity, and during winter or rainy seasons, when people would crowd together in their homes. The disease tended to spread slowly through partially immune communities, but could become endemic in densely populated regions, even in a population with up to 80% vaccination rates.28

Historically, the diagnosis of smallpox was largely based on the characteristic clinical findings, particularly the rash. In some

cases, such clinical diagnosis could be problematic. Prodromal smallpox is difficult to differentiate from other febrile syndromes. The early rash of smallpox has been commonly mistaken for varicella and other viral exanthems (e.g., adenovirus), as well as conditions such as erythema multiforme that can cause febrile illness with rash. Flat-type and hemorrhagic smallpox may be difficult to differentiate clinically from other fulminant infectious syndromes presenting with shock and disseminated intravascular coagulation.

Monkeypox, although not classified as a category A threat by the U.S. CDC, is another Orthopox virus closely related to small- pox that occurs naturally in equatorial Africa and presents with a very similar rash. Descriptions of human monkeypox outbreaks

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 463

Figure 29.5. Flat-type smallpox in an unvaccinated woman on the sixth day of rash. Extensive flat lesions (A and B) and systemic toxicity with a fatal outcome were typical. Reproduced with permission from reference 27. See color plate.

in sub-Saharan Africa reveal a disease that could be clinically indistinguishable from smallpox with the exception of a gener- ally lower case fatality rate (11% in nonimmunized people) and notable enlargement of cervical and inguinal lymph nodes man- ifesting 1–2 days before the rash in 90% of cases.33 An outbreak involving 81 human cases of monkeypox occurred in 2003 in the United States due to exposure to exotic pets (such as Gambian rats) imported from West Africa. These cases demonstrated only localized lesions and mild disease, with no deaths or secondary transmission occurring among humans.34

Early, specific diagnosis is paramount to ensuring appro- priate patient isolation and preventing the spread of smallpox. Definitive diagnosis historically required isolation of the virus and characterization of its growth on chicken egg chorioal- lantoic membrane or in cell culture. More recently, the U.S. CDC, via its Laboratory Response Network, has published guide- lines for laboratory diagnosis of acute, generalized vesicular or pustular rash illnesses. These guidelines rely heavily on non- specific Orthopoxvirus PCR testing, as well as specific variola PCR to identify probable cases, with confirmatory testing then performed at national level laboratories under Biosafety Level 4 (BSL-4) conditions. Acceptable specimens for this protocol include: vesicular “touch-prep,” vesicle roof, vesicular swab or impression slide, and biopsy specimens. Other specimens that could be useful in the prevesicular rash phase of illness include serum or pharyngeal swabs. Providers who collect or process

Figure 29.6. Early hemorrhagic-type smallpox with cutaneous signs of hemorrhagic diathesis. Death usually intervened before the com- plete evolution of pox lesions. Reproduced with permission from Herrlich A, Munz E, Rodenwaldt E. Die pocken; Erreger, Epidemiolo- gie und klinisches Bild. 2nd ed. Stuttgart, Germany: Thieme; 1967. See color plate.

specimens should only do so under the direction of public health officials, should be vaccinated against smallpox, and should exer- cise both contact and airborne precautions.35

There are no proven specific therapies for symptomatic smallpox. Parenteral cidofovir is an antiviral drug (licensed for use in cytomegalovirus retinopathy) that shows in vitro activity against a broad range of poxviruses and potential in vivo ben- efit in animal studies of poxvirus infections. Additional studies are under way to determine whether parenteral or oral cidofovir analogues, as well as other drugs such as ST-246, might be effi- cacious in the treatment of human Orthopoxvirus infections.36

Ocular smallpox may benefit from application of topical antivi- rals such as trifluridine or idoxuridine. Aggressive supportive care is the cornerstone of successful management of smallpox disease, however, and should include maintenance of hydration and nutrition, pain control, and prevention and management of secondary infections.

Infection control within healthcare facilities could repre- sent a significant challenge; smallpox patients should be iso- lated under contact and airborne precautions. Caregivers should be immunized and wear appropriate personal protective equip- ment regardless of their immunization status. Patients should be considered infectious until all scabs separate. Across the United States and elsewhere, communities are currently strug- gling with development of smallpox and other contagious infec- tious disease response plans. The CDC maintains planning guid- ance for states and communities on their bioterrorism website (http://emergency.cdc.gov/agent/smallpox/prep). Victims of an attack using weaponized smallpox, as well as contacts of known smallpox cases, should be immunized and monitored for at least 17 days following the last known exposure regardless of their vaccination status; at the onset of fever they should be immedi- ately isolated using droplet and airborne precautions. Isolation should continue until smallpox is either ruled out or confirmed, and, if confirmed, until all scabs separate.

Two vaccines against smallpox are licensed in the United States: DryvaxTM (Wyeth),37 a live, dried, calf-lymph-derived, lyophilized vaccinia virus product last manufactured in 1982, and ACAM-2000TM (Acambis), a cell culture–derived prepara- tion derived from the same vaccinia virus strain and licensed

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

464 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

in 2007. It is administered via intradermal inoculation using a bifurcated needle – a process known as scarification. The typical reaction to the vaccine includes appearance of a pruritic vesicle at the inoculation site 5–7 days following administration. Local ery- thema, induration, local pain, fatigue, axillary lymphadenopathy, and mild systemic symptoms including fever, malaise, headache, and myalgias are common. Over the ensuing several days the vesicle progresses to form a pustule, then a 3–10 mm scab that sloughs within 1–2 weeks, leaving a permanent scar. The lesion contains live vaccinia virus and spread of infection via contact is possible until the scab has separated.

Historically, serious adverse reactions have occurred in approximately 1 per 1,000 patients immunized, with 1–5 in 10,000 immunizations leading to life-threatening complications, and 1 in 1 million leading to death. Serious reactions are approxi- mately 10 times more common in those being immunized for the first time than in those undergoing reimmunization. Inadvertent autoinoculation of the virus to distant skin sites, as well as trans- fer of virus to contacts, has occurred historically in approximately 6 per 10,000 vaccinees; ocular vaccinia is the most common form of inadvertent inoculation and can result in permanent corneal scarring. Generalized vaccinia results from systemic spread of the virus producing lesions removed from the primary vaccination site and occurs in 3 per 10,000 vaccinees. Postvaccinial encephali- tis is seen in approximately 1 per 100,000 primary vaccinees and has a 25% mortality rate, with another 25% developing perma- nent neurological sequelae. Fetal vaccinia is a rare (<50 reported cases) but often fatal complication of maternal vaccination, most commonly reported in the third trimester of pregnancy. Approx- imately 1 in 10,000 primary vaccinees during modern time U.S. DoD and civilian vaccination efforts experienced acute myoperi- carditis – a rarely reported adverse event in previous U.S. immu- nizations campaigns. Eczema vaccinatum (generalized cuta- neous spread of vaccinia in patients with eczema), a potentially lethal complication, can occur when patients with a history of eczema (a contraindication to vaccination) are inadvertently vac- cinated. Progressive vaccinia is the systemic spread of vaccinia virus in immunocompromised individuals; this is seen in 1 in 1 million primary vaccinees and is almost uniformly fatal.38–40

The U.S. CDC’s Advisory Committee on Immunization Prac- tices recommends that laboratory workers who directly handle live, unattenuated Orthopoxvirus cultures or infected animals, as well as select healthcare workers who are members of smallpox response teams, receive the vaccine. The U.S. DoD has elected to immunize many healthcare workers, as well as personnel deploy- ing to areas of the world perceived to be at increased risk from smallpox if it were used as a biological weapon.41 Vaccination with a verified clinical “take” (vesicle with scar formation) within the past 3 years is considered to render a person immune to nat- ural variola.

Preexposure vaccination is contraindicated in persons with the following conditions: immunosuppression (including those taking immunosuppressing drugs such as corticosteroids or alkylating agents), human immunodeficiency virus infection, clinical evidence or a history of eczema or other chronic exfolia- tive skin disorders, pregnancy or breastfeeding, and age younger than 1 year. Additionally, the presence of household, sexual, or other close physical contacts with the aforementioned conditions would constitute a contraindication to vaccination of a potential recipient. There are no absolute contraindications to vaccination after bona-fide exposure to variola. Vaccination after exposure to weaponized smallpox or a case of smallpox may prevent or

ameliorate disease if given promptly. Vaccination is likely to be most effective if given within 24 hours, but may still be somewhat effective up to 7 days after exposure. Newer smallpox vaccine can- didates (grown using modern cell culture technology) are being studied with the goal of replacing aging stocks of calf-lymph- derived vaccine.42 Attenuated live vaccines such as “Modified Vaccinia Ankara,” as well as killed virus and protein component vaccines are being explored to develop safer vaccines with fewer side effects and contraindications.43

A formulation of intravenous Vaccinia Immune Globulin (VIG) has been licensed for treatment of vaccinia adverse reac- tions and is available through the Centers for Disease Control. VIG is indicated for treating certain complications due to vac- cinia vaccine, including generalized vaccinia with systemic ill- ness, ocular vaccinia without keratitis, eczema vaccinatum, and progressive vaccinia. The dose for prophylaxis or treatment is 100 mg/kg (for the intravenous formulation). Additionally, cid- ofovir and ST-246 may be beneficial in treating vaccinia adverse events, although the efficacy of both drugs requires further study and both should be administered under the auspices of a compas- sionate use protocol. Limited data suggest that VIG may also be of value in the postexposure prophylaxis against smallpox when given within the first week after exposure, and concurrently with vaccination. Concomitant administration of VIG may be par- ticularly useful for pregnant and eczematous persons in such circumstances.44,45

Questions often arise as to the duration of effective immu- nity postvaccination. There is no definitive answer to this ques- tion; however, there is evidence that some protective immu- nity remains many years after vaccination particularly if one has received more than one dose during a lifetime.

Plague

Yersinia pestis, the causative agent of plague, is a rod-shaped, non- motile, nonsporulating, Gram-negative bacterium of the family Enterobacteriaceae. Plague is principally a zoonotic disease of rodents. Fleas that live on the rodents can transmit the bacteria to humans via biting. These exposed persons may then contract the bubonic form of plague within the lymphatic distribution after the fleabite. The bubonic form may progress to the septicemic or pneumonic forms, discussed in detail later. Pneumonic plague would likely be the predominant form encountered following the purposeful aerosol dissemination of a weaponized form of Yersinia pestis. All human populations are thought to be suscep- tible to plague, and recovery from the disease is followed by only temporary immunity. Y. pestis can remain viable in water, moist soil, and grain for several weeks. It can also remain viable for some time in dry sputum, flea feces, and cadavers, but is killed within several hours on exposure to sunlight.

The United States examined Y. pestis as a potential biological warfare agent in the 1950s and 1960s before the offensive biowar- fare program was terminated; other countries are suspected of weaponizing this organism as well. The former Soviet Union had more than 10 institutes and thousands of scientists who worked with Y. pestis. During WW II, Unit 731, a battalion of the Japanese Army, reportedly released Y. pestis infected fleas from aircraft over Chinese cities, but this method of dissemination proved cumbersome and unpredictable. The United States and Soviet Union developed a more reliable and effective method of aerosolizing the organism.5 Interest in the terrorist potential of plague was brought to light in 1995 when Larry Wayne Harris

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 465

Figure 29.7. A femoral bubo (A) is the most common site of an ery- thematous, tender, swollen, lymph node in patients with plague. The next most common lymph node regions involved are the inguinal, axil- lary (B), and cervical areas. Bubo location is a function of the region of the body in which an infected flea inoculates the plague bacilli. Photographs: Courtesy of Kenneth L Gage, PhD, Centers for Disease Control and Prevention Laboratory, Fort Collins, Colorado. See color plate.

was arrested in Ohio for the illicit procurement of a Y. pestis culture through the mail.19

The contagious nature of pneumonic plague makes it par- ticularly dangerous as a biological weapon. Plague may present as three distinct clinical syndromes in humans: bubonic, sep- ticemic, and pneumonic plague. The bubonic form begins after an incubation period of 2–10 days, with acute and fulminant onset of nonspecific symptoms, including high fever, malaise, headache, myalgias, and sometimes nausea and vomiting. Up to 50% of patients have abdominal pain. Simultaneous with or shortly after the onset of these nonspecific symptoms, the bubo develops – a swollen, very painful, infected lymph node (Fig- ures 29.7a and 29.7b). Buboes are normally seen in the groin region (femoral or inguinal lymph nodes) because the legs are the most commonly flea-bitten part of the adult human body. The liver and spleen of bubonic plague victims are often ten-

der and palpable. One quarter of patients will have a pustule, vesicle, eschar, or papule (containing leukocytes and bacteria) in the lymphatic drainage of the bubo, and presumably repre- senting the site of the inoculating fleabite. Secondary septicemia is common, and greater than 80% of blood cultures are posi- tive for the organism in patients with bubonic plague; however, only approximately 25% of bubonic plague patients progress to clinical septicemia.46

Among those who do progress to secondary septicemia, as well as those presenting septicemic but without lymphadenopa- thy (primary septicemia), symptoms are similar to those seen in other Gram-negative septicemias: high fever, chills, malaise, hypotension, nausea, vomiting, and diarrhea. Plague septicemia, somewhat distinct from that caused by other bacteria, is also characterized by thromboses in the acral vessels, with necro- sis, gangrene, and disseminated intravascular coagulation (DIC). Black necrotic appendages and more proximal purpuric lesions caused by endotoxemia are often present, and organisms in the bloodstream can gain access to the central nervous system, lungs, and elsewhere (Figures 29.8a and 29.8b). Plague meningitis occurs in approximately 6% of septicemic and pneumonic cases.

Pneumonic plague is an infection of the lungs due to either inhalation of organisms (primary pneumonic plague) or spread to the lungs from septicemia (secondary pneumonic plague). After an incubation period varying from 1 to 6 days for pri- mary pneumonic plague (usually 2–4 days, and presumably dose dependent), onset is acute and often fulminant. The first signs of illness include high fever, chills, headache, malaise, and myal- gias, followed within 24 hours by a cough with bloody sputum. Although bloody sputum is characteristic, it can sometimes be watery or, less commonly, purulent. Gastrointestinal symptoms, including nausea, vomiting, diarrhea, and abdominal pain, may be present. Rarely, a cervical bubo results from an inhalational exposure. Radiographic findings are variable but chest x-ray most commonly shows bilateral infiltrates, which may be patchy or consolidated. Plague pneumonia progresses rapidly, resulting in dyspnea, stridor, and cyanosis. The disease terminates with respi- ratory failure, and circulatory collapse. In humans, the mortality rate of untreated bubonic plague is approximately 60% (reduced to <5% with the prompt administration of effective therapies), whereas in untreated pneumonic plague the mortality rate is nearly 100%, and survival is unlikely if treatment is delayed beyond 18–24 hours of the onset of symptoms.46

Nonspecific laboratory findings seen in patients with plague include leukocytosis, with a predominance of polymorphonu- clear cells. One also often finds increased fibrin split products in the blood indicative of a low-grade DIC. The serum blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, and bilirubin may also be elevated, consis- tent with multiorgan failure.47 Nonetheless, a prompt diagnosis must often be based primarily on clinical suspicion. The pre- sentation of large numbers of previously healthy patients with sudden, severe, rapidly progressive pneumonia with hemopt- ysis strongly suggests plague. A presumptive diagnosis can be made microscopically by identification of the coccobacillus in Gram, Wright, Giemsa, or Wayson’s stained smears from lymph node needle aspirate, sputum, blood, or cerebrospinal fluid sam- ples. When available, immunofluorescence staining can be very useful. Definitive diagnosis relies on culturing the organism from blood, sputum, cerebrospinal fluid, or bubo aspirates. The organism grows slowly at normal incubation temperatures and may be misidentified by automated systems because of delayed

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

466 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

Figure 29.8. (A) This patient developed bubonic plague that progressed to the sep- ticemic and pneumonic forms after the causative organism, Y. pestis, disseminated from his buboes into his bloodstream. (B) Note the necrosis of tissue involving the tip of the nose, the fingers and the toes. This is due to thrombosis of distal arterioles and is a known complication of septicemic plague. Photographs: Courtesy of Kenneth L Gage, PhD, Centers for Disease Control and Preven- tion Laboratory, Fort Collins, Colorado. See color plate.

biochemical reactions. It can be cultured on blood agar, Mac- Conkey agar, or infusion broth. Most naturally occurring strains of Y. pestis produce an F1-antigen in vivo, which can be detected in serum samples by immunoassay. A fourfold rise in antibody titer in patient serum is retrospectively diagnostic. PCR (using specific primers), although not sufficiently developed and eval- uated for routine use, is a very sensitive and specific technique, able to identify as few as 10 organisms per milliliter. Most clinical

assays can be performed in BSL-2 laboratories, whereas pro- cedures producing aerosols or yielding significant quantities of organisms require BSL-3 containment.47

Standard precautions are adequate when caring for bubonic plague patients. Suspected pneumonic plague cases require strict isolation with droplet precautions for at least 48 hours follow- ing the initiation of antibiotic therapy or until sputum cultures are negative in confirmed cases. If competent vectors (fleas) and reservoirs (rodents) are present in the environment, measures must be taken to prevent disease from becoming enzootic. These measures might include but are not limited to the use of flea insecticides, rodent control measures (after or during flea con- trol), and flea barriers for patient care areas.

Streptomycin, gentamicin, doxycycline, and chlorampheni- col are highly effective in the treatment of plague, if therapy is initiated early. Streptomycin is generally not available in many countries in modern times but gentamicin, doxycycline, and chloramphenicol are acceptable alternatives. Results obtained from animal studies indicate that quinolone antibiotics, such as ciprofloxacin and ofloxacin, may also be effective. Chloram- phenicol is recommended for the treatment of plague menin- gitis.48 Usual supportive therapy includes intravenous adminis- tration of crystalloids and hemodynamic monitoring. Although low-grade DIC may occur, clinically significant hemorrhage is uncommon, as is the need to treat with heparin. Endotoxic shock is common, but pressor agents are rarely needed. Finally, buboes rarely require any form of local care, but instead recede with systemic antibiotic therapy. In fact, incision and drainage poses a risk to others in contact with the patient; aspiration is recom- mended for diagnostic purposes and may provide symptomatic relief.

At this time of this writing, no vaccine is available for the prophylaxis of plague. A licensed, killed whole cell vaccine was available in the United States from 1946 until November 1998; it offered protection against bubonic plague, but would likely have been ineffective against aerosolized Y. pestis. An F1-V anti- gen (fusion protein) vaccine is in development at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). It protects mice against an inhalational challenge, and is under- going testing in primates.

Persons having face-to-face contact (within 2 m) with pneu- monic plague victims, or persons possibly exposed to a plague aerosol during a biological attack, should receive antibiotic pro- phylaxis for at least 7 days following the cessation of exposure. Doxycycline (100 mg orally twice daily) is the recommended choice of antibiotic for prophylaxis because of its oral bioavail- ability and relative lack of toxicity. Ciprofloxacin (500 mg orally twice daily) has also been shown effective in preventing dis- ease in exposed mice. Tetracycline (500 mg orally four times daily) and chloramphenicol (25 mg/kg orally four times daily) are acceptable alternatives. Contacts of bubonic plague patients should be observed for symptoms for at least 1 week. Should such symptoms occur, antibiotic therapy should be instituted pending the results of diagnostic studies.

Botulism

Botulism is a toxin-mediated disease (rather than a true infec- tion), which occurs following exposure to one of seven related botulinum neurotoxins (A-G) produced by certain strains of Clostridium botulinum and closely related bacteria. These organisms are anaerobic spore-forming Gram-positive bacilli

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 467

ubiquitously found in soil. The neurotoxins they produce are the most toxic substances known, with an LD50 (for type A toxin) of approximately 0.001 µg/kg. On a weight basis, this is approximately 15,000 times more lethal than VX, the most potent chemical warfare agent. The botulinum neurotoxins func- tion at the presynaptic nerve terminal, preventing the release of acetylcholine, interrupting neuronal transmission at cholinergic autonomic (muscarinic) and motor (nicotinic) receptors, and thereby leading to a generalized flaccid paralysis and autonomic dysfunction. Binding of toxin within the presynaptic neuron is permanent; recovery is thus predicated on development of new axons, which may require several months.

Only types A, B, and E toxins are significant causes of nat- urally occurring human botulism. Exposure to type C toxin appears to be a prevalent cause of botulism among poultry and other avian species, whereas types C and D are associated with disease in cattle. Neither type C nor D appears to affect humans in nature, although all serotypes have the potential to cause human disease. A single small outbreak of botulism due to type G toxin was reported in Argentina.49 More recently, several cases of neonatal botulism due to type F toxin associated with C. baratii have been described,50 and a few cases of infant botulism (type E) have been associated with C. butyricum.51

Sinister use of botulinum toxin might involve its aerosoliza- tion or the intentional contamination of food and water sup- plies. Iraq weaponized botulinum toxin for aerosol delivery and declared such weapons under the auspices of the Biological Weapons Convention in the aftermath of the 1990–1991 Gulf War. The Japanese doomsday cult Aum Shinrykyo made sev- eral unsuccessful attempts to use botulinum toxin as a weapon prior to their sarin attack on the Tokyo subway system. Natu- rally occurring botulism, however, is generally acquired in one of three ways.

1) Food-borne botulism occurs as a result of the consump- tion of improperly canned food. As C. botulinum is ubiq- uitous in soil, contamination of food with small amounts of soil provides the nidus of bacteria. Failure to sterilize food during canning allows for bacterial survival. The can- ning process then provides for the strict anaerobic environ- ment necessary for bacterial proliferation and toxin produc- tion. Subsequent heating or cooking (such as might occur after opening the previously canned goods) may kill bacteria but does not destroy the toxin. Consuming contaminated canned goods, then, allows for the gastrointestinal absorp- tion and hematogenous circulation of toxin, which ulti- mately reaches its target at the peripheral cholinergic synapse. Although types A and B toxin are traditionally associated with food-borne outbreaks, type E toxin is particularly asso- ciated with botulism resulting from improperly canned fish products.

2) “Wound botulism” occurs when soil contaminates a wound and is contained within an anaerobic pocket beneath the skin. C. botulinum proliferates in this environment and produces toxin. A large number of wound botulism cases have been associated with the injection of “black-tar” heroin.52

3) Infant botulism occurs when infants ingest dirt or sub- stances (e.g., honey) heavily contaminated with C. botulinum spores.53 Under normal circumstances, the acidity of the stomach would destroy these spores, but the relatively weak acid levels of the neonatal stomach are thought to permit some spores to survive and transit the intestinal tract, where

the relative lack of competing bowel flora allow them to colo- nize and germinate in the large intestine and begin to produce toxin, which is then absorbed.

Regardless of the original route of acquisition of botulinum toxin (via any of the three aforementioned routes or via inhalation fol- lowing a deliberate aerosol attack), the final common pathway is the same. Following exposure to botulinum toxin, a latent period ranging from 24 hours to several days occurs before clinical man- ifestations develop. Signs and symptoms initially involve cranial nerve dysfunction, manifested as bulbar palsies, ptosis, photo- phobia, and blurred vision caused by a difficulty in accommoda- tion. The autonomic effects of botulism may include dry mouth, ileus, constipation, and urinary retention. Nausea and vomiting may occur as nonspecific sequelae of an ileus. Hence the com- bination of neurological and gastrointestinal symptoms should lead clinicians to suspect botulism. Symptoms then progress to include dysarthria, dysphonia, and dysphagia. Finally, a descend- ing symmetrical flaccid paralysis develops and, in the absence of ventilatory support, death results from respiratory muscle failure.

The diagnosis of botulism is principally clinical. The extreme potency of the toxin is such that the LD50 is below the threshold of human immune response. Detectable antibody production is thus absent in cases of human botulism, and diagnostic assays using a search for antibodies are fruitless. For this reason, clinical botulism does not confer immunity to subsequent intoxications. It is possible to detect toxin in clinical or environmental samples by using enzyme-linked immunosorbent assays, but the mouse neutralization bioassay remains the gold standard for botulinum neurotoxin detection. In cases of food-borne botulism, then, it is critical to obtain implicated food for testing in such assays.

Clinically, a solitary case of botulism must be differentiated from other uncommon neurological disorders such as myas- thenia gravis, tick paralysis, Guillain–Barré syndrome, Eaton– Lambert syndrome, and others. The presence of multiple casu- alties with similar symptoms clustered in time and space should make elucidation of a botulism outbreak relatively easy. In the setting of terrorism, botulism might superficially be confused with nerve agent intoxication, owing to the preponderance of neuromuscular symptomatology in both conditions. The two are easy to differentiate, however, when one recalls that organophos- phate nerve agents inhibit acetylcholinesterase, leading to exces- sive cholinergic activity at neuronal synapses, in contrast to the lack of cholinergic activity seen in botulism. The paralysis of nerve agents, then, is spastic, rather than flaccid, and autonomic hyperarousal is seen.

Supportive care, including meticulous attention to ventila- tory support, remains the mainstay of botulism management. Patients may require such support for several months, making the management of a large-scale botulism outbreak especially problematic in terms of medical resources. Unlike other types of public health emergencies such as pandemic influenza, hypoxia is generally not a concern; therefore, large supplies of oxygen are less critical than ventilation resources. Recognition of the utility of tracheostomy and improvements in mechanical ventilation have reduced the mortality rate from isolated cases of botulism in the United States from greater than 60% prior to 1950 to less than 5% in modern times. Attention to hydration status, as well as bowel and bladder care and cognizance of the need to prevent decubitus ulcers and deep vein thromboses, play a large role in patient outcome. When antibiotics are prescribed in

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

468 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

the treatment of wound botulism no aminoglycosides or clin- damycin should be administered because of their weak pharma- cological effects as neuromuscular blocking agents. This effect worsens paralysis acutely and may precipitate respiratory arrest.

A licensed bivalent (type A and B) botulinum antitoxin is available through the U.S. CDC, as is a separate investigational type E antitoxin. Although administration of antitoxin is unlikely to reverse disease (which is mediated by toxin already taken up in presynaptic nerve terminals and thus inaccessible to circulat- ing antitoxin), it may prevent progression when administered to exposed persons. Botulinum antitoxin is prepared from equine serum; a test dose should thus be administered prior to therapy. Patients reacting to a test dose require desensitization. An inves- tigational heptavalent (type A-G) despeciated (Fab2) antitoxin, also produced in horses, is available through USAMRIID on a compassionate use protocol.54 Administration of a test dose is also required with this product. Finally, a human-derived anti- botulinum immune globulin (BabyBIG R©) is licensed specifi- cally for the treatment of infant botulism due to type A or B toxin.55

Although no licensed vaccine is available to protect against botulism, considerable experience exists with an investigational pentavalent (types A–E) vaccine, produced by the Michigan Department of Public Health, and available through the CDC. A theoretical concern, given the expansion of indications for therapeutic use of botulinum toxins, surrounds the possibility that vaccine recipients may be immune to the beneficial effects of such treatment. Of note, therapeutic botulinum toxin is pro- duced from type A (Botox R©) or type B (Myobloc R©) toxin.

Viral Hemorrhagic Fevers

The term “viral hemorrhagic fever” (VHF) is a clinically descrip- tive one, referring to a group of widely heterologous diseases caused by lipid-enveloped, single-stranded RNA viruses of four taxonomic families: the arenaviruses, bunyaviruses, filoviruses, and flaviviruses. Although “brand name recognition” (i.e., the notoriety of diseases such as Ebola fever and the consequent ability of these diseases to capture public attention and create concern) is somewhat responsible for their inclusion among the category A agents, actual weaponization of these agents would likely be problematic, owing to difficulties in mass production, and rapid inactivation of the various causative viruses by envi- ronmental heat, desiccation, and ultraviolet light.56 Nonetheless, several have been examined as weapons candidates.

The VHFs would be expected to present as acute febrile ill- nesses with a host of nonspecific features such as malaise, fatigue, nausea, vomiting, diarrhea, abdominal pain, and headache. Hypotension and shock can rapidly ensue and death occurs fre- quently among patients with certain VHFs. Vascular involve- ment constitutes the defining characteristic of this broad group of infectious diseases and can manifest as hypotension, flushing, edema, petechiae, bruising, and bleeding. Such findings can vary from subtle to overt in their presentation.

The pathogenesis of the VHFs is complex and variable (Fig- ure 29.9). Although some hemorrhagic fever viruses cause vascu- lar damage and DIC through direct endothelial infection, others result in immune complex deposition, thereby activating com- plement and the inflammatory cascades. Vascular endothelial damage, however, is the final common pathway that results in vascular leak, with secondary hypotension, edema, and hemor- rhage, often resulting in shock and end-organ failure. Although

the VHFs share common manifestations, many clinical find- ings may vary among the different diseases and among patients with the same disease. Lassa fever, caused by an Old World are- navirus, typically presents with edema, without hemorrhage, whereas diseases due to the New World arenaviruses (the agents of Argentinean, Bolivian, and Venezuelan hemorrhagic fevers) are notable for prominent petechiae, purpura, ecchymoses, and mucosal hemorrhage.

The hantaviruses are members of the bunyavirus family. Hemorrhagic fever with renal syndrome (HFRS), usually caused by Old World (and occasionally New World) hantaviruses, begins with a nonspecific prodrome, followed by the development of facial edema. A morbilliform eruption, flushing of the upper body, and dermatographism may appear. Hemorrhagic mani- festations may range from subtle petechiae to massive hemor- rhage. Congo-Crimean hemorrhagic fever (CCHF), caused by another bunyavirus, is often characterized by severe ecchymoses and hemorrhage, although some cases may present only nonspe- cific, minor hemorrhagic manifestations (Figure 29.10).

The filoviral fevers, Ebola and Marburg, are characterized by an acute severe prodrome, followed by a papular exanthem that rapidly progresses into large, coalescent papules, purpura, and ecchymoses. Patients often develop gross hemorrhage and shock. Mortality rates range from 23% in some Marburg disease outbreaks to as high as 90% for certain Ebola outbreaks. Most of these deaths occur during the second week of illness.

Flavivirus infections also produce manifestations that are quite variable. Dengue fever typically presents as a nonspecific febrile illness, sometimes accompanied by a diffuse morbilliform rash. Although infection due to one of the four dengue virus serotypes confers lifelong immunity to that particular serotype, reinfection with another (heterologous) strain may result in “immune amplification.” This leads to a more severe clinical outcome, termed dengue hemorrhagic fever or dengue shock syndrome. Severe ecchymoses and hemorrhage can occur in cases of dengue hemorrhagic fever. Two other flaviviral hemorrhagic fevers, Omsk hemorrhagic fever and Kyasanur Forest disease, can also feature a wide range of hemorrhagic manifestations.

Clinical diagnosis is essential in the case of the VHFs due to their highly contagious nature. Because the endemic range of the various causative viruses is often unique and limited, naturally occurring cases can frequently be suspected on geographical and epidemiological grounds, and a detailed travel history should be sought when VHF is suspected. Clinical laboratory findings of proteinuria, thrombocytopenia, leukopenia, elevated serum transaminase levels, and abnormal coagulation studies may be sought but are nonspecific. Specific etiological diagnosis by serological testing or viral isolation would require forwarding clinical specimens to a limited number of laboratories with high-level biosafety containment capabilities. In fact, many of the hemorrhagic fever viruses (the filoviruses and arenaviruses, as well as CCHF, Omsk, and Kyasanur Forest) are assigned to BSL-4 laboratories. Within the United States for example, clinical specimens potentially containing these viruses should thus be handled only at the CDC or USAMRIID, both of which possess BSL-4 laboratories.

Supportive care is the mainstay of therapy in most cases of VHF, and many severely affected individuals will require heroic and intensive support. Vigorous fluid resuscitation, as well as vasoactive agent administration and inotropic support may be necessary; the provision of such modalities is especially prob- lematic in developing nations where many of these diseases are

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 469

A B

Infected macrophages

Recruitment of naive macrophages

Chemokines

Virus particles

Dendritic cells infected

Fibrin mesh

Vessel

Lymphocyte apoptosis

Increased endothelial porosity and edema

Spleen

Adrenal gland

Infection/necrosis of hepatocytes — dysfunction, decreased production of clotting factors and albumin

Infection of adrenal cortical cells causes impaired synthesis of steroid-synthesizing enzymes

Hypotension

Costimulatory molecules

Release of soluble factors

Peripheral lymph nodes

Macular rash

Liver

Small abrasions

Figure 29.9. Model of VHF pathogenesis. (A) Virus spreads from the initial infection site to regional lymph nodes, liver, and spleen. At these sites, the virus infects tissue macrophages (including Kupffer cells) and dendritic cells. Soluble factors released from virus-infected monocytes and macrophages act locally and systemically. Release of chemokines from these virus-infected cells recruits additional macrophages to sites of infection, making more target cells available for viral exploitation and further amplifying the dysregulated host response. Although none of these viruses infect lymphocytes, the rapid loss of these cells by apoptosis is a prominent feature of disease. The direct interaction of lymphocytes with viral proteins cannot be discounted as having a role in their destruction, but the marked loss of lymphocytes is likely to result from a combination of factors, including viral infection of dendritic cells and release of soluble factors from virus-infected monocytes and macrophages. For example, viral infection of dendritic cells impairs their function by interfering with the upregulation of costimulatory molecules, which are important in providing rescue signals to T lymphocytes. Additionally, release of soluble factors from infected monocytes and macrophages results in deletion of lymphocytes, both directly by release of mediators such as nitric oxide, and indirectly by contributing to upregulation of proapoptotic proteins such as Fas and tumor necrosis factor–related apoptosis-inducing ligand. The coagulation abnormalities vary in nature and magnitude among the VHFs. For example, Ebola virus induces the overexpression of tissue factor that results in activation of the clotting pathway and the formation of fibrin in the vasculature. As another example, coagulation disorders are less marked in Lassa fever, and impairment of endothelial function contributes to edema, which seems to be a more prominent finding in Lassa fever than in other VHFs. (B) The hemodynamic and coagulation disorders common among all of the VHFs are exacerbated by infection of hepatocytes and adrenal cortical cells. Infection of hepatocytes impairs synthesis of important clotting factors. At the same time, reduced synthesis of albumin by hepatocytes results in a reduced plasma osmotic pressure and contributes to edema. Impaired secretion of steroid-synthesizing enzymes by VHF-infected adrenal cortical cells leads to hypotension and sodium loss with hypovolemia. Macular rashes are often seen in VHFs. Reproduced with permission from: Geisbert TW, Jahrling PB. Exotic emerging viral diseases: progress and challenges. Nat Med. 2004;10(12 suppl):S110–121. See color plate.

endemic. Sedative and anxiolytic therapy, pain control, anticon- vulsant therapy, antibacterial therapy (for secondary infections), mechanical ventilation, and renal dialysis are often required as well. Because coagulopathy is an integral component of the pathogenesis of many VHF cases, particular attention should be paid to clotting studies, and blood products (e.g., red blood cells, platelets, and clotting factors) should be provided as clinically indicated. As with any case of significant coagulopathy, intra- muscular injections and anticoagulating drugs (such as aspirin) should be avoided.

At the time of this writing, intravenous ribavirin is being studied in the treatment of many of the VHFs, and shows promise in cases of Lassa fever, Rift Valley fever, CCHF, and HFRS.57 As of

this writing, ribavirin is not yet FDA-approved for the treatment of any VHF and should thus be used under an IND protocol. Antisense oligomers have shown considerable promise in treating Ebola in a nonhuman primate model.58 The 17-D yellow fever vaccine is the only U.S. FDA-approved immunization available against a hemorrhagic fever virus.59 Vaccine candidates effective against Rift Valley fever, Argentine hemorrhagic fever, HFRS, Lassa, Marburg, and Ebola fevers are in various stages of study or development.

Patients with certain VHFs may present infection control and public health challenges because many are highly conta- gious and are commonly transmitted to healthcare workers. The arenaviruses, CCHF, and the filoviruses are communicable,

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

470 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

Figure 29.10. Ecchymosis associated with late-stage CCHF infection 1 week following development of clinical signs and symptoms. Ecchy- mosis indicates significant impairment of the patient’s coagulation system and loss of vascular integrity. Photograph: Courtesy of Dr. Sadegh Chinikar, Pasteur Institute of Iran, Tehran, Iran. See color plate.

primarily by contact with blood and body fluids. Specific infec- tion control guidelines for hospitalized patients with these dis- eases include contact precautions, augmented in some circum- stances with airborne precautions, with special attention to dis- posal of body waste. Patients with hemorrhagic fever with renal syndrome and those with flavivirus infections can be managed using standard precautions.

Tularemia

Tularemia, a plaguelike zoonotic illness that occasionally affects humans, is caused by the Gram-negative facultative intracellular coccobacillus, Francisella tularensis. First discovered in ground squirrels in Tulare County, California in 1911, two biotypes of F. tularensis are now known. F. tularensis tularensis, the causative agent of “type A” tularemia, is found only in temperate areas of North America, where it is typically transmitted by ticks among lagomorph reservoirs (hence the moniker “rabbit fever”), although many other wild animals may serve as reservoirs and many insects as vectors. Humans are accidental “dead end” hosts who cannot transmit disease. Natural infection in humans occurs through inoculation by an infected arthropod, contact with con- taminated animal tissue, inhalation of aerosolized bacterium, or ingestion of contaminated water or meat. Approximately half of all naturally occurring U.S. cases originate in Missouri, Arkansas, or Oklahoma.60

The number of reported cases of tularemia in the United States has decreased from several thousand per year prior to 1950 to less than 200 per year over the past decade. A diminishing fondness for rabbit hunting may be responsible in part for this finding. Occupational exposure and environmental factors are major risk factors for disease acquisition, and tularemia generally affects more men than women, likely owing to its association with hunting. Naturally occurring tularemia occurs in two seasonal peaks: during the summer (when ticks are active) and the winter (during hunting season).

F. tularensis tularensis is an organism of extraordinarily high virulence, and as few as 10 cells constitute the rabbit lethal dose (LD50) and human infectious dose (ID50). F. tularensis palearc- tica, the causative agent of “type B” tularemia, on the other

hand, is a low-virulence organism found in Europe and the for- mer Soviet Union. The rabbit LD50 of this organism is on the order of 10 million cells. Although these differences have epi- demiological and vaccine development implications, immunity against one biotype appears to be cross-protective.

Widespread waterborne outbreaks of tularemia in the for- mer Soviet Union prior to and during the WWII led to a more thorough study of the bacterium and its potential as a biologi- cal weapon. Its high infectivity, ability to produce severe or fatal human illness, relative heartiness and ease of dissemination, and nonspecific clinical presentation apparently argued convincingly for such a role. In fact, the United States developed tularemia aerosols during human exposure experiments, and by the 1950s, had stockpiled tularemia as one of the first entries in its biological arsenal. Some, including Ken Alibek, a former Soviet biological weapons scientist, have alleged that tularemia was intentionally deployed by the Soviets at the Battle for Stalingrad in 1942– 1943.61 Unusual epidemiology and an overwhelming prepon- derance of pneumonic disease, which occurred during a large outbreak between the Don and Volga rivers during that period,62

lend credence to these allegations, although others dispute a sin- ister explanation for the outbreak.63 Finally, Alibek also detailed the Soviet Union’s attempts to engineer drug resistance into F. tularensis weapons during the 1990s.

Tularemia presents in at least six different clinical forms, all heralded by the onset of a nonspecific flulike syndrome. The terms ulceroglandular, glandular, oculoglandular, pharyngeal, typhoidal, and pneumonic, describe these clinical presentations. In many ways, however, glandular and oculoglandular disease can be thought of as variants of the ulceroglandular form, and the six clinical syndromes can be more simply thought of as constituting two broader clinical pictures, analogous in many ways to plague. Ulceroglandular tularemia is similar in many regards to the bubonic form of plague (or, in some respects, to the cutaneous form of anthrax). Pneumonic tularemia, on the other hand, is likely to be the predominant presentation following an intentional aerosolized release and can be thought of as somewhat analogous to the pneumonic form of plague (or the inhalational form of anthrax).

Ulceroglandular tularemia results from arthropod bites or from exposure of skin and mucous membranes to the hides and meat of infected animals. Onset of fever and systemic symptoms (which may include chills, headache, cough, myalgia, chest pain, vomiting, arthralgia, sore throat, and abdominal pain) occur following a 3–6 day incubation period.64 Within 48–72 hours an erythematous maculopapular lesion develops at the site of inoculation and soon ulcerates. This characteristic chancrelike ulcer is typically 0.4–3.0 cm in diameter with raised edges. Bac- teria present at the ulcer site, subsequently gain access to lym- phatic vessels and travel to regional lymph nodes, producing the lymphadenitis, which, in combination with the ulcer, consti- tutes the hallmark dyad of ulceroglandular tularemia. The lym- phadenopathy of ulceroglandular tularemia is often pronounced and persistent. Involved lymph nodes can grow as large as 10 cm in diameter, and frequently become fluctuant whether treated with appropriate antibiotics or not. From the lymph node, bac- teria may secondarily gain access to the systemic circulation and ultimately seed the liver, spleen, and other distant organs.

Pneumonic tularemia results from inhalation of aerosolized bacteria but may also occur secondarily following seeding of the lungs in complicated cases of ulceroglandular and typhoidal tularemia. Aerosolization of bacteria can occur intentionally,

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 471

through the actions of belligerents or terrorists, but may occur naturally in circumstances in which infected blood and other animal products are mishandled. Modern day reports attribute cases of inhalational (pneumonic) tularemia to aerosolization of rabbits by lawnmowers.65 Symptoms of pneumonic tularemia include cough (which may be productive), dyspnea, and pleuritic chest pain. Pleural effusions, cavitary lesions, bronchopleural fis- tulae, and pulmonary calcifications occur less commonly.

F. tularensis may be cultured from the blood of infected indi- viduals, but such culturing should only be attempted by experi- enced technicians in laboratories equipped with BSL-3 safety systems. Testimony to the extreme infectivity of F. tularensis in laboratory settings, in fact, can be obtained by considering that this agent was, far and away, the most common cause of laboratory-acquired infection at Fort Detrick, Maryland (home to the U.S. Army Medical Research and Materiel Command) prior to the immunization of laboratory workers.66 Identification of F. tularensis may also be accomplished by microscopic exam- ination of secretions or biopsy specimens by using direct fluo- rescent antibody or immunohistochemical staining techniques. Although such fluorescence-labeled antibody assays are used in the rapid diagnosis of tularemia at many U.S. state public health laboratories, diagnosis in most clinical settings is best made using bacterial agglutination or enzyme-linked immunosorbent assays. Detectable levels of agglutinating antibodies against F. tularensis typically appear in blood within approximately 7 days of infec- tion, although higher levels and improved diagnostic sensitivity may be obtained by sampling 2 weeks following suspected infec- tion. Moreover, the serological response may be blunted by the administration of antibiotics, making diagnosis more problem- atic. Due to cross-reactivity of F. tularensis with Brucellae, Proteus OX19, and Yersinia organisms, acute infection should ideally be diagnosed only in the presence of a fourfold or greater increase in titer following an acute infection.67

As with plague, streptomycin (1 g intramuscularly twice daily) has traditionally been considered the drug of choice in the treatment of all forms of tularemia, and a review of the literature reveals a 97% cure rate with no relapses when using strepto- mycin.68 Patients treated with streptomycin usually demonstrate a clinical response within 48 hours. Gentamicin (5 mg/kg daily intramuscularly or intravenously) was an acceptable alternative in this same review, and, given the difficulty in procuring Strep- tomycin in many countries, has become a widely used therapy for this condition. Both should be given for a minimum of 10 days. Ciprofloxacin (400 mg intravenously twice daily) given for 10 days is a potential alternative to aminoglycoside therapy. Bacte- riostatic drugs such as chloramphenicol (15 mg/kg intravenously four times daily) and doxycycline (100 mg intravenously twice daily) are effective, but should be administered for at least 14– 21 days to prevent relapse. Postexposure prophylaxis of persons thought to have been exposed to tularemia may be accomplished using oral doxycycline (100 mg twice daily) or ciprofloxacin (500 mg twice daily) for 14 days.

The prevention of tularemia among hunters and other per- sons handling animals, animal hides, or carcasses is best achieved by the wearing of gloves, attention to good hand hygiene, and avoidance of mucosal exposure. BSL-3 precautions, including face masks, rubber gloves, and biological containment hoods should be used by laboratory technicians working with cultures or potentially infectious clinical material. In the event of a lab- oratory spill or surface contamination, decontamination can be accomplished with ordinary bleach solution. Although over half

a century of experience exists with investigational tularemia vac- cines, none are licensed in the United States at the time of this writing.69

RECOMMENDATIONS FOR FUR THER RESEARCH

Barriers to Research

Developing useful preventative measures, therapeutics, and diag- nostics for the myriad of potential agents that could be used as biological weapons is a monumental task, and optimal coun- termeasures for many of the dozens of potential threat agents contained within the U.S. CDC’s and other governments’ prior- itized lists are currently lacking. In fact, for many of these agents there is an incomplete understanding of the pathogenesis of dis- ease. Compounding the challenges associated with known threat agents is the realization that rogue nations or terrorist groups may be covertly developing new biological weapons. Moreover, organ- isms can be selected for antibiotic or vaccine resistance, normally benign organisms can be modified to evade host defenses or pro- duce toxins, and chemokines, cytokines, or other biomodulators could be designed for use as weapons. Significant obstacles to the development of an effective biological warfare medical counter- measures research program include

1) The threat is poorly understood or characterized. Those who wish to use biological agents as weapons are likely to keep their arsenals secret, and biological programs, which do not require the massive infrastructure that chemical and nuclear programs do, are much easier to camouflage. As such, intelligence agencies face a daunting task in discov- ery. The U.S. anthrax attacks in 2001 caught the nation by surprise. Another example is that the extent of the former Soviet program was unknown until former soviet scientists came forward at the close of the cold war. They revealed, for example, that the Soviets had stockpiled a smallpox weapon and had developed a Marburg virus weapon.9 Even natu- rally occurring new and emerging infections, such as Nipah, severe acute respiratory syndrome, and avian influenza regu- larly catch the medical intelligence community unprepared. It is a challenge to determine how to prioritize research to match such diverse and constantly changing threats.

2) Rare diseases are difficult to study. Many of the known threats are difficult to characterize adequately, study, and replicate the resultant human disease. For example, there are no small- pox victims available to test new therapeutic drugs, as natu- ral disease has been eliminated from the human population. Human monkeypox, which exists in tropical Africa, may serve as a viable surrogate for smallpox, but this model has not yet been validated. Although Burkholderia mallei occa- sionally causes disease in humans, it is too rare to allow orga- nization of clinical therapeutic trials. Additionally, naturally occurring human glanders may not resemble the disease that would result from intentional aerosolization of the organ- ism. Consequently, animal models of disease, when available, must play a vital role in any therapeutics research. To address the problem of FDA approval of therapeutics for such rare and dangerous diseases, in 2002 the U.S. FDA enacted the “animal rule.” This legislation allows for approval of “certain new drug and biological products based on animal data when adequate and well-controlled efficacy studies in humans can- not be ethically conducted.”70

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

472 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

3) Resources are limited. Basic scientific preclinical research into novel therapeutic compounds or vaccines can take decades and often fails to result in the discovery of candidate prod- ucts despite massive expenditures of human resources and money. Of those candidate products entering Phase I testing, only 8% are ultimately approved for use in humans. Between 2000 and 2002 it cost 1.7 billion dollars on average in the United States to bring a new drug from discovery to mar- ket.71 Within the United States, private funding for biologi- cal warfare medical countermeasures is virtually nonexistent. The biopharmaceutical industry is in the business of produc- ing medical solutions with a high likelihood of profit. Novel or improved antimicrobials have potential value in treating common infectious diseases as well as biological terrorism threat agents; thus, some privately funded research into these products may ultimately benefit medical response. Nonethe- less, prophylactic and therapeutic agents specific to biolog- ical threat organisms typically have no market other than the government. Moreover, when government does invest in such products, it may do so in the form of single (or limited) large purchases instead of providing the continuing market demand that drives private investors. In the United States, government funding for bioterrorism agent medical coun- termeasures has increased over the past decade, as will be detailed later, but resources remain limited, and thus expen- ditures must be prioritized.

4) Highly specialized laboratory facilities are required. Many of the biological threat agents, including those that are trans- missible via aerosol and have high lethality, can be safely studied only within high-containment laboratories. At the time of this writing, there are six operational BSL-4 (the highest level of biocontainment) laboratories in the United States: at the CDC in Atlanta; USAMRIID in Frederick, Mary- land; the Southwest Foundation for Biomedical Research in San Antonio, Texas; the University of Texas at Galveston; Georgia State University in Atlanta; and Virginia Common- wealth University in Richmond. The National Institute of Allergy and Infectious Diseases is funding construction of four additional BSL-4 laboratories: at Boston, Massachusetts; Galveston, Texas; Fort Detrick, Maryland; and at the Rocky Mountain Laboratory in Hamilton, Montana. Despite fund- ing for new biosafety laboratories, construction, in some communities, has been slowed or even curtailed over public concern for security of the pathogens to be held within.73

Limited space, like limited funding, forces the research com- munity to prioritize research.

5) Unique legal issues are involved. Although the 1925 Geneva Gas Protocol prohibited only the “practice” of bacteriological warfare, the 1972 BWC places much more stringent limita- tions on the handling and manipulation of biological agents. Under the terms of the BWC, production, storage, and trans- fer of agents are regulated. Such regulation creates potential conundrums for scientists performing defensive work and developing medical countermeasures. For example, enhanc- ing the aerosolization of agents would be considered “offen- sive” work and is prohibited by the BWC; however, to test a candidate vaccine in animals, those animals must be exposed to agent. Moreover, for such experiments to yield optimal information, challenge of such animals should ideally involve aerosolized agent. For this to happen, small quantities of high-purity agent must be produced, forcing scientists to walk a legal tightrope. As more BSL-4 laboratories prolif-

erate, concerns regarding biosecurity and consequent legal issues are only likely to increase.

Despite these formidable barriers, the past decade has seen a dramatic shift in emphasis on preparedness for biologi- cal weapons events and emerging infectious disease outbreaks within the United States and other countries. In the U.S., tens of billions of dollars have been allocated and a rapidly evolv- ing massive reorganization of federal preparedness and response organizations has been undertaken in an attempt to develop a sound solution to this very complex problem. In the last half of the 20th century, the military performed the vast majority of research in the United States to enhance preparedness for a biological attack. Recent world events, however, have increased civilian awareness of the threat of biological weapons, and pub- licly identified a “critical need for cutting-edge technology that can quickly and effectively detect, analyze, facilitate interdic- tion of, defend against, defeat, and mitigate, the consequences of weapons of mass destruction (WMD).”70 As an example, the United States has developed parallel, although somewhat com- plimentary, WMD medical countermeasure research programs led by DoD and the Department of Homeland Security (DHS). To understand current biological warfare medical countermea- sures research strategy and progress within the United States, it is useful to review these two programs.

U.S. CIV ILIAN BIOLOGICAL WARFARE MEDICAL COUNTERMEASURES RESEARCH

1) Organizational Structure. Within the U.S. government, the Secretary of the DHS is responsible for developing a strate- gic chemical, biological, radiological, and nuclear (CBRN) threat assessment. DHS issues Material Threat Determina- tions (MTDs) for those CBRN threat agents thought to present a material threat to U.S. security. DHS then per- forms a population threat assessment (PTA) on each iden- tified threat agent to determine the number of individuals who could be exposed in what are believed to be plausible, high-consequence attack or outbreak scenarios. The Depart- ment of Health and Human Services (HHS) leads efforts to “research, develop, evaluate, and acquire public health emergency medical countermeasures to prevent or mitigate the health effects of CBRN threats facing the U.S. civil- ian population,”74 a responsibility delegated to the Assis- tant Secretary for Preparedness and Response (ASPR) at the time of this writing. The ASPR oversees the HHS Pub- lic Health Emergency Medical Countermeasures Enterprise (HHS PHEMCE), which is assigned to develop and acquire medical countermeasures for the list of CBRN threats deter- mined by DHS. The HHS PHEMCE is composed of members from three HHS agencies (CDC, the FDA, and the National Institutes of Health), with ex officio membership from DoD, DHS, the Department of Veterans Affairs, and other agen- cies. After analyzing MTD and PTA information for each threat agent, PHEMCE performs its own medical and pub- lic health consequence assessments, which include modeling the effects of medical countermeasures. Based on their find- ings, PHEMCE identifies and prioritizes near-, mid-, and long-term development and acquisition programs for med- ical countermeasures. These priorities are communicated to the National Institute of Allergy and Infectious Diseases,

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 473

Table 29.6: U.S. Biodefense Research Priorities

Near-Term Mid-Term Long-Term (FY 2007–2008) (FY 2009–2013) (FY 2014–2023)

Civilian Priorities 1. New antibiotics for treatment of: B. anthracis B. mallei B. pseudomallei R. prowazekii F. tularensis

2. Next generation vaccines: Anthrax Smallpox

1. Broad spectrum antibiotics 2. Point-of-care, rapid diagnostics 3. Anthrax antitoxin 4. Filovirus countermeasures 5. Smallpox antivirals

Broad spectrum antivirals effective against: Ebola Junin Marburg Smallpox

Military Priorities 1. Yersinia pestis vaccine 2. Recombinant botulinum (A/B)

toxin vaccine 3. Multiagent vaccine platforms 4. Demonstration of

immunotherapeutics for filoviruses, bacteria, and toxins

5. Licensure of intravenous therapeutics for smallpox

6. New oral therapeutics for smallpox

1. Continued development of: Y. pestis vaccine Recombinant botulinum (A/B) toxin

vaccine Multiagent vaccine platforms

2. Licensure of oral smallpox therapeutics 3. Licensure of an antisepsis drug for

filovirus shock 4. Advanced development of monoclonal

antibody for filovirus infection 5. Advanced development of botulinum

toxin small molecule therapy

1. Licensure of: Botulinum (A/B) vaccine Y. pestis vaccine Filovirus vaccine(s)

2. Alternate delivery methods for vaccines and immunogens

3. Development (and licensure) of vaccines for: VEE, EEE, WEE filoviruses

4. Development and licensure of multiagent vaccines against multiple BW threats

5. Licensure of novel therapies using antisense or similar technologies

which uses them to determine the allocation of federal funds to research potential biological weapons medical counter- measures. In 2004 the BioShield program was created to acquire medical countermeasures for the U.S. CDC Strategic National Stockpile.75 A $5.6 billion “Special Reserve Fund” was appropriated to DHS for use through 2013, with HHS designated as the acquisition agent. Finally, the Pandemic and All-Hazards Preparedness Act76 of 2007 created the Biomedi- cal Advanced Research and Development Authority to select promising candidate medical countermeasures for funding through the late-stage research and development phase.

2) Funding. Between fiscal years 2001 and 2008 almost $40 billion was allocated for civilian biodefense in the United States.77 Perhaps a quarter of this funding has furthered research that could lead to new medical countermeasures or diagnostics for biological agents. Some $3.4 billion of this total has been allocated through Project BioShield for procurement of countermeasures for the Strategic National Stockpile.

3) Priorities. To date, DHS has issued MTDs and PTAs for the following biological threat agents: Bacillus anthracis, Mar- burg virus, botulinum toxins, multidrug-resistant Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Rickettsia prowazekii, Ebola virus, Variola virus, Francisella tularensis, Junin virus, and Yersinia pestis. Thus, it is only for these 12 threat agents that PHEMCE can currently pri- oritize medical countermeasure research. PHEMCE’s stated priority is to focus development of medical countermeasures on postexposure prophylaxis and postexposure treatment. Preventive measures such as vaccines and pre-exposure treat- ments are considered primarily for threats of “potential catas- trophic consequence.”78 Thus, although Project BioShield has resulted in the acquisition of large amounts of smallpox and anthrax vaccines,79 and although it continues to fund

research into next-generation vaccines, the HHS deployment strategy, for the most part, reserves these vaccines for use fol- lowing an initial attack or disease outbreak. Thus far, few U.S. civilians outside of the research and public emergency response communities have been vaccinated against anthrax or smallpox.

The PHEMCE strategy is divided into near-term (fiscal years 2007–2008), mid-term (2009–2013), and long-term (past 2013) programs; the goals of these programs are outlined in Table 29.6. The PHEMCE implementation plan states the HHS intention is to promote research to address emerging, enhanced, and advanced threats by adding flexibility into research programs for traditional agents.

U.S. MILITAR Y BIOLOGICAL WARFARE MEDICAL COUNTERMEASURES RESEARCH

1) Organizational Structure. Within the U.S. DoD, biological defense research, development, and acquisition programs are overseen within the Office of the Secretary of Defense by the Assistant to the Secretary of Defense for Nuclear and Chemical and Biological Defense Programs. DoD’s biode- fense research requirements are developed by a separate and independent organization, the Joint Requirements Office for Chemical, Biological, Radiological, and Nuclear Defense (JRO-CBRND), under the Chairman of the Joint Chiefs of Staff. The JRO-CBRND coordinates with the combatant commands and the individual military branches to develop a joint set of CBRN requirements that fit into an overarching CBRN defense architecture and strategic plan for implemen- tation. Finally, the Defense Threat Reduction Agency’s Joint Science and Technology Office for Chemical and Biological

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

474 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

Defense manages the funding for DoD biodefense research projects meeting the requirements and priorities set forth by the JRO-CBRND. The U.S. Army Acquisition Executive oversees product acquisition via the Joint Program Executive Office for Chemical and Biological Defense.

2) Funding. The Chemical and Biological Defense Program budget request for fiscal year 2008 was $1.57 billion. Several hundred million dollars of this is earmarked for biological medical countermeasures research. Both DoD and civilian researchers can apply for project funding from either DoD or civilian sources.

3) Priorities. In contrast to the civilian research program, DoD’s biodefense medical countermeasures research embraces pre- ventive measures such as vaccines (Table 29.6). Although the public may be somewhat unwilling to accept the perceived risks of vaccines and other preventative treatments for what are thought to be rare, unlikely, or nonlethal threat events, the military recognizes unique value in prevention for “preserv- ing the fighting strength” of its personnel. In this regard, DoD is pursuing a vaccine for Venezuelan Equine Encephalitis virus, a nonlethal threat agent that has the potential to inca- pacitate, realizing that a military incapable of responding, even temporarily, could have dire consequences for national security. The DoD biodefense research budget, however, is challenged to fund vaccine research from discovery to acqui- sition, and purely preventive vaccines (i.e., not useful for postexposure prevention or ameliorization of disease, such as vaccines for Yersinia pestis) may not meet criteria for HHS funding.

CONCLUSION

Biological warfare has been a threat to humanity since ancient times and crude attempts at bioterrorism have been a growing concern for several decades. A renewed appreciation of the sinis- ter potential of biological weapons has resulted from the glasnost that followed the end of the cold war and the migration of ex- Soviet scientists to the west. Similarly, an escalation of terrorist capabilities in the area of CBRN weapons was brought to public attention with Aum Shinrykyo’s use of sarin in the Tokyo subway system, and the subsequent use of anthrax-contaminated let- ters in the United States in October 2001. As a result of this latter event, occurring as it did, on the heels of the September 11 terror- ist attacks in New York and Washington, U.S. federal efforts in the area of biodefense have taken on a new sense of importance and urgency. Such efforts have evolved to include a wide spectrum of surveillance initiatives, security and biosecurity measures, coun- termeasures research and development, intelligence gathering, public health preparedness, and education. Other countries have initiated similar programs. Over time, these efforts have become more organized, better supported, and more focused.

REFERENCES

1. Derbes VJ. De Mussis and the great plague of 1348. A forgotten episode of bacteriological warfare. JAMA. 1966;196(1):59–62.

2. Parkman F. The Conspiracy of Pontiac and the Indian War After the Conquest of Canada. Vol 2. Boston: Little, Brown, and Co; 1901.

3. Hugh-Jones M. Wickham Steed and German biological warfare research. Intell Natl Secur. 1992;7:379–402.

4. Harris S. Japanese biological warfare research on humans: a case study of microbiology and ethics. Ann NY Acad Sci. 1992;666:21– 52.

5. Christopher GW, Cieslak TJ, Pavlin JA, Eitzen EM Jr. Biolog- ical warfare. A historical perspective. JAMA. 1997;278(5):412– 417.

6. Sims N. The Diplomacy of Biological Disarmament. Vol 18. New York: St. Martin’s Press; 1988.

7. US Department of the Army. US Army Activity in the US Biolog- ical Warfare Programs. Washington DC: US Department of the Army; February 24, 1977;2. Publication DTIC B193427L.

8. Vorobjev A, Cherkassky B, Stepanov A, Kyuregyan Y, Fjedorov M. Key problems of controlling especially dangerous infections. Paper presented at: Proceedings of an international symposium: Severe infection diseases: epidemiology, express-diagnostics and prevention. 1997; Kirov, Russia.

9. Alibek K, Handelman S. Biohazard: The Chilling True Story of the Largest Covert Biological Weapons Program in the World, Told From the Inside by the Man Who Ran It. 1st ed. New York: Random House; 1999.

10. Torok TJ, Tauxe RV, Wise RP, et al. A large community out- break of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA. 1997;278(5):389–395.

11. Kolavic SA, Kimura A, Simons SL, Slutsker L, Barth S, Haley CE. An outbreak of Shigella dysenteriae type 2 among labo- ratory workers due to intentional food contamination. JAMA. 1997;278(5):396–398.

12. Jernigan DB, Raghunathan PL, Bell BP, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemio- logic findings. Emerg Infect Dis. 2002;8(10):1019–1028.

13. 7 CFR Part 331, 9 CFR Part 121, 42 CFR Part 73. 14. Atlas R. Biological weapons pose challenge for microbiology

community. ASM News 1998;64:383–389. 15. NATO Handbook on the Medical Aspects of NBC Defensive Opera-

tions (AmedP-6) Departments of the Army, Navy, and Air Force; February 1996.

16. Thermos J, Quarles M. JPBDS underway with USS The Sullivans. Chem Bio Def Q. 2006;3(1):6–7.

17. Bioterrorism Alleging Use of Anthrax and Interim Guidelines for Management – United States, 1998. Centers for Disease Control and Prevention; 1999.

18. Health Aspects of Chemical and Biological Weapons. Geneva: World Health Organization; 1970.

19. Carus W. Bioterrorism and Biocrimes: The Illicit Use of Biological Agents in the 20th Century. Washington, DC: National Defense University; 1999.

20. Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM. Public health assessment of potential biological terrorism agents. Emerg Infect Dis. 2002;8(2):225–230.

21. Jernigan JA, Stephens DS, Ashford DA, et al. Bioterrorism- related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis. 2001;7(6):933–944.

22. Update: Interim recommendations for antimicrobial prophy- laxis for children and breastfeeding mothers and treatment of children with anthrax. MMWR. 2001;50(45):1014–1016.

23. Updated recommendations for antimicrobial prophylaxis among asymptomatic pregnant women after exposure to Bacil- lus anthracis. MMWR. 2001;50(43):960.

24. Inglesby TV, O’Toole T, Henderson DA, et al. Anthrax as a biological weapon, 2002: updated recommendations for man- agement. JAMA. 2002;287(17):2236–2252.

25. Department of Health and Human Services. Biological prod- ucts; bacterial vaccines and toxoids; implementation of efficacy review; anthrax vaccine adsorbed; final order. Fed Reg. 2005; 70(242):75180–75198.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

BI O LO G I C A L EV E N TS ■ 475

26. Use of anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices. MMWR. 2002;51(45):1024–1026.

27. Migone T-S et al. Raxibacumab for the treatment of inhalational anthrax. N Engl J Med. 2009 Jul 9; 361:135.

28. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva: World Health Organization; 1988.

29. Henderson DA, Inglesby TV, Bartlett JG, et al. Smallpox as a bio- logical weapon: medical and public health management. Work- ing Group on Civilian Biodefense. JAMA. 1999;281(22):2127– 2137.

30. Smith JA, Casey CG, Tierney BC. The ocular complications of smallpox and smallpox immunization. Arch Ophthalmol. 2004;122(9):1407; author reply 1407–1408.

31. Eeckels R, Vincent J, Seynhaeve V. Bone lesions due to smallpox. Arch Dis Child. 1964;39:591–597.

32. Wehrle PF, Posch J, Richter KH, Henderson DA. An airborne outbreak of smallpox in a German hospital and its significance with respect to other recent outbreaks in Europe. Bull World Health Organ. 1970;43(5):669–679.

33. Jezek Z, Szczeniowski M, Paluku KM, Mutombo M. Human monkeypox: clinical features of 282 patients. J Infect Dis. 1987;156(2):293–298.

34. Update: multistate outbreak of monkeypox–Illinois, Indi- ana, Kansas, Missouri, Ohio, and Wisconsin, 2003. MMWR. 2003;52(27):642–646.

35. Acute, Generalized Vesicular or Pustular Rash Illness Testing Pro- tocol in the United States. Centers for Disease Control and Pre- vention; 2006.

36. Buller RM, Owens G, Schriewer J, Melman L, Beadle JR, Hostetler KY. Efficacy of oral active ether lipid analogs of cid- ofovir in a lethal mousepox model. Virology. 2004;318(2):474– 481.

37. Smallpox Vaccine. Dried, Calf Lymph Type. Dryvax. Dried Smallpox Vaccine. Package Insert. Philadelphia: Wyeth Phar- maceuticals, Inc.; June 2007.

38. Casey CG, Iskander JK, Roper MH, et al. Adverse events asso- ciated with smallpox vaccination in the United States, January– October 2003. JAMA. 2005;294(21):2734–2743.

39. Smallpox vaccination and adverse reactions: guidance for clini- cians. Ann Pharmacother. 2003;37(3):467–468.

40. Grabenstein JD, Winkenwerder W Jr. US military smallpox vac- cination program experience. JAMA. 2003;289(24):3278–3282.

41. From the Centers for Disease Control and Prevention. Supple- mental recommendations on adverse events following smallpox vaccine in the pre-event vaccination program: recommenda- tions of the Advisory Committee on Immunization Practices. JAMA. 2003;289(16):2064.

42. Parrino J, Graham BS. Smallpox vaccines: Past, present, and future. J Allergy Clin Immunol. 2006;118(6):1320–1326.

43. Parrino J, McCurdy LH, Larkin BD, et al. Safety, immunogenic- ity and efficacy of modified vaccinia Ankara (MVA) against Dryvax challenge in vaccinia-naive and vaccinia-immune indi- viduals. Vaccine. 2007;25(8):1513–1525.

44. Hopkins RJ, Lane JM. Clinical efficacy of intramuscular vac- cinia immune globulin: a literature review. Clin Infect Dis. 2004;39(6):819–826.

45. Wittek R. Vaccinia immune globulin: current policies, pre- paredness, and product safety and efficacy. Intl J Infect Dis. 2006;10(3):193–201.

46. Inglesby TV, Dennis DT, Henderson DA, et al. Plague as a biolog- ical weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA. 2000;283(17):2281–2290.

47. Perry RD, Fetherston JD. Yersinia pestis–etiologic agent of plague. Clin Microbiol Rev. 1997;10(1):35–66.

48. Boulanger LL, Ettestad P, Fogarty JD, Dennis DT, Romig D, Mertz G. Gentamicin and tetracyclines for the treatment of human plague: review of 75 cases in new Mexico, 1985–1999. Clin Infect Dis. 2004;38(5):663–669.

49. Sonnabend O, Sonnabend W, Heinzle R, Sigrist T, Dirnhofer R, Krech U. Isolation of Clostridium botulinum type G and identification of type G botulinum toxin in humans: report of five sudden unexpected deaths. J Infect Dis. 1981;143(1):22–27.

50. Barash JR, Tang TW, Arnon SS. First case of infant botulism caused by Clostridium baratii type F in California. J Clin Micro- biol. 2005;43(8):4280–4282.

51. Aureli P, Fenicia L, Pasolini B, Gianfranceschi M, McCroskey LM, Hatheway CL. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J Infect Dis. 1986;154(2):207–211.

52. Passaro DJ, Werner SB, McGee J, MacKenzie WR, Vugia DJ. Wound botulism associated with black tar heroin among inject- ing drug users. JAMA. 1998;279(11):859–863.

53. Spika JS, Shaffer N, Hargrett-Bean N, Collin S, MacDonald KL, Blake PA. Risk factors for infant botulism in the United States. Am J Dis Child. 1989;143(7):828–832.

54. Hibbs RG, Weber JT, Corwin A, et al. Experience with the use of an investigational F(ab)2 heptavalent botulism immune globu- lin of equine origin during an outbreak of type E botulism in Egypt. Clin Infect Dis. 1996;23(2):337–340.

55. Arnon SS, Schechter R, Maslanka SE, Jewell NP, Hatheway CL. Human botulism immune globulin for the treatment of infant botulism. N Engl J Med. 2006;354(5):462–471.

56. Borio L, Inglesby T, Peters CJ, et al. Hemorrhagic fever viruses as biological weapons: medical and public health management. JAMA. 2002;287:2391–2405.

57. Snell NJ. Ribavirin–current status of a broad spectrum antiviral agent. Expert Opin Pharmacother. 2001;2:1317–1324.

58. Warfield KL, Panchal RG, Aman MJ, Bavari S. Antisense treat- ments for biothreat agents. Curr Opin Mol Ther. 2006;8:92–103.

59. Cetron MS, Marfin AA, Julian KG, et al. Yellow fever vaccine. Recommendations of the Advisory Committee on Immuniza- tion Practices (ACIP). MMWR. 2002;51(RR-17):1–11.

60. Centers for Disease Control. Summary of notifiable diseases, United States, 2005. MMWR. 2007;54:20–30.

61. Alibek K. Biohazard. New York: Random House; 1999:29–38. 62. Rogozin II. Tularemia prevention during the Second World War.

Zh Mikrobiol Epidemiol Immunobiol. 1970;47:23–26. 63. Croddy E, Krcalova S. Tularemia, biological warfare, and the

battle for Stalingrad (1942–1943). Mil Med. 2001;166:837–838. 64. Evans ME, Gregory DW, Schaffner W, McGee ZA. Tularemia: a

30-year experience with 88 cases. Medicine. 1985;64:251–269. 65. Feldman KA, Enscore RE, Lathrop SL, et al. An outbreak of

primary pneumonic tularemia on Martha’s Vineyard. N Engl J Med. 2001;345:1601–1606.

66. Rusnak JM, Kortepeter MG, Hawley RJ, et al. Risk of occupation- ally acquired illnesses from biological threat agents in unvacci- nated laboratory workers. Biosecur Bioterror. 2004;2:281–293.

67. Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon, medical and public health management. JAMA. 2001;285:2763–2773.

68. Enderlin G, Morales L, Jacobs RF, Cross JT. Streptomycin and alternative agents for the treatment of tularemia: review of the literature. Clin Infect Dis. 1994;19:42–47.

69. Sandstrom G. The tularemia vaccine. J Chem Tech Biotechnol. 1994;59:315–320.

70. The White House. National Strategy to Combat Weapons of Mass Destruction. 2002.

71. Food and Drug Administration. Challenge and Opportunity on the Critical Path to New Medical Products, 2004. Available at:

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .

476 ■ RO B E RT G. DA R L I N G, JO N B. WO O D S, A N D TH E O D O R E J. CI E S L A K

http://www.fda.gov/oc/initiatives/criticalpath/whitepaper.html. Accessed January 21, 2009.

72. Gronvall GK, Fitzgerald J, Chamberlain A, Inglesby TV, O’Toole T. High-containment biodefense research laboratories: meet- ing report and center recommendations. Biosecur Bioterror. 2007;5(1):75–85.

73. Lawler A. Biodefense labs. Boston University under fire for pathogen ishap. Science. 2005;307(5709):501.

74. Homeland Security Presidential Directive/HSPD-18: U.S. Depart- ment of Homeland Security; 2007.

75. The Project Bioshield Act; 2004.

76. The Pandemic and All-Hazards Preparedness Act; 2007. 77. Franco C, Deitch S. Billions for biodefense: federal agen-

cy biodefense funding, FY2007-FY2008. Biosecur Bioterror. 2007; 5(2):117–133.

78. Office of the Assistant Secretary for Preparedness and Response. HHS Public Health Emergency Medical Countermeasures Enter- prise implementation plan for chemical, biological, radiological and nuclear threats. Notice. 2007.

79. Russell PK. Project BioShield: what it is, why it is needed, and its accomplishments so far. Clin Infect Dis. 2007;45(Suppl 1):S68– 72.

C op

yr ig

ht ©

2 00

9. C

am br

id ge

U ni

ve rs

ity P

re ss

. A ll

rig ht

s re

se rv

ed .