Anthrax: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, treatment, and prophylaxis

Last updated July 28, 2010

Agent and Pathogenesis
Epidemiology
Anthrax as a Biological Weapon
Clinical Syndromes and Differential Diagnosis
Clinical Laboratory Testing
Treatment and Postexposure Prophylaxis
Anthrax Vaccination
Management of Exposure Events
Hospital Infection Control (Including Autopsies and Burial)
Environmental Testing and Decontamination
Public Health Reporting and Case Definitions
Images
References

Agent and Pathogenesis

Agent

Key microbiological characteristics of Bacillus anthracis follow (see References: CDC 2001: Basic laboratory protocols for the presumptive identification of Bacillus anthracis, Martin 2010: Bacillus anthracis [anthrax]):

Vegetative cell: large, gram-positive bacillus (1.0 to 1.5 mcm x 3.0 to 5.0 mcm); "jointed bamboo-rod" appearance
Endospore: oval, central-to-subterminal, does not usually swell (1.0 x 1.5 mcm); CO₂ levels within the body inhibit sporulation
Forms long chains of vegetative cells in vitro; single cells or short chains of two to four cells in direct clinical samples
Readily forms spores in the presence of oxygen
Aerobic or facultatively anaerobic
Nonmotile
Catalase-positive
Nonhemolytic growth on sheep blood agar (SBA)
Rapid growth on SBA; comma-shaped projections may give "Medusa head" or "comet tail" appearance on SBA
Colonies have been described as having a "ground glass" or "curled hair" appearance and have the consistency of beaten egg whites
Colonies are 2 to 5 mm in diameter after 16 to 18 hours of incubation
Forms mucoid capsule when grown on agar with sodium bicarbonate and incubated in CO₂-enriched atmosphere; capsule can be visualized with India ink preparation
Susceptible to lysis by gamma phage

Spores germinate and form vegetative cells in environments rich in nutrients (eg, glucose, amino acids, nucleosides). Vegetative bacteria generally survive poorly outside of mammalian hosts. Conversely, vegetative cells form spores when nutrients in the environment are exhausted. Spores are protected by a morphologically complex protein coat (see References: Giorno 2007). The exosporium may restrict dispersal and thereby increase the probability of a lethal dose for the grazing animal (see References: Hugh-Jones 2009). Spores have been shown to have heat-resistance characteristics similar to other Bacillus species, and can survive in the environment for more than 40 years (see References: Manchee 1990, Montville 2005).

Pathogenesis

Virulence factors

The primary virulence factors produced by B anthracis are plasmid coded and include:

A poly-D-glutamic acid capsule (coded for on plasmid pXO2) that inhibits phagocytosis of vegetative bacilli
Three exotoxins that combine to produce two binary toxins:

Protective antigen (PA) is a binding protein that permits entry of toxin into host cells via endocyte formation. Once in the endocyte, PA toxin forms a pore, which creates a small passageway in the endosomal membrane that allows the enzymatic components of the toxin to enter the cytoplasm.
Edema factor (EF) is a calmodulin-dependent adenylate cyclase. EF combines with PA to form edema toxin (coded by the cya gene of plasmid pXO1).

Edema toxin converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP); high intracellular levels of cAMP lead to impaired maintenance of water homeostasis and characteristic edema (see References: Kumar 2002).
Edema toxin also inhibits neutrophil function and stimulates production or release of multiple inflammatory mediators, including neurokinins, prostanoids, and histamine (see References: Tessier 2007).

Lethal factor (LF) is a zinc metalloprotease. LF combines with PA to form lethal toxin (coded by the lef gene on pXO1).

Lethal toxin is thought to stimulate overproduction of cytokines (eg, tumor necrosis factor [TNF] alpha and interleukin [IL] 1-beta), which leads to lysis of macrophages. Rapid release of inflammatory mediators also may contribute to the sudden death that can occur with anthrax.
Lethal toxin has been shown to cause endothelial cell apoptosis and endothelial barrier dysfunction, which may contribute to vascular destruction (see References: Kirby 2004, Warfel 2005).

Virulence of B anthracis appears to be related to clonality and to the numbers of copies of the pXO1 and pXO2 plasmids within each bacterial cell (see References: Coker 2003). The complete genomic sequence of B anthracis (Ames strain) has been analyzed; several chromosomally encoded potential virulence factors were identified, including hemolysin, phospholipases, and iron acquisition proteins (see References: Read 2003).

Anthrax toxin genes were identified in a naturally occurring B cereus isolate obtained from a patient with an illness similar to inhalational anthrax (see References: Hoffmaster 2004). More recently, anthrax-like illnesses were identified in two metalworkers who had inhalation exposure to B cereus that contained B anthracis toxin genes (see References: Avashia 2007).

Cutaneous anthrax

The pathogenesis of cutaneous anthrax involves the following process:

Endospores are introduced through the skin (usually via preexisting skin lesions or abrasions).
Low-level germination at the site of introduction produces localized necrosis with eschar formation and soft-tissue or mucosal edema (which can be massive in some cases). Epithelial damage appears to be required for germination of spores. Germination begins 1 to 3 hours after inoculation, but spore germination by itself is not sufficient to produce infection in undamaged skin (see References: Bischof 2007).
Endospores often are phagocytosed by macrophages and carried to regional lymph nodes, causing painful lymphadenopathy and lymphangitis.
Hematogenous spread with resultant toxemia can occur, although such spread is not common with appropriate antibiotic therapy.
The infective dose for cutaneous anthrax is not known.

Inhalational anthrax

Pathogenesis of inhalational anthrax involves the following steps (see References: Abramova 1993, Hanna 1998):

Endospores are introduced into the body via inhalation. Endospores are 1 mcm x 1.5 mcm in size (see References: CDC 2001: Basic laboratory protocols for the presumptive identification of Bacillus anthracis) and are, therefore, able to reach the alveoli (ie, <5 mcm).
Endospores are phagocytosed by macrophages and carried to regional lymph nodes. They also appear to be taken up by lung epithelial cells (see References: Russell 2008).
The endospores then germinate inside macrophages and become vegetative cells, which leave the macrophages and multiply in the lymphatic system.
Bacteria enter the bloodstream and lead to septic shock and toxemia; hematogenous spread can lead to hemorrhagic meningitis.
Regional hemorrhagic lymphadenitis of mediastinal and peribronchial lymph nodes causes hemorrhagic mediastinitis (see References: Abramova 1993). A widened mediastinum may be noted on chest radiograph or enlarged lymph nodes may be directly visualized on chest computed tomography.
Pulmonary lymphatic drainage can be blocked, leading to pulmonary edema.
Pleural effusions are common and may be massive. B anthracis bacilli, bacillary fragments, and antigens can be noted with immunohistochemistry (IHC) testing of pleural effusions (see References: Guarner 2003).
True pneumonia rarely occurs, although a focal, hemorrhagic, necrotizing pneumonic lesion (similar to the Gohn complex of tuberculosis) may be seen (see References: Abramova 1993). Intraalveolar edema, focal areas of hyaline membrane formation, and interstitial mononuclear inflammation may be noted (see References: Guarner 2003).
Compression of the lungs and septic shock are the major causes of death.
The median infective dose (ID₅₀) for inhalational anthrax is estimated at 8,000 to 50,000 spores (see References: Franz 1997), although the minimum infective dose may be considerably less. On the basis of experimental studies involving primates, the US Department of Defense (DoD) has estimated that the median lethal dose (LD₅₀) for inhalational anthrax in humans from weapons-grade anthrax is 2,500 to 55,000 spores (see References: DIA 1986).
Extrapolation of dose-response curves involving cynomolgous monkeys suggest that the LD₁₀ (dose at which 10% of the population is expected to die) in humans following exposure to airborne anthrax spores may be as low as 50 to 98 spores, the LD₅ (5% fatalities) may be only 14 to 28 spores, and the LD₁ (1% fatalities) may be only 1 to 3 spores (see References: Peters 2002).
Mathematical modeling of airborne anthrax infection based on observations from the US Postal Service experience during the 2001 anthrax outbreak (and an assumption that 10,000 people may have been exposed) suggests that exposures ranged from 18 to 863 spores and may have been as low as 2 to 9 spores (see References: Fennelly 2004).
The dose-response relationship for inhaled B anthracis is highly uncertain. However, a review of animal and human studies, including those that documented spore exposures insufficient to cause inhalational anthrax, suggests that an exposure threshold may exist (see References: Coleman 2008).
Animal data have suggested that anthrax spores might be able to survive in the lungs for longer than 60 days; in one study, live spores were detected in the lymph nodes of a monkey 100 days after exposure (see References: Henderson 1956).

Gastrointestional anthrax

The pathogenesis of gastrointestinal anthrax is not clear, since this condition is relatively rare. Historically, illness has been thought to result from ingestion of B anthracis spores; however, recently experts have postulated that illness predominantly results from ingestion of large numbers of vegetative cells (such as may be found in poorly cooked meat from infected animals) (see References: Inglesby 2002).

Two forms of gastrointestinal anthrax have been recognized: oropharyngeal and abdominal.
In oropharyngeal anthrax, the portal of entry is the oral or pharyngeal mucosa. A mucosal ulcer occurs initially, followed by regional lymphadenopathy and localized edema.
In abdominal anthrax, the portal of entry often is the terminal ileum or cecum. Intestinal lesions occur and are followed by regional lymphadenopathy. Edema of the bowel wall and ascites (sometimes massive) may be present.
Hematogenous spread with resultant toxemia can occur.
The infective dose for gastrointestinal anthrax is not known. In animal models (guinea pigs, rabbits, and rhesus monkeys), investigators failed to induce infection following oral challenge with 10⁸ spores; these animals are all thought to be more susceptible to anthrax than humans (see References: Beatty 2003).

Epidemiology

Reservoir

B anthracis is found in soil in many areas of the world. Ecologic factors (such as abundant rainfall following a period of drought) may enhance spore density in soil, although the exact influence of such factors remains poorly understood.

The organism generally exists in the endospore form in nature; germination of spores outside an animal host may occur when the following conditions are met (see References: WHO 1998):

Temperature between 8°C and 45°C
pH between 5 and 9
Relative humidity >95%
Presence of adequate nutrients

Endospores are resistant to drying, heat, ultraviolet light, gamma radiation, and some disinfectants. Spores can persist in soil for decades, as illustrated by biological warfare experiments during World War II on the Scottish island of Gruinard (see References: Manchee 1990). During 1943 and 1944, an estimated 4 x 10¹⁴anthrax spores were dispersed on the island through explosive means. Spores were still detectable more than 40 years later. Disinfection of the island was finally completed in 1987, using a combination of seawater and formaldehyde.

Anthrax in Animals

Most mammals are susceptible to anthrax, but it is predominantly a disease of livestock. Livestock or other herbivores (eg, cattle, sheep, goats, pigs, bison, water buffalo) acquire infection from consuming contaminated soil or feed. Anthrax in animals is hyperendemic or endemic in the following areas of the world (see References: WHOCC):

Most areas of the Middle East
Most areas of equatorial Africa
Mexico and Central America
Chile, Argentina, Peru, and Bolivia
Certain Southeast Asian countries (eg, Myanmar, Vietnam, Cambodia, Thailand)
Papua New Guinea
China
Some Mediterranean countries

In most of the rest of the world, anthrax occurs only sporadically. In the United States, outbreaks in animals have occurred since 1990 in the Midwest (Kansas, Minnesota, Missouri, Nebraska, North Dakota, South Dakota), in the West (California, Nevada), and in Texas and Oklahoma (see References: MBAH 2006, WHOCC). Outbreaks also have recently occurred in Saskatchewan and Manitoba, Canada, affecting more than 800 animals (see References: APHIS 2006).

Anthrax has been reported as the cause of death among chimpanzees in Ivory Coast (see References: Leendertz 2004) and chimpanzees and a gorilla in Cameroon (see References: Leendertz 2006). Investigators postulated that the chimps became ill either from consuming an infected animal or drinking contaminated water. Isolates from the wild apes in both outbreaks showed that the strains were clearly different from those of any previously described. The isolates established a new "forest anthrax cluster," termed "F," suggesting that B anthracis is a far less homogeneous species than currently believed (see References: Leendertz 2006).

Anthrax also has been reported in cheetahs following consumption of infected meat (see References: Good 2008).

Anthrax spores were detected in two of six species of raptors (road-side hawks and chimango caracaras [a bird of prey that is in the falcon family]) in central Argentina, suggesting that scavenger and nonscavenger bird species may influence anthrax epidemiology in some countries (see References: Saggese 2007).

Modes of Transmission

Illness in humans most commonly occurs following exposure to infected animals or contaminated animal products; such exposures include:

Contact with infected tissues of dead animals (eg, butchering, preparing contaminated meat), which generally leads to cutaneous anthrax
Consumption of contaminated undercooked meat, which can lead to gastrointestinal anthrax
Contact with contaminated hair, wool, or hides (particularly during processing) or contact with products made from them, which can lead to either inhalational or cutaneous anthrax. Animal hair from endemic regions continues to represent an occupational risk for modern woolworkers (see References: Wattiau 2008).

Cases following laboratory exposure have been recognized (see References: Brachman 1980, CDC 2002: Suspected cutaneous anthrax in a laboratory worker—Texas, 2002). Person-to-person transmission of B anthracis has been reported rarely with cutaneous anthrax, but has not been recognized with gastrointestinal or inhalational disease (see References: Weber 2001, Weber 2002).

Anthrax in Humans—United States

Approximately 130 human cases of anthrax occurred annually in the United States during the early 1900s. The incidence has gradually declined over time, with typically fewer than 10 cases reported each year since the early 1960s (see References: CDC 1995).
About 95% of naturally occurring cases in the United States are cutaneous and 5% are inhalational. Only one case of gastrointestinal infection has been recognized in this country (see References: Brachman 1980, Ramer 2010).
Only 18 cases of naturally occurring inhalational anthrax were reported in the United States during the 20th century (see References: Brachman 1980). All but three were associated with industrial exposures; two of the remaining cases were laboratory-acquired, and the source of exposure for the third case remains unknown.
Between 1990 and 2000, only two cases of naturally occurring anthrax were reported in the United States (one in 1992 and one in 2000); both patients had cutaneous disease. The latter case occurred in North Dakota and resulted from agricultural exposure (see References: CDC 2001: Human anthrax associated with an epizootic among livestock—North Dakota, 2000).
Since 2006, three cases of anthrax have resulted from direct occupational association with djembe drums made from untreated West African animal hides. Two cases were of cutaneous anthrax (see References: CDC 2008), and one case was inhalational (see References: CDC 2006, Walsh 2007). This was the first new case of naturally occurring inhalational anthrax in the United States since 1976. In response to this case, the Centers for Disease Control and Prevention (CDC) developed a document on safety issues related to anthrax and animal hides (see References: CDC: Q & A: anthrax and animal hide drums).
In December 2009, a New Hampshire woman developed gastrointestinal anthrax after attending a drum circle gathering. Two animal hide drums and environmental samples from the building later tested positive for the same strain of B anthracis that infected the woman. She is believed to have swallowed airborne anthrax spores released from the drums during the gathering (see References: CDC 2010: Gastrointestinal anthrax after an animal-hide drumming event—New Hampshire and Massachusetts, 2009).

Anthrax in Humans—Global Perspective

An estimated 2,000 to 20,000 human cases of anthrax occur globally each year (see References: Brachman 1984).
Human cases generally follow disease occurrence in ruminants and are most prevalent in Africa, the Middle East, and parts of Southeast Asia.
Most cases are cutaneous.
Viable anthrax spores recently were detected in goat hair fibers, airborne dust, and unprocessed wastewater in a Belgian factory that scours wool and goat hair. Although no definitive clinical cases of anthrax were recorded among this unvaccinated workforce, evidence of asymptomatic B anthracis infection was found in approximately 10% of the employees (see References: Wattiau 2009).
Two fatal cases of inhalational anthrax recently occurred in the United Kingdom, one in Scotland in 2006 and one in England in 2008. Both patients were drum makers (see References: Anaraki 2008).
A recent systematic review of MEDLINE (1996-2005) and selected journal indexes (1900-1966) for inhalational anthrax cases worldwide between 1900 and 2005 identified 82 cases of inhalational anthrax (see References: Holty 2006: Systematic review: a century of inhalational anthrax cases from 1900 to 2005). Cases from the 1979 Sverdlovsk outbreak in the former Soviet Union (see section below on weaponized anthrax) were excluded from analysis because symptoms, treatment, and disease progression were not described.

Seventy-one of the 82 identified cases were naturally occurring, and 11 were part of the 2001 bioterrorism outbreak in the United States.
Among naturally occurring cases, most involved exposure to contaminated wool, goat hair, or animal hides.

An outbreak of anthrax was first observed among injection-drug users in Scotland in December 2009 (see References: Ramsay 2010). Since then, 31 confirmed cases and 11 deaths among heroin users have been reported from three countries (Scotland, England, and Germany) (see References: Christie 2010). The patients reported injecting, smoking, and/or snorting heroin. The symptoms at presentation varied greatly, were inconsistent, and were not typical of cutaneous, inhalational, or gastrointestinal anthrax (see References: Booth 2010).

Outbreaks of Naturally Occurring Disease

Outbreaks have been reported in industrial settings where animal products are processed and in agricultural settings following consumption of or exposure to ill animals. Notable examples of outbreaks include the following:

A major outbreak involving nearly 10,000 cases and 182 deaths (most of them cutaneous infection) occurred in Zimbabwe during the late 1970s and early 1980s (see References: Davies 1982). An epizootic in cattle occurred at that time in the same area.
An outbreak involving 9 cases (5 inhalational and 4 cutaneous) occurred in 1957 in the United States in a New Hampshire goat-hair processing plant (see References: Brachman 1960, Plotkin 1960). This was the last recognized outbreak of naturally occurring infection in this country.
An outbreak of oropharyngeal anthrax involving 24 cases occurred in Thailand in 1982 following consumption of contaminated meat (see References: Sirisanthana 1984). Oropharyngeal disease is an unusual manifestation of infection, which makes this outbreak of particular interest.

Anthrax as a Biological Weapon

Aerosol Release of Anthrax Spores

Aerosol release of weaponized spores is the most likely mechanism for use of anthrax as a biological weapon (see References: Inglesby 2002). Although there is no formal definition of weaponized anthrax, weaponization for aerosol release generally involves:

Use of small particles
A high concentration of spores
Treatment to reduce clumping
Neutralization of the electrical charge
Use of antimicrobial-resistant strains or genetic modification of the organism to increase virulence or escape vaccine protection

The impact of a large aerosol release of weaponized anthrax remains unknown; however, scenarios have been hypothesized, including:

A 1970 World Health Organization (WHO) report estimated that an aerosol release of 50 kg of dried powder containing 6 x 10¹⁵ anthrax spores over a city of 5 million people in an economically developed country (such as the United States) would produce 250,000 incapacitating illnesses and up to 100,000 deaths (see References: WHO 1970).
A 1993 Office of Technology Assessment (OTA) study estimated that up to 3 million deaths could occur following the release of 100 kg of B anthracis (see References: OTA 1993).

Experience with aerosol spraying of B thuringiensis in Canada to control the European gypsy moth demonstrated the following pertinent findings (see References: Levin 2003):

Approximately 5 to 6 hours after the spray application began, indoor concentrations of airborne B thuringiensis actually exceeded outdoor concentrations, suggesting that the organisms entered homes and buildings after the aerosol release.
Although most of the particles were relatively large, particles with a medium diameter of 2 to 7 microns were detected both inside and outside of homes. The authors estimated that, at 5 to 6 hours after spraying, adults in the spray zone inhaled approximately 203 spores per hour.
Nasal swabs from asthmatic children in the spray zone were collected after spraying; Bacillus species with genetic patterns consistent with B thuringiensis were identified in 76.6% of isolates obtained from nasal passages.

The findings from this study suggest that it is technologically feasible to disseminate biological agents from aircraft; however, the applicability of this information to an intentional aerosol release of B anthracis is unknown. These data also raise concerns about indoor air safety should an intentional release of a biological agent occur.

Contamination of Food or Water

Deliberate contamination of food or water with anthrax spores also is a possibility. During World War II, the Japanese reportedly impregnated chocolate with anthrax to kill Chinese children. The apartheid government of South Africa also experimented with anthrax in chocolate (see References: Sirisanthana 2002).

Even though contamination of a water supply is unlikely owing to the large volume of water involved and the chlorination process, contamination of smaller water sources is theoretically feasible since spore counts remain stable in water for at least several days following inoculation (see References: Beatty 2003). Since B anthracis spores are not destroyed by pasteurization, contamination of milk is another theoretical possibility (see References: Perdue 2003).

Historical Perspective

Although anthrax has not been responsible for the massive number of deaths associated with cholera, plague, or smallpox, it has played a prominent role in the history of infectious diseases. Anthrax was the first disease for which a microbial origin was definitively established. Robert Koch identified the bacterium that causes anthrax in 1877 (see References: Martin 2010: Bacillus anthracis [anthrax], Purcell 2007). Anthrax also was the first disease for which an effective live bacterial vaccine was developed; Louis Pasteur developed this vaccine in 1881 and tested it in domesticated animals. Additionally, inhalational anthrax among wool and animal hide processors introduced the concept of occupational infectious disease risk.

Intentional spread of anthrax dates back to World War I, when German operatives infected horses and cattle with B anthracis while they were awaiting shipment overseas. During World War II, both the Axis and the Allies had biowarfare programs that involved anthrax (see References: Martin 2010: Anthrax as an agent of bioterrorism). The US military has been concerned about anthrax as a potential biological weapon for many years because of its infectiousness via the aerosol route and the associated high mortality rate for untreated inhalational anthrax (see References: Purcell 2007). B anthracis is readily accessible and easy to grow; in the spore form it is stable, easily stored, and portable. Spores may be dispersed or sprayed as a powder or liquid. Thus, anthrax remains the agent of greatest concern for bioterrorism (see References: Martin 2010: Anthrax as an agent of bioterrorism).

In 1972, more than 140 countries signed the Biological and Toxin Weapons Convention, which called for termination of all offensive biological weapons research and development and destruction of existing biological weapons stocks. However, the former Soviet Union continued to expand its biological weapons program (which included weaponization of anthrax) throughout the 1980s and early 1990s.

After the demise of the Soviet Union, many of the scientists who worked in the biological weapons program left the country. The status of those scientists remains unknown; however, Iraq, Iran, Syria, Libya, and North Korea actively have recruited such experts (see References: Henderson 1999). These countries and others have been suspected of ongoing development of offensive bioweapons programs. Reports from the past few years suggest that at least three countries have offensive biological weapons programs and at least an additional six have research programs with possible production of offensive weapons (see References: MIIS).

Weaponized anthrax has caused two outbreaks of disease (key points from each are outlined in the sections below). In addition, in July 1993, the religious group Aum Shinrikyo aerosolized a liquid suspension of B anthracis from the roof of an eight-story building in the Kameido district of Tokyo, but the release did not cause any human cases. Factors contributing to failure of this bioterrorist act included use of an attenuated B anthracis strain, low spore concentration, ineffective dispersal, a clogged spray device, and inactivation of spores by sunlight (see References: Takahashi 2004).

Sverdlovsk, USSR—1979

This outbreak in Sverdlovsk in the Union of the Soviet Socialist Republics (now Ekaterinburg, Russia) resulted from accidental release of anthrax spores from a military microbiological facility, Compound 19, where weaponized anthrax was being produced (see References: Dembek 2007, Meselson 1994).
Seventy-seven human cases were reported and 66 of the patients died, for a case-fatality rate of 86%; 75 cases were inhalational and two were cutaneous (one on the back of the neck and one on the shoulder). A subsequent statistical analysis of available data suggests that 250 cases with 100 fatalities actually may have occurred (see References: Brookmeyer 2001).
The mean incubation period was 9 to 10 days (range, 2 to 43 days), and the mean time between illness onset and death was 3 days.
Mean patient age for male cases was 42 years and for female cases was 55 years, and no cases occurred in children.
The geographic distribution of human and animal cases indicated that the outbreak was confined to a narrow zone, downwind from a point of origin in Sverdlovsk (approximately 4 km for humans and 40 km for animals). Historical meteorological data, combined with this case distribution, identified Compound 19 as the point of origin. These data also showed that the release most likely took place on April 2, 1979.
Approximately 2 weeks after the presumed date of exposure, a vaccination campaign of 59,000 eligible residents was begun; an estimated 80% of the target population received at least one dose of vaccine. Prophylactic antibiotics also were provided to both suspected and confirmed cases.
Investigators postulated that the weight of spores released as aerosol "could have been as little as a few milligrams or as much as nearly a gram."
Recent modeling studies suggest that the incubation period for anthrax is dose-dependent. The authors postulate that the relatively long incubation period for cases associated with Sverdlovsk was related to the level of exposure (see References: Wilkening 2006).

United States—2001

An outbreak of cutaneous/inhalational anthrax occurred in the United States in 2001.
The outbreak predominantly involved direct exposure to mail that was deliberately contaminated with anthrax spores. Several contaminated letters were sent to members of Congress and media outlets, and one was reported to contain 2 g of powder, with 100 billion to 1 trillion anthrax spores per gram (see References: Inglesby 2002).
The following features were noted in an epidemiologic report that summarized the outbreak findings (see References: Jernigan 2002):

Twenty-two cases (11 inhalational and 11 cutaneous) were identified. Five of the patients with inhalational anthrax died, for a case-fatality rate of 45% among that group.
Cases occurred in residents of seven states along the East Coast (Connecticut, Florida, Maryland, New Jersey, New York, Pennsylvania, and Virginia), with illness onsets between September 22 and November 16, 2001.
Four contaminated letters were recovered; all four were mailed in or around Trenton, New Jersey. Two were postmarked September 18, 2001, and two were postmarked October 9, 2001.
Twenty of the patients were either mail handlers or were exposed to work sites where mail was handled or received; 1 of these cases was a 7-month-old infant. The remaining 2 cases had no apparent association with contaminated mail. These persons likely became exposed through cross-contamination of bulk mail that passed through contaminated mail facilities (see References: Griffith 2003, Holtz 2003).
Illness in the 7-month-old infant with cutaneous anthrax was complicated by quick progression to severe microangiopathic hemolytic anemia despite early antibiotic treatment. The source is thought to be the workplace of the infant's mother, which the infant visited the day before symptom onset. One possible exposure scenario, according to the authors, is that spores on the hands of someone in the workplace who lifted or held the child may have contacted an exposed or possibly abraded area of the child's skin (see References: Freedman 2002).
B anthracis isolates were obtained from the 4 contaminated letters, 17 clinical specimens from cases, and 106 environmental samples collected along the mail path of the contaminated letters; all were identical by molecular subtyping.

Following recognition of anthrax cases in postal workers, air sampling was conducted before and during activation of a contaminated mail-sorting machine at a Washington, DC, postal facility. This testing (which was conducted several weeks after the contaminated mail passed through the machine) demonstrated that a mail-sorting machine can remain contaminated for many days after processing mail contaminated with B anthracis (see References: Dull 2002).
The outbreak demonstrated several important points about weaponized anthrax:

Mail can be an effective vehicle for disseminating anthrax spores.
Cross-contamination of mail likely can occur within postal facilities.
Persons who handle or process unopened contaminated mail are at risk of acquiring anthrax.
Substantial environmental contamination can occur in facilities handling contaminated mail or in offices where contaminated mail is opened.

Following the outbreak, a case of cutaneous anthrax occurred in a laboratory worker in Texas who was working at a private laboratory that was processing environmental samples from the CDC investigations (see References: CDC 2002: Suspected cutaneous anthrax in a laboratory worker—Texas, 2002).
A 1-year health assessment of adult anthrax survivors demonstrated that survivors continued to report moderate to severe health complaints affecting multiple organ systems and had significantly greater overall psychological distress compared with US referent populations (see References: Reissman 2004). Fifty-three percent had not returned to work since their infection.
The alleged perpetrator and the source of the anthrax in this outbreak have been identified. Bruce Ivins, the scientist named by Federal Bureau of Investigation (FBI) investigators as the sole orchestrator of the attack, committed suicide before he was charged. Ivins worked at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) and had access to the spores used in the attack. Sequence analyses of anthrax strains allowed investigators to trace the spores to two flasks, one at USAMRIID that was under Ivin's charge and one flask from another laboratory. Several questions still remain, including the basis on which the FBI ruled out spores from the other flask and ruled out other individuals who had access to the spores. Many researchers in the scientific community remain skeptical of the FBI statements, in light of the circumstantial nature of the the evidence. The FBI closed the case in February 2010, in spite of the remaining unanswered questions (see References: Bhattacharjee 2008, Warrick 2010). The original Ames strain came from a laboratory in College Station, Texas (rather than Ames, Iowa). Several distinct Ames strains have been identified.
As a result of this outbreak, the US Postal Service has deployed an autonomous biodetection system (BDS) for anthrax in mail-processing systems across the United States (see References: CDC 2004: Responding to detection of aerosolized Bacillus anthracis by autonomous detection systems in the workplace).

Clinical Syndromes and Differential Diagnosis

The three primary forms of anthrax correspond to the route of exposure: cutaneous, inhalational, and gastrointestinal (see References: HPA 2007). A fourth form, anthrax meningitis, may occur as a complication of cutaneous, inhalational, or gastrointestinal anthrax, or it may be the presenting form.

Clinical Features of Cutaneous Anthrax
Feature	Characteristics
Incubation period	1-7 days (more commonly 2-5 days; may be as long as 12 days)
Signs and symptoms^a	—Initial lesion is small papule or vesicle, which may be pruritic. —By second day, papule ulcerates with central necrosis and drying. —Painless, localized, nonpitting edema surrounds ulcerated area. —Fine vesicles may encircle ulcer; these enlarge over next 1-2 days and may discharge serosanguineous fluid. —After 1-2 days, painless black eschar forms over ulcerated area. —Eschar sloughs off after 12-14 days. —Lesions resolve without complications or scarring in 80%-90% of patients. —Extensive nonpitting edema, lymphangitis, and painful lymphadenopathy may occur. —Malignant edema is rare complication and is characterized by severe edema, multiple bullae, and shock.^b —Fever and malaise are common.^a —Lesions tend to occur on exposed areas of body (eg, hands, arms, face, neck). —One outbreak in Thailand demonstrated the following cutaneous findings for 13 patients with cutaneous anthrax^c: ~Eschar (85%) ~Blister (92%) ~Ulcer (23%) ~Edema around lesion (77%) ~Lymphadenopathy (100%)
Case-fatality rate	—Currently <1%^a,g (most patients recover with appropriate antimicrobial therapy) —In preantibiotic era, case-fatality rates of about 20% were reported.^d —A literature review of pediatric anthrax cases identified between 1900 and 2005 demonstrated an overall mortality rate for cutaneous disease of 14% (5 of 37 cases).^e Not all cases in this report received antimicrobial therapy.
Laboratory findings	—Gram stain of lesion may reveal gram-positive rods; neutrophils are uncommon. —WBC count often is normal or may be slightly elevated.^a —Histologic examination shows necrosis, edema, and lymphocytic infiltrate.^f
Abbreviation: WBC, white blood cell. ^aSee References: Gold 1955. ^bSee References: Amadi 1974. ^cSee References: Kunanusont 1990. ^dSee References: Smyth 1941. ^eSee References: Bravata 2007. ^fSee References: Dixon 1999. ^gSee References: Doganay 2009.

Clinical Features of Inhalational Anthrax
Feature	Characteristics
Incubation period	1-43 days (usually 1-6 days)^a
Signs and symptoms	—Illness may be biphasic, with an initial prodrome (which includes symptoms such as fever, malaise, fatigue, anorexia) followed by sudden increase in fever, severe respiratory distress, diaphoresis, and shock, if left untreated. —Symptoms for 10 patients with inhalational anthrax identified during the 2001 US outbreak^b-c: ~Fever, chills (100%) (7 were febrile on initial presentation) ~Sweats, often drenching (70%) ~Fatigue, malaise, lethargy (100%) ~Cough (minimally or nonproductive) (90%) ~Nausea or vomiting (90%) ~Dyspnea (80%) ~Chest discomfort or pleuritic pain (70%) ~Myalgias (60%) ~Headache (50%) ~Confusion (40%) ~Abdominal pain (30%) ~Sore throat (20%) ~Rhinorrhea (10%) —In the 2001 US outbreak, no evidence of a mild form of inhalational anthrax was detected through follow-up serologic testing of exposed persons.^b —A systematic review of 82 inhalational anthrax cases reported between 1900 and 2005 found that the most common symptoms or findings on admission included the following^d: ~Abnormal lung findings (80%) ~Fever or chills (67%) ~Tachycardia (66%) ~Fatigue or malaise (64%) ~Cough (62%) ~Dyspnea (52%) ~All 26 patients who had chest radiography had abnormal findings, including pleural effusion (69%) or widened mediastinum (54%).
Case-fatality rate	—Sverdlovsk outbreak: 86%^a —US outbreak: 45%^c (lower observed CFR in the US outbreak likely was due to early diagnosis and aggressive therapy) —In a systematic review of 82 cases of inhalational anthrax, the overall CFR was 85%; however, patients in the US 2001 outbreak who received early antibiotic therapy (ie, during the prodromal phase [<4.7 days after illness onset]) had a CFR of 40% and those who received antibiotics >4.7 days after illness onset had a CFR of 75%.^d —A literature review of pediatric anthrax cases identified between 1900 and 2005 demonstrated an overall mortality rate for inhalational disease of 60% (3 of 5 cases).^e Not all cases in this report received antimicrobial therapy.
Laboratory findings	Findings for 10 patients with inhalational anthrax identified during 2001 US outbreak^c: —Median WBC count at presentation was 9,800/mm³(range, 7,500/mm³ to 13,300/mm³) —Differential WBC count >70% neutrophils (70%) —Neutrophil band forms present (4 of 5; 80%) —Peak WBC during illness was 26,400/mm³(range, 11,900/mm³ to 49,600/mm³) —Elevated transaminases (SGOT or SGPT) >40 (90%) —Hypoxemia^f (60%) —Metabolic acidosis (20%) —Abnormal chest radiograph (100%): ~Mediastinal widening (70%) ~Infiltrates, consolidation (70%) ~Pleural effusion (80%) —Abnormal CT scan (8 of 8; 100%): ~Mediastinal lymphadenopathy, widening (7 of 8; 88%) ~Pleural effusion (8 of 8; 100%) ~Infiltrates, consolidation (6 of 8; 75%)
Abbreviations: CFR, case-fatality rate; CT, computed tomography; SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase; WBC, white blood cell. ^aSee References: Meselson 1994. ^bSee References: Baggett 2005. ^cSee References: Jernigan 2001. ^dSee References: Holty 2006 : Systematic review: a century of inhalational anthrax cases from 1900 to 2005. ^eSee References: Bravata 2007. ^fAlveolar-arterial oxygen gradient >30 Hg on room air; O₂ saturation <94%.

Clinical Features of Gastrointestinal Anthrax
Feature	Characteristics
Incubation period	1-7 days (usually 2-5 days)
Signs and symptoms	—One outbreak of GI anthrax in Uganda demonstrated the following findings in 143 patients^a: ~Fever (may be low-grade) (70%) ~Abdominal tenderness (85%) ~Diarrhea (80%; bloody in only 5%) ~Vomiting (may be coffee-ground or blood-tinged) (90%) ~Headache (100%) ~Pharyngeal edema (10%) —Ascites may develop 2-4 days after onset (fluid may be clear or purulent)^b and in rare instances GI anthrax cases may present with progressive ascites without other classic symptoms.^c —Ulcerations can occur anywhere along the GI tract and may cause hemorrhage, obstruction, or perforation.^d —If the patient survives, symptoms last about 2 wk —One outbreak of oropharyngeal anthrax in Thailand demonstrated the following findings for 24 patients^e: ~Neck swelling (100%) ~Fever (96%) ~Sore throat, dysphagia (63%) ~Mouth or pharyngeal ulcerative or necrotic lesions (100%) (pseudomembranes also were noted in some patients) ~Respiratory distress (25%) ~Hoarseness (8%) ~Sensation of a "lump in throat" (8%) ~Diarrhea (4%) ~Bleeding from the mouth (4%)
Case-fatality rate	—Rate for GI anthrax is between 25% and 60%.^f,j In outbreaks where patients received antibiotic therapy, rates have ranged from 0% to 29%.^g —A literature review of pediatric anthrax cases identified between 1900 and 2005 demonstrated an overall mortality rate for gastrointestinal disease of 65% (13 of 20 cases).^h Not all cases in this report received antimicrobial therapy. —In Thai outbreak of oropharyngeal disease, rate was 13%.^e In another report of 6 cases of pharyngeal anthrax, rate was 50%.ⁱ
Laboratory findings	—Gram stain of peritoneal fluid or oropharyngeal ulcers may demonstrate gram-positive rods. —Median WBC count for 13 patients with oropharyngeal anthrax in Thailand outbreak was 15,635/mm³ (range, 5,100/mm³ to 30,570/mm³). Mean percentage of neutrophils was 79.6% (range, 73% to 91%).^e —B anthracis has been cultured from oropharyngeal swabs and stool specimens in patients with GI anthrax.^g
Abbreviations: GI, gastrointestinal; WBC, white blood cell. ^aSee References: Ndyabahinduka 1984. ^bSee References: Dixon 1999. ^cSee References: Hatami 2010. ^dSee References: Sirisanthana 2002. ^eSee References: Sirisanthana 1984. ^fSee References: CDC 2001: Investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. ^gSee References: Beatty 2003. ^hSee References: Bravata 2007. ⁱSee References: Doganay 1986. ^jSee References: Doganay 2009.

Clinical Features of Anthrax Meningitis
Feature	Characteristics
Incubation period	Varies according to primary source of infection.
Signs and symptomsa^a-d	—May occur as complication of cutaneous, inhalational, or gastrointestinal anthrax, and symptoms of primary site of infection usually will be present; however, meningitis may be the presenting illness. —Characteristic features of bacterial meningitis usually present (eg, fever, nuchal rigidity, headache, change in mental status, seizures). —Nausea and/or vomiting are common. —Hemorrhagic meningoencephalitis is a characteristic presentation.
Case-fatality rate^e	—Illness fatal in >90% of cases. —A review of human anthrax in Turkey from 1990 to 2007 identified 5 cases of anthrax meningitis; 100% of cases died despite antibiotic and supportive therapy.^f —A literature review of pediatric anthrax cases identified between 1900 and 2005 demonstrated an overall mortality rate for meningoencephalitis of 100% (6 of 6 cases).^g Not all cases in this report received antimicrobial therapy. —Death usually occurs 1 to 6 days after illness onset, and 75% of patients die within 24 hr of presentation.
Laboratory findings	—Gram stain of CSF reveals many gram-positive rods. —CSF is usually bloody. —Report of 2 cases demonstrated elevated WBC counts in both patients at presentation (24,000 with 84% neutrophils and 18,000 with 90% neutrophils).^b —CT or MRI of head may show focal intracerebral hemorrhage, subarachnoid hemorrhage, diffuse cerebral edema, intraventricular hemorrhage, and/or leptomeningeal enhancement.^d
Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography; MRI, magnetic resonance imaging; WBC, white blood cell. ^aSee References: Dixon 1999. ^bSee References: Rangel 1975. ^cSee References: Meyer 2003. ^dSee References: Sejvar 2005. ^eSee References: Lanska 2002. ^fSee References: Doganay 2009. ^gSee References: Bravata 2007.

Differential Diagnosis

The differential diagnosis for anthrax depends upon the clinical syndrome (cutaneous, inhalational, gastrointestinal, or meningeal). Other diagnoses to consider are outlined in the tables below.

Differential Diagnosis for Cutaneous Anthrax
(Note:* Two key features that distinguish cutaneous anthrax from other conditions in differential diagnosis are painlessness of the lesion and the relatively large extent of associated edema.)*
Diagnosis^a-c	Distinguishing Features
Ecthyma gangrenosum	—Usually in neutropenic patients with Pseudomonas aeruginosa bacteremia —Edema usually not present
Ulceroglandular tularemia (Francisella tularensis)	—Clinical course usually indolent; disease often self-limited —Systemic toxicity uncommon
Bubonic plague (Yersinia pestis)	—Systemic toxicity common —Extremely tender regional lymphadenopathy present —Ulceration and eschar formation usually absent
Staphylococcal or streptococcal cellulitis	—May be history of trauma or preexisting lesion at site of infection —Eschar formation does not occur —Usually painful
Necrotizing soft tissue infections (particularly Group A Streptococcus and Clostridium species)	—Severe systemic toxicity often present —Early in course, pain usually more severe than clinical findings would indicate
Bite of brown recluse spider (Loxosceles reclusa)^d	—Brown recluse spiders prefer warm temperatures and are not native to northern half of United States —Spiders tend to hide in barns, woodpiles, and similar places —Bite usually causes painful blister that progresses to necrosis (unlike anthrax, which is painless) —Edema generally absent
Rickettsialpox (Rickettsia akari)	—Initial presentation involves painless papule that forms black eschar —Generalized maculopapular rash appears 2-3 days later
Scrub typhus (Orientia tsutsugamushi; formerly Rickettsia tsutsugamushi)	—Zoonotic infection from chigger bites; occurs in endemic areas (Asia and Western Pacific) —Often associated with generalized maculopapular rash
Orf (orf virus, a parapox virus)	—Occurs in farm workers —Characterized by pustule that progresses to weeping nodule —Eschar formation does not occur —Edema usually absent
Necrotic herpes simplex infection	—More likely to occur in immunocompromised host
^aSee References: Dixon 1999. ^bSee References: Swartz 2001. ^cSee References: Bell 2002. ^dSee References: Nelson 2002.

Differential Diagnosis for Inhalational Anthrax
(Note: Features that distinguish inhalational anthrax from other conditions in differential diagnosis include presence of widened mediastinum and pleural effusions on chest radiograph or CT scan with minimal evidence of pneumonia.)
Diagnosis^a,b	Distinguishing Features
Pneumonic plague (Yersinia pestis)	—Hemoptysis relatively common with pneumonic plague, but rare with inhalational anthrax
Tularemia (Francisella tularensis)	—Clinical course usually indolent, lasting weeks —Less likely to be fulminant
Community-acquired bacterial pneumonia —Mycoplasmal pneumonia (Mycoplasma pneumoniae) —Pneumonia caused by Chlamydia pneumoniae —Legionnaires' disease (Legionella pneumophila or other Legionella species) —Psittacosis (Chlamydia psittaci) —Other bacterial agents (eg, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis)	—Rarely as fulminant as inhalational anthrax —Legionellosis and many other bacterial agents (S aureus, S pneumoniae, H influenzae, K pneumoniae, M catarrhalis) usually occur in persons with underlying pulmonary or other disease or in elderly —Bird exposure with psittacosis —Gram stain of sputum may be useful —Community outbreaks caused by other etiologic agents not likely to be as explosive as pneumonic plague outbreak —Outbreaks of S pneumoniae usually institutional —Community outbreaks of Legionnaires' disease often involve exposure to cooling towers
Viral pneumonia —Influenza —Hantavirus —RSV —CMV	—Influenza generally seasonal (October-March in United States) or involves history of recent cruise ship travel or travel to tropics —Exposure to mice infected with hantavirus or their urine or feces —RSV usually occurs in children (although may be cause of pneumonia in elderly); tends to be seasonal (winter/spring) —CMV usually occurs in immunocompromised patients
—Q fever (Coxiella burnetii)	—Exposure to infected parturient cats, cattle, sheep, goats —Severe pneumonia not prominent feature
Abbreviations: CMV, cytomegalovirus; CT, computed tomography; RSV, respiratory syncytial virus. ^aSee References: Dixon 1999. ^bSee References: Bell 2002.

Differential Diagnosis for Gastrointestinal Anthrax
Diagnosis	Distinguishing Features
Abdominal Subtype^a
Typhoid fever (Salmonella typhi)	—Ascites usually not present —Other clinical features may be similar
Intestinal tularemia (Francisella tularensis)	—Illness often less severe than that seen with gastrointestinal anthrax —Ascites not present —Less likely to resemble acute abdomen —Fever may be less prominent
Bacillary dysentery (Shigella dysenteriae)	—Ascites usually not present —Other clinical features may be similar
Acute bacterial gastroenteritis caused by other agents (eg, Campylobacter jejuni, Shiga toxin–producing Escherichia coli, Yersinia enterocolitica)	—Illness often less severe than that seen with gastrointestinal anthrax —Ascites not present —Less likely to resemble acute abdomen —Fever may be less prominent —Hemolytic uremic syndrome may occur with infection caused by Shiga toxin–producing E coli
Bacterial peritonitis	—Gastrointestinal symptoms (nausea, vomiting, gastrointestinal bleeding, diarrhea) not prominent features —Tends to occur in persons with underlying medical conditions (eg, alcoholism, other liver disease)
Acute abdomen (eg, appendicitis)^b	—Anthrax generally begins with vague systemic symptoms rather than abdominal pain —Ascites is relatively common with gastrointestinal anthrax and less common with appendicitis and similar conditions
Oropharyngeal Subtype
Diphtheria (Corynebacterium diphtheriae)	—Primarily occurs in nonimmune children under 15 yr of age —Pharyngeal membrane is prominent feature; ulcerative or necrotic lesions generally not present —Removal of pharyngeal membrane often causes bleeding of submucosa
Pharyngeal tularemia (Francisella tularensis)	—Neck swelling usually absent —Exudative pharyngitis common; ulcerative lesions may occur
Streptococcal pharyngitis (Streptococcus pyogenes)	—Exudative pharyngitis most prominent feature; necrotic ulcers generally absent —Neck edema usually absent, although cervical lymphadenopathy may be prominent
Infectious mononucleosis	—Most common in young adults —Splenomegaly commonly occurs —Neck edema usually absent, although cervical lymphadenopathy may be prominent
Enteroviral vesicular pharyngitis (coxsackievirus)	—Small vesicles noted on soft palate, uvula, or anterior tonsillar pillars —Generally occurs in children —Neck edema usually absent
Acute herpetic pharyngitis (herpes simplex virus)	—Vesicles, shallow ulcers may be noted, but lesions usually not necrotic —Neck edema usually absent, although cervical lymphadenopathy may be prominent
Anaerobic pharyngitis (Vincent's angina)	—Purulent exudate covers posterior pharynx —Tonsillar abscesses may occur —Neck edema usually absent
Yersinia enterocolitica pharyngitis	—Exudative pharyngitis most prominent feature —Neck edema usually absent —Cervical adenopathy, abdominal pain may occur
^aSee References: Dixon 1999. ^bSee References: Kanafani 2003.

Differential Diagnosis for Anthrax Meningitis
Diagnosis	Distinguishing Features
Subarachnoid hemorrhage	—Fever not usually prominent feature —Can be distinguished by CT without contrast^a
Bacterial meningitis from other causes	—Meningitis not usually hemorrhagic as seen with anthrax meningitis —CSF Gram stain may be useful in diagnosis
Aseptic meningitis	—Meningitis not hemorrhagic —CSF does not show characteristic gram-positive bacilli —CSF usually demonstrates lymphocytosis
Encephalitis	—CSF findings may be variable, depending on etiology —CSF Gram stain may be useful in diagnosis
Abbreviations: CSF, cerebrospinal fluid; CT, computed tomography. ^aSee References: Dixon 1999.

Staging of Inhalational Anthrax

Historically, inhalational anthrax has been divided into a prodromal phase and a fulminant phase. The table below outlines a proposed staging scheme (see References: Lucey 2005) that adds an intermediate stage in which symptoms are clearly worsening but the illness may still be treated successfully.

Proposed Staging of Inhalational Anthrax
Stage	Comments
1: Asymptomatic	—Usually <1 wk after exposure and rarely >1 mo
2: Early—Prodromal	—Nonspecific malaise, myalgias, low-grade fever, mild headache, nausea, general "flu-like" prodromal illness
3: Intermediate—Progressive	—Blood cultures positive in <24 hr —Mediastinal lymphadenopathy —Pleural effusions that are often hemorrhagic and large and require repeated drainage —Findings may include: high fever, dyspnea, confusion or syncope, increasing nausea/vomiting —Patients at this stage can still be cured with antibiotics and intensive support
4: Late—Fulminant	—Respiratory failure requiring intubation, sepsis, meningitis, end-organ hypoperfusion (ie, "shock") —Cure less likely at this stage —Future therapies for this stage may require inhibitors of both anthrax toxin and systemic inflammatory response, in addition to antibiotics and intensive care
Adapted from Lucey 2005 (see References).

Distinguishing Inhalational Anthrax from Influenza-Like Illness (ILI) and Community-Acquired Pneumonia (CAP)

Early symptoms of inhalational anthrax, ILI, and CAP are similar and include fever, chills, myalgias, fatigue, malaise, and cough. However, several features can be used to distinguish these illnesses. One study of symptomatic patients with possible exposure to anthrax found that the presence of nonheadache neurologic symptoms (eg, dizziness, confusion), dyspnea, and upper gastrointestinal tract symptoms (eg, nausea, vomiting) were more suggestive of anthrax, whereas rhinorrhea and sore throat were far more common in patients with viral illnesses (see References: Hupert 2003).

Another study reviewed the CDC guidelines for inhalational anthrax during the 2001 outbreak and found that the guidelines would have missed 10 of the 11 cases (see References: Mayer 2003). The authors found that the modifications to the CDC guidelines shown below in italics would have led to recognition of 8 of the 11 cases.

Fever
Sweats
Fatigue
Cough
Chest discomfort, pleuritic pain
Nausea, vomiting
Headache
Dyspnea
Myalgias
Abdominal pain
Confusion
Fever (low grade: mean temperature, 38°C)
Tachycardia (mean heart rate, 121 beats per minute)
Clinical presentation consistent with inhalational anthrax when five or more of the above symptoms are present, in addition to tachycardia and fever

Howell and colleagues have suggested that use of this revised screening protocol may incur lower medical costs than the screening protocol proposed by Hupert and coworkers (outlined in the first paragragh of this section) and may be similar in its sensitivity to detect anthrax cases (although the numbers of anthrax cases in the comparison study were small) (see References: Howell 2004, Hupert 2003, Mayer 2003).

Another study examined the clinical features of the 2001 inhalational anthrax cases and compared them with those of ILI cases seen in an ambulatory clinic and of hospitalized patients with CAP. On the basis of these comparisons, the authors developed scoring systems for distinguishing ILI and CAP from inhalational anthrax (see References: Kuehnert 2003).

The scoring system for comparing inhalational anthrax with ILI included the following features. Patients with a score of 4 or more were more likely to have inhalational anthrax (sensitivity, 100%; specificity, 96.1%) than those with lower scores.

Low serum albumin (2 points)
Tachycardia (2 points)
No nasal symptoms (2 points)
No myalgias or arthralgias (1 point)
Low serum sodium level (1 point)
No headache (1 point)
High hematocrit or hemoglobin level (1 point)

The scoring system for comparing inhalational anthrax with CAP included the following features. Patients with a score of 2 or more were more likely to have inhalational anthrax (sensitivity, 100%; specificity, 48%) than those with lower scores. When the score was increased to 3 or more, the sensitivity dropped to 82% and the specificity increased to 81%.

Elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (1 point)
Low serum sodium level (1 point)
Normal white blood cell count (1 point)
Nausea and vomiting (1 point)
Tachycardia (1 point)

A third study, which compared 47 historical anthrax cases (including 11 with bioterrorism-related anthrax) with 376 controls with CAP or ILI, found that the most accurate predictor of anthrax was mediastinal widening or pleural effusion on chest radiograph (see References: Kyriacou 2004).

A report from the CDC included the following table, which compared the clinical features of 10 patients with inhalational anthrax to patients with laboratory-confirmed influenza.

Symptoms and Signs of Inhalational Anthrax, Laboratory-Confirmed Influenza, and Influenza-Like Illness (ILI) from Other Causes
	Inhalational Anthrax^a (%)	Laboratory-Confirmed Influenza (%)	ILI from Other Causes (%)
Elevated temperature	70	68-77	40-73
Fever or chills	100	83-90	75-89
Fatigue/malaise	100	75-94	62-94
Cough	90	84-93	72-80
Shortness of breath	80	6	6
Chest discomfort/pain	60	35	23
Headache	50	84-91	74-89
Myalgias	50	67-94	73-94
Sore throat	20	64-84	64-84
Rhinorrhea	10	79	68
Nausea or vomiting	80	12	12
Abdominal pain	30	22	22
^aN=10. Adapted from CDC 2001. Considerations for distinguishing influenza-like illness from inhalational anthrax (see References).

A literature review of 42 patients who had atypical anthrax presentations (ie, patients with confirmed anthrax infection who did not have known cutaneous, gastrointestinal, or inhalational ports of entry) revealed that these patients were significantly less likely to have cough, chest pain, or abnormal lung findings, even though they most likely had inhalational anthrax (see References: Holty 2006: Anthrax: a systematic review of atypical presentations).

A 2004 study used a decision-analytic model to assess the best treatment strategy for patients presenting with ILI in settings with varying probabilities for inhalational anthrax (see References: Fine 2004). The authors concluded that, for inhalational anthrax probabilities between 0.1% and 2%, when the sensitivity of blood culture exceeds 95%, the best strategy is to treat with a short course of empiric ciprofloxacin until blood culture results are available. During influenza season, the best strategy involves rapid testing for influenza followed by empiric treatment for anthrax pending blood culture results for those who test negative for influenza.

Pediatric Considerations

Reports in the literature support the following observations about anthrax in children.

Inhalational anthrax is uncommon in children. For example, none of the cases in the Sverdlovsk inhalational anthrax outbreak occurred in children (see References: Meselson 1994), and reports of inhalational disease among children are rare.
Naturally occurring cutaneous anthrax also is uncommon in children, probably because children have less opportunity for exposure to infected animals.
Other modes of transmission (such as person-to-person through skin-to-skin contact or transmission via fomites) may be more common for young children who acquire cutaneous anthrax (see References: Freedman 2002).
The skin lesions described for children who have cutaneous anthrax are usually similar to those seen in adults. Progression to severe systemic disease can occur (see References: Freedman 2002).
Anthrax meningitis has been reported in children and may be the presenting feature (see References: Rangel 1975, Tabatabaie 1993).
A review of 73 cases (5 inhalational, 22 gastrointestinal, 37 cutaneous, 6 primary meningoencephalitis, and 3 atypical) in children from 1900 to 2005 noted that children with inhalational anthrax lacked nonheadache neurologic symptoms, a key distinguishing finding (see References: Bravata 2007).
Another review of 62 pediatric cases of anthrax (2 inhalational, 20 gastrointestinal, 37 cutaneous, and 3 atypical) between 1966 and 2005 suggests that infected children may manifest different symptoms than do infected adults and that difficulties in diagnosing the disease in children may lead to delays in care (see References: AHRQ 2006). Children with gastrointestinal anthrax have two distinct clinical presentations, similar to adults; however, children with inhalational anthrax may have atypical presentations, including meningoencephalitis. Clinicians and public health officials must recognize the broad spectrum of potential presentations for timely diagnosis and detection.

Anthrax During Pregnancy

A report summarized two cases of anthrax in pregnant women; the features of these cases are outlined in the table below (see References: Kadanali 2003).

Summary of Clinical Features for Two Pregnant Women with Anthrax
Patient Age	Weeks of Gestation at Illness Onset	Clinical Presentation of Anthrax	Outcome of Pregnancy
33	32	Cutaneous anthrax (treated with penicillin G and prednisone)	—Infant delivered preterm at 35 wk —No evidence of congenital infection
29	33	Cutaneous anthrax (treated with procaine penicillin)	—Infant delivered preterm at 34 wk —No evidence of congenital infection

Clinical Laboratory Testing

Specimen Collection and Transport

When the diagnosis of anthrax is being considered, the hospital clinical laboratory should be alerted because some laboratories will not further identify Bacillus species unless specifically requested. The following table outlines the collection of laboratory specimens for diagnosis of anthrax.

Collection and Transport of Laboratory Specimens for the Diagnosis of Anthrax^a-d
Type of Illness	Specimen Collection^e and Transport	Comments^f,g,h
Cutaneous anthrax^g	—All stages: Collect 2 swabs, one for Gram stain and culture, and 1 for PCR. —Vesicular stage: Perform Gram stain, culture, and PCR of fluids from unroofed vesicle (soak two dry sterile swabs in vesicular fluid). Note: Gram stain is most sensitive during vesicular stage. —Eschar stage: Perform Gram stain, culture, and PCR of ulcer base or edge of eschar without removing it. —Ulcer (no vesicle or eschar present): swab base of ulcer with pre-moistened sterile saline —A punch biopsy for IHC testing and a second biopsy for culture, Gram stain, PCR, and frozen-tissue IHC if patient has not received antibiotics should be obtained on all patients with suspected cutaneous anthrax. Include skin adjacent to papule or vesicle. If vesicles and eschars are both present, separate biopsies should be obtained. —Serum: collect acute serum within first 7 days of symptom onset, and convalescent serum 14-35 days after symptom onset. —Collect blood for culture and PCR with evidence of systemic involvement.	—Swabs: Moisten with sterile saline or water; transport in sterile tube at at 2^o-8^oC. Transport swabs for PCR only at –70^oC. Do not use transport medium. —Tissue, fresh, >5 mm³: Store and transport at 2^o-8^oC (<2 hr) or frozen at –70^oC (>2 hr). —Tissue, preserved in 10% buffered formalin, 1.0 cm³: Store and transport at room temperature. —Biopsy of lesions for histopathology, preserved in 10% buffered formalin, 0.3 mm diameter: Store and transport at room temperature. —Freeze serum after separation at –20^oC or colder, ship on dry ice. Ship part of sample (>1.0 mL) and retain part in case of shipping problems.^h —Obtain blood for culture per local protocol. Collect blood for PCR in EDTA (purple top) tube. Ship at room temperature (<2 hr transport time) or 2-8^oC (>2 hr transport time).
Inhalational anthrax^g	—If sputum is being produced, collect sputum specimen for Gram stain and culture (note: inhalational anthrax does not usually result in sputum production). —Obtain blood for smear, culture, and PCR. —If a pleural effusion is present, collect a specimen for culture, Gram stain, and PCR. —Collect CSF if meningeal signs are present or meningitis is suspected for culture, Gram stain, and PCR. —Serum: Collect acute serum within first 7 days of symptom onset, and convalescent serum 14-35 days after symptom onset. —Biopsy material: Bronchial or pleural biopsy material can be evaluated if available.	—Sputum: transport at room temperature in sterile, screw-capped container (<1 hr transport time) or at 2^o-8^oC (>1 hr transport time). —Blood cultures: Obtain appropriate blood volume, number, and timing of sets per laboratory protocol; transport at room temperature. —Blood for PCR: 10 mL in EDTA (purple top) tube (for pediatric patients, collect volumes allowable). Transport directly to laboratory at room temperature (2-8^oC if transport > 2 hr). —Pleural fluid: Collect >1.0 mL in sterile container; store and transport at 2-8^oC. —CSF: Transport directly to laboratory at room temperature, or 2-8^oC if transport >2 hr. —Transport serum or citrated plasma (separated and removed from clot) at 2-8^oC (transport <2 hr) or freeze at –20^oC or colder (transport >2 hr); ship on dry ice. Ship part of sample (>1.0 mL) and retain part in case of shipping problems.^h —Preserve biopsies in 10% buffered formalin, and transport at room temperature.
Gastrointestinal anthrax^g	—Obtain stool specimen for culture (>5.0 g). —Obtain rectal swab from patients unable to produce stool (insert swab 1 in. beyond anal sphincter). —Obtain blood for smear and culture (and possibly PCR testing). —Blood cultures most likely to yield B anthracis if taken 2-8 days postexposure and prior to administration of antibiotics. —If ascites is present, obtain a specimen for Gram stain and culture (and possibly PCR testing).	—Stool: Transport in sterile container unpreserved (<1 hr transport time) or at 2^o-8^oC in Cary-Blair medium or equivalent (>1 hr transport time); specimen >5.0 g. —Blood: Transport at room temperature.
Anthrax meningitis	—Obtain CSF specimen for Gram stain, culture, and PCR. —Obtain blood for Gram stain, culture, and PCR.	—See comments above for collection and transport of blood and CSF for Gram stain, culture, and PCR.
Abbreviations: CSF, cerebrospinal fluid; EDTA, ethylenediaminetetraacetic acid; IHC, immunohistochemistry; PCR, polymerase chain reaction. ^aSee References: CDC/ASM/APHL 2002. ^bSee References: CDC 2001: Investigation of bioterrorism-related anthrax and interim guidelines for clinical evaluation of persons with possible anthrax. ^cSee References: Bell 2002. ^dSee References: CDC: Algorithm for laboratory diagnosis of anthrax. ^eSerologic testing may be useful in certain situations for retrospective diagnosis, since antibodies take several weeks to develop. Serum should be collected during the acute illness, and 14, 28, 42, and 60 days after onset (see References: CDC 2001: Clinical diagnosis and management of anthrax—lessons learned). ^fConsult with testing laboratory for specific instructions. ^gSee References: CDC: Recommended specimens for microbiology and pathology for diagnosis: inhalation, cutaneous, and gastrointestinal anthrax. ^hSee References: CDC. Anthrax: collecting, preparing, and shipping serum samples to CDC for serology testing.

Guidelines have been published for packing and shipping of infectious substances, diagnostic specimens, and biological agents from suspected acts of bioterrorism (see References: ASM 2008). Chain of custody should be documented for material that may constitute evidence of criminal activity.

B anthracis is classified under WHO risk group 4. Isolates that are reasonably suspected to contain B anthracis must be transported as "infectious substances." International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including infectious substances. Once B anthracis is identified, isolates and specimens are regulated as select agents and are subject to additional transport requirements (see below).

Laboratory Biosafety and Biosecurity Information

Most clinical and environmental screening can be conducted under biosafety level 2 (BSL-2) conditions. The B anthracis cells present in clinical samples are primarily vegetative, which are not easily transmitted to laboratory workers. However, most hospital laboratories are not sufficiently staffed, trained, or equipped for environmental testing. The most recent case of anthrax in a laboratory worker illustrates this potential risk (see References: CDC 2002: Suspected cutaneous anthrax in a laboratory worker—Texas, 2002).
Anthrax spores may remain viable after standard DNA purification procedures (see References: Rantakokko-Jalava 2003). Experimental heat treatment of anthrax spores at 121^oC for 45 minutes appears to eliminate viability without significantly affecting polymerase chain reaction (PCR) efficiency (see References: Fasanella 2003). Spores also can be irradiated with gamma rays to inactivate them without affecting PCR results (see References: Dauphin 2008).
Inadvertent exposure to B anthracis spores occurred in 2004 in a California laboratory when workers used a suspension from a contract laboratory that was supposed to contain nonviable vegetative cells but actually contained viable B anthracis spores (see References: CDC 2005: Inadvertent laboratory exposure to Bacillus anthracis—California, 2004). Following this report, the CDC stated that "Research laboratory workers should assume that all inactivated B anthracis suspension materials are infectious until inactivation is adequately confirmed."
Biosafety level 3 (BSL-3) practices are required for "production quantities or concentrations of cultures, or activities with a high potential for aerosols," including suspect powders.
BSL-2 and BSL-3 criteria and practices are described elsewhere (see References: CDC 2007).
B anthracis is classified as a select agent and therefore is regulated under 42 CFR part 73 (Possession, Use, and Transfer of Select Agents and Toxins), which was published in final form in the Federal Register in March 2005 (see References: HHS 2005). As specified in the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 CFR part 73 provides requirements for laboratories that handle select agents (including registration, security risk assessments, safety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications). These new requirements went into effect on February 7, 2003, and override earlier government requirements regarding possession and transfer of select agents. Select agents are biological agents designated by the US government to be major threats to public health and safety. A current list of select agents is published on the CDC Web site under information about the Select Agent Program (see References: CDC/APHIS 2008). In addition, the CDC has published additional guidelines for enhancing laboratory security for laboratories working with select agents (see References: CDC 2002: Laboratory security and emergency response guidance for laboratories working with select agents).

The Laboratory Response Network (LRN)

The LRN is a national network of approximately 150 laboratories. The network includes the following types of labs: federal, state and local public health, military, food testing, environmental, veterinary, and international (located in Canada, the United Kingdom, and Australia) (see References: CDC: Facts about the Laboratory Response Network, CDC: The Laboratory Response Network).

The LRN structure for bioterrorism designates laboratories as either national, reference, or sentinel. Designation depends on the types of tests a laboratory can perform and how it handles infectious agents to protect workers and the public.

National laboratories have unique resources to handle highly infectious agents and the ability to identify specific agent strains.
Reference laboratories, sometimes referred to as "confirmatory reference," can perform tests to detect and confirm the presence of a threat agent. These laboratories ensure a timely local response in the event of a terrorist incident. Rather than having to rely on confirmation from laboratories at the CDC, reference laboratories are capable of producing conclusive results; this allows local authorities to respond quickly to emergencies. These are mostly state or local public health laboratories with BSL-3 containment facilities that have been given access to nonpublic testing protocols and reagents.
Sentinel laboratories represent the thousands of hospital-based labs that are on the front lines. Sentinel laboratories have direct contact with patients. In an unannounced or covert terrorist attack, patients provide specimens during routine patient care. Sentinel laboratories could be the first facility to spot a suspicious specimen. A sentinel laboratory’s responsibility is to refer a suspicious sample to the right reference laboratory. These laboratories generally have at least BSL-2 containment.

Standard Tests for Detection of B anthracis

Direct examination of bacterial micromorphology may demonstrate broad encapsulated gram-positive rods (approved for LRN sentinel [formerly level A] laboratories). The laboratory must be notified that anthrax is being considered, because the laboratory may regard gram-positive rods as potential contaminants or not work them up beyond identifying them as Bacillus species.

Gram stain can be performed on appropriate clinical specimens (eg, vesicular fluid, swabs from cutaneous lesions, cerebrospinal fluid [CSF], pleural fluid) (see References: Bell 2002, Inglesby 2002). Two positive Gram stain findings, one from CSF and one buffy-coat smear, have been reported in cases of inhalational anthrax (see References: Jernigan 2001).
The sensitivity of the Gram stain and other biological stains (eg, H&E, silver stain) for direct detection of anthrax cells in infected tissues or blood has not been established. However, the organism appears to be present in large numbers in advanced cases, which suggests the probable utility of direct smears in those presenting with advanced disease (see References: CDC 2001: Clinical diagnosis and management of anthrax—lessons learned, Inglesby 2002). In cutaneous anthrax, B anthracis cells are most likely to be seen by Gram stain during the vesicular stage of disease (see References: CDC/ASM/APHL 2002).
Buffy-coat smears may be positive in patients with bacteremia. The specific usefulness of buffy coat for early diagnosis of bacteremia has been out of favor owing to early reports of low predictive value, but its use for anthrax has not been adequately studied (see References: Coppen 1981). PCR of buffy-coats has been successfully used for diagnosis of other bloodstream infections both before and after antibiotic treatment (see References: Michelow 2002, Newcombe 1996). The PCR detection limit in simulated blood specimens was 400 colony-forming units (CFU)/mL (see References: Rantakokko-Jalava 2003).
Endospores and free spores can be seen in cultures and generally are not seen in direct microscopic examination of patient samples (see References: CDC 2001: Clinical diagnosis and management of anthrax—lessons learned).
India ink staining (sentinel laboratories) or M'Fadyean stain (reference laboratories) can be performed for capsule visualization directly on clinical specimens (see References: CDC: Approved tests for the detection of Bacillus anthracis in the Laboratory Response Network).

Culture of clinical specimens is the "gold standard" for diagnosis of anthrax (initial isolation approved for LRN sentinel laboratories) (see References: CDC/ASM/APHL 2002, CDC: Approved tests for the detection of Bacillus anthracis in the Laboratory Response Network).

Culture should be performed using standard 5% SBA. After 15 to 24 hours of incubation at 35°C to 37°C, colonies are 2 to 5 mm in diameter, flat or slightly convex, irregularly round, and slightly irregular or have a wavy border and a ground-glass appearance. Colonies typically have a tenacious consistency (ie, teasing with an inoculating loop causes the colony to stand up like beaten egg white).
Motility is measured by wet mount (working in a BSL-2 cabinet) or by motility test medium.
Spores are visualized by Gram stain (sentinel laboratories), wet mount, or malachite green (reference laboratories) from cultures incubated for 18 to 24 hours at 35°C to 37°C without enhanced CO₂. Spores are oval, are central to subterminal, and do not appreciably swell the mother cell.
India ink for demonstration of capsule can be used on cultures grown on media supplemented with sodium bicarbonate.
Suspicious isolates should be forwarded to an LRN reference laboratory for confirmatory identification.
Blood culture sensitivity is compromised when samples are collected after administration of even one or two doses of antibiotics; therefore, cultures should be obtained before antibiotic therapy is initiated. If antibiotics have been administered, alternate diagnostic methods should be considered.

Stepwise Identification and Confirmation

Presumptive identification of anthrax in clinical specimens is based on Gram stain morphology and detection of a capsule (see References: CDC/ASM/APHL 2002). Note: The absence of evidence for a capsule does not rule out B anthracis.

Large gram-positive rods in short chains of two to four cells: Possible Bacillus species
India ink test positive (evidence of encapsulation): Presumptive B anthracis
Other tests conducted at LRN laboratories may provide additional direct evidence of anthrax; definitive identification is by culture

Standard identification of isolates is based on classical biochemical and morphologic characteristics (see References: CDC/ASM/APHL 2002, Sneath 1986). A stepwise protocol for identification of B anthracis is as follows:

Gram-positive broad spore-forming rods: Bacillus species
Spores are nonswelling and oval-shaped, and colonies have a ground-glass appearance: Bacillus morphology group 1 (includes B anthracis, B cereus, B thuringiensis, and B cereus var mycoides)
Nonmotile: B anthracis and B cereus var mycoides
Nonhemolytic: presumptive B anthracis (weak hemolysis may be observed under areas of confluent growth in aging colonies and should not be confused with beta-hemolysis)

Standard confirmatory testing (performed by LRN reference and national laboratories) includes the following tests (see References: CDC: Algorithm for laboratory diagnosis of anthrax, CDC: Approved tests for the detection of Bacillus anthracis in the Laboratory Response Network):

Lysis by gamma phage (provides confirmatory testing when demonstrated concomitantly with the presence of a capsule). Performance characteristics have been described in detail (see References: Abshire 2005).
Direct immunofluorescent assays (DFA) for cell-wall-associated polysaccharide and capsule produced by vegetative cells (demonstration of both antigens provides confirmatory identification) (see References: De 2002).
Additional test procedures for visualizing spores by LRN reference laboratories include wet mount and malachite green stain.
PCR-based assays for rapid identification of B anthracis:

A real-time PCR assay for rapid identification of B anthracis was evaluated by the CDC during the 2001 anthrax outbreak (see References: Hoffmaster 2002: Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis, Hoffmaster 2002: Supplement to above). Sensitivity and specificity were reported as 100%. PCR testing for anthrax is available through the LRN.
Real-time PCR assays capable of detecing multiple agents have been developed and are able to detect B anthracis, Francisella tularensis, and Yersinia pestis in a single assay without any cross-reactivity. The assay may prove useful as a rapid tool for detection of category A bioterrorism agents (see References: Skottman 2007). Commercial ready-to-use reagent systems have been developed for use in PCR assays (see References: Sohni 2008).
PCR primers that distinguish B anthracis from related Bacillus species have been developed. The primers may be useful for developing an efficient diagnostic tool for rapid identification (see References: Kim 2008). A restriction site insertion–PCR (RSI-PCR) method can distinguish B cereus group strains closely related to B anthracis from B anthracis strains (see References: Gierczynski 2007).

IHC is a sensitive and specific method for detection of B anthracis in affected tissues that uses antibody directed against cell-wall and capsule components. This test is unaffected by prior administration of antibiotics or formalin fixation (see References: Guarner 2003, Shieh 2003). IHC is not widely available, but requests for testing can be made through the LRN.
Molecular subtyping (multilocus variable-number tandem repeat analysis [MLVA]), is used by the CDC and others for strain identification and tracking (see References: CDC: Approved tests for the detection of Bacillus anthracis in the Laboratory Response Network, Hoffmaster 2002: Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States, Keim 1999, Keim 2000). Applications of single nucleotide polymorphism (SNP) and MLVA also have been used to discriminate closely related B anthracis isolates during outbreaks in animals. The combined SNP-MLVA analysis may hold promise for use in forensic investigations (see References: Kenefic 2008).

Antimicrobial susceptibility testing (performed at LRN reference laboratories) should be performed on clinical isolates.

National Committee for Clinical Laboratory Standards (NCCLS) standard protocols for broth microdilution have been used with staphylococcal breakpoints.
Concerns about penicillin use hinge on potential inducible penicillinases and poor penetration into macrophages (see References: Bell 2002).

Serologic testing can be used for retrospective diagnosis.

During the 2001 anthrax outbreak, the CDC developed, optimized, and qualified an enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to B anthracis PA (see References: Quinn 2002). The diagnostic sensitivity of the assay was 97.8% and the diagnostic specificity was 97.6%; a competitive inhibition anti-PA IgG ELISA enhanced the diagnostic specificity to 100%.
Serum should be collected at 0 to 7 days for acute-phase testing and at 14 to 28 days for convalescent-phase testing.
Development of measurable antibodies in recent cases required 10 to 16 days after onset of overt disease, but peak IgG levels may not be seen until 40 days after symptom onset (see References: Bell 2002).
Requests for serologic testing can be made through the LRN.
The Anthrax QuickELISA, a simplified lateral-flow immunochromatographic assay for anthrax antibody, has received approval from the Food and Drug Administration (FDA). The test, developed by Immunetics in collaboration with the CDC, has a diagnostic sensitivity and specificity of 100%, and is commercially available. Positive results on paired sera (-/+ or +/+) should be sent to the CDC for quantitative confirmation (see References: Biagini 2006, CDC 2004: CDC collaboration yields new test for anthrax. Stephenson 2004).

Other tests for detection of B anthracis infection:

A skin test for delayed-type hypersensitivity to anthrax antigen has been widely used in the former Soviet Union since 1962 for retrospective diagnosis of human and animal anthrax infection and for vaccine evaluation. An 81.5% positivity rate has been reported during the first 3 days of disease; this increases to 97% to 99% within the next 2 to 3 weeks. Anthraxin, as the product is called, is not available in the United States and has not been evaluated in this country (see References: Pfisterer 1991, Shlyakhov 1996, WHO 1998).

Antimicrobial Susceptibility Studies

Several studies have examined antimicrobial susceptibilities of B anthracis strains to various antibiotics. Data from four such studies are shown in the table below. Naturally occurring ciprofloxacin or doxycycline resistance has not been described, but isolates resistant to each of these antibiotics have been acquired in vitro (see References: Brook 2001, Price 2003). Because resistance can be induced relatively rapidly in vitro, close monitoring of patients treated for anthrax is important (see References: Athamna 2004).

MICs of Various Antibiotics for Bacillus anthracis Isolates as Identified in Four Studies
	Study^a
	Mohammed 2002		Coker 2002		Cavallo 2002		Turnbull 2004
Antibiotic	MIC range^b	S-I-R (%)^c	MIC range^b	S-I-R (#)^c	MIC range^b	S-I-R (%)^c	MIC range^d	S-I-R (%)^c
Amoxicillin					0.125-16	88.5-0-11.5
Amoxicillin–clavulanic acid							0.016-0.5	100-0-0
Azithromycin							1-12	26-64-10
Aztreonam					1->128	0-0-100
Cefaclor			0.125-0.75	25-0-0
Cefotaxime							3-32	1-0-99
Cefoxitin					1-64	74-15.3-10.7
Ceftriaxone	4-32	22-78-0			4-64	1-100-0
Cefuroxime			6-48	1-19-5
Cephalexin			0.38-2	25-0-0
Cephalothin					0.125-32	83.2-12.2-24.6
Chloramphenicol	2-8	100-0-0			1-4	100-0-0
Ciprofloxacin	0.03-0.12^e	100-0-0^e	0.032-0.38	25-0-0	0.03-0.5	100-0-0	0.032-0.094	100-0-0
Clindamycin	<0.5-1	94-6-0			0.125-1	100-0-0
Doxycycline			0.094-0.38	25-0-0	0.125-0.25	100-0-0
Erythromycin	0.5-1^f	3-97-0^f			0.5-4	95.4-4.6-0	0.5-4	15-85-0
Gatifloxacin					0.125-0.125	100-0-0
Gentamicin					0.125-0.5	100-0-0	0.064-0.5	100-0-0
Imipenem					0.125-2	100-0-0
Levofloxacin					0.03-1	100-0-0
Nalidixic acid					0.125-32	94.8-4.2-1
Ofloxacin					0.06-2	99-1-0
Penicillin	<0.06 -128	97-0-3	<0.016-0.5	22-0-3	0.125-16	88.5-0-11.5	<0.016->32	97-0-3
Pefloxacin					0.03-1	100-0-0
Piperacillin					0.25-32	99-1-0
Rifampin	<0.25-0.5	100-0-0			0.125-0.5	100-0-0
Streptomycin					0.5-2	100-0-0
Teicoplanin					0.125-0.5	100-0-0
Tetracycline	0.03-0.06	100-0-0					0.016-0.094	100-0-0
Tobramycin			0.25-1.5	25-0-0
Vancomycin	0.5-2	100-0-0			0.25-2	100-0-0	0.75-5	99-1-0
Abbreviation: MIC, minimum inhibitory concentration. ^aSee References: Cavallo 2002, Coker 2002, Mohammed 2002, Turnbull 2004. ^bMIC values in mcg/mL. ^cCategorical interpretation: susceptible (S), intermediate (I), and resistant (R); expressed as percent of isolates (%) or number of isolates (#), per author preference. (Note: Standard interpretive criteria for B anthracis have not been established.) ^dMIC determination by Etest. ^eA subset of 20 isolates were tested and found to be susceptible to clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin. ^fA subset of 20 isolates were also tested to clarithromycin (MIC range: 0.12-0.25 mcg/mL), azithromycin (MIC range: 1-2 mcg/mL), and doxycycline (MICs = <0.015 mcg/mL).

Multiple new antifolate compounds (multiple 2, 4-diaminopyrimidine derivatives, a class of compounds with dihydrophthalazine side chains) have demonstrated in vitro activity against trimethoprim (TMP)–resistant B anthracis and have the potential to become the basis of clinically important antibacterial therapies (see References: Barrow 2007).

Tests for Exposure

Nasal swab cultures were used in the 2001 US outbreak as an epidemiologic tool to assess exposure to inhalational anthrax. In one study of 625 persons potentially exposed to anthrax spores in the Hart Senate Building, nasal swabs were positive in 28 (4.5%) (see References: Hsu 2002).

All positive swabs were identified from persons who were in the immediate exposure area during the hour after the contaminated letter was opened. Seventy-one persons were in the exposure area during this time; therefore, the percentage of positive swabs among this group was 39% (28 of 71). Swabs from the 71 persons who were in the immediate area were obtained the day of exposure.
Swabs collected from persons other than those in the immediate area were collected 4 days after exposure; all were negative.
All exposed persons were placed on antimicrobial therapy, and repeat nasal swaps obtained 7 days later were negative. Results of serologic testing at 7, 21, and 42 days after exposure were negative for all exposed persons.

Another study at a worksite where a contaminated letter was opened demonstrated that two (<1%) of 1,076 nasal swabs taken from potentially exposed persons were positive; however, these swabs were obtained about 13 days after exposure (see References: Traeger 2002). Similarly, all nasal swabs collected 9 to 10 days after exposure from 3,110 postal employees at a Washington, DC, postal facility that handled contaminated mail were negative (see References: Dewan 2002). These findings, along with the findings from nasal swab testing of persons exposed in the Hart Senate Building, suggest that early collection of nasal swabs results in a higher yield of positive tests.

Because the sensitivity, specificity, and predictive value of nasal swab cultures are not known, nasal swabs are not recommended for use in the clinical setting. According to the CDC (see References: CDC 2001: Interim guidelines for investigation of and response to Bacillus anthracis exposures), collection of nasal swabs is not indicated to:

Diagnose anthrax
Determine risk of exposure and the need for antimicrobial prophylaxis
Determine when antimicrobial prophylaxis should be stopped
Supplement random environmental sampling

Although nasal swabs should not be used to determine the need for antimicrobial prophylaxis, if a swab is performed for some reason and is positive, then the patient should receive a course of postexposure antibiotics, since a positive nasal swab indicates exposure to aerosolized B anthracis (see References: Inglesby 2002). Methods for collection, labeling, transport, and processing of swabs have been published (see References: CDC/ASM/APHL 2002).

Serologic testing does not appear to be a useful tool for assessing asymptomatic exposure to B anthracis (see References: Dewan 2002, Hsu 2002, Traeger 2002).

Treatment and Postexposure Prophylaxis

Anthrax countermeasures include antibiotics (for treatment and postexposure prophylaxis [PEP]), antibodies, antitoxin agents, and vaccines. The current status of these countermeasures is summarized elsewhere (see References: Bouzianas 2009).

The tables below review current treatment recommendations for clinical disease caused by B anthracis.

Protocol for Treatment of Cutaneous Anthrax^a-c
Patient Category	Initial Therapy (Oral)^d	Duration^e
Adults	Ciprofloxacin: 500 mg PO twice daily or Doxycycline: 100 mg PO twice daily	60 days
Children	Ciprofloxacin: 10-15 mg/kg PO twice daily (maximum daily dose, 1 g) or Doxycycline^f: >8 yr and >45 kg: same as adult >8 yr and <45 kg: 2.2 mg/kg PO twice daily <8 yr: 2.2 mg/kg PO twice daily	60 days
Pregnant women^g	Same as for nonpregnant adults (high death rate from the infection outweighs risk posed by antimicrobial agent)	60 days
Immunocompromised persons	Same as for nonimmunocompromised persons and children	60 days
Abbreviation: PO, orally. ^aThese treatment recommendations were made during the US 2001 anthrax outbreak. In other settings, antimicrobial susceptibility testing should be used to guide therapy decisions. ^bCutaneous anthrax cases with signs of systemic involvement, extensive edema, or lesions on the head or neck require intravenous therapy, and a multidrug approach is recommended (see table below: Protocol for Treatment of Inhalational and Gastrointestinal Anthrax). ^cTreatment of cutaneous anthrax does not prevent the evolution of the skin lesions; however, it usually will prevent progression to systemic disease (see References: Inglesby 2002). ^dCiprofloxacin or doxycycline should be considered first-line therapy. Amoxicillin (500 mg orally 3 times daily for adults or 80 mg/kg/day divided every 8 hr for children) is an option for completion of therapy after clinical improvement. Oral amoxicillin dose is based on need to achieve appropriate minimum inhibitory concentration. ^eIn cases of naturally occurring cutaneous anthrax, previous recommendations have indicated that treatment for 7 to 10 days is adequate; however, in the setting where inhalational exposure also is likely, treatment should be continued for 60 days. ^fThe American Academy of Pediatrics recommends treatment of young children with tetracyclines for serious infections (eg, Rocky Mountain spotted fever). ^gAlthough tetracyclines and ciprofloxacin are not recommended for pregnant women, their use may be indicated for life-threatening illness. Adverse effects on developing teeth and bones are dose-related; therefore, doxycycline might be used for a short time (7-14 days) before 6 months of gestation. Adapted from CDC 2001: Investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001 (see References).

Protocol for Treatment of Inhalational and Gastrointestinal Anthrax^a
Patient Category	Initial IV Therapy^b,c	Oral Regimens (continue therapy for 60 days [IV and PO combined])
Adults	Ciprofloxacin: 400 mg twice daily or Doxycycline: 100 mg twice daily^e and One or two additional antimicrobials (agents with in vitro activity include rifampin, vancomycin, penicillin, ampicillin, chloramphenicol, imipenem, clindamycin, and clarithromycin)^f	Patients should be treated with IV therapy initially.^d Treatment can be switched to oral therapy when clinically appropriate: Ciprofloxacin: 500 mg PO twice daily or Doxycycline: 100 mg PO twice daily
Children	Ciprofloxacin: 10-15 mg/kg twice daily (maximum daily dose, 1 g)^g or Doxycycline^e,h: >8 yr and >45 kg: same as adult >8 yr and <45 kg: 2.2 mg/kg twice daily <8 yr: 2.2 mg/kg twice daily and One or two additional antimicrobials (see agents listed under therapy for adults)^f	Patients should be treated with IV therapy initially.^d Treatment can be switched to oral therapy when clinically appropriate: Ciprofloxacin: 10-15 mg/kg PO twice daily (maximum daily dose, 1 g) or Doxycycline^h: >8 yr and >45 kg: same as adult >8 yr and <45 kg: 2.2 mg/kg PO twice daily <8 yr: 2.2 mg/kg PO twice daily
Pregnant womenⁱ	Same as for nonpregnant adults (high death rate from the infection outweighs risk posed by antimicrobial agent)	Patients should be treated with IV therapy initially.^d Treatment can be switched to oral therapy when clinically appropriate. Oral therapy regimens are the same as for nonpregnant adults.
Immunocompromised persons	Same as for nonimmunocompromised persons and children.	Same as for nonimmunocompromised persons and children.
Abbreviations: IV, intravenously; PO, orally. ^aMeningitis involvement must be assumed in all systemic infections. Antibiotic selection must consider penetration across blood-brain barrier (see References: Stern 2008). These treatment recommendations were made during US 2001 anthrax outbreak. In other situations, antimicrobial susceptibility testing should be used to guide therapy decisions. ^bCiprofloxacin or doxycycline should be considered an essential part of first-line therapy for inhalational anthrax. ^cSteroids may be considered an adjunct therapy for patients with severe edema (see References: Doust 1968) and for meningitis based on experience with bacterial meningitis of other causes. ^dInitial therapy may be altered based on clinical course of patient; one or two antimicrobial agents (eg, ciprofloxacin or doxycycline) may be adequate as patient improves. ^eIf meningitis is suspected, doxycycline may be less optimal because of poor central nervous system penetration. ^fBecause of concerns of constitutive and inducible beta-lactamases in B anthracis isolates, penicillin and ampicillin should not be used alone. Consultation with an infectious disease specialist is advised. Other agents with in vitro activity include tetracycline, linezolid, macrolides, aminoglycosides, and cefazolin (see References: Inglesby 2002). B anthracis strains are naturally resistant to sulfamethoxazole, trimethoprim, cefuroxime, cefotaxime sodium, aztreonam, and ceftazidime (see References: Inglesby 2002). ^gIf IV ciprofloxacin is not available, oral ciprofloxacin may be acceptable because it is rapidly and well absorbed from gastrointestinal tract with no substantial loss by first-pass metabolism. Maximum serum concentrations are attained 1-2 hr after oral dosing but may not be achieved if vomiting or ileus is present. ^hThe American Academy of Pediatrics recommends treatment of young children with tetracyclines for serious infections (eg, Rocky Mountain spotted fever). ⁱAlthough tetracyclines are not recommended for pregnant women, their use may be indicated for life-threatening illness. Adverse effects on developing teeth and bones are dose-related; therefore, doxycycline might be used for a short time (7-14 days) before 6 months of gestation. Adapted from CDC 2001: Investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001, Stern 2008 (see References).

A systematic review of inhalational anthrax cases identified between 1900 and 2005 reported the following observations with regard to treatment (see References: Holty 2006: Systematic review: a century of inhalational anthrax cases from 1900 to 2005).

Initiation of therapy with antibiotics or anthrax antiserum during the prodromal phase was associated with improved survival.
Patients who progressed to the fulminant phase, regardless of therapy, had a very high case-fatality rate (97%).
Multidrug antibiotic therapy and pleural fluid drainage were associated with decreased mortality; however, since these modalities were predominantly used during the 2001 US anthrax outbreak, other confounding factors (eg, differences in patient characteristics, anthrax exposure, supportive care, antibiotic efficacy) may have contributed to enhanced overall survival.

Because mortality for inhalational anthrax remains high despite use of antibiotics, potential adjuvant therapies are being studied. Examples include gamma and alpha/beta interferon and adefovir (see References: Gold 2004, Shen 2004). As noted above, drainage of pleural fluid (through repeated thoracentesis or chest tube drainage) may enhance survival in cases of inhalational anthrax (see References: Holty 2006: Systematic review: a century of inhalational anthrax cases from 1900 to 2005).

Raxibacumab is a human monoclonal antibody directed against PA. The efficacy of raxibacumab for the treatment of inhalational anthrax has been evaluated in rabbits and monkeys. Following inhalational challenge, the survival rate was significantly higher among rabbits that received a 40 mg/kg dose of raxibacumab (44%; 8 of 18) than among rabbits that received placebo (0%; 0 of 18). Treated monkeys also had significantly increased survival (64%; 9 of 14) compared with untreated monkeys (0%, 0 of 12) (see References: Migone 2009).

Treatment of Anthrax Meningitis

Anthrax meningitis is treated in similar fashion to inhalational anthrax, although initial treatment should include an intravenous (IV) fluoroquinolone and not doxycycline, because doxycycline has poor central nervous system (CNS) penetration. In addition to an IV fluoroquinolone, one or two other agents that have good CNS penetration and activity against B anthracis should be added (eg, penicillin, ampicillin, meropenem, vancomycin, rifampin) (see References: Sejvar 2005). Case reports suggest that adding corticosteroids may be of benefit in the management of cerebral edema/inflammation (see References: Sejvar 2005).

The optimal duration of therapy is not known, but treatment should be continued for 10 to 14 days or as long as is clinically indicated.

Postexposure Prophylaxis

The CDC currently recommends 60 days of oral antimicrobial therapy in combination with a three-dose series of anthrax vaccine adsorbed (AVA) for PEP following potential inhalational exposure to aerosolized B anthracis spores (see References: Stern 2008).

Antimicrobial therapy should be continued for at least 60 days for the following persons:

Those exposed to an air space known to be contaminated with aerosolized B anthracis
Those exposed to an air space known to be the source of an inhalational anthrax case
Those along the transit path of an envelope or other vehicle containing B anthracis that may have been aerosolized (eg, a postal sorting facility in which an envelope containing B anthracis was processed)
Unvaccinated laboratory workers exposed to confirmed B anthracis cultures in situations where aerosolization is suspected

Antimicrobial prophylaxis is not indicated for the following:

Prevention of cutaneous anthrax
Autopsy personnel examining bodies infected with anthrax when appropriate isolation precautions and procedures are followed
Hospital personnel caring for patients with anthrax
Persons who routinely open or handle mail (in the absence of a suspicious letter or credible threat)

The decision to prescribe PEP to asymptomatic persons in the setting of an outbreak of inhalational anthrax should be based on the likelihood of exposure and not on nasal swab testing (see the Clinical Laboratory Testing: Tests for Exposure section above).

In the event of a mass exposure, local and state public health agencies would rapidly make antibiotics available to the exposed population (see the section below on Mass Exposure Events).

Since experience with gastrointestinal anthrax is limited, currently there are no recommendations for using PEP in the setting of gastrointestinal exposure, such as in a foodborne outbreak or intentional contaminaton of a food source. However, if public health officials determine that the risk of B anthracis infection is high, it may be reasonable to consider using PEP in the setting of gastrointestinal exposure (see References: CDC 2000: Human ingestion of Bacillus anthracis-contaminated meat—Minnesota, August 2000).

The FDA has approved several antimicrobial agents for use as anthrax PEP (see References: FDA 2001, FDA: Levaquin [levofloxacin] information for inhalational anthrax, Meyerhoff 2004).

Ciprofloxacin
Doxycycline
Penicillin G procaine
Levofloxacin

Analysis of published reports suggests that development of antibiotic resistance may be less likely to occur with doxycycline than with fluoroquinolones. In addition, doxycycline is several times less expensive than most fluoroquinolones and appears in clinical studies to have similar efficacy in most scenarios (see References: Brouillard 2006).

Other antimicrobial agents, including clindamycin, chloramphenical, rifampin, vancomycin, and other fluoroquinolones, may be considered for off-label use in patients unable to tolerate FDA-approved antimicrobial agents for PEP (see References: Stern 2008). Athamna and colleagues found that the combination of rifampin and clindamycin demonstrated a synergistic effect in vitro against two strains of B anthracis (see References: Athamna 2005). A number of other combinations were either indifferent or antagonistic.

The CDC recommendations for PEP to prevent inhalational anthrax (those issued during the 2001 bioterrorism anthrax attack as well as later modifications) are outlined in the table below.

Initial CDC Recommendations for PEP for Prevention of Inhalational Anthrax Following Exposure to Bacillus anthracis
Patient Category	Initial Therapy	Duration^a
Adults (including immunocompromised patients)	Ciprofloxacin: 500 mg PO twice daily or Doxycycline: 100 mg PO twice daily or Levofloxacin: 500 mg PO once daily [Note: Levofloxacin is FDA-approved for PEP for inhalational anthrax in adults >18 years; however, data on the safety of using levofloxacin beyond 28 days are limited. Therefore, levofloxacin is recommended as a second-line PEP agent, to be reserved for instances in which medical issues call for its use (see References: FDA: Levaquin [levofloxacin] information for inhalational anthrax, Stern 2008).]	60 days
Pregnant women and breastfeeding mothers	Ciprofloxacin: 500 mg PO twice daily (first-line oral agent for PEP in pregnant women.) or Doxycycline: 100 mg PO twice daily (In pregnant women, doxycycline should be used only during the third trimester.) [Note: Amoxicillin, 500 mg orally three times daily, may be used if isolate involved in exposure is determined to be susceptible to penicillin.^b-d]	60 days
Children (including immunocompromised patients)	Ciprofloxacin: 10-15 mg/kg PO twice daily (maximum daily dose, 1 g) or Doxycycline: >8 yr and >45 kg: same as adult >8 yr and <45 kg: 2.2 mg/kg PO twice daily <8 yr: 2.2 mg/kg PO twice daily [Note: Amoxicillin, 80 mg/kg/day divided every 8 hr not to exceed 500 mg/dose, may be used if the isolate involved in exposure is determined to be susceptible to penicillin^c] or Levofloxacin: 500 mg PO once daily for children >50 kg, or 8 mg/kg twice daily (not to exceed 250 mg per dose) for children <50 kg. [Note: In May 2008, the FDA approved the use of levofloxacin for PEP for inhalational anthrax in children. As noted above for adults, data on the safety of using levofloxacin beyond 28 days are limited. In addition, levofloxacin may cause an increase in musculoskeletal adverse events in children (see References: FDA: Levaquin [levofloxacin] information for inhalational anthrax).]	60 days
Abbreviation: FDA, Food and Drug Administration; PEP, postexposure prophylaxis; PO, orally. ^aRecommended in combination with a 3-dose series of anthrax vaccine adsorbed (AVA) (BioThrax [BioPort Corp, Lansing, MI]) administered at time zero, 2 weeks, and 4 weeks. AVA is not FDA-approved for PEP and therefore would be available under an Investigational New Drug (IND) protocol (see References: Stern 2008). After the 2001 attack, exposed persons were subsequently given the option to take an additional 40 days of antibiotics with or without anthrax vaccine; see comments below. Other antimicrobial agents, including clindamycin, chloramphenical, rifampin, vancomycin, and other fluoroquinolones, may be considered for off-label use in patients unable to tolerate FDA-approved antimicrobial agents for PEP (see References: Stern 2008). ^bSee comments below from American College of Obstetricians and Gynecologists regarding use of amoxicillin. ^cAmoxicillin is not approved by the FDA for PEP or treatment of anthrax; however, the CDC has indicated that it could be used for pregnant women or children for PEP if the isolate is determined to be susceptible. In such situations, amoxicillin could be provided under an IND or under an Emergency Use Authorization in a declared emergency (see References: CDC 2001: Interim recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax, CDC 2001: Updated recommendations for antimicrobial prophylaxis among asymptomatic pregnant women after exposure to Bacillus anthracis, Stern 2008). A study to evaluate the pharmacokinetics of amoxicillin during pregnancy and postpartum found that drug concentrations adequate to prevent anthrax may be difficult to achieve during pregnancy and in the postpartum period (see References: Andrew 2007). Amoxicillin given to pediatric patients <40 kg should yield adequate time above the minimum inhibitory concentration for susceptible B anthracis (0.5 mcg/mL) over most of the dosing interval (see References: Alexander 2008) ^dThe American Academy of Pediatrics considers ciprofloxacin and tetracyclines to usually be compatible with breastfeeding because the amount of either drug absorbed by infants is small, but little is known about the safety of long-term use. Therefore, amoxicillin may be considered an alternative for breastfeeding mothers if the isolate causing exposure is known to be susceptible to penicillin. Alternatively, mothers could consider expressing and discarding breast milk during therapy with ciprofloxacin or doxycycline and resuming breastfeeding after therapy is complete. Adapted from CDC 2001: Investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001, Stern 2008 (see References).

More than 10,000 people were placed on PEP during the 2001 anthrax outbreak; no cases of anthrax occurred among this group (see References: CDC 2001: CDC responds: an update on treatment options for postal and other workers exposed to anthrax).

A follow-up study of those persons who were offered antimicrobial prophylaxis demonstrated that 5,343 took at least one dose of antimicrobial therapy (see References: Shepard 2002). Of this group, 3,032 (57%) reported adverse events during the first 60 days of therapy. Gastrointestinal complaints (nausea, vomiting, diarrhea, stomach pain) were reported by 44% of those with adverse events and neurologic symptoms (headache, dizziness, light-headedness, fainting, and seizures) were reported by 33%. Fewer than half of respondents (2,712 [44%]) reported taking antimicrobial prophylaxis for at least 60 days. Of the 2,631 persons who stopped therapy before 60 days, 43% stopped because of adverse events, 25% stopped because of a low perceived risk of anthrax, and 7% stopped because of concern about long-term side effects of prolonged antimicrobial therapy.
A statistical model was used to estimate the number of anthrax cases prevented among about 5,000 persons placed on prophylaxis who were potentially exposed to airborne anthrax spores at one of three locations (the media center in Florida, the two postal facilities in New Jersey, and the postal facility in Washington, DC). The model suggested that about nine cases were prevented through the use of postexposure antibiotics (see References: Brookmeyer 2002).
Another model-based study explored the impact of initial response time, anthrax incubation period, and antibiotic effectiveness on hospital surge after a large-scale release of anthrax spores over a major urban area. If an antibiotic prophylaxis campaign was begun within 2 days after the exposure event and completed within 48 hours, approximately 87% of exposed persons would be protected from illness (assuming a 95% attack rate and 90% antibiotic effectiveness). On average, each additional day of delay in initiating the campaign (beyond 2 days) would result in 5.2% to 6.5% additional hospitalizations in the exposed population, whereas every additional day needed to complete the campaign would result in 2.4% to 2.9% additional hospitalizations. The authors concluded that commencement of the prophylaxis campaign (no more than 3 days) and antibiotic effectiveness (greater than 90%) are the parameters with the greatest preventive impact (see References: Hupert 2009).

The American College of Obstetricians and Gynecologists (ACOG) has recommended the following for anthrax prophylaxis in pregnant women (see References: ACOG):

Prophylactic treatment should be limited to women who have been exposed to confirmed environmental contamination or a high-risk source as determined by public health authorities.
Pregnant women who have been exposed to anthrax should be started on a 60-day course of ciprofloxacin.
Therapy should be switched to amoxicillin if the strain is found to be penicillin-sensitive.

According to ACOG, doxycycline use in pregnant women generally should be avoided because it can cause problems in fetuses, including staining of teeth and impeded bone growth; however, doxycycline should be used for exposed pregnant women who are allergic to amoxicillin and ciprofloxacin, since the risk of anthrax outweighs any potential risks to the fetus from doxycycline.

A national poll conducted by the Harvard School of Public Health in December 2009 revealed that 89% of adults probably would follow public health recommendations to obtain prophylactic antibiotics at a dispensing site. However, of those, only 57% said they would start taking the antibiotics immediately, while 39% said they would wait before taking them (in most cases to see if they had been exposed). In response to a fictional scenario of an anthrax attack, more than 80% of adults said they would be worried about becoming seriously ill or dying (see Feb 19, 2010, CIDRAP News story).

New Therapeutic Approaches

In addition to available treatment protocols, a variety of promising new therapeutic approaches for treatment of anthrax are being researched; many involve use of monoclonal antibodies (see References: Borio 2005, Bouzianas 2009, Migone 2009). An alternative to monoclonal antibodies is antisera from previously vaccinated persons undergoing serial plasmapheresis. Hyperimmune plasma and immune globulin isolated in this way could potentially serve as the basis for new therapeutic treatments (see References: Pittman 2006).

The Department of Health and Human Services (HHS) awarded a contract to Human Genome Sciences, Inc (HGS) of Rockville, Maryland, to provide the US government with 10 grams of ABthrax (raxibacumab), a human monoclonal antibody for treating anthrax (see Dec 19, 2005, CIDRAP News story). In June 2006, HHS announced that it would purchase 20,000 treatment courses of ABthrax (see References: HHS 2006: HHS to acquire new anthrax therapeutic treatment for stockpile). In February 2009, HGS began delivery of the first 20,000 doses of ABThrax (see References: HGS 2009).
Similarly, HHS has contracted with Cangene (a company based in Winnipeg, Manitoba) to supply anthrax immune globulin (AIG) for preliminary efficacy testing. The company describes AIG as a hyperimmune product for treating or preventing inhalational anthrax. In July 2006, HHS announced that it will purchase 10,000 treatment courses of AIG from Cangene (see References: HHS 2006: HHS to acquire anthrax immune globulin for stockpile).

Anthrax Vaccination

Current Anthrax Vaccine

AVA (BioThrax), manufactured by BioPort, a subsidiary of Emergent BioSolutions, Inc in Lansing, Michigan, is the only licensed human anthrax vaccine in the United States (see References: Emergent BioSolutions: BioThrax package insert).
Most AVA vaccine has been used by the US military; it is not available to the general public (except through public health officials during an anthrax emergency as part of the Strategic National Stockpile).
AVA is prepared from cell-free filtrates of microaerophilic cultures of an avirulent, nonencapsulated strain of B anthracis. The final product contains no live or dead bacteria.
The final product is formulated to contain 1.2 mg/mL aluminum, added as aluminum hydroxide in 0.85% sodium chloride.
The final product is formulated to contain 25 mcg/mL benzethonium chloride and 100 mcg/mL formaldehyde, added as preservatives.

Vaccine Efficacy

Randomized controlled trials on the clinical effectiveness, immunogenicity, and safety of anthrax vaccines were recently reviewed. The authors concluded that vaccines based on anthrax antigens are immunogenic in most vaccinees with few adverse events, although limited data were available (see References: Donegan 2009).

The efficacy of AVA is based on several animal studies, one controlled human trial, and immunogenicity data from humans and other mammals (see References: Brachman 1962, Gladstone 1946, Mahlandt 1966, Turnbull 1986). One study demonstrated that vaccination of adults induced an immune response in 83% of vaccinees 2 weeks after the first dose and in 91% of vaccinees who received two or more doses (see References: Turnbull 1986). Approximately 95% of vaccinees seroconverted after three doses. However, the correlation between antibody titer and protection against infection has not been defined.
Analysis of serum from vaccinees and clinical anthrax patients shows that vaccination with three AVA injections and anthrax infection both elicited anti-PA, IgG1, IgG2, and IgG3 subclass responses (see References: Semenova 2007). One study suggests that AVA-induced antibodies to PA are sufficient to neutralize toxin activity and that AVA-induced LF and EF antibodies do not contribute to anthrax toxin neutralization in humans (see References: Taft 2008).
Duration of efficacy is unknown, although data from animal studies suggest that it may be 1 to 2 years after two doses. Studies of military personnel vaccinated during the 1990-1991 Gulf War indicate that antigen-specific T-cell recall responses present in the circulation are comparable in magnitude to those of tetanus-diphtheria toxoids (see References: Allen 2006).
Anthrax vaccines intended for animals should not be used in humans.

Recommendations for Use

Preexposure vaccination

Preexposure immunization with AVA is licensed for individuals aged 18 to 65 who have a high likelihood of coming into contact with B anthracis (see References: CDC 2010: Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices [ACIP], 2009). The vaccine is not licensed for pregnant women. The vaccine also is not licensed for children because no studies have been conducted in the pediatric population; however, the vaccine probably is safe and efficacious in children, based on experience with other inactive vaccines (see References: Inglesby 2002).

Preexposure vaccination is indicated for the following groups (see References: CDC 2010: Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices [ACIP], 2009):

Laboratory personnel engaged in work involving production of quantities or concentrations of B anthracis cultures
Laboratory personnel handling environmental specimens (especially powders) and performing confirmatory testing for B anthracis in LRN reference and national laboratories
Workers who will be making repeated entries into known B anthracis-spore-contaminated areas after a terrorist attack
Persons who come in contact in the workplace with imported animal hides, furs, bone meal, wool, animal hair, or bristles in areas where current standards and restrictions are insufficient to prevent exposure to anthrax spores
Veterinarians and other high-risk persons handling potentially infected animals in areas of the world that have a high incidence of anthrax cases (Note: The incidence of anthrax in the United States is low; therefore, routine vaccination of US veterinarians is not recommended)
Military personnel and other select populations who have a risk for exposure to weaponized B anthracis. After a lapse due to legal action, the DoD has resumed mandatory vaccinations for military personnel, essential DoD civilians, contractors stationed in the Middle East and South Korea, and units involved in homeland bioterrorism defense (see References: DoD 2006, and see Oct 19, 2006, CIDRAP News story).

Laboratory workers using standard BSL-2 practices are not considered by the Advisory Committee on Immunization Practices (ACIP) to be at increased risk for exposure to B anthracis spores.

Postexposure vaccination

ACIP guidelines state that anthrax vaccine can be used in combination with antibiotics following inhalational exposure to anthrax (see References: CDC 2010: Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices [ACIP], 2009):

Exposed persons should receive a three-dose regimen of vaccine subcutaneously (0, 2, and 4 weeks following exposure) and at least a 60-day course of antimicrobial therapy. Persons who do not receive vaccine also should take antimicrobial therapy for at least 60 days.
Anthrax vaccine is not currently licensed for postexposure use, so it must be given under an investigational new drug (IND) application with the FDA.

Due to problems of noncompliance and side effects associated with prolonged antibiotic treatment, investigators have studied whether a short course of antibiotics combined with vaccination could provide protection after exposure. Monkeys were exposed to an aerosol dose (1,600 LD₅₀) of B anthracis spores (see References: Vietri 2006). One group of animals received only ciprofloxacin (an initial loading dose of 250 mg of ciprofloxacin within 2 hours after exposure followed by 125 mg twice daily for 14 days). Another group of animals received ciprofloxacin (as described) and was vaccinated (0.5 ml of AVA on day 0 within 2 hours after exposure, and on days 14 and 28). In contrast to the untreated control group, all animals in both the ciprofloxacin-only and the ciprofloxacin-plus-vaccine groups survived during the 14 days of antibiotic prophylaxis. However, once the antibiotics were discontinued, only four of nine animals (44%) in the ciprofloxacin-only group survived compared with 10 of 10 animals (100%) in the ciprofloxacin-plus-vaccine group. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax.

Dosage, Route of Administration, and Vaccination Schedule

Each vaccine dose is 0.5 mL. The current vaccination schedule consists of a primary series of two intramuscular injections followed by three booster intramuscular injections as outlined in the table below. In December 2008, the FDA approved a change in the primary series from three doses subcutaneously (0, 2, and 4 weeks) to two doses intramuscularly (0 and 4 weeks). The ACIP supported this change in February 2009 (see Feb 25, 2009, CIDRAP News story). The change is based on a randomized, phase 4 trial, which found that a three-dose intramuscular schedule provided similar immunologic priming by month 7 compared with a four-dose subcutaneous regimen. An intramuscular route also significantly reduced the occurrence of injection-site adverse events (see References: Marano 2008).

To maintain immunity among those with anticipated ongoing exposure, the manufacturer recommends an annual booster injection using the same dosage and route after completion of the primary and booster series.

Schedule for Anthrax Vaccination^a
Primary series (2 injections)	Week 0 Week 4
Booster series (3 injections)^b	6 mo after primary series 12 mo after primary series 18 mo after primary series
^aAll doses are 0.5 mL and should be given intramuscularly. ^bAdditional boosters to be given annually (same route and dose) for those at ongoing risk of exposure.

Contraindications and Precautions

The main contraindication is a history of anaphylaxis after receiving a dose of AVA or any of the vaccine components.
History of anthrax disease may increase the potential for severe local adverse reactions.
If AVA is administered to immunocompromised persons, including those receiving immunosuppressive therapy, the immune response may be diminished.
AVA is labeled as Pregnancy Category D (see References: Emergent BioSolutions: BioThrax package insert). According to the manufacturer's package insert, "Pregnant women should not be vaccinated against anthrax unless the potential benefits of vaccination have been determined to outweigh the potential risk to the fetus."
Few studies have been published regarding the use of anthrax vaccine in pregnant women.

A study among US army women demonstrated that anthrax vaccine had no effect on subsequent pregnancies, birth rates, or adverse birth outcomes, although the power to detect adverse birth outcomes in the cohort was limited (see References: Wiesen 2002).
A retrospective cohort study evaluated birth defects in relation to maternal anthrax vaccination for infants born to US military service women from 1998 through 2004. Among 115,169 infants born to military women during this period, 37,140 were born to women ever vaccinated, and 3,465 were born to women vaccinated in the first trimester of pregnancy. Birth defects were slightly more common among infants born to women vaccinated during the first trimester compared with infants born to women vaccinated beyond the first trimester; however, this finding was not statistically significant. An analysis of infants born to women vaccinated in the first trimester and infants born to women who were never vaccinated found that birth defects were slightly higher (odds ratio = 1.2; 95% confidence interval = 1.02 to 1.42) in the vaccinated group (see References: Ryan 2008).

Adverse Reactions

Injection site adverse reactions are the most common and occur in more than half of vaccine recipients. These include warmth, tenderness, itching, erythema, induration, edema, and nodule formation. Most local adverse reactions are mild or moderate in severity.
Systemic reactions (fever, chills, myalgia, nausea) following vaccination may occur in 10% to 25% of recipients.
One report identified five cases of new-onset rheumatoid arthritis (RA) temporally related to anthrax vaccine. The most recent occurred in a 42-year-old man who experienced knee and interphalangeal joint stiffness and pain 5 days after receiving the first dose of anthrax vaccine. Serologic and radiologic examinations revealed mild degenerative changes in his hands and knees bilaterally (see References: Vasudev 2006). It is unclear at this time whether or not these case reports represent a true association between anthrax vaccination and RA, since this issue has not been systematically evaluated.

Postlicensure Adverse Event Reporting

Between 1998 and 2008, the Vaccine Adverse Event Reporting System (VAERS) received 6,015 reports of adverse events following anthrax vaccination (see References: CDC 2010: Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices[ACIP], 2009). The most frequent adverse events that occurred were arthralgia (n = 1,036, 17%), headache (981, 16%), pruritus (878, 15%), pain (824, 14%), injection-site erythema (753, 13%), fever (655, 11%), erythema (626, 10%), injection-site pain (613, 10%), rash (606, 10%), and myalgia (583, 10%). A total of 600 (10%) serious adverse events were reported.
Review of anthrax vaccination data from a 1967 to 1972 study and medical literature from 1955 to 2005 suggests that women have at least twice the risk of having a vaccine reaction compared with men. The age-adjusted relative risk for injection site reaction was 2.78 (95% confidence interval, 2.29 to 3.38) (see References: McNeil 2007: Short-term reactogenicity and gender effect of anthrax vaccine: analysis of a 1967-1972 study and review of the 1955-2005 medical literature).
Between December 15, 1997, and April 12, 2000, 425,976 service members received 1,620,793 doses of AVA. As of April 7, 2000, 428 VAERS reports had been received through the DoD (see References: CDC 2000: Surveillance for adverse events associated with anthrax vaccine—US Department of Defense, 1998-2000). Of these reports, 311 (72.7%) involved systemic reactions, 78 (18.2%) were mild or moderate local reactions, and 39 (9.1%) were large or complicated local reactions. As of March 21, 2000, a panel of civilian scientific and medical experts had not identified any pattern of adverse events among 674 reports that had been reviewed. Comparative analysis of immunization records in the military VAERS reports and the Defense Medical Surveillance System suggests that the information contained in both sets of records is similar for anthrax and for nonanthrax vaccine immunizations (see References: McNeil 2007: A comparative assessment of immunization records in the Defense Medical Surveillance System and the Vaccine Adverse Event Reporting System).
Some reports have suggested an association between optic neuritis and anthrax vaccination (or receipt of other vaccines). However, a matched case-control study among US military personnel from Jan 1, 1998, through Dec 31, 2003, revealed no significant associations between optic neuritis and anthrax vaccination (see References: Payne 2006).

The Institute of Medicine's Committee to Assess the Safety and Efficacy of Anthrax Vaccine found no evidence that people receiving the anthrax vaccine are at increased risk of life-threatening or permanently disabling adverse events. An FDA review of VAERS reports also found no causal relationship between anthrax vaccination deaths and other serious adverse events (other than some serious injection site and allergic reactions). Similarly, the HHS Anthrax Vaccine Expert Committee concluded that there was not a high frequency or an unusual pattern of serious or other medically important adverse events. A recent review of 4,753 anthrax vaccine–related VAERS reports from January 1, 1990 through January 16, 2007 confirmed these previous findings (see References: Niu 2009).

Development of New Vaccines

Research into new anthrax vaccines is ongoing. Most vaccines that have been studied utilize recombinant technology or employ new adjuvants to increase the immune response.

Current human anthrax vaccines consist of supernatant material from cultures of a toxigenic, nonencapsulated strain of B anthracis. Second-generation vaccines using recombinant PA (rPA) are being developed as an alternative, as much of the immunogenicity of the current vaccine arises from this protein component (see References: Rhie 2005).
Emergent BioSolutions (maker of the current anthrax vaccine) is studying a new candidate vaccine (AV7909), which comprises AVA in combination with the immunostimulatory compound CPG 7909 as an adjuvant (see References: Emergent BioSolutions 2008).
One study suggests that oral, live, attenuated, spore-based vaccines could offer long-lasting protection against anthrax in humans (see References: Aloni-Grinstein 2005). The investigators administered a previously described live, attenuated, nontoxinogenic, nonencapsulated B anthracis spore vaccine orally to guinea pigs and found that protective immunity correlated with development of a threshold level of PA neutralizing antibody titers. Protective immunity also appeared to be long-lasting.
Several animal studies have shown that the presence of EF in vaccine preparations can promote protective immunity against anthrax, and inclusion of EF in multicomponent subunit vaccines is a consideration (see References: Quesnel-Hellmann 2006, Zeng 2006).
Combination vaccines such as one against anthrax and plague may represent the next generation of vaccines against potential agents of bioterrorism (see References: DuBois 2007).

Management of Exposure Events

Potential Types of Exposures

The most likely methods of exposure to weaponized anthrax spores include:

Localized exposure to a white powder (such as a contaminated letter or package sent through the mail)
Contamination of a closed air supply (such as the ventilation system of a building)
Broad contamination of outdoor air (such as release of anthrax spores via a crop duster or similar aircraft)
Contamination of a commercial food or beverage source (which would cause gastrointestinal disease)

Only exposure to a white powder would be recognized at the time of the event. The other methods of exposure most likely would not be recognized until cases of disease occurred (unless indoor or outdoor air-monitoring systems are in place and capable of detecting B anthracis spores with some degree of sensitivity).

Handling of Suspicious Packages

According to the US Postal Service, suspicious letters or packages should not be opened (see References: US Postal Service). A letter or package should be considered suspicious if it:

Has any powdery substance on the outside
Is unexpected or from someone unfamiliar to you
Has excessive postage, a handwritten or poorly typed address, an incorrect title or a title with no name, or misspellings of common words
Has no return address or one that can't be verified as legitimate
Is of unusual weight, given its size, or is lopsided or oddly shaped
Has an unusual amount of tape
Is marked with restrictive endorsements, such as "Personal" or "Confidential"
Has a strange odor or stain

During the 2001 anthrax attacks, cases occurred among persons who opened mail and among persons who merely handled contaminated mail; therefore, suspicious packages or letters should be handled as little as possible. Forensic analysis has indicated that nearly 8 x 10⁶ CFU were removed from the most highly cross-contaminated piece of mail found (see References: Beecher 2006). The CDC has published specific recommendations regarding handling suspicious packages or letters (see References: CDC 2001: Investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001):

Do not shake or empty the contents of a suspicious package or envelope.
Do not carry the package or envelope, show it to others, or allow others to examine it.
Put the package or envelope on a stable surface; do not sniff, touch, taste, or look closely at it or any contents that may have spilled.
Alert others in the area about the suspicious package or envelope. Leave the area, close doors, and take actions to prevent others from entering the area. If possible, shut off the ventilation system.
Wash hands with soap and water to prevent spreading potentially infectious material to face or skin. Seek additional instructions for exposed or potentially exposed persons.
If at work, notify a supervisor, a security officer, or a law enforcement official. If at home, contact the local law enforcement agency (ie, police).
If possible, create a list of persons who were in the room or area when the suspicious letter or package was recognized and a list of persons who also may have handled the package or letter. Give the list to both the local public health authorities and law enforcement officials.

If a patient reports exposure to a suspicious package that contains an unknown white powder, further evaluation should be undertaken by public health and law enforcement officials.

Recommendations for testing the package contents for B anthracis should be made on the basis of a risk assessment conducted by public health and law enforcement officials.
If the patient is considered at risk and is asymptomatic, then antimicrobial prophylaxis should be initiated pending results of microbiological testing of the package contents.
If the patient has symptoms compatible with anthrax, then appropriate antimicrobial treatment should be administered.

A prospective longitudinal study of 124 subjects who may have been exposed to B anthracis during the anthrax attack in the US Capitol demonstrated that the significance of low-level exposure should not be underestimated (see References: Doolan 2007). The authors’ conclusion is based on the following:

Spore exposure primed immune responses in a dose-dependent manner and may have enhanced vaccine boost and recall responses.
Immune responses were detected among subjects inside the defined exposure zone as well as subjects outside the zone, implying more extensive spore migration than had been predicted.
Despite the fact that subjects received PEP with antibiotics, spore inhalation provoked immune system stimulation consistent with subclinical infection (and possibly antibiotic-aborted infection after germination of viable spores); greater levels of exposure corresponded to more complete immune responses.

Mass Exposure Events

In the event of a mass exposure, rapid delivery of prophylactic antibiotics to the exposed population would be critical to prevent illness and death (see References: Wein 2003). A simulation analysis suggested that postattack antibiotic therapy and vaccination of exposed individuals represents the most cost-effective strategy for a small-scale attack (see References: Schmitt 2007).
In emergency situations involving a bioterrorist release, state governments can request antibiotic and medical supplies from the Strategic National Stockpile through the CDC; the CDC is ready to rapidly deploy the stockpile as needed and can deliver initial supplies within several hours.
State and local health departments have bioterrorism preparedness plans in place to provide points of distribution for mass antibiotic prophylaxis against anthrax and other biological agents as needed. During the 2001 anthrax outbreak, the New York City Department of Health and Mental Hygiene activated its incident command system and put its antibiotic distribution plan into effect. Lessons learned from this experience were published (see References: Blank 2003).
One analysis suggests that the critical determinant of mortality after an anthrax bioterrorism event is local dispensing capacity (see References: Bravata 2006). Modeling suggests a higher mortality among sites with low dispensing capacities, compared with those with high dispensing capacities. Doubling local inventories at high dispensing sites makes stockpiling fivefold more cost-effective than at low dispensing sites.
Mass prophylaxis campaigns carry the risk of overwhelming emergency health services owing to visits for actual or perceived medication-related adverse events (see References: Hupert 2007). Modeling suggets that the length of a mass prophylaxis campaign (eg, 10 days vs 2 days) plays an importantrole in determining the subsequent intensity in emergency servicesuse due to real or suspected adverse events.

Surveillance During Exposure Events or for Early Detection of Outbreaks

Early disease outbreak recognition may significantly modify the outcome of a biological attack. A list has been developed of potential epidemiologic clues or "red flags" for an unusual event. Although these clues may be associated with natural outbreaks and bioterrorism events, their occurrence should heighten suspicion. Potential clues include the following (see References: Dembek 2007):

Highly unusual event with large numbers of casualties
Higher morbidity or mortality than is expected
Uncommon disease
Unusual disease manifestation
Lower attack rates in protected persons
Point-source outbreak
Multiple epidemics
Downwind plume pattern
Dead animals
Reverse or simultaneous spread of human and animal cases
Direct evidence

A review of the accidental release of aerosolized anthrax spores in Sverdlovsk showed that the resulting outbreak had many characteristics of an unusual event. Of the clues listed above, the first four were present in the outbreak, as were the sixth (point-source outbreak) and eighth and ninth (downwind plume pattern and dead animals). Despite concealment of information and confiscation of records by the Soviet military and government, public health response measures were implemented within 10 days of the event. The public health response is estimated to have prevented an additional 14% of fatalities.

Several reports have examined surveillance approaches for anthrax either in the setting of a known exposure or as an early population-based detection tool.

During 2003, the Connecticut Public Health Department (CPHD) implemented gram-positive rod surveillance for early anthrax detection (see References: Begier 2005). The CPHD reported that this laboratory-based surveillance system is a tool that could provide early detection of even a single case of inhalational anthrax.
During the 2001 anthrax outbreak, the New York City Department of Health and Mental Hygiene established the Cutaneous Anthrax Rapid Referral System for rapid referral and early diagnosis of anthrax cases (see References: Redd 2005). This system functioned to efficiently assess patients but also provided a mechanism for rapid centralized reporting, which could be a good surveillance model in the setting of a known mass exposure to anthrax.
Syndromic surveillance may be a valuable tool for early detection of anthrax cases in the setting of a mass exposure where relatively large numbers of cases would be expected to occur (see References: CDC 2005: Syndromic surveillance: reports from a national conference, 2004, Nordin 2005).
A recent review found evidence from the Sverdlovsk outbreak that livestock (such as cattle, sheep, and goats) can provide early warning of a bioterrorist event caused by B anthracis. In addition to livestock, cats and dogs might serve as markers for ongoing exposure risk following an anthrax bioterrorist attack (see References: Rabinowitz 2006).

Hospital Infection Control (Including Autopsies and Burial)

Isolation Precautions

Although people with inhalational anthrax may have had contamination of hair and clothing at the time of their exposure, residual contamination at the time of medical presentation does not appear to be of concern. Also, no secondary cases occurred among household contacts of the inhalational cases in the 2001 US outbreak.
Standard Precautions are considered adequate for patients with inhalational and gastrointestinal anthrax, since person-to-person transmission for these forms of disease has not been reported.
Most sources also recommend Standard Precautions for cases of cutaneous anthrax (see References: APIC/CDC 1999, Inglesby 2002). However, person-to-person transmission rarely has been reported for patients with cutaneous anthrax; therefore, several sources have recommended that Contact Precautions be followed for patients who have draining cutaneous lesions (see References: Swartz 2001, Weber 2001). Contact Precautions include the following:

Place the patient in a private room, or, if a private room is not available, place the patient in a room with a patient who has an active infection with the same pathogen (ie, cohort). When a private room is not available and cohorting is not possible, a spatial separation of at least 3 ft should be maintained between the infected patient and other patients and visitors.
Gloves should be worn when entering the room and removed before leaving the room; hands should be washed with an antimicrobial agent or a waterless hand washing agent immediately after removing gloves, and clean hands should not touch potentially contaminated items or environmental surfaces.
Gowns should be worn when entering the room if it is anticipated that clothing will have substantial contact with the patient, environmental surfaces, or items in the room; the gown should be removed before leaving the patient's environment.
Patient transport should be limited to essential purposes only; if the patient is transported out of the room, precautions should be maintained.
Noncritical patient-care equipment should be dedicated whenever possible. If equipment cannot be dedicated, then it should be adequately cleaned and disinfected between patients.

Soiled dressings should be incinerated or autoclaved.
A study on the use of hand-hygiene agents to remove B atrophaeus (a surrogate of B anthracis) from contaminated hands demonstrated that use of a waterless hand rub containing ethyl alcohol was not effective in removing spores (see References: Weber 2003). Conversely, hand washing with soap and water, 2% chlorhexidine gluconate, or chlorine-containing towels reduced the level of spore contamination.

Cleaning Surfaces and Instruments

According to the CDC, the following procedures should be followed when cleaning surfaces following spills or when cleaning instruments (see References: CDC 2001: Basic laboratory protocols for the presumptive identification of Bacillus anthracis):

Commercially available bleach or 0.5% hypochlorite solution (a 1:10 dilution of household bleach) is considered adequate for cleaning.
Contaminated items (eg, pipettes, needles, loops, microscope slides) should be immersed in decontamination solution until autoclaving.
Work surfaces should be wiped down before and after use with decontamination solution.
Spills involving fresh cultures or samples known to have low concentration of spores should be flooded with decontamination solution and soaked for 5 minutes before cleanup.
Spills that involve samples with high concentration of spores, involve organic matter, or occur in areas of lower than room temperature (eg, refrigerators, freezers) should be exposed to decontamination solution for at least 1 hour before cleanup.
Personnel involved in the cleanup of any spill should wear gloves, safety glasses, and a laboratory coat or gown during the cleanup process.
Respiratory protection should be considered for spills in which a substantial aerosolization is suspected.

Personal Protective Equipment for Workers

Information regarding personal protective equipment for first responders and others with potential occupational exposure to anthrax spores can be found on the following Web pages (see References):

CDC: Protecting investigators performing environmental sampling for Bacillus anthracis: personal protective equipment
CDC: Interim recommendations for the selection and use of protective clothing and respirators against biological agents
OSHA: Anthrax prevention and controls

Other Issues

One study examined biocide inactiviation of B anthracis spores in the presence of food residues (see References: Hilgren 2007). The presence of food residues had only a minimal effect on peroxyacetic acid and H₂0₂ sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residue. Decontamination procedures should be adjusted to address situations for food-soiled surfaces.

Autopsy Practices

Instruments used in autopsies should be autoclaved or incinerated.
Guidelines from the CDC indicate that Standard Precautions should be used for postmortem care. These include using a surgical scrub suit, surgical cap, impervious gown or apron with full sleeve coverage, a form of eye protection (eg, goggles or face shield), shoe covers, and double surgical gloves with an interposed layer of cut-proof synthetic mesh (see References: CDC 2004: Medical examiners, coroners, and biologic terrorism: a guidebook for surveillance and case management).
In addition, autopsy personnel should wear N-95 respirators during all autopsies, regardless of suspected or known pathogens. Powered air-purifying respirators equipped with N-95 or high-efficiency particulate air (HEPA) filters should be considered.

Burial

Contact with corpses should be limited to trained personnel, and routine precautions should be implemented when transporting corpses.
According to the CDC, cremation is the preferred disposition method. If cremation is not possible, bodies should be "properly secured in a sealed container (eg, a Ziegler case or other hermetically sealed casket) to reduce the potential risk of pathogen transmission" (see References: CDC 2004: Medical examiners, coroners, and biologic terrorism: a guidebook for surveillance and case management).
According to WHO guidelines (see References: WHO 1998), "In fatal cases, postmortem should be discouraged; cremation is preferable to burial where local custom permits. It is advisable for the body to be placed in an impervious body bag for transport from the place of death and preferably the body should not be extracted from the bag. Where only burial is permitted, the bagged body should be placed in a hermetically sealed coffin and buried without re-opening."
An example of the type of system that can be used to seal remains prior to placing them in a casket for burial is the BioSeal System, produced by Barrier Products, LLC (see References). This system utilizes a poly-aluminum foil–extruded laminate material that, when used with a heat sealer, will provide level 1 containment for all gases, fluids, vapors, and odors associated with the transport and storage of human and animal remains.

Environmental Testing and Decontamination

Environmental Testing as a Detection Tool

Autonomous detection systems (ADSs) have been developed to detect B anthracis spores in the environment (indoor or outdoor) as the first signal of an aerosol release. Examples include the following:

Biohazard Detection System (BDS): The BDS is a fully automated air-sampling system consisting of an aerosol collector, PCR cartridge based on Cepheid GeneXpert technology, and controller computer. It has been deployed in mail processing and distribution centers across the United States. Positive BDS signals will be confirmed by an LRN reference or national laboratory (see References: CDC 2004: Responding to detection of aerosolized Bacillus anthracis by autonomous detection systems in the workplace, NALC).

According to the CDC guidelines, when a positive BDS signal occurs, the following immediate response is appropriate:

Stop work activities.
Stop and secure any potential aerosol-generating equipment.
Turn off heating, ventilation, and air conditioning (HVAC) units serving the production or processing area (leave local exhaust ventilation on machines turned on).
Notify local and federal law enforcement and public health officials.
Immediately account for all workers to ensure their evacuation.
Gather personal identification and contact information.

Depending on the level of potential exposure, decontamination of employees (with removal of clothing and washing exposed areas or showering at the site) may be necessary.
The decision to begin postexposure antibiotic prophylaxis should be made on the basis of risk of exposure, threat assessment, validity of preliminary laboratory testing, and logistics of initiating an intervention.

Autonomous Pathogen Detection System (APDS): Consists of an aerosol collector, flow-through PCR subsystem with sequential injection analysis, and a multianalyte flow-cytometry subsystem for PCR product detection (see References: Hindson 2004, LLNL 2002).
Anthrax Smoke Detector (ASD): An automated system designed by the National Aeronautics and Space Administration (NASA) to detect changes in airborne bacterial spore concentrations, including spores of B anthracis. The system measures dipicolinic acid (DPA), a chemical marker of bacterial spores. The ASD is intended as a relatively low-cost screening tool and requires that positive signals must be confirmed by B anthracis–specific assays (see References: Lester 2004). A recent test revealed that the device had a detection limit of 16 spores/L when 250 L of air was sampled (see References: Yung 2007).
Handheld Advanced Nucleic Acid Analyzer (HANAA). A real-time PCR analyzer using a miniaturized thermal cycling process (see References: LLNL 2002).
Mobile laboratory systems (used primarily by the military): Automatic Biological Agent Testing System (ABATS), PortalShield, Joint Biological Point Detection System (JBPDS), and Biological Integrated Detection System (BIDS) (see References: Fitch 2003).

Environmental Testing Following a Release

Environmental testing for B anthracis should be conducted by public health or law enforcement officials in the context of an epidemiologic or criminal investigation. Sampling should be guided by available epidemiologic data whenever possible (see References: Teshale 2002) and should be performed in accordance with the most recently recommended sampling methods and protocols.

Criteria for performing directed sampling of environmental surfaces as specified by the CDC include the following (see References: CDC 2001: Interim guidelines for investigation of and response to Bacillus anthracis exposures):

To identify a site or source of B anthracis exposure that resulted in a case(s) of anthrax
To trace the route of an exposure vehicle (eg, powder-containing letter)
To obtain the B anthracis strain when isolates from patients are not available
To guide cleanup activities in a contaminated area or building
To assess biosafety procedures in laboratories processing B anthracis specimens

ASTM International (formerly the American Society for Testing and Material Standards), a voluntary standards development organization, has published a standard on practices for "bulk sample collection and swab sample collection of visible powders suspected of being biological agents from nonporous surfaces." A recent collaborative study validated the methods in the standard and showed that high levels of B anthracis and B thuringiensis spores can be recovered from surfaces by both dry and wet swab sampling methods (see References: Locascio 2007).

Environmental sampling of a US postal facility in Washington, DC, during the 2001 anthrax outbreak demonstrated the following (see References: Sanderson 2002):

Sampling using wipes or HEPA vacuum socks provided a better yield than sampling with wet or dry swabs.
Dry swabs generally should not be used for sampling, since yields are so low.
Wet swabs may be useful in certain situations (eg, crevices, inside machinery, other areas difficult to reach by wipe or HEPA vacuum samples).
Wipes are preferable for sampling areas with light dust.
HEPA vacuum socks should be used to sample surfaces with heavy dust.

One study evaluated four swab materials (cotton, macrofoam, polyester, and rayon) and methods of processing to determine optimal spore recovery. Results demonstrated that premoistened cotton and macrofoam swabs were the most efficient (see References: Rose 2004). In a study of polyester-rayon wipes, sonication extraction improved recovery of spores from wipes used for cleaning surfaces. The wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel surface area and 105 CFU per unit of painted wallboard (see References: Brown 2007). In a study of recovery efficiencies of anthrax spores, contact plates performed better than other methods for flat, nonporous, nonabsorbent surfaces, with recovery rates of 28% to 54%. Contact plates also performed the best on flat, porous, absorbent surfaces, although recoveries were low (less than 7%). For moistened devices (wipes, swabs, and sample collection and recovery devices), wipes were generally the best (see References: Frawley 2008).

Official procedures for testing food and water have not been published, but protocols reportedly have been developed by USAMRIID (see References: Higgins 1999) and are under development elsewhere.

A number of commercial test kits for environmental sampling are available. A study of three such systems, BioThreat Alert (BTA), BioWarfare Agent Detection Devices (BADD), and SMART II, found a minimum detection limit of 10⁵ spores, far above the level of 10² spores desired by first-responders (see References: King 2003). The specificities of these systems have not been independently evaluated.

Decontamination of Persons Exposed to Anthrax

According to guidelines from the Association for Professionals in Infection Control and Epidemiology (APIC) and the CDC (see References: APIC/CDC 1999):

Patients should remove contaminated clothing and store in labeled, plastic bags.
Clothing should be handled as little as possible to avoid agitation.
Patients should shower thoroughly with soap and water.

Decontamination of Environments

Concerns regarding environmental contamination involve both primary and secondary aerosolization.

Primary aerosolization occurs when the spores are first made airborne. This is the period when the risk of inhalation is the greatest. The risks of primary aerosolization depend on how long spores remain airborne and how far they travel before falling to the ground or other surfaces. Meteorological conditions and aerobiological properties of the dispersed aerosol will influence the duration and scale of the risk (see References: HPA 2007).
Secondary aerosolization involves resuspension of spores into the air after they have initially settled on environmental surfaces. The risks posed by secondary aerosolization have not been defined and are dependent on a number of variables (eg, concentration of spores in the environment, type of powder used in the suspension, level of activity in the contaminated area, type of environmental surface involved).

Determining the extent of remediation necessary for contaminated environments remains controversial. Ideally, the remediation effort should be timely and cost-effective and protect the public health (see References: Martin 2010: Anthrax as an agent of bioterrorism). A process for determining what level of contamination is acceptable has been proposed as follows (see References: Price 2009):

Decide on the level of risk that is acceptable.
Convert the risk to an airborne spore concentration, via an assumed dose-response curve.
Convert the airborne spore concentration to a surface concentration, using an assumed resuspension rate.
Convert the surface concentration to a probability that any single sample is positive for B anthracis, using a sampling effectiveness curve.
Find the number of samples needed (for the single-sample probability above) such that if all the samples are negative, then the building or area is "safe" with a specified certainty.

During the 2001 anthrax attacks, the Environmental Protection Agency (EPA) assessed the potential for secondary aerosolization inside the Hart office building by conducting environmental sampling under semiquiescent (minimal activity) conditions and under simulated active office conditions (see References: Weis 2002).

Findings demonstrated that viable B anthracis spores could be reaerosolized during simulated active office conditions.
CFU levels detected through air sampling demonstrated as much as a 65-fold increase under the active office conditions compared with the semiquiescent conditions.
These findings support the need for environmental decontamination and protection of decontamination workers following release of high concentrations of anthrax spores into indoor environments.

During the 2001 US anthrax outbreak, several buildings underwent environmental decontamination to eliminate the risk of potential secondary aerosolization. In the setting of a bioterrorism attack, the EPA is charged with directing cleanup activities and providing the necessary technical expertise to guide such efforts. The EPA used several methods and technologies to decontaminate buildings during the 2001 outbreak (ie, chlorine dioxide, decontamination foam, ethylene oxide). Cleanup plans generally involve the following:

Assess the size and type of the potentially contaminated area.
Assess how the contamination was delivered.
Conduct sampling to determine the level of contamination.
Determine the microbiocide to be used and methods of delivery for decontamination.
Carry out decontamination procedures.
Conduct environmental sampling after decontamination to ensure that anthrax spores have been removed or killed and that the area is safe to reoccupy.

Until the recent anthrax attack, experience with decontamination of buildings after contamination with weapons-grade anthrax spores was limited. Questions regarding the best methods for decontamination in such situations still remain (see References: Spotts Whitney 2003). In addition, reasonable standards for cleanup effectiveness remain to be established (see References: Canter 2005). Paraformaldehyde (with B subtilis as the indicator) has been used to decontaminate laboratories.

The chlorine dioxide fumigation approach used after the 2001 postal anthrax attacks is expensive and can preclude reoccupation of contaminated buildings for many years. Wein and colleagues compared this approach to a strategy of HEPA vacuuming, HEPA air cleaners, and vaccination of building occupants (see References: Wein 2005). They found in a simulated outdoor release in lower Manhattan that the HEPA/vaccine method would require less time, cost less, and reduce solid waste problems associated with disposal of contaminated carpets and upholstery. However, the details of the massive cleanup operation modeled by the study depend on many factors related to the attack itself, which obviously cannot be accurately predicted. Furthermore, vaccination of reoccupants may not be a practical or acceptable approach.

Vaporized hydrogen peroxide (Vaprox) may represent a new method for decontamination of buildings or other enclosed areas. The EPA has granted vaporized hydrogen peroxide an emergency exemption for the specific use of anthrax decontamination. Available data showed that the gas significantly reduced bacterial spore populations under specific conditions (see References: EPA 2007).

Several hypochlorite-containing household products on the market were found to be effective in decontaminating milk or similar food products contaminated by spores to allow safe disposal (see References: Black 2008). A combination of high temperature (90°C to 95°C) and hydrogen peroxide also could be used to inactivate B anthracis spores (see References: Xu 2008).

Gaps in Decontamination Policy and Practice

The environmental decontamination response following the 2001 postal anthrax attacks required hundreds of millions of dollars in direct costs, and some facilities were closed for more than 2 years (see References: Franco 2010). Although the attack represents the worst case of bioterrorism in US history, it is considered to be small in scale. Recently, an analysis was undertaken to identify current gaps in decontamination policy and practice at the federal level that must be addressed to achieve a successful response following a large-scale attack. Identified gaps include:

Unclear roles and responsibilities for federal agencies involved in a decontamination response
Lack of a coordinated, sustained, and adequately funded research program for biological decontamination
Limited technologies and methods for sampling, testing, and analysis of contamination
Scientific uncertainty about biological agent properties, decontamination methods, and risks to human health
Absence of decontamination standards
Shortage of trained personnel to carry out decontamination response

Public Health Reporting and Case Definitions

Anthrax cases should be reported immediately to local or state health departments, according to disease reporting rules.

The anthrax case definition for public health surveillance is as follows (see References: CDC 1997, CDC 2001: CDC update: CDC case definition of anthrax and summary of confirmed cases):

Clinical Description

An illness with acute onset characterized by several distinct clinical forms, including the following:

Cutaneous: a skin lesion evolving during a period of 2 to 6 days from a papule, through a vesicle, to a depressed black eschar
Inhalational: a brief prodrome resembling a viral respiratory illness, followed by development of hypoxemia and dyspnea, with radiographic evidence of mediastinal widening
Gastrointestinal:

Abdominal (or intestinal) subtype: severe abdominal distress followed by fever and signs of septicemia
Oropharyngeal subtype: mucosal lesion in the oral cavity or oropharynx, cervical adenopathy and edema, and fever

Confirmed Cases

The CDC defines a confirmed case of anthrax as:

A clinically compatible case of cutaneous, inhalational, or gastrointestinal illness that is laboratory-confirmed by isolation of B anthracis from an affected tissue or site, or other laboratory evidence of B anthracis infection based on at least two supportive laboratory tests.

Images

Cutaneous anthrax images (CDC)
Cutaneous anthrax infection (Roche KJ, Chang MW, Lazarus H. N Engl J Med 2001;345[22]:1611)
Histopathology, microbiology, skin lesions, chest x-ray findings (Public Health Image Library—type "anthrax" in the search field)
Inhalational anthrax images (CDC)
Inhalational anthrax images: histopathology (Armed Forces Institute of Pathology)
Inhalational anthrax images: microbiology (Armed Forces Institute of Pathology)

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