Last updated May 1, 2013
Key microbiological characteristics of Bacillus anthracis follow (ASM 2013):
- 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 cell wall (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
- Nonhemolytic growth on sheep blood agar (SBA)
- Rapid growth on SBA; colonies are 2 to 5 mm in diameter after 16 to 18 hours of incubation, are flat or slightly convex, are irregularly round, are gray to white, and have a "ground glass" appearance
- Colonies may exhibit a comma-shaped projection or a "Medusa head" appearance caused by chains of bacilli growing out from the edge of colonies
- When lifted using an inoculating loop, colonies show a tenacity that allows them to stand upright with the consistency of beaten egg whites
- 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 (Davison 2005)
B anthracis strains from around the world have been subtyped using molecular techniques such as variable number tandem repeat (VNTR) analysis. On the basis of such testing, all known anthrax strains can be classified into three different clades (A, B, and C) (Pilo 2011).
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 exposed to air and nutrients in the environment are exhausted. Spores are protected by a morphologically complex protein coat (Giorno 2007). The exosporium may restrict dispersal and thereby increase the probability of a lethal dose for the grazing animal (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 (Manchee 1990, Montville 2005).
The primary virulence factorsproduced by B anthracis are plasmid coded and include:
- A poly-D-glutamic acid (PGA) capsule (coded for on plasmid pXO2) that inhibits phagocytosis of vegetative bacilli. In mice PGA increases the toxicity of lethal toxin (LT), intensifying the toxemia in the fulminant stage of infection (Jang 2011).
- 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 (Kumar 2002).
- Edema toxin also inhibits neutrophil function and stimulates production or release of multiple inflammatory mediators, including neurokinins, prostanoids, and histamine (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.
- Lethal toxin has been shown to cause endothelial cell apoptosis and endothelial barrier dysfunction, which may contribute to vascular destruction (Kirby 2004, Warfel 2005). It has also been shown to reduce myocardial function (Moayeri 2009, Sweeney 2010).
During early stages of infection (prodromal stage), LT and edema toxin (ET) may coordinate to incapacitate neutrophils and macrophages, which allows for a systemic infection to occur (Liu 2010, Cote 2011). During the fulminant stage of infection (see Staging of Inhalational Anthrax for additional description) the combined vascular impacts of LT and ET lead to shock (Guichard 2011, Hicks 2011).
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 (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 (Read 2003).
B anthracis toxin genes are located on plasmids, and translocation of the plasmids has been identified in a naturally occurring B cereus isolate obtained from a patient with an illness similar to inhalational anthrax (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 (Avashia 2007). In another situation, a Bacillus strain, with a higher similarity to B thuringiensis and B cereus than to B anthracis, was isolated from a chimpanzee in Cote d'Ivoire that died with clinical signs of anthrax (Klee 2010).
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 (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.
- Endospores are introduced into the body via inhalation. Endospores are 1 mcm x 1.5 mcm in size (ASM 2013) 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 (Russell 2008).
- The endospores then germinate inside macrophages and become vegetative cells, which leave the macrophages and multiply in the lymphatic system.
- Regional hemorrhagic lymphadenitis of mediastinal and peribronchial lymph nodes causes hemorrhagic mediastinitis (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 (Guarner 2003).
- True pneumonia rarely occurs, although a focal, hemorrhagic, necrotizing pneumonic lesion (similar to the Gohn complex of tuberculosis) may be seen (Abramova 1993). Intraalveolar edema, focal areas of hyaline membrane formation, and interstitial mononuclear inflammation may be noted (Guarner 2003).
- Bacteria enter the bloodstream and lead to septic shock and toxemia; hematogenous spread can lead to hemorrhagic meningitis.
- Compression of the lungs and septic shock are the major causes of death.
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, more 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) (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 median infective dose (ID50) for inhalational anthrax is estimated at 8,000 to 50,000 spores (Franz 1997), although the minimum infective dose may be considerably lower. 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 (DIA 1986).
- Extrapolation of dose-response curves involving cynomolgus 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 (Peters 2002).
- In rabbits, the LD50 for the Ames strain of B anthracis is 5.18 x 104 colony forming units (CFU) (95% confidence interval, 6.14 x 103 to 7.27 x 105 CFU). Rabbits exposed to less than 2.06 x 103 CFU of B anthracis survived for the duration of the study (21 days) (EPA 2011).
- 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 (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 (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 (Henderson 1956).
- 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 (Beatty 2003).
- A rare case of gastrointestinal anthrax was identified in New Hampshire following an exposure to infected animal hides. Environmental sampling data from this case suggested that the infective dose can be very low (CDC 2009: Gastrointestinal anthrax after an animal-hide drumming event).
- The infective dose for cutaneous anthrax is not known.
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 (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 (Manchee 1990). During 1943 and 1944, an estimated 4 x 1014 anthrax 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.
Frequent outbreaks of anthrax caused the death of 1.5 million deer in northern Russia from 1897 to 1925 (Revich 2011). Because anthrax spores can survive in permafrost for approximately 100 years, researchers have expressed concern that thawing of the permafrost in Siberia will expose extensive animal burial grounds that contain the remains of animals that died from anthrax (Revich 2011). These researchers have suggested that careful monitoring of permafrost conditions in areas where anthrax outbreaks occurred is warranted, since it is unclear if further warming will lead to release of viable spores.
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 (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
- Some Mediterranean countries
In most of the rest of the world, anthrax in animals 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 (MBAH 2006, WHOCC). Outbreaks also occurred in 2006 in Saskatchewan and Manitoba, Canada, affecting more than 800 animals (APHIS 2006). Other notable points about anthrax in animals include the following.
- Anthrax has been reported as the cause of death among chimpanzees in Ivory Coast (Leendertz 2004) and chimpanzees and a gorilla in Cameroon (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 (Leendertz 2006).
- Anthrax also has been reported in cheetahs following consumption of infected meat (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 (Saggese 2007).
- During an outbreak investigation of anthrax in white-tailed deer in Texas in 2005, investigators found that necrophilic flies that were feeding on animals that died from anthrax and tested positive for B anthracis. This raises the possibility that such flies may play a role in the dispersion of B anthracis during animal outbreaks (Blackburn 2010).
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 (Wattiau 2008).
- Consumption of contaminated illicit drugs (through injection and possibly through smoking or snorting)
Cases following laboratory exposure have been recognized (Brachman 1980, CDC 2002: Suspected cutaneous anthrax in a laboratory worker). Person-to-person transmission of B anthracis has been reported rarely with cutaneous anthrax, but has not been recognized with gastrointestinal or inhalational disease (Weber 2001, Weber 2002).
- 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 (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 (Brachman 1980, Ramer 2010).
- Only 18 cases of naturally occurring inhalational anthrax were reported in the United States during the 20th century (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 (CDC 2001: Human anthrax associated with an epizootic among livestock).
- 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 (CDC 2008), and one case was inhalational (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 (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 (CDC 2010: Gastrointestinal anthrax after an animal-hide drumming event).
- A molecular analysis of B anthracis isolates from recent anthrax cases in the United States found that the New York, Connecticut, and New Hampshire isolates all were of the same lineage (although the New Hampshire isolates were of a slightly different genotype) (Marston 2011). However, limited information is available on the molecular subtypes of B anthracis that circulate in West Africa, so the isolates could not be traced back to that region of the world. These findings illustrate the value of acquiring and subtyping isolates from around the world so that accurate predictions can be made about isolate origins.
- An estimated 2,000 to 20,000 human cases of anthrax occur globally each year (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 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 (Wattiau 2009). These workers had positive serologic tests for anti-PA immunoglobulin G (IgG). A second serologic survey was conducted in the same facility a year later. All who were seropositive in the first study remained seropositive. In addition, five persons seroconverted between the two periods and none had symptoms compatible with anthrax (Kissling 2011). These results suggest that repeated exposure to low doses of anthrax spores can cause an anti-PA IgG response to B anthracis in the absence of clinical disease and support the concept of a dose-response association between exposure and illness.
- Two fatal cases of inhalational anthrax occurred in the United Kingdom, one in Scotland in 2006 and one in England in 2008. Both patients were drum makers (Anaraki 2008).
- A 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 (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 Use as a Biological Weapon) 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 (Ramsay 2010) and subsequently spread to several other countries.
- In Scotland 119 cases and 14 deaths were recognized, with an additional 5 cases in the England and 2 in Germany.
- The patients reported injecting, smoking, and/or snorting heroin. Although B anthracis was not identified in any heroin samples, epidemiologic evidence implicated heroin as the source of contamination (HPS 2011). The strain of B anthracis was identical in all cases and represented a new introduction into the United Kingdom; it was related to Trans-Eurasian (TEA) B anthracis strains previously identified in goats in Turkey.
- The symptoms at presentation varied greatly, were inconsistent, and were not typical of cutaneous, inhalational, or gastrointestinal anthrax (Booth 2010). Most patients presented with soft-tissue infections, localized swelling, or symptoms similar to necrotizing fasciitis. Among confirmed cases, swelling was the most common symptom, followed by pain, malaise, and fever. Some patients presented with only generalized symptoms suggesting systemic infection (HPS 2011). In one case, a man presented with acute abdominal pain, leading to peritonitis, then septic shock and death (Johns 2011).
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 (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 (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 (Sirisanthana 1984). Oropharyngeal disease is an unusual manifestation of infection, which makes this outbreak of particular interest.
Aerosol release of weaponized spores is the most likely mechanism for use of anthrax as a biological weapon (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 spores 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 (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 (OTA 1993).
Experience with aerosol spraying of B thuringiensis in Canada to control the European gypsy moth demonstrated the following pertinent findings (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 outdoor release of a biological agent occur.
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 (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 (Beatty 2003). Since B anthracis spores are not destroyed by pasteurization, contamination of milk is another theoretical possibility (Perdue 2003).
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 (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 (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 (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 (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 (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 (MIIS).
Weaponization of anthrax spores 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 (Takahashi 2004).
- 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 (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 (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."
- 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 (Wilkening 2006).
- 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 (Inglesby 2002).
- The following features were noted in an epidemiologic report that summarized the outbreak findings (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; one of these cases was a 7-month-old infant. The remaining two 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 (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 was 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 (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 (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 (CDC 2002: Suspected cutaneous anthrax in a laboratory worker).
- 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 (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 factors continue to be discussed, 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. Some scientists remain skeptical of the FBI statements, in light of the circumstantial nature of the the evidence. The FBI closed the case on February 19, 2010, in spite of the remaining unanswered questions (Bhattacharjee 2008, Warrick 2010).
- In response to this skepticism, the FBI requested that the National Research Council (NRC) of the National Academy of Sciences (NAS 2011) launch an independent review of the scientific approaches used and the conclusions reached during the FBI investigation. On Feb 15, 2011, the NRC completed its review; in its report, the NRC ruled that the genetic analyses conducted by the FBI were scientifically sound but the results could not by themselves conclusively link the anthrax strain to the flask (designated "RMR-1029") in Ivins' custody (NRC 2011). "Spores in the mailed letters and in RMR-1029 … share a number of genetic similarities consistent with the FBI finding that the spores in the letters were derived from RMR-1029," the NAS said in a press release (NAS 2011). "However, the committee found that other possible explanations for the similarities—such as independent, parallel evolution—were not definitively explored during the investigation." The FBI replied in a statement, "The FBI has long maintained that while science played a significant role, it was the totality of the investigative process that determined the outcome of the anthrax case" (FBI 2011).
- In March 2011, scientists involved in the anthrax investigation reported how, through whole-genome sequencing and comparative genomics, they were able to identify four mutations in spore material from letter samples. The mutations were related to a regulatory protein involved in sporulation and were found only in spores linked to the B anthracis in Ivins' custody (Rasko 2011). Only 8 of the 947 isolates they studied had all four mutations, and these all came directly or indirectly from the "parent" strain from Ivins' lab (see Feb 15, 2011, CIDRAP News story).
- 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 (CDC 2004: Responding to detection of aerosolized Bacillus anthracis by autonomous detection systems in the workplace).
- A summary of public health actions during this attack was compiled by the Trust for America's Health on the 10-year anniversary of the attacks (TFAH 2011).
In October 2011, the bipartisan WMD (weapons of mass destruction) Terrorism Research Center published a bio-response report card on the level of preparedness in the United States for responding to various levels of biological attacks (WMD Center 2011). According to that report, the United States is moderately well prepared for small-scale biological incidents but is seriously unprepared for a large-scale incident.
- As part of the preparedness for a large-scale incident, the US Postal Service is evaluating distributing medical countermeasures along its mail routes (HHS 2011: HHS preparedness grants help cities plan for anthrax attacks).
- Since its inception, project BioShield has been focused on ensuring that effective medical countermeasures are available in the event of a bioterrorism incident. Some experts question if the financial investments in BioShield have been worth it, with the limited number of acquisitions that made it to the Strategic National Stockpile (SNS). However, these acquisitions probably would not have happened without the influx of BioShield funding (Cohen 2011).
The three primary forms of anthrax correspond to the route of exposure: cutaneous, inhalational, and gastrointestinal (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. These four clinical forms are described in the tables below.
Anthrax also has been recognized in injecting drug users following the use of contaminated heroin (mostly from injection, but snorting and smoking also may play a role). Such patients may have localized findings at injection sites (including presentations similar to necrotizing fasciitis), or they may present with relatively nonspecific findings, such as malaise and fever (HPS 2011).
Clinical Features of Cutaneous Anthrax
1-7 days (more commonly 2-5 days; may be as long as 12 days)
Signs and symptomsa
—Initial lesion is small papule or vesicle, which may be pruritic.
—Currently <1%a,g (most patients recover with appropriate antimicrobial therapy)
—Gram stain of lesion may reveal gram-positive rods; neutrophils are uncommon.
Clinical Features of Inhalational Anthrax
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.
—Sverdlovsk outbreak: 86%a
Findings for 10 patients with inhalational anthrax identified during 2001 US outbreakc:
Clinical Features of Gastrointestinal Anthrax
1-7 days (usually 2-5 days)
Signs and symptoms
—One outbreak of GI anthrax in Uganda demonstrated the following findings in 143 patientsa:
—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
—Gram stain of peritoneal fluid or oropharyngeal ulcers may demonstrate gram-positive rods.
Clinical Features of Anthrax Meningitis
Varies according to primary source of infection.
Signs and symptomsaa-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.
—Illness fatal in >90% of cases.
—Gram stain of CSF reveals many gram-positive rods.
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.)
B megaterium, B pumilus, B cereus cutaneous infections may resemble cutaneous anthrax (Duncan 2011).
—Usually in neutropenic patients with Pseudomonas aeruginosa bacteremia
Ulceroglandular tularemia (Francisella tularensis)
—Clinical course usually indolent; disease often self-limited
Bubonic plague (Yersinia pestis)
—Systemic toxicity common
Staphylococcal or streptococcal cellulitis
—May be history of trauma or preexisting lesion at site of infection
Necrotizing soft tissue infections (particularly Group A Streptococcus and Clostridium species)
—Severe systemic toxicity often present
Bite of brown recluse spider (Loxosceles reclusa)d
—Brown recluse spiders prefer warm temperatures and are not native to northern half of United States
Rickettsialpox (Rickettsia akari)
—Initial presentation involves painless papule that forms black eschar
Scrub typhus (Orientia tsutsugamushi; formerly Rickettsia tsutsugamushi)
—Zoonotic infection from chigger bites; occurs in endemic areas (Asia and Western Pacific)
Orf (orf virus, a parapox virus)
—Occurs in farm workers
Necrotic herpes simplex infection
—More likely to occur in immunocompromised host
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.)
Pneumonic plague (Yersinia pestis)
—Hemoptysis relatively common with pneumonic plague, but rare with inhalational anthrax
Tularemia (Francisella tularensis)
—Clinical course usually indolent, lasting weeks
Community-acquired bacterial pneumonia
—Rarely as fulminant as inhalational anthrax
—Influenza generally seasonal (October-March in United States) or involves history of recent cruise ship travel or travel to tropics
—Q fever (Coxiella burnetii)
—Exposure to infected parturient cats, cattle, sheep, goats
Differential Diagnosis for Gastrointestinal Anthrax
Typhoid fever (Salmonella typhi)
—Ascites usually not present
Intestinal tularemia (Francisella tularensis)
—Illness often less severe than that seen with gastrointestinal anthrax
Bacillary dysentery (Shigella dysenteriae)
—Ascites usually not present
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
—Gastrointestinal symptoms (nausea, vomiting, gastrointestinal bleeding, diarrhea) not prominent features
Acute abdomen (eg, appendicitis)b
—Anthrax generally begins with vague systemic symptoms rather than abdominal pain
Diphtheria (Corynebacterium diphtheriae)
—Primarily occurs in nonimmune children under 15 yr of age
Pharyngeal tularemia (Francisella tularensis)
—Neck swelling usually absent
Streptococcal pharyngitis (Streptococcus pyogenes)
—Exudative pharyngitis most prominent feature; necrotic ulcers generally absent
—Most common in young adults
Enteroviral vesicular pharyngitis (coxsackievirus)
—Small vesicles noted on soft palate, uvula, or anterior tonsillar pillars
Acute herpetic pharyngitis (herpes simplex virus)
—Vesicles, shallow ulcers may be noted, but lesions usually not necrotic
Anaerobic pharyngitis (Vincent's angina)
—Purulent exudate covers posterior pharynx
Yersinia enterocolitica pharyngitis
—Exudative pharyngitis most prominent feature
Differential Diagnosis for Anthrax Meningitis
—Fever not usually prominent feature
Bacterial meningitis from other causes
—Meningitis not usually hemorrhagic as seen with anthrax meningitis
—Meningitis not hemorrhagic
—CSF findings may be variable, depending on etiology
Historically, inhalational anthrax has been divided into a prodromal phase and a fulminant phase. The table below outlines a proposed staging scheme (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
—Usually <1 wk after exposure and rarely >1 mo
—Nonspecific malaise, myalgias, low-grade fever, mild headache, nausea, general "flu-like" prodromal illness
—Blood cultures positive in <24 hr
—Respiratory failure requiring intubation, sepsis, meningitis, end-organ hypoperfusion (ie, "shock")
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 (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 (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.
- Chest discomfort, pleuritic pain
- Nausea, vomiting
- Abdominal pain
- 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) (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 (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 (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 Anthraxa (%)
Laboratory-Confirmed Influenza (%)
ILI from Other Causes (%)
Fever or chills
Shortness of breath
Nausea or vomiting
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 (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 (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.
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 (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 (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 (Freedman 2002).
- Anthrax meningitis has been reported in children and may be the presenting feature (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 (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 (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.
A report summarized two cases of anthrax in pregnant women; the features of these cases are outlined in the table below (Kadanali 2003).
Summary of Clinical Features for Two Pregnant Women with Anthrax
Weeks of Gestation at Illness Onset
Clinical Presentation of Anthrax
Outcome of Pregnancy
Cutaneous anthrax (treated with penicillin G and prednisone)
—Infant delivered preterm at 35 wk
Cutaneous anthrax (treated with procaine penicillin)
—Infant delivered preterm at 34 wk
- Cutaneous anthrax images (CDC)
- Cutaneous anthrax infection (Roche KJ, Chang MW, Lazarus H. N Engl J Med 2001;345:1611)
- Histopathology, microbiology, skin lesions, chest x-ray findings (Public Health Image Library—type "anthrax" in the search field)
Specimen Collection and Transport
The Laboratory Response Network (LRN)
Standard Tests for Detection of B anthracis
Antimicrobial Susceptibility Studies
Tests for Exposure
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 American Society for Microbiology (ASM), in collaboration with the CDC and the Association of Public Health Laboratories (APHL), has developed sentinel laboratory guidelines to enable clinical laboratories to perform microbiological analyses to rule out B anthracis (ASM 2013). When anthrax is suspected or B anthracis cannot be ruled out, sentinel laboratories should contact their Laboratory Response Network (LRN) reference laboratory for specimen collection consultation, and isolates should be referred for confirmation testing. The following table outlines the collection of laboratory specimens for diagnosis of anthrax.
Collection and Transport of Laboratory Specimens for the Diagnosis of Anthraxa-d
Type of Illness
Specimen Collectione and Transport
—All stages: Collect 2 swabs, one for Gram stain and culture, and 1 for PCR.
—Swabs: Moisten with sterile saline or water; transport in sterile tube at 2o-8oC. Transport swabs for PCR only at –70oC. Do not use transport medium.
—Obtain blood prior to antibiotic therapy for smear, culture, and PCR.
—Sputum: transport at room temperature in sterile, screw-capped container (<1 hr transport time) or at 2o-8oC (>1 hr transport time).
—Collect blood for Gram stain, culture, and PCR. Blood cultures are most likely to yield B anthracis if taken 2-8 days postexposure and prior to administration of antibiotics.
—Stool: Transport in sterile container unpreserved (<1 hr transport time) or at 2o-8oC in Cary-Blair medium or equivalent (>1 hr transport time); specimen >5.0 g.
—Collect CSF specimen for Gram stain, culture, and PCR.
—Blood cultures: Obtain appropriate blood volume, number, and timing of sets per laboratory protocol; transport at room temperature (<2 hr transport) or 2-8oC (>2 hr transport).
Guidelines have been published for packing and shipping of infectious substances, diagnostic specimens, and biological agents from suspected acts of bioterrorism (ASM 2012).
- B anthracis may be present in blood, skin-lesion exudates, cerebrospinal fluid, pleural fluid, sputum, and rarely, in urine and feces. The primary hazards to laboratory personnel are: direct and indirect contact of broken skin with cultures and contaminated laboratory surfaces, accidental parenteral inoculation, and, rarely, exposure to infectious aerosols. To avoid production of aerosols, work with infectious organisms should be done in a biosafety cabinet. In addition, all centrifugation should be done using aerosol-tight rotors that are opened within the biosafety cabinet after each run (CDC 2007).
- Because B anthracis cells present in clinical samples are primarily vegetative and not easily transmitted to clinical laboratory workers, BSL-2 practices, containment equipment, and facilities are recommended for activities using clinical materials and diagnostic quantities of infectious cultures. BSL-2 practices, containment equipment, and facilities are recommended for studies using experimentally infected laboratory rodents (CDC 2007).
- BSL-3 practices, containment equipment, and facilities are recommended for work involving production quantities or high concentrations of cultures, screening environmental samples (especially powders) from anthrax-contaminated locations, and activities with a high potential for aerosol production. Workers who frequently centrifuge B anthracis suspensions should use autoclavable aerosol-tight rotors (CDC 2007).
- Hospital laboratories are not sufficiently staffed, trained, or equipped for environmental sample testing. This testing should be performed at the nearest LRN reference laboratory under BSL-3 conditions.
- Anthrax spores may remain viable after standard DNA purification procedures (Rantakokko-Jalava 2003). Experimental heat treatment of anthrax spores at 121oC for 45 minutes appears to eliminate viability without significantly affecting polymerase chain reaction (PCR) efficiency (Fasanella 2003). Spores also can be irradiated with gamma rays to inactivate them without affecting PCR results (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 (CDC 2005: Inadvertent laboratory exposure to Bacillus anthracis). 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."
- B anthracis is classified under WHO risk group 3. Cultures of B anthracis must be transported as "Category A infectious substances." The US Department of Transportation regulations and International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including infectious substances (DOT and IATA 2012).
- 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 and amended in October 2012 (HHS 2012). 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, biosafety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications). Effective April 3, 2013, B anthracis will be considered a Tier 1 agent and subject to additional security requirements. (HHS 2012) 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 (CDC/APHIS 2008). In addition, the CDC has published additional guidelines for enhancing laboratory security for laboratories working with select agents (CDC 2002: Laboratory security and emergency response guidance for laboratories working with select agents).
The LRN is a network of more than 150 national and international laboratories. The network includes federal, state, and local public health, military, food testing, environmental, veterinary, and international laboratories (CDC: Facts about the Laboratory Response Network, CDC: Laboratory Response Network [The]).
The LRN structure for bioterrorism designates laboratories as sentinel, reference, or national. Designation depends on the types of tests a laboratory can perform and how it handles infectious agents to protect workers and the public.
- Sentinel laboratories, formally called "level A laboratories,"represent an estimated 25,000 hospital-based laboratories that have direct contact with patients. In an unannounced or covert terrorist attack, sentinel laboratories could be the first facilities to encounter suspicious specimens. These laboratories generally have at least BSL-2 containment capabilities. A sentinel laboratory's responsibility is to rule out B anthracis or refer a suspicious sample to the nearest LRN reference laboratory. Sentinel laboratories use the ASM Sentinel Level Clinical Microbiology Laboratory Guidelines to rule out microorganisms that might be suspected as agents of bioterrorism (ASM: Sentinel level clinical microbiology laboratory guidelines).
- 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 but also include military, international, veterinary, agriculture, and food- and water-testing laboratories. Reference laboratories operate with BSL-3 containment facilities that have been given access to nonpublic testing protocols and reagents. One of the roles of the LRN reference laboratories is to provide guidance, training, outreach, and communications to the sentinel laboratories in their jurisdictions.
- National laboratories have unique resources to handle highly infectious agents and the ability to identify specific agent strains through molecular characterization methods. These laboratories also are responsible for methods development, bioforensics, and select-agent activity.
The ASM Sentinel Level Clinical Microbiology Laboratory Guidelines for Bacillus anthracis (ASM 2013) are the approved clinical laboratory procedures to isolate and rule out B anthracis. Current standard tests for detection of B anthracis are outlined in this document.When B anthracis cannot be ruled out, sentinel laboratories should contact their LRN reference laboratory and isolates should be referred for confirmation testing.
- 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 (Hoffmaster 2002: Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis, Hoffmaster 2002: Supplement to above).
- Real-time PCR assays capable of detecing multiple agents have been developed for detection of 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 (Skottman 2007). Commercial ready-to-use reagent systems have been developed for use in PCR assays (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 (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 (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 (Guarner 2003, Shieh 2003). IHC is not widely available, but requests for testing can be made by contacting an LRN reference laboratory prior to specimen collection.
- Molecular subtyping(multilocus variable-number tandem repeat analysis [MLVA]), is used by the CDC and others for strain identification and tracking (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 (Kenefic 2008).
Serologic testing for anti-PA antibodies 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 (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 confirmed 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 (Bell 2002).
- Requests for serologic testing can be made by contacting an LRN reference laboratory prior to specimen collection.
- 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 (Biagini 2006, CDC 2004:CDC collaboration yields new test for anthrax, Stephenson 2004).
Other tests for detection of B anthracis infection:
- Testing for toxins (particularly LF) in the serum has been used in an outbreak setting to identify acute cutaneous infections that were culture negative due to prior antibiotic use (Boyer 2011). Investigators demonstrated that toxins can be detected in the serum relatively early in the clinical course before antibodies to PA are detectable. They concluded that although skin lesions remain relatively localized, in many cases of cutaneous anthrax toxins are secreted into the blood in sufficient quantity to be measured during the acute phase of infection; therefore, toxin testing (if available) may be a useful tool to confirm infection when B anthracis cannot be cultured or visualized by staining methods.
- 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 (Pfisterer 1991, Shlyakhov 1996, WHO 1998).
Antimicrobial susceptibility testing should be performed on clinical isolates at LRN reference laboratories or the CDC. Clinical Laboratory Standards Institute (CLSI) protocols for broth microdilution have been used with staphylococcal breakpoints (Coker 2002).
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 (Brook 2001, Price 2003). Because resistance can be induced relatively rapidly in vitro, close monitoring of patients treated for anthrax is important (Athamna 2004).
MICs of Various Antibiotics for Bacillus anthracis Isolates as Identified in Four Studies
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 (Barrow 2007).
Antimicrobial susceptibility testing (AST) requires time for the growth of the organism. A rapid AST for B anthracis recently has been described (Weigel 2010). With this test, a broth microdilution method is combined with a real-time quantitative PCR method to yield comparable results in less than 6 hours.
Nasal swab cultures were used in the 2001 US outbreak as an epidemiologic tool to assess exposure to aerosolized B anthracis spores. In one study of 625 persons potentially exposed to anthrax spores in the Hart Senate Building, nasal swabs were positive in 28 (4.5%) (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 (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 (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 (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 (Inglesby 2002). Methods for collection, labeling, transport, and processing of swabs have been published (ASM 2012).
Serologic testing does not appear to be a useful tool for assessing asymptomatic exposure to B anthracis unless it is being used as part of an epidemiologic study (Dewan 2002, Hsu 2002, Traeger 2002).
Anthrax countermeasures include antibiotics (for treatment and postexposure prophylaxis [PEP]), antibodies, antitoxin agents, and vaccines. The tables below review available treatment recommendations for clinical disease caused by B anthracis.
Protocol for Treatment of Cutaneous Anthraxa-c
Initial Therapy (Oral)d
Ciprofloxacin: 500 mg PO twice daily
Ciprofloxacin: 10-15 mg/kg PO twice daily (maximum daily dose, 1 g)
Same as for nonpregnant adults (high death rate from the infection outweighs risk posed by antimicrobial agent)
Same as for nonimmunocompromised persons and children
Protocol for Treatment of Inhalational and Gastrointestinal Anthraxa
Initial IV Therapyb,c
Oral Regimens (continue therapy for 60 days [IV and PO combined])
Ciprofloxacin: 400 mg twice daily
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
Ciprofloxacin: 10-15 mg/kg twice daily (maximum daily dose, 1 g)g
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)
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.
Same as for nonimmunocompromised persons and children.
Same as for nonimmunocompromised persons and children.
A systematic review of inhalational anthrax cases identified between 1900 and 2005 reported the following observations with regard to treatment (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 (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 (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) (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) (Sejvar 2005). Case reports suggest that adding corticosteroids may be of benefit in the management of cerebral edema/inflammation (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.
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 (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 Tests for Exposure in the Clinical Laboratory Testing section).
In the event of a mass exposure, local and state public health agencies would rapidly make antibiotics available to the exposed population (see the discussion of mass exposure in the Management of Exposure Events section).
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 contamination 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 (CDC 2000:Human ingestion of Bacillus anthracis-contaminated meat).
- Penicillin G procaine
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 (Brouillard 2006). An in vitro analysis of five antibiotics found that doxycycline was more effective against spore-forming B anthracis and least effective against vegetative-phase B anthracis. For vegetative-phase B anthracis, meropenem was the most effective antibiotic (Louie 2011: Impact of spores on the comparative efficacies of five antibiotics for the treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model). 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 (Stern 2008). In an in vitro analysis, linezolid was found to work as well as ciprofloxacin and it also prevented toxin production (Louie 2011: Differential effect of linezolid and ciprofloxacin on toxin production by Bacillus anthracis in an in vitro pharmacodynamic system). Athamna and colleagues found that the combination of rifampin and clindamycin demonstrated a synergistic effect in vitro against two strains of B anthracis (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
Adults (including immunocompromised patients)
Ciprofloxacin: 500 mg PO twice daily
Pregnant women and breastfeeding mothers
Ciprofloxacin: 500 mg PO twice daily (first-line oral agent for PEP in pregnant women.)
Children (including immunocompromised patients)
Ciprofloxacin: 10-15 mg/kg PO twice daily (maximum daily dose, 1 g)
More than 10,000 people were placed on PEP during the 2001 anthrax outbreak; no cases of anthrax occurred among this group (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 (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 (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 (Hupert 2009).
The American College of Obstetricians and Gynecologists (ACOG) has recommended the following for anthrax prophylaxis in pregnant women (ACOG 2002):
- 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.
Results of a recent nationally representative survey found that 89% of participants would be very likely (65%) or somewhat likely (24%) to follow public health recommendations to obtain postexposure prophylaxis medication for themselves within 48 hours of an anthrax attack, and 91% indicated that they would be very or somewhat likely to obtain medication for their children (SteelFisher 2011). Ninety percent of participants also believed that inhalational exposure to anthrax spores can lead to a serious illness or death.
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 (Chen 2011, Froude 2011, Artenstein 2012)). 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 (Pittman 2006).
- In 2005, 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 (HHS 2006: HHS to acquire new anthrax therapeutic treatment for stockpile). In April 2009, HGS finished delivery of the first 20,000 doses of ABThrax (HGS 2012). HHS then placed a second order for an additional 45,000 doses of ABthrax, which is expected to be delivered by the end of 2012 (HGS 2009).
- HHS also has contracted with Elusys Therapeutics to evaluate its anthrax antitoxin (humanized monoclonal antibody against PA), known as Anthim, for potential usage in the SNS (HHS 2011: Novel anthrax vaccine and antitoxin being developed with federal support).
- 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 (HHS 2006: HHS to acquire anthrax immune globulin for stockpile). Cangene completed shipment of AIG to the SNS in the fourth quarter of 2011 (Cangene 2011).
Current Anthrax Vaccine
Recommendations for Use
Dosage, Route of Administration, and Vaccination Schedule
Contraindications and Precautions
Postlicensure Adverse Event Reporting
Development of New Vaccines
- 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 (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 SNS).
- 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.
One group reviewed randomized controlled trials on the clinical effectiveness, immunogenicity, and safety of anthrax vaccines. The authors concluded that vaccines based on anthrax antigens are immunogenic in most vaccinees with few adverse events, although limited data were available (Donegan 2009).
- The efficacy of AVA is based on several animal studies, one controlled human trial, and immunogenicity data from humans and other mammals (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 (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 (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 (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 (Allen 2006).
- Anthrax vaccines intended for animals should not be used in humans.
Preexposure immunization with AVA is licensed for individuals aged 18 to 65 who have a high likelihood of coming into contact with B anthracis (CDC 2010: Use of anthrax vaccine in the United States). 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 (Inglesby 2002).
Preexposure vaccination is indicated for the following groups (CDC 2010: Use of anthrax vaccine in the United States):
- 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 (DoD 2006, and see Oct 19, 2006, CIDRAP News story).
- Vaccination is the best defense currently available for first responders in the event of an anthrax attack with an antibiotic resistant strain (Zink 2011). The Department of Homeland Security and the CDC are developing a program to provide anthrax vaccines that are nearing expiration to first responders. Approximately two million doses of vaccine in the SNS expire annually. A pilot program involving two federal agencies or departments and two state or local jurisdictions is being developed to assess the feasibility of this program (Polk 2012).
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.
ACIP guidelines state that anthrax vaccine can be used in combination with antibiotics following inhalational exposure to anthrax (CDC 2010: Use of anthrax vaccine in the United States):
- 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 in any age-group, so it must be given under an emergency use authorization for adults. The use of AVA in children under 18 years of age has never been studied and therefore would need to occur as part of an investigational new drug (IND) application with the FDA.
Use of AVA in children under an IND raises a number of complex clinical, ethical, and regulatory issues, since the vaccine has not been studied in children. The National Biodefense Science Board (NBSB) developed a position paper on this issue in 2011. They recommended that, if the ethical considerations can be adequately addressed, HHS should develop a plan for and conduct a pre-event study of AVA in children, to include a research IND (NBSB 2011).
Because of 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 (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.
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 (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 Vaccinationa
Primary series (2 injections)
Booster series (3 injections)b
6 mo after primary series
12 mo after primary series
18 mo after primary series
- 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 (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 (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 (Ryan 2008).
- 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 (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.
- Between 1998 and 2008, the Vaccine Adverse Event Reporting System (VAERS) received 6,015 reports of adverse events following anthrax vaccination (CDC 2010: Use of anthrax vaccine in the United States). 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) (McNeil 2007: Short-term reactogenicity and gender effect of anthrax vaccine).
- 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 (CDC 2000: Surveillance for adverse events associated with anthrax vaccine). 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 (McNeil 2007: A comparative assessment of immunization records in the Defense Medical Surveillance System and the Vaccine Adverse Event Reporting System).
- To evaluate the potential for AVA to contribute to disability in US service members, a cohort of persons who had received AVA was identified. Between December 15, 1997, and February 15, 2005, a total of 439,059 service members received at least one dose of AVA (Sulsky 2011). There was no difference in risk of disability among these service members when compared with service members who did not receive AVA.
- 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 (Payne 2006).
- LRN workers who were vaccinated with AVA did not show any change in physical or mental functional status when compared with LRN workers who were not vaccinated (Stewart 2012). These workers were followed for 30 months for any health-related quality-of-life issues postvaccination.
- Some investigators have proposed that AVA (and possibly other vaccines) may be associated with the development of type 1 diabetes. But a retrospective cohort study among military personnel did not find any evidence of an increased risk for the disease among those who received AVA vaccine or five other vaccines commonly administered to military personnel (Duderstadt 2012).
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 review of 4,753 anthrax vaccine–related VAERS reports from January 1, 1990 through January 16, 2007 confirmed these previous findings (Niu 2009).
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 (Rhie 2005). The Biomedical Advanced Research and Development Authority (BARDA), which is within the Office of the Assistant Secretary for Preparedness and Response in HHS, is funding the development of an rPA nasal spray vaccine made by Vaxin (HHS 2011: Novel anthrax vaccine and antitoxin being developed with federal support).
- 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 (Emergent BioSolutions 2008). This vaccine increased the immune response and reduced the time to peak immune response in human subjects compared with AVA alone (Rynkiewicz 2011).
- Combination vaccines such as one against anthrax and plague may represent the next generation of vaccines against potential agents of bioterrorism (DuBois 2007).
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).
According to the US Postal Service, suspicious letters or packages should not be opened (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 (Beecher 2006). The CDC has published specific recommendations regarding handling suspicious packages or letters (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 (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.
- In the event of a mass exposure, rapid delivery of prophylactic antibiotics to the exposed population would be critical to prevent illness and death (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 (Schmitt 2007).
- In emergency situations involving a bioterrorist release, state governments can request antibiotic and medical supplies from the SNS through the CDC; the CDC is ready to rapidly deploy the stockpile as needed and can deliver initial supplies within several hours.
- In locations with a high risk of an attack, several additional strategies can be used to enhance rapid access to appropriate medical countermeasures (IOM 2011). These include forward-deployed (positioning of medical countermeasures geographically near where they may be used), cached (storage of medical countermeasures at specific locations where they will be used such as worksites or hospitals), and predispensed (storage of medical countermeasures by intended users such as home storage) countermeasures. Each of these strategies has benefits and costs, which should be evaluated for each location. The IOM has provided a framework to help officials perform such an evaluation (IOM 2011). HHS is considering how to incorporate this work into its preparedness efforts (HHS 2011: Statement by Dr. Nicole Lurie).
- 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 (Blank 2003).
- One analysis suggests that the critical determinant of mortality after an anthrax bioterrorism event is local dispensing capacity (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 (Hupert 2007). Modeling suggest 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.
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 (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 (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 (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 (CDC 2005: Syndromic surveillance: reports from a national conference, 2004, Nordin 2005).
- One 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 (Rabinowitz 2006).
- 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 (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 (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 (Weber 2003). Conversely, hand washing with soap and water, 2% chlorhexidine gluconate, or chlorine-containing towels reduced the level of spore contamination.
According to the CDC, the following procedures should be followed when cleaning surfaces following spills or when cleaning instruments (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.
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:
- 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
- One study examined biocide inactiviation of B anthracis spores in the presence of food residues (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.
- 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 (CDC 2004: Medical examiners, coroners, and biologic terrorism).
- 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.
- 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" (CDC 2004: Medical examiners, coroners, and biologic terrorism).
- According to WHO guidelines (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 Systems, produced by Barrier Products, LLC. 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 as a Detection Tool
Environmental Testing Following a Release
Decontamination of Persons Exposed to Anthrax
Decontamination of Environment
Gaps in Decontamination Policy and Practice
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 (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 (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 be confirmed by B anthracis–specific assays (Lester 2004). One test revealed that the device had a detection limit of 16 spores/L when 250 L of air was sampled (Yung 2007).
- Handheld Advanced Nucleic Acid Analyzer (HANAA). A real-time PCR analyzer using a miniaturized thermal cycling process (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) (Fitch 2003).
Environmental testing for B anthracis should be conducted by public health or law enforcement officials in the context of an environmental, epidemiologic, or criminal investigation. Sampling should be guided by available data whenever possible (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 (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 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 (Locascio 2007).
Environmental sampling of a US postal facility in Washington, DC, during the 2001 anthrax outbreak demonstrated the following (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 (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 (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 (Frawley 2008).
Official procedures for testing food and waterhave not been published, but protocols reportedly have been developed by USAMRIID (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 (King 2003). The specificities of these systems have not been independently evaluated.
According to guidelines from the Association for Professionals in Infection Control and Epidemiology (APIC) and the CDC (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.
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 (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 (Martin 2010: Anthrax as an agent of bioterrorism). A process for determining what level of contamination is acceptable has been proposed as follows (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 (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.
A recent review of decontamination technologies and strategies following an anthrax release identified the following as key considerations when determining the appropriate decontamination strategy (Campbell 2012):
- The identity and characteristics of the biological agent (including its environmental persistence and ability to aerosolize), the mode of delivery (release), and the nature of spread
- Boundaries (ie, exclusion zones) that restrict and control access to contaminated areas (ie, hot zones)
- Results of environmental sampling, including extent and magnitude of contamination
- Analysis of epidemiologic evidence
- Health risks posed by the agent and its susceptibility to medical countermeasures; balancing risk mitigation through reducing exposure versus large-scale prophylaxis programs
- Clearance goals and clearance criteria to meet the goals
- Nature of the sites and items to be decontaminated
- Prioritization of facilities, areas, and infrastructure from the results of characterization and according to possible national security considerations
- Projected timelines to complete decontamination, if performed
- Public perception, such as acceptance of proposed decontamination technologies
- Quantities of estimated waste and suitable staging and storage areas for waste
Until the 2001 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 (Spotts Whitney 2003). In addition, reasonable standards for cleanup effectiveness remain to be established (Canter 2005). Using Bayesian statistical methods can improve the decision making process for anthrax clean up, as it helps to overcome gaps in empirical data (Linkov 2011).
The EPA has tested seven sporicidal products to evaluate their efficacy in inactivating B anthracis spores, and all products successfully inactivated the pathogen in at least one test (EPA 2010). In these tests gaseous chlorine dioxide and vapor-phase hydrogen peroxide performed the best under the most stringent test conditions. Paraformaldehyde has been used to decontaminate laboratories and postal equipment. There are four fumigants that can potentially be used for neutralization of anthrax spores: gaseous chlorine dioxide, vapor-phase hydrogen peroxide, paraformaldehyde, and methyl bromide (Campbell 2012). An EPA exemption is required to use methyl bromide; thus, it has largely been phased out of use.
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 (Black 2008). A combination of high temperature (90°C to 95°C) and hydrogen peroxide also could be used to inactivate B anthracis spores (Xu 2008).
The waste generated in response to anthrax incidents has been problematic, owing to the challenges of identifying locations willing to take such waste, procedures for disposing of waste, and procedures for minimizing risks associated with placing the waste in a landfill (EPA 2012). Decisions regarding waste management need to be made within a comprehensive recovery framework (Lesperance 2011: Developing a regional recovery framework). While there are several research questions that need to be addressed, the most pressing waste management issue is the classification of the waste, which will dictate waste management plans (Lesperance 2011: Challenges in disposing of anthrax waste).
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 (Franco 2010). It cost $320 million to clean up seven contaminated buildings after the 2001 postal anthrax attack (Schmitt 2012). 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 (Franco 2010). 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
Anthrax cases should be reported immediately to local or state health departments, according to disease reporting rules.
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
- 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
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.
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