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Table of Contents:
Taxonomy Information
  1. Species:
    1. Bacillus anthracis (Shafazand et al., 1999, Website 15):
      1. Common Name: Anthrax, Woolsorter's disease, and Charbon
      2. GenBank Taxonomy No.: 1392
      3. Description: The anthrax bacterium is named Bacillus anthracis because it is rod-shaped when viewed under a microscope. The name "Bacillus" comes from the Latin word "baculus," meaning "rod." Anthrax is derived from the Greek word for coal, the characteristic color and appearance of the eschar in cutaneous anthrax. Anthrax is also known as charbon (pronounced shar-bawn), which is French for coal. Pulmonary anthrax is also known as woolsorter's disease. This is because people who sorted the wool of animals had contracted it in this way(Shafazand et al., 1999).
      4. Variant(s):
Lifecycle Information
  1. Bacillus anthracis lifecycle
    1. Stage Information:
      1. Vegetative Phase:
        • Size: The non-flagellated vegetative cell is large (1-8 micrometers long, 1-1.5 micrometers wide). Spore size is approximately 1 micrometer.
        • Shape: Vegetative cells are rod-shaped. The chains of virulent forms of the bacteria are usually surrounded by a capsule.
        • Picture(s):
          • SEM Images of Vegetative and Spore Stages (Website 37)



            Description: Scanning Electron Micrograph of Bacillus anthracis spore and vegetative stages. Bacillus anthracis is a Gram-positive, encapsulated, spore-forming, zoonotic, rod prokaryote. Magnification: x700.
        • Description: Spores ingested by herbivores germinate within the host to produce the vegetative forms; these multiply and express their virulence factors, killing the host (ref76, Figure 1). The vegetative form is square ended and capsulated. Sporulating cells carry elliptic, centrally located spores. Bacilli shed by the dying or dead animal will sporulate on contact with air. Sporulation requires the presence of free oxygen, and the efficiency of the process is influenced by the environmental conditions. The proportion of cells that reach the ultimate stage, a dormant spore, is variable. The various steps of this cycle and current knowledge of the molecular mechanisms they involve are reviewed(Mock and Fouet, 2001).
      2. Intense Growth Phase:
        • Description: This is the phase where the bacteria have intense growth(Website 36).
      3. Stationary Phase:
        • Description: The growth of B. anthracis is stationary(Website 36).
      4. Sporulation Phase:
        • Size: Endospores are 1 micrometer by 1.5 micrometers in size and are able to reach the alveoli (ie, less than 5 micrometers).
        • Shape: Endospores are nonswelling and oval-shaped.. Endospore is located central-to-subterminal and does not usually swell; carbon dioxide levels within the body inhibit sporulation. Forms long chains in vitro giving them a bamboo appearance; single cells or short chains in direct clinical samples.
        • Picture(s):
          • Scanning Electron Micrograph of Spores in Lung (Website 37)



            Description: Photocomposite of Bacillus anthracis spores in lung bronchiole.Magnification: x43(lung), x1,410(bacteria).
        • Description: The SPORE is a particular stage in the life cycle of the ANTHRAX organism; during this stage it goes into STASIS and remains dormant until something happens in its natural environment(Website 35). Dormant spores are highly resistant to adverse environmental conditions including heat, ultraviolet and ionizing radiation, pressure, and chemical agents. They are able to survive for long periods in contaminated soils and thus account for the ecological cycle of the organism. In a suitable environment, spores reestablish vegetative growth. However the bacilli are poor survivors, and it is unclear whether existence of a complete cycle, from germination to resporulation, occurs outside the host. Indeed the particular properties of B. anthracis, compared with those of other Bacillus cereus-group bacilli sharing the same ecological niche, are consistent with a life cycle that almost exclusively takes place in the mammalian host(Mock and Fouet, 2001).
    2. Progression Information:
      1. :
      2. :
      3. :
        • From Stage: Stationary Phase
        • To Stage: Sporulation Phase
        • Description: The rate of sporulation by the shed vegetative cells and the proportion which succeed in sporulating are influenced in a complex manner by the environmental conditions into which they fall. Temperature, pH, water, oxygen availability, sunlight, organic matter and the presence of certain cations such as Mn++ are among the many influcencing factors. The spore forms are highly resistant ot adverse environmental conditions(Turnbull et al., 2002).
      4. :
    3. Description: Bacillus anthracis is Gram-positive, non-motile, aerobic, facultative anaerobic, spore-forming, bacterium(Mock et al., 2001, Website 2, Shafazand et al., 1999).
Genome Summary
  1. Genome of Ames Strain(Website 38, Read et al., 2002)
    1. Chromosome Information
      1. GenBank Accession Number: NC_003997
      2. Size: 5227293 bp(Website 19).
      3. Description: Bacillus anthracis strain Ames, complete genome(Website 19).
  2. Genome of Pasteur Strain(Read et al., 2002)
    1. Description: Plasmid pXO2 from the Pasteur strain has been sequenced.
    2. Plasmid pX02(Mock et al., 2001, Website 1, Okinaka et al., 1999, Dixon et al., 2000, Website 27)
      1. GenBank Accession Number: NC_002146
      2. Size: 96,231 bp in length(Mock et al., 2001).
      3. Gene Count: 85 open reading frames(Mock et al., 2001).
      4. Description: Plasmid pXO2 (60Mda) carries genes required for the synthesis of an antiphagocytic poly-gamma-D-glutamic acid capsule which inhibits phagocytosis of vegetative cells. Plasmid pXO2 carries: three genes required for capsule synthesis (capB, capC, and capA), a gene associated with capsule degradation (dep), and a trans-acting regulatory gene (acpA)(Pannucci et al., 2002, Mock et al., 2001, Website 1, Okinaka et al., 1999, Dixon et al., 2000).
  3. Genome of Sterne Strain(Read et al., 2002)
    1. Description: Plasmid pXO1 has been isolated from the Sterne strain (Read et al. 2002).
    2. Plasmid pXO1(Mock et al., 2001, Website 1, Okinaka et al., 1999, Leppla et al., 1995, Dixon et al., 2000, Pannucci et al., 2002, Bradley et al., 2001, Website 28)
      1. GenBank Accession Number: NC_001496
      2. Size: 181,654 bp in length(Okinaka et al., 1999).
      3. Description: Plasmid pX01 contains 143 ORFs, covering ~61% of the DNA. Plasmid pXO1 (110-Mda) contains genes required for synthesis of the anthrax toxin proteins: cya which encodes edema factor (EF), lef which encodes lethal factor (LF), and pagA which encodes protective antigen (PA). Plasmid pXO1 also harbors two trans-acting regulatory genes [atxA the toxin gene transactivator], pagR [the negative regulator of pagA], a gene encoding a type I topoisomerase (topA); a resolvase and a transposase, an operon containing three genes (gerXC, -A and -B) whose functions appear to affect germination. The virulence genes of pXO1 are organized in a manner similar to pathogenicity islands (PAIs) located on the chromosomes of other bacterial pathogens (Mock et al., 2001)(Mock et al., 2001, Website 1, Okinaka et al., 1999, Dixon et al., 2000).
  4. Genome of Florida isolate of Ames Strain(Read et al., 2002)
    1. Chromosome Information(Website 21)
      1. GenBank Accession Number: NC_003995
      2. Size: 5093554 bp(Website 21).
      3. Description: Bacillus anthracis A2012, unfinished sequence, whole genome shotgun sequencing project(Read et al., 2002).
    2. Plasmid pXO1(Website 29)
      1. GenBank Accession Number: NC_003980
      2. Size: 181677 bp(Read et al., 2002).
      3. Description: Bacillus anthracis str. A2012 plasmid pXO1, complete sequence(Website 29).
    3. Plasmid pXO2(Website 30)
      1. GenBank Accession Number: NC_003981
      2. Size: 94829 bp(Read et al., 2002).
      3. Description: Bacillus anthracis str. A2012 plasmid pXO2, complete sequence(Website 30).
Biosafety Information
  1. Biosafety information for Bacillus anthracis
    1. Level: Biosafety Level 2(Website 2, Website 3).
    2. Precautions: Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities using clinical materials and diagnostic quantities of infectious cultures. Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended for studies utilizing experimentally infected laboratory rodents. The B. anthracis cells present in clinical samples are primarily vegetative, which are not easily transmitted to laboratory workers. However, most hospital laboratories are not sufficiently staffed, trained, or equipped for environmental testing. The recent case of cutaneous anthrax in a laboratory worker illustrates this potential. The agent may be present in blood, skin lesion exudates, cerebrospinal fluid, pleural fluid, sputum, and rarely, in urine and feces. Direct and indirect contact of the intact and broken skin with cultures and contaminated laboratory surfaces, accidental parenteral inoculation, and rarely, exposure to infectious aerosols are the primary hazards to laboratory personnel(Website 3, Website 2).
    3. Disposal: Because sporulation of B. anthracis requires oxygen and therefore does not occur inside a closed carcass, regulations in most countries forbid postmortem examination of animals when anthrax is suspected. If heat treatment or incineration of the contaminated material is possible, this should be done in preference to chemical decontamination and disinfection. For certain materials or animal by-products, irradiation with gamma rays or particle bombardment or fumigation with a gaseous disinfectant such as ethylene oxide may be appropriate. In fatal human cases, postmortem should be discouraged; cremation is preferable to burial where local custom permits. Bedding and contaminated materials should be bagged and incinerated, autoclaved (121+1 degree C core temperature for 30 min) or fumigated as appropriate. Whether room fumigation is necessary will depend on the perceived level of contamination in the room where the patient died. Immersion in 4% formaldehyde (10% formalin) for >12 hours is an alternative but full penetration of the fluids must be ensured. Disinfection of contaminated surfaces involves a three-step approach aimed at (i) preliminary disinfection, (ii) cleaning, and (iii) final disinfection. Preliminary disinfection: One of the following disinfectants may be used in amounts of 1-1.5 liters per square meter for an exposure time of 2 hours: 10% formaldehyde (approximately 30% formalin), 4% glutaraldehyde (pH 8.0-8.5), or a high pressure cleaner may be used but, to avoid spreading the contamination, the pressure should not exceed 10 bar. Cleaning: Where practical, cleaning of all surfaces should be done by straightforward washing and scrubbing using ample hot water. The operator should wear protective clothing, face and hands included. Cleaning should be continued till the original colors and surfaces are restored and the waste water is free of dirt particles. At the end of the process, residual water should be removed and the surfaces dried. High pressure cleaners are again discouraged because of the greater potential to spread the contamination through aerosols. If used, however, the water jet should be applied at a pressure of 80-100 bar delivering 13-15 litres/minute. Final disinfection: One of the following disinfectants should be applied at a rate of 0.4 liters per square meter for an exposure time of at least 2 hours: 10% formaldehyde (approximately 30% formalin), 4% glutaraldehyde (pH 8.0-8.5), 3% hydrogen peroxide, or 1% peracetic acid(Website 6).
  2. Biosafety information for Bacillus anthracis
    1. Level: Biosafety Level 3(Website 2, Website 3).
    2. Precautions: Biosafety Level 3 practices, containment equipment, and facilities are recommended for work involving production quantities or concentrations of cultures, and for activities with a high potential for aerosol production including suspect powders. The agent may be present in blood, skin lesion exudates, cerebrospinal fluid, pleural fluid, sputum, and rarely, in urine and feces. Direct and indirect contact of the intact and broken skin with cultures and contaminated laboratory surfaces, accidental parenteral inoculation, and rarely, exposure to infectious aerosols are the primary hazards to laboratory personnel(Website 3).
    3. Disposal: Because sporulation of B. anthracis requires oxygen and therefore does not occur inside a closed carcass, regulations in most countries forbid postmortem examination of animals when anthrax is suspected. If heat treatment or incineration of the contaminated material is possible, this should be done in preference to chemical decontamination and disinfection. For certain materials or animal by-products, irradiation with gamma rays or particle bombardment or fumigation with a gaseous disinfectant such as ethylene oxide may be appropriate. In fatal human cases, postmortem should be discouraged; cremation is preferable to burial where local custom permits. Bedding and contaminated materials should be bagged and incinerated, autoclaved (121+1 degree C core temperature for 30 min) or fumigated as appropriate. Whether room fumigation is necessary will depend on the perceived level of contamination in the room where the patient died. Immersion in 4% formaldehyde (10% formalin) for >12 hours is an alternative but full penetration of the fluids must be ensured. Disinfection of contaminated surfaces involves a three-step approach aimed at (i) preliminary disinfection, (ii) cleaning, and (iii) final disinfection. Preliminary disinfection: One of the following disinfectants may be used in amounts of 1-1.5 liters per square meter for an exposure time of 2 hours: 10% formaldehyde (approximately 30% formalin), 4% glutaraldehyde (pH 8.0-8.5), or a high pressure cleaner may be used but, to avoid spreading the contamination, the pressure should not exceed 10 bar. Cleaning: Where practical, cleaning of all surfaces should be done by straightforward washing and scrubbing using ample hot water. The operator should wear protective clothing, face and hands included. Cleaning should be continued till the original colors and surfaces are restored and the waste water is free of dirt particles. At the end of the process, residual water should be removed and the surfaces dried. High pressure cleaners are again discouraged because of the greater potential to spread the contamination through aerosols. If used, however, the water jet should be applied at a pressure of 80-100 bar delivering 13-15 litres/minute. Final disinfection: One of the following disinfectants should be applied at a rate of 0.4 liters per square meter for an exposure time of at least 2 hours: 10% formaldehyde (approximately 30% formalin), 4% glutaraldehyde (pH 8.0-8.5), 3% hydrogen peroxide, or 1% peracetic acid(Website 6).
Culturing Information
  1. General Culturing Information (Inglesby et al., 2002, Website 2, Shafazand et al., 1999, Andrews et al., 2002, Website 6, Website 3):
    1. Description: Spores germinate when they enter an environment rich in amino acids, nucleosides, and glucose, such as that found in the blood or tissues of an animal or human host. The rapidly multiplying vegetative bacilli will only form spores after local nutrients are exhausted, such as when anthrax-infected body fluids are exposed to ambient air(Inglesby et al., 2002).
    2. Medium: Rapid growth on sheep blood agar (SBA); Grows well on blood agar plates within 18-24 hours. Growth on SBA or equivalent medium forms spores which are not encapsulated. SBA can be used for cutaneous swab , stool, and sputum specimens. For primary growth media, B. anthracis grows well on SBA but does not grow on MacConkey agar (MAC).MacConkey agar can be used for cutaneous swab and stool samples. Phenyl ethyl alcohol agar (PEA) can be used for stool samples. Chocolate agar (CA) can be used for sputum samples. Tryptic soy broth (TSB) can be used for wet mount procedure.Conventional broth or agar MIC (minimum inhibitory concentration) determinations currently appear to be the most reliable methods for determining susceptibility of B. anthracis(Shafazand et al., 1999, Andrews et al., 2002, Website 6, Website 10, Website 2).
    3. Optimal Temperature: The optimal growth temperature for the organism is 35 degrees celcius. Spores grow readily on all ordinary laboratory media at 37 degrees celcius. The organism generally exists in the endospore form in nature; germination of spores outside an animal host may occur when the temperature is between 8 degrees celcius and 45 degrees celcius and presence of adequate nutrients(Shafazand et al., 1999, Inglesby et al., 2002, Website 2).
    4. Upper Temperature: When grown above 45 degrees celcius, the bacteria becomes attenuated or avirulent due to loss of the capsule(Shafazand et al., 1999).
    5. Lower Temperature: 12 degrees celcius(Shafazand et al., 1999).
    6. Optimal Humidity: Germination of spores outside an animal host may occur when the relative humidity is >95%(Website 2).
    7. Optimal pH: Germination of spores outside an animal host may occur when the pH between 5 and 9(Website 2).
    8. Upper pH: pH 7.4(Shafazand et al., 1999).
    9. Lower pH: pH 7.0(Shafazand et al., 1999).
    10. Note: Comma-shaped projections may give "Medusa head" appearance on Sheep Blood Agar. Colonies have been described as having a ground-glass appearance and the consistency of beaten-egg whites. Colonies are 2 to 5 mm in diameter after 16 to 18 hours of incubation. Forms mucoid capsule when grown on agar with sodium bicarbonate and incubated in carbon dioxide -enriched atmosphere; capsule can be visualized with India ink preparation. On culture, plated colonies are usually large (4 to 5 mm), opaque, and irregular, with characteristic comet tail protrusions. Disturbed sections of the colony often stand up like "beaten egg whites". Oxygen is needed for sporulation but not germination of spores. Spores grow in culture plates, soil, and tissue of dead animals. They do not form in the blood or tissues of infected living animals(Website 2, Shafazand et al., 1999, Website 3, Website 10).
Epidemiology Information:
  1. Outbreak Locations:
    1. Anthrax in animals is hyperendemic or endemic in the following areas of the world: most areas of the Middle East, most areas of equatorial Africa, Mexico, Central Africa, Chile, Argentina, Peru, Bolivia, certain Southeast Asian countries (e.g. Burma (Myanmar), Vietnam, Cambodia, Thailand), Papua New Guinea, China, and some Mediterranean countries. In most of the rest of the world, anthrax occurs only sporadically. In the United States (US), outbreaks in animals have occurred since 1990 in the Midwest (Kansas, Nebraska, North Dakota, South Dakota, Missouri); in the West (California, Nevada); and in Texas and Oklahoma. In the US, the microorganism remains endemic in the soil of Texas, Oklahoma, and the lower Mississippi valley(Website 2, Shafazand et al., 1999).
    2. An estimated 2,000 to 20,000 human cases of anthrax occur globally each year(Website 2).
    3. Early descriptions of anthrax date to 3,500 years ago; anthrax may have been responsible for two of the plagues that afflicted Egypt in 1491 BC(Shafazand et al., 1999).
    4. A major outbreak involving nearly 10,000 cases (most of them cutaneous infection) occurred in Zimbabwe during the late 1970s and early 1980s. An epizootic outbreak in cattle occurred at that time in the same area(Website 2).
    5. An outbreak involving nine cases (five inhalational and four cutaneous) occurred in 1957 in the United States in a New Hampshire goat-hair processing plant. This was the last recognized outbreak of naturally occurring infection in this country(Website 2).
    6. An outbreak of oropharyngeal anthrax involving 24 cases occurred in Thailand in 1982 following consumption of contaminated meat. Oropharyngeal disease is an unusual manisfestation of infection, which makes this outbreak of particular interest(Website 2).
    7. In 1941, the British conducted limited experiments by realeasing spores of anthrax on Gruinard Island near Scotland. During 1943 and 1944, an estimated 4 x 10e14 anthrax spores were dispersed on the island through explosive means. Spores were still detectable more than 40 years later. The viable anthrax spores persisted until the island was decontaminated with formaldehyde and seawater in 1986(Shafazand et al., 1999, Website 2).
    8. The US experimented with biological weapons including anthrax spores in 1950s and 1960s(Shafazand et al., 1999).
    9. In 1979, a large epidemic of anthrax occurred in the former Soviet Union at Sverdlosk, an industrial city of 1.2 million people just east of the Ural Mountains. The accidental release of Bacillus anthracis spores from a nearby military facility was responsible for the epidemic, which caused at least 77 clinical cases and 66 deaths(Shafazand et al., 1999, Website 2, Website 31).
    10. Between 1984 and 1993, only three cases of cutaneous anthrax were reported to the Centers for Disease Control. A fatal case occurred in 1976; when a home craftsman died of inhalational anthrax after working with yarn imported form Pakistan. Approximately 130 cases occurred annually in the US in early 1900s. The incidence has gradually declined over time, with less than 10 cases reported each year since the early 1960s(Shafazand et al., 1999, Website 2).
    11. About 95% of naturally occurring cases in the United States (US) are cutaneous and 5% are inhalational. Gastrointestinal infection has not been recognized in the country. Only 18 cases of naturally occurring inhalational anthrax were reported in the US during the 20th century, with the most recent case in 1976. 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. Since 1990, only two cases of naturally occurring infection have been reported in the US (one in 1992 and one in 2000); both patients had cutaneous disease. The latter case occurred in North Dakota and resulted from agricultural exposure(Website 2).
    12. In 2001, an outbreak in the United States involved direct exposure to mail that was deliberately contaminated with anthrax spores. At least five contaminated letters were sent and one was reported to contain 2g of powder, with 100 billion to 1 trillion anthrax spores per gram. Twenty-two confirmed or suspect cases of anthrax infection resulted. Eleven of these were inhalational cases, of whom 5 died; 11 were cutaneous cases (7 confirmed, 4 suspected)(Website 2, Inglesby et al., 2002).
  2. Transmission Information:
    1. From: Grazing Herbivores (at lifecycle stage: Vegetative Phase, Intense Growth Phase) , To: Homo sapiens (at lifecycle stage: Vegetative Phase, Intense Growth Phase) (Dixon et al., 1999, Website 2, Website 7)
      Mechanism: Exposure to infected animals or contaminated animal products. Anthrax is predominantly a disease of animals. Contact with infected tissues of dead animals (e.g. butchering, preparing contaminated meat), which generally leads to cutaneous anthrax, consumption of contaminated undercooked meat, which can lead to gastrointestinal or oropharyngeal 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(Website 2).
    2. From: Humans. (at lifecycle stage: Vegetative Phase, Intense Growth Phase) , To: Humans. (at lifecycle stage: Vegetative Phase, Intense Growth Phase) (Website 2)
      Mechanism: The agent may be present in blood, skin lesion exudates, cerebrospinal fluid, pleural fluid, sputum, and rarely, in urine and feces. Reported rarely with cutaneous anthrax, but has not been recognized with gastrointestinal or inhalational disease(Website 2, Dixon et al., 1999, Website 3).
  3. Environmental Reservoir:
    1. Contaminated environment(Website 35, Website 7, Inglesby et al., 2002, Website 2, Shafazand et al., 1999):
      1. Description: Contaminated environment reservoir includes soil, air, or animal carcass. B. anthracis spores remain prevalent in soil samples throughout the world and cause anthrax cases among herbivores annually. 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 (Inglesby et al., 2002; Shafazand et al., 1999; Website 2).
      2. Survival: The results of studies of agricultural outbreaks have suggested that conditions for multiplication are favorable when the soil pH is >6.0 and rich in organic matter. Major changes in the soil microenvironment, such as drought or rainfall, enhance the sporulation. Spores germinate and form vegetative cells in environments rich in nutrients (e.g. glucose, amino acids, nucleosides). Vegetative bacteria have poor survival outside of an animal or human host; colony counts decline to being undetectable within 24 hours following inoculation into water. Conversely, vegetative cells form spores when nutrients in the environment are exhausted. Spores have been shown to survive in the environment for decades (more than 40 years). The environmentally hardy properties of the spore allow it to survive for decades in ambient conditions. Endospores are resistant to drying, heat, ultraviolet light, gamma radiation, and some disinfectants (Inglesby et al., 2002; Website 2).
  4. Intentional Releases:
    1. Intentional Release Information(Website 2):
      1. Description: Aerosol release of weaponized spores is the most likely mechanism for use of anthrax as a biological weapon. Although there is no formal definition of weaponized anthrax, weaponization generally involves: small particle size, high concentration of spores, treatment to reduce clumping, neutralization of the electrical charge, and use of antimicrobial-resistant strains by genetic modification of the organism to increase virulence or escape vaccine protection(Website 2).
      2. Emergency Contact: Anthrax is a notifiable disease in all 50 states and is Federally reportable. According to bioterrorism guidelines put forth by Centers for Disease Control (CDC), any case of inhalational (pulmonary) anthrax in the United States should also be reported to the Federal Bureau of Investigation, as it is assumed that inhalational anthrax is so rare in the United States that any case must be due an intentional release. Immediately notify the local or state public health department, local hospital epidemiologist, and local or state public health laboratory. If you are at home, then report the incident to local police. If you are at work, then report the incident to local police, and notify your building security official or an available supervisor.The Laboratory Response Network (LRN) has been developed in the United States to coordinate clinical diagnostic testing for bioterrorism events. LRN has been established through a collaboration of the Association of Public Health Laboratories and the CDC. The network is organized into four laboratory levels (A, B, C, and D). The LRN can be accessed by contacting state public health laboratories. Level A laboratories can perform standard initial tests to rule out (but not definitively identify) B. anthracis, and include Clinical Laboratory Improvement Act (CLIA)-certified clinical laboratories with BSL-2 safety practices. Level B laboratories have core capacity for agent isolation and confirmatory testing and include most state public health laboratories. Level C laboratories have advanced capacity for rapid identification and include selected public health, federal, and academic laboratories. Level D laboratories have the highest level of containment (BSL-4) and expertise in the diagnosis of rare and dangerous biologic agents and include specialized Federal laboratories(Website 5, Inglesby et al., 2002, Website 2, Website 10, Website 23).
      3. Delivery Mechanism: Aerosolized spores may be delivered by missiles, bomblets, artillery fires, point release, or airborne line release. Contamination of food and water may be used. Mail can also be an effective vehicle for disseminating anthrax spores(Pannucci et al., 2002, Website 2, Website 7).
      4. Containment: A suspicious, unopened letter or package should be marked with a threatening message such as "anthrax": Do not shake or empty the contents of any suspicious envelope or package. PLACE the envelope or package in a plastic bag or some other type of container to prevent leakage of contents. If you do not have any container, then COVER the envelope or package with anything (e.g., clothing, paper, trash can, etc.) and do not remove this cover. Then LEAVE the room and CLOSE the door, or section off the area to prevent others from entering (i.e., keep others away). WASH your hands with soap and water to prevent spreading any powder to your face. Envelope with powder and powder spills out onto surface: DO NOT try to CLEAN UP the powder. COVER the spilled contents immediately with anything (e.g., clothing, paper, trash can, etc.) and do not remove this cover! Then LEAVE the room and CLOSE the door, or section off the area to prevent others from entering (i.e., keep others away). WASH your hands with soap and water to prevent spreading any powder to your face.REMOVE heavily contaminated clothing as soon as possible and place in a plastic bag, or some other container that can be sealed. This clothing bag should be given to the emergency responders for proper handling. SHOWER with soap and water as soon as possible. Do Not Use Bleach Or Other Disinfectant On Your Skin. Question of room contamination by aerosolization: for example: small device triggered, warning that air handling system is contaminated, or warning that a biological agent released in a public space. Turn off local fans or ventilation units in the area. LEAVE area immediately. CLOSE the door, or section off the area to prevent others from entering (i.e., keep others away). SHUT down air handling system in the building, if possible. If possible, list all people who were in the room or area, especially those who had actual contact with the powder. Give this list to both the local public health authorities so that proper instructions can be given for medical follow-up, and to law enforcement officials for further investigation(Website 23, Website 2).
Diagnostic Tests Information
  1. Organism Detection Test:
    1. Gram Staining (Website 2, Shafazand et al., 1999):
      1. Description: Direct examination of bacterial micromorphology may demonstrate broad encapsulated gram-positive rods (approved for Level A laboratories). Gram stain can be performed on appropriate clinical specimens (eg, vesicular fluid, swabs from cutaneous lesions, cerebrospinal fluid (CSF), pleural fluid, peritoneal fluid, sputum or oropharyngeal ulcers). Two positive Gram stain findings, one from CSF and buffy-coat smear, recently were reported in cases of inhalational anthrax. Gram's stains of the vesicular fluid may show rare leukocytes and Gram-positive rods. The organism appears to be present in large numbers in advanced cases, which suggests the probable utility of direct smears in those presenting with advanced disease. Endospores and free spores can be seen in cultures and generally are not seen in direct microscopic examination of patient samples(Website 2, Shafazand et al., 1999, Website 10).
      2. False Negative: The sensitivity of the Gram stain and other biologic stains (eg, H&E, silver stain) for direct detection of anthrax cells in infected tissues or blood has not been established(Website 2).
    2. Buffy-coat Smears using Light Microscopy (Website 2):
      1. Description: Buffy-coat smears may be positive in patients with bacteremia. The specific usefulness of buffy coat for early diagnosis of bacteremia has been out of favor owing to early reports of low predictive value, but its use for anthrax has not been adequately studied(Website 2).
    3. India ink staining (Website 2, Website 10):
      1. Description: India ink staining can be performed for capsule visualization directly on clinical specimens. Used to improve visualization of encapsulated B. anthracis in clinical samples such as blood, blood culture bottles, or cerebrospinal fluid. The capsule will appear as a well-defined clear zone around the cells(Website 2).
      2. False Negative: A negative result should not be used to rule out B. anthracis. Interpretation of results requires trained/experienced staff(Website 10).
    4. Direct immunofluorescent assays (DFA) (Website 2):
      1. Description: Direct immunofluorescent assays (DFA) for cell-wall-associated polysaccharide and capsule produced by vegetative cells (demonstration of both antigens provides confirmatory identification). Included in Standard confirmatory testing (performed by Level B or C laboratories)(Website 2).
    5. Biochemical tests to differentiate B anthracis :
      1. Description: B. anthracis is characterized by the absence of hemolysis on sheep blood agar, lack of motility, absence of salicin fermentation, gelatin hydrolysis, and lack of growth on phenylethyl alcohol medium. Susceptible to lysis by gamma phage (provides confirmatory testing when demonstrated concomitantly with the presence of a capsule). Included in standard confirmatory testing (performed by Level B or C laboratories)(Shafazand et al., 1999, Website 2).
    6. Nasal swabs :
      1. Description: Nasal swab cultures were used in the 2001 United States outbreak as an epidemiologic tool to assess exposure to inhalational anthrax; however, nasal swabs are not recommended for use in the clinical setting. According to CDC, 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, or 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(Website 2).
    7. Culture of clinical specimens :
      1. Description: Culture of clinical specimens is the "gold standard" for diagnosis of anthrax (initial isolation approved for Level A laboratories). Culture a stool and blood sample for diagnosis of gastrointestinal anthrax. Culture should be performed using standard 5% sheep blood agar. Motility is measured by wet mount (working in a BSL-2 cabinet) or by motility test medium. Spores are visualized by Gram stain from cultures incubated without CO2. India ink for demonstration of capsule can be used on cultures grown on media supplemented with sodium bicarbonate. Suspicious isolates should be forwarded to a Level B or C laboratory for confirmatory identification. Blood culture sensitivity is compromised when samples are collected after administration of even one or two doses of antibiotics; therefore, cultures should be obtained before antibiotic therapy is initiated. If antibiotics have been administered, alternate diagnostic methods should be considered. The most useful microbiologic test is the standard blood culture, which should show growth in 6 to 24 hours but cultures must be obtained before initiation of antibiotic therapy(Website 2, Inglesby et al., 2002).
    8. Additional test procedures for visualizing spores :
      1. Description: Standard confirmatory testing performed by Level B or C laboratories include wet mount and malachite green stain(Website 2).
    9. Antimicrobial susceptibility testing :
      1. Description: Performed at Level C and D laboratories, should be performed on clinical isolates. National Committee for Clinical Laboratory Standards (NCCLS) standard protocols for broth microdilution have been used with staphylococcal breakpoints. Concerns about penicillin use hinge on potential inducible penicillinases and poor penetration into macrophages(Website 2).
    10. Chest Radiograph :
      1. Description: Widened mediastinum, infiltrates, pleural effusion(Website 2, Inglesby et al., 2002).
    11. Thoracentesis :
      1. Description: Hemorrhagic pleural effusions(Inglesby et al., 2002).
    12. Chest computed tomographic scan :
      1. Description: Hyperdense hilar and mediastinal nodes, mediastinal edema, infiltrates, pleural effusion(Inglesby et al., 2002).
  2. Immunoassay Test:
    1. A two-step enzyme-linked immunosorbent assay (ELISA) (Website 2):
      1. Description: A two-step enzyme-linked immunosorbent assay (ELISA), which measures antibody directed against protective antigen (PA). Serologic testing (performed at Level D laboratories) can be used for retrospective diagnosis. Development of measurable antibodies in recent cases required 10 to 16 days after onset of overt disease, but peak IgG levels may not be seen until 40 days after symptom onset. Serum should be collected during acute illness, and 14, 28, 42, and 60 days after onset. Requests for serologic testing can be made through the LRN or by contacting CDC(Website 2).
      2. False Positive: The first-stage assay has been reported to be 80% specific; the specificity increases after competitive blocking by PA. This test is still considered investigational(Website 2).
      3. False Negative: The first-stage assay has been reported to be 98.6% sensitive. This test is still considered investigational(Website 2).
    2. Time-resolved fluorescence (TRF) assay :
      1. Description: Used for rapid detection of B anthracis antigens. Is under investigation at level C and D laboratories(Website 2).
      2. False Positive: The specificity of these tests is generally unknown or unpublished owing to the scarcity of cases. In addition, access to certain detection protocols is controlled through the Laboratory Response Network(Website 2).
      3. False Negative: The sensitivity of these tests is generally unknown or unpublished owing to the scarcity of cases. In addition, access to certain detection protocols is controlled through the Laboratory Response Network(Website 2).
    3. Immunohistochemistry (IHC) (Website 2):
      1. Description: A sensitive and specific method for detection of B anthracis in affected tissues utilizing antibody directed against cell wall and capsule components. This test is unaffected by prior administration of antibiotics or formalin fixation. IHC used in pathologic examinations of experimentally infected animals and in recent human cases was more sensitive than standard staining methods. IHC is not widely available, but requests for testing can be made through the LRN. Generally performed by Level C or D laboratories. Consider punch biopsy for immunohistochemical testing if the patient has received antibiotics or has a negative Gram stain and culture, despite high index of suspicion for anthrax(Website 2).
      2. False Positive: The specificity of these tests is generally unknown or unpublished owing to the scarcity of cases. In addition, access to certain detection protocols is controlled through the Laboratory Response Network(Website 2).
      3. False Negative: The sensitivity of these tests is generally unknown or unpublished owing to the scarcity of cases. In addition, access to certain detection protocols is controlled through the Laboratory Response Network(Website 2).
    4. Commercial/Investigational anthrax-specific tests for environmental sampling. :
      1. Description: "Smart ticket" is commercially available. Detection technology is Embedded flow immunochromatography. The test, targets spore antigen. Turnaround time is <5 minutes. Currently used on surfaces and powders. NO published validation. Detection limits are unknown. Centers for Disease Control does not have enough scientific data to recommend the use of these assays. Until validation testing is complete and guidelines for effective use are developed, PCR- or immune-based assay results for B anthracis should not be used alone, but should be confirmed with samples analyzed by culture methods to make public health decisions(Website 2, Website 9).
    5. Commercial/Investigational anthrax-specific tests for environmental sampling. :
      1. Description: "Guardian bio-threat alert test-strips" are commercially available. Detection technology is Lateral flow immunochromatography. The test target is single, unspecified. Turnaround time is <15 minutes. Currently used in the environment. NO published validation. Detection limits are unknown. Until validation testing is complete and guidelines for effective use are developed, PCR- or immune-based assay results for B anthracis should not be used alone, but should be confirmed with samples analyzed by culture methods to make public health decisions(Website 2, Website 9).
    6. The Canary :
      1. Description: The Canary, which is being developed at the Massachusetts Institute of Technology Lincoln Laboratory, is an innovative example of the devices that detect pathogens based on unique surface molecules. The sensors consist of B cells of the immune system that have been genetically altered to emit light when their calcium levels change(Website 12).
    7. The Cyranose detection system :
      1. Description: Anthrax spores are packed full of dipicolinic acid (DPA). Molecules that fluoresce when bound to DPA have shown promise in chemically based anthrax detectors. The Cyranose detection system made by Cyrano Sciences in Pasadena, Calif., could possibly "smell" the presence of DPA in an air sample laced with anthrax spores(Website 12).
    8. Nano-scale Electronic Chips for DNA Detection :
      1. Description: Researchers at Northwestern University in Evanston, Illinois, report creating simple nano-scale electronic chips that can detect DNA from anthrax and other organisms in minutes. An unusual salt concentration-dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. The chips appear to be vastly more sensitive than other high-speed techniques. And, unlike many such tests, they don't rely on the polymerase chain reaction. The method was used to detect target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of ~ 100,000:1(Website 25).
  3. Nucleic Acid Detection Test: