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Anthrax information for laboratory personnel

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Anthrax Information for Laboratory Word Version

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This document provides background information and guidance to clinical laboratory personnel in recognizing Bacillus anthracis in a clinical specimen. It is NOT intended to provide training for laboratory identification of B. anthracis.

Clinical laboratory personnel will most likely be the first ones to perform preliminary testing on clinical specimens from patients who may have been intentionally exposed to the organism, and will play a critical role in facilitating rapid identification of B. anthracis.

Laboratory confirmation of B. anthracis should be performed at the Washington State Department of Health Public Health Laboratory.

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Any confirmed or suspected isolate of B. anthracis must be reported to your local health department or Washington State Department of Health (DOH) IMMEDIATELY.

The Washington State Public Health Laboratory is available for consultation or testing 24 hours per day and can be reached through the DOH, Communicable Disease Epidemiology 24-hour emergency number: 1-877-539-4344.

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Bacillus anthracis

• A large, facultative anaerobe, gram-positive, non-motile, encapsulated, spore-forming bacillus
• Forms ground-glass colonies sometimes with comma-shaped protrusions ("Medusa head" colonies)
• Non-hemolytic (weak hemolysis may be observed under areas of confluent growth in aging cultures and should NOT be confused with real ß-hemolysis)

Human Infection

• Occurs naturally in the environment, but transmission to humans is usually through occupational contact with infected animals or animal products
• Naturally-occurring anthrax usually involves the skin (cutaneous anthrax); rarely involves the lungs (inhalation anthrax) or gastrointestinal tract (gastrointestinal anthrax)
• Cutaneous anthrax occurs when the bacterium (vegetative cells) or spores enter a cut or abrasion on the skin, such as when handling contaminated wool, hides, leather, or hair products (especially goat hair) from infected animals.
• Inhalation anthrax results from inhaling B. anthracis spores, and is the most likely illness following an intentional aerosol release of B. anthracis.
• Person-to-person transmission of inhalational anthrax has not been documented.
• Expedient laboratory diagnosis of the initial cases of anthrax in a bioterrorist incident is essential to identifying other cases or exposures for early treatment or prophylaxis

Clinical Presentation of Inhalational Anthrax

• Incubation period of 1-6 days, may be up to 60 days (depending on number of inhaled spores)
• Disease onset is gradual and nonspecific:
  • Initial - fever, malaise, fatigue, nonproductive cough, and mild chest discomfort
  • Initial symptoms may be followed by short period of improvement (hours to days)
  • Abrupt development of severe symptoms: respiratory distress with labored breathing, sweating, high pitched breathing sounds, and bluish discoloration of skin
• Shock and death usually occur within 24-36 hours after onset of respiratory distress,
• Mortality approaches 100% despite aggressive treatment after onset of severe symptoms

HANDLING LABORATORY SPECIMENS (possible B. anthracis)

• Risk to lab personnel from handling clinical lab specimens with B. anthracis is low, but it is important to minimize possible exposures to personnel as well as prevent contamination of the lab. Standard lab practices are sufficient. If B. anthracis is suspected, these precautions should be followed:
  • Wear gloves and protective gowns when handling clinical specimens
  • Wash immediately with soap and water if there is direct contact with a clinical or lab specimen
  • Avoid splashing or creating aerosols
  • Perform lab tests in an annually certified Class II Biological Safety Cabinet; if that is not possible, then use standard lab protective eyewear and a mask
  • Blood cultures should be maintained in a closed system (blood culture bottles)
  • Keep culture plates covered at all times; minimize exposure when extracting specimens for testing
  • Work on a smooth surface that can be cleaned easily and wipe with bleach regularly.
• If lab or clinical specimen material is spilled or splashed onto lab personnel:
  • Remove outer clothing carefully while still in the lab and place in a labeled, plastic bag
  • Remove rest of clothing in the locker room and place in a labeled, plastic bag
  • Shower thoroughly with soap and water in the locker room
  • Inform your supervisor and physician
• If exposure to contaminated sharps occurs:
  • Follow standard reporting procedures for sharps exposures
  • Thoroughly irrigate site with soap and water and apply a disinfectant solution such as a 0.5% hypochlorite solution. DO NOT SCRUB AREA.
  • Promptly begin prophylaxis for cutaneous anthrax
  • Recommended treatment for cutaneous exposure: prophylaxis with Ciprofloxacin 500 mg by mouth twice a day for 7-10 days or Doxycycline 100 mg by mouth twice a day for 7-10 days.
  • Notify the State Department of Health (DOH), Public Health Laboratory (PHL)

   

ROLE OF THE CLINICAL LABORATORY

• Perform laboratory tests for presumptive identification of B. anthracis on clinical specimens
• Raise your index of suspicion for B. anthracis when the clinical picture (provided by the clinician) involves a rapidly progressive respiratory illness of unknown cause in a previously healthy person
• Refer any suspected isolates IMMEDIATELY to the DOH, PHL

PRESUMPTIVE IDENTIFCATION OF Bacillus anthracis

• Direct smears from clinical specimens
  • Encapsulated broad rods in short chains, 2-4 cells. India Ink will demonstrate capsule (Gram stain will not)
  • B. anthracis will not usually be present in clinical specimens until late in course of the disease
• Smears from sheep blood agar or other routine nutrient medium
  • Non-encapsulated broad rods in long chains
  • Encapsulated bacilli will only grow in nutrient agar supplemented with 0.8% sodium bicarbonate in the presence of 5% CO2 (Note: this procedure is performed in Level B laboratories)

   

Gram Stain Morphology of B. anthracis

• Broad, gram-positive rod: 1-1.5 x 3-5 µ
• Oval, central to subterminal spores: 1 x 1.5 µ with no significant swelling of cell
• Spores usually are NOT present in clinical specimens unless exposed to atmospheric O2

Colonial characteristics of B. anthracis

• Bacillus anthracis can be isolated primarily from blood, sputum, CSF, vesicular fluid or eschar, and stool (if gastrointestinal anthrax).
• After incubation on a blood agar plate for 15-24 hours at 35-37o C, well isolated colonies are 2-5 mm in diameter; heavily inoculated areas may show growth in 6-8 hours
• Gray-white, flat or slightly convex colonies are irregularly round, with edges that slightly undulate, and have "ground glass" appearance
• Often have comma-shaped protrusions from colony edge ("Medusa head" colonies)
• Tenacious consistency (when teased with a loop, the growth will stand up like a beaten egg white)
• Non-hemolytic (weak hemolysis may be observed under areas of confluent growth in aging cultures and should NOT be confused with real ß-hemolysis) )
• Will not grow on MacConkey agar
• Non-motile

Presumptive Identification key for Bacillus anthracis

• Non-hemolytic
• Non-motile
• Encapsulated (requires India ink to visualize the capsule)
• Gram-positive, sporeforming rod

If B. anthracis is suspected

• The health care provider, and the local and State DOH should be notified immediately
• Do not perform further tests once you have reason to suspect B. anthracis. The specimen should be transported to the DOH as directed (see Packaging and Transporting Protocol)
• Level B, or C laboratories (State DOH) will perform the following presumptive and confirmatory tests:
  • lysis by gamma phage
  • capsule detection (by DFA)
  • detection of cell-wall polysaccharide antigen by DFA

DECONTAMINATION
Effective sporicidal decontamination solutions

• Commercially-available bleach, 0.5% hypochlorite (a 1:10 dilution of household bleach)
• Rinse off the concentrated bleach to avoid its caustic effects

Surfaces and non-sterilizable equipment

• Work surfaces should be wiped before and after use with a sporicidal decontamination solution
• Routinely clean non-sterilizable equipment with a decontamination solution

Contaminated instruments (pipettes, needles, loops, micro slides)

• Soak in a decontamination solution until autoclaving

Accidental spills of material known or suspected to be contaminated with B. anthracis

• For contamination involving fresh clinical samples:
  • Flood with a decontamination solution
  • Soak five minutes before cleaning up
• For contamination involving lab samples, such as culture plates or blood cultures, or spills occurring in areas that are below room temperature:
  • Gently cover spill, then liberally apply decontamination solution
  • Soak for one hour before cleaning up
  • Any materials soiled during the clean-up must be autoclaved or incinerated

DISPOSAL

• Incinerate or steam sterilize cultures, infected material, and suspect material

PACKAGING and TRANSPORTING PROTOCOL
Packaging and labeling specimens is the same as for any infectious substance

• If the specimen is a dry powder or paper material, place it in a plastic zip-lock bag, place biohazard label, and follow steps 1-4 (see diagram)
• If the specimen is a clinical specimen, place biohazard label on the specimen receptacle, wrap the receptacle with an absorbent material, and follow steps 1-4 (see diagram)
  1. Place the bag or specimen receptacle into a leak proof container with a tight cover that is labeled "biohazard."
  2. Place this container into a second leak proof container with a tight cover that is labeled "biohazard." The size of the second container should be no larger than a one-gallon paint can.
     • For a clinical specimen, an ice pack (not ice) should be placed in the second container to keep the specimen cold
     • If the specimen is not a clinical specimen, but is paper or powder, the ice pack should be omitted
  3. Place the second container into a third leak proof container with a tight cover that is labeled "biohazard." The third container should be no larger than a five-gallon paint can.
  4. Both containers should meet state and federal regulations for transport of hazardous material, and be properly labeled.

Transporting specimens to the DOH Public Health Lab

• Will be coordinated with the DOH Public Health Lab at 1-877-539-4344
• Local FBI personnel may be utilized to transport specimens if bioterrorism is suspected
• In cases where the specimen is shipped by commercial carrier, ship according to State and Federal shipping regulations

HELPFUL WEBSITES

Links to external resources are provided as a public service and do not imply endorsement by the Washington State Department of Health.

REFERENCES

1. Laboratory Protocols for bioterrorism response laboratories for the identification of Bacillus anthracis. CDC BT public web site
2. Inglesby TV, Henderson DA, Barlett JG, Ascher MS, et al. Anthrax as a biological weapon: Medical and public health management (consensus statement). JAMA, May 12, 1999;281(18):1735-1745.
3. No authors listed. Biological warfare and terrorism: the military and public health response. U.S. Army, Public Health Training Network, Centers for Disease Control, Food and Drug Administration, Satellite broadcast, September 21-23, 1999.

This material has been developed by the Washington State Department of Health in collaboration with the Centers for Disease Control and Prevention. Reuse or reproduction of this material is authorized.
Information updated May 11, 2001.

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