Advice to Laboratory Personnel

These guidelines provide background information and guidance to clinical laboratory personnel in recognizing Bacillus anthracis in a clinical specimen. They are NOT
intended to provide training for laboratory identification of B. anthracis. Clinical lab 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 State Public Health Laboratory.

Any suspected isolate of B. anthracis must be reported to the State Public Health Laboratory IMMEDIATELY. The State Public Health Laboratory is available for
consultation or testing 24 hours per day and can be reached through the Department
of Health Communicable Disease Epidemiology 24-hour emergency number. Following
an appropriate consultation with the State Public Health Lab regarding a suspected
isolate of B. anthracis, communication should then be established with the local FBI
field office for possible law enforcement involvement.

A. 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:

• 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 I 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 the supervisor and physician
    
    
• If exposure to contaminated sharps occurs:

• Follow standard reporting procedures for sharps exposures

• Thoroughly irrigate site with soap and 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 14 days or Doxycycline 100 mg by mouth twice a day for 14 days.
  

• Notify the State Department of Health (SDOH) and the State Public Health Laboratory (SPHL).

B. Role of the clinical laboratory

• Perform laboratory tests to rule out 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 one is unable to rule out immediately to the SDOH and SPHL

C. Presumptive identification of Bacillus anthracis

• Direct smears from clinical specimens
  

• Encapsulated broad rods in short chains, 2-4 cells. Gram stain can demonstrate clear zones (capsule) around rods. An India ink stain should be used to further visualize the capsule microscopically.
  

• B. anthracis will not usually be present in clinical specimens until late in the course of the disease
  

• Smears from sheep blood agar or other routine nutrient medium
  

• Non-encapsulated broad rods in long chains
  

• When grown on nutrient agar in presence of 5% CO2 or other basal media supplemented with 0.8% sodium bicarbonate, virulent strains will yield heavily encapsulated rods (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 m
  

• Oval, central to subterminal spores: 1 x 1.5 m with no significant swelling of cell
  

• Spores usually 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, spore-forming rod
  
  

  If B. anthracis is suspected
  

• The health care provider, local law enforcement, 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 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 D.
  

D. Decontamination

• Effective sporicidal decontamination solutions approved for hospital use
  

• Commercially-available bleach, 0.5% hypochlorite (a 1:10 dilution of household bleach); may be corrosive to some surfaces
  

• 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 is performed
  
  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

E. Disposal

Incinerate or steam-sterilize cultures, infected material, and suspect material.

F. 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, and place biohazard label (see diagram)

If the specimen is a clinical specimen, place biohazard label on the specimen receptacle, wrap the receptacle with an absorbent material (see diagram)

Place the bag or specimen receptacle into a leak-proof container with a tight cover that is labeled "biohazard".

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.

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.

Both containers should meet state and federal regulations for transport of hazardous material, and be properly labeled.

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