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 Public Health - Seattle & King County (206-296-4774; after hours 206-726-2128) and referred to the WA 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 DOH, Communicable Disease Epidemiology 24-hour emergency number (206- 361-2914).

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:
	>	IFollow 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 Public Health - Seattle & King County (206-296-4774; after hours 206-726-2128).

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 WA DOH Public Health Lab and report to Public Health - Seattle & King County (206-296-4774; after hours 206-726-2128).

Presumptive Identification 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 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, 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 WAState Public Health Labas 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

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, and place biohazard label
	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
	If the specimen is not a clinical specimen, but is paper or powder, the ice pack should be omitted
	Both containers should meet state and federal regulations for transport of hazardous material, and be properly labeled.

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