Many facilities in communities around the country have received anthrax threat letters. Most were empty envelopes; some have contained powdery substances. The purpose of these guidelines is to recommend procedures for handling such incidents.

1. Anthrax organisms can cause infection in the skin, gastrointestinal system, or the lungs. To do, so the organism must be rubbed into abraded skin, swallowed, or inhaled as a fine, aerosolized mist. Disease can be prevented after exposure to the anthrax spores by early treatment with the appropriate antibiotics. Anthrax is not spread from one person to another person.

2. For anthrax to be effective as a covert agent, it must be aerosolized into very small particles. This is difficult to do, and requires a great deal of technical skill and special equipment. If these small particles are inhaled, life-threatening lung infection can occur, but prompt recognition and treatment are effective.

Initial therapy and Duration

Adults (including pregnant women and immmunocompromised)

Ciprofloxacin 500 mg po BID Or Doxycycline 100 mg po BID 60 days

Children ^1,

Ciprofloxacin 15-20 mg/kg po Q12 hrs Or Doxycycline:

>8 yrs and >45 kg: 100 mg po BID, >8 yrs and = 45 kg: 2.2 mg/kg po BID,

= 8 yrs: 2.2 mg/kg po BID 60 days

? Confirm the diagnosis by obtaining the appropriate laboratory specimens based on the clinical form of anthrax that is suspected (inhalational, gastrointestinal, or cutaneous).

- Inhalational anthrax: blood, CSF (if meningeal signs are present); chest X-ray

- Gastrointestinal anthrax: blood

- Cutaneous anthrax: vesicular fluid and blood

a. Presumptive identification criteria (level A LRN laboratory)

1. From clinical samples, such as blood, CSF, or skin lesion (vesicular fluid or eschar) material: encapsulated Gram-positive rods

2. From growth on sheep blood agar: large Gram-positive rods

3. Non-motile

4. Non-hemolytic on sheep blood agar

Additional LRN level B laboratory criteria for confirmation of B. anthracis are available through State Public Health Laboratories and involve:

b. Confirmatory criteria for identification of B. anthracis (level B LRN laboratory)

1. Capsule production (visualization of capsule), and

2. Lysis by gamma-phage, or

3. Direct fluorescent antibody assays (DFA)

 

Rapid screening assays, such as nucleic acid signatures and antigen detection, which can be performed directly on clinical specimens and environmental samples, are being made available for restricted use in LRN B and C level laboratories.

Inhalational anthrax: A brief prodrome resembling a viral respiratory illness followed by development of hypoxia and dyspnea, with radiographic evidence of mediastinal widening. This, the most lethal, form of anthrax results from inspiration of 8,000-40,000 spores of B. anthracis. The incubation of inhalational anthrax among humans is unclear, but it is reported to range between 1 and 7 days possibly ranging up to 60 days. Host factors, dose of exposure and chemoprophylaxis may play a role. Initial symptoms include sore throat, mild fever, muscle aches and malaise. These may progress to respiratory failure and shock. Meningitis frequently develops. Case-fatality estimates for inhalational anthrax are based on incomplete information regarding exposed populations and infected populations in the few case series and studies that have been published. However, case-fatality is extremely high, even with all possible supportive care including appropriate antibiotics. Records of industrially acquired inhalational anthrax in the United Kingdom before antibiotics were available reveal that 97% of cases were fatal. With antibiotic treatment the fatality rate is estimated to be at least 75%. Estimates of the impact of the delay in post-exposure prophylaxis or treatment on survival are not known.

Gastrointestinal anthrax: Severe abdominal distress followed by fever and signs of septicemia. This form of anthrax usually follows the consumption of raw or undercooked contaminated meat and is considered to have an incubation period of 1-7 days. An oropharyngeal and an abdominal form of the disease have been described in this category. Involvement of the pharynx is usually characterized by lesions at the base of the tongue, sore throat, dysphagia, fever, and regional lymphadenopathy. Lower bowel inflammation usually causes nausea, loss of appetite, vomiting and fever, followed by abdominal pain, vomiting blood, and bloody diarrhea. The case-fatality rate is estimated to be 25-60%, the effect of early antibiotic treatment on that case-fatality rate is not defined.

Cutaneous anthrax: A skin lesion evolving from a papule, through a vesicular stage, to a depressed black eschar. This is the most common naturally occurring type of infection (>95%) and usually occurs after skin contact with contaminated meat, wool, hides, or leather from infected animals. Incubation period ranges from 1-12 days. Skin infection begins as a small papule, progresses to a vesicle in 1-2 days followed by a necrotic ulcer. The lesion is usually painless, but patients also may have fever, malaise, headache and regional lymphadenopathy. The case fatality rate for cutaneous anthrax is 20% without, and less than 1% with, antibiotic treatment.

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.

- 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 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)

• Perform laboratory tests for 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

? 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).

• 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 35-37^o 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

• 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

• 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

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

• Soak in a decontamination solution until autoclaving is performed

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

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

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.

• Will be coordinated with the DOH Public Health Lab at [state telephone number]
• 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

H. References for laboratory guidelines