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CAP Home > CAP Reference Resources and Publications > CAP TODAY > CAP Today Archive 2001 > CDC protocol for anthrax cases
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CDC protocol for anthrax cases

November 2001

The Centers for Disease Control and Prevention has asked that all clinical laboratories familiarize themselves with the CDC protocol for anthrax cases. The full protocol can be found at www.bt.cdc.gov/Agent/Anthrax/Anthracis20010417.pdf. CAPTODAY presents here the portion of the protocol devoted to laboratory procedures.

Laboratory procedures for the identification of Bacillus anthracis

The procedures described below function to rule out presumptively identified B. anthracis in clinical specimens or isolates. These procedures should be performed in microbiology laboratories that utilize Biological Safety Level 2 (BSL 2) practices. Laboratory coats and gloves shall be worn when processing specimens and performing tests. Safety glasses or eye shields are recommended. Any activities that bring hands in contact with mucosal surfaces (for example, eating, drinking, smoking, or applying makeup) are prohibited. Hands should be washed before leaving the laboratory. Anthrax vaccination is not required.

Disclaimer: Names of vendors or manufacturers are provided as examples of suitable product sources and inclusion does not imply endorsement by the Centers for Disease Control and Prevention, the U.S. Public Health Service, Department of Health and Human Services, the U.S. Army, or the Federal Bureau of Investigation.

Handling samples

For safety considerations, analysis of samples for biological threat agents is performed within a certified Class II biological safety cabinet (BSC). Procedures requiring removal of items from a BSC, such as slides for microscopy, should follow published microbiological practices and precautions. When using a BSC, ensure that the cabinet does not contain unnecessary items that will interfere with proper airflow and function. As for any procedure involving infectious materials, standard personal protective gear should be used, such as latex gloves and laboratory coats, or disposable over-garments. Additional respiratory protection should also be considered with materials or analytical procedures determined to be potentially hazardous outside the BSC. Once a biological agent has been identified, modifications in handling of samples can then be considered.

Decontamination

Commercially available household bleach solutions contain 5.25% hypochlorite and, when diluted 1:10, are effective in routine decontamination of surfaces and instruments after working with B. anthracis. Contaminated items such as pipettes, needles, loops, and microscope slides should be immersed in decontamination solution until autoclaving. Work surfaces, such as a biological safety cabinet, should be wiped down before and after use with decontamination solution. The method of decontamination of a spill depends on the nature of the spill. Spills involving fresh cultures or samples known to have low concentrations of spores should be flooded with decontamination solution and soaked for five minutes before cleanup. Spills that involve samples with high concentrations of spores, involve organic matter, or occur in areas of lower than room temperature (refrigerators, freezers) should be exposed to decontamination solution for at least one hour before cleanup. Personnel involved in the cleanup of any spill should wear gloves, safety glasses, and a laboratory coat or gown during the cleanup process. Respiratory protection should be considered for spills in which a substantial aerosolization is suspected.

Collection of clinical specimens

Materials
Sporicidal disinfectant
Sputum cup
Sterile cotton swabs
Blood culture collection kit
Stool collection cup

Cutaneous anthrax
Vesicular stage: The organism is best demonstrated in this stage. Soak two dry sterile swabs in vesicular fluid from a previously unopened vesicle.

Eschar stage: Rotate two swabs beneath the edge of the eschar without removing the eschar.

Gastrointestinal anthrax
If the patient is able to produce a stool specimen, stool cultures should be performed. Obtain a stool culture.

In later stages of disease, blood cultures will yield the organism, especially if specimens are obtained prior to antibiotic treatment.

Inhalation anthrax
If respiratory symptoms are present and sputum is being produced, obtain a specimen for culture and smear.

In later stages of disease (two to eight days post exposure), blood cultures may yield the organism, especially if specimens are drawn before antibiotic treatment.

Materials needed for processing clinical specimens

  • 5% sheep blood agar plates [SBA] (BD Bioscience or Remel Inc. or equivalent)
  • MacConkey agar plates (BD Bioscience or Remel Inc. or equivalent)
  • Phenyl ethyl alcohol agar (PEA) plates (for stool specimens) (BD Bioscience or Remel Inc. or equivalent)
  • Trypticase soy broth (BD Bioscience or Remel Inc. or equivalent)
  • Clean glass microscope slides
  • Sterile cotton swabs (commercially available specimen transport swabs for aerobic culture are preferred)
  • Disposable bacteriologic inoculation loops
  • Clinical centrifuge with appropriate biocontainment tube holders
  • Sporicidal disinfectant (0.5% sodium hypochlorite or 0.5% calcium hypochlorite)

Isolation from clinical specimens

Sputum specimens
Inoculate three routine media for sputum specimens (i.e. SBA, MacConkey agar, broth enrichment).

Blood specimens
Routine blood culture methods are sufficient.

There may be enough organisms in the blood to see them on direct smears by Gram stain. B. anthracis appears as short chains of two to four cells that are encapsulated as evidenced by clear zones around the bacilli. The presence of large encapsulated gram-positive rods in the blood is strongly presumptive for B. anthracis identification.

If blood culture bottle is positive, perform a Gram stain directly and observe for encapsulated rods. These blood cultures should also be subcultured to SBA and MacConkey agar plates.

Swab specimens
Use one swab to inoculate three standard media for surface wounds (e.g. SBA, MacConkey agar, or broth enrichment).

Prepare a smear for Gram staining with the second swab.

Stool specimens
Routine stool culture methods are sufficient (e.g. SBA, MacConkey agar, or PEA plates).

Do not use CVA or hectone agar plates.

CSF specimens
If a clinical centrifuge with appropriate biocontainment tube holders is available, centrifuge the CSF specimen at 1500 x g for 15 minutes.

Collect the sediment and prepare a smear for Gram staining.

Inoculate the remainder of the sediment onto SBA and broth enrichment media (tryptic soy broth or thioglycollate).

Incubation and examination of cultures

Cultures should be incubated at 35-37°C under ambient conditions.

Cultures should be examined within 18-24 hours of incubation. Growth of B. anthracis may be observed as early as eight hours after inoculation.

Differential tests for the presumptive identification of B. anthracis

Colony characteristics of B. anthracis
After incubation of SBA plates for 15-24 hours at 35-37°C, well-isolated colonies of B. anthracis are 2-5 mm in diameter. The flat or slightly convex colonies are irregularly round, with edges that are slightly undulate (irregular, wavy border), and have a ground-glass appearance. There are often comma-shaped projections from the colony edge, producing the "Medusa head" colony.

Colonies on SBA usually have a tenacious consistency. When teased with a loop, the growth will stand up like beaten egg white. In contrast to colonies of B. cereus and B. thuringiensis, colonies of B. anthracis are not ß-hemolytic. However, weak hemolysis may be observed under areas of confluent growth in aging cultures and should not be confused with ß-hemolysis.

When examining primary growth media, it is important to compare the extent of growth on SBA plates with that on MacConkey agar plates. B. anthracis grows well on SBA, but does not grow on MacConkey agar. B. anthracis grows rapidly; heavily inoculated areas may show growth within six to eight hours and individual colonies may be detected within 12-15 hours. This trait can be used to isolate B. anthracis from mixed cultures containing slower-growing organisms.

Gram stain morphology of B. anthracis

Procedure
Perform Gram stain by usual procedures.

Interpretation of results
B. anthracis is a large gram-positive rod (1-1.5 x 3-5 µm) that forms oval, central-to-subterminal spores (1 x 1.5 µm) on SBA that do not cause significant swelling of the cell. Spores are not present in clinical samples unless exposed to atmospheric levels of CO2; CO2 levels within the body inhibit sporulation. Vegetative cells seen on Gram stain of blood and impression smears are in short chains of two to four cells that are encapsulated. However, cells from growth on SBA under ambient conditions are not encapsulated and occur as long chains of bacilli. When grown on nutrient agar in the presence of 5% CO2 or on other basal media supplemented with 0.8% sodium bicarbonate, virulent strains will yield heavily encapsulated bacilli. The capsule can be visualized microscopically using India ink.

India ink staining of clinical samples (blood and CSF) for capsule

Purpose
India ink is useful for improving visualization of encapsulated B. anthracis in clinical samples such as blood, blood culture bottles, or cerebrospinal fluid.

Materials
Microscope slides
Cover glasses
India ink (Bactidrop, Remel Inc., catalog # 21-518 or equivalent).
Microscope with 100x oil immersion objective

Controls

  • Control strains
  • Positive control: Klebsiella pneumoniae on SBA or equivalent
    Negative control: E. coli ATCC 25922 or equivalent
  • Method controls: Perform the test with suspensions of fresh cultures of the control strains. Control strains should be assayed on each day of testing.
  • Resolving an out-of-control result: Check the purity and identity of the control strains and repeat the test.

Procedure
For the controls, transfer a small amount of growth (1 mm diameter) from each control SBA plate into 0.5 mL whole EDTA-treated blood or serum. Mix.

For the unknowns, take 100 µL of sample (blood, CSF).

Transfer 5-10 µL of unknown or control to a slide, place a cover glass on the drop, and then add 5-10 µL of India ink to the edge of the cover glass.

After the ink diffuses, view the cells using 100x oil immersion objective with oil on top of the cover glass.

Interpretation of results

The capsule will appear as a well-defined clear zone around the cells for the positive control. No zone should be present in the negative control.

Motility test: Wet mount or motility medium

Purpose
This test determines the motility of suspect isolates. B. anthracis is a nonmotile species. This characteristic is unusual among Bacillus species and is therefore useful in the preliminary identification of B. anthracis isolates. Two methods are given: the wet mount and the tubed motility test.

Materials

  • For wet mount procedure
  • Precleaned microscope slides
    Cover glasses
    Sterile distilled water
    Disposable bacteriologic inoculating loop
    Light microscope with 40x objective and 10x eyepiece
    Sterile glass tube

  • For tubed motility test
  • Tubed motility media (Remel Inc., catalog # 06-1408 or equivalent),
    5mL per tube
    Sterile disposable 1µL inoculating loop or needle

Controls

  • Control strains
  • Positive control: Pseudomonas aeruginosa ATCC 35032
    or equivalent.
    Negative control: Acinetobacter spp. ATCC 49139 or equivalent.
  • Method controls: Perform the test with fresh cultures of the control strains using the same method as with unknowns. Control strains should be assayed on each day of testing.
  • Resolving an out-of-control result: Check the purity and identity of the control strains and repeat the test.

Procedures
Wet mount and motility

  • Wet mount procedure
  • Deliver two drops (approximately 0.1 mL) of sterile distilled water into
    the sterile glass tube.
    Using the inoculating loop, sample a suspicious colony from a 12-20-
    hour culture and suspend the growth in the water. (Alternatively, a
    loopful of medium from a fresh broth culture can be used.)
    Transfer one drop of the suspension to the microscope slide and
    overlay with the cover glass.
    Examine the slide under the microscope using the 40x objective
    (total magnification = 400x).
    Discard slides in 0.5% hypochlorite solution.
  • Motility test medium procedure
  • Using the sterile inoculating needle, remove some growth from an
    isolated, suspicious colony of an 18-24-hour culture.
    Inoculate the motility tube by carefully stabbing the needle 3-4 cm
    into the medium and then drawing the needle directly back out so
    that a single line of inoculum can be observed.
    Incubate the tube aerobically at 35-37°C for 18-24 hours.

Interpretation of results
For wet mount: Motile organisms can be observed moving randomly throughout the suspension. Nonmotile organisms either fail to move or move with Brownian motion.

For motility test medium: Nonmotile organisms, such as B. anthracis, will form a single line of growth that does not deviate from the original inoculum stab. Motile organisms will form a diffuse growth zone around the inoculum stab.

Presumptive identification key for B. anthracis

  • From clinical samples, such as blood, CSF, or lesion material: encapsulated gram-positive rods.
  • Gram-positive, broad rod, spore-positive: Bacillus species.
  • Spores are nonswelling and oval shaped; ground glass appearance of colonies: Bacillus morphology group 1 (includes B. anthracis, B. cereus, B. thuringiensis, and B. cereus var. mycoides)
  • Nonmotile: B. anthracis and B. cereus var. mycoides
  • Nonhemolytic: presumptive B. anthracis

Actions if a presumptive B. anthracis colony is identified and suspected as a bioterrorist threat agent

Preserve original specimens pursuant to a potential criminal investigation.

Contact local FBI, state public health laboratory, and state public health department.

Local FBI agents will forward isolates to a state health department laboratory as is necessary. Consultation with a state health department laboratory is strongly encouraged as soon as B. anthracis is suspected as a bioterrorist threat agent.

Listed vendors American Type Cell Culture [ATCC],
800-638-6597

Remel Inc.,
800-255-6730

Becton-Dickinson Bioscience [BD],
800-675-0908

   
 

 

 

   
 
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