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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 Cutaneous anthrax Eschar stage: Rotate two swabs beneath the edge of the eschar without removing the eschar. Gastrointestinal anthrax In later stages of disease, blood cultures will yield the organism, especially if specimens are obtained prior to antibiotic treatment. Inhalation anthrax 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
Isolation from clinical specimens Sputum specimens Blood specimens 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 Prepare a smear for Gram staining with the second swab. Stool specimens Do not use CVA or hectone agar plates. CSF specimens 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 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 Interpretation of results India ink staining of clinical samples (blood and CSF) for capsule Purpose Materials Controls
Procedure 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 Materials
Controls
Procedures
Interpretation of results 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
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], Remel Inc., Becton-Dickinson Bioscience [BD], |
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