Collecting and Sending Samples to Laboratories

Overview

Anthrax is caused by Bacillus anthracis bacteria. Culturing B. anthracis from clinical specimens is the gold standard for diagnosing anthrax. Depending on the form of disease, B. anthracis can be cultured from the following specimens:

• Ascites fluid
• Biopsy tissue
• Blood
• Cerebrospinal fluid (CSF)
• Pleural fluid
• Rectal swab (if gastrointestinal anthrax is suspected)
• Skin lesion fluid

Healthcare providers in the United States rarely see a patient with anthrax. CDC clinical guidance and case definitions are available to help doctors:

• Diagnose anthrax
• Take patient histories to determine how they may have been exposed
• Order necessary diagnostic tests

Call CDC's Emergency Operations Center at 770-488-7100 for an anthrax testing consultation.

Specimen collection overview

To accurately diagnose anthrax, samples ideally should be taken before the patient starts antibiotic treatment. The type of samples collected for testing will depend on the exposure and/or symptoms the patient has. Common types of samples collected for anthrax testing are summarized below.

Blood, Plasma, and Serum

Blood samples can help diagnose inhalation and gastrointestinal anthrax, especially if a patient shows signs of systemic infection. Blood will be tested using real-time polymerase chain reaction (PCR) to confirm an anthrax diagnosis.

• Collect 10 mL of blood in EDTA or Sodium Citrate (purple or blue top tubes) when testing for anthrax.
• For pediatric cases, collect volumes allowable.
• Samples for blood culture and real-time PCR should be shipped using cold packs.

Plasma is the preferred sample for anthrax lethal factor (LF) toxin testing for all types of anthrax. Samples for LF toxin testing may be collected from zero to 18 days after suspected exposure or the onset of symptoms; earlier collection is preferred.

When testing for anthrax LF toxin:

• Collect 10 mL of plasma using EDTA purple top tube.
• Keep the specimen on ice for 30 minutes then centrifuge it.
• Plasma should be shipped using dry ice.

Serum can be used to test for an immune response to anthrax and to test for anthrax LF toxin. For serological testing:

• Two samples should be collected, the first seven days after symptom onset, and the second collected 14-35 days after symptom onset.
• Ideally, the second sample should be taken two weeks after the first.
• Samples for testing may be collected from zero to 18 days after suspected exposure or the onset of symptoms.
• Earlier sample collection is preferred because of gradual declines in toxin levels during antimicrobial therapy.

Be sure to label specimens appropriately so that labs can process serum specimen using Biosafety Level (BSL)-2 practices (see Biosafety in Microbiological and Biological Laboratories). Do not send blood culture bottles or whole blood.

Tissue Biopsies

Cutaneous anthrax: A full thickness biopsy of a papule or vesicle, including adjacent skin, can be used to diagnose cutaneous anthrax. This biopsy should be taken preferably before the patient has started antibiotic treatment.

If the patient has not started antibiotics or antibiotics have been started within 24 hours, do both of the following:

• Take a full thickness punch biopsy specimen for culture and real-time PCR
• Take one full thickness punch biopsy sample from the papule or vesicle (include adjacent skin) for histopathology, special stains, and IHC.

If the patient is on antibiotics at the time of presentation:

• Take one full thickness punch biopsy sample from the papule or vesicle (include adjacent skin) for histopathology, special stains, and IHC.
• Place the sample into 10% buffered formalin for fixation. Do not freeze.

Testing from this biopsy may include histopathology, special stains, and immunohistochemistry (IHC).

Inhalation anthrax: A bronchial or pleural biopsy should be obtained for patients with symptoms of inhalation anthrax.

If the biopsy samples are formalin-fixed, they should be shipped to CDC at room temperature. Do not freeze. If the biopsy samples are fresh-frozen, they should be stored at -70°C and shipped to CDC using dry ice. Biopsy specimens should be accompanied by supporting documents, including:

• A brief clinical history and description of the lesion
• A chronology of the presentation of the lesion(s)
• A description of the treatment course (with duration and dosage)
• The date of biopsy in relation to the initiation of antibiotic treatment
• A photograph, digital image, or diagram of the biopsy sites

Testing from this biopsy may include histopathology, special stains, and IHC.

For additional instructions on pathology testing, visit the Infectious Diseases Pathology Branch website.

Lesion swabs

Cutaneous anthrax: Always collect two separate swabs, one for real-time PCR, and one for culture. The specific location of swab sampling will depend on the stage of the lesion.

• Vesicular stage: In a sterile manner, collect vesicular fluid on sterile dry swabs from previously unopened vesicles.
• Eschar stage: Collect the eschar material by carefully lifting the eschar's outer edge; inserting a sterile moist swab (pre-moistened with sterile saline); and rotating the swab slowly for 2-3 seconds beneath the edge of the eschar before removing it.
• Ulcer stage: If no vesicle or eschar is present, swab the base of the ulcer using a sterile moist swab (pre-moistened with sterile saline).

For samples intended for culture or both culture and real-time PCR, ship samples using cold packs and store at 2 to 8°C. For samples intended for real-time PCR testing only, ship on dry ice and store at -70°C.

Gastrointestinal anthrax: Always collect two separate swabs, one for real-time PCR, and one for culture. The specific location of swab sampling will depend on the stage of the lesion.

• Oropharyngeal: Use a swab pre-moistened with sterile saline to aseptically swab the surface and edge of suspected lesions. Lesions may be present in the oropharynx or buccal cavity or on the tongue, tonsils, or posterior pharyngeal wall.
• Rectal: Use a sterile dry swab to collect a rectal swab.

For samples intended for culture or both culture and real-time PCR, ship samples using cold packs and store at 2 to 8°C. For real-time PCR testing only, ship on dry ice and store at -70°C.

Collecting pleural or ascites fluid collections for inhalation or gastrointestinal anthrax

• Collect more than 1 mL of pleural fluid into a sterile container for culture, real-time polymerase chain reaction (PCR), and anthrax LF toxin testing.
• For optimum testing results, pleural fluid specimens should be stored at 2-8°C for no more than 24 hours.
• Ascites fluid can be obtained for patients with symptoms of gastrointestinal or inhalation anthrax.
• Collect the appropriate volume of ascites fluid according to local hospital protocol, as well as what's needed for culture, PCR, and anthrax LF toxin testing.

Submitting specimens

CDC accepts specimens from state public health laboratories and federal agencies. Private healthcare providers and institutions should submit specimens to the local state or territorial health department laboratory (state, county, city) for processing.

Shipping samples

Samples that could contain B. anthracis must be packaged and shipped according to specific instructions to ensure they arrive safely, intact, and uncontaminated.

Resources

• Printable fact sheet: Collecting, preparing, and shipping serum specimens to CDC for serology testing
• Submitting Specimens to CDC Infectious Diseases Laboratories
• Bacillus anthracis Detection in Clinical Specimens
• Bacillus anthracis Identification
• Bacillus anthracis Identification and Antimicrobial Susceptibility Testing (AST)
• Bacillus anthracis Serology
• Bacillus anthracis Study
• Infectious Diseases Pathology Branch (IDPB)