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Recommended Specimens for Microbiology and Pathology for Diagnosis: Inhalation, Cutaneous, and Gastrointestinal Anthrax

Specimens should be collected from any patient being evaluated for inhalation, cutaneous, or gastrointestinal Bacillus anthracis infection. This includes cases of known exposure or high risk of exposure (including bioterrorism events where the agent is unconfirmed), or cases with a clear epidemiologic link to exposure with presence of inhalation, cutaneous, or gastrointestinal anthrax symptoms, or cases where there is evidence of symptoms compatible with anthrax, but with no diagnosis.

Culturing B. anthracis from clinical specimens remains the gold standard for diagnosing anthrax. In systemic infections, organisms can easily be cultured from the blood, and B. anthracis may be cultured from other clinical specimens including skin lesion exudates, pleural fluid, cerebrospinal fluid (CSF), and stool if collected prior to antimicrobial therapy. If anthrax is suspected, diagnostic specimens including blood cultures should be obtained prior to starting antimicrobial therapy. Culture will likely be negative if specimens are obtained after antimicrobial therapy has been initiated, regardless of the form of disease. Antigen or molecular testing methods are less likely to yield positive results the longer the samples are obtained after the start of antimicrobial therapy.

List of recommended biological specimens for laboratory testing for suspected cases of anthrax, sorted by clinical form of presentation

CDC recommends that all of the following specimens be collected for testing, as available. For diagnostic testing of patients with suspected cutaneous anthrax:

  1. For vesicular lesions, two swabs of vesicular fluid from an unopened vesicle, one for culture and the second for real-time polymerase chain reaction (PCR).
  2. For eschars, the edge should be lifted and two swab samples rotated underneath and submitted, one for culture and the second for real-time PCR.
  3. For ulcers, the base of the lesion should be sampled with two saline moistened swabs and submitted, one for culture and the second for real-time PCR.
  4. Blood cultures obtained prior to antimicrobial therapy, if the patient has evidence of systemic symptoms.
  5. A full thickness punch biopsy of a papule or vesicle including adjacent skin should be obtained from all patients with a lesion being evaluated for cutaneous anthrax, to be submitted in 10% formalin for histopathology, special stains, and immunohistochemistry (IHC).
  6. In patients not on antibiotic therapy or on therapy for <24 hours, a second biopsy specimen should be submitted for culture and real-time PCR.
  7. Acute serum samples for testing for anthrax lethal toxin, and acute and convalescent serum samples for serologic testing.

For diagnostic testing of patients with suspected inhalation anthrax

  1. Blood cultures obtained prior to antimicrobial therapy.
  2. Pleural fluid, if present, for culture, real-time PCR, and testing for anthrax lethal toxin.
  3. Pleural and/or bronchial biopsies for IHC.
  4. CSF, in patients with meningeal signs, for culture and real-time PCR.
  5. Acute serum samples for testing for anthrax lethal toxin, and acute and convalescent serum samples for serologic testing.
  6. Autopsy tissues from fatal cases for histopathology, special stains, and IHC.

For diagnostic testing of patients with suspected gastrointestinal anthrax

  1. Blood cultures obtained prior to antimicrobial therapy.
  2. Ascites fluid for culture and real-time PCR.
  3. Stool or rectal swab for culture and real-time PCR.
  4. Oropharyngeal lesion, if present, for culture and real-time PCR.
  5. Acute and convalescent serum samples for serologic testing.
  6. Autopsy tissues from fatal cases for histopathology, special stains, and IHC.

List of recommended biological specimens for laboratory testing for suspected cases of anthrax, sorted by specimen

Swabs of Lesion
Cutaneous

  1. The specific location/sampling of the swab would depend on the stage of the lesion.
    1. Vesicular stage: Aseptically collect vesicular fluid on sterile dry swabs from previously unopened vesicles.

Note: Anthrax bacilli are most likely to be seen by Gram stain in the vesicular stage.

      1. Eschar stage: Collect eschar material by carefully lifting the eschar’s outer edge; insert a sterile dry swab, then slowly rotate for 2-3 seconds beneath the edge of the eschar without removing it.
      2. Ulcer: If no vesicle or eschar is present, swab the base of the ulcer using a sterile moist swab (pre-moistened with sterile saline).
    1. Regardless of the stage of the lesion, collect 2 separate swabs.
      1. 1 swab for culture
      2. 1 swab for real-time polymerase chain reaction (PCR)
  1. Submission
    1. Specimens intended for culture, or both culture and real-time PCR, should be shipped using cold packs and stored at 2 to 8°C.
    2. Specimens intended for real-time PCR testing only may be shipped on dry ice and stored at -70°C.

Gastrointestinal

  1. The specific location/sampling of the swab would depend on the clinical type or presentation of gastrointestinal anthrax suspected: oropharyngeal or intestinal.
    1. Oropharyngeal type: Using a sterile moist swab (pre-moistened with sterile saline), aseptically swab surface and edges of suspected lesions in the oropharynx or buccal cavity, or on the tongue, tonsils or posterior pharyngeal wall, for culture and real-time PCR.
    2. Intestinal type: Using sterile dry swabs, obtain a rectal swab for culture and real-time PCR. An aseptically collected stool sample may be obtained in addition to or instead of a rectal swab.
  2. Regardless of the clinical type or presentation collect 2 separate swabs.
      1. 1 swab for culture
      2. 1 swab for real-time PCR
    1. Submission
      1. Specimens intended for culture, or both culture and real-time PCR, should be stored at 2 to 8°C and shipped using cold packs.
      2. Specimens intended for real-time PCR testing only may be stored at -70°C and shipped on dry ice.

Biopsy 
Cutaneous

  1. A skin biopsy should be obtained on every patient with a lesion being evaluated for cutaneous anthrax, preferably before the initiation of antibiotic treatment.
    1. If the patient is on antimicrobial therapy at the time of presentation, obtain one full thickness punch biopsy sample from papule or vesicle and include adjacent skin; place into 10% buffered formalin for histopathology, special stains and immunohistochemistry (IHC).
    2. If the patient is not yet receiving antibiotics or if antibiotic therapy has been initiated in the proceeding 24 hours, obtain a second full thickness punch biopsy specimen for culture, PCR and IHC.
    3. Do not attempt to split one sample for items (i) and (ii) above, separate samples should be obtained.
    4. Biopsies should be taken from both vesicle and eschar, if present.
    5. Biopsy specimens should be accompanied by a brief history, description, chronology of lesion(s), specific treatment, and date of biopsy in relation to antibiotic treatment.
    6. A photograph, digital image, or diagram of the biopsy sites relative to the lesion(s) should be sent.

    More specific guidelines on collection of these specimens are provided in Shieh et al. American Journal of Pathology, Nov 2003, Vol 163, No. 5, Page 1908, Column 2.

  2. Formalin fixed samples should be shipped to CDC at room temperature; if the specimen is fresh-frozen store at -70°C and ship to CDC using dry ice.

Inhalation

  1. A bronchial or pleural biopsy may be obtained from patients with signs and symptoms of inhalation anthrax.
  2. Formalin fixed samples should be shipped to CDC at room temperature; if the specimen is fresh-frozen store at -70°C and ship to CDC using dry ice.

Serum (process using BSL2 practices) 
Cutaneous, Inhalation, and Gastrointestinal

  1. Acute serum specimens should ALWAYS be collected within the first 7 days of symptom onset or as soon as possible after known exposure.Convalescent serum specimens should be collected 14 to 35 days after the acute serum specimen has been obtained.
  2. Even if diagnosis of anthrax is confirmed by isolation of B. anthracis from clinical specimens, collect a convalescent serum sample 14-35 days after symptom onset. Testing of serum can be used to determine if the body has developed an immune response to anthrax, which can be used to guide treatment with anthrax antitoxins.
  3. Both acute and convalescent serum specimens should be obtained from a minimum of 10 mL blood, yielding ~ 5 mL of sera (laboratories may be testing for multiple potential etiologies).
  4. Separate serum from clot; sera should be frozen immediately following separation and stored frozen at -20°C or colder, and should be shipped frozen on dry ice to CDC, in appropriately labeled plastic screw cap vials.
  5. Do NOT send blood culture bottles or whole blood.
  6. See Collecting, Preparing, and Shipping Serum Samples to CDC for Serology Testing for shipping information.

Blood 
Cutaneous, Inhalation, and Gastrointestinal
Cutaneous Anthrax: If the patient has evidence of systemic symptoms, specimens for blood culture should be obtained.

  1. For culture, collect appropriate blood volume and number of sets per local hospital laboratory protocol, prior to initiation of antimicrobial therapy.
  2. For PCR, collect 10 mL blood in EDTA or Sodium Citrate (purple or blue top tubes).  For pediatric cases collect volumes allowable.
  3. Blood specimens intended for culture and real-time PCR should be transported directly to the laboratory on cold packs.

Pleural Fluid 
Inhalation

  1. For culture, real-time PCR, and anthrax lethal toxin testing, collect >1 mL of a pleural fluid into a sterile container. Store at 4°C for no more than 24h.
  2. Pleural fluid specimens should be stored at 2 to 8°C and shipped using cold packs.

Cerebrospinal Fluid (CSF) 
Anthrax Meningitis

  1. Meningitis has been reported in association with cutaneous, inhalation, and gastrointestinal anthrax cases.
  2. For culture and real-time PCR: If meningeal signs are present or meningitis is suspected, collect > 1 ml CSF into a sterile container.
  3. CSF specimens intended for culture and real-time PCR should be transported directly to the laboratory on cold packs.

Ascites Fluid 
Gastrointestinal

  1. If the patient has clinical symptoms compatible with gastrointestinal anthrax with presence of ascites, specimens should be obtained for culture, real-time PCR, and anthrax lethal toxin testing.
  2. Collect appropriate volume of ascites fluid per local hospital protocol, prior to initiation of antimicrobial therapy.

Autopsy Tissues 
Inhalation

  1. Collection of tissue specimens: The preferred specimens would be a minimum of 8 blocks and fixed tissue representing different pulmonary sites listed below.
    1. Hilar lung with regional lymph nodes, bronchi, and trachea
    2. Peripheral pulmonary parenchyma from both lungs
    3. Specimens should be included from the major organs, particularly any organs showing significant gross or microscopic pathology.
  2. Submission of Specimens
    1. Paraffin-embedded tissue blocks: In general, this is the preferred specimen and is especially important to submit in cases where tissues have been in formalin for a significant time.  Prolonged fixation (>2 weeks) may interfere with some immunohistochemical and molecular diagnostic assays.
    2. Wet tissue:  If available, CDC also highly recommends that unprocessed tissues in 10% neutral buffered formalin be submitted in addition to paraffin blocks. 
  3. Submission of supporting electronic or hard-copy documentation
  4. Copy of the pathology report (preliminary or final)
  5. Clinical history, treatment and pertinent laboratory reports 
 
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