Anthrax, a serious infectious disease caused by Bacillus anthracis, necessitates accurate and timely laboratory diagnosis for effective patient management and public health response. Culturing B. anthracis from clinical specimens remains the gold standard method for confirming anthrax. The type of specimen required for lab diagnosis varies depending on the clinical presentation and suspected form of anthrax. Specimens can range from easily accessible samples like skin lesion fluid to more invasive collections such as cerebrospinal fluid or biopsy tissues.
For healthcare providers in English-speaking regions, especially where anthrax cases are infrequent, navigating the diagnostic process can be challenging. This guide provides a detailed overview of Anthrax Lab Diagnosis, focusing on specimen collection techniques and considerations for optimal testing outcomes, aligning with international best practices and guidelines for accuracy and reliability. Consulting resources like the CDC clinical guidance and case definitions is crucial for clinicians to effectively diagnose anthrax, obtain thorough patient histories to understand potential exposure routes, and order the appropriate diagnostic tests. For immediate consultation on anthrax testing, the CDC’s Emergency Operations Center is a valuable resource.
Specimen Collection: An Overview
Optimal anthrax lab diagnosis hinges on collecting appropriate specimens, ideally before initiating antibiotic treatment. Antibiotic administration can reduce bacterial load and potentially impact culture results. The specific types of samples required are dictated by the suspected form of anthrax and the patient’s presenting symptoms. The following sections detail the recommended specimen collection procedures for various sample types.
Detailed Specimen Collection by Sample Type
Blood, Plasma, and Serum
Blood samples are particularly useful in diagnosing systemic anthrax forms, such as inhalation and gastrointestinal anthrax, where bloodstream invasion is likely. Real-time Polymerase Chain Reaction (PCR) testing on blood samples is a rapid and sensitive method for confirming the presence of Bacillus anthracis.
For PCR testing:
- Collect 10 mL of blood into either EDTA (purple top tube) or Sodium Citrate (blue top tube) vacutainer.
- For pediatric patients, collect the maximum allowable volume appropriate for their age and size.
- Maintain cold chain integrity by shipping samples with cold packs to preserve sample quality for PCR analysis.
Plasma is the preferred matrix for Anthrax Lethal Factor (LF) toxin detection, irrespective of the anthrax form. LF toxin testing is most effective when performed within 18 days of suspected exposure or symptom onset, with earlier collection being more advantageous due to toxin level decline during antibiotic therapy.
For LF toxin testing in plasma:
- Collect 10 mL of plasma using an EDTA purple top tube.
- Immediately after collection, place the specimen on ice for 30 minutes, followed by centrifugation to separate plasma.
- Ship plasma samples frozen on dry ice to ensure toxin stability during transport.
Serum samples serve dual purposes: assessing the immune response to anthrax and detecting Anthrax LF toxin. Serological testing requires paired serum samples to demonstrate a rise in antibody titers, indicative of recent infection.
For serological testing:
- Collect two serum samples. The first sample should be drawn within seven days of symptom onset, and the second sample should be collected 14 to 35 days after symptom onset, ideally two weeks after the first sample.
- For LF toxin testing in serum, samples can be collected within 18 days of suspected exposure or symptom onset, similar to plasma collection guidelines. Earlier collection is favored due to the potential decrease in toxin levels with antimicrobial treatment.
When processing serum specimens, laboratories should adhere to Biosafety Level (BSL)-2 practices, as detailed in the CDC’s “Biosafety in Microbiological and Biological Laboratories” guidelines. It is crucial to clearly label specimens for appropriate lab handling. Whole blood or blood culture bottles should not be submitted for serum testing.
Tissue Biopsies
Tissue biopsies are essential for diagnosing cutaneous and inhalation anthrax, providing material for culture, PCR, histopathology, and immunohistochemistry (IHC).
Cutaneous Anthrax: For suspected cutaneous anthrax, a full-thickness biopsy of a papule or vesicle, including adjacent skin, is recommended. Biopsy collection should ideally precede antibiotic treatment.
For patients not yet on antibiotics or treated within 24 hours:
- Obtain a full-thickness punch biopsy for both culture and real-time PCR analysis.
- Collect a second full-thickness punch biopsy sample from the same lesion site, including adjacent skin, for histopathology, special stains, and IHC studies.
For patients already receiving antibiotics at presentation:
- Collect a full-thickness punch biopsy sample from the papule or vesicle (including adjacent skin) for histopathology, special stains, and IHC.
- Fix this sample in 10% buffered formalin. Freezing formalin-fixed tissue is contraindicated.
Histopathological examination, special stains, and IHC on biopsy specimens aid in visualizing Bacillus anthracis and characterizing tissue pathology associated with anthrax infection.
Inhalation Anthrax: In cases of suspected inhalation anthrax, bronchial or pleural biopsies are necessary to obtain lung tissue for comprehensive analysis.
Formalin-fixed biopsy samples should be shipped to the CDC at room temperature. Fresh-frozen biopsy samples must be stored at -70°C and shipped on dry ice. Accompanying documentation is critical for biopsy specimens and should include:
- A concise clinical history and lesion description.
- A chronological timeline of lesion development.
- Details of antibiotic treatment, including drug, dosage, and duration.
- The biopsy date relative to antibiotic treatment initiation.
- A photograph, digital image, or diagram illustrating biopsy sites for anatomical context.
Further guidance on pathology testing can be found on the Infectious Diseases Pathology Branch website.
Lesion Swabs
Lesion swabs are the primary collection method for cutaneous and gastrointestinal anthrax diagnosis, especially when vesicular or eschar lesions are present.
Cutaneous Anthrax Swabs: Always collect two separate swabs: one for real-time PCR and another for culture. Swab collection technique varies based on the lesion stage:
- Vesicular Stage: Using sterile technique, collect vesicular fluid from unopened vesicles with sterile dry swabs.
- Eschar Stage: Carefully lift the eschar’s outer edge and insert a sterile swab pre-moistened with sterile saline. Rotate the swab gently for 2-3 seconds beneath the eschar edge to collect eschar material.
- Ulcer Stage: If no vesicle or eschar is present, swab the ulcer base using a sterile swab pre-moistened with sterile saline.
For culture or combined culture and PCR, ship swabs with cold packs and store at 2 to 8°C. For PCR testing alone, ship swabs on dry ice and store at -70°C.
Gastrointestinal Anthrax Swabs: Collect two swabs (one for PCR, one for culture) for gastrointestinal anthrax diagnosis.
- Oropharyngeal Swabs: Use a sterile saline-moistened swab to sample the surface and edges of suspected lesions in the oropharynx, buccal cavity, tongue, tonsils, or posterior pharyngeal wall.
- Rectal Swabs: Collect rectal swabs using sterile dry swabs.
Shipping and storage conditions for gastrointestinal anthrax swabs are identical to those for cutaneous anthrax swabs, depending on whether the samples are intended for culture, PCR, or both.
Collecting Pleural or Ascites Fluid
Pleural fluid and ascites fluid collections are crucial for diagnosing inhalation and gastrointestinal anthrax, respectively, particularly when systemic involvement is evident.
- Collect more than 1 mL of pleural fluid or ascites fluid in a sterile container for culture, real-time PCR, and anthrax LF toxin testing.
- Pleural fluid specimens should ideally be stored at 2-8°C and processed within 24 hours for optimal test results.
- Ascites fluid volume should be collected according to local hospital protocols while ensuring sufficient volume for culture, PCR, and LF toxin assays.
Submitting Specimens
Pre-Shipment Consultation
Prior to shipping any specimens potentially containing B. anthracis, it is mandatory to consult with and obtain authorization from your state health department. Contact the CDC Emergency Operations Center for an anthrax testing consultation before submission.
The Laboratory Response Network (LRN) plays a vital role in anthrax diagnostic confirmation, offering critical support to clinical care providers and public health departments. Timely confirmation through the LRN is essential for effective anthrax response.
Specimen submission to CDC is generally facilitated through state public health laboratories and federal agencies. Private healthcare providers and institutions should route specimens through their local state or territorial health department laboratory for initial processing and onward submission if necessary.
Shipping Procedures
Packaging and shipping samples suspected of containing B. anthracis must strictly adhere to specific regulations to ensure safe, secure, and uncontaminated transport. Detailed packaging and shipping instructions are available in the “Anthrax Specimen Packaging and Shipping” guidelines.
Resources
- CDC Clinical Guidance
- CDC Case Definitions
- Biosafety in Microbiological and Biological Laboratories
- Infectious Diseases Pathology Branch
- State and Territorial Health Department Websites
- Anthrax Specimen Packaging and Shipping
