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Microbiology

Specimen sources

This section covers the crucial topic of BSL-3 pathogens and Select Agents related to bioterrorism, specifically focusing on specimen sources. This is a critical area for Medical Laboratory Scientists, as it involves working with potentially deadly organisms and requires meticulous attention to safety and procedure

General Principles

  • Safety First: This is paramount. All procedures must be performed in a BSL-3 laboratory with strict adherence to safety protocols, including the use of appropriate personal protective equipment (PPE) such as respirators, gloves, gowns, and face shields. All work should be performed in a certified biosafety cabinet (BSC)
  • Chain of Custody: Maintaining a clear and unbroken chain of custody for all specimens is crucial, especially in the context of bioterrorism. This ensures traceability and accountability
  • Communication: Clear communication with healthcare providers, public health officials, and the laboratory team is essential
  • Reporting: Suspected Select Agent or BSL-3 pathogen isolates must be reported to the appropriate authorities (e.g., local and state health departments, CDC) immediately
  • Decontamination: All work surfaces, equipment, and waste must be thoroughly decontaminated using appropriate disinfectants (e.g., bleach) before and after use
  • Training: Personnel must receive thorough training in the handling, identification, and safety procedures related to these agents
  • Laboratory Response Network (LRN): Laboratories should be part of the LRN, a network of laboratories that are equipped and trained to respond to biological threats

Specimen Sources and Potential Agents

Let’s break down the common specimen sources and the specific BSL-3 pathogens and Select Agents that might be encountered in each:

Blood

  • Potential Agents
    • Bacillus anthracis (Anthrax) - Bacillus anthracis causes anthrax
    • Brucella spp. (Brucellosis) - Brucella species cause brucellosis (undulant fever)
    • Francisella tularensis (Tularemia) - Francisella tularensis causes tularemia (rabbit fever)
    • Yersinia pestis (Plague) - Yersinia pestis causes plague
    • Burkholderia mallei (Glanders) - Burkholderia mallei causes glanders
    • Burkholderia pseudomallei (Melioidosis) - Burkholderia pseudomallei causes melioidosis
  • Clinical Context
    • Anthrax: Blood samples are typically collected in cases of systemic anthrax infection
    • Brucellosis: Blood cultures are often used to diagnose brucellosis
    • Tularemia: Blood cultures may be used to diagnose tularemia, especially in cases of bacteremia
    • Plague: Blood cultures are crucial for diagnosing septicemic plague
    • Glanders/Melioidosis: Blood cultures are crucial for diagnosing glanders and melioidosis, especially in cases of bacteremia
  • Laboratory Procedures
    • Collection: Collect blood in appropriate blood culture bottles
    • Processing: Blood cultures should be handled in a BSC
    • Incubation: Incubate blood culture bottles in an automated blood culture system
    • Gram Stain: Perform Gram stains on positive blood culture bottles to look for characteristic morphology
    • Culture: Subculture positive blood cultures onto appropriate media (e.g., blood agar, chocolate agar)
    • Identification: Perform biochemical tests and/or molecular methods (e.g., PCR) for identification

Sputum

  • Potential Agents
    • Bacillus anthracis (Anthrax) - Bacillus anthracis causes inhalational anthrax
    • Francisella tularensis (Tularemia) - Francisella tularensis causes pneumonic tularemia
    • Yersinia pestis (Plague) - Yersinia pestis causes pneumonic plague
  • Clinical Context
    • Inhalational Anthrax: Sputum samples are collected from patients with inhalational anthrax
    • Pneumonic Tularemia: Sputum is collected from patients with pneumonic tularemia
    • Pneumonic Plague: Sputum is collected from patients with pneumonic plague
  • Laboratory Procedures
    • Collection: Collect sputum samples in a sterile container. Induce sputum if necessary
    • Processing: Handle sputum samples in a BSC
    • Gram Stain: Perform Gram stains to assess bacterial morphology and cellular content
    • Culture: Culture on appropriate media (e.g., blood agar, chocolate agar)
    • Microscopy: Direct fluorescent antibody (DFA) or other specialized stains may be used
    • Identification: Perform biochemical tests and/or molecular methods (e.g., PCR) for identification

Tissue

  • Potential Agents
    • Bacillus anthracis (Anthrax) - Bacillus anthracis causes cutaneous anthrax
    • Francisella tularensis (Tularemia) - Francisella tularensis causes ulceroglandular tularemia
    • Yersinia pestis (Plague) - Yersinia pestis can be found in tissue from bubonic plague
    • Burkholderia mallei (Glanders) - Burkholderia mallei causes glanders
    • Burkholderia pseudomallei (Melioidosis) - Burkholderia pseudomallei can be found in tissue from melioidosis
  • Clinical Context
    • Cutaneous Anthrax: Tissue samples from skin lesions are essential
    • Ulceroglandular Tularemia: Tissue samples from ulcers or lymph nodes may be collected
    • Bubonic Plague: Tissue samples from buboes (swollen lymph nodes) may be taken
    • Glanders/Melioidosis: Tissue samples from skin lesions, abscesses, or other infected sites
  • Laboratory Procedures
    • Collection: Tissue samples must be collected using appropriate sterile techniques
    • Processing: Handle tissue samples in a BSC
    • Smears: Prepare Gram stains and other special stains (e.g., Giemsa) from tissue samples
    • Culture: Culture on appropriate media (e.g., blood agar, chocolate agar)
    • Histopathology: Tissue may be submitted for histopathological examination
    • Identification: Perform biochemical tests and/or molecular methods (e.g., PCR) for identification

Lymph Node Aspirate/Biopsy

  • Potential Agents
    • Francisella tularensis (Tularemia) - Francisella tularensis causes ulceroglandular tularemia and glandular tularemia
    • Yersinia pestis (Plague) - Yersinia pestis causes bubonic plague
    • Burkholderia mallei (Glanders) - Burkholderia mallei causes glanders
  • Clinical Context
    • Ulceroglandular/Glandular Tularemia: Aspirates or biopsies of lymph nodes are often collected
    • Bubonic Plague: Aspirates or biopsies of buboes are crucial for diagnosis
    • Glanders: Aspirates or biopsies of lymph nodes or other affected tissues
  • Laboratory Procedures
    • Collection: Lymph node aspirates or biopsies must be collected using appropriate sterile techniques
    • Processing: Handle aspirates and tissue samples in a BSC
    • Smears: Prepare Gram stains and other special stains (e.g., Giemsa)
    • Culture: Culture on appropriate media (e.g., blood agar, chocolate agar)
    • Histopathology: Tissue may be submitted for histopathological examination
    • Identification: Perform biochemical tests and/or molecular methods (e.g., PCR) for identification

Key Considerations for Specific Agents

Let’s highlight some key aspects specific to each of these organisms:

  • Bacillus anthracis
    • Morphology: Large, Gram-positive rods with a characteristic “bamboo cane” appearance in chains. Non-hemolytic on blood agar
    • Testing: Presumptive identification with Gram stain and colonial morphology. Confirmation with PCR or fluorescent antibody testing
    • Safety: Spores are highly resistant and can aerosolize
  • Brucella: spp
    • Morphology: Small, Gram-negative coccobacilli
    • Testing: Slow-growing and fastidious. Requires prolonged incubation
    • Safety: Highly infectious through aerosols and skin contact
  • Francisella tularensis
    • Morphology: Small, Gram-negative coccobacilli
    • Testing: Fastidious; requires enriched media (e.g., cysteine-containing media)
    • Safety: Highly infectious via aerosols and skin contact
  • Yersinia pestis
    • Morphology: Gram-negative coccobacilli
    • Testing: Requires specific media
    • Safety: Highly infectious via aerosols and through contact with infected animals
  • Burkholderia mallei
    • Morphology: Gram-negative bacilli
    • Testing: Slow-growing and fastidious
    • Safety: Highly infectious through aerosols and skin contact
  • Burkholderia pseudomallei
    • Morphology: Gram-negative bacilli
    • Testing: Can mimic other organisms
    • Safety: Highly infectious through aerosols and skin contact

Important Notes

  • Reporting: Always report suspected cases to public health authorities immediately, following your laboratory’s protocols
  • Training: Ensure all personnel are thoroughly trained in the handling, identification, and safety procedures for these agents
  • Consultation: Consult with your laboratory supervisor, infectious disease specialists, and public health officials if you suspect a Select Agent

Key Terms

  • BSL-3 (Biosafety Level 3): A containment level for laboratories working with infectious agents that can cause serious or potentially lethal disease through inhalation. BSL-3 labs require specific engineering controls, such as a dedicated ventilation system, sealed windows and doors, and the use of a biosafety cabinet for all manipulations
  • Select Agent: A biological agent or toxin that has the potential to pose a severe threat to public health and safety, animal or plant health, or to the safety of animal or plant products. These agents are regulated by the federal government, with strict requirements for handling, storage, and reporting
  • Aerosolization: The process by which a liquid or solid is converted into a fine mist or suspension in the air. Many BSL-3 pathogens can be transmitted via aerosols, making this a critical hazard in the laboratory
  • Chain of Custody: A documented process that tracks the movement of a specimen from collection to disposal, ensuring its integrity and providing a clear record of who handled it and when. Essential for legal and epidemiological purposes, especially in bioterrorism investigations
  • Biosafety Cabinet (BSC): A ventilated enclosure designed to provide a physical barrier to protect the user and the environment from exposure to infectious agents. BSCs are classified by performance characteristics and are a critical component of BSL-3 laboratories
  • Inhalational Anthrax: A severe form of anthrax caused by the inhalation of Bacillus anthracis spores. It is often fatal if not treated promptly
  • Pneumonic Plague: A severe form of plague caused by Yersinia pestis that affects the lungs. It is highly contagious and can be spread through respiratory droplets
  • Ulceroglandular Tularemia: A common form of tularemia caused by Francisella tularensis, characterized by an ulcer at the site of entry and swelling of the regional lymph nodes
  • Spore: A dormant, highly resistant form of bacteria, such as Bacillus anthracis, that can survive harsh environmental conditions and can be easily aerosolized, making it a significant concern in laboratory settings
  • Laboratory Response Network (LRN): A national network of laboratories (including clinical, state, and federal laboratories) equipped and trained to respond to biological and chemical threats. The LRN plays a crucial role in the rapid detection and identification of Select Agents