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Microbiology

BSL-3 Pathogens

This area of clinical microbiology deals with the identification, handling, and management of highly dangerous microorganisms that pose a threat to public health, particularly in the context of potential bioterrorism attacks. The core focus is on ensuring rapid and accurate identification, implementing stringent safety protocols, and coordinating a response to minimize harm

Specimen Sources

  • Critical Importance: The source of the specimen dictates the potential agents that might be present and guides the diagnostic approach
  • Common Specimen Types
    • Blood: Potential agents include Bacillus anthracis, Brucella spp., Francisella tularensis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei
    • Sputum: Potential agents include Bacillus anthracis, Francisella tularensis, Yersinia pestis
    • Tissue: Potential agents include Bacillus anthracis, Francisella tularensis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei
    • Lymph Node Aspirate/Biopsy: Potential agents include Francisella tularensis, Yersinia pestis, Burkholderia mallei
  • Collection and Handling
    • Sterile Technique: Strict adherence to sterile collection techniques is essential
    • Biosafety Cabinet (BSC): All specimen handling and processing must be performed within a certified BSL-3 BSC
    • Chain of Custody: Maintaining an unbroken chain of custody is critical for legal, epidemiological, and security purposes
    • Proper Transport: Specimens must be transported promptly and under appropriate conditions to the laboratory

Colony Morphology and Rapid Tests for Presumptive Identification

  • Initial Assessment: Colony morphology on selective and differential media, along with rapid tests, provide the first clues for identification. These are presumptive and require confirmation
  • Key Organisms and Characteristics
    • Bacillus anthracis
      • Colony Morphology: Non-hemolytic, large, grayish-white, “ground glass” or “frosted glass” appearance, irregular edges (“medusa head”), tenacious consistency
      • Rapid Tests: Gram stain (large, Gram-positive rods in chains), “string of pearls” test (positive), Catalase (positive), Motility (non-motile), Gamma Phage Lysis (positive), DFA
    • Yersinia pestis
      • Colony Morphology: Small, grayish-white or translucent, non-hemolytic, “fried egg” appearance
      • Rapid Tests: Gram stain (small, Gram-negative coccobacilli with bipolar staining), Catalase (positive), Oxidase (negative), Urease (negative), Motility (non-motile), Rapid Antigen Tests, DFA
    • Brucella spp.
      • Colony Morphology: Small, translucent to slightly opaque, smooth, non-hemolytic, slow-growing
      • Rapid Tests: Gram stain (small, Gram-negative coccobacilli), Catalase (positive), Oxidase (variable), Urease (positive), H2S production (positive), Motility (non-motile), Rapid Agglutination Tests, DFA
    • Francisella tularensis
      • Colony Morphology: Very small, grayish-white or translucent, non-hemolytic, slow-growing, requires enriched media (cysteine-containing media)
      • Rapid Tests: Gram stain (small, Gram-negative coccobacilli), Catalase (negative), Oxidase (negative), Motility (non-motile), DFA, PCR
    • Burkholderia mallei
      • Colony Morphology: Slow-growing, Gram-negative bacilli
      • Rapid Tests: Gram stain, biochemical tests, and molecular methods (e.g., PCR)
    • Burkholderia pseudomallei
      • Colony Morphology: Gram-negative bacilli
      • Rapid Tests: Gram stain, biochemical tests, and molecular methods (e.g., PCR)
  • Confirmation: Presumptive identification must be confirmed by definitive methods (e.g., biochemical tests, molecular methods)

Role of the Regional Laboratory and Laboratory Response Network (LRN)

  • LRN Structure: A national network of laboratories (clinical, state/local, regional, and national) coordinated by the CDC
  • Regional Laboratory
    • Central Hub: Serves as a critical link between clinical laboratories and national laboratories
    • Key Responsibilities
      • Surge capacity
      • Confirmatory testing
      • Training
      • Surveillance
      • Communication
      • Quality assurance
      • Reagent and supply management
      • Equipment maintenance
      • Collaboration
      • Select Agent expertise
    • Capabilities: BSL-3 lab, advanced diagnostic testing (PCR, sequencing), culture and identification capabilities, expert personnel
  • Clinical Laboratory
    • First Line of Defense: Often the first point of contact for patients with potential bioterrorism-related illnesses
    • Key Responsibilities
      • Awareness of potential agents
      • Proper specimen collection and handling
      • Initial screening tests
      • Reporting suspect findings
      • Communication with healthcare providers and public health authorities
      • Training
      • Participation in LRN activities
  • Response Process: Suspicion/Detection -> Notification -> Investigation -> Specimen Submission -> Initial Testing -> Confirmatory Testing -> Identification and Reporting -> Public Health Response -> Communication

Organism Pathogenicity

  • Understanding the Threat: Knowledge of pathogenicity is essential for risk assessment, biosafety, diagnosis, and treatment
  • Key Components
    • Etiology: The causative agent (e.g., Bacillus anthracis, Yersinia pestis)
    • Transmission: The mechanisms by which the organism spreads (e.g., inhalation, contact, ingestion, arthropod vector)
    • Virulence Mechanisms: Factors and processes that enable the organism to cause disease (e.g., spore formation, capsule, toxins, intracellular survival, immune evasion)
  • Pathogenicity of Specific Organisms (Examples)
    • Bacillus anthracis
      • Etiology: Gram-positive, spore-forming bacterium
      • Transmission: Spores (cutaneous, inhalation, gastrointestinal)
      • Virulence: Spores, capsule, toxins (protective antigen, lethal factor, edema factor)
    • Yersinia pestis
      • Etiology: Gram-negative, non-motile bacterium
      • Transmission: Flea bites (bubonic), inhalation (pneumonic)
      • Virulence: Type III Secretion System (T3SS), Yops, capsule
    • Brucella spp.
      • Etiology: Gram-negative coccobacilli
      • Transmission: Contact, ingestion, inhalation
      • Virulence: Intracellular survival, LPS
    • Francisella tularensis
      • Etiology: Gram-negative coccobacillus
      • Transmission: Tick bites, contact, inhalation, ingestion
      • Virulence: Intracellular survival, capsule
    • Burkholderia mallei
      • Etiology: Gram-negative bacillus
      • Transmission: Contact, inhalation
      • Virulence: Adherence, invasion, Type III Secretion System (T3SS)
    • Burkholderia pseudomallei
      • Etiology: Gram-negative bacillus
      • Transmission: Contact, inhalation, ingestion
      • Virulence: Adherence, invasion, Type III and Type VI Secretion Systems (T3SS & T6SS), capsule

General Principles and Safety

  • BSL-3 Laboratory: All procedures must be performed in a certified BSL-3 laboratory with strict adherence to safety protocols
  • Personal Protective Equipment (PPE): Mandatory use of gloves, gowns, respiratory protection, and eye protection
  • Biosafety Cabinet (BSC): All manipulations must be performed within a BSC
  • Training: Thorough training in BSL-3 practices, handling of Select Agents, PPE use, and emergency procedures is crucial
  • Decontamination: Strict adherence to decontamination procedures for work surfaces, equipment, and waste
  • Chain of Custody: Maintaining a meticulous chain of custody is essential
  • Reporting: Immediate reporting of suspected Select Agent isolates to the appropriate authorities
  • SOPs: Strict adherence to detailed standard operating procedures