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Microbiology

Molecular Methods

Antigen detection and molecular methods have become increasingly important in the diagnosis of gastrointestinal (GI) infections, providing rapid and sensitive alternatives or adjuncts to traditional culture-based techniques. These advanced methods are particularly valuable when:

  • Rapid Diagnosis is Critical: For example, Clostridioides difficile infections
  • Low Bacterial Load: Some pathogens may be present in small numbers
  • Antibiotic Therapy: The diagnosis can be difficult when a patient has already received antibiotics
  • Outbreak Investigations: Quick identification of pathogens
  • Public Health Surveillance: These methods are used

General Principles

  • Rapid Results: These methods provide results within hours
  • High Sensitivity and Specificity: These methods can detect very small amounts of pathogens
  • Targeted Approach: Focus on specific pathogens
  • Limitations
    • Cost: Can be more expensive
    • Specialized Equipment and Training: Requires specialized equipment
    • May Not Provide Antibiotic Susceptibility Information: Not all pathogens have antibiotic information
    • Does not always provide information about the live organism

Antigen Detection Methods

Clostridioides difficile (formerly Clostridium difficile)

  • Purpose: Rapid detection of C. difficile toxins
  • Principle: Antibodies react with the toxin (toxin A and/or toxin B)
  • Types of Tests
    • Enzyme Immunoassays (EIAs): Simple, relatively inexpensive. However, EIA tests have variable sensitivity
    • Immunochromatographic Assays (Lateral Flow Assays): Rapid, easy to perform, and can be used at the point of care (POC)
  • Procedure
    • Specimen Preparation: Stool is prepared, usually by mixing it with a diluent
    • Testing: Follow the manufacturer’s instructions
    • Result Interpretation: A positive result indicates the presence of C. difficile toxin
  • Advantages
    • Rapid results
    • Easy to perform
  • Disadvantages
    • Lower sensitivity than molecular methods
    • Some tests detect only toxin A, or both A and B

Molecular Methods

Clostridioides difficile (formerly Clostridium difficile)

  • Purpose: To detect C. difficile DNA or toxin genes
  • Method
    • PCR: Amplifies specific regions of the C. difficile genome
    • Real-time PCR (qPCR): Quantifies the amount of DNA
  • Procedure
    • Specimen Preparation: DNA is extracted
    • Amplification: PCR is performed
    • Detection and result interpretation
  • Advantages
    • High sensitivity and specificity
    • Rapid results
  • Disadvantages
    • More expensive
    • Requires specialized equipment
  • Clinical Application: The most sensitive method

Shiga Toxin-Producing Escherichia coli (STEC)

  • Purpose: To detect Shiga toxin genes
  • Method
    • PCR: Amplifies the Shiga toxin genes (stx1 and stx2)
  • Procedure
    • Specimen Preparation: DNA is extracted from the stool
    • Amplification: PCR is performed
    • Detection and result interpretation
  • Advantages
    • High sensitivity and specificity
    • Rapid results
  • Disadvantages
    • Cost
    • Requires specialized equipment
  • Clinical Application: Detects E. coli that produce Shiga toxin

Additional Considerations

  • Specimen Quality: Proper collection is essential
  • Combination of Tests: Combining several tests
  • Test Limitations: All tests have limitations
  • Prompt Communication: Results must be communicated
  • Other tests
    • Salmonella
    • Shigella

Key Terms

  • Gastrointestinal (GI): Pertaining to the stomach and intestines
  • Antigen: A substance that triggers an immune response
  • Antibody: A protein produced by the immune system that recognizes and binds to a specific antigen
  • Antigen Detection Test: A test that detects the presence of a specific antigen
  • Enzyme Immunoassay (EIA): Test that uses enzymes
  • Immunochromatographic Assay (Lateral Flow Assay): Simple test
  • Clostridioides difficile: (formerly Clostridium difficile): Bacteria that causes diarrhea
  • PCR (Polymerase Chain Reaction): Amplifies a specific DNA region
  • Real-Time PCR (qPCR): Used to quantify PCR products
  • Shiga Toxin-Producing Escherichia coli (STEC): Produces toxins
  • Escherichia coli: Gram-negative bacteria
  • Shiga toxin: A toxin
  • Toxin A and/or Toxin B: Toxins produced by C. difficile
  • Nucleic Acid Extraction: The process of isolating DNA or RNA from a sample
  • Sensitivity: Ability to correctly identify a pathogen
  • Specificity: Ability to correctly identify the absence of a pathogen
  • False Positive: A test result that indicates the presence of a pathogen when it is not actually present
  • False Negative: A test result that indicates the absence of a pathogen when it is actually present
  • Antimicrobial Therapy: The use of antibiotics or antifungals to treat infections
  • Amplification: The process of making multiple copies of a DNA or RNA sequence
  • Target: The specific DNA or RNA sequence that is amplified by PCR