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Microbiology

Antigens & Molecular

Antigen detection and molecular methods have become increasingly important tools in the diagnosis of CSF infections, offering rapid and sensitive alternatives or adjuncts to traditional culture-based techniques. These advanced methods are particularly valuable in situations where time is critical, antimicrobial therapy is needed quickly, or culture results are negative or delayed

Antigen Detection Tests

  • Purpose: Rapidly detect specific bacterial or fungal antigens directly in CSF
  • Principle: Use antibodies that specifically bind to antigens (specific components of the pathogen)
  • Benefits
    • Rapid Results: Results available in minutes to hours
    • High Specificity: Antibodies are highly specific, minimizing false-positive results
    • Can Detect Organisms Even if Non-Viable: Can detect antigens even in the presence of prior antibiotic therapy
    • Easy to Perform: Often simpler and less technically demanding than culture
  • Types of Tests
    • Latex Agglutination: Latex beads coated with antibodies against specific antigens. If the antigen is present, the beads will agglutinate (clump together)
    • Enzyme Immunoassays (EIAs): Antibodies are conjugated to an enzyme that produces a color change when the antigen is present
    • Immunochromatographic Assays (Lateral Flow Assays): Antibodies migrate along a strip and bind to the antigen, producing a visible line (similar to a pregnancy test)
  • Targeted Pathogens
    • Streptococcus pneumoniae: Pneumococcal capsular antigen
    • Haemophilus influenzae: type b: Capsular polysaccharide antigen
    • Neisseria meningitidis: Capsular antigens (A, B, C, Y, W-135)
    • Cryptococcus neoformans: Cryptococcal capsular antigen (CrAg)
    • Group B Streptococcus: Capsular antigen
    • Other pathogens: Tests are available for Staphylococcus aureus and Listeria monocytogenes
  • Limitations
    • Sensitivity: Lower sensitivity than molecular methods
    • Limited Number of Targets: Available for only a limited number of pathogens
    • Specificity: Can sometimes have false-positive results
    • May Not Provide Antibiotic Susceptibility: Only detects the antigen; not the viability of the organism
  • Procedure
    • Specimen Preparation: CSF is prepared, usually by centrifugation to remove cells and debris
    • Test Procedure: The test is performed according to the manufacturer’s instructions
    • Result Interpretation: Positive results indicate the presence of the antigen. Negative results do not rule out infection

Molecular Methods

  • Purpose: Rapidly detect specific DNA or RNA sequences of pathogens directly in CSF
  • Principle: Amplify target DNA or RNA sequences to detect the presence of the organism
  • Benefits
    • High Sensitivity and Specificity: PCR is very sensitive, detecting very small amounts of pathogen
    • Rapid Results: Results available within hours
    • Can Detect Organisms Even if Non-Culturable: Detects the genetic material, even if the organism is non-viable or inhibited by antibiotics
    • Multiplexing: Multiple pathogens can be detected in a single test
    • Can Provide Quantitative Results: Some methods (e.g., real-time PCR) can provide the approximate amount of DNA present
  • Types of Tests
    • Polymerase Chain Reaction (PCR)
      • Most Common Method: Amplifies specific DNA sequences of pathogens
      • Procedure
        • DNA is extracted from the CSF
        • Specific primers (short DNA sequences) are designed to bind to a unique region of the pathogen’s DNA
        • PCR amplification: the DNA is amplified (copied) in cycles, creating millions of copies of the target sequence
        • Detection: The amplified DNA is then detected using various methods (gel electrophoresis, real-time PCR)
    • Real-Time PCR (qPCR)
      • Quantitative Results: Measures the amount of DNA produced in each cycle, allowing for quantification of the pathogen
      • Procedure
        • Similar to PCR, but uses fluorescent probes or dyes to detect and quantify the amplified DNA in real-time
        • Can provide the viral load
    • Reverse Transcription PCR (RT-PCR)
      • Detects RNA: Used to detect RNA viruses (e.g., enteroviruses, herpes simplex virus)
      • Procedure
        • RNA is converted to DNA (reverse transcription)
        • PCR is then performed on the newly synthesized DNA
    • Multiplex PCR
      • Multiple Targets Simultaneously: Detects multiple pathogens in a single reaction (e.g., bacterial meningitis panel)
      • Procedure
        • Multiple primer sets are used to amplify different DNA or RNA targets in the same reaction
        • Different detection methods are used to distinguish the amplified products
    • Next Generation Sequencing (NGS)
      • Advanced Method: Used to identify pathogens when conventional methods are non-diagnostic
      • Procedure
        • Extract nucleic acids from the sample
        • Sequencing libraries are prepared and sequenced
        • Sequencing data are analyzed to identify pathogens
  • Targeted Pathogens
    • Bacteria
      • Streptococcus pneumoniae
      • Haemophilus influenzae
      • Neisseria meningitidis
      • Listeria monocytogenes
      • Escherichia coli
      • Other Enterobacteriaceae
    • Viruses
      • Enteroviruses (e.g., poliovirus, coxsackievirus)
      • Herpes simplex virus (HSV)
      • Varicella-zoster virus (VZV)
      • Cytomegalovirus (CMV)
      • West Nile virus
    • Fungi
      • Cryptococcus neoformans
    • Parasites
      • Naegleria fowleri
  • Limitations
    • Cost: More expensive than traditional culture
    • Requires Specialized Equipment and Training
    • Cannot Always Provide Antibiotic Susceptibility Information: Molecular tests can identify the presence of genetic markers for resistance
    • Primer Design: Requires knowledge of the pathogen’s genome to design primers
    • May Detect Non-Viable Organisms: Can detect DNA from dead organisms, which may not be clinically significant
  • Procedure
    • Specimen Preparation: CSF is prepared, usually by centrifugation
    • Nucleic Acid Extraction: DNA or RNA is extracted from the CSF
    • Amplification: PCR or RT-PCR is performed
    • Detection and Result Interpretation: The amplified DNA is detected, and results are interpreted

Role in Diagnosis and Management

  • Rapid Diagnosis: Enables quick confirmation of the presence of a pathogen, allowing for rapid initiation of appropriate antimicrobial therapy, or supportive care
  • Improved Outcomes: Early and targeted therapy improves patient outcomes
  • Identification of Non-Culturable Organisms: Can identify pathogens when culture is negative due to prior antibiotic use or fastidious organisms (e.g., Listeria monocytogenes)
  • Multiplex Testing: Allows for simultaneous detection of multiple pathogens, which is particularly useful in cases of suspected mixed infections, or when clinical presentation is unclear
  • Monitoring Treatment Response: Quantitative PCR can be used to monitor the response to antiviral therapy (e.g., HSV encephalitis)
  • Antimicrobial Stewardship: Guides antibiotic choices and helps with de-escalation

Key Terms

  • Antigen: A substance (e.g., protein, polysaccharide) 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
  • Latex Agglutination: A type of antigen detection test
  • Enzyme Immunoassay (EIA): A type of antigen detection test
  • Immunochromatographic Assay (Lateral Flow Assay): A type of antigen detection test
  • Molecular Method: A method that detects the presence of specific DNA or RNA sequences
  • Polymerase Chain Reaction (PCR): A molecular method to amplify specific DNA sequences
  • Primer: A short DNA sequence that binds to a specific region of DNA
  • Real-Time PCR (qPCR): A PCR method that quantifies DNA during amplification
  • Reverse Transcription PCR (RT-PCR): A PCR method that detects RNA
  • Multiplex PCR: A PCR method that detects multiple targets simultaneously
  • Nucleic Acid Extraction: The process of isolating DNA or RNA from a sample
  • Sensitivity: The ability of a test to correctly identify a pathogen
  • Specificity: The ability of a test 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
  • Viral Load: The amount of virus present in a sample
  • 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
  • Quellung Reaction: (capsular swelling). Serotyping
  • Enriched Media: Culture media containing extra nutrients to support the growth of fastidious microorganisms
  • Fastidious Organism: A microorganism with complex nutritional requirements