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Microbiology

Direct Detection

This section covers direct detection of viral pathogens. Direct detection methods aim to identify the virus itself, either by detecting viral particles, viral antigens, or viral nucleic acids, rather than detecting the host’s immune response (e.g., antibodies)

Direct Detection of Viral Pathogens

  • Definition: Direct detection methods are laboratory techniques used to identify the presence of a virus in a clinical specimen by detecting viral components (e.g., viral particles, antigens, or nucleic acids)
  • Advantages
    • Rapid turnaround time compared to viral culture
    • High sensitivity and specificity for many assays
    • Can detect non-cultivable viruses
    • Can provide quantitative data (viral load)

Microscopy

  • Electron Microscopy (EM)
    • Principle: Uses a beam of electrons to visualize viral particles in clinical specimens
    • Procedure: Specimen is prepared and examined under high magnification
    • Applications: Detection of viruses that are difficult to culture, identification of novel viruses, and research purposes
    • Advantages: Can visualize viral morphology, detect multiple viruses simultaneously
    • Disadvantages: Requires specialized equipment and trained personnel, low sensitivity, labor-intensive
  • Light Microscopy
    • Principle: Uses visible light to visualize viral inclusions or cytopathic effects in infected cells
    • Procedure: Infected cells are stained and examined under a light microscope
    • Applications: Detection of viral inclusions in tissue biopsies or cytologic specimens
    • Advantages: Simple and inexpensive
    • Disadvantages: Low sensitivity, requires skilled interpretation

Antigen Detection Assays

  • Principle: Uses antibodies to detect viral antigens in clinical specimens
  • Types
    • Enzyme Immunoassay (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA)
      • Principle: An enzyme-labeled antibody binds to viral antigen, and the enzyme activity is measured to quantify the amount of antigen present
      • Procedure: Specimen is incubated with antibody, washed, and then incubated with an enzyme-labeled secondary antibody. A substrate is added, and the resulting color change is measured
      • Applications: Detection of respiratory viruses (e.g., influenza, RSV), hepatitis viruses (e.g., HBsAg), and HIV (e.g., p24 antigen)
      • Advantages: High throughput, relatively inexpensive
      • Disadvantages: Lower sensitivity compared to molecular methods
    • Immunofluorescence Assay (IFA)
      • Principle: An antibody labeled with a fluorescent dye binds to viral antigen, and the complex is visualized under a fluorescent microscope
      • Procedure: Specimen is incubated with antibody, washed, and then examined under a fluorescent microscope
      • Applications: Detection of respiratory viruses (e.g., influenza, RSV), herpes simplex virus (HSV), and varicella-zoster virus (VZV)
      • Advantages: Can detect multiple antigens simultaneously, relatively rapid
      • Disadvantages: Requires a fluorescent microscope and trained personnel, subjective interpretation
    • Lateral Flow Immunoassay (Rapid Antigen Test)
      • Principle: A sample migrates along a membrane containing antibodies specific for the target antigen. If the antigen is present, it binds to the antibodies, resulting in a visible colored line
      • Procedure: Specimen is applied to the test device, and the results are read visually after a specified time
      • Applications: Rapid detection of respiratory viruses (e.g., influenza, RSV, SARS-CoV-2)
      • Advantages: Rapid turnaround time, simple to perform, no specialized equipment required
      • Disadvantages: Lower sensitivity compared to other methods, qualitative results only

Nucleic Acid Amplification Tests (NAATs)

  • Principle: Amplifies viral nucleic acids (DNA or RNA) to detectable levels
  • Types
    • Polymerase Chain Reaction (PCR)
      • Principle: Uses DNA polymerase to amplify a specific DNA sequence
      • Procedure: Specimen is mixed with primers, DNA polymerase, and nucleotides, and subjected to repeated cycles of heating and cooling to amplify the target DNA sequence
      • Applications: Detection and quantification of a wide range of viruses, including HIV, hepatitis viruses, herpesviruses, and respiratory viruses
      • Advantages: High sensitivity and specificity, can provide quantitative data (viral load)
      • Disadvantages: Susceptible to contamination, requires specialized equipment and trained personnel
    • Reverse Transcription PCR (RT-PCR)
      • Principle: Uses reverse transcriptase to convert RNA into DNA, followed by PCR amplification
      • Procedure: RNA is extracted from the specimen, converted to DNA using reverse transcriptase, and then amplified using PCR
      • Applications: Detection and quantification of RNA viruses, such as HIV, hepatitis viruses, and respiratory viruses
      • Advantages: High sensitivity and specificity, can provide quantitative data (viral load)
      • Disadvantages: Susceptible to contamination, requires specialized equipment and trained personnel
    • Real-Time PCR (qPCR)
      • Principle: Measures the amount of amplified DNA or RNA in real time during the PCR reaction
      • Procedure: Similar to PCR or RT-PCR, but includes a fluorescent dye or probe that binds to the amplified DNA or RNA, allowing for quantification
      • Applications: Detection and quantification of a wide range of viruses, including HIV, hepatitis viruses, herpesviruses, and respiratory viruses
      • Advantages: High sensitivity and specificity, rapid turnaround time, quantitative results, reduced risk of contamination
      • Disadvantages: Requires specialized equipment and trained personnel, more expensive than conventional PCR
    • Transcription-Mediated Amplification (TMA)
      • Principle: Uses RNA polymerase and reverse transcriptase to amplify RNA targets
      • Procedure: RNA is amplified using RNA polymerase and reverse transcriptase in an isothermal reaction
      • Applications: Detection of Chlamydia trachomatis and Neisseria gonorrhoeae, as well as some viruses
      • Advantages: High sensitivity and specificity, isothermal reaction (no need for thermal cycling)
      • Disadvantages: Requires specialized equipment and reagents, less widely used than PCR
    • Loop-Mediated Isothermal Amplification (LAMP)
      • Principle: Amplifies DNA using a DNA polymerase and a set of four to six primers that recognize six to eight distinct regions on the target DNA
      • Procedure: DNA is amplified in an isothermal reaction using a DNA polymerase and a set of specific primers
      • Applications: Detection of various viruses, including SARS-CoV-2
      • Advantages: Rapid turnaround time, simple to perform, isothermal reaction
      • Disadvantages: Requires careful primer design, less flexible than PCR

Viral Culture

  • Principle: Growing viruses in cell culture to detect their presence and identify them
  • Procedure: Clinical specimen is inoculated into susceptible cell cultures, and the cells are monitored for cytopathic effects (CPE) or viral antigens
  • Applications: Isolation and identification of viruses, susceptibility testing, and research purposes
  • Advantages: Can isolate live virus, useful for studying viral properties
  • Disadvantages: Slow turnaround time, requires specialized equipment and trained personnel, not all viruses can be cultured

Next-Generation Sequencing (NGS)

  • Principle: High-throughput sequencing of nucleic acids to identify and characterize viruses
  • Procedure: Viral nucleic acids are extracted from the specimen, amplified, and sequenced using NGS platforms
  • Applications: Identification of novel viruses, characterization of viral genomes, detection of drug resistance mutations, and surveillance of viral populations
  • Advantages: Can detect multiple viruses simultaneously, provides comprehensive genomic information
  • Disadvantages: Requires specialized equipment and bioinformatics expertise, expensive, long turnaround time

Key Terms

  • Direct Detection: Identifying a virus by detecting viral components (particles, antigens, nucleic acids)
  • Antigen: A substance that triggers an immune response, typically a protein on the surface of a virus
  • Antibody: A protein produced by the immune system that binds to a specific antigen
  • Nucleic Acid: DNA or RNA, the genetic material of viruses
  • PCR (Polymerase Chain Reaction): A molecular technique used to amplify DNA
  • RT-PCR (Reverse Transcription PCR): A molecular technique used to amplify RNA
  • Real-Time PCR (qPCR): A PCR technique that measures the amount of amplified DNA or RNA in real time
  • Viral Load: The quantity of virus present in a specific amount of a patient’s body fluid (e.g., copies/mL)
  • Cytopathic Effect (CPE): Visible changes in cells caused by viral infection
  • Sensitivity: The ability of a test to correctly identify individuals with the infection
  • Specificity: The ability of a test to correctly identify individuals without the infection
  • Turnaround Time: The time it takes to complete a test and report the results
  • Isothermal Amplification: Nucleic acid amplification at a constant temperature
  • Primer: A short sequence of nucleic acid that serves as a starting point for DNA synthesis
  • Probe: A labeled sequence of nucleic acid that is used to detect a specific target sequence
  • ELISA (Enzyme-Linked Immunosorbent Assay): A type of immunoassay that uses an enzyme-labeled antibody to detect antigens
  • IFA (Immunofluorescence Assay): A type of immunoassay that uses a fluorescent-labeled antibody to detect antigens