AIDS/HIV Testing Methodology (www.ubio.in)
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HIV testing methodologies: an Introduction

HIV testing methodologies: an Introduction

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AIDS/HIV Testing Methodology (www.ubio.in) Presentation Transcript

  • 1.
    • HIV testing
    • Methods and Procedures
  • 2.
    • diagnosis of infection
      • acute, recent, established or late stage disease
    • prognostic markers
    • monitoring of ARV therapies
      • immunological and virological markers
      • toxicities
    • diagnosis of opportunistic infections
    • drug resistance testing
    • Laboratory Tests
  • 3.
    • Typical Primary HIV-1 Infection
    symptoms HIV-1 p24 antigen 0 1 2 3 4 5 6 / 2 4 6 8 10 weeks years HIV antibodies Time following infection HIV viral load HIV proviral DNA symptoms ‘ window’ period 1° infection
  • 4.
    • Types of HIV Assays
    DIAGNOSIS Virus Detection & Quantification Antibody Antigen Detection RNA modified Ag Viral Culture, phenotyping CD4+ ARV Resistance – genotyping ARV Sensitivity EIA Simple, rapid tests Immunoblots Incident assays MANAGEMENT DNA (RNA)
  • 5.
    • HIV Test Spectrum
    DNA PCR RNA PCR p24 Ag 3rd gen ELISA 1st gen ELISA Detuned ELISA 1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr early recent / established advanced
  • 6.
    • HIV p24 antigen– serology
      • In isolation or Ab/Ag Combo test
      • Diagnosis of primary infection viraemia
    • Virus culture/isolation
    • Nucleic acid detection (NAT)
      • Clinical uses Proviral DNA vs. plasma RNA(viral load) resolution of inconclusive serology/neonatal
      • subtyping
      • drug resistance monitoring
    • Direct Virus Detection
  • 7.
    • Principle of Immunoassays
    SOLID PHASE ANTIGEN SAMPLE ANTIBODY ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR
  • 8.
    • EIAs including
      • rapid, simple assays
      • particle agglutination
      • dot/blot
      • Western blot
      • Antigen and Antibody/Antigen (Ab/Ag)
      • Incidence assays
    • Direct Virus Detection
    • Available Assays
  • 9.
    • Dot/Blot Assays
  • 10.
    • Particle Agglutination Assays
  • 11.
    • technically more difficult
    • visual interpretation
    • lack standardisation
      • Performance
      • Interpretation
    • ‘ Gold Standard’ for confirmation
    • Western Blot
  • 12.
    • Difficulties in interpretation
    • Limitations - ‘window period’
    • antibodies appear within 3-4 weeks
      • Direct detection for early detection
      • HIV p24 antigen
      • DNA/RNA (NAT)
    • Combo test = earlier detection
    • Primary infection + therapy = delayed antibody response
    • Incident populations – ‘at risk’
    • Antibody Testing: Limitations
  • 13.
    • Detection of Ag & Ab in a single test
    • utility in primary infection – pre-seroconversion ‘window period’
    • Useful for blood banks
    • Automated platforms available
    • Ag/Ab Combo Tests
    Ag & Ab
  • 14.
    • Chance of false reactivity
    • Might need confirmation tests
    • Replacement by other assays (especially in the USA)
    • More expensive than Antibody assays
    • Ag/Ab Assays: Limitations
  • 15.
    • Detect HIV-1 & HIV-2
    • Cannot differentiate between HIV types
    • Procedural control using anti human IgG
    • Can test using whole blood, serum or plasma
    • Widely available
    • No additional reagents required
    • Storage at room temperature
    • 15 minutes to get result
    • Simple Assays: HIV Determine
  • 16.
    • Detects HIV-1 and HIV-2
    • Will differentiate between types 1 and 2
    • Procedural control using anti-human IgG
    • Serum/plasma only
    • Need additional reagents (included in kit)
    • Requires refrigerated storage
    • ‘ Immunoconcentration’ principle
    • 15 minutes to result
    • BioRad HIV-1/2 Multispot
  • 17.
    • Strategy I : Test all samples with one EIA
    • Strategy II: Strategy I with all reactives retested in a more specific test with different principle and/or antigen.
    • Strategy III: Strategy II with reactives tested in a third test differing from the first two tests.
    • Transfusion Safety
      • Strategy I
    • Surveillance
      • >10% Strategy I, < 10% Strategy II
    • Diagnosis
      • >10% Strategy II, < 10% Strategy III
    • WHO Test Strategies
  • 18.
    • Use strategy for confirmation
      • Use combination of affordable and simple assays
      • Use tests with different principles
      • Use different antigen preparations
    • Use two or three types of ELISAs or rapid tests
    • diagnosis confirmed by second sample
    • Use direct detection of virus (PCR) for final confirmation
    • Important: Always use a QC sample
    • WHO Test Guidelines
  • 19.
    • www.ubio.in