HIV testing Methods and Procedures

HIV testing

Methods and Procedures

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

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

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

Typical Primary HIV-1 Infection

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)

Types of HIV Assays

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

HIV Test Spectrum

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

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

Principle of Immunoassays SOLID PHASE ANTIGEN SAMPLE ANTIBODY ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR

Principle of Immunoassays

EIAs including rapid, simple assays particle agglutination dot/blot Western blot Antigen and Antibody/Antigen (Ab/Ag) Incidence assays Direct Virus Detection Available Assays

EIAs including

rapid, simple assays

particle agglutination

dot/blot

Western blot

Antigen and Antibody/Antigen (Ab/Ag)

Incidence assays

Direct Virus Detection

Available Assays

Dot/Blot Assays

Dot/Blot Assays

Particle Agglutination Assays

Particle Agglutination Assays

technically more difficult visual interpretation lack standardisation Performance Interpretation ‘ Gold Standard’ for confirmation Western Blot

technically more difficult

visual interpretation

lack standardisation

Performance

Interpretation

‘ Gold Standard’ for confirmation

Western Blot

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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