AIDS/HIV Testing Methodology ()

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HIV testing

Methods and Procedures

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diagnosis of infectionacute, recent, established or late stage disease

prognostic markersmonitoring of ARV therapies

immunological and virological markerstoxicities

diagnosis of opportunistic infectionsdrug 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 10weeks years

HIV antibodies

Time following infection

HIV viral load

HIV proviral DNA

symptoms

‘window’period

1° infection


Types of HIV Assays

DIAGNOSIS

Virus Detection & Quantification

Antibody

Antigen

Detection

RNAmodified Ag

Viral Culture, phenotyping

CD4+

ARV Resistance – genotypingARV Sensitivity

EIASimple, rapid tests Immunoblots

Incident assays

MANAGEMENT

DNA(RNA)


HIV Test Spectrum

DNA PCRDNA PCRRNA PCRRNA PCR

p24 Agp24 Ag3rd gen ELISA1st gen ELISA

Detuned ELISA1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

early recent / established advanced


HIV p24 antigen– serologyIn 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/neonatalsubtypingdrug resistance monitoring

Direct Virus Detection


Principle of Immunoassays

SOLID PHASESOLID PHASESOLID PHASESOLID PHASE

ANTIGEN

SAMPLE ANTIBODY

ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR


EIAs including rapid, simple assaysparticle agglutinationdot/blotWestern blotAntigen and Antibody/Antigen (Ab/Ag)Incidence assays

Direct Virus Detection

Available Assays


Dot/Blot Assays


Particle Agglutination Assays


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 detectionHIV p24 antigenDNA/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 & AbAg & AbAg & AbAg & Ab


Chance of false reactivityMight need confirmation testsReplacement 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


Detects HIV-1 and HIV-2Will differentiate between types 1 and 2Procedural control using anti-human IgGSerum/plasma only Need additional reagents (included in kit)Requires refrigerated storage ‘Immunoconcentration’ principle15 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


Use strategy for confirmationUse combination of affordable and simple assaysUse tests with different principlesUse 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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Technology

AIDS/HIV Testing Methodology ()