HIV Testing

CDC power point edited by M. Myers

HIV Testing

CDC power point edited by M. Myers

MessageMessage

There are numbers of tests

They should be used in combination (strategies)

Combinations must be consistent

There are numbers of tests

They should be used in combination (strategies)

Combinations must be consistent

Laboratory TestsLaboratory 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

diagnosis of infectionacute, recent, established or late

stage disease prognostic markers monitoring of ARV therapies

immunological and virological markers

toxicities diagnosis of opportunistic infections drug resistance testing

‘typical’ primary HIV-1 infection‘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

DIAGNOSIS

Virus Detection

Antibody

Antigen

Detection

+

HIV Assays: Methodologies FOR THE DIAGNOSIS (DETECTION)

EIASimple, rapid

tests Immunoblots

Incident assays

MANAGEMENT

DNA(RNA)

HIV Testing -Direct Detection of VirusHIV Testing -Direct Detection of Virus HIV antigen– serology

- In isolation - 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

HIV antigen– serology - In isolation - 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

Available AssaysAvailable Assays

EIAs including rapid, simple particle agglutination, dot/blotWestern blotAntigen & Ab/AgIncidence assays

Direct Virus Detection

EIAs including rapid, simple particle agglutination, dot/blotWestern blotAntigen & Ab/AgIncidence assays

Direct Virus Detection

Particle AgglutinationParticle Agglutination

Western BlotWestern Blot

Expensive – $ 80 - 100 technically more difficult visual interpretation lack standardisation

- performance- interpretation- indeterminate reactions –

resolution of ?? ‘Gold Standard’ for confirmation

Expensive – $ 80 - 100 technically more difficult visual interpretation lack standardisation

- performance- interpretation- indeterminate reactions –

resolution of ?? ‘Gold Standard’ for confirmation

Antibody testing limitationsAntibody testing limitations

Difficulties in interpretation

Limitations - ‘window period’

antibodies appear within 3-4 weeksDirect detection – HIV p24 antigen or

DNA/RNA (NAT) – pre-antibodyCombo test = earlier detection

Primary infection + therapy = delayed antibody response

Difficulties in interpretation

Limitations - ‘window period’

antibodies appear within 3-4 weeksDirect detection – HIV p24 antigen or

DNA/RNA (NAT) – pre-antibodyCombo test = earlier detection

Primary infection + therapy = delayed antibody response

Ag/Ab Combo testsAg/Ab Combo tests

Detection of Ag & Ab in a single test

utility in primary infection – pre-seroconversion ‘window period’

Incident populations – ‘at risk’ Blood bank Automated platforms available

Detection of Ag & Ab in a single test

utility in primary infection – pre-seroconversion ‘window period’

Incident populations – ‘at risk’ Blood bank Automated platforms availableAg & AbAg & AbAg & AbAg & Ab

Ab & Ag

Issues with Combo AssaysIssues with Combo Assays Testing strategies False reactivity rates Confirmation strategies Replacement of other assays

(especially in the USA) Cost Legal issues

Testing strategies False reactivity rates Confirmation strategies Replacement of other assays

(especially in the USA) Cost Legal issues

What about simple assays?What about simple assays?

HIV Determine testHIV Determine test

Detect HIV-1 & HIV-2 Cannot differentiate Procedural control – anti Hu IgG Whole blood or serum/plasma Widely available No additional reagents required Room temperature storage 15 minutes to result

Detect HIV-1 & HIV-2 Cannot differentiate Procedural control – anti Hu IgG Whole blood or serum/plasma Widely available No additional reagents required Room temperature storage 15 minutes to result

BioRad HIV-1/2 MultispotBioRad HIV-1/2 Multispot Detects HIV-1 and HIV-2 Will differentiate 1 and 2 Procedural control – anti-Hu IgG Serum / plasma only Additional reagents (included) Requires refrigerated storage ‘Immunoconcentration’ principle 15 minutes to result

Detects HIV-1 and HIV-2 Will differentiate 1 and 2 Procedural control – anti-Hu IgG Serum / plasma only Additional reagents (included) Requires refrigerated storage ‘Immunoconcentration’ principle 15 minutes to result

WHO Recommended StrategiesWHO Recommended 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.

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.

WHO Recommended Testing StrategiesWHO Recommended Testing Strategies

Transfusion safety

Surveillance

Diagnosis Risk factors No risk factors

Transfusion safety

Surveillance

Diagnosis Risk factors No risk factors

Strategy I

>10% I <10% II

Strategy II >10% II <10% III

Strategy I

>10% I <10% II

Strategy II >10% II <10% III

Testing Strategies Testing Strategies

AIM: To develop the logic used in establishing the use of HIV tests (testing strategies)

AIM: To develop the logic used in establishing the use of HIV tests (testing strategies)

Objectives of Testing StrategiesObjectives of Testing Strategies To achieve the correct diagnosis in the most

efficient manner To maintain consistency in testing To know the predictive value of the testing

process To develop baseline data for assessing

changes To deliver useful results

To achieve the correct diagnosis in the most efficient manner

To maintain consistency in testing To know the predictive value of the testing

process To develop baseline data for assessing

changes To deliver useful results

Aims in Developing HIV Testing StrategiesAims in Developing HIV Testing Strategies

To arrive at the correct sero-diagnosisTo arrive at the correct sero-diagnosis To minimise total testing; thus costTo minimise total testing; thus cost Minimise samples classed as indeterminate Minimise samples classed as indeterminate

or dual reactorsor dual reactors Detect HIV-1 negative but HIV-2 positiveDetect HIV-1 negative but HIV-2 positive Follow likely seroconverters (HIV-1 or -2)Follow likely seroconverters (HIV-1 or -2)

To arrive at the correct sero-diagnosisTo arrive at the correct sero-diagnosis To minimise total testing; thus costTo minimise total testing; thus cost Minimise samples classed as indeterminate Minimise samples classed as indeterminate

or dual reactorsor dual reactors Detect HIV-1 negative but HIV-2 positiveDetect HIV-1 negative but HIV-2 positive Follow likely seroconverters (HIV-1 or -2)Follow likely seroconverters (HIV-1 or -2)

Screening AssaysScreening Assays

Are used to detect antibody-- specific or nonspecific

Are designed to handle large numbers of samples with rapid throughput

Must be high performance

Should include a full range of HIV antigens

Are used to detect antibody-- specific or nonspecific

Are designed to handle large numbers of samples with rapid throughput

Must be high performance

Should include a full range of HIV antigens

Serological Testing StrategySerological Testing Strategy

NEG SCREENING TEST, highly sensitiveSCREENING TEST, highly sensitive

POSSUPPLEMENTAL TEST, SUPPLEMENTAL TEST,

highly sensitive & higher highly sensitive & higher specificity specificity

ADDITIONALADDITIONALTESTSTESTS

REACTIVE

NEGNEG

POS

IND

NEG

IND

POINT OF REPORTING

NEGHIV1/2

SCREEN

POS HIV-1 WB

ADDITIONALTESTS

REACTIVE

NEG

NEG

INDPOS

IND

HIV Testing StrategyHIV Testing StrategySCREENIN

G

SUPPLEMENTAL

Supplemental AssaysSupplemental Assays

Range of assays that further define

sero-status

High Performance (higher specificity)

Range of assays that further define

sero-status

High Performance (higher specificity)

The Use of Screening AssaysThe Use of Screening Assays

Define samples as negative for a given analyte

Enable high throughput

Define samples as negative for a given analyte

Enable high throughput

Predictive Values

Positive Predictive ValuesPositive Predictive Values::The likelihood of a sample identified as a The likelihood of a sample identified as a reactive by a test being truly POSITIVE for reactive by a test being truly POSITIVE for the analyte used as the basis of the test.the analyte used as the basis of the test.

PPV =True Positives

True Positives + False ReactivesX 100%

Predictive Values

Negative Predictive ValuesNegative Predictive Values::

The likelihood that a sample identified as a non-The likelihood that a sample identified as a non-reactive by a test is truly NEGATIVE for the reactive by a test is truly NEGATIVE for the analyte used as the basis of the test.analyte used as the basis of the test.

NPV =True Negatives

True Negatives + False Negatives

X 100%

WHO Recommended StrategiesWHO Recommended 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.

WHO Recommended Testing StrategiesWHO Recommended Testing Strategies

Transfusion safety

Surveillance

Diagnosis Risk factors No risk factors

Transfusion safety

Surveillance

Diagnosis Risk factors No risk factors

Strategy I

>10% I <10% II

Strategy II >10% II <10% III

Strategy I

>10% I <10% II

Strategy II >10% II <10% III

WHO GuidelinesWHO GuidelinesOther possibilities strategy for confirmation

combination of affordable & simple assaysdifferent test principlesdifferent antigen preparations

two or three ELISAs or rapid tests diagnosis confirmed by second sample detection of virus (PCR) antigen detection (limited lab.facilities) Always use a QC sample

Other possibilities strategy for confirmation

combination of affordable & simple assaysdifferent test principlesdifferent antigen preparations

two or three ELISAs or rapid tests diagnosis confirmed by second sample detection of virus (PCR) antigen detection (limited lab.facilities) Always use a QC sample

Cost of HIV TestingCost of HIV Testing

comparative costs ELISA (Ab only) - $2 per test

EIA (Ab/Ag combo) - $3.50

rapid test - $10-20 per test

Western blot $80 - 100

p24 antigen $30

PCR - qualitative $80 - 100

PCR - quantitative (viral load) $90 – 150*

DNA sequencing (resistance) $400 – 700

comparative costs ELISA (Ab only) - $2 per test

EIA (Ab/Ag combo) - $3.50

rapid test - $10-20 per test

Western blot $80 - 100

p24 antigen $30

PCR - qualitative $80 - 100

PCR - quantitative (viral load) $90 – 150*

DNA sequencing (resistance) $400 – 700

Summary of Testing StrategiesSummary of Testing Strategies

-Screening test x1

Screening test x2

Supplemental test

Other tests

NEG

R

- NEG-

- NEG+

POS

Eliminates laboratory

error

RR or R-