3 update onhivtesting

Update on HIV Testing

[A Very Fast]

Message

There are numbers of tests

They should be used in combination (strategies)

Combinations must be consistent

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

‘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 & Quantification

Antibody

Antigen

Detection

RNAmodified Ag

Viral Culture, phenotyping

CD4+

ARV Resistance – genotypingARV Sensitivity

HIV Assays: Methodologies FOR THE DIAGNOSIS (DETECTION) & MANAGEMENT OF HIV

EIASimple, rapid

tests Immunoblots

Incident assays

MANAGEMENT

DNA(RNA)

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

Spectrum Spectrum of anti-HIV of anti-HIV

testing testing

HIV Testing -Direct Detection of Virus 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

Principle of Immunoassays

SOLID PHASESOLID PHASESOLID PHASESOLID PHASE

ANTIGEN

SAMPLE ANTIBODY

ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR

Available Assays

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

Direct Virus Detection

Particle Agglutination

Western Blot

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

- performance - interpretation - indeterminate reactions –

resolution of ??

‘Gold Standard’ for confirmation

Antibody testing limitations

Difficulties in interpretation

Limitations - ‘window period’

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

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

Primary infection + therapy = delayed antibody response

Ag/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 availableAg & AbAg & AbAg & AbAg & Ab

Ab & Ag

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

(especially in the USA) Cost Legal issues

Conclusions HIV-1/2 Ag/Ab combo assays perform well

Differences in limit of detection of Ag (140 - <25 pg/mL)

May shorten window period by 3-5 days PHI detection without indication

Issues associated with introduction Strategies and confirmatory algorithms

ARV therapy effects seroconversion events

What about simple assays?

HIV 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

BioRad 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

WHO 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 Strategies

Transfusion safety

Surveillance

� Diagnosis� Risk factors� No risk factors

Strategy I

>10% I <10% II

� Strategy II� >10% II� <10% III

Testing Strategies

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

Objectives 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

Aims 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)

Screening 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

Ab + Ag AbAg

Ab +AgAb +AgAb +AgAb +Ag AbAgAbAg

Ab +AgAb +AgAb +AgAb +Ag

Ab +AgAb +Ag

Ab +AgAb +AgAb +AgAb +AgAb +AgAb +Ag

AbAgAbAg

AbAgAbAg

AbAgAbAg

AbAgAbAgAbAgAbAgAbAgAbAgAbAgAbAg

Serological 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 StrategySCREENIN

G

SUPPLEMENTAL

Supplemental Assays

Range of assays that further define

sero-status

High Performance (higher specificity)

The Use of Screening Assays

Define samples as negative for a given analyte

Enable high throughput

Assay Selection depends on:

laboratory infrastructure access to reference laboratory desired characteristics of the test equipment performance time shelf life and stability of reagents price technical skills of personnel support (technical, kit supply, etc)

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%

Negative Predictive ValueNon-reactive test, prevalence 2%

99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98

99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98

99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98

Sp

eci

fic

ity

Sensitivity

60

70

90

60 70 80 90 95 98 99 %

Positive Predictive ValueReactive test, prevalence 2%

4.95 4.954.95 4.95

6.49 6.496.49 6.49

9.43 9.43 9.439.43 9.43 9.43

17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24 17.24

29.41 29.41 29.41 29.41 29.41 29.41 29.4129.41 29.41 29.41 29.41 29.41 29.41 29.41

51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02 51.02

67.57 67.57 67.57 67.57 67.57 67.57 67.57 67.57 67.57 67.57 67.57 67.57

67.5767.57

Sensitivity

Sp

ecif

icit

y

60

70

80

90

95

98

99

60 70 80 90 95 98 99 %

Cut-off valueCut-off value

XX

1 1

Fre

qu

ency

Fre

qu

ency

3SD2SD

1SD

2 2 3 3

Cross reactivity

Positive Predictive ValueAssays used separately

Assay 1Sens 99.0%Spec 99.5%

PPV

28.4%

80.2%

98.0%


PPV

66.4%

95.3%

99.6%

Prevalence

0.2%

2.0%

20.0%

Positive Predictive ValueAssays used sequentially


PPV

28.4%

80.2%

98.0%


PPV

99.75%

99.97%

99.99%

Prevalence

0.2%

2.0%

20.0%

Assay 1 Assay 2 Assays 1+2Assay 1 Assay 2 Assays 1+2

PPV vs Prevalence

(in sequence)

Sensitivity 99.0% 99.0% Specificity 99.5% 99.9%

Prevalence

0.2%

2.0%

20.0%

28.4%

80.2%

66.4% 99.75%

98.0%

80.2% 99.97%

99.6% 99.99%

PPV PPV PPV

Why Follow a Strategy?

The Importance of Maintaining a Strategy

Consistency of laboratory records Consistency of results Clarity of results to doctors Maintaining data base to assess

performances Avoiding common false reactivity Avoiding technical errors Reducing costs

WHO 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 Strategies

Transfusion safety

Surveillance

� Diagnosis� Risk factors� No risk factors

Strategy I

>10% I <10% II

� Strategy II� >10% II� <10% III

WHO GuidelinesOther possibilities strategy for confirmation

combination of affordable & simple assays different test principles different 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 Testing

comparative costs (AUD$) 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 Strategies

-Screening test x1

Screening test x2

Supplemental test

Other tests

NEG

R

- NEG-

- NEG+

POS

Eliminates laboratory

error

RR or R-

Message

There are numbers of tests

They should be used in combination (strategies)

Combinations must be consistent

Documents

3 update onhivtesting