HIV Infection Laboratory diagnosis and monitoring 2006 · Laboratory diagnosis and monitoring 2006 Philip Cunningham NSW State Reference Laboratory for HIV/AIDS St Vincent’s Hospital

NIPH/NCHADS Phnom PenhKingdom of Cambodia 2006

HIV Infection HIV Infection

Laboratory diagnosis and Laboratory diagnosis and monitoring 2006monitoring 2006

Philip Cunningham

NSW State Reference Laboratory for HIV/AIDS

St Vincent’s Hospital Sydney


HIV laboratory testsHIV laboratory tests

diagnosisdiagnosis of infectionof infectionacute, recent, established or late stage disease

monitoringmonitoring of ARV therapiesof ARV therapiesimmunological and virological markerspharmacologytoxicities surrogate prognostic markershost factors

diagnosis of opportunistic infectionsdiagnosis of opportunistic infections


‘‘typicaltypical’’ primary HIVprimary HIV--1 infection1 infection

symptoms

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10weeks yearsTime following infection

HIV viral load

HIV proviral DNA

symptoms

‘‘windowwindow’’periodperiod

1° infection

HIV antibodies

Limit of detection


HIV Testing HIV Testing Virus Specific AntibodiesVirus Specific Antibodies

Screening test Screening test –– ELISA, EIA, particle agglutinationELISA, EIA, particle agglutinationReference (confirmatory) testing Reference (confirmatory) testing –– 22ndnd EIA, western blotEIA, western blotSupplemental testing Supplemental testing –– resolve discordant testsresolve discordant testsSerum rapid testsSerum rapid testsSurveillance testing Surveillance testing –– monitoring incidencemonitoring incidence

Testing strategy dictated by populationTesting strategy dictated by populationsensitivity sensitivity vsvs specificityspecificity


ELISA ELISA –– EEnzyme nzyme LLinked inked IImmunosorbent mmunosorbent AAssayssay

detect HIVdetect HIV--1 and HIV1 and HIV--2 responses2 responsesHIV specific antibody and HIV antigen may be HIV specific antibody and HIV antigen may be detected in detected in ‘‘44thth’’ generation testsgeneration testssensitive, specific and reproduciblesensitive, specific and reproduciblefalse reactions possible false reactions possible –– but lowbut lowsuitable for large number of tests suitable for large number of tests –– automatedautomatedMedicare listed November 2005Medicare listed November 2005

Screening widely availableScreening widely availableConfirmation in reference laboratoriesConfirmation in reference laboratories


Antibody/antigen comboAntibody/antigen combo

HIVHIV--1/2 Ag/1/2 Ag/AbAb combo assays detect both antibodies (HIVcombo assays detect both antibodies (HIV--1+2) 1+2) and viral antigen (HIV p24) in single testand viral antigen (HIV p24) in single testResult in 60 minutesResult in 60 minutes

Reduction in window period by 3Reduction in window period by 3--5 days5 daysAcute infection detection without indicationReports of increases in cases identified

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

Issues associated with introductionIssues associated with introductionStrategies and confirmatory algorithmsCost and legal


HIV HIV AbAb/Ag Combo strategy/Ag Combo strategyHIV-1/2 Ab/Ag combo

(x1)

Non reactive (Ag/Ab) Reactive (Ab/Ag)

Negative Non-reactive (Ab/Ag)(x2)

Repeat-reactive (Ab/Ag)(x2)

Negative

HIV-1 western blot (Ab)

Genescreen (Ab only) (x2)

Positive Negative/Indeterminate

Additional Tests HIV-1 p24 antigen

DNA PCR HIV-2

Negative

Indeterminate


Serology of primary Serology of primary HIVHIV--1 infection1 infection

Antibody responses to Antibody responses to immunogenic proteins immunogenic proteins occur in typical series occur in typical series -- diagnosticdiagnostic

All viral proteins are All viral proteins are detected detected –– increasing increasing in intensityin intensity


Effect of antiretroviral Effect of antiretroviral therapytherapy

Antigenic stimulus is Antigenic stimulus is reducedreduced

EnvEnvelope responses elope responses preserved preserved –– high antigenic high antigenic diversity to glycoprotein diversity to glycoprotein --less redundancyless redundancy

Responses to Responses to gaggag discrete discrete structural proteins are lost structural proteins are lost -- redundancy redundancy

DonDon’’t reach positive status t reach positive status –– underreporting underreporting


Advanced HIV infectionAdvanced HIV infection

Low level antibody test Low level antibody test reactivity reactivity Indeterminate profile Indeterminate profile ––pattern typical of late stagepattern typical of late stageRetain glycoprotein Retain glycoprotein reactivityreactivityLate presentersLate presenters

helpfulhelpfulCD4 lymphocyte countCD4 lymphocyte countHIV HIV proviralproviral DNA PCRDNA PCRHIV viral loadHIV viral load


DNA PCRRNA PCR

p24 Ag3rd gen ELISA1st gen ELISA

Detuned ELISA

1wk 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

acute established late


Window periodWindow period

antibodies appear within 3antibodies appear within 3--4 weeks4 weeks

Antibody testing strategies have limitations Antibody testing strategies have limitations –– 100% 100% people seroconvert within 12 weeks people seroconvert within 12 weeks

More sensitive testing strategies including direct More sensitive testing strategies including direct detection of virus (NAT) and 3detection of virus (NAT) and 3rdrd / 4/ 4thth generation generation immunoassays will reduce window period to 6 immunoassays will reduce window period to 6 weeksweeks

HAART therapy during primary infection = delayed HAART therapy during primary infection = delayed antibody responseantibody response


HIV TestingHIV TestingDirect Detection of VirusDirect Detection of Virus

p24 antigen detection – serologyp24 only assays – qualitative and quantitativep24 in combination with antibody Serum

Virus isolation - cultureNucleic acid detection - (NAT))

HIV DNA or RNA ?DNA qualitative – proviral (cellular)

resolution of inconclusive serologydiagnosis in infants - maternal antibodies

RNA quantitative – serial viral loaddrug resistance monitoring subtyping


Pooled NAT testing in a Pooled NAT testing in a blood donor settingblood donor setting

24 x negativeresults

Re-test ALLIndividual samples


Residual risk and NATResidual risk and NAT

BBVBBV PrePre--NAT NAT (<1997)(<1997)

PostPost--NATNAT(>2000)(>2000)

HIVHIV 1 in 1,250,0001 in 1,250,000 1 in 4,800,0001 in 4,800,000

HCVHCV 1 in 230,0001 in 230,000 1 in 3,100,0001 in 3,100,000

HBVHBV 1 in 150,0001 in 150,000 1 in 1,000,0001 in 1,000,000


High risk screeningHigh risk screening

6 x negativeresults

Re-test ALLIndividual samples

? Utility in high risk collection centres and high case load primary care centres? Ampliscreen HIV test v1.5HIV Ag-Ab combo test negative samples pooled and NAT tested every 3 days

Cohen M, Busch M etal South Carolina and San Fransisco


POC testing POC testing –– rapid testsrapid tests

Use of rapid HIV testing strategiesUse of rapid HIV testing strategiesHigh sensitivity >99%High specificity >99%Good reproducibility †

Unprocessed sample type – capillary bloodLittle laboratory equipment requiredNo need for constant water / electricity supplyFew steps – rapid to performVisual interpretationStorage at room temperature









Predictive values for rapid HIV testsPredictive values for rapid HIV tests

HIV HIV prevalenceprevalence

0.1%0.1% 1%1% 5%5% 10%10% 30%30%

NPV single NPV single testtest

100%100% 100%100% 99.9%99.9% 99.9%99.9% 99.6%99.6%

PPV single PPV single testtest

9%9% 50%50% 84%84% 92%92% 98%98%

PPV two PPV two teststests

91%91% 99%99% 99.8%99.8% 99.9%99.9% 100%100%

WHO/CDC rapid testing guidelines 2004WHO/CDC rapid testing guidelines 2004

Assuming 99% sensitivity and 99% specificityAssuming 99% sensitivity and 99% specificity


Rapid testing strategyRapid testing strategy

Blood Sample

Test 1

Test 2

Negative

Result given

Reactive

Discordant

Retest after 6 weeksReferral lab

Positive result

Retest to confirmor Result given

Negative

Result given

Test 2 – may be ELISA or PA

WHO/CDC rapid testing guidelines 2004


POC testing issuesPOC testing issues

Regulatory / licensing issuesRegulatory / licensing issuesEnvironmental factorsEnvironmental factorsTraining Training Test counseling Test counseling Performance characteristicsPerformance characteristicsCostCostUtility in certain settingsUtility in certain settings

Monitoring established Monitoring established infections infections

and treatmentand treatment


Monitoring effect of ARV TherapyMonitoring effect of ARV Therapy

viral loadviral loadTest of choice

when to start therapywhen to change therapyrelative benefit of different regimens

CD4+ lymphocyte countCD4+ lymphocyte countclinical examinationclinical examinationdrug resistance testingdrug resistance testing


Monitoring effect of ARV TherapyMonitoring effect of ARV Therapy

goals of therapygoals of therapyviral load BLD (50 or 400 copies)viral load BLD (50 or 400 copies)

also clinical benefit survivalVL < 5000 copies

CD4 > 300CD4 > 300--500500prevent resistance prevent resistance


Quantitative Viral Load AssaysQuantitative Viral Load Assays

Widely available commercial tests include:Widely available commercial tests include:Roche Amplicor HIV-1 Monitor® (PCR)Bayer Quantiplex HIV-RNA (bDNA)Nuclisens nucleic acid sequence base amplification (NASBA)

Emerging new testsEmerging new testsReal time systems – advantages – extended analytical rangeEasyQ - BiomerieuxTaqMan - RocheRealTime - Abbott


HIV viral load testsHIV viral load testsManufacturer Principle Results Availability Analytical range

Roche RT-PCR (gag)(COBAS HIV MONITOR v1.5)

copies/mL(6 hours to result)

Widely <50 – 100,000<400 – 750,000

Bayer HIV Branched DNA 3.0 (bDNA) (pol)

copies./mL(results 2x less than Roche)

(36 hours to result)

NSW, Vic <50 – 800,000

Biomerieux HIV-1 QT NASBA (gag)

Copies/mL(6 hours to result)

NSW <400 – 1,000,000<80 – 500,000

Roche Real time (Taqman)(gag)

Copies/mL(4-6 hours to result)

New (no sites)

<40 – 10,000,000

Biomerieux EasyQ HIV-1 real time TMA (gag)

Copies/mL & IU/mL(4-5 hours to results)

New (no sites)

<40 – 10,000,000

Abbott Celera RealtimePCR m2000 (polintegrase)

copies/mL Evaluation <40 – 10,000,000

Artus Realtime PCR –Rotorgene

Copies/mL Evaluation <40 – 10,000,000


Interpretation of VL ResultsInterpretation of VL Results

different viral strains different viral strains -- subtypessubtypesday to day variabilityday to day variabilityinterinter--laboratory variabilitylaboratory variabilityvariability between assaysvariability between assaysinterinter--current infectionscurrent infectionsrecent vaccinationrecent vaccinationspecimen quality specimen quality -- transport laboratory factorstransport laboratory factorsbaseline baseline -- 2 x tests 2 x tests -- 4 weeks apart4 weeks apart


VLT Assay variabilityVLT Assay variabilityPCR is exponential PCR is exponential –– log transform copy numberslog transform copy numbers0.25 log0.25 log(10)(10) variation is normal variation is normal (between labs same method)(between labs same method)

5.95889,1405.45288,1205.70500,000

5.03106,7004.5233,7404.7860,000

4.2517,8003.755,6004.0010,000

3.031,0702.533402.78600

1.95881.45301.7050

HIVHIV--1 RNA copy1 RNA copy Acceptable differenceAcceptable difference


VLT Assay variabilityVLT Assay variabilityPCR is exponential PCR is exponential –– log transform copy numberslog transform copy numbers0.20 log0.20 log(10)(10) variation is normalvariation is normal (within lab)(within lab)

5.90794,3305.50316,2305.70500,000

4.9895,5004.5838,0204.7860,000

4.2015,8503.806,3104.0010,000

2.989542.583802.78600

1.90791.50321.7050

HIVHIV--1 RNA copy1 RNA copy Acceptable differenceAcceptable difference


Undetectable by the most sensitive testUndetectable by the most sensitive testH

IV R

NA

(cop

ies/

mL)

10,000

1,000

100

10

1

100,000

0 10 20 30 40 50 60 70

LLD Standard VLT

LLD Ultrasensitive

Weeks on Therapy

20 weeks earlier

Resistance testingResistance testing


Recommendations for resistance Recommendations for resistance testingtesting

1.1. Treatment naTreatment naïïve patients with acute or recent ve patients with acute or recent infection infection virologicvirologic failure during therapyfailure during therapy

2.2. Therapy failure including suboptimal viral Therapy failure including suboptimal viral suppression after initiation of ARV suppression after initiation of ARV

3.3. Pregnant HIV infected women and Pregnant HIV infected women and paediatricpaediatric cases cases with detectable virus load, when therapy change is with detectable virus load, when therapy change is consideredconsidered

4.4. ‘‘sourcesource’’ patient when post exposure prophylaxis is patient when post exposure prophylaxis is consideredconsidered


ConsiderationsConsiderations

drug nadrug naïïve patients with chronic infection in ve patients with chronic infection in who treatment is to be startedwho treatment is to be started

Recommend testing the earliest stored sample if suspicion of resistance is high or prevalence of resistance in the population exceeds 10%


How is it done?How is it done?

Genotypic resistanceGenotypic resistanceDNA sequencing – mutation detectionInterpretation issues - consensusSubtype determination possible$200 - $400

Phenotypic resistancePhenotypic resistanceVirus exposed to drugMolecular based methods>$1000


Breakthrough of HIV Breakthrough of HIV resistanceresistance


DNA sequencing and Virtual Phenotype

-Computer assisted interpretation-Issues with update in rapidly evolving virus-Online updates-In house sequencing advantages



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HIV Infection Laboratory diagnosis and monitoring 2006 · Laboratory diagnosis and monitoring 2006 Philip Cunningham NSW State Reference Laboratory for HIV/AIDS St Vincent’s Hospital