New Technologies in New Technologies in Molecular Diagnosis: HIVMolecular Diagnosis: HIV

NSW State Reference Laboratory for HIV/AIDS & Molecular Diagnostic Medicine Laboratory, SydPath

St Vincent’s Hospital Sydney

Trends in molecular diagnosticsTrends in molecular diagnosticsDetection of target Detection of target genes of interestgenes of interest

QuantificationQuantification

Infectious diseasesInfectious diseasesHIVHepatitis C & BTB / MAC CytomegalovirusHerpes simplexVaricella zosterCT/GCHPV

Profiling mutations Profiling mutations associated with disease associated with disease outcomeoutcome

Hepatitis C genotypeHIV drug resistance genotypeHost genetic factorsThrombophiliaCyP450 – drug metabolismHLA type

Key developmentsKey developments

TechnologyTechnology

Uptake in diagnostic arena Alternative methods to PCR – SDA, TMA, LCR, NASBA, bDNAAvailability of Analyte Specific Reagents (ASR)Trend to real time or kinetic formatsAutomationContamination and inhibitor control

? What is driving commercial NAT ? What is driving commercial NAT platform developmentplatform development

Reduce sources of errorReduce sources of error

Reduce tedious processesReduce tedious processes

Improve time to resultImprove time to result

Improved analytical rangeImproved analytical range

Improve limit of detectionImprove limit of detection

Improve specificityImprove specificity

Pre amplification Pre amplification -- extractionextraction

•Volumetric errors amplified•Tedious – manual, repetitive•Specimen integrity

Post amplification & detectionPost amplification & detectionEndpoint detectionEndpoint detection

•Volumetric error•Fragment size vs. probe hybridisation•Time to result•Automation calibration issues•Result calculations

Signal amplification Signal amplification -- bDNAbDNA

TMA PCR bDNA

Target RNA or DNA

Add primers & enzymes

Add primers & enzymes

Copies(RNA) Copies

(DNA)

1 detection probe per copy

1 detection probe per amplified copy

Multiple detection probes per target

Add probes and branched DNA

Virus, bacterium or cell

Comparison of Amplification Methods

HIV, HBV, HCV, CMVStandard curveAmplify signal of label – no amplicon issuesOvernightHigh throughputLimited extraction

Kinetic / real time product Kinetic / real time product detectiondetection

Monitoring in Real timeMonitoring in Real time

Agarose Gel Blotting FRET

Real time PCRReal time PCR

Real Time PCR with 5Real Time PCR with 5’’ Nuclease AssayNuclease AssayProduct detection during amplificationProduct detection during amplification

Fluorescence EmissionQuenched

||||||||||||| ||||||||||||

R Q

hγ

R

|||||||||||||||||||||| |||||

Q

Fluorescence Emission

Detected

hγ

Q

||||||||||||||||||||||||||||||||||

R

Primer Probe

HIV viral load testsHIV viral load tests

<40 – 10,000,000New / evaluation

copies/mLCelera RealtimePCR m2000 (polintegrase)

Abbott

<40 – 10,000,000New Copies/mL & IU/mL(4-5 hours to results)

EasyQ HIV-1 real time TMA (gag)

Biomerieux

<40 – 10,000,000EvaluationCopies/mLRealtime PCR –Rotorgene

Artus

<40 – 10,000,000New Copies/mL(4-6 hours to result)

Real time (Taqman)(gag)

Roche

<400 – 1,000,000<80 – 500,000

NSWCopies/mL(6 hours to result)

HIV-1 QT NASBA (gag)

Biomerieux

<50 – 800,000NSW, Viccopies./mL(results 2x less than Roche)

(36 hours to result)

HIV Branched DNA 3.0 (bDNA) (pol)

Bayer

<50 – 100,000<400 – 750,000

Widelycopies/mL(6 hours to result)

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

Roche

Analytical rangeAvailabilityResultsPrincipleManufacturer

Selected ApplicationsSelected Applications

Primary HIV diagnosis & enhanced surveillancePrimary HIV diagnosis & enhanced surveillance

Infant HIV diagnosisInfant HIV diagnosis

HIV treatment & progressionHIV treatment & progressionmonitoringmonitoring

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 antibodiesacute infection (pre-seroconversion)

RNA quantitative – monitoring / serial viral loaddrug resistance monitoring subtyping – treatment and surveillance

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

MinipoolMinipool NAT testing in blood NAT testing in blood donorsdonors

1 2 3 4

1 2 3 4

4 mini pools of 6 samples each4 mini pools combined and tested(total = 24 samples in single test)

MinipoolMinipool NAT testing in blood NAT testing in blood donorsdonors

1 2 3 4

1Individual retesting of each mini-pool members to identify the positive sample

1

Enhanced surveillance of acuteEnhanced surveillance of acuteprimary HIV infectionprimary HIV infection

Mini pools of 6 samples each

Re-test ALLMini pool members

to identify the positive sample

Eligible samples referred from high case load primary care practices screened NEGATIVE by standard diagnostic

serology tests

HIV drug resistance testingHIV drug resistance testing

Breakthrough of Breakthrough of resistanceresistance

DNA sequencingDNA sequencing

Neonatal HIV diagnosisNeonatal HIV diagnosisSerologic assays

Maternal antibodies persist up to 18 months postpartumAntibody tests not helpful in newborn diagnosisSero-reversion (pos → neg) in serial samplesHIV-1 p24 antigen limited value – complexed by Ab

Virologic assaysVirus culture from PBMCMaternal HIV-1 RNA in obstetric setting is useful in predicting risk of perinatal transmission HIV DNA and RNA useful in infant

Detection in infant is diagnostic for perinatal HIV infectionUseful in timing of transmission (in utero, intrapartum, post partum)Monitoring response to therapy in infected infant

Neonatal DiagnosisNeonatal DiagnosisQualitative HIV DNA PCRQualitative HIV DNA PCR

Detects HIV proviral DNA in peripheral blood mononuclear cells

Most often recommended as preferred virologic test

Sensitivity varies from 50% in the first month to >96% after 1 month (Zaman MM etal Clin Infect Dis 2002; 34:417-18)

Meta analysis of 96 studies using DNA PCR in infants reported 91.6% median sensitivity and 100% median specificity in early diagnosis (Owens DK etal JAMA 1996;275:1342-48)

38% (29-46% 90%CI) were detectable at 48hrs

93% (76-97% 09%CI) detectable at 14 days

Positive 48h(likely intrauterineInfection - early)

1 2 3 4 5 6 7 8 9 10 11 12

48h

14d1-2mo

Positive 14d(likely intrapartum

Infection - late)

3-6mo

HIV RNA/CD4

? considercease ZDV px

aggressive ARV

13 14 15

HIV infection reasonablyexcluded in non-breast fed infant

if negative in 2 or more≥≥1month and 1month and ≥≥4months4months

6-12mo

>2 negative HIV Abtests (<1month apart)Loss Ab/ neg DNA =

un-infected>15-18mo

Infant still Ab+ at12mo – retest 15-18mo

HIV Ab+ >18mo =

HIV infection

Months post partum

HIV laboratory test quiz?HIV laboratory test quiz?

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++----++HIV DNAHIV DNAHIV RNAHIV RNAHIV AgHIV AgHIV HIV AbAb Established HIV

infection on ARV therapyRecent primary Infection

BFP, maternal Abin uninfected infant

Acute infectionVery early acute infectionFalse positive HIV RNA viral load