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
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Q
Fluorescence Emission
Detected
hγ
Q
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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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++++----++++++--
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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