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 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)

Widely <40 – 10,000,000

Biomerieux EasyQ HIV-1 real time TMA (gag)

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

1 lab <40 – 10,000,000

Abbott Celera Realtime PCR m2000 (pol integrase)

copies/mL Widely <40 – 10,000,000

Artus Realtime PCR –Rotorgene

Copies/mL Unregistered <40 – 10,000,000

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 Ag EIA3rd/4th gen ELISA

1st gen ELISAIncidence EIA

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

Minipool NAT testing in blood Minipool 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)

1 2 3 4

1

1

HIV drug resistance testingHIV drug resistance testing

Breakthrough of Breakthrough of resistanceresistance

DNA sequencingDNA sequencing

Neonatal HIV diagnosisNeonatal HIV diagnosis

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

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 Ab tests (<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

CASE STUDYCASE STUDY

gp160

gp120

p68

p55/51

gp41

p40

p34

p24

p18

α-huIgG

BA,RIBA,RIHIV serology• HIV-1/2 Ab/Ag 4th gen EIA – reactive • HIV-1/2 3rd generation EIA - reactive• HIV-1/2 rapid test (X2) – reactive• HIV-1 western blot – POSITIVE

HIV direct detection• HIV-1 p24 antigen – not detected• HIV proviral DNA PCR – not detected• HIV RNA (gag) – PCR – not detected

N P

BA,RIBA,RI

30 30 y/o African male Ghanay/o African male GhanaImmigration detention centre NSWImmigration detention centre NSWHeterosexualHeterosexualExposure Exposure FSW 1 year agoFSW 1 year agoCurrently 1Currently 1°° syphilissyphilisRecurrent episodes malaria Recurrent episodes malaria –– treated as outpatient treated as outpatient several times over last few yearsseveral times over last few years

What next?What next?Follow up sample and detailed history!Follow up sample and detailed history!HIV serology testing strategies HIV serology testing strategies • Supplementary EIAs

CD4 lymphocyte count CD4 lymphocyte count • 480 (normal)

HIVHIV--2 serology 2 serology • Negative to specific gp 36 antigens

HIV RNA HIV RNA (pol)(pol)• Not detected by bDNA RNA test

HIV culture HIV culture -- virus isolationvirus isolation• CD8 depleted, IL2 enriched, PHA stimulated

PBMC co-culture

gp140

gp105

p68

p56

gp36

p34

p26

p16