Fundamentals of DiagnosisMolecular Testing in 2004

Philip Cunningham

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

St Vincent’s Hospital Sydney

Molecular Diagnostics

Diagnosis

Monitoring

Key developments

Technology

– Uptake in diagnostic arena – lab workflow and design issues– Alternative methods ‘other than PCR’– Availability of ASR– Real time or kinetic formats– Automation– Contamination control

Key developments

Diagnosis

– Herpes simplex (types 1&2)

– Cytomegalovirus - active infection

– Human papillomavirus (HPV) diagnosis –♀♂

– HIV primary infection

– Chlamydia trachomatis & Neisseria gonorrhoeae

Key developments

Monitoring

– Cytomegalovirus - response to therapy / relapse

– HIV RNA quantification – viral load testing

– HIV drug resistance testing

– Hepatitis C RNA quantification and genotype

– Improved technology –sensitivity / specificity / efficiency

Nucleic Acid Tests (NAT)

Target amplification– Polymerase chain reaction – PCR – real time + endpoint– Nucleic acid sequence base amplification – NASBA– Transcription Mediated Amplification – TMA– Strand Displacement Amplification - SDA

Signal amplification– ‘Branched’ DNA – bDNA– Hybrid Capture & detection

Polymerase chain reaction (PCR)

Endpoint product detection

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

Roche Amplicor HIV MONITOR Test

Endpoint PCR technique to amplify viral RNA targetHIV-1 gag geneVersion 1.5 primers = improved subtype detection6 hours to reportableAmpliLink softwareMost common assay in Australiastandard assay 400 – 750,000 cpy/mLultrasensitive assay 50 -100,000 cpy/mLInternal quantitation standard

NucliSens HIV QT - NASBA

Boom method for NA extraction – pure yeildssRNA amplified NASBA (AMV RT, T7 RNA pol, RNAseH)HIV-1 gag gene4 internal quantitationstandards80-1,000,000 cpy/mLLabor intensiveHIV-1 group M subtypes A, B, C equally detected; E,F,G variable

HIV-1 Quantiplex (bDNA) Assay

No viral purification or nucleic acid amplificationViral RNA captured, labeled and detectedHIV-1 pol geneStandard curve50-500,000 cpy/mLOvernight assayHIV-1 group M subtypes A-F equally detected

Kinetic / real time product detection

NucliSens EasyQ System

Analyser

Dell computer with windows XP based intuitive software for data reduction and result reporting

CentrifugeThermocycler

What are Molecular Beacons?

Quencher

FluorescentDye

Loop

Stem

DNA probes with a stem-loop structureand two modified ends

20-25 loop sequence complementary to the target sequence for hybridization

6-7 base pair stem sequences

Different labels for different targets(wild type and calibrator)

Molecular Beacons

Detection of RNA with molecular beacon

target RNA

R Q

Q

Fluorescence signal increases with increasing RNA levels

Real-time Detection in NASBA

oligo P2

RT RT

T7 RNAPanti-sense

RNA

RNase Holigo P1

oligo P1

RNase H &oligo P2

sense RNA

T7 RNA polymerase

Target specific molecular beacons

Reverse Transcriptase

Reverse Transcriptase

Molecular beacon hybridization

Qa

Qb

Qc

Logsignal

Log copies

End-point measurement(WT and 3 calibrators)

Fluo

resc

ence

Time

WT

Qd

Kinetic measurement(WT and 1 calibrator)

NucliSens HIV-1 QT NucliSens EasyQ HIV-1

Calibrators & Quantitation

NucliSens EasyQ System

Analyser

CentrifugeThermocycler

Roche lightcycler – real time PCR

Dia

gnos

tics

RQ

5’5’ nuclease activity

Anneal

RQ

R

Q

hט

R3’

3’

5’

Extend

Herpes simplex PCR

HSV-1/2 PCR

Differentiates HSV 1 and 2Performed 3 x per weekMore sensitive than cultureDetects pre-vesicular stage (prodrome / inflammation)Self-collection possibleSterile dry swab (cold transport)Medicare

Cytomegalovirus

Current Diagnostic Current Diagnostic Techniques for CMVTechniques for CMV

• Serology (IgM, IgG)

• Histology

• Culture

• Shell Vial Culture (HELF monolayer + IF)

• Antigenemia (pp65 Antigen IF in PB leukocytes)

• Nucleic acid detection (NAT)

DNA

ImmediateEarly antigen

Late mRNA

Early antigen

Virus

DNA replication blockby antiviral therapy

Late antigen

RNA & antigen detection (UL123), during latent and active infection

1. Detection of intracellular CMV pp67 mRNA (UL65)

2. Detection of pp65 (UL83) antigen in blood (antigenemia)

3. Changes in intracellular DNA copies

DNA detection in cellsduring latent and active infection

Latency Active infection

4. Changes in shedded DNAin plasma & serum

Infection of new cells

Potential UtilitiesPotential UtilitiesTransplantation • Replacement of culture & antigenemia• Pre-emptive treatment strategies

AIDS• Rapid confirmation of CMV disease• identification of high risk patients = therapy• CSF – CNS involvement• Replacement of culture

Anti-natal / Pre-natal Screening• Confirmation of serology

CMV Nucleic Acid Tests

mRNA detection – intermediate transcript– Test performed on leukocytes– CMV is a ubiquitous DNA virus, persistent in leukocytes (latent)– Latent or abortive infection clinically not relevant– Active infection is the deciding factor for initiation of anti-CMV

therapy– exclusively expressed during active replication

CMV DNA quantification– Plasma / serum = lytic disease > shedding– Monitor fold changes in serial samples– Response to therapy– Prediction of reactivation – pre-emptive therapy

Monitoring Therapeutic Efficacy AIDS patient with relapse of disease

100

1,000

10,000

100,000

0 10 20 30 40We e ks

CM

V D

NA

cop

ies/

ml H C S

NucliSens pp67+

HIV therapy

DiseaseAntigenemia

CMV therapy

CMV retinitis cryptococcosis

cut-off

weak+

GCV / PFA

CMV ulcer

maintenance therapyGCV

2 NRTIGCV pellets implant

+ -

+

-

-

+

+ - -+

HIV study Bonn, Germany, poster G0-24 at 7th Int. CMV workshop Brighton

HPV infection diagnosis

Known Risk Factors for Cervical Cancer

HPV positivity and specific typePersistent HPV infectionViral load (HPV)Cytological confirmation of SILSexual behaviorEpithelial location and characteristicsGenetic factors

Prevalence of HPV Genotypes in Invasive Cancers

Prevalence of HPV Genotypes in Invasive Cancers

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0N u m b e r o f In v a s iv e C a n c e r s

P 2 9 1H P V 6

H P V 1 1H P V 5 5

H P V 2 6P 2 3 8 AW 1 3 B

H P V 5 1H P V 6 8

H P V 3 9H P V 5 9H P V 3 5H P V 5 6

H P V 5 8H P V 5 2H P V 3 3H P V 3 1

H P V 4 5H P V 1 8H P V 1 6

Bosch, et al. JNCI 1995

Clavel C et al. Br J Cancer 2001; 89(12):1616-23

HPV High Risk TypesHPV High Risk Types

Digene HPV DNA Test uses RNA Probe cocktails to detect carcinogenic HPV types

High Risk– 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68

Anal intraepithelial neoplasia - AIN

Squamous intraepithelial lesions (SIL) are associated with HPV

SIL are precursor to anal cancer

Anal HPV infection and anal SIL are common in HIV+ MSM

Primary HIV infection

‘typical’ primary HIV-1 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

‘window’period

1° infection

‘typical’ primary HIV-1 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

‘window’period

1° infection

HIV antibodies

DNA PCRDNA PCRRNA PCRRNA PCR

p24 Agp24 Ag

3rd 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

acute recent / established advanced

Spectrum Spectrum of HIV of HIV

testingtesting

HIV Ab/Ag combo

Direct Virus Detection - HIV

Precedes Antibody response

DNA (provirus) PCR (+/-) 5-8 daysp24 antigen – serology 3-5 days

RNA (quantitation - viral load) Virus culture

HIV-1 proviral DNA PCR

Qualitative PCR5-8 days before antibodiesResolution of inconclusive serologyDiagnosis of neonatesLimitations – non-B subtypes

Quantitative Assay – Potential Clinical utility

– Prognostic marker for disease progression– Monitor viral load in patients with

undetectable plasma HIV-1 RNA– Monitor efficacy of therapy targeting

virus reservoir

Monitoring HIV infection

Kinetics of HIV-therapyHigh prevalence Full resistant Full replication Low prevalence

Full resistant Full replication

Baseline

Efficacy pre-existing mutations

“Turnover”

Low prevalence Replication compromised

100% inhibition replication incompetent prevalence zero

A

B

C

B

* Additional mutations-Resistance

-Replication

*

Algorithm for assessment of antiviral therapy

Resistant virus

Assay for drug resistance

Change drug combination

Change therapy

Combination therapy

Initial response

< 1 log in HIV-1 RNA

>1 log in HIV-1 RNA > 1-2 log in HIV-1 RNA

Yes Poor compliance ? No

Patient education Lack of drug efficacy? (due to patient’s sub-optimum pharmacokinetic parameters)

Continue therapy

Yes No

Modify/change therapy

Continue therapy

Healthy Years Active Disease Chronic Disease

Predisposition to illness:(genetic testing)

Enhanced monitoring:HIV viral load, resistance

testing

Personalized Medicine: HIV

Latent Disease

Diagnose and intervene

before illnessoccurs:

HIV serology,viral load andCD4 cell count

Disease Prognosis/Therapy Selection:HIV viral load, CD4 cell count, and drug resistance testing

Medical Intervention:HAART

Highly Active Antiretroviral Therapyinfection

(proviral DNA, genetic testing)

(proviral DNA)

Hepatitis C infection management

HCV-RNA levels

Personalized Medicine HCV Infection

106

HCV Qual

confirm viremia

viral load

Relapse

HCVgenotype 1 (or 4)

Wk 0

Quantity & Strainof Virus

HCV QuantGenotype

12 week response

HCV Quant

48 wks

Pegylated IFN treatment

response at end of

treatment

HCV Qual

72 wks

response at end of follow-up

HCV Qual

SVR

undetectable

prolonged follow-up

HCV Qual

years

Personalized MedicineHCV Infection

HCV-RNA levels

106

HCV Qual

confirm viremia

viral load

Wk 0 48 wksHCVgenotype 1 (or 4)

72 wksundetectable

Pegylated IFN treatment

Quantity & Strainof Virus

HCV QuantGenotype

12 week response

HCV Quant

Stop Rx at week 12

104 IU/mL

< 2 log drop

Week 12 Quantitative Assay(Early Virologic Response)

Personalized MedicineHCV Infection – Peg-IFN + Ribavirin

All patients(n=453)

SVR3%

NPV=97%NPV=97%

Fried, et al. NEJM 2002; 347:975Fried, et al. NEJM 2002; 347:975--982982

86%

14%

EVR*EVR*

No EVR*No EVR*

Week 12Response (EVR)

SVR65%

Week 72 Response (SVR)

Week 12 Quantitative Assay