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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
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Molecular Diagnostics
Diagnosis
Monitoring
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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
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Key developments
Diagnosis
– Herpes simplex (types 1&2)
– Cytomegalovirus - active infection
– Human papillomavirus (HPV) diagnosis –♀♂
– HIV primary infection
– Chlamydia trachomatis & Neisseria gonorrhoeae
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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
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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
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Polymerase chain reaction (PCR)
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Endpoint product detection
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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
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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
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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
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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
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Kinetic / real time product detection
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NucliSens EasyQ System
Analyser
Dell computer with windows XP based intuitive software for data reduction and result reporting
CentrifugeThermocycler
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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)
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Molecular Beacons
Detection of RNA with molecular beacon
target RNA
R Q
Q
Fluorescence signal increases with increasing RNA levels
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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
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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
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NucliSens EasyQ System
Analyser
CentrifugeThermocycler
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Roche lightcycler – real time PCR
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Dia
gnos
tics
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RQ
5’5’ nuclease activity
Anneal
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RQ
R
Q
hט
R3’
3’
5’
Extend
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Herpes simplex PCR
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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
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Cytomegalovirus
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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)
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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
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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
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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
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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
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HPV infection diagnosis
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Known Risk Factors for Cervical Cancer
HPV positivity and specific typePersistent HPV infectionViral load (HPV)Cytological confirmation of SILSexual behaviorEpithelial location and characteristicsGenetic factors
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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
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Clavel C et al. Br J Cancer 2001; 89(12):1616-23
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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
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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
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Primary HIV infection
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‘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
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‘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
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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
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Direct Virus Detection - HIV
Precedes Antibody response
DNA (provirus) PCR (+/-) 5-8 daysp24 antigen – serology 3-5 days
RNA (quantitation - viral load) Virus culture
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HIV-1 proviral DNA PCR
Qualitative PCR5-8 days before antibodiesResolution of inconclusive serologyDiagnosis of neonatesLimitations – non-B subtypes
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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
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Monitoring HIV infection
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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
*
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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
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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)
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Hepatitis C infection management
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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
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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)
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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