Jean-Michel PAWLOTSKY Slide 2 How to Use Virological Tools for
the Optimal Management of Chronic Hepatitis C ? Prof. Jean-Michel
Pawlotsky, MD, PhD French National Reference Center for Hepatitis
B, C and delta, Department of Virology & INSERM U841, Henri
Mondor Hospital University of Paris 12 Crteil, France Slide 3
Virological Markers in HCV Treatment Monitoring HCV genotypeHCV
genotype Tailor ribavirin dose and treatment durationTailor
ribavirin dose and treatment duration HCV RNA presence and levelHCV
RNA presence and level Decide to treatDecide to treat Assess
virological responsesAssess virological responses Tailor treatment
duration to the actual virological responseTailor treatment
duration to the actual virological response Slide 4 I HCV
Virological Tools Slide 5 HCV Genotype Determination Slide 6 HCV
Genotypes (Simmonds P., et al. Hepatology 2005;42:962-73) Slide 7
HCV Genotype Determination Molecular methods (genotyping):Molecular
methods (genotyping): Direct sequence analysisDirect sequence
analysis Reverse hybridization: line probe assay (Inno-LiPA)Reverse
hybridization: line probe assay (Inno-LiPA) Serological methods
(serotyping)Serological methods (serotyping) Competitive
ELISACompetitive ELISA Slide 8 HCV Genotype Determination HCV
genome Slide 9 HCV Genotype Determination HCV genome NS5B Slide 10
HCV Genotype Determination HCV genome NS5B 5 noncoding region Slide
11 Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006)
491 patients, HCV genotype 1 (local method) Slide 12 Subtyping in
the 5NC Region (Chevaliez S., et al., AASLD 2006) 491 patients, HCV
genotype 1 (local method) NS5B sequence analysis Slide 13 Subtyping
in the 5NC Region (Chevaliez S., et al., AASLD 2006) 491 patients,
HCV genotype 1 (local method) 1a (n=226, 46%) 1b (n=245, 50%) other
1 (n=11, 2%) 1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) NS5B
sequence analysis non-1 (n=9, 2%) 2l (n=1) 3a (n=2) 6 (n=6) Slide
14 Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006)
NS5B 1a (n=226) 5NC region 1a (n=180) 1b (n=39) 1nd (n=7) Slide 15
Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006) NS5B
1a (n=226) NS5B 1b (n=245) 5NC region 1a (n=180) 1b (n=39) 1nd
(n=7) 5NC region 1b (n=229) 1a (n=8) 1nd (n=8) Slide 16 7%
Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006) NS5B
1a (n=226) NS5B 1b (n=245) 5NC region 1a (n=180) 1b (n=39) 1nd
(n=7) 5NC region 1b (n=229) 1a (n=8) 1nd (n=8) 20% Slide 17
Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006) NS5B
1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) 5NC region 1b (n=3)
1nd (n=1) 1a (n=2) 1nd (n=1) 1a (n=1) 1a (n=2) 1b (n=1) Slide 18
Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006) NS5B
1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) 5NC region 1b (n=3)
1nd (n=1) 1a (n=2) 1nd (n=1) 1a (n=1) 1a (n=2) 1b (n=1) 100% Slide
19 Subtyping in the 5NC Region (Chevaliez S., et al., AASLD 2006)
NS5B 2l (n=1) 3a (n=2) 6 (n=6) 5NC region 1 (n=1) 3a (n=2) 1a (n=1)
1b (n=5) Slide 20 Subtyping in the 5NC Region (Chevaliez S., et
al., AASLD 2006) NS5B 2l (n=1) 3a (n=2) 6 (n=6) 5NC region 1 (n=1)
3a (n=2) 1a (n=1) 1b (n=5) ? Slide 21 Subtyping in the 5NC Region
(Chevaliez S., et al., AASLD 2006) NS5B 2l (n=1) 3a (n=2) 6 (n=6)
5NC region 1 (n=1) 3a (n=2) 1a (n=1) 1b (n=5) ? ? Slide 22 Current
LiPA Assay 2a/b 3a 4a 1b 1a 5a 6a Marker CC CC AC AC
3456789101112131415161718192021 Band positions 5NC region probes
Slide 23 New LiPA Assay 3 18 17 15 19 20 21 23. AC Marker CC CC AC
AC 4 8 7 5 6 9 10 11 12 13 14 25. 1 a 26. 1 b 24. 6 c-l 16 2a/b 3a
4a 6a 1b 61a 5NC region probes core region probes Slide 24
GenotypeSubtype 1 a, b 2 a, b, c 3 a, b, c, k 4 a, b, c, d, e, f, h
5a 6 a, c-l New LiPA Assay Slide 25 Detection and Quantification of
HCV RNA Slide 26 Ranges of Linear Quantification of HCV RNA Assays
(IU/ml) 10 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Cobas Amplicor HCV
Monitor v2.0 Versant HCV RNA 3.0 (bDNA) Untreated hepatitis C Slide
27 Advantages of Real-Time PCR Quantification Improved
sensitivityImproved sensitivity No carryover contaminationNo
carryover contamination Extended dynamic range of
quantificationExtended dynamic range of quantification Precision
and reproducibilityPrecision and reproducibility Slide 28 Real-Time
PCR Platforms for HCV RNA Roche Molecular Systems:Roche Molecular
Systems: Cobas Taqman HCV TestCobas Taqman HCV Test Amplification:
Cobas Taqman Amplification: Cobas Taqman Extraction: Cobas
Ampliprep (CAP/CTM)Extraction: Cobas Ampliprep (CAP/CTM) Abbott
DiagnosticAbbott Diagnostic Abbott Real-Time HCV AssayAbbott
Real-Time HCV Assay Amplification: m2000 RT SystemAmplification:
m2000 RT System Extraction: m2000 SP (m2000 Real-Time PCR
System)Extraction: m2000 SP (m2000 Real-Time PCR System) Slide 29
Ranges of Linear Quantification of HCV RNA Assays (IU/ml) 10 10 2
10 3 10 4 10 5 10 6 10 7 10 8 Cobas Amplicor HCV Monitor v2.0
Versant HCV RNA 3.0 (bDNA) Untreated hepatitis C Slide 30 Ranges of
Linear Quantification of HCV RNA Assays (IU/ml) 10 10 2 10 3 10 4
10 5 10 6 10 7 10 8 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA
3.0 (bDNA) Untreated hepatitis C Cobas TaqMan HCV Test (Roche)
Abbott Real-Time HCV Assay (Abbott) Slide 31 Roche Real-Time PCR
Cobas Ampliprep Cobas Taqman Cobas Ampliprep /Cobas Taqman
(CAP/CTM) platform Slide 32 Calibration of CAP/CTM HCV AcroMetrix
HCV RNA Panel r = 0.9981; p < 0.0001 HCV RNA level in CAP/CTM
(Log 10 IU/ml) 6 5 4 3 2 1 0 7 8 Expected HCV RNA level (Log 10
IU/ml) 012345678 (Chevaliez et al., Hepatology 2007; in press)
Slide 33 Overestimation of HCV RNA Levels in CAP/CTM vs bDNA -1.5
-0.5 0 0.5 1.0 1.5 Genotype 1 Genotype 2 Genotype 3 Genotype 4
Genotype 5 Difference between HCV RNA levels measured in CAP/CTM
and in bDNA (in Log 10 UI/ml) (Chevaliez et al., Hepatology 2007;
in press) Slide 34 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 HCV RNA (Log 10
IU/ml) Dilutions undiluted 1/5 1/25 1/1251/625 1/3125
1/156251/78125 HCV genotype 1 1.0 2.0 3.0 4.0 5.0 6.0 7.08.0 HCV
RNA (Log 10 IU/ml) Dilutions undiluted 1/5 1/25 1/1251/625 1/3125
1/15625 1/78125 HCV genotype 2 Overestimation of HCV RNA Levels in
CAP/CTM vs bDNA (Chevaliez et al., Hepatology 2007; in press) Slide
35 Genotype 1 (n=29) Genotype 2 (n=27) Genotype 3 (n=29) Genotype 4
(n=30) Genotype 5 (n=9) Genotype 6 (n=2) -1.5 1.0 - 1. 0 1.50.0 0.5
-0.5 Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2
and 4 (Chevaliez et al., Hepatology 2007; in press) Slide 36
Genotype 1 (n=29) Genotype 2 (n=27) Genotype 3 (n=29) Genotype 4
(n=30) Genotype 5 (n=9) Genotype 6 (n=2) -1.5 1.0 - 1. 0 1.50.0 0.5
-0.5 Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2
and 4 (Chevaliez et al., Hepatology 2007; in press) 15% 30% Slide
37 Abbott Real-Time PCR m2000 RT Slide 38 (Chevaliez et al., in
progress) Abbott Real-Time PCR Slide 39 II Treatment Algorithms
Slide 40 HCV Genotype Determination Type1Types 2, 3 Types 4, 5, 6
Slide 41 HCV GENOTYPE 1 Slide 42 Assessment of liver disease
severity Slide 43 HCV GENOTYPE 1 Assessment of liver disease
severity Good prognosis Follow-up without treatment Slide 44 HCV
GENOTYPE 1 Assessment of liver disease severity Bad prognosis Good
prognosis Follow-up without treatment Peginterferon + ribavirin
1.0-1.2 g qd 48 weeks Slide 45 HCV GENOTYPE 1 HCV RNA
quantification at baseline and at week 12 Assessment of liver
disease severity Bad prognosis Good prognosis Follow-up without
treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks Slide 46
HCV GENOTYPE 1 < 2 log HCV RNA decrease HCV RNA quantification
at baseline and at week 12 Assessment of liver disease severity Bad
prognosis Good prognosis Follow-up without treatment Peginterferon
+ ribavirin 1.0-1.2 g qd 48 weeks Stop treatment OR continue to
slow liver disease progression Slide 47 HCV GENOTYPE 1 2 log HCV
RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA
decrease HCV RNA quantification at baseline and at week 12
Assessment of liver disease severity Bad prognosis Good prognosis
Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd
48 weeks Stop treatment OR continue to slow liver disease
progression Slide 48 HCV GENOTYPE 1 2 log HCV RNA decrease or HCV
RNA (-) Continue treatment < 2 log HCV RNA decrease HCV RNA
quantification at baseline and at week 12 Assessment of liver
disease severity Bad prognosis Good prognosis Follow-up without
treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA
quantification at week 24 Stop treatment OR continue to slow liver
disease progression Slide 49 HCV GENOTYPE 1 2 log HCV RNA decrease
or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease HCV
RNA quantification at baseline and at week 12 Assessment of liver
disease severity Bad prognosis Good prognosis Follow-up without
treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA
quantification at week 24 HCV RNA 50 IU/ml Stop treatment OR
continue to slow liver disease progression Stop treatment OR
continue to slow liver disease progression Slide 50 HCV GENOTYPE 1
2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log
HCV RNA decrease HCV RNA quantification at baseline and at week 12
Assessment of liver disease severity Bad prognosis Good prognosis
Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd
48 weeks HCV RNA quantification at week 24 HCV RNASlide 51 HCV
GENOTYPE 1 2 log HCV RNA decrease or HCV RNA (-) Continue treatment
< 2 log HCV RNA decrease HCV RNA quantification at baseline and
at week 12 Assessment of liver disease severity Bad prognosis Good
prognosis Follow-up without treatment HCV RNA detection at the end
of treatment and 24 weeks later Peginterferon + ribavirin 1.0-1.2 g
qd 48 weeks HCV RNA quantification at week 24 HCV RNASlide 52 HCV
GENOTYPE 1 2 log HCV RNA decrease or HCV RNA (-) Continue treatment
< 2 log HCV RNA decrease End-of-treatment virological response
Sustained virological response HCV RNA quantification at baseline
and at week 12 Assessment of liver disease severity Bad prognosis
Good prognosis Follow-up without treatment HCV RNA detection at the
end of treatment and 24 weeks later Peginterferon + ribavirin
1.0-1.2 g qd 48 weeks HCV RNA quantification at week 24 HCV
RNASlide 53 HCV GENOTYPES 2 or 3 Slide 54 Peginterferon + ribavirin
0.8 g qd 24 weeks HCV GENOTYPES 2 or 3 Slide 55 Peginterferon +
ribavirin 0.8 g qd 24 weeks HCV RNA detection at the end of
treatment and 24 weeks later HCV GENOTYPES 2 or 3 Slide 56
Peginterferon + ribavirin 0.8 g qd 24 weeks End-of-treatment
virological response Sustained virological response HCV RNA
detection at the end of treatment and 24 weeks later HCV GENOTYPES
2 or 3 Slide 57 HCV GENOTYPES 4, 5 or 6 Slide 58 Liver biopsy or
serological markers of disease severity HCV GENOTYPES 4, 5 or 6
Slide 59 Liver biopsy or serological markers of disease severity
Good prognosis Follow-up without treatment HCV GENOTYPES 4, 5 or 6
Slide 60 Liver biopsy or serological markers of disease severity
Bad prognosis Good prognosis Follow-up without treatment
Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV GENOTYPES 4, 5
or 6 Slide 61 Liver biopsy or serological markers of disease
severity Bad prognosis Good prognosis Follow-up without treatment
Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA detection
at the end of treatment and 24 weeks later HCV GENOTYPES 4, 5 or 6
Slide 62 Liver biopsy or serological markers of disease severity
Bad prognosis Good prognosis Follow-up without treatment
Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks End-of-treatment
virological response Sustained virological response HCV RNA
detection at the end of treatment and 24 weeks later HCV GENOTYPES
4, 5 or 6 Slide 63 III Future Challenges Slide 64 Future Challenges
Better tailor treatment duration:Better tailor treatment duration:
Treat shorter when no more is neededTreat shorter when no more is
needed Treat longer when more is neededTreat longer when more is
needed Slide 65 Shorter Treatment Slide 66 16 weeks 24 weeks 0 20
40 60 80 100 65% 82% 65% 71% % SVR 60% 70% 86% 77%
n=346n=303n=333n=327 Genotype 2 Genotype 3 p=
