1. HCV Diagnostic
Strategies & Monitoring
Prof. Jean-Michel Pawlotsky, MD, PhD
National Reference Center for Viral
Hepatitis B, C and delta
Department of Virology&INSERM U955
Henri Mondor Hospital
University of East Paris
Créteil, France

2. I
HCV Markers and Kinetics

3. HCV Markers
• HCV genotype :
• Intrinsic characteristic of the infecting HCV strain.
• HCV RNA :
• Marker of HCV replication.
• Total HCV core Ag :
• Surrogate marker of HCV replication.
• Anti-HCV antibodies :
• Marker of past or present infection.

4. Kinetics of HCV Markers
Spontaneous resolution
HCV RNA
HCV core Ag
anti-HCV
antibodies
ALT
Infection Acute
hepatitis

5. Kinetics of HCV Markers
Persistent infection
HCV RNA
HCV core Ag
anti-HCV
antibodies
ALT
Infection Acute Chronic hepatitis
hepatitis

6. II
HCV Virological Tools

7. Classical HCV Virological Tests
• SerologicalAssays
• ELISA tests for anti-HCVantibodydetection
• Molecular tests
• HCVgenotypingassays
• HCV RNA quantification assays

8. New HCV Virological Tests
• New serologicaltests
• Quantitativecoreantigendetection (ELISA)
• Molecular tests
• Real-time PCR assays for HCV RNA level
quantification
• Ultra-deeppyrosequencing (resistance)

9. Anti-HCVantibodydetection

10. Anti-HCVAntibodyDetection
• Based on ELISA
• Easy to use, automated
• 3rd-generation tests available
• ADVIA Centaur (Siemens)
• VITROS ECi (Ortho-ClinicalDiagnostics)
• AXSYM HCV 3.0 (Abbott)
• CobasElecsysModular HCV (Roche)
• INNOTEST HCV Ab IV (Innogenetics)
• Monolisaanti-HCV Plus version 2 (Bio-Rad)

11. “Combo“ Tests (Ag + Ab)
• Based on ELISA
• Commerciallyavailable
• Reduce the serologicalwindowduring
acute infection by 20-30 days
• No benefit in the diagnostic setting
• No benefit in blood screening in the
context of Nucleicacidtesting (NAT)

12. HCV core Ag quantification

13. HCV CoreAntigen Quantification
(Bouvier-Alias M. et al., Hepatology 2002;36:211-8)

14. Architect HCV Ag Assay
RVR (G1b) SVR (G1b)
Core Ag
RNA
Relapser(G1b) NR (G1a)
(Ross M. et al., J ClinMicrobiol 2010;48:1161-8)

15. HCV Core Ag Quantification
• HCV core Ag quantification canbeused as a
surrogate marker of HCV replication in the
monitoring of antiviral therapy
• However, HCV core Ag
assayslacksensitivitycompared to HCV RNA
level quantification by real-time PCR (LLD
equivalent to 500-3000 IU/mLaccording to the
HCV genotype)

16. HCVGenotypedetermination

17. HCV Genotypes

18. HCV GenotypeDetermination
• Molecularmethods:
• Direct sequenceanalysis
– Home-made : NS5B or E1 regions,
– Commercial : 5’ noncodingregion (Trugene HCV 5’NC
Genotyping Kit, Bayer HealthCare) or NS5B (Trugene
HCV NS5B Genotyping Kit, Bayer HealthCare)
• Real-time PCR withgenotype-specificprimers
and probes
• Reverse hybridization of PCR products:
– Line Probe Assay (INNO-LiPA HCV II, Innogenetics)
• Serologicalmethods: serotypingassay

19. Versant®
HCV Genotype 2.0
Assay (INNO-LiPA)

20. HCV Genotype 1 Subtype
Determination
Sequence 1st Generation of 2nd Generation of
RealTime HCV
Analysis of the Line Probe Assay Line Probe Assay
Genotype II
5’NCR (LiPA 1.0) (LiPA 2.0)
GT 1a 77.6% 70.5% 97.5% 93.2%
(n=237) (n=184) (n=167) (n=231) (n=220)
GT 1b 90.5% 91.3% 96.2% 88.6%
(n=263) (n=238) (n=240) (n=253) (n=233)
(Chevaliez et al., PLoS One 2009;4:e820)

21. Interest of Genotype 1
Subtyping in Practice
• Peg-IFN and ribavirintherapy:
• No practicalinterest for clinicaldecisions
• Triple combinationtherapywith Pis:
• Modest differencebetween 1a and 1b
• Differentresistance profiles
• No practicalinterest for clinicaldecisions
• IFN-freeregimens
• Possibly important

22. HCV RNA Quantification

23. Linear Ranges of Quantification
10 102 103 104 105 106 107 108
CobasAmplicor
HCV Monitor v2.0
SuperQuant
LCx HCV
RNA Assay
Versant HCV RNA
3.0 (bDNA)
(Chevaliez et al., Gastroenterology 2012;142:1303-13)

24. Linear Ranges of Quantification
10 102 103 104 105 106 107 108
CobasAmplicor
HCV Monitor v2.0
SuperQuant
LCx HCV
RNA Assay
Versant HCV RNA
3.0 (bDNA)
CTM HCV test v2.0
(Roche)
CAP/CTM HCV
test, v2.0 (Roche)
RealTime™ HCV
(Abbott)
Artus HCV QS-
RGQ (Qiagen)
Versant HCV RNA
1.0 (kPCR, Siemens)
(Chevaliez et al., Gastroenterology 2012;142:1303-13)

25. Real-Time PCR Platforms
CAP-CTM96 (ROCHE)
kPCR (SIEMENS)
m2000SP-m2000RT (ABBOTT)

26. CAP/CTM HCV Assay
8
7
Genotype 1 (n=29)
6
Genotype 2 (n=27)
Genotype 3 (n=29)
5
Genotype 4 (n=30)
Genotype 5 (n=9)
4 Genotype 6 (n=2)
r = 0.889; p < 0.0001
3
3 4 5 6 7 8
HCV RNA level in Versant HCV 3.0 Assay bDNA
(Log10 UI/mL)
(Chevaliez et al., Hepatology 2007;46:22-31)

27. Underestimation of HCV RNA Levels
by CAP/CTM in Genotypes 2 and 4
1.5
1.0
Genotype 1 (n=29)
0.5 Genotype 2 (n=27)
Genotype 3 (n=29)
0.0
Genotype 4 (n=30)
-0.5 Genotype 5 (n=9)
Genotype 6 (n=2)
-1.0
-1.5
15%
30%
(Chevaliez et al., Hepatology 2007;46:22-31)

28. Lack of HCV RNA Detection by
CAP/CTM in Genotype 4
CAP/CTM bDNA 3.0 Real-Time PCR
Patient Genotype
(Roche) (Siemens) (Abbott)
Undetectable
A 4h 5.4 log IU/ml 5.0 log IU/ml
<12 IU/ml
Undetectable
B 4l 6.0 log IU/ml 5.7 log IU/ml
<12 IU/ml
(Chevaliez et al., Hepatology 2008;49:1397-8)

29. Genotype 4 Quantification
with CAP/CTM v2.0 (Roche)
8
HCV RNA level in CAP/CTM48 v2.0
Genotype 4a (n=43)
Genotype 4c (n=4)
7
Genotype 4d (n=34)
Genotype 4e (n=9)
(Log10 IU/mL)
6 Genotype 4f (n=9)
Genotype 4g (n=2)
Genotype 4h (n=5)
5 Genotype 4k (n=4)
Genotype 4n (n=1)
4 Genotype 4r (n=8)
Genotype 4t (n=3)
r = 0.9581; p < 0.0001
3
3 4 5 6 7 8
HCV RNA level in Versant HCV 3.0bDNA Assay
(Log10 IU/mL)
(Chevaliez et al., J ClinMicrobiol 2013; in press)

30. CAP/CTM v1.0 vs v2.0 (Roche)
CAP/CTM96 v1.0 CAP/CTM96 v2.0 m2000SP/m2000RT bDNA 3.0
(Log10 IU/mL) (Log10 IU/mL) (Log10 IU/mL) (Log10 IU/mL)
Patient 1 (4h) <1.08 5.8 5.4 5.0
Patient 2 (4l) <1.08 6.3 6.0 5.7
Patient 3 (4) <1.08 6.7 6.5 6,2
Patient 4 (4k) <1.08 5.4 5.7 5.8
(Chevaliez et al., J ClinMicrobiol 2013; in press)

31. Abbott Real-Time PCR
8.0
R=0,9658, p<0.0001
7.0
m2000sp (Abbott)
HCV genotype 1 (n=43)
6.0
HCV genotype 2 (n=11)
HCV genotype 3 (n=19)
5.0 HCV genotype 4 (n=17)
HCV genotype 5 (n=5)
4.0
3.0
3.0 4.0 5.0 6.0 7.0 8.0
bDNA 3.0
(Chevaliez et al., J Clin Microbiol 2009;47:1726-32)

32. Clinical Achievements of
Real-Time PCR Assays
• Replace qualitative viral genome detection
assays
• Accurately quantify a broad range of viral
levels observed in clinical practice:
• High pretreatment levels
• Low levels during antiviral treatment
• Efficiently monitors viral kinetics (early
assessment of virologic responses to therapy)

33. HCV Resistance Testing

34. Quasispecies Distribution
of Viral Populations
Major viral
population
Intermediate viral
populations
Minor viral
populations

35. Viral SequenceAnalysis Tools
Major viral population
detected by direct
sequencing
Intermediate viral
populations detected by
cloning and sequencing
Minor viral populations
detected by ultra-
sensitive techniques
such as ultra-deep
sequencing

36. Available NGS Techniques
Technology Number of Number of
Maximum
Sequencing (template single reads nucleotides
Manufacturer Type sequence Accuracy
device preparation/ per run* per run*
length* (bp)
NGS chemistry) (x 106) (Gb)
High
5500 800 9 75
throughput
emPCR/ligation 99.6-99.8%
Applied High
5500xl 1600 15 75
Biosystems throughput
Ion Torrent emPCR/RTsequ
Long reads 6.2 >1 >400 99.97%
(ChiP 316) encing
High
MiSeq 3.4 >1 150
throughput
Genome High
Solid 320 95 150
Analyzer IIx throughput
Illumina capture/reversib 96.7-100%
le terminator High
HiSeq 1000 1500 300 100
throughput
High
HiSeq 2000 3000 600 100
throughput
GS Junior Long reads 0.1 0.035 400 99%
emPCR/pyroseq
454 /Roche
uencing
GS FLX+ Long reads 1 0.7 1000 97.4-99.9%
Single
PacBio/Gen-
PacBio RS molecule/RTseq Long reads 0.035 0.045 1200 99.99%
Probe
uencing
(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)

37. Available NGS Techniques
Technology Number of Number of
Maximum
Sequencing (template single reads nucleotides
Manufacturer Type sequence Accuracy
device preparation/ per run* per run*
length* (bp)
NGS chemistry) (x 106) (Gb)
High
5500 800 9 75
throughput
emPCR/ligation 99.6-99.8%
Applied High
5500xl 1600 15 75
Biosystems throughput
Ion Torrent emPCR/RTsequ
Long reads 6.2 >1 >400 99.97%
(ChiP 316) encing
High
MiSeq 3.4 >1 150
throughput
Genome High
Solid 320 95 150
Analyzer IIx throughput
Illumina capture/reversib 96.7-100%
le terminator High
HiSeq 1000 1500 300 100
throughput
High
HiSeq 2000 3000 600 100
throughput
GS Junior Long reads 0.1 0.035 400 99%
emPCR/pyroseq
454 /Roche
uencing
GS FLX+ Long reads 1 0.7 1000 97.4-99.9%
Single
PacBio/Gen-
PacBio RS molecule/RTseq Long reads 0.035 0.045 1200 99.99%
Probe
uencing
(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)

38. Ultra-DeepPyrosequencing
Data collection
RT
Viral genome
PCR amplification
extraction fromserum emPCRPyrosequenci
ng
Analysis
% of each
mutations
PyroClass©. PyroMute©, PyroDyn©, PyroLink© are protected under IDDN
(Rodriguez C. et al., in revision)

39. Pre-existingHCV
ResistantVariants by UDPS
Response
genotype
subtype
Patient
pegIFN
V36 T54 Q80 R155 A156 D168 I170
IL28B
HCV
RBV
TVR
V55A
A/M A/S R/K K/T/Q S/T/V A/V/T/H A/T
Pt-1 CT 1a NR - 90.0% - - 0.1% 0.4% 0.1% 0.5%
Pt-2 CT 1a NR - - - - 0.1% 1.1% - 0.2%
Pt-3 CT 1b RR - - - - 0.5% 0.5% - 0.2%
Pt-4 TT 1b RR - 29.4% - - - 1.3% - 0.1%
Pt-5 CT 1a RR - - - - 0.1% 2.9% 0.1% -
Pt-6 CT 1b RR 4.2% - - - 0.1% 0.1% 0.1% 0.1%
Pt-7 CT 1a SVR - 11.1% - 0.7% - 0.3% - 0.3%
Pt-8 CT 1a SVR - - - - 0.1% 0.5% 0.1% -
Pt-9 CC 1a SVR - - - - 0.6% 1.8% - -
Pt-10 CC 1a SVR - - - - 0.6% - - 0.1%
Pt-11 TT 1a RR - - 100.0% 0.1% 6.0% 3.2% 0.1% 0.3%
Pt-12 CT 1b SVR - - - - - 0.3% - 0.1%
Pt-13 CT 1b SVR - - - - 0.2% 0.2% - 0.8%
Pt-14 TT 1b NR - - - - 0.1% 0.2% - 0.1%
Pt-15 CT 1b SVR - - - - 0.4% 0.2% 0.1% 0.1%
Pt-16 CT 1a SVR - - 1.3% 0.5% 7.8% 0.2% 0.1% 0.1%
Pt-17 CT 1a SVR - 47.4% - - 0.1% 0.4% 0.1% 0.1%
Pt-18 CT 1b SVR - 20.0% - - 0.1% 0.4% 0.1% 0.1%
SVR: sustained virological response; RR: response-relapse; NR: non-response *SNP rs12979860
(Chevaliez S., et al., manuscript in preparation)

40. Treatment Failure-PROVE2
H28Q+R155K
100%
H28Q+R155K+S54T+Y52C
% of variants in the quasispecies
80% H28Q+R155K+S54T+Y52C+V36M+H5
7L+P96H
60%
8
40%
HCV RNA(Log10 IU/mL)
6
20%
0% 4
0
2
29
0
57
Days of therapy
85
Viral populations
*PyroLink®
(Chevaliez S., et al., EASL 2011)

41. Treatment Failure-PROVE2
100% H28Q+R155K
% of variants in the quasispecies
H28Q+R155K+S54T+Y52C
% of mutations in the whole quasispecies
80% H28Q+R155K+S54T+Y52C+V36M+H57
L+P96H
V36M+R155K+H57L
60% R155K
40% 8
HCV RNA (Log10 IU/mL)
HCV RNA (Log10 IU/mL)
20%
6
0%
0
4
29
57
85
2
182
595
0
Days of treatment
Days of therapy
686
*PyroLink® 903 Viral populations
(Chevaliez S., et al., EASL 2011)

42. UDPS in HCV Resistance
• Novel technologies for the study of HCV
resistance, such as ultra-
deeppyrosequecing,willbring new
insights intoitsmolecularmechanisms
andmay have clinical utility in the future

43. III
Practical Use

44. Standard-of-Care for HCV
Genotype non-1
Pegylated
IFN- + Ribavirin

45. Standard-of-Care (EU label)
Genotype 2,3 Genotype 4, 5, 6
PegIFN+ PegIFN+
ribavirin ribavirin
24 weeks 48 weeks

46. Virological Monitoring
PegIFN-ribavirin
0 4 8 12 24 36 48 60 72
Weeks of treatment

47. On-Treatment Virologic
Responses
-2 log
LLOD
Baseline Week 4 Week 8 Week 12

48. On-
TreatmentVirologicRespons
es
-2 log
LLOD
Baseline Week 4 Week 8 Week 12

49. On-
TreatmentVirologicRespons
es
-2 log
Delayed VR
72 weeks
RVR
24 weeks EVR
48 weeks LLOD
Baseline Week 4 Week 8 Week 12

50. 24 vs 48 Weeks in Genotype 2/3
Patients Without an RVR (N-Core)
p=0.19 p=0.14 p=0.02
80
73%
70
61% 63%
60
SVR24 (% of patients)
52% 52% 54%
50 Peg-IFN alfa-2a/RBV
24 weeks
40
Peg-IFN alfa-2a/RBV
48 weeks
30
20
10
49 57 49 51 49 46
95 93 95 81 95 63
0
ITT PP Study
(n=188) (n=176) completers
(n=153)
(Cheinquer et al., AASLD 2012)

51. New Standard-of-Care
for HCV Genotype 1
Telaprevir
Boceprevir
Pegylated
+
Ribavirin
IFN-

52. Virological Monitoring
PegIFN-ribavirin
0 4 8 12 24 36 48 60 72
Weeks of treatment

53. Virological Monitoring
Telaprevir
PegIFN-ribavirin
0 4 8 12 24 36 48 60 72
Weeks of treatment

54. Response-GuidedTherapy
Peg-IFN + Ribavirin + Telaprevir
Treatment-naive
or responder-
relapser
Partial responder
or null-responder
W0 W4 W12 W24 W36 W48 W72

55. Response-GuidedTherapy
Peg-IFN + Ribavirin + Telaprevir
eRVR: undetectable HCV RNA atweeks 4 and 12
Follow-up: 24 weeks
Treatment-naive
or responder- TVR + PR PR HCV RNA detectableatweeks 4 and/or 12 but ≤1000 UI/mL
relapser
PR Follow-up: 24 weeks
Partial responder
or null-responder
W0 W4 W12 W24 W36 W48 W72

56. Response-GuidedTherapy
Peg-IFN + Ribavirin + Telaprevir
eRVR: undetectable HCV RNA atweeks 4 and 12
Follow-up: 24 weeks
Treatment-naive
or responder- TVR + PR PR HCV RNA detectableatweeks 4 and/or 12 but ≤1000 UI/mL
relapser
PR Follow-up: 24 weeks
Partial responder
TVR + PR PR Follow-up: 24 weeks
or null-responder
W0 W4 W12 W24 W36 W48 W72

57. FutilityRules
Peg-IFN + Ribavirin + Telaprevir
• HCV RNA >1000 IU/mLatW4 or W12
• HCV RNA detectable (>10-25 IU/mL)
atW24

58. FutilityRulewithTelaprevir
• Retrospectiveanalysis of ADVANCE, ILLUMINATE and REALIZE
data (n=903 treatment-naive and 266 treatmentexperienced)
• Likelihood of an SVR
 HCV RNA atweek 4 >1000 IU/mL (2.1%): SVR: 0%
 HCV RNA atweek 4 =100-1000 IU/mL (2.0%):SVR: 22%
 HCV RNA atweek 12 >1000 IU/mL (1.5%): SVR: 0%
 HCV RNA atweek 12 =100-1000 IU/mL (1.6%): SVR: 15%
• Conclusion: A futilityrule of greaterthan 1000 IU/mLatweek 4 and
atweek 12 identifies treatment-naïve or -experienced patients
unlikely to achieve an SVR
(Jacobson et al., EASL 2012)

59. Futility Rule with Telaprevir
HCV RNA Profiles in Patients with HCV RNA >1000 IU/mL at week 4
(Jacobson et al., EASL 2012)

60. Virological Monitoring
Boceprevir
Telaprevir
PegIFN-ribavirin
0 4 8 12 24 36 48 60 72
Weeks of treatment

61. Response-GuidedTherapy
Peg-IFN + Ribavirin + Boceprevir
Treatmentnaive
patients
(excluding F4)
Treatment-
experienced
patients
(excludingnull-
responders and
F4)
F4 patients and
null-responders

62. Response-GuidedTherapy
Peg-IFN + Ribavirin + Boceprevir
W0 W4 W8 W12 W24 W28 W36 W48
Undetectable HCV RNA atweek 8
Boceprevir
Boceprevir+ PegIFN/RBV
Treatmentnaive
PegIFN/
patients RBV Detectable HCV RNA atweek 8
(excluding F4)
Boceprevir + PegIFN/RBV PegIFN/RBV
Treatment-
experienced
patients
(excludingnull-
responders and
F4)
F4 patients and
null-responders

63. Response-GuidedTherapy
Peg-IFN + Ribavirin + Boceprevir
W0 W4 W8 W12 W24 W28 W36 W48
Undetectable HCV RNA atweek 8
Boceprevir
Boceprevir+ PegIFN/RBV
Treatmentnaive
PegIFN/
patients RBV Detectable HCV RNA atweek 8
(excluding F4)
Boceprevir + PegIFN/RBV PegIFN/RBV
Treatment- W0 W4 W12 W24 W36 W48
experienced Boceprevir
patients
(excludingnull- PegIFN
Boceprevir+ PegIFN/RBV PegIFN/RBV
RBV
responders and
F4)
F4 patients and
null-responders

64. Response-GuidedTherapy
Peg-IFN + Ribavirin + Boceprevir
W0 W4 W8 W12 W24 W28 W36 W48
Undetectable HCV RNA atweek 8
Boceprevir
Boceprevir+ PegIFN/RBV
Treatmentnaive
PegIFN/
patients RBV Detectable HCV RNA atweek 8
(excluding F4)
Boceprevir + PegIFN/RBV PegIFN/RBV
Treatment- W0 W4 W12 W24 W36 W48
experienced Boceprevir
patients
(excludingnull- PegIFN
Boceprevir+ PegIFN/RBV PegIFN/RBV
RBV
responders and
F4)
W0 W4 W12 W24 W48
F4 patients and Boceprevir
null-responders
PegIFN
RBV Boceprevir + PegIFN/RBV

65. FutilityRules
Peg-IFN + Ribavirin + Boceprevir
• HCV RNA ≥100 IU/mLatW12
• HCV RNA detectable (>10-25 IU/mL)
atW24

66. FutilityRuleswithBoceprevir
• Treatment-naïve and -experienced patients
• In SPRINT-2
 None of the 65 patients with an HCV RNA >100 IU/mL at week 12
achieved an SVR
 49 out of 79 patients (62%) with detectable HCV RNA <100
IU/mL at week 12 subsequently became HCV RNA undetectable
and 43% achieved an SVR
• In RESPOND-2
 Only 1 patient with an HCV RNA >100 IU/mL at week 12
achieved an SVR
 5 out of 6 patients (83%) with detectable HCV RNA <100 IU/mL
at week 12 subsequently achieved an SVR
(Jacobson et al., Hepatology 2012;56:567-75)

67. TreatmentFailures on Triple
Combinationwith a DAA
• Due to an inadequateresponseto Peg-
IFN and ribavirin
• Results in
uncontrolledoutgrowthofresistantHCV
variantsselected by the
proteaseinhibitor
(Pawlotsky JM. Hepatology 2011;53:1742-51)

68. SVR According to Lead-in
(SPRINT-2, non-black)
100
90 82% 82%
% of patients with SVR
80
70
60 <1 log HCV RNA
50 decrease
39%
40 ≥1 log HCV RNA
29% decrease
30
20
10
0
BOC/RGT BOC/PR48
(Poordad et al., N Engl J Med 2011;364:1185-206)

69. HCV Resistance Testing
• Prior to therapy:
• There is no indication for resistancetestingatbaseline
• All patients shouldbeconsidered as harboringminor viral
populations that are resistant to telaprevir and boceprevir
• In case of treatmentfailure:
• There is no indication for resistancetestingduring and
aftertherapy, as the resultwill have no impact on
treatmentdecisions
• Proteaseinhibitor-resistant viral populations have been enriched
in every patient treatedwithtelaprevir or boceprevirwhodid not
clear infection
• Resistance testingisrequired in clinical trials and
global surveillance studies (research setting)