1. Hepatitis C Molecular Diagnosis in the Era of DAAs July 22, 2016, Tehran Ali Namvar Ph.D of Molecular Genetics Iranian Comprehensive Hemophilia Care Center (ICHCC)-Molecular Diagnostics Division 1

2. Hepatitis C Molecular Diagnosis in the Era of DAAs Outline: Viral Load Monitoring on DAAs Therapy Hepatitis C Genotyping method Drug Resistance Test Summary 2

3. Viral Load Monitoring on DAAs Therapy Nucleic acid testing is particularly important in viral hepatitis : 1. Confirm diagnosis 2. Assess infection activity and the risk of transmission 3. Define the virus genotype & Drug Resistance 4. Last but not least, to monitor and guide antiviral therapy. 3

4. Viral Load Monitoring on DAAs Therapy Nucleic acid testing for detection of viruses causing hepatitis may be based on different methods. By far the most important today is quantitative real- time PCR. Amplification is confirmed and quantified by hybridization with specific fluorescent probes. However, if quantification is not needed, conventional PCR may be run. In addition to real-time PCR, alternative techniques are still being used: 1- Transcription-mediated amplification (TMA) 2- Branched DNA (bDNA) 4

5. Currently available hepatitis C Tests Qualitative & Quantitaive: Test NameMethodClinical Significance Limit Of Detection Hepatitis C Viral RNA, Qualitative Transcriptio n-Mediated Amplificatio n or RT-PCR Qualitative RNA by TMA is useful in confirming HCV infection and to assess response to therapy Down to 5 IU/ml – 99 % Down to 1 IU/ml – 55-75 % Hepatitis C Viral RNA, Quantitative Real-Time Polymerase Chain Reaction Or Brancbed DNA Amplificatiio n Useful in monitoring therapy and/or disease progression. Down to 12 IU/ml – 95 % 5

6. Viral Load Monitoring on DAAs Therapy Historically, hepatitis C virus (HCV) RNA levels have been a predictive on-therapy marker of treatment outcome. HCV RNA threshold values at select time points have been used to guide decisions regarding the continuation or halting of interferon-containing therapy, based on the likelihood of treatment success. Interferon- free, DAA-only treatment regimens are replacing interferon-containing regimens as the standard of care. As a result, it has become essential to reevaluate he utility of HCV RNA levels in predicting treatment outcome and in guiding clinical decision making. 6

7. Viral Load Monitoring on DAAs Therapy For each viral load assay, a linear range exists that encompasses the upper and lower limits of quantification (ULOQ and LLOQ, respectively). 7

8. Viral Load Monitoring on DAAs Therapy 8

9. Commercially available quantitative real-time PCR-based hepatitis C virus RNA assays 9

10. Viral Load Monitoring on DAAs Therapy Predictive Ability of HCV RNA at Week 4 for SVR12 Among patients treated with sofosbuvir + ledipasvir ± ribavirin in the ION-1, -2, and -3 trials, 268/1504 (18%) and 5/1504 (0.3%) patients had detected or quantifiable (≥25 U/mL, Roche v2.0) HCV RNA at week 4. Detactable for HCV RNA at week 4 in these trials underscore the importance of continued therapy for patients who fail to achieve undetectable viral loads during treatment, as likelihood of SVR12 is still high. 10

11. Viral Load Monitoring on DAAs Therapy Table: Hepatitis C Virus (HCV) RNA Levels in Interferon-Free HCV Direct- Acting Antiviral Registrational Trials(FDA-2016) 11

12. Viral Load Monitoring on DAAs Therapy Table: Hepatitis C Virus (HCV) RNA Levels in Interferon-Free HCV Direct- Acting Antiviral Registrational Trials(FDA-2016) 12

13. Viral Load Monitoring on DAAs Therapy Predictive Ability of HCV RNA at end of treatment(EOT) for SVR12 Highly potent DAA regimens have been shown to have undetectable HCV RNA at end of treatment. Studies have shown that low levels of quantifiable HCV RNA on therapy and even at the end of treatment should not be considered indicative of non adherence and do not preclude achieving an SVR (Sidharthan, Sreetha, et al. NIH – 2015 ). Detectable virus at EOT in patients who achieve SVR12 may be explained by a role of the host immune system that persists after cessation of therapy 13

14. Viral Load Monitoring on DAAs Therapy HCV Viral load kinetic Table: Patients with quantifiable hepatitis C virus (HCV) RNA at end of treatment (EOT) achieve sustained virologic response after 12 weeks of treatment( Sidharthan, Sreetha, et al. NIH – 2015) 14

15. Viral Load Monitoring on DAAs Therapy HCV Viral load kinetic Table: Patients with quantifiable hepatitis C virus (HCV) RNA at end of treatment (EOT) achieve sustained virologic response after 12 weeks of treatment( Sidharthan, Sreetha, et al. NIH – 2015) 15

16. Hepatitis C Genotyping Method The new HCV therapies have specific indications, including durations and drug combinations appropriate for use for treatment of a particular patient, which vary according to HCV genotype. Subtyping of HCV genotypes has also proven important due to the lower barrier to resistance of genotype 1a isolates compared to genotype 1b for multiple classes of DAAs. A wide variety of genotyping methods are used, including PCR amplification followed by strip-based reverse hybridization, PCR followed by Sanger sequencing, and real-time PCR. 16

17. Hepatitis C Genotyping Method To date, there have been no perfect genotyping tests. While sequencing can offer excellent resolution of HCVgenotype, it can be time-consuming and labor- intensive, it requires skilled technologists and is costly, and results are not standardized. The 5’-NTR is the preferred part of the HCV genome for genotyping. It is sufficiently diverse between subtypes for differentiation and is highly conserved within subtypes and genotypes limiting ‘background noise’. 17

18. Hepatitis C Genotyping Method 18

19. Drug Resistance Test DAAs interrupt HCV replication by targeting specific HCV proteins, such as the NS5A protein, NS5B polymerase, and NS3/4A protease.6 DAAs that inhibit NS3/4A (simeprevir, paritaprevir), NS5A (daclatasvir, ledipasvir, ombitasvir), and NS5B (sofosbuvir, dasabuvir) have been approved for treatment of HCV infection and are available in fixed-dose combinations. The high mutation rate of the HCV genome, combined with selective pressure from ongoing therapy, can lead to selection of HCV variants that are resistant to DAAs 19

20. Drug Resistance Test While resistance to HCV therapies is a concern, unlike those in human immunodeficiency virus (HIV), some baseline HCV drug resistance mutations are often not clearly related to treatment outcome. Currently, there is no regulatory-agency approved assay for the determination of HCV antiviral drug resistance and testing is largely performed in specialized settings with self-validated, laboratory-developed, sequencing- based assays. 20

21. Summary Studies have shown that low levels of quantifiable HCV RNA on therapy and even at the end of treatment should not be considered indicative of non adherence and do not preclude achieving an SVR The new HCV therapies have specific indications, including durations and drug combinations appropriate for use for treatment of a particular patient, which vary according to HCV genotype. While resistance to HCV therapies is a concern, unlike those in human immunodeficiency virus (HIV), some baseline HCV drug resistance mutations are often not clearly related to treatment outcome. 21

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