Hepatitis C Virus is a single stranded RNA virus belonging to the Flaviviridae family and genus Hepacivirus.

Chronic HCV infection is globally a major cause of liver disease including liver cirrhosis and hepatocellular carcinoma.It is estimated that more than 150 million individuals are persistently infected worldwide ¹. In comparison with HIV and HBV where treatment goal is lifelong suppression of the virus, HCV is completely curable and treatment goal is viral clearance from plasma and liver. Over the past decade or so, there have been discoveries of certain novel molecules which interfere with the HCV life-cycle by targeting specific steps in the cycle thus making more options available for antiviral therapy. These are known as directly acting antiviral (DAA) drugs. In an HCV infected individual, due to the highly replicative nature (1012 virions/day) of the virus, large numbers of viral variants are formed daily (quasispecies), some of which are responsible for drug resistance. It has been observed that the patients who do not show sustained virological response (undetectable HCV RNA 24 weeks after treatment cessation - SVR) have more of these resistant variants ². Thus resistance determination for HCV is critical to treatment outcome.

The resistant variants can occur naturally and can exist in the patient even before antiviral treatment is initiated. Further, as the drugs are ineffective on these variants, due to selection or enrichment, these resistant variants increase in numbers as the drugs act on the sensitive variants. Also, drug resistance may emerge during treatment with antiviral drugs. Improper treatment, non-compliance etc may result in drug resistance and consequent treatment failure.

Antiviral drugs

Currently, available DAA drugs approved or in development are classified on the basis of the mechanism of action and molecular targets.

When antiviral treatment is initiated in an individual there is selection or enrichment of resistant variants with all known antiviral drugs. Single nucleotide changes leading to codon change and consequent change in resultant amino acid can confer resistance. It may lead to decreased binding of the drug leading to decreased inhibition of viral replication. It should be noted that drug resistance often results in reduced ‘viral fitness’ i.e. these variants are less fit than the wild type variants ³. A wide range of frequency of resistance mutations have been found with non nucleoside analogs in vitro as well as in vivo. Nucleoside analogs have demonstrated selection of resistant variants in vitro but they have not yet been observed in patients with HCV.

Why drug resistance determination?

As pointed out earlier resistant variants get selected during antiviral treatment as potential antiviral therapy eliminates sensitive variants uncovering the resistant ones which expand. Frequent monitoring of HCV RNA levels can detect treatment failure and resistance. HCV RNA levels fall from initial baseline values after treatment is initiated, however in a period of time viral breakthrough may occur due to increased resistant variants which have been uncovered. There can be a continued replication of the virus in the presence of a drug which leads to a further evolution of the viral population. Long term follow up from patients with drug resistance indicate that the drug resistant viral populations may return to pre treatment levels in many patients ⁵. Thus a combination drug regimen of Pegylated interferon α/ribavirin with specific antiviral agents selected after seeing the HCV- drug resistance profile eliminates all viral variants with non overlapping resistance. Lack of cross resistance between Peg-IFN α/ ribavirin and antiviral drugs provides an opportunity for the elimination of resistant variants. The motto should be to maximize drug response, minimize drug resistance. The regimen characteristics which increase the likelihood of achieving SVR are shorter, combination, adherence friendly regimens with minimal drug interactions, good tolerability providing potent viral suppression while overcoming resistance.

Tests to determine HCV drug resistance

Sequence analysis and phenotype analysis are used to identify resistance or discover new resistance pathways. Sequence analysis will detect specific amino acid substitutions relative to a pre-treatment or standard reference sequence that are known to decrease susceptibility to different antiviral agents. There are known resistance mutations associated with NS5B polymerase nucleoside and non-nucleoside inhibitors. This type of analysis can also help to identify novel drug resistance pathways associated with treatment failure. The current routine genotypic analysis practice for HCV drug resistance is direct sequencing by Sanger method. It can be directly performed on clinical specimens like plasma to show appropriate sensitivity after forming a consensus sequence of the dominant HCV variants present in the clinical specimen. As HCV resistance pathways differ between different viral genotypes and subtypes, genotype specific PCR primers are used to ensure amplification of target genes like NS3, NS5B or NS5A. However minor variants present at low frequencies (≤20%) are not detected by this method. Next generation sequencing (N.G.S.) can detect even minor variants present at a much lower frequency ^4, generatingthe whole HCV genome to accurately and simultaneously determine HCV genotypes, Resistance Associated Variants (RAV), quasi-species diversity and to allow comprehensive viral strain analysis.

Testing for NS3 and NS5A RAVs is recommended for retreatment of cirrhotic patients or other patients who require re-treatment urgently when there is a history of failure to NS5A inhibitor containing regime. Monitoring resistance for persistence of these RAVs will lead to better management of second line therapy.

Since there is no efficient infection system for most HCV strains, phenotypic analysis by a viral infection model cannot be performed. Recombinant replicon based shuttle vector assays, cell based viral enzyme assays or biochemical enzyme assays can be used. So far such systems have been mainly used for research purposes only and not for patient management as available systems are limited to only a few HCV subtypes.
 

References

1. Messina, Jane P et al. “Global Distribution and Prevalence of Hepatitis C Virus Genotypes.” Hepatology (Baltimore, Md.) 61 (2015): 77–87. PMC. Web. 18 July 2017.
2. Naggie S. Management of Hepatitis C Virus Infection: The Basics. Topics in antiviral medicine. 2012;20(5):154-161.
3. Cooper, C. L. (2010). Viral response to specifically targeted antiviral therapy for hepatitis C and the implications for treatment success. Canadian Journal of Gastroenterology, 24(6), 385–390.
4. Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W, Ansari MA et.al Comparison of Next-Generation Sequencing Technologies for Comprehensive Ass essment of Full-Length Hepatitis C Viral Genomes. J Clin Microbiol. 2016 Oct;54(10):2470-84)

Contributed by Dr Shreeraj Talwadekar, MD Research Scientist, R&D, SRL Limited, Mumbai; Dr B R Das, PhD Advisor & Mentor R&D, Molecular Pathology and Clinical Research Services, SRL Limited 

Disclaimer-The information and views set out in this article are those of the author(s) and do not necessarily reflect the official opinion of M3 India. Neither M3 India nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein.