Hepatitis C Virus (HCV) Genotyping with NS5A Drug Resistance

The Hepatitis C Virus (HCV) Genotyping with NS5A Drug Resistance assay detects NS5 (nonstructural protein 5A) mutations and polymorphisms in HCV genotypes 1a, 1b, 2 and 3 that are associated with resistance to direct-acting antivirals: daclatasvir (Daklinza^®), elbasvir, ledipasvir, ombitasvir, pibrentasvir, velpatasvir and other antiviral combination therapies. The assay is intended to be used for patients with HCV viral loads who are being screened prior to treatment with the direct-acting HCV antivirals, or during treatment with these antivirals when drug resistance is suspected.

About HCV

HCV infection is the most common chronic bloodborne infection in the U.S., with approximately 3.2 million people chronically infected and an additional 17,000 (approx.) new infections acquired annually. Multiple drugs approved to treat HCV target the NS5A protein. Specific mutations in these genes have been implicated in resistance to various HCV antiviral drugs including: daclatasvir (Daklinza^®), elbasvir (in Zepatier^®), ledipasvir (in Harvoni^®), ombitasvir (in Viekira Pak^®, Viekira XR^® and Technivie^®), pibrentasvir (in Mavyret^®), and velpatasvir (in Epclusa^® and Vosevi^®). Multiple mutations causing resistance to these FDA-approved HCV inhibitors have been defined, and the levels of increased resistance for each mutation have been calculated using HCV replicons or reporter constructs. Some of the mutations cause resistance to multiple NS5A inhibitors. Knowledge of these mutations is important in patients since an incomplete suppression of viral replication by an ineffective drug combination could prevent a sustained viral response (“cure”), and could readily support the development of antiviral drug resistance.

This test includes two separate assays.

The HCV NS5A Genotyping assay includes extraction of nucleic acid followed by amplification. The amplified DNA is converted to single-stranded DNA via exonuclease digestion and is then combined with a signal buffer containing ferrocene-labeled signal probes that are specific to the different types/subtypes. The mixture of amplified sample and signal buffer is loaded onto the eSensor cartridge, which contains single-stranded oligonucleotide capture probes bound to gold-plated electrodes. The presence of each target is determined by voltammetry, which generates specific electrical signals from the ferrocene-labeled signal probe.

The HCV NS5A Drug Resistance assay utilizes RT-PCR amplification with primers in highly conserved viral genomic regions to amplify HCV genotypes 1a, 1b, 2 and 3. The fragments are purified and sequenced using sequencing primers from conserved regions of the fragments. The sequence is compared to a database of mutations associated with antiviral resistance. This test has not been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.