Real World SOF/VEL/VOX Retreatment Outcomes and Viral Resistance Analysis for HCV Patients With Prior Failure to DAA Therapy

David A. Smith; Daniel Bradshaw; Jean L Mbisa; Carmen F. Manso; David F Bibby; Joshua B Singer; Emma C Thomson; Ana da Silva Filipe; Elihu Aranday-Cortes; M. Azim Ansari; Anthony Brown; Emma Hudson; Jennifer Benselin; Brendan Healy; Phil Troke; John McLauchlan; Eleanor Barnes; William L. Irving


J Viral Hepat. 2021;28(9):1256-1264. 

In This Article


Patients and Samples

This study included 215 individuals from across England who did not achieve SVR with previous interferon-free DAA treatment. Blood samples were taken prior to retreatment and samples sent to Oxford and Glasgow for HCV whole-genome sequencing (WGS). Clinical, demographical and treatment outcome data were collected from patients, who were enrolled in HCV Research UK (n = 37), following informed consent and ethical approval.[14] For patients not enrolled in HCV Research UK, whole-genome HCV sequences and anonymised clinical data were provided by Public Health England (PHE), collected as part of the Virus Reference Department's HCV antiviral resistance testing service. This provides a clinical service for National Health Service Trusts, which send patients' blood samples for HCV genotyping and resistance testing by WGS and receive results in real time to inform clinical management. Approval for the use of data from this service was granted under Regulation 3 of the Health Service (Control of Patient Information) Regulations 2002. For retreatment, SVR has defined a minimum of 12 weeks after the end of treatment.

This project has been supported by the Hepatitis C Trust an organisation which campaigns for issues affecting patients with HCV in the UK. A patient representative was present at all STOP-HCV steering group committee meetings where the plans for this study were discussed in detail with direct patient involvement in the planning at all stages. In addition, both HCV Research UK and Public Health England have a Patient and Public Involvement Strategy and engage with patients and the public about how the data collected by them is used.

Whole Genome Sequencing of HCV

Whole HCV genome sequencing was conducted at three sites to define HCV subtype and identify RAS: (i) The MRC-University of Glasgow Centre for Virus Research (CVR), (ii) The Peter Medawar Building for Pathogen Research, University of Oxford, and (iii) The Virus Reference Department, National Infection Service, Public Health England. These sites have previously collaborated to ensure uniform standards for HCV sequencing.[15] Briefly, RNA was isolated from patient plasma samples and reverse transcribed to produce cDNA. An Illumina sequencing library was generated and enriched for HCV viral sequences using specific oligonucleotide probes. Illumina sequencing reads were then processed and mapped to the closest HCV reference genome. Precise details of how the sequencing was performed at each site are described in the supplementary materials.

Resistance-associated Substitution Calling Using HCV-GLUE

HCV subtypes were assigned and NS3, NS5A and NS5B RAS relevant to each genotype were identified (15% of reads cut off) using HCV-GLUE and RAS definitions provided by PHE[16] (Supplementary Table 3). A RAS was considered relevant if there is in-vitro evidence of a >50 Fold change in EC50 and/or in-vivo evidence that the RAS is treatment emergent or associated with treatment failure. HCV-GLUE is a resource created as part of the STOP-HCV consortium based on the GLUE (Genes Linked by Underlying Evolution) platform.[17] HCV-GLUE uses the ICCT HCV reference sequences and collates publicly available HCV sequences, to construct phylogenies using RAxML to assign sequences to HCV clades. It also contains a database of HCV RAS created by PHE[16] and this is used to identify RAS relevant to the HCV genotype and subtype.[18]

Statistical Analysis

All statistical analysis was performed using custom code and R version 3.6.2. Logistic regression was used for the analysis of association between categorical variables.