Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination

Abstract

BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes.

Methods: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools.

Results: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis.

Conclusion: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

Keywords: HCV; RT-PCR; Sanger sequencing; direct-acting antivirals; genotyping; in house method.

Figure 1

Patient data summary. (A) Age of patients at time of serum‐sample receipt from HCV‐UK. Date of birth data was not available for three patients. Frequency values (n) are shown above columns. (B)The primary suspected route of HCV acquisition for patients in the cohort were detailed. It was not possible to distinguish between patients for whom no data was provided and those where no specific transmission route was suspected. (C) Map of Africa highlighting the country of birth of patients in this study. The number of patients born in each country is shown in brackets. HCV, Hepatitis C virus.

Figure 2

Proportion of SSA HCV subtypes with known or potentially reduced susceptibility to NS5A inhibitors. Radar plots illustrating known or potential reduced efficacy of NS5A inhibitors for treatment of SSA HCV samples. Samples grouped by HCV subtype and clustered by genotype (Gt1, blue; Gt2, green; Gt3, yellow; Gt4, orange; Gt5, red). Six samples could not be assigned to a single subtype and are shown as duplexed subtypes. Subtypes assessed for their combined predicted response to treatment with a given inhibitor class (0%, all samples fully susceptible; 100%, all samples possessing at least one known or uncharacterized RAS applicable to that drug class). DCV, Daclatasvir; EBR, elbasvir; HCV, Hepatitis C virus; LDV, ledipasvir; OBV, ombitasvir; PIB, pibrentasvir; SSA, sub‐Saharan Africa; VEL, velpatasvir.