Next Generation Sequencing Redefines Reinfection and Relapse in Acute HCV Patients Urging a Review of Current Guidelines of Management of HCV Infection

Background In hepatitis C virus (HCV) infected patients, the virus circulates as a mixture of closely related but distinct genomes called quasispecies. The hypervariable region-1 (HVR-1) is the most heterogeneous region of the HCV genome and is an excellent target for sequence analysis to distinguish between different variants. We studied the dynamics of quasispecies in pre- and post-treatment samples taken from patients who failed standard of care therapy in a rare HIV-infected cohort of 160 patients with acute HCV. Methods A group of 16 patients failed to respond to treatment. A 220 bp region of the E2 envelope gene including (HVR-1) was amplified using nested RT-PCR using a combination of genotype-specific primers. PCR products were sequenced by direct sequencing (DS), clonal analysis (CA) and next generation sequencing (NGS). Phylogenetic trees were constructed using the maximum likelihood (ML) method. Findings Using DS, in the 16 patients that failed treatment (6 relapsers, 6 null responders and 4 partial responders), 44% of patients had evidence of a new infection post-treatment. However, NGS results revealed that such new infection representing new dominance of a pre-existing minority strain that was not detected by DS. Only one (6%) had completely new strains, which were presumed to represent re-infection. The emergence of new viral strains in HCV patients with treatment failure may most commonly be attributed to new dominance of pre-existing minority variants rather than re-infection. NGS could become an important screening tool at baseline for decision making when treating HCV-infected patients to identify mixed infection, particularly in the context of treatment decisions involving genotype-specific direct-acting antiviral agents.