Next‐generation sequencing sheds light on the natural history of hepatitis C infection in patients who fail treatment

Abstract

High rates of sexually transmitted infection and reinfection with hepatitis C virus (HCV) have recently been reported in human immunodeficiency virus (HIV)-infected men who have sex with men and reinfection has also been described in monoinfected injecting drug users. The diagnosis of reinfection has traditionally been based on direct Sanger sequencing of samples pre- and posttreatment, but not on more sensitive deep sequencing techniques. We studied viral quasispecies dynamics in patients who failed standard of care therapy in a high-risk HIV-infected cohort of patients with early HCV infection to determine whether treatment failure was associated with reinfection or recrudescence of preexisting infection. Paired sequences (pre- and posttreatment) were analyzed. The HCV E2 hypervariable region-1 was amplified using nested reverse-transcription polymerase chain reaction (RT-PCR) with indexed genotype-specific primers and the same products were sequenced using both Sanger and 454 pyrosequencing approaches. Of 99 HIV-infected patients with acute HCV treated with 24-48 weeks of pegylated interferon alpha and ribavirin, 15 failed to achieve a sustained virological response (six relapsed, six had a null response, and three had a partial response). Using direct sequencing, 10/15 patients (66%) had evidence of a previously undetected strain posttreatment; in many studies, this is interpreted as reinfection. However, pyrosequencing revealed that 15/15 (100%) of patients had evidence of persisting infection; 6/15 (40%) patients had evidence of a previously undetected variant present in the posttreatment sample in addition to a variant that was detected at baseline. This could represent superinfection or a limitation of the sensitivity of pyrosequencing. Conclusion: In this high-risk group, the emergence of new viral strains following treatment failure is most commonly associated with emerging dominance of preexisting minority variants rather than reinfection. Superinfection may occur in this cohort but reinfection is overestimated by Sanger sequencing. (Hepatology 2015;61:88–97)

Diagnostic and Therapeutic Implications in a Case of Mixed Hepatitis C Virus (HCV) Infection

“…The literature reports include frequent occurrence of superinfections in patients with an IDU history (3,6), and reinfections in patients with late virological relapse after virological response achievement (10,20,21). However, a reinfection is very unlikely to occur with any virus at all during a 2-drug treatment cycle, which, in addition, was conducted well enough to lead to a systemic vascular resistance (SVR) for the subtype 4d virus.…”

“…However, methods to detect minority populations within the circulating quasi-species are not yet of widespread use; thus, the overall prevalence of HCV MI may be underestimated (2). Although the next generation sequencing (NGS) technologies offer a more sensitive method to detect minority populations within the circulating quasi-species, allowing distinguishing coinfections and superinfections from reinfections, the latter may be overestimated by standard sequencing due to the poor sensitivity in detection of minor variants (2,9,10). Due to the relative genotype specificity of the new anti-HCV drugs, some concerns are voiced about the effectiveness of treatment in HCV MI, but the question is not yet addressed by guidelines, and no reports are issued on the topic.…”

HCV reinfection among HIV‐positive men who have sex with men: A pragmatic approach

“…In a recent edition of HEPATOLOGY, Abdelrahman et al 1 describe the use of next-generation sequencing to investigate acute hepatitis C virus (HCV) treatment failures among human immunodeficiency virus (HIV)-infected individuals. They show that among individuals with a null response, partial response, or relapse during HCV treatment with peg-interferon alpha and ribavirin, the dominant strain of virus posttreatment was frequently a nondominant strain only detectable at low levels pretreatment.…”

Causes of treatment failure for hepatitis C in the era of direct-acting antiviral therapy

“…-Superinfection: Variants are identified post-treatment with a clear phylogenetic relation with pre-treatment variants (persistent infection) besides variants unrelated to original isolates (reinfection). Recently, the need to discuss the issue of virological failure using NGS was clearly demonstrated (45). Through the study of quasispecies dynamics using conventional, clonal, deep sequencing in patients with a high risk for reinfection (homosexuals co-infected with HCV and HIV), differentiation between persistent infection and reinfection was attempted.…”