Late Relapse and Reinfection in HCV Patients Treated with Direct-Acting Antiviral (DAA) Drugs

Abstract

The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1-6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.

Keywords: HCV; HCV late relapse; HCV reinfection; NGS; hepatitis C virus; next-generation sequencing; phylogenetic.

Figure 1

Patient HCV RNA viral load over time. The sampling times T0, T1, and T2 are indicated. The period and type of treatment are indicated in the red box. DAS: Dasabuvir; EBR: Elbasvir; GLE: Glecaprevir; GZR: Grazoprevir; OMB: Ombitasvir; PAR: Paritaprevir; PIB: Pibrentasvir; RBV Ribavirina; RTV: Ritonavir; SIM: Simeprevir; SOF: Sofosbuvir.

Figure 2

HCV NS5B phylogenetic tree. The maximum-likelihood method with the transition-free rate model and empirical codon frequencies was employed to build the NS5B phylogenetic tree. The HCV GT4d sequence (DQ418786.1) was selected as an out-group. Reference sequences (GT1a: EF407457.1; GT1b: EU781827.1; GT3a: X76918.1) were retrieved from GenBank. Sanger sequences for all patient samples were included and indicated as Patient_N-T0, Patient_N-T1, and Patient_N-T2. The bar represents the genetic distance (substitution per nucleotide position). Bootstrap analysis with 1000 replicates was performed. Each patient is represented by a different color: Pt1_T0 and Pt1_T1 are depicted in light and dark blue, respectively; Pt2_T0 and Pt2_T1 are depicted in light and dark red, respectively; Pt3_T0, Pt3_T1, and Pt3_T2 are depicted in light green, olive green, and dark olive green, respectively; Pt4_T0 and Pt4_T1 are depicted in light and dark grey, respectively; Pt5_T0 and Pt5_T1 are depicted in light and dark violet, respectively; Pt6_T0 and Pt6_T1 are depicted in light and dark brown, respectively. The orange, blue, and green circled areas represent sequences clustering with GT1a, GT1b, and GT3a, respectively.

Figure 3

Three-dimensional plots illustrating genetic distances of quasispecies sequences with reference sequences representing GT1a (EF407457.1), GT1b (EU781827.1), and GT3a (X76918.1). Genetic distances between quasispecies sequences at T0, T1, and T2 are shown in orange, blue, and grey, respectively.

Figure 4

Graphic representing the frequency of nucleotide and amino acid substitutions detected at each tested time point for the six patients (Pt1–Pt6).