Reassessment of Genotype 1 Hepatitis C Virus Subtype Misclassification by LiPA 2.0: Implications for Direct-Acting Antiviral Treatment

Abstract

The accuracy of LiPA 2.0 for hepatitis C virus 1 (HCV-1) subtype classification was analyzed. LiPA 2.0 genotype results from 101 HCV-1-infected patients were compared to genotype findings determined by direct core sequencing. Eleven (11%) samples were misclassified. Given the influence of the HCV-1-subtype in the anti-HCV therapy response, an alternative classification method is warranted.T he effect of the hepatitis C virus (HCV) genotype 1 subtype on the response to treatment can be dramatic for some direct antiviral agent (DAA)-containing regimens (1)(2)(3)(4)(5)(6)(7)(8)(9). This is the case for simeprevir, a DAA recently approved for clinical use, that in combination with pegylated interferon and ribavirin (peg-IFN/ RBV) has a lower sustained virological response (SVR) rate for subtype 1a than for 1b (2). Simeprevir plus peg-IFN/RBV treatment achieves rates of SVR similar to those for peg-IFN/RBV treatment in HCV-1a-infected patients who present the viral variation Q80K. This variation is frequent in the HCV-1a subtype, but it is not found in HCV-1b (2, 5). Similarly, daclatasvir plus peg-IFN/RBV treatment also achieves higher SVR rates among HCV1b-infected patients than among HCV-1a-infected individuals (4). For interferon-free regimens, DAA combinations such as asunaprevir plus daclatasvir may only be indicated for subtype 1b, because of high relapse rates observed among patients with subtype 1a (3, 4). Because of these, accurate subtyping of HCV genotype 1-infected patients is needed for those who are candidates for DAAs.Sequencing of specific HCV genome regions with subsequent phylogenetic analysis is considered the gold standard of HCV genotyping (10). However, it includes several complex techniques and is time consuming. For these reasons, LiPA 2.0 is currently one of the most widely used genotyping assays. However, several studies have reported HCV genotyping errors using this commercial method (11-13). Those studies were not designed to evaluate the frequency of HCV-1 subtyping misclassifications, and they included few HCV-1-infected patients. Therefore, due to the high prevalence of HCV-1 infections in western countries and the importance of correct subtyping in clinical decisions, the accuracy of this method for HCV-1 subtyping needs to be evaluated in larger samples. For these reasons, we aimed to evaluate the accuracy of LiPA 2.0 for classifying HCV-1 by subtypes.This study was performed according to the ethical guidelines of the 1975 Declaration of Helsinki, and it was approved by the ethics committee of the Hospital Universitario de Valme (Seville, Spain).The first 110 consecutive patients starting from October 2001, who received a course of peg-IFN/RBV treatment in our hospital and who were infected with HCV-1, according to the LiPA 2.0 determination, were selected. All of them were Caucasian with a median age (quartile 1 to quartile 3) of 42 years (range, 39 to 46 years). Among the patients, 82 (81%) were male, and 48 (47.5%) were infected with HIV.To determine the HCV-1 subtype, we analyzed...

NS3 genomic sequencing and phylogenetic analysis as alternative to a commercially available assay to reliably determine hepatitis C virus subtypes 1a and 1b

“…In fact, an HCV genotype 4-infected patient was erroneously genotyped as HCV-1 by LiPA 2.0 herein and may have received a combination based on dasabuvir, a drug efficient in HCV-1 but not indicated for genotype 4 [ 1 – 3 ]. This finding is in agreement with that previously reported by Guelfo and colleagues, who also observed a patient erroneously genotyped by LiPA [ 4 ]. Therefore, although the concordance between NS3 sequencing and LiPA could mathematically be classified as “substancial”, it is not acceptable in the clinical practice.…”

“…Second, in a considerable proportion of individuals, HCV-1 subtypes were misclassified. Although the proportions of both HCV-1 misclassification, as well as indeterminate calls vary considerably between reports [ 4 – 9 , 16 , 19 ], the misclassification rate of 14% observed in this study is in accordance with a number of previously published studies [ 4 , 7 – 9 ]. Since the misclassification of HCV-1 subtype might lead to inadequate DAA choice or no testing of RASs, sequencing should be considered for HCV-1 infected patients.…”

“…The Versant HCV 2.0 reverse hybridization line-probe assay (LiPA 2.0) represents a widely used commercially available assay to determine HCV genotype. However, a number of studies have detected an important proportion of discrepancies in HCV-1 subtype determination when compared to genomic sequencing methods including HCV core, E1 and NS5B regions [ 4 – 9 ]. To date, there is no data on comparisons with the NS3 region, which could simultaneously provide information on resistance-associated substitutions (RASs).…”

Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000‐2015 period (the GEHEP 005 study)

“…Despite the epidemiological characteristics of GEHEP 005 and having included a large population of patients with chronic hepatitis C from many hospitals all over Spain to ensure the representativeness and external validity of the present study, we believe that this study has some limitations. Apart from the limitations of retrospective studies, we also found some related to the methods mainly employed herein for HCV genotyping; this limitation refers to the correct viral subtype determination of genotype 1, especially when comparing commercial to reference methods based on sequencing and phylogenetic analyses of the NS5B region of HCV . In this regard, the inclusion of HCV genotyping by sequencing‐based technologies of different viral genome regions may notably improve viral subtype knowledge, which is still important for choosing the most appropriate treatment regimen and for determining resistance‐associated substitutions (RAS).…”

“…we also found some related to the methods mainly employed herein for HCV genotyping; this limitation refers to the correct viral subtype determination of genotype 1, especially when comparing commercial to reference methods based on sequencing and phylogenetic analyses of the NS5B region of HCV. [34][35][36][37][38][39][40] In this regard, the inclusion of HCV genotyping by sequencing-based technologies of different viral genome regions may notably improve viral subtype knowledge, which is still important for choosing the most appropriate treatment regimen and for determining resistance-associated substitutions (RAS). A final limitation is that our study lacks immigration data.…”