Hepatitis C virus gene sequencing as a tool for precise genotyping in the era of new direct antiviral agents
Authors: Francesca Ceccherini Silberstein, Velia Chiara Di Maio, Marianna Aragri, Marco Ciotti, Valeria Cento, Carlo Federico Perno
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. To the Editor: The large majority of new direct antiviral agents (DAAs) against hepatitis C virus (HCV) are not pangenotypic and show major differences in response rates and resistance patterns, depending on HCV genotype and/or subtype (i.e., 1a/1b).1 Therefore, in view of the (exceptionally) high cost of these treatments, the accurate assessment of HCV genotype/subtype before treatment initiation is mandatory to reduce the risk of failure driven by inappropriate genotype characterization.2 Several commercial assays are available for HCV genotyping, all targeting the 5' noncoding‐region of genotypes 1‐6. In addition, two assays (Abbott RealTime HCV Genotype II and Versant HCV genotyping 2.0) also target the nonstructural (NS)5b and core genes, respectively, allowing a potentially more precise subtype discrimination within genotypes.3 Nevertheless, in daily clinical practice, indeterminate, mixed, and unspecified subtype results are frequently observed. As an example, a recent article suggests that one of the commercial assays currently available may fail a precise determination of HCV genotype in 9%‐10% of cases examined (N = 1,052).3 In order to define the extent of this unmet clinical need, we reanalyzed genotype data by phylogenetic analysis of 343 HCV‐infected patient candidates to DAA treatment who performed a genotypic resistance test between 2011 and 2014. To confirm the appropriate genotype allocation, HCV sequencing was performed by homemade protocols, specific for each genotype, on NS3 protease (95% samples), together with/in alternative to NS5A (9%) and/or NS5B (14%). When more than one genomic region per patient was analyzed (N = 52), sequencing results were 100% concordant with one another, thus confirming the specific genotype/subtype assignment. Conversely, HCV sequencing and commercial assays were concordant in 91.8% of cases (Table 1): HCV sequencing identified 4 of 343 genotypes discordant with the assignment given by commercial assays (commercial⃯sequencing: 1a⃯2c; 1b⃯2c; 1b⃯4d; 2a/2c⃯1b) and 10 of 343 discordant subtype cases (commercial⃯sequencing: 1a⃯1g [N = 2]; 1a⃯1b [N = 3]; 1b⃯1a [N = 5]). Of interest, 1 discordant patient found to be infected with HCV 1g, carrying baseline NS3 mutation T54S, rapidly failed a boceprevir‐containing regimen.4 Table 1 - Number of HCV‐Infected Patients With at Least One Available NS3 or NS5A or NS5B Sequence Patients (N) Patients (%) Genotype/subtype confirmed 315 91.84 Genotype 1 with no subtypea 6 1.75 Discordant genotypesb 4 1.17 Genotype 1 with discordant subtypec 10 2.91 Mixed genotypesd 8 2.33 Total 343 100 aPerformed by using Abbott assay in 2013‐2014 (N = 6).bPerformed by Versant LiPA assay (N = 2) and unknown methodology (N = 2).cOne patient showed indeterminate result with Abbott assay in 2013 and genotype 1a in 1999, with unknown methodology. Another patient infected with HCV 1g showed HCV 1a result by both Abbott and Versant LiPA assays.4 The other 8 patients showed discordant results with Versant LiPA assay (N = 5) or Trugene (N = 3).dPerformed by using Abbott‐assay (N = 2), LiPA assay (N = 1), Trugene (N = 2), and unknown methodology (N =3). Notably, all patients with a previous result of mixed or indeterminate HCV genotype or subtype by commercial assays (14 of 343; 4.1%) were instead precisely resolved by HCV sequencing. As a whole, 28 of 343 (8.2%) patients achieved the correct genotyping assignment by sequencing. The overall percentage of genotype/subtype resolved by direct sequencing in the current study is similar to what has been found in other recent publications.3 Our results emphasize the importance of dedicating time and effort for a proper genotype/subtype assignment before starting therapy. HCV sequencing allows precise subtype/genotype assignment, along with evaluation of natural resistance, thus reducing the risk of failure, especially in difficult‐to‐treat patients. The relatively low cost of sequencing (compared to therapy) should encourage studies aimed at better defining the advantage of its use at therapy baseline.