Recombinant hepatitis C viruses that might hamper accurate genotype classification and choice of treatment with direct‐acting agents, southeastern France
Authors: Philippe Colson, Patrick Borentain, Catherine Dhiver, Serge Benhaim, René Gerolami, Catherine Tamalet
Journal: Hepatology (2015)

Potential conflict of interest: Dr. Dhiver consults for Gilead, ViiV, and MSD. To the Editor: We read with interest the article by Hedskog et al. on characterization of intergenotypic recombinant hepatitis C viruses (HCV) and their potential impact on genotype determination and subsequent response to genotype‐dependent combination therapies based on direct‐acting agents (DAAs).1 In this work, 2.5% of HCV classified genotype 2 based on 5′ untranslated region (UTR) sequencing or INNO‐LiPA were revealed as being HCV‐1 based on nonstructural (NS)5b‐polymerase gene analysis. We searched in our IHU clinical microbiology laboratory sequence database for such discordance between genotype determined based on phylogeny of 5′ UTR and NS3‐protease or NS5b regions.2 Between May 5, 2000 and March 26, 2015, 5,683 HCV sequences (>200‐nucleotide‐long) were obtained in routine clinical practice from blood samples of 3,759 patients. They included 1,251 5′UTR, 1,668 NS3, and 2,764 NS5b sequences. 5′UTR/NS3 or 5′UTR/NS5b sequences were concurrently obtained from a same sample for 145 and 446 patients, respectively. These sequences were compared by BLAST and phylogeny to reference sequences of HCV genotypes that included recombinants identified by Hedskog et al.1 In 35 cases, genotype was 2 according to 5′ UTR phylogeny, and a NS3 or NS5b sequence was concurrently obtained in 9 and 28 cases, respectively. For 2 patients, genotype was 1 according to NS3 gene phylogeny and HCV NS3 sequence was clustered with a HCV 2k/1b recombinant (Fig. 1; Supporting Information). Hence, in our cohort, such discrepancy regarding HCV genotype determination involved 5.7% of 5′UTR sequences of genotype 2. Such misclassifications of 5′UTR sequences for HCV 2k/1b recombinant strains were also reported recently in Belgium.5 We further performed NS3 phylogeny reconstruction with sequences from our database for which a HCV 2/1 recombinant was the best BLAST hit and identified sequences that were clustered with 2k/1b or 2b/1b recombinants for 7 patients, who included those 2 we formerly identified as infected with HCV 2k/1b recombinant strains (Supporting Information). Although coinfections by different HCV cannot be completely ruled out here because clonal full‐length genomes were not generated in routine clinical practice, our data suggest that HCV‐2/1 recombinants circulate in our geographical area and, in addition to previous reports of inaccurate 5′UTR‐based HCV classification,2 that this genome region might be inadequate in few cases to guide DAA‐based HCV treatments.Figure 1: Phylogenetic trees based on 230‐ and 468‐nucleotide‐long fragments from the 5′UTR (A) and NS3 protease encoding gene (B) of the HCV genome, respectively. Phylogeny reconstruction included HCV sequences obtained in our laboratory as previously described (3) and stored in our IHU HIV‐hepatitis virus sequence database (indicated by identification numbers and MrsTim [for Marseille‐Timone]), and sequences from a recently updated set of reference sequences of HCV genotypes (labeled with Ref., genotype and subtype, and GenBank accession number).4 Sequences from recombinant HCV are in boldface. Nucleotide alignments were performed using the MUSCLE software (http://www.drive5.com/muscle/). The evolutionary history was inferred in the MEGA6 software (http://www.megasoftware.net/) using the Neighbor‐Joining method. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the scale bar indicates the number of nucleotide substitutions per site. These evolutionary distances were computed using the Kimura two‐parameter method. Bootstrap values > 50% are labeled on the tree. Some branches were collapsed to allow a better legibility of the tree. Abbreviations: Mrs, Marseille; Tim, CHU Timone; Ref., reference.