Unique NS5b Hepatitis C Virus Gene Sequence Consensus Database Is Essential for Standardization of Genotype Determinations in Multicenter Epidemiological Studies

Abstract

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.In a previous multicenter study designed to assess the applicability of hepatitis C virus (HCV) genotyping methods (5), we found a wide heterogeneity of results depending on both the laboratories and the genotyping methods. We conducted a new multicenter study of genotype determination based on HCV NS5b sequencing in order to further evaluate the performances of the laboratories involved in the previous study and to define a consensus method for further epidemiological studies.The panel included 12 samples collected from HCV-infected blood donors and selected as a subset of subtypes currently found in Europe. Each HCV-positive sample was characterized by its viral load and by its HCV genotype determined with a method based on the NS5b region sequence analysis (7). The genotype of each sample was determined by comparison (with a neighbor-joining phylogenetic tree [3]) of its sequence with the same region of a selection of 92 genomes obtained from GenBank (17 genomes of genotype 1, 30 of genotype 2, 10 of genotype 3, 25 of genotype 4, 4 of genotype 5a, and 6 of genotype 6) and selected for representing the main genotypes encountered in France. Genotypes were classified according to the nomenclature proposed by Simmonds et al. (9). The nucleic acid sequences obtained in the laboratory which characterized the panel were considered the reference sequences. The characteristics of these samples are given in Table 1.Each laboratory could use the NS5b genotyping method of its choice. However, the database of the 92 selected reference sequences was proposed to the laboratories. Twelve laboratories (A to L) participated in this study. Ten used the same primers (7). Two laboratories (J and L) used different sets of primers (2, 6). Four interpreted the results with the recommended database, while eight used their own sequence database. The result was considered exact when both the genotype and the subtype were correctly identified. An incomplete result was defined as an exact genotype result with an unidentified subtype. A correct genotype associated with an incorrect subtype was interpreted as a misclassification. The accuracy was defined as the percentage of correct results (exact genotype and subtype) among the 12 samples. The protocol stated that nucleotide sequences of all misclassified samples obtained in any laboratory should be sent to the organizing committee and compared to all sequences obtained by the other laboratories in order to define the cause of the misclassification.The accuracy ranged from 66.7% to 100% (Table 2). Four laboratories correctly identified all samples; four gave an incorrect result for a unique sample (three provided incomplete genotypes, and one misclassified one ...

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