The gold standard for HCV genotyping remains PCR amplification followed by sequencing of one of the phylogenetically informative coding regions of the HCV genome, such as NS5B or core/E1, and comparison with the consensus sequences in GenBank or the Los Alamos hepatitis C virus databases (14
). We have developed a novel microarray-based assay for identifying the HCV genotype and subtype and evaluated it in comparison with the phylogenetic analysis of the NS5B region as a reference method. The new NS5B microarray assay and NS5B sequencing were in almost complete agreement.
The assay relies on hybridization of a 380-nt NS5B fragment with oligonucleotides specific for HCV genotypes and subtypes immobilized on a biochip. The reliable identification of each individual genotype and subtype required the design of several oligonucleotides for each of them, in consequence of the variability of the NS5B region. The results were interpreted using an original algorithm that included preliminary processing of the hybridization signal intensities from the biochip elements and comparison of signals from elements within the sets of genotype-specific probes and then from sets of subtype-specific probes.
The new method enabled us to determine all six HCV genotypes with a sensitivity of approximately 2.0 × 102
IU/ml of HCV RNA. This analytical performance using biochip-based genotyping and subtyping is comparable to that of commercially available assays (50
), including the new generation of line probe assays (49
The new method was tested on 345 HCV-positive samples. The results were 100% concordant for the genotype and 99.7% concordant for the subtype with the results obtained by direct sequencing of the NS5B segment. The accuracy and reliability of the assay make it suitable for large-scale genotyping and subtyping projects.
Hybridization on the biochip correctly identified HCV isolates of subtypes 1a, 1b, 1e, 2a, 2b, 2c, 2i, 2k, 3a, 4a, 4c, 4d, 4f, 4k, 4p, 4r, and 5a. It failed to identify subtypes 1d, 2j, 2l, and 4h. This could be because there are fewer of these NS5B sequences in GenBank and other databases, which resulted in less accurate selection of subtype-specific probes. However, these subtypes are very infrequent in Europe—2.9% for 2l, 0.9% for 2j, and 1% for 4h (30
). However, the hybridization on the microarray and NS5B sequencing were in 100% agreement for identifying the most widespread and clinically relevant subtypes, such as 1a, 1b, 4a, 4d, and 3a. The only limitation of the study is that not many samples of HCV genotype 6 were tested because this is very rare in France.
No mixed infections were encountered during the evaluation. Testing the analytical mixed samples revealed that the method is able to detect two different genotypes within the sample if the concentration of the minor genotype constitutes 20% or more of the total HCV RNAs.
Some recent studies have shown that HCV subtypes can predict the response to standard treatment regimens that include pegylated interferon and ribavirin. One French multicenter study of 597 treated patients showed that subtypes 1b, 4a, and 4d were independent predictors of SVR (16
). A recent study also demonstrated that patients infected with HCV subtype 1b had a higher antiviral response than did patients infected with HCV subtype 1a (29
). Another study of 1,532 patients infected with HCV genotype 4 showed that subtype 4a was more sensitive to anti-HCV treatment than was subtype 4d (36
). Moreover, the development of new specific inhibitors of HCV enzymes whose antiviral responses and resistance profiles may be determined by the HCV subtype may require identification of the subtype prior to treatment (7
). Several HCV inhibitors appear to act selectively against certain HCV genotype 1 subtypes, both in vitro
and in vivo
. Differences in the activities of NS3/4A protease inhibitors (telaprevir and boceprevir) against different subtypes have been reported. There is evidence that the selection of resistant variants and virus breakthrough is more frequent in patients infected with subtype 1b than in those harboring subtype 1a (13
). The antiviral activities of nucleoside analogs of polymerase inhibitors are similar regardless of the HCV subtype, while nonnucleoside inhibitors are more active against subtype 1b than against subtype 1a (18
). These findings suggest that the antiviral activity of new anti-HCV agents may also vary with the subtypes of genotypes other than 1. It is therefore essential to accurately discriminate between subtypes in order to tailor anti-HCV treatment schedules with HCV protease and polymerase inhibitors. There are few methods presently available other than direct sequencing of NS5B and core/E1 segments for identifying numerous subtypes. One of the commercially available methods, the INNO-LiPA v.2, discriminates better between subtypes 1a and 1b than does the previous version, INNO-LiPA v.1, but does not discriminate between subtypes 4a, 4c, and 4d (4
). Our new method correctly identified HCV subtypes 1a and 1b in more than 99% of samples; it also identified subtypes 4a and 4d.
All the experimental microarray-based methods for HCV genotyping use immobilized oligonucleotides from the 5′ untranslated region of the HCV genome. They can therefore identify only a small number of subtypes (1a, 1b, 2a/2b/2c, 3a, 3b, and 6a), although their determination of genotypes is reported to be almost 100% (6
). In this work, use of probes complementary to subtype-specific sequences of the NS5B region enabled us to identify more than 20 HCV subtypes in the specimens tested. Other methods, such as real-time PCR, can identify a limited number of subtypes and genotypes (1a, 1b, 2a, 2b, 2c, 3, 4, 5, and 6) (23
). Only the clip sequencing method can, in theory, discriminate as many subtypes as can our procedure (35
In conclusion, this new approach to analyzing the NS5B region of HCV based on hybridization with a low-density microarray is a promising tool for rapidly, sensitively, and accurately identifying viral genotype and subtype. It provides clinicians with the information needed for the choice of a correct individual treatment of hepatitis C. In addition, the performance of the new procedure and the range of identifiable genotypes and subtypes make it suitable for epidemiological surveys.