Nucleotide sequence analysis is the current “gold standard” for identifying different HCV genotypes and subtypes but is generally regarded as not practical for routine clinical laboratory settings (8
). Therefore, a variety of surrogate HCV typing procedures has been proposed during recent years, mainly based upon amplification of the viral sequences by PCR, using either type-specific primers (15
), analysis of PCR products by hybridization with genotype-specific probes (21
), or restriction fragment length polymorphism (1
). The underlying assumption of all these assays that the region (e.g., 5′NCR, core, or NS5B) analyzed is representative of the whole HCV genome is generally supported by the very consistent typing results which have been obtained so far using assays based on sequence analysis of different regions of the HCV genome (8
In this study, we have combined diagnostic HCV RNA detection with the widely distributed Roche AMPLICOR HCV assay and direct sequencing of the already available HCV 5′NCR PCR product by a newly developed commercial system (26
; TRUGENE HCV 5′NC
genotyping kit manual). Following this approach, HCV genotyping including analysis of the sequence data was possible in about 5 h. Genotyping by DEIA, besides requiring an additional nested PCR with primers from the HCV core region, requires an overnight incubation with the biotinylated hybridization probes and, therefore, cannot be accomplished in less than 15 h. Comparison of the results obtained by the TRUGENE HCV 5′NC
genotyping kit with those of the already well-established DEIA (24
) indicated an identical HCV subtype assignment in 91% of all cases. Direct sequencing of the 183-bp 5′NCR fragment in our study, however, was not able to completely resolve all existing HCV genotypes and subtypes. This failure in three cases was attributable to an inadequate subtype assignment by the current version of the GeneLibrarian module, as shown by phylogenetic analysis of the obtained 5′NCR sequences. Possibilities to improve the overall performance of the TRUGENE system might, therefore, comprise the inclusion of more HCV 5′NCR sequences in the GeneLibrarian database, the modification of the general algorithm used for sequence analysis, and the search for modes of HCV subtype assignment other than by a simple alignment of sequences. The remaining discrepancies recorded in this study between TRUGENE HCV 5′NC
genotyping kit and DEIA could not be resolved by a more-sophisticated sequence analysis system and are the result of the high sequence conservation of HCV 5′NCR (13
). For instance, subtypes 1a and 1b in the 5′NCR fragment analyzed by TRUGENE HCV 5′NC
genotyping kit differ in only 1 nt in position 99, and subtype 2b only differs from 2a by 2 nt at positions 124 and 164. A reliable discrimination of subtypes 2a and 2c isolates is also not possible based upon sequence analysis of the 5′NCR alone (20
). Consequently, failures in correct assignment of all HCV subtypes similar to those observed in this study had already been demonstrated in several comparative evaluations of HCV genotyping procedures based solely on the analysis of the 5′NCR (3
), indicating that less-conserved parts of the HCV genome, such as the core (used by DEIA) or NS5B, are more suited for the identification of all existing HCV subtypes.
These inherent disadvantages of the 5′NCR for HCV typing are, however, not crucial for clinical purposes. From the point of view of a clinician, currently it is not necessary to know precisely the subtype of the HCV strain present but to achieve a reliable discrimination between HCV genotypes 1 and non-1 types. According to the most-recent therapeutic recommendations, highly viremic patients chronically infected with HCV genotype 1 isolates should be treated with alpha-interferon and ribavirin for at least 1 year, whereas for infections with non-1 types an initial course of 6 months is sufficient (6
). Since with the TRUGENE HCV 5′NC
genotyping kit no misclassifications between HCV genotypes 1 and non-1 types occurred, the typing results obtained with this system are, in general, reliable for clinical practice.
Taken together, the newly developed TRUGENE HCV 5′NC genotyping kit and the MicroGene Clipper sequencing platform turned out to be a convenient analytical system that provides clinically valid HCV typing results in about 5 h. The assay might be an attractive option for HCV genotyping in laboratories that already use the Roche AMPLICOR HCV test for diagnostic RT-PCR.