Our experience with direct sequence analysis of the Amplicor HCV PCR product indicates that this approach is an efficient means of genotyping HCV. It does not require an additional specimen-processing step and utilizes products obtained from a single, nonnested amplification reaction, thus eliminating the delays and expense involved in performing additional amplifications. In addition, the direct sequencing of amplification products provides more detailed sequence information than genotyping assays based upon either hybridization or restriction analysis. This additional information could prove to be quite useful in the detection of novel viral types, several of which may have been already observed in this and other studies (31
Using this method, we were able to successfully sequence and classify 89.4% of those 416 specimens demonstrating a positive HCV RNA test result. Of those 44 of 416 (10.6%) HCV RNA-positive specimens which could not be classified based on the 5′UTR, the majority corresponded to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2.0 assay (Fig. ). Despite the fact that there were detectable levels of amplification products found in these reactions, these results suggest the lack of sufficient quantities of amplified material as the primary cause of direct sequencing failure. In turn, the most likely explanation for lack of amplification products is limited quantities of viral RNA present in the original specimen. We could have performed nested amplification to generate sufficient product for sequencing, but this procedure would add delays, expense, and the risk of carryover contamination to the procedure (24
). Ultimately, we decided against further amplification, since the sensitivity of the procedure already exceeded that of the NS-5 sequencing protocol (see below).
We attempted to minimize the occurrence of failed or uninterpretable sequencing reactions through the use of the same amplification target for both detection and genotyping. Compared to the current 5′UTR protocol, NS-5 amplification, analysis, and sequencing demonstrated a high failure rate. Failure rates for NS-5 amplification and genotyping were found to be 26.5% higher than those established for the 5′UTR, a total of 58 of 219 specimens which were tested by both methods. This discrepancy is likely due in large part to a reduction in amplification efficiency resulting from differential primer binding efficiencies. Although the primers utilized in the NS-5 amplifications are thought to bind highly conserved sequences (9
), primer-target mismatching within this region is still likely because of inherently high sequence variability. In our experience, PCR amplifications targeting the 5′UTR are consistently more likely to detect HCV, consistent with the data presented in this study. Viral quantitation data also suggest a correlation between low viral titer and failure to amplify or generate usable sequence information from the NS-5 region (data not shown).
Disadvantages of genotyping schemes based on the 5′UTR may also ironically be related to its high sequence conservation. Although amplification and genotyping of HCV based upon the 5′UTR appears to be quite sensitive and efficient, the higher level of conservation found within this region can make discrimination of all genotypes and subtypes difficult (25
). In fact, genotypes 7, 8, and 9 cannot reliably be distinguished from genotype 1 based upon the 5′UTR and would therefore be misclassified as genotype 1 if this region alone was used for classification (31
). In other situations, subtype distinctions cannot be made; such is the case between subtypes 2a and 2c. There are also examples in which only one or two minor nucleotide changes distinguish unique subtypes. An example of this situation is illustrated by the minor differences seen in the 5′UTR sequences of subtypes 1a and 1b. A single base change at position −99 (A/G) is the only change within the 5′UTR differentiating these two subtypes, thus leading to poor statistical support for their discrimination by phylogenetic analysis of this region alone (data not shown). In addition, based on analysis of other regions, it has been shown that this nucleotide difference is not absolute, and interpretations based upon this nucleotide position (−99) can lead to errors in the determination of viral subtype (25
). The occurrence of both type 1a and 1b variants was supported by the results of this study. We found that 3 of 87 (3.4%) of the subtype 1a specimens were actually subtyped as 1b by NS-5 sequence analysis, while 12 of 48 (25%) of the subtype 1b specimens were shown to contain subtype 1a sequences by NS-5 analysis. Another possible explanation for these results could have been preferential amplification of different subtypes from a mixed population. However, the clarity of the NS-5 sequencing data obtained from these samples suggests the presence of single viral subtypes (data not shown). Finally, although other studies have suggested that viral recombination appears to be a rare occurrence in HCV (4
), further sequencing studies of these isolates could provide evidence for recombination in these specimens.
Despite the inability to completely resolve all existing HCV genotypes and subtypes, the use of this 5′UTR genotyping method provided a sensitive and efficient means of HCV genotyping in a clinical setting, especially in light of studies which show that the clinically relevant distinction is between genotype 1 and non-type 1 (7
). The use of a commercially available 5′UTR assay designed for the detection of HCV in clinical specimens provides the user with a sensitive, standardized amplification protocol specifically designed for large-volume testing and rapid turnaround time. In addition to these features, this method utilizes uracil N
-glycosylase as an amplicon carryover prevention method. Together with the use of dedicated work areas and a nested sequencing primer, this method provides a reliable means of carryover control in a high-volume testing facility and still provides an acceptable template for direct sequence analysis. For these reasons, in addition to those mentioned above, we have adopted the 5′UTR direct sequencing approach to HCV genotyping as our routine diagnostic procedure.