Clinical laboratories are under increased pressure to perform HCV genotyping because of the importance of the HCV genotype as a predictor of treatment response. Nucleotide sequence analysis is the “gold standard” for the identification of different HCV types and subtypes but, in the absence of commercially available methods, was considered impractical for many clinical laboratories. Therefore, a number of surrogate typing methods were developed, including LiPA, subtype-specific RT-PCR, DNA restriction fragment length polymorphism, heteroduplex mobility analysis, melting curve analysis, and serologic genotyping. The most widely used of these surrogate typing methods in clinical laboratories is the LiPA.
We compared the HCV genotyping results obtained with a commercially available sequencing system with those obtained with the LiPA. We found a very high level of agreement (99.5%) between the results of the two methods at the genotype level, but only moderate agreement (68.2%) at the subtype level was seen when a large number of clinical specimens representing all of the major types and subtypes of HCV were used. The lack of agreement between the subtype calls is not surprising considering that both methods target the 5′NC region. The 5′NC region is among the most highly conserved regions of the viral genome, and in several cases, only one or two nucleotide changes distinguish unique subtypes (23
By using sequence analysis of the less-conserved NS5B region as the gold standard, we found that the accuracies of LiPA and TRUGENE 5′NC genotype calls were 98.6 and 98%, respectively, but were only 85.8 and 91.9% accurate, respectively, at the subtype level. Our findings are consistent with other studies that have compared genotyping results from analyses of different subgenomic regions (3
). These data suggest that clinical laboratories should not call HCV subtypes from analysis of the 5′NC regardless of the method employed because of the inherent inaccuracy of the calls. There are no recognized subtype-specific differences in disease progression or response to therapy that would warrant these designations.
Our results are similar to two previously published comparisons of the TRUGENE HCV 5′NC kit and the LiPA (2
). Both studies found a high level of concordance between the results of the two methods at the genotype level and that neither method could reliably discriminate subtypes of HCV. A third study compared the results of the TRUGENE assay with a core region microwell hybridization assay and found an overall concordance of 91% between the results of the two genotyping methods, with no genotype miscalls with the TRUGENE assay (18
). We extend and expand the observations made in these previous studies. Ours is the first study to critically evaluate the sensitivities, abilities to detected mixed genotype infections, and laboratory costs of these methods. Our study is also the first evaluation of the TRUGENE method with specimens obtained from patients in the United States. In addition, we identified two distinct HCV strains that were assigned different genotypes on the basis of sequence analysis of different subgenomic regions.
In practice, sequence analysis of a single subgenomic region is used to genotype HCV because full genome sequencing is impractical in a clinical setting. The underlying assumption is that the region analyzed is representative of the entire HCV genome. The assumption is supported by studies that have compared genotype assignments based on the sequencing of different regions of the HCV genome (10
). However, it would be challenged if recombination between different HCV genotypes in patients with coinfections occurred during replication, producing viable hybrid viruses.
A naturally occurring intergenotypic recombinant of HCV was recently identified in St. Petersburg, Russia (9
). This virus was found to belong to two different genotypes, 2 and 1, by sequence analysis of the 5′NC region and NS5B regions, respectively. The crossover point for this virus was mapped within the NS2 region. This recombinant virus now accounts for 5% of the HCV infections in St. Petersburg. Recombination may play a role in creating genetic diversity in HCV that may be important in understanding the natural history and treatment of hepatitis C.
We also found two distinct strains of HCV classified as type 2 by analysis of the 5′NC region that were clearly type 1 by analysis of the NS5B region. These sequencing results were reproducible, and little or no polymorphism at key sites within these regions was observed in the electropherograms. Consequently, sample mislabeling or mixed-genotype infections, with selective amplification of one type, are unlikely explanations for our observations. These viruses, like the St. Petersburg strains, may also be examples of hybrids that were produced by separate intertype recombination events. However, further sequence analysis and mapping of the crossover junction are required to prove that these two strains are indeed hybrid viruses. Discrepancy of HCV genotypes as determined by analysis of partial NS5 (genotype 3a) and core sequences (genotype 1a) were also reported for two Honduran HCV strains (28
). However, the low frequency of discordant results at the type level does not justify sequencing of multiple regions for routine genotype assignments in most patient populations.
We used the AMPLICOR and AMPLICOR MONITOR tests to generate the 5′NC amplicons for genotyping. These tests are widely used in clinical laboratories to detect and quantitate HCV RNA, and the amplicons from these tests can used for genotyping in both the LiPA and the TRUGENE 5′NC test. However, genotypes can be determined from specimens with lower viral loads if the qualitative HCV RNA test is used to generate the amplicons for genotyping. The qualitative RNA test is carried through more thermal cycles than the quantitative test, and as a result, more PCR product accumulates for a given viral load. Genotypes can be determined from samples with viral loads as low as 103 IU/ml when amplicons from the qualitative test are used. However, the genotyping methods will likely fail for specimens with viral loads of <105 IU/ml when amplicons from the quantitative test are used.
The reported prevalence of HCV mixed-genotype infections varies widely from 0 to more than 20% and appears to be influenced by both the patient population studied and the genotyping method employed (6
). Reliable detection of mixed-genotype infections may have broad applications in studies of epidemiology, natural history, pathogenesis, and treatment of hepatitis C. PCR amplification, followed by cloning of the PCR products and sequence analysis of the individual clones, is the only way to establish with certainty that a patient is infected with more than one genotype of HCV but is impractical in a clinical setting. We demonstrated that both the LiPA and the TRUGENE 5′NC method can detect mixtures of two different HCV genotypes. Mixtures of genotypes were recognized as long as the minority population represented at least 5% of the total virus population with the LiPA and at least 10% of the total virus population with the TRUGENE 5′NC method. Although we showed that both methods could reliably detect mixtures of different genotypes prepared in the laboratory, neither method provided convincing evidence of mixed infections in any of the clinical specimens tested.
The LiPA identified four clinical specimens that potentially contained mixtures of genotypes 1a and 1b. All four specimens shared the same unusual pattern of reactivity with the probes in the LiPA (lines 1, 2, 3, 4, 5, and 6). This pattern of probe reactivity was not one of the 58 reactivity patterns predicted by Stuyver et al. (25
) on the basis of their analysis of 448 sequences present in several large databases. Probes 5 and 6 are used to detect a subtype-defining A/G polymorphism at position −99. Sequencing of the 5′NC region demonstrated that three of these viruses had both G and A at that position. This apparent G/A polymorphism could result from natural genome divergence during replication (quasispecies), base misincorporation during RT-PCR, or mixed-genotype infection. Chen and Weck (3
) recently provided evidence that the A/G polymorphism at position −99 cannot differentiate subtype 1a from subtype 1b. We found no evidence of mixed infection when the NS5B region was sequenced, and all viruses were subtyped as 1a, with no polymorphisms at key sites in this region. Furthermore, a recent update of the LiPA interpretation chart issued by the manufacturer after completion of this study identifies specimens with this pattern of reactivity as genotype 1 without any subtype designation.
In our laboratory, we estimated the direct costs per test (reagents and labor) of the LiPA and the TRUGENE 5′NC sequencing method for HCV genotyping to be comparable at approximately $100. These costs do not include the capital equipment costs, which are substantially greater for the sequencing method. However, in laboratories already using the TRUGENE HIV-1 genotyping assay, these costs could be avoided because the equipment required for HCV genotyping is the same. The Centers for Medicare and Medicaid Services recently decided to reimburse laboratories that bill under the HCV genotyping CPT code (87902) at the same rate as human immunodeficiency virus type 1 resistance genotyping (87901). In most regions of the United States, the Medicare reimbursement for CPT code 87901 is sufficient to cover the costs associated with HCV genotyping methods.
The two methods provide reliable HCV genotyping results at the type level and have similar analytical sensitivities and abilities to recognize mixed infections. The TRUGENE 5′NC method is more technically complex to perform than LiPA, but the more detailed information provided by the direct sequencing method could prove valuable in the detection of new viral types or in epidemiological investigations. In addition, the TRUGENE 5′NC system can be easily updated as new HCV sequence information becomes available and the platform offers users the option of generating sequence information from other regions of the HCV genome.