A variety of laboratory-developed and commercial assays are used for HCV genotyping. The methods include nucleic acid sequencing, reverse hybridization, subtype-specific PCR, DNA fragment length polymorphism, heteroduplex mobility analysis, melting curve analysis and serological genotyping. The FDA has not cleared any of these methods for clinical diagnostic use.
A commercially available reverse hybridization line probe assay is the most commonly used method for geno-typing HCV among clinical laboratories participating in the HCV proficiency-testing surveys of the College of American Pathologists. This reverse hybridization assay was developed by Innogenetics to genotype HCV and is now marketed as the Versant HCV Genotype Assay by Bayer. In this line probe assay (LiPA), biotinylated PCR products from the 5' UTR are hybridized under stringent conditions with 19 type- and subtype-specific oligonucleotide probes attached to a nitrocellulose strip. Hybridized PCR products are detected with a streptavidin-alkaline phosphatase conjugate. The second-generation assay discriminates among genotypes 1a, 1b, 2a/c, 2b, 3a, 3b, 3c, 4a-h, 5a, and 6a.77 The results from the Versant HCV Genotype LiPA Assay correlate well with results obtained by direct sequencing assays of the 5' UTR and other genes in published evaluations, but may not distinguish between genotypes 1a and 1b in 5% to 10% of cases, and does not distinguish between genotypes 2a and 2c.32,59,78,79 The LiPA is the most common method used in clinical laboratories for HCV genotyping because it can be used with amplicons from both the qualitative and quantitative Amplicor HCV tests, and is easy to perform and interpret. Mixed genotype infections are easily recognized as unusual patterns of hybridization with the typing probes. However, the LiPA requires a considerable amount of ampli-con for typing, and the assay may regularly fail when the viral load is less than 104 copies/ml.
Sequence analysis of amplified subgenomic sequences is the most definitive way to genotype HCV strains. Genotyping schemes based on sequencing variable genes such as E1,C,and NS5B provide enough resolution to determine types and subtypes.26-28 The 5' UTR is too highly conserved to discriminate all subtypes reliably.59 Genotyping methods targeting highly variable regions have higher failure rates due to primer mismatches and failed amplification reactions. Sequencing reactions can be performed directly on PCR products or on cloned amplicons. Mixed infections with multiple genotypes may be missed with direct sequence analysis. Definitive detection of mixed infections requires analysis of a large number of clones. Cloning may, however, emphasize artifactual nucleotide substitutions introduced by the DNA polymerase during amplification or by selection during the cloning procedure,80 and is generally not practical for the clinical laboratory.
A standardized direct sequencing system has been recently developed for clinical use by Visible Genetics (Suwanee, GA) and now marketed by Bayer. The Trugene HCV 5'NC genotyping kit targets the 5' UTR (nt 96 to 282) and employs proprietary single-tube chemistry that is robust and highly sensitive. This method can be used with the 244 bp amplicon generated by either the Roche Amplicor HCV or Amplicor HCV Monitor tests as the sequencing template after a column purification step.81 The sequencing chemistry produces bidirectional sequences. The software acquires the sequence data in real time, and each pair of forward and reverse sequences is combined. A reference sequence library module contains approximately 200 sequences from the six major genotypes and 24 subtypes of HCV. The software automatically aligns the patient HCV sequence with the reference sequences in the library and reports type, subtype, and closest isolate determinations. The Trugene HCV 5'NC genotyping system is a rapid and reliable method for determining HCV genotypes but, like all approaches targeting the conserved 5' UTR, cannot reliably distinguish all HCV subtypes.36,48,81,82
The practice of using sequence analysis of a single subgenomic region for HCV genotyping has recently been challenged by the description of a naturally occurring intergenotypic recombinant of two HCV genotypes.83 This virus was found in patients in St Petersburg, Russia, and was assigned to two different genotypes, 2 and 1, by sequence analysis of the 5' UTR and the NS5B region, respectively. The crossover point for the genome was mapped within the NS2 region. The extent to which such viruses occur in other patient populations is largely unknown.
A DNA EIA (Sorin Biomedica, Saluggia, Italy) for HCV genotyping is based on hybridization of denatured ampli-con from the core region to genotype-specific probes that are bound to the wells of a microtiter plate. Mouse monoclonal antibodies to double-stranded DNA are used to detect the hybrids. The results of the DNA EIA were highly concordant with the results of other genotyping methods in two evaluations.79,81
A variety of lab oratory-developed methods have been used to genotype HCV, including subtype-specific PCR,25 primer-specific and mispair extension analysis,84 nested restriction site-specific PCR,85 restriction fragment length polymorphism,86 heteroduplex mobility analysis,87 and melting-curve analysis with fluorescence resonance energy transfer probes.88,89
Genotype-specific antibodies directed against the immunodominant epitopes in NS4 have been used to develop serotyping or serological genotyping tests. Two serological genotyping tests are commercially available. An NS4 recombinant immunoblot assay (Chiron Corporation, Emeryville, CA) uses synthetic proteins from the NS4 and core regions to discriminate among HCV genotypes 1, 2, and 3.90 The competition enzyme-linked immunoassay (Murex Diagnostics Ltd, Dartford, UK) uses eight branched synthetic peptides to detect genotype-specific anti-NS4 antibodies to discriminate among HCV genotypes 1 to 6.14 The high degree of cross reactivity among genotypes to these synthetic peptides necessitates absorbing the cross-reacting antibodies with an excess of heterologous pep-tides in solution prior to use. Both of these serologic assays lack sensitivity and specificity as compared with direct sequencing or the Versant HCV Genotype LiPA Assay for HCV genotyping; however serological genotyping is inexpensive, is simple to perform, and lends itself well to large epidemiological studies. It is the only way to determine the genotype of a virus in patients with low-level viremia or who have cleared their infection, as well as for specimens in which the RNA has been destroyed by improper handling.
Was this article helpful?