Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis

Abstract

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixedgenotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5 untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n ‫؍‬ 312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n ‫؍‬ 190), 12.8% type 2 (n ‫؍‬ 40), 20.2% type 3 (n ‫؍‬ 63), 0.3% type 4 (n ‫؍‬ 1), and 0.3% other (n ‫؍‬ 1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.Infection by hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide (19). The overall prevalence of HCV infection in the United States is 1.8%, with most of the patients unaware of their infection and at risk for developing cirrhosis and hepatocellular carcinoma (15,21). HCV is a positive-sense, single-stranded RNA virus that displays extensive genetic heterogeneity (1). At least six major HCV genotypes comprising numerous, more closely related subtypes have been identified (26). HCV genotypes display significant differences in their global distribution and prevalence, making genotyping a useful method for determining the source of HCV transmission in an infected localized population (11). Furthermore, in addition to viral load and liver histology, the genotype of the infecting HCV strain appears to be an important determinant of the severity and aggressiveness of liver infection, as well as patient response to antiviral therapy (26). Consequently, several methods for genotyping HCV have been developed, including direct DNA sequencing (2, 5, 6), typespecific PCR (17), restriction fragment length polymorphism (16), line probe assays (22, 23), primer-specific and mispair extension analysis (7,8), heteroduplex mobility analysis by temperature gradient capillary electrophoresis (14)...

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

“…The latter hypothesis is very unlikely given that they display very high divergence rates, and this would imply that these sequences converged toward an existing genotype. In the multiple-infection hypothesis, the competition between two genotypes would necessarily lead to the extinction of the less fit one over time, which is in accordance with the observation that cases of mixed infection in serum are very rare (40,41). Therefore, the use of lymphocytes as hidden reservoirs could be a means for the less fit viruses to maintain their population and persist within an independent reservoir in a chronically infected patient (Fig.…”

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

“…For both reverse transcription and PCR reactions, 5′UTR universal primers: forward KY80 (5-GCA GAA AGC GTC TAG CCA TGG CGT-3) and reverse KY78 (5-CTC GCA AGC ACC CTA TCA GGC AGT-3) were used [34]. HCV PCR products (nucleotides [nt] 9 to 252; 244 bp) were tested on 1.5% agarose gel electrophoresis.…”

Computer-aided identification of polymorphism sets diagnostic for groups of bacterial and viral genetic variants

“…Current HCV genotyping methods such as the line probe assay (INNO-LiPA) [33], the real-time PCR-based Abbott HCV analyte-specific reagent (ASR) and COBAS TaqMan48 HCV tests [34,35], restriction fragment length polymorphism analysis [36] and primer extension methods [37] primarily target the 5' non-coding region (5'-NCR). A drawback of targeting the 5'-NCR is that some subtypes, such as 1a and 1b, 1b and 6a, or 2a and 2c, remain indistinguishable in a small number of cases due to the conserved nature of this region [38,39].…”