Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Clinical evaluation of a newly developed automated massively parallel sequencing assay for hepatitis C virus genotyping and detection of resistance-association variants. Comparison with a line probe assay.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. Recently, HCV was classified into 6 major genotypes (GTs) and 67 subtypes (STs). Efficient genotyping has become an essential tool for prognosis and indicating suitable treatment, prior to starting therapy in all HCV-infected individuals. The widely used genotyping assays have limitation with regard to genotype accuracy. This study was a comparative evaluation of exact HCV genotyping in a newly developed automated-massively parallel sequencing (MPS) system, versus the established Line probe assay 2.0 (LiPA), substantiated by Sanger sequencing, using 120 previously identified-HCV RNA positive specimens. LiPA gave identical genotypes in the majority of samples tested with MPS. However, as much as 25% of LiPA did not identify subtypes, whereas MPS did, and 0.83% of results were incompatible. Interestingly, only MPS could identify mixed infections in the remaining cases (1.67%). In addition, MPS could detect Resistance-Associated Variants (RAVs) simultaneously in GT1 in 56.82% of the specimens, which were known to affect drug resistance in the HCV NS3/NS4A and NS5A genomic regions. MPS can thus be deemed beneficial for guiding decisions on HCV therapy and saving costs in the long term when compared to traditional methods.

6 HCV genotyping 9G test and its comparison with VERSANT HCV genotype 2.0 assay (LiPA) for the hepatitis C virus genotyping.

In this article, we describe the 6 HCV Genotyping 9G test and its evaluation by using clinical samples and plasmid DNA standards. In tests with 981 plasmid DNA standards, the 6 HCV Genotyping 9G test showed higher than 92.5% sensitivity and 99.4% specificity. The 6 HCV Genotyping 9G test was compared with the VERSANT HCV Genotype 2.0 assay (LiPA 2.0) for detection and discrimination of HCV genotypes in clinical samples. The results of both tests were verified by genomic sequencing. The 6 HCV Genotyping 9G test demonstrated a 100% agreement with the sequencing results, which was higher than LiPA 2.0. These results indicate that the 6 HCV Genotyping 9G test can be a reliable, sensitive, and accurate diagnostic tool for the correct identification of HCV genotypes in clinical specimens. 6 HCV Genotyping 9G test can genotype six HCV types in 1 PCR in 30min after PCR amplification. The 6 HCV Genotyping 9G test, thus provide critical information to physicians and assist them to apply accurate drug regimen for the effective hepatitis C treatment.

6 HCV Genotyping 9G test for HCV 1a, 1b, 2, 3, 4 and 6 (6a, 6f, 6i and 6n) with high accuracy.

According to EASL guidelines and WHO recommendations, the accurate detection of HCV genotypes such as HCV 1a, HCV1b, HCV 2, HCV 3, HCV 4, and HCV 6 (6a, 6f, 6i, 6n) is crucial for the efficient treatment of hepatitis C. HCV Genotyping 9G test allows simultaneous genotyping of HCV 1a, 1b, 2, 3, 4, and 6 (6a, 6f, 6i, and 6n) in clinical samples in 30min. The performance of the test was evaluated by comparison with sequence analysis. Serum samples (n=152) from HCV-infected patients (n=110) and healthy individuals (n=42) were processed under blinded codes. The k coefficient (kappa) values indicated high agreement between the HCV Genotyping 9G test and sequencing. The sensitivity and specificity of the test were 99.1% and 99.7%, respectively. The results indicate that HCV Genotyping 9G test is rapid, reliable, sensitive, and accurate for screening and genotyping of HCV in the clinical specimens.

HBV/4DR 9G test and its comparison with INNO-LiPA HBV multi-DR test for the detection of drug-resistant Hepatitis B virus.

A significant proportion of patients with chronic Hepatitis B infection require antiviral therapy during their life time. The Antiviral therapy with lamivudine or adefovir or telbivudine has shown to be a major risk factor for selection of resistance. Eighty percent of patients showed a development of lamivudine-resistant strains after five years of treatment with lamivudine alone. Adefovir and telbivudine inhibit HBV with very high efficacy and have moderate incidences of drug resistance. Entecavir and tenofovir have been shown to have a higher barrier to resistance with rates of less than 1.5% after five years of treatment. The rtA181V, rtM204V/I, rtN236T and, rtM250V are high prevalent mutations found in the drug-resistant HBV strains. Therefore, for accurate treatment of HBV-infected patients, it is important to discriminate the drug-resistant HBV strains by using simple and accurate detection method. In this study, we describe the HBV/4DR 9G test and its evaluation by using clinical samples and plasmid DNA standards with a range of HBV mutation sites. In tests with 384 plasmid DNA standards, the HBV/4DR 9G test showed higher than 95% sensitivity and 98% specificity. The HBV/4DR 9G test was compared with the INNO-LiPA HBV Multi DR test for detection of drug-resistant HBV strains only in clinical samples. The plasma samples were collected from patients suspected with HBV drug-resistant strain infection. The results of both tests were cross-checked with the HBV DNA sequence analysis. The HBV/4DR 9G test demonstrated a good agreement with the sequencing results as compared to the INNO-LiPA HBV Multi-DR test. These results indicate that the HBV/4DR 9G test can be a reliable, sensitive, and accurate diagnostic tool for the detection of drug-resistant genotypes of HBV in clinical specimens. HBV/4DR 9G test can genotype 4 drug resistant HBV strains in 1 PCR. The HBV/4DR 9G test will help to minimize the risk of HBV patients from liver cancer.