Multicenter clinical evaluation of the new 3rd generation assay for detection of antibodies against hepatitis C virus on the VIDAS(®) system.

BACKGROUND: Detection of antibodies (anti-HCV) against hepatitis C virus (HCV) is indispensable for screening and diagnosis of viral hepatitis and for the viral safety of blood, tissue or organ donations. It gains additional importance by the new HCV drugs which improve the therapeutic possibilities dramatically. OBJECTIVE: To evaluate the performance of a newly developed immune assay for anti-HCV based on the well-established VIDAS platform. STUDY DESIGN: The assay was evaluated with samples from anti-HCV negative blood donors and from patients with or without HCV markers in six centres in France, Spain and Egypt. The status of the samples was determined by using CE-marked immune assays (Architect, AxSym, Prism, Vitros), two immunoblots (RIBA, Inno-Lia) and/or HCV RNA results. RESULTS: Specificity was 99.67% in 10,320 French blood donors without anti-HCV, 99.5% in 200 anti-HCV negative hospitalized European patients and 99.0% in 198 negative patients from Egypt. Sensitivity was 99.7% in 1054 patients pretested positive by other assays; 345 patients with known genotype had genotype 1-6; 61 patients were co-infected with HIV. VIDAS was reactive in 78% of 91 patients with uncertain or very weak anti-HCV. It became on average positive at day 37 with seroconversion panels. CONCLUSIONS: This multicentric, international study with >12,000 samples show that the new VIDAS anti-HCV assay is very suitable for screening and confirmation of HCV infection. Sensitivity, specificity and recognition of seroconversion compare favorably with well-established CE-marked tests and help to clarify discrepant results obtained with other assays.

Animal models for the study of HBV infection and the evaluation of new anti-HBV strategies.

BACKGROUND: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), in study models of HBV infection. METHOD: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models. RESULTS: beta-L-Fd4C-TP exhibited a more potent inhibitory effect on the RT activity of the DHBV polymerase than other cytidine analogs (lamivudine-TP, ddC-TP, beta-L-FddC-TP). In primary duck hepatocyte cultures, beta-L-Fd4C exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral cccDNA. In vivo treatment with beta-L-Fd4C in infected ducklings and woodchucks, induced a greater suppression of viremia and intrahepatic viral DNA synthesis than with lamivudine. However, covalently closed circular DNA persistence explained the relapse of viral replication after treatment withdrawal. Viral spread was strongly reduced in the case of early therapeutical intervention, but the number of infected cells did not decline when therapy was started during chronic infection. Liver histology analysis showed a decrease in the inflammatory activity of chronic hepatitis while no ultrastructural modification of liver cells was observed in electron microscopy studies. Furthermore, in human primary hepatocyte cultures, beta-L-Fd4C induced a significant inhibition of HBV DNA synthesis. CONCLUSION: beta-L-Fd4C is a potent inhibitor of hepadnavirus RT and inhibits viral DNA synthesis in hepatocytes both in vitro and in vivo. These experimental studies allowed as to show that beta-L-Fd4C is a promising anti-HBV agent. Combination therapy should be evaluated to eradicate viral infection.

Duck hepatitis B virus polymerase gene mutants associated with resistance to lamivudine have a decreased replication capacity in vitro and in vivo.

BACKGROUND/AIMS: Hepatitis B virus mutants of the polymerase gene are frequently selected during lamivudine therapy for chronic hepatitis B. To study the biology of these mutants, we analyzed their replication capacity in the duck hepatitis B virus (DHBV) infection. METHODS: The B and C domain polymerase mutants corresponding to the clinical isolates were engineered by site directed mutagenesis in the DHBV genome in different expression vectors. RESULTS: The study of the enzymatic activity of the mutated viral polymerase polypeptides analyzed in a cell free system demonstrated a lower priming activity and a decreased capacity of elongation of viral minus strand DNA that was consistent with the lower replication capacity of these mutants in transfected leghorn male hepatoma cells compared to wild type genome. These mutants had a lower replication capacity in primary hepatocytes and in in vivo transfected ducklings. Although resistant to lamivudine, these mutants remained sensitive to PMEA. CONCLUSION: YMDD mutants of the DHBV reverse transcriptase have a decreased replication capacity both in vitro and in vivo, and are not cross-resistant to PMEA. These results may be important to design new antiviral strategies to combat the replication of the lamivudine resistant viral strains.

Evolution of hepatitis B virus polymerase gene sequence during famciclovir therapy for chronic hepatitis B.

Prolonged administration of nucleoside analogues for chronic hepatitis B may result in the emergence of hepatitis B viral polymerase mutants. To gain insight into the mechanism involved in the virus's resistance to famciclovir, the amino acid sequences of the terminal protein and reverse-transcriptase (RT) domains of the viral polymerase were determined during therapy among 28 patients. The antiviral response was independent of viral genotypes, and nonresponse to famciclovir was associated with a complex variability of the RT domain. No mutation in the YMDD motif was observed, whereas an L528M mutation was clearly selected by famciclovir treatment in 2 patients, as well as 14 novel mutations in 7 patients. Clone sequence analysis of the RT domains of patients undergoing retreatment with famciclovir and/or lamivudine showed the selection of a preexisting drug-resistant mutant in one case and indicated that sequential antiviral therapy may allow the rapid selection of resistant strains.

Transient selection of a hepatitis B virus polymerase gene mutant associated with a decreased replication capacity and famciclovir resistance.

Prolonged therapy for chronic hepatitis B (HBV) with nucleoside analogs may result in the emergence of HBV mutants resistant to antivirals. Here, we describe the transient selection of an HBV polymerase gene mutant that was associated with viral persistence in an immune competent patient treated with famciclovir. Viral polymerase gene sequence was analyzed directly on polymerase chain reaction (PCR) products and also after cloning. The results showed the transient selection of a V542I mutant in the C domain of the viral polymerase. This mutation was associated with a stop codon at amino acid position 199 in the overlapping S gene. The mutated sequence was subcloned in a vector expressing the entire HBV pregenome to study its replication capacity after transient transfection in cultured hepatoma cells. The results showed that the V542I mutant has a decreased replication capacity compared with wild type virus and does not produce HBsAg. The sensitivity of the V542I mutant to penciclovir, the active metabolite of famciclovir, was further studied in tissue culture. This mutant was shown to be resistant to penciclovir, but remained sensitive to lamivudine, as was subsequently observed in vivo. These findings indicate that a prolonged administration of famciclovir may allow for the selection of HBV polymerase gene mutants in immune competent patients. The impaired replication capacity of this V542I mutant may have contributed to the absence of outgrowth of this viral strain in vivo. The study of the in vitro sensitivity of HBV polymerase mutants to nucleoside analogs will be important to design new anti-HBV strategies.