title: Evaluation of a "home-made" method to determine viral genotype and characterize mutations conferring drug resistance in chronic hepatitis B patients.
abstract: We compared a home-made sequencing system to analyze plasma samples from patients with chronic HBV infection with the commercial TRUGENE(®) HBV Genotyping Assay. A PCR and sequencing protocol based on published primers was applied to detect the viral genotypes as well as the major patterns of point mutations leading to resistance to lamivudine, adefovir and entecavir. For the determination of HBV genotypes the obtained sequences were aligned with a database created within the RIDOM TraceEdit program and publicly available reference sequences. Our results showed perfect correlation with the commercial system, with types D (72%) and A (22%) being the most frequent genotypes. The resistance loci were also reliably detected with mostly combined L180M and M204V/I mutations as the local patterns. M204I mutations were more frequent in genotype D, M204V in genotype A isolates. G173L mutations were not found. The only genotype C isolate tested revealed a different pattern (E263D and I269L). These data speak for the usability of this rapid amplification and sequencing approach for routine genotyping of HBV isolates and simultaneous determination of the drug resistance profile of the dominant viral species.
authors: Laboratorio Aziendale di Microbiologia e Virologia, Azienda Sanitaria dell'Alto Adige, Bolzano, Italy.
authors: Elisabetta
authors: E
authors: Pagani
authors: Valentina
authors: V
authors: Pasquetto
authors: Chiara
authors: C
authors: Cemin
authors: Patrizia
authors: P
authors: Rossi
authors: Marina
authors: M
authors: Crovatto
authors: Graziella
authors: G
authors: Gava
authors: Martina
authors: M
authors: Felder
authors: Hartwig P
authors: HP
authors: Huemer
authors: Clara
authors: C
authors: Larcher
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D024882:Drug Resistance, Viral / Q000235:genetics*; D005814:Genes, Viral; D005838:Genotype; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000821:virology*; D006801:Humans; D007558:Italy; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D016037:Single-Blind Method; D014766:Viremia / Q000097:blood / Q000821:virology*; D014773:Virology / Q000379:methods*
chemical_list: D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic