title: Assessment of selective real-time PCR for quantitation of lamivudine and adefovir hepatitis B virus-resistant strains and comparison with direct sequencing and line probe assays.
abstract: A selective real-time PCR (sPCR) assay has been developed to detect the rtM204V/I and rtN236T mutations of hepatitis B virus (HBV) associated with resistance to lamivudine and adefovir. Using mixtures of mutant and wild-type plasmids, this sPCR was able to detect 0.1% of mutated strain in a total plasmid population of 10(5) copies and was more sensitive in detecting resistant strains than the line probe INNO-LiPA-DR-v2 assay and a direct sequencing assay. The comparison of these methods on 20 clinical specimens from treated patients confirmed the plasmid results: the three methods were concordant for the detection of the mutant strains in 72% of the cases and the discrepant results were caused mainly by the sequencing assay's lack of sensitivity. The line probe assay was more sensitive for detecting mutations than sPCR when the viral load was less than 10(4) copies/ml; conversely, the sPCR provided a more sensitive detection when the viral load was greater than 10(4) copies/ml. Although difficult to perform in clinical practice, sPCR appears to be a reliable technique for detecting and quantifying quasi-species resistant to lamivudine (LAM) and adefovir (ADV) and can be useful to gain a better understanding of the natural history of antiviral resistance during the treatment of chronic hepatitis B (CHB).
authors.affiliation: Unit of Virus Host Cell Interactions, CNRS-EMBL-UJF-UMR 5233, University Hospital and University of Joseph Fourier, Grenoble, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lupo
authors.forename: Sylvie
authors.initials: S
authors.lastname: Larrat
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Hilleret
authors.forename: Raphaële
authors.initials: R
authors.lastname: Germi
authors.forename: Véronique
authors.initials: V
authors.lastname: Boyer
authors.forename: Sandrine
authors.initials: S
authors.lastname: Nicod
authors.forename: Gérard
authors.initials: G
authors.lastname: Barguès
authors.forename: Vincent
authors.initials: V
authors.lastname: Leroy
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Seigneurin
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Zarski
authors.forename: Patrice
authors.initials: P
authors.lastname: Morand
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000032:analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology; D009154:Mutation; D063065:Organophosphonates / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D063065:Organophosphonates; D019259:Lamivudine; C053001:adefovir; D000225:Adenine