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

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 105 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 104 copies/ml; conversely, the sPCR provided a more sensitive detection when the viral load was greater than 104 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).