BACKGROUND: Lamivudine (LAM)-resistant hepatitis B virus (HBV) with mutations in the polymerase region frequently appears after long-term use of LAM. Several methods allowing detection of mutant strains (YIDD, YVDD) have been reported, but they have no quantitative characteristics. In this study, we explored a unique approach for quantification of each mutant strain. METHODS: A method for detection and quantification of wild and mutant strains was developed using realtime polymerase chain reaction and type-specific minor groove binder (MGB) probes, and tested in patients with chronic hepatitis B before and after additive treatment with adefovir dipivoxil (ADV). RESULTS: A good correlation was confirmed in HBV DNA quantity obtained between the YMDD-specific MBG probe assay and Amplicor HBV Monitor assay results (P < 0.001), linear between 3 and 9 log copies/mL serum. Of 109 samples from patients with chronic hepatitis B tested by both these assays and conventional direct sequencing, 90 (88.2%) showed identical results. The assays successfully detected and quantified a single type of mutant in three of four patients with additive ADV treatment, and also two coexisting mutant types (YIDD and YVDD) in the remaining patient. CONCLUSIONS: Our specific and sensitive method for detection and quantification of HBV DNA with the wild-type YMDD motif and its two mutant forms (YIDD and YVDD) appears to be clinically useful, especially in patients with multiple mutant HBV infections.
BACKGROUND:Lamivudine (LAM)-resistant hepatitis B virus (HBV) with mutations in the polymerase region frequently appears after long-term use of LAM. Several methods allowing detection of mutant strains (YIDD, YVDD) have been reported, but they have no quantitative characteristics. In this study, we explored a unique approach for quantification of each mutant strain. METHODS: A method for detection and quantification of wild and mutant strains was developed using realtime polymerase chain reaction and type-specific minor groove binder (MGB) probes, and tested in patients with chronic hepatitis B before and after additive treatment with adefovir dipivoxil (ADV). RESULTS: A good correlation was confirmed in HBV DNA quantity obtained between the YMDD-specific MBG probe assay and Amplicor HBV Monitor assay results (P < 0.001), linear between 3 and 9 log copies/mL serum. Of 109 samples from patients with chronic hepatitis B tested by both these assays and conventional direct sequencing, 90 (88.2%) showed identical results. The assays successfully detected and quantified a single type of mutant in three of four patients with additive ADV treatment, and also two coexisting mutant types (YIDD and YVDD) in the remaining patient. CONCLUSIONS: Our specific and sensitive method for detection and quantification of HBV DNA with the wild-type YMDD motif and its two mutant forms (YIDD and YVDD) appears to be clinically useful, especially in patients with multiple mutant HBV infections.