Annals of Clinical Biochemistry > Volume 45, Number 1 > Pp. 59-64

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Original Article

Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B

Shigeru Yoshida ¹ , Shuhei Hige ³, Miyuki Yoshida ², Naoki Yamashita ², Shin-ichi Fujisawa ², Kaori Sato ², Tadashiro Kitamura ², Masaharu Nishimura ⁴, Makoto Chuma ³, Masahiro Asaka ³ and Hitoshi Chiba ¹

¹ Department of Health Sciences, Hokkaido University School of Medicine, North-12, West-5, Sapporo 060-0812; ² Department of Clinical Laboratory, Hokkaido University Hospital, Sapporo 060-8648; ³ Gastroenterology and Hematology Section, Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638; ⁴ First Department of Medicine, Hokkaido University School of Medicine, Sapporo 060-8638, Japan

Corresponding author: Dr Shigeru Yoshida. Email: shiyoshi{at}med.hokudai.ac.jp

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.

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