Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B.
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.
Yoshida
Shigeru
S
Department of Health Sciences, Hokkaido University School of Medicine, North-12 West-5, Sapporo, Japan. shiyoshi@med.hokudai.ac.jp
Hige
Shuhei
S
Yoshida
Miyuki
M
Yamashita
Naoki
N
Fujisawa
Shin-ichi
S
Sato
Kaori
K
Kitamura
Tadashiro
T
Nishimura
Masaharu
M
Chuma
Makoto
M
Asaka
Masahiro
M
Chiba
Hitoshi
H
D001483:Base Sequence; D015342:DNA Probes / Q000032:analysis* / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology* / Q000627:therapeutic use; D009154:Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*
D015342:DNA Probes; D019259:Lamivudine; D019914:Taq Polymerase