Early Detection of Lamivudine Resistant Mutant in Chronic Hepatitis B Patients Using a Novel Sensitive Method
Background and aims: It has been reported that prolonged lamivudine therapy for chronic hepatitis type B (CH-B) patients induces lamivudine resistant YMDD motif mutants in high rate, resulting in breakthrough hepatitis frequently. To clarify the clinical significance of YMDD motif mutant, we tried to develop a novel sensitive method to detect YMDD motif mutant and have done follow-up study of CH-B patients using this method.
Materials and methods: Twenty-one cases of CH-B patients and 11 cases of cirrhotic patients who received lamivudine or lamivudine with interferon (IFN) therapy were enrolled in this study. Furthermore, eighteen anti-HBe antibody positive non-lamivudine treated CH-B patients and eighteen HBe antigen positive non-limivudine treated (H-B patients) were also studied. Blood sampling was done before and serially during lamivudine and IFN therapy. Lamivudine resistant mutants were examined by a novel sensitive method using peptide nucleic acid (PNA)-mediated PCR clamping with RFLP.
Results:Our study demonstrated that PNA-mediated PCR clamping worked effectively under the following conditions: PNA primer concentration of 50 pmol, PCR primer 25 pmol, PCR annealing temperature 60°C, PNA annealing and inhibition temperature 76°C. To enhance the detectability of YMDD mutants, fragments amplified from wild-type sequences were digested with NdeI after the first PCR. Then, we assessed the sensitivity of this method using mixture of wild-type and mutants type HBV and compared the results with those of the conventional RFLP assay. The detection limit of YMDD motif mutant was as little as 0.01-0.001% of mutant viruses coexsisting in 105-109copies of wild type viruses using this method. However, it was around 10% by the conventional RFLP. YMDD motif mutant was detected 7 months before breakthrough in a cirrhotic patient, which was 7 months earlier than using the conventional RFLP assay. YMDD motif mutants were also detected in 4 of 18 anti-HBe antibody positive non-lamivudine treated CH-B patients: YMDD+YVDD in 2 patients and YMDD+YIDD in 2 patients, howerer, these mutants were not detected in eighteen
In eAg-positive chronic hepatitis patients, the rate of normalization of serum ALT at the end of treatment is 64%. In eAg-negative patients, it is 57%. In cases without YMDD motif mutant, a higher rate of normalized serum ALT was seen.
PNA-mediated PCR clamping with RFLP method is very sensitive for the detection of YMDD motif mutants.
The rate of emergence of YMDD motif mutant and the rate of flare-up hepatitis is higher in eAg-negative patients. In cirrhotic patients, the emergence of YMDD motif mutants was earlier than in chronic hepatitis patients, but flare-up hepatitis was mild.
KeywordsCirrhotic Patient Peptide Nucleic Acid HBeAg Positive Patient Lamivudine Therapy Lamivudine Treatment
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