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Early Detection of Lamivudine Resistant Mutant in Chronic Hepatitis B Patients Using a Novel Sensitive Method

  • Takeshi Okanoue
  • Kohjiro Mori
  • Toshihiko Kirishima
  • Masahito Minami
Conference paper

Abstract

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.

Keywords

Cirrhotic Patient Peptide Nucleic Acid HBeAg Positive Patient Lamivudine Therapy Lamivudine Treatment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Kirishima T, Okanoue T, Daimon Y, et al. Detection of YMDD motif mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment. J Hepatol 2002;37:259–265.PubMedCrossRefGoogle Scholar
  2. 2.
    Orum H, Nielsen PE, Egholm M, et al. Single base pain mutation analysis by PNA directed PCR clamping. Nucl Acids Res 1991;21:5332–5336.CrossRefGoogle Scholar

Copyright information

© Springer Japan 2004

Authors and Affiliations

  • Takeshi Okanoue
    • 1
  • Kohjiro Mori
    • 1
  • Toshihiko Kirishima
    • 1
  • Masahito Minami
    • 1
  1. 1.Third Department of Internal MedicineKyoto Prefectural University of MedicineKyotoJapan

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