Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment.
OBJECTIVE
The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant.


METHODS
We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients).


RESULTS
We could detect as little as 0.01-0.001% of mutant viruses coexisting in 10(5)-10(9) copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients.


CONCLUSIONS
We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.
Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Japan.
Toshihiko
T
Kirishima
Takeshi
T
Okanoue
Yukiko
Y
Daimon
Yoshito
Y
Itoh
Hideki
H
Nakamura
Atsuhiro
A
Morita
Tetsuya
T
Toyama
Masahito
M
Minami
D000328:Adult; D000368:Aged; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000032:analysis; D024882:Drug Resistance, Viral / Q000235:genetics; D005260:Female; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy*; D006801:Humans; D019259:Lamivudine / Q000008:administration & dosage*; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D018894:Reverse Transcriptase Inhibitors / Q000008:administration & dosage*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine