[Evaluation of different methods in monitoring YMDD motif mutations associated with lamivudine resistance].
OBJECTIVE
To evaluate the three different methods in monitoring the lamivudine-resistant HBV mutants in lamivudine-treated patients with chronic hepatitis B.


METHODS
The sensitivity and specialty of melting curve assay and polymerase chain reaction microplate nucleotide hybridization-enzyme linked immunosorbent assay (PCRmnh-ELISA) were compared with those of mismatch polymerase chain reaction-restriction fragment length polymorphism (mPCR-RFLP) and sequence analysis, through detection of HBV YMDD mutants in 44 serums from chronic hepatitis B patients receiving lamivudine monotherapy at the time of viral breakthrough.


RESULTS
mPCR-RFLP assay was more sensitive (10(4) copies/ml) than both PCRmnh-ELISA (10(5) copies/ml) and melting curve assay (10(6) copies/ml). 26 YMDD mutants and 18 wild-types were determined by the means of mPCR-RFLP. Among the 26 mutants, only 16 and 18 mutants were found by melting curve assay and PCRmnh-ELISA, respectively. Whereas, out of the 18 wild-types, 2 and 13 mutants were detected by melting curve assay and PCRmnh-ELISA, respectively. To confirm the different results determined by the three methods in 16 samples, sequence analysis was conducted and showed that the rate of consistency with sequencing was 93.8% by mPCR-RFLP, 43.8% by melting curve, and 18.8% by PCRmnh-ELISA, respectively (chi2=18.7, P<0.01).


CONCLUSIONS
The mPCR-RFLP assay is reliable to monitor HBV YMDD mutations. Melting curve assay and PCRmnh-ELISA should be further improved to increase their sensitivity and specialty.
Hu
Ying-ying
YY
Fuzhou Municipal Infectious Hospital, Fuzhou 350025, China.
Jiang
Jia-ji
JJ
Li
Dian
D
Lin
Cai-wen
CW
Li
Qin-guang
QG
Chen
Yi
Y
D000328:Adult; D000998:Antiviral Agents / Q000627:therapeutic use*; D004279:DNA, Viral / Q000097:blood; D024882:Drug Resistance, Viral; D005260:Female; D015684:Gene Products, pol / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
D000998:Antiviral Agents; D004279:DNA, Viral; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D019259:Lamivudine