Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine is a ssociated with the appearance in the circulation of HBV variants with mutat ions in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the polymerase gene. Fluorometric real-time PCR with the LightCycler assay was used for the detection of resistant variants. Differences in the hybridizat ion melting curve kinetics of probes bound to the sequences encoding the wi ld-type or the mutant YMDD motifs (YIDD or YVDD in which the methionine res idue is altered to an isoleucine or a valine, respectively) distinguished t he single-base changes responsible for the resistance phenotype, The LightC ycler probe hybridization assay was applied to 40 serum specimens from 19 p atients, and the results were correlated with the nucleotide sequences dete rmined for the corresponding PCR products. All three variants could be iden tified in the specimens. PCR clones obtained front four patients early in t he course and prior to lamivudine therapy were investigated for the appeara nce of YIDD and YVDD variants with the LightCycler assay, In one patient, a transient appearance of the YIDD variant was observed 6 weeks into therapy , Subsequently, after II months of lamivudine therapy, the YVDD variant eme rged in that patient.