RESUMO
The emergence of entecavir (ETV) resistance is rare, particularly in a longitudinal study. The aim of the present study was to characterize the evolution of ETV-resistant variants during antiviral therapy using entecavir monotherapy followed by ETV-adefovir dipivoxil (ADV) combination therapy. The study included a prospective cohort of 53 consecutive chronic hepatitis B (CHB) patients. During the 60-month period of ETV therapy, 2 patients exhibited ETV resistance and their medical records were comprehensively reviewed. A total of 25 consecutive serum samples were regularly collected from the 2 patients. All the samples were used to characterize the evolution of the polymerase gene mutations using pyrosequencing. The linkage of the variants was analyzed from 87 reverse transcriptase sequences of 3 selective samples using clone sequencing. The 2 patients presented with viral breakthrough during ETV monotherapy. In patient A, the rtL180M, rtS202G and rtM204V mutant variants were detected using pyrosequencing prior to virological breakthrough. Although the viral load declined following the administration of ADV, the ETV-resistant variants were persistently dominant in the viral populations. In patient B, the rtL180M, rtM204I and rtM204V mutants were present in ~70, 30 and 10% of the viral populations, respectively, at the time of study entry. In addition, rtT184F was present in ~20% of the viral population during virological breakthrough, at month 24. The rtL180M, rtT184F and rtM204V were predominant during the combination treatment. Clonal analysis further revealed that the rtS202G or rtT184F was in all cases co-localized with rtL180M and rtM204V in any single virus isolate clone. The results of the present study indicate that the addition of ADV therapy with ETV for treating ETV-resistant mutation may not inhibit the replication of ETV-resistant variants that developed previously in lamivudine-treated CHB patients.
RESUMO
OBJECTIVE: To evaluate the quality and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance (HBV DRT). METHODS: Serial dilutions of the International Standard for HBV DNA were used to test the detection limit of the PCR for HBV DRT. Plasmids containing the either a wild-type (WT) copy or one of 10 mutant (MT) copies of the HBV RT gene were used to prepare a series of samples with various mutation ratios. To construct the linear relationship between the true mutation rate and the detected mutation rate, each sample was repeated at least 10 times. A total of 102 clinical samples were analyzed by Sanger sequencing and retested by the PCR for HBV DRT to determine the concordance of these two methods. RESULTS: The lower detection limit of the PCR for HBV DRT was 50 IU/ml. Except for the RT236 MT, the correlation between the true mutation rate and the detected mutation rate for the other nine resistance-related mutation sites were excellent, with R² more than 0.98 (P less than 0.001). Among the 102 clinical samples, four were not amplified successfully by PCR. The results were significantly different between the PCR for HBV DRT method and the Sanger sequencing method (x² = 71.2, P less than 0.001), and concordance was observed for 897/969 (92.6%) amino acid positions in 98 samples. Concordant results were achieved in 46/98 (46.9%) samples at all 10 mutation sites. For detection of a single mutation site, concordance rates ranged from 71.5% to 100% at the 10 mutation sites, respectively. Analysis of discordant samples showed that in 87.5% (63/72), Sanger sequencing detected WT and the PCR for HBV DRT detected WT/MT. In 5.6% (4/72) of samples, Sanger sequencing detected WT/MT and the PCR for HBV DRT detected WT. In the remaining 6.9% (5/72) of samples, Sanger sequencing detected WT but PCR for HBV DRT detected MT. CONCLUSION: The PCR for HBV DRT showed high sensitivity and accuracy in detecting antiviral drug-resistant mutations. The method is superior to Sanger sequencing for detecting minor mutations and can be used for early detection of a resistance mutation.
