Evolution of entecavir-resistant hepatitis B virus during entecavir and adefovir dipivoxil combination therapy

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.