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Little is known about the prevalence and pattern of hepatitis B virus (HBV) mutations in HIV/HBV co-infected individuals on long-term lamivudine (3TC) therapy.HBV polymerase/envelope/basal core promoter/pre-core sequences from 81 HIV–HBV co-infected persons who received at least 6 months 3TC were compared to HBV reference sequences. Host and viral characteristics associated with HBV mutations were determined.HBV viraemia was detected in 53 persons (65%) and was associated with lower CD4 cell count nadir and higher HIV RNA at the time of testing but not with 3TC duration. Known 3TC-resistant mutations occurred in 50% and 94% of viremic patients with < 2 years and > 4 years 3TC, respectively. The CD4 cell count at testing was significantly higher in those with 3TC-resistant mutations. The triple polymerase mutant (rtL173V, rtL180M, rtM204V), which behaves as a vaccine escape mutant in vitro, occurred in 17% of viraemic patients. Polymerase mutations that may confer resistance to other anti-HBV agents were also detected.In HIV–HBV co-infected patients, greater immunocompromise is associated with continued HBV viraemia while on 3TC, and development of 3TC-resistant mutations are inevitable with prolonged 3TC use. These mutant viruses may limit future therapeutic options due to cross-resistance or may produce HBV vaccine escape mutants. Thus, timing and selection of antiretroviral therapy is critical in this population.