Patients and MethodsPatients This is a follow-up study of our previous controlled trial which evaluated the efficacy of a 12-month course of lamivudine, administered as either monotherapy (100 mg/day), or in combination with interferon (5 MU three times a week; cumulative dose, 780 MU), in Caucasian patients from the Apulia region in southern Italy. All patients were anti-HBe positive, negative for hepatitis C and D virus (HCV and HDV) and human immunodeficiency virus (HIV) infections and similar with respect to age, sex, alanine transaminase and HBV-DNA levels at baseline.[12] In the initial cohort of patients, therapy had to be discontinued per protocol after 1 year of treatment in all cases; of the 50 patients enrolled in the initial trial, five patients who developed a YMDD mutant during the first course of therapy were judged to be unsuitable for lamivudine re-treatment; they showed a virological breakthrough before month 12 and YMDD mutants were detected by sequence analysis. They were excluded because re-treatment in this setting is ineffective due to the prompt reappearance of viral resistance. Nine more patients from the initial cohort were enrolled in experimental clinical trials using entecavir or adefovir dipivoxil and were also excluded from the present study. The remaining 36 agreed to be re-treated with lamivudine when the disease relapsed after therapy discontinuation; none had experienced a virological breakthrough or had developed YMDD mutants during the previous course of therapy. Lamivudine Re-TreatmentAfter an initial evaluation, the 36 patients were administered lamivudine orally at a dose of 100 mg once daily. Patients were seen in the out-patient clinic and had blood drawn at baseline and routinely every 3 months throughout treatment. According to recent guidelines, lamivudine was continued thereafter as long as there was evidence of biochemical and virological response.[6] The initial protocol called for therapy to continue for 5 years. Normal alanine transaminase values and undetectable HBV-DNA (< 0.5 pg/mL) were used as criteria for response. Breakthrough was defined as the reappearance of serum HBV-DNA by molecular hybridization during treatment after an initial virological response, and biochemical breakthrough was defined as an increase in alanine transaminase activity. For patients with virological and biochemical breakthrough, re-treatment had to be stopped whenever alanine transaminase levels reached > 10 times normal, or persistently abnormal alanine transaminase values were found; lamivudine was continued for another 6-9 months in the case of a lower or sporadic increase in alanine transaminase levels. All patients were treated as part of a two-centre, open-label, prospective trial of long-term therapy of hepatitis B. The protocol was approved by the ethics committees at the two centres, and all patients gave written informed consent. Laboratory and Virological TestingRoutine laboratory tests, performed on each clinical visit, included serum alanine and aspartate transaminase, serum direct and total bilirubin, albumin and complete blood counts. HBV, HDV, HCV and HIV serological markers were detected by commercial enzyme immunoassays (Abbott Laboratories, North Chicago, IL, USA; Sorin Biomedica, Saluggia, Italy; Ortho Diagnostic Systems, Raritan, NJ, USA). Serum HBV-DNA was detected by a sandwich capture hybridization assay (Digene Diagnostics, Hybrid Capture II, Abbott Laboratories, North Chicago, IL, USA), which has a lower detection limit of 0.5 pg/mL (1.42 x 105 copies/mL). Virological resistance was evaluated by direct sequencing of the polymerase gene at baseline and during treatment in patients with breakthrough, as described previously.[12] With this method, the lower detection limit of a minor viral population is about 20% of the total population.[14] In addition, at baseline, the HBV polymerase region was amplified and analysed by means of the line probe assay (INNO LiPA HBV-DR, Innogenetics, Ghent, Belgium), which identifies 5% of a specific variant within a mixed viral population.[15] Statistical AnalysisData were analysed using the SPSS statistical package (version 6.1.3; SPSS Inc., Chicago, IL, USA). Continuous variables were checked for normality using the Shapiro-Wilks test. If normally distributed, Student's t-test was used to evaluate differences between mean values. Otherwise, the Mann-Whitney U-test was carried out to test differences between median values. Pearson's chi-squared test (or Fisher's exact test, when appropriate) was used for categorical variables. A P value of less than 0.05 was considered to be significant. Kaplan-Meier statistics were used to evaluate differences in the cumulative risk of breakthrough occurrence between the two groups of patients, sorted according to the previous course of treatment.
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