No baseline serum samples were available for sequencing in the remaining
18 patients. The recently published nomenclature for amino acid positions in the HBV polymerase gene was used.16 With nested polymerase chain reaction (PCR) using the primers 252 (5′-AGACTCGTGGTGGACTTCTCT-3′) and 1309 (5′-AGAATGTTTGCTCCAGACC-3′) as external primers and 377 (5′-GGATGTGTCTGCGGCGTTT-3′) and 998 (5′ACGTTGACAGACTTTCCAATC-3′) as internal primers, a PCR product bridging region from codon rt88 to codon rt282 was amplified. The PCR products were separated on 2% Y-27632 price agarose gel (NuSieve 3:1; FMC, Rockland, ME), eluted with Gene-Clean (Bio 101, Vista, CA), and directly sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) with an automated sequencer (ABI-Prism; Applied Biosystems). The derived sequences of both strands of the amplification products
were investigated for all mutations described Epigenetics inhibitor to be associated with resistance against LAM and telbivudine as rtV173L, rtL180M, and rtM204I/V/S, against entecavir as rtT184G, rtS202I, and rtM250V, and against ADV as rtA181V/T and rtN236T. HBV genotypes were determined by sequence alignment of the overlapping HBsAg with HBV sequences derived from GenBank.17 For statistical data analysis, SPSS software for Windows, release 11.0 (Chicago, IL), was used. HBV DNA initially measured in copies/mL was converted to the base 10 logarithmic scale, with a lower limit of detection of 400 copies/mL, corresponding to 2.6 log10. For the primary endpoint, the time until the HBV DNA level reached <400 copies/mL was estimated using Kaplan-Meier methodology, and time to event subgroup comparisons were performed using the Log-Rank test. Categorical data were analyzed with the two-sided Fisher exact test. To determine the influence of baseline HBV DNA levels, the cohort
was divided into patients with high and low HBV DNA levels, using a threshold of 107 copies/mL. Baseline characteristics of the 131 eligible patients are shown in Table 1. In total, 101 of these patients received TDF for at least 12 months and the median 上海皓元医药股份有限公司 time on TDF treatment was 23 months (range, 6–60 months). TDF treatment resulted in a decrease from a mean of 7.6 ± 1.4 log10 copies/mL to undetectable HBV DNA levels in 79% of all patients during the observation period. To assess the long-term efficacy of TDF treatment, the probability of achieving undetectable HBV DNA levels was calculated for all timepoints of TDF treatment by Kaplan-Meier analysis (Fig. 1). A Kaplan-Meier analysis, which describes a binary outcome, could be applied because during the observation period no reincrease of HBV DNA levels after suppression to undetectable levels was observed in any of the patients.