Detection And Pilot Study Of Entecavir Resistant Mutations In Chronic Hepatitis B Patients With Entecavir-refractory

BackgroundChronic hepatitis B is one of the main threatens to global health. Over one third of the chronic hepatitis B virus (HBV) carriers are in China. Anti-HBV therapy is the core treatment of chronic hepatitis B. Among the available medications, Nucleoside Analogs (NA) are one of the main stream treatment drugs currently. Entecavir (ETV) is one of those novel NAs, with strong effect on the inhibition of HBV replication, has been saled in the market since 2006 and applied in clinic gradually. However, the problem of ETV resistance has gained more attention concomitant with applied time continued and patients larged. The study about genic and clinic characteristics of ETV mutation is few, and majority of them was reported by western investigators. So it is necessary to detect and analyze patients who have underwent treatment failure of ETV in China, and it may provide keys and evidences for basic research and clinic application. Recent research has shown that ETV resistance mutation required at least three substitutions consisting of LVDr-encoding substitutions M204V and L180M plus an additional ETVr-encoding change which were not assured, and the typies of substitution replacement may be multiplex. Therefore, the methods of Real-time PCR and RFLP are not feasible while PCR-sequencing is fittest.Objectives1. To design a kind of PCR which may contain longer base pairs of HBV reverse transcriptase (RT) and detect resistance mutations of reverse transcriptase cooperated with directing sequencing and cloning sequencing.2. Detect and analyze genic and clinic characteristics of patients who have underwent treatment failure of ETV in China and provide keys and evidences for basic research and clinic application.Methods1. Four primers for nested PCR ( ntPCR) were designed for amplifying RT region of HBV, and the usability and sensitivity were checked up by HBV recombinant plasmids and sixty serum samples from chronic hepatitis B patients with positive HBV DNA. Establish ntPCR-directing sequencing and ntPCR-cloning-sequencing methods to detect mutations of HBV.2. Twenty serum samples of chronic hepatitis B patients who underwent ETV refractory were obtained in Beijing Ditan hospital from January to December in 2008. All patients had taken ETV canonically (0.5 mg QD ETV for patients not taken LVD in the past, 1.0 mg QD ETV for LVD-refractory patients).All samples were detected by PCR-directing sequencing and three of them were detected by PCR-cloning-sequencing additionally. Patients were devided into two groups: patients not attained completed viral response (6/20) and patients underwent virologic breakthrough(14/20). The positive rate of mutations and genotype of the two groups were analyzed by statistical methods.Results1. The successful amplification of almost full-length RT regions(nt146-1051, rt5～rt318)of HBV which had contained all known mutation substitutions and the full-length of HBsAg-coding was confirmed by agarose gelelect rophoresis and sequencing. The amplified target fragments were confirmed by direct sequencing.2. Results for patients with ETV-refractory2.1 Results of PCR-directing sequencing:⑴RT regions: 11/20 ETV-refractory patients were ETV resistance positive. rtM204V+ rtL180M+rtT184L was the main type in RT regions(8/11), rtM204V+rtL180M+rtS202G secondly(3/11), rt250 mutation was not deceted.⑵HBsAg-coding regions: sP127T were detected in 2/20 ETV-refractory patients, it had been reported to associated with immune escape.2.2 Results of PCR-cloning-sequencing: samples of 1, 5 and 9 were analyzed by PCR-cloning-sequencing. The disadvantage of PCR-directing sequencing could been improved by its repetitious detections.2.3 All patients with ETV resistance mutations came from group of virologic breakthrough, no ETV mutations was deteced in patients without completed viral response. There is statistica difference between the two groups.2.4 Among 11 patients with ETV mutations, 10 of them were LVD-refractory in the past. The mean time of ETV therapy was (19.43±2.72)month.2.5 3 samples were detected ETV mutation among 6 samples which were B genotype, 8/14 C genotype. There is no difference between them.Conclusions1. Established a new nt-PCR to amplify almost full-length RT regions(nt146-1051, rt5～rt318)of HBV which had contained all known mutation substitutions and the full-length of HBsAg-coding. The nt-PCR combined with sequencing could detect mutations of P gene RT regions and S gene, it also could be used to analyze HBV genotype.2. rtM204V+rtL180M+rtT184L mutations were the main type in genotype B and C ETV resistance patients.3. The incidence of ETV resistance mutation was more often in virologic breakthrough group and patients with LVD refractory in the past.4. There may be no statistical difference in ETV resistance rate between genotype B and C patients. We need more samples to approve it.