Character Of HBV Polymerase Gene Mutations And Preliminary Observation On The Efficacy Of Domestic Adefovir Dipivoxil In Patients With Lamivudine Resistance

IntrodutionInfection of Hepatitis B virus is one of the most serious global public health problems .It was supposed that there were 2 billion people infected with the hepatitis B virus (HBV) all over the world .HBV infection was associated with chronic hepatitis B liver cirrhosis and hepatocellular carcinoma. The main strategy of chronic hepatitis B therapy is anti-viral treatment.Lamivudine, an oral nucleoside, was the most popular drug used, which can inhibit HBV replication and markedly reduce serum HBV DNA levels . But the clinical benefit of this therapy has often been eroded by the emergence of resistant mutant strains , leading to hepatitis B recurrence and deterioration .In such situation ,adefovir dipivoxil was adopted alternatively .The serum HBV DNA polymerase gene was analyzed by sequence detection via polymerase chain reaction (PCR) in order to investigate the relationship between HBV DNA polymerase gene mutation and lamivudine resistance, Patients with YMDD mutation were chosed to receive the treatment of domestic adefovir dipivoxil.Materials and methods1. Patients :60 patients who were diagnosed as chronic Hepatitis B frominpatients and outpatients in the first affiliated hospital of Zhejiang Universtity from August 2005 to April 2006 were invited to the research. All patients had received lamivudine treatment (100mg/d)more than six months and all of them were consistent with the criterion of clinical drug resistance . (l)The rebound of serum HBV DNA> 105copies/ml after the serum HBV DNA loss.(2)The patients whose HBV DNA load rebound excluding poor compliance. (3)The patients whose ALT level increase excluding other reasons.2. Samples: Serum of venous blood of these 60 patients were collected and HBV YMDD domain were purified with polymerase chain reaction(PCR). The products of reaction were sequenced with Mega.BACE? 500 automatic sequencer (America).Patients who had YMDD mutation were offered domestic adefovir dipivoxil. To avoid syndrome caused by urgently stop taking lamivudine, patients were guided to take adefovir dipivoxil(10mg qd) combined with lamivudine(100mg qd) for 8 weeks, then take adefovir dipivoxil alone. Every 4 weeks patients took laboratory tests include liver function, HbsAg, HbsAb, HbeAg, HbeAb, HbcAb, HBV DNA, blood routine, urine routine. Before treatment and in 12 and 24 weeks during treatment , patients had a detection which include fibrosis index, AFP, electrolyte, BUS of liver and gall and spleen. The rate of normalization of ALT, the HBV DNA loss (HBV DNA<104copies/ml was regarded as HBV DNA loss) , the HBeAg loss and the HBeAb seroconversion were analysed . We also investigated the impact of some factors such as the history of HBV, sex and age to effect adefovir dipivoxil treatment. Statistics method : Data were statisticed with SPSS 10.0. Data were shown as mean ± standard deviation.Results1. Types of HBV polymerase gene mutationAmong 60 cases there were 48 patients who had YMDD mutation.The mutation type were rtL180M/M204V, rtM204I, rtL180M/M204I. Among them, 20 patients (20/48 41.7%)had rtL180M/M204V mutation which played the most important role inYMDD mutation. In addition, 16 patients (16/48 33.3%) had rtM204I mutation and 12 patients (12/48 25%) had rtL180M/M204V mutation. There were 2 patients with rtL180M in the other 12 patients. All 20 patients with rtM204V mutation also had rtL180M mutation;the rate of this kind of combinatorial mutation was 100%. Among 28 patients with rtM204I mutation, only 12 patients also had rtL180M mutation;the occurrence of this kind of mutation was 42.8%. Among 32 patients with rtL180M mutation, 20 patients had rtM204V mutation, and 12 patients had rtM204I mutation. The combinatorial mutation rate was 62.5% and 37.5% respectively.2. The efficacy of domestic adefovir dipivoxil in 48 cases of YMDD mutationWe had made a further observation of liver function, HBV DNA, HBeAg andHBeAb in these 48 patients who had YMDD mutation at the point of 12 weeks and 24 weeks during treatment. At the point of 12 weeks and 24 weeks, the rate of normalization ALT was 60.4% (29/48) ,87.5% (42/48) .the rate of HBV DNA loss was 29.2% (14/48) ,77.1% (37/48) .the rate of HBeAg loss was 8.3% (4/48) , 20.8% (10/48) .the rate of HBeAg seroconversion 0% (4/48) ,8.3% (4/48) .At the point of 12 weeks and 24 weeks, the median of liver function is ALT 77.9 ±32.5u/L, 47.9±15.3u/L, AST 53.95 ± 16.9u/L, 35.6±9.8u/L, TB16.8 ± 4.4umol/L, 15.5±4.2umol/L, the median HBV DNA 5.01+0.88 loglOcopies/ml, 4.05 ± 0.79 loglOcopies/ml o Compared with baseline,there was significant difference( PO.05).The possible factors related with Analysis adefovir dipivoxil treatment were analysed in our study. We collected the corresponding information to assess the effect factors which was associated the efficacy of domestic adefovir dipivox. We found that the family history of HBV and age had greatly impact on the effect of therapy. In 48 cases of patients received domestic adefovir dipivox 24 weeks who had YMDD mutation, 34 patients had a family history of HBV, while 14 patients did not have such history . A statistic difference was observed between those two groups. Patients without history of HBV would obtain better efficacy of treatment, that is to say, the high rate of normalization of ALT level, HBV DNA and HBeAg loss, HBeAgseroconversion. We also found the index of rate of normalization of ALT level, HBV DNA and HBeAg loss, HBeAb seroconversion were significantly increased in patients under 45 years old than above 45 years old . Sex seemed have no response to the efficacy of adefovir dipivoxil.. Though there had a raise of index in female group(ll cases), there has no statistic significance between male(37 cases) and female.Conclusion1. In the chronic hepatitis B who are lamivudine clinic resistance, about 80% patients have YMDD mutations and other types and sites mutations, such as rtL180M/M204V, rtM204I, rtL180M/M204I, et al.2. Adefovir Dipivoxil administration was associated with a significantly efficacy in terms of improvement of liver function, decrease of hepatitis B viral DNA load and an increase of seroconversion.3. Familial history and age may effect the efficacy of Adefovir Dipivoxil antiviral treatments. Patients with negative familial history with YMDD mutations may have better efficacy than those with positive familial history patients (PO.05 ) . Compared with the patients in older than 45 years old, the patients under 45 years old have significantly benefit efficacy (P<0.05) .4. Adefovir Dipivoxil administration have some advantages in treatment, such as less side effects, lower occurrence rate of adverse case and resistance .