The Detection And Analysis Of The YMDD Mutation Of Treatment Chronic Hepatitis B By Lamivudine

Background Chronic hepatitis B antiviral treatment is increasingly important today, what is the treatment of selected conditions? What is the evaluation of the efficacy of the indicators? What is the stopping signal? The most reliable indicator is the HBVDNA. Studies have shown that HBVDNA level of patients with acute exacerbation of chronic hepatitis B occurs is an independent predictor of decompensation. Treatment indications now are in accordance with an updated version of "Chronic hepatitis B prevention and treatment guidelines in2010." However, due to historical reasons, a considerable portion of patients in the treatment of the past, did not follow this guide specification medication, the prevalence of overmedication,not strictly testifying, and so on,leading to poor response, emergence of YMDD mutations, and other adverse consequences. Clinicians in the admissions of such patients are very difficult, one is to clearly answer to the causes of poor, mainly the detection of the YMDD mutant, the second is to give specific remedies based on the presence of YMDD mutations. In fact, taking what means of detection, both timely and accurately to find the poor response, but also to reduce the economic burden of patients, perhaps, is the direction of efforts. Existing means of detection HBVDNA and YMDD mutation are very much, and they have both advantages and disadvantages. Therefore, how to choose an appropriate method is worthy of exploring.Objectives Part of cases used by the two methods of detection, PCR-inverting dot blot hybridization and PCR fluorescence were detected the indicators such as YIDD, YVDD, rtL180M,rtM204V, rtM204I. And then compare the accuracy of the test results, the errors, the costs, the time used and the convenience in order to compare, to judge the advantages and disadvantages of the two methods. To study the proportion of YMDD mutation in the poor response and to analyze the baseline value, including HBVDNA and ALT levels in order to identify the intrinsic link.Methods1) Case selection and specimen collection:the case selection criteria:All patients were diagnosed as chronic hepatitis B, positive HBVDNA and accepted lamivudine alone for more than24weeks, then review HBVDNA at24weeks compared with baseline values which lowed Hog10IU/mL but not down to the lower limit of detection. We collected160cases of chronic HBV infection who are in the Department of Infectious Diseases of the First Affiliated Hospital of Anhui Medical University and of the First People’s Hospital of Huainan City January2008-December2011in patient department or clinic diagnosed Chronic hepatitis B who have accepted lamivudine alone and have been in poor response.2) Detect the YMDD mutant and mutation using the PCR fluorescence method and PCR reverse dot blot hybridization method respectively from randomly selected38cases.Then analyze the data, compare the advantages and disadvantages of the detection methods, and lead to the conclusion which method is more sensitive and convenient,more suitable for the primary hospitals for YMDD mutation detection.3) Treatment of viral DNA template:the classic proteinase K digestion-phenol-chloroform method was used.4) The HBVDNA and ALT levels on baseline before lamivudine therapy were analyzed to identify their inner relationship.Results1) Randomly selected38cases of YMDD mutations, respectively, using the PCR fluorescence method and PCR reverse dot blot hybridization method for genotyping of YMDD mutants and mutation detection, found no significant difference in genotype B and C in YMDD mutation probability. The main variants are rtL180M+rtM204V, there are20cases (52.63%).2)The PCR fluorescence method was used,there was35in positive,and the sensitivity was92.11%.While PCR reverse dot blot hybridization method was uesd,there was34in positive,and the sensitivity was89.47%.The results were statistically analyzed and found that the difference was not significant.3) PCR reverse dot blot method requires relatively expensive fluorescent PCR instrument and a number of fluorescent probes, so is high cost; PCR fluorescence method can not only detect mutations, but also precise quantitative the proportion of mutation strains in the virus population, in addition to similarto accuracy, specificity, less time-consuming, but has the highlight price advantage, so be suitable for the laboratory whose detection precision requirements are not too high.4) In160cases of poor response,62cases (38.75%)had only wild strains exist,and no mutants to be found, while37cases (23.13%) had neither YMDD wild strain nor mutants.61cases (38.13%) had been determined as YMDD mutation cases,in which26cases (16.25%) were detected as YVDD mutation,22cases (13.75%) were detected as YIDD mutation, the YVDD and YIDD mutation co-exist in5cases (3.13%),mixed infections of mutant and wild strains in8cases (5%).5) With time prolonged, the probability of variation was significantly increased. Do a comparative analysis of the mutation rate and the level of ALT levels and HBVDNA on baseline, we can find that the higher the HBVDNA level and the lower the ALT on baseline, the higher the chances of the YMDD mutations.Conclusions Although the effect of lamivudine for chronic hepatitis B were proved to be effective and safe by numerous tests, but with the course of the extension, variation problem becomes more serious. If you plan to determine the YMDD mutation as early as possible in order to take positive interventions, accurate means of detection is essential. In many detection methods, PCR fluorescence method is undoubtedly a time-saving, convenient, low cost, high accuracy, high specifical detection methods, more suitable for the primary clinical use. However, due to the small sample size of this experiment, compared with PCR reverse dot blot hybridization method, the accuracy is of no significant difference in PCR fluorescence method. We will expand the sample size, to do further research on the advantages and disadvantages of both.