The Strategies To Improve The Sensitivity Of Polymerase Chain Reaction For The Detection Of Hepatitis B Virus P Gene And HBV Genotypic Drug Resistance Monitoring During Adefovir Therapy

Antiviral treatment is the primary measure to improve the prognosis of patients with chronic hepatitis B.Antiviral therapy is the only option to control and prevent progression of disease in patients with chronic HBV infection.Nucleos(t)ide analogues have been proven to be effective in controlling the disease and perhaps decreasing the incidence of cirrhosis and cirohepatocellular carcinoma.However,the major problem of long-term Nucleos(t)ide analogues therapy is the occurrence of drug resistance.Drug resistance can emerge during the process of treatment,which leads to impaired sensitivity towards the appropriate antiviral therapy.Therefore,monitoring viral genotypic resistance in patients appears very important in guiding clinical administration,including switching for or adding on other nucleos(t)ide analogues. HBV genome mutation has been proven to be directly related to drug resistance.The emergence of HBV resistance mutations can be identified by genotype detection and all the new variation sites may be found by sequencing PCR products.In order to identify potential genotypic resistance,HBV DNA sequence obtained from virologic breakthrough patients should be compared to wild type sequence as well as the sequence before treatment,so as to conform the mutant sites with drug resistance.For a long time,the issue of false negative of PCR has been ignored.Many factors may be involved in false-negative results during PCR operation.To amplify the target sequences successfully,PCR procedure needs complex interplay of many factors,such as template requirements,primer,dNTPs,and DNA polymerase buffer and so on.Since HBV P gene sequence is a non-conservative region,false negative results would happen due to the mismatch between primers and their binding targets.Therefore,improving the sensitivity of PCR in detecting of HBV P gene is the premise to understand virus mutation,which is of great importance in monitoring drug resistance.In this study, comprehensive stratigies had been used to improve the sensitivity of PCR for the detection of HBV P gene.With the help of these strategies,we optimized a sensitive PCR procedure for amplifying the region encoding lamividine,adefovir and enticavir resistant substitutions.Taking advantages of this method,we also observed dynamic changes of HBV genotype drug resistance in patients treated with adefovir by DNA sequence analysis.PartⅠThe Strategies to Improve the Sensitivity of Polymerase Chain Reaction for the detection of Hepatitis B Virus P GeneObjective1.To improve the sensitivity of PCR for the detection of HBV P gene region.2.To find out the relationship between HBV genotypic resistance and clinical resistance.Methods1.Five strategies were applied to reduce the blight of the mismatch between primers and templates:(1) Comparing the recovery of HBV DNA by 7 methods for isolation of template HBV DNA,(2) optimizing PCR conditions,(3)Selecting the best primers,(4)Lowering annealing temperature,(5) To reduce the risk of 3'-terminal mismatch between primers and templates 3'-terminus shifted bases degeneracy primers were used.With the mentioned optimizing strategies,82 cases of chronic hepatitis B patients' serum samples were amplified again and the positive PCR products were sequenced.2.Serum HBV DNA from 372 cases of lamivudine resistance in different therapy stages were isolated and amplified.Then the positive PCR products were sequenced for monitoring HBV genotypic resistance.Results1.Among the 7 methods for isolation of template,four methods(serum boiling, protease plus Phenol:Chloroform:Isoamyl Alcohol with ethanol sedimentation,protease plus Phenol:Chloroform:Isoamyl Alcohol without ethanol sedimentation,DNA isolation kit) of template preparation were shown to be positive for HBV DNA isolation,with the recovery of 75.2%,13.8%21.9%and 31.0%respectively.HBV DNA cannot be detected when the templates are prepared as follows:alkaline-denatured,cleaved with protease and boiling,and without any preparation.Five to seven minutes serum boiling duration of the samples was the best choice to get maximum contents of template DNA for PCR.2.Under the conditions of this study,optimized PCR was identified as:using serum boiling for isolation of template HBV DNA,38 amplification cycles,consisting of 5 min at 94℃for predenaturation,30 s at 94℃for denaturing,30 s at 60℃for annealing,30 s at 72℃for extension,and 5 min at 72℃for final extension after the last cycle,were carried out in a 50μl final volume containing 5μl template,5μl 10×PCR buffer,1μl 4×dNTP(10mmol/L),12.5pmol prototype primers,1U Taq DNA polymerase.3.The sensitivity of PCR in detecting HBV P gene increased from 81.7%(67/82) to 92.7%(76/82)(P＜0.05) by using 3'-terminus shifted bases degeneracy primers and decreased the annealing temperature.4.Average administrated time of lamivudine in 372 patients is 30.35±6.82 months.339 samples from the 372 different serum samples were PCR positive for the detection of HBV P gene,and the positive rate was 91.1%.All positive amplicons were sequenced successfully and then compared to wild strains of YMDD gene sequence. YMDD mutants were detected in 193 strains(51.2%),of which M204I,M204V, M204V/I were 104(28.0%),68(18.3%) and 18(4.84%) respectively.Moreover,CVDD, YMDH and YMHD/YIHD mutant were also detected with 1 strain(0.27%) respectively.Conclusion1.Our results strongly suggested the method of HBV DNA isolation could be one of the factors to improve the sensitivity of PCR especially in quantitative analysis.With many advantages such as high recovery,less time and material consuming and much convenience,serum boiling isolation method may be the best choice of template preparation for PCR.2.Comprehensive methods including choosing primers,modifying primers, optimizing PCR conditions and decreasing the annealing temperature should be used to improve the sensitivity of PCR for the detection of variable region of HBV DNA.3.Higher proportion of HBV YMDD mutation exists in Lamivudine resistant patients.The common resistance mutations are M240I,M204V and M204V/I.PartⅡHBV Genotypic Drug Resistance Monitoring during Adefovir TherapyObjective1.To survey whether there is natural gene variation associated with drug resistant in patients suffered chronic hepatitis B and when it becomes predominant strains.2.To understand the relationship between genotype resistance and clinical resistance.3.To analyze the risk factors of genotypic drug resistance.MethodsA total of 56 naive patients treated with Hepsera^?(adefovir dipivoxil 10 mg,p.o, once daily) in this study were evaluated for antiviral efficacy and resistance.Samples at baseline,4^th week,12^th week,24^th week,36^th week and at 48^th(52^nd) week intervals during treatment were collected.Liver function,serum markers of HBV and HBV DNA level were tested for all samples.Using the optimized PCR procedure for amplifying the region encoding lamividine,adefovir and enticavir resistant substitutions,available isolates were amplified.To monitor for resistance,dynamic changes of HBV genotype drug resistance were monitored by DNA sequence analysis.ResultsIn the 56 patients,adefovir treatment resulted in patients of non-virologic response and virologic breakthroughs at week 48 were 2 and 12,respectively.The percentage of drug resistant patients was 25%(14/56).Sequencing showed that except for 1 case was found in the rt233 ATA-GTA subtitution,predominant strains of other cases were consistent with wild strains.However,at the end of 48-week treatment,the patient with variant strain acquired chemistry and virologic response.Other 4 patients were detected to have non-predominant variations,but the patients appeared in excellent virologic response.Fourteen cases of patients with non-virological response or virologic breakthrough were not found in any related substitution at both baseline and 48 weeks of treatment.Logistic regression showed that gender,age,baseline HBeAg status, baseline HBV DNA level and baseline ALT level were not effective predictors of adefovir resistance.ConclusionIdentified gene variation of adefovir resistance may not be the only reason for non-virological response or breakthrough.The mechanism of drag resistance needs more research.