提高乙型肝炎病毒P基因区PCR检测阳性率的策略及阿德福韦酯治疗中HBV基因型耐药临床监测研究
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摘要
抗病毒治疗是改善慢性乙型肝炎患者预后、提高生活质量的主要措施。核苷(酸)类似物的临床应用极大地改善了病人的病情。理论上,所有口服核苷(酸)类似物在治疗过程中均可出现耐药性,HBV对核苷类抗病毒药物产生的耐药性是阻碍其临床应用、降低其疗效的主要原因。因此在慢性HBV感染抗病毒治疗中进行耐药基因监测对于指导临床用药,包括撤换或增加其他核苷(酸)类似物十分重要,倍受临床重视。HBV基因变异已经被证明与HBV的耐药性有直接的因果关系。HBV变异的出现可通过基因型检测来诊断,而PCR产物直接测序的方法能够鉴定已知的并发现新的变异位点。为了鉴定潜在的基因型耐药,在病毒学突破时分离提取的HBV DNA序列及其推导出的氨基酸序列需与患者治疗前标本的序列和野毒株序列相对比以确定导致对某种抗病毒药物耐药的位点替换。
长期以来,人们对PCR假阴性问题认识不足。在实际应用中有许多因素可能造成假阴性结果,要成功地得到靶序列的扩增,PCR要求模板、引物、dNTP、DNA聚合酶和缓冲液之间的无数复杂相互作用。由于HBV DNA的P基因区是非保守区,检测时可能会由于引物结合区的碱基的变异而出现假阴性结果。因此提高HBVP基因区PCR的检测阳性率,是了解病毒变异情况的前提,它对在慢性HBV感染抗病毒治疗中进行耐药基因监测具有十分重要的意义。本研究采用一些综合措施提高了HBV DNA P基因区PCR检测的阳性率。在此基础上,建立了包含有拉米夫定、阿德福韦及恩替卡韦耐药相关突变位点碱基P基因区的PCR检测方法,并以阿德福韦酯治疗乙肝病人为研究对象,扩增病人不同治疗观察点血清HBVDNA,进行DNA序列分析,动态监测HBV基因型耐药变化。
第一部分提高乙型肝炎病毒P基因区PCR检测阳性率的策略
目的提高HBV DNA P基因区PCR检测的阳性率,了解病毒基因型耐药与临床耐药之间相互关系。
方法(1)通过采用①比较7种HBV DNA抽提技术的DNA抽提得率,②优化PCR反应条件,③选择最佳引物,④降低退火温度,⑤3′末端碱基游移兼并引物以减少引物与模板引物结合区的错配对PCR的影响等五种方法,对常规PCR检测HBV DNA P基因区阴性的病人血清再进行PCR检测。(2)以372例拉米夫定治疗失败乙肝病人为研究对象,扩增病人血清HBV DNA,进行DNA序列分析,监测HBV基因型耐药变化。
结果①在7种抽提方法中血清直接沸水浴、经典的蛋白酶裂解+酚/氯仿抽提法、蛋白酶裂解+酚/氯仿抽提法省缺乙醇沉淀以及柱抽提法的HBV DNA抽提得率分别为75.2%、13.8%、21.9%、31.0%;用碱变性裂解和蛋白酶裂解后沸水浴所得上清,以及血清直接作为模板,PCR后不能得到阳性扩增条带;沸水浴时间在5～7分钟能获得最多的PCR产物。②本研究条件下,优化的PCR反应条件确定为:采用直接沸水浴法抽提血清HBV DNA;模板上样量5μl,10×PCR缓冲液5μl,MgCl_2(25mmol/L)3μl,4×dNTP(10mmol/L)1μl,上、下游引物各12.5pmol,Taq DNA聚合酶1U,用去离子水补充至总体积50μl;PCR反应过程:94℃预变性5分钟;94℃变性30秒,53℃退火30秒,72℃延伸45秒,共38循环;72℃再延伸5分钟。③通过降低退火温度及减少3′末端错配,PCR检测阳性率由81.7%(67/82)增加至92.7%(76/82)(P＜0.05)。④372例病人拉米夫定平均治疗时间为30.35±6.82月,来自不同病人的372份血清标本中P基因区PCR阳性339份,阳性率91.1%;所有阳性标本均测序成功。测序结果经与野生株YMDD基序基因比对后,共检出YMDD变异193株(51.2%),其中M240I 104株(28.0%),M204V 68株(18.3%),M204V/I混合变异株18株(4.84%),CVDD(半胱氨酸-缬氨酸-天门冬氨酸-天门冬氨酸)1株(0.27%),YMDH(酪氨酸-蛋氨酸-天门冬氨酸-组氨酸)1株(0.27%),YMHD/YIHD(酪氨酸-异亮氨酸-天门冬氨酸-组冬氨酸)混合变异1株(0.27%)。
结论①核酸抽提方法选择不当能直接影响基因检测的灵敏度,导致基因定量准确性下降。血清直接沸水浴法HBV DNA得率高、操作简便、省时、经济,值得推荐。②综合采用优化PCR反应条件,选择引物,降低退火温度以及应用3′末端碱基游移兼并引物等方法可提高基因高突变区PCR检测阳性率。③拉米夫定治疗失败乙肝病人存在较高比例HBV YMDD基序变异,最常见变异为M240I、M204V和M204V/I混合变异株。
第二部分阿德福韦酯治疗中HBV基因型耐药临床监测研究
目的观察阿德福韦酯治疗慢性乙肝病人是否存在相关基因自然变异和变异病毒株处于优势地位的发生时间,以期了解基因型耐药与临床耐药之间相互关系,并对基因型耐药发生的相关危险因素进行分析。
方法56例口服贺维力(阿德福韦酯片10mg,每日一次)病人,在治疗前(基线)、治疗第4周、第12周、第24周、第36周、第48(或52)周分别采集血清标本。标本进行肝功能全套、HBV血清标志物检测,HBV DNA定量检测。HBV DNA阳性者,采用已建立的包含有拉米夫定、阿德福韦及恩替卡韦等已知耐药相关突变位点碱基HBV P基因区的PCR检测方法,动态监测病毒基因型耐药变化。
结果①阿德福韦治疗48周时,56例病人中出现病毒学无应答病人2例,病毒学突破病人12例,共占被研究人数25%(14/56)。②56例病人基线血清均获得PCR阳性结果,并测序成功。测序结果显示:除1例发生rt233A ATA-GTA的碱基置换外,所有病例基线优势病毒株均与野生株一致,48周治疗结束时,该病人获得完全生化学和病毒学应答;基线时,4例病人存在相关位点的氨基酸变异非优势株,但病人未出现病毒学无应答或病毒学突破。③出现病毒学无应答或病毒学突破的14例病人,不论在基线还是在治疗48周后均未检测到已知相关位点碱基变异。④Logistic逐步回归表明,患者性别、年龄、HBeAg状态、基线HBV DNA水平和基线ALT水平都不是有效预测阿德福韦耐药的因素。
结论已知相关耐药基因变异不是阿德福韦治疗病人发生病毒学无应答或病毒学突破唯一原因,其发生机制有待进一步研究。
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 Gene
Objective
1.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.
Methods
1.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.
Results
1.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.
Conclusion
1.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 Therapy
Objective
1.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.
Methods
A 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.
Results
In 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.
Conclusion
Identified 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.
长期以来,人们对PCR假阴性问题认识不足。在实际应用中有许多因素可能造成假阴性结果,要成功地得到靶序列的扩增,PCR要求模板、引物、dNTP、DNA聚合酶和缓冲液之间的无数复杂相互作用。由于HBV DNA的P基因区是非保守区,检测时可能会由于引物结合区的碱基的变异而出现假阴性结果。因此提高HBVP基因区PCR的检测阳性率,是了解病毒变异情况的前提,它对在慢性HBV感染抗病毒治疗中进行耐药基因监测具有十分重要的意义。本研究采用一些综合措施提高了HBV DNA P基因区PCR检测的阳性率。在此基础上,建立了包含有拉米夫定、阿德福韦及恩替卡韦耐药相关突变位点碱基P基因区的PCR检测方法,并以阿德福韦酯治疗乙肝病人为研究对象,扩增病人不同治疗观察点血清HBVDNA,进行DNA序列分析,动态监测HBV基因型耐药变化。
第一部分提高乙型肝炎病毒P基因区PCR检测阳性率的策略
目的提高HBV DNA P基因区PCR检测的阳性率,了解病毒基因型耐药与临床耐药之间相互关系。
方法(1)通过采用①比较7种HBV DNA抽提技术的DNA抽提得率,②优化PCR反应条件,③选择最佳引物,④降低退火温度,⑤3′末端碱基游移兼并引物以减少引物与模板引物结合区的错配对PCR的影响等五种方法,对常规PCR检测HBV DNA P基因区阴性的病人血清再进行PCR检测。(2)以372例拉米夫定治疗失败乙肝病人为研究对象,扩增病人血清HBV DNA,进行DNA序列分析,监测HBV基因型耐药变化。
结果①在7种抽提方法中血清直接沸水浴、经典的蛋白酶裂解+酚/氯仿抽提法、蛋白酶裂解+酚/氯仿抽提法省缺乙醇沉淀以及柱抽提法的HBV DNA抽提得率分别为75.2%、13.8%、21.9%、31.0%;用碱变性裂解和蛋白酶裂解后沸水浴所得上清,以及血清直接作为模板,PCR后不能得到阳性扩增条带;沸水浴时间在5～7分钟能获得最多的PCR产物。②本研究条件下,优化的PCR反应条件确定为:采用直接沸水浴法抽提血清HBV DNA;模板上样量5μl,10×PCR缓冲液5μl,MgCl_2(25mmol/L)3μl,4×dNTP(10mmol/L)1μl,上、下游引物各12.5pmol,Taq DNA聚合酶1U,用去离子水补充至总体积50μl;PCR反应过程:94℃预变性5分钟;94℃变性30秒,53℃退火30秒,72℃延伸45秒,共38循环;72℃再延伸5分钟。③通过降低退火温度及减少3′末端错配,PCR检测阳性率由81.7%(67/82)增加至92.7%(76/82)(P＜0.05)。④372例病人拉米夫定平均治疗时间为30.35±6.82月,来自不同病人的372份血清标本中P基因区PCR阳性339份,阳性率91.1%;所有阳性标本均测序成功。测序结果经与野生株YMDD基序基因比对后,共检出YMDD变异193株(51.2%),其中M240I 104株(28.0%),M204V 68株(18.3%),M204V/I混合变异株18株(4.84%),CVDD(半胱氨酸-缬氨酸-天门冬氨酸-天门冬氨酸)1株(0.27%),YMDH(酪氨酸-蛋氨酸-天门冬氨酸-组氨酸)1株(0.27%),YMHD/YIHD(酪氨酸-异亮氨酸-天门冬氨酸-组冬氨酸)混合变异1株(0.27%)。
结论①核酸抽提方法选择不当能直接影响基因检测的灵敏度,导致基因定量准确性下降。血清直接沸水浴法HBV DNA得率高、操作简便、省时、经济,值得推荐。②综合采用优化PCR反应条件,选择引物,降低退火温度以及应用3′末端碱基游移兼并引物等方法可提高基因高突变区PCR检测阳性率。③拉米夫定治疗失败乙肝病人存在较高比例HBV YMDD基序变异,最常见变异为M240I、M204V和M204V/I混合变异株。
第二部分阿德福韦酯治疗中HBV基因型耐药临床监测研究
目的观察阿德福韦酯治疗慢性乙肝病人是否存在相关基因自然变异和变异病毒株处于优势地位的发生时间,以期了解基因型耐药与临床耐药之间相互关系,并对基因型耐药发生的相关危险因素进行分析。
方法56例口服贺维力(阿德福韦酯片10mg,每日一次)病人,在治疗前(基线)、治疗第4周、第12周、第24周、第36周、第48(或52)周分别采集血清标本。标本进行肝功能全套、HBV血清标志物检测,HBV DNA定量检测。HBV DNA阳性者,采用已建立的包含有拉米夫定、阿德福韦及恩替卡韦等已知耐药相关突变位点碱基HBV P基因区的PCR检测方法,动态监测病毒基因型耐药变化。
结果①阿德福韦治疗48周时,56例病人中出现病毒学无应答病人2例,病毒学突破病人12例,共占被研究人数25%(14/56)。②56例病人基线血清均获得PCR阳性结果,并测序成功。测序结果显示:除1例发生rt233A ATA-GTA的碱基置换外,所有病例基线优势病毒株均与野生株一致,48周治疗结束时,该病人获得完全生化学和病毒学应答;基线时,4例病人存在相关位点的氨基酸变异非优势株,但病人未出现病毒学无应答或病毒学突破。③出现病毒学无应答或病毒学突破的14例病人,不论在基线还是在治疗48周后均未检测到已知相关位点碱基变异。④Logistic逐步回归表明,患者性别、年龄、HBeAg状态、基线HBV DNA水平和基线ALT水平都不是有效预测阿德福韦耐药的因素。
结论已知相关耐药基因变异不是阿德福韦治疗病人发生病毒学无应答或病毒学突破唯一原因,其发生机制有待进一步研究。
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 Gene
Objective
1.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.
Methods
1.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.
Results
1.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.
Conclusion
1.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 Therapy
Objective
1.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.
Methods
A 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.
Results
In 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.
Conclusion
Identified 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.
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