Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B

INTRODUCTION It is estimated that 350 million individuals are chronically infected with hepatitis B virus (HBV) and that more than 1 million will die of liver cirrhosis and hepatocellular carcinoma (HCC) each year[1-3]. Lamivudine is an effective antiviral agent for patients with chronic hepatitis B and advanced liver diseases[4]. However, long-term lamivudine monotherapy induces emergence of lamivudine-resistant HBV mutants in some patients chronically infected with HBV[4,5]. Resist…     

Detection of YMDD mutants using universal template real-time PCR

AIM: To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection. METHODS: We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions (reaction V and reaction I) and total hepatitis B viruses were detected in another reaction for control (reaction C). Results were determined by deltaCt < 3.5 (deltaCt = Ct of reaction V or I - Ct of reaction C). Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing. RESULTS: As many as 1000 copies per milliliter of wide-type plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples. CONCLUSION: This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.     

A novel method based on ligase detection reaction for low abundant YIDD mutants detection in hepatitis B virus

A novel method based on ligase detection reaction (LDR) coupled with polymerase chain reaction (PCR) was used in this study to detect low abundant YIDD mutants in hepatitis B virus (HBV), which was able to detect 10² mutants from 10⁸ wild type copies in plasmids and 10³ from 10⁷ in clinical serum samples. Direct sequencing and microarray hybridization were selected for sensitivity comparison together with this assay. By direct sequencing, 6 of 50 clinical specimens infected with HBV were found to be YIDD and the others to be YMDD, while by microarray hybridization, 28 specimens were detected to contain both YMDD and YIDD, and the others contained YMDD only, and with this assay, 31 specimens containing both YMDD and YIDD were detected with the others containing YMDD only, which indicated that the assay was relatively more sensitive.     

基因芯片高通量检测乙型肝炎病毒拉米夫定耐药株及对其突变热点的分析

目的 通过大规模、多位点检测深圳地区拉米夫定耐药株，进一步了解拉米夫定耐药突变的各种分布状况。方法 用基因芯片法对552份乙型肝炎患者血清进行检测，得出l92份拉米夫定耐药突变标本，再对192份耐药标本检测结果进行分析。结果 192份耐药标本中，191份YMDD突变，124份528位点突变，9份555位点突变。YMDD突变中88％为：YVDD、528位点同时突变；YIDD单独突变；YIDD、528位点同时突变。YMDD突变密码子91％为：GTG、ATT；9％为：ATA、ATC。结论 552位点(YMDD)突变为核心突变，528、555位点的突变为协同突变。YVDD突变总是与528位点同时出现；YIDD突变则表现为多样化。YMDD突变密码子约有9％为少见密码子ATA、ATC，这可能是传统聚合酶链反应法检测YMDD突变阳性率较低的原因。     

慢性乙型肝炎病毒感染者病毒YMDD的自然变异

目的 了解慢性HBV感染患者外周血HBV YMDD自然变异情况及其影响因素.方法 采用引物特异性实时荧光PCR法检测慢性HBV感染者外周血HBV YMDD变异情况,并对影响YMDD自然变异检出率的可能因素进行单因素及多因素分析.根据不同资料分别采用χ~2检验、Fisher's确切概率法、t检验、秩和检验及Logistic回归分析进行统计学处理. 结果在196例未经抗病毒治疗的慢性HBV感染者中,检出存在YMDD自然变异株感染者21例(10.70%),其中YVDD阳性20例,YIDD阳性例1变;变异毒株占总病毒株超过50%者1例,25%～500者5例,9%～25%者15例.B基因型HBV感染病例中YMDD变异株的检出率(20.00%,12/60)显著高于C基因型HBV感染病例(7.38%,9/122),χ~2=6.28,P<0.05.患者性别、年龄、HBeAg状态、HBVDNA载量、疾病状态、病毒感染时间对YMDD自然变异株的检出率无显著影响. 结论 在未经抗病毒治疗的慢性HBV感染者中存在HBV YMDD自然变异;YMDD自然变异的发生率与患者性别、年龄、HBeAg状态,HBV DNA载量、疾病状态、感染时间无显著相关性.B基因型较C基因型HBV更易出现YMDD自然变异.     

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

INTRODUCTIONHepatitis B virus (HBV) is one of the most common infectious diseases in the world. More than 300 million people worldwide are estimated to have chronic HBV infection. Ten percent of these patients will die as a direct consequence of persistent viral infection[1]. Nucleoside analogue therapy allows safe, long-term suppression of HBV and is a major milestone in the treatment of chronic hepatitis B. Lamivudine, the f irst of these agents approved worldwide, effectively supp…     

基因芯片和聚合酶链反应-限制性片段长度多态性法检测拉米夫定耐药的慢性乙型肝炎患者YMDD变异

目的对基因芯片和聚合酶链反应-限制性片段长度多态性(PCR-RFLP)两种方法检测YMDD变异进行比较。方法取40例临床考虑拉米夫定耐药的患者血清，分别用基因芯片和PCR-RFLP法检测YMDD基序变异情况。随机抽取8份血清标本进行测序验证两种方法的准确性。结果40例患者血清标本中，用基因芯片法检测出YMDD变异阳性标本27例，其中，YIDD变异4例，YVDD变异16例，YVDD／YMDD共存12例；YIDD与YVDD混合变异7例。用PCR-RFLP法检测出变异阳性标本21例。在变异株中，YIDD变异和YVDD变异各9例，YIDD／YVDD混合变异3例，明显低于基因芯片的检出率。结论1、用基因芯片能快速灵敏地检测YMDD变异情况，并能检测到患者体内变异株与野生株混合存在形式；2、基因芯片能同时检测两种YMDD变异株和YMDD野生株，优于PCR-RFLP法，适用于临床检测乙型肝炎病毒YMDD变异。     

三种检测乙型肝炎病毒YMDD自然变异方法的比较

目的筛选一种特异性及灵敏性较高的检测乙型肝炎病毒YMDD自然变异的方法。方法通过分子克隆的方法,构建YMDD、YVDD及YIDD三种质粒;通过检测不同比例混合的质粒,比较PCR产物直接测序法、单探针特异性荧光PCR技术及引物特异性荧光PCR技术三种检测方法。结果经酶切鉴定及直接测序鉴定,显示三种质粒构建成功;当待测模板占总模板的比例大于20%时,直接测序法才能准确检测;当突变株占总毒株的比例占50/51时,单探针特异性荧光PCR法才能判定该样品为YMDD变异型;当突变株占总毒株的比例高于1/11时,引物特异性荧光PCR技术非特异结合的影响可忽略,具有很好的特异性,同时具有较高的灵敏性,检测突变株占总毒株的最低比例可达到1/11。结论引物特异性荧光PCR技术特异性及灵敏性较高,适合用于乙型肝炎病毒YMDD自然突变株的检测。     

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM： To construct eukaryotic expression plasmids of full-length Hepatitis B Virus （HBV） genotype C genome, which contain lamivudine-resistant mutants （YIDD, YVDD） or wild-type strain （YMDD）, and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg）] of the recombinant plasmids in HepG2 cells. METHODS： Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 （＋）, between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.
RESULTS： Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.
CONCLUSION： Eukaryotic expression plasmids pcDNA3.1 （＋）-HBV/YIDD, pcDNA3.1 （＋）-HBV/YVDD or pcDNA3.1 （＋）-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.     

拉米夫定治疗中乙型肝炎病毒YMDD野毒株和变异株的变化

目的 观察慢性乙型病毒性肝炎(CHB)患者应用拉米夫定治疗前后,YMDD野毒株及变异株的动态变化,并分析其临床意义。方法 取5例CHB患者治疗前与治疗48周的10份血清标本,先用PCR法扩增包括YMDD基序的乙型肝炎病毒(HBV)部分核苷酸序列,然后进行DNA序列测定,同时分别进行克隆,每份标本随机挑选20～24株单克隆,用实时荧光PCR法检测YMDD野毒株及其变异株。结果 5例CHB患者治疗前PCR产物直接测序结果未检出YMDD变异,治疗48周时有4例检出YMDD变异,但对每株克隆的分别检测显示：治疗前YMDD变异株(YIDD／YvDD)所占比率分别为0％、9．5％、0％、4．5％、5．6％;治疗48周时所占比率分别为100％、100％、65％、100％、0％。其中1例患者治疗前检出YIDD变异株,而治疗48周时YVDD变异株则变成优势株。治疗52周时,4例YMDD变异患者中2例HBVDNA和血清丙氨酸转氨酶(ALT)突破,1例患者ALT突破,但HBVDNA为阴性。结论 YMDD变异株在拉米夫定治疗前的血清中已存在,在服用拉米夫定后,由于选择性抑制了野毒株,使YMDD变异株由弱势株变成优势株,部分患者可导致拉米夫定临床耐药。YVDD变异株可能比YIDD变异株的复制能力强。     

Emergence of YMDD motif mutant of hepatitis B virus during short-term lamivudine therapy in South Korea

BACKGROUND/AIMS: The emergence of a YMDD mutant resistant to lamivudine therapy has been reported in patients with hepatitis B treated with long-term lamivudine therapy. However, it is not well known whether the YMDD mutant could be detected early in lamivudine therapy in hepatitis B virus (HBV) endemic areas. The aim of this study was to investigate the emergence of the YMDD mutant during short-term lamivudine therapy in South Korea. METHODS: We prospectively investigated the emergence of the YMDD mutant by the nested PCR assay using restriction fragment length polymorphism in 28 patients with chronic hepatitis B who were treated with 100 mg of lamivudine daily for 12 weeks. RESULTS: The YMDD mutant was detected in 17 (60.7%) out of 28 patients at week 12, and the only type of mutation found was the YIDD mutation. When we carried out the nested PCR serially in five patients, YIDD mutants were detected as early as 2 weeks by the nested PCR assay. The nested PCR results were in concordance with DNA sequencing in one patient's serial samples. CONCLUSIONS: YMDD mutants in HBV were detected within a few weeks during lamivudine therapy in South Korea, which suggests that the YMDD mutant may exist even before lamivudine therapy in HBV endemic areas.     

Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers

BACKGROUND/AIM: It is well documented that long-term lamivudine treatment induces emergence of lamivudine-resistant hepatitis B virus (HBV), namely, YMDD motif mutation in some patients chronically infected with HBV. We previously reported that there were no YMDD mutant viruses in patients with chronic hepatitis B who were not treated with lamivudine. In this series, we examined mutations in the YMDD motif gene in asymptomatic carriers who maintained normal ALT values for 1 year or more. METHODS: Serum samples obtained from 18 patients chronically infected with HBV who consulted our university were used. None of these patients had any experience of using antiviral agents. For detection of mutant viruses, a kit developed in our laboratory was used. RESULTS: Mutations were detected in five of 18 samples: YMDD+YIDD in three samples and YMDD+YVDD+YIDD in two samples. All of these five samples were positive for anit-HBe. In five samples in which mutations were observed, sequencing was carried out following subcloning. CONCLUSION: The present study demonstrates that YMDD mutant viruses are present in lamivudine-untreated asymptomatic hepatitis B virus carriers as well.     

拉米夫定治疗慢性乙型肝炎过程中HBV YMDD变异及基因芯片检测

建立乙型肝炎病毒变异基因诊断芯片对拉米夫定治疗慢性乙型肝炎过程中出现的肝炎病毒P基因区YMDD变异进行快速准确的检测方法。设计特异性寡核苷酸探针数组 ,特殊处理芯片载体。用点样法制备乙型肝炎病毒变异基因诊断芯片。在本院住院治疗病人中选择 3 0例服用拉米夫定后 ,可能出现YMDD变异的病人进行基因芯片杂交检测分析 ;同时用PCR直接测序法对上述 3 0例病人血清标本进行双盲HBVDNA聚合酶活性区域测序对照。 3 0例服药后HBVDNA反跳病人中 ,基因芯片测得HBVYMDD变异 2 1例 ,其中YVDD变异 11例。YIDD变异 10例。HBVDNAPCR直接序列测定结果与基因芯片检测结果完全一致。乙型肝炎病毒变异基因诊断芯片可以同时检测YVDD、YIDD变异 ,同PCR直接测序法比较 ,准确率达 10 0 % ,无假阳性     

YMDD耐药变异与HLA等位基因多态性的相关性

目的：初步探讨慢性乙型肝炎(CHB)患者拉米夫定治疗中YMDD变异与HLA-A,B,DRB1各位点等位基因分布频率的相关性．方法：对142例CHB患者,采用荧光标记杂交双探针PCR融解曲线法(FH-PCR-MC)检测血浆HBV YMDD变异；对其中56例患者的外周血白细胞,采用序列特异性引物/聚合酶链式反应(PCR-SSP)技术检测人类白细胞表面抗原等位基因(HLA-A-B,DRB1)分型．结果：在用拉米夫定治疗的142例CHB患者中,YMDD变异率为56．3％．HLA-B~*58和DRB1~*03等位基因分布频率在YMDD变异组与YMDD野生组比较有显著性降低(0．013 vs 0．094,P=0．036；0．000 vs 0．063,P=0．024)；HLA-A~*30等位基因分布频率在YIDD组明显增高,与YVDD组比较差异显著(0．158 vs 0．024,P=0．034)；HLA-A~*33等位基因分布频率在YVDD变异组明显增高,与YIDD变异组比较差异显著(0．119 vs 0．000,P=0．028)．结论：YMDD耐药变异与HLA等位基因多态性有一定相关性．携有HLA-B~*58和DRB1~*03等位基因的个体感染的HBV可能不易发生YMDD变异；携有HLA-A~*30等位基因的个体感染的HBV可能易发生YIDD变异：携有HLA-A~*33等位基因的个体感染的HBV可能易发生YVDD变异．     

慢性乙型肝炎患者拉米夫定治疗24周时YMDD变异对疗效的影响

目的 使用基因芯片方法检测乙型肝炎病毒(HBV)YMDD变异的发生情况,研究YMDD变异发生与肝功能损伤和HBV复制水平指标之间的关系。方法 120例以常规剂量(100mg/d)口服拉米夫定的慢性乙型肝炎患者,治疗前和治疗第24周抽取血清检测丙氨酸氨基转移酶(ALT)、HBV DNA(定量)水平,对24周HBV DNA阳性的17例患者血清样本,以基因芯片方法检测其治疗前和治疗24周时血清中YMDD变异是否存在,并分析该变异发生和ALT、HBV DNA的关系。结果 (1)120例入选患者治疗24周时有17例患者HBV DNA仍为阳性,除外治疗前已存在的1例感染变异病毒,基因芯片共检出7例变异,变异率为5.8％。其中纯变异2例,YIDD变异1例,YVDD变异1例,混合变异5例,其中YMDD/YVDD变异3例,YVDD/YMDD变异2例。(2)变异组在治疗前和治疗24周时ALT定量水平与非变异组相比,差异无显著性,P>0.05。(3)变异组在治疗前和治疗24周时HBV DNA定量水平与非变异组相比,差异无显著性,P>0.05。结论 在拉米夫定治疗过程中,YMDD基因变异发生对肝脏炎症活动度和病毒复制的抑制作用无显著影响。     

Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment

BACKGROUND/AIMS: The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant.METHODS: We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients).RESULTS: We could detect as little as 0.01-0.001% of mutant viruses coexisting in 10(5)-10(9) copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients.CONCLUSIONS: We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.     

Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization

AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system.The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and Rl10 or TAMRA labeled acyclo-terminator was added on the 3‘ end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed. RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9 %), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDDphenotype (7.1%). CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.     

拉米夫定耐药突变相关慢性重型乙型肝炎27例分析

目的 研究在拉米夫定治疗慢性乙型肝炎的过程中出现拉米夫定耐药突变相关的慢性重型乙型肝炎的临床特点.方法 回顾性分析27例在拉米夫定治疗过程中出现耐药突变的慢性重型乙型肝炎病例,分析其临床特征,用基因芯片法或基因测序法检测YMDD突变类型,对8例施行肝移植术后的离体肝组织分析其病理特点.采用χ2检验进行统计学分析.结果 27例拉米夫定耐药患者的YMDD突变类型分别为:YVDD突变5例,YVDD突变+L180M突变2例,YIDD突变13例,YIDD+L180M突变4例,YVDD突变+YIDD突变1例,YVDD突变+Y1DD突变+L180M突变2例,单独L180M突变未检出.根据治疗前是否存在肝硬化,分为肝硬化组和非肝硬化组.与肝硬化组相比,非肝硬化组慢性重型乙型肝炎的发生率低,预后好,年龄小,HBeAg阳性率高.8例离体肝组织病理分析显示两种主要病理类型,一种主要表现为活动性肝硬化,另一种为大块或亚大块坏死,肝脏显著萎缩.结论 肝硬化为拉米夫定耐药突变相关的慢性重型乙型肝炎的高危因素.拉米夫定耐药突变相关的慢性重型乙型肝炎可能存在两种发病机制.