基因芯片技术检测在乙型肝炎病毒基因突变分析中的应用

目的:探讨DNA芯片在乙型肝炎病毒基因突变分析中的应用及临床意义。方法:用基因多态性检测芯片检测HBV DNA阳性慢性乙型肝炎(CHB)患者血清前C区1896/1814位点、基本核心启动子区(BCP区)1762/1764位点及P区528/552位点突变情况,并与血清转氨酶、血清HBV DNA复制程度、血清e系统进行对比分析。结果:未接受抗病毒药物拉米夫定治疗的12例HBsAg( )、HBeAg( )、HBeAb(-)的患者血清中未检测到病毒变异,其他患者中未检测到P区基因变异;30例接受拉米夫定治疗48-96周后的HBV DNA阳性患者中,有19例患者检测到YMDD变异,并出现血清转氨酶升高,HBV DNA复制再度活跃。结论:前C区、BCP区突变较多的出现在HBeAb( )的患者中并与HBeAg的阴性表型相关,BCP区突变与HBeAg/HBeAb血清学转换密切相关,而P区528/552位点突变更多的出现在接受拉米夫定治疗后的患者中,与拉米夫定耐药相关且加重肝损害;基因芯片技术检测乙型肝炎病毒多位点变异对临床判断病情具有一定的参考意义。     

乙型肝炎病毒YMDD及e抗原相关多重变异及其临床意义

目的 研究拉米夫定治疗慢性乙型肝炎期间HBVYMDD基序、影响HBeAg分泌的多重变异情况与临床的关系。方法 采用基因芯片技术对拉米夫定治疗9～30个月的慢性乙型肝炎患者进行YMDD基序、G1896A、A1814C、A1762T和G1764A(BCP双突变)单碱基变异检测。结果 102例慢性乙型肝炎患者拉米夫定平均治疗18个月时,22例发生YMDD变异,其中8例发生多重变异,包括G1896A3例、A1814C2例、G1896A A1814C、BCP双突变、BCP双突变 G1896A多重变异各1例,单纯YMDD变异和前5例联合变异均为HBeAg阳性,而后3例多重变异则为HBeAg阴性,其中1例多重变异继续治疗3个月后转变为单纯YMDD野生株阳性,同时伴有HBeAg的复阳。结论 拉米夫定治疗过程中存在YMDD及HBeAg相关多重变异的优势病毒株可能是HBVDNA复阳、同时伴有HBeAg阴转的原因之一,拉米夫定治疗过程中,HBeAg阴性时应监测其可能的相关变异。     

拉米夫定治疗前后乙型肝炎病毒P区基因的动态变化

目的研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒P基因区的变异情况。方法从5例慢性乙型肝炎患者拉米夫定治疗前和治疗12个月的血清标本中，扩增目的片段，阳性结果双酶切后克隆至JM105感受态细胞。每份标本随机挑取20个阳性克隆，以错配聚合酶链反应一限制性片段长度多态性分析法检测YMDD基因序列，出现变异者进行双向测序。结果5例慢性乙型肝炎患者在拉米夫定治疗12个月后，其中2例HBVDNA为阴性，2例HBVDNA转阴后又转阳，测序结果提示出现M5521变异；1例HBVDNA始终阳性，和治疗前一样存在D553G的变异。结论D553G变异可能是慢性乙型肝炎患者拉米夫定治疗无效的原因之一，拉米夫定治疗后出现的乙型肝炎病毒基因组P区YMDD变异是抗病毒药物诱导的结果。     

乙型肝炎病毒基因型、YMDD变异与拉米夫定治疗后HBV DNA反弹关系的研究

目的探讨HBV基因型、YMDD变异与拉米夫定抗病毒治疗后HBV DNA反弹的关系。方法应用多引物对巢式PCR法、PCR-序列分析法检测拉米夫定治疗的27例乙型肝炎患者和19例从未用过抗病毒治疗的患者HBV基因型和P区（YMDD）的突变位点。结果在27例HBV DNA反弹的患者中，13例（48．15％）检出YMDD变异，而对照人群无YMDD变异（P〈0．05）。YMDD变异的位点为rtM204V／I（C区）±rtL180M（B区）；在治疗组YMDD变异的患者中，B、C基因型构成比（46．15％和59．26％）与对照组（53．85％和68．42％）比较无显著性差异（P〉0．05）。结论YMDD变异是拉米夫定治疗后出现耐药导致HBV DNA反弹的主要原因；YMDD变异的常见位点依然为rtM204V／I（C区）±rtL180M（B区）；YMDD变异在B、C基因型病人中无差别。     

拉米夫定治疗慢乙肝的长期疗效及安全性评价

研究拉米夫定（lamivudine)对慢性乙型肝炎（慢乙肝）的长期疗效和安全性。49例患者均用拉米夫定100mg/d，疗程1－2年。疗效评估包括临床症状和体征、肝功能、HBV复制的指标。治疗3－6个月时，HBV－DNA阴转率为87．8％－75．5％，2年时降至40．9％。疗程结束时HBeAg的血清转换率为11．8％。6个月时ALT复常率为79．6％。疗程结束时ALT复常率为59．1％。拉米夫定治疗6－9个月后可出现YMDD变异，随着疗程的延长YMDD变异率升高，YMDD变异后病情急性发作者，多出现HBeAg的血清转换。拉米夫定能有效抑制HBV复制，迅速降低血清中HBV－DNA水平，提高HBeAg血清转换率，长期服用耐受性好，但疗程越长YMDD变异率越高。     

乙型肝炎病毒多聚酶变异与拉米夫定治疗反应

目的 探讨乙型肝炎病毒（HBV）多聚酶酪氨酸－蛋氨酸－天氧氨酸－天冬氨酸（YMDD）基序变异对拉米夫定抗病毒疗效的影响。方法 对19例拉米夫定治疗（100mg/d）48周时血清病毒核酸阳性的慢性乙型肝炎患者,采用聚合酶链反应（ PCR）产物直接测序技术,检测其血清中HBV多取和酶（YMDD）变异情况,并观察其血清HBV DNA和丙氨酸转氨酶（ALT）水平。结果 在检出YMDD变异株的10 例患者（4例为YV^550DD型,均伴L^526→M突变,6例为YI^550DD型）,至52周为止,共5例出现HBV DNA突发（分枝DNA信号扩增法）,其中2例伴ALT再高。追踪其治疗前血清均未发生上述变异。而在未检出YMDD变异的9例患者中,1例出现DNA突发后又阴转,而另1例治疗过程中HBV DNA未阴转、ALT始终波动于正常值上下的患者,发现多聚酶其他部位的变异^473→R,D^477→N,D^480→N,L^491→M)治疗前后相同。结论 乙型肝炎病毒多聚酶（ YMDD）变异与药物诱导相关,发生变异者较无变异者疗效降低;在拉米夫定治疗过程中,该变异的检测将有助于其疗效监测。     

拉米夫定治疗前后乙型肝炎病毒YMDD变异的相关因素分析

目的 了解遵义地区HBV基因型以及拉米夫定治疗前后发生YMDD变异的相关因素,及早进行拉米夫定疗效及耐药的预测. 方法 53例慢性乙型肝炎患者分别在口服拉米夫定前及治疗后3、6、12、18、24个月进行血清HBV DNA定量、乙型肝炎标志物、ALT、AST、总胆红素,白蛋白的检测.同时在接受拉米夫定治疗前采用基因测序法检测HBV基因型及YMDD变异株,治疗后HBV DNA定量下降又反弹升高,且血清HBV DNA＞1×104拷贝/ml时,再次进行YMDD变异株检测.率的比较用卡方检验及确切概率法,两组均数之间比较采用独立样本t检验,有序变量之间的比较采用秩和检验.结果 遵义地区的HBV基因型由B、C及B+C基因型构成.拉米夫定治疗后18例检出YMDD变异株,用药1年和2年的变异率分别为15.1%和34.0%.HBV突变类型有rtL180M/M204V、rtL180M/M204I、rtM204I和rtL180M四种,其中C区rtM204V全部合并rtL180M突变(100%),C基因型中rtL180M/M204V联合突变及rtL180M/M204I联合突变明显高于B基因型(77.8%比25.0%及22.2%比12.5%);C基因型中未见点突变,而rtM204I、rtL180M的点突变仅见于B基因型.YMDD变异与未变异组性别、民族、乙型肝炎家族史及HBeAg情况差异无统计学意义(P＞0.05),病程≥2年组和年龄＜35岁组变异率明显升高(X2值分别为4.707和5.853,P值均＜0.05).不同HBV DNA滴度患者YMDD变异率差异无统计学意义(X2=0.801,P＞0.05),但HBV DNA＜105拷贝/ml者未发现YMDD变异.结论 拉米夫定治疗后YMDD变异可能与HBV基因型及P基因突变类型有关,并随治疗时间的延长而增加.为了减少YMDD变异的发生,应选用病程短、HBV DNA水平较低、肝损害较重的患者进行拉米夫定治疗,有条件的应检测HBV基因型.     

慢性乙型肝炎前C区G1896A基因变异对HBV复制及疾病严重程度的影响

目的研究乙型肝炎病毒(HBV)前C区G1896A变异对乙型肝炎病毒复制及疾病严重程度的影响。方法选取43例慢性乙型肝炎及49例乙型肝炎肝硬化患者为研究对象。采用PCR技术检测HBV前C区G1896A位点变异及乙型肝炎基因分型。同时检测HBeAg、HBV DNA定量、肝功能血清生化指标。结果(1)发生HBV前C区G1896A变异患者的年龄大于野生株感染者(P0.01),发生肝硬化的可能性更大(P0.01)。(2)随着变异株感染率的增加,HBeAg阴性率较野生株组增加(P0.01),基因C型较野生株组增加(P0.05),HBV DNA水平降低差异意义(P0.05)。结论 HBV前C区G1896A变异与HBeAg的状态,HBV DNA的复制及疾病严重程度相关。     

乙肝患者拉米夫定治疗过程中YMDD耐药突变株的变化

目的探讨乙肝患者拉米夫定治疗过程中YMDD变异与HBV基因型、HBV DNA含量及治疗时间的关系。方法采用实时PCR法和基因测序法分别对107例拉米夫定治疗的慢乙肝患者进行HBV DNA含量和HBV基因分型、YMDD变异检测。结果 107例慢乙肝患者中,B型19例（17.8%）,C型81例（75.7%）,B、C混合型7例（6.5%）,未发现A、D、E、F、G、H等其他基因型;共25例（23.4%）发生YMDD变异,其中B型、C型和B、C混合型YMDD变异的发生率分别为26.3%、22.2%、28.6%。各基因型患者YMDD变异的发生率及Y IDD、YVDD的变异类型均无统计学差异（P〉0.05）。血清HBV定量水平低、中、高度组产生YMDD变异株的时间有统计学差异（P〈0.05）。结论 YMDD变异与HBV基因型无明显相关性;治疗前血清HBV DNA含量越高,拉米夫定治疗期间发生YMDD变异的时间越早;出现YMDD变异患者肝组织内处于不同的炎症及纤维化状态。     

RtL164V,一个可能与拉米夫定耐药相关的突变

目的 进一步了解HBV P基因结构与拉米夫定的疗效关系.方法 一步法扩增HBVP基因后进行测序,对比拉米夫定治疗应答,无应答和突破患者治疗前后血清HBV P基因结构的变化.结果 在无应答和突破患者中,2例B基因型和1例C基因型转变为B、C混合型HBV感染;1例患者血清HBV发生B→C基因型转换.3组中部分患者治疗前血清中检出YMDD变异株HBV,所有8例治疗无应答和突破患者在治疗过程中均出现YMDD变异.所有无应答患者治疗前后和突破患者突破时的血清HBV均出现rtL164V变异,而在应答者中未见.突破和无应答患者逆转录酶(rt)保守区还出现rt191L、rtK168R、rtH2ML、rtS256C 4个氨基酸替代.结论 YMDD基因序列变异不是产生拉米夫定临床耐药的惟一原因,rtL164V变异可能是一个新的与拉米夫定耐药相关的突变.     

Evolution of wild type and mutants of the YMDD motif of hepatitis B virus polymerase during lamivudine therapy

BACKGROUND AND AIM: Long-term lamivudine treatment for chronic hepatitis B virus (HBV) infection induces the emergence of lamivudine resistant HBV YMDD mutant strains. The aim of the present study was to observe the clone evolution of YMDD wild type and mutant strains in pretreatment and post-treatment samples during lamivudine therapy and analyze their clinical significance. METHODS: Ten serum samples (five before and five after 48 weeks of therapy) obtained from five patients chronically infected with HBV and treated with lamivudine were studied. Part of the HBV polymerase gene flanking the YMDD motif was sequenced after polymerase chain reaction (PCR) amplification. Meanwhile, 20-24 clones were selected at random from each sample and YMDD wild type and mutant strains were detected by real-time fluorimetry PCR established in our laboratory. RESULTS: The YMDD mutants were not detectable in all five patients before treatment and were found in four patients after 48 weeks of therapy by sequencing directly on PCR products. Analysis of individual clones showed that the ratios of mutant strains in each of the five patients were 0, 9.5, 0, 4.5 and 5.6%, respectively, before therapy and 100, 100, 65, 100 and 0%, respectively, after 48 weeks of therapy. M552I was detected before therapy in one patient but M552V became the domain strain after therapy. Until 52 weeks of therapy, serum HBV DNA and alanine aminotransferase (ALT) breakthrough were found in two of the four patients with YMDD mutations. The fifth patient experienced breakthrough of ALT but HBV DNA remained undetectable. CONCLUSIONS: The mutant strains of YMDD motif of HBV polymerase could be found in patients before lamivudine treatment, indicating that antiviral therapy allows the rapid selection of resistant strains. The replication ability of the M552V mutant strain might be stronger than that of the M552I mutant strain.     

Extended lamivudine retreatment for chronic hepatitis B: maintenance of viral suppression after discontinuation of therapy.

In patients with chronic hepatitis B, brief lamivudine therapy suppresses hepatitis B virus (HBV) DNA but results infrequently in sustained losses of virus replication posttreatment. We evaluated treatment response and its posttreatment durability during up to 18 months of lamivudine therapy (100 mg/d) in 24 patients who had hepatitis B e antigen (HBeAg) despite 1 to 3 months of prior therapy. Therapy was to be stopped after HBeAg loss or seroconversion (acquisition of antibody to HBeAg); posttreatment monitoring continued for 6 months. During therapy, which was well tolerated, HBV DNA became undetectable in all evaluable patients, accompanied by reduced alanine transaminase (ALT) activity. The cumulative 18-month confirmed loss of HBeAg during therapy was 9 of 24 (38%) and seroconversion was 5 of 24 (21%). Therapy was discontinued after HBeAg loss/seroconversion in 7 patients, and HBeAg status was maintained in all. Four of the patients with HBeAg responses lost HBsAg at least once. In 10 (43%) of 23 patients tested, we identified HBV polymerase YMDD mutations, 3 with detectable HBV DNA (2 with ALT elevations) and 7 without virological/biochemical breakthrough. In conclusion, up to 18 months of lamivudine therapy was well tolerated, suppressed HBV replication consistently, and tripled the frequency of HBeAg losses observed during brief-duration therapy; HBeAg loss/seroconversion remained durable posttreatment. The emergence of YMDD-variant HBV was relatively common but occurred typically without reappearance of detectable HBV DNA or ALT elevation. Our observations suggest that lamivudine can be stopped after confirmed HBeAg loss or seroconversion.     

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.     

Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy

A study in Chinese patients with chronic hepatitis B showed that treatment with lamivudine for 1 year significantly improves liver histology and enhances hepatitis B e antigen (HBeAg) seroconversion compared with placebo. Fifty-eight patients from this 1-year study have received long-term treatment with lamivudine 100 mg; the outcome of 3 years of lamivudine is reported here. Before treatment, all patients had detectable HBeAg. HBeAg seroconversion (HBeAg-negative, anti-HBe-positive), hepatitis B virus (HBV)-DNA suppression, alanine transaminase (ALT) normalization, emergence of YMDD variant HBV, liver histology, and long-term safety were assessed. After 3 years of continuous treatment with lamivudine 100 mg daily, 40% (23 of 58) of patients achieved HBeAg seroconversion. In patients with baseline serum ALT >2 x upper limit of normal (ULN), the rate of HBeAg seroconversion was 65% (17 of 26). Median serum HBV-DNA concentrations were below the level of detection, and median ALT concentrations were within the normal range throughout 3 years of treatment. YMDD variant HBV emerged in 33 of 58 (57%) patients during the 3 years, of whom 9 (27%) achieved HBeAg seroconversion (6 after emergence of YMDD variant HBV). ALT levels and histologic scores after emergence of YMDD variant HBV did not show major deterioration. Lamivudine was well tolerated during 3 years of therapy. In conclusion, these data in Chinese patients with chronic hepatitis B show enhanced seroconversion rates with extended lamivudine treatment. Up to two thirds of patients with moderately elevated pretreatment ALT achieved HBeAg seroconversion after 3 years of therapy.     

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.     

Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance

BACKGROUND/AIMS: Hepatitis B mutant strains of virus emerging during treatment with the nucleoside analog lamivudine are being increasingly recognized. In the majority of lamivudine-resistant isolates the mutations have been reported to occur within the YMDD motif of the viral polymerase, either as a single mutation M552I or as M552V concomitant with L528M. We analyzed the time course and genetic succession pattern during the emergence of lamivudine resistance. METHODS: Seven patients with breakthrough viremia in the setting of chronic hepatitis (n=5) or recurrent HBV after liver transplantation (n=2) were investigated. Pre- and post-breakthrough serum samples were evaluated by single- or second-round PCR amplification and sequencing analysis. RESULTS: Genotypic succession of the virus populations was observed to occur from M552I to M552I/L528M (n=2) and from L528M to M552V/L528M (n=1). The double mutations M552I/L528M (n=4) or M552V/L528M (n=2) were found in six out of seven patients, and represented the stable virus populations throughout the follow-up period. Breakthrough viremia was not associated with the single L528M mutation. The mean duration of uninterrupted treatment with lamivudine until breakthrough was 422 days (range 182-642) and was longer in the setting of chronic hepatitis B than in recurrent hepatitis B after liver transplantation. HBV DNA levels after breakthrough were lower than pretreatment levels in the majority of patients with chronic hepatitis but higher after liver transplantation. CONCLUSION: Our observations show that the virus populations conferring resistance to lamivudine can evolve from single to double mutations at amino acid 552 and 528 of the HBV polymerase, and that M552I/ L528M or M552V/L528M seem to be the predominant mutations arising during long-term antiviral therapy with lamivudine.