乙型肝炎病毒耐拉米夫定多聚酶基因变异检测方法研究

目的 建立简便、准确、实用的检测乙型肝炎病毒耐拉米夫定多聚酶(P)基因变异的方法。方法 根据HBV基因序列，设计5只寡核苷酸引物，用巢式聚合酶链反应(nested PCR)分别扩增HBVP基因B区和C区片段，产物用NdeⅠ或NIa Ⅲ酶切，琼脂糖胶电泳，分析酶切产物长度多态性(RFLP)，建立检测P基因变异的方法。对30例长期服用拉米夫定的慢性乙型肝炎(慢乙肝)患者检测YMDD基序及526位点变异，16例未用拉米夫定的慢乙肝患者为对照。4份PCR产物作克隆测序以验证方法的准确、可靠。结果 所建的巢式PCR-RFLP方法操作简便、快速，从模板提取到酶切后电泳分析仅需11h；灵敏度高，可检测到10^3拷贝／ml的HBVDNA；结果准确，4份经酶切分别判断为野毒株或变异株的标本经测序证实。30例用拉米夫定的慢乙肝患者中，发现单纯YMDD变异8例(26．7％)，YMDD联合L526M变异3例(10．0％)，16例对照未检出上述位点的变异。结论 本方法简便、准确，适合较大样本检测。可用于临床筛检常见拉米夫定耐药性HBVP基因变异。     

拉米夫定治疗慢性乙型肝炎HBV DNA阴转疗效影响因素研究

目的探讨拉米夫定治疗慢性乙型肝炎患者疗效的影响因素。方法对拉米夫定治疗的慢乙肝202例和乙型肝炎肝硬化（简称乙肝肝硬化）55例患者，采用聚合酶链反应及错配聚合酶链反应限制性片段长度多态性分析的方法检测HBV DNA及乙型肝炎病毒（HBV）聚合酶YMDD变异，应用SPSS统计软件对基线ALT、AST、TBIL、白蛋白、球蛋白、HBeAg（高、中、低）、HBV DNA等共10个变量作为处理因素进行统计学检验。结果随治疗时间的延长，慢乙肝和肝硬化者HBV DNA的阴转率于3、6、12、18、24、30个月分另1为87．62％、95．04％、86．63％、82．65％、75．43％、77．27％和94．54％、98．18％、81-81％、74．35％、73．91％、63．63％，时序检验及Cox回归分析结果表明：联合干扰素、基线ALT水平较高、HBV DNA水平较低和基线白蛋白水平较低，与HBV DNA转阴率高相关。结论联合干扰素、基线ALT水平较高、HBV DNA水平较低和基线白蛋白水平较低可作为HBV DNA转阴的预测因素。     

乙型肝炎病毒基因型与拉米夫定治疗疗效关系的研究

目的探讨乙型肝炎病毒(HBV)基因型与拉米夫定治疗疗效的关系及其机理。方法对拉米夫定治疗的228例慢性乙型肝炎患者的临床资料进行收集和整理,分析HBV基因型与拉米夫定治疗疗效的关系;并对各HBV基因型影响拉米夫定疗效的可能因素进行探讨。结果HBV基因型B型对拉米夫定治疗的应答率为40.6%,C基因型的应答率为21.2%,两组应答率差异有显著性(P<0.05);B基因型的HBeAg的血清转换率为26.3%,C基因型为12.4%,统计学方法处理有显著性差异(P<0.05)。拉米夫定治疗期间的YMDD变异发生率B基因型组为13.2%,C基因型组为37.4%,两组差异有显著性统计学意义(P<0.05)。结论拉米夫定治疗慢性乙型肝炎患者,HBVB基因型的疗效优于C基因型,其疗效机理可能与拉米夫定治疗后C基因型容易发生YMDD变异有关。     

限制性片段长度多态性分析拉米夫定治疗引起乙型肝炎病毒耐药基因变异的研究

目的　应用聚合酶链反应限制性片段长度多态性分析方法 (PCRRFLP) ,研究拉米夫定治疗引起慢性乙型肝炎患者血清中乙型肝炎病毒耐药基因 (HBVDNA)变异。方法　收集 2 4 0例用拉米夫定治疗 5 2～ 78周慢性乙型肝炎患者的血清标本 ,应用PCRRFLP方法扩增HBV 5 5 2 ,5 2 8两个基因位点片段 ,用限制性内切酶NdeⅠ ,NlaⅢ酶切分析 ,同时测定HBVDNA含量 ,并用DNA序列分析测定 3例患者HBVDNAP区基因序列 (1例野毒株 ,1例M5 5 2V伴L5 2 8M变异 ,1例M5 5 2I变异 )。结果　 2 4 0例患者应用拉米夫定治疗 5 2～ 78周后 ,5 1例血清HBVDNA出现YMDD变异 ,其中M5 5 2V变异 38例 ,M5 5 2I变异 13例 ,L5 2 8M和M5 5 2V同时变异 2 6例 ,L5 2 8M和M5 5 2I同时变异 1例。与定量PCR比较 ,可以测定YMDD变异的最低含量为 1× 10 4 copies mlHBVDNA序列分析结果与RLFP测定一致。结论　PCRRLFP方法是一种快速、简便的检测HBVDNA聚合酶变异的方法 ,可以用来筛查大量的血清标本 ,适宜作为临床用来观察拉米夫定治疗慢性乙型肝炎患者检测HBV变异的方法     

Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus

The purpose of this prospective study was to evaluate the efficacy and safety of adefovir dipivoxil with or without ongoing lamivudine in decompensated lamivudine-resistant chronic hepatitis B patients. Forty-six hepatitis B e antigen (HBeAg)-positive patients with decompensated liver function and lamivudine-resistant hepatitis B virus (HBV) were assigned to adefovir dipivoxil monotherapy (n=18) or combination therapy with ongoing lamivudine (n=28) according to their own preference. After 24 weeks of treatment, 83% of monotherapy and 86% of combination therapy showed serum HBV DNA below detection limit (<0.5 pg/mL). Alanine aminotransferase (ALT) normalized in 78% and 82% respectively. Median Child-Pugh-Turcotte (CPT) score or Model for End-Stage Liver Disease (MELD) score reduced significantly by 3 or 5 point in monotherapy and 2 or 2 point in combination therapy respectively. There were no significant differences in rate of undetectable serum HBV DNA, median change of ALT and median reduction of CPT or MELD scores between the two groups. In conclusion, both adefovir dipivoxil monotherapy and combination therapy with ongoing lamivudine result in comparable virologic, biochemical, and clinical improvements in HBeAg-positive patients with decompensated liver function and lamivudine-resistant HBV. Combination with lamivudine showed no additional benefit over monotherapy during 24 weeks of treatment in these patients.     

Long-term incidence and predictors of hepatitis B surface antigen loss after discontinuing nucleoside analogues in noncirrhotic chronic hepatitis B patients

ObjectiveTo investigate the long-term incidence and predictors for hepatitis B surface antigen (HBsAg) loss after nucleoside analogue therapy.MethodsThe study included 411 noncirrhotic chronic hepatitis B patients (148 hepatitis B e antigen (HBeAg)-positive and 263 HBeAg-negative patients) who were treated with lamivudine (n = 110) or entecavir (n = 301) with posttreatment follow-up of at least 12 months.ResultsIn HBeAg-positive patients, the 8-year cumulative rates of virologic relapse, clinical relapse and HBsAg loss were 55.6%, 47.7% and 19.6%, respectively. In HBeAg-negative patients, the rates were 69.3%, 58.9% and 33.1%, respectively. Cox regression analysis showed that hepatitis B virus genotype C and lower end-of-treatment HBsAg levels were independent predictors of HBsAg loss in HBeAg-positive and -negative patients. The 5-year HBsAg loss rate was 47.3% in HBeAg-positive patients with end-of-treatment HBsAg levels <300 IU/mL, while the 8-year HBsAg loss rate was 69.3% in HBeAg-negative patients with end-of-treatment HBsAg levels <200 IU/mL. Five patients experienced hepatitis flares with decompensation after stopping nucleoside analogue therapy, and one died after retreatment. Of the 48 patients who developed off-therapy HBsAg loss, two developed hepatocellular carcinoma.ConclusionsThe rate of HBsAg loss was relatively high and the rate of hepatic events was low in noncirrhotic patients who discontinued nucleoside analogue therapy.     

Molecular biology of hepatitis B virus: effect of nucleotide substitutions on the clinical features of chronic hepatitis B

Hepatitis B virus (HBV) is a major cause of liver disease worldwide. It is covered with envelope (surface antigen) proteins with the nucleocapsid (core antigen) inside. In the nucleocapsid, there is an incomplete double-stranded DNA and a DNA polymerase. Four genes, S, C, X, and P, are encoded, and these partially overlap. Mutations have been reported in each gene and in their promoter regions, and these mutations can change the efficiency of HBV replication and the clinical course of patients. In this article, we review the relationship between the molecular biology of HBV and its clinical outcome.     

乙型肝炎病毒感染时的C基因热点变异

为了解乙型肝炎病毒感染在不同病变时的Ｃ基因热点变异，对９１名感染者以限制片段长度多态性技术检测前Ｃ终２８和Ｃ区Ｌ９７点突变。在急性乙型肝炎和慢性无症状携带者几无突变、在慢性持续性肝炎中罕见，而在慢性活动性肝炎和活动性肝硬变中分别达８０％和７８％，肝细胞癌中为６１％。ＨＢｅＡｇ（＋）的慢性活动性肝炎和活动性肝硬变１６例中，混合有终２８变异１１例，抗－ＨＢｅ（＋）病例中亦有野毒株混合。Ｃ区密码９７亮氨酸变异（Ｌ９７）亦见混合。野毒株和变异株在病程中有消长，Ｃ基因的热点变异与病变活动性密切相关。第一军医大学南方医院     

Hepatitis B surface antigen levels at 6 months after treatment can predict the efficacy of lamivudine-adefovir combination therapy in patients with lamivudine-resistant chronic hepatitis B

Background/Aims

Quantitation of hepatitis B surface antigen (HBsAg) is an increasingly popular method to determine the treatment response in chronic hepatitis B (CHB) patients. The clinical value of HBsAg level measurement during rescue therapy for lamivudine (LMV)-resistant CHB patients have not been evaluated to date. Therefore, this study investigated the correlation between HBsAg level and treatment response in LMV-resistant CHB patients treated with adefovir (ADV) add-on therapy.

Methods

LMV-resistant CHB patients treated with LMV-ADV combination therapy for over 2 years were included. HBsAg levels were measured at 6 month intervals until 1 year, and annually thereafter. Treatment response was assessed by determining the virological response (VR, undetectable HBV DNA levels) during treatment.

Results

Fifty patients were included, of which 40 showed a VR. HBsAg levels were not different significantly at baseline (4.0 vs. 3.6 Log10 IU/mL, P=0.072). However, the HBsAg level decreased after 6 months of treatment in patients with a VR and became different significantly between the groups thereafter (3.9 vs. 3.3 at 6 months, P=0.002; 3.8 vs. 3.2 at 1 year, P=0.004; 3.9 vs. 3.2 at 2 years, P=0.008; 3.7 vs. 3.1 at 3 years, P =0.020).

Conclusions

The HBsAg level at 6 months after treatment can help predict treatment response.     

拉米夫定耐药后的抗乙型肝炎病毒治疗

目的探讨拉米夫定耐药后慢性乙型肝炎患者采用α2b干扰素或α2b干扰素加α1胸腺肽进行后续抗病毒的疗效。方法共66例拉米夫定耐药患者,分治疗组A、治疗组B和对照组。治疗组A26例,单用α2b干扰素治疗1个月后停用拉米夫定,然后继续单用α2b干扰素治疗5个月;治疗组B10例,α2b干扰素和α1胸腺肽联合治疗1个月后停用拉米夫定,然后继续两药联合治疗5个月;对照组30例,直接停用拉米夫定,不用其他任何抗病毒药。定期进行血清肝功和病毒学指标检测。结果治疗组A和治疗组B的HBVDNA阴转率和HBeAg/HBeAb转换率均明显高于对照组。结论慢性乙型肝炎患者拉米夫定耐药后采用α2b干扰素或α2b干扰素加α1胸腺肽进行后续抗病毒治疗可能是有益的。     

Hepatitis B virus mutations associated with in situ expression of hepatitis B core antigen, viral load and prognosis in chronic hepatitis B patients

In this retrospective study, we investigated the prevalence and significance of mutations in part of the hepatitis B virus (HBV) x gene, and tried to clarify their relationship with clinicopathological or histopathological characteristics and prognosis in patients with chronic hepatitis B (CHB). A total of 83 consecutive CHB patients (1986-1994) were chosen for the present study. Sequence analysis was performed using polymerase chain reaction (PCR) and the direct sequencing method. The histological activity index was described using Scheuer scores. Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by fluorescence quantitative real-time PCR. For the prognostic study, all the patients were followed up using clinical and laboratory data. Mutation at nt1726-1730 correlated significantly with decreased expression of HBcAg in situ (P = 0.006) and with lower HBV DNA levels in the liver (P = 0.004). In particular, the CTGAC mutation showed the strongest decrease of the viral load (P = 0.007). By contrast, nt1762/1764 mutation correlated with increased HBcAg (P = 0.005) and higher HBV DNA levels (P = 0.006). The mutants with the wild-type of nt1726-1730 or nt1762/1764 mutation were more prevalent in hepatocellular carcinoma (HCC) patients than in CHB patients. Although the mutations did not correlate with cirrhosis, the frequency of nt1762/1764 mutation in patients with hepatocarcinogenesis was significantly higher than in those without hepatocarcinogenesis (P = 0.011). Mutations at nt1726-1730 and nt1762/1764 are associated with in situ expression of HBcAg and viral load. Higher HBV DNA levels in the liver may be associated with hepatocarcinogenesis. Mutation at nt1762/1764 remarkably increases the risk of hepatocarcinogenesis.     

拉米夫定治疗中乙型肝炎病毒基因序列变异特点及其准种研究

目的研究拉米夫定治疗中乙型肝炎病毒（HBV）逆转录酶区核苷酸序列变异、特点、含量及其与基因型和病毒载量的关系。方法普通DNA测序法检测拉米夫定治疗的117份慢乙肝患者血清HBV逆转录酶区基因序列及其基因型;其中99份用TaqMan法定量HBVDNA;64份用焦磷酸测序（Pyrosequencing）检测YMDD基序中碱基的频率。结果HBVYMDD变异组中C型43例,B型10例,A／B混合型1例;YMDD不变异组中C型54例,B型8例,D型1例,HBVDNA含量平均对数值在变异组和不变异组分别为：6．5699和6．6165;YMDD变异与其基因型及病毒载量无统计学意义;rtL180M位点变异与rtM204I／V位点变异高度相关;并且HBV野生株与变异株均同时存在。结论结合两种测序方法可以用来研究拉米夫定治疗中HBV基因序列变异情况及对YMDD耐药株进行定量。     

山东省人群乙型肝炎病毒＂a＂抗原决定簇突变分析

目的 了解目前山东省社区人群乙型肝炎病毒（HBV）＂a＂抗原决定簇突变率和突变形式,探讨乙肝疫苗（HepB）接种对＂a＂抗原决定簇突变的影响.方法 在全省1～59岁社区人群中通过多阶段随机抽样确定调查对象,通过询问（15岁以上）或查阅接种记录（14岁以下）了解调查对象HepB免疫史;采集血标本,酶联免疫吸附方法 检测血清乙肝表面抗原（HBsAg）,阳性者提取血清DNA,采用巢式PCR方法 扩增HBV S基因,测序后与标准序列进行比较.结果 共对7601人进行调查和血标本采集,得到HBsAg阳性标本239份（3.14%）,可用于HBV DNA提取206份,扩增HBV S基因并成功测序102份.15份血清标本检测到13种HBV＂a＂抗原决定簇突变,突变率为14.70%（15/102）.新生儿普种HepB前、后出生调查对象间,以及有、无HepB免疫史调查对象间＂a＂抗原决定簇突变率差异均无统计学意义.结论 目前山东社区人群中＂a＂抗原决定簇突变率较低且突变位点比较分散;未发现HepB接种对人群＂a＂抗原决定簇突变产生影响.     

拉米夫定耐药的慢性乙肝患者联合干扰素或苦参素治疗疗效观察

目的 观察拉米夫定耐药的慢性乙型肝炎患者联合干扰素或苦参素治疗的效果。方法 40例患者在继续应用拉米夫定的前提下，A组14例联合应用干扰素a-2b3MU IM每日1次，30d，然后隔日1次，共计6个月。B组15例联合苦参素，苦参素60mg，IM每日1次，3个月，然后改为口服0．2g每日3次3个月。C组11例继续单用拉米夫定100mg每日1次口服。疗程结束后，观察乙肝病毒血清学指标HBVDNA、HBeAg阴转及HBeAg／anti-HBe转换，肝功能(ALT)恢复情况。结果 联合干扰素治疗组，HBVDNA阴转率为35．71％(5／14)；联合苦参素治疗组HBVDNA阴转率为13．33％(2，15)，ALT复常率分别为85．71％(12／14)、86．67％(13／15)。C组无HBVDNA及HBeAg阴转，ALT复常率为36．36％(4／11)。结论 对拉米夫定耐药的慢性乙型肝炎患者，联合干扰素或苦参素治疗后，可以提高拉米夫定疗效，抑制病毒复制，促进肝功能恢复。     

拉米夫定治疗慢性乙型肝炎2年临床疗效

目的 研究拉米夫定治疗慢性乙型肝炎的临床疗效和安全性。方法 选取72例慢性乙型肝炎病人,第一阶段为随机、双盲、安慰剂对照的研究共12周,分为拉米夫定组（n=54）和安慰剂组（n=18）;第二阶段为开放研究,所有病人均服用拉米夫定100mg/d至104周。观察指标包括临床症状、肝功、乙型肝炎病毒（HBV）血清标志物、HBV DNA和病毒YMDD变异等。结果 拉米夫定治疗12周时,HBV DNA阴转率显著高于安慰剂组（61％对6％,P＜0.01）,ALT持续复常率也高于安慰剂组（65％对11％,P＜0.05）;治疗52周时,两组病人总的HBV DNA阴转率为785,ALT持续复常率为39％,HBeAg阴转率和血清转换率分别为8.2%和6.1%;治疗104周时,两组病人总的HBV DNA阴转率36％,ALT持续复常率为33％,HBeAg阴转率和血清转换率分别为12.2%和6.1%。两组病人总的YMDD变异率在52周时为13.7%,104周时为39.7%。第一阶段拉米夫定和安慰剂组不良瓜在的差异不显著（P＞0.05）,治疗期间未发生与药物有关的严重不良反应。结论 拉米夫定100mg/d可以迅速降低血清HBV DNA和ALT水平,安全性良好。但应严密监测以及时发现YMDD病毒变异引起的HBV DNA反跳。     

慢性HBV C基因启动子变异的检测

目的 了解慢性乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子变异的情况以及对病毒血清学的影响。方法 应用错配聚合酶链反应特异性扩增含有ＨＢＶ Ｃ基因启动子的片段,扩增产物用限制性内切酶Ｂｃｌ Ｉ酶切,琼脂糖凝胶电泳后,观察酶切后的限制性片段长度多态性（ＲＦＬＰ）图谱,分析Ｃ基因启动子区段的核苷酸（ｎｔ）１７６２由碱基Ａ→Ｔ和（ｎｔ）１７６４碱基由Ｇ→Ａ的变异,并经直接测序分析证实ＲＦＬＰ结果。结果 １１０例慢     

Precore and core promoter mutations of the hepatitis B virus gene in chronic genotype C-infected children

The precore (G1896A) and core promoter (A1762T, G1764A) mutations of the hepatitis B virus gene are known to be associated with changes in immunologic phase or the progression to complicated liver disease in adults. We analyzed these mutations in chronically HBV-infected children. Serum was collected from 37 children with chronic HBV infection from March 2005 to September 2008. HBV DNA extraction and nested PCR were followed by sequencing of the PCR products. The children were 6.7 ± 4.6 yr old. All of 37 children had HBV genotype C. Of the cohort, 31 (83.8%) were HBeAg-positive and 6 (16.2%) were HBeAg-negative; the former group comprised 18 (48.6%) who were in the immune-tolerance phase (ITP) and 13 (35.2%) in the immune-clearance phase (ICP). Most of the patients had HBV DNA levels of > 1.0 × 10(8) copies/mL. In the ITP group, only 1 (5.5%) had core promoter mutations, and none had the precore mutation. In the ICP group, only 2 (15.4%) had core promoter mutations; the remaining 6 patients had HBV DNA levels of < 2.0 × 10(3) copies/mL and no core promoter/precore mutations. The very low incidence of the precore/core promoter gene mutation, in children, suggests that these mutations may be the result of life-long chronic HBV infection.     

慢性乙型肝炎抗病毒序贯治疗方案疗效的对比研究

目的　建立抗病毒序贯治疗方案 ,并采用对比研究评价其治疗慢性乙型肝炎的效果。方法　 74例慢性乙型肝炎患者分成 3组。抗病毒序贯治疗组 30例 ,接受日达仙治疗 8周 ,1 6mg 次 ,皮下注射 ,2次 周 ,于第 5周起加用α 干扰素 5 0 0MU 次 ,肌内注射 ,隔日 1次 ,疗程 6个月 ;HBeAg阴转 2个月或α IFN结束后 ,使用拉米夫定 ,10 0mg d ,用至 18个月以上。IFN联合日达仙组 14例 ,接受日达仙和α 干扰素治疗 ,用法同抗病毒序贯治疗组 ,疗程为 6个月。拉米夫定组 30例 ,接受拉米夫定治疗 ,用法同抗病毒序贯治疗组 ,疗程 18个月以上。结果　抗病毒序贯治疗组、α 干扰素 (α IFN)联合日达仙组和拉米夫定组的短期ALT复常和HBeAg阴转率 (有效率 )分别为 76 7%、78 6 %和 13 3%。抗病毒序贯治疗组与α IFN联合日达仙组的效果相当 ,均高于拉米夫定组 ,差异有显著意义 (P <0 0 1)。持续有效率分别为 76 7%、5 7 1%和 16 7%。抗病毒序贯治疗组高于α IFN联合日达仙组和拉米夫定组 ,治疗费用仅约为IFN联合日达仙组的 6 0 %。抗病毒序贯治疗组和α IFN联合日达仙组中肝损敏感期出现率为 4 7 7% ,出现时间为IFN治疗开始后的 2～ 8周 ,较文献中单用IFN治疗引起的肝损敏感期的出现时间 (6～ 8周 )早。结论　抗