拉米夫定耐药慢性乙型肝炎患者HBVP区基因突变模式与基因型的关系

目的分析慢性乙型肝炎患者在拉米夫定（LAM）治疗过程中出现耐药后,HBVP区基因突变模式及基因分型的关系。方法对2008年9月至2010年6月在我院就诊的107例临床诊断为LAM耐药慢性乙型肝炎患者进行HBVP区及基因分型测定。结果107例患者的基因突变模式为8种,100％均发生YMDD序列突变,其中单位点突变43例,其余均为联合突变;107例患者主要以B（25．2％）和C（73．8％）基因为优势,1例为B和c混合基因,且c基因以rtM204＋rtL180M模式为主,占60．7％（48／79）,B基因以rtM204突变模式为主,占66．7％（18／27）;rtM204和rtM204＋rtL180M突变模式的B和C基因比较两者差异有统计学意义（x2分别为8．4和7．2,P〈0．01）。结论YMDD基序突变是LAM耐药后HBVP区基因突变的主要模式,不同基因型决定了与耐药相关的变异出现形式。     

Mutational patterns of hepatitis B virus genome and clinical outcomes after emergence of drug-resistant variants during lamivudine therapy: analyses of the polymerase gene and full-length sequences

It remains unclear whether mutational patterns of the hepatitis B virus (HBV) genome are associated with the development of severe hepatitis after the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) variants during lamivudine treatment. Thirty patients with chronic hepatitis B who had YMDD variants during lamivudine therapy and were followed up subsequently while receiving lamivudine alone for at least 6 months were examined retrospectively. The lamivudine resistant mutations in the HBV polymerase gene were detected by a line probe assay, and the full-length sequences of HBV DNA were determined in some patients. Between months 5 and 33 of therapy, mutations from methionine to isoleucine at rt204 (rtM204I) were detected in 18 patients, and mutations from methionine to valine at rt204 (rtM204V) were detected in 12. The rtM204V mutations were always accompanied by mutations from leucine to methionine at rt180 (rtL180M), while rtM204I mutations were not. Baseline characteristics, alanine aminotransferase (ALT) levels, and HBV DNA levels within 6 months after the emergence of YMDD variants did not differ significantly between patients with rtM204I alone and those with rtL180M/rtM204V. No specific mutation was identified on full-length sequence analysis in three patients with a hepatitis flare. During long term follow-up, the addition of rtL180M to rtM204I was found in four patients 7-31 months after detecting the change at rt204 and was linked to increased ALT levels. In conclusion, mutational patterns of HBV DNA at the time of emergence of YMDD variants were apparently unrelated to the clinical outcomes in Japanese patients with chronic hepatitis B during lamivudine therapy.     

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。     

Association of lamivudine‐resistant mutational patterns with the antiviral effect of adefovir in patients with chronic hepatitis B

Adefovir has a potent antiviral activity as a rescue treatment against lamivudine‐resistant strains. The aim of this study was to assess the patterns of lamivudine‐resistant mutations and their influence on the virologic response to adefovir rescue therapy in patients with lamivudine‐resistant chronic hepatitis B. Sixty‐seven patients with lamivudine‐resistant chronic hepatitis B were treated with adefovir monotherapy. Baseline blood samples were analyzed for lamivudine‐resistant mutations via restriction fragment mass polymorphism. Virologic responses, ALT normalization and loss of HBeAg were assessed. Serum HBV DNA levels were measured using real‐time PCR at baseline and 24 weeks of adefovir therapy. Of the 67 patients with chronic hepatitis B, 65 patients (97%) had lamivudine‐resistant mutations in the YMDD motif [27 (41%) rtM204I, 22 (34%) rtM204V, and 16 (25%) rtM204I/V]. In addition to the YMDD mutations, the rtL180M, rtL80I, and rtV173L mutations were also present in 78%, 43%, and 11% of patients, respectively. The rtM204V mutation always accompanied rtL180M, and rtL80I was always observed in conjunction with rtM204I. Decrease in mean serum HBV did not differ between patients carrying the rtM204I versus rtM204V mutant at week 24 (?3.3 vs. ?3.3 log₁₀ copies/ml, respectively; P = 0.303). The presence of the rtL180M, rtL80I, and rtV173L did not significantly affect viral load reduction during adefovir administration. These results demonstrate that the rtL80I mutant is co‐selected with rtM204I as a compensatory mutation in the same manner as rtL180M with rtM204V, and that adefovir shows similar antiviral efficacy against all of the evaluated patterns of lamivudine‐resistant HBV mutations. J. Med. Virol. 81:417–424, 2009. © 2009 Wiley‐Liss, Inc.     

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。     

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

BACKGROUND: In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. RESULTS: In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. CONCLUSION: The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.     

三联叠加疗法对慢性乙型肝炎患者乙型肝炎病毒复制和变异的影响

目的观察疗效确切的抗病毒药物和免疫调节剂,叠加伍用对慢性乙型肝炎病人HBV复制和变异的影响.方法应用拉米夫定、干扰素α-2b、黄芪注射液三联叠加疗法,对慢性乙型肝炎病人进行抗病毒治疗,并与单独应用拉米夫定的抗HBV效果进行比较评价.检测HBVDNA阴转率和HBeAg-抗HBe血清转换率;血清HBVDNA浓度;HBVDNA的YMDD变异率和前C区变异率.结果A组(叠加用药组)与B组(拉米夫定单药组)比较,HBVDNA阴转率分别在第12周、36周及48周差异有显著性(P<0.05),HBeAg阴转率在第36周、48周差异有显著性(P<0.05),抗HBe阳转率仅在第48周差异有显著性(P<0.05);两组患者血清HBVDNA浓度比较,治疗12周时降低程度差异有显著性(P<0.05),治疗第36周和48周时差异有非常显著性(P<0.01);治疗后12周,A组出现前C区BCP变异;疗后24周、36周和48周,两组均出现YMDD和前C区变异.结论与拉米夫定单独用药比较,三联叠加疗法可增加慢性乙型肝炎患者HBVDNA阴转率和HBeAg-抗HBe血清转换率,更显著降低血清HBVDNA浓度,并减少YMDD变异,故其抗HBV疗效优于拉米夫定单独用药.     

在慢性乙型肝炎治疗过程中HBsAg水平与HBV DNA病毒复制的相关性分析

目的： 病毒载量的测定常被用来诊断和监测慢性乙型肝炎的疗效。文献报道采用全自动微粒子化学发光免疫反应测定HBsAg水平可作为替代标记。本研究旨在进一步分析慢性乙型肝炎治疗过程中血清HBsAg水平和HBV DNA水平的相关性。方法：本研究选取47例慢性乙型肝炎的患者［男性35人,女性12人,平均年龄(35±8)岁］。聚乙二醇化干扰素α-2a单一治疗患者（18例）、聚乙二醇化干扰素α-2a +拉米夫定联合治疗患者（14例）、拉米夫定单一治疗患者（15例）治疗48周并留取0、4、8、24、48、72周的血清。用TaqMan PCR测定HBV DNA水平,全自动微粒子化学发光免疫反应测定HBsAg水平。结果：18例聚乙二醇化干扰素α-2a单一治疗的患者和14例聚乙二醇化干扰素α-2a +拉米夫定联合治疗的患者可观察到HBsAg滴度随HBV DNA复制水平的一致变化,呈显著相关（r=0.83,P<0.01）。而在15例拉米夫定单一治疗的患者则未观察到HBsAg滴度随HBV DNA复制水平一致的变化趋势,但无相关性。聚乙二醇化干扰素α-2a单一治疗、聚乙二醇化干扰素α-2a+拉米夫定联合治疗的患者HBsAg水平中位数下降幅度均明显优于拉米夫定单一治疗的患者（P<0.05）。结论：在以干扰素为基础的慢性乙型肝炎治疗期间,血清HBsAg水平与HBV DNA 水平显著相关,动态HBsAg定量监测对治疗有一定的指导意义,可作为监测HBV疗效的替代标记。     

Clinical characteristics and chronicity of acute hepatitis B induced by lamivudine-resistant strains

Whether resistant hepatitis B virus (HBV) strains are transmissible and can lead to chronic infection remains to be studied. The aim of this study was to investigate the clinical characteristics of patients with acute hepatitis B caused by lamivudine (LAM)‐resistant strains. Sera were collected from 234 Chinese patients with acute hepatitis B. LAM‐resistance mutations were identified by direct polymerase chain reaction (PCR) sequencing. LAM‐resistant HBV variants were detected in 11 of the 234 (4.7%) patients. Among these patients, six harbored the rtM204I mutation, two harbored the rtL180M + rtM204I mutations, one harbored the rtM204I + rtM204V mutations, one harbored the rtL80I + rtM204I mutations, and one harbored the rtV173L + rtL180M + rtM204V mutations. Three patients were infected with genotype B HBV and eight patients were infected with genotype C HBV. Two patients infected with viruses with LAM‐resistance mutations developed severe acute hepatitis. One patient developed chronic hepatitis B. This patient was infected with genotype C HBV that had LAM‐resistance mutations (rtL180M + rtM204I). The patient was diagnosed with an occult hepatitis B virus infection based on the presence of HBV DNA in the liver and the absence of detectable hepatitis B surface antigen (HBsAg) in the serum. LAM‐resistant HBV strains in China are transmissible, can cause acute hepatitis B, and can even progress to chronic infection in China. J. Med. Virol. 84:1558–1561, 2012. © 2012 Wiley Periodicals, Inc.     

乙肝病毒YMDD变异与乙型肝炎病毒大蛋白相关性的探讨

目的 通过检测乙型肝炎病毒YMDD变异患者血清中的乙型肝炎病毒大蛋白（Hepatitis B Virus Large Surface Protein,LHBs）,探讨乙型肝炎病毒大蛋白（LHBs）的检测与YMDD变异的关系.方法 选取经过拉米夫定治疗后的患者,进行YMDD变异检测,再对所有患者进行HBVDNA和乙型肝炎病毒大蛋白（LHBs）的检测;YMDD变异和HBV DNA检测采用荧光定量PCR法,LHBs检测采用酶联免疫法（ELISA）.结果 65例YMDD变异患者中,HBV DNA拷贝数对数值与LHBs吸光度（A值）呈良好的相关关系（r=0.961）;75例非变异患者中,HBV DNA拷贝数对数值与LHBs吸光度（A值）呈良好的相关关系（r=0.954）.结论 在YMDD变异患者和非变异患者中LHBs吸光度与HBV DNA拷贝数均具有良好的正相关性,表明患者体内LHBs与病毒复制程度密切相关,而且不受乙肝病毒YMDD变异的影响.     

Loss of hepatitis B surface antigen from the serum of patients with chronic hepatitis treated with lamivudine

Although loss of hepatitis B e antigen (HBeAg) from the serum is sought by treatment with lamivudine, clearance of hepatitis B surface antigen (HBsAg) is the eventual goal of any antiviral therapy. In a single hepatology center in the Metropolitan Tokyo, 486 patients with chronic hepatitis B were followed up for longer than 3 years after they started treatment with lamivudine. HBsAg disappeared from the serum in 17 (3.5%). Age >or=50 years and low HBsAg levels (hemagglutination titer or=50 years at the start of lamivudine was the only factor predicting the loss of HBsAg (hazard ratio: 2.96 [95% confidence interval: 1.14-7.68], P = 0.028). By the method of Kaplan-Meier performed on the 486 patients, the loss of HBsAg was estimated to occur in 3% and 13% of patients, respectively, who had received lamivudine therapy for 5 and 10 years. These results indicate that loss of HBsAg occurs in a minority (3.5%) of patients with chronic hepatitis B who receive lamivudine therapy and more frequently in those with lower HBsAg titers and older ages at the start of treatment.     

HBV rtA181位点突变的病毒学特点及临床耐药研究

目的 回顾性研究HBV rtA181位点突变模式与临床核苷（酸）类似物耐药的关系及挽救治疗效果.方法 核苷（酸）类似物治疗过程中发现HBV rtA181位点突变的45例慢性乙型肝炎或肝硬化患者,检测其血清HBV DNA水平,PCR产物直接测序检测HBV变异模式及基因型,收集突变前用药史及突变后的挽救治疗方案及疗效相关资料.结果 HBV rtA181位点共发现三种突变形式,其突变率分别为rtA181T 66.67％ （30/45）,rtA181V 31.11％ （14/45）,rtA181S 2.22％ （1/45）,rtA181T多见于HBV C基因型患者（P＜0.05）;涉及rtA181的HBV突变模式共发现13种,多位点联合突变率为57.78％ （26/45）,其中rtA181与rt236位点联合突变率为40.00％ （18/45）,与rt204位点联合突变率为22.2％ （10/45）,多位点突变患者较单位点突变患者有病毒载量分布更高的趋势,但差异无统计学意义（P＞0.05）,多位点突变与基因型及突变形式无关（P＞0.05）;发生rtA181突变的患者97.78％ （44/45）有拉米夫定（LAM）和（或）阿德福韦酯（ADV）治疗史,另外1例有替比夫定（LdT）治疗史.其后的挽救治疗采用加用或换用恩替卡韦（ETV）、LAM联合ADV以及LdT联合ADV均能在一个月内将HBV DNA降低2个lg值,继续随访效果最显著的方法为加用或换用ETV.结论 HBVrtA181位点突变模式多样,与LAM、ADV或LdT长期单药或序贯治疗有关,加用或换用ETV可取得显著的挽救治疗效果.     

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。     

Quantitative detection of hepatitis B surface antigen by chemiluminescent microparticle immunoassay during lamivudine treatment of chronic hepatitis B virus carriers

The usefulness of fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) for monitoring serum levels of hepatitis B virus (HBV) during antiviral therapy remains unclear. Using this assay, hepatitis B surface antigen (HBsAg) was measured in 20 patients with chronic hepatitis B before and during lamivudine treatment. At the start of therapy, 12 patients had detectable hepatitis B e antigen (HBeAg) and 8 did not. The median serum HBV DNA level and HBsAg concentration (25th-75th centile) were 7.2 (6.1-7.8) log genome equivalents/ml and 3,932 (1,585-12,330) IU/ml, respectively. The HBsAg concentration was significantly higher in HBeAg positive than in HBeAg negative patients (P=0.031). There was a significant correlation between the HBsAg concentration and HBV DNA level (r=0.490, P=0.027). The HBsAg concentration negatively correlated with patient age (r=-0.395, P=0.085). After the start of lamivudine therapy, HBV DNA levels fell rapidly in all patients. Serum HBsAg concentrations also fell in most patients, but to a lesser extent. When drug-resistant variants emerged, serum HBsAg usually increased before biochemical breakthrough. Although HBV DNA was elevated persistently after the emergence of drug-resistant variants, the increase in HBsAg was transient. In some patients, the increase in HBsAg preceded the increase in HBV DNA. Monitoring of serum HBsAg concentrations with the use of Architect HBsAg QT, in addition to measurement of HBV DNA levels, is helpful for evaluating the response to lamivudine treatment and for the early detection of drug-resistant strains.     

Lamivudine resistance mutations in European patients with hepatitis B and patients co-infected with HIV and hepatitis B

Evaluation of resistance pattern in patients with chronic hepatitis B. Retrospective study of hepatitis B virus (HBV) resistance mutations in patients found viraemic after first-line treatment. HBV viral load was determined by a real-time polymerase chain reaction and the substitutions in HBV-DNA were studied by polymerase sequencing test. First line treatment had failed in 12 out of 33 patients (36%) receiving anti-HBV drugs. The 12 patients with persistent viraemia were all lamivudine (LAM) experienced and 7 had a polymerase sequencing test available. LAM substitution mutations L180M + M204V/I were found in six out of seven cases, with an accompanying V173L mutation in three cases. These mutations were also related with changes in HBsAg. The use of potent drugs in the first line anti-HBV therapy may reduce the resistance mutations in the future.     

Sequence analysis of pre-S/surface and pre-core/core promoter genes of hepatitis B virus in chronic hepatitis C patients with occult HBV infection

Although occult hepatitis B virus (HBV) infection in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and has been reported to be common in patients with chronic hepatitis C, the related molecular mechanisms remain unknown. With the polymerase chain reaction, serum HBV DNA was sought in 100 HBsAg-negative patients with chronic hepatitis C virus (HCV)-infection. In those with occult HBV infection, possible genomic variability of HBV was evaluated by amplification and direct sequencing of pre-S, surface, and pre-core/core promoter genes. In total, 10 of the 100 patients (10%) had detectable serum HBV DNA, documenting an occult HBV infection. A deletion mutant in the pre-S gene was found in one patient and mutations of the a determinant of HBsAg were observed in 2. In addition, a novel core promoter mutant (a dinucleotide substitution: T-to-C at nucleotide 1,802 and T-to-G at nucleotide 1,803, T1802C/T1803G) was found frequently in patients with occult HBV infection as compared to sex- and age-matched HBsAg-positive patients (80 vs. 10%, P < 0.001). In conclusion, the data suggest occult HBV infection is not uncommon in chronic hepatitis C patients in Taiwan, and a novel core promoter mutant may be associated with the absence of circulating HBsAg in these patients.