YMDD自然变异毒株流行病学调查

目的 了解YMDD自然变异的发生情况及影响YMDD自然变异的因素.方法 依托国家食品药品监督管理局注册药物临床试验"阿德福韦酯片治疗成人慢性乙型肝炎的多中心、随机、双盲、对照临床研究(Ⅲ期)"开展.采用乙型肝炎病毒(HBV)拉米夫定耐药荧光PCR检测试剂盒及实时荧光PCR基因型分型试剂盒,对该临床研究筛选期183例半年内未经抗病毒治疗的慢性乙型肝炎(CHB)患者血清标本进行检测,并通过Logistic逐步回归对影响YMDD自然变异检出率的可能因素进行多因素分析.结果 183例CHB患者中,检出YMDD自然变异毒株者40例(21.86%),其中YVDD阳性36例,YIDD阳性3例,YVDD和YIDD同时阳性者1例;变异毒株占总病毒量50%以上者6例,占25%～50%者9例,占9%～25%者25例.B基因型CHB患者中YMDD自然变异检测阳性的比例高于C基因型(31.67%对17.65%,P=0.038 2),患者性别、年龄、HBeAg状态、血清HBV DNA载量及丙氨酸氨基转移酶(ALT)水平对YMDD自然变异检出率无显著影响(P＞0.05).结论 在CHB患者中存在YMDD自然变异毒株;YMDD自然变异的发生率与患者性别、年龄、HBeAg状态、HBV DNA水平、ALT水平无关.B基因型较C基因型更易出现YMDD自然变异.     

未接受抗病毒治疗的慢性乙型肝炎患者多聚酶P基因YMDD变异检测

目的 了解未经拉米夫定及干扰素抗病毒治疗的慢性乙型肝炎患者中HBV多聚酶YMDD变异情况。方法 应用错配PCR扩增方法检测病人血清HBV的YMDD位点，选择65例病史超过半年以上、肝功能异常、HBV DNA阳性的慢性乙型肝炎患者。结果 在65例患者中，YMDD变异阳性6例(9．07％)，阴性59例，6例变异中2例为YIDD阳性，其中1例为YMDD野毒株和变异株混合存在，4例为YYDD阳性，其中1例为YMDD野毒株和变异株混合存在。结论 本检测方法简便、实用，在未经抗病毒治疗的慢性乙型肝炎患者中可存在YMDD变异株，其与野生株一样是自然存在的。     

乙肝肝硬化发生HBV YMDD自然变异的意义探讨

目的 探讨乙肝肝硬化情况下,乙肝病毒发生YMDD自然变异的几率及对肝硬化发展的影响.方法 对106例未应用过抗病毒药物的乙肝肝硬化病例,检测其YMDD变异,乙肝病毒载量,HBVM,肝功;根据YMDD变异情况,分为YMDD变异组与未变异组,进行对比分析.结果 106例乙肝肝硬化患者中检出YMDD自然变异株者28例(26.41%),发生YMDD变异组与未变异组比较,在性别、HBeAg、病毒载量方面未显示有差异性,而肝功能指标中ALT及AST高,二组有显著性差异.结论 在乙肝肝硬化患者中存在着HBV YMDD自然变异株,发生YMDD变异的患者肝内炎症稍重.     

急性乙型肝炎患者血清HBV基因耐药变异及相关因素分析

目的了解急性乙型肝炎(AHB)患者感染HBV基因耐药相关变异性,并对变异相关因素HBV DNA、HBeAg、ALT、AST及TBil进行分析。方法收集AHB患者123例,提取血清HBV DNA对反转录酶(RT)基因区核苷(酸)类似物变异耐药突变10个位点进行检测,同时进行HBV DNA载量、HBeAg、ALT和AST检测。采用焦磷酸测序技术(pyrosequencing)进行DNA测序,数据分析采用PSQ-96MA型焦磷酸测序仪配套的分析软件进行。结果 123例患者中9例发生变异,检出率为7.32%,病毒以变异/未变异共存形式存在。变异位点和变异形式均以204V/I和181V为主,相关耐药主要为拉米夫定、替比夫定和阿德福韦酯。发生变异者与未发生变异者比较,HBV DNA载量、ALT、AST、TBil及HBeAg阴性率差异均无统计学意义(P>0.05)。结论急性HBV感染者体内存在HBV核苷(酸)类似物耐药相关突变,自然变异与HBVDNA载量、ALT、HBeAg、TBil及AST无显著相关性。     

未抗病毒治疗的慢性乙型肝炎患者YMDD变异分析

我们对近1年内未经抗病毒治疗的25例慢性乙型肝炎患者进行血清乙型肝炎病毒聚合酶P区变异(YMDD)变异检测，以探讨未经抗病毒治疗的慢性乙型肝炎患者YMDD变异情况及其与HBV基因型的相关性。     

未经治疗的慢性乙型肝炎患者乙型肝炎病毒耐药变异、基因型和血清型研究

目的 研究未经核苷(酸)类似物(NA)治疗的慢性乙型肝炎患者HBV耐药变异、基因型、基因亚型和血清型特点.方法 从北京大学附属医院收集97例未经NA治疗的慢性乙型肝炎患者血清,用半巢式聚合酶链反应-直接测序法获得HBV全长逆转录酶区序列,用生物信息学技术筛查该区内11个经典耐药变异位点并鉴定基因型、基因亚型和血清型.用统计分析软件SPSS11.0进行t检验和χ~2检验. 结果 HBV在11个经典耐药变异位点上均为野生型氨基酸;B基因型和C基因型分别占36.1%(35/97)和63.9%(62/97),前者均属B2亚型,后者C2亚型占91.9%(57/62),C1亚型占6.5%(4/62),1例未能分出亚型.已知出生地的患者中,71.9%(23/32) B基因型感染者出生于我国南方地区,81.6%(40/49) C基因型感染者出生于北方地区,基因型地域分布特点明显,χ~2=23.19,P<0.01.血清型为adr者占60.8%(59/97),与C基因型相关;为adw者占38.1%(37/97),与B基因型相关,χ~2=87.83,P<0.01.结论 未经NA治疗的慢性乙型肝炎患者体内野毒株为优势株,其基因型、基因亚型和血清型与患者出生地有关.     

慢性HBV携带者YMDD自然变异的追踪研究

目的 探讨慢性HBV携带者YMDD自然变异随时间的推移发生变化的情况.方法 随机选择未经抗病毒治疗的慢性HBV携带者103例,分别在开始、1年、2年时观察血清HBV DNA、HBV血清学标志物、肝功能、YMDD变异的变化情况.结果 在开始检测的103例慢性HBV携带者中,YMDD阳性27例,观察2年后4例转为非活动性H...     

不同HBV感染转归人群外周血PD-1拷贝数分布研究*

目的 调查HBV感染不同转归人群外周血程序性死亡受体-1（PD-1）基因拷贝数（CN）分布频率的差异。方法 在300例HBV感染恢复者、437例慢性乙型肝炎（CHB）、249例肝硬化（LC）和123例肝细胞癌（HCC）患者,采用AccuCopy法检测外周血PD-1基因拷贝数。选择5个可能影响慢性HBV感染结局的指标,即HBeAg滴度、年龄、性别、HBV DNA和PD-1拷贝数,行Logistic回归分析,以发现影响感染结局的指标。结果 HBV感染恢复者、CHB、LC和HCC组外周血PD-1单倍体检出率分别为11.7%、9.6%、6.0%和10.6%,多倍体检出率分别为89.3%、90.4%,94%和89.4%,显示LC组多倍体检出率显著高于HBV感染恢复组或HCC组 （P<0.05）;以CHB组作为估计参数,经Logistic回归分析发现年龄和血清HBV DNA载量为影响疾病转归的独立危险因素。结论 本研究结果提示影响HBV感染转归的因素还是病毒本身和患者年龄,而外周血PD-1基因拷贝数变异检测的意义还有待于进一步研究。     

FCGR3A和FCGR3B基因拷贝数变异对HBV感染转归的影响

目的 探讨FCGR3A和FCGR3B基因拷贝数变异与HBV感染后不同转归和疾病进展的相关性。方法 收集2014年1月—2018年12月安徽医科大学第一附属医院和第二附属医院感染病科住院和门诊841例慢性HBV感染者和296例自限性HBV感染者外周血标本,并将慢性HBV感染者根据病情进展程度进一步分为慢性乙型肝炎（CHB）组、肝硬化（LC）组、肝细胞癌（HCC）组。采用AccuCopy技术定量分析两组患者外周血FCGR3A和FCGR3B基因拷贝数变异。计量资料两组间比较采用独立样本t检验,多组间比较采用单因素方差分析和Kruskal-Wallis H检验;计数资料组间比较采用χ²检验。同时采用χ²检验分析FCGR3基因CNV在不同组间的分布差异。应用年龄和性别校正的logistic回归模型综合分析CNV对HBV感染慢性化的影响。结果 慢性HBV感染组与自限性HBV感染组的FCGR3A、FCGR3B 拷贝数变异频率分布差异均有统计学意义（χ²值分别为11.406、19.143,P值均<0.05）。在慢性HBV感染后疾病...     

拉米夫定耐药变异株在急性HBV感染者中的流行调查

目的:调查YMDD变异株在急性HBV感染(AHB)中的流行情况,为指导AHB的防治提供依据.方法:收集HBV感染者321例(HBV DNA≥1.0×107copies/L),其中未开始治疗的AHB患者100例和拉米夫定治疗(100 mg/d)不同时期的CHB患者221例.采集外周血标本,用荧光标记杂交双探针PCR融解曲线法(FH-PCR-MC)检测血清HBV YMDD及其变异,统计分析HBVYMDD变异在两组中的分布.结果:在AHB组,只检出HBV YMDD野生型,未检出变异型.而在CHB组,YMDD变异检出率为63.4%,其中YIDD占52.1%,YVDD占37.9%,YIDD+YVDD混合变异占10.0%:1年内、1-2年、2-3年、3-4年和4年以上疗程YMDD变异率分别为45%,66%,77%,75%和40%,YMDD变异检出率在两组中差异显著(X2=112.3,P=0.00).结论:目前在用拉米夫定治疗的CHB患者中仍有较高比率的YMDD变异株存在,但尚未发现该变异株在AHB患者中流行.     

Clinical characteristics and distribution of hepatitis B virus genotypes in Guangxi Zhuang population

AIM: To investigate the distribution of HBV genotypes and their YMDD mutations in Guangxi Zhuang population, China, and to study the relationship between HBV genotypes and clinical types of HB, ALT, HBV DNA, HBe system as well as the curative effect of Lamivudine (LAM) on hepatitis B. METHODS: A total of 156 cases were randomly chosen as study subjects from 317 patients with chronic hepatitis B (CHB). HBV genotypes were determined by PCR-microcosmic nucleic acid cross-ELISA. YMDD mutations were detected by microcosmic nucleic acid cross-nucleic acid quantitative determination. HBV DNA was detected by fluorescence ratio PCR analysis. LAM was given to 81 cases and its curative effect was observed by measuring ALT, HBV DNA load, HBeAg, and HBeAg/HBeAb conversion rate. RESULTS: HBV genotypes B, C, D, and non-classified genotypes were found in Guangxi Zhuang population, accounting for 25.6%, 47.4%, 58.3%, and 16.0%, respectively. Seventy-four cases were CD-, CB-, BD-mixed genotypes (47.7%). Forty-six (29.5%) cases had YMDD mutations. Genotype B was mostly found in mild and moderate CHB patients. Genotypes C, D and mixed genotype mostly occurred in severe CHB cases. Genotypes D and CD HBV-infected patients had higher ALT and HBV DNA than patients with other types of HBV infection. There was no significant difference among the genotypes in YMDD mutations, clinical types, ALT and HBV DNA level. Non-classified types geno had a significantly lower positive rate of HBeAg than other genotypes (X2=12.841,P<0.05). There was no significant difference in ALT recovery rate, HBV DNA load, HBeAg, and HBeAg/HBeAb conversion rate, 48 wk after LAM treatment between groups of genotypes D, CD, and non-classified type. CONCLUSION: Genotypes B, C, and D, non-classified and mixed genotype of HBV are identified in the Guangxi Zhuang population. Variations in genotypes are associated with clinical severity and serum ALT levels, but not with YMDD mutation or HBV DNA load. Therapeutic effects of LAM on clinical parameters are not influenced by differences in genotypes. Further studies are needed to gain an in-depth understanding of the relationship between HBV genotypes and serum HBeAb and HBeAg.     

YMDD motif mutations in chronic hepatitis B antiviral treatment naïve patients: a multi-center study

ObjectiveThis study aimed to determine the natural prevalence of variants of tyrosinemethionine- aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels.MethodsA total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA.ResultsYMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level.ConclusionMutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.     

乙型肝炎病毒基因型与拉米夫定疗效关系的研究

探讨乙型肝炎病毒(HBV)基因型与拉米夫定治疗慢性乙型肝炎(CHB)疗效的关系。采用PCR、核酸杂交和酶联显色技术对CHB进行HBV基因分型,观察123例(B型93例和C型30例)CHB患者拉米夫定治疗1年后肝功能、病毒学指标和YMDD变异的变化。ALT复常率为92.47%,HBeAg阴转率为27.96%,HBVDNA阴转率为82.80%,有效应答率为89.25%,与C基因型相比差异有显著性(P<0.05或P<0.005)。B型YMDD变异的发生率为9.68%,显著低于C型的26.67%(P<0.05)。B型对拉米夫定的抗病毒疗效高于C型,YMDD变异的发生率则低于C型。HBV基因型是影响拉米夫定疗效和变异的重要因素之一。     

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.     

拉米夫定对湖北地区乙型肝炎病毒基因型的影响

目的研究拉米夫定对湖北地区乙型肝炎病毒基因型的影响及其临床意义。方法采用多对型特异性引物-聚合酶链反应检测160例慢性乙型肝炎患者血清HBV基因型;采用基因芯片技术对86例拉米夫定治疗18个月的慢性乙型肝炎患者进行酪氨酸—蛋氨酸—天冬氨酸—天冬氨酸(YMDD)基序、G1896A、A1814C、A1792T和G1764A单碱基变异检测。结果160例慢性乙型肝炎患者,B基因型127例(79%),C基因型24例(15%),BC混合型9例(6%),未发现A、D和E基因型。拉米夫定治疗的86例患者中,17例(19.7%)发生YMDD变异,B型14例,C型2例,BC混合基因型1例,其中6例发生多重变异,包括B型4例,C型2例;HBeAg/HBeAb血清转换率B型41例(68.3%)高于C型8例(40%)(P<0.05)。另外17例患者,经拉米夫定治疗后,HBVDNA仍阳性,亦未发现YMDD变异株。结论湖北地区HBV存在B、C和BC混合基因型,B型为本地区优势基因型,B型在拉米夫定治疗中更易发生YMDD变异;未变异者,血清HBeAg/HBeAb转换率高。     

Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection

BACKGROUND/AIMS: End-stage renal disease patients on chronic hemodialysis are at risk for both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Although the prevalence is unknown in hemodialysis patients, occult HBV infection is frequent in subjects with chronic HCV infection. We aimed to investigate (1) the prevalence and clinical impact of occult HBV infection in hemodialysis patients with chronic HCV infection, and (2) the frequency of YMDD variants (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) in this setting. METHODS: Thirty-three anti-HCV and HCV-RNA-positive, HBsAg-negative hemodialysis patients (mean age 36.9+/-10.4 years, 22 male) were admitted to this study. HBV-DNA (Innogenetics kit) and HCV-RNA (Cobas Amplicor HCV kit) were investigated by polymerase chain reaction technique (PCR). YMDD mutation was studied in all HBV-DNA-positive patients by the BOOM method. RESULTS: HBV-DNA was detected in 12 of 33 patients (36.4%) by PCR. Their mean age was 33.0+/-9.0 years. Age, dialysis period (years) and biochemical parameters were not significantly different in patients with and without occult HBV infection. YMDD variants were identified in six of 12 (50%) patients with occult HBV infection. CONCLUSIONS: Occult HBV infection is frequent in hemodialysis patients with chronic HCV infection. YMDD variants are common in this setting.     

HBeAg阳性和阴性慢性乙型肝炎患者YMDD的变异

目的研究YMDD变异在HBeAg阳性和HBeAg阴性乙型肝炎病毒（HBV）感染者中发生的情况.方法对我科接受拉米夫定治疗的247例慢性乙型肝炎患者进行定期随访,检测肝功能和HBV病毒学指标,利用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）检测YMDD变异,利用实时定量PCR检测HBV DNA定量以及利用Abbott试剂检测HBV标志物,对比分析HBeAg阳性和HBeAg阴性患者中YMDD变异的发生情况.结果247例患者中共有42例出现YMDD变异,YMDD变异的累积发生率随着时间的延长逐年增加.与治疗前HBeAg阴性患者相比,治疗前HBeAg阳性患者YMDD变异的发生率明显高于治疗前HBeAg阴性患者.结论HBeAg阳性患者YMDD变异年累积发生率高于HBeAg阴性患者.     

HBeAg阳性患者中乙型肝炎病毒基因型分布及YMDD变异位点分析

目的通过研究乙型肝炎患者HBV基因型的分布及YMDD变异位点的分布特点,了解两者之间的关系,探讨临床意义。方法选取在我院门诊和住院部就诊的HBeAg阳性并经拉米夫定治疗两年以上的慢性乙型肝炎患者137例,用实时荧光PCR方法检测HBV DNA分型情况,用LDR方法检测YMDD位点变异情况。结果 137例患者中21例未能检测出基因型,其中男性17例,女性4例;其余116例标本确定了感染病毒的基因型,包括男性94例,女性22例;其中A型2例(1.72%)、B型19例(16.38%)、C型91例(78.45%)、D型4例(3.45%);分型成功率为84.67%(116/137)。116例标本中有41例患者的感染病毒中检测到了YMDD变异位点,变异发生率为35.35%。其中B基因型中检出7例(36.84%);C基因型中检出33例(36.26%);D基因型中检出1例(25.0%);A基因型中未检测出变异位点。B、C基因型中变异位点的检出率高于D型,但各基因型之间差异无统计学意义(χ2=1.392,P>0.05)。在41例变异中,其中I突变占60.9%,V突变占31.7%,I/V共生突变占7.4%。结论兰州大学第一医院所在地区患者中HBV基因型主要以C型为主,其次是B型,也有少量D、A型存在;B、C基因型的YMDD位点发生突变的几率相近。突变几率与基因型无关。     

A 7‐year study of lamivudine therapy for hepatitis B virus e antigen‐positive chronic hepatitis B patients in China

OBJECTIVE: To evaluate the long‐term efficacy and safety of lamivudine treatment for chronic hepatitis B and the impact of emergence of YMDD mutation of hepatitis B virus (HBV). METHODS: A total of 429 patients with serum HBsAg, HBeAg and HBV DNA positive were randomized to receive either lamivudine 100 mg daily or a placebo in a 3:1 ratio for the first 12 weeks. Thereafter, all patients were administered with lamivudine 100 mg/d for 5 years and followed up for 2 years. RESULTS: After 12 weeks of the lamivudine treatment, serum HBV DNA levels decreased rapidly and HBV DNA negativity (<1.6 pg/mL) was 92.2%, whereas it was only 14.1% (P < 0.01) in the placebo group. At the end of 5 years, serum HBV DNA continued to be substantially suppressed. The loss of HBeAg and seroconversion were significantly correlated with baseline alanine aminotransferase (ALT) levels, in patients with baseline ALT > 2 × upper limits of normal, the loss of HBeAg was 54% and seroconversion rate was 50%, respectively. YMDD mutation developed in 70.8% of the patients at years 5. In YMDD mutant patients, HBV DNA levels were increased moderately and with mild to moderate elevations of ALT. ALT flares (ALT > 5ULN) occurred in 22 patients, 16 with YMDD variants and six with non‐variants. One year durability of seroconversion after stopping lamivudine was 80%. CONCLUSION: Lamivudine is effective and tolerable for chronic hepatitis B.