利用基因芯片分析拉米夫定治疗过程中HBV DNA的基因变异

依据乙型肝炎病毒(Hepatitis B virus; HBV)聚合酶基因序列研制HBV基因芯片,此芯片可分析HBV的7个基因型、4种血清型和HBV聚合酶基因rtV173、rtL180、rtM204和rtV207位点的突变.利用此芯片对A、B两组共计45例拉米夫定治疗12个月的患者进行服药前和服药后3、6、9、12个月的动态检测,其中C基因型39例,且血清型均为adr;B基因型6例,其血清型均为adw.在完成全程检测的38例患者中,17例ALT升高的A组出现1例拉米夫定耐药变异株,而21例ALT正常的B组出现4例变异株,且所有变异株均为rtM204 V/rtL180M,其中2例野生株和变异株共存.rtM204V变异最早在服药6个月时出现,随后出现rtL180M变异.10份PCR产物测序分析表明,芯片检测结果与测序结果基本一致,仅在rtL173位点出现1例差异.进一步分析HBV DNA变异与HBV DNA含量、ALT水平和HBeAg血清转换率的相关性,初步结果表明变异株的出现与治疗过程中的DNA反弹呈正相关,而与起始HBV DNA水平、ALT值无关联.HBV基因芯片可初步用于HBV DNA 检测,可能是临床追踪评价抗病毒治疗效果的较好方法之一.     

拉米夫定联合阿德福韦酯治疗乙型肝炎的疗效及对HBV耐药基因突变的影响

目的探讨拉米夫定联合阿德福韦酯治疗慢性乙型肝炎的疗效,并利用反向点杂交技术检测其对HBV基因耐药突变的影响。方法156例慢性乙型肝炎患者随机分为2组：对照组70例采用拉米夫定治疗,治疗组86例采用拉米夫定联合阿德福韦酯治疗。采用实时荧光定量PCR和ELISA检测2组治疗前和治疗后48周的HBV-DNA载量和HBeAg并采用PCR-反向点杂交技术（PCR-RDB）检测2组治疗48周后的HBV耐药基因突变情况。结果对照组及治疗组在经过48周治疗后HBV-DNA载量较治疗前都明显下降（P 〈0. 05）,治疗组HBV-DNA载量明显低于对照组（P〈0.05）。治疗组经过48周治疗后HBeAg的阴转率为54.9%,明显高于对照组15.0% （P 〈0.05）。对照组44例未出现耐药突变,25例拉米夫定耐药突变中rtL180M突变6例,rtM204V/I突变11例,rtL180M ＋ rtM204V/I混合突变8例;阿德福韦酯HN236T耐药突变1例。治疗组77例未出现耐药突变;5例拉米夫定耐药突变中rtL180M突变1例,rtM204V/I突变2例,rtL180M ＋ rtM204V/I混合突变2例;阿德福韦酯耐药突变中rtN236T突变1例;拉米夫定和阿德福韦酯交叉耐药rtN236T ＋ rtM204V/I混合突变3例。对照组耐药突变率为37. 1%（26/70）明显髙于治疗组的10.5%（9/86）（P〈0.05）。结论拉米夫定联合阿德福韦酯对治疗慢性乙型肝炎方面有效并在减少HBV耐药基因突变方面具有一定的作用。     

乙型肝炎病毒RT 区变异及其与基因型分布的关系

目的:探讨大样本乙型肝炎病毒(HBV)感染患者RT区耐药位点变异的流行情况,及各耐药位点变异与HBV基因型的关系。方法:采用P区测序法对1117例慢性乙型肝炎患者的血清病毒进行P区测序、进化树分型。结果:RT区耐药位点变异发生率与基因型关系密切,在基因型C患者中的变异发生率远远高于基因型B患者(P=0.000)。Rt180、rtM204V、rtM204I、rt181、rt213位点变异均与基因型C有关(P<0.05)。主要的三种变异类型rt180+rtM204V、rtM204I、rt180+rtM204I间基因型分布存在显著差异(P=0.003)。不同HBeAg状态下,耐药变异的发生有显著差异(P=0.020),特别是rt181和rt236位点变异。结论:HBV基因型影响RT区耐药变异发生率及变异类型,且耐药变异发生率也与HBeAg状态有关。     

乙型肝炎病毒RT区变异及其与基因型分布的关系

目的：探讨大样本乙型肝炎病毒（HBV）感染患者RT区耐药位点变异的流行情况,及各耐药位点变异与HBV基因型的关系。方法：采用P区测序法对1117例慢性乙型肝炎患者的血清病毒进行P区测序、进化树分型。结果：RT区耐药位点变异发生率与基因型关系密切,在基因型C患者中的变异发生率远远高于基因型B患者（P=O．000）。Rt180、rtM204V、rtM204I、rt181、rt213位点变异均与基因型c有关（P〈O．05）。主要的三种变异类型rt180＋rtM204V、rtM204I、rt180＋rtM204I间基因型分布存在显薯差异（P=0．003）。不同HBeAg状态下,耐药变并的发生有显著差并（P=O．020）,特别是rt181和rt236位点变畀。结论：HBV基因型影响RT区耐药变异发生率及变异类型。且耐药变异发生率也与HBeAg状态有关。     

山东省潍坊市慢性乙型肝炎患者HBVP区基因耐药突变检测及相关因素分析

探讨慢性乙型肝炎(Chronic hepatitis B,CHB)患者多聚酶基因逆转录保守区(P区)位点突变情况。选择212例行核苷(酸)类似物抗病毒治疗的CHB患者,采用PCR产物直接测序法检测HBV P基因区耐药变异位点,同时检测其HBV基因型。结果表明,HBV的P基因区突变位点有173、180、18l、184、204、236和250,主要的耐药位点为204和180,分别占35.8%和23.5%。180位点在不同年龄组之间比较均有显著差异,204位点在30岁以下组与41~50岁组、51~60岁组间比较有显著差异(P0.05,P0.01);180位点联合204位点突变率为66.6%,l81位点联合236位点突变率为23.3%;HBV C基因型患者年龄明显大于B基因型患者(P0.01)。M204V/I多以联合L180M突变的形式存在,突变率与年龄有关,HBV基因型和HBV P区耐药位点的检测对CHB患者的治疗和病情预后具有重要的临床意义。     

黑龙江地区400例乙型肝炎病毒基因分型与耐药突变研究

目的:运用基因芯片技术分析黑龙江地区乙型肝炎病毒(HBV)基因型分布特征及基因耐药变异情况。方法:随机选择2012年11月至2015年11月本医院乙型肝炎患者血清样本400例,应用PCR-反向点杂交的基因芯片技术对样本血清中HBV基因型及常见4类抗病毒药物耐药相关的多个位点进行检测,并进行数据分析。结果:400例样本中基因型分布以C型为主占83.25%(333例),B型7.25%(29例)、D型0.25%(1例)及混合基因型2.75%(11例);耐药突变位点检出188例,总耐药率为45.19%,其中突变位点236T(4.61%)提示阿德福韦酯单项耐药,耐药率为5.82%(10例),与拉米夫定耐药相关的为126例,突变位点以rt204I和(rt180M+rt240V)为主,显著高于其他抗病毒类药物,耐药风险较高。结论:黑龙江地区乙型肝炎基因分型以C为主,B型和其它混合型较少,且更容易对拉米夫定产生耐药。     

2010年舟山某医院拉米夫定无良好应答乙肝患者耐药监测分析

目的观察2010年舟山某医院拉米夫定单药治疗无良好应答的乙肝患者其基因型和P区耐药位点突变的关系。方法随机选取舟山某医院就诊的124例拉米夫定单药治疗无良好应答的本地乙肝患者,男64例,女60例,平均年龄(46.92±15.16)岁,用实时荧光定量PCR检测其基因型和HBV-DNA,并用毛细管电泳测序技术对P区进行突变位点检测。结果 124例患者35例(28.23%)B型和89例(71.77%)C基因型。C型HBV-DNA为(5.83±0.91)log10 copies/mL,高于B型的(5.07±1.13)log10 copies/mL,差异具有统计学意义(t=3.55,P<0.01)。拉米夫定在B基因型中的耐药率为45.71%(16/35),低于C基因型的65.17%(58/89),差异具有统计学意义(2χ=3.951,P<0.05)。拉米夫定耐药株在B基因型中rtM204 I/V/S位点突变率为68.75%(11/16),高于C基因型的37.93%(22/58),差异具有统计学意义(2χ=4.821,P<0.05)。结论对于本地区拉米夫定治疗无良好应答的C基因乙肝患者,应加强拉米夫定的耐药位点筛查,而拉米夫定治疗无良好应答的B基因乙肝患者应加强rtM204 I/V/S位点检测。     

汉族和维吾尔族乙型肝炎病毒耐药基因的相关临床研究

目的:利用基因芯片技术研究伊犁地区汉、维两个民族乙型肝炎病毒(HBV)耐药基因的差异性。方法:收集2014年1月-2015年12月我院收治的汉族及维吾尔族(以下简称"维族")慢性HBV感染患者各50例,患者均经基因芯片技术筛选确诊存在天然HBV耐药基因(耐核苷酸类药物),观察HBV耐药基因分型特点及对核苷酸类药物的耐药突变位点的情况。结果:汉族HBV感染患者的耐药基因型集中于B、C型,且以C型为主,而维族以D型为主,两组各基因型比较,差异均有统计学意义(P0.05)。汉族患者在rt204位点变异明显,而维族在rtn236t、rta181v/t+rtn236t位点变异明显,差异均有统计学意义(P0.05)。结论:在新疆伊犁地区天然HBV病毒耐药基因患者中,汉族及维族耐药基因分型及耐药基因突变位点均存在显著差异,临床可通过基因芯片技术筛选变异靶点,选择合适的敏感药物。     

河南省乙型肝炎病毒分型特征及小S蛋白主要亲水区变异特点分析北大核心CSCD

为了解河南省乙型肝炎(乙肝)HBV基因型分布及其主蛋白抗原主要亲水区(MHR)氨基酸(aa)位点变异情况。本研究采集河南省2012年HBV流行病学调查的部分HBsAg和HBeAg阳性的血清样本,提取HBV DNA并进行序列扩增,测序得到s基因序列,利用Mega6.0软件比较分析。共得到HBVS基因序列50条,基因型分布B型为16.0%(8/50)、C型为84.0%(42/50)。血清型分布中,adrq+为HBV主要流行血清型,流行率为84.0%。S基因MHR aa位点变异中,T126变异率最高,为14.0%。HBV MHR变异株总流行率为24.0%(12/50),其中B型的突变率为37.5%(3/8),C型的突变率为21.4%(9/42)。河南省乙肝基因型分布以C型为主,B型次之。血清型主要为adrq+为主,adw2次之。HBV MHR aa位点存在变异,应在今后的计划免疫和HBIG治疗中给予重视。     

Hepatitis B genotype G and high frequency of lamivudine-resistance mutations among human immunodeficiency virus/hepatitis B virus co-infected patients in Brazil

In this study, we evaluated the hepatitis B virus (HBV) genotype distribution and HBV genomic mutations among a group of human immunodeficiency virus-HBV co-infected patients from an AIDS outpatient clinic in S?o Paulo. HBV serological markers were detected by commercially available enzyme immunoassay kits. HBV DNA was detected using in-house nested polymerase chain reaction and quantified by Cobas Amplicor. HBV genotypes and mutations in the basal core promoter (BCP)/pre-core/core regions and surface/polymerase genes were determined by sequencing. Among the 59 patients included in this study, 55 reported prior use of lamivudine (LAM) or tenofovir. HBV DNA was detected in 16/22 patients, with a genotype distribution of A (n = 12,75%), G (n = 2,13%), D (n = 1,6%) and F (n = 1,6%). The sequence data of the two patients infected with genotype G strongly suggested co-infection with genotype A. In 10 patients with viremia, LAM-resistance mutations in the polymerase gene (rtL180M + rtM204V and rtV173L + rtL180M + rtM204V) were found, accompanied by changes in the envelope gene (sI195M, sW196L and sI195M/sE164D). Mutations in the BCP and pre-core regions were identified in four patients. In conclusion, genotype G, which is rarely seen in Brazil, was observed in the group of patients included in our study. A high prevalence of mutations associated with LAM-resistance and mutations associated with anti-HBs resistance were also found among these patients.     

Long-Term Hepatitis B Virus (HBV) Response to Lamivudine-Containing Highly Active Antiretroviral Therapy in HIV-HBV Co-Infected Patients in Thailand

BACKGROUND: Approximately 4 million of people are co-infected with HIV and Hepatitis B virus (HBV). In resource-limited settings, the majority of HIV-infected patients initiate first-line highly active antiretroviral therapy containing lamivudine (3TC-containing-HAART) and long-term virological response of HBV to lamivudine-containing HAART in co-infected patients is not well known. METHODOLOGY/PRINCIPAL FINDING: HIV-HBV co-infected patients enrolled in the PHPT cohort (ClinicalTrials.gov NCT00433030) and initiating a 3TC-containing-HAART regimen were included. HBV-DNA, HIV-RNA, CD4+ T-cell counts and alanine transaminase were measured at baseline, 3 months, 12 months and then every 6 months up to 5 years. Kaplan-Meier analysis was used to estimate the cumulative rates of patients who achieved and maintained HBV-DNA suppression. Of 30 co-infected patients, 19 were positive for HBe antigen (HBeAg). At initiation of 3TC-containing-HAART, median HBV DNA and HIV RNA levels were 7.35 log(10) IU/mL and 4.47 log(10) copies/mL, respectively. At 12 months, 67% of patients achieved HBV DNA suppression: 100% of HBeAg-negative patients and 47% of HBeAg-positive. Seventy-three percent of patients had HIV RNA below 50 copies/mL. The cumulative rates of maintained HBV-DNA suppression among the 23 patients who achieved HBV-DNA suppression were 91%, 87%, and 80% at 1, 2, and 4 years respectively. Of 17 patients who maintained HBV-DNA suppression while still on 3TC, 4 (24%) lost HBsAg and 7 of 8 (88%) HBeAg-positive patients lost HBeAg at their last visit (median duration, 59 months). HBV breakthrough was observed only in HBeAg-positive patients and 6 of 7 patients presenting HBV breakthrough had the rtM204I/V mutations associated with 3TC resistance along with rtL180M and/or rtV173L. CONCLUSIONS: All HBeAg-negative patients and 63% of HBeAg-positive HIV-HBV co-infected patients achieved long-term HBV DNA suppression while on 3TC-containing-HAART. This study provides information useful for the management of co-infected patients in resource-limited countries where the vast majority of co-infected patients are currently receiving 3TC.     

Plant growth promotion and Penicillium citrinum

Background

Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results

Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 × 10⁷ and 6.9 × 10⁷ copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion

A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.     

Molecular mechanisms of adefovir sensitivity and resistance in HBV polymerase mutants: a molecular dynamics study

Molecular modeling studies of adefovir diphosphate with the wild type and the mutant HBV polymerase-DNA complex demonstrated that the increase in adefovir sensitivity toward HBV polymerase mutants (rtL180M, rtM204V/I, rtL180M-M204V/I) is a result of increased van der Waals interaction and is supplemented by the decreased affinity of natural substrate toward the mutant HBV polymerase. In the case of rtN236T mutant, loss of two hydrogen bonds accompanied by significant decrease in electrostatic interactions is observed, which explains the observed decrease in drug sensitivity and binding affinity of adefovir diphosphate toward the rtN236T mutant HBV polymerase.     

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).     

Molecular Epidemiology and Genotyping of Hepatitis B Virus of HBsAg-Positive Patients in Oman

Background

Hepatitis B virus (HBV) infection is a major global health burden with distinct geographic public health significance. Oman is a country with intermediate HBV carrier prevalence; however, little is known about the incidence of HBV variants in circulation. We investigated the HBV genotype distribution, the occurrence of antiviral resistance, and HBV surface antigen (HBsAg) escape mutations in HBsAg-positive patients in Oman.

Methods

Serum samples were collected from 179 chronically HBV-infected patients enrolled in various gastroenterology clinics in Oman. HBV genotypes were determined by sequencing and phylogenetic analysis. Mutations in the HBV polymerase and the HBsAg gene were characterized by mutational analysis.

Results

HBV genotypes D (130/170; 76.47%) and A (32/170; 18.28%) are predominant in Oman. The HBV genotypes C and E were less frequent (each 1.18%), while the HBV genotypes B, G, F, and H were not detected. Four patients revealed HBV genotype mixtures (HBV-A/D and D/C). The analyses of vaccine escape mutations yield that 148/170 (87.06%) HBV sequences were wild type. 22/170 (12.94%) HBV sequences showed mutations in the “a” determinant of the HBsAg domain. Two patients showed the described HBV vaccine escape mutation sP120T. 8/146 (5.48%) HBV isolates harbored mutations in the HBV polymerase known to confer resistance against antiviral therapy. Especially the lamivudine resistance mutations rtL180M/rtM204V and rtM204I were detected.

Conclusion

This study shows the distribution of HBV genotypes, therapy resistance, and vaccine escape mutations in HBV-infected patients in Oman. Our findings will have a major impact on therapy management and diagnostics of chronic HBV infections in Oman to control HBV infection in this intermediate HBV-endemic country.