扬州地区2013年—2016年乙型肝炎病毒逆转录酶区基因突变与多位点变异基因耐药的特征及其对策

目的:研究扬州地区经核苷(酸)类似物治疗疗效不佳的慢性乙型肝炎(CHB)患者发生乙型肝炎病毒(HBV)耐药基因突变的特征、多位点变异的发生率、影响因素及其对策。方法:选取2013年1月—2016年12月间就诊并经核苷(酸)类似物治疗6月以上且HBV-DNA阳性患者182例资料,采用核酸扩增技术,通过测序和检测耐药突变的变异特点,分析核苷(酸)类药物对HBV耐药相关位点,并比较近4年多位点变异的发生率有无统计学差异。结果:182例患者中,检出基因耐药103例,未检出79例;在11个经典耐药位点中,检测到8个耐药位点,未检出rtA194T、rtI233V、rtM250V等3个位点的变异;其中单个位点突变以rtM204I的突变率为最高(65.31%),其次为rtA181T;2个位点突变以rtL180M合并rtM204I突变为主(占42.86%),3个及其以上突变主要以rtL180M及rtM204V(或rtM204I)突变的基础上合并rtI169T、rtT184A、rtA181C、rtA181V、rtS202S、rtV173L等位点的突变;比较2013年—2016年4年间,3个及其以上位点突变的年发生率经比较其差异无统计学意义(P>0.05)。结论:扬州地区CHB患者接受核苷(酸)类药物治疗中,发生耐药变异的相关位点种类多,模式复杂;3个及其以上位点突变(多位点变异)与初治未使用高基因屏障核苷类似物及拉米夫定、阿德福韦酯、恩替卡韦不规则联用或序贯治疗相关;规范、优化核苷(酸)类药物抗HBV治疗能有效控制多位点变异的发生率,治疗中应密切监测耐药相关位点的变异。     

拉米夫定耐药慢性乙型肝炎患者HBV多聚酶区基因突变分析

目的探讨拉米夫定（LAM）耐药慢性乙型肝炎（CHB）患者多聚酶区序列突变特点。方法收集63例接受拉米夫定治疗且耐药的CHB患者的临床资料,采用PCR产物直接测序法检测HBVP基因多聚酶区序列耐药变异。结果 63例诊断为耐药的患者中,51例患者检测到LAM相关的HBV多聚酶区基因突变,其中rtM204V/I变异46例（73.0%）,rtL180M变异25例（39.7%）,rtV173L/M变异5例（7.9%）,rtQ214E变异1例（1.6%）,rtS213T变异2例（2.%）12,rtV207L/M/I变异2例（3.2%）,rtA181T变异3例（4.8%）,rtT184I/S/M变异1例（1.6%）。结论对拉米夫定治疗患者的耐药检测除HBV多聚酶区常见的rtLl80M和rtM204V/I位点变异外,还应考虑其他位点的耐药变异。     

长期应用恩替卡韦治疗的慢性乙型肝炎患者耐药发生情况及原因分析:8年随访研究

目的:了解慢性乙型肝炎患者长期应用恩替卡韦(ETV)治疗的耐药发生情况并探讨耐药原因。方法:对2003-2007年3月参加ETV剂量和疗效的随机双盲Ⅱ期临床试验、ETV与拉米夫定(LAM)治疗慢性乙型肝炎的随机双盲对照Ⅲ期临床试验、ETV治疗LAM失效的慢性乙型肝炎Ⅲ期临床试验及其后续ETV开放治疗临床试验的63例慢性乙型肝炎患者继续给予治疗和随访,计算截至2011年3月的累积耐药发生率,对临床耐药患者进行乙型肝炎病毒(HBV)逆转录酶区(RT)序列突变检测,并分析耐药原因。结果:63例患者中3例治疗2年后失访;1例治疗2.5年时转其他中心治疗;其他59例中16例用药4～6年,43例用药7～8年,随访4～8年,中位随访时间7年。13例患者发生临床耐药,累积耐药发生率为20.5%。9例耐药患者进行了HBV RT序列突变检测,其中5例检测出LAM耐药位点L180M和M204V突变和1～2个ETV耐药位点突变,符合ETV耐药特点;3例仅检出LAM耐药位点突变;1例检出2个ETV耐药位点突变和ADF耐药位点A181T突变。LAM经治、治疗前高病毒载量、治疗6个月病毒学应答不佳、治疗依从性差可能与耐药的发生有关。结论:长期应用恩替卡韦治疗慢性乙型肝炎耐药率较低。对治疗初期应答不佳患者,加强监测和提高治疗依从性可减少耐药的发生。     

基于替诺福韦的挽救性疗法治疗核苷/核苷酸类似物耐药乙型肝炎患者长期疗效的回顾性研究

摘 要 目的：研究长期采用基于替诺福韦酯（TDF）的挽救性疗法治疗核苷/核苷酸类似物耐药乙型肝炎患者的临床疗效。方法：采用回顾性研究方法,收集40例对核苷/核苷酸类似物耐药的乙型肝炎患者,在接受以TDF为基础的挽救治疗后的随访期间对患者进行分析,随访时长中位数为45个月。对病毒应答、乙型肝炎表面抗原水平的变化以及治疗期间的病毒学突破等因素进行了分析。结果：在以TDF为基础的挽救治疗0.5～4年,血清未检测到HBV DNA水平的患者的比例分别为68%, 78%,85%,88%,83%,81%,88%,100 %。TDF+LAM组与TDF+ETV组两组的病毒应答率无差异。与ADV耐药或ETV耐药HBV患者相比,在2年和3年时,LAM耐药HBV患者的乙肝表面抗原水平的平均减少量较大(P<0.05）。有2位ADV耐药HBV患者在基线时就发生了病毒学突破。结论：以TDF为基础的长期挽救治疗对接受LAM+ADV治疗、ETV+ADV治疗或ETV单独治疗失败的患者具有高效的病毒抑制效果。     

石家庄地区乙型肝炎病毒基因分型及P区耐药基因突变分析

目的:分析石家庄地区乙型肝炎病毒(HBV)基因型分布以及核苷(酸)类似物耐药基因突变状况。 方法:对2011年1月-2013年12月在石家庄市五医院就诊的遵医嘱口服核苷(酸)类似物6个月以上的354例慢性乙型肝炎患者进行HBV P区基因序列测定。结果:HBV B型为17例(4.8%),C型为337例(95.2%);共检出突变182例,在B型中检测出有10例突变(58.8%),在C型中检测出有172例突变(51.1%),两组间突变率无显著差别;182例HBV P区存在突变的病例中以rtL180M+rtM204I/V/S联合突变最多,占突变比例24.2%;其次是rtM204I/V/S单位点突变,占突变比例20.3%,这两种突变模式的比例达到44.5%;第3位是rtL180M+rtM204I/V/S 再加上其他一个或多个位点的突变,占突变比例12.6%。结论:石家庄地区HBV基因型以C型为主;核苷(酸)类似物耐药基因突变以拉米夫定和替比夫定耐药最常见。     

恩替卡韦联合阿德福韦酯治疗双重耐药慢性乙型肝炎22例

目的 观察恩替卡韦(ETV)与阿德福韦酯(ADV) 联合治疗对拉米夫定(LAM)与ADV双重耐药的慢性乙型肝炎(CHB)的临床疗效. 方法 22例对LAM与ADV双重耐药的CHB患者中,14例行肝脏穿刺病检为G_1-3S_1-3,直接改用ETV(0.5 mg)联合ADV(10 mg)治疗;8例行肝脏穿刺病检为G1S0,停药观察,待丙氨酸氨基转移酶(ALT)反弹后再改用上述联合治疗. 以HBV-DNA定量、乙肝血清免疫学指标及肝功能作为观察指标来判断6和12个月内的抗病毒疗效. 结果14例直接更换药物者中,6个月内完全应答者4例,不完全应答者8例,无应答2例;12个月内完全应答者6例,不完全应答者7例,无应答者1例. 8例停药后ALT反弹再治疗者中,3例3个月内完全应答,2例6个月内完全应答,12个月内全部达到完全应答 . 结论 对LAM与ADV双重耐药的CHB患者,采用ETV联合ADV治疗取得较好的疗效.     

核苷(酸)类似物耐药相关位点分析

目的:分析慢性乙型肝炎患者HBV逆转录酶基因与核苷(酸)类似物耐药相关位点的变异情况与临床耐药的关系。方法:分析不同核苷(酸)类似物的耐药相关突变情况及不同核苷(酸)类似物耐药的突变形式。结果:拉米夫定耐药突变中以M204V/I最常见,可伴随L180M突变,阿德福韦耐药中以N236T±A181位碱基替换为主;恩替卡韦耐药突变发生在拉米夫定耐药基础上;替比夫定的耐药突变为M204I;替诺福韦的变异位点以194位碱基替换为主(rtA194T)。结论:检测HBV逆转录酶基因多位点耐药相关突变,有助于临床及时发现乙型肝炎患者是否存在HBV耐药,从而合理进行抗病毒治疗。     

替比夫定联合阿德福韦酯治疗青年高病毒载量HBeAg阳性慢性乙型肝炎的临床观察

目的 对比观察替比夫定（LdT）联合阿德福韦酯（ADV）治疗青年高病毒载量乙型肝炎e抗原（HBeAg）阳性慢性乙型病毒性肝炎（CHB）的疗效。方法 106例HBeAg阳性、HBV DNA≥10⁷ 拷贝/mL的青年CHB初治患者分为替比夫定（LdT）联合阿德福韦酯（ADV）联合治疗组（54例）和恩替卡韦（ETV）对照组（52例）。联合治疗组口服LdT 600 mg/d+ADV 10 mg/d,1次/d;对照组口服ETV 0.5 mg/d,1次/d。总疗程48周,观察两组患者治疗12、24、36、48周时乙型病毒性（HBV）DNA阴转率、HBeAg血清学转换率及丙氨酸氨基转移酶（ALT）的复常率。结果 治疗后,两组患者均取得较好的疗效,获得较高的HBV DNA转阴率和ALT复常率。在治疗第12、24、36、48周时两组的HBV DNA阴转率及ALT复常率比较,差异均无统计学意义;但联合治疗组HBeAg血清学转换率24周后明显高于ETV对照组（33.3% vs 13.5%,χ²=5.804、P=0.016）,差异有统计学意义,且36周和48周统计学差异更加显著（42.6% vs 15.4%,χ²=9.477、P=0.002;48.1% vs 19.2%,χ²=9.877、P=0.002）。结论 替比夫定联合阿德福韦酯治疗青年高病毒载量的初治HBeAg阳性CHB患者,不仅获得较高的病毒学应答率和肝功能复常率,与恩替卡韦相比还能获得更高的HBeAg血清转换率。     

重度慢性乙型肝炎患者的病毒耐药突变检测

目的：探讨石家庄地区重度慢性乙型肝炎患者HBV耐药突变的状况。方法对51例2012年1月至2013年12月在石家庄市五医院就诊的重度慢性乙型肝炎患者进行HBV P区基因序列测定。结果51例重度慢性乙型肝炎患者中,25例存在HBV P区发生突变,突变率为49·．0％。突变率在HBeAg阳性组和阴性组之间无显著差别（ P ＞0．05）。 HBV P区突变组血清HBV DNA水平显著高于未突变组（ P ＜0．05）,ALT、AST和TBIL在突变组也显著升高（ P ＜0．05）。对25例存在HBV P区基因突变患者进行突变模式分析,居于前四位的突变模式的总和占所有突变的72％。突变模式主要为rtM204I／V单独突变以及联合rtL180M和（或）其他突变位点、rtA181V／T单独突变以及联合rtN236T或rtM250V／L突变。结论重度慢性乙型肝炎患者服用拉米夫定及阿德福韦酯等进行抗病毒治疗过程中,应实时监测HBV P区耐药突变状况,及时调整用药。     

拉米夫定治疗慢性乙型肝炎患者YMDD变异及其伴随变异的初步研究

目的 探讨拉米夫定治疗慢性乙型肝炎过程中HBV P基因YMDD变异及其伴随变异的分布情况.方法:以聚合酶链反应(PCR)扩增93例慢性乙型肝炎患者血清HBV P区,对PCR产物直接测序,检测YMDD及相关变异.结果:93份血清标本中,发生YMDD变异者为60例.变异及伴随变异:1型(rtL180M rtM204Ⅴ)变异26例(43%),2型(rtM204Ⅰ)14例(23.3%),rtL180M rtM204Ⅰ变异8例(13.3%),rtV173L rtL180M rtM204Ⅴ变异8例(13.3%),rtV173L rtM204Ⅰ变异和单纯rtM204Ⅴ部分变异各2例(3.3%).结论:拉米夫定治疗慢性乙型肝炎过程中,部分忠者可发生HBV YMDD变异和(或)其它位点伴随变异.     

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-na?ve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.     

Sexual Behavior and HBV Infection Among Noninjecting Cocaine Users (NICUs)

The aim is to estimate HBV prevalence and the associated risks among noninjecting cocaine users (NICUs). In 2002–2003, a total of 824 NICUs from Buenos Aires (Argentina) and Montevideo (Uruguay) were interviewed using a structured questionnaire. Serologic tests were carried out for Human Immunodeficiency Virus (HIV), hepatitis B (HBV), syphilis, and others. The population was divided into two serologic groups: HBV-infected and seronegative group. Univariate and binary logistic model were developed. The results seem to indicate that, among NICUs, HBV is transmitted through sexual contact. Prevention measures, including vaccine, are needed in order to control and minimize risks. The study's limitations are noted.     

新型核苷膦酸酯类化合物的合成及其抗乙肝病毒活性研究

目的设计合成新型核苷膦酸酯类化合物,并进行体外抗乙肝病毒活性评价。方法以不同取代的硫酚与2-氨基-9-[2-[二(2,2,2-三氟乙氧基)膦酰甲氧基]乙基]-6-氯嘌呤进行烃化反应合成目标化合物,化合物结构经1H-NMR和FAB-MS谱确证。采用HepG2.2215细胞进行体外抗乙肝病毒活性评价。结果与结论设计合成了9个核苷膦酸酯类新化合物,这些化合物均有一定的抗乙肝病毒活性。化合物4a、4b的活性强于拉米夫定、阿德福韦酯。苯环上取代基的类型显著影响核苷膦酸酯类化合物抗乙肝病毒活性。     

Inhibition of hepatitis B virus (HBV) replication by pyrimidines bearing an acyclic moiety: effect on wild-type and mutant HBV

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The main clinical limitation of a current antiviral drug for HBV, lamivudine, is the emergence of drug-resistant viral strains upon prolonged therapy. A group of 5-, 6-, or 5,6-substituted acyclic pyrimidine nucleosides with a 1-[(2-hydroxyethoxy)methyl] moiety were synthesized and evaluated for antiviral activities. The target compounds were prepared by the reaction of silylated uracils possessing a variety of substituents at the C-5 or C-6 positions or both with 1,3-dioxolane in the presence of potassium iodide and chlorotrimethylsilane by a convenient and single-step synthesis. Among the compounds tested, 5-chloro and 5-bromo analogues possessing an acyclic glycosyl moiety were the most effective and selective antiviral agents in the in vitro assays against wild-type duck HBV (EC50=0.4-2.2 and 3.7-18.5 microM, respectively) and human HBV-containing 2.2.15 cells (EC50=4.5-45.4 and 18.5-37.7 microM, respectively). These compounds were also found to retain sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and double mutations (L180M/M204V). The compounds investigated did not show cytotoxicity to host HepG2 and Vero cells, up to the highest concentration tested. The results presented here confirm and accentuate the potential of acyclic pyrimidine nucleosides as anti-HBV agents and extend our previous observations. We herein report the capability of acyclic pyrimidine nucleosides to inhibit the replication of both wild-type and drug-resistant mutant HBV.     

体外免疫法制备猪脾乙肝特异性转移因子

以乙肝疫苗为抗原,猪脾细胞为效应细胞,进行体外免疫反应,成功地制备出猪脾乙肝特异性转移因子PS_（HBV）-TF.抗原特异性皮肤试验证实,该PS_（HBV）-TF具有转移抗乙肝病毒特异性细胞免疫功能.     

Breaking B and T cell tolerance using cationic lipid--DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV

Cationic lipid DNA complexes (CLDC), referred to here as JVRS-100, were evaluated as an adjuvant for hepatitis B surface antigen (HBsAg) for eliciting B and T cell responses in transgenic mice expressing hepatitis B virus (HBV). To confirm the immunogenicity of HBsAg+JVRS-1000, a study was conducted in C57BL/6 mice, the genetic background of the HBV transgenic mice used in the study. HBsAg+JVRS-100 elicited a T cell response and B cell response as evidenced by interferon-gamma (IFN-γ) secretion by re-stimulated splenocytes and anti-HBsAg IgG induction, respectively, whereas, HBsAg only elicited a B cell response. In HBV transgenic mice, HBsAg did not elicit either T or B cell responses, unlike the HBsAg+JVRS-100 that elicited both. Energix-B vaccine did perform better than the HBsAg by eliciting a B cell response in the transgenic mice, but it did not perform as HBsAg+JVRS-100 since it did not elicit a T cell response. The response by HBsAg+JVRS-100 was not sufficient to cause destruction of infected liver cells, but it did suppress HBV DNA non-cytolytically. From these results, JVRS-100 might be considered for further development as an adjuvant for HBV therapeutic vaccines.     

The prevalence of viral hepatitis (HAV, HBV and HCV) in the Christchurch community

AIM: To determine the prevalence of hepatitis A (HAV), hepatitis B (HBV) and hepatitis C (HCV) in adults randomly selected from the Christchurch community. METHODS: A list of names was randomly generated from the Christchurch electoral roll and subjects were sequentially contacted and invited to participate. A blood sample was taken and tested for hepatitis A (IgG anti-HAV antibody), hepatitis B (HBsAg and anti-HBc) and HCV (anti-HCV antibody) using Abbott Elisa kits. Subjects positive for HBsAg were also tested for HBeAg/HBV DNA. Those positive for anti-HBc were tested for anti-HBs. HCV antibody positive samples were tested for HCV RNA using PCR. RESULTS: 1064 subjects (30.3% of those invited) participated in the study. The prevalence of HAV antibodies was 27.9%, and increased with age. The overall prevalence of HBV markers was 42/1064 (4.2%), and of these 0.3% were HBsAg positive and 3.9% were considered immune. No gender or ethnic differences in these proportions were observed. The seroprevalence of HVC antibody was 3/1064 (0.3%), two of whom were also PCR positive for HCV RNA. CONCLUSION: In the Christchurch community there was a high prevalence of antibodies to HAV, which increased with age. The prevalence of HBsAg and antibody to HCV were both low at 0.3%.     

Chitosan-modified poly(D,L-lactide-co-glycolide) nanospheres for plasmid DNA delivery and HBV gene-silencing

Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles (NS) formulated using the biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA) for plasmid DNA (pDNA) delivery. A cationic polymer, Chitosan (CHS), was incorporated in the PLGA matrix to improve pDNA loading efficiency and cellular uptake ability. PLGA-CHS NS were prepared by a spontaneous emulsion diffusion (SED) method, and various formulation factors were investigated. Spherical nanoparticles with particle size of around 60 nm were obtained under optimum formulation condition. The effectiveness of pDNA-loaded PLGA-CHS nanoparticles in expressing the indicative enhanced Green Fluorescent Protein (eGFP) and in slicing Hepatitis B virus (HBV) gene were examined in HepG2.2.15 cells. CHS-modified PLGA NS exhibited much higher loading efficiency than unmodified PLGA NS. CHS-PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. EGFP expression studies by observation with confocal leaser scanning microscopy (CLSM) indicated that pDNA-loaded CHS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The corresponding results showed that the HBV gene-silencing efficiency of CHS-PLGA NS was higher than those of plain-PLGA NS and naked pDNA. Thus, CHS-PLGA NS containing pDNA could provide an effective pDNA delivery system in vitro, showing that such an approach could be useful in the treatment of viral diseases in vivo.