ADV初治耐药与LAM耐药者再耐药时HBV-RT区的变异特征

目的:研究阿德福韦酯(adefovir,ADV)初治耐药和ADV用于挽救治疗拉米夫定(lamivudine,LAM)耐药者再耐药时HBV毒株RT区变异特征的差异性.方法:我院就诊的ADV初治耐药者73例,ADV用于挽救治疗LAM耐药者后再耐药者34例,共计107例,在HBVDNA突破时采用PCR方法扩增HBV-RT区基因,对PCR产物直接测序,用Chromas2.0软件分析HBV-RT区基因的核苷酸和氨基酸差异、变异模式,同时使用Clustalx1.81.msw进行基因型分析.结果:在107例患者中,存在rtA181V变异36例,rtA181T变异43例,rtN236T变异47例,rtM204I/V±rtL180M变异20例,其他相关变异如rtA181S/C、rtH/N238S/T/R/A/K、rtV214I/A、rtQ215H共12例.ADV初治组检测到rtA181V/T变异27例(36.99%)、rtN236T变异14例(19.18%)、rtA181V/T+rtN236T变异28例(38.36%),rtA181T/V和rtN236T以外的变异4例(5.48%).其中发生rtA181V和rtA181T变异患者分别有33.3%、64.5%合并rtN236T变异(P=0.0091);ADV挽救治疗LAM耐药者再耐药,检测到rtA181V/T变异21例(61.76%)、rtN236T变异2例(5.88%)、rtA181V/T+rtN236T变异3例(8.82%),rtA181T/V和rtN236T以外的变异8例(23.53%).其中发生rtA181V患者25%合并rtN236T变异,而rtA181T变异患者未检测到rtN236T变异(P=0.2311).20例(58.83%)患者合并rtM204I/V变异,这20例患者中12例(35.29%)合并rtA181V/T和/或rtN236T变异的多重耐药变异.有12例未检测到rtA181T/V和rtN236T变异,但检查到rtA181S/C(2例)、rtV214I/A(3例)、rtQ215H(1例)、rtH/N238S/T/R/A/K(6例)等ADV耐药相关变异,其中ADV挽救治疗LAM耐药患者8例,ADV初治患者4例,两组间存在统计学差异(P=0.0169).结论:ADV初治耐药主要发生rtA181V/T和rtN236T变异,ADV挽救治疗LAM耐药再耐药主要发生rtA181V/T变异,且后者耐药模式更复杂.     

阿德福韦酯相关性HBV潜在耐药变异位点研究进展

目前阿德福韦酯仍是我国临床治疗慢性乙型肝炎的一线抗病毒药物之一。但是,长期接受阿德福韦酯治疗可能导致HBV耐药性变异,目前公认的与之相关的HBV耐药变异位点为rtA181V/T和rtN236T。此外,尚有报道rtQ215S/P/H、rtV214A、rtN238H、rtI233V、rtN118H、rtM207V、rtA200V和rtL180M等位点变异与HBV对阿德福韦酯耐药相关,但其重要性尚须进一步明确。     

乙型肝炎病毒阿德福韦酯耐药相关变异的研究进展

目前口服核苷酸类似物阿德福韦酯(adefovir dipivoxil,ADV)仍是我国临床治疗慢性乙型肝炎(chronic hepatitis B,CHB)的主要抗病毒药物之一。但是,长期服用ADV可能导致乙型肝炎病毒(hepatitis B virus,HBV)产生耐药变异。经典的ADV耐药变异形式为rt A181V和rt N236T,新鉴定的ADV原发耐药变异为rt A181S、rt E218G和rt N236V。此外,rt A181T、rt V214A、rt Q215H/P/S、rt L217R/P、rt I233V和rt N238T/D/H等位点变异与HBV对ADV耐药仍存在争议,尚需进一步明确。     

慢性乙型肝炎患者HBV基因型分布及其耐药基因突变分析*

目的 探讨慢性乙型肝炎（CHB）患者HBV基因型及其耐药突变发生情况。方法 纳入240例接受核苷（酸）类似物单药或联合或序贯治疗的CHB患者,采用PCR扩增HBV逆转录（RT）区和序列测定鉴定耐药基因突变,采用HBV S基因测序法鉴定基因型。结果 在35例单用拉米夫定治疗的CHB患者中,发生耐药突变14例（40.0%）,突变位点为rtL80I/V、rtVl73L、rtLl80M、rtM204V/I和rtV207I,23例单用阿德福韦治疗者发生耐药突变11例（47.8%）,突变位点为rtAl81T/V、rtS213T/N、rtV214A、rtQ215S/H/P、rtl233V、rtN236T、rtP237H和rtN/H238A/K/D/S,70例单用恩替卡韦治疗者发生耐药突变10例（14.3%）,突变位点为rtM204I,12例单用替比夫定治疗者发生耐药突变5例（41.7%）,突变位点为rtI169T、rtL180M、rtT184G/S/A/I/L/F、rtS202I/G、rtM204V和rtM250V/I/L,100例接受联合或序贯治疗者发生耐药突变51例（51.0%）,突变位点为rtA194T,恩替卡韦治疗患者耐药突变发生率最低（P<0.05）;240例CHB患者中,HBV基因B型21例（8.8%）、C型216例（90.0）和D型3例（1.2%）;在发生耐药突变的91例患者中,B型6例（6.6%）、C型83例（91.2%）和D型2例（2.2%,x²=1.22,P>0.05）;在发生耐药突变的6例B型感染者中有2例（33.3%）和83例C型感染者中有15例（18.1%）发生了多重耐药突变。结论 检测CHB患者感染HBV基因型并及时获得耐药突变基因分布,将有助于指导临床治疗。     

HBV反转录酶区rtA181S变异与阿德福韦酯耐药相关性研究

目的：分析HBV反转录酶（RT）区rtA181S变异与阿德福韦酯（ADV）耐药的相关性。方法应用直接测序法筛选分析大样本慢性HBV感染者rtA181S变异的检出频率,并对1例接受ADV单药治疗失败的慢性乙型肝炎患者血清中HBV RT区基因进行克隆测序,分析相关变异形式。用XhoⅠ和SphⅠ双酶切pGEM-Teasy RT及pTriEx-HBV（C）载体后再连接,构建1.1倍HBV野生株和耐药株的重组质粒,转染人肝癌细胞系HepG2细胞,5 h后分别加入不同浓度的 ADV（0、0.033、0.100、0.330、1.000、3.300μmol/L）。隔天换药,4 d后收集细胞上清,采用实时荧光定量PCR法检测不同药物浓度作用下细胞培养上清中的HBV DNA载量,并分析其表型耐药特点。结果9830例慢性HBV感染者的12000个血清样本中,有46例样本检出rtA181S变异（单独或与其他耐药变异联合出现）,在653例中检出经典的rtN236T/A181V变异。其中随访的1例在接受ADV治疗19个月后出现了病毒学和生化学突破,直接测序检出rtA181S＋N236T变异,克隆测序分析显示在24个克隆中11个（45.83%）为野生型,6个（25.00%）为rtN236T变异型,5个（20.83%）为rtA181S变异型,1个（4.16%）为rtA181V变异型,1个（4.16%）为rtA181S＋N236T变异型。在体外实验中,rtN236T、rtA181S和rtA181S＋N236T变异株的相对复制力分别是野生株的91.35%、29.90%和68.53%。表型耐药分析显示rtN236T、rtA181S和rtA181S＋N236T变异株对ADV的灵敏性分别为野生株的1/4.41、1/3.05和1/5.43。结论 rtA181S是一种ADV耐药相关变异,可单独或联合其他变异引起患者耐药。但与经典ADV耐药变异相比,rtA181S变异引起的ADV耐药相对较弱,临床检出率较低。     

LAM耐药后ADV序贯治疗再耐药HBV变异株的动态变化

目的:观察阿德福韦酯(ADV)单药序贯治疗拉米夫定(LAM)耐药慢性乙型肝炎(CHB)出现再耐药患者HBV变异株的动态变化.方法:28例初始LAM治疗的CHB患者, 出现LAM耐药后换用ADV单药序贯治疗再次出现不充分病毒学应答或病毒学突破. 采用实时荧光定量PCR检测DNA, 分析HBV DNA动态变化; 采用直接PCR产物测序法检测耐药变异,分析变异模式变化. 对其中2例患者系列血清进行基因克隆分析其HBV耐药株动态变化.结果:28例入选患者HBV DNA载量随耐药变异株的消长而波动: 基线为(7.65±1.04)Log10copies/mL, LAM疗程中最低值中位数为3.68 Log10copies/mL; 出现病毒学突破或/和耐药变异时为(6.87±1.16) Log10copies/mL, 患者换用ADV后最低值中位数为3.78 Log10copies/mL; 患者出现耐药变异模式改变或再次病毒学突破时为(6.04±0.93) Log10copies/mL. 治疗基线及两次病毒突破3个时点HBV DNA载量均数逐渐下降, 且两两之间差异均有统计学意义. 28例患者LAM治疗后24例发生rtM204I/V伴或不伴rtL180M等变异, 4例无耐药变异; 换用ADV单药序贯治疗出现病毒学突破后, 进行测序检测耐药发现: LAM耐药变异13例(包括3例多重耐药), ADV耐药变异11例(包括3例多重耐药), 无耐药变异7例. 其中11例ADV耐药患者: rtA181V变异6例、rtA181T变异3例、rtN236T变异和rtA181V+rtN236T联合变异各1例. 对2例患者血清TA克隆发现ADV耐药准种出现和LAM耐药准种消失的过程, 同时发现对ADV耐药的rtI233M变异和对替诺福韦耐药的rtA194T变异.结论:ADV单药序贯治疗LAM耐药CHB出现再耐药患者HBV变异株动态变化存在5种形式, 且克隆分析可观察耐药株动态变化过程和发现预存的耐药准种.     

拉米夫定耐药后阿德福韦酯治疗应答欠佳患者乙型肝炎病毒耐药变异模式

目的 观察拉米夫定(LAM)耐药后单用或联合阿德福韦酯(ADV)治疗应答欠佳患者HBV耐药变异模式. 方法收集15例LAM耐药后采用ADV治疗病毒学应答欠佳患者的血清,对HBV聚合酶逆转录区进行聚合酶链反应、扩增、克隆、测序,分析与耐药相关的变异模式.组间HBV DNA水平比较采用t检验.结果 单用ADV组检测出A181T+N236T、A181V和A181T 等模式的ADV耐药变异,而联合治疗组中主要检测出M204V+L180M、M204V+L180M+L229V、M204I+L80I和M204V+L180M+V207I等LAM耐药变异模式.另外,在联合治疗组的3份血清中,20%的克隆上同时出现对LAM和恩替卡韦耐药的变异,分别为M204I+L80I+T184I(2/10)、M204V+L180M+T184S(2/10)和M204V+L180M+G173L+S202G(2/10).两组中各有1份未检测出已知耐药变异的血清,在它们的测序结果中发现所有克隆均出现1269L变异,且其中单用组中所有克隆均出现P109S变异.联合治疗组和单用ADV组患者血清HBV DNA水平分别为(3.86±0.85)log10拷贝/ml和(5.71±0.94)log10拷贝/ml,差异有统计学意义(t=3.947,P<0.01).结论 LAM耐药后ADV治疗病毒学应答欠佳的患者中,单用ADV治疗容易筛选出A181T+N236T和A181V/T等ADV耐药变异模式,而联合ADV治疗组仍以M204V+L180M、M204V+L180M+L229V、M204I+L80I和M204V+L180M+V207I等LAM耐药变异模式为主.联合治疗时由于LAM的持续使用可选择出恩替卡韦耐药变异模式T184I/S和S202G;对于部分患者,1269L和P109S变异可能影响ADV治疗应答.     

江苏部分地区慢性乙型肝炎病毒感染者天然耐药的调查

目的 通过连续监测江苏部分地区乙型肝炎病毒（HBV）感染者发生天然耐药基因变异的频度及耐药位点的分布特点,为合理选择抗病毒治疗的药物提供依据.方法 回顾性分析2004～2010年未服用核苷类似物抗病毒药物的352例慢性乙型肝炎（CHB）患者临床资料.应用焦磷酸测序技术及双脱氧链终止法基因测序的方法进行耐药基因的检测.结果 352例CHB患者中,345例基因测序成功,7例测序失败.8例有耐药基因突变,天然耐药突变检出率为2.32％.主要耐药位点为rtV173L、rtL180M、rtA181T、rtM204V、rtV207L、rtS213T、rtV214I、rtN236T.2004～2006年间145例CHB患者均未检测出耐药突变,2007、2008、2009及2010年耐药突变的检出率分别为2.63％、3.92％、4.69％和4％.多因素Logistic回归分析显示耐药突变的发生与感染者年龄相关.结论 江苏部分地区CHB患者有天然耐药基因突变,耐药突变检出率为2.32％.2004～2010年耐药突变检出率呈上升趋势.年龄超过30岁是耐药突变发生的相关因素.     

乙型肝炎病毒RT区原发性耐药变异的研究

目的探讨核苷(酸)类似物(NA)治疗前乙型肝炎病毒(HBV)RT区原发性耐药变异的发生情况。方法采用PCR产物直接测序技术对61例未接受NA治疗的慢性HBV感染者进行HBV RT区序列测定,分析NA治疗前HBV RT区原发性耐药变异的发生率。结果通过HBV基因扩增、PCR产物直接测序后,共获得61个HBV RT区基因序列,采用HBV RT区基因进化树基因分型共分出B型42例(42/61,68.85%),C型19例(19/61,31.15%),未检出其他基因型。通过核苷酸序列及氨基酸序列分析,对NA常见的10个耐药变异位点(rtI169T、rtV173L、rtL180M、rtA181V/T、rtT184G/S/A/I、rtA194T、rtS202I/G、rtM204I/V、rtN236T、rtM250V)进行比对后,61例患者中共检出HBV RT区变异3例(rtM204I 2例,rtM204I+L180M 1例),自然耐药变异的检出率为4.92%。结论慢性HBV感染者存在少量RT区原发性耐药变异位点,与原发性无应答有关。     

HBV PRt204+180以外位点变异与拉米夫定耐药关系探讨

目的 探讨慢性乙型肝炎患者HBV P区常见耐药位点Rt204±180以外位点变异与拉米夫定耐药关系.方法 收集27例接受拉米夫定初治患者治疗基线和获得病毒学应答后出现病毒学反弹的血清标本,采用PCR产物直接测序技术,检测治疗基线和病毒学反弹时HBV DNA的P区变异位点,对位点变异与拉米夫定耐药关系进行分析.结果 HBV P区的rtA181V、rtM171R、rtN/H238T/A和rtI187V变异不伴随有P区rtM204V/1±rtL180M耐药位点的变异.结论 HBV P区的rtA181V、rtM171R、rtN/H238T/A和rtI187V变异可能是拉米夫定耐药的新变异位点.     

Virological response to adefovir monotherapy and the risk of adefovir resistance

AIM:To evaluate virological response to adefovir(ADV) monotherapy and emergence of ADV-resistant mutations in lamivudine(LAM)-resistant chronic hepatitis B patients.METHODS:Seventy-seven patients with documented LAM resistance who were treated with 10 mg/d ADV for>96 wk were analyzed for ADV resistance.RESULTS:At week 48 and 96,eight(10%)and 14(18%)of 77 LAM-resistant patients developed the ADV-resistant strain(rtA181V/T and/or rtN236T mutations),respectively.Hepatitis B virus(HBV)DNA levels during therapy ...     

Efficacy of switching to telbivudine plus adefovir in suboptimal responders to lamivudine plus adefovir

AIM:To examine the efficacy of telbivudine(LdT)+adefovir(ADV)vs continuation of lamivudine(LAM)+ADV in patients with LAM-resistant chronic hepatitis B(CHB)who show a suboptimal response to LAM+ADV.METHODS:This was a randomized,active-control,open-label,single-center,parallel trial.All eligible patients were enrolled in this study in Severance Hospital,Yonsei University College of Medicine,Seoul,South Korea,between March 2010 and March 2011.Hepatitis Be antigen(HBeAg)-positive CHB patients whose serum hepatitis B virus(HBV)DNA remained detectable despite at least 6 mo of LAM+ADV therapy were included.Enrolled patients were randomized to either switching to LdT(600 mg/d orally)plus ADV(10 mg/d orally)(LdT+ADV group)or to continuation with LAM(100 mg/d orally)plus ADV(10 mg/d orally)(LAM+ADV group),and were followed for 48 wk.One hundred and six patients completed the 48-wk treatment period.Serum HBV DNA,HBeAg status,liver biochemistry and safety were monitored at baseline and week 12,24,36 and 48.RESULTS:The duration of prior LAM+ADV treatment was 18.3(LdT+ADV)and 14.9 mo(LAM+ADV),respectively(P=0.131).No difference was seen in baseline serum HBV DNA between the two groups[3.66(LdT+ADV)vs 3.76(LAM+ADV)log10IU/mL,P=0.729].At week 48,although there was no significant difference in the mean reduction of serum HBV DNA from baseline between LdT+ADV group and LAM+ADV group(-0.81 vs-0.47 log10IU/mL,P=0.167),more patients in the LdT+ADV group had undetectable HBV DNA levels compared to those in the LAM+ADV group(30.2%vs 11.5%,P=0.019).Three patients with LdT+ADV treatment and 2 patients with LAM+ADV treatment achieved HBeAg loss.The patients in both groups tolerated the treatment well without serious adverse events.The proportion of patients with estimated glomerular filtration rate≥90 mL/min per 1.73 m2in the LdT+ADV group increased from 49.1%(26/53)at baseline to 58.5%(31/53)at week 48,while that in the LAM+ADV group decreased from 37.7%(20/53)at baseline to 30.2%(16/53)at week 48.CONCLUSION:The switch to LdT+ADV in s     

阿德福韦酯片的稳定性

目的 研究阿德福韦酯片的稳定性并测算其有效期.方法 根据《新药临床前研究指导原则汇编》,在模拟市售包装条件下,通过室温贮存12个月,对阿德福韦酯片的性状、含量测定等项指标进行定期考察.结果 在考察期内,各项指标均符合规定.结论 本品常温放置12个月,其质量稳定,故有效期定为12个月.     

阿德福韦酯相关肾脏损害

目的:探讨阿德福韦酯引起的肾脏损害临床病理特点。方法:回顾性分析4例因慢性乙型肝炎长期使用阿德福韦酯出现肾脏损害患者的临床病理资料。结果:4例均为男性患者,年龄34～44岁,3例慢性乙型肝炎病史10年,1例慢性乙型肝炎病史20年,使用阿德福韦酯治疗3～6年。肾脏疾病病程3～23月,患者血压均正常。4例血清肌酐(SCr)均轻度升高(116.7～158.3μmol/L),1例出现低磷血症,2例伴低尿酸血症,2例少量蛋白尿(0.54g/24h和0.57g/24h),1例蛋白尿0.79～1.72g/24h,1例尿蛋白阴性,1例尿糖阳性,4例患者均无镜下血尿,肝功能正常,无低白蛋白血症和高脂血症。肾活检病理示3例患者光镜下肾小球节段轻度系膜增生,1例合并IgA肾病。4例患者肾小管间质病变均较轻,见小灶性近端肾小管上皮细胞刷状缘脱落,其中3例伴慢性病变轻～中度,灶性肾小管萎缩、基膜增厚。电镜下均见肾小管上皮细胞胞质内线粒体外形不规则、变形、体积增大。药物停用后,随访3～25月,3例患者SCr恢复正常,1例SCr稳定,血磷恢复正常,所有患者尿蛋白阴性。结论:长期使用阿德福韦酯可导致肾脏损害,患者表现轻度肾功能下降,部分患者血清磷、尿酸减低,伴肾小管功能损伤。阿德福韦酯肾脏损害可单独出现,也可和原发性肾脏疾病同时出现。形态学主要表现肾小管损伤,肾小管上皮细胞刷状缘脱落,电镜下肾小管上皮细胞胞质内异常增大和变形的线粒体。     

Telbivudine in combination with adefovir versus adefovir monotherapy in HBeAg-positive, lamivudine-resistant chronic hepatitis B

Purpose

Lamivudine (LAM) resistance is common on lamivudine monotherapy for chronic hepatitis B. This study examined the safety and efficacy of telbivudine (LDT) given with adefovir (ADV) versus ADV monotherapy in patients with chronic, lamivudine-resistant HBV infection.

Methods

An open-label, 96?week study with planned recruitment of 150 HBeAg-positive, lamivudine-experienced Asian patients with a confirmed YMDD resistance mutation, randomized 1:1 to receive ADV alone or with LDT. The study was terminated early due to difficulty in enrolling monotherapy patients. At termination, 42 patients had received study medication for 8?C61?weeks. Due to incomplete enrolment, summary statistics only were prepared, without significance testing.

Results

A total of 42 patients underwent rescue therapy (switch to ADV or LDT?+?ADV; n?=?21 per group). Median treatment duration was 48?weeks in both groups. HBV DNA changes from baseline were greater in the LDT?+?ADV arm at all time points (Week 48: ?7.4 log₁₀ vs. ?4.9 log₁₀ copies/ml), and serum DNA was undetectable (<300?copies/mL) at week 48 in 38.5% (5/13) on LDT?+?ADV versus 0% (0/9) on ADV monotherapy Two patients (9.6%) on ADV monotherapy experienced virologic breakthrough without evidence of ADV resistance, but none on LDT?+?ADV; and no confirmed ADV resistance was observed in any on-treatment sample. HBeAg loss occurred in three patients on LDT?+?ADV and one patient on ADV monotherapy through week 48. Safety profiles were similar between the arms.

Conclusion

LDT?+?ADV combination treatment showed better outcomes against lamivudine resistant HBV than ADV alone, with a similar safety profile.     

Increased risk of adefovir resistance in patients with lamivudine-resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy

Although adefovir dipivoxil (ADV) has a unique profile of delayed and infrequent resistance in treatment-na?ve chronic hepatitis B patients, the association of ADV resistance with previous lamivudine (LAM) resistance is not well understood. We compared the emergence of the ADV-resistant mutations rtA181V/T and rtN236T between LAM-resistant patients and treatment-na?ve patients at 48 weeks of ADV monotherapy. Fifty-seven LAM-resistant patients and 38 treatment-na?ve patients were treated with 10 mg/d ADV for more than 48 weeks. Both baseline and 48-week blood samples were analyzed for ADV-resistant mutations via restriction fragment mass polymorphism analysis. Antiviral responses were evaluated according to changes in serum HBV DNA (measured via real-time polymerase chain reaction) and alanine aminotransferase (ALT) levels and loss of hepatitis B e antigen (HBeAg). After 48 weeks, 10 (18%) of the 57 LAM-resistant patients were found to have developed ADV-resistant mutations, whereas none of the 38 treatment-na?ve patients developed such mutations (P < .01). Among LAM-resistant patients, the reduction in serum HBV DNA levels was significantly lower in patients with ADV-resistant mutations than in those without such mutations (-1.04 vs. -2.63 log10 copies/mL) (P = .01). However, the rates of serum ALT normalization (60% vs. 55%) and HBeAg loss (14% vs. 21%) were not significantly different between the 2 groups (P > .05). In conclusion, the emergence of the rtA181V/T and rtN236T mutations was more common in LAM-resistant patients than in treatment-na?ve patients after 48 weeks of ADV therapy and was associated with reduced antiviral efficacy to drug treatment.