恩替卡韦、拉米夫定治疗HBeAg阴性乙型肝炎相关慢加急性肝衰竭12周疗效比较

目的 比较恩替卡韦（ETV）与拉米夫定（LAM）治疗HBeAg阴性乙型肝炎相关慢加急性肝衰竭（HBV-ACLF） 12周的疗效及安全性.方法 98例HBV DNA阳性且无核苷类似物抗病毒治疗史的HBeAg阴性HBV-ACLF随机分为ETV组、LAM组各49例,在内科综合治疗基础上分别予ETV（0.5 mgqd）或LAM （0.1,qd）抗病毒.比较12周时组间死亡率、临床好转率、完全病毒学应答率和不良事件的差异.结果 ①ETV组、LAM组的基线特征相似.②12周时ETV组死亡率低于LAM组（28.6％ vs 48.9％,P＜0.05）;临床好转率呈高于LAM组趋势（65.3％ vs 48.3％,P=0.067）,且主要来自基线终末期肝病模型（MELD）评分≤30患者（75.6％vs52.5％,P＜0.05）;完全病毒学应答率高于LAM组（94.3％vs 72％,P＜0.05）.③ETV组、LAM组间均无患者因药物不良事件而终止治疗.结论 ETV治疗HBeAg阴性HBV-ACLF能较LAM更强地抑制HBV复制,降低短期病死率,且安全性相当.     

慢性乙型肝炎患者阿德福韦酯耐药的变异检测

目的 通过检测乙型肝炎基因rtA181V和rtN236T位点来探讨阿德福韦酯耐药与HBV变异的相关性.方法 选取95例符合2012年《慢性乙型肝炎防治指南》诊断标准、且无其他疾病感染或其他疾病造成的肝功异常的患者.对所选患者进行阿德福韦酯口服治疗,每人10mg/d治疗64周,在治疗第16、32、48、64周时分别对HBV DNA含量,ALT水平,HBsAg、抗-HBs、HBeAg、抗-HBe和抗-HBc进行检测.治疗结束后,选取有效但HBV DNA未转阴患者、治疗无效以及出现病毒学突破的患者,观察rtA181V、rtN236位点是否发生变异来进行分析研究.结果 通过检测结果可以显示出患者接受一段时间治疗后其突变率逐渐增高,即rtA181V在16、32、48、64周后变异率为7.3％、16.8％、22.1％、32.6％,rtN236T变异率为9.5％、20％ 、36.8％、47.4％.结论 在治疗初期虽然阿德福韦酯的耐药率很低,但随着治疗时间的延长其耐药率越来越高,因此目前治疗乙型肝炎耐药最好的方法是阿德福韦酯与其他核苷类似物联合用药.     

阿德福韦酯单药治疗的慢乙肝患者聚合酶RT区变异位点及临床意义

目的 通过对慢性乙型肝炎（CHB）患者血清HBV聚合酶逆转录（RT）区基因序列分析,探讨阿德福韦酯（ADV）单药抗病毒治疗失败,聚合酶RT区变异位点特点及临床意义.方法 采用回顾性分析方法,对接受阿德福韦单药抗病毒治疗出现病毒学突破,经聚合酶RT基因测序,证实存在基因突变的44例患者的聚合酶RT区变异位点、临床特点及随访资料进行分析.结果 44例阿德福韦酯单药治疗患者,至出现RT区变异最短治疗时间8个月,最长5年,平均（32.2＆#177;6.7）个月.出现病毒学突破后,病毒水平波动在103拷贝/ml～108拷贝/ml之间;变异位点A181T 11例（25.0％）、A181V 8例（18.2％）、A181T/N236T 10例（22.7％）、A181V/N236T 6例（13.6％）、N236T9例（20.4％）;出现生化学突破40例（90.9％）,ALT指标波动在37 IU/L～ 946 IU/L;挽救治疗采用联合拉米夫定23例、联合恩替卡韦6例,联合替比夫定1例,改单药拉米夫定4例,改单药恩替卡韦10例.预后出现疾病重症化3例,经挽救治疗病情得到恢复41例.结论 ①阿德福韦单药耐药RT区A181T（A181T/N236T）变异约占半数（47.7％）,随访2年,其肝癌发生率呈现高于非A181T患者趋势.②条件允许,初始治疗应首选HBV抑制作用强、耐药发生率低的药物,避免疾病重症化或肝癌等不良事件发生.     

宁夏地区慢性乙型肝炎患者乙肝病毒基因分型与耐药突变分析

目的:了解宁夏地区慢性乙型肝炎(CHB)患者的优势基因型和耐药突变模式的流行情况,为个体化医疗提供依据。方法:选取2016-08-2017-12本院收治的CHB患者血液样本226例,应用化学发光微粒子免疫分析法定量检测乙肝血清学标志物[乙肝表面抗原(HBsAg)、乙肝表面抗体(HBsAb)、乙肝e抗原(HBeAg)、乙肝e抗体(HBeAb)、乙肝核心抗体(HBcAb)],荧光定量PCR检测样本HBV-DNA载量,直接测序法检测突变位点并分析基因型及耐药情况。结果:226例样本中C基因型168例占74.34%,B基因型22例占9.74%,D基因型和混合型均18例,各占7.96%。41例发生耐药变异,变异率为18.1%,以单点突变rtM204I/V为主,突变率为30.5%。多位点变异13例,包含2例对拉米夫定、替比夫定和恩替卡韦均耐药的特殊突变。B基因型rt184和rt204位点突变率高于C基因型,rt213位点突变率低于D基因型;D基因型rt214位点突变率高于C基因型(P0.05)。结论:本地区CHB患者中,C基因型占有绝对优势,以rtM204突变模式为主,较其它基因型更易发生拉米夫定耐药。及时了解HBV基因型及耐药突变体的流行信息有重要临床价值。     

拉米夫定与阿德福韦酯初始联合治疗HBeAg阳性慢性乙型肝炎96周疗效观察

目的 观察拉米夫定(LAM)和阿德福韦酯(ADV)初始联合治疗HBeAg阳性的慢性乙型肝炎(CHB)的长期疗效与安全性.方法 自首都医科大学附属北京地坛医院肝病中心2009年2月-2009年5月开始初始抗病毒治疗的HBeAg阳性CHB患者中,选择ETV单药治疗患者、LAM和ADV联合治疗患者各25例进行前瞻性观察;比较治疗96周时两组患者在病毒学应答率、生化学应答率及免疫学应答率等方面有无差异.结果 经过96周抗病毒治疗,取得完全病毒学应答的患者在ETV组为19/22例(86.4％),而联合治疗组仅有9/21例(42.9％),二者差异显著(x2=8.953,P=0.003);在肝脏生化学应答方面两组均取得明显改善;两组的HBeAg阴转、HBe血清学转换、HBsAg阴转等免疫控制指标均无明显变化.两组患者在为期96周的治疗随访过程中,均未发生病毒学突破或HBV DNA反跳.两组患者在为期96周的治疗随访过程中,未发现任何不良反应.两组患者的肾功能在基线及96周时均处于正常范围,且治疗前后无显著性差异.结论 在HBeAg阳性CHB初始抗病毒治疗患者中,应用ETV单药治疗组的96周病毒学应答率显著高于LAM和ADV联合治疗组.     

核苷(酸)类似物治疗的慢性乙型肝炎患者HBV RT区突变序列分析

目的 分析使用过核苷(酸)类似物(NA)治疗的慢性乙型肝炎患者体内HBV RT区基因耐药特点.方法 收集229例接受过核苷类似物治疗的慢性乙型肝炎患者的血清,应用snPCR法扩增HBV全长RT区,纯化snPCR产物直接测序后分析11个经典耐药位点的氨基酸变异情况.结果 229例患者中基因B和C型各占14.41％ (33/229)和85.59％ (196/229).且C型患者较B型更易发生耐药变异(x2=2.95,P＜0.05).共有63例检出HBV耐药变异,在rtI169、rtT184、rtA194和rtS202位点上均未检出耐药变异.在63个耐药变异株中rtM204V/I变异株检出率最高(40/63,63.49％),共有11种耐药突变模式.其中rtM204I以与rtL80I/V或rtL180M联合变异为主,而rtM204V变异常伴有rtL180M变异.结论 使用过核苷(酸)类似物(NA)治疗的慢性乙型肝炎患者体内HBV RT区基因耐药呈现出复杂的变异类型,rtM204V/I突变最为常见.     

HBV核心启动子A1762T/G1764A双突变与慢加急性肝衰竭关系的研究

目的 分析HBV BCP A1762T/G1764A双突变与慢加急性肝衰竭（Acute on chronic liver failure,ACLF）之间的相关性.方法 对166名HBV慢性感染后处于疾病不同阶段的患者进行HBV前BCP A1762T/G1764A双突变检测,比较不同患者之间突变率的差异.结果 慢性肝炎（CHB）45人,肝硬化（LC）45人,ACLF 49人,肝细胞癌（HCC）27人,各组A1762T/G1764A双突变率分别为40.0%（18/45）、84.4%（38/45）、73.5%（36/49）、92.6%（25/27）.但是,以CHB为基础的ACLF患者和以LC为基础的ACLF患者A1762T/G1764A双突变率差异无统计学意义[（81.3%）vs.（69.7%）,P=0.502].HBeAg阳性者和HBeAg阴性者BCP双突变率差异无统计学意义（P=0.735）.A1762T/G1764A双突变者HBV DNA水平（10g）为5.68±1.36,阴性者HBV DNA水平（log）为6.14±1.81,差异无统计学意义（P=0.075）.结论 A1762T/G1764A双突变与HBV感染后疾病进展相关,不具备ACLF特异性;A1762T/G1764A联合突变者HBV DNA水平及HBeAg状态也与非联合突变者差异无统计学意义.     

联合免疫失败过程中乙型肝炎病毒S基因突变的母婴配对研究

 目的：探讨在当前联合免疫方案下,发生乙肝母婴传播免疫预防失败过程中乙型肝炎病毒（HBV）S 基因的突变特征。方法：选择15例分娩免疫失败婴儿的孕妇及其联合免疫前的新生儿和免疫后的7月龄婴儿,将母亲、新生儿和婴儿分别配对,对其外周血中HBV的S基因（包括前 S和S）分2个片段进行PCR扩增测序,对比母亲、新生儿及婴儿3组间HBV 基因型、前 S和S基因突变率及突变位点的不同。结果：(1) 新生儿和婴儿体内HBV的基因型与母亲完全相同。(2) 3组2个片段的突变率差异均无统计学意义（P＞0.05）;同源树簇集分析中,母亲与所分娩的新生儿和婴儿体内 HBV 均形成各自独立的簇集。(3)突变位点：母亲-新生儿（13对）: 7例新生儿与母亲体内的HBV存在不同的突变位点（共15个位点）;新生儿-婴儿（13对）：3例婴儿与新生儿体内的HBV存在不同的突变位点,即nt273A→A/G、nt512C→C/T和nt1139C→A（共3个位点）,前2个位于S区的“a”决定簇之外,后1个则在与X编码框重叠的区域;婴儿-母亲（15对）：有9例婴儿与母亲体内的HBV存在不同的突变位点（共25个位点）,但仅1例是母亲、新生儿和免疫后婴儿均存在不同的突变位点。结论：（1）新生儿及免疫后婴儿体内的HBV均源自于母亲;（2）HBV的 S基因在联合免疫前和免疫后均可发生突变,主要发生在联合免疫前,是否与免疫失败有关尚需进一步研究。     

HBV基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系

 目的：探讨乙型肝炎病毒（HBV）基因型、前S/S基因突变与HBV母婴传播免疫预防失败的关系。
方法：选择血清HBsAg阳性且HBV DNA定量≥1×1010 IU/L的孕妇及其新生儿,以新生儿是否发生免疫预防失败将孕妇分为免疫预防失败组（15例）和免疫预防成功组(45例),采用PCR扩增直接测序法对2组孕妇血中HBV进行基因分型和前S/S基因突变的检测,对比分析2组的不同。
结果：（1）基因型：2组孕妇血中HBV的基因型均为B和C型,均以B型为主,2组基因型分布相比,差异无统计学意义（P>0.05）。（2）突变率：HBV 前S/S基因2个片段的突变率在免疫预防失败组与免疫预防成功组间相比,差异均无统计学意义（P>0.05）;而B基因型与C基因型间相比,差异均有统计学意义（P<0.05）;但在同一基因型内,2个片段的突变率在免疫预防失败组与免疫预防成功组相比,差异均无统计学意义（P>0.05）。对HBV 前S/S基因2个片段的遗传树分析也显示不同基因型的基因突变率不同,但同一基因型的突变率在2组间差异无统计学意义（P>0.05）。（3）突变热点：在免疫失败组的4个病例中发现4个突变热点：529G-A、530A-G、826A-G1和166het-dupC各1例;在免疫成功组的6个病例中发现3个突变热点：A530T 1例,A530G 2例, T531C 3例。
结论：（1）不同HBV基因型间前S/S基因突变率不同。（2）HBV前S/S基因突变普遍存在,但并不是每个突变都与母婴传播免疫失败有关,仅分析基因突变率对研究免疫失败并无意义,寻找与免疫失败有关的特异性突变位点可能更有意义。     

慢性乙型肝炎患者HBV耐药模式及rtA181变异准种分析

目的分析慢性乙型肝炎患者HBV逆转录酶区耐药变异情况及rtA181变异准种的分布。方法提取患者血清中HBVDNA,PCR扩增逆转录酶区域,产物测序后经软件比对分析耐药变异情况和基因型;对部分rtA181变异标本进行克隆后测序分析准种分布。结果 489例标本中,检出明确耐药变异265例,在B、C基因型中分布比例存在差异（56.6%vs43.0%,P=0.022）。拉米夫定相关耐药138例（52.1%）,以M204I、M204I/V＋L180M±L80I/V变异为主;阿德福韦相关耐药35例（13.2%）,以N236T＋A181T/V较为多见;拉米夫定＋阿德福韦相关耐药70例（26.4%）,几乎都和A181有关。rtA181准种分析发现1例位于同一病毒株的多耐药组合,且未发现单一A181T变异的病毒准种。结论 HBV耐药变异主要表现为M204和A181相关变异,耐药模式复杂;检测HBV逆转录酶区的变异有助于临床及时发现和确认耐药情况,指导临床合理进行抗病毒用药。     

Multidrug‐resistant hepatitis B virus resulting from sequential monotherapy with lamivudine,adefovir, and entecavir: Clonal evolution during lamivudine plus adefovir therapy

Whether multidrug‐resistant (MDR) hepatitis B virus (HBV) harbors mutations co‐located in the same HBV clones that confer reduced sensitivity to antiviral therapy remains uncertain. This study investigated the evolution of MDR HBV strains developed from sequential monotherapy with lamivudine (LAM), adefovir (ADV), and entecavir (ETV) during LAM plus ADV salvage therapy. Sera were obtained from six patients who had developed sequential resistance to LAM, ADV, and ETV before and during LAM plus ADV therapy. The HBV genomes from each patient were amplified, cloned, and sequenced. Among 6 sets of 20 clones obtained before salvage therapy, all clones harbored the rtM204V mutation, and ETV‐resistant mutations were detected with the rtM204V in 108 clones. The rtA181 mutation was not detected at baseline, but emerged in five patients during therapy. Among 9 sets of 20 clones obtained during salvage therapy, 39 clones harbored rtA181T/V ± rtN236T mutations, which were detected in the absence of rtM204 and ETV‐resistant mutations in 37 clones (94.9%). Only two clones (5.1%) harbored both rtA181T/V and ETV‐resistant mutations. The rtA181T/V mutation emerged after reversion from ETV‐resistant mutants to wild‐type HBV. Five patients achieved a partial virologic response to LAM plus ADV therapy. In conclusion, the majority of MDR mutations existed in different genomes. Suboptimal response to LAM plus ADV therapy may not result from the co‐localization of MDR HBV mutations in the same genome, but instead the low antiviral potency of these drugs. Thus, more potent antiviral drug combinations may be an effective salvage therapy for patients infected with MDR HBV. J. Med. Virol. 85:55–64, 2012. © 2012 Wiley Periodicals, Inc.     

基因芯片法检测HBV多位点变异和基因分型的临床价值

目的运用基因芯片技术检测临床乙肝患者血清中HBV病毒的相关耐药基因突变及基因分型,评价该方法在监测HBV耐药及基因分型中的作用。方法在使用不同核苷类药物的260例乙肝患者血清中,采用寡核苷酸探针芯片检测其携带的HBV相关耐药基因变异情况及基因型。并随机挑取40例第2次扩增产物进行DNA序列分析。结果经基因芯片检测到拉米呋啶耐药31例（19．4％）,阿德福韦酯耐药6例（6％）。260例患者中C基因型180例（69．2％）,B型59例（22．7％）,BC混合型21例（8．1％）。结论基因芯片法可同时检测针对拉米呋啶、阿德福韦酯、恩替卡韦、替比呋啶的多重耐药基因位点以及基因分型,与基因测序法比较,结果完全一致,是一种可靠的检测方法,可为临床抗病毒药物的科学选择提供参考。     

Do different lamivudine‐resistant hepatitis B genotypes carry the same risk of entecavir resistance?

Entecavir switch is one of the treatment options for lamivudine‐resistant hepatitis B (HBV) patients in Asia. This study examined the outcome of patients with different baseline resistance genotypes in a cohort study. In this study, 14 patients with chronic HBV were treated with entecavir 1 mg/day for 5 years. Enrolment criteria include: documented lamivudine resistant mutations, treatment with adefovir 10 mg/day for at least 24 weeks, and Child‐Pugh score <7. Most had previous failed adefovir therapy and compensated cirrhosis of the liver. Clinical outcomes, liver biochemistries, and HBV DNA were monitored regularly. Patients with virologic breakthrough were rescued with add‐on adefovir. At the end of the treatment period, the mean HBV DNA fell from 5.92 × 10⁶ (baseline) to 3.67 × 10¹ IU/ml. The presence of a HBV polymerase rtM204V mutation at the baseline was found to be the major risk factor for adverse outcomes. Compared to the patients with the rtM204I mutant, patients with the rtM204V mutant had increased risk of virologic breakthrough (80% vs. 0%, P = 0.010) requiring add‐on adefovir, slower virologic responses (log rank test, P = 0.0011), failure to reach undetectable HBV DNA levels (60% vs. 0%, P = 0.045), and higher risk of entecavir‐resistance (60% vs. 0%, P = 0.045). All the patients with rtM204I and rtA181 mutants had undetectable HBV DNA from 18th month. In summary, lamivudine‐resistant HBV patients with the rtM204V mutation have the highest risk of developing entecavir resistance, and entecavir monotherapy should be avoided. Those with the rtM204I and rtA181V mutations may have lower risks, but regular surveillance for viral breakthrough is required. J. Med. Virol. 85:26–33, 2012. © 2012 Wiley Periodicals, Inc.     

In vitro drug susceptibility analysis of hepatitis B virus clinical quasispecies populations

Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC(50)) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC(50). In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.     

Telbivudine and adefovir combination therapy for patients with chronic lamivudine-resistant hepatitis B virus infections

We evaluated second-line salvage therapy with adefovir + telbivudine (group 1), adefovir followed by adefovir + telbivudine (group 2), or lamivudine + adefovir followed by adefovir + telbivudine (group 3) in hepatitis B patients with an inadequate virologic response to lamivudine treatment. Simple linear regression analysis showed that for each additional month of treatment, the most significant reduction in viral load occurred in group 1 (HBV DNA [Log₁₀ IU/mL]: group 1, ?0.149; group 2, -0.081; group 3, ?0.123). Generalized estimating equation analysis revealed that compared to group 1, hepatitis B virus (HBV) DNA levels were 1.203 and 0.443 Log₁₀ IU/mL higher in groups 2 and 3, respectively. Overall, a significant reduction in viral load (?0.060 Log₁₀ IU/mL) was observed for each additional month of treatment. Adefovir + telbivudine treatment resulted in a significant reduction in HBV DNA levels. Moreover, telbivudine treatment resulted in a significant reduction in viral load (?0.050 Log₁₀ IU/mL) compared to lamivudine treatment after the emergence of lamivudine resistance.     

Snapshot on drug‐resistance rate and profiles in patients with chronic hepatitis B receiving nucleos(t)ide analogues in clinical practice

While the selection of complex HBV drug‐resistance patterns on therapeutic failure can compromise the efficacy of anti‐HBV therapies, recent data show that patients failing treatment without drug‐resistance have a rate of virological success close to drug‐naive patients. The goal of this study is defining, in clinical practice, the burden of drug‐resistance mutations in a cohort of patients treated with anti‐HBV drugs. Prevalence and patterns of drug‐resistance were analyzed by RT‐sequencing in 204 patients infected chronically: 148 experiencing virological rebound (defined as an increase in serum HBV‐DNA > 20 IU/ml after achieving virological success [HBV‐DNA < 20 IU/ml]), and 56 null/partial responders (always detectable serum HBV‐DNA [>20 IU/ml] within 48 weeks of therapy). The highest rate of drug‐resistance was observed in patients experiencing virological rebound (prevalence, 79.1%). Conversely, almost half (46.4%) null/partial responders have no evidence of drug‐resistance. The rate of drug‐resistance was higher in patients treated with lamivudine (76.8% [109/142]) and telbivudine (83.3% [5/6]), followed by adefovir (62.5% [15/24]), and entecavir (52.2% [12/23]). Complex mutational patterns characterized by the co‐presence of rtM204V/I‐rtA181T/V (impairing the efficacy of all anti‐HBV drugs) were detected in four patients (2.7%) with virological rebound. Drug‐resistance is the main cause of failure to therapy in patients experiencing virological rebound, supporting the need of rapid switch to anti‐HBV drugs with higher genetic barrier and potency (entecavir/tenofovir). Conversely, nearly half of null/partial responders shows no evidence of drug‐resistance mutations, maintaining high chance of achieving therapeutic success with the same class of drug. In this setting, genotypic resistance may help in selecting patients still carrying wild‐type viruses, that may take major benefits from antiviral treatment. J. Med. Virol. 85: 996–1004, 2013. © 2013 Wiley Periodicals, Inc.