国产丙型肝炎病毒核心抗原检测试剂盒的临床应用评价

丙型肝炎是由HCV感染引起的一种全球性传染病,主要通过输血和注射途径传播,在我国感染率为3％ ～ 5％[1];极易转为慢性,与肝硬化和原发性肝癌的关系十分密切.因此,丙型肝炎的早期诊断对于筛查HCV的传染源、指导临床治疗和预后判断有重要意义.     

丙型肝炎病毒核心抗原检测技术及其临床意义

丙型肝炎病毒（HCV）感染是在世界范围内引起输血后肝炎的主要病因之一。目前中国HCV感染的诊断和筛查主要采用基于酶免疫分析法（EIA）的HCV抗体检测技术,但该方法存在较长的窗口期,残余风险高。核酸检测（NAT）的应用可显著缩短窗口期,但成本高昂。HCV核心抗原（HCVcAg）作为HCV感染的标志几乎与核糖核酸（RNA）同时出现,且浓度呈现动态相关,可用于HCV感染的检测。与NAT相比,HCV核心抗原的检测成本相对较低,对实验室条件要求不高。目前多种基于不同方法的HCVcAg检测试剂盒已投入市场。该文对已有的几种HCV核心抗原检测技术在其原理、检测效果以及临床意义等方面进行综述。     

乙型肝炎病毒核心相关抗原的监测及临床意义

监测乙型肝炎病毒核心相关抗原(HBcrAg),能有效预测核苷(酸)类药物抗病毒治疗后产生的病毒学反弹及耐药风险,具有重要的临床实用价值。     

酶联免疫吸附测定法检测丙型肝炎病毒核心抗原

自1989年丙型肝炎病毒（HCV）基因克隆成功之后，HCV感染的实验室诊断得以推广，出现多种检测方法，包括多聚酶反应（PCR）检测HCV RNA（定性和定量）及酶联免疫吸附测定（ELISA）法测抗HCV抗体（HCVIgG和HCVIgM抗体）目前已可检测HCV感染者血清HCV核心抗原（HCVcAg）。本文综述用ELISA检测HCVcAg的研究。     

慢性乙型肝炎患者肝细胞内乙型肝炎核心抗原阳性的临床意义

目的探讨CHB患者肝组织HBcAg阳性的意义。方法对200例CHB患者应用荧光聚合酶链反应（FQ-PCR）法精确定量检测血清HBV DNA含量。患者均检测血清中HBeAg含量,同时进行肝活组织检查,应用免疫组织化学技术检测HBcAg情况,并进行相关性分析。结果按测定血清HBV DNA水平,分为A组（＜3 log10拷贝/ml）20例,B组（≥3 log10拷贝/ml-＜5 log10拷贝/ml）13例,C组（≥5 log10拷贝/ml～＜6 log10拷贝/ml）24例,D组（≥6 log10拷贝/ml～＜8 log10拷贝/ml）116例,E组（≥8 log10拷贝/ml）27例。肝组织HBcAg阳性者175例,占87．5％,A组HBcAg阳性率55．0％（11/20）,B组53．8％（7/13）,C组75．0％（18/24）,D组96．6％（112/116）,E组100．0％（27/27）,HBcAg阳性率与血清HBV DNA水平之间呈显著正相关（r=0．80,P＜0．01）。血清HBV DNA水平高低与HBeAg阳性率之间呈显著正相关（r=0．47,P＜0．01）。其中20例HBV DNA阴性者中（A组）,HBeAg阳性者5例（25％）,HBcAg阳性者11例（55％）；15例HBV DNA阴性且HBeAg阴性者中有7例HBcAg阳性,占46．7％。结论CHB患者肝组织HBcAg阳性能更可靠地反映肝细胞内HBV复制状态。检测肝组织内HBcAg对CHB患者疗效评价和对治疗反应性的预测更具有临床意义。     

乙型肝炎病毒核心抗原在慢性乙型肝炎病毒感染者肝细胞内的分布及临床意义

目的 探讨HBcAg在慢性HBV感染者肝细胞内的分布情况及其临床意义.方法 对41例慢性HBV感染者进行肝组织穿刺,用免疫荧光组织化学技术在激光共聚焦显微镜下观察肝细胞内HBcAg的分布情况.计量资料采用Kruskal Wallis检验,计数资料采用卡方检验.结果 41例慢性HBV感染者中,36例肝细胞内HBcAg阳性表达,阳性率为87.8%,其中23例肝功能中度异常,10例肝功能轻度异常,3例肝功能正常.HBcAg有膜、胞质与核三种形式表达.在23例肝功能中度异常者中,6例呈明显膜型表达,无明显胞质型及核型,17例以胞质型与膜型混合表达,未发现核型表达.在10例肝功能轻度异常者中,以单纯胞质型为主,未见膜型与核型表达.在3例肝功能正常者中,以胞质型表达与少许核型表达为主,未见膜型表达.HBcAg表达类型与肝功能之间差异有统计学意义(χ2=10.60,P＜0.01).结论 慢性HBV感染者肝组织损伤与HBcAg的表达有直接联系,提示膜型表达的HBcAg是肝脏免疫损伤过程中的靶抗原.

Abstract：

Objective To explore the distribution and clinical significance of hepatitis B core antigen( HBcAg) in the hepatocytes of chronic hepatitis B virus infected patients. Methods Paraffin sections were made from 41 liver biopsy samples obtained from chronic hepatitis B virus infected patients. The immuno-fluorescence confocal technique was utilized to analyze the expression level and localization of HBcAg in hepatocytes. The data were analyzed by using Kruskal Wallis test and chisquare test. Results HBcAg expression were detected in 36 (87. 8%) patients, among whom 23 cases had moderate abnormal liver function, 10 with mild abnormal liver function and 3 with normal liver function. Among the cases with moderate abnormal liver function, 6 cases showed the simple membrane-type HBcAg expression, 17 cases showed mixed cytosolic-type and membrane-type HBcAg expression without the nuclear-type expression. Twelve cases with mild abnormal liver function only showed simple cytosolic-type HBcAg expression without membrane-type or nuclear-type expression. In the three patients with normal liver function, HBcAg was expressed in cytoplasm and nuclear but not on membrane. The correlation between HBcAg expression pattern and liver function was statistically significant (χ2 =10. 60, P＜0.01). Conclusion HBcAg expression is directly correlated with liver injury in chronic hepatitis B virus infected patients, which indicates that membrane expressed HBcAg is the target antigen during the immuno-attack of liver.     

乙型肝炎病毒和丙型肝炎病毒不同模式重叠感染患者临床特征分析

目的 了解HBV和HCV不同模式重叠感染患者临床特征的差异.方法 回顾分析中山大学附属第三医院1999年5月至2010年5月HCV和HBV重叠感染患者186例.统计分析不同病毒学模式重叠感染患者的人口学、流行病学、实验室检查和病理学表现,数据处理采用t检验和x2检验等.结果 186例HBV和HCV重叠感染患者中,HBV...     

乙型肝炎核心相关抗原对慢性乙型肝炎病毒感染诊疗及预后评价的作用

未行治疗的慢性乙型肝炎(chronic hepatitis B,CHB)有进展至肝纤维化、肝硬化甚至肝癌的风险,严重威胁人类的生命健康。准确地评价肝炎所处免疫状态及肝内炎症进展程度,是选择合适的治疗方案和有效控制患者病情的关键。寻找可靠、有效的血清学指标对CHB诊疗及预后评价有重要作用。乙型肝炎核心相关抗原是一种无创的...     

丙型肝炎核心抗原检测在丙型肝炎诊断中的意义

目的了解丙型肝炎核心抗原（HCV-cAg）检测方法的敏感性及特异性,确定具有临床意义的S/CO值,探讨其在丙型肝炎诊断中的意义。方法使用ELASA方法检测丙型肝炎核心抗原,RT-PCR检测HCV RNA定量,观察不同S/CO值所对应的HCV RNA定量之间的关系,以HCV RNA为诊断金标准,列四格表做诊断实验。结果 HCV-cAg抗原检测的敏感性为87.05%,特异性为76.67%,阳性预测值为96.53%,阴性预测值为44.23%。结论 （1）随着HCV-cAg的S/CO值逐渐增大,其与HCV RNA阳性符合率明显增高,随着HCV-cAg的S/CO值减小,其与HCV RNA阴性符合率明显增高;（2）S/CO值=2可以作为临床判断HCV感染病毒血症存在的一个标准;（3）本实验的敏感性和特异性较好,检测方法简单,可以作为丙型肝炎临床诊断的补充试验及筛查。     

乙型肝炎核心抗原为载体进行丙型肝炎混合性治疗疫苗的实验

目的 以HBcAg为载体进行丙型肝炎混合性治疗疫苗的实验研究。方法 利用DNA重组技术将HCVT表位（131～140位氨基酸）和（1445～1453位氨基酸）分别插入HBcAg el—loop处，构建病毒颗粒样抗原表达载体pTrc-core-T1和pTrc-core-T2，并在大肠埃希菌DH5α中表达，蔗糖密度梯度离心提取纯化表达产物HBcAg—T1和HBcAg—T2。以T1和T2表位肽混合物、HBcAg—T1和HBcAg-T2混合蛋白免疫Balb／C小鼠，并设空白对照，适时行抑瘤实验；流式细胞仪检测CD4^＋、CD8^＋、IL-4及IFN-γ、IL-5，ELISA法检测IL-12；并行细胞杀伤实验，以观察混合蛋白的免疫原性。结果 PCR法鉴定质粒pTrc—core—T1和pTrc—core—T2正确。抑瘤实验中HBcAg—T1和HBcAg—T2混合蛋白免疫组仅1只小鼠形成瘤块，直径0．1cm，低于对照组的平均直径1．3cm、T1T2混合肽免疫组的0．9cm；混合蛋白免疫组脾细胞内CD8^＋T细胞占（20．21±2．01）％，高于混合肽免疫组的（15．33±1．45）％和空白对照组的（5．09±1．66）％（P〈0．01）；3组内脾细胞内IFN-γ阳性细胞百分比分别为（1．58±0．05）％、（0．88±0．02）％和（0．53±0．03）％（P〈0．01）；血清中IL-5较混合表位肽组有所下降（P〈0．01）；而ELISA检测混合蛋白组IL-12高于T1T2混合表位肽免疫组；混合蛋白免疫组小鼠HCV特异细胞毒性T淋巴细胞（CTL）活性明显高于T1T2混合表位肽免疫组（P〈0．01）。结论 HBcA乎T1和HBcAg～T2混合蛋白诱导出高水平的细胞免疫应答，可作为HCV治疗性疫苗的候选。     

Hepatitis B virus core and core-related antigen quantitation in Chinese patients with chronic genotype B and C hepatitis B virus infection

BACKGROUND AND AIMS: Hepatitis B virus (HBV) core-related antigen (HBcrAg) and HBV core antigen (HBcAg) assays were developed for the measurement of serum HBV load. The aim of this study was to assess the clinical utility of these assays in Chinese patients with chronic genotype B and C HBV infection. METHODS: One hundred and ninety-three chronic hepatitis B patients were enrolled. Serum HBcrAg and HBcAg were measured by chemiluminescence enzyme immunoassay, and HBV-DNA was measured by using a sensitive polymerase chain reaction assay. The data were analyzed in patients with HBV genotype B (HBV/B) and genotype C (HBV/C). The HBcrAg/HBcAg ratio was calculated and compared between patients with and without hepatitis B e antigen (HBeAg). RESULTS: The concentrations of HBcrAg and HBcAg showed significant positive correlation with the HBV-DNA concentration in both HBV/B (r = 0.79, P < 0.001, and r = 0.77, P < 0.001, respectively) and HBV/C (r = 0.87, P < 0.001, and r = 0.90, P < 0.001, respectively). The cut-off for a positive HBcAg corresponded to approximately 4.5 log copies/mL, and that for a positive HBcrAg result corresponded to 3-4 log copies/mL. The HBcrAg/HBcAg ratio was higher in patients with HBeAg than in those without HBeAg. CONCLUSIONS: The HBcrAg assay and HBcAg assay are clinically useful in viral quantitation of HBV/B and HBV/C. A combination of these assays would be a valuable tool for analyzing the clinical status of HBV infection.     

Hepatitis C virus antigens enzyme immunoassay for one-step diagnosis of hepatitis C virus coinfection in human immunodeficiency virus infected individuals

BACKGROUND Current diagnosis of hepatitis C virus(HCV) infection requires two sequential steps: testing for anti-HCV followed by HCV RNA PCR to confirm viremia. We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA) for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera: 10 without HCV or HIV infection;54 with viremic HCV monoinfection; 38 with viremic HCV/HIV coinfection; and45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding, it was 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test. In five sera with HCV infection, HCV RNA was as low as50-59 IU/mL, and four out of five tested positive for HCV-Ags EIA. Likewise, it was also 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection, regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL. It is highly sensitive and specific in the setting of HIV coinfection, regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.     

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on

AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells.METHODS: The recombinant adenoviruses AdXC,Ad-X and Ad-C expressing HBV X-HCV C fusion gene,HBVX gene and HCV C gene were constructed,respectively.Hepatoma cells were infected with different recombinant adenoviruses.MTT,colonyforming experiment,FCM,TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells.RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells.Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells.FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of GIS phase in the HepG2 cell cycle.The apoptosis index of the Ad-XC,Ad-X,Ad-C group cells was significantly lower than that of the AdO and control group cells.Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in AdXC infected cells.Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells,but not in control mice.CONCLUSION: Ad-XC,Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro.The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C.Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.     

乙型肝炎病毒核心抗原核酸疫苗对猕猴的体液和细胞免疫原性观察

目的：观察乙型肝炎病毒核心抗原（HBcAg）核酸疫苗对猕猴的体液和细胞免疫原性。方法：实验组和对照组猕猴分别肌内注射HBcAg核酸疫苗（pJW4303/HBc)和对照质粒（pJW4303)。采用酶联免疫试验检测接种前后猕猴血清中抗-HBc、抗-HBc IgG亚类和猕猴外周血单个核细胞（PBMC）培养上清中干扰素-γ（IFN-γ）及白介素-4（IL-4）水平。采用^3H-TdR掺入法检测猕猴PBMC HBcAg特异性增殖反应。结果：实验组猕猴接种该核酸疫苗后均产生了较强的抗-HBc反应。实验组猕猴抗-HBc IgG亚类以IgG2为主,PBMC培养上清中IFN-γ水平显著增高,提示为Th1型免疫应答。实验组猕猴的PBMC抗原特异性增殖反应明显高于对照组。结论：HBcAg核酸疫苗对猕猴具有良好的体液和细胞免疫原性。     

新型乙型肝炎病毒核心区核酸疫苗的免疫原性研究

目的观察新型乙型肝炎病毒(HBV)核心抗原(HBcAg)核酸疫苗的免疫原性。方法应用新型人体应用载体质粒pSW389l构建HBcAg核酸疫苗(pSW3891／HBc)，对照组和实验组Balb／c小鼠分别以基因枪法免疫对照载体质粒(PSW3891)和HBcAg核酸疫苗，采用酶联免疫吸附试验检测抗HBc，乳酸脱氢酶(LDH)释放测定法检测小鼠HBcAg特异性cTL杀伤活性。结果HBcAg核酸疫苗可在体外293T细胞中高效表达，免疫小鼠后可产生高滴度抗HBc(1：97200)，免疫鼠脾细胞HBc特异性CTL杀伤活性达73．25％。结论新型HBcAg核酸疫苗在Balb／c小鼠实验中表现出良好的体液和细胞免疫原性。     

人乙型肝炎免疫球蛋白定量清除新生儿乙型肝炎病毒表面抗原的研究

目的 根据新生儿出生时HBsAg和HBV DNA的载量,调整人乙型肝炎免疫球蛋白使用量,以期更有效地阻断HBV的母婴传播. 方法 收集出生2h内静脉血HBsAg阳性新生儿资料125例.分为研究组64例,对照组61例,研究组根据新生儿出生时HBsAg感染量调整乙型肝炎免疫球蛋白使用量,与对照组比较新生儿12个月龄以上治疗效果.计量资料采用非正态分布采用秩和检验,计数资料采用x2检验.结果 2组新生儿出生时HBsAg和HBV DNA检测值的差异均无统计学意义(p 值均＞0.05).研究组出生HBV感染新生几64例,12月龄以上成功清除HBsAg者53例,成功清除率为82.8％,感染11例(1.2％).对照组出生HBV感染新生儿61例,12月龄以上成功清除HBsAg者35例,成功清除率为57.4％,感染26例(3.1％).2组出生HBV感染新生儿12个月龄以上清除HBsAg效果比较,x2=9.696,P＜0.05,差异有统计学意义.结论 根据新生儿的HBsAg感染量调整使用乙型肝炎免疫球蛋白,可提高乙型肝炎母婴传播的阻断成功率.     

HBsAg阴性或抗-HBc阳性者肺癌术后辅助化学治疗中乙型肝炎病毒的再激活

目的 探讨HBsAg阴性或抗-HBc阳性者肺癌术后辅助化学治疗中HBV的再激活及其相关危险因素.方法 回顾性分析2003年1月到2011年12月接受辅助化学治疗的3280例肺癌术后患者,所有入组患者进行HBV血清学标志物和生物化学检测,并接受以顺铂为基础的辅助化学治疗方案.数据比较行x2检验.结果 367例HBsAg阴性或抗-HBc阳性肺癌术后患者中,14例(3.81％)进展为乙型肝炎.单因素分析表明,患者年龄≥70岁(x2=13.003,P=0.019)、肝脏CT检查为脂肪肝或早期肝硬化(x2=11.225,P=0.026)和使用糖皮质激素累计剂量超过150mg(x2=7.008,P=0.033)是辅助化学治疗中HBV再激活的相关因素;而性别、基础辅助化学治疗方案与HBV的再激活无关联.结论 肺癌术后的辅助化学治疗中,HBsAg阴性或抗-HBc阳性者有一定比例可以发生HBV的再激活.