探讨三种检测方法在丙型肝炎诊断中的应用价值

目的探讨ELISA法检测丙型肝炎病毒核心抗原(HCVcAg)、病毒抗体(抗-HCV)及RT-PCR法检测丙型肝炎病毒RNA(HCV-RNA)3种方法在丙型肝炎诊断的应用价值。方法采用HCVcAg ELISA试剂盒,抗-HCV ELISA试剂盒及HCV-RNA PCR试剂盒,对临床200例疑似丙肝病毒感染的样本进行HCVcAg、抗-HCV和HCV-RNA检测。结果 HCVcAg阳性检出率为42%;HCV-RNA阳性检出率最高,为61%;抗-HCV阳性检出率为52%。Kappa检验示3种检测方法结果阳性吻合度基本一致。HCVcAg阳性检出率随着HCV病毒含量的升高而升高。结论 3种方法中,RT-PCR检测HCV-RNA仍是判断丙肝感染最准确方法。HCV核心抗原检测可以有效缩短窗口期,联合运用抗-HCV和HCVcAg或抗-HCV和HCV-RNA,能有效降低单独使用抗-HCV检测的漏检风险。HCVcAg可作为HCV抗体常规检测的补充指标,提高检出率。     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

目的 探讨丙型肝炎病毒核心抗原酶联免疫吸附法在血液透析患者HCV感染检测中的应用.方法 对本院250名血液透析患者,用第三代酶联免疫吸附试剂盒检测抗.HCV抗体、酶联免疫吸附法检测HCV核心总抗原,用巢式RT-PCR法对阳性标本进行确认.结果 250例血液透析患者中抗-HCV抗体阳性43例(阳性率17.2%),HCV-cAg阳性45例(阳性率18%),经配对x2检验,P=0.839,差异无统计学意义;43例抗-HCV抗体阳性中RT-PCR检测结果 为36例HCV-RNA阳性,其中32例HCV-cAg也呈阳性,另外4例HCV-cAg阴性;45例HCV-cAg阳性标本中HCV-RNA结果 全阳性;13例核心抗原阳性但抗体阴性的患者样本中也含有HCV-RNA,抗-HCV抗体的漏检率为23.2%(13/56);核心抗原阳性标本测出病毒载量是(49258±28682)拷贝/ml,高于抗-HCV抗体阳性组(23938±10780)拷贝/ml(P<0.05);4例核心抗原阴性但抗-HCV抗体及RT-PCR均阳性标本的病毒载量是(306±161)拷贝/ml,明显低于HCV核心抗原阳性组(P<0.001).结论 HCV核心总抗原酶联免疫吸附试验将有益于由于免疫抑制状态、窗口期更长的血液透析患者,与抗-HCV的检测结果 具有互补性;HCV-cAg能很好反映HCV RNA,相对于PCR来说,HCV-cAg检测既具有成本效益好又可减少人力资源,具有广阔的临床应用前景.     

酶联法抗-HCV和HCV-cAg联检对丙型肝炎实验室诊断的应用价值

目的 探讨联合丙型肝炎病毒核心抗原(HCV-cAg)及抗-HCV抗体酶联免疫检测对丙型肝炎实验室诊断的应用价值.方法 289份血清标本按抗-HCV阳性、弱阳性、阴性及HCV-cAg阳性、弱阳性及阴性分组,用PCR扩增法检测其HCV RNA含量.结果 单项抗-HCV阳性及弱阳性标本其HCV RNA检测阳性符合率分别为87...     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

丙型肝炎病毒抗体阳性人群中抗-HCV、HCV-cAg与HCV-RNA结果的相关性及其联合检测在临床应用价值的评估

目的探讨丙型肝炎病毒抗体阳性人群中抗-HCV、HCV-cAg与HCV-RNA结果的相关性及其联合检测在临床应用的价值,同时界定雅培I2000全自动化学发光仪检测抗-HCV真阳性95%时标本S/CO值的临界点。方法选取全自动化学发光仪检测的抗-HCV阳性血清样本179例(包括11例灰区标本),利用化学发光法检测HCV-cAg,利用RT-PCR法检测HCV-RNA,采用SPSS16.0软件对抗-HCVS/CO值绘制受试者操作特性(ROC)曲线,得到95%真阳性时抗-HCV结果的临界值(S/CO)。再对抗-HCV、HCV-cAg与HCV-RNA进行相关性分析。结果对179例样本进行抗-HCV、HCV-RNA及HCV-cAg的检测,其中HCV-RNA阳性43例,HCV-cAg阳性42例。在43例HCV-RNA阳性的标本中,抗-HCV的阳性检出率为93.02%,HCV-cAg阳性检出率为95.35%。抗-HCV灵敏度为93.02%,特异性为5.88%;HCV-cAg灵敏度为95.35%,特异性为99.26%。且HCV-cAg及抗-HCV阳性率随着HCV病毒含量升高而增高,RNA病毒含量越高,HCV-cAg及抗-HCV的阳性率也越高,差异具有统计学意义(P<0.05)。HCV-cAg联合抗-HCV检测与HCV-RNA的阳性符合率为100%,高于单独检测HCV-cAg或抗-HCV的阳性符合率95.35%、93.02%。以HCV-RNA检测为金标准,当抗-HCV的S/CO值>4.42时,真阳性率可达到95%。结论HCV-cAg与HCV-RNA检测的阳性符合率高达95.35%,故HCV-cAg可作为丙型肝炎早期诊断的特异性指标。为保证标本真阳性率达到95%,建议使用美国雅培I2000仪器进行丙肝抗体检测时,若抗-HCV结果(S/CO)小于4.42的标本,抗-HCV、HCV-cAg与HCV-RNA联合检测可有效降低丙型肝炎窗口期的漏检率,为丙型肝炎的早期发现、早期诊断、早期治疗提供有力证据。     

化学发光法和酶联免疫法作为筛选试验测定丙肝抗体的评价

目的比较化学发光法(CLIA)和酶联免疫法(EIA)测抗-HCV对诊断HCV感染的检出率,分析CLIA测定抗-HCV作为HCV感染初筛试验的重要临床意义。方法分别用CLIA、EIA方法检测所有临床标本中的抗-HCV,选取抗-HCV初筛阳性样本进行HCV RNA确认试验。结果 6461例样本中,EIA和CLIA测抗-HCV阳性率分别为2.86%和3.17%;确认试验中,CLIA法抗-HCV阳性与HCV RNA的符合率为97.1%,显著高于EIA组(91.9%)。EIA法测抗-HCV,S:CO>5.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为69.7%;CLIA法测抗-HCV,S:CO>2.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为92.9%。有31例样本CLIA法抗-HCV和HCV RNA阳性,而EIA法抗-HCV阴性;有4例样本EIA法检测为阳性,而CLIA法和PCR确认试验均为阴性。结论CLIA法测抗-HCV作为HCV感染初筛实验,与传统EIA相比,特异性和阳性预测值更高,有效降低了假阳性率,有利于临床医生对HCV感染患者的早期诊断和治疗。     

抗-HCV和HCV-RNA检测及其与ALT的相关性分析

目的探讨化学免疫发光法（CLIA）定量检测抗-HCV和FQ-PCR法检测HCV-RNA含量与丙氨酸氨基转移酶（ALT）水平的相关性。方法用CLIA定量筛选抗-HCV阳性的100例病人标本,以荧光定量PCR法检测HCV-RNA含量和酶速率法检测ALT浓度水平,并对所得数据进行统计分析。结果在100份抗-HCV阳性标本中,检出HCV-RNA阳性者76例,阳性率为76%。随着抗-HCV的S/CO值增高,HCV-RNA检出率增高较明显;ALT水平与HCV-RNA含量无显著相关性（P〉0.05）,但ALT异常率与HCV-RNA含量呈正相关。结论在HCV诊断与疗效观察中,血清抗HCV、HCV-RNA和ALT指标各有利弊,3者有机结合能正确诊断和预测肝脏损伤及评价疗效。     

丙型肝炎病毒不同基因型NS3蛋白的抗原异质性分析

目的探讨不同基因型丙型肝炎病毒(HCV)NS3蛋白的抗原特性及其用于抗-HCV检测的意义。方法分别构建和表达含有HCV1型和6型NS3基因片段的重组质粒和重组蛋白，以EIJSA法和Western blot分析不同基因型重组蛋白与已知抗-HCV阳性血清的抗原反应性。结果HCV1型和6型NS3重组蛋白氨基酸序列的同源性为83．2％；85份抗-HCV阳性血清以此不同基因型HCV NS3单片段抗原检测，阳性检出率分别为61．2％(1型)和58．8％(6型)，其中有7份标本以NS3-1型抗原检测阴性，但可被NS3-6型抗原检出，反之，有9份血清以NS3-6型重组蛋白检测为阴性，而NS3．1型检测阳性；54份大学生体检血清和39份阴性质控血清以此两种抗原检测均为阴性。结论HCV1型和6型NS3重组蛋白存在抗原异质性，在发展HCV抗体检测试剂时需考虑加入不同基因型的NS3抗原。     

丙型肝炎病毒抗原检测方法的建立

目的以特异性单克隆抗体为基础建立丙型肝炎病毒(HCV)抗原检测的酶联免疫吸附(ELISA)法,探索从血浆或血清中检测HCV抗原的可能性.方法利用我们制备的抗-HCV核心及NS3区单克隆抗体(McAbs),进行多种交叉组合模式的分析,确立实验室模式,并测定348份义务献血员血样,确定此方法的Cutofff值,并分析146份抗-HCV阳性及225份抗-HCV阴性血浆,阳性结果用套式PCR试剂盒确证.结果构建了以抗-HCV核心区单抗C39及NS3区单抗C7-6为包被抗体,以C39及NS3区C7-57为标记抗体的夹心ELISA检测模型,以C7为抗原,其检出灵敏度为5ng/ml,其Cutoff值为阴性对照均值±0.25.146份抗-HCV阳性样本中,11份为抗原反应阳性,225份抗-HCV阴性样本中,16份为抗原阳性,这些阳性样本经PCR检测后,23份为HCVRNA阳性.结论用单抗构建的HCV抗原检测方法分别从抗-HCV阳性及阴性样本中检测出HCV抗原反应阳性样本,并经PCR确证,表明直接从血浆样本中检测HCV抗原是可能的,这将对于HCV和基础研究及控制HCV的传播有重要意义.     

丙型肝炎实验室诊断研究进展

经过十余年的深入研究,已建立起了较为完整的丙型肝炎病毒（HCV）感染诊断系统,包括血清学诊断,基因诊断,基因分型,基因变异,血清学分型和肝脏组织活检诊断等,现概述如下. 1血清学诊断（抗HCV检测） HCV感染的血清学诊断主要是检测HCV抗体.抗-HCV酶免疫法（EIA）适用于高危人群筛查,也可用于HCV感染者的初筛.但抗-HCV阴转与否不能作为抗病毒疗效的指标.用第三代EIA法检测丙型肝炎患者,其敏感度和特异度可达99%,因此,不需要用重组免疫印迹法（RIBA）验证[1].但一些透析、免疫功能缺陷和自身免疫性疾病患者可出现抗-HCV假阳性,因此,HCV RNA检测有助于确诊这些患者是否合并感染HCV.     

Hepatitis B and D genomes in hepatitis B surface antigen negative patients with chronic hepatitis C

Hepatitis B and hepatitis D viral genomes were tested by nested polymerase chain reaction in the serum and liver of 69 hepatitis B surface antigen (HBsAg) negative, anti-hepatitis C virus (HCV) positive patients (47 with HCV RNA and 22 without HCV RNA). Serum hepatitis B virus (HBV) DNA-was detected in 49% of the patients with HCV-RNA and in 64% of those without HCV-RNA. Furthermore, intrahepatic HBV-DNA was found in four of five (80%) of the biopsies analysed. Delta genome was found in 72% and 73%, respectively, of the anti-HCV positive patients with or without HCV-RNA. In addition, intrahepatic delta virus genome was detected in another four liver biopsies studied. In the group of patients with HCV-RNA, the simultaneous presence of hepatitis B and D genomes was statistically higher in transfused patients than in drug addicts, or in those with an unknown infection route (P < 0.001). These results show a high percentage of B and D genomes in HBsAg negative patients with anti-HCV, irrespective of the presence or absence of the HCV genome. However, the clinical implications of this finding should be examined in future studies. © 1995 Wiley-Liss, inc.     

Evaluation of a total hepatitis C virus (HCV) core antigen assay for the detection of antigenaemia in anti-HCV positive individuals

A new, sensitive enzyme immunoassay has been developed for detecting and quantifying total hepatitis C virus (HCV) core antigen in anti-HCV positive or negative sera ("trak-C", Ortho Clinical Diagnostics, Raritan, NJ). The purpose of this study was to evaluate the performance of trak-C as an additional laboratory diagnostic marker of viraemia. The performance was compared to HCV-RNA detection in the "screening" of sera from a large heterogeneous population of hospitalised patients and outpatients. Six hundred and eighteen anti-HCV negative sera, 405 anti-HCV positive/HCV-RNA negative sera, 604 anti-HCV positive/HCV-RNA positive sera and 67 anti-HCV negative sera containing antigens or antibodies potentially interfering with the performance of the assay were analysed. Supplemental HCV antibody testing was performed using a commercial strip immunoblot assay. HCV-RNA was investigated using a qualitative commercial assay. A quantitative commercial RT-PCR was used for the analysis of selected samples. Sensitivity and specificity values were 94.7 and 100%, respectively. The latter was also confirmed when anti-HCV negative samples containing potentially interfering antigens/antibodies were examined. Sensitivity below 100% was probably due to an antigenaemia below the detection limit of trak-C. Besides, because 65.6% of HCV-RNA positive/trak-C negative samples presented specific antibodies against all four RIBA antigens, the hypothesis was raised that, in some cases, the dissociation step efficiency could be sub-optimal. In conclusion, trak-C seems suitable for identifying HCV infection on large based populations. It is a rapid to perform, reliable and specific assay that can be adapted to any laboratory setting.     

Serum hepatitis C virus (HCV)-RNA and response to alpha-interferon in anti-HCV positive chronic hepatitis.

Hepatitis C virus (HCV) replication was assessed before and during alpha-interferon (IFN) treatment in 22 anti-HCV positive patients with posttransfusion or sporadic chronic hepatitis (CH). Eleven patients were "responders" and 11 patients "non-responders" to IFN. Thirteen anti-HCV negative healthy subjects and five anti-HCV negative patients with autoimmune CH served as controls. Serum HCV-RNA was detected by the polymerase chain reaction (PCR) in all untreated anti-HCV positive patients but in none of the anti-HCV negative subjects. PCR primers from the 5'-noncoding (NC) region were more sensitive than primers from a non-structural (NS5) region in detecting HCV-RNA (21/22, 95% vs. 7/22, 32%, respectively). Positive strand HCV-RNA titre and positivity rate for the negative strand were similar in responders and non-responders before IFN treatment, as well as anti-c100-3 titre by enzyme-linked immunosorbent assay (ELISA), and anti-5-1-1, anti-c33c, anti-c22 positivity rate by immunoblot assay (RIBA). HCV-RNA positivity by both NC and NS primers was more frequent before IFN among responders. During IFN treatment, serum HCV-RNA was detectable, mostly at low titres, in 1 (NC positive) of the 11 responders and in 9 (4 NS positive and 5 NC positive) of the 11 non-responders. Among the four non-responders who were NS positive during IFN, three were NC positive before IFN. Serum HCV-RNA was always found in our post-transfusion or sporadic anti-HCV positive patients with CH. Viraemia generally decreased during IFN treatment, but no available HCV markers clearly distinguished responders from non-responders before IFN treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of combined detection of hepatitis C virus core antigen and antibodies to reduce the serological window-phase

OBJECTIVES: In this study, we aimed at evaluating the performances of a combined assay for the detection of hepatitis C virus core antigen and antibodies and comparing this test with conventional third generation Elisa. MATERIAL AND METHODS: Two hundred forty-one samples were included in this study and tested by Monolisa HCV Ag-Ab ULTRA, Biorad and compared to Monolisa Anti-HCV Plus. A comparative study was performed on a HCV seroconversion panel (Monolisa anti-HCV Plus, Biorad; Innotest HCV Ab IV, Innogenetics and Murex anti-HCV, Abbott). False positive samples were detected with western blot assay (INNO-LIA HCV Ab III, Innogenetics). Two anti-HCV negative haemodialysis patients with rise in ALT have been tested for RNA detection (Amplicor v2.0, Roche). RESULTS: Results obtained with Biorad Ag-Ab were in agreement with third generation ELISA on HCV seroconversion panel. From anti-HCV negative patients, four samples were found low positive with HCV Ag-Ab. Two anti-HCV negative haemodialysis patients/HCV RNA positive were also negative with HCV Ag-Ab and 13 low positive samples with Biorad Ab were found negative with Ag-Ab. CONCLUSION: The HCV Ag-Ab assay has a high specificity and sensitivity comparatively to conventional ELISA; but in our study we don't prove the reduction of the "serologic window" for detection of anti-HCV antibodies.     

丙型肝炎病毒感染相关自身抗体(抗-GOR)的测定及其临床意义

本文以人工合成肽GOR作为包被抗原建立ELISA法检测患者血清中HCV感染相关自身抗体(抗-GOR)。检测581份各类临床标本,其中急、慢性NANBH的抗-GOR阳性率分别为19.2％和25.7％,而属于甲、乙型急慢性肝炎、SLE等均在10％以下。抗-GOR总检出率低于抗-HCV,但两者间具有互补性,且抗-GOR效价与HCV核心抗体有很好的相关性。以上结果表明:①GOR抗体的出现与HCV感染有关。②同时检测抗-GOR和抗-HCV可提高HCV感染的诊断率。③HCV感染的致病可能是通过HCV核心蛋白和自身蛋白联合触发而诱导机体产生自身抗体即抗-GOR。     

Surrogate markers are not useful for identification of HCV carriers in chronic hemodialysis patients.

Several diagnostic hepatitis C assays have been developed for the detection of antibodies to different antigens of the virus. This virus is the major cause of non-A, non-B hepatitis. Seventy-nine patients undergoing chronic hemodialysis and/or hemofiltration were tested for the presence of anti-HCV antibodies (anti-C-100-3 antibodies and anti-core antibodies), anti-hepatitis B core antibodies (anti-HBc), and aminotransferases (ALT). Seven patients were positive by one or more of the anti-HCV enzyme linked immunoassays (EIAs), while HCV-RNA was detectable in only four patients. These four patients had at least one, but not necessarily the same, positive anti-HCV EIA. HCV-RNA was not detected in patients who had no antibodies as determined by all six anti-HCV EIAs. All patients with a marker for HCV infection had persistent normal levels of transaminases. Three patients had elevated ALT values without a marker for HCV infection and suffered from hepatitis B virus infection. Anti-HBc was detected in 27/72 patients without any marker and in four patients with a marker of HCV infection. However, HCV-RNA was detectable in only one of these four anti-HBc positive patients. It is concluded that surrogate markers (anti-HBc and serum transaminases) are not useful for identification of HCV carriers in chronic hemodialysis patients.     

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.