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E1抗体检测及临床应用

目的：研究丙型肝炎患者血清中HCV E1抗体，确定HCV E1抗原在抗体检测中的应用价值。方法：应用酶联免疫吸附测定（ELISA）方法检测80份卫生部第3代HCV血清参比品，821例职业献血员血清和720例临床肝炎患者血清中E1抗体。结果：用E1抗原单包板检查80份卫生部血清参比品的阳性符合率为70％、阴性符合率为100％；从821例职业献血员血清样品中检出E1抗体阳性率为1．9％；从720例临床肝炎患者血清中检出E1抗体阳性率为68％。大部分E1抗体阳性血清，同时也能和HCV的Core、NS3抗原及NS5A抗原呈阳性反应，但有个别血清只对E1抗原呈阳性反应。用市购HCV抗ELISA检测试剂盒检测为阴性的218例肝炎科门诊患者、813例献血员和848例一般人群血清，用E1抗原单包板复检，检出的阴性率分别为1．4％、1．1％和0．9％。在3例患者血清学转变的追踪研究中，HCV E1抗体都同现最早。结论：用E1工程蛋白检测E1抗体具有较高的灵敏度和特异性。E1抗体在HCV感染患者中普遍存在而且早出现，在临床诊断上是有意义的。     

检测丙型肝炎病毒实验研究

目的探讨丙型肝炎患者血清丙型肝炎病毒抗体（抗-HCV）结果与丙型肝炎病毒核糖核酸（HCV—RNA）定量的相关性。方法采用酶联免疫吸咐实验（ELISA）和实时荧光定量PCR（FQ—PCR）技术,对621例同时做抗-HCV和HCV—RNA的丙型肝炎患者检测。结果621份标本中有115份HCV—RNA含量高于1000拷贝／ml,124份抗-HCV阳性。经统计分析,两者有明显的相关性。结论血清抗-HCV与血清HCV—RNA之间有较好的一致性;抗-HCV检测和HCV—RNA检测均有一定的局限性,同时检测抗-HCV和HCV-RNA可提高丙型肝炎患者的检出率．为其诊断和治疗提供帮助。     

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.     

丙型肝炎病毒核心抗原检测用于献血者筛查价值的探讨

目的应用酶联免疫吸附技术筛查献血员中丙型肝炎病毒核心抗原（HCV—cAg）和抗体（HCV—Ab），了解丙型肝炎病毒核心抗原筛查献血员应用价值。方法对我站2004年8-12月间的3972份献血者血清标本进行抗-HCV初、复检和HCV—cAg ELISA检测，将ELISA法阳性的25份血清标本，再做RT-PCR检测证实。结果3972份血清标本检测中，有10份仅初检抗-HCV阳性样本，经HCVRNA检测阳性有l份，12份仅复检抗-HCV阳性样本，经HCVRNA检测阳性有1份，HCV—cAg检测阳性有3份，经HCVRNA检测阳性有2份。结论HCV—cAg ELISA检测技术的敏感性与HCVRNA技术类似，但成本明显降低，可与HCV抗体联合检测应用献血员筛查。     

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.     

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。     

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...     

实时荧光定量HCV RNA与ALT检测在丙肝筛查中临床价值的研究

目的 探讨抗-HCV抗体阳性时,实时荧光定量HCV RNA与ALT检测在丙肝筛查中临床价值的研究.方法 回顾选择2019年1月至2019年12月首都医科大学附属房山区良乡医院收治的患者109例为研究对象,均留取外周静脉血,测定血清抗-HCV抗体、HCV RNA,丙氨酸氨基转移酶(ALT)水平.结果 在收集的109例抗-HCV抗体阳性患者样本中,以HCV RNA为金标准作为丙型肝炎的诊断,HCV RNA阳性患者占20.2％(22/109),HCV RNA阴性患者占79.8％(87/109),两组样本经独立样本配对t检验,P值为0.006,差异有统计学意义(P<0.05),以HCV RNA检测为金标准,用SPSS 23.0软件对抗-HCV的S/CO值及ALT进行ROC曲线处理,HCV抗体S/CO值对丙型肝炎的ROC曲线下面积(AUC)为0.851,ALT对丙型肝炎的ROC曲线下面积(AUC)为0.831.结论 HCV RNA阳性患者ALT平均值明显高于HCV RNA阴性患者的ALT平均值.抗-HCV的S/CO值及ALT对于丙型肝炎的筛查都有一定的价值,且抗-HCV抗体优于ALT的筛查.临床上,患者筛查丙型肝炎抗体阳性时,可同时进行ALT的检测,可为丙型肝炎早期诊断提供依据,以免延误治疗时机.     

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。     

游离丙型肝炎病毒核心抗原检测的临床价值探讨

目的:探讨游离丙型肝炎病毒(HCV)核心抗原检测在HCV感染诊断中的价值。方法:采用荧光定量PCR法检测HCV-RNA、ELISA法同步检测抗-HCV和游离HCV核心抗原。结果:191例HCV感染者HCV-RNA的检出率为71·2%(136/191);抗-HCV的检出率为97·4%(186/191);游离HCV核心抗原的检出率为33·0%(63/191)。其中有2例经抗病毒治疗的患者HCV-RNA和抗-HCV检测均阴性,但游离HCV核心抗原检测阳性;另有1例患者抗-HCV阴性,而游离HCV核心抗原阳性,经HCV-RNA证实为HCV感染。27例非HCV感染者HCV-RNA、抗-HCV和游离HCV核心抗原检测结果均为阴性。结论:HCV核心抗原检测作为抗-HCV检验的补充试验对HCV感染的诊断具有重要价值。     

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

Abstract

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (?5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

Evolving strategy for HCV testing in an Italian tertiary care hospital

BackgroundDiagnostic tests for hepatitis C virus (HCV) infection should be adapted according to the clinical status of the patient.ObjectivesWe exploited the application of different HCV diagnostic algorithms in a tertiary care hospital practice.Study designThe laboratory clinical reports to the medical orders for HCV testing during three years were clustered by different combinations of assays for anti-HCV antibodies (HCV Ab) (screening and confirmatory), HCV nucleic acid (HCV-RNA), HCV core antigen (HCV Ag). The latter was the first-line assay in acute HCV infections requiring a rapid assessment of the infectious state.ResultsThe majority (91.9%) of the 2726 subjects whose samples were analyzed were inpatients. Most of the patients/subjects were tested for clinical suspicion of viral hepatitis (49.2%), or occupational accident to health care professionals (20.0%). On 66% of samples HCV Ag test alone was performed and resulted positive in 116 cases (6%), while it was detected in 50.3% of anti-HCV positive samples. The agreement between HCV Ag and HCV-RNA was very high (k = 0.97); HCV Ag positivity rates increased according to the signal of the HCV Ab screening test.ConclusionsThe use of different testing strategies according to the patients’ history and clinical status allowed a significant reduction of the number of tests performed and the time needed to provide a diagnostic response useful for patients’ management without compromising the overall diagnostic accuracy for HCV infection.     

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)     

Immunohistochemical detection of the NS4 antigen of hepatitis C virus and its relation to histopathology.

The immunohistochemical localization of the hepatitis C virus (HCV) nonstructural antigen 4 (NS4) was investigated in formalin-fixed human liver biopsy samples taken from 10 patients who were anti-HCV positive. NS4 was detected within the cytoplasm of hepatocytes in all HCV-positive patients studied, but not in the mononuclear cell infiltrates, bile duct epithelium, or endothelial cells. A high proportion of hepatocytes appeared positive, but the staining intensity was variable. After a coded histological evaluation of the liver tissue, the pattern of liver injury was shown to have no significant correlation with antigen-positive hepatocytes, and no direct relationship was observed between the distribution of antigen-positive hepatocytes and areas of hepatocyte necrosis. The staining pattern was considered to be specific because liver samples from patients chronically infected with hepatitis B virus or from uninfected individuals were negative. Furthermore, no staining was noted when either preimmune rabbit serum or anti-NS4 adsorbed against the specific synthetic peptide was substituted for the primary antibody.     

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.     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

Hepatitis C virus nonstructural 3 protein: recombinant NS3 protein of the Thai isolates as an antigen in a diagnostic assay

Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.