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。     

核酸检测技术在血液筛查中的应用研究

目的大样本、多方法调查深圳地区无偿献血人群中乙肝、丙肝和艾滋病病毒血清学阴性者的核酸阳性率,探讨在我国血液筛查中引进核酸扩增技术的必要性,了解和分析献血者血清学阴性核酸阳性感染状况。方法采用大样本数调查,应用ROCHE PCR-ELISA、PCR-微流芯片、实时荧光PCR方法和CHIRON TMA（转录依赖的扩增技术）多种方法对血清学检测阴性的献血者进行HBV DNA、HCV RNA和HIV-1RNA检测,对乙肝阳性献血者追踪检测ALT和乙肝两对半标志物,对丙肝核酸阳性献血者追踪检测ALT及抗-HCV及HBV DNA和HCV RNA病毒载量。结果共对141288人份血样进行了检测,检出HBsAg（-）、HBV DNA阳性28例,总阳性率为0.020%,其中21例为anti-HBc阳性,占0.015%。HIV-1RNA未检出阳性,17例HBsAg（-）、HBV DNA阳性样本追踪发现,9例发生了血清转换现象,4例呈窗口期特征,所有追踪的HBV DNA阳性献血者ALT检测结果正常。1例anti-HCV（-）、HCV RNA阳性献血者追踪发现为典型窗口期献血,ALT显著升高。结论应采用高灵敏度的核酸扩增技术筛查血液中的乙肝和丙肝病毒,可提高血液安全。     

海洛因静脉吸毒者HBV和HCV感染状况的调查

乙型肝炎病毒(HBV)与丙型肝炎病毒(HCV)在不同人群中的感染率不尽相同,国外报告证实静脉吸毒者是HBV和HCV的高危人群,国内个别地区也有报导,本文对94例海洛因静脉吸毒者进行了调查,现将结果报告如下:1 材料与方法1.1 对象 吸毒组:男性69例,女性25例,均为广州某戒毒所收客的吸毒者,年龄18～45岁,吸毒时间1个月至3年,均无肝炎病史,无乙肝疫苗免疫注射史,吸毒方式为静脉注射.对照组:男性62例,女性38例,均为广州医学院教职工,年龄在20～55岁.1.2 血清学检测方法 用ELISA法检测血清中的HBsAg,HBsAb,HBeAg,HBeAb,HBcAb及抗HCV,试剂购自上海科华实业生物技术有限公司.双份测定阳性的判为阳性,出现任何1项HBV感染指标即为HBV感染.     

某艾滋病治疗示范区人免疫缺陷病毒感染者合并隐匿性乙型肝炎病毒感染的调查分析

目的 调查分析某艾滋病治疗示范区人免疫缺陷病毒（HIV）-1感染者中隐匿性乙型肝炎病毒（HBV）感染的情况及其影响因素.方法 采集某艾滋病治疗示范区97例经血感染HIV-1的感染者的血浆,采用酶联免疫吸附试验（ELISA）检测乙型肝炎表面抗原与抗体（HBsAg与抗HBs）、乙型肝炎e抗原与抗体（HBeAg与抗Hbe）、乙型肝炎核心抗体（抗HBc）及丙型肝炎抗体（抗HCV）;采用吸附柱法抽提HBV DNA;采用巢式聚合酶链反应（PCR）法检测HBV S区;采用流式细胞仪计数CD4＋T淋巴细胞.HBsAg阴性PCR阳性结果 者为合并隐匿性HBV感染者.合并隐匿性HBV感染者为实验组,未合并隐匿性HBV感染者为对照组.结果 97例HIV感染者中HBsAg阴性者92例（94.85%）.92例HBsAg阴性者中合并隐匿性HBV感染者27例（29.35%）,抗HCV阳性者73例（79.35%）.合并隐匿性HBV感染者和未合并HBV感染者CD4＋T淋巴细胞数、单独抗HBc阳性率分别为（212.11±133.1）和（318.9±172.2）cells/mm3、62.96%和18.46%,以上两指标两组比较差异均有统计学意义（P＜0.01）,两组间年龄、性别、是否合并HCV感染及抗HBs阳性率比较差异无统计学意义（P＞0.05）.结论 经有偿献血途径感染HIV者中存在隐匿性HBV感染;HIV阳性合并隐匿性HBV感染者中易出现单纯抗HBc阳性;CD4＋T淋巴细胞数低的HIV感染者更容易合并隐匿性HBV感染.     

献血人群隐匿性乙型肝炎病毒感染的流行病学和血清学研究

目的了解广州地区献血人群隐匿性乙型肝炎病毒感染（OBI）的流行病学和血清学情况。方法对广州地区199631例无偿献血者标本同时用ELISA法检测HBsAg、紫外-乳酸脱氢酶法检测ALT、核酸扩增技术（NAT）联合检测HBV/HCV/HIV及HBV单项鉴别试验,对HBsAg阴性HBV DNA阳性者进行随访,用荧光定量PCR检测病毒载量,用ELISA法检测乙肝两对半。结果 199631例标本中共检出104例HBsAg阴性HBV DNA阳性者,经随访有54例为OBI,OBI检出率为0.027%,年龄以46～55岁组检出率最高（P〈0.01）,外地身份证的献血者检出率高于广州市身份证者（P〈0.01）,OBI检出率与性别和献血次数无关（P〉0.05）。104例HBsAg阴性HBV DNA阳性的标本ALT均正常,病毒载量均〈1000IU/ml,平均值为162IU/ml。随访标本中,除6例ALT异常外其余均正常,54例OBI标本病毒载量均〈1000IU/ml,平均值为122IU/ml,乙肝两对半中抗-HBc阳性率明显高于其他项目（P〈0.01）。结论 HBsAg阴性献血者中存在OBI,有必要在献血者中开展核酸检测。     

慢性乙型肝炎患者HBV cccDNA定量方法的建立及应用

目的 建立慢性乙型肝炎患者HBV cccDNA荧光定量检测方法 ,并检测慢性乙型肝炎患者血清中HBV cccDNA含量.方法 根据HBV DNA结构特点,于HBV cicada缺口两侧高度保守区域设计巢式PCR引物和探针,并优化PCR反应条件;选取175例慢性乙型肝炎患者,提取抗病毒治疗前后血清DNA,以不降解质粒的ATP依赖的DNA酶(PSAD)进行酶切,进行rcDNA定量检测;分析影响血清HBV cccDNA检出率的相关因素,探讨血清HBV cccDNA载量与慢性乙型肝炎患者临床特征的关系.结果 慢性乙型肝炎患者血清中可检测到HBV cccDNA.血清HBV cccDNA检出率与血清HBV DNA载量相关.HBeAg阳性慢性乙肝患者血清HBV cccDNA载量高于HBeAg阴性慢性乙肝患者.结论 本方法 具有较好的敏感性,可用于HBV cccDNA的检测.     

肝细胞癌病理学研究的新进展

1 原位PCR检测石蜡包埋肝组织中丙型肝炎病毒 HCV是单股正链RNA病毒,Negro等用原位杂交研究发现HCV共存于人肝组织细胞核及细胞质中,但主要位于胞质中。原位PCR检测肝组织HCV感染的敏感性比原位杂交高40％-50％,血清HCV阳性患者的肝组织检出率高达86％”。尤其对HBsAg阴性的癌旁组织进行检测可使50％原因不明的肝癌得到诊断。存档多年的蜡块也可进行检测。     

HBV、HCV合并感染患者血清抗丙肝抗体分片段与HCVRNA测定的意义

目的 探讨乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）合并感染患者与单HCV感染者血清抗HCV抗体分片段阳性率以及HCV RNA阳性率是否有显著性差异以及检测意义。方法 对76例同时感染HBV和HCV患者进行抗HCV抗体分片段以及HCV RNA检测,同时从同期仅感染HCV患者中随机抽取163例作为对照组进行以上检测。结果 HBV、HCV合并感染患者与单HCV感染者比较：抗HCV抗体分片段相同片段间比较差异无统计学意义,但其S/CO值比较差异有统计学意义（P〈0.01）;相同片段组合间除抗-C＋抗-NS3组合阳性率HBV、HCV合并感染患者比仅HCV感染患者显著要低（P〈0.05）外,其余各组合间比较差异无统计学意义（P〉0.05）;HBV、HCV合并感染患者HCV RNA阳性率比仅HCV感染患者低（P〈0.01或P〈0.05）。结论 HBV、HCV合并感染时HBV对HCV复制存在抑制作用,从而导致HCV RNA含量显著降低,合并感染与单独感染间抗HCV抗体分片段无显著性差异,但由于HCV复制受到抑制从而导致抗HCV抗体分片段含量降低,这对研究HBV、HCV合并感染时患者体内HBV对HCV间的抑制作用有一定意义。     

荧光定量PCR法检测乙型肝炎病毒DNA

目的：了解不同乙型肝炎病毒(HBV)感染患者血清HBV-DNA含量与乙肝血清免疫标志物的关系并探讨其临床意义。方法：用荧光定量聚合酶链反应(FQ-PCR)方法定量检测375例不同临床类型HBV感染者及70例正常人血清HBV-DNA含量。结果：血清HBsAg阳性组与HBeAg阳性组HBV-DNA的检出率及含量均明显高于HBsAg阴性组和HBeAg阴性组，且HBeAg即使阴性，不论HBeAb阳性组或阴性组均仍有较高的HBV-DNA检出率。不同临床类型HBV感染者中均有HBV-DNA的检出。结论：定量PCR可真实反映HBV感染、复制及病程变化，对乙型肝炎临床诊断及治疗均有一定的临床意义。     

1995至2000年北京地区散发性急性肝炎患者肝炎病毒抗体的检测与分析

目的 了解北京市地区散发性肝炎患者甲型、乙型、丙型和戊型肝炎病毒感染型别分布及重叠感染。方法 用EIA法检测1995斫2月至2000年12月北京地区散发性急性肝炎患者抗－HAVIgM、HBsAg／抗－HBcIgM、抗－HCVIgM/IgG和抗－HEVIgM/IgG。结果 214例散发性急性肝炎患者血清,抗甲、乙、内和戊型肝炎病毒IgM总阳性数155例,戊型肝炎76例。有9名患者检出2种肝炎病毒抗原或抗体阳性,其中在3名肝硬化患者和2名静脉吸毒者同时检测到HBV和HCV抗原或抗体,1例HBsAg阳性者检测到抗－HAVIgM,3例－HCVIgG阳性者中分别检测到2例抗－HBVIgM和1例抗－HEVIgM。肝炎病毒重叠感染的9名患者年龄在31岁至49岁之间。结论 北京地区散发性急性肝炎78％是由消化道传染的甲戊型肝炎病毒引起,戊型肝炎在四种肝炎中位居首位,其次为甲型肝炎、乙型肝炎和丙型肝炎。肝炎病毒重叠感染多见乙、丙型肝炎病毒合并感染或慢性乙、丙型肝炎患者合并甲型或戊型肝炎病毒感染。在我国对散发性病毒性肝炎的预防应引起高度重视。     

The role of core antigen detection in management of hepatitis C: a critical review.

Several assays in research format and two commercial assays for the detection of hepatitis C virus (HCV) core protein or HCV core antigen have been developed in recent years. In order to elucidate the role and significance of HCV core antigen detection in the diagnosis and management of hepatitis C, we reviewed 56 studies published in peer-reviewed journals until September 2004. Evaluations in transfusion settings showed that the HCV core antigen assay detects HCV infection, similarly as nucleic acid techniques (NAT), between 40 and 50 days earlier than the current third generation HCV antibody screening assays. HCV core antigen levels closely track HCV RNA dynamics, and allow clinical monitoring of a patient's therapy, independently of HCV genotype, however, mainly in the samples with HCV RNA levels above 20,000 IU/ml. Considering the lower sensitivity of HCV core antigen detection in comparison to NAT, the HCV core antigen assay is not practical for the determination of the end of treatment response and sustained viral response, but could be useful for the determination of early viral response in the pegylated interferon-alpha and ribavirin treated patients infected with HCV genotype 1. The HCV core antigen detection is a viable tool for study of hepatitis C pathogenesis. The HCV core antigen can be used as a marker of HCV replication in anti-HCV positive individuals in the areas of the world that cannot afford NAT and/or in the settings that are not equipped or competent to perform HCV RNA testing. Because the manufacturer of HCV core antigen assays recently stopped an active marketing of these assays in several countries, it will, unfortunately and probably, never be possible to determine the actual potential and usefulness of HCV core antigen testing in the management of hepatitis C.     

Utility of HCV core antigen ELISA in the screening for hepatitis C virus infection in patients on hemodialysis

An enzyme immuno assay for hepatitis C core antigen was recently developed and its performance was compared with that of the hepatitis C virus (HCV) RNA in the screening of HCV infection in patients on hemodialysis. One hundred and eleven chronic renal failure patients undergoing haemodialysis between May 2003 and October 2004 were included in the study. All the patients were tested for anti HCV antibody, core antigen and RNA. Fifteen patients were anti HCV antibody positive, three patients were positive for HCV core antigen and RNA, three patients were positive for HCV RNA, while two patients were positive only for core antigen but negative for RNA. In anti HCV antibody positive patients, the core antigen was negative while the viral RNA continued to be present. Hence, relying solely on a single HCV core antigen assay may not be useful for a definite diagnosis of early HCV infection. The sensitivity and specificity of the assay were 60% and 83% respectively, while the positive predictive value was 14.3%, negative predictive value was 97.7% and the efficiency was 81.9%.     

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users

BackgroundHepatitis C virus (HCV) core antigen is a serological marker of current HCV infection.ObjectivesThe aim of this study was mainly to evaluate the performance characteristics of the ARCITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users.Study designA total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test.ResultsHCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9–94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959.ConclusionsHCV core antigen testing may be reliably used to identify current HCV infection.     

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.     

Performance of an automated system for quantitation of hepatitis C virus core antigen

The performance of an automated system for the HCV core antigen assay using the Lumispot LS-2000 automated analyzer was evaluated against that of the COBAS AMPLICOR HCV MONITOR Test, version 2.0 (COBAS HCM-2) for the testing of sera from 155 chronic hepatitis C patients. The within-run coefficient of variations (CVs) and the between-day CVs were <9.6 and <8.4%, respectively. The analytical detection limit of the HCV core antigen assay was 5.0 fmol/l and it was linear up to at least 45000 fmol/l. No blood elements interfered with the assay. HCV core antigen levels were significantly correlated with HCV RNA levels in both serogroup 1 and serogroup 2 (r=0.829, P<0.001). It is estimated that 100 fmol/l of HCV core antigen level was equivalent to approximately 30000 IU/ml of HCV RNA level. Three sera had HCV core antigen levels below the detection limit of the assay and their HCV RNA levels as determined by COBAS HCM-2 assay were very low at 1000, 1100 and 1700 IU/ml, respectively. In six IFN responders among seven patients, HCV core antigen levels were in parallel with HCV RNA levels. In conclusion, since this assay demonstrated good reproducibility, a favorable dynamic range and adequate sensitivity, it may be useful as an alternative direct marker of viral level monitoring in patients with hepatitis C.     

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。     

Tattooing as a risk of hepatitis C virus infection.

The association of hepatitis C virus (HCV) infection and tattooing was studied in 87 tattooed and 126 tattoo free healthy young men who did not engage in intravenous drug use or multiple sexual activity. Antibody against HCV (anti-HCV) was tested in serum specimens by enzyme immunoassay with C100-3, NS3, and core antigens; 11 of the 87 (12.6%) tattooed and 3 of the 126 (2.4%) tattoo free subjects were positive for anti-HCV (odds ratio = 5.9, 95% CI = 1.6-22.0). A relationship was demonstrated by an increased risk for HCV infection with an increasing number of tattooed site (P(trend) = 0.002). All but one of the 87 tattooed subjects had been infected by hepatitis B virus (HBV) and 25 were carriers of hepatitis B surface antigen (HBsAg). None of the 25 HBsAg carriers was positive for anti-HCV whereas 11 of the 62 HBsAg non-carriers had anti-HCV, suggesting a negative association between the HBsAg carriage and the long lasting anti-HCV (P = 0.02, Fisher's exact). The status of the tattooer was also an important determinant for HCV infection; the risk was higher if tattooing was done by a non-professional friend than by a professional tattooist. Tattooing, probably with improperly sterilized needles, can clearly pose an increased risk for HCV infection in Taiwan. This study indicates the need for legal standards for hygienic tattooing as part of preventive measures for the control of parenterally transmitted infections.     

Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection

To evaluate whether a new enzyme immunoassay developed for the simultaneous detection of hepatitis C virus (HCV) core antigen (Ag) and anti-HCV antibodies (anti-HCV Ab) (Monolisa HCV Ag/Ab ULTRA; Bio-Rad) could improve the early detection of HCV infection, we compared its sensitivity to that of anti-HCV, HCV core Ag, and HCV RNA assays. The populations studied included 12 blood donor samples positive for HCV RNA and HCV core Ag but negative for anti-HCV antibodies and 23 hemodialysis patients who developed anti-HCV Ab (seroconversion) during the follow-up. From these 23 individuals, 83 samples sequentially collected prior to seroconversion and 108 samples collected after seroconversion were tested. Six of 12 blood donations were positive by the HCV Ag/Ab assay. In the hemodialysis cohort, the 24 HCV RNA-negative samples were negative by the HCV Ag/Ab assay and 23 of the 59 HCV RNA-positive samples (39%) were positive. The HCV Ag/Ab assay detected HCV infection on average 21.6 days before the most sensitive antibody assay. The HCV Ag/Ab assay did not detect HCV infection as early as the HCV RNA assay (mean delay, 30.3 days) or HCV Ag assay (mean delays, 27.9, and 16.3 days by the HCV core Ag quantification assay and the HCV Ag blood screening assay, respectively). This new assay provides a notable improvement for the early detection of HCV infection during the so-called window period compared with anti-HCV Ab assays and could be a useful alternative to HCV RNA detection or HCV core Ag assays for diagnosis or blood screening when nucleic acid technologies or HCV core Ag detection are not implemented.