Detection of hepatitis C core antigen in serum or plasma as a marker of hepatitis C viraemia in the serological window-phase

A new immunoassay for the detection of hepatitis C core antigen (HCVcoreAg) in peripheral blood during serological window-phase was evaluated among healthy blood donors, commercially available hepatitis C virus (HCV) seroconversion panels and in-house specimens from individuals undergoing seroconversion. Among 1964 low-risk blood donor samples, seven samples were initially reactive but only one was repeat reactive. Reactivity of this specimen was not confirmable by neutralization with specific anti-HCV core antibody, and the sample was negative for HCV RNA by polymerase chain reaction (PCR). The specificity of the HCVcoreAg enzyme-linked immunosorbent assay (ELISA) was 99.95%. In seven commercially available HCV seroconversion panels, HCVcoreAg appeared 23-46 days earlier than anti-HCV antibody by third generation assay. Additional testing with specimens from patients undergoing anti-HCV seroconversion indicated that HCVcoreAg becomes undetectable by the present test format soon after the onset of antibody. This test may be considered as an alternative to nucleic amplification techniques (NAT) for blood donor HCV screening. Additional development of technology for detecting HCVcoreAg may be useful for patient diagnosis and therapy monitoring.     

Clinical performance of the new rRoche COBAS TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum

We evaluated the Roche COBAS TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported linear dynamic range of 3.0x10(1) to 2.0x10(8) HCV RNA IU/ml, and a reported lower limit of detection (LLD) of 10 IU/ml. Calculation of the HCV RNA titre is based upon an external standard curve in the presence of an internal control. Intra-assay and inter-assay variation were small in reference panel members with HCV RNA > or =100 IU/ml. Genotype performance and quantitative correlation between the TaqMan HPS and the bDNA (VERSANT HCV 3.0 assay; Bayer Diagnostics), assessed in 59 patient samples, were good for HCV genotype 1 but poor for genotypes 2, 3 and 4. For genotypes 2, 3 and 4, values obtained from the TaqMan HPS were in general 0.5 log lower than those from the bDNA. Sensitivity was poor in low viral titre samples of genotypes 1, 2, 3 and 4. The LLD (95%) was estimated at 41 HCV RNA IU/ml for genotype 4. The TaqMan HPS underestimates HCV RNA at all levels in plasma and serum from HCV-infected individuals, and the LLD should be reconsidered. This is clinically relevant because underestimation of HCV RNA levels during therapy may lead physicians into making incorrect treatment decisions.     

丙型肝炎病毒总抗原检测用于病程监测的研究

目的探讨丙型肝炎病毒(HCV)总抗原检测方法在丙肝病程监测方面的临床意义。方法对来本院就诊的40位丙肝患者于治疗前、治疗1个月时、治疗3个月时、治疗6个月(停药)时,停药6个月后等不同时期进行采血,收集血清或血浆标本,用抗-HCV检测试剂盒(酶联免疫法)、HCV核酸(RNA)扩增(PCR)荧光定量检测试剂盒、HCV总抗原检测试剂盒(酶联免疫法)进行检测。结果从患者确认感染丙肝到治疗结束抗-HCV检测均呈阳性,而HCV-RNA检测和HCV总抗原检测会随着病程的变化而变化。本次共检测了189例标本(40位患者不同时期标本总例数),其中HCV-RNA阳性51例,该51例阳性标本中,HCV总抗原检测阳性44例,阳性检出率为86.27%;138例HCV-RNA阴性标本,有3例HCV总抗原检测为阳性(2.2%)。2种方法比较,差异无统计学意义(χ2=1.6,P>0.05)。HCV总抗原检测其OD值会随着病程的变化而相应改变,可以较好地反应丙肝患者的病程状况。结论 HCV总抗原检测方法在丙肝病程监测方面具有很好的临床意义,适合在缺少荧光定量PCR检测能力的中小医院使用,可在一定程度上替代HCV-RNA检测,对抗-HCV阳性患者作进一步的验证检测或补充,更好地应用于丙肝患者的病程监测。     

Utility of a commercial quantitative hepatitis C virus core antigen assay in a diagnostic laboratory setting

In this study, the utility and impact of hepatitis C virus (HCV) core antigen (Cag) detection via a commercial assay have been evaluated in diagnostic laboratory conditions. In a total of 272 samples from 226 individuals, HCV RNA was detected in 81.3% and anti-HCV antibody prevalence was 86.4%. HCV Cag reactivity was identified in 59.9% of the samples and in 75.8% with detectable RNA. The sensitivity and specificity of HCV Cag assay have been calculated as 75.8% and 95.1%, respectively, and agreement between HCV RNA and HCV Cag was moderate (κ = 0.554). HCV Cag and RNA levels were highly correlated (r = 0.915 and 0.937). A viral load threshold of 10³ IU/mL has been recognized, above which the correlation with RNA became statistically significant and sensitivity increased to 90.9%. Detection and quantification of HCV core antigen have been observed as a strong alternative to nucleic acid testing for HCV monitorization.     

新型二聚体突变荧光引物定量PCR方法的建立及其在检测HCV中的应用

目的 开发新型二聚体突变荧光引物技术,建立一种能广泛应用于临床检测HCv的实时PCR方法.方法 构建重组质粒pMD18-T-HCV 5′NCR作为标准品,设计二聚体突变荧光引物,优化定量PCR体系,并进行方法学评价.将本法应用于临床确诊的30份HCV阳性患者、30份其他病毒性肝炎患者和30份健康志愿者血清标本的检测,定量结果与商品化TaqMan HCv定量试剂盒的定量结果进行比较.结果 建立了利用二聚体突变荧光引物的实时PCR方法,检测的线性范围为20～109IU/ml;批内CV在1.37%～4.59%之间,批间CV在1.58%～4.81%之间;对所有其他病毒性肝炎患者和健康志愿者血清HCV的检测结果均阴性,检测特异性为100%(60/60);对临床确诊的HCV感染患者血清标本全部检出HCV阳性,定量结果与商品化TaqMan HCV定量试剂盒的定量结果具有很好的相关性,相关系数R2=0.9501.结论 建立的以二聚体突变荧光引物为平台的HCV实时PCR检测方法,具有快速、价廉、准确、结果可靠等特点,可为HCV感染的诊断、治疗监测和流行病学调查提供较好的技术支持.     

Performance of a conventional enzyme immunoassay for hepatitis C virus core antigen in the early phases of hepatitis C infection

There are periods within the early phase of hepatitis C virus (HCV) infection in which the anti-HCV antibody test is unable to confirm HCV viremia. To reduce the risk of transmitting HCV through transfusions, we developed a simple and highly sensitive enzyme immunoassay (EIA) which detects the core antigen of HCV (HCVcAg). This assay employed a conventional colorimetric EIA system, and was based on a two-step sandwich assay, using a 96- well microplate. The reproducibility of the results was very high. When the cutoff values were set to 30 fmol of recombinant HCVcAg/L, as determined by the distribution of healthy subject sera (n=223), 99.6% of healthy subject sera and 100% of hepatitis B patient sera (n=50) were negative for HCVcAg. The clinical performance of this EIA was examined using 14 commercially available seroconversion panels. In every panel, HCVcAg could be detected at points preceding the seroconversion of anti-HCV antibodies. The points at which HCVcAg was detected were the same as those at which it was detected by an AMPLICOR HCV Monitor test. The EIA's window period for detecting the HCVcAg in all panels was on average 26 days shorter than that of the anti-HCV antibody test. In three panels where the first sample is negative for HCV RNA, the window period was shortened 50 days by this EIA for HCVcAg. There was a positive correlation between the concentration of HCVcAg and HCV RNA in anti-HCV antibody negative specimens. This assay was simpler to perform than assays based on gene amplification technology for the detection of HCV RNA, and the window period was shortened to that of the AMPLICOR HCV Monitor test. Thus, the EIA for HCVcAg would be useful in screening seroconverting donors and could reduce the residual risk of secondary HCV infections through transfusions.     

The use of polymerase chain reaction in plasma pools for the concomitant detection of hepatitis C virus and HIV type 1 RNA

BACKGROUND: Detection of viral nucleic acids might increase blood transfusion safety through the detection of recently infected blood donors during the preseroconversion window period. Individual screening is difficult to apply, because of technical and financial constraints. STUDY DESIGN AND METHODS: A polymerase chain reaction (PCR)-based assay including a polyethylene glycol precipitation step was developed for the concomitant detection of hepatitis C virus (HCV) and HIV type 1 (HIV-1) RNA in plasma pools corresponding to 50 blood donations by the use of commercial assays. RESULTS: The assay had a sensitivity of less than 33 copies per mL for HCV RNA and 1000 copies per mL for HIV-1 RNA for each individual sample included in the pool. The eight preseroconversion samples with HCV RNA between 1,250 and 762,000 copies per mL were all detected when 100-microL aliquots from the samples were introduced into 5-mL pools of 50 blood donations. CONCLUSIONS: A PCR- based pooling assay associating a prepurification step with polyethylene glycol allows for the screening of blood donations for HCV and HIV-1 RNA without marked loss of sensitivity from that seen with commercially available assays. This procedure might increase blood safety through systematic screening of blood donations at relatively low cost.     

丙型肝炎病毒载量与核心抗原及丙氨酸转氨酶检测的比较研究

目的研究实时荧光定量PCR定量检测丙型肝炎病毒(HCV)RNA载量与HCV核心抗原和丙氨酸氨基转移酶(ALT)检测的相关性及符合性。方法逆转录-PCR荧光探针法定量检测94例怀疑HCV感染患者的血清HCV RNA,同时用酶联免疫吸附试验(ELISA)检测HCV核心抗原,全自动生化分析仪检测ALT水平。结果 94份样本中HCV RNA阳性率为56.4%(53/94),核心抗原阳性率为53.2%(50/94),经统计学分析,两种方法的阳性率差异无统计学意义(P>0.05),符合率为84.9%(45/53);ALT异常率为62.3%(33/53),并随HCV RNA载量的升高而增加。结论 HCV RNA定量检测及HCV核心抗原检测结合ALT结果分析有助于临床了解HCV在体内的复制水平和肝脏的炎性反应状态,指导临床用药及观察疗效。     

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV

BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. STUDY DESIGN AND METHODS: Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing with a maximum pool size of 40 serum samples. RESULTS: Three donations out of 251,737 were HCV RNA positive and anti-HCV negative. ALT levels of these donations were 271, 32, and 10 U per L. The HCV infection of a fourth HCV RNA-positive donor could not be identified by routine, second-generation HCV EIA (Abbott Diagnostika). In this case, two previous donations were also HCV RNA positive, and three second-generation test systems (Abbott) could not detect anti-HCV, whereas third-generation anti-HCV screening assays detected antibody with different sensitivity. The first HCV RNA-positive donation was identified only by the HCV ELISA 3.0 (Ortho Diagnostic Systems). The results of confirmatory assays like RIBA HCV 3.0 (Ortho) and Matrix (Abbott) indicate a restricted immune response to NS3 only. CONCLUSION: HCV RNA detection by PCR can be carried out routinely in blood donor screening without significant delay of release of the components. The residual risk of transmission can be reduced by identification of early infection, which can lead to an improved safety of blood components. RNA screening can also be advantageous in cases of incomplete or lack of antibody response to HCV.     

HCVRNA定量及HCV核心抗原和丙氨酸氨基转移酶结果分析

目的分析丙型肝炎病毒(HCV)RNA定量检测结果与HCV核心抗原及丙氨酸氨基转移酶(ALT)检测结果的相关性。方法收集涪陵监狱卫生所94例住院和门诊患者血清,采用逆转录聚合酶链反应荧光探针法定量检测标本中的HCV RNA,同时采用酶联免疫吸附试验检测HCV核心抗原,采用全自动生化分析仪检测ALT水平。结果 94例样本中HCV RNA阳性率为56.4%(53/94),HCV核心抗原阳性率为53.2%(50/94),经统计学分析,两种方法的阳性率差异无统计学意义(P>0.05),符合率为84.9%(45/53);ALT异常率为62.3%(33/53),随着HCV RNA含量的升高而增加。结论 HCV RNA定量检测结合HCV核心抗原及ALT检测,可帮助临床了解HCV在体内的复制水平及肝脏的炎性反应状态,以指导临床用药及疗效观察。     

抗-HCV结果分析及灰区范围设置的探讨

目的分析酶联免疫吸附试验(ELISA)筛查丙型肝炎病毒抗体(抗-HCV)结果,探讨实验室ELISA中灰区范围设置的必要性。方法回顾性分析34 942例患者抗-HCV筛查结果,比较抗-HCV与HCV-RNA及临床确诊丙型肝炎患者间的关系;以初筛S/CO值在0.4~2.0范围内的标本为灰区样本,进行不同厂家试剂复检及HCV-RNA检测,探讨设置抗-HCV检测灰区范围的必要性。结果抗-HCV筛查阳性率0.61%;31~50岁阳性率最高;男性高于女性,两者比较差异有统计学意义(P0.05)。S/CO≥10时抗-HCV与HCV-RNA检测结果符合程度高,S/CO≥3.8时抗-HCV与临床确诊丙型肝炎符合程度高。灰区样本的阳性率0.38%,双试剂双孔复检后阳性率0.20%和0.05%。结论抗-HCV筛查阳性率在不同地域、性别及年龄段存在差异;S/CO值越大,HCVRNA阳性率越高,与临床丙型肝炎的确诊符合程度越高,而抗-HCV筛查落在灰区范围的样本应复检并检测RNA,以减少实验室漏检或假阳性结果的产生。