从基因分型看血液透析及肾移植患者丙型肝炎病毒感染状况

采用国产和美国Ｏｒｔｈｏ公司第２代抗丙型肝炎病毒（ＨＣＶ）试剂对１００例维持性血透及肾移植患者进行血清丙型肝炎病毒抗体（抗－ＨＣＶ）对比检测。阳性标本用聚合酶键反应（ＰＣＲ）法检测ＨＣＶＲＮＡ并采用型特异的ＨＣＶ亚基因探针对其非结构蛋白ＮＳＳ区扩增产物进行了杂交基因分型。结果表明，这组病人中抗－ＨＣＶ阳性率为４１％，肾移植术后再透析者达５６．５２％，且与透析时间、输血次数、受血量成正相关；国产抗－ＨＣＶ试剂同美国Ｏｒｔｈｏ公司试剂比较阳性符合率达９１．４３％；抗－ＨＣＶ阳性患者中有３１．４３％（１１／３２）血清ＨＣＶＲＮＡＮＳ５阳性；透析患者中，ＨＣＶ基因型各型均有，以混合型为主，占６３．６４％。     

丙型肝炎病毒核心区抗体分型方法的研究

用丙型肝炎病毒核心区２个型特异性俣成肽作为包被抗原，建立了ＥＬＩＳＡ法，对血清中抗－ＨＣＶ进行分型。在１２１份抗－ＨＣＶ阳性血清中检出ＣＰ９阳性率为８３．４７％，在１０１份ＣＰ９阳性血清中进行核心区抗体分型，其中血清１型１７．８２％，血清２型２５．７４％、１．２混合型７．９２％。     

尿中丙型肝炎病毒抗体的检测

尿中丙型肝炎病毒抗体的检测孟庆松王玫王民玉抗丙型肝炎病毒（ＨＣＶ）抗体是丙型肝炎病毒感染的血清学指征。我们用第二代酶联免疫试剂（ＥＬＩＳＡ）对８２份献血员血清抗－ＨＣＶ阳性的献血员尿标本用同一批试剂盒同时检测抗－ＨＣＶ。８２份血清标本抗－ＨＣＶ全部阳...     

戊型肝炎病人和实验感染戊型肝炎病毒的猕猴血清抗－HEV ORF2和ORF3的动态变化

应用人工合成的戊型肝炎病毒（ＨＥＶ）基因组开放读码框架２（ＯＲＦ２）和３（ＯＲＦ３）两段多肽，分别建立了酶联免疫试验（ＥＩＡ），检测８５份实验感染ＨＥＶ的猕猴和１４３份戊型肝炎（简称戊肝）病人血清，结果两组抗－ＨＥＶＯＲＦ２阳性率分别为７０．８９％和６８．５３％，均显著高于抗－ＨＥＶＯＲＦ３（分别为４０．０３％和５７．３４％），且前者阳转时间较早，持续阳性时间也较长。虽然多数（９７．３％）抗－ＨＥＶＯＲＦ３阳性血清，抗－ＨＥＶＯＲＦ２也为阳性，但有少数血清（２．７％）抗－ＨＥＶＯＲＦ２为阴性。上述结果提示，ＨＥＶ基因组ＯＲＦ２和ＯＲＦ３编码的蛋白含有不同的抗原表位，可引起宿主不同的免疫反应。因此，在制备抗－ＨＥＶＥＩＡ试剂时，应联合应用ＯＲＦ２和ＯＲＦ３多肽，以提高其灵敏度和特异性。     

聚合酶链反应鉴定丙型肝炎病毒母婴传播的状态及其意义

采用多种方法，动态检测了１１例丙型肝炎病毒（ＨＣＶ）感染的孕妇所生的婴儿血抗－ＨＣＶ和ＨＣＶＲＮＡ。发现用合成肽酶联免疫吸附试验（ｓｐＥＬＩＳＡ）检测婴儿抗－ＨＣＶ阳性率（２３．５２％）显著低于第二代重组抗原ＥＬＩＳＡ（２ｎｄＥＬＩＳＡ）（４１．１８％）（Ｐ＜０．０５）；用２ｎｄＥＬＩＳＡ检测，６例婴儿脐血和静脉血抗－ＨＣＶ阳性，５例持续１～５月阴转，１例阳性持续１３个月。经重组免疫印迹试验（ＲＩＢＡ）鉴定，４例阳性，２例可疑阳性。用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测ＨＣＶＲＮＡ，５例阳性，３例于生后１～６个月自然阴转，２例持续阳性分别达９个月和１３个月。提示检测抗－ＨＣＶ判断ＨＣＶ母婴传播的状态受到婴儿抗－ＨＣＶ产生水平低下、母体抗－ＨＣＶ的被动输入和不同检测方法的影响，用ＲＴ－ＰＣＲ检测ＨＣＶＲＮＡ是判断母婴传播更可靠的指标。     

套式PCR检测HBV与HCV垂直传播的比较研究

用套式ＰＣＲ法检测了２７例ＨＢｓＡｇ阳性母亲的新生儿脐带血ＨＢＶ ＤＮＡ，阳性３例（１１．１％），同时检测了７例抗－ＨＣＶ阳性母亲的新生儿脐带血ＨＣＶ ＲＮＡ及抗－ＨＣＶ，结果ＨＣＶ ＲＮＡ无１例阳性，抗－ＨＣＶ全部阳性，因此认为婴儿体内抗－ＨＣＶ是一种由母体被动获得的抗体，不能作为ＨＣＶ感染的客观指标。     

戊型肝炎病人和实验感染戊型肝炎病毒的猕猴血清抗——H…

应用人工合成的戊型肝炎病毒（ＨＥＶ）基因组开放读码框架２（ＯＲＦ２）和３（ＯＲＦ３）两段多肽，分别建立了酶联免疫试验（ＥＩＡ），检测８５份实验感染ＨＥＶ的猕猴和１４３份戊型肝炎（简称戊肝）病人血清，结果两组抗－ＨＥＶ ＯＲＦ２阳性率分别为７０．８９％和６８．５３％，均显著高于抗－ＨＥＶ ＯＲＦ３，且前者阳转时间较早，持续阳性时间也较长。虽然多数（９７．３％）抗－ＨＥＶ ＯＲＦ３阳性血清，抗－ＨＥＶ     

肝病患者血清抗HCV及HCVRNA的检测与意义

利用第２代抗ＨＣＶＥＬＩＳＡ试剂和ＰＣＲ技术对１８６例肝病患者进行了抗ＨＣＶ和ＨＣＶＲＮＡ检测。６３例患者（３３．８７％）抗ＨＣＶ和／或ＨＣＶＲＮＡ阳性，表明本组肝病患者存在较严重的ＨＣＶ感染，ＨＣＶ标志物（ＨＣＶｍ）阳性肝病患者较ＨＣＶｍ阴性肝病患者有更高的输血，手术，接种和拔牙等血液暴露史（Ｐ〈０．０５或Ｐ〈０．０１）。６３例ＨＣＶ感染者中，３４例抗ＨＣＶ和ＨＣＶＲＮＡ同时阳性，另有患者表现为     

国产丙型肝炎病毒NS3抗原的质量检定

目的为了研究我国丙型肝炎病毒（ＨＣＶ）诊断试剂用抗原的质量，加快改进丙型肝炎病毒抗体诊断试剂。方法特选国内“八五”攻关课题研究出的丙型肝炎ＮＳ３抗原和目前我国的主要使用的两种国外丙型肝炎ＮＳ３抗原，建立了单片段检测ＮＳ３抗体的试剂，对ＮＳ３抗原质量利用我国抗－ＨＣＶ第二代国家检验参考品以及中国药品生物制品检定所最近收集的部分血清样品，对其抗原性进行了比较研究。结果发现我国生产单位对ＮＳ３抗原的纯化条件及在试剂中的组装条件的研究尚嫌薄弱，使得在利用这些ＮＳ３抗原组成诊断试剂检测时出现了检出率较低及假阳性等问题。结论提示国内有关单位应加强对这方面的研究。     

利用双扩增聚合酶链反应检测非甲非乙型肝炎患者血清中丙型肝炎病毒RNA

利用丙型肝炎病毒（ＨＣＶ）５’－端序列合成两对引物，建立了灵敏、特异的ＨＣＶＲＮＡ双扩增聚合酶链反应检测方法。用此方法及第二代Ａｂｂｏｔｔ酶联抗－ＨＣＶ检测试剂盒，检测了４４例非甲非乙型肝炎患者血清及１０名抗－ＨＣＶ阴性健康人。在４４例患者中，４１例（９３％）ＨＣＶＲＮＡ阳性，３６例（８２％）抗－ＨＣＶ阳性，３３例（７５％）ＨＣＶＲＮＡ、抗－ＨＣＶ全部阳性。３例ＨＣＶＲＮＡ阴性，但抗－ＨＣＶ阳性，另外，有８例抗－ＨＣＶ阴性，ＨＣＶＲＮＡ阳性。１０名健康人ＨＣＶＲＮＡ均为阴性。结果表明，大部分（９２％）抗－ＨＣＶ阳性患者带有ＨＣＶ，但为了检测所有病毒血症患者，抗－ＨＣＶ检测是不够的，利用双扩增ＰＣＲ方法检测ＨＣＶＲＮＡ对于抗－ＨＣＶ阴性患者的诊断是非常有用的。     

Comparison of HCV core antigen and anti-HCV with HCV RNA results

Background

The measurement of anti-HCV antibodies using immunological methods and the confirmation of viral nuclear acid based on molecular methods is important in diagnosis and follow-up of the HCV infection.

Objectives

In this study, we aimed to analyse HCV core Antigen positivity among anti-HCV antibody positive sera to determine the significance of testing of HCV core Ag for the laboratory diagnosis of HCV infection, by considering the correlation between serum HCV core Ag and HCV RNA levels.

Methods

115 patients suspected of having hepatitis C and who were positive for anti-HCV antibody were investigated using chemiluminescent and molecular methods. Anti-HCV antibody, HCV core Ag and HCV RNA levels were detected by the Vitros ECiQ immunodiagnostic system, Architect i2000 system and RT-PCR, respectively.

Results

The sensitivity, specificity, positive and negative predictive values and accuracy rate of HCV core Antigen assay were detected as 86.5%(83/96), 100%(19/19), 100%(83/83), 59.4%(19/32), 88.7%(102/115) respectively.

Conclusion

HCV core Ag assay could be used for diagnosis of HCV infection as it is easy to perform, cost-effective, has high specificity and positive predictive value. However, it should be kept in mind that it may have lack of sensitivity and negative predictive value.     

A European multicentre study on the comparison of HCV viral loads between VERIS HCV assay and COBAS® TaqMan® HCV Test and RealTime HCV Assay

BackgroundBeckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic System^¥ for HCV viral load monitoring.ObjectivesEvaluate the clinical performance of the new quantitative VERIS HCV Assay.Study designComparison was performed on 279 plasma specimens from HCV infected patients tested with the VERIS HCV Assay and COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test and 369 specimens tested with the VERIS HCV Assay and RealTime HCV Assay. Patient monitoring sample results from four time points were also compared.ResultsThe average bias between the VERIS HCV Assay and the COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test was 0.04 log₁₀ IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log₁₀ IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000 IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test between −0.42 and −0.22 log₁₀ IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log₁₀ IU/mL. Patient monitoring of HCV viral load over time demonstrated similar levels between VERIS HCV Assay results and COBAS^® Ampliprep/COBAS^® Taqman^® HCV Test (52 samples from 13 patients) and RealTime HCV Assay (112 samples from 28 patients).ConclusionsVERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring.     

Serotyping and genotyping of hepatitis C virus (HCV) strains in chronic HCV infection

Hepatitis C virus (HCV) genotypes can be established by methods based on PCR typing and serological typing. The accuracy of these methods depends on their sensitivity and specificity. These should be compared with the reference method, direct sequencing, and analysis of viral genomes. Among the serologic methods recently developed, the performance of a new serotyping assay (RIBA HCV 3.0 SIA, Chiron corporation, Emeryville) was assessed using a panel of 147 well-characterized French isolates from chronic hepatitis C patients. Definitive genotypes of the isolates were established by direct sequencing in 5′ NC and in some cases in NS-5B. HCV serotypes 1, 2, and 3 were determined by measuring type specific antibodies to core and NS-4 derived peptide antigens. Of the 147 sera, serotypic-specific antibodies were detected in 136 (sensitivity, 92.5%). The specificity of the RIBA SIA HCV serotyping assay was 92.6% (including samples with mixed results); without these, the specificity was 80.1%. Analysis of the 28 discrepant samples showed that (1) a different serotype was found in 18 samples including five for genotype 1, three for genotype 2, two for genotype 3, five for genotype 4, and three for genotype 5, and that (2) ten patients showed a reactivity with mixed serotypes, one had circulating antibodies to type 1 or 2, and nine had circulating antibodies to type 1 or 3. In summary, except for genotypes 4 and 5, the results of the test were well correlated (85.7%) with those of direct sequence genotyping. The former test is rapid and does not require the strict HCV RNA storage and preservation conditions of the latter. This new method may thus be considered as an alternative for HCV typing. However, although it is convenient, its lower sensitivity compared to the molecular typing method and the discrepant results limit its routine use in a clinical context. J. Med. Virol. 52:391–395, 1997. © 1997 Wiley-Liss, Inc.     

Patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus recover genotype cross-reactive neutralising antibodies to HCV during antiretroviral therapy

When severely immunodeficient HIV/HCV co-infected patients are treated with antiretroviral therapy, it is important to know whether HCV-specific antibody responses recover and whether antibody profiles predict the occurrence of HCV-associated immune restoration disease (IRD). In 50 HIV/HCV co-infected patients, we found that antibody reactivity and titres of neutralising antibodies (nAb) to JFH-1 (HCV genotype 2a virus) increased over 48 weeks of therapy. Development of HCV IRD was associated with elevated reactivity to JFH-1 before and during the first 12 weeks of therapy. Individual analyses of HCV IRD and non-HCV IRD patients revealed a lack of an association between nAb responses and HCV viral loads. These results showed that increased HCV-specific antibody levels during therapy were associated with CD4⁺ T-cell recovery. Whilst genotype cross-reactive antibody responses may identify co-infected patients at risk of developing HCV IRD, neutralising antibodies to JFH-1 were not involved in suppression of HCV replication during therapy.     

鼠抗丙型肝炎病毒ns－5区单克隆抗体的研究

用ＨＣＶ非结构区ｎｓ－５合成多肽抗原交联后免疫小鼠，成功地建立了３株抗ｎｓ－５单克隆抗体（ＭｃＡｂ），经检测这３株ＭｃＡｂ属于同一位点，与其它区域无明显交叉反应，具有高度特异性。ＭｃＡｂ的研制为检测ｎｓ－５抗原奠定了一定基础。     

Comparative evaluation of the Geenius HCV supplemental assay and Inno-LIA HCV score assay in detecting anti-HCV antibodies

ObjectiveThis study compared two assays aimed at confirming the presence of anti-HCV antibodies (Ab) after a positive screening: Geenius HCV supplemental assay (Bio-Rad, Marne la Coquette, France) and the Inno-LIA HCV score assay (Fujirebio, Les Ulis, France).Material and methodsA total of 180 archived samples were investigated including 119 samples collected at different stages of HCV infection in 25 hemodialyzed patients who underwent HCV seroconversion, 14 samples from 4 commercial seroconversion panels, 47 Ab positive/HCV-RNA positive blood donations of which 7 showing an single reactivity in confirmatory assays. Samples were investigated and results were interpreted with the two assays according to the manufacturers’ instructions.ResultsOverall, Geenius and Inno-LIA were concordant for 84% (151/180) samples: 38 negative, 17 indeterminate and 96 positive. Of the 29 discrepant results, 26 were overclassified with Inno-LIA. HCV seroconversion was detected with Inno-LIA 4 and 7 days prior to Geenius in two panels. The high positive rate observed with Inno-LIA (64%) compared to Geenius (54%) was mainly due to low reactivities considered positive according to interpretation criteria, which could affect specificity.ConclusionAlthough HCV supplemental assays are not recommended for the diagnostic of HCV infection, which is primarily based on HCV-RNA testing, both assays are suitable as second line anti-HCV tests when Ab screening is positive and RNA testing cannot be performed. Moreover, Geenius system provides an objective result in less than 30 minutes, which is compatible when a rapid diagnostic is required.     

首次及重复感染HCV后抗体及HCV RNA的动态观察

本文报道对首次感染ＨＣＶ的１４人与重复输入含ＨＣＶ血的１１位感染者进行长达一年半的动态观察，总结出输入大量含ＨＣＶ血后导致的感染者在自然状态下ＨＣＶ ＲＮＡ及抗体变化的规律。７／８再次输入４００ｍｌ以上含ＨＣＶ血的感染者，其自身存在的高滴度抗体在受血后１～３个月下降０．５～１．０ＯＤ值。首次输出含ＨＣＶ血的感染者中，１１／１４的感染者３个月内ＡＬＴ高于参考值的３倍以上，而再次输入含ＨＣＶ血的患者中     

HCV in peripheral blood mononuclear cells are predominantly carried on the surface of cells in HIV/HCV co-infected individuals

HCV replication in extra-hepatic reservoirs has been suggested to occur in many tissues including PBMCs. A recent study showed evidence for compartmentalization and evolution of HCV in PBMCs. However, the cells that support HCV replication in PBMCs have not been identified. In this study we have fractionated the PBMC from HIV/HCV co-infected patients into T, monocytes, B and NK cells, and most of the HCV was located in CD3-cell fractions. Protease treatment of PBMCs to remove cell surface receptors resulted in the loss of HCV RNA suggesting that most of the HCV is present on the cell surface. PBMCs were treated by freeze-thaw nuclease method that would protect the HCV RNA in the virus but not the intracellular viral RNA. Data from this analysis support the conclusion that most of HCV is present on the cell surface. Even though the presence of minus strand RNA in PBMCs suggests that a low level HCV replication takes place within the PBMCs of HIV/HCV co-infected individuals, HCV in PBMC is present mainly on the surface of non-T cells, mostly on NK, monocytes and B cells. These results suggest a unique pathogenic role of NK, monocyte and B cells as carriers of HCV.     

Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA

BackgroundThe diagnosis of HCV relies on the detection of viral RNA.ObjectiveTo evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS^® Ampliprep/COBAS^® Taqman™ (CAPCTM) HCV Test version 2.0.Study designThe limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6 log IU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples.ResultsThe limit of detection was 6.1 IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11 log IU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4 log IU/ml. Passing-Bablok regression analysis gave: VERIS log IU/ml = −0.33 + [1.04× CAPCTM] log IU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06 log IU/ml. The two assays were well correlated (ρ = 0.92, p < 0.001) and Bland-Altman analysis gave biases of 0.12, log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4.ConclusionThe VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples.