病毒核酸检测在献血者血液筛查中的应用

目的　建立献血者血液混合核酸检测方法 ,调查北京现有检测体系下血液的残余风险度 ,评估核酸检测 (NAT)的必要性和可行性。方法　用世界卫生组织标准品对国产丙型肝炎病毒(HCV)和人免疫缺陷病毒 (HIV)荧光 聚合酶链反应核酸扩增检测试剂进行灵敏度、重复性和精密度试验 ;对 2 0 0 2年 2～ 10月 34373份常规血清学检测 (ALT、HBsAg、抗 HCV、抗 HIV、梅毒抗体 )合格的献血者血样进行HCVRNA和HIV 1RNA核酸扩增分析。采取 2 4人份混合血样测定 ,超离心浓缩病毒 ,Roche核酸提取柱提取病毒核酸。结果　扩增系统能 10 0 %检出 5 0IU/mlHCV及 5 0IU/mlHIV 1标准品核酸 (n =16 ) ;常规血清学检测合格的献血者血液中 ,没有检出HCV或HIVNAT阳性。结论　该核酸检测体系适用于献血者血液病毒筛查 ;北京市血液的病毒安全性已有相当高的保障。为更准确地评估NAT检测项目的可行性和必要性 ,检测标本量尚待增加。     

应用加样仪进行核酸扩增技术(NAT)的样本汇集

目的　探讨在STAR2 0 0 0加样仪上实现核酸扩增技术样本汇集、样本留档和条码的管理等准备工作。方法　应用STAR2 0 0 0控制语言C+ + 及组件 ,编制控制程序控制加样仪进行两级汇集 ,留档及条码打印 ,同时进行全自动核酸提取及扩增和分析。结果　用国际标准核酸质控品考评及实际应用 ,表明应用STAR2 0 0 0进行全自动汇集 ,全自动核酸提取及扩增和检测可检出HBVDNA、HCVRNA和HIV1RNA分别为 2 0、10IU/ml和 2 0Copy/ml,通过对 6 0 2 4例合格样本共 2 5 1个汇集池分析 ,检出 6例HBVDNA阳性样本 ,阳性检出率为 0 .0 99%。HCVRNA和HIV1RNA未检出阳性。结论　STAR 2 0 0 0加样仪可用于血液核酸筛查的样本汇集及条码管理工作。     

丙型肝炎病毒核酸检测的国家标准物质的研制

目的研制国内用于丙型肝炎病毒核酸(HCVRNA)扩增检测的血清标准物质。方法用HCVRNA阴性血浆将阳性血浆稀释至含量约300000拷贝数/ml,然后进行真空冷冻干燥。检测方法采用实时荧光定量聚合酶链反应方法和罗氏公司的PCR内标定量方法。制备标准品含量通过与国际标准(世界卫生组织属下的国家生物标准研究所标准物质编号:96/790)比对获得。结果该标准物质定值为:(1·29±0·24)×105IU/ml。其稳定性实验表明室温20~25℃(相对湿度20%~50%)14d、37℃(相对湿度60%~80%)7d均稳定。长期监测结果表明:2~8℃6个月及-20℃保存两年的含量均无明显变化。均一性检测结果:瓶间不精密度为3·53%。结论该制备物可以作为用于核酸扩增检测的丙型肝炎病毒核酸国家标准物质。     

某国产NAT试剂盒应用于血液筛查的评价与应用

目的 评估、验证某国产血液NAT试剂盒用于血液筛查的技术性能指标. 方法 对某国产血液NAT试剂盒进行了灵敏度、特异性、重复性、抗干扰能力和抗污染能力测试,并将其初步用于血液筛查. 结果 乙型肝炎病毒(hepatitis B virus,HBV)DNA、丙型肝炎病毒(hepatitis C virus,HCV)RNA、人类免疫缺陷病毒(human immunodeficiency virus,HIV) RNA三者检测灵敏度分别为不高于50IU/mL、50IU/mL与100IU/mL;特异性、重复性、抗污染能力能满足血站血液核酸检测需求;混检模式下,HBV DNA、HCVRNA、HIV RNA三者的检出基本不受脂肪血颗粒的影响,但在单检模式下会导致HIV RNA的漏检,溶血对HBV DNA HCVRNA、HIV RNA的检出均有明显干扰. 结论 某国产NAT 检测分析系统的灵敏度、特异性、重复性、抗污染能力等指标均达《血站核酸检测质量控制指南》要求,脂肪血、溶血样本能明显干扰HBV DNA、HCVRNA、HIV RNA的检出能力.     

核酸血液筛查试剂内标质控效果研究

目的竞争性内标对核酸检测灵敏度的影响程度及方式,探寻采用适度的内标质控浓度,用于有效控制核酸检测过程。方法使用竞争性混合内标试剂对不同浓度的HBV、HCV、HIV阳性标本检测,HBV内标浓度<103IU/ml,HCVt HIV内标浓度为(200—300)IU/ml。结果HBV内标浓度<5IU/ml检出率<95%,浓度>10IU/ml后检出率≥95%,HCV、HIV内标浓度<100IU/ml检出率<95%,≥100IU/ml检出率≥95%。结论内标(内参照)把质量控制从每批阳性质控提高到每管阳性质控的水平,能有效减少假阴性检测结果,竞争性内标结构和待检物几乎完全相同,更能真实反应情况。     

应用核酸扩增技术对血液中HBV、HCV和HIV-1检测

目的探讨采用核酸扩增技术(NAT)对血液进行HCV、HBV和HIV1核酸检测的可行性。方法采用手工法将血清学检测HBsAg、抗HCV、抗HIV1/2和TPHA呈阴性,以及ALT<40U的血浆标本进行48份×50μl混合;超速离心浓缩病毒后提取病毒核酸,采用RocheCOBASAmplicor分析系统对微量血浆混合标本进行HBVDNA、HCVRNA和HIV1RNA的检测;阳性反应的混合标本进行交叉混合和单份确认检测。结果18621份血清学检测合格的血液中,发现5份HBsAg阴性、HBVDNA呈阳性的血液,阳性率为0.027%(5/18621)。经进一步随访6～20月显示,2名献血者为HBV低水平感染,1例为HBV血清转换窗口期感染。结论NAT检测能够发现HBV低水平和窗口期感染的标本。     

不同保存条件对丙型肝炎病毒核酸稳定性的影响

目的探讨不同温度和时间条件下保存对血液中丙型肝炎病毒核酸(HCV RNA)稳定性的影响。方法采集12名丙型肝炎患者4 ml的外周血,分装、保存在不同温度及相同温度下的不同时间,利用实时荧光定量PCR检测其不同时间点血浆HCV RNA的水平(log IU/ml),并与该血液标本的起始HCV RNA水平做比较分析。结果当血液标本中的HCV RNA起始水平≥105IU/ml时,在25℃放置6 h后转至4℃保存18 h(a)、25℃放置12 h后转至4℃保存12 h(b)及4℃放置24 h(c)等3种保存条件,24 h时内检测到血液标本的HCV RNA水平105IU/ml组分别为(5.15±0.16)、(5.15±0.15)及(5.16±0.16)IU/ml(P>0.05),107 IU/ml组分别为(7.45±0.21)、(7.44±0.23)及(7.45±0.24)IU/ml(P>0.05);当血液标本中HCV RNA起始水平在临界范围[(3.38±0.14)IU/ml]时,在条件a、b保存24 h血液标本的HCV RNA含量分别为(2.28±1.52)IU/ml、0(均为P<0.01),而条件c保存24 h血液标本的HCV RNA含量为(3.28±0.20)IU/ml(P>0.05);当血液标本中HCV RNA起始水平为103 IU/ml时,在4℃保存48 h时HCV RNA含量为(2.31±1.54)IU/ml(P<0.01)。结论当血液中HCVRNA起始浓度较低时,HCV RNA的稳定性较差,短时常温保存都会造成病毒核酸的降解。     

科华V2.2核酸检测系统灵敏度验证

目的验证血液筛查实验室科华V2.2核酸检测系统(简称V2.2系统)的灵敏度,观察其检出弱反应核酸样品的能力。方法采用6份样品合并(简称6混样)检测模式检测系列浓度HBV DNA、HCV RNA、HIV RNA标准品,验证V2.2系统的灵敏度。用V2.2系统与科华V2.1核酸检测系统(8份样品合并检测模式,简称V2.1系统)对HBV DNA弱反应样品同时进行混样检测,并进行卡方检验。选取无偿献血者血浆样品3 000例,用两系统平行检测,并进行卡方检验。结果 V2.2系统对HBV DNA载量为15、30、40、60 IU/m L标准品的检出率分别为53.5%(16/30)、70%(21/30)、90.0%(27/30)、93.3%(28/30);对HCV RNA载量为150、300、400 IU/m L标准品的检出率分别为83.3%(25/30)、100%(30/30)、100%(30/30);对HIV RNA载量为150、300、400 IU/m L标准品的检出率分别为83.3%(25/30)、96.7%(29/30)、96.7%(29/30)。V2.2系统对HBV DNA弱反应标本的检出率为27.86%(39/140),V2.1系统的检出率为10.71%(15/140),差异有统计学意义(χ~2=13.215,P0.05)。平行试验中V2.2系统的反应性检出率为0.07%(2/3 000),V2.1系统的反应性检出率为0.1%(3/3000),差异无统计学意义(χ~2=0.667,P0.05)。结论 V2.2系统检测HCV RNA/HIV RNA的灵敏度与厂商提供的理论值相符合,HBV DNA的灵敏度与理论值存在一定差距。V2.2系统较V2.1系统检出HBV DNA弱反应性样品的能力高。     

应用NASBA技术对献血者血浆/血清进行HCVRNA检测

目的　探讨对献血者混合血浆标本检测HCVRNA的必要性和可行性。方法　每 5 0份血浆混合为一组 ,用NucliSensExtractor提取核酸 ,以NASBA技术扩增HCVRNA和用ECL检测扩增产物。标本贮存 ,核酸提取、扩增、检测过程设立系统对照和阳性对照。结果　在抗 HCVELISA阴性的献血者 10 0 0 0人中发现 2例HCVRNA阳性。结论　用NASBA技术对混合血浆进行HCVRNA检测可有效控制输血后丙型肝炎的发生。     

血站实验室溶血、脂血比色卡的建立及探讨

目的探讨血站实验室溶血、脂血目测比色卡的建立过程,保证实验室检测标本质量,确保基于PCR方法的核酸检测结果准确。方法用Roche Cobas s201全自动核酸检测系统以Pools of 1模式检测血红蛋白水平分别为0、50、150、250、500mg/dL 5个梯度的溶血标本及三酰甘油水平分别为0、800、1 700、2 500、3 300mg/dL 5个梯度的脂血标本对3倍最低检出限(LOD)HBV DNA、HCV RNA和HIV RNA检出率的影响情况;对不同水平溶血、脂血标本拍照、制作比色卡。结果 0、50、150、250、500mg/dL 5个水平梯度的溶血标本对HBV DNA、HCV RNA和HIV RNA 3个项目的核酸检测检出率均无影响(P0.05);0、800、1 700、2 500mg/dL 4个水平梯度的脂血标本对HBV DNA、HCV RNA和HIV RNA 3个项目的核酸检测检出率无影响(P0.05),3 300mg/dL的脂血标本对HBV DNA、HCV RNA两个项目的检出率无影响(P0.05),但对HIV RNA的检出率有影响(P0.05)。结论该实验室的无偿献血者人群标本,血红蛋白水平≤500mg/dL所对应的比色卡溶血标本、三酰甘油水平≤2 500mg/dL所对应的比色卡脂血标本对核酸检测结果无影响,可接收。     

Screening of blood donations by hepatitis C virus polymerase chain reaction (HCV-PCR) improves safety of blood products by window period reduction

Introduction of the nucleic acid amplification technique (NAT) as a screening test for blood donors to detect HCV RNA became mandatory on 1 April 1999. Few automated commercial systems are available for HCV RNA detection at the moment. The Cobas Amplicor HCV 2.0 System is able to perform fully automated amplification and detection of nucleic acids. A concentration of 98 IU HCV RNA/ml can be detected by the Cobas Amplicor HCV 2.0 System (n = 233, in 100% of the cases). With a pool size of 40 donor samples, the guidelines of the Paul-Ehrlich-Institute concerning sensitivity (5,000 IU HCV RNA per mL in a single donation) were followed. One whole blood donation was identified as HCV-RNA positive (anti-HCV IgG negative, GPT < 30 U/L) during a period of 5 months. No false positive test results could be observed. The internal control and the run control are primarily helpful to monitor methodological problems.     

全自动血液核酸筛查及阳性献血员追踪的研究

目的 建立献血者全自动核酸检测方法，探讨在我国血液筛查中引进全自动核酸筛查方法的可行性。方法 在酶联免疫吸附实验（ELISA）筛查血液基础上，选用全自动汇集仪进行血样汇集（24人份），在全自动核酸提取仪上提取样本核酸，应用聚合酶链反应（PCR），在COBAS AMPLICOR进行扩增和检测结果，用国际标准核酸质控品考评检出限量，对阳性献血者追踪检测。结果经考评及常规应用表明，全自动汇集、全自动核酸提取及扩增和检测95％的检出限量HBV DNA、HCV RNA和HIV-1 RNA分别为38．9、17．4IU／ml和20．6拷贝／ml，95％的可信限分别为[21，323]、[10．5，342]和[12，300]。通过对16512个样本共688汇集池分析，HBVDNA阳性8例，阳性率为0．048％，其中7例为Anti-HBc阳性，其余1例亦转换为阳性。HCV RNA和HIV-1 RNA未检出阳性，6例HBV DNA阳性样本追踪发现，3例发生了血清转换现象。结论本实验结果认为全自动血样汇集，全自动核酸提取扩增和检测方法可应用于血液的筛查工作。     

Effect of viral nucleic acid testing on contamination frequency of manufacturing plasma pools

BACKGROUND: In Europe, all plasma pools used for manufacturing of plasma derivatives must be tested negative for hepatitis C virus (HCV) RNA by nucleic acid amplification techniques (NAT) with a defined minimal sensitivity. For a subset of pools, quantitative B19V DNA NAT is also mandatory. NAT for further viral targets was introduced by most of the manufacturers on a voluntary basis. The contamination frequency of plasma pools with HCV RNA, human immunodeficiency virus (HIV)-1 RNA, and hepatitis B virus (HBV) DNA was investigated with representative pools before and after introduction of NAT. STUDY DESIGN AND METHODS: A total of 873 pools from 1996 and 331 pools from 2006 were analyzed for the detection of HCV RNA, HIV RNA, and HBV DNA with an automated multiplex NAT system. The pools were obtained from different manufacturers and the source material was of European and US origin. RESULTS: HCV RNA, HIV-1 RNA, and HBV DNA were detectable in plasma pools from 1996 with the following frequencies: 17.8 percent (HCV RNA), 0.8 percent (HIV-1 RNA), and 0.5 percent (HBV DNA). Viral genome concentrations were up to 3 x 10(4) IU HCV RNA per mL and 7 x 10(3) IU HIV RNA per mL, whereas HBV DNA was below the quantitation limit of the quantitative NAT assay. Among the pools from 2006, one pool (0.3%) was found HCV RNA-positive at low titer (<10 IU/mL), whereas no HIV RNA or HBV DNA was detectable in any pool. CONCLUSION: The results imply that the introduction of NAT systems for the detection of viral genomes has largely reduced the contamination frequency and viral loads of manufacturing plasma pools and thereby improved the safety margin for human medicinal products manufactured from human plasma. Even with NAT, however, low-titer contamination may occur, which will be coped with by virus inactivation steps included into the manufacturing of plasma derivatives.     

Preanalytical stability of HIV-1 and HCV RNA: impact of storage and plasma separation from cells on blood donation testing by NAT

Background: The aim of this study was to evaluate the optimal preanalytical conditions prior to nucleic acid amplification technology (NAT) for human immunodeficiency virus‐1 (HIV‐1) or Hepatitis C virus (HCV) RNA in pools of 96 plasma specimens with regard to storage temperature, time and plasma separation in a blood donation environment. Study design and methods: Changes in viral nucleic acid concentration of HIV‐1 and HCV were observed for 5 days according to the Paul‐Ehrlich‐Institute's (PEI) guidelines that demand 95%‐detection limit of at least 10 000 IU mL^?1 for HIV‐1 RNA and 5000 IU mL^?1 for HCV RNA within a single donor blood specimen. Ninety‐five per cent detection limits of HIV‐1 RNA over 3 days after storage at either 5 or 21 °C were evaluated by using standardised HIV‐1 RNA‐positive plasma. Results: HCV RNA in whole blood samples proved to be more stable than HIV‐1 RNA. Whole blood storage at 21 °C was shown to decrease the detectability of HIV‐1 RNA even after only 18 h. Plasma samples once used for NAT at time 18 h did not alter viral stability up to 48 h after donation. Ninety‐five per cent detection limits of HIV‐1 RNA were securely below 10 000 IU mL^?1 for 24 h after whole blood storage at 5 °C. Conclusions: These results may lead to a discussion around the most suitable preanalytical conditions in blood donation environments. Contrary to the current PEI guidelines that allow storage of whole blood specimens up to 18 h at 21 °C, these results suggest that immediate storage in a 5 °C container after blood donation is more suitable and would permit storage of whole blood up to 24 h prior to the separation of plasma from cells.     

Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems

BACKGROUND: The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY DESIGN AND METHODS: Serial dilutions of both the EUROHEP standard (3,800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organization (WHO) international standard (100,000 IU/mL; HCV genotype 1) were made in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serologic tests. Serial dilutions of plasma (2 mL) were used for extraction of HCV RNA with an automated version of a nucleic acid isolation method (NucliSens Extractor, Organon Teknika). HCV RNA was co-extracted from 2 mL of plasma, together with 84 copies of an in vitro-synthesized single-strand RNA serving as internal extraction control (IC) to monitor the efficiency of extraction and PCR. Amplification and detection of both HCV RNA and IC RNA were performed with an automated PCR system and a qualitative HCV assay (COBAS Amplicor 2.0 HCV, Roche Diagnostics). RESULTS: A cutoff value of 16 geq per mL (10/10 runs [100% hit rate]) was found by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mL (10/10 runs [100% hit rate]). The IC had a cutoff value of approximately 17.5 copies per mL (6/6 runs [100% hit rate]). Forty-two copies per mL of IC RNA were found in 282 of 284 runs (99% hit rate). The negative controls (ESDEP plasma) were negative in all experiments. Experiments with pool sizes of 12, 24, 48, and 96 using serial dilutions of the WHO international standard revealed a cutoff value of 8 IU per mL (100% hit rate). The EUROHEP standard and the WHO international standard were detected with a 50 percent detection endpoint of 5.2 geq per mL and 1.5 IU per mL, respectively. CONCLUSION: This test system (NucliSens Extractor, and the COBAS Amplicor 2.0 HCV assay) revealed a high sensitivity for HCV RNA; considering the proposed requirements for sensitivity of NAT assays for the detection of HCV RNA in donor plasma, pool sizes of about 400 donors are possible. These endpoint results indicated that 1 IU is equal to about 3.4 geq.     

Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia.

The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1$ were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.     

一种国产血液核酸检测系统的性能验证研究

目的对一种国产血液核酸检测系统在本实验室进行献血者乙型肝病毒脱氧核糖核酸(HBV DNA)、丙型肝炎病毒核糖核酸(HCV RNA)、人类免疫缺陷病毒核糖核酸(HIV RNA)检测的性能进行研究,确定该系统是否稳定、准确、可靠。方法根据美国临床实验室标准化协会(CLSI)的相关文件要求,对一种国产血液核酸检测系统HBV DNA、HCV RNA、HIV RNA项目的检测灵敏度、准确度、精密度、抗交叉污染、抗干扰及稳定性进行验证。结果该国产血液核酸检测系统HBV DNA、HCV RNA、HIV RNA的95%检出限分别为4.87(3.84~8.22)IU/mL、7.29(5.68~13.89)IU/mL、30.22(22.90~60.17)IU/mL;对16例阳性样本和32例阴性样本进行混样检测,结果均为反应性,拆分检测阴性样本和阳性样本的检测符合率均为100%;HBV DNA、HCV RNA、HIV RNA阳性样本重复检测的变异系数(CV)分别为4.29%、1.51%、3.73%;将10个阴性样本和10个阳性样本进行阴阳交叉排列单检结果无交叉污染;低浓度HBV(10 IU/mL)、HCV(10 IU/mL)、HIV(40 IU/mL)样本在溶血样本[血红蛋白(Hb)含量为12 g/L]和脂肪样本[甘油三酯(TG)含量为6.13 mmol/L]中的检出均无显著影响;当溶血样本中Hb含量提高至24 g/L、脂肪样本中TG含量提高至11.85 mmol/L时,低浓度样本的检出会受到影响;经过2017年仪器设备的日常核酸检测情况分析,该国产血液核酸检测系统稳定性良好。结论该国产血液核酸检测系统的检测灵敏度、准确度、精密度和抗交叉污染等均达到生产商的检测性能的要求,在标本Hb≤12 g/L以及TG≤6.13 mmol/L时,对低浓度HBV DNA、HCV RNA、HIV RNA的检测无显著影响,满足本实验室检测需求。     

多重定量PCR法同步检测HBV、HCV和HIV-1在血液筛查中的优化及应用

目的 初步探讨多重定量聚合酶链反应（PCR）同步检测乙型肝炎病毒（HBV） DNA,丙型肝炎病毒（HCV） RNA及人类免疫缺陷病毒（HIV）-1 RNA在血液筛查中的应用前景.方法 选择2012年8月至12月,于孝感市中心血站志愿献血的合格献血者血样中,经2次酶联免疫吸附法（ELISA）检测HBV表面抗原（HBsAg）、抗HCV及抗HIV-1,检测结果均呈阴性的4 800份血样为研究对象.采用全自动核酸混合提取仪对该4 800份血样进行核酸提取,然后利用多重定量PCR方法对血样中HBV、HCV及HIV-1进行同步扩增检测.采用中国药品和生物制品检定所提供的HBV DNA、HCV RNA及HIV-1 RNA标准参考品,检测多重定量PCR的灵敏度,并与单重定量PCR的灵敏度进行比较.在HBV、HCV及HIV-1 3者中任意1种病毒基因组浓度较高的条件下,对多重定量PCR检测另2种低浓度病毒基因组的能力进行评估.结果 增加多重定量PCR中c-MMLV逆转录酶和Hot Taq酶的用量,并适量加入单链结合蛋白（SSB）,可使其扩增效率提升至单重定量PCR扩增水平.本组4 800份血样中,经多重定量PCR检测出3份HBV DNA阳性样品,ELISA漏检率为0.062 5％,未发现HCV RNA和HIV-1 RNA阳性样品;多重定量PCR检测HBV DNA、HCV RNA及HIV-1 RNA在95％置信区间的灵敏度浓度分别为115 IU/mL、376 IU /mL和232 IU /mL;单重定量PCR检测HBV DNA、HCV RNA和HIV-1 RNA在95％置信区间的灵敏度浓度分别为51 IU /mL、94 IU /mL和78 IU/mL.结论 本研究初步建立了对献血者血液同时进行HBV DNA、HCV RNA及HIV-1 RNA检测的多重定量PCR检测方法;该检测体系经过进一步优化后,有望应用于临床大规模血液病毒筛查.     

Second hepatitis C virus transmission by blood components since introduction of mandatory NAT screening in Germany

Background

Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999.

Study Design and Methods

When a repeat donor who had tested negative for anti-HCV tested positive for HCV RNA by NAT in a minipool (MP) of eight, a look-back procedure was initiated. Qualitative, quantitative and genotyping assays were used to investigate the titers of the quarantined fresh frozen plasma (FFP) from the donor and a serum sample from the recipient of the pooled platelet concentrate (PPC). Amplified products of 5'UTR and HVR1 were used for sequence comparison to characterize the HCV genomic identity of donor and recipient samples.

Results

All NAT tests utilized in this procedure were able to detect a low HCV RNA titer (~15 IU/ml) in the FFP from the donation. Dilution of FFP by factor 8 was performed to mimic an MP, and the detection rate correlated well with the claimed sensitivity of the tests. Analysis of donor and recipient samples revealed genotype 3a viral transmission confirmed by sequence analysis.

Conclusion

This TT HCV case could have been prevented by individual donation (ID) NAT. However, a low titer blood donation in the window period (WP) is very rare. Residual risk calculation for TT HCV in the WP revealed that, compared to MP-NAT testing, ID-NAT would improve blood safety only marginally.