两种核酸血液筛查系统降低HBV残余风险的比较

Comparison of two nucleic acid testing systems for reducing residual risk in blood donations

目的评估不同原理核酸检测(NAT)血液筛查系统在降低输血传播HBV残余风险中的作用。方法对6 889人(次)无偿献血者血液标本做常规血清学检测,应用转录介导的扩增(TMA)技术(Ultrio试剂)对所有血样平行单样本定性检测HBV/HCV/HIV,对血清学无反应性标本5 165例采用PCR-荧光探针法(MPX试剂)以6人份混样模式做NAT。追踪2个NAT平台得到的反应性标本,罗氏CAP/CTM核酸定量检测系统做核酸鉴别试验,罗氏电化学发光做乙肝血清学5项检测,QPCR法测定HBV病毒载量,巢氏-PCR方法做HBV DNA基因分型,统计分析各项检测指标之间关联及差异。结果 TMA初筛检出HBV DNA单独反应性标本12例[反应率0.17%(12/6 889),鉴别试验阳性7例(占TMA初筛阳性数的58.3%)],PCR-荧光探针法拆分后检出HBV DNA单独反应性9例[0.17%(9/5 165)],2种NAT平台检测HBV DNA均为阳性4例,合计检出17例反应性标本。该2种原理的NAT检测系统对HBV DNA检测结果的一致性差(TMA vs QPCR,K<0),检出率明显不同(TMA vs QPCR,P<0.05)。阳性标本乙肝血清学5项检测:HBs Ag、HBe Ag均为阴性(0/16),抗-HBc阳性12例(12/16),抗-HBs阳性6例(6/16),抗-HBe阳性3例(3/16)。HBV DNA定量阳性4例,含量均<5 IU/m L。献血者追踪结果:TMA检出HBV DNA单独反应性3例(3/10),鉴别试验HBV DNA阳性3例,MPX检出HBV DNA单独阳性5例(5/10),2种NAT平台检测均为阳性者3例,合计检出5例;10例追踪标本的HBs Ag、HBe Ag仍均为阴性,抗-HBc阳性8例(新转阳3例),抗-HBs阳性5例(新转阳1例),抗-HBe阳性2例(新转阳1例),HBV DNA定量阳性4例(新检出1例),含量均<5 IU/m L。巢氏-PCR S区序列阳性标本做HBV基因分型:B型占66.7%(6/9),C型占33.3%(3/9)。结论 TMA与PCR-荧光定量法均能有效降低输血残余风险,但针对检出限附近低病毒载量标本均有部分漏检,血站在条件具备时可使用更高灵敏度的新试剂。

Objective To assess the functions of two different NAT systems in reducing the residual risk of HBV transmission in blood donors. Methods The HBV / HCV / HIV NAT yield rate was estimated by testing 6 889 donor samples concurrently on the PROCLEIX ULTRIO（ transcription-mediated amplification,Ultrio） assay as individual donor samples with the TIGRIS and the routine serological testing was performed in parallel processing. 5 165 seronegative samples were further tested in pools of 6 with the cobas s 201（ real time PCR,MPX）. Discrimination tests,serological test of these NAT yield samples and the follow-up donations were undertaken,followed by a molecular analysis for detection of the viral load and genotyping. Results Among 12 potential HBV-yield cases（ Ultrio-reactive specimens） initially detected by TMA,7 were tested reactive（ occupied 58. 3% of all specimens tested by TMA）. Nine reactive cases were detected by PCR- fluorescent probe.Either system detected 4 identical HBV NAT yield samples. Both NAT systems had bad concordance and were significantly different for detecting HBV DNA（ TMA vs QPCR,K 〈0,Fisher＇s exact test： = 0. 019,P〈0. 05）. The serological test results showed： 12 anti-HBc positive,6 anti-HBs positive and 3 anti-HBe positive. 4 cases showed the presence of HBV DNA by quantitation PCR and the viral load were less than 5IU / Ml. Ten donors were followed up,from which,3 HBV-yield cases were detected by TMA,5 HBV-yield cases by Ultrio,and 3 identical HBV NAT yield samples were detected by two NAT systems. All ten followed up specimens were negative for HBs Ag and HBe Ag. Eight anti-HBc positive cases included 3 new cases,1 seroconverted case in 5 anti-HBs positive cases and 2 anti-HBe positive cases. Among the four cases with the HBV DNA detected by quantitation PCR and the viral load were less than 5IU / Ml,one new case was discovered. Twelve cases underwent nested-PCR,among which,6 were found to have B genotypes（ 66. 7%） and 3 had C genotypes（ 33. 3%）,out of 9 nested-PCR positive samples. Conclusion Both systems had sufficient analytical sensitivity for decrease the residual risk,but the DNA levels in certain samples were too low to be detected.Hence,blood banks should use more sensitive assays if possible.