核酸检测非重复反应性的HBsAg阴性血液HBV感染的确认  

Confirmationof HBV infections in blood donors with HBsAg-negative and non-reproducible nucleic acid testing( NAT) reactivity

作　　者：邓雪莲[1] 李婷婷[2] 郭笑寒 周磊[1] 臧亮[1] 梁晓华[1] Daniel Candotti DENG Xuelian;LI Tingting;GUO Xiaohan;ZHOU Lei;ZANG Liang;LIANG Xiaohua;CAN-DOTTI Daniel(Dalian Blood Center,Dalian 116001,China;South Medical University;National Institute of Blood Transfusion（INTS）.)

机构地区：[1]大连市血液中心,辽宁大连116001 [2]南方医科大学 [3]法国国家输血研究所输血相关病毒参考实验室

出　　处：《中国输血杂志》2018年第9期962-967,共6页Chinese Journal of Blood Transfusion

基　　金：中国输血协会威高科研基金资助(CSBT-WG-2017-01)

摘　　要：目的对单个标本核酸检测呈初始反应性（Initial reactive,IR）但鉴别试验为无反应性,即所谓的核酸检测非重复反应性（NAT non-reproducible reactivity,NAT NRR）,且HBsAg阴性的献血者进行HBV感染的确认。方法采用Ultrio plus（盖立复）进行单个标本核酸检测（Individual donation nucleic acid testing,ID-NAT）。应用超高速离心技术（25 0000 g×3 h）与靶向HBV基因BCP/PC、PreCore/Core、pre-S/S和S区段的高灵敏度巢式PCR检测相结合的超离体系对12mL NAT NRR血浆标本进行HBV DNA确认,以获得HBV基因序列作为HBV DNA确认的标准;同时与采用2次联检和1次鉴别试验的推荐流程的确认结果进行比较。NAT NRR标本补充电化学发光法（ECL,罗氏）的HBsAg,抗-HBc和抗-HBc检测。结果 2016年1月至2018年2月间共筛查标本77 556份,检出HBsAg-/NAT NRR标本85份（0. 11%）,其中有79份标本进行了确认试验。超离体系确认出HBV感染标本43份,推荐流程确认出32份。配对比较显示超离体系明显优于推荐流程（McNemar test,P〈0. 05）：有48份（60. 7%）标本至少被1种流程确认,其中27份（34. 2%）标本同时被2种流程确认,16份（20. 2%）标本单独被超离体系确认,超离体系未能确认的5份（6. 3%）标本被推荐流程确认。HBV感染确认组和未确认组间抗-HBc＋（92%和80%）和抗-HBs＋（42%和55%）的占比无统计学差异,但确认组抗-HBs定量值的分布（中位数22 IU/mL,范围10-1 000 IU/mL）明显低于未确认组（P〈0. 05）。在HBV感染确认组中鉴别出血清学阳性型隐匿性乙型肝炎病毒感染（Occult Hepatitis B virus infection,OBI）献血者45位（94%）、血清学阴性OBI献血者1位,2位献血者因未能跟踪而疑似HBV窗口期感染。结论加大核酸提取的血浆体积并结合以高灵敏度巢式PCR的方法能够确认60%的HBsAg-/NAT NRR标本存在HBV DNA。NAT NRR现象与病毒载量极低的OBI有关,抗-HBc检测可有助于此类HBV感�Objective To confirm the HBV infection statusin HBsAg-negative blood donors with non-reproducible reactivity（ NRR） in nucleic acid testing（ ID-NAT）.That is,to further confirm samples that are initially reactive in NAT tests but turned out to be non-reactive in other screening tests. Methods HBV DNA was screened initially with multiplex Ultrio Plus NAT assay（ Grifols Ltd）. HBV DNA was investigated in NAT NRR samples by using optimized nested PCRs targeting BCP/PC,PreCore/Core,pre-S/S,and S regionsfollowing viral particle concentrationfrom 12 mLplasma by ultracentrifugation（ UC）. Successful sequencing of HBV-specific ampliconsprovided definitive confirmation of HBV infection. Results were compared to those obtained with the recommended procedure（ RP） routinely used for HBV NAT confirmation,where ID-NATought to be repeatedtwice withone extra cycleof discriminatory assay. HBsAg,anti-HBc and anti-HBs antibodies were tested with Electrochemiluminescence（ ECL; Roche）.ResultsEighty-five（ 0. 11%） HBsAg-negative/NAT NRRdonations were identified among 77,556 donations screened between January 2016 and February 2018. Out of the 79 NAT NRR donations investigated further,43（ 54. 4%） were confirmed HBV-DNA-positive after UC and sequencing compared to 32（ 40. 5%） confirmed bythe RP（ McNemar test,P〈0. 05）. Overall,48（ 60. 7%） NAT NRR donations were confirmed HBV-DNA-positive by at least one workflow type. Twenty-seven（ 34. 2%） donations were confirmed bytwo screening protocols,16（ 20. 2%） were confirmed with UC alone,and 5（ 6. 3%） RPconfirmed donations remain ambiguous by UC. Similar anti-HBc（ 92% vs 80%） and anti-HBs（ 42% vs 55%） reactivity rates were observed in confirmed and non-confirmed HBV DNA reactive donations. Anti-HBs levels（ median： 22 IU/mL,range：10—1 000 IU/mL） were significantly lowerin the confirmed HBV DNA positive than in non-confirmeddonations（ P〈0. 05）. Forty-five of NRR donors were identified carrying seropositive occult hepatiti

关 键 词：血液筛查 核酸检测 非重复反应性 隐匿性HBV感染 血液安全 确认试验 

分 类 号：R446.11[医药卫生—诊断学] R373.21[医药卫生—临床医学]