献血者中低水平HBsAg乙肝病毒感染者分子生物学特征研究

Molecular characteristics of hepatitis B infection with low level HBsAg and nucleic acid testing non-reactive results in blood donors

目的了解核酸检测初筛阴性献血者中低水平乙肝表面抗原(HBsAg)乙肝病毒(HBV)感染情况并探讨其分子生物学特征。方法从本中心2017~2018年100363(人)份无偿献血者血样中,收集HBsAg酶联免疫试验(ELISA)阳性核酸检测(NAT)阴性献血者标本,用化学发光微粒子免疫法CMIA复检HBsAg,用电化学发光免疫法(ECLI)定量检测乙肝两对半,再做NAT单人份复检,采用巢式聚合酶链反应(PCR)扩增病毒BCP/PC和S区,用荧光定量PCR(qPCR)测定病毒载量。结果本中心2017~2018年HBV DNA(-)的低水平HBsAg献血者(标本)的检出率0.054%(60/100363),其中HBsAg ELISA试剂1检出比例90%(54/60),试剂2检出比例95%(57/60);60例中有33个可做HBV基因分型的标本,B型占87.9%(29/33),其中3.4%(1/29)为血清型ayw1、96.6%(28/29)血清型adw2;C型占12.1%(4/33),均为血清型adqr+。巢式PCR扩增:在B型S基因区发现影响HBsAg检测Q101R、Q129H、T131I、M133L/T、F134L、G145R、V168A、L175S和V177A变异株,在BCP/PC区发现减少HBV复制的高频突变C1799G(87.5%,21/24)。qPCR测得病毒载量中位数49.6(0~628)IU/ml。结论ELISA检测低水平HBsAg且NAT阴性献血者标本中存在少数未能被目前ELISA方法检出的HBV。在献血者血液筛查中应用灵敏度和特异性更高的HBsAg和NAT检测方法可提高检出HBV突变株的能力。

Objective To investigate HBV infection with low level of HBsAg and nucleic acid testing(NAT)non-reactive results in blood donors,and analyze molecular characteristics.Methods Low level HBsAg but NAT-nonreactive samples were collected and tested for HBsAg by Abbott chemiluminescent microparticle immunoassay(CMIA).,HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc were further detected by Roche electrochemiluminescence immunoassay(ECLI).BCP/PC and S regions were also amplified by Nested-PCRs and qPCR for HBV DNA quantity were adopted simultaneously.Results Of 100363 donations,60(0.054%)low level HBsAg and NAT-nonreactive blood samples were enrolled the study.In which,54/60(90%)and 57/60(95%)were WanTai HBsAg ELISA and DiaSorin HBsAg ELISA reactive respectively.Of 33 cases genotyped,genotype B were 87.9%(29/33),including adw296.6%(28/29)and adw13.4%(1/29),C was observed in 4(12.1%)with sero-type adrq+.Mutations in S gene of genotype B such as Q101R,Q129H,T131I,M133L/T,F134L,G145R,V168A,L175S and V177A were observed as notable mutations,which can affect HBsAg diagnosis.A high frequency mutation C1799G(87.5%,21/24)were detected in BCP/PC and would reduce the replication of virus.The median viral load measured by qPCR was 49.6(0~628)IU/mL.Conclusion A small part of donations with low-level HBsAg and NAT-nonreactive can not be deferred by one isolated ELISA screening assay.It is necessary to apply more sensitive and specific HBsAg assays and NAT in blood screening,and improve the ability to detected mutants.