【作者】 杨生义；

【导师】 李文凡；

【作者基本信息】 兰州大学 ， 内科学， 2010， 硕士

【摘要】 目的：了解我国健康献血员隐匿性乙肝病毒感染者的流行率。探讨国内血站对献血员乙型肝炎病毒表面抗原(HBsAg)筛查的安全性和可靠性。进一步改进献血员HBV筛查方法,减少HBV的漏检率；分析隐匿性乙肝感染者病毒S区,探讨引起隐匿性感染的原因。方法：1.收集经中心血站依据国家卫生部制定的血库安全的要求双筛HBsAg阴性、抗-HCV阴性、抗-HIV阴性、梅毒螺旋体抗体阴性、谷丙转氨酶(ALT<40IU／m1)阴性的合格献血员血浆2～3ml。-20℃保存；2.雅培Architect 12000电化学发光仪检测(?)HBsAg定量；Nested-PCR扩增乙肝病毒S区、C区、X区；3.Nested-PCR扩增隐匿性乙肝病毒感染者病毒PreS-S区,PCR产物直接测序并与标准病毒序列对比分析病毒株变异。结果：1.经雅培Architect 12000复检健康献血员中HBsAg阳性率为6.5%(64／983)；扩增病毒S、C、X区,60.9%(39／64)至少两个区扩增阳性；健康献血员隐匿性HBV感染率为4.0%(39／983)。2.漏检的样本HBsAg滴度定量,中位数为0.17IU/ml (0.05-1.14 IU/ml); HBsAg阳性的标本60.9%(39／64)病毒扩增阳性,9.1%(25／64)病毒扩增阴性；HBsAg定量：阳性组中位数为0.15IU/ml (0.05-0.96IU/ml),阴性组中位数为0.18IU/ml (0.05-1.14IU/ml),病毒扩增阳性组与阴性组之间HBsAg定量、性别比例和转氨酶水平均无统计学意义。3.隐匿性感染者病毒载量均很低,约50～1000拷贝／ml；病毒PreS-S区段扩增阳性率35.9%(14／39)；其中60.9%(39／64)至少两个区扩增阳性,29.6%(19／64)S区扩增阳性,64.1%(41／64)C区扩增阳性,65.6%(42／64)X区扩增阳性。35.9%(14／39)扩增出病毒PreS-S区段；4.测序结果峰图均一,未见混合感染。将S基因翻译成氨基酸后,与GenBank中各种基因型标准株的S区蛋白对比,并没有发现常见的已有文献证实可导致HBsAg阴性或分泌量降低的“a”决定簇变异。可见少数可能与HBsAg阴性相关的S区氨基酸位点突变：S45D, N68D, Q129R, F134N, V180S, V190I。结论：目前经血站常规检测HBsAg为阴性的合格血液,用敏感性更高的试剂仍能检测出HBsAg及HBV DNA。临床用血存在导致乙肝感染的风险。HBsAg分泌量处于判断界限附近时,即使是较为精确的电化学发光法检测技术,对于样本病毒阳性和阴性也难于作出判定。造成OBI的主要原因可能由于HBV低水平复制,检测试剂的灵敏度不够；病毒变异导致HBsAg的检测失败。更多还原

【Abstract】 ObjectiveTo investigate the prevalence of the presons with hepatitis B virus among blood donors, and explore the safety and reliability of hepatitis B surface antigen screening to blood donors in domestic blood bank. To analysis the PreS-S section in hepatitis B virus to explore the cause of occult infection.Methods1.To collect the plasma screened by domestic blood banks, which is from the blood donors with HBsAg negative, anti-HCV and anti-HIV negative, anti-TP, and alanine aminotransferase (ALT<40 U/ml) negative in normal, and store it in-20℃.2.HBsAg quantitative should be tested with Abbott Architect 12000 electrochemiluminescence and Nested-PCR amplify HBV S, C and X region, to defined as occult HBV infection.3.The Nested-PCR should be used to amplify the PreS-S region of virus from the persons with occult hepatitis B virus infection and PCR products can sequenced directly while contrasting with the standard virus strain and analyzing the variation of virus strain together with it.Results1.6.5% of health donors (64/983) was positive for HBsAg determined through Abbott Architect 12000 chemiluminescence apparatus,60.9%(39/64) of samples was positive in two districts for Nested-PCR HBV S, C, Xregion. the prevalence of occult hepatitis B Virus infection in healthy blood donors was 4.0%(39/983).2.Undetected samples of HBsAg titer, has a median of 0.17IU/ml (0.05-1.14 IU/ml), of which HBsAg positive specimens 60.9%(39/64) virus expands positive, and 9.1% (25/64) virus does negative; HBsAg quantitative:positive group with a median of 0.15IU/ml (0.05-0.96IU/ml) and negative group with a median of 0.18IU/ml (0.05-1.14IU/ml). There are no statistical significances on the HBsAg quantitative between the positive group of virus expansion and the negative group, sex ratio and transaminase level.3.Those with occult infection have low virus load, about 50～1000 copies/ml; virus PreS-S section expands at a positive rate of 35.9%(14/39), of which 60.9%(39/64), at least two district, expands positive, so does the 29.6%(19/64) S region,64.1%(41/64) C region, and the 65.6%(42/64) X region.35.9%(14/39) expands out of the virus PreS-S region.4.Sequencing results peak map shows a average situation without mixed infection. The "a" determinant mutation is not found, which has been proved to be the cause of HBsAg negative or the lowering of secretion volume by existing documents, so the mutation may happen to a small number of amino acid site in S-area relating to HBsAg negative:S45D, N68D, Q129R, F134N, V180S, V190I.ConclusionsAt present, the HBsAg and HBV DNA still can be found from the negative blood in the required standard with more sensitive reagents, which is tested routinely by the Blood Bank, so the risk leading to hepatitis B infection exists in the clinical use of blood. It is difficult to discriminate between the samples of virus in positive and negative when the HBsAg secretory volume can not be justified because of minute amounts, even using highly precise electrochemiluminescence testing technology. The main reason leading to OBI may be the low level replication of HBV, the lack of detection reagent sensitivity and the failure on HBsAg testing led by virus variation.更多还原