核酸检测技术在血液筛查中的应用研究

目的大样本、多方法调查深圳地区无偿献血人群中乙肝、丙肝和艾滋病病毒血清学阴性者的核酸阳性率,探讨在我国血液筛查中引进核酸扩增技术的必要性,了解和分析献血者血清学阴性核酸阳性感染状况。方法采用大样本数调查,应用ROCHE PCR-ELISA、PCR-微流芯片、实时荧光PCR方法和CHIRON TMA（转录依赖的扩增技术）多种方法对血清学检测阴性的献血者进行HBV DNA、HCV RNA和HIV-1RNA检测,对乙肝阳性献血者追踪检测ALT和乙肝两对半标志物,对丙肝核酸阳性献血者追踪检测ALT及抗-HCV及HBV DNA和HCV RNA病毒载量。结果共对141288人份血样进行了检测,检出HBsAg（-）、HBV DNA阳性28例,总阳性率为0.020%,其中21例为anti-HBc阳性,占0.015%。HIV-1RNA未检出阳性,17例HBsAg（-）、HBV DNA阳性样本追踪发现,9例发生了血清转换现象,4例呈窗口期特征,所有追踪的HBV DNA阳性献血者ALT检测结果正常。1例anti-HCV（-）、HCV RNA阳性献血者追踪发现为典型窗口期献血,ALT显著升高。结论应采用高灵敏度的核酸扩增技术筛查血液中的乙肝和丙肝病毒,可提高血液安全。     

绍兴市无偿献血者中隐匿性HBV感染的基因型别及突变类型分析

目的 了解绍兴市无偿献血者隐匿性乙肝病毒感染情况及病毒的分子生物学特征.方法 应用ELISA的方法对绍兴市中心血站8692例无偿献血者进行筛查,对HBsAg阴性标本进行HBV DNA检测,从而检出隐匿性乙肝病毒感染者,再对阳性标本进行序列分析和氨基酸突变分析,从而了解绍兴市无偿献血者隐匿性乙肝病毒感染情况和病毒分子生物学特征,探讨隐匿性乙肝病毒感染的可能机制.结果 在总共8692例无偿献血者标本中,共计H BsAg（-）8644例,在8644例HBsAg（-）标本中,有8例HBV DNA阳性,隐匿性HBV感染比例为0.92‰（8/8692）,其中基因C型6例（75％）,基因B型2例（25％）;氨基酸突变分析显示有7株OBI病毒株在“a”表位发生特异性突变.结论 绍兴市无偿献血者中存在一定比例隐匿性乙肝病毒感染,隐匿性乙肝病毒感染与病毒基因突变有相关性.     

国产HBV DNA荧光定量PCR试剂检测准确性的初步分析

目的 评价国产HBV DNA荧光定量PCR试剂检测结果的准确性.方法 美国Roche公司COBAS TaqMan HBV Test对标本的HBV DNA定量为标准,评价国产HBV荧光定量PCR试剂(PG)对不同滴度的标本的检测准确性.结果 在病毒载量分别为10~1、10~2、10~3、10~4、10~5、10~6、10~7(IU/mL)范围内的标本中,国产试剂检测结果与Roche定量结果的相关性分别为:-0.08011、-0.05056、0.105642、0.312181、0.908046、0.866175、-0.23295;结果为阴性(低于检测范围)的比例分别为:60%、30%、33.3%、8.3%、0、0、0.对于病毒载量高于10~7(IU/ml)的标本,国产试剂检测结果全部低于美国Roche定量结果,至少低一个数量级.结论 国产HBV荧光定量PCR试剂线性范围较窄,仅对病毒载量在10~5～10~6(IU/ml)范围的标本检测结果准确,对于低病毒载量,尤其是低于10~3(IU/ml)的标本,结果不可靠,假阴性率较高.而对于病毒载量高于10~7(IU/ml)的标本检测结果也欠准确.因此,建议对病毒载量低于10~4(IU/ml)的标本采用美国Roche试剂检测,对于高于10~7(IU/ml)建议稀释标本后重新检测.     

孕产妇血清和乳汁中HBV DNA检测比较

目的探讨乙肝血清学阳性孕产妇血清和乳汁中HBV DNA阳性率及病毒载量的关系,以指导母乳喂养。方法采用荧光定量PCR方法检测乙肝血清学阳性孕产妇血清（168例）及乳汁（43例）中HBV DNA载量。其中对42例孕产妇的血清和乳汁同时进行了HBV DNA检测。结果乙肝血清学阳性孕产妇血清和乳汁中HBV DNA阳性率分别为65.63%（168/256）和36.44%（43/118）（P〈0.01）;病毒载量的对数值分别为5.82±1.52和4.42±1.26（P〈0.05）。42例配对的孕产妇阳性率分别为59.52%（25/42）和30.95%（13/42）（P〈0.01）;病毒载量的对数值分别为4.87±1.04和3.96±0.89（P〈0.05）。结论乙肝血清学阳性孕产妇血清中HBV DNA阳性率及病毒载量均高于乳汁中的阳性率和含量,对于乳汁阴性的哺乳期妇女可以进行母乳喂养。     

低载量隐匿性乙型肝炎病毒检测和序列分析

目的对低载量隐匿性乙型肝炎病毒进行Q-PCR定量检测和BCP序列分析。方法采用诺华公司NAT方法筛查35332份献血者HBV DNA,有18例确认为HBsAg-/HBV DNA＋,48例为可疑HBV DNA＋血样,使用Q-PCR和Nested-PCR方法对48例可疑血样进行定量检测和BCP区域扩增和测序;并与野毒株BCP序列作比对分析。结果从48例可疑HBV DNA＋血样中成功应用Q-PCR定量检出HBV DNA阳性6例,均为隐匿性乙型肝炎（OBI）,并获得6例BCP序列,发现在TA富含区的变异相对较多,1例样品发生缺失变异,6例OBI血样共有14个变异位点在BCP区域。结论本方法能对可疑标本进行检测和定量,通过BCP区域的检测进行OBI的确认。     

乙型肝炎病毒前S1抗原检测的临床意义

探讨乙型肝炎病毒前s1抗原（Pre-S1）检测在乙型肝炎病毒诊断中的临床意义。采用酶联免疫吸附试验（ELISA）和荧光定量聚合酶链反应技术（fluorescenee quantitative PCR,FQ-PCR）对650份HBV-M不同阳性模式及40份HBV—M全阴性模式血清标本进行乙型肝炎病毒Pre-S1、乙肝五项和HBV—DNA检测,并对三种检测结果进行统计学分析。在650份HBV—M不同阳性模式标本中,在119份大三阳标本中Pre—S1阳性检出率92．4％,HBV-DNA阳性检出率100％,在186份小三阳标本中Pre—SI阳性检出率42．5％,HBV—DNA阳性检出率63．4％,在21例HBsAg（＋）和HBcAb（＋）阳性组中Pres1阳性检出率47．6％,HBV．DNA阳性检出率66．7％;在297例HBsAb（＋）标本中Pre—S1阳性检出率0．4％,HBV-DNA阳性检出率0％,在268例HBV—DNA阳性的标本中Pre-S1阳性检出率79．3％。在40份HBV—M全阴模式中Pre-S1阳性检出率0％,HBV-DNA阳性检出率0％。Pre—S1在大三阳、小三阳及HBV-DNA阳性组阳性检出率明显高于阴性组（P〈0．01）,Pre—S1检测可补充和完善乙肝“两对半”检测的不足,尤其对HBeAg阴性或变异的HBV感染者能更好的反映病毒的复制状态和传染性。     

隐匿性乙型肝炎病毒感染检测的实验研究

目的 了解乙型肝炎病毒表面抗原（HBsAg）阴性个体HBV DNA检出情况,探讨检测乙型肝炎病毒表面大蛋白（HBLP）用于筛查临床隐匿性HBV感染的血清学检测策略.方法 根据日常检测乙型肝炎病毒血清学模式（HBVM）结果,将HBsAg阴性标本分为乙型肝炎病毒表面抗体（HBsAb）阳性和阴性两大类,除外HBVM抗原抗体同时阳性等特殊模式,从临床检测备份管随机收集各1000份共计2000份血清标本,双份分装-20℃冻存;用多管混合的方法实施HBV DNA定量分析,筛选出阳性标本;HBV DNA阳性标本行HBLP检测,再经美国超敏MONOLISA HBsAg ULTRA试剂复检HBsAg.结果 1000份HBsAb阳性标本未检出HBV DNA阳性;1000份HBsAb阴性标本共检出19例HBV DNA阳性;19份HBV DNA阳性标本经HBLP检测和美国超敏试剂复检HBsAg均呈阳性.19份HBV DNA定量分析结果显示：大于500拷贝/ml 2例,400～500拷贝/ml 3例,300～400拷贝/ml 3例,200～300拷贝/ml 7例,100～200拷贝/ml 4例.结论 用国产ELISA试剂常规检测HBsAg漏检标本多来自HBsAb阴性个体;漏检个体HBLP结果可能阳性,检测HBLP有利于筛查隐匿性HBV感染;本研究为积极寻找临床隐匿性HBV感染的检测策略提供了血清学参考.     

衢州市无偿献血者中隐匿性HBV感染的基因型别及突变类型分析

目的 了解衢州市无偿献血者隐匿性乙肝病毒感染情况及病毒的分子生物学特征.方法 对衢州市中心血站24 178例无偿献血者血液用北京万泰和Sorin公司生产的ELlSA试剂盒进行HBV的初检和复检,对HBsAg阴性标本进行HBV DNA检测,从而检出隐匿性乙肝病毒感染者,再对HBV DNA 阳性标本进行 S 基因片段序列分析和氨基酸突变分析,从而了解本市无偿献血者隐匿性乙肝病毒感染情况和病毒分子生物学特征,探讨隐匿性乙肝病毒感染的可能机制.结果 24 178例无偿献血者标本中,158例HBsAg阳性,24 020例HBsAg阴性标本中,15例HBV DNA阳性,隐匿性HBV感染比例为0.62‰（15/24 020）,其中基因C型9例（60％）,基因B型6例（40％）;S基因“a”表位氨基酸突变分析显示有11例隐匿性HBV感染病毒株在“a”表位发生突变.结论 衢州市无偿献血者中存在一定比例隐匿性乙肝病毒感染,隐匿性乙肝病毒感染与病毒基因突变有相关性.     

临床中隐匿性HBV感染的病毒血清标志物和T淋巴细胞亚群特征

目的 分析隐匿性乙型肝炎病毒感染(occult HBV infection, OBI)患者的病毒血清学标志物特征及外周T淋巴细胞亚群情况。方法 将2020年5月至2021年12月就诊于首都医科大学附属北京佑安医院血清HBsAg阴性、HBV DNA阳性的292例OBI患者作为OBI组,240例同期入院的HBsAg和HBV DNA持续阳性(>6个月)的慢性HBV感染患者为HBsAg阳性组,收集录入入组532例患者的基本病历资料和实验室检查数据,如HBV血清学标志物、血清HBV DNA载量、肝功能指标等;在OBI组中T淋巴细胞亚群检测数据完整的48例患者作为OBI亚组,同时根据性别和年龄在HBsAg阳性组中匹配48例HBsAg阳性患者作为HBsAg阳性亚组,20例同期入院体检的健康人作为健康对照组,收集此116例患者的外周血T淋巴细胞亚群数量和比值的临床数据。结果 OBI患者的主要血清学模式是：HBsAg阴性、抗-HBs阴性、HBeAg阴性、抗-HBe阳性、抗-HBc阳性,占总OBI组的48.97%;抗-HBs阳性率为45.21%,抗-HBe阳性率为85.62%,都明显高于HBsAg阳...     

丽水地区无偿献血者中隐匿性乙型肝炎病毒感染调查与分析

目的 隐匿性乙型肝炎感染一直是困扰献血中健康血清筛查的一个重要因素,通过高度灵敏的Nest-PCR检测方法对献血者中的OBI进行调查,了解该地区隐匿性HBV感染情况和基因分析.方法 共收集10080例献血者血浆,进行进口雅培HBsAg试剂和北京万泰抗-HBc及抗-HBs试剂平行检测,采用高灵敏度的Nest-PCR进行核酸检测和基因调取.结果 10080例无偿献血者中,共检出雅培（超敏）HBsAg阳性108例（阳性率1.07％）,抗-HBC单独阳性767例（阳性率7.67％）.10080例献血者中筛查出25例血浆HBsAg检测阴性HBV DNA检测阳性的隐匿性HBV携带者.隐匿性HBV感染者的发生率为0.25％.HBV基因C型12例（48％）,基因B型13例（52％）,未发现其他基因型.基因B型和基因C型之间没有明显的统计区别（P＞0.05）,序列分析显示其中5例在HBsAg“a”表位（aa124～aa147）存在突变（20％）.结论 我国无偿献血中存在较高比例的隐匿性乙型肝炎感染,且不同地区的基因型与突变情况存在差异.     

Characterization of occult hepatitis B virus infection from blood donors in China

Prevalence and characteristics of occult hepatitis B virus (HBV) infection (OBI) of genotypes B and C prevalent in China have not been extensively explored. Characterization of OBI strains obtained from Chinese blood donors was based on clinical and serological analyses, follow-up testing, and sequence analyses. Twenty-eight samples from 165,371 HBV surface antigen (HBsAg)-negative plasmas were confirmed HBsAg negative and DNA positive(HBsAg(-)/DNA(+)), of which 22 were classified as OBIs and 6 as window period infections. The OBI incidence was 1:7,517 in blood donors, whose ages ranged between 20 and 45 years (median, 28 years). OBI donors had normal alanine aminotransferase (ALT) levels and low viral loads ranging between unquantifiable amounts and 178 IU/ml (median, 14 IU/ml). Sequences from 21 basic core promoter/precore (BCP/PC) regions, five whole genomes, and two additional pre-S/S regions from OBI strains were compared to genotypes B and C HBsAg(+) reference strains. Eighty-six percent (6/7) of OBI strains were genotype C. Deletions, insertions, stop codons, and substitutions were detected in 15/21 (71%) core regulatory elements of OBI strains. Critical mutations were found in the core proteins of 5/5 OBI strains in parallel with random substitutions in pre-S/S proteins from 6/7 (86%) OBI strains. Critical mutations in core regulatory elements and core proteins might affect OBI genotype B and C strain replication. That there were few S protein substitutions suggests a minor role of the host immune defenses in OBI occurrence.     

广州市从业人员HBV及ALT检测结果分析

目的了解广州市从业人员乙型肝炎病毒（HBV）及丙氨酸氨基转移酶（ALT）的检测情况,为制定防控措施提供科学依据。方法对2009年在广州市疾病预防控制中心健康体检的从业人员的血清标本进行乙型肝炎病毒表面抗原（HBsAg）、乙型肝炎病毒e抗原（HBeAg）、ALT检测。结果从业人员的HBsAg阳性率为4.51%（10368/229738）,HBeAg阳性率为1.30%（2998/229738）,ALT异常率为0.92%（2125/229738）;30～岁组HBsAg阳性率最高（5.15%）,〈30岁组HBeAg阳性率最高（1.61%）;不同年龄组间的HBsAg和HBeAg阳性率比较差异有统计学意义（P〈0.001）;男性的HBsAg、HBeAg阳性率及ALT异常率比女性高,差异均有统计学意义（P〈0.001）。结论广州市从业人员HBV感染状况较我国一般人群携带率低,实行预防性健康体检、健康教育和疫苗接种等措施可逐步降低HBV感染。     

Hepatitis B virus (HBV) infection and recombination between HBV genotypes D and E in asymptomatic blood donors from Khartoum, Sudan

Sudan is a highly endemic area for hepatitis B virus (HBV), and >5% of blood donors are chronically infected. To examine potential strategies to improve HBV blood safety, 404 replacement donor samples previously screened for HBV surface antigen (HBsAg) were tested for antibody to HBV core (anti-HBc), anti-surface antigen (anti-HBs), and HBV DNA. Of 145 anti-HBc-containing samples (36%) identified, 16 retested were HBsAg positive (11%). Anti-HBs was detected in 43/77 (56%) anti-HBc-reactive samples. Six samples were HBsAg(-)/anti-HBc(+)/anti-HBs(+) and contained HBV DNA, meeting the definition of occult HBV infection (OBI). OBIs had low HBV DNA loads (<10 IU/ml) and were genotype B (n = 1) or genotype D (n = 5). Pre-S/S and/or whole genome sequences were obtained from 47 randomly selected HBsAg-positive donors added to the previous 16. Genotype E was identified in 27 strains (57.5%), genotype D in 19 strains (40.5%), and genotype A2 in 1 strain (2%). Two outlier strains within genotype D ultimately were identified as recombinants of genotypes D and E with identical recombination points, suggesting circulating, infectious, recombinant strains. Anti-HBc screening does not appear to be a sustainable blood safety strategy because of the cost and the negative impact on the Sudanese blood supply, even when reduced by anti-HBs testing. Being at the junction between two main African HBV genotypes, genetic recombination occurred and became part of the molecular epidemiology of HBV in Sudan.     

深圳地区2003—2012年献血者HBsAg／NAT检测结果情况分析

目的分析深圳地区2003—2012年期间献血者HBV感染的流行走势情况。方法所有献血者血样均使用进口与国产酶联免疫试剂,以及血液病毒核酸检测（NAT）方法筛查HBsAg和HBVDNA,采用X^2检验进行统计分析献血者感染HBV的流行趋势。结果深圳地区2003—2012年采血量稳步增长,HBsAg阳性检出率总体呈下降趋势。HBsAg＋／NAT＋与HBsAg＋／NAT-检出率结果相当（1．2：1）;献血者中HBsAg-／HBVDNA＋阳性率呈升高趋势。不同年龄组献血者HBV感染率差异无统计学意义（P〉0．05）。结论酶联免疫与NAT方法为重要的互补检测手段,能进一步确保输血安全,而隐匿性乙型肝炎病毒感染呈升高态势,应引起高度重视。     

聚乙二醇干扰素α-2a联合双环醇治疗高转氨酶水平慢性乙型肝炎疗效初探

目的 探讨慢性乙型肝炎患者接受聚乙二醇干扰素-2a(派罗欣)±核苷(酸)类似物(NUC)治疗前和治疗早期转氨酶明显升高者,联合双环醇(百赛诺)治疗的疗效.方法 收集HBeAg阳性/阴性慢性乙型肝炎患者,给予派罗欣180 μg,每周一次,皮下注射,疗程48周以上,治疗结束后随访26周.HBV DNA≥1×108拷贝/ml者联合NUC(阿德福韦或恩替卡韦).治疗前ALT＞ 500 U/L或派罗欣治疗第一针后ALT＞ 300 U/L者联用双环醇25 mg,每日3次,治疗1～2个月.治疗前2 × ULN＜ ALT ＜300 U/L或治疗后ALT＜ 300 U/L者单用抗病毒治疗.比较两组患者的治疗应答和不良反应状况.结果总计54例患者(HBeAg阳性44例,HBeAg阴性10例)完成治疗及随访,其中20例联用双环醇(联合治疗组),34例未联用双环醇(对照组).结果 显示双环醇联合治疗组于治疗后ALT水平逐周下降,4周后有90％( 18/20)患者ALT＜ 200 U/L,对照组为85.3％ (29/34例).随访26周时两组ALT复常率分别为80％(16/20)和85.3％ (29/34);病毒学应答率相仿,联合治疗组较对照组HBsAg血清学转换率有明显增高(P =0.044),分别为30％ (6/20)和11.8％(4/34).结论 双环醇可明显缓解干扰素治疗诱导的转氨酶升高反应,确保干扰素治疗顺利进行,且不影响其抗病毒疗效.     

Occult hepatitis B virus infection in Korean patients with isolated anti-HBc

Serological detection of isolated anti-hepatitis B core antibody (anti-HBc) can occur in various scenarios, but the most clinically relevant situation is occult hepatitis B virus (HBV) infection (OBI). The purpose of this study was to evaluate the prevalence and clinical relevance of isolated anti-HBc and of OBI with isolated anti-HBc from an unselected hospital population. A total of 14,253 patients referred for hepatitis B surface antigen (HBsAg)/anti-HBs testing were classified into either the Health Promotion Center (HPC) group or the patient group. For patients who were negative for both HBsAg and anti-HBs, an anti-HBc test was additionally performed. An HBV DNA real-time PCR test was performed on isolated anti-HBc patients, and their demographic and clinical data were reviewed. The measured prevalence of isolated anti-HBc and OBI in isolated anti-HBc patients was 5.9 % and 4.7 %, respectively. Prevalence was higher in males, elderly people, and the patient group than in females, the younger, and the HPC group, respectively. In most cases, the levels of HBV DNA load were very low (less than 200 IU/mL), and most cases of OBI presented without liver disease or history of HBV infection. The prevalence of isolated anti-HBc and OBI are affected by the methods of detection, subject population, and constituent factors such as sex and age. Although detection of HBV DNA does not always indicate infectivity, highly sensitive standardized HBV DNA tests are needed in clinical settings to exclude the possibility of OBI, especially in the advanced age group.     

乙型肝炎免疫球蛋白阻断乙型肝炎病毒母婴传播的临床研究

目的探讨乙型肝炎（乙肝）表面抗原（HBsAg）阳性孕妇及其新生儿采用乙肝免疫球蛋白（HBIG）阻断乙型肝炎病毒（HBV）母婴垂直传播的效果。方法将136例HBsAg（＋）的孕妇分为观察组（72例）和对照组（64例）,观察组孕妇于孕28、32与36周分别注射乙型肝炎免疫球蛋白（HBIG）,双阳性注射400IU,单阳性注射200IU;对照组只作随访及常规产检。两组的新生儿在出生6h内、第1、6个月时分别注射乙肝疫苗（HBvac）10μg、5μg、5μg;观察组新生儿在出生6h内臀部肌内注射HBIG 100IU。分别检测两组新生儿及6月龄婴儿血清中HBsAg、乙型肝炎表面抗体（HBsAb）及HBV DNA。结果观察组新生儿HBsAg和HBV DNA阳性率较对照组低,差异有统计学意义（P〈0．05和P〈0．01）。观察组6月龄婴儿HBsAb阳性率较对照组高,而HBV DNA阳性率较对照组低,差异也均有统计学意义（P〈0．01和P〈O．05）。结论HBsAg（＋）的孕妇应用HBIG可有效阻断HBV母婴传播,而新生儿出生时应用HBIG和HBvac联合免疫,可明显提高6月龄婴儿HBsAb阳性率。     

Detection of hepatitis B virus DNA in blood units with anti-HBc as the only positive serological marker

Serum samples from 10,629 blood donors were screened for hepatitis B virus (HBV) serological markers (HBsAg, anti-HBs, anti-HBc, anti-HBc IgM), anti-HCV, anti-HIV1/2 and ALT. Seventy five (0.7%) blood donors were found HBsAg-positive, 1,543 (14.5%) were carrying both anti-HBc and anti-HBs. whereas 507 (4.8%) samples were positive only for anti-HBc. Among the group of 507 anti-HBc positive samples, 303 were obtained from regular volunteer blood donors who were studied in two separate time intervals of at least 6 months' duration, and 204 were from first-time blood donors. The possibility of post-transfusion hepatitis B after donation of these 507 blood units was studied by determining the presence of HBV DNA as a marker of viral replication and infectivity. HBV DNA was detected by two methods (i) a chemiluminescent molecular hybridization assay, (ii) polymerase chain reaction (PCR) followed by DNA enzyme immunoassay (DEIA). Six out of 507 samples exhibited HBV DNA results in the gray zone of the hybridization assay, but were confirmed as negative by PCR DEIA. The other 501 samples were HBV DNA-negative by both methods, although 36 of them had increased ALT levels. No cases of post-transfusion hepatitis B were reported during the year in which these 501 blood units were provided. These results show that blood units which were positive only for anti-HBc, with normal ALT and were HBV DNA-negative may be considered not infectious for hepatitis B. Gray zone results of HBV DNA using hybridization quantitative assay must be confirmed as positive or negative by a more sensitive method such as PCR. Blood units which are anti-HBc-positive, with increased ALT levels and are HBV DNA-negative, which appear to not be related to HBV replication and infectivity, may be not safe for donation because of the potential existence of other as yet unknown, hepatotropic viruses.