乙型肝炎病毒表面大蛋白检测用于筛查隐匿性HBV感染

目的 用血清学方法检测乙型肝炎病毒表面大蛋白(HBLP)来筛查隐匿性HBV感染,探讨临床隐匿性HBV感染的血清学检测策略.方法 在日常工作中从临床榆测备份管随机收集2000份用国产ELISA试剂检测HBsAg阴性结果的血清标本,双份分装-20℃冻存,单份标本实施HBLP检测,HBLP阳性标本增加另外两种共三种国产ELISA试剂双份复查HBsAg;HBLP阳性标本再经美国超灵敏MONOLISA HBsAg ULTRA试剂双份检测HBsAg,并行双份DNA定量分析、结果取均值.结果 2000份HBsAg阴性标本共检出15例HBLP阳性:HBLP阳性标本用三种国产ELISA试剂双份复查HBsAg结果一致阴性;通过美国超灵敏MONOLISA HBsAS ULTRA试剂复查HBsAg结果全部阳性;前述15份标本HBV DNA定量分析结果显示均小于500拷贝/ml,其中400～500拷贝/ml 1例,300～400拷贝/ml 3例,200～300拷贝/ml 5例,100～200拷贝/ml 4例,小于100拷贝/ml 2例.结论 用国产常规ELISA试剂检测HBsAg普遍存在漏检,漏检标本HBLP结果可能阳性,检测HBLP有利于筛查出隐匿性H BV感染,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.     

隐匿性乙型肝炎病毒感染检测的实验研究

Objective To see the HBV DNA detection instance in the HBsAg negative people and to study the serological method detection strategy for detecting hepatitis B virus large surface protein (HBLP) to filtrate the occult HBV infection.Methods The HBsAg negative serum samples were divided into HBsAb negative and positive two species according to the hepatitis B virus markers (HBVM) in daily work excepting the special HBVM modes.Totsl 2000 stochastic serum samples with 1000 HBsAb negative results and 1000 HBsAb positive results were collected from the copy tubes to detect HBVM with national ELISA reagent kits and put them-20℃ frostily.Mixed samples(8 × 30μl)were analyzed with fluorescence quantitative PCR (FO-PCR)and filtrated the individual positive samples.The filtrated samples were doubly tested again with American MONOLISA HBsAg ULTRA reagents.Results No HBV DNA positive results were found out from the 1000 HBsAb positive samples and 19 cases HBV DNA positive results were found out from the 1000 HBsAb negative samples.On these 19 samples,the HBsAg results from the American MONOLISA HBsAg ULTRA reagents were all positive and the HBLP results were all positive,too.The 19 HBV DNA quantitative results were divided into 2 cases more than 500 copies/ml,3 cases between 400-500 copies/m1,3 cases between 300-400 copies/ml,7 cases between 200-300 copies/ml and 4 cases between 100-200 copies/ml.Conclusion The leaked samples tested HBsAg with national reagents are mostly from the HBsAb negative people.HBLP results may be positive on these samples and detecting HBLP marker is propitious to filtrate the occult HBV infection.This study provided a kind of serological reference for actively searching for the detecting strategy in occult HBV infection field.     

隐匿性乙型肝炎病毒感染检测的实验研究

Objective To see the HBV DNA detection instance in the HBsAg negative people and to study the serological method detection strategy for detecting hepatitis B virus large surface protein (HBLP) to filtrate the occult HBV infection.Methods The HBsAg negative serum samples were divided into HBsAb negative and positive two species according to the hepatitis B virus markers (HBVM) in daily work excepting the special HBVM modes.Totsl 2000 stochastic serum samples with 1000 HBsAb negative results and 1000 HBsAb positive results were collected from the copy tubes to detect HBVM with national ELISA reagent kits and put them-20℃ frostily.Mixed samples(8 × 30μl)were analyzed with fluorescence quantitative PCR (FO-PCR)and filtrated the individual positive samples.The filtrated samples were doubly tested again with American MONOLISA HBsAg ULTRA reagents.Results No HBV DNA positive results were found out from the 1000 HBsAb positive samples and 19 cases HBV DNA positive results were found out from the 1000 HBsAb negative samples.On these 19 samples,the HBsAg results from the American MONOLISA HBsAg ULTRA reagents were all positive and the HBLP results were all positive,too.The 19 HBV DNA quantitative results were divided into 2 cases more than 500 copies/ml,3 cases between 400-500 copies/m1,3 cases between 300-400 copies/ml,7 cases between 200-300 copies/ml and 4 cases between 100-200 copies/ml.Conclusion The leaked samples tested HBsAg with national reagents are mostly from the HBsAb negative people.HBLP results may be positive on these samples and detecting HBLP marker is propitious to filtrate the occult HBV infection.This study provided a kind of serological reference for actively searching for the detecting strategy in occult HBV infection field.     

乙型肝炎特殊血清学表现模式的探讨

目的 探讨乙型肝炎病毒（HBV）特殊血清学表现模式。方法 A试剂检测到的乙肝特殊模式标本用B、C两种试剂重检；对HBeAg阳性、HBsAg阴性标本用倍比稀释和二步法重做HBsAg；以聚合酶链反应（PCR）检测乙肝特殊模式的HBV DNA。结果 A试剂检测到的乙肝特殊模式10种，145例；分为“12”同时阳性和“3”阳性、“1”阴性两个模式组；用B、C两种试剂复检的结果同A试剂相比存在较大差异；用倍比稀释和二步法检测“35”模式的HBsAg，阳性率分别提高到75．0％和80．0％，与其HBV DNA的阳性率（74．0％）相一致。结论 不同厂家试剂对乙肝特殊血清学模式的检测结果存在差异。“35”模式标本一步法试剂检测HBsAg漏检率高，用倍比稀释或二步法重检可提高HBsAg的阳性率，应结合HBV DNA的检测结果作出综合判断。     

抗-HBc单项阳性患者中的隐匿性HBV感染

目的 研究抗-HBc单项强阳性患者中的隐匿性HBV感染发生率并分析发生原因.方法 收集183例血清学检测抗-HBc单项强阳性（A≤0.1）患者血清标本,采用实时定量PCR进行HBV DNA含量检测.对于DNA定量阳性的标本,PCR扩增HBV pre-S/S区基因,并进行克隆测序.结果 183例血清标本中3例HBV DNA定量结果大于103拷贝/ml,占1.6%.这3例标本中有2例得到pre-S/S区测序结果,2例标本均存在S基因＂a＂决定簇内Q129R/P点突变,且突变株与野生型共存.结论 抗-HBc单项阳性患者中存在隐匿性HBV感染,HBsAg血清免疫学方法的漏检与HBV S基因突变有关,同时与循环中HBsAg量低于检测限也有一定关系.HBV隐匿感染不仅可以造成临床诊断失误,更为严重的是献血员HBV隐匿感染造成血液的污染.     

乙肝两种血清学模式HBV DNA与HBV M定量结果的关系

近年来有关乙型肝炎病毒核酸(HBV DNA)与乙型肝炎病毒血清学标志物(HBV M)的定性评价已有报道[1-3],我们对HBsAg阳性感染者的HBV DNA和HBV M进行定量测定,并对两种常见阳性模式(HBsAg/HBeAg/抗-HBc,HBsAg/抗-HBe/抗-HBc)的HBV DNA与HBV M结果进行定量分析,揭示两种模式HBV DNA与HBV M之间的关系。1材料和方法1.1材料收集HBsAg阳性血清标本共225份。HBV DNA采用荧光定量PCR测定,仪器为PE7000型PCR仪及上海申友试剂;HBV M采用微粒子酶免法,仪器为AXSYM免疫分析仪及配套试剂。1.2方法对225份血清标本进行HB…     

隐匿性乙型肝炎病毒感染的检测探讨

目前临床隐匿性乙型肝炎病毒(HBV)感染的筛查主要依靠分子生物学手段~[1],尚无简便、经济、适合大批量常规筛查的方法,如ELJSA.本实验旨在了解HBV表面抗原(HBsAg)阴性个体的HBVDNA检出情况,探讨HBV表面大蛋白(HBLP)用于常规筛查隐匿性HBV感染的可行性,积极寻找临床筛查隐匿性HBV感染的检测策略.     

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.     

隐匿性乙型肝炎病毒感染的检测探讨

目前临床隐匿性乙型肝炎病毒(HBV)感染的筛查主要依靠分子生物学手段[1],尚无简便、经济、适合大批量常规筛查的方法,如ELJSA.本实验旨在了解HBV表面抗原(HBsAg)阴性个体的HBVDNA检出情况,探讨HBV表面大蛋白(HBLP)用于常规筛查隐匿性HBV感染的可行性,积极寻找临床筛查隐匿性HBV感染的检测策略.     

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。     

Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.

Methods

Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.

Results

Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.

Conclusions

Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.     

HBV-DNA阳性育龄妇女HBV血清标志物、前S1抗原的检测分析

目的：探讨HBV—DNA阳性育龄妇女病毒复制与HBV标志物及前S1抗原的关系。方法：对1643例慢性乙型肝炎育龄妇女采用荧光定量PCR法检测血清HBV—DNA,酶联免疫吸附法（ELISA）检测HBV标志物和前S1抗原。结果：HBV—DNA阳性育龄妇女432例。其中,HBsAg阴性者占19．44％,前S1抗原总阳性率55．09％,且随着HBV—DNA载量增加,前S1抗原阳性率也升高。结论：采用HBV—DNA、HBV标志物、前S1抗原联检,才能更准确提供育龄妇女HBV感染诊疗依据,有效控制HBV感染率。     

Does Hepatitis B Virus Prenatal Transmission Result in Postnatal Immunoprophylaxis Failure?

The objective of this work was to evaluate whether postnatal hepatitis B immunization failure in children is caused by prenatal infections. A prospective study was conducted from October 2006 to September 2008. Fetal samples from HBsAg-positive mothers were retrieved by either amniocentesis or cordocentesis (percutaneous umbilical blood sampling [PUBS]). Hepatitis B virus (HBV) serologic markers (HBVM) and quantitative HBV DNA assays were performed to assess prenatal infection. All neonates were given combined HBV immunoprophylaxis after delivery. The newborns were followed up with HBV serologic testing at 1 year old. For the 252 pregnant women recruited, 16 fetuses were found to be HBV DNA positive, with all HBV DNA levels under 10(4) copies/ml. HBsAg and HBV DNA detected in the uterus were uncommon and were expressed at low levels. In contract to the case with prenatal statuses, neonatal serologies were more similar to their mothers'. The response rate of vaccination was 95%. Six children for whom immunoprophylaxis failed were born to HBeAg-positive mothers with high HBV DNA levels (>10(8) copies/ml), but only one of them was found to be positive for intrauterine HBV DNA (8.5 × 10(2) copies/ml). The presence of intrauterine hepatitis B antigen and DNA does not indicate postnatal HBV infection and vaccination failure.     

中国北方地区隐源性肝炎及乙型肝炎表面抗原阴性肝癌患者中隐匿性HBV感染流行状况分析

目的调查中国北方地区隐源性肝炎及乙肝表面抗原（HBsAg）阴性肝癌患者中隐匿性乙型肝炎病毒（HBV）感染的流行状况。方法收集393个受试者的血清,其中包括隐源性慢性肝炎患者215名、HBsAg阴性肝癌患者178名。使用巢式PCR的方法检测血清中的HBV-DNA,同时使用实时定量PCR的方法检测HBV—DNA的载量。结果在隐源性慢性肝炎患者、HBsAg阴性肝癌患者中隐匿性HBV感染流行率分别为23．7％（51／215）和68．5％（122／178）。在IgGanti—HBc阳性者中,隐匿性HBV感染率较高。所有隐匿性感染者的病毒载量均较低（〈10^5拷贝／ml）。结论在中国北方隐源性肝炎及肝癌患者中,隐匿性HBV感染率较高。因此,对隐匿性HBV感染应给及足够的重视,避免因输血及器官移植造成HBV的传播。     

Genotype, phylogenetic analysis, and transmission pattern of occult hepatitis B virus (HBV) infection in families of asymptomatic HBsAg carriers

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the serum in absence of hepatitis B surface antigen (HBsAg). Studies were conducted to screen for occult HBV infection among family members of HBV carriers, incidentally detected positive for HBV infection with a view to assess the pattern of virus transmission among them. Nested PCR assay, employing independent sets of primers to surface and core genes, was used for detection of HBV DNA in serum samples from 28 index cases with asymptomatic HBV infection, and in serum samples from 72 HBsAg negative/anti-HBc positive family members. HBV DNA was detected in 15 HBsAg negative family members of 10 HBsAg positive index patients and was studied in detail. Direct sequencing of S gene region of 25 isolates (10 index cases and 15 contacts) and phylogenetic analysis with data base sequences revealed that genotypes A, C, and D and subtype adw2, adr, and ayw3 were present among them. Evidence of transmission from outside family sources was found in addition to intrafamilial transmission among individuals with occult infection. Mutations in the major hydrophilic loop (MHL) of the S gene region were also detected, including the 'vaccine escape' mutation G145R in three cases. Although majority of the occult infection was associated with low viral load, 3/15 (20%) cases were with higher viral load and potential infectivity. These cases are especially notable in diagnostic, blood banking, and transplantation services.     

Occult hepatitis B in persons infected with HIV is associated with low CD4 counts and resolves during antiretroviral therapy

Occult hepatitis B virus (HBV) is defined by the presence of plasma HBV DNA in individuals with HBV core antibodies (anti‐HBc), but without HBV surface antigen (HBsAg). The prevalence of occult HBV in HIV‐infected patients remains controversial, and the risk factors, clinical significance and effect of highly active antiretroviral therapy (HAART) are unknown. The aim of this study was to determine prevalence, risk factors, and clinical significance of occult HBV in HIV‐infected patients and to evaluate the effect of HAART. Plasma HBV DNA levels were determined in 191 HIV positive, antiretroviral naïve patients, who were anti‐HBc positive and HBsAg negative. Quantitative HBV DNA was determined using a Taqman real‐time nested PCR. Additionally, plasma HIV RNA levels, CD4 cell counts, anti‐HBs‐antibodies, anti‐HCV‐antibodies, ALT, AST, and γGT were determined. Occult HBV (a plasma HBV DNA level >50 copies/ml) was detected in 9/191 (4.7%) of the patients. Among 45 anti‐HBs‐negative patients (isolated anti‐HBc positive), the prevalence was 11.1%. Patients with occult HBV had significantly lower CD4 count compared to anti‐HBc‐positive/HBsAg negative/HBV DNA‐negative patients (105 ± 157 (median ± SD) vs. 323 ± 299 cells/mm³, P = 0.019). When HAART (including lamivudine) was initiated in the patients with occult HBV, HBV DNA was no longer detectable in any of the patients during 3 years of follow‐up. In conclusion, occult HBV was associated with low CD4 counts and may be viewed as opportunistic reactivation of HBV that resolves as a consequence of HAART induced immune reconstitution and/or the effect of lamivudine. J. Med. Virol. 81:441–445, 2009. © 2009 Wiley‐Liss, Inc.     

High prevalence of occult hepatitis B virus infection in Taiwanese intravenous drug users

The epidemiology and impact of occult HBV infection in intravenous drug users remain largely unknown. The aim of the study was to investigate the prevalence of occult HBV infection among intravenous drug users in Taiwan. Molecular assays were used to determine the level of serum HBV DNA and the genotype in 304 intravenous drug users negative for both HBsAg and anti-HCV. Of 304 intravenous drug users, 125 (41.1%) were positive for serum HBV DNA. The genotype distribution of HBV was as follows: B, 55 (44%); C, 29 (23%); and mixed B and C infections, 41 (33%). The mean and median serum HBV DNA levels in 125 intravenous drug users with occult HBV infection were 4.0 +/- 0.6 and 4.0 log(10) copies/ml, respectively. The mean serum HBV DNA level in carriers with mixed genotype B and C infections was significantly higher than those infected with HBV genotype B or genotype C alone (mean, 4.2 +/- 0.6 log(10) vs. 3.9 +/- 0.5 log(10), and 3.9 +/- 0.7 log(10) copies/ml, P = 0.01 and 0.05, respectively). The amino acid sequence determination of HBV surface gene in 20 intravenous drug users with occult HBV infection selected at random showed no mutation of amino acid at codon 145. In conclusion, the prevalence of occult HBV infection and mixed HBV genotype infections are not uncommon in intravenous drug users residing in an HBV endemic areas. In addition, intravenous drug users with occult mixed genotype B and C infections have significantly higher viral loads than those with occult infection of single HBV genotype.     

Occult hepatitis B virus infection in patients with leprosy

Leprosy patients may present with immune system impairment and have a higher hepatitis B virus (HBV) seroprevalence, justifying the investigation of occult HBV infection in these individuals. The aim of this study was to verify the frequency and the clinical factors associated with occult HBV infection in leprosy patients. Between 2015 and 2016, leprosy patients from a reference center in Brazil were interviewed to assess clinical data. Blood samples were collected for the screening of HBV serological markers using enzyme-linked immunosorbent assay. Patients with negative hepatitis B surface antigen (HBsAg) that had positive anti-HBc and/or anti-HBs were selected for HBV DNA detection using real-time polymerase chain reaction. SPSS was used for data analysis. Among 114 selected patients, six were identified with occult infection (5.3%) and five of them with multibacillary leprosy. Three patients with occult infection had a history of a type 2 reaction (P = 0.072; OR, 4.97; 95% CI, 0.87-28.52). Only two patients with occult infection had isolated anti-HBc, while three had isolated anti-HBs, including those with the highest HBV DNA titers. In conclusion, in leprosy patients with negative HBsAg and positive anti-HBc and/or anti-HBs, occult HBV infection occurs in 5.3% and can be found even in patients with isolated anti-HBs.