上海地区无偿献血者乙肝病毒核酸检测分析

目的了解无偿献血者乙肝病毒核酸筛查(NAT)阳性人群特点,为血液安全策略提供参考。方法无偿献血者血液经Murex和科华HBsAg ELISA试剂检测,结果为阴性的血液使用cobas TaqScreen MPX试剂进行HBV DNA,HCV RNA,HIV RNA 3项联合核酸检测。对于MPX反应性标本,使用COBAS AmpliPrep/TaqMan进行核酸鉴别试验,同时使用罗氏ECL电化学发光检测系统进行乙肝补充血清学试验。结果 2011年11月1日～2012年1月31日3个月共有献血者86 375人(次),其中有63 351人(次)为初次献血者,HBsAg反应性为1.04%,23 024人(次)为重复献血者,HBsAg反应性为0.46%,两者差异有统计学意义(χ2=63.63,P0.05)。84 990份HBsAg、抗-HCV、抗-HIV1/2阴性血液进行MPX核酸检测,共发现52例(0.060%)HBV DNA阳性,均为低拷贝,含量为(20～200)IU/ml间,其中32例(0.051%)来自初次献血者,20例(0.087%)来自重复献血者,两者比例差异无统计学意义(χ2=3.65,P0.05),没有发现HCV RNA与HIV RNA阳性。结论重复献血者HBsAg反应性比率低于初次献血者;HBsAg阴性献血者HBV DNA阳性率为0.060%,重复献血者HBV DNA阳性率与初次献血者比较,两者差异无统计学意义;开展HBV核酸检测能够进一步保障血液安全。     

单独抗-HBs阳性献血者HBV DNA阳性原因分析

目的分析乙肝血清学仅抗-HBs(+)的献血者HBV DNA(+)的原因。方法对ELISA法HBsAg(-)/HBV DNA(+)献血者标本进行化学发光法乙肝血清学(HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc)检测和HBV DNA定量检测,对乙肝血清学中仅抗-HBs阳性者进行随访。将化学发光法乙肝血清学均阴性或仅HBsAg阳性者定义为ELISA法乙肝窗口期者,同样进行随访,作为对照。结果 2010年6月—2018年5月期间共检出23例单独抗-HBs(+)且HBV DNA(+),对其中4名献血者进行了随访:有1例随访时出现抗-HBc,并且抗-HBs数值显著上升,HBV DNA检测阴性;其余3例的乙肝血清学模式不变,且抗-HBs变化不大,HBV DNA检测结果或阴或阳。作为对照的7例窗口期献血者经随访均发生乙肝血清学模式的改变,其中6例出现抗-HBs/抗-HBc,1例只出现抗-HBs;HBV DNA检测均转阴。结论单独抗-HBs(+)的献血者HBV DNA(+)可能为乙肝疫苗注射后的突破感染,也可能为隐匿性乙肝感染。     

HBV DNA PCR检测在HBsAg阴性献血人群中的应用

目的 探讨HBsAg阴性献血者HBV DNA榆测的应用价值,评估核酸检测的必要性.方法 采用PCR检测HBsAg阴性献血者HBV DNA.采用8人份混合血样测定,超离心浓缩病毒,磁珠法提取病毒核酸.如HBV DNA为阳性,则进一步检测乙型肝炎病毒血清标志物5项.结果 HBVDNA检测限量为25 U/ml,23 225份标本中有4份为HBV DNA阳性,检出率为0.17‰.进一步检测其他HBV感染的血清学指标,发现这4份标本中有2份为抗HBe和抗HBc阳性,1份为抗HBc阳性,1份为抗HBs、抗HBc阳性.对HBV DNA的定量测定表明,其含量在50～200 U/ml.结论 现行的2次酶联免疫技术的血液筛查存在HBV漏检,有必要在现有的血液筛查模式中增加抗HBc检测,或增加病毒核酸筛查.     

Two HBV DNA+/HBsAg− blood donors identified by HBV NAT in Shenzhen, China

Background

In order to further improve blood safety, mini-pool (MP) nucleic acid testing (NAT) was implemented to screen samples negative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), anti-human immunodeficiency virus (anti-HIV), syphilis (anti-Treponemal antibody) and with normal ALT.

Study design and methods

From August 2006 to February 2008, 41,301 donations were screened using commercial HIV/HCV RNA and HBV DNA Real-Time PCR NAT assays in pools of 8. Reactive pools were re-tested as individual samples using the appropriate screening test and confirmed using an alternate commercial NAT assay. Donors reactive on both NAT assays were considered ‘confirmed’ positive for the virus concerned and recalled for additional follow-up testing and counseling.

Results

Of the 41,301 samples screened, no HIV or HCV RNA-positive/seronegative donations were detected but two HBV DNA positive/HBsAg negative blood donors (Donors 1 and 2) were identified. Their respective hepatitis immunological markers were: Donor 1 - anti-HBc positive/anti-HBe positive/HBeAg negative/ALT normal and HBV DNA viral load of 112 IU/ml; Donor 2 - anti-HBc positive/anti-HBe negative/HBeAg negative/ALT normal and HBV DNA viral load 2750 IU/ml.

Conclusions

MP NAT identified two HBsAg negative donors with presumed occult infection but no HIV or HCV seronegative/NAT positive (yield) donors. The HBV yield rate of 1 in 20,650 (95%CI - 1 in 5663 to 1 in 75,303) is comparatively high, exceeds the predicted rate based on previous modeling for the population and demonstrates the incremental blood safety value of NAT in countries where HBV is highly epidemic. The low viral load of the two yield samples underscores the importance of optimizing the sensitivity of the HBV NAT assay selected for screening.     

Molecular genotyping of Indian blood group system antigens in Indian blood donors

Background

Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, In^a and In^b. The In^a /In^b polymorphism arises from 252C?>?G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles.

True HIV seroprevalence in Indian blood donors

The National AIDS Control Organization (NACO), the apex body for controlling AIDS in India, projected that HIV seroprevalence would increase from 7/1000 in 1995 to 21.2/1000 in 1997. A high incidence (8.2%) of HIV was observed in blood donors. This study was carried out to find out the true HIV positivity in Indian blood donors. Blood donors from our centre were followed for more than 5 years to determine the true HIV seroprevalence and our result was compared with similar studies from India. Voluntary and relative blood donors who visited the SGPGIMS, Lucknow, since 1993 to June 1998 were included. They were screened for HIV 1/2 by ELISA kits (WHO approved). First-time HIV-positive samples were preserved frozen for further study (stage-I). They were repeated in duplicate and retested with other kits. If found positive, the sample was labelled as ELISA positive (stage-II). ELISA-positive samples were confirmed by Western Blot (WB) at stage-III. A total of 65 288 donors were included and 834 (12.8/1000) were reactive at stage-I. But 1.1/1000 donors were found to be ELISA positive at stage-II, and 0.28/1000 donors were positive by WB at stage-III. The 'seropositivity' rate from the NACO was significantly (P < 0.001) higher than our study. There were five similar Indian studies and seropositivity rate varied from 0.72/1000 (using ELISA and WB) to 5.5/1000 (using ELISA alone). The 'seropositivity' rate from the NACO was significantly (P < 0.001) higher than all these studies. HIV seroprevalence in the present study is lower (P < 0.001) than other Indian figures. The present and other studies confirmed that the projected HIV seroprevalence (82/1000) in Indian blood donors was high. The NACO result was based on one-time ELISA screening reports from zonal blood testing centres which also receive samples from paid donors donating in commercial blood banks. The HIV prevalence of blood donors (and national prevalence) is to be reassessed.     

HBsAg阴性献血者人群HBV感染的检测和输血残余风险分析

目的 了解献血人群乙型肝炎病毒(HBV)感染状况和血液经酶免疫法(EIA)筛查乙型肝炎病毒表面抗原(HBsAg)后经血传播HBV感染的残余风险.方法 采用国产和进口两种EIA试剂对献血者血液进行HBsAg筛查,罗氏诊断COBAS Ampliscreens NAT血筛系统检测EIA检测合格标本中HBV DNA,对HBV DNA阳性标本进行半套式PCR检测,并对PCR扩增产物进行测序和病毒基因亚型分析.结果 共筛查1998～2008年的献血者232 305例,发现HBsAg阳性2 999例,阳性率为1.3%;对2002～2007年EIA检测合格的113 639例献血者血液标本进行NAT检测,检测出13份HBV DNA阳性、HBsAg阴性的献血者血液,HBV残余风险高达1.1/10 000.结论 EIA筛查后血液安全性有了很好的保障,经血传播HBV残余风险依然处于较高的水平,NAT应用对提高血液安全,降低输血传播HBV残余风险意义重大.     

兰州地区献血者人群乙型肝炎病毒感染及S区基因分析

目的了解兰州地区无偿献血者乙肝病毒的感染情况,分析其S区基因的特征,探明本地区实行核酸筛查的安全性和可靠性,为HBV感染的监测策略提供重要依据,持续改进血液筛查方法,降低经输血传播HBV的风险。方法对2015年1月—2016年12月收集到献血者标本采用ELISA进行HBsAg筛查,将HBsAg(+)标本进行中和试验,对中和试验阳性和HBsAg(-)/HBV DNA(+)标本进行核酸提取,然后按照HBV/HCV/HIV-1 3项目分项做核酸病毒检测,再对HBV核酸检测阳性标本做S区基因分型,运用Mega 5.0将HBV S基因分型的结果做系统进化树,并分析其S区的氨基酸序列的变异。结果本次共筛查108 244份血液标本,HBsAg阳性率为0.34%;NAT检测108 045份,共有HBV DNA反应性标本32份,流行率为1∶3 376 (32/108 045);中和试验有63份标本检测,其中40份为阳性,中和阳性率为63.5%。40份中和试验为阳性的标本中,26份标本成功测通S区基因,其分型结果为C型26例;32份HBV DNA反应性标本中,11份成功测通S区基因序列,其分型结果为C型9例、D型2例。分析ELISA与NAT之间在S区的氨基酸序列的差异性,发现主要的突变位点是sV126 L/T/I,sT148L/V,sC149F/V,sA159G/D,sR160D/K和sW163/G/R,而sV168A/K位点可能是本地区的潜在突变位点。结论兰州地区献血者HBV S区流行株C和D,以C基因型为主,其氨基酸序列存在多个位点的变异,这些变异受基因型的影响,这些变异序列为血液筛查策略和筛查试剂的选择提供了依据。     

西宁地区无偿献血者抗-HIV检测结果分析

目的对青海西宁地区2008~2013年无偿献血者抗HIV检测结果分析,改进献血招募采集工作,杜绝经输血传播HIV的发生,保证临床输血安全。方法抗HIV检测采用ELISA方法,初筛阳性采用免疫印迹法(WB)进行确认。对检测结果进行统计并分析。结果共检测无偿献血者标本201 652例次,WB确认HIV感染者54例,阳性率为0.027%,感染者中以男性为主,占90.7%,以21~<41岁年龄段为高发年龄段,占68.5%。ELISA试验S/CO值与确证试验的阳性符合率成正比,单种ELISA试剂和2种ELISA试剂检测阳性结果与确证结果阳性符合率分别为0%和87%,54例感染者都为抗HIV1型,抗HIV的特异带gp160、gp120、P24、P17均100%出现。结论献血者中HIV感染整体处于低流行平稳趋势,无职业和自由职业者为主要感染人群,感染者从高危人群向社会各种人群传播,呈现感染人群多样化,流行形势复杂化的特点。实施和加强防治艾滋病知识在献血活动中的宣传,进一步强化招募各环节管理,不断提高检测水平,最大限度筛除高危人群献血,确保血液安全。     

HBsAg阴性献血者血清(浆)HBV DNA检测的意义

目的　明确HBsAg阴性献血者血清 (浆 )HBVDNA检测的应用价值。 方法　采用聚合酶链反应 (PCR)检测HBsAg阴性献血者血清 (浆 )HBVDNA。如HBVDNA为阳性 ,则进一步检测乙肝“两对半”血清学指标。结果　 5 0 0份标本中有 1 4份为HBVDNA阳性 ,检出率为 2 .8%。进一步检测其它HBV感染的血清学指标 ,发现这 1 4份标本中有 5份表现为抗 HBs、抗 HBe和抗 HBc一项或两项阳性。对HBVDNA的定量测定表明 ,其含量在 1 0 4～ 1 0 6拷贝数 /ml。结论　为尽可能减少输血后HBV感染的发生 ,有必要采用PCR方法检测HBsAg阴性献血者血清 (浆 )HBVDNA     

Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: implications for future HBV screening policy

BACKGROUND: Studies showing a significant correlation between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) deoxyribonucleic acid (DNA) levels have focused on the HBV seroconversion window period. STUDY DESIGN AND METHODS: HBsAg levels relative to HBV DNA results in 200 HBsAg-positive, anti-hepatitis B core antigen (HBc)-reactive blood donations were analyzed using quantitative polymerase chain reaction (PCR) (detection limit 400 copies/mL), two research PCR assays with increasing sensitivities (65 copies/mL and 1.3 copies/mL, respectively), and a quantitative HBsAg assay; HBsAg and HBV DNA levels were correlated with HBV serologic profiles; and the potential for replacing HBsAg screening with nucleic acid testing (NAT) was analyzed. RESULTS: Serologic profiles for over 90 percent of the donor samples were consistent with chronic HBV infection. Correlation between HBsAg and HBV DNA concentrations was weak (correlation coefficient = 0.33). Thirty-six percent (72/200) of donor samples had DNA levels under 400 copies per mL. Retesting of the 72 samples by more sensitive PCR assays showed that 60 out of 200 (30%) were positive by PCR with sensitivity of 65 copies per mL, whereas 6 out of 200 (3%) required PCR sensitivity of 1.3 copies per mL for positivity. Three percent (6/200) were negative by all three NAT assays. CONCLUSIONS: HBV DNA levels in HBsAg-positive, anti-HBc-reactive blood donations can be extremely low. About 6 percent of donations would be negative by current minipool HBV NAT methods. About 3 percent of donations would remain undetected by sensitive single-donor NAT. These results indicate caution in any consideration of dropping HBsAg screening.     

Significance of detecting anti‐HBc among Egyptian male blood donors negative for HBsAg*

Background: Screening for hepatitis B virus surface antigen (HBsAg) reduces the risk of transfusion‐transmitted hepatitis B viral (HBV) infection. However, the absence of HBsAg in the blood of apparently healthy individuals may not be sufficient to ensure the lack of circulating HBV. Blood containing anti‐hepatitis B core antibody (anti‐HBc) without detectable presence of HBsAg might be infectious; therefore, screening for anti‐HBc has been implemented in some countries resulting in a decrease in the risk of post‐transfusion HBV infection. Aim: To study the seroprevalence of anti‐HBc. The relationship between anti‐HBc positivity and the presence of circulating HBV among healthy blood donors negative for HBsAg will be helpful to decide whether supplemental testing may bring additional safety to blood products. Material and methods: A total of 1026 serum samples collected from HBsAg‐negative Egyptian healthy male donors were tested for the presence of anti‐HBc (both IgM and IgG types) using the competitive enzyme‐linked immunosorbent assay technique. Anti‐HBc‐positive samples were subjected to real‐time polymerase chain reaction to confirm the presence of HBV DNA. Results: Of the 1026 samples tested, 80 (7·8%) blood samples were found to be reactive to anti‐HBc. Of those, HBV DNA was detected in five of the samples (6·25%). The levels of detected viraemia were variable among the five donors. Conclusion: This study shows the insufficient effectiveness of HBsAg screening in protecting blood recipients from HBV infection. Inclusion of anti‐HBc testing should be considered in the screening of blood donors.     

Efficacy of hepatitis B virus (HBV) DNA screening and characterization of acute and occult HBV infections among blood donors from Madrid, Spain

BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI).
STUDY DESIGN AND METHODS: From January 2005 to December 2006, a total of 383,267 blood units were screened for hepatitis B surface antigen (HBsAg) and HBV DNA in two transfusion centers in Madrid, using either individual-donation nucleic acid testing (ID-NAT) or minipool (MP-NAT) of eight donations (MP8). Samples positive for HBV DNA and negative for HBsAg were confirmed by a second molecular test, the viral DNA was quantified, and a genome fragment including the region encoding the major hydrophilic region (MHR) of HBsAg was sequenced.
RESULTS: The overall yield of HBV DNA–positive, HBsAg-negative units was 1 in 21,282 (18 cases), higher when using ID-NAT than MP8-NAT (1:9862 vs. 1:51,011; p < 0.01). Four donations (1/95,817) were collected during the infectious pre-HBsAg WP, one during an early recovery stage, and the remaining 13 (1/29,482) were OBIs, six of whom had no detectable antibody to HBsAg. Low-level Genotype D HBV DNA was detected in all OBI cases; the frequencies of this genotype and MHR amino acid substitutions were significantly higher than reported from unselected Spanish HBsAg carriers. Donors with OBI had normal aminotransferase levels and were significantly older than donors carrying HBsAg.
CONCLUSIONS: Blood donors in the WP and with OBI are not uncommon in Madrid and are detected at a higher frequency with ID-NAT than MP-NAT.     

献血者HBV DNA存在的确认与S蛋白变异特征分析

目的对HBsAg阴性和阳性献血者血样HBV DNA存在的确认并分析隐匿性乙型肝炎病毒S区变异特征。方法使用EIA/NAT方法筛查深圳地区19 397份无偿献血者血样,把109例乙肝不合格样品分成3类(HBs Ag+/NAT+、HBs Ag+/NAT-、HBs Ag-/NAT+),通过跟踪检测,确认为OBI毒株10例、HBV窗口期感染期3例和5例缺失追踪的HBs Ag-/HBV DNA+样品,采用荧光定量聚合酶链反应(QPCR)测定HBV病毒载量,应用NestedPCR技术扩增S基因片段并测定序列,与B/C基因型HBs Ag+/HBV DNA+阳性野毒株序列比对。结果深圳市无偿献血者经乙肝表面抗原胶体金快速试纸筛查后的HBs Ag阳性检出率为0.34%(66/19 397);隐匿性乙型肝炎病毒感染(OBI)的流行率范围为1∶1 939-1∶1 293,HBV窗口期感染流行率范围为1∶6 465-1∶2 424;10例OBI样品其病毒载量介于不能定量至112.0 IU/m L(中位数98.5 IU/m L)。10例OBI样本在S蛋白区(nt215-710)出现随机变异,OBI样品S区氨基酸置换率显著高于野毒株(P0.000 1),有4、2、3个OBI样品分别在CTL表位21-29、86-96、172-180出现L21S(2)、K/R24E(1)、I25M(1)、L88P(2)、S172F/L(2)、V178T(1)变异;OBI非CTL表位免疫区的氨基酸置换率亦显著高于野毒株(P0.05);其中1个OBI样品在nt636发生缺失变异。结论深圳献血者OBI流行率有增高趋势,OBI发生机制与乙型肝炎病毒的S蛋白区变异,特别是免疫活性区的变异密切相关。     

Evaluation of serologic screening of blood donors in India reveals a lack of correlation between anti-HBc titer and PCR-amplified HBV DNA

BACKGROUND: Transfusion associated-HBV (TAHBV) is estimated at approximately 1.5 percent in postsurgical recipients and 50 percent or more in multiple-transfusion recipients in India. Not transfusing blood with high-titer anti-HBc, which reportedly correlates with the presence of HBV DNA, helped reduce TAHBV in Japan. This study tested anti-HBc-reactive donors for PCR-amplified HBV DNA and its correlation with anti-HBc titers. STUDY DESIGN AND METHODS: In total, 30,853 donors from Cohort 1 (24,694 in 2001) and Cohort 2 (6159 in 2000) were screened for anti-HBc and anti-HBs. Amplification of HBV DNA was performed on an unselected subset of 147 out of 3304 anti-HBc-only sera from Cohort 1 and 230 out of 6159 from Cohort 2. Correlation of anti-HBc titers in DNA positive (n = 48), DNA negative (n = 40), anti-HBs reactive (n = 44), and HBsAg reactive (n = 45) donors was by Fisher's exact test. RESULTS: In Cohort 1, 2673 (10.82%) donors were reactive for anti-HBc, of whom 1038 (4.20%) were anti-HBc only. HBV DNA was detected in 40 out of 147 (27.21%) and 48 out of 230 (20.87%) donors with anti-HBc only from the two cohorts. Anti-HBc titers detected no significant difference between the first three groups. CONCLUSION: Cryptic HBV infection was observed in approximately 25 percent of anti-HBc-only donors. No correlation was established between HBc titers and presence of HBV DNA.     

血液HBV DNA全自动检测及基因分型分析

目的建立血液HBVDNA标本汇集、核酸提取、扩增及检测的全自动筛查模式,对阳性标本进行基因分型和血清学追踪检测,为血液HBVDNA自动化筛查提供科学依据。方法在酶联免疫吸附试验(ELISA)筛查血液基础上,应用STAR2000加样仪自编程序进行全自动血样汇集(24人份),在MPLC仪上全自动提取标本核酸,应用PCR方法在COBASAMPLICOR进行扩增和检测分析结果,用国际标准核酸质控品考评检出限量,对阳性标本进行基因分型并追踪血清转换过程。结果全自动汇集、全自动核酸提取和扩增及检测HBVDNA95%检出限量为38.9IU/ml,95%CI为(21323)。通过对16512个标本共688个汇集池分析,HBVDNA阳性8例,阳性率为0.049%。其中C型3人,B型2人,D型1人,2人未能确定基因型,6例阳性标本追踪发现,3例发生了血清转换现象。结论HBVDNA全自动核酸检测方法可应用于24人份血液混样核酸筛查。