丙型肝炎病毒分片段抗体检测蛋白质芯片质控参比品的建立及质量验证

建立能同时检测丙型肝炎病毒(HCV)嵌合抗体、核心抗体和NS3、NS4、NS5抗体的蛋白质芯片质控参比品,对质控合格的芯片进行质量验证。用3种HCV EIA试剂分别检测从3家医院收集的丙型肝炎病毒感染患血清及其他非HCV感染患血清,从3种EIA试剂同时阴性或阳性的血样中挑取阳性和阴性血清,然后用RNA hyb PCR试剂进行检测,从中再选取部分样本用RIBA3．0进行检测,确定HCV分片段抗体检测蛋白质芯片质控参比品。经质检合格的芯片用中国药品生物制品检定所的HCV参比品进行检定。通过490例临床标本的检测对芯片的质量进行进一步的验证。从收集的240份丙型肝炎病毒感染患血清及其他非HCV感染患血清筛选出30份血样(15份阳性,15份阴性)作为HCV分片段抗体检测蛋白芯片质控参比品。中国药品生物制品检定所的80份HCV参比品检定结果表明,混合抗体阳性检出率为39／40,阴性符合率为40／40,总符合率为98．7％;核心抗体阳性检出率为27／40,阴性符合率为40／40;NS3抗体阳性检出率为26／40,阴性符合率为39／40;NS4抗体阳性检出率为19／40,阴性符合率为40／40;NS5抗体阳性检出率2／40,阴性符合率为40／40。490例临床标本的检测结果表明,对于194例HCV阳性标本,蛋白质芯片混合抗体与ELISA的符合率达99．5％,分片段抗体符合率达97．4％,两种方法检测结果不符的标本经RIBA试剂确认,蛋白质芯片与RIBA试剂的符合率高度一致。对于296例各种HCV抗体阴性标本,蛋白质芯片检测结果全部为阴性。以上结果表明,制备的丙型肝炎病毒分片段抗体检测蛋白质芯片质控参比品可用于芯片生产的质量控制,经质控合格的芯片符合国家标准的要求,可用于临床检测。     

NASBA荧光分子信标技术定量检测丙型肝炎病毒

建立NASBA荧光分子信标探针检测技术,并对国家HCV标准品、人工构建HCVRNA野生株及HCV抗体阳性不同人群进行检测。实验结果:该方法检测HCV的灵敏度为103拷贝ml血清,阴性参比品的符合率为100%;检测的线性范围为103拷贝～109拷贝ml血清;精密性(CV值)小于6%,在HCV抗体阳性人群中HCVRNA的检出率在45%～65%之间。结论:该方法在HCVRNA临床定量检测中具有良好的灵敏度、特异性、重复性与实用性。     

吸毒人群中HIV/HCV核酸和抗体关系的分析

研究了高危人群中HIV/HCV核酸和抗体的关系.从新疆地区采集吸毒人群的血样,并对其进行HIV/HCV核酸和抗体的检测.320例吸毒人员血浆样品中HCV抗体阳性为80.3%,HIV抗体阳性率为41.9%,HIV和HCV共感染者为38.3%.HIV RNA与抗体的总符合率为98.8%,在186例HIV抗体阴性样品中可能有2例为HIV感染的窗口期.HCV抗体和HCV RNA的阳性符合率为92.6%,HCV RNA与HCV抗体的总符合率为90.0%,以上结果说明在HIV/HCV的高流行区进行HIV/HCV核酸检测可以发现病毒感染的窗口期,而约8%的HCV抗体阳性样品为病毒核酸阴性,也值得进一步研究.     

重组HIV-1抗原的应用及评价

将纯化的基因重组HIV-1P24和融合蛋白P24-gp41包被微孔板,用ELISA分别检测正常人血清和HIV-Ab国家参比品（Panel),rP24对20份HIV-Ab阳性Panel的检出率为95%（19/20）,对20份HIV-Ab阳性Panel通过率为90%（18/20）,P24-gp41对20份HIV-Ab阳性Panel检出率为90%（18/20）,对20份HIV-Ab阴性Panel的通过率为60%（12/20）;rP24和P24-gP41对正常人血清的检测结果均为阴性,结果表明研制的P24具有较高的敏感性和特异性,可作为组份抗原用于HIV抗体诊断试剂盒的生产。     

吸毒人群中HIV/HCV核酸和抗体关系的分析

研究了高危人群中HIV/HCV核酸和抗体的关系。从新疆地区采集吸毒人群的血样,并对其进行HIV/ HCV核酸和抗体的检测。320例吸毒人员血浆样品中HCV抗体阳性为80．3％,HIV抗体阳性率为41．9％,HIV 和HCV共感染者为38．3％。HIV RNA与抗体的总符合率为98．8%,在186例HIV抗体阴性样品中可能有2例 为HIV感染的窗口期。HCV抗体和HCV RNA的阳性符合率为92．6％,HCV RNA与HCV抗体的总符合率为 90．0％,以上结果说明在HIV／HCV的高流行区进行HIV／HCV核酸检测可以发现病毒感染的窗口期,而约8% 的HCV抗体阳性样品为病毒核酸阴性,也值得进一步研究。     

江苏省300例艾滋病人群联合检测 HCV、TP、HIV的结果分析

摘要 目的：探究江苏省2017年至2019年度艾滋病哨点监测人群丙型肝炎病毒（Hepatitis C virus,HCV）、梅毒螺旋体（Treponema Pallidum,TP）以及人类免疫缺陷病毒（Treponema Pallidum,HIV）感染状况,以期为制定对应的疾患干预手段提供临床数据支撑。方法：严格按照《全国艾滋病哨点监测实施方案操作手册（2012版）》对入组的900例个体开展相关问卷调查,并对入组对象进行HCV、TP以及HIV感染血清学检测,分别统计3年总体感染情况、年度感染情况、不同年龄段人群感染情况以及合并感染情况。结果：（1）性工作者HCV、TP以及HIV感染率分别为3.00 %、4.00 %和9.00 %,吸毒人群感染率分别为13.00 %、73.00 %和13.00 %,孕产妇感染率分别为1.00 %、0.67 %和1.33 %,比较显示,吸毒人群HCV、TP以及HIV感染率明显高于其余两类人群（P<0.05）;（2）就各年度不同人群HCV、TP以及HIV感染率开展年度横向比较显示,不同年度不同人群的HCV、TP以及HIV感染率差异无统计学意义（P>0.05）;（3）针对不同年龄段不同人群的HCV、TP以及HIV感染率开展比较显示,吸毒人群中≥50岁HCV和HIV感染率明显高于其他年龄段（P<0.05）,性工作者中18~20岁HIV感染率明显高于其他年龄段（P<0.05）,其余差异无统计学意义（P>0.05）;（4）合并感染情况分析显示合并TP以及HIV感染率要高于其他合并感染。结论：吸毒人群是江苏省HCV、TP以及HIV感染高发群体,以≥50岁年龄段感染率最高,总体来看HCV、TP以及HIV感染率呈现逐年降低趋势,但仍应该加强对上述疾患的防控工作。     

化学发光微粒子免疫法和梅毒螺旋体颗粒凝集试验检测梅毒抗体的比较

目的评价化学发光微粒子免疫法（chemiluminescence microparticle immunoassay, CMIA）检测临床血清标本梅毒螺旋体抗体的敏感性和特异性。方法用梅毒螺旋体颗粒凝集试验（TVeponema Pallidum particie agglutination test,TPPA）法作为对照标准,采用CMIA法检测2012年11月到12月1200例住院患者的血清标本,并用卡方检验评价两种检测方法对同一个样本的化验结果的一致性。结果1200例血清标本中用CMIA法检出阳性率为11.3%,TPPA法检出阳性率为10. 9% ,以TPPA为标准,CMIA法敏感性为96. 9%,特异性为99. 2%,其中CMIA法检测血清S/CO值〉 4. 00的110例,用TPPA确认107例阳性,阳性预测值（PPV）为97.3%;CMIA法S/C0值在1.0-9.0,TPPA可出现阴性结果。结论CMIA法可替代TPPA法进行梅毒螺旋体抗体检测,对于CMIA法检测S/C0值1. 0-4.0的需进一步复检。     

利用寡核苷酸微阵列技术对HBV、HCV、HIV、HAV、GBV-C/HGV和B19进行同时监测的初步研究

探讨研制能同时检测HBV、HCV、HIV、HAV、GBV-C/HGV和B19的微阵列监控芯片。根据病毒公开发表序列,序列比对,得出保守区域,设计病毒的特异性检测探针,同时设置阴性、阳性参照探针,制备监控微阵列。利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与微阵列上的探针杂交,清洗、扫描后进行结果分析。通过对质粒或模式分子的检测以及经HBV、HCV、HIV临床标本的验证,发现该微阵列监控芯片具有良好的特异性。其对质粒的检测灵敏度可达102病毒拷贝数,对临床标本的检测灵敏度可达103病毒拷贝数。此外,该微阵列监控芯片可检测出病毒混合感染血清。为微阵列监控芯片应用于此六种血液病毒的检测打下一定的基础。     

四种丙型肝炎病毒抗体试剂检测方法的性能评估

对血液筛查的四种丙型肝炎病毒(Hepatitis C virus,HCV)抗体试剂检测方法的性能进行评估。方法采用协作标定方式,应用四种检测方法(间接ELISA法、双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法)16种HCV抗体试剂,对经确证筛选出的70份(HCV抗体阳性35份、阴性35份)血浆样本进行检测,通过分析阳性、阴性符合率,评估四种方法 HCV抗体试剂的性能。结果间接ELISA法试剂检测阳性符合率为88.6%(31/35)~94.3%(33/35),双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法试剂检测阳性符合率为91.4%(32/35)~94.3%(33/35),四种检测方法 HCV抗体试剂阳性符合率之间的差异无统计学意义(P0.05);间接ELISA法和间接CLIA法试剂弱阳性样本检出率为11.4%(4/35)~31.4%(11/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂弱阳性样本检出率为5.7%(2/35)~11.4%(4/35),四种检测方法 HCV抗体试剂对弱阳性样本的检出率之间的差异有统计学意义(P0.05);间接ELISA法试剂检测阴性符合率分别为94.3%(33/35)~100%(35/35),间接CLIA法试剂阴性符合率分别为94.3%(33/35)~97.1%(34/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂阴性符合率均为97.1%(34/35),四种检测方法 HCV抗体试剂阴性符合率之间的差异无统计学意义(P0.05)。结论四种检测方法 HCV抗体试剂的性能基本一致,不同方法各有优缺点,建议应用多种检测方法对弱阳性样本进行确认检测。     

电化学发光法检测梅毒螺旋体特异性抗体的应用研究

目的对罗氏电化学发光免疫法(ECLIA)检测梅毒螺旋体特异性抗体的临床价值进行评估。方法收集梅毒疑似病例血清标本132份,分别用ECLIA、梅毒螺旋体明胶凝集试验(TPPA)和免疫印迹法(WB)进行检测,以WB为金标准,计算并比较ECLIA和TPPA的灵敏度和特异性,进而比较化学发光免疫法检测低S/CO值和高S/CO值的灵敏度和特异性差异。结果针对132份血清标本,ECLIA敏感性为100.00%,特异性为83.33%,阳性预测值为96.43%,阴性预测值为100.00%,总符合率为96.97%。TPPA敏感性为93.52%,特异性为87.50%,阳性预测值为97.12%,阴性预测值为75.00%,总符合率为92.42%。ECLIA检测1≤S/CO3组与S/CO≥3组的敏感性均为100.00%,特异性分别为86.96%和95.24%,结论 ECLIA检测具有较高的敏感性,适合临床大样本筛查,对S/CO值低的标本应结合TPPA、WB及临床资料确诊。     

丙型肝炎病毒分片段抗体检测蛋白质芯片的制备及临床评价

为了制备丙型肝炎病毒分片段抗体检测蛋白质芯片,并对其临床应用价值进行评价,将基因工程表达的丙型肝炎病毒分片段抗原,点至经特殊处理的玻片上,制成蛋白质芯片.收集来自三家临床单位用于临床验证的905份血清标本.分别用丙肝病毒分片段抗体检测蛋白质芯片、ELISA丙肝病毒抗体检测试剂进行检测.部分样本同时采用进口RIBA抗体检测试剂进行了检测,分别比较蛋白质芯片法与ELISA法以及RIBA试剂的符合率.结果表明：a.905份血清标本,ELISA法检出阳性294份,阴性611份.阳性标本用蛋白质芯片法检测,融合抗原292份显示阳性结果、2份阴性结果,根据蛋白质芯片的核心抗原,以及NS3, NS4,NS5分片段抗原综合判断确定阳性样本288份阳性,阴性样本2份,4份样本结果不确定.ELISA法检出的611份阴性标本用两种蛋白质芯片法检测,检出阴性均为611份.两种蛋白质芯片法与ELISA法的阳性符合率分别为99.3%和98.9%,与ELISA法的阴性符合率均为100%.用RIBA 试剂检测6份ELISA法为阳性,蛋白质芯片法为非阳性的样本,结果均为非阳性.b.290份经 RIBA试剂确认的阳性标本104份,单片段阳性标本66份,阴性标本120份,用蛋白质芯片法检测,检出阳性标本103份,单片段阳性标本61份,阴性标本126份,二者具有很高的符合率（P＞0.01）.丙型肝炎病毒分片段抗体检测蛋白质芯片,检测灵敏度和特异性高于ELISA法,对血清样本的确认程度与进口的RIBA试剂高度一致,具有操作简便,费用低廉的特点,是一种新型、高效的体外诊断试剂.     

Usefulness of a Fourth Generation ELISA Assay for the Reliable Identification of HCV Infection in HIV-Positive Adults from Gabon (Central Africa)

Background/Objectives

Guidelines for optimized HCV screening are urgently required in Africa, especially for patients infected with HIV, who sometimes show false positive or false negative reactivity in anti-HCV antibody assays. Here, we assessed the usefulness of a fourth-generation HCV Ag-Ab ELISA for the identification of active HCV infection in HIV-positive patients.

Methods

This cross-sectional study was conducted between 03/2010 and 01/2013 and included 762 Gabonese HIV-positive adult patients. The results of ELISA (Monolisa HCV Ag-Ab ULTRA, Bio-Rad) were compared with those obtained by RT-PCR (gold standard). The optimal ELISA signal-to-cutoff (S/CO) ratio to identify patients with active hepatitis C (positive HCV RNA) was determined. Specimens were further tested by the INNO-LIA HCV Score assay (Innogenetics) and the Architect HCV Ag kit (Abbott) to define the best diagnostic strategy.

Results

Sixty-seven patients tested positive for HCV (S/CO ratio ≥ 1) by ELISA. Of these, 47 (70.1%) tested positive for HCV RNA. The optimal S/CO associated with active HCV infection was 1.7. At this threshold, the sensitivity of ELISA was 97.9% (95% confidence interval (CI) 90.0–99.9%), its specificity was 91.3% (95% CI 85.0–95.5%), and HCV seroprevalence rate was 7.3% (56/762) (95% CI 5.6–9.4%). Among 57 HCV-seropositive patients with available INNO-LIA results, false reactivity was identified in 14 (24.6%), resolved HCV infection in two (3.5%), possible acute HCV infections in nine (15.8%) and likely chronic HCV infections in 32 (56.1%) patients. HCV core Ag was undetectable in 14/15 (93.3%) specimens that tested negative for HCV RNA whereas it was quantified in 34 (out of 39, 87.2%) samples that tested positive for HCV RNA.

Conclusions

Our study provides comprehensive guidance for HCV testing in Gabon, and will help greatly clinicians to improve case definitions for both the notification and surveillance of HCV in patients co-infected with HIV.     

Dot-ELISA法检测血液及唾液HIV抗体的应用评价

评价人类免疫缺陷病毒1+2型抗体检测试剂盒(Dot-ELISA法)检测血清和唾液样本的临床性能。采用对照试验研究,选取背景清晰的研究对象200例,采集同一研究对象的血清和唾液样本,应用万泰生物药业公司生产的人类免疫缺陷病毒1+2型抗体检测试剂盒作为考核试剂,法国生物梅里埃公司生产的人类免疫缺陷病毒抗体诊断试剂盒(ELISA法)作为参考试剂,考核试剂检测结果与参考试剂及研究对象背景进行比较分析。考核试剂检测血清HIV抗体与参考试剂相比较,阳性符合率100%,阴性符合率100%,总符合率100%,Kappa值1.00,一致性为最强;考核试剂检测唾液HIV抗体与参考试剂检测结果相比较,阳性符合率98.78%,阴性符合率100%,总符合率99.50%,Kappa值0.99,一致性为最强。Dot-ELISA法人类免疫缺陷病毒1+2型抗体检测试剂盒对血清及唾液样本检测性能优越,适合HIV抗体快速筛查。     

MicroRNAs expressed by human cytomegalovirus

The detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling. Samples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity. From 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p < 0.05) was observed between two incubation protocols. The detection of LSD-specific antibodies from bulk milk samples (pools of 2–10 individuals) came along with a reduced sensitivity over the sample of individual animals. Results show that the detection of capripoxvirus specific antibodies in milk samples using the commercially available ELISA from IDvet is feasible and can represent a helpful tool for LSDV monitoring programs.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Immunodiagnosis of Dengue Virus Infection Using Saliva

Keeping in view the complications and the case fatality associated with dengue virus, several serologic tests have been developed. However, the major drawback of these serologic tests is the need for a venous blood sample obtained by invasive venipuncture. As a noninvasive alternative, saliva provides a body fluid that contains antibodies of diagnostic importance. Hence, the detection of DEN-specific IgM and IgG antibodies in serum and saliva from 80 patients was compared. Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases. None of the specimens from the healthy controls showed the presence of IgM or IgG antibodies. The detection of both IgG and IgM antibodies in saliva correlated well with the serum IgG and IgM detection by the ELISA test (r = 0.6322 and r = 0.4227). Detection of salivary IgM antibodies by ELISA showed 100% sensitivity, 70% specificity, 90.9% positive predictive value, and 100% negative predictive value. The detection of IgG in saliva proved to be a promising tool as the sensitivity, specificity, positive predictive value, and negative predictive value were found out to be 93.3%, 100%, 100%, and 83.3%, respectively. Thus, from this study we conclude that the detection of DEN-specific salivary IgG and IgM antibodies are useful markers for dengue infection.     

Hepatitis B and C in the hemodialysis unit of Tocantins,Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of tile on hemodialysis were associated with HBV and HCV positivity respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes la and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.