胶体金免疫层析法检测新型冠状病毒IgM/IgG抗体的临床评价与应用

新型冠状病毒(SARS-CoV-2)感染暴发流行已成为全球公共卫生事件,急需快速有效的现场诊断试剂。为探讨SARS-CoV-2病毒IgM/IgG抗体胶体金免疫层析法检测效果及临床应用价值,本研究选取304例新冠肺炎临床诊断病例、114例SARS-CoV-2核酸检测阴性的正常人(138)及其他发热伴呼吸道症状病例(64),采用胶体金法对采自上述病例的血浆或血清标本进行IgM和IgG抗体检测,并选取部分病例进行了全血和血浆或血清标本的检测同源性比较,进一步对304例临床诊断病例的病毒核酸、IgM/IgG抗体的时间分布进行了分析。结果显示,304临床诊断病例中,SARS-CoV-2核酸检测阳性病例为105例,胶体金法检测SARS-CoV-2 IgM和IgG抗体的敏感性分别为76.2%(80/105)和86.6%(91/105),IgM/IgG抗体阳性总体符合率为96.1%(101/105);核酸、抗体均为阴性者为73例;其余126例临床诊断病例中,IgM阳性率为69.2%(87/126),IgG阳性率为98.3%(125/126),IgM/IgG总体符合率为100%(126/126)。健康人和其他发热病人中,IgM和IgG检测特异性分别为99%(200/202)和98%(198/202)。同源全血与血浆或血清标本抗体检测结果的总符合率为99%,显示两者具有高度一致性。本研究提示,胶体金法检测SARS-CoV-2抗体有较好的敏感性及特异性,可用于临床辅助诊断和流行病学调查等,具有较广泛的应用场景,在新冠肺炎疫情防控中具有一定的应用价值。     

石家庄地区肺炎支原体感染的流行病学调查

目的:了解石家庄地区肺炎支原体感染的血清流行病学情况。方法:选择2011年3月~2012年2月我院住院和门诊收治的急性呼吸道感染患者1902例为研究对象,采用间接免疫荧光法(IFA)检测其血清肺炎支原体IgM抗体,并分析其流行病学资料。结果:1902例血清标本中,284例(14.93%)肺炎支原体IgM抗体阳性,男性和女性的阳性率无显著差异。肺炎支原体抗体阳性的患者主要分布于0~15岁年龄段,阳性检出率最高的年龄组为0~6岁,占21.26%(132/621)。各个季节均有肺炎支原体感染阳性患者,感染率无显著差异性,秋(80例)、冬(96例)两季的阳性感染率高于春(56例)、夏(52例)两季。患者100%出现发热症状,95.77%出现咳嗽。结论:石家庄地区肺炎支原体感染的主要人群为未成年人,无季节性和性别差异,以发热和咳嗽为最主要的临床症状。     

儿科呼吸道疾病中肺炎支原体感染的结果分析

目的探讨儿科肺炎支原体（MP）感染特点,辅助临床医师早期诊断,合理用药。方法测定我院一年来2013例儿科呼吸道疾病患儿的肺炎支原体抗体（IgM）。结果2013例呼吸道感染患儿,检出肺炎支原体抗体（IgM）阳性者769例,占38.2%,769例阳性分别表现为肺炎369例（48%）,支气管炎238例（31%）,咽炎92例（12%）,哮喘70例（9%）。其中,肺炎组与各组相比较,具有统计学意义。MP IgM感染的检出率明显高于其他各组（P〈0.01）。结论MP感染是患儿不可忽视的病原体,检测患儿血清MP抗体能够及早诊断,指导治疗。     

肺炎支原体肺炎患儿血清免疫球蛋白变化及其临床意义

目的 探讨肺炎支原体肺炎(MPP)患儿血清免疫球蛋白变化的临床意义.方法 随机抽取2010年6月~2012年6月在我院儿科就诊的50例肺炎支原体肺炎患儿和50例普通肺炎患儿,分为支原体感染组(MPP 组)和非支原体感染组(nMPP组),同时选取同期在我院体检的健康儿童50例作为对照组,分别对3组儿童的血清免疫球蛋白水平进行检测,比较其差异.同时根据症状轻重对支原体感染组患儿的血清免疫球蛋白水平进行比较.结果 MPP组和nMPP组血清免疫球蛋白IgA、IgM、IgG水平均高于对照组;MPP组IgM、IgG水平均高于nMPP组;重症组IgA、IgM、IgG水平均高于轻症组,以上差异均有统计学意义(均P<0.05).结论 体液免疫紊乱在MPP中发挥了重要作用,免疫球蛋白水平的检测为MPP的诊断和发病程度的判断提供一定的临床依据.     

尿素解离法降低新型冠状病毒IgM和IgG检测结果假阳性的效果评价

探讨引起新型冠状病毒(SARS-CoV-2)免疫球蛋白M(IgM)和免疫球蛋白G(IgG)抗体检测结果假阳性的干扰因素,改良检测方法,完善实验室检测方案。本研究收集2020年1月2日至2020年3月5日就诊于川北医学院附属医院及南充市中心医院的门诊及住院患者血清样本共74份,其中19例为SARS-CoV-2核酸检测确诊阳性,10例为其他呼吸道病毒IgM抗体阳性,10例肝炎病毒抗体IgM阳性,20例类风湿因子IgM阳性,15例抗核抗体阳性。采用胶体金免疫层析法(试剂A、试剂B)分别对研究对象血清进行SARS-CoV-2 IgM和IgG抗体检测,并对结果为SARS-CoV-2 IgM或SARS-CoV-2 IgG阳性的病例进行分析,发现造成检测结果假阳性的可能因素。再采用合适浓度的尿素对检测为阳性结果的血清及3例SARS-CoV-2 IgM阳性的COVID-19早期患者血清进行解离,解离后分别重新测定SARS-CoV-2 IgM、IgG抗体。采用SPSS19.0统计软件对数据进行统计学分析。结果显示,19例COVID-19确诊患者血清中,试剂A检出SARS-CoV-2 IgM、IgG抗体阳性数为15例及18例,试剂B检出SARS-CoV-2 IgM、IgG抗体阳性数均为12例;20例类风湿因子IgM阳性患者血清中,试剂A检出SARS-CoV-2 IgM、IgG抗体阳性数为16例及14例;15例高滴度ANA阳性患者血清中,试剂B检出4例SARS-CoV-2 IgG抗体阳性。尿素解离浓度为2 mol/L时,试剂A检出的RF-IgM阳性血清中的16例SARS-CoV-2 IgM抗体有14例转阴,14例SARS-CoV-2 IgG抗体有13例转阴,而试剂A在COVID-19确诊患者血清中检出的SARS-CoV-2 IgM、IgG抗体均未出现阴转;尿素解离浓度为4 mol/L时,试剂B检出的ANA阳性血清中的4例SARS-CoV-2 IgG抗体全部转阴,而在COVID-19确诊患者血清中检出的的SARS-CoV-2 IgM、IgG抗体均未出现阴转。另外,经过尿素解离后,试剂A和试剂B在3例COVID-19早期患者血清中检出的SARS-CoV-2 IgM抗体也都未出现阴转。本研究提示,IgM型类风湿因子易造成试剂A检测血清SARS-CoV-2 IgM、IgG结果的假阳性;高滴度的ANA抗体也会引起试剂B检测血清SARS-CoV-2 IgG结果的假阳性。对检测结果假阳性的样本,采取尿素解离方案,能有效降低检测过程中假阳性发生的概率;尿素解离法对COVID-19患者发病早期标本检测灵敏度的影响尚待进一步深入研究。     

内蒙地区发热患者血清中冠状病毒抗体的检测

目的分析内蒙地区发热患者中冠状病毒的感染情况。方法以SARS冠状病毒感染Vero细胞涂片为冠状病毒抗原片,用间接免疫荧光法分别检测55例发热患者和68例正常人血清中冠状病毒的IgG、IgM抗体。结果发热患者血清中冠状病毒IgG抗体和IgM抗体阳性率分别为29.1%(16/55)和10.9%(6/55),而正常人血清中只检测到2.9%(2/68)的IgG抗体,且未检测到IgM抗体,2组患者的IgG和IgM抗体阳性率比较差异均有显著性;随机选取7例患者的IgG阳性血清进行SRAS冠状病毒的特异性抗体封闭实验,结果有6例血清仍为阳性,有1例血清转为阴性,说明冠状病毒IgG抗体阳性血清中85.7%为普通冠状病毒特异性,14.3%为SARS冠状病毒特异性。结论普通冠状病毒是内蒙地区发热患者的主要病原体之一,部分患者还存在SARS冠状病毒的既往感染。     

巨细胞病毒抗原片的制备及其在快速诊断中初步应用

本文报道了用巨细胞病毒(CMV,AD_(169)株)感染的人胚肺细胞(HL)制备CMV抗原片。在这种抗原片上进行的间接免疫酶组化法(IPA)初步应用于CMV的快速诊断,并与补体结合试验(CFT)和免疫荧光技术(IFA)进行了比较。检测103份血清中抗CMV IgG抗体和18例双份血清中抗CMV IgG和IgM抗体的结果表明,本方法是特异的,其结果与CFT和IFA的结果一致。这种方法简便、快速、灵敏,不需要特殊的设备,可广泛用于临床诊断和流行病学调查。     

石家庄地区肺炎支原体感染的流行病学调查

目的：了解石家庄地区肺炎支原体感染的血清流行病学情况。方法：选择2011年3月-2012年2月我院住院和门诊收治的急性呼吸道感染患者1902例为研究对象,采用间接免疫荧光法（IFA）检测其血清肺炎支原体IgM抗体,并分析其流行病学资料。结果：1902例血清标本中,284例（14．93％）肺炎支原体IgM抗体阳性,男性和女性的阳性率无显著差异。肺炎支原体抗体阳性的患者主要分布于0～15岁年龄段,阳性检出率最高的年龄组为0～6岁,占21．26％（132／621）。各个季节均有肺炎支原体感染阳性患者,感染率无显著差异性,秋（80例）、冬（96例）两季的阳性感染率高于春（56例）、夏（52例）两季。患者100％出现发热症状,95．77％出现咳嗽。结论：石家庄地区肺炎支原体感染的主要人群为未成年人,无季节性和性别差异,以发热和咳嗽为最主要的临床症状。     

严重急性呼吸综合征患者血清特异性抗体消涨规律的长期追踪研究

目的:追踪检测SARS冠状病毒(SARS-CoV)抗体在严重急性呼吸综合征(SARS)患者血清中的产生及其转归规律,为SARS诊断及防治提供依据。方法:对41例临床诊断SARS患者的血清进行了连续3年的检测,分别应用间接免疫荧光(IFA)检测患者血清特异性IgG抗体平均滴度,应用双抗原夹心ELISA法检测患者血清核衣壳蛋白(N蛋白)抗体的平均滴度,绘制消涨曲线,得出消涨规律。结果:应用IFA检测患者血清特异性IgG抗体与应用双抗原夹心ELISA法检测N蛋白抗体所得到的消涨规律不同,前者测得康复者血清IgG抗体滴度维持在较低水平,但后者检测35例康复者血清N蛋白抗体仍维持在较高水平。结论:SARS-CoV的N蛋白是免疫原性较强的抗原,感染3年后仍存在高滴度抗体;抗原夹心ELISA检测SARS-CoV N蛋白抗体的灵敏度较IFA方法高。     

啮齿类动物中汉坦病毒感染血清学和病原学检测方法比较研究

致病性汉坦病毒的宿主主要为啮齿类动物,其病毒感染状况是人间疫情发生的关键影响因素,可通过检测宿主动物标本中病毒基因组RNA、蛋白抗原及特异性抗体而进行监测。本研究利用367份鼠肺及鼠血标本,对双抗原夹心ELISA(ELISA)、实时荧光RT-PCR(RT-PCR)和免疫荧光(IFA)等三种分别检测抗体、核酸和抗原的方法进行比较评估。ELISA法检出抗体阳性鼠血标本46份,阳性率为12.53%;RT-PCR法检出病毒RNA阳性鼠肺标本28份,阳性率为7.63%;IFA检出抗原阳性鼠肺标本24份,阳性率为6.54%。宿主动物组织标本中检出汉坦病毒RNA和(或)结构蛋白抗原的标本,对应的血液标本中可检出病毒特异性抗体,100%(24/24)IFA检测阳性标本和89.3%(25/28)RT-PCR检测阳性标本对应血标本ELISA抗体检测阳性,反之亦然,检出抗体的标本基本包含了可检出抗原和RNA的标本。RT-PCR与IFA检测结果差异无显著性(χa2=0.64,P0.05),一致性检验Kappa系数为0.71,一致性高(Z=13.66,P0.05),首先对血标本开展基于ELISA的特异性抗体检测,可显著缩小RT-PCR或IFA法检测病毒RNA或抗原的范围(χb2=12.04,χc2=20.05,P0.05)。本研究为宿主动物汉坦病毒感染实验室监测方案优化提供了有益的依据。     

Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2). We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA) using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA), we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6%). Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3%) of the 64 patients.     

肺炎衣原体感染与青少年I型糖尿病的相关性研究

目的：探讨肺炎衣原体感染与青少年I型糖尿病的相关性,为I型糖尿病的的临床治疗提供参考依据。方法：选择2010年12月．2012年6月间石家庄地区各医院收治的49例青少年T1DM患者为观察组,及同期50例健康人作为对照组,应用即时指尖血免疫测定仪分析受试者HbAlc水平;应用RT—PCR技术检测血液中CpnDNA;应用ELISA方法检测受试者血清中Cpn特异性抗体水平,对CpnDNA的检出情况及HbAlc水平与CpnDNA和特异性抗体水平的相关性进行统计学分析。结果：观察组CpnDNA的栓出率为46．9％,显著高于对照组（P〈0．05）;观察组Cpn抗体阳性率显著高于对照组（P〈0．05）,且观察组再次感染或慢性感染Cpn的百分率显著高于对照组（p／0．05）;HbAlc与IgG／IgA抗体水平显著相关,血糖控制较差0qbAlc〉9％）的糖尿病患者CpnIgG／IgA抗体阳性率与血糖控制较好的患者（HbA1c〈7％）相比显著升高（P〈0．05）。结论：与健康对照相比,青少年T1DM患者更容易感染Cpn,且更容易由急性感染状态进展为慢性感染形式,良好的血糖可能降低患者发生与代谢控制有关的慢性并发症。     

检测巨细胞病毒(CMV)DNA及其即刻早期抗原、pp65和pp67在肾移植受者术后巨细胞病毒感染早期诊断中的价值

目的探讨检测巨细胞病毒(CMV)DNA及其即刻早期抗原(IE)、巨细胞病毒pp65和pp67抗体对肾移植受者术后巨细胞病毒感染早期诊断的临床应用价值。方法按肾移植术受者术后3个月外周血是否出现CMV抗原,将71例患者分为CMV感染组(56例)和CMV未感染组(15例),肾移植术受者手术前和术后第1个月每周检查1次,第2、3个月每2周检查1次外周血巨细胞病毒pp65和巨细胞病毒pp67、即刻早期抗原(immediate early antigen,IE),巨细胞病毒DNA和IgM、IgG,共8次;以监测与分析评价肾移植术受者手术前后各项指标变化。结果肾移植术前71例肾移植受者PP65、PP67、IE、CMV DNA均为阴性;肾移植术后CMV感染组的pp65、pp67、IE、CMV DNA阳性率分别为67.8%(38/56)、66.1%(37/56)、64.2%(36/56)和48.2%(27/56),CMV未感染组4项指标值分别为0%、0%、13.3%(2/15)、和0%,两组差异均有统计学意义(P均0.01)。肾移植术后CMV感染组(56例)和CMV未感染组(15例)CMV IgG均为阳性,而IgM阳性率在CMV感染组仅为3.5%(2/56),在CMV未感染组为0%,IgM表达率在CMV感染组和未感染组无统计学差异(P0.05)。观察期内感染组与未感染组相比,术后CMV pp65,pp67,CMV DNA和IE指标出现阳性的例数及阳性出现的具体时间均有显著性差别(P均0.01),而IgM和IgG则均无显著性差别(P均0.05)。结论肾移植术后患者外周血CMV DNA,IE,pp65和pp67抗原检测阳性与其术后巨细胞病毒感染相关。检测CMV DNA、IE、pp65和pp67抗原可能更早更准确反映器官移植术后CMV活动性感染。而CMV IgG和IgM不能作为肾移植后患者CMV感染的诊断指标。     

Persistent Low Toxoplasma IgG Avidity Is Common in Pregnancy: Experience from Antenatal Testing in Norway

The parasite Toxoplasma gondii might harm the fetus if a woman is infected during pregnancy. IgG seroconversion and significant increase in IgG antibody amount in pregnancy indicates maternal infection. Presence of toxoplasma immunoglobulin M (IgM), immunoglobulin G (IgG) and low IgG avidity in a single serum sample indicates possible maternal infection, but positive toxoplasma IgM and low IgG avidity may persist for months and even years. We aimed to evaluate avidity development during pregnancy in a retrospective study. Serial blood samples from 176 pregnant women admitted to Oslo University Hospital 1993–2013 for amniocentesis because of suspected toxoplasma infection were included. Data were obtained from journals and laboratory records. The avidity method used was based on Platelia Toxo IgG assay. Mean maternal age at first serology was 29.9 years (SD 5.2, range 18–42). In 37 (21%) women only the avidity increased from low to high in < 3 months. In 139 (79%) the IgG avidity remained below the high threshold ≥ 3 months and within this group 74 (42%) women had stable low IgG avidity during the observation period. Median gestational age at first test was 10.6 weeks (range 4.6–28.7). Fetal infection was detected in four children, but none among children whose mother had stable low IgG avidity. The first antenatal toxoplasma serology should ideally be collected in early pregnancy and if stable values of toxoplasma IgM and low IgG-avidity are detected in a second sample after three to four weeks, the need for amniocentesis can be questioned.     

Evaluation of the indirect and IgM‐capture anti‐human cytomegalovirus IgM ELISA methods as confirmed by cytomegalovirus IgG avidity

Primary cytomegalovirus (CMV) infection during pregnancy often results in congenital CMV infection with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for 3 months after primary infection. Here, we evaluated and compared the performance of CMV IgM and IgG avidity assays. Because sensitivity and specificity reportedly differ between CMV IgM kits, CMV IgM detection was compared between the two commercially available ELISA kits that are most commonly used in Japan. Sera for CMV IgM were first screened using a traditional indirect ELISA kit. Selected samples were then tested for CMV IgM and CMV AI using a CMV IgM‐capture ELISA kit and a CMV IgG avidity assay, respectively. The rate of concordance between the IgM kits was 89% (42/47), indicating the absence of any significant difference. Most of the CMV IgM‐positive plasma samples showed high CMV IgG AI; however, 18 commercially available plasma samples with low CMV IgG AI were all CMV IgM‐positive. One plausible explanation for this discrepancy is that the duration of low IgG AI is shorter than that of IgM positivity. Alternatively, CMV IgM tests may generate pseudo‐positive readouts in cases of congenital infection. Nevertheless, our study confirms that CMV IgG AI can be a reliable indicator of CMV primary infection.     

Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40-80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.     

Performance of the immunoglobulin G avidity and enzyme immunoassay IgG/IgM screening tests for differentiation of the clinical spectrum of toxoplasmosis

Toxoplasmosis has been well known as an important human infection to consider especially in pregnant women. Although many serologic methods are available, the diagnosis of toxoplasmosis can be extremely difficult. The presence of increased levels of Toxoplasma-specific IgG antibodies indicates an infection, but it does not differentiate between a recent and past infection. The purpose of our study was to compare the performance of the ELISA T. gondii IgG/IgM test, a widely used enzyme-linked immunosorbent assay, to the ELISA IgG avidity method. One hundred and four serum samples (from 38 males and 66 females) were tested and evaluated from symptomatic patients (chorioretinitis, lymphadenopathy), and from women in their first trimester of pregnancy who were suspected of having toxoplasmosis. The high IgG avidity and ELISA IgG antibody levels were in agreement for 51 of the specimens (49.0%). Thirty-eight discrepant (borderline) results from the IgG avidity method were positive for IgM (3 specimens) and IgG (37 specimens). Interestingly, out of the eight serum samples that were positive for both IgG and IgM antibodies, two samples were low IgG avidity, and three samples were borderline. There was no statistically significant relation observed between the results of the IgG avidity method and the ELISA IgG test, and the IgG avidity method and ELISA IgM test (chi2 = 1.987; p = 0.370 and chi2 = 2.152; p = 0.341, respectively). The IgG avidity method was considered easy to perform and an acceptable approach for the differentiation of discrepant results (recent/chronic) and for the current detection of T. gondii antibodies. We concluded that the determination of IgG avidity is a helpful tool for the diagnosis of the ocular form of toxoplasmosis and it is a safe method for screening this disease in the first trimester of pregnancy.     

肺炎支原体实验室检测方法研究进展

肺炎支原体(Mycoplosma pneumonia,MP)为人类非典型肺炎的病原体,是引起呼吸道感染的重要病原体。但是支原体肺炎与其他病原体感染的肺炎,在临床症状、影像学上并无特异性差别,且其对一般治疗肺炎、上呼吸道感染的药物有耐药性,因此肺炎支原体及时、准确的实验室检测对于支原体肺炎的诊断治疗显得尤为重要。目前MP的实验室检测方法不断推陈出新,但各种方法均有其优势与不足,临床可选择两种不同的方法同时检测。比如:血清学抗体的检测结合MP快速培养药敏的方法;血清学抗体的检测结合PCR的方法,不同方法相互补充为临床的早期诊断、治疗提供依据。而MP药敏试验的检测和耐药机制的研究对于临床用药方案的选择,减少耐药株的产生和流行具有重要意义。