快速胶体染料试纸条法检测弓形虫病IgM抗体的研究

目的　研制一种检测弓形虫病Ig M抗体的快速胶体染料试纸条法(DDIA) ,与国外进口试剂盒进行比较,以评价其临床应用价值。方法　以本实验室筛选的胶体染料D- 1标记兔抗人Ig M,以此标记物与弓形虫病人血清中的抗弓形虫Ig M抗体结合,用点有弓形虫可溶性抗原的硝酸纤维膜通过层析捕获染料标记兔抗人Ig M与弓形虫Ig M抗体的复合物,采用正交试验确定DDIA的最佳检测条件;对2 5份弓形虫Ig M抗体阳性血清和5 0份正常人血清进行检测;同时对弓形虫病流行病学筛查时的84份血清进行检测并与进口试剂盒的检测结果进行比较。结果　DDIA的最佳检测条件为:弓形虫可溶性抗原的点膜浓度为1mg/ m l,血清用量为10μl和兔抗人Ig M胶体染料检测液作1∶2 0稀释时的检测带清晰且背景较浅;2 5份弓形虫Ig M抗体阳性血清均为阳性,5 0份正常人血清有3份为Ig M抗体阳性;DDIA与进口试剂盒的总符合率为97.6 % ,其中阳性和阴性符合率分别为10 0 .0 % (35 / 35 )和95 .9% (47/ 4 9)。结论　DDIA简便快速,有较好的敏感性和特异性,同时与进口试剂盒有较高的符合率,表明该法具有较高的弓形虫病诊断价值。     

Specific anti-Toxoplasma gondii antibodies produced in inbred mice differring in their natural resistance to toxoplasmosis

The laboratory diagnostics of T. gondii infections both in humans and animals relies mainly upon detection of specific antibodies. We studied the influence of the host genetic factors on the level and repertoire of antibodies produced in Toxoplasma infection employing as an experimental model two inbred mouse strains with different innate susceptibility to toxoplasmosis. Using polyvalent antigen preparations in ELISA and microagglutination we found no differences in the antibody levels in both strains. By comparison of the antibody profiles in immunoblot we determined mouse strain-specific and common toxoplasmosis markers.     

Importance of high IgG anti-Toxoplasma gondii titers and PCR detection of T. gondii DNA in peripheral blood samples for the diagnosis of AIDS-related cerebral toxoplasmosis: a case-control study

BackgroundCerebral toxoplasmosis (CT) continues to cause significant morbidity and mortality in human immunodeficiency virus (HIV)-infected patients in Brazil. In clinical practice, the initial diagnosis is usually presumptive and alternative diagnosis tools are necessary. Our objective was to evaluate whether the detection of high titers of IgG anti-Toxoplasma gondii and T. gondii DNA in blood samples are associated with the diagnosis of CT.MethodsIn this case-control study we included 192 patients with HIV-1 infection: 64 patients with presumptive CT (cases) and 128 patients with other diseases (controls). Blood samples to perform indirect immunofluorescense reaction (IFI) to detect anti-T. gondii IgG antibodies and polymerase chain reaction (PCR) were collected before or within the first three days of anti-Toxoplasma therapy. Two multivariate logistic regression models were performed: one including the variable qualitative serology and another including quantitative serology.ResultsIn the first model, positive IgG anti-T. gondii (OR 4.7, 95% CI 1.2-18.3; p = 0.027) and a positive T. gondii PCR result (OR 132, 95% CI 35-505; p < 0.001) were associated with the diagnosis. In the second model, IgG anti-T. gondii titres ≥ 1:1024 (OR 7.6, 95% CI 2.3-25.1; p = 0.001) and a positive T. gondii PCR result (OR 147, 95% CI 35-613; p < 0.001) were associated with the diagnosis.ConclusionsQuantitative serology and molecular diagnosis in peripheral blood samples were independently associated with the diagnosis of CT in HIV-infected patients. These diagnostic tools can contribute to a timely diagnosis of CT in settings where Toxoplasma infection is common in the general population.     

The prevalence of anti-Toxoplasma gondii antibodies among abattoir workers in Lublin

Abattoir workers are occupationally exposed to Toxoplasma gondii by the contact with raw meat. One hundred and seven abattoir workers from the Meat Factory in Lublin were examined for the presence of anti-Toxoplasma antibodies. Sixty-one blood donors were also tested as the reference group. Sera from workers and referents were tested by direct agglutination with 2-mercaptoethanol (DA-2ME), and also by ELFA IgG and ELFA IgM tests (Vidas Toxo IgM, Vidas Toxo IgG, bioMerieux, France). In the workers group, out of 107 tested sera, 70 were found positive (65.4%). The highest percentage of seropositive results was found in the Cured Meat Division--76.2%. In the Meat Production Division 66.6% of seropositive results were found, and in the Slaughter Division--46.1%. Three persons with the presence of IgM antibodies were found in the Cured Meat Division. In the reference group, 34 out of 61 sera (55.7%) were positive. The difference in seropositivity between Cured Meat Division workers and reference group was statistically significant (p < 0.05). The high percentage of seropositive reactions among the workers of Cured Meat Division and the presence of persons in early stage of invasion suggest an increased risk of exposure to T. gondii in this section.     

The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats

The enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Toxoplasma gondii antibodies in 1258 small ruminants (732 sheep and 526 goats) sampled from 28 different locations in the three ecological zones of Ghana. The animals sampled had an overall seroprevalence of 30.5% (384 of the total). Sheep had a higher overall prevalence (33.2%) compared to the goats (26.8%). Animals sampled from the Coastal Savannah and the Forest zones had prevalences of 39.4% and 39.1%, respectively, which were significantly higher (P<0.01) than the prevalence recorded for the drier Guinea Savannah zone (20%). Prevalence of antibodies in female animals (35.8%) was significantly higher (P<0.01) than that for males (21.1%). Significant differences were also observed between breeds and age groups. The ELISA was found to be both highly sensitive (92%) and specific (91%) when compared to the IFAT, which was used as a reference test.     

两种抗原检测弓形虫病IgM的效果

目的观察弓形虫P30重组抗原及可溶性抗原检测弓形虫病IgM的效果。方法建立斑点免疫金渗滤法（DIGFA）。采用2种抗原分别包被在两条硝酸纤维素膜（NC）上,与待检血清中的相应IgM抗体结合,通过金标记兔抗人IgM直接显色,以检测弓形虫病IgM。2种抗原的DIGFA分别检测弓形虫IgM抗体阳性、类风湿、支原体肺炎、血吸虫病人和正常人血清。结果两种抗原建立的DIGFA检测试剂共检测5种血清样本166份,其中弓形虫感染IgM抗体阳性病人血清38份,弓形虫P30DIGFA敏感性为92.1%（35/38）,可溶性抗原DIGFA敏感性为81.6%（31/38）,差异无统计学意义（P〉0.05）。正常人群对照血清63份,检测全部阴性,特异性为100%。检测类风湿病人血清30份、血吸虫病人血清25份及支原体肺炎病人血清10份,仅类风湿病人血清出现阳性交叉反应,其中弓形虫P30DIGFA阳性2例,可溶性抗原DIGFA阳性3例。结论弓形虫P30重组抗原检测弓形虫病人血清IgM的DIGFA试剂可用于临床早期或急性期弓形虫病的诊断。     

Seroprevalence of IgG and IgM anti-Toxoplasma antibodies in HIV/AIDS patients, northern Iran

ObjectiveTo determine the seroprevalence of anti-Toxoplasma gondii (T. gondii) IgG and IgM antibodies in HIV/AIDS patients and uninfected subjects.MethodsThis cross sectional survey was carried out on 78 healthy and 62 HIV⁺/AIDS individuals in northern Iran between September 2007 and October 2008. Five mL of blood samples were collected from each person in case and control groups. Determination of CD4+ counts was performed by flow cytometry. The serum separated from blood samples was evaluated by conventional ELISA technique to determine the presence of antibodies to T. gondii.ResultsForty eight out of 62 (77.4%) HIV/AIDS serum samples were found positive for anti-T. gondii IgG antibody, compared with 59 among 78 (75.6%) HIV negative samples from the same area (P > 0.05). Six out of 62 (9.7%) HIV+/AIDS patients showed anti-T. gondii IgM antibody in their serum samples, compared with 7 among 78 (9%) HIV negative samples (P > 0.05). The mean of CD4+ counts in HIV⁺/AIDS was (430.8±182.3) cells/μL and in control group was (871.0±243.3)% cells/μL (P<0.01). CD4+ estimation in 5 (11.1%) of HIV+/AIDS patients was <200 cells/μL (P < 0.0001).ConclusionsSeroprevalence of latent toxoplasmosis in HIV patients is high, therefore the prevention of toxoplasmic encephalitis, administration of primary prophylaxis with co-trimoxazole to all HIV⁺/AIDS patients are necessary.     

不孕妇女抗弓形虫抗体抗精子抗体及IFN-γ的检测与分析

目的 了解不孕妇女血清抗弓形虫IgG抗体(ATAb)、抗精子抗体(AsAb)及其外周血细胞培养上清中γ-干扰素(IFN-γ)的水平,探讨ATAb、AsAb与不孕的关系以及与IFN-γ的相关性. 方法 应用酶联免疫吸附试验分别检测并比较182例不孕妇女及94例正常生育妇女血清ATAb、AsAb,同时测定并比较其外周血细胞培养上清中IFN-γ水平. 结果 182例不孕妇女血清ATAb、AsAb阳性率分别为15.38%(28/182)、18.13%(33/182),与正常生育组妇女血清ATAb、AsAb阳性率6.38%(6/94)、5.32%(5/94)比较,差异均有显著性 (χ2 ATAb=4.65,P＜0.05;χ2 AsAb=8.57,P＜0.01);不孕妇女外周血细胞培养上清中IFN-γ水平为(0.970±0.493) μg/L,显著高于正常生育妇女组(0.531±0.274) μg/L (P＜0.01). 结论 弓形虫感染与不孕有一定关系,AsAb是与不孕有密切联系的重要免疫因素.不孕症患者体内IFN-γ水平异常增高,可能通过影响机体生殖免疫功能而导致不孕.     

Enzyme immunoassay for IgG and IgM antibodies against dsDNA and ssDNA

Two enzyme-linked immunosorbent assays (ELISAs) for the detection of DNA antibodies are presented, one using lambda-phage DNA for the detection of dsDNA antibodies, the other using denatured calf thymus DNA for ssDNA antibodies. It was shown that only dsDNA antibodies of the IgG class were specific for SLE. IgM-dsDNA antibodies or ssDNA antibodies were not SLE-specific. Of 62 SLE patients 58% were positive for IgG-dsDNA antibodies. When only those patients with active disease were considered, 88% were positive. There was a positive correlation between the levels of IgG-dsDNA antibodies and the incidence of nephritis. When the ELISA for IgG-dsDNA antibodies was compared with the filter assay, the ELISA showed a considerably higher sensitivity.     

Multicenter evaluation of a novel quantification method for rubella and toxoplasmosis antibodies

Summary Antibody quantification by EIA is possible without a standard curve. Following the so-called alpha method only one test dilution is used, the resulting absorbance is corrected and the IU/ml will be calculated by means of a mathematical formula. This new kind of a single point measurement was evaluated in seven independent laboratories by comparison with commercial EIAs using a standard curve or a titer calibration line. For the quantification of IgG against rubella virus this study comprised 1,480 individual samples and three comparison EIAs. For IgG againstToxoplasma gondii a total of 743 samples was evaluated in two comparison tests. The results obtained by the alpha method show a precision and accuracy more than sufficient for routine testings. Also the technical expenses and reagent costs were reduced. Prerequisites and limitations are discussed against the background of the problem of immune status definition.

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Mehrfacherprobung einer neuartigen Antikörperquantifizierung bei Röteln und Toxoplasmose

Zusammenfassung Die Antikörperquantifizierung ist im EIA auch ohne Standardkurve möglich. Bei der sogenannten Alpha-Methode wird nur eine Testverdünnung benötigt, das Meßsignal korrigiert und dann über eine Rechenformel die IU/ml ermittelt. Diese neue Einpunktquantifizierung wurde in sieben unabhängigen Arbeitsgruppen durch Vergleich mit kommerziellen EIA's bewertet, die eine Standardkurve oder Titerbezugsgerade verwenden. Zur IgG-Quantifizierung bei Rubella wurden insgesamt 1480 einzelne Untersuchungsproben und drei Vergleichs-EIA's eingesetzt. Beim IgG gegenToxoplasma gondii waren es über alles 743 Proben und zwei Vergleichstests. Die mittels Alpha-Methode erzielten Ergebnisse besaßen eine voll praxistaugliche Präzision und Richtigkeit. Zusätzlich wurden der technische Aufwand und die Reagenzienkosten reduziert. Voraussetzungen und Einschränkungen werden vor dem problematischen Hintergrund einer Immunstatusdefinition diskutiert.

     

Measurement of antinuclear antibodies by multiplex immunoassay: a prospective, multicenter clinical evaluation

OBJECTIVE: We conducted a prospective, multicenter evaluation of autoantibody testing by multiplex immunoassay in patients with known or suspected connective tissue diseases (CTD). We evaluated agreement between multiplex immunoassay and enzyme immunoassay (EIA) and assessed the diagnostic utility of autoantibody profiles. METHODS: Samples from 908 patients with suspected CTD seen in rheumatology clinics were collected prospectively at 3 tertiary care centers. Diagnoses were established according to recognized classification criteria. Tests for autoantibodies were obtained by multiplex immunoassay and by EIA. The results of the multiplex immunoassay were analyzed using a previously validated interpretative algorithm, MDSS (Medical Decision Support Software), that suggests possible disease associations based on the pattern of results for the autoantibodies. RESULTS: The median patient age was 49.7 years; 83% were female. The most common diagnoses were rheumatoid arthritis in 352 patients and systemic lupus erythematosus (SLE) in 332 patients. Agreement between multiplex and EIA testing ranged from a high of 99% (95% CI 98% to 100%) for Jo-1 to a low of 79% (95% CI 76% to 82%) for antinuclear antibodies. The MDSS algorithm suggested an appropriate disease association in 75% to 100% of patients with SLE. The results varied depending on the disease and the autoantibodies present. CONCLUSION: These results suggest that patterns of autoantibodies detected by multiplex immunoassay testing, when analyzed by an interpretative algorithm, are useful in the evaluation of patients with CTD in situations of high disease prevalence. Further testing is necessary to determine its utility in settings of low disease prevalence.     

几种弓形虫IgM酶联免疫吸附试验的建立及评估

目的　建立和评估几种弓形虫ELISA -IgM的检测方法。 方法　应用酶联免疫吸附试验 (ELISA)的原理 ,建立四种检测方法 ,并进行比较。结果　检测 373例孕妇及正常献血员血清标本 ,方法Ⅰ、方法Ⅱ、方法Ⅲ、方法Ⅳ的阳性率分别为3 8%、3 2 %、2 9%、2 4 %。结论　利用抗人IgM、弓形虫抗原和抗体建立的捕获法具有快速、敏感、特异的特点 ,适合于育龄妇女的筛查和早期诊断。     

Serologic evidence for acute toxoplasmosis in polymyositis-dermatomyositis. Increased frequency of specific anti-toxoplasma IgM antibodies

Several case reports have suggested an association between acquired toxoplasmosis and polymyositis-dermatomyositis. Because the presence of anti-Toxoplasma IgM antibodies suggests recent infection, 58 patients with polymyositis-dermatomyositis (from two medical centers) were examined for the presence of IgM antibodies using a specific indirect immunofluorescent technique. Serum samples were also examined for antibodies using the Sabin-Feldman dye test and complement fixation methods. Of 58 patients with polymyositis-dermatomyositis, 29 (50 percent) had positive Sabin-Feldman dye test results and 14 (24 percent) had positive IgM immunofluorescent findings. This is higher than the expected frequency. None of the patients with negative Sabin-Feldman dye test results had IgM immunofluorescent antibodies. Furthermore, IgM immunofluorescent antibodies were associated with the presence and titer of both Sabin-Feldman dye test and complement fixation antibodies. Evidence that the presence of antinuclear antibody and rheumatoid factor did not influence these results is presented. Patients with muscular dystrophy and systemic lupus erythematosus (with or without myositis) did not have an increased frequency of anti-Toxoplasma IgM immunofluorescent antibodies.     

Detection of specific IgM antibodies for the diagnosis of Mycoplasma pneumoniae infections: a clinical evaluation

The diagnostic value of detection of specific IgM antibodies was analysed in Mycoplasma pneumoniae infections. In a retrospective clinical and serological study, M. pneumoniae IgM antibodies were determined by a mu-capture ELISA using enzyme-labelled antigen. The study group consisted of 91 patients with significantly raised titers in paired sera or a single high titer of complement fixation antibodies. About 40% of the patients had been treated with antibiotics ineffective against M. pneumoniae infections prior to admission to hospital. Treatment with erythromycin or tetracycline was shown to give a shorter period of fever compared to if no or ineffective therapy was given. Specific IgM antibodies were detected in about 80% of sera sampled 9 days or more after onset of symptoms. In sera sampled at 7-8 days after onset IgM antibodies were found in about 40% of the sera but only occasionally in sera sampled earlier. In the age group 0-20 years 88% of the patients developed an IgM response. In the higher ages (greater than 60 years) a significantly lower rate of IgM responders was observed.     

Toxoplasma gondii: detection of antibodies in human saliva and serum

Saliva samples from 27 patients with a recent toxoplasma infection were tested for specific IgG, IgM and IgA antibodies to Toxoplasma gondii. Thirteen of the 27 saliva samples were positive for IgG anti-T. gondii by direct agglutination and 8 of the 27 were positive for IgM anti-T. gondii by an immunosorbent agglutination assay. Twenty of the 27 saliva samples were positive for IgG antibody on toxoplasma immunoblots with three major immunodominant antigens; 38, 30 and 35 kDa. IgA results on toxoplasma immunoblots were positive for all three groups tested, recently infected patients, chronically infected and seronegative adults without distinguishing between them. The 35 and 43 kDa antigens were the most frequently detected proteins. IgM in saliva gave negative or very weak reactions. None of the eight seronegative or the 17 chronically infected adults gave positive results in any of the tests performed to detect IgG or IgM in saliva. Serial saliva and serum samples from a laboratory-infected patient were collected and tested for toxoplasma-specific IgG, IgM and IgA. IgG in saliva was detected by 27 days post infection (p.i.) and was negative by 81 days p.i.; it detected mainly the 38 and 30 kDa antigens. IgM in saliva was detected by 11 days p.i. and was negative by 81 days p.i., with no reaction on immunoblots.     

Indirect immunofluorescence test for detection of IgM antibodies to cytomegalovirus.

A technique for the detection of IgM antibodies to cytomegalovirus (CMV) by immunofluorescence was developed. In order to prevent interference by rheumatoid factor in the sera, IgG was removed by prior immunoabsorption with antiserum to gamma Fc. Sera from 63 patients with a rise in titer of antibody to CMV, indicated by complement fixation, were IgM-positive, but yielded negative results on the Paul-Bunnell-Davidsohn test for infectious mononucleosis. Convalescent-phase serum samples from 20 patients with a seroconversion to herpes simplex virus and from 20 patients with a seroconversion to varicella-zoster virus also had no IgM antibodies to CMV. Sera from six of 10 patients with infectious mononucleosis and two of 100 normal blood donors were positive for IgM antibodies to CMV. In 38 of 63 patients, the diagnosis of CMV infection could be made several weeks earlier by the immunofluorescence test than by the complement-fixation test. IgM antibodies to CMV persisted for more than two months after onset of symptoms of the infection.     

结核抗体lgG/lgM检测在肺结核和肺外结核中的诊断价值

目的 探讨异种血清抗体检测技术在结核病诊断中的应用价值。方法 采用IgG/IgM抗体试剂盒分别检测102例结核病患者(包括73例肺结核和29例肺外结核)、223例其他肺部疾病患者和100例对照者结核感染情况,以临床诊断为标准评价该方法的敏感度和特异性,同时分别与痰菌培养及痰涂片平行检验的结果作比较,统计学分析采用χ²检验。结果 结核抗体IgG/IgM检测结核病患者的敏感度为74.51%、特异度为91.64%。结核抗体IgG/IgM检测肺结核和肺外结核的敏感度分别为82.19%、55.17%,肺内和肺外结核的敏感度差异有统计学意义(P＜0.05),结核抗体lgG/IgM检测结核患者阳性检出率明显高于痰培养法和痰涂片法(P<0.05)。102例结核病患者年龄段分组分析,少年组和老年组检出率分别为58.33%、36%,远低于青年组和中年组的96.15%和89.74%。不同年龄组间进行卡方比较分析显示,P＜0.05,差异有统计学意义。425例标本中,共发现8例非结核分枝杆菌,其中6例胞内分枝杆菌, 2例脓肿分枝杆菌,lgG/lgM抗体检测均为阴性。结论 IgG/IgM血清抗体检测肺内、外结核具有快速方便、经济和较高的敏感度,适合用于临床结核筛查。