Dissecting the IgM antibody response during the acute and latent phase of toxoplasmosis.

A major problem in anti-toxoplasma IgM serology is the occurrence of clinically non-relevant (CNR) IgM in sera of latently infected (LI) individuals. The susceptibility for CNR IgM of the Toxo ISAGA IgM, Platelia Toxo IgM and Vidas Toxo IgM assays was determined using sera of LI individuals with an IgM titer in the Abbott IMx Toxo IgM assay. The specificity of CNR-IgM and IgM antibodies in sera of acutely infected (AI) individuals was compared by immunoblotting.Seven/19 samples with CNR IgM antibodies were found positive in the ISAGA IgM, compared with 16/19 and 17/19 with the Vidas and Platelia Toxo IgM assays, respectively. In contrast, immunoblotting allowed a distinction between CNR IgM and AI IgM antibodies of the 30 sera studied.Clearly, IgM assays are susceptible to CNR IgM antibodies. In cases of doubt, immunoblotting is of value as confirmation method. Because CNR IgM antibodies are specific for toxoplasma, it will be difficult to improve IgM ELISAs in such a way that CNR IgM antibodies will not interfere with the analysis.     

Value of a specific IgA assay on cord blood for the diagnosis of congenital toxoplasmosis

Congenital toxoplasmosis (CT) is a severe disease which must be recognized without delay to enable effective treatment. Detection of specific IgG and IgM antibodies in cord blood is not adequate for early diagnosis of CT. The diagnostic value of specific IgA detection in cord blood was assessed by testing cord blood samples from 41 infants with CT and 155 infants without CT. Each IgA assay was performed on serum diluted to 1 : 20 and 1 : 100. IgA values were compared with IgM values determined using the ISAgA^® test. Among the 41 CT infants, 38 had IgA positive response at the 1 : 20 dilution, 30 had IgA positive response at the 1 : 100 dilution, and 26 had IgM positive response. Among the 155 infants without CT, nine had IgA at the 1 : 20 dilution and two had IgA at the 1 : 100 dilution. Rate of true positives was higher for IgA (93%) than for IgM (63%). Diluting to 1 : 20 significantly improved sensitivity (93%) as compared with diluting to 1 : 100 (73%) and is therefore recommended for IgA cord blood assays with the reagent used in this study, even if it is associated with a lower specificity (94% instead of 99%). Our data suggest that IgA assays are helpful for the early diagnosis of CT.     

Evaluation of the second generation IMx Toxo IgG antibody assay for detection of antibodies to Toxoplasma gondii in human sera

For an evaluation of the Abbott Imx Toxo IgG second generation, antibodies to Toxoplasma gondii were detected by Abbott Imx Toxo IgG and IgM, Vidas Toxo IgG and Toxo IgM (bioMérieux, France) with immunofluorescence assay verified by the dye-test for IgG, and immunosorbent agglutination assay (ISAGA) for IgM as references. The study included 507 serum samples collected over one month in two laboratories, 32 samples from HIV-infected patients, and 70 serial samples from 23 women surveyed for seroconversion or persistent IgM. After exclusion of nine equivocal results from the 507 samples, the sensitivity and specificity, respectively, were 100% (156/156) and 100% (342/342) for the Imx Toxo IgG and 98.1% (153/156) and 100% (342/342) for the Vidas Toxo IgG. Of the 32 HIV-infected patient samples, 7 gave false positive results with Imx Toxo IgG. This was because the samples had been heated. In 5 of the 70 serial samples, Imx Toxo IgG gave positive results earlier than Vidas Toxo IgG and in two cases earlier than IgM antibody assays. In this study Imx Toxo IgG second generation showed an increase in sensitivity and specificity in comparison with data reported previously for the first generation. J. Clin. Lab. Anal. 11:214–219, 1997. © 1997 Wiley-Liss, Inc.     

The post-natal serodiagnosis of congenital toxoplasmosis

A total of 20 cases of suspected congenital toxoplasmosis, investigated in the post-natal period, were reviewed. Maternal and infant sera were examined by the dye test, latex agglutination test, immunoglobulin M immunosorbent agglutination assay (ISAGA) and double sandwich enzyme-liked immunosorbent assay (DS-ELISA). Ten children were found not to be infected. The diagnosis of congenital toxoplasmosis was confirmed in 10 infants by the persistence of specific antibody at the age of 12 months, with or without clinical signs of infection. Of these 10 cases of congenital infection, three had toxoplasma specific IgM detected by ISAGA and a further three showed positive reactions by ISAGA and DS-ELISA at the time of investigation. The ISAGA was found to be superior to DS-ELISA for the diagnosis of congenital toxoplasmosis in the post-natal period. However, four infants subsequently demonstrated to be congenitally infected did not have specific IgM detectable by ISAGA or DS-ELISA and there remains a need for improved diagnostic methods for this condition.     

The confusing diversity of IgM tests in the diagnosis of Toxoplasma infections: efforts towards an optimal strategy

IgM antibodies are indicative for a recent infection, thus the detection of this isotype is of essential significance particularly in the diagnosis of infections with Toxoplasma gondii during pregnancy (primary infection, seroconversion). Numerous serological tests and test kits (e.g. indirect immunofluorescent assay/IFAT, enzyme-linked immunosorbent assay/ELISA, Immunosorbent agglutination assay/ISAGA, Westernblot/WB) using different antigens and antigen preparations are provided by numerous companies. The sensitivities of such tests, however, variy considerably: The serological test results of four pregnant women with seroconversions and of three newborns from mothers with seroconversions are presented: VIDAS M and ISAGA M from one company yielded false negative results in three pregnant women whereas ISAGA M from another company could detect specific IgM. However, examination of the cord blood of the three newborns unanimously revealed IgM-negative results. Thus, our diagnostic strategy for pregnant women includes IIFT (or SFT) as basic test and ISAGA M (Toxotool I from Innogenetics) as well as IgG avidity test as additional tests; the serological diagnosis of suspected congenital infection comprises IFAT (or SFT), ISAGA M (from Innogenetics) and IgM/IgG Westernblot.     

Antibiodies of the IgG, IgM, and IgA classes in newborn and adult sera reactive with gram-negative bacteria

Umbilical cord serum and adult serum antibodies reactive with heat-stable somatic antigens of Gram-negative bacteria (Neisseria gonorrhoeae, Escherichia coli, and Salmonella typhosa) were assayed by using an indirect fluorescent antibody test. Reactive IgG, IgM, and IgA antibodies were identified by using fluoresceinconjugated antisera specific for these immunoglobulin classes.IgG antibody titers in cord serum approximated those found in the corresponding maternal sera. IgM and IgA antibodies were present in adult sera but were not demonstrable or were present only in small amounts in cord sera. The presence of IgG and IgM antibodies reactive with Gram-negative bacteria was confirmed by the testing of purified 7S and 19S fractions. In addition, both IgG and IgM reactivities were inhibited by the prior incubation of serum with purified specific lipopolysaccharide preparations.The ubiquity and magnitude of these natural IgG antibodies in the sera of both adults and neonates have apparently eluded detection in previous studies. The use of bactericidal and agglutination tests, which are apparently more sensitive to the presence of IgM than to IgG antibodies, may account for the failure of previous studies to detect adult and cord IgG antibodies reactive with somatic antigens of Gram-negative bacteria. The presence of these IgG antibodies may be correlated with the resistance to infection demonstrated by most newborns as they are challenged by the septic extrauterine environment.     

Automated time-resolved immunofluorometric assay for Toxoplasma gondii-specific IgM and IgA antibodies: study of more than 130,000 filter-paper blood-spot samples from newborns

BACKGROUND: To screen for congenital toxoplasmosis, we developed a time-resolved immunofluorometric assay for the simultaneous detection of Toxoplasma gondii-specific IgM and IgA in filter-paper samples collected from newborns 4-7 days after birth. METHODS: The assay was performed on the AutoDELFIA, and results were calculated based on the ToxoM WHO Third International Reference Serum. Comparison with an in-house mu-capture immunoassay was carried out retrospectively on filter-paper samples from children with confirmed congenital toxoplasmosis. Prospectively the assay was compared with a mu-capture immunoassay on 68 394 samples and a commercially available assay on another 69,467 samples. Before serum was requested from the newborn, positive samples were tested for IgA and IgM separately and in an IgM-immunosorbent agglutination assay developed for filter-paper samples. RESULTS: Intra- and interassay variations (CVs) were 8% and 16%, respectively. The cutoff of 5 units/mL produced a 0.5% retest rate. The assay detected 13 of 18 (72%) samples from newborns diagnosed with congenital toxoplasmosis in the retrospective study. Prospectively, the assay identified 24 newborns who were later diagnosed with congenital toxoplasmosis. Results for all 24 cases were positive by the respective comparison method. No cases were detected solely by the IgA antibodies in the sample. CONCLUSION: Neonatal screening for congenital toxoplasmosis can be automated by use of purified europium-labeled antigen for detection of T. gondii-specific IgM and IgA eluted from filter-paper samples.     

IgG avidity assay firms up the diagnosis of acute toxoplasmosis on the first serum sample in immunocompetent pregnant women

Rapid diagnosis of acute toxoplasmosis during pregnancy permits timely treatment and prevents or attenuates congenital toxoplasmosis. Specific IgM antibodies to Toxoplasma as marker of acute infection are often poorly informative, meaning that a complementary technique is needed to reach a diagnosis on the first sample. Here we evaluated 2 commercial kits designed to assist with the diagnosis of acute toxoplasmosis: Platelia Toxo IgG Avidity Complementary Reagents and Platelia Toxo IgA, both from BIO-RAD (Marnes La Coquette, France). We tested 2 groups of subjects: 36 patients with acute toxoplasmosis and 55 patients with chronic toxoplasmosis. The IgG avidity test had a sensitivity of 100% (36/36), a specificity of 92.7% (51/55), a positive predictive value of 90%, and a negative predictive value of 100%. Among the immunocompetent women population, the avidity test had perfect sensitivity and specificity, and positive and negative predictive values of 100%. The IgA test had a sensitivity of 88.8% (32/36) and a specificity of 85.4% (47/55), and positive and negative predictive values of 80% and 92.1%, respectively. When the 2 tests were combined, there was only 1 case in which the diagnosis of chronic toxoplasmosis could not be confirmed. The IgG avidity test can therefore be used to rapidly distinguish between chronic and acute infection on the first sample from a pregnant woman, provided there is no underlying immunodepression and no ongoing antitoxoplasmic treatment. In these 2 situations, the results must be interpreted with care, and other serologic markers, including IgA, should be tested. Determination of a pregnant woman's status on a first serum sample allows therapeutic and preventive management to be started without delay.     

Immunosorbent agglutination assay: comparison with other methods of indirect detection of anti-Toxoplasma gondii IgM

Results obtained from the use of ISAGA method onto 180 serum samples in order to estimate its sensibility, specificity and reliability in detection of IgM anti-Toxoplasma gondii are referred. A comparison between ISAGA and ELISA-IgM or ISAGA and AD-GF/AD-2ME shows that the results coincide and that specificity and sensibility make it reliable in indirect detection of acquired or congenital toxoplasmosis.     

A study of IgM antibodies in diagnosis of acute infection by Toxoplasma gondii in Spain

A study of two ELISA methods (Eti-toxok-M, Sorin; Vidas Toxo IgM, bioMerieux) and one agglutination (Toxo Isaga, bioMerieux) was carried for the detection of specific IgM antibodies to Toxoplasma gondii in Southern Spain in different population groups. For diagnosis of acute toxoplasmosis ELISA methods showed 100% sensitivity, 99.4% specificity, 12% predictive value positive and 100% predictive value negative. Toxo Isaga showed 100% sensitivity, 99.5% specificity, 15% predictive value positive and 100% predictive value negative.     

Simultaneous detection of specific serum IgM and IgA antibodies for rapid serodiagnosis of congenital or acquired cytomegalovirus infection

Three hundred and seventy-one serum samples from 170 children with congenital or natally or post-natally acquired cytomegalovirus (CMV) infections, demonstrated by virus isolation from urine and/or saliva, were tested for specific IgM and IgA antibodies by an indirect enzyme immunoassay.Among infants with congenital CMV infection, IgM was detected more frequently than IgA (P < 0·001). In sera obtained 1 month post-birth, the IgA was more frequently raised (P < 0·05) than the IgM. Of 24 infants with perinatally-acquired CMV infection, 22 had IgM; of these, 41·7% had also IgA. Among 46 children with CMV-associated acute diseases, 26·1% of which showed no IgM or IgA, IgA was detected more frequently but not significantly (P > 0·05) than IgM. Among 53 children with recurrent or chronic cytomegaloviral diseases, IgA was detected in 11 (20·7%) and IgM in two (3·8%) patients (P < 0·001).The simultaneous detection of specific IgM and IgA antibodies is better than only IgM for rapid serological diagnosis of both congenital, and post-natally acquired CMV infections.     

Contribution of the Toxoplasma ICT IgG IgM® test in determining the immune status of pregnant women against toxoplasmosis

BackgroundAn immunochromatography technology (ICT) rapid diagnostic test, the Toxoplasma ICT IgG‐IgM^®, was recently developed. Our aim was to study its contribution to establish accurately the Toxoplasma immune status in Tunisian pregnant women using Western blot (WB) Toxo II IgG^® as a reference technique.MethodsThirty‐nine sera were selected for the study from among 2,615 which were already tested by IgG and IgM ELISA. They displayed equivocal IgG titres (4.4–9 IU/ml) in absence of IgM (19 sera) or IgM anti‐Toxoplasma antibodies in absence of IgG (titre <4.4 IU/ml) (20 sera). All these sera were additionally tested by WB Toxo II IgG^®.ResultsImmunochromatography technology Sensitivity in the detection either of low IgG titres in absence of IgM or of specific anti‐Toxoplasma IgM was 100%. Only one serum with equivocal IgG titre by ELISA and negative with Toxo II IgG^® test revealed positive in ICT. However, this serum showed a P30 band in WB analysis. On the other hand, three sera positive in ELISA IgM and negative in ELISA IgG revealed positive in ICT and negative in WB Toxo II IgG^®, the reference test.ConclusionResults confirm the high sensitivity of Toxoplasma ICT IgG‐IgM^® in detecting both specific anti‐Toxoplasma IgG and IgM, and highlight the usefulness of this rapid test as a first or second‐line Toxoplasma serological test in pregnant women.     

Enzyme-linked immunosorbent assay for determination of antibodies to xanthine oxidase

An enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG, IgA and IgM antibodies to xanthine oxidase. The method used xanthine oxidase to coat sample wells on microtitre plates. The anti-xanthine oxidase concentrations were determined by reference to standard curves constructed by coating plates with anti-IgG, anti-IgA and anti-IgM to capture antibodies of different classes in standard human serum. The standard curves for IgG, IgA and IgM had a working range of 0 to about 60 ng/ml, and all results with commercial quality control serum fell within expected ranges. The coefficients of variation (CV) for within-batch precision (n = 30) and between-batch precision (n = 20) for IgG and IgM were about 9% and 12% respectively. The detection limit was 2 ng/ml. The ELISA was applied to assay serum samples of 110 Chinese and 110 European healthy subjects. A positively-skewed distribution in their anti-xanthine oxidase IgG and IgM levels was observed.     

Antibodies to histo-blood group substances A and B: agglutination titers, Ig class, and IgG subclasses in healthy persons of different age categories

Isotypes and IgG subclasses of ABO antibodies from sera of 235 healthy blood donors were determined by an enzyme-linked immunosorbent assay (ELISA). Synthetic A and B trisaccharide-bovine serum albumin glycoconjugates were used for coating and monoclonal antibodies for the detection of heavy chain isotypes. Hemagglutination titers were determined in addition. Blood donors were between 20 and 67 years old, and at least 10 sera per 10-year age category and ABO blood group were included in this study. Antibody concentrations were expressed as a percentage of an internal standard, and sera with subclass-restricted anti-A and/or anti-B (anti-A/B) responses were used to normalize the ELISA values of IgG subclasses. A good correlation between the sum of the four subclasses and the total anti-A/B IgG values (rs = 0.81 for anti-A and 0.84 for anti-B) was obtained. IgG1 and IgG2 were the most predominant subclasses, but were found in various proportions in different individuals. Donor-to-donor variation exceeded age-related changes for all measured parameters. The correlation of anti-A IgM, IgG, IgA, and their sum with the agglutination titers was significant and revealed rs values of 0.70, 0.65, 0.65, and 0.80, respectively. For anti-B as well, the correlation of ELISA values with the agglutination titer was best when all three isotypes were added. We conclude that anti-A/B IgA, together with IgM and IgG, substantially contributes to the agglutination reaction. Potentially autoreactive antibodies were detected in sera of blood groups A, B, and AB.     

Sequential determination of IgG subclasses and IgA specific antibodies in primary and reactivating toxoplasmosis

During the course of T. gondii infection, we have analysed serum IgG and IgA antibodies responses in 50 immunocompetent with acquired infection and 19 immunocompromised patients with evidence of reactivated toxoplasmosis. Using an ELISA, IgG1, IgG2, IgG3 and IgA antibodies were found in sera of all patients, whereas IgG4 antibodies were usually not detectable. In immunocompetent patients, the predominant antibody isotype was IgG1 at the different stages of infection, presumably in relation with a T-cell control of humoral response during toxoplasmosis. In immunocompromised hosts (kidney or bone marrow transplanted and HIV infected patients), a sequential study was performed on serum samples taken before and after reactivation had occurred. The isotypic distribution of antibodies was similar to that observed in immunocompetent patients, but differences between groups of immunocompromised patients were detected when the kinetics of the antibody response was considered. The IgG and IgA antibody rise was lower in HIV1 infected patients with clinical toxoplasmosis; whatever was the peak antibody value, clinical symptoms appeared earlier in patients with a slower antibody response. This presumably reveals a functional T-cell abnormality, which may rely to the defective containment of the parasite in these patients.     

Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Complete congenital heart block (CCHB) is associated with anti-Ro/SS-A and anti-La/SS-B antibodies. Calreticulin, a calcium-binding, multifunctional protein of the endoplasmic reticulum with C-terminal KDEL-sequence, is not part of the Ro/SS-A ribonucleoprotein complex. In this study anti-calreticulin autoantibody responses in serum samples from 18 infants with CCHB, their mothers and in a control group of 11 anti-Ro/SS-A or anti-La/SS-B positive infants without heart block and their mothers were analysed. Specific enzyme-linked immunosorbent assays were performed. Nine out of 18 sera with CCHB contained IgG anti-calreticulin antibodies. Four sera of those with IgG antibodies also had IgM antibodies. One serum contained anti-calreticulin IgM antibodies only. In the non-CCHB group two sera were positive for IgG and one serum was positive for IgM anti-calreticulin antibodies. Sera of healthy infants were negative both for anti-IgG and anti-IgM calreticulin antibodies. Calreticulin is involved in calcium storage and therefore anti-calreticulin antibodies might influence the development of CCHB. The new finding of IgM autoantibodies and the observed differences in antibody response in infants and mothers support the hypothesis of a fetally mediated and passively acquired autoimmune disease.     

不同分娩方式对脐血抵抗素及新生儿免疫功能的影响

目的探讨不同分娩方式对脐血抵抗素浓度、新生儿免疫功能的影响;以及抵抗素与新生儿免疫功能的关系。方法2008年1月至2008年9月滕州市中心人民医院产科足月单胎分娩产妇,其中经阴道自然分娩60例,择期剖宫产50例,采用酶联免疫(ELISA)法检测脐血抵抗素含量,采用速率散射法免疫浊度测定系统检测脐血IgG、IgA、IgM、C3、C4;酶标法测定T细胞亚群,同时行血常规化验。结果阴道分娩脐血抵抗素浓度、免疫球蛋白水平和细胞免疫水平高于剖宫产分娩。结论不同分娩方式对脐血抵抗素与新生儿免疫功能有影响,阴道分娩可促进新生儿免疫功能的增加。     

Analysis of antibodies to Aspergillus fumigatus antigens by class-specific enzyme-linked immunosorbent assay in patients with pulmonary aspergillosis

Antibodies to two commercial extracts of Aspergillus fumigatus were determined by a class-specific enzyme-linked immunosorbent assay (ELISA) in 203 serum samples from 139 patients with various pulmonary diseases. In 22 patients with aspergilloma immunoglobulin (Ig)M, IgA, and especially IgG antibodies were found, whereas in 50 patients with allergic alveolitis IgG antibody was most frequent, IgM occurring rarely. One patient with allergic bronchopulmonary aspergillosis demonstrated IgG and IgA antibodies. Of 20 cases with bronchial asthma, 10% reacted against A. fumigatus in immunodiffusion as well as in ELISA. Of 46 cases with carcinoma, tuberculosis, and miscellaneous pulmonary diseases, 17% were positive by immunodiffusion and 26% demonstrated antibodies usually IgG, by ELISA. Of 100 healthy blood donors, none had Aspergillus antibodies of the IgG class, whereas 3% were positive in the IgM and 3% in the IgA assay. The ELISA proved to be sensitive and useful in the follow-up of patients with aspergilloma after operation.     

The prevalence of toxoplasmosis in Imam Reza Hospital blood bank samples,Tehran, Iran

BackgroundThe prevalence of toxoplasma gondii (T.g) infection in blood donors has been poorly studied. The aim of this study was to assess the prevalence of acute and chronic toxoplasmosis in blood products.MethodsA total of 223 blood products (101 fresh frozen plasma (FFP) and 122 packed cells (PC)) in Imam Reza hospital blood bank, Tehran, Iran were tested for specific T.g antibodies (IgG and IgM) by ELISA method. Positive IgG anti-T.g samples were further tested for IgM anti-T.g. A positive IgG test with the negative and positive IgM test was interpreted as a chronic and acute toxoplasmosis respectively.ResultsOf 223 samples 38.6% and 0.45% were positive for IgG anti-T.g and IgM anti-T.g levels respectively. Therefore, one and 85 samples were involved acute and chronic toxoplasmosis respectively. Twenty-six of fresh frozen plasma samples were positive for IgG anti-T.g and one of them was positive for IgM anti-T.g. Sixty packed cell samples were positive for IgG anti-T.g.ConclusionsOur study showed that there were chronic and acute toxoplasmosis in blood products and the prevalence of toxoplasmosis especially chronic form was high. Therefore screening of blood for T.g antibodies may be considered.     

Evaluation of three commercially available kits for serological diagnosis of dengue haemorrhagic fever

Seventy one acute phase serum samples collected during an epidemic of dengue hemorrhagic fever were tested by immunoblot, a rapid immunochromatographic assay and Dengue Duo ELISA for presence of anti dengue IgM and IgG antibodies. A concordance of 81.7% and 76.1% was seen between the three tests for the detection of anti-dengue IgM antibodies and IgG antibodies respectively. The rapid test takes only five minutes, can be easily carried out in most laboratories and compares well with the ELISA and the immunoblot.