Gondii IgA Research Articles

Comparison of immunological and molecular methods for laboratory diagnosis of ocular toxoplasmosis in blood, serum and tears in Brazil.

Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients' clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.

Screening for the Detection of Toxoplasma gondii IgG, IgM and IgA in Females of Reproductive Age from Western Romania.

Toxoplasma gondii, a zoonotic protozoan parasite, has the capacity to infect the fetus if the pregnant woman primarily acquires the infection during pregnancy. We evaluated the prevalence of T. gondii IgG, IgM and IgA antibodies in women of reproductive age residing in Western Romania. We also assessed the value of adding a T. gondii IgA test to the serologic panel for the diagnosis of toxoplasmosis, including the detection of a recently acquired infection. Serologic testing to demonstrate the presence of T. gondii IgG antibodies was conducted in 1317 females aged 15-45 years. T. gondii IgM and IgA antibody tests were performed in those with detectable IgG antibodies and IgG avidity test was performed if IgM and/or IgA screening test results were positive. T. gondii IgG were detected in 607 (46.09%; 95%CI: 43.41-48.79) of 1317 study participants and IgG seroprevalence tended to increase with age from 35.44% (95%CI: 29.89-41.30) in age group 15-24 years to 62.85% (95%CI: 56.57-68.82) in age group 35-45 years, showing a significant age-associated increase (p < 0.001). Of the 607 persons with detectable T. gondii IgG antibodies, T. gondii IgM antibodies were demonstrated in 8.90% (95%CI: 6.88-11.43), T. gondii IgA in 1.65% (95%CI: 0.90-3.01) and both T. gondii IgM and IgA in 0.99% (95%CI: 0.45-2.14). The prevalence of IgA antibodies tended to decrease with increasing avidity, from 75% (95%CI: 19.41-99.37) in samples with low avidity to 11.76% (95%CI: 4.44-23.87) in those with high avidity (p = 0.01). Of the study participants who were positive for both T. gondii IgM and IgA antibodies, 66.67% had low or equivocal IgG avidity test results compared to 6.25% who tested positive for IgM, were negative for IgA and in whom low or equivocal IgG avidity test results were noted (p = 0.001). This study indicates that in Western Romania, T. gondii IgG seroprevalence is high in females of reproductive age and T. gondii IgA antibodies may be rarely detected during a serologic screening. However, in individuals with demonstrable T. gondii IgG and IgM antibodies, testing for T. gondii IgA may improve the rate for the detection of a recently acquired toxoplasmosis.

Diagnosis of Congenital Toxoplasmosis: No Benefit of IgA Antibody Detection by Platelia ELISA in a Tricentric Evaluation.

The diagnostic accuracy of a commercial Toxoplasma gondii IgA antibody enzyme-linked immunosorbent assay (ELISA) was evaluated in the context of routine practice on 289 newborns with congenital toxoplasmosis (CT) and 220 healthy controls. The performance of this assay was compared to that of the current gold-standard test for anti-Toxoplasma IgM detection, an immunosorbent agglutination assay (ISAGA). IgM and IgA sensitivity and specificity were assessed in cord and postnatal samples. The sensitivity of IgA detection by ELISA on all serum and peripheral blood samples was 60.56% and 56.52%, respectively, which is low compared with the sensitivity of IgM detection by ISAGA (73.26% on serum samples, 82.35% on peripheral blood). Adding the T. gondii IgA antibody ELISA to the diagnostic panel did not significantly increase the overall performance of the serological diagnosis based on IgM detection.

Application and expression of Toxoplasma gondii surface antigen 2 (SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia coli strain

The gene encoding surface antigen 2 (SAG2) or rhoptry protein 2 (ROP2) of Toxoplasma gondii was cloned into the plasmid pGEX-4T-1 and subsequently expressed in Escherichia coli as a glutathione-s-transferase (GST) fusion protein. The characteristics of purified GST-SAG2 or GST-ROP2 were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The specific IgG of a panel of serum samples provided by the National Institute for the Control of Pharmaceutical and Biological Products were tested with commercial ELISA and the lateral flow immunoassay (LFIA) based on GST-SAG2, GST-ROP2 or GST-SAG2+ROP2. A total of 1096 sera and saliva samples from pregnant women were tested by GST-SAG2+ROP2-LFIA. In total, 20 T. gondii IgM positive sera (1.82%), 81 T. gondii IgG positive sera (7.4%) and 23 T. gondii IgA positive saliva (2.1%) were finally confirmed. The SAG2+ROP2 specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunised with GST-SAG2+ROP2. The results indicate that GST-SAG2+ROP2 protein can be used as an antigen for diagnosing T. gondii infection and provide a strategy for development of subunit vaccines for protection against T. gondii infection.

Assessment of the value of detecting specific IgA antibodies for the diagnosis of a recently acquired primary Toxoplasma infection

To assess the value of detecting IgA antibodies for the diagnosis of a recently acquired primary Toxoplasma infection. IgA antibodies were screened in sera from 87 women with different serological profiles of Toxoplasma gondii IgM and IgG antibodies and Toxoplasma-specific IgG avidity. The IgM and IgG antibodies and the IgG avidity were measured with an automated Vitek Immuno Diagnostic Assay System (VIDAS). Anti-T.gondii IgA was measured with Platelia Toxo IgA TMB kits. All 12 sera obtained from women with clinical and/or serological evidence of a recently acquired Toxoplasma infection were positive for IgA. In 42 serum samples obtained more than 6 months after T. gondii infection from women with no clinical evidence of infection, but who had a positive IgM test and a high IgG avidity index, the IgA-enzyme linked immunosorbent assay (ELISA) test results were positive, negative, and doubtful in 16 (38.1%), 23 (54.8%), and 3 (7.1%) sera, respectively. In eight women, IgA was detected in sera collected more than 9 months after the onset of infection. The IgA test result was also positive in 11 of 12 sera (91.7%) obtained from women with no clinical evidence of toxoplasmosis, but who had a positive IgM test and a borderline IgG avidity index. The IgA-ELISA was negative in 21 sera obtained more than 2 years after the onset of T. gondii infection from women with no clinical evidence of toxoplasmosis, but who had a negative IgM test and a positive IgG test. These results show that IgA is not a dependable marker for a recently acquired primary Toxoplasma infection.

Detection of clinical-stage specific molecular Toxoplasma gondii gene patterns in patients with toxoplasmic lymphadenitis

Three cases of symptomatic toxoplasmic lymphadenitis, together with a serologic profile of recent infection, are described, for which quantitative real-time PCR (LightCycler PCR) targeting different parasite genes was designed, in order to quantify Toxoplasma gondii DNA in acute and follow-up blood specimens. Similar parasite gene kinetics and DNA concentrations were observed in the patients studied. However, the profile of each target gene investigated was different. While the level of B1 DNA remained elevated for the entire time of observation, irrespective of clinical and serologic resolution, the SAG-1 gene was detected at the end of acute symptomatic disease, overlapping with a strong anti-T. gondii IgA antibody response, and persisting for over 3 months after infection and clinical recovery. With respect to the two bradyzoite genes investigated (SAG-4 and MAG-1), levels peaked during the symptomatic phase, but did not fall until 2 or 3 months of follow up. The real-time PCR assay with new alternative targets to the B1 gene may have potential for monitoring the clinical outcome of disease and for providing molecular information regarding the actual state of infection.

Value of the comparative enzyme-linked immunofiltration assay for early neonatal diagnosis of congenital Toxoplasma infection.

The transplacental transfer of specific maternal IgG antibodies makes the diagnosis of congenital Toxoplasma infection quite difficult in the neonate. The enzyme-linked immunofiltration assay (ELIFA), comparing at delivery the immunologic profile of the mother's antibody response and that of her child, allows discrimination between IgG antibodies of maternal origin and IgGs synthesized by the fetus. To evaluate the diagnostic reliability of the comparative ELIFA for diagnosing congenital Toxoplasma infection as well as the reliability of testing for IgM- and IgA-specific antibodies in cord blood. From November, 1991, to December, 1995, an ELIFA was prospectively performed at delivery on blood samples obtained from 227 women with primary Toxoplasma infection during pregnancy and from their infants. For each child the ELIFA result was evaluated in relation to the serologic follow-up: disappearance of specific anti-Toxoplasma gondii IgG antibodies in the absence of treatment before 12 months of age indicating an uninfected child, as opposed to persistence beyond 12 months of age indicative of a congenital infection. Of 227 children 139 were lost to follow-up. Among the 88 children available for follow up, the ELIFA was negative in 70 infants, 69 of whom were confirmed to be uninfected. Thirteen of these 69 cord blood ELIFA-negative samples were positive for anti-T. gondii IgM and/or IgA detected by means of a conventional immunosorbent agglutination assay. Of the remaining 18 children (representing 75% of all new cases of congenital toxoplasmosis diagnosed during the study period at our institution), the ELIFA was positive in 16, negative in 1 and inconclusive in 1. The ELIFA test is a valuable tool for diagnosing congenital T. gondii infection and in differentiating between true neonatal infection and cord blood contamination. In our experience the diagnostic sensitivity of the ELIFA test was 94.1% and the specificity was 98.6%. The cord blood was contaminated by specific maternal anti-T. gondii IgA and/or IgM in as many as 20% of the cases.

Serologic evaluation of patients with primary and recurrent ocular toxoplasmosis for evidence of recent infection

PURPOSE: To identify the frequency of recently acquired vs chronic systemic Toxoplasma gondii infections in patients with ocular toxoplasmosis. METHODS: Serum samples from 22 patients with primary ocular toxoplasmosis (not from scars) and 42 patients with recurrent ocular toxoplasmosis were tested for the presence of anti– T. gondii IgM, IgG, and IgA antibodies and compared with samples from 24 patients with other causes of uveitis. Intraocular production of anti– T. gondii IgG and IgA, and the presence of T. gondii DNA was determined in patients and control subjects from whom ocular fluid was available. RESULTS: Serologic evidence of recently acquired infection was found for 11 (50%) of 22 patients with primary ocular toxoplasmosis and for one (2%) of 42 with recurrent ocular toxoplasmosis. In the uveitis control group, anti– T. gondii IgM antibodies could be detected in two (8%) of 24 patients, but anti– T. gondii IgA antibodies were not detectable. Patients with primary ocular toxoplasmosis and serologic markers of recently acquired systemic infection were significantly older than those with chronic infection ( P = .008). Intraocular production of anti– T. gondii IgG was more frequently noted in patients with recurrent than primary ocular toxoplasmosis (81% vs 41%; P < .001), but intraocular T. gondii DNA was more frequently found in patients with primary ocular toxoplasmosis than in those with recurrent ocular toxoplasmosis (37% vs 4%; P < .01). CONCLUSIONS: Primary ocular toxoplasmosis can be seen in either recently acquired or chronic T. gondii infection. Patients with ocular disease and recently acquired infection were older and more likely to have T. gondii DNA in intraocular fluids.

Serological diagnosis of toxoplasmosis: usefulness of IgA detection and IgG avidity determination in a patient with a persistent IgM antibody response to toxoplasma gondii

We report the detection of specific IgA antibodies and the determination of IgG avidity in sequential serum samples from a patient exhibiting significant levels of Toxoplasma-specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. IgM antibodies were detected by an indirect immunofluorescence test and by three commercial enzyme-linked immunosorbent assays (ELISA). Anti-T. gondii IgA was quantified by the alpha-capture ELISA technique using a commercial kit. As defined by the manufacturer of the IgA ELISA test used, most patients with acute toxoplasmosis have antibody levels > 40 arbitrary units per ml (AU/mL). At this cut-off level, the patient still had a positive ELISA result (45 AU/mL) in a serum sample taken one year after the beginning of clinical manifestations. The IgG avidity-ELISA test was performed with the Falcon assay screening test (F.A.S.T.(R)) - ELISA system. Avidity indices compatible with a recent Toxoplasma infection were found only in serum samples taken during the first 5 months after the onset of the clinical symptoms of toxoplasmosis. These results show that the interpretation of positive IgM results as indicative of recently acquired toxoplasmosis requires additional laboratory confirmation either by other tests or by the demonstration of a significant rise in the antibody titers in sequential serum samples.

Positive evidence that anti- Toxoplasma gondii IgA antibody exists in the intestinal tract of infected cats and exerts protective activity against the infection

The presence of anti- Toxoplasma gondii IgA antibody in the feces and intestinal tract of cats infected with this parasite was demonstrated using an immunoblotting assay. Cats ( n = 5) were inoculated orally with T. gondii cysts and supernatants of feces and washings of the intestinal tract were assayed for secretory IgA specific for the parasite. The secretory IgA detected recognized tachyzoite antigens of m.w. 24, 34, 38 and 43 kDa and one sporozoite antigen of m.w. 24 kDa. No reactivity was shown against bradyzoites or enteroepithelial stage parasites. Tachyzoites preincubated with washings of the intestinal tract of infected cats showed decreased activity in penetration of feline fibroblast cells, as compared to tachyzoites preincubated with similar derived from non-infected cats. The addition of either anti-cat IgA or anti-cat IgG to the washings had no effect on the inhibitory activity which reduced the parasite's cell-penetration activity. However, the addition of both anti-cat IgA and anti-cat IgG to the washings diminished the inhibitory activity. These results suggest that anti- T. gondii antibodies of both classes, secretory IgA and IgG, exist in the intestinal tract of infected cats and these may be capable of preventing infection.