Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay

We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of acute toxoplasmosis that used the recombinant granule antigen GRA6-GST as diagnostic antigen for the detection of IgG antibodies to Toxoplasma gondii in human sera. A total of 431 sera obtained from 336 patients with acute and chronic toxoplasmosis and from patients who were not infected with T. gondii were tested. Sera from patients with acute T. gondii infection, chronic infection, and no infection showed different absorbance values. For discrimination between the presence and the absence of acute toxoplasmosis the assay reached a specificity of 99.6%. Only one of the sera without significant anti-T. gondii. IgM antibodies showed a positive reaction to rGRA6-GST. The assay showed good intra- and interassay reproducibility (CV 6%/14%). We included a glutathione S-transferase (GST)-IgG enzyme immunoassay as a control assay in this study. Only 7 (4%) of 159 random sample sera reacted positively with GST. Received: 22 November 1997 / Accepted: 26 March 1998     

Unspecific Reactivity of IgM Directed Against the Low-Molecular-Weight Antigen of<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis>

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.     

Value and limitations of toxoplasmosis serology in HIV patients

Antibody titers to Toxoplasma gondii were studied in 62 AIDS patients with active toxoplasmosis (cerebral in 42, pulmonary in 10 and ocular in 10), confirmed by biopsy or by imaging techniques with a therapeutic test, and in 1,499 HIV-positive patients. The purpose of this study was to evaluate the value of antibody assays for the diagnosis of active infection and the prevalence of toxoplasmosis in HIV-positive individuals. IgG antibodies to Toxoplasma were found in 61 of the 62 AIDS patients, but not in one patient with pulmonary toxoplasmosis, with no significant differences in mean titers obtained by dye test, indirect immunofluorescence and sensitized agglutination. Twenty patients (31.7%) had dye test titers of 400 IU/ml or more; three patients had IgM antibodies. Thirteen (38%) of the 34 patients who had serial antibody assays exhibited a rise in IgG titers with no detectable production of IgM antibodies. Antibodies to Toxoplasma were found in 75% of the 1,499 HIV-positive subjects, a proportion which is not significantly different from that seen in HIV-negative controls; however, HIV-positive subjects were significantly more likely than controls to have high titers (greater than or equal to 500 IU in 18.7% of patients versus 9.2% of controls, p less than 0.001). A follow-up study in 177 HIV-positive patients with antibodies to Toxoplasma showed an annual reactivation rate of 12%; in five of 30 patients, the rise in antibody titers occurred concomitantly with or a few months before clinical toxoplasmosis; 25 patients remained asymptomatic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a Commercial IgG/IgM Western Blot Assay for Early Postnatal Diagnosis of Congenital Toxoplasmosis

The aim of this study was to evaluate a commercial Western blot IgG/IgM assay for use in the early serological diagnosis of congenital toxoplasmosis. This assay compares the immunological profile of mother and infant and allows differentiation between passive transmitted maternal antibodies and newly synthesized antibodies of the infant within the first 3 months of life. Over a 6-year period (1995–2001), the sera from 169 mothers and their 175 offspring (6 had twins) were examined for specific anti-Toxoplasma gondii IgG, IgM and IgA antibodies with an enzyme-linked immunosorbent assay or an immunosorbent agglutination assay. All mothers had primary Toxoplasma infection during pregnancy. Serological and clinical follow-up of the infants during the first year of life confirmed 36 cases of congenital toxoplasmosis. In 139 cases, infection could be ruled out. Three hundred fifty-one paired samples from 175 mother-child pairs were tested retrospectively for IgG and IgM patterns by Toxoplasma Western blot IgG/IgM (LDBIO Diagnostics, France). The results of conventional serological analysis (immunosorbent agglutination assay or enzyme-linked immunosorbent assay) to detect IgM or IgA were compared with the results of the Toxoplasma Western blot IgG/IgM on samples obtained within the first 3 months of life. The performance of the combination of the two methods was also assessed. At birth, the sensitivity values of conventional serological analysis and the Toxoplasma Western blot were 52% and 67%, with specificity values being 99% and 96%, respectively. Combination of the Western blot and conventional serological analysis increased the sensitivity at birth to 78% and within the first 3 months of life to 85%. Overall, the combination of both methods detected 94% of congenital infections. Therefore, this commercial Western blot represents a useful tool for early postnatal diagnosis of congenital toxoplasmosis. Electronic Publication     

Value of specific immunoglobulin a detection by two immunocapture assays in the diagnosis of toxoplasmosis

The diagnosis ofToxoplasma gondii infection is currently based on immunological tests, but tests for IgG and IgM antibodies alone are often insufficient to assess the risk of active disease, especially during pregnancy and in immunodeficient subjects. The supplementary diagnostic value of testing for antitoxoplasmic IgA in cases of acute, chronic, congenital and reactivated toxoplasmosis, relative to classical immunological tests, was evaluated using two immunocapture tests, one based on tachyzoite agglutination and the other on an immunoenzymatic complex recognizing the membrane protein P30 ofToxoplasma gondii. A total of 4,541 sera from 395 uninfected subjects, 468 immunized subjects with chronic infection, 117 subjects with acute infection and 403 children, 103 of whom had congenital toxoplasmosis, was tested. Specific IgA tests were negative in the nonimmune population, but tests for this immunoglobulin subtype became positive very rapidly during primary infection, and IgA disappeared more rapidly than IgM. In the children infected in utero, specific IgA was detected more frequently than IgM. In contrast, in a population of HIV-seropositive subjects with clinical toxoplasmosis, tests for IgA were poorly sensitive. The two tests for specific IgA produced similar results, except in the early stages of primary infection, in which immunoenzymatic testing for anti-P30 IgA was less sensitive than the agglutination method.     

Antibody response of HIV-infected patients to latent,cerebral and recently acquired toxoplasmosis

The aim of this longitudinal study with 626 HIV-infected patients was to evaluate the capability of serological tests in diagnosing the presence of Toxoplasma gondii infection in HIV-infected patients, as well as the potential impact of various treatment regimes on serological results. Low IgG antibody levels and stable or declining titres predominated. IgM positivity occurred in ten patients (one seroconversion, seven latent, two cerebral toxoplasmosis). Complement fixation test (CFT) titres ≥1:32 imply that the relative risk of cerebral toxoplasmosis is 6.84 (95% confidence interval [CI] 1.44–32.5) but with a predictive value of only 14.0% (95% CI 5.3–27.9). Values of specific antibodies are not biassed by antiretroviral treatment and/or prophylaxis for toxoplasmosis, and the detection of specific antibodies is very useful in the identification of T. gondii infection in the HIV-infected population, but the role of serology in predicting the clinical manifestation of T. gondii infection is limited.     

Estimation of the avidity of immunoglobulin G for routine diagnosis of chronicToxoplasma gondii infection in pregnant women

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies toToxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated forToxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt lowavidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n=493) containing both IgG and IgMToxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of >35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (<3 months) infectious incident. Values of <35% require repeat testing four weeks later to confirm the patient's status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.     

Toxoplasma gondii surface antigen-1 in sera of HIV-infected patients as an indicator of reactivated toxoplasmosis

The major surface antigen from the proliferative form ofToxoplasma gondii (P-30 or SAG-1) was chosen as a target for exploration ofToxoplasma gondii reactivation in sera from immunocompromised patients. Samples were obtained from 37 HIV-infected subjects with lymphocyte levels of CD4+ <200/mm³. The prevalence of IgG antibodies toToxoplasma gondii was 64.9 %. Ten patients had clinical symptoms of reactivated toxoplasmosis; eight of these hadToxoplasma encephalitis. The SAG-1 epitopes were found as circulating antigen in five cases with an immunocapture enzyme immunoassay (EIA). The EIA was improved with an IgG1 monoclonal antibody to SAG-1 and a streptavidinbiotin amplification. The sensitivity, specificity and positive predictive value were 30, 92 and 60 %, respectively. The SAG-1 levels were compared with different biological parameters such as HIV p24 antigen, ₂ microglobulin, CD4+ cell count and IgG antibodies toToxoplasma gondii. The levels of SAG-1 in these patients were significantly higher than those in the 75 healthy control persons with or without a chronicToxoplasma gondii infection. Therefore, SAG-1 may be involved as a marker of reactivated toxoplasmosis in HIV-infected patients.     

Onset of ocular complications in congenital toxoplasmosis associated with immunoglobulin M antibodies toToxoplasma gondii

Four patients with congenital toxoplasmosis serologically diagnosed by the Sabin-Feldman test (SFT) and the IgM-indirect fluorescent antibody test (IgM-IFAT) in the first year of life presented with eye disease between the age of 21 months and ten years. Repeated serological testing revealed increasing levels of specific antibodies as measured by the SFT. IgM antibodies toToxoplasma gondii were detected in all four patients by the immunosorbent agglutination assay, in two by the IgM-IFAT and in three by the IgM-indirect haemagglutination test. Findings suggest that specific IgM antibodies reappear at the time of reactivation of congenital toxoplasmosis later in life, or possibly persist for an extraordinary long period (up to ten years).     

Recognition of recombinantToxoplasma gondii antigens by human sera in an ELISA

We developed an enzyme-linked immunosorbent assay (ELISA) that uses one of two recombinant polypeptides, termed H4/GST and H11/GST, as diagnostic antigens for the detection of antibodies toToxoplasma gondii in human sera. A total of 59 sera from humans with acute toxoplasmosis, 194 sera from patients with chronic toxoplasmosis, and 151 sera from subjects who were not infected withT. gondii were examined. In all, 68% of the sera from humans with acute toxoplasmosis reacted positively with one or both recombinantT. gondii antigens. By contrast, only 14% of those from patients with chronic toxoplasmosis recognized H4/GST or H11/GST. None of the sera from humans who were not infected withT. gondii, including patients with echinococcosis, entamoebosis, toxocarosis, trichinellosis, glandular fever, or rheumatoid arthritis, recognized H4/GST or H11/GST.This publication is dedicated to Professor J. Eckert (Zürich) on the occasion of his 60th birthday     

Performance of a Western Blot Assay to Compare Mother and Newborn Anti-Toxoplasma Antibodies for the Early Neonatal Diagnosis of Congenital Toxoplasmosis

 The aim of this study was to retrospectively evaluate the performance of a Western blot assay to compare mother and newborn anti-Toxoplasma gondii antibodies for the early neonatal diagnosis of congenital toxoplasmosis. Since specific anti-Toxoplasma IgM or IgA is detected inconstantly at birth in the neonate, the diagnosis of congenital toxoplasmosis is often delayed until 6–9 months, after IgG titers have been observed persistently. In this study, 81 paired samples from 60 mother/child pairs were tested for IgG and IgM patterns. All mothers had (or were strongly suspected to have) acquired toxoplasmosis during pregnancy. Specific IgM and IgA were simultaneously detected by immunocapture tests, and IgG was titrated. A serological and clinical follow-up of infants was conducted during the first year of life until the diagnosis of congenital toxoplasmosis could be either confirmed or ruled out. Seventeen of the 60 newborns were congenitally infected. Specific IgM or IgA was detected by immunocapture at birth in 76.5% and 70.6% of cord sera from infected neonates, respectively, with an equal specificity of 77.5%. Comparative Western blot allowed the detection of neosynthesized IgG and IgM in the cord blood of 50% and 78.6% of infected infants, respectively, with a specificity of 100%. The combination of IgA and IgM immunocapture tests, the analysis of IgG and IgM Western blot patterns, and the combination of both techniques allowed the detection of 94%, 94%, and 100% of cases within the first 3 months of life, respectively. In conclusion, Western blotting seems to be a useful complementary tool for the early postnatal diagnosis of congenital toxoplasmosis.     

The probable relation between Toxoplasma gondii and Parkinson's disease

Parkinson's disease (PD), a chronic progressive neurodegenerative disorder, has a mainly unknown multifactorial etiology. Neuroinflammatory mechanisms might contribute to the cascade of events leading to neuronal degeneration. Toxoplasmosis can be associated with various neuropsychiatric disorders. The most commonly affected central nervous system (CNS) region in toxoplasmosis is the cerebral hemisphere, followed by the basal ganglia, cerebellum and brain stem. Therefore, in this study, we aimed to investigate the possible association between Toxoplasma infection and PD by evaluating the serum anti-Toxoplasma gondii IgG antibodies. There were no difference between the socioeconomic status of the patients and control subjects and magnetic resonance images of the patients were normal. Serum anti-T. gondii IgG levels were measured using ELISA. There was no statistically significant differences among the patients and control subjects with respect to age (66.01 ± 12.14 years, 62.42 ± 5.93 years, p = 0.089; respectively) and gender. The sero-positivity rate for anti-T. gondii IgG antibodies in PD patients and control groups were 42.3 and 22.5%, respectively, and they were statistically significant (p = 0.006). These results suggest that Toxoplasma infection may be involved in the pathogenetic mechanisms of PD. If confirmed, this hypothesis would represent a valuable advancement in care of patients with Parkinson's disease.     

Toxoplasmosis in heart transplant recipients

In cardiac transplant recipients, infection withToxoplasma gondii may be transmitted with the transplanted organ to immunosuppressed recipients or may be due to reactivation under immunosuppression in cases of pretransplant infection. In the present study the incidence of infection withToxoplasma gondii and the clinical presentation of the infection in 121 consecutive heart transplant recipients were investigated. Data on IgG and IgM antibodies forToxoplasma gondii measured by a semiquantitative microparticle immunoassay of donors and recipients were collected prospectively in 121 patients. Infection withToxoplasma gondii was defined as IgM seroconversion with proven pretransplant seronegativity (primary infection) or at least a fourfold increase of IgG antibodies (reactivation). Infection withToxoplasma gondii occurred in 16 of 121 patients (13%), whereas overt clinical disease occurred in 5 of 121 patients (4%). Organ-transmitted infection was more frequent (11/18, 61%) and more often associated with acute disease than reactivation of latent infection (5/69 patients, 7%) (p < 0.01), although one case ofToxoplasma retinochoroiditis occurred in a patient with recrudescence of latent pretransplant infection. Treatment with pyrimethamine and sulfadiazine was efficient in all patients with acute disease and in controlling disease in patients with evidence of acute infection.     

Detection of toxoplasmosis in patients with end-stage renal disease by enzyme-linked immunosorbent assay and polymerase chain reaction methods

Toxoplasmosis caused by Toxoplasma gondii is an opportunistic infection. In healthy individuals, the infection is largely asymptomatic, but in immunocompromised people the parasite can become widely disseminated, causing severe toxoplasmosis. In patients undergoing haemodialysis, the phagocytic process shows a highly significant impairment. Therefore, this study aimed to investigate toxoplasmosis in patients with end-stage renal disease (ESRD) undergoing haemodialysis in Ahvaz hospitals, southwest of Iran. A total of 280 patients and 100 healthy subjects participated in this study. The presence of serum IgM and IgG antibodies against T. gondii was detected by ELISA and the presence of Toxoplasma parasites in whole blood was evaluated by GRA6 PCR. Anti-T. gondii IgG antibodies were detected in 82 (29.3 %) haemodialysis patients and 26 (26 %) controls. In addition, anti-T. gondii IgM antibodies were detected in 7.9 % of patients and in 4 % of controls. For both the antibodies, the differences were statistically significant (P?<?0.05). PCR was performed with DNA extracted from blood samples of all patients and controls. PCR gave positive results with four of the 280 blood samples from patients but none for the control blood samples. The results revealed a high percentage of positivity for Toxoplasma antibodies in patients with ESRD undergoing haemodialysis and also confirmed the parasite in whole blood, indicating disseminated infection in these patients. Patients undergoing dialysis have a higher rate of active infection with Toxoplasma likely due to reactivation of a chronic infection. Thus, parasitological examinations of ESRD patients should be periodically carried out for monitoring and evaluating the possible dissemination of toxoplasmosis during haemodialysis.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibody profile in HIV-infected pregnant women and the risk of congenital toxoplasmosis

The purpose of this study was to investigate the antibodies to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected pregnant women and to determine the association between serological profile and the risk of congenital toxoplasmosis. The study, conducted in a public maternity ward from May 2002 to April 2005, included all HIV-infected women who delivered live infants during the 36 months, and, as a control group, all HIV-negative women that delivered live infants in the first 12 months of the study. Antibodies to T. gondii were detected in 1,624 of 2,421 HIV-negative women (67%; 95% confidence interval [CI] 65–69%) and in 121 of 168 HIV-infected patients (72%; 95% CI 65–79%). A total of 547 HIV-negative and 103 HIV-infected patients were tested at delivery and had positive T. gondii-specific IgG. In HIV-negative women, the median of the specific IgG concentration was 79 (interquartile range 38–160), and in HIV-infected patients, it was 283 (interquartile range 94–704) (P < 0.001). In the group of co-infected women, the only infant with congenital toxoplasmosis was born to a mother with acute toxoplasmosis infection acquired during pregnancy who did not have a high specific IgG concentration or a positive result for specific IgM. We concluded that high T. gondii-specific IgG values were much more frequent among HIV-infected pregnant women, but it did not translate into an increased risk of maternal–fetal transmission of toxoplasmosis.     

Detection of specific antibodies toToxoplasma gondii by a competitive enzyme immunoassay using a monoclonal antibody against the P30 antigen

An inhibition EIA using a monoclonal antibody against the major P30Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody toToxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100 % and 97 % respectively. This test was found to be as sensitive as the dye test.     

Neospora spp. and Toxoplasma gondii antibodies in horses in the Czech Republic

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had ≥50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.     

Analysis of the antibodies anti-<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> by ELISA based on two diagnostic antigens: rSAG1 and rBAG1

Schizophrenia is a serious neuropsychiatric disease of uncertain etiology. Previous studies have demonstrated that antibodies to Toxoplasma gondii infection are associated with an increased risk of schizophrenia. The objective of this study was to analyze anti-T. gondii antibodies in 477 Chinese schizophrenia patients using an enzyme-linked immunosorbent assay (ELISA) based on recombinant surface antigen 1 (rSAG1), recombinant bradyzoite antigen 1 (rBAG1) and the soluble tachyzoite antigens (STAg) of T. gondii RH strain. Results showed that among the sero-positives (IgG and/or IgM) for T. gondii infection examined in schizophrenia patients, sero-positive samples for rSAG1, rBAG1 and STAg were 20.5% (98/477), 20.5% (98/477) and 23.5% (112/477) respectively, while compared to 210 blood donors, sero-positive (IgG and/or IgM) samples for these antigens (rSAG1, rBAG1 and STAg) were only 5.7% (12/210), 6.2% (13/210) and 5.7% (12/210), respectively. Furthermore, when IgG antibody reaction in the schizophrenia sera was compared with the rBAG1 and rSAG1, results demonstrated that beside the cases which can be detected by both rSAG1 and rBAG1, some sero-positive for T. gondii in schizophrenia sera can only be detected either by rSAG1 or rBAG1. This phenomenon was also observed in the detection of IgM with rSAG1 and rBAG1. 5.9% (28/477) of cases of schizophrenia which are positive for IgG or IgM by rSAG1 are negative for STAg, while 9.2% (44/477) of the schizophrenia cases which are positive for IgG or IgM by rBAG1 are negative for STAg. Although STAg can also be used to diagnose T. gondii infection from schizophrenia patients, it may not actually indicate the infection as some positive samples may be mistakenly considered to be negative. In conclusion, our results demonstrate that the sero-positive rate for T. gondii in the Chinese schizophrenia patients was higher than blood donors. More importantly, our results provide evidence that the combination of rSAG1 and rBAG1 antigens in the diagnosis of T. gondii infection could closely reflect the actual infection of this parasite in schizophrenia patients.     

Performance of European laboratories testing serum samples forToxoplasma gondii

One hundred twenty-nine European laboratories participated in a collaborative, multicentre study designed to evaluate the overall reliability of different serological techniques for diagnosis ofToxoplasma gondii infection. Five freeze-dried reference sera were distributed to each laboratory, each of which analysed the sera with its routine methods. The enzyme-linked immunosorbent assay was the technique used most frequently, followed by the immunofluorescent antibody technique. Only nine laboratories performed the Sabin-Feldman dye test. In general, there was good concordance between qualitative results, but for sera with low concentrations ofToxoplasma gondii-specific IgG antibodies, some false-negative results were found. For specific IgM and IgA antibodies, the immunosorbent agglutination assay proved the most sensitive. The present study demonstrates the need for regular assessment of laboratory serodiagnosis ofToxoplasma gondii infection.     

Seroepidemiology of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in pet dogs and cats in Beijing,China

Sera from 534 pet dogs and 335 pet cats from Beijing (China) were tested for anti-Toxoplasma gondii antibodies using an enzyme-linked immunosorbent assay or the latex agglutination test. The seropositivity by year, season, sex and age was analysed. Overall, 128 dogs (24.0%) and 50 cats (14.9%) had antibodies to T. gondii. When analysed by season, the highest seroprevalence was found in spring for dogs (31.3%) and cats (25.1%), and the differences in seroprevalence by season was statistically significant in cats (P<0.01) but not in dogs. The seroprevalence in male dogs (23.7%) and cats (15.1%) were slightly higher than their female counterparts (18.0% in dogs and 12.3% in cats). There was no obvious pattern of seropositivity or significant difference in different age groups in dogs or cats; nonetheless, a high proportion of dogs at 4 years of age were positive to T. gondii (31.8%) while cats with relatively high seropositivity rates were at 1 or 3.4 years of age (13.14%).