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     

Prevalence of latent toxoplasmosis and serological diagnosis of active infection in HIV-positive patients

The seroprevalence of latentToxoplasma gondii infection was determined in a cohort of 715 HIV-positive patients followed up at an HIV outpatient clinic. Using indirect immunofluorescence and direct agglutination assays for detecting IgG, the prevalence of anti-Toxoplasma gondii antibodies was shown to be 50 %. During a four-year period, clinically apparent acute toxoplasmosis occurred in 47 patients (43 with cerebral, 3 with ocular and 1 with bone marrow toxoplasmosis) among the 360 patients positive for anti-Toxoplasma gondii IgG and in one patient (with cerebral toxoplasmosis) among the 355 patients who were serologically negative. A significant rise in IgG levels could be shown during acute toxoplasmosis episodes in only 30 % of patients, compared with 3 % of patients without active toxoplasmosis. During acute toxoplasmosis, IgM antibodies were detected in only two patients (6 %) by an immunosorbent agglutination assay and in one (3 %) by an enzymatic immunocapture assay. Specific IgA was detected by a non-enzymatic immunocapture assay in six patients (18 %) during acute episodes. The very high predictive value (99.7 %) of a negative IgG test remains the best serological parameter for excluding an acute episode of toxoplasmosis in HIV-positive patients.     

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.     

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.     

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.     

Molecular Markers in Acute and Chronic Phases of Human Toxoplasmosis: Determination of Immunoglobulin G Avidity by Western Blotting

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.     

Susceptibility of heat shock protein 70.1-deficient C57BL/6 J,wild-type C57BL/6 J and A/J mice to Trypanosoma congolense infection

Toxoplasma gondii is a protozoan parasite with a worldwide distribution. In both sheep and humans, if the parasite is encountered during pregnancy, fetal infection and abortion can occur. Therefore, Toxoplasma infection in sheep has a major economic impact upon sheep farming. Clinically, there is a need to distinguish recent (acute) infections from longstanding (chronic) infections. However, current serological techniques, such as detection of anti-T. gondii IgG, cannot discriminate between acute and chronic infections. Increasing immunoglobulin avidity is a good determining factor of how recent an infection is. In this study, we describe the application and validation of a T. gondii IgG avidity ELISA, based on the use of an affinity-purified, native T. gondii P30 antigen. The assay was used to examine sera from eight sheep experimentally infected with T. gondii and found that all seroconverted within 21 days post-infection (p.i.), beginning with avidities that were initially low but that increased over time, with all sheep reaching high IgG avidity within 10 weeks p.i. In addition, sera from clinically healthy but T. gondii-seropositive lambs and ewes and seropositive ewes with a history of abortion were also subjected to a preliminary serological investigation. High IgG avidities were found in 80% of the seropositive lambs, in 90% of the clinically healthy ewes and in 97% of the ewes with abortion problems. These findings indicate that the animals had most likely contacted the parasite a longer time ago.     

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.     

Detection of circulating antigens in immunocompromised patients during reactivation of chronic toxoplasmosis

One hundred and fifteen sera from nineteen patients undergoing bone marrow transplant and four recipients of kidney transplant who showed serological or clinical reactivation of chronic toxoplasmosis were tested forToxoplasma gondii circulating antigen (TCA) by enzyme-linked immunosorbent assay using antitoxoplasma IgG antibody coupled to alkaline phosphatase. Seven bone marrow transplant patients and one kidney transplant recipient were TCA positive either before or at the time of antitoxoplasma IgG antibody increase. TCA continued to be detected in one patient with neurological toxoplasmosis until his death. In the other patients, TCA disappeared when IgG antibodies rose, probably due to the formation of immunocomplexes consisting of TCA and immunoglobulins. In the TCA seronegative patients, the presence of circulating immunocomplexes or TCA kinetics of short duration may explain these results. Patients receiving immunosuppressive therapy should be tested for TCA.     

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.     

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.     

Use of MAG1 recombinant antigen for diagnosis of Toxoplasma gondii infection in humans

This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.     

Immunoglobulin G Avidity in Diagnosis of Toxoplasmic Lymphadenopathy and Ocular Toxoplasmosis

Traditional serological techniques have some limitations in evaluating the duration of Toxoplasma gondii infection in pregnant women, patients with lymphadenopathy, and older children suspected of having congenital toxoplasmosis. In these three groups of patients, two variants of T. gondii immunoglobulin G (IgG) avidity tests were used: an EIA Kit (Labsystems) and a noncommercial enzyme-linked immunosorbent assay specially elaborated in the laboratory. The avidity of specific IgG in sera from 23 patients with a known recently acquired infection (mainly pregnant women) was low (less than 30%), whereas that in sera from 19 patients with toxoplasmic lymphadenopathy of 3 weeks to 6 months in duration (mean, 8.3 weeks) covered a large range (between 0.2 and 57.8%; mean, 25.7%); high avidity results were observed for 10 of 19 patients (52.6%). The large range of IgG avidity in patients with toxoplasmic lymphadenopathy suggests various durations of infection in these patients, with a tendency for a chronic phase of toxoplasmosis. According to the avidity marker, five patients with lymphadenopathy for less than 3 months did not have a recent Toxoplasma infection. In 6 of 19 patients with lymphadenopathy (31.6%), low IgG avidity values persisted until 5 months after the first serological examination. In all four patients with a documented chronic course of Toxoplasma infection (6 months to 8 years after the first positive serology), high IgG avidity values were observed. Among sera from 10 children and young immunocompetent adults suspected of having ocular reactivation of congenital toxoplasmosis, all had high IgG avidity values (over 40%), suggesting congenitally acquired ocular infection rather than noncongenital infection. In conclusion, the avidity of IgG is a valuable marker of recent toxoplasmosis in pregnant women, suggests the duration of invasion in patients with lymphadenopathy, and may be helpful for differentiation between reactivation of congenital infection and recently acquired ocular toxoplasmosis in immunocompetent patients. A low IgG avidity does not always identify a recent case of toxoplasmosis, but a high IgG avidity can exclude primary infections of less than 5 months’ duration.     

Evaluation of serological markers for the immunodiagnosis of acute acquired toxoplasmosis

The detection of specific IgM antibodies has been the most frequently used serological marker for diagnosing recent toxoplasmosis. However, the persistence of specific IgM antibodies in some patients and the use of tests with a low specificity have complicated the interpretation of serological results when toxoplasmosis is suspected. The purpose of the present study was to determine the value of newer serological techniques in the diagnosis of acute acquired toxoplasmosis. Sixty-four sera, 31 from patients with Toxoplasma gondii infection and 33 from patients with latent infection, were tested. Anti-T. gondii IgA was measured by two antibody capture ELISA tests (Platelia Toxo IgA and ETI-TOXOK A) and an automated direct ELISA (IMx Toxo IgA); all three assays detected antibody levels compatible with a recent infection in sera from all 31 patients with acute toxoplasmosis. However, significant levels of IgA were also detected with high frequency by all three assays in sera from patients with latent infection. IgE antibodies detected by IgE immunosorbent agglutination assay (ISAGA) were present in 26 (84%) of 31 patients with acute toxoplasmosis and in sera from two subjects with latent infection taken >1 year after the beginning of the clinical symptoms of infection. Thirty (97%) of 31 patients with a recent T. gondii infection and 15 (45%) of 33 subjects with latent infection had an AC/HS pattern compatible with acute toxoplasmosis. The avidity of T. gondii IgG was evaluated by two methods. One method was based on the titration of each serum sample and calculation of the titres, in the absence and presence of urea, in relation to a defined cut-off value. In the other method, a single serum dilution was used and the absorbances of the reactions in the presence and absence of urea were compared. The titration method was more sensitive for diagnosing recent primary infection; all 31 sera from patients with acute toxoplasmosis had avidity indices compatible with acute toxoplasmosis by the titration method, whereas with the single dilution method, sera from four patients had equivocal results. In the 33 individuals with latent infection, similar results were obtained with the two avidity methods; only one serum sample had a non-compatible avidity value with the titration method. The results obtained in the present study show that the current serological markers used for diagnosing acute acquired toxoplasmosis have significant limitations. The data suggest that determination of the avidity of T. gondii-specific IgG by the titration method in patients with detectable IgM antibodies defines most accurately the stage of infection by T. gondii.     

Peptide Microarray Analysis of In Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples (n = 184) were collected from patients presenting with acute toxoplasmosis (n = 21), latent T. gondii infection (n = 53), and inactive ocular toxoplasmosis (n = 10) and from seropositive forest workers (n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers (n = 75) and patients (n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis.     

Seroprevalence and risk factors of Toxoplasma gondii infection in invasive raccoons (Procyon lotor) in Central Europe

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis in warm-blooded animals. Most mammals, including humans, can become intermediate host, resulting in subclinical infection or even death. Generally, there is limited information on the epidemiology of T. gondii of game species in Germany. As omnivores, raccoons, which are particularly widespread and abundant in Germany, are particularly exposed to infection the parasite. Here, we report the seroprevalence of T. gondii antibodies from 15 study sites located in Luxembourg and Germany. Using the indirect modified agglutination test (MAT), 170 (37.4%; 95% CI: 33.0–41.9) out of 454 raccoons were surveyed to be T. gondii seropositive. While values ranged from 19.0% to 53.3%, there was no significant difference in seroprevalence between study areas. Animal weight had a strong influence on the presence of T. gondii antibodies in raccoon sera, with heavier animals more likely to be seropositive. Our results show that T. gondii infection is widespread in central European raccoons, suggesting a high degree of ecosystem circulation of the parasite.

     

Induction of tolerance toToxoplasma gondii in newborn mice by maternal antibody

To develop an animal model for analysing the suppressed immune response toToxoplasma gondii in newborn humans with congenital toxoplasmosis, newborn mice from chronically infected mothers were inoculated intraperitoneally with bradyzoites of an avirulent strain. The newborn mice with maternalToxoplasma antibodies showed a marked delay in the production ofToxoplasma antibodies when infected after birth. Many mice (11/13; 85%) developed a state of tolerance toT. gondii after disappearance of the maternal antibody, demonstrable by the absence ofToxoplasma antibody in their sera despite the fact that they were infected. The duration of tolerance differed between individuals, with two mice showing the longest tolerant state of 8 weeks. This murine model might be suitable for analysing the mechanism of suppressed immune response toT. gondii that has been observed in many human cases of congenital toxoplasmosis.     

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.     

Secretion of Toxoplasma gondii-specific antibody in vitro by peripheral blood mononuclear cells as a new marker of acute toxoplasmosis.

Antigen-specific antibody secretion in vitro by peripheral blood mononuclear cells (PBMC) reflects an in vivo stimulation of the immune system by the antigen. Primary infection of immunocompetent patients with T. gondii causes an acute infection followed by chronic toxoplasmosis. We examined in vitro anti-Toxoplasma antibody production by PBMC during the acute and chronic phases of toxoplasmosis. PBMC from patients with acute or chronic toxoplasmosis and seronegative subjects were cultured for up to 6 days. Anti-Toxoplasma antibodies were assayed in supernatants by ELISA and immunoblotting. Anti-Toxoplasma antibodies were detected in supernatants of PBMC from 29 pregnant women who seroconverted during gestation. PBMC from 17 patients who had chronic toxoplasmosis and PBMC from 10 seronegative healthy controls did not secrete Toxoplasma-specific antibodies. This in vitro antibody secretion was spontaneous, active and transient since it disappeared between 11 and 24 weeks after seroconversion. Anti-Toxoplasma antibody secretion by PBMC from patients with acute toxoplasmosis is consistent with an in vivo stimulation of the immune system by T. gondii antigens. Our results represent a new approach for studying the immunological response during T. gondii infection and could have important implications for the diagnosis of acute and re-activated toxoplasmosis.     

Flow cytometry-based algorithm to analyze the anti-fixed Toxoplasma gondii tachyzoites IgM and IgG reactivity and diagnose human acute toxoplasmosis

In the present study we evaluated the performance of a flow cytometry-based algorithm as a new serological approach to detect antibodies to T. gondii and specific IgG avidity to diagnose acute toxoplasmosis. The results showed that using FC-AFTA-IgM assay, all serum samples from patients with acute toxoplasmosis demonstrated seropositivity, whereas 90% of patients with chronic infection and 100% of non-infected individuals presented negative results. Thus, only 10% of patients with chronic toxoplasmosis showed residual IgM, in contrast with other methodologies used to diagnosis acute toxoplasmosis. On the order hand, FC-AFTA-IgG assay as well as FC-AFTA-IgG subclasses is unlikely to discriminate acute from chronic toxoplasmosis. We have also evaluated the performance of FC-AFTA-IgG avidity as a tool to exclude chronic toxoplasmosis in patients with positive FC-AFTA-IgM. Our data showed an excellent performance of FC-AFTA-IgG avidity employing the cut-off of 60% for Avidity Index (AI) with sensitivity and specificity of 100%. All serum samples from patients presenting acute toxoplasmosis showed low avidity index (AI≤60%), whereas all chronic patients showed high avidity index (AI>60%). The outstanding performance indexes of this novel flow cytometry-based algorithm support its use as a non-conventional alternative serological approach to diagnose human acute toxoplasmosis.