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PubMed1996

Predictive value of Toxoplasma gondii antibody titres on the occurrence of toxoplasmic encephalitis in HIV-infected patients. ANRS 005/ACTG 154 Trial Group.

OBJECTIVE: To study the predictive value of anti-Toxoplasma gondii antibody titres for the occurrence of toxoplasmic encephalitis (TE) in HIV-infected patients. METHODS: Data from the placebo arm of a trial of primary prophylaxis for TE (ANRS 005/ACTG 154) were analysed. Patients included had CD4+ cell counts < 200 x 10(6)/l and a positive Toxoplasma serology. Immunoglobulin (Ig) G and IgM Toxoplasma antibody titres at entry were retrospectively determined by enzyme-linked immunosorbent assay and agglutination on serum samples in a single laboratory. Incidence of TE was estimated by Kaplan-Meier method and a Cox model was used to study the predictive value of antibody titres, adjusted for other covariates. RESULTS: All 164 patients studied were positive for IgG antibodies and one had IgM antibodies. After a mean follow-up of 16 months, 31 cases of TE were documented. One-year incidence of TE was significantly higher in patients with IgG titres > or = 150 IU/ml (23.7%) than in patients with titres < 150 IU/ml (7.7%; relative risk, 3.1; P < 0.003). IgG titres remained significantly associated with the occurrence of TE (relative risk, 3.3; P < 0.005) in the Cox model. Predictive value of IgG titres did not differ according to baseline CD4+ cell counts. CONCLUSIONS: In patients with CD4+ cell counts < 200 x 10(6)/l, IgG anti-Toxoplasma antibody titre is a prognostic factor of occurrence of TE, with a higher risk for titres > or = 150 IU/ml. This finding should reinforce the recommendation of specific prophylaxis in these patients.

AIDS-Related Opportunistic Infections↗

PubMed1999

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Animals↗

PubMed2000

Neonatal screening for congenital toxoplasmosis in the Poznań region of Poland by analysis of Toxoplasma gondii-specific IgM antibodies eluted from filter paper blood spots.

OBJECTIVES: The aims of the study were to determine the prevalence of congenital toxoplasmosis at birth in the Poznań region of Poland, the value of the serologic examination of filter paper blood specimens collected from newborns for the diagnosis of congenital Toxoplasma infection and the duration of anti-Toxoplasma-specific IgM antibodies in infants' sera. MATERIALS AND METHODS: All neonates born in the maternity wards of the University Hospital of Gynaecology and Obstetrics in Poznań and in 10 selected obstetrics wards in the district hospitals were included. Blood samples were collected on filter paper cards, between the first and sixth day of life, screened for anti-Toxoplasma-specific IgM antibodies by an immunocapture enzyme-linked immunosorbent assay and if positive further analyzed for specific IgG and IgA antibodies. RESULTS: Between June, 1996, and October, 1998, filter paper samples from 27,516 liveborn infants were tested, which constituted approximately 75% of all births and 83% of liveborn neonates from the Poznań region. Anti-T. gondii-specific IgM antibodies were found in 13 newborns, equivalent to a prevalence of Toxoplasma-specific IgM in newborns of 1 per 2,117 liveborn children (0.47 per 1,000) or 1 per 870 children (1.15 per 1,000) born to seronegative women at risk of primary T. gondii infection during pregnancy. We identified two congenitally infected infants who were IgM-negative at birth, had a classic triad of clinical symptoms during the first year of life and had high levels of specific IgG. The birth prevalence of congenital toxoplasmosis in the Poznań region was at least 1 per 1,834 live births (0.55 per 1,000) or 1 per 754 live neonates born to seronegative women (1.33 per 1,000). The sensitivity of the IgM assay on eluate from filter paper was not more than 86.7%, and the mean duration of IgM detectable by enzyme-linked immunosorbent assay in serum samples was the first 4.8 weeks of life. CONCLUSION: In Poland the screening for congenital toxoplasmosis detecting one case per each 2,000 live births could be considered for inclusion in existing national neonatal screening programs for phenylketonuria and congenital hypothyroidism.

Animals↗

PubMed1987

Toxoplasma antibody titers in renal transplant recipients. Pretransplant evaluation and posttransplant follow-up of 73 patients.

In 73 consecutive kidney transplant recipients, anti-Toxoplasma antibodies were determined before transplantation and during a 3-year follow-up after transplantation. In 9 patients, antibody titers increased significantly after transplantation. Antibody titers to various viral antigens determined in parallel remained unchanged, suggesting that the anti-Toxoplasma antibody increase was not due to polyclonal nonspecific stimulation. In 2 of the 24 pretransplant seronegative patients, acquired toxoplasmosis was diagnosed serologically after transplantation, with the observation of a strong IgM and IgG antibody response. The incidence of toxoplasmosis in this group of patients was not found to be significantly different from that in a normal population, suggesting that transmission of Toxoplasma from the transplanted kidney may not be a significant mode of contamination. Among the 49 patients who were seropositive before transplantation, reactivation of toxoplasmosis was suspected in 7 cases on the basis of a significant increase in IgG antibodies. Reactivation occurred more frequently in patients treated with azathioprine and antithymocyte globulin, and a direct relationship between administration of steroids and antibody increase was demonstrated in three patients. Although toxoplasmosis has occasionally been reported as a major infectious problem in kidney transplant recipients, our clinical and serological data show that the potential risk of developing Toxoplasma infection is low since none of the patients with either acquired or reactivated toxoplasmosis developed clinical disease.

Adolescent↗

PubMed2006

Evaluation of antigenic variations between two virulent toxoplasma strains.

Toxoplasma gondii infection in humans is routinely assessed by serological means. Here, the authors attempted to compare the response of different Toxoplasma strains to serological tests and to evaluate the antigenic profiles of the RH and RH Ankara (TRH) strains with Western blotting. Anti-Toxoplasma IgG antibodies of 72 patients were examined with the indirect immunofluorescence antibody (IFA) test, ELISA and Western blotting (WB) by using antigen from both strains. Antigenic variations between strains did not affect IFA and ELISA test results, but qualitative and quantitative differences between the WB patterns were observed. A number of bands with molecular masses varying between 17 and 105 kDa were detected in WB. Fourteen different bands were obtained with the assay performed with RH strain antigen. An additional four bands were observed with TRH strain antigen. Also, an 80 kDa band was observed to stain darker in the blot with TRH strain antigen, whereas with RH strain antigen 30 and 38 kDa bands were darker. The results showed that strain-specific polymorphism in tachyzoite antigens of different Toxoplasma strains is important in the evaluation of WB but not in conventional serological analyses such as ELISA and IFA.

Animals↗

PubMed1993

Enhancement by recombinant human interleukin 2 of host resistance to Toxoplasma gondii infection in pregnant mice.

The lymphokine production by pregnant mice infected with a lethal dose of Toxoplasma gondii was evaluated in comparison with that by virgin mice infected with a sublethal dose of this protozoan parasite. Splenocytes taken from mice before and on the day after infection produced considerable amounts of IL-2 in response to concanavalin A (Con A) stimulation, but the titers rapidly declined in both pregnant and virgin mice as infection progressed. A trace amount or undetectable level of IL-2 was produced by splenocytes from acutely infected mice when stimulated with Toxoplasma lysate antigen (TLA). In contrast to the kinetics of IL-2 production, the levels of IFN-gamma produced by splenocytes cultured with Con A or TLA increased steadily in the later stage of infection in both pregnant and virgin mice. Thus, the response to Con A or TLA of splenocytes to produce IL-2 and IFN-gamma differed strikingly in acute toxoplasmosis in mice. The administration of rHuIL-2 resulted in a significant decrease in the mortality of pregnant mice infected with a lethal dose of Toxoplasma. The combination of rHuIL-2 and rMuIFN-gamma increased the survival rate slightly but not significantly compared with pregnant mice receiving either rHuIL-2 or rMuIFN-gamma. Moreover, exogenously administered rHuIL-2 enhanced the production of both IFN-alpha and IFN-gamma in the bloodstreams of pregnant mice, in accordance with the decreased mortality. These results indicate that IL-2 may play a significant role in modulating the host defense against Toxoplasma infection in pregnant mice.

Acute Disease↗

PubMed2005

Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2.

Toxoplasma gondii crosses non-permissive biological barriers such as the intestine, the blood-brain barrier and the placenta thereby gaining access to tissues where it most commonly causes severe pathology. Herein we show that in the process of migration Toxoplasma initially concentrates around intercellular junctions and probably uses a paracellular pathway to transmigrate across biological barriers. Parasite transmigration required viable and actively motile parasites. Interestingly, the integrity of host cell barriers was not altered during parasite transmigration. As intercellular adhesion molecule 1 (ICAM-1) is upregulated on cellular barriers during Toxoplasma infection, we investigated the role of this receptor in parasite transmigration. Soluble human ICAM-1 and ICAM-1 antibodies inhibited transmigration of parasites across cellular barriers implicating this receptor in the process of transmigration. Furthermore, human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. These findings reveal that Toxoplasma exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues.

Animals↗

PubMed2006

Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water.

AIMS: To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water. METHODS AND RESULTS: The method incorporated concentration of water samples by Al(2)(SO(4))(3)-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples. CONCLUSIONS: The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.

Animals↗

PubMed1996

[Anti-toxoplasma antibody levels in the main-land raccoon dog, Nyctereutes procyonoides viverrinus, in southeastern Kanagawa prefecture].

Naturally-occurring toxoplasma infection has been observed for years in various kinds of wild animals. However, the infection situation has not been well characterized due to a lack of research. In present study, latex fixation tests were conducted for 109 main-land raccoon dogs (67 males and 42 females) all of which were kept in Kanazawa Zoo in Yokohama City, Kanagawa Prefecture between 1991-1994 to measure levels of toxoplasma antibodies. The test results indicated 20 positive samples out of the total 109, an 18.3% positive rate. This demonstrates that toxoplasma infection exists in the raccoon dog population in Kanagawa Prefecture, and that the raccoon dog could be a source of spread of toxoplasma infection. We believe there is significant reason to continue the investigation as a part of a prophylaxis for zoonotic diseases.

Animals↗

PubMed1993

Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker.

A system for stable transformation of Toxoplasma gondii tachyzoites was developed that exploited the susceptibility of Toxoplasma to chloramphenicol. Introduction of the chloramphenicol acetyltransferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection resulted in parasites stably expressing CAT. A construct incorporating the tandemly repeated gene, B1, targeted efficiently to its homologous chromosomal locus. Knockout of the single-copy gene, ROP1, was also successful. Stable transformation should permit the identification and analysis of Toxoplasma genes important in the interaction of this opportunistic parasite with its host.

Animals↗

PubMed2000

Anti-toxoplasma activities of 24 quinolones and fluoroquinolones in vitro: prediction of activity by molecular topology and virtual computational techniques.

The apicoplast, a plastid-like organelle of Toxoplasma gondii, is thought to be a unique drug target for quinolones. In this study, we assessed the in vitro activity of quinolones against T. gondii and developed new quantitative structure-activity relationship models able to predict this activity. The anti-Toxoplasma activities of 24 quinolones were examined by means of linear discriminant analysis (LDA) using topological indices as structural descriptors. In parallel, in vitro 50% inhibitory concentrations (IC(50)s) were determined in tissue culture. A multilinear regression (MLR) analysis was then performed to establish a model capable of classifying quinolones by in vitro activity. LDA and MLR analysis were applied to virtual structures to identify the influence of each atom or substituent of the quinolone ring on anti-Toxoplasma activity. LDA predicted that 20 of the 24 quinolones would be active against T. gondii. This was confirmed in vitro for most of the quinolones. Trovafloxacin, grepafloxacin, gatifloxacin, and moxifloxacin were the quinolones most potent against T. gondii, with IC(50)s of 0.4, 2.4, 4.1, and 5.1 mg/liter, respectively. Using MLR analysis, a good correlation was found between measured and predicted IC(50)s (r(2) = 0.87, cross-validation r(2) = 0.74). MLR analysis showed that the carboxylic group at position C-3 of the quinolone ring was not essential for anti-Toxoplasma activity. In contrast, activity was totally dependent on the presence of a fluorine at position C-6 and was enhanced by the presence of a methyl group at C-5 or an azabicyclohexane at C-7. A nucleophilic substituent at C-8 was essential for the activity of gatifloxacin and moxifloxacin.

4-Quinolones↗

PubMed2004

Evaluation of a strategy for Toxoplasma gondii oocyst detection in water.

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.

Animals↗

PubMed2002

Cellular immune responses to recombinant antigens in pregnant women chronically infected with Toxoplasma gondii.

The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4(+). Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-gamma) into the culture supernatant (4,343 +/- 2,536 pg/ml). Cells from 12 patients released IFN-gamma after culture with rGRA1 (130 +/- 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 +/- 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 +/- 374 pg/ml). Intensity of IFN-gamma production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r = 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.

Animals↗

PubMed1981

Effects of muramyl dipeptide treatment on resistance to infection with Toxoplasma gondii in mice.

Studies were carried out to determine whether treatment of mice with the synthetic adjuvant muramyl dipeptide afforded any resistance to infection with the obligate intracellular protozoan Toxoplasma gondii. Marked resistance to lethal challenge infection was observed in CBA but not C57BL/6 mice pretreated with muramyl dipeptide. In CBA mice, a single muramyl dipeptide treatment administered 14, 7, or 4 days before Toxoplasma challenge did not afford protection, whereas mice treated at -1 day were highly resistant. Additional studies carried out to investigate the mechanisms underlying the enhanced resistance to Toxoplasma in muramyl dipeptide-treated mice failed to reveal either enhanced cytolytic antibodies to the parasite or evidence that peritoneal macrophages from treated mice were activated as determined in vitro by their microbicidal capacity for Toxoplasma or cytotoxic capacity for tumor target cells.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1985

Lymphokine activation of J774G8 cells and mouse peritoneal macrophages challenged with Toxoplasma gondii.

In vitro activation of macrophage cell line J774G8 and mouse peritoneal macrophages resulted in oxygen-dependent and oxygen-independent killing of intracellular Toxoplasma gondii. Activation was characterized by oxygen-dependent killing detectable by enhanced lysosome fusion and digestion of T. gondii. The toxoplasmacidal activity of activated J774G8 cells and peritoneal macrophages was prevented by adding the oxygen intermediate scavengers catalase or superoxide dismutase during culture. Activated J774G8 cells and peritoneal macrophages also inhibited replication of those Toxoplasma organisms which survived the initial microbicidal activity. The inhibition of Toxoplasma replication was not significantly affected by exogenous catalase or superoxide dismutase. Peritoneal macrophages from Toxoplasma-immune mice showed similar microbicidal and inhibitory responses, supporting the model that activation leads to destruction of intracellular parasites by two different mechanisms.

Animals↗

PubMed1972

A role for activated macrophages in resistance to infection with Toxoplasma.

Activated macrophages from mice which were chronically infected with Toxoplasma gondii or Besnoitia jellisoni, or which had received Freund complete adjuvant, had an enhanced capacity to to kill intracellular Toxoplasma. Enhanced killing by activated macrophages was demonstrated by decreased incorporation of isotopically labeled uridine by intracellular Toxoplasma and by inhibition of plaque formation. The latter resulted from lack of proliferation of the intracellular Toxoplasma which is normally followed by destruction of the host cell (macrophage) and secondary invasion and destruction of fibroblast monolayers.

Animals↗

PubMed2001

Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP.

Toxoplasma gondii is an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-gamma) is the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain. Microglia are important IFN-gamma-activated effector cells controlling the growth of T. gondii in the brain via a nitric oxide (NO)-mediated mechanism. IFN-gamma can also activate astrocytes to inhibit the growth of T. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasma mechanisms. In this study we investigated the role of IGTP, a recently identified IFN-gamma-regulated gene, in IFN-gamma inhibition of T. gondii in murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-gamma, and then tested for anti-Toxoplasma activity. In wild-type astrocytes T. gondii growth was significantly inhibited by IFN-gamma, whereas in astrocytes from IGTP-deficient mice IFN-gamma did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-gamma induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-gamma-induced inhibition of T. gondii in murine astrocytes.

Animals↗

PubMed2002

Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice.

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.

Animals↗