Hervé Pelloux

Genotype of 86 Toxoplasma gondii Isolates Associated with Human Congenital Toxoplasmosis, and Correlation with Clinical Findings

To study the influence of Toxoplasma gondii genotypes on the severity of human congenital toxoplasmosis (asymptomatic, benign, or severe infection or newborn or fetal death), 8 microsatellite markers were used to analyze 86 T. gondii isolates collected from patients with congenital toxoplasmosis. Seventy-four different genotypes were detected, some identical genotypes originating probably from the same source of contamination. The 3 less polymorphic microsatellite markers associated with 6 isoenzymatic markers allowed a classification of isolates into the 3 classical types and detected atypical genotypes. Whatever the clinical findings, type II isolates were largely predominant (84.88% in the whole collection and 96.49% in 57 consecutive cases). Type I and atypical isolates were not found in asymptomatic or benign congenital toxoplasmosis. However, in 4 cases in which children were not infected despite isolation of T. gondii from placenta, only type I isolates were found.

Prevention of toxoplasmosis in transplant patients

Toxoplasmosis is a life-threatening opportunistic infection that affects haematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients. Its incidence in these patients is closely related to the prevalence of toxoplasmosis in the general population, which is high in Europe. In SOT recipients, toxoplasmosis results mainly from transmission of the parasite with the transplanted organ from a Toxoplasma-seropositive donor to a Toxoplasma-seronegative recipient. This risk is high in cases of transplantation of organs that are recognized sites of encystation of the parasite, e.g. the heart, and is markedly lower in other SOT recipients. Clinical symptoms usually occur within the first 3 months after transplantation, sometimes as early as 2 weeks post transplant, and involve febrile myocarditis, encephalitis or pneumonitis. In HSCT recipients, the major risk of toxoplasmosis results from the reactivation of a pre-transplant latent infection in seropositive recipients. The median point of disease onset is estimated at 2 months post transplant, with <10% of cases occurring before 30 days and 15-20% later than day 100. Toxoplasmosis usually manifests as encephalitis or pneumonitis, and frequently disseminates with multiple organ involvement. Diagnosis of toxoplasmosis is based on the demonstration of parasites or parasitic DNA in blood, bone marrow, cerebrospinal fluid, bronchoalveolar lavage fluid or biopsy specimens, and serological tests do not often contribute to the diagnosis. For prevention of toxoplasmosis, serological screening of donors and recipients before transplantation allows the identification of patients at higher risk of toxoplasmosis, i.e. seropositive HSCT recipients and mismatched (seropositive donor/seronegative recipients) SOT recipients. Preventing toxoplasmosis disease in those patients presently relies on prophylaxis via prescription of co-trimoxazole.

Strategy for Diagnosis of Congenital Toxoplasmosis: Evaluation of Methods Comparing Mothers and Newborns and Standard Methods for Postnatal Detection of Immunoglobulin G, M, and A Antibodies

ABSTRACT In a study involving 14 laboratories supported by the European Community Biomed 2 program, we evaluated immunologic methods for the postnatal diagnosis of congenital toxoplasmosis (CT). Among babies born to mothers who seroconverted to positivity for toxoplasmosis during pregnancy, we analyzed 55 babies with CT on the basis of persistent anti-Toxoplasma immunoglobulin G (IgG) at 1 year of life and 50 control babies without anti-Toxoplasma IgG at 1 year of life in the absence of curative treatment with pyrimethamine-sulfonamides. We tested in-house methods such as the enzyme-linked immunofiltration assay (ELIFA) or Immunoblotting (IB) for the detection of IgG or IgM; these methods allowed comparison of the immunologic profiles of the mothers and the infants. We compared ELIFA and IB with a commercial enzyme immunoassay (EIA) or in-house immunosorbent agglutination assay (ISAGA) for the detection of IgM or IgA. The performances of combinations of methods were also assessed. A cumulative sensitivity of 98% during a 1-year follow-up was obtained with the ELIFA plus ISAGA combination. Only one case of CT was missed by the ELIFA plus ISAGA combination, whereas three cases were missed by the IB plus ISAGA combination, even though 48% of patients with CT were treated with pyrimethamine-sulfonamides, which are known to inhibit antibody neosynthesis. A similar performance was obtained with either ELIFA or IB in combination with EIA. The difference in performance between ELIFA plus ISAGA and IB plus ISAGA was not statistically significant (P = 0.31), and we conclude that both combinations of tests can be used for the diagnosis of CT in newborns.

Why prevent, diagnose and treat congenital toxoplasmosis?

Evidence that prevention, diagnosis and treatment of toxoplasmosis is beneficial developed as follows: anti-parasitic agents abrogate Toxoplasma gondii tachyzoite growth, preventing destruction of infected, cultured, mammalian cells and cure active infections in experimental animals, including primates. They treat active infections in persons who are immune-compromised, limit destruction of retina by replicating parasites and thereby treat ocular toxoplasmosis and treat active infection in the fetus and infant. Outcomes of untreated congenital toxoplasmosis include adverse ocular and neurologic sequelae described in different countries and decades. Better outcomes are associated with treatment of infected infants throughout their first year of life. Shorter intervals between diagnosis and treatment in utero improve outcomes. A French approach for diagnosis and treatment of congenital toxoplasmosis in the fetus and infant can prevent toxoplasmosis and limit adverse sequelae. In addition, new data demonstrate that this French approach results in favorable outcomes with some early gestation infections. A standardized approach to diagnosis and treatment during gestation has not yet been applied generally in the USA. Nonetheless, a small, similar experience confirms that this French approach is feasible, safe, and results in favorable outcomes in the National Collaborative Chicago-based Congenital Toxoplasmosis Study cohort. Prompt diagnosis, prevention and treatment reduce adverse sequelae of congenital toxoplasmosis.

Determination of anti–Toxoplasma gondii immunoglobulin G avidity: adaptation to the vidas system (bioMérieux)

The determination of specific anti-Toxoplasma gondii IgG avidity has been proposed to improve the determination of the date of toxoplasmic seroconversion in pregnant women. In this study, we adapted this serological technique to the Vidas system (bioMérieux) using 6 M urea as the dissociating agent. We studied 356 sera, including 42 sequential sera from sero-conversions in pregnant women. Our results show that the test is easy to use, and that an avidity index higher than 0.300 allows the exclusion of a recent infection acquired less than 4 months before serum sampling.

Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites

Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACis such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ∼370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.

Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii.

Abstract The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M, IgA, IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3–12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.

Host Cell Subversion by Toxoplasma GRA16, an Exported Dense Granule Protein that Targets the Host Cell Nucleus and Alters Gene Expression

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation

Toxoplasma gondii secretes a novel dense granule protein, GRA24, that traffics from the vacuole to the host cell nucleus where it prolongs p38a activation and correlates with proinflammatory cytokine production.

Diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of DNA detection and serological techniques.

BACKGROUND The biological diagnosis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) is based on the detection of Toxoplasma gondii DNA in blood specimens or other samples. Serological testing is used mainly to define the immunity status of the patient before HSCT. The aim of our study was to examine the performance of polymerase chain reaction (PCR) and serological techniques in the diagnosis of toxoplasmosis after HSCT. METHODS Seventy patients underwent allogeneic HSCT from September 2004 through September 2006. DNA was detected by PCR, and immunoglobulin G and immunoglobulin M were detected by enzyme-linked immunosorbent assay. RESULTS The results of immunoglobulin G detection before allogeneic HSCT were positive in 40 (57.1%) of the patients and negative in 30 (42.9%). After HSCT, 57 patients (81.4%) had test results that were negative for immunoglobulin M and had negative results of DNA detection, without toxoplasmosis infection. Four patients (5.7%) had at least 4 samples with positive PCR results and/or test results positive for immunoglobulin M against T. gondii; toxoplasmosis was then confirmed by clinical symptoms. Nine patients (12.9%) with positive PCR results and 1 or 2 samples with test results negative for immunoglobulin M were considered to have asymptomatic T. gondii infection. Reactivation of latent infection was the cause of toxoplasmosis in 3 of the 4 patients, and toxoplasmosis occurred as a primary infection in 1 patient. The detection of specific anti-T. gondii immunoglobulin M was the only biological evidence of toxoplasmosis in 2 patients, and samples were positive for immunoglobulin M before PCR was performed in 1 patient. CONCLUSIONS Thus, after HSCT, all patients were at risk for toxoplasmosis; all patients who receive HSCTs should be followed up with biological testing that combines PCR and serological techniques.