[Cell-host-parasite interactions: biodiversity, pathogenesis, environment]. / Interactions cellules-hôtes-parasites: biodiversité, pathogénie, environnement.

The apicomplexan Toxoplasma gondii, an obligate intracellular parasite, can infect humans and a wide range of vertebrates leading to toxoplasmosis. This generally benign affection can causes severe life-threatening disease, particularly in immunocompromised patients and in children with congenital toxoplasmosis. Our research team works on cell-host-parasite interactions by studying biodiversity, pathogenic mechanisms and environment. We search to identify prognostic factors of disease and markers of resistance. This project is an integral part of our Research Institute (IFR53) which receives support from the Toxoplasma Biological Resource Center for constituting a bank of well characterized toxoplasma isolates for genotyping, clinical and epidemiological data. The involvement of metalloproteinases implicated during monocytic cell invasion and identification of ABC transporter proteins in T. gondii, factors implicated in resistance, need to be explored.

Population pharmacokinetics of pyrimethamine and sulfadoxine in children with congenital toxoplasmosis.

AIMS: To develop a population pharmacokinetic model for pyrimethamine (PYR) and sulfadoxine (SDX) in children with congenital toxoplasmosis. METHODS: Children were treated with PYR (1.25 mg kg(-1)) and SDX (25 mg kg(-1)) (Fansidar) plus folinic acid (Lederfoline) 5 mg). Plasma concentrations, available from a therapeutic drug monitoring database, were determined by high-performance liquid chromatography. Population pharmacokinetic analysis was performed using a nonlinear mixed effects model. RESULTS: Eighty-nine children, aged 1 week to 14 years and weighing 2.9-59 kg, were available for evaluation. Both PYR and SDX concentration-time profiles were best described by a one-compartment open model. Volume of plasma distribution (V) and clearance (CL) were significantly related to body weight (BW) using an allometric function. Typical CL and V estimates (95% confidence interval), for a child weighing 11 kg were 5.50 (5.28, 5.73) l day(-1) and 36 (33, 39) l for PYR and 0.26 (0.25, 0.27) l day(-1) and 2.1 (1.9, 2.3) l for SDX. For BW between 3.5 and 60 kg, plasma half-lives were predicted to vary from 4.0 to 5.2 days for PYR, and from 5.0 to 7.5 days for SDX. CONCLUSION: This study indicated that body weight influences PYR and SDX pharmacokinetics in children. To optimize PYR/SDX combination treatment in congenital toxoplasmosis, short dosing intervals in very young low-wight children are probably appropriate.

Activation of the cellular mitogen-activated protein kinase pathways ERK, P38 and JNK during Toxoplasma gondii invasion.

Host cell invasion is essential for the pathogenicity of the obligate intracellular protozoan parasite Toxoplasma gondii. In the present study, we evaluated the ability of T. gondii tachyzoites to trigger phosphorylation of the different mitogen-activated protein kinases (MAPK) in human monocytic cells THP1. Kinetic experiments show that the peak of extracellular-signal-regulated kinase (ERK1/2), P38 and cjun-NH2 terminal kinase (JNKs) phosphorylation occurs between 10 and 60 min. The use of specific inhibitors of ERK1/2, P38 and JNK1/2 phosphorylation indicates the specificity of MAPKs phosphorylation during invasion. Signaling through cellular and parasite mitogen-activated protein (MAP) kinase pathways appears to be critical for T. gondii invasion.

Clinical value of specific immunoglobulin E detection by enzyme-linked immunosorbent assay in cases of acquired and congenital toxoplasmosis.

The clinical value of immunoenzymatic (enzyme-linked immunosorbent assay) detection of anti-Toxoplasma immunoglobulin E (IgE) was assessed by studying 2,036 sera from 792 subjects, comprising seronegative controls and subjects with acute, active, reactivated, or congenital toxoplasmosis. Included were nonimmunized adults; pregnant women with recently acquired infection (acute toxoplasmosis); immunocompetent subjects with recently acquired severe infection (active toxoplasmosis) expressed as fever, adenopathies, splenomegaly, pneumonia, meningitis, or disseminated infection; subjects-some of them immunocompromised-whose previously moderate IgG antibody levels rose, suggesting a reactivation of quiescent toxoplasmosis; and infants born to seroconverted mothers and evaluated for diagnosis of congenital infection and therapeutic management. Specific IgE antibodies were never detected in seronegative subjects. They were present in 85.7% of asymptomatic seroconverters and in 100% of seroconverters with overt toxoplasmosis, following two different kinetics: in the former, the specific IgE titer generally presented a brief peak 2 to 3 months postinfection and then fell rapidly, whereas specific IgE persisted at a very high titer for several months in the latter. IgE emerged concomitantly with the increase in IgG during toxoplasmic reactivation. For neonatal diagnosis of congenital toxoplasmosis, IgE was less informative than IgM and IgA (sensitivities, 59.5, 64.3, and 76.2%, respectively) and had a specificity of 91.9%. Nevertheless, simultaneous measurement of the three isotypes at birth improved the diagnostic yield to 81% relative to the combination of IgA and IgM. Emergence of specific IgE during postnatal treatment for congenital toxoplasmosis is a sign of poor adherence or inadequate dosing.

Use of Fourier-transform infrared spectroscopy for typing of Candida albicans strains isolated in intensive care units.

Comparative studies of Candida albicans strains are essential for proving cross-infections in epidemiological investigations. Typing of C. albicans strains is mainly based on genotypic methods. Fourier-transform infrared (FTIR) spectroscopy is described in this study as a novel phenotypic approach to the typing of C. albicans. The first step in the approach was the standardization of sample preparation (culture conditions and sampling parameters) and acquisition and classification parameters (spectral acquisition, spectral window selection, classification algorithm, and heterogeneity threshold). The second step consisted of validating the established parameters with a set of 79 strains of C. albicans isolated over 4 months from nine patients hospitalized in two intensive care units. Strains were isolated from multiple anatomical sites with repeated sampling. FTIR spectroscopy results were compared to randomly amplified polymorphic DNA (RAPD) results; this analysis showed that the amplification patterns of strains isolated from a given patient were identical and that different patients had different profiles. FTIR spectroscopy data were analyzed by hierarchical clustering performed with the second-derivative spectra. This classification revealed nine groups, one per patient. Only one spectrum out of 79 was misclassified by the FTIR spectroscopy method. RAPD and FTIR spectroscopy results were in good agreement, showing that, when nosocomial candidiasis transmission is suspected and urgent information is needed, this technique may be useful as a quick identification tool to give solid clues before confirmation by a genotypic method.

Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells.

The coccidian Toxoplasma gondiiis an obligate intracellular parasite which can infect all cell types. Among the cytokines elicited by the immune response to Toxoplasma, tumor necrosis factor alpha (TNF-alpha) acts synergistically with gamma interferon (IFN-gamma) and plays a major role in host cell resistance. We have previously reported that TNF-alpha production induced by IFN-gamma/LPS decreases after T. gondii infection of human myelomonocytic THP-1 cells. Here, we investigated the regulation of TNF-alpha and its specific receptors during T. gondii infection of THP-1 cells. We found that TNF-alpha production was regulated at a post-transcriptional level and that TNF receptor expression was regulated at a pretranscriptional level. The TNF receptor I shedding and the fall in TNF-alpha levels observed after T. gondii infection would thus be induced by a parasite component with serine protease activity. These findings indicate that T. gondii participates not only in controlling the cytotoxic effects of TNF-alpha during the infection process, but also in signal transduction mediated mainly by TNF receptor I.

Preconception seroconversion and maternal seronegativity at delivery do not rule out the risk of congenital toxoplasmosis.

We describe two unusual cases of congenital toxoplasmosis, one occurring after preconception maternal infection with cervical adenopathies and the other occurring after maternal infection at the very end of pregnancy with maternal seronegativity at delivery. These documented cases of congenital toxoplasmosis demonstrate the value of extending the serologic monitoring period during pregnancy, according to the individual clinical context.

[Prevention of congenital toxoplasmosis in France. Risk assessment. Results and perspectives of prenatal screening and newborn follow up]. / La prévention de la toxoplasmose congénitale en France. Evaluation des risques. Résultats et perspectives du dépistage anténatal et du suivi du nouveau-né.

In France, a national program for the prevention of congenital toxoplasmosis has been set up 25 years ago. This program is here presented and discussed in details. It is based on a decision tree well defined, with pre and/or per gravidic serological screening with several different tests, completed, if necessary, by ultrasounds examinations of the fetus, biomolecular tests (PCR) on amniotic fluid, and by clinical, biological, and radiological surveillance of neo-nates. The purpose of this prevention program is to: 1/identify nonimmune young women and limit their contamination risk during pregnancy by appropriate counseling on hygiene and diet; 2/screen and treat per gravidic toxoplasmosis as early as possible so as to prevent or limit transmission to the fetus and its consequences. 3/in utero diagnose and treat infestation of the fetus; 4/diagnose and treat asymptomatic congenital toxoplasmosis in neonates, to prevent risks of reactivation and late complications, especially ocular. Such a prevention program has a cost validated by the prevalence of acquired toxoplasmosis in adults in France (over 50% of the population) and by the yearly incidence of congenital toxoplasmosis (at least 0.1% of births according to the best hypothesis). These 6 to 700 congenital toxoplasmosis cases per year may be compared to the 6 to 7,000 per gravidic seroconversions which could lead to fetal contamination if no preventive measures are taken. Nevertheless, as it is often the case in the field of prevention, it is very difficult to statistically assess the efficacy of this program even though several arguments show that it allows to eliminate the most serious toxoplasmosis, sources of serious handicaps at birth, and to limit the frequency of late complications (especially retino-choroiditis) of asymptomatic infections in neonates. The position of European countries varies as to prevention of congenital toxoplasmosis. Some countries (Austria, Belgium) have national prevention programs similar to the French one, whereas others have set up only limited programs or set up no systematic prevention. These differences may be accounted for by the different frequencies of toxoplasmic risk. It seems mandatory to forget all dogmatism and not to stick to a strictly statistical approach for a disease with not only medical but also social and human consequences.

Membrane potential changes after infection of monocytes by Toxoplasma gondii.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii.

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.

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.

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.

Calmodulin distribution and the actomyosin cytoskeleton in Toxoplasma gondii.

The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actin-myosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca(2+)-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosin-CaM complex and the steps of a signaling pathway for parasite motility.

Involvement of tumor necrosis factor-alpha during infection of human monocytic cells by Toxoplasma gondii.

Although the involvement of tumor necrosis factor-alpha (TNF-alpha) during Toxoplasma gondii infection has largely been described in mouse models, only a few studies in human models have been reported. We demonstrated the role of TNF-alpha during the process of invasion by T. gondii in human monocytic cells. Cotreatment of cells with interferon-gamma (IFN-gamma) and lipolysaccharide (LPS) during T. gondii infection induced TNF-alpha production and decreased the number of parasitized cells (invasion index). Neutralization of the production of TNF-alpha using a specific monoclonal antibody or pentoxifylline enhanced the invasion index. The relationship between TNF-alpha production and protection of monocytic cells against T. gondii invasion was confirmed by treatment of infected cultures with exogenous TNF-alpha. Thus, in contrast to the results obtained in murine models but in accordance with those observed in other human models, the present study shows for the first time in human monocytic cells that T. gondii does not induce any TNF-alpha secretion and inhibits TNF-alpha production induced by IFN-gamma/LPS.

Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay.

We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94. 5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.

Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in colombian patients with acute and chronic ocular toxoplasmosis.

We studied the frequency of specific anti-Toxoplasma IgM, IgA and IgE antibodies in serum of 28 immunocompetent Colombian patients, selected by ophthalmologists and with lesions that were compatible with ocular toxoplasmosis. Patients were classified in three groups: (i) group 1 consisted of ten patients with a first episode; (ii) group 2, with seven patients with a recurrence and (iii) group 3, consisted of eleven patients with chronic chorioretinal lesion without uveitis. We found that 10/28 (35%) of Colombian patients with ocular toxoplasmosis possessed at least one serological marker for Toxoplasma infection different from IgG. In group 1 (first episode), we found simultaneous presence of specific IgM plus IgA plus IgE in 1/10 (10%). In group 2 (recurrences) in 1/7 (14%) we found IgM and IgA test positives and in 1/7 (14%) we found IgM and IgE tests positives. In group 3 (toxoplasmic chorioretinal scar) the IgA serological test was positive in 2/11 (18%). These results show that serum IgM or IgA or IgE can be present during recurrences.

Diagnosis of congenital toxoplasmosis in the neonatal period: A multicenter evaluation.

OBJECTIVE: To evaluate different laboratory tests used to diagnose congenital toxoplasmosis in the neonatal period. STUDY DESIGN: A retrospective multicenter study of 294 pregnant women who experienced seroconversion for Toxoplasma gondii and subsequently delivered live-born infants. Fetal infection was assessed via specific IgM and IgA antibodies (cord and neonatal blood) and detection of T gondii in placenta and cord blood by mouse inoculation. RESULTS: Ninety-three (32%) of the 294 infants were congenitally infected. The sensitivity of IgA in cord blood and in neonatal blood was 64% and 66%; the sensitivity of IgM was 41% and 42%, respectively. Mouse inoculation of the placenta and cord blood had sensitivities of 45% and 16%. Positive results of the serologic tests in congenitally infected children correlated significantly with the gestational age at the time of maternal infection but was not significantly influenced by the administration of specific antiparasitic treatment during pregnancy. CONCLUSION: Specific T gondii IgA antibody is a more sensitive test than IgM for detecting congenital toxoplasmosis in the neonatal period. The overall specificity is better for serologic tests performed on neonatal blood than for those on cord blood. Neonatal screening with IgM or IgA antibodies will not detect the majority of children with congenital toxoplasmosis when the maternal infection occurred before the 20th week of pregnancy.