Serotyping of Toxoplasma gondii: striking homogeneous pattern between symptomatic and asymptomatic infections within Europe and South America.

Field isolates of Toxoplasma gondii in Europe and North America have been grouped into three clonal lineages that display different virulence in mice. Whether the genetic structure of the parasite is related to clinical expression in humans has not yet been demonstrated. We developed an enzyme-linked immunosorbent assay which uses lineage-specific, polymorphic polypeptides derived from the dense granule antigens, GRA5 and GRA6. Our goal was to compare serotypical patterns observed in asymptomatic versus symptomatic (ocular disease and severe infection in human immunodeficiency virus (HIV)-positive patients) infections among patients from Europe and South America. Independent of the clinical presentation of the disease, serotypes differed according to geographical origin, with a homogeneous distribution of serotype II in Europe and of serotypes I and III in South America. We conclude that GRA5-GRA6 serotyping is an interesting tool to study serotype prevalence in populations but it is not an accurate marker of pathogenicity of Toxoplasma infection in humans.

Artesunate-erythropoietin combination for murine cerebral malaria treatment.

Cerebral malaria is the most severe and rapidly fatal complication of Plasmodium falciparum infection. Despite appropriate anti-malarial treatment using quinine or artemisinin derivatives, 10-20% of mortality still occurs during the acute phase. To improve cerebral malaria outcome, adjunctive therapies are clearly needed. Most experiments in this area have been dedicated to immuno-modulation with various successes. Since erythropoietin has been shown to be highly effective in human ischemic stroke and in murine cerebral malaria, we addressed the issue of cerebral malaria outcome improvement by erythropoietin-artesunate drug combination. Compared to the previous study using erythropoietin high doses at the early beginning of the disease, erythropoietin treatment was decreased by six-fold and delayed to the pre-mortem phase. We studied effects on survival and on clinical recovery of the drug combination given from day 6 to day 8 post-infection to CBA/J mice infected by Plasmodium berghei ANKA. We showed that the artesunate-erythropoietin drug combination led to clinical recovery 24 h earlier for surviving mice, and to increase in the global survival rate compared to artesunate monotherapy (p<0.01). Since erythropoietin has no effect on parasite clearance, it could be stated that this drug combination is efficient and that erythropoietin could be a lead for the implementation of a new adjunctive therapy during the acute phase of cerebral malaria.

Multicenter proficiency study for detection of Toxoplasma gondii in amniotic fluid by nucleic acid amplification methods.

UNLABELLED: A proficiency panel was designed to assess the performance of nucleic acid amplification technologies for the detection of Toxoplasma gondii in amniotic fluid. METHODS: The proficiency panel consisted of five lyophilised coded samples in a range of concentration between 5 to 1000 parasites/ml and a negative control. The distribution also included a questionnaire on the applied methods. RESULTS: Thirty-three laboratories in 17 countries participated and returned a total of 38 data sets. The percentage of data sets achieving correct results on all panel samples was 42.1%, whereas two or more incorrect or equivocal results were reported for 36.8%. The lowest concentration (5 parasites/ml) was not identified correctly in 15 (39.5%) data sets. False positive results were reported by two laboratories both of which had not included a step in their procedure to rule out contamination. In 32 (84.2%) data sets an "in-house" method was used, and in 6 (15.8%) sets a commercial assay was applied. CONCLUSIONS: Overall, the results of this study demonstrate the need for improvements in both sensitivity and specificity of molecular detection methods of T. gondii and for the development of international reference materials to help laboratories with the development and validation of their assays.

Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America).

Isolates of Toxoplasma gondii, which is responsible for a wide range of clinical manifestations are grouped into three clonal lineages of different virulence in mice. However, it is not clear whether this genotypic pattern is associated with the clinical profile of the disease in humans nor is the geographical distribution of the genotypes known. This is mainly due to difficulties in obtaining parasitic DNA from patients. The available data are therefore limited and originate from acute or congenital infections or from animals. A non-invasive assay is needed to address issues of strain type, geographical distribution and severity of clinical toxoplasmosis. To serotype T. gondii strains, we have developed an enzyme-linked immunosorbent assay (ELISA) that uses polymorphic polypeptides specific to the three clonal lineages and derived from two dense granule antigens, GRA5 and GRA6. Two hundred and fifty-two sera from chronically infected pregnant women from three different European countries and Colombia were investigated. The analysis of genotype-specific antibody response showed a homogeneous type II distribution in the European samples compared with types I and III but no type II in the Colombian population. Our data concord with those obtained from the genotyping of other isolates from Europe and South America. We demonstrated that, despite some limitation due to antigen and/or antibody specificity, serotyping is a promising assay to investigate the relationship between type of strain and severity of the disease.

Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites.

The 14-3-3 protein was shown to be present into the parasitophorous vacuole of Toxoplasma gondii-infected human monocyte cells and in the excreted/secreted antigens (ESA). The ESA 14-3-3 protein migrates electrophoretically as the cytosol and the main membranous 14-3-3 isoforms. The excretion/secretion of 14-3-3 was not sensitive to cycloheximide, a protein synthesis inhibitor, even at a concentration which inhibited the production of 14-3-3 inside the tachyzoites. Recombinant 14-3-3/GST protein was used to test the presence of 14-3-3 antibodies in different human sera. A positive immunoreactivity was observed with sera corresponding to acute toxoplasmosis and a possible involvement of 14-3-3 in host immunity is discussed.

Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form.

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.

Migration and maturation of human dendritic cells infected with Toxoplasma gondii depend on parasite strain type.

Migration and maturation of human dendritic cells derived from CD34+ progenitor cells (DC) infected by Toxoplasma gondii were studied in an in vitro model. We demonstrated that infection with virulent type I strains RH and ENT or type II low virulent strains PRU and CAL induced DC migration towards MIP-3beta. However, type II strains induced a higher percentage of migrating cells than that induced by type I strains or positive controls (chemical allergen or lipopolysaccharides). Type II strains produced soluble factors responsible of the high migration whereas heat killed tachyzoites did not induced a migration higher than positive controls. We also demonstrated that infection by virulent strains and not by type II stains or heat killed tachyzoites triggers DC maturation. A soluble factor released by type II strains was responsible of the absence of DC maturation. Taken together, these results demonstrated that the interference of T. gondii in the behaviour of DC functions is related to the strain types and can be supported by secretion of soluble factors by the parasite.

Recombinant human erythropoietin prevents the death of mice during cerebral malaria.

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women.

An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.

Progesterone fails to modulate Toxoplasma gondii replication in the RAW 264.7 murine macrophage cell line.

Cell mediated immunity is very important for host defence against intracellular pathogens and many studies have shown the role of the production of nitric oxide (NO) by interferon (IFN)-gamma/lipopolysaccharide (LPS)-activated macrophages. As the progesterone level increases during pregnancy in mammals, and as previous studies have shown that progesterone inhibits inducible nitric oxide synthase (iNOS) expression and NO production, we aimed to investigate whether progesterone might modulate intracellular replication of Toxoplasma gondii in macrophages. Our results showed that progesterone does not influence T. gondii replication in non-activated RAW 264.7 cells, and although progesterone inhibits NO production induced by IFN-gamma/LPS, we observed that it fails to restore the growth of T. gondii blocked by IFN-gamma/LPS. After discussing the reasons for this apparent discrepancy, we concluded that progesterone has no direct effect on the macrophage response. The real effect of the sex steroids in T. gondii infection and their implication in clinical toxoplasmosis therefore need to be investigated further to involve wider mechanisms of the immune system.