Daniel Ajzenberg

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.

Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups.

Previous studies on Toxoplasma gondii population structure, based essentially on multilocus restriction fragment length polymorphism analysis or on multilocus enzyme electrophoresis, indicated that T. gondii comprises three clonal lineages. These studies showed a weak polymorphism of the markers (2-4 alleles by locus). In this study, we used eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3-16 alleles detected, these markers can be considered as the most discriminating multilocus single-copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologically related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages that can be equated to discrete typing units, but there is some evidence of occasional genetic exchange that could explain why one of these discrete typing units is less clearly individualised than the other.

Severe Acquired Toxoplasmosis in Immunocompetent Adult Patients in French Guiana

ABSTRACT The most common presentation of symptomatic postnatally acquired toxoplasmosis in immunocompetent patients is painless cervical adenopathy. Acute visceral manifestations are associated in rare cases. We report 16 cases of severe primary toxoplasmosis diagnosed in French Guiana during a 6.5-year period. All of the subjects were immunocompetent adults hospitalized with clinical presentations consisting of a marked, nonspecific infectious syndrome accompanied by an altered general status with at least one visceral localization, mainly pulmonary involvement (14 cases). Acute toxoplasmosis was diagnosed according to the results of serological tests suggestive of recent primary infection and the absence of an alternative etiology. Recovery was rapid following specific antitoxoplasmosis treatment. Thirteen of the 16 patients had consumed game in the 2 weeks before the onset of the symptoms, and in eight cases the game was considered to have been undercooked. Toxoplasma strains, which were virulent in mice, were isolated from three patients. Microsatellite analysis showed that all of these isolates exhibited an atypical multilocus genotype, with one allele found only for isolates of this region.

Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages

Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.

Fatal Outbreak of Human Toxoplasmosis along the Maroni River: Epidemiological, Clinical, and Parasitological Aspects

BACKGROUND Well-documented outbreaks of human toxoplasmosis infection are infrequently reported. Here, we describe a community outbreak of multivisceral toxoplasmosis that occurred in Patam, a Surinamese village near the French Guianan border. METHODS From the end of December 2003 through the middle of January 2004, 5 adult patients in Patam, including 2 pregnant women, were initially hospitalized for multivisceral toxoplasmosis. A French-Surinamese epidemiological investigation was conducted in the village; inquiries and clinical examinations were performed, and blood and environmental samples were obtained. For all serologically confirmed cases of toxoplasmosis, molecular analysis and mouse inoculations were performed for diagnosis and genetic characterization of Toxoplasma gondii. RESULTS The hospitalized patients, who did not have any immunodeficiencies, presented with an infectious disease with multivisceral involvement. Serological examination confirmed acute toxoplasmosis. One adult died, and a neonate and a fetus with congenital toxoplasmosis also died. During the investigation, 4 additional acute cases of toxoplasmosis were diagnosed among the 33 villagers. Only 3 inhabitants had serological evidence of previous T. gondii infection. In total, we reported 11 cases of toxoplasmosis: 8 multivisceral cases in immunocompetent adults, resulting in 1 death; 2 cases of lethal congenital toxoplasmosis in a neonate and a fetus; and 1 symptomatic case in a child. Molecular analysis demonstrated that identical isolates of only 1 atypical strain were responsible for at least 5 of the 11 cases of toxoplasmosis in the outbreak. No epidemiological sources could be linked to this severe community-wide outbreak of toxoplasmosis. CONCLUSION This report is in agreement with the particular features of toxoplasmosis involving atypical strains that were recently described in French Guiana.

Congenital Toxoplasmosis and Reinfection during Pregnancy: Case Report, Strain Characterization, Experimental Model of Reinfection, and Review

We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.

Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana.

From 1998 through 2006, 44 cases of severe primary toxoplasmosis were observed in French Guiana in immunocompetent adults. Toxoplasma gondii isolates exhibited an atypical multilocus genotype. Severe disease in humans may result from poor host adaptation to neotropical zoonotic strains of T. gondii circulating in a forest-based cycle.

Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR.

Different transmission routes, including the ingestion of undercooked meat, can result in Toxoplasma gondii infection in humans. The development of effective prevention strategies is hampered by a lack of quantitative information on the contamination level of different types of meat. Therefore, we developed a method for detection and quantification of T. gondii. The method involved preparation of crude DNA extract from hundred gram samples of meat, magnetic capture of T. gondii DNA and, quantitative real-time PCR targeting the T. gondii 529-bp repeat element. The detection limit of this assay was approximately 230 tachyzoites per 100 g of meat sample. There was a linear relation between the number of parasites added to the samples and Cp-values. Results obtained with the PCR method were comparable to bioassay results for experimentally infected pigs, and to serological findings for sheep. In addition, the T. gondii in 50% of the positive sheep samples could be genotyped by sequencing of the GRA6 gene, after isolation of the gene by magnetic capture. Two subtypes of GRA6 type II were identified in the 16 samples from sheep. For seven samples, the identification of T. gondii as type II was confirmed by microsatellite typing. The PCR method can be used as an alternative to bioassay for detection and genotyping of T. gondii, and to quantify the organism in meat samples of various sources.

An innovative survey underlining the significant level of contamination by Toxoplasma gondii of ovine meat consumed in France

Consumption of sheep meat presents a risk of human contamination by Toxoplasma gondii. A nationwide study was conducted in France to evaluate the prevalence of Toxoplasma in fresh ovine meat. A sampling procedure was established to guarantee the representativity of consumption. As is the case for meat consumed, half of the samples were from France and half were imported from other countries. Animals were selected according to their age, as lamb (<12months) represents 90% of the meat consumed. Available data for French samples allowed the selection of 16 districts distributed in seven areas according to their density of production. Diaphragms and hearts from 433 sheep were collected. Diaphragms were collected from 398 imported carcasses. Fluids from hearts and diaphragms were tested serologically. All hearts were bioassayed in mice and parasite isolates were genotyped using PCR-restriction fragment length polymorphism and microsatellite markers. Prevalence estimates were calculated, taking into account uneven distribution of production and age. For French meat, the effect of area, age and their interactions was evaluated. The overall estimate of Toxoplasma seroprevalence was 17.7% (11.6-31.5%) for lambs and 89% (73.5-100%) for adults (P<0.0001). No significant difference was observed between imported and French meat. In France, seroprevalence in lambs showed an increasing North-western to Southern gradient. The proportion of French carcasses carrying live parasites according to bioassay results was estimated at 5.4% (3-7.5%) (45 genotype II; one genotype III). This study offers an accurate drawing of the toxoplasmosis pattern amongst sheep consumed in France and a model for a zoonosis hazard control survey.

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

ABSTRACT We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.