Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

A ring trial to harmonize Toxoplasma gondii microsatellite typing: comparative analysis of results and recommendations for optimization.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

Spinal cord toxoplasmosis in a young immunocompetent patient.

We report a case of spinal cord toxoplasmosis occurring as a primary infection in a 31-year-old immunocompetent man. Exhaustive immunologic and genetic investigations did not identify any immunodeficiency. The causative agent was a typical type 2 strain. In cases of spinal cord lesions, toxoplasmosis should be considered, even in an immunocompetent patient.

Epidemiological review of Toxoplasma gondii infection in humans and animals in Portugal.

Toxoplasmosis is a worldwide zoonosis. However, data from Portugal are limited and a considerable part of the literature is in Portuguese. Currently, the rate of congenital infection in Portugal is unknown, and almost nothing is known of sequelae of congenital toxoplasmosis. There is no recent general population-based serological survey of Toxoplasma gondii in humans in Portugal. In addition, there is little information on genetic characteristics of T. gondii in animals and humans. In the present paper, we review prevalence, clinical spectrum and epidemiology of T. gondii in humans and animals in Portugal. This knowledge should be useful to biologists, public health workers, physicians and veterinarians.

Toxoplasma seroconversion with negative or transient immunoglobulin M in pregnant women: myth or reality? A French multicenter retrospective study.

Classically, Toxoplasma infection is associated with high levels of specific IgM antibody and a rise in specific IgG levels 1 to 3 weeks later. Atypical IgG seroconversion, without IgM detection or with transient IgM levels, has been described during serologic follow-up of seronegative pregnant women and raises difficulties in interpreting the results. To evaluate the frequency and the characteristics of these atypical cases of seroconversion, an investigation was conducted within the French National Reference Center for Toxoplasmosis, from which 26 cases collected from 12 laboratories belonging to the network were identified. The aim of this work was to retrospectively analyze the results of serologic testing, the treatments administered, and the results of prenatal and postnatal follow-up for these women. In each case, IgG antibodies were detected using both screening and confirmatory tests. IgM antibodies were not detected in 15 cases, and the levels were equivocal or low-positive in 11 cases. The IgG avidity results were low in 16 cases and high in one case. Most of the pregnant women (22/26) were treated with spiramycin from the time that IgG antibodies appeared until delivery. Amniotic fluid was analyzed for Toxoplasma gondii DNA by PCR in 11/26 cases, and the results were negative in all cases. Congenital toxoplasmosis was ruled out in 12/26 newborns. There was no abnormality observed at birth for 10 newborns and no information available for 4 newborns. In conclusion, when the interpretation of serological results is so difficult, it seems cautious to initiate treatment by spiramycin and to follow the pregnant women and their newborns.

Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France.

We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.

Diversity of Toxoplasma gondii strains at the global level and its determinants.

The population structure of Toxoplasma gondii is characterized by contrasting geographic patterns of strain diversity at different spatial scales: global, regional and even local scales in some regions. The determinants of this diversity pattern and its possible evolutionary mechanisms are still largely unexplored. This review will focus on three main dichotomies observed in the population structure of the parasite: (1) domestic versus wild, (2) South America versus the rest of the world and (3) intercontinental clonal lineages versus regional or local clonal lineages. Here, the impact in terms of public health of this remarkably contrasting geographic diversity of T. gondii populations is discussed, with emphasis on the role of globalization of exchanges that could lead to rapid evolution of T. gondii population spatial structure and new challenges in a One Health context.

[Seroprevalence of Toxoplasmosis among Pregnant Women in Benin: Meta-Analysis and Meta-Regression]. / Séroprévalence de la toxoplasmose chez les femmes enceintes au Bénin : méta-analyse et métarégression.

To assess the seroprevalence of toxoplasmosis among pregnant women in Benin, we conducted a meta-analysis using the PRISMA criteria. Al research published between 1990 and 2018 on toxoplasmosis among pregnant women Benin were eligible. A total of five databases were investigated, and the extracted data were subjected to a meta-analysis under R 3.1 using both random effect model and fixed effect model. The overall prevalence of toxoplasma-specific IgG among pregnant women was 47% (CI 95%: 40-53) and that of specific IgM was 2% (CI 95%: 1-3). The infection rate in urban areas (52%) was significantly higher than in rural areas (33%). The two main risk factors identified by the various eligible studies were the age of the pregnant women and the consumption of raw vegetables. We show that toxoplasmosis is endemic in pregnant women in Benin, implying that primary prevention measures must be put in place by the competent authorities to control this infection.

Afin d'évaluer le niveau de l'infection toxoplasmique chez les femmes enceintes au Bénin, nous avons effectué une méta-analyse selon le protocole PRISMA. Étaient éligibles tous les articles de recherche publiés entre 1990 et 2018 sur la toxoplasmose chez les femmes enceintes en consultation prénatale au Bénin. Au total, cinq bases de données ont été consultées, puis les données extraites ont été soumises à une méta-analyse sous R 3.1 selon les modèles à effet aléatoire et à effet fixe. La séroprévalence de la toxoplasmose chez la femme enceinte était de 47 % (IC 95 % : 40­53) pour les IgG et de 2 % (IC 95 % : 1­3) pour les IgM spécifiques. Le taux d'infection en milieu urbain (52 %) était significativement plus élevé qu'en milieu rural (33 %). Deux principaux facteurs de risque associés à la toxoplasmose ont été identifiés par les différentes études éligibles : l'âge des gestantes et la consommation de crudités. Nous montrons ainsi que la toxoplasmose est endémique chez les femmes enceintes au Bénin, impliquant que des mesures de prévention primaire soient mises en place par les autorités compétentes pour contrôler cette infection.

Atypical Toxoplasma gondii strain from a free-living jaguar (Panthera onca) in French Guiana.

Like domestic cats, wild felids are involved in the complete infective cycle of Toxoplasma gondii because they can host in their gastrointestinal tract sexually mature parasites and shed infective oocysts in their feces. We report, to our knowledge, the first isolation and molecular characterization of a T. gondii strain from the heart tissue of a free-living jaguar (Panthera onca) in French Guiana. Sequencing at six polymorphic markers indicated that the jaguar isolate had an atypical genotype, including an allele at TgM-A previously found only in isolates from South America, and an allele at GRA6, which was previously reported only in Californian sea otter isolates. These findings are consistent with the recent description of atypical T. gondii strains involved in severe toxoplasmoses in immunocompetent patients in French Guiana that seemed to be linked to a neotropical forest-based cycle involving wild cats and their prey.

Toxoplasma gondii, "new" genotypes and virulence.

Toxoplasma gondii has been described as a parasite with a low genetic diversity and a clonal population structure. The three main clonal lineages designated as type I, II or III largely predominate in Europe and North America. But strains not related to these main lineages circulate, notably, in other continents. They possess a shuffled combination of alleles that typify the three clonal types and unique polymorphisms detected by multilocus analysis. The population structure of Toxoplasma in these continents is also characterized by a higher genetic diversity associated with a lower linkage desequilibrium suggesting a role for genetic exchange. Due to their genomic diversity, it is difficult to draw global conclusions about their virulence. However, most of them are virulent in mice at isolation. Several reports also suggest a higher pathogenicity in humans and an association with ocular toxoplasmosis or severe cases of acquired toxoplasmosis in immunocompetent patients.

Rapid genotyping of Toxoplasma gondii by pyrosequencing.

Most human infections with the protozoan parasite Toxoplasma gondii are asymptomatic, but severe symptoms can occur in immunocompromised patients, in developing foetuses, and in ocular infections in immunocompetent individuals. The majority of T. gondii strains can be divided into three main lineages, denoted types I, II and III, which are known to cause different clinical presentations. Simple molecular methods with the capacity to discriminate rapidly among strains may help to predict the course of infection and influence the choice of treatment. In the present study, real-time PCR followed by pyrosequencing was used to discriminate among types I, II and III by analysis of two single nucleotide polymorphisms in the GRA6 gene. Twenty-one isolates of T. gondii characterised previously were analysed. Three different GRA6 alleles detected by the pyrosequencing technique identified types I, II and III isolates correctly, while four atypical isolates possessed either the GRA6 allele 1 or the GRA6 allele 3. Reproducibility was 100%, and typeability, when including atypical strains, was 81%. It was also possible to discriminate a mixture of two genotypes. The method was used to identify GRA6 type II in blood and lung tissue from an allogeneic transplant recipient with toxoplasmosis.

Mouse-virulent Toxoplasma gondii isolated from feral cats on Mona Island, Puerto Rico.

Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts. Samples of serum, feces, and tissues from cats from Mona, a remote island off the coast of Puerto Rico, were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test and found in 16 of 19 (84.2%) of cats, with titers of 1:10 in 2, 1:80 in 1, 1:160 in 4, 1:320 in 3, and 1:1,280 or higher in 6. Tissues of 19 of the 20 cats were bioassayed in mice for T. gondii infection. Toxoplasma gondii was isolated from tissues of 12 cats: from the hearts of 9, skeletal muscle of 10, and brain of 1 cat. All infected mice from 10 of 12 isolates died of acute toxoplasmosis during primary infection. Genotyping of these 12 T. gondii isolates (designated (TgCatPr 1-12) by 10 multilocus PCR-RFLP markers, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico, and the 6 multilocus microsatellite markers TUB2, W35, TgM-A, B18, B17, and M33, revealed 7 genotypes; 5 isolates had Type I alleles at all loci except at 1 microsatellite locus, and the remainder were atypical. The latter isolates of T. gondii were different biologically and phenotypically from the feline isolates from the rest of the Americas. One isolate (TgCatPr 12) was a mixed infection with 2 genotypes.

Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors.

The study was conducted to determine the prevalence and risk factors of toxoplasmosis in goats in Southern and central Ethiopia between October 2005 and May 2006. A total of 641 goats sera were tested using Modified Direct Agglutination Test (MAT), of which 480 (74.8% CI: 71.3, 78.2) were found to be positive. The highest prevalence was recorded in South Omo zone (82%) while the lowest was observed in East Shewa zone (62.2%). The study revealed that goats raised in southern Ethiopia are at a greater risk of acquiring T. gondii infection (OR = 2.55, CI: 1.726, 3.776; p = 0.000) than those which are raised in central Ethiopia. The prevalence of anti T. gondii antibody was significantly higher in older goats than in kids (OR = 2.33, CI: 1.490, 3.655; p < 0.0002) and in females than in males (p < 0.0007; OR = 0.68, CI: 0.542, 0.849). No significant difference was observed among goats kept under various husbandry practices. The high prevalence of toxoplasmosis in Ethiopian goats suggests a high risk of human infections. Further epidemiological investigation, isolation and genotyping of T. gondii are planned.

Geographical distribution of Toxoplasma gondii genotypes in Asia: A link with neighboring continents.

Defining the pattern of genetic diversity of Toxoplasma gondii is important to understand its worldwide distribution. During the last decades, a large number of studies have been published on Toxoplasma genotypes circulating in Europe, in North and South America. Two continents are still largely unexplored, Africa and, to a less extent, Asia. In this last continent, an increasing number of publications reported genotypes circulating in diverse provinces of China, but very few data are available for other Asian countries. After a systematic database search, 47 papers related to T. gondii genotypes in Asia were analyzed. Genetic characterization of DNA was performed by microsatellite markers, or more usually by a multiplex PCR using 11 PCR-RFLP markers, allowing data comparison to draw a first global picture of the population structure of this parasite throughout Asia. Overall, 390 isolates or DNA extracts were completely typed by PCR-RFLP and/or microsatellite marker methods, revealing 36 different PCR-RFLP or equivalent microsatellite genotypes: 15 genotypes identified by a ToxoDB number and 21 atypical or unique genotypes. The most common genotype found in Asia is the genotype ToxoDB#9 (Chinese 1). The clonal types I, II and II variant, and III were also commonly found in Asia. The geographical distribution of these genotypes across Asia may reflect either a continuum with Europe for the western part of Asia (presence of Type II), or the circulation of strains through animal migration or human activities between Africa and the Southwestern part of Asia (Africa 1 genotype in Turkey or ToxoDB#20 both I Sri-Lanka and in Ethiopia or Egypt). Although there are some indications of a genetic population structure in Southeast Asian countries different from the rest of Asia, more studies in this tropical part of Asia will be necessary for a region which represent as well as Africa one of the missing links of the T. gondii genetic diversity.

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis.

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.

Genetic diversity, clonality and sexuality in Toxoplasma gondii.

The majority of Toxoplasma gondii strains from a variety of human and animal sources have been grouped into three highly clonal but closely related lineages. The low occurrence of nucleotide differences among the three predominant lineages and their unusual dimorphic allelic composition suggest that they have arisen from a recent common ancestry. Less than 1% of the previously studied strains contain unique genotypes and high divergence of DNA sequence, and therefore are considered 'exotic' or 'atypical' strains. The seemingly low genetic diversity in T. gondii may have been underestimated because most parasite strains in previous studies were collected from human patients and domestic animals in North America and Europe. To investigate the genetic diversity of T. gondii, we analysed parasite strains isolated from remote geographical regions by multilocus microsatellite sequencing and phylogenetic analysis. The genetic diversity indices, the molecular analysis of microsatellite genotypes and the constructed phylogram considered together suggest that the global T. gondii population is highly diversified and not characteristic of a clonal organism. The most parsimonious hypothesis is that T. gondii presents a complex population structure with a mix of clonal and sexual propagation as a function of the environmental conditions. The comparison between domestic strains data on one hand and wild strains data on the other hand is in favour of more frequent sexual recombinations in wild environment even though Toxoplasma subpopulation in human and domestic animals is largely clonal.

Isoenzymic characterization of seven strains of Toxoplasma gondii by isoelectrofocusing in polyacrylamide gels.

Toxoplasma gondii strains are usually defined by biological parameters such as pathogenicity in mice. A characterization of toxoplasma strains by biochemical techniques has not been reported. In this study, extracts of tachyzoites of 7 toxoplasma strains were compared on the basis of their isoenzyme patterns for 39 enzymes by means of isoelectrofocusing in polyacrylamide gels. Eighteen enzymes gave clear and reproducible bands. Of these, 14 had identical electrophoretic patterns for all strains. Two different isoenzyme types were found for the enzymes aspartate aminotransferase, glutathione reductase, glucose phosphate isomerase, and amylase. This allowed the description of 3 isoenzyme pattern groups among the 7 toxoplasma strains. The possible relationship between biological behavior and isoenzyme pattern groups is discussed.

Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice.

Human African trypanosomiasis (HAT) or sleeping sickness is a major public health problem in 36 sub-Saharan African countries and is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. About 25,000 new cases of the disease are reported annually, and around 50 million people are classed as at risk of contracting the disease. Until now; the only effective drug available for treatment of advanced HAT was the trypanocide melarsoprol. The mortality rate of melarsoprol treated patients is 1-5%. Megazol is a nitroimidazole derivative shown to be effective in vitro against T. b. brucei with an EC50 of 0.01 micrograms.ml-1. When this compound was tested for its in vivo activity in T. b. brucei infected Swiss mice, it was shown to cure the acute disease. However, megazol alone did not cause cure of mice carrying a subacute infection with involvement of the central nervous system (CNS). Combined suramin and megazol treatment did prove effective and the mice were shown to have remission without further relapse from the CNS. The study of three megazol derivatives is also described here. Substitution of a bromine, methyl or trifluoromethyl moiety at the 4 position of the imidazole ring abolished trypanocidal activity both in vivo and in vitro. Intermediates of megazol synthesis (imidazole sulfoxide and imidazole sulfone) were also tested, but were shown not to be active. It is thought that megazol trypanocidal effect may be due to the triggering of radical production by the compound, which have toxic effects on the trypanosomes metabolism. In depth study of megazol is needed to fully elucidate its pharmacokinetics and to precisely pin down its mode of action.