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.

Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R² = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods.

Toxoplasmosis is one of the most important foodborne diseases in humans, potentially acquired by ingesting unpasteurized goat milk. This study examined the role of goat milk as a source of infection of Toxoplasma gondii for humans in Algeria. Sera, blood, and milk samples collected from 106 female goats were tested for the presence of antibodies against T. gondii and its DNA, using indirect ELISA and PCR, respectively. Multiplex PCR was performed using 15 microsatellite markers to determine the clonal type of the T. gondii DNA detected. Seropositive results were found in 51 she-goats (48.11%). T. gondii DNA was detected in 16 (15.09%) and 15 (14.15%) blood and milk samples, respectively. In total, 15 (29.41%) out of 51-seropositive goats were PCR-positive for blood, while only 6 of them (6/15, 40%) showed the presence of T. gondii DNA in their milk. A fair correlation was found between indirect ELISA and PCR assays for T. gondii detection in milk (K = 0.2243) and blood (K = 0.28300), with a substantial difference in the screening ability of the tests (G2 = 38.96, p < 0.0001). The genotyping of samples could not be completed, but showed the absence of type I and type III lineages in goats from the Mila region, northeastern Algeria. The Algerian goat population is highly exposed to T. gondii, with a potentially increased risk of parasite transmission to humans via milk consumption.

Background: Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis, a widespread zoonotic disease. Despite the clinical significance of this zoonotic parasite, little is known regarding its prevalence in South Africa, particularly in wildlife. Considering the possible presence of the parasite in wildlife species and the popularity of South African game meat, in a ‘One Health context’, the consumption of undercooked game meat by people could represent a public health issue. The objective of this study was to determine the prevalence of T. gondii and any genotypes in impala from the Greater Kruger region destined for game meat. Methods: Serum and seven different tissue samples were collected from 138 impala ( Aepyceros melampus ) from the Timbavati Private Nature Reserve in South Africa. The seroprevalence of T. gondii was determined using the Modified Agglutination Test (MAT). The presence of T. gondii DNA within the impala tissues and possible tissue tropism were determined using a quantitative PCR (qPCR). For strong qPCR-positive samples, T. gondii DNA was genotyped using a panel of 15 microsatellite markers. Results: The seroprevalence was determined to be 8.7%. The qPCR identified T. gondii DNA in at least one tissue type of 7.2% of the impala. The T. gondii DNA was detected in the brain and tongue samples from two impala respectively, and were genotyped as belonging to the Africa 4 lineage. To place the two genotypes identified in this study within the broader context of the genetic diversity of T. gondii in Africa, a genetic tree was constructed using all African strains genotyped with 15 microsatellite markers. Conclusion: These results shed light on serological versus molecular techniques in determining infection of T. gondii in impala, and also point to possible tissue tropism during infection. The results identify Africa 4 strains circulating in South African wildlife intended for human consumption, and the importance of genotype and phenotype characterisation to assess the potential public health risks.

Toxoplasmosis is a food- and waterborne zoonosis of a great importance, ranked as the fourth most important foodborne parasitosis in the world. The objective of this study was to determine the seroprevalence and molecular prevalence of Toxoplasma gondii in wild boar (Sus scrofa) and to identify genotypes circulating in Croatia. A total of 103 wild boars from four hunting areas in Croatia were screened. Cardiac fluid samples were tested for anti-T. gondii antibodies by MAT, while heart samples were tested for T. gondii DNA using qPCR. The seroprevalence in the cardiac fluid reached 54.4%, while 19.4% of the heart samples were positive by qPCR. The highest seroprevalence was detected in the hunting ground Visocica with 95.6%. Partial microsatellite genotyping was achieved for 2/20 qPCR-positive heart samples suggesting type II strain of T. gondii. Relatively high seroprevalence and detection of T. gondii DNA in tested samples highlights the risk of human infection through consumption of undercooked meat. Further studies focusing on parasite detection in wild boar meat, especially across different climates, are needed to understand regional differences. Expanding research to other wildlife species is also important to clarify their role in the spread of T. gondii.