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.

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.

Human toxoplasmosis: a systematic review for genetic diversity of Toxoplasma gondii in clinical samples.

Toxoplasma gondii (T. gondii) as an obligate intracellular protozoan with a worldwide distribution can infect virtually all warm-blooded animals and humans. This study aims to provide a summary of the available data on genotypes of T. gondii in human. Five databases including MEDLINE in PubMed, Scopus, Science Direct, Web of Science and Google Scholar were searched for the T. gondii genotyping in human during 1995-August 2017. Next, we screened all the articles based on the inclusion and exclusion criteria. Overall, 26 studies were eligible regarding genotyping T. gondii in human samples. In clonal genotyping, 167 out of 286 cases (58%) were infected with type II. Genetic characterisation of T. gondii isolates displayed that type II was the most predominant genotype in human with the prevalence of 64.3%, 62.1% and 41.7% in patients with AIDS, congenital and ocular toxoplasmosis, respectively. In ToxoDB genotyping, most individuals were infected with genotypes #9 and #65 (21.2%). Based on these results, genotype profile of T. gondii isolates is different throughout the world. The strains in Asian and African countries are characterised by low genetic diversity, while in North and South America a wide diversity of this parasite is found. In countries without any data (e.g. Australia, Western and Southern Africa and Western Asia), identification of T. gondii genotypes might discover higher genetic diversity.

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.

Three new cases of human infection with Dirofilaria repens, one pulmonary and two subcutaneous, in the Egyptian governorate of Assiut.

Human dirofilariasis caused by Dirofilaria repens is a parasitic infection, currently considered to be an emerging zoonosis, that has been observed in many areas of the Old World. Dogs are the main 'reservoir' host. In humans, D. repens can cause inflammatory nodules, most commonly in the subcutaneous tissues or in the subconjunctival space. Lung involvement, which is the most common of the non-cutaneous and non-ocular manifestations, is frequently mis-diagnosed as a primary or metastatic lung tumour. Two new subcutaneous cases and one new pulmonary case, all observed in the Egyptian governorate of Assiut, are described here. This represents the first report of human pulmonary dirofilariasis caused by D. repens on the African continent. All the worms collected from the cases, including a living worm from the left lung of the pulmonary case, were identified morphologically, and the identity of the lung parasite was confirmed by PCR.