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...

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Published in:	European journal of clinical microbiology & infectious diseases Vol. 43; no. 2; pp. 355 - 371
Main Authors:	Joeres, M., Maksimov, P., Höper, D., Calvelage, S., Calero-Bernal, R., Fernández-Escobar, M., Koudela, B., Blaga, R., Vrhovec, M. Globokar, Stollberg, K., Bier, N., Sotiraki, S., Sroka, J., Piotrowska, W., Kodym, P., Basso, W., Conraths, F. J., Mercier, A., Galal, L., Dardé, M. L., Balea, A., Spano, F., Schulze, C., Peters, M., Scuda, N., Lundén, A., Davidson, R. K., Terland, R., Waap, H., de Bruin, E., Vatta, P., Caccio, S., Ortega-Mora, L. M., Jokelainen, P., Schares, G.
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Springer Berlin Heidelberg 01-02-2024 Springer Nature B.V Springer Verlag
Subjects:	Biomedical and Life Sciences Biomedicine Deoxyribonucleic acid Dilution DNA DNA, Protozoan DNA, Protozoan - genetics Female Gene sequencing Genetic variability Genetic Variation Genomes Genotype Genotypes Genotyping Haplotypes High resolution High-Throughput Nucleotide Sequencing Humans Internal Medicine Life Sciences Medical Microbiology Multiplex Polymerase Chain Reaction Next-generation sequencing Nucleotides Original Original Article Polymorphism, Restriction Fragment Length Pregnancy Protozoa Sensitivity analysis Single-nucleotide polymorphism Strains (organisms) Toxoplasma Toxoplasma - genetics Toxoplasma gondii Whole genome sequencing Intra-genotype variability Discriminatory power Typing Highly polymorphic regions Multilocus sequence typing Toxoplasmosis
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 PMCID: PMC10822014
ISSN:	0934-9723 1435-4373 1435-4373
DOI:	10.1007/s10096-023-04721-7