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

• 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
• ISSN: 0934-9723; 1435-4373; 1435-4373
• DOI: 10.1007/s10096-023-04721-7

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.