Authors

AA Khedr³; LF Valenzuela-Moreno⁵; CP Rico-Torres²; H Caballero-Ortega⁴; Z Koutsogiannis⁶; PW Denny¹;  ¹ Durham University, UK;  ² Instituto Nacional de Pediatría, Mexico;  ³ New Valley University, UK;  ⁴ Programa de Doctorado en Ciencias de la Producción y de la Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, CDMX, México., Mexico;  ⁵ Laboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría, CDMX, Mexico;  ⁶ Centre for Discovery Brain Sciences, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Hugh Robson Building, UK

Discussion

Foodborne protozoan parasites, including Toxoplasma gondii, Sarcocystis spp., and Cryptosporidium spp., remain major contributors to global gastrointestinal and systemic disease. Their environmentally resistant stages, broad host ranges, and complex transmission cycles complicate surveillance and outbreak investigations, necessitating the development of a portable, rapid molecular surveillance platform for simultaneous detection and high-resolution genotyping of foodborne protozoan pathogens. We developed a Nanopore-based multi-locus sequence typing (MLST) workflow using Oxford Nanopore Technologies, employing Toxoplasma gondii as a representative model to establish a portable, high-resolution genotyping framework for foodborne protozoan surveillance.  Initial validation was performed using cultured reference lineages, Type I (RH strain) and Type II (ME49 strain), targeting six loci (SAG2, SAG3, ROP5, ROP8, ROP17, and ROP21). Multi-locus variant analysis revealed distinct strain-specific SNP and INDEL profiles, enabling clear discrimination between Type I and Type II lineages. Notably, long-read sequencing enabled resolution of the paralogous ROP5 locus, allowing assessment of sequence polymorphisms and repeat copy number variation, a key determinant of strain-specific virulence. Field validation using isolates from Mexico demonstrated that ROP5 effectively differentiated atypical strains, highlighting its utility as both a genotyping and virulence-associated marker. Ongoing analyses are expanding the genotyping panel to include additional virulence-associated loci (ROP17, ROP18, GRA7, and GRA15) to further characterize recombinant and geographically diverse isolates. Future development of the platform will expand its diagnostic capacity through the integration of nested and multiplex PCR assays targeting additional apicomplexan parasites within complex biological matrices, including Cryptosporidium spp. and Sarcocystis spp., thereby enabling comprehensive, field-deployable surveillance of foodborne protozoan pathogens.