PARAsite Detection, Isolation and Evaluation

In Europe, protozoa of the genera Cryptosporidium and Giardia are particularly relevant, and have been associated with large outbreaks linked to contaminated water and food. Outbreak investigation and source attribution remain difficult, also due to the lack of standardized typing methods. The PARADISE project aims to deliver informative genotyping schemes derived from whole genome sequence (WGS) data and innovative enrichment strategies applicable to food matrices for the zoonotic pathogens Cryptosporidium parvum and Giardia duodenalis.

The PARADISE project successfully delivered informative genotyping schemes derived from whole genome sequence (WGS) data and innovative enrichment strategies applicable to food matrices for the zoonotic pathogens Cryptosporidium parvum and Giardia duodenalis.The main achievements were: The generation of about 130 WGS from C. parvum (humans and ruminants), 50 WGS from G. duodenalis assemblage B (mostly from humans) and about 70 WGS from G. duodenalis assemblage A (humans and other mammals). Comparative genomic studies are progressing and will shed light on the population structure, virulence and evolution of these parasites, and provide insights for future functional studies. The applicability of metagenomics approaches for the detection of parasites in various biological, water and food samples was critically evaluated in silico and experimentally. The analyses show that efficient detection can also be achieved in the presence of excess background material of eukaryotic origin (e.g., a vegetable matrix), but that confirmation of the results obtained by k-mer or mapping procedure is essential to avoid false positives. These efforts have been paralleled by the refinement of analytical workflows for the analysis of complex data that took into account the specific biology of parasitic pathogens. EFSAshort forEuropean Food Safety Authority and ECDCshort forEuropean Centre for Disease Prevention and Control can have an interest towards this work, as it demonstrates full applicability of genomics and metagenomics approaches to detect and characterize these important foodborne pathogens. Ongoing collaborations with European Union Reference Laboratories for bacterial and viral pathogens will helping fostering the use of WGS for parasitic pathogens New multi-locus sequence typing (MLST) schemes for both C. parvum and G. duodenalis assemblage B have been designed and tested on hundreds of isolates from different hosts and different geographic origin. The schemes demonstrated a high discriminatory power and represent valuable tools for outbreak investigations and epidemiological studies. Standard Operating Procedures (SOPs) are available and ring tests among Consortium partners showed efficient implementation of the typing schemes. Adoption of the two MLSTs by Reference Laboratories will address the specific need for robust molecular tying methods recently highlighted by EFSAshort forEuropean Food Safety Authority. A post-DNA extraction capture system for Cryptosporidium, based on probes targeting the 18S rDNA and the gp60 genes, have been designed and extensively characterized, and demonstrated to have high sensitivity in inter-laboratory studies. This technique will allow investigation of DNA extracts from samples from outbreaks of unknown aetiology. The pre-DNA enrichment strategies based on nanobody and aptamer technologies culminated in the selection of nanobodies with high specificity towards Giardia.  These reagents could be used on matrices (food, water) contaminated with low number of parasites, whose detection is relevant for risk assessment.