Background. Nowadays, most of the C. parvum and C. hominis epidemiological studies are based on gp60 gene subtyping using the Sanger sequencing (SgS) method. Unfortunately, SgS presents the limitation of being unable to detect mixed infections. Next-Generation Sequencing (NGS) seems to be an interesting solution to overcome SgS limits. Thus, the aim of our study was to (i) evaluate the reliability of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the genetic diversity of the parasite and the frequency of mixed infections, (iii) assess NGS usefulness in Cryptosporidium sp. outbreak investigations, and (iv) assess an interpretation threshold of sequencing data. Methods. 108 DNA extracts from positive samples were sequenced by NGS. Among them, two samples were used to validate the reliability of the subtyping obtained by NGS and its capacity to detect DNA mixtures. In parallel, 106 samples from French outbreaks were used to expose NGS to epidemic samples. Results. NGS proved suitable for Cryptosporidium sp. subtyping at the gp60 gene locus, bringing more genetic information compared to SgS, especially by working on many samples simultaneously and detecting more diversity. Conclusions. This study confirms the usefulness of NGS applied to C. hominis and C. parvum epidemiological studies, especially aimed at detecting minority variants.

Intestinal parasitic diseases affect millions of people worldwide. Numerous commercial molecular methods detecting digestive parasites have been developed recently, including multiplex PCR assays able to identify multiple parasites at once. Several studies have demonstrated that the efficacy of these molecular methods is dependent on the specific protocols employed at each stage of the process including pretreatment, extraction and amplification. However, previous studies have exclusively focused on one of these steps, without considering the others. The objective of the present study was to evaluate the performances of molecular tools for Cryptosporidium parvum detection in stool samples, considering all steps of the process simultaneously. 30 distinct combinations of protocols were evaluated corresponding to three pre-treatment methods, four DNA extraction techniques and six DNA amplification assays. The performances of these combinations were evaluated in terms of detection limit. We showed that different combinations yielded varying results. The FTD® Stool Parasite technique proved to be the most effective, achieving 100 % detection. Manual extraction methods demonstrated excellent outcomes, although they are time-consuming. The optimal approach for detecting C. parvum DNA is a combination of mechanical pretreatment, the Nuclisens® Easymag® extraction method, and the FTD® Stool Parasite DNA amplification method. This work shows that the molecular diagnosis should consider all stages. A PCR method may not be effective with an unsuitable extraction technique, but can yield optimal results with an appropriate one.