*4.2. Detection Limit Assays*

Serial ten-fold oocyst dilutions (105-1 oocysts/gram of stool) were performed using a negative liquid stool human matrix. Each corresponding dilution was confirmed by counting oocysts microscopically both on Kova slides (Labellians, Nemours, France) and using Crypto-Cel FITC (Cellabs, Sydney, Australia) staining according to the manufacturer's instructions. Limits of detection were estimated considering the positivity of at least one of the three tested replicates per condition. *Cryptosporidium* negativity of the matrix was based on both microscopy and PCR investigations from the method described by Valeix et al. 2020.
