Routine and outbreak monitoring of Cryptosporidium using a molecular diagnostic method.

Cryptosporidiosis is a significant public health disease associated with various water matrices. In response to the increase of outbreaks reported in several countries and the limitations of conventional detection methods, this study presents and validates a quantitative molecular protocol based on qPCR for the detection of Cryptosporidium spp. and Cryptosporidium parvum in different water matrices. Our method includes high-volume water filtration, thermal-mechanic-enzymatic cell disruption and an optimized qPCR assay, resulting in a highly sensitive and reproducible method. We evaluated the sensitivity, specificity, turbidity interference, and performance compared to the conventional IMS-IM method. Validation over 27 months demonstrated robust performance, with recovery rates up to 55.1 %, detection of as few as 9 oocysts per sample and a cost reduction of over 35 % compared to traditional methods. The method was successfully applied during recent outbreak investigations and offers a reliable, rapid, and scalable alternative for environmental Cryptosporidium monitoring, with strong potential for integration into regulatory frameworks.