Development of a centrifugation-based method for the qualitative molecular detection of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in surface water samples

Workflow for waterborne parasites DNA detection in surface water. Screening for environmental surveillance in monitoring and resource-limited settings. Zoonotic protozoa and microsporidia are waterborne pathogens with implications for animal and human health. Their resistant stages can persist in freshwater. Standard reference methods for water analysis are often limited to specific parasites, require large water volumes, and depends on expensive equipment. Practical alternatives are needed to facilitate parasite detection in water. This study aimed to develop a pellet-based workflow to allow the DNA detection of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi from the same surface water sample. Three organism-specific spiked controls and eight environmental samples from four freshwater sites, collected over two months, were processed by centrifugation to concentrate the pellet fraction, while the supernatant recovered post-centrifugation was filtered through membranes. The samples were collected as part of a broader ongoing year-long monitoring project. DNA was extracted from both fractions and analysed by qualitative PCR assays. Pellets yielded positive results whereas no positivity was detected in filter membranes, suggesting pellet fractions as the most suitable for downstream molecular analysis. This qualitative pellet-based processing approach was designed to provide an assessment and a practical screening strategy for environmental surveillance.