Wastewater-based epidemiology for waterborne protozoa: a comparison of analytical methods for detecting Cryptosporidium spp.

Cryptosporidium spp., Giardia intestinalis, and Toxoplasma gondii are waterborne pathogens of public health significance. Wastewater-based epidemiology (WBE) has been proposed as an epidemiological tool for monitoring parasites. Although several studies have used standardized methods to detect these parasites in drinking and surface water, they are not always suitable for wastewater analysis. In this study, two different extraction protocols and five concentration methods for Cryptosporidium spp. were evaluated using wastewater spiked with C. parvum oocysts. The aluminium-based adsorption-precipitation method, combined with three freeze-thaw cycles and a magnetic-beads-based nucleic acid extraction, proved to be the most efficient, achieving qPCR detection limits of 1.29 × 10⁴ oocysts/L for the selected molecular targets. Levels of Cryptosporidium spp., C. parvum, C. hominis, G. intestinalis, and T. gondii were assessed in wastewater samples from a hospital sewer and a wastewater treatment plant (WWTP) using the optimized procedure. A higher prevalence of T. gondii (46.67 %) was observed in hospital wastewater, followed by C. parvum and C. hominis (13.33 %), with levels ranging from 2.56 to 4.04 Log₁₀ genome copies (gc)/L, 4.86 to 5.17 Log₁₀ gc/L and 4.77 to 5.16 Log₁₀ gc/L, respectively. In samples from the analysed WWTP, the prevalence of C. parvum and T. gondii was similar (52.38 % and 47.62 %, respectively), with levels ranging from 3.60 to 4.87 Log₁₀ gc/L and from 2.49 to 3.77 Log₁₀ gc/L, respectively. Our study provides a feasible protocol for the analysis of Cryptosporidium and Toxoplasma, supporting the use of WBE for the surveillance of parasites transmitted through contaminated water and food.