Evaluation of Molecular-based Methods for the Detection and Quantification of Cryptosporidium spp. in Wastewater

Cryptosporidium , a eukaryotic protozoan parasite, poses a significant public health risk as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is estimated to be largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Unlike clinical surveillance, wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized methods for Cryptosporidium spp. detection in wastewater challenges the comparability of studies. Additionally, the use of standard methods for the detection and quantification of Cryptosporidium spp. in surface waters may not be appropriate for wastewater samples due to the more complex composition of the wastewater matrix. Thus, in this study, we evaluated methods for concentrating Cryptosporidium oocysts from wastewater, extracting DNA, and detecting its genetic markers, using wastewater seeded with C. parvum oocysts. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome particles. All methods except the Nanotrap Microbiome particles were conducted with or without additional purification via immunomagnetic separation. For DNA extractions, we tested the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit, while assessing the impact of bead beating and freeze-thaw cycles on DNA yield. Genetic detection was performed using qPCR targeting the Cryptosporidium 18S rRNA gene. Our results indicated that centrifugation yielded the highest oocyst recoveries (39-77%), followed by the Nanotrap Microbiome particles (24%), electronegative filtration with a phosphate buffered saline with 20% Tween 80 elution (22%), and Envirocheck HV capsule filtration (13%). IMS was found to be unsuitable due to interference from the wastewater matrix, significantly reducing recovery rates to 0.03 to 4%. DNA yields were highest with bead-beating pretreatment with either the DNeasy Powersoil Pro kit (314 gc/μL DNA) or the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA yields to under 92 gc/μL DNA, likely through DNA degradation. This study is amongst the first to compare different concentration methods, including Nanotrap Microbiome particles, and DNA extraction methods that can be utilized for Cryptosporidium spp. wastewater surveillance, highlighting the importance of method selection for accurate detection and quantification.