High-Resolution Molecular Surveillance of Giardia duodenalis and Cryptosporidium spp. in Surface and Wastewater Using Oxford Nanopore Sequencing
Giardia duodenalis and Cryptosporidium spp. are major waterborne pathogens, yet environmental genotyping remains limited by low-resolution methods. We developed new primers targeting the Cryptosporidium 18S rRNA gene and applied a multilocus strategy for Giardia (gdh, tpi, bg) coupled with Oxford Nanopore (ONT) sequencing to characterize 102 surface- and wastewater samples from the Pasto River basin (Colombia). Giardia was detected in 84.8–95% of samples, predominantly assemblages A (43.0–66.6%) and B (40.2–73.5%), with frequent multi-assemblage co-occurrence (75.0–89.3%). Cryptosporidium was identified in 85.1% (57/67) of positives, comprising six species dominated by C. canis, C. parvum, and C. andersoni, with multi-species detection in 98.2% of samples. Co-detection of both protozoa occurred in 34 samples. These results demonstrate high genotypic complexity in environmental waters and support long-amplicon Nanopore sequencing as a high-resolution tool for molecular surveillance under a One Health framework.