Occurrence, Diversity, and Building Occupancy Association of Human microRNAs in Wastewater

Abstract Wastewater-based epidemiology (WBE) relies on biomarkers to detect infectious viruses and synthetic opioids in communities. Expanding the number of biomarkers would broaden surveillance capability to noncommunicable diseases and other human health issues. MicroRNAs (miRNAs), noncoding, cellular regulatory molecules, represent a potential new class of WBE biomarkers. This study evaluated miRNA occurrence and site specificity in wastewater from three hospitals, three community centers, and three wastewater treatment facilities (WWTFs) using Next-Generation Sequencing (NGS) and targeted microarrays. NGS generated 3.9 to 7 million reads per sample and identified 486 unique miRNAs across 42 samples. Nonmetric Multidimensional Scaling showed that NGS-derived miRNA profiles clustered by building occupancy type. Technical duplicates were more similar than samples collected across days, demonstrating reproducibility. The TaqMan Array Card and urine exposome panel detected 463 of 752 and 67 of 87 miRNAs, respectively. Sixty-three miRNAs overlapped between NGS and TAC. Microarray data identified 89 miRNAs unique to hospital sewers and 69 unique to community centers; 94% and 96% of these unique miRNAs were causally associated with diseases. These findings demonstrate the feasibility of miRNA detection in wastewater and suggest location-specific miRNA patterns may provide complementary biological insight for WBE applications, potentially helping to identify noncommunicable disease states.