Simultaneous detection and quantification of multiple pathogen targets in wastewater.

Wastewater-based epidemiology has emerged as a critical tool for public health surveillance, building on decades of environmental surveillance work for pathogens such as poliovirus. Work to date has been limited to monitoring a single pathogen or small numbers of pathogens in targeted studies; however, few studies consider simultaneous quantitative analysis of a wide variety of pathogens, which could greatly increase the utility of wastewater surveillance. We developed a novel quantitative multi-pathogen surveillance approach (35 pathogen targets including bacteria, viruses, protozoa, and helminths) using TaqMan Array Cards (TAC) and applied the method on concentrated wastewater samples collected at four wastewater treatment plants in Atlanta, GA from February to October of 2020. From sewersheds serving approximately 2 million people, we detected a wide range of targets including many we expected to find in wastewater (e.g., enterotoxigenic E. coli and Giardia in 97% of 29 samples at stable concentrations) as well as unexpected targets including Strongyloides stercoralis (a human threadworm rarely observed in the USA). Other notable detections included SARS-CoV-2, but also several pathogen targets that are not commonly included in wastewater surveillance like Acanthamoeba spp., Balantidium coli, Entamoeba histolytica, astrovirus, norovirus, and sapovirus. Our data suggest broad utility in expanding the scope of enteric pathogen surveillance in wastewaters, with potential for application in a variety of settings where pathogen quantification in fecal waste streams can inform public health surveillance and selection of control measures to limit infections.