Simultaneous detection and quantification of multiple enteric 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, simultaneous analysis of a wide variety of pathogens would greatly increase the utility of wastewater surveillance. We developed a novel quantitative multi-pathogen surveillance approach (33 pathogen targets including bacteria, viruses, protozoa, and helminths) using TaqMan Array Cards (RT-qPCR) 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 stercolaris (i.e., human threadworm, a neglected tropical disease rarely observed in clinical settings 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.