Influence of Sampling Frequency on Wastewater-based Public Health Assessments of Opioid Consumption and SARS-CoV-2 Infections

Wastewater-based epidemiology (WBE) has emerged as a powerful tool for community health assessment, using wastewater-borne biological and chemical markers as analytical targets. This study investigates the critical influence of sampling frequency on the resultant estimates of opioid consumption and the prevalence of SARS-CoV-2 infections at the neighborhood level using common WBE biomarkers including fentanyl, norfentanyl, and the SARS-CoV-2 N1 gene as targets. The goal was to assess sampling methodologies that include the impact of the day of the week and of the sampling frequency. Wastewater samples were collected two or three times per week over the course of five months (n=525) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) or reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for target chemical or molecular indicators of interest. Results showed no statistically significant differences for days of the week (i.e., Tuesday vs. Thursday vs. Saturday) for 24-hour composite samples analyzed for fentanyl or SARS-CoV-2; however, concentrations of the human metabolite of fentanyl, norfentanyl, were statistically different between Tuesday and Saturday (p < 0.05). When data were aggregated either by Tuesday/Thursday or Tuesday/Thursday/Saturday to examine sensitivity to sampling frequency, data were not statistically different except for the Tuesday/Thursday weekly average and Saturday for norfentanyl (p < 0.05). These results highlight how sample collection and data handling methodologies can impact wastewater-derived public health assessments. Care should be taken when selecting an approach to the sampling frequency based on the public health concerns under investigation.