Improving correlation of wastewater SARS-CoV-2 gene copy numbers with COVID-19 public health cases using readily available biomarkers

The COVID-19 (coronavirus disease 2019) pandemic has highlighted the potential role that wastewater-based epidemiology can play in assessing aggregate community health. However, efforts to translate SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) gene copy numbers obtained from wastewater samples into meaningful community health indicators are nascent. In this study, SARS-CoV-2 nucleocapsid (N) genes (N1 and N2) were quantified weekly using reverse transcriptase droplet digital PCR from two municipal wastewater treatment plants for 6 months. Four biomarkers [ammonium, biological oxygen demand (BOD), creatinine and human mitochondrial gene NADH dehydrogenase subunit 5] were quantified and used to normalize SARS-CoV-2 gene copy numbers. These were correlated to daily new case data and 1-, 2- and 3-week cumulative case data. Over the course of the study, the strongest correlations were observed with a 1-day case data lag. However, early measurements were strongly correlated with a 5-day case data lag. This indicates that in the early stages of the pandemic, the wastewater samples may have indicated active COVID-19 cases before clinical indications. Mitochondrial and creatinine normalization methods showed the strongest correlations throughout the study, indicating that human-specific biomarkers were better at normalizing wastewater data than ammonium or BOD. Granger causality tests supported this observation and showed that gene copies in wastewater could be predictive of new cases in a sewershed.