Effects of Inhibition on Accurate Quantification of Viral RNA during Wastewater-Based Surveillance

Abstract Wastewater-based surveillance (WBS) during the COVID-19 pandemic has been a crucial tool for understanding the prevalence of SARS-CoV-2 in communities. Despite the strong correlation between viral RNA levels detected in wastewater and clinical metrics, accurate viral RNA quantification in wastewater using reverse transcriptase (RT) quantitative polymerase chain reaction (qPCR) remains a challenge, especially at sites where there is potential matrix inhibition affecting the assay. In this study, model environmental RT-qPCR inhibitors, humic and tannic acids, were spiked into uninhibited wastewater extracts causing inhibition of the RT step based on comparison of selected RNA and DNA targets affecting quantification in both RT-qPCR and digital RT-PCR platforms. Wastewater from a municipal treatment plant that experienced frequent inhibition was also examined to determine the possible source of inhibition and its impact on the viral quantification. RT inhibition in the wastewater samples caused a significant underestimation of viral RNA, emphasizing the need for RT-specific inhibition controls in WBS. Further investigation suggested that the rotary drum thickener (RDT) filtrate return was a possible source of these effects, with the inhibition effects ceasing when the RDT filtrate was diverted. Understanding how inhibition of RT-qPCR occurs in wastewater is critical to avoiding underestimation of the viral RNA in wastewater and has implications in WBS for public health decisions.