ABSTRACT
Wastewater-based epidemiology has become a powerful surveillance tool for monitoring the pandemic of COVID-19. Although it is promising to quantitatively correlate the SARS-CoV-2 RNA concentration in wastewater with the incidence of community infection, there is still no consensus on whether the viral nucleic acid concentration in sewage should be normalized against the abundance of endogenous biomarkers and which biomarker should be used as a reference for the normalization. Here, several candidate endogenous reference biomarkers for normalization of SARS-CoV-2 signal in municipal sewage were evaluated. The human fecal indicator virus (crAssphage) is a promising candidate of endogenous reference biomarker for data normalization of both DNA and RNA viruses for its intrinsic viral nature and high and stable content in sewage. Without constructing standard curves, the relative quantification of sewage viral nucleic acid against the abundance of the reference biomarker can be used to correlate with community COVID-19 incidence, which was proved via mimic experiments by spiking pseudovirus of different concentrations in sewage samples. Dilution of pseudovirus-seeded wastewater did not affect the relative abundance of viral nucleic acid, demonstrating that relative quantification can overcome the sewage dilution effects caused by the greywater input, precipitation and/or groundwater infiltration. The process of concentration, recovery and detection of the endogenous biomarker was consistent with that of SARS-CoV-2 RNA. Thus, it is necessary to co-quantify the endogenous biomarker because it can be not only an internal reference for data normalization, but also a process control.
ABSTRACT
Wastewater-based epidemiology (WBE) is expected to become a powerful tool to monitor the dissemination of SARS-CoV-2 at the community level, which has attracted the attention of scholars all over the world. However, there is not yet a standard protocol to guide its implementation. In this paper, we proposed a comprehensive technical and theoretical framework of relative quantification via qPCR for determining the virus abundance in wastewater and estimating the infection ratio in corresponding communities, which is expected to achieve horizontal and vertical comparability of the data using a human-specific biomarker as the internal reference. Critical factors affecting the virus detectability and the estimation of infection ratio include virus concentration methods, lag-period, per capita virus shedding amount, sewage generation rate, temperature-related decay kinetics of virus/biomarker in wastewater, and hydraulic retention time (HRT), etc. Theoretical simulation shows that the main factors affecting the detectability of virus in sewage are per capita virus shedding amount and sewage generation rate. While the decay of SARS-CoV-2 RNA in sewage is a relatively slow process, which may have limited impact on its detection. Under the ideal condition of high per capita virus shedding amount and low sewage generation rate, it is expected to detect a single infected person within 400,000 people.