Evaluation of SARS-CoV-2 concentrations in wastewater and river water samples

Abstract

There are only a few established methods to determine the concentration of encapsulated viruses, such as SARS-CoV-2, in water matrices, limiting the application of wastewater-based epidemiology (WBE)-an important tool for public health research. The present study compared four methods that are commonly used to concentrate non-encapsulated enteric viruses for determining SARS-CoV-2 concentration in wastewater and wastewater-enriched river water samples. The four methods tested were electronegative membrane with Mg⁺² addition, aluminum hydroxide-based precipitation, polyethylene glycol (PEG) 8000 precipitation, and ultrafiltration (with porosity of 10 and 50 kDa). Prior to the concentration step, filtration or centrifugation was performed to remove suspended particles from the samples (pretreatment). To evaluate the recovery efficiency (%), samples of SARS-CoV-2 from nasopharyngeal swabs obtained from RT-qPCR-positive patients were used as spiked samples. The second part of the analysis involved the quantification of the SARS-CoV-2 copy number in analytes without SARS-CoV-2-spiked samples. Among the tested methods, pretreatment via centrifugation followed by ultrafiltration with a 50-kDa cut-off was found the most efficient method for wastewater samples with spiked samples (54.3 or 113.01% efficiency). For the wastewater-enriched river samples with spiked samples, pretreatment via centrifugation followed by filtration using an electronegative membrane was the most efficient method (110.8% and 95.9% for N1 and N2 markers, respectively). However, ultrafiltration of the raw river water samples using 10 or 50 kDa cut-off filters and PEG 8000 precipitation showed the best concentration efficiency based on copy number, regardless of the pretreatment approach or sample type (values ranging from 3 × 10⁵ to 6.7 × 10³). The effectiveness of the concentration method can vary depending on the type of sample and concentration method. We consider that this study will contribute to more widespread use of WBE for the environmental surveillance of SARS-CoV-2.

Keywords: COVID-19; Molecular diagnosis; PEG concentration; Ultrafiltration; Wastewater surveillance; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Overview of the methodology applied for the evaluation of different concentration methods for detecting SARS-CoV-2 in river and wastewater samples. Created with BioRender.com.

Fig. 2

Wastewater samples not subjected to pretreatment (filtration or centrifugation) after concentration using the PEG 8000 method: (A) before and (B) after using the OneStep PCR Inhibitor Removal Kit™.