Detection of SARS-CoV-2 in wastewater is influenced by sampling time, concentration method, and target analyzed

Abstract

The detection of SARS-CoV in wastewater has been proposed as a tool for monitoring COVID-19 at the community level. Although many reports have been published about detecting viral RNA in wastewater and its presence has been linked to infected people, appropriate analytical methodologies to use this approach have not yet been established. In this study, we compared ultrafiltration, polyethylene glycol precipitation, flocculation using AlCl3, and flocculation with skim milk for the recovery of SARS-CoV-2, using RNA from patients with positive diagnoses for COVID-19 and Pseudomonas phage φ6 as the control. We also evaluated the primers for detecting the E, RdRp, and N genes of the virus, as well as different storage times. Differences in the recovery efficiencies were evident with the different concentration methods, the best being ultrafiltration and precipitation with aluminum, which had recovery rates of 42.0% and 30.0%, respectively, when virus was present at high levels. Significant differences were found between the recoveries using wastewater and deionized water and between different storage times, with better recoveries for 6 and 12 h samplings. The E gene was the only one detected in all the samples analyzed. The results show that although this approach can provide important data for studying the pandemic, clear protocols are necessary for investigations to be comparable. HIGHTLIGHTS RNA of SARS-CoV-2 was used to evaluate the recovery of different methods in wastewater. The utility of phage φ6 of Pseudomonas as a control for evaluation of recovery methods of SARS-CoV-2 from wastewater was demonstrated. Differences due to the target selected for the detection of SARS was demonstrated. The results show that it is possible to detect SARS RNA in wastewater, however its epidemiological usefulness will depend on the correct validation of the technique used for detection.

Improved methods for the detection and quantification of SARS-CoV-2 RNA in wastewater

“…However, the combination of different control viruses and methods used by other groups, makes difficult to compare recovery efficiencies. The data got in this study showed low recovery and a great variability for both methods, 8.37% ± 5.88 n = 43 (method 4); 6.97% ± 6.51 n = 20 (method 5), in comparison with other data found in the bibliography relating to other control viruses, such as 1.6–2.6% for Murine Norovirus, 26.7–64.7% for MHV, 73 ± 50% for F-specific RNA, 42% and 30% for Pseudomonas phage φ6 20 , 27 , 36 . Even when results using the same control virus and method were compared, such as virus recovery showed in this work using method 5 (6.97% ± 6.51), data from Randazzo et al 26 (10% ± 2.1%) and data from Pérez-Cataluña et al 38 (9.0 ± 2.2%), there are other factors which could affect the results, such as the type of sample or the kits used for the RNA extraction and the RT-qPCR quantification.…”

“…The presence of organic matter limits the volume to be load, because could cause the filters to collapse. Indeed, Pino et al 36 showed differences in virus recovery using wastewater and deionized water, suggesting a negative effect of the presence of pollutants in wastewater analysis. Other studies have demonstrated that the removal of the solid can reduce the virus recovery, since the virus is retained in the pellet 33 , 37 .…”

A rapid, high-throughput, and sensitive PEG-precipitation method for SARS-CoV-2 wastewater surveillance

“…Similarly, PEG-precipitation presented higher recovery efficiency than AlCl 3 flocculation ( Perez-Cataluna et al, 2021 ), and skimmed milk flocculation ( Pino et al, 2021 ) in other studies comparing virus concentration methods. However, this result disagrees with our previous study, which found that AlCl 3 flocculation outperformed PEG-precipitation using SARS-CoV-2 spiked wastewater for method evaluation ( Zheng et al, 2022a ).…”

The Inhibition and Variability of Two Different RT-qPCR Assays Used for Quantifying SARS-CoV-2 RNA in Wastewater

“…Despite the simple concept of WBE, producing high-quality, accurate and reproducible data is challenging and quality issues can at any stage of the process. Sample collection- and processing-related quality issues, however, are not the focus of this study and are reviewed in detail by Polo et al, ( 2020 ), Pino et al, ( 2021 ) and Ahmed et al, ( 2022 ). The challenges of producing high-quality WBE data, however, continue beyond quantification of SARS-CoV-2.…”