Assessment of virus concentration methods for detecting SARS-CoV-2 IN wastewater

Abstract

Wastewater-based epidemiology has been described as a valuable tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a community. However, there is no consensus on the best concentration method to allow reliable detection of SARS-CoV-2 in this matrix, considering different laboratory facilities. This study compares two viral concentration methods, ultracentrifugation (ULT) and skimmed-milk flocculation (SMF), for detecting SARS-CoV-2 in wastewater samples. The analytical sensitivity (limits of detection and quantification [LoD/LoQ]) of both methods was evaluated using a bovine respiratory syncytial virus (BRSV) as a surrogate. Three different approaches were conducted to establish LoD of each method based on the assays on the standard curve (ALoDsc), on the dilution of internal control (ALoDiC), and the processing steps (PLoD). For PLoD, ULT method had the lowest value (1.86 × 10³ genome copy/microliter [GC/µL]) when compared to the SMF method (1.26 × 10⁷ GC/µL). The LoQ determination showed a mean value of 1.55 × 10⁵ GC/µL and 3.56 × 10⁸ GC/µL to ULT and SMF, respectively. The detection of SARSCoV-2 in naturally contaminated wastewater revealed 100% (12/12) and 25% (3/12) of detection using ULT and SMF with quantification ranging from 5.2 to 7.2 log10 genome copy/liter (GC/L) and 5.06 to 5.46 log10 GC/L, respectively. The detection success rate of BRSV used as an internal control process was 100% (12/12) for ULT and 67% (8/12) for SMF, with an efficiency recovery rate ranging from 12 to 38% and 0.1 to 5%, respectively. Our data consolidates the importance of assessing the methods used; however, further analysis should be carried out to improve low-cost concentration methodologies, essential for use in low-income and developing countries.

Keywords: BRSV; Limit of detection; Recovery efficiency; SARS-CoV-2; Skimmed-milk flocculation; Ultracentrifugation.

Fig. 1

Box plot of bovine respiratory syncytial virus detection and recovery results for evaluation between viral concentration methods. The raw data are shown in A. The other figures represent the distribution of results after processing the data with the ALoDsc (B), ALoDic (C), and PLoD (D)

Fig. 2

Box plot of SARS-CoV-2 detection and quantification results for comparative analysis, demonstrating a statistically significant difference between the concentration methods (p = 0.0088)