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

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.

Evaluation of low-cost viral concentration methods in wastewaters: Implications for SARS-CoV-2 pandemic surveillances.

In the pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) many strategies have been performed in order to control viral spread in the population and known the real-time situation about the number of infected persons. In this sense, Wastewater Based Epidemiology (WBE) has been applied as an excellent tool to evaluate the virus circulation in a population. In order to obtain reliable results, three low-cost viral concentration methods were evaluated in this study, polyethylene glycol (PEG) precipitation, skimmed milk flocculation (SM) and Aluminum polychloride flocculation, for Pseudomonas aeruginosa bacteriophage PP7 as a surrogate for non-enveloped viruses and Bovine Coronavirus (BCoV) as a surrogate for enveloped virus, with emphasis for SARS- CoV-2. Our results suggest that PEG precipitation for viral concentration, for both enveloped and non-enveloped virus from wastewater is an appropriate approach since it was more sensitive compared to SM flocculation and Aluminum polychloride flocculation. This methodology can be used for WBE studies in order to follow the epidemiology of the SARS-CoV-2 pandemic, mainly in developing countries where the economic resources are frequently limited.