We are excited as a consortium as HAZMAT has published our REPARES joint review paper. This review, titled ‘Monitoring antibiotic resistance genes in wastewater environments: the challenges of filling a gap in the One-Health cycle,’ delves deeply into threats, strides, issues, and future opportunities in the monitoring of antibiotic resistance genes in wastewater environments. 

a b s t r a c ta r t i c l e i n f o
Article history:
Received 10 July 2021
Received in revised form 21 August 2021
Accepted 5 September 2021
Available online 10 September 2021
Editor: Warish Ahmed
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing.

Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for
such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and
mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage.

Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or
500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared.

The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). 

⁎ Corresponding author.
E-mail address: (D.G. Weissbrodt).
1 Shared senior authorship.
0048-9697/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (
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