A rapid and simple protocol for concentration of SARS-CoV-2 from sewage

Abstract

The aim of this study was to set up a simple protocol to concentrate SARS-CoV-2 from sewage, which can be implemented in laboratories with minimal equipment resources. The method avoids the need for extensive purification steps and reduces the concentration of potential inhibitors of RT-qPCR contained in sewage. The concentration method consists of a single step, in which a small volume (40 mL) of sewage sample is incubated with polyaluminum chloride (PAC)(0.00045 N Al 3+ final concentration). Virus particles adsorbed to the precipitate are collected by low-speed centrifugation, after which the recovered pellet is resuspended with a saline buffer. PAC concentrated samples are stable for at least one week at 4 °C. Therefore, they may be sent refrigerated to a diagnosis center for RNA extraction and RT-qPCR for SARS-CoV-2 RNA detection if the lab does not have such capabilities. The PAC concentration method produced an average shift of 4.5-units in quantification cycle (Cq) values compared to non-concentrated samples, indicating a 25-fold increase in detection sensitivity. The lower detection limit corresponded approximately to 100 viral copies per ml. Kappa index indicated substantial agreement between PAC and polyethylene glycol (PEG) precipitation protocols (k = 0.688, CI 0.457-0.919). This low-cost concentration protocol could be useful to aid in the monitoring of community circulation of SARS-CoV-2, especially in low- and middle-income countries, which do not have massive access to support from specialized labs for sewage surveillance.

SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan

“…Previous studies showed that composite samples, which were collected by flow-weighting for 24 h, were more detectable than grab samples for SARS-CoV-2 RNA in wastewater ( Gerrity et al, 2021 ; Monteiro et al, 2022 ). However, grab sampling had been performed for SARS-CoV-2 RNA detection ( Randazzo et al, 2020 ; Carrillo-Reyes et al, 2021 ; Kitamura et al, 2021 ; Street et al, 2021 ; Wehrendt et al, 2021 ), and correlated with COVID-19 cases ( Kitamura et al, 2021 ; Street et al, 2021 ). While grab samples have the limitation of low sensitivity, they could be a useful sampling method because they have the advantage of being collected quickly, do not require automated equipment, and were able to reflect COVID-19 cases in our study.…”

“…In addition, hepatitis E virus was detectable in raw sewage when 1%–4% of residents in a WWTP basin were infected ( Miura et al, 2016 ). Monitoring the prevalence of SARS-CoV-2 in wastewater is also considered to be an effective approach for predicting the COVID-19 epidemic following the detection of SARS-CoV-2 RNA in sewage in several countries ( Ahmed et al, 2020a ; La Rosa et al, 2020b ; Wang et al, 2020 ; Wurtzer et al, 2020 , 2022 ; Albastaki et al, 2021 ; Wehrendt et al, 2021 ; Kevill et al, 2022 ). In some of these studies, the RNA copy number of SARS-CoV-2 in the sewage was correlated with the number of COVID-19 clinical cases ( Medema et al, 2020a ; Carrillo-Reyes et al, 2021 ; Nagarkar et al, 2021 ; Street et al, 2021 ; Wurtz et al, 2021 ; Monteiro et al, 2022 ).…”

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022

“…Jara in Mar del Plata in portable coolers, according to the WHO biosafety recommendations. In order to compare protocols, some of the General Pueyrredon wastewater water samples were concentrated using the polyaluminum chloride flocculation protocol described by Wehrendt et al ( 23 ).…”

“…The samples from Pinamar taken in 2022 were concentrated according to the Wehrendt et al protocol ( 23 ). For this step, 250 μL of each sample was used to perform the RNA purification using the TRIzol™ reagent (Invitrogen™, Waltham, MA, USA) protocol, followed by RNA isolation using QIAamp R Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.…”

Presentation_1.PPTX

“…In addition, hepatitis E virus was detectable in raw sewage when 1%-4% of residents in a WWTP basin were infected (Miura et al, 2016). Monitoring the prevalence of SARS-CoV-2 in wastewater is also considered to be an effective approach for predicting the COVID-19 epidemic following the detection of SARS-CoV-2 RNA in sewage in several countries (Ahmed et al, 2020a;La Rosa et al, 2020b;Wang et al, 2020;Wurtzer et al, 2020Wurtzer et al, , 2022Albastaki et al, 2021;Wehrendt et al, 2021;Kevill et al, 2022). In some of these studies, the RNA copy number of SARS-CoV-2 in the sewage was correlated with the number of COVID-19 clinical cases (Medema et al, 2020a;Carrillo-Reyes et al, 2021;Nagarkar et al, 2021;Street et al, 2021;Wurtz et al, 2021;Monteiro et al, 2022).…”