key: cord-0986072-a70mqafh
authors: Bertrand, Isabelle; Challant, Julie; Jeulin, Hélène; Hartard, Cédric; Mathieu, Laurence; Lopez, Séverine; Group Obépine, Scientific Interest; Schvoerer, Evelyne; Courtois, Sophie; Gantzer, Christophe
title: Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: comparison of ultrafiltration- and protein precipitation-based methods
date: 2021-01-31
journal: Int J Hyg Environ Health
DOI: 10.1016/j.ijheh.2021.113692
sha: fea350aa9ae9bb29fd15754c5d9da0760505fb92
doc_id: 986072
cord_uid: a70mqafh

The aim of the present study was to develop a simple, sensitive, and specific approach to quantifying the SARS-CoV-2 genome in wastewater and to evaluate this approach as a means of epidemiological surveillance. Twelve wastewater samples were collected from a metropolitan area in north-eastern France during April and May 2020. In addition to the quantification of the SARS-CoV-2 genome, F-specific RNA phages of genogroup II (FRNAPH GGII), naturally present in wastewater, were used as an internal process control for the viral concentration and processing of RT-PCR inhibitors. A concentration method was required to allow the quantification of the SARS-CoV-2 genome over the longest possible period. A procedure combining ultrafiltration, phenol-chloroform-isoamyl alcohol purification, and the additional purification of the RNA extracts was chosen for the quantification of the SARS-CoV-2 genome in 100-mL wastewater samples. At the same time, the COVID-19 outbreak was evaluated through patients from the neighbouring University Hospital of Nancy, France. A regular decrease in the concentration of the SARS-CoV-2 genome from ∼10(4) gc/L to ∼10(2) gc/L of wastewater was observed over the eight weeks of the study, during which the population was placed under lockdown. The SARS-CoV-2 genome was even undetectable during one week in the second half of May and present but non-quantifiable in the last sample (28 May). A concordant circulation in the human community was highlighted by virological diagnosis using respiratory samples, which showed a decrease in the number of COVID-19 cases from 677 to 52 per week over the same period. The environmental surveillance of COVID-19 using a reliable viral quantification procedure to test wastewater is a key approach. The real-time detection of viral genomes can allow us to predict and monitor the circulation of SARS-CoV-2 in clinical settings and survey the entire urban human population.

The VTB4-Fph GGII set was used on 2 µL of RNA in a 20-µL reaction volume with final 185 concentrations of 1 µM for each primer and 0.3 µM for the probe. A StepOnePlus Real-Time PCR 186 System (Applied Biosystems™) was used for these three real-time RT-PCR assays. For the RdRp_IP4 187 and E sets, the RT step was performed at 50 °C for 20 min and PCR amplification was performed at 188 95°C for 2 min, followed by 50 cycles of 15 s at 95°C and 30 s at 58 °C. For the VTB4-Fph GGII set 189 the RT step was performed at 50 °C for 30 min and PCR amplification was performed at 95°C for 2 190 min, followed by 45 cycles of 15 s at 95°C and 40 s at 58 °C. Negative and positive controls were 191 included in each experiment. Quantification was carried out using standard curve ranges. RNA 192 extracted from patients who had tested positive for SARS-CoV-2 was quantified using ddRT-PCR 193 with the E set, as described below. Quantified RNA was then used to obtain the standard curve for 194 both RdRp_IP4 and E genes. The nCoV-ALL-Control plasmid (Eurofins genomic) was also used for 195 the standard curve of the E gene. This plasmid containing ampicillin resistance gene was 196 maintained in TOP10 chemically competent E. coli (Invitrogen) and quantified using Qubit 4 197 fluorometer (Invitrogen). At last, RNA extracted from GA phage suspension was used to obtain the 198 standard curve for FRNAPH GGII. RNA extracted from GA phage suspension was quantified by 199 ddRT-PCR using VTB4-Fph the GGII set as described below. The standard curves ranged from 200 1 × 10 -1 to 1 × 10 4 gc/reaction for the RdRp_IP4 and E genes of SARS-CoV-2 and from 2.8 × 10 -1 to 201 2.8 × 10 4 gc/reaction for the GA phage. The limit of detection (LoD) was 1 gc/RT-qPCR reaction for 202 the RdRp and E genes. The limit of quantification (LoQ) ranged from 1 to 10 gc/RT-qPCR reaction 203 for the RdRp gene and reached 1 gc/RT-qPCR reaction for the E gene. When we take the analytical 208 GA phage. Amplifications were carried out in a 20-µL reaction mixture containing 5 µL of RNA and 209 15 µL of One-Step RT-ddPCR™ Kit for Probes (Bio-Rad). The reaction mix contained 0.9 μM of each 210 primer and 0.3 μM of the probe. The samples were placed in the droplet generator using 70 µL of 211 generator oil to generate up to 20,000 droplets per sample. The resulting picolitre droplet 212 emulsions (40 µL) were transferred to a Veriti 96-Well Thermal Cycler (Applied Biosystems). After 213 amplification, the plate was transferred to the QX100TM Droplet Reader (Bio-Rad) and 214 QuantaSoft™ Software (Bio-Rad) was used to measure the number of positive droplets per well. 215 Droplets were designated positive or negative based on their fluorescence amplitude, using 216 thresholding. The starting concentration of each target RNA molecule was then calculated, by 217 modelling a Poisson distribution. 218 For each sample and each targeted virus (SARS-CoV-2 and FRNAPH GGII) the recovery rate was 219 calculated as follows: recovery rate = genome copies (gc) in 5 mL unconcentrated sample 220 × 20 × 100 / gc in 100 mL of the concentrate. The high concentration of the FRNAPGH GGII 221 genome allowed for the determination of the recovery rate in all wastewater samples. It was also 222 possible to determine the recovery rate for SARS-CoV-2 in 5 of the 12 samples. 223 2.6. Estimation of RT-qPCR inhibition 224 The estimation of RT-qPCR inhibition in the wastewater samples was based on the concentrations 225 232 of the data and the homogeneity of variances could not be met, two non-parametric tests were 233 used. A Kruskal-Wallis test was performed on k-independent samples (k>3) to compare the viral 234 genome copy values obtained using the different concentration and purification methods. A 235 Wilcoxon signed-rank test was performed on paired samples to compare the recovery rates 236 following the two methods and the genome concentrations of the two targeted genes. P values < 237 0.05 were considered statistically significant. 241 The epidemiological surveillance of SARS-CoV-2 by quantifying its genome in wastewater requires 242 reliable methods of concentration and detection. Beginning our study in a region that was highly 243 impacted by COVID-19 allowed us to estimate recovery rates for SARS-CoV-2. 244 FRNAPH are usually present in wastewater at concentrations that are relatively stable over time 245 and around the world (Lucena et al., 2003) . We obtained just under 2.5 × 10 7 gc/L which was high 246 enough to allow us to evaluate both recovery rates and PCR inhibition problems. Using these 247 bacteriophages, the presence of PCR inhibitors was detected at even a low volume (5 mL) of 248 unconcentrated wastewater. Indeed, the PCR inhibition varied between 88% and 100% in 249 undiluted RNA extracts (n=5) and between 14% and 42% in 1/10 diluted RNA extracts (n=5). 250 Following phenol-chloroform purification, the PCR inhibitors had been completely removed from 5 251 mL of wastewater. In these five wastewater samples, the level of contamination by SARS-CoV-2 252 also enabled the quantification of the virus in 5 mL unconcentrated samples and showed that, 253 following phenol-chloroform purification, the concentrations of SARS-CoV-2 genome detected in 257 2011; Hartard et al., 2015) . The phenol-chloroform purification method was systematically applied 258 in the following experiments. A complementary method for the removal of PCR inhibitors in RNA 259 extracts was tested during the comparison of the concentration methods (as described below). 260 Two concentration procedures, based on protein precipitation using PEG 6 000 and on 261 ultrafiltration using the Centricon® 70-Plus 100 kD device, respectively, were compared. Recovery 262 rates for both FRNAPH GGII and SARS-CoV-2 could be determined (Table 1) precipitation. It also led to a decrease in the standard deviation in both concentration methods. 272 The concentration values obtained for FRNAPH GGII using the two concentration methods, with or 273 without the use of the PCR inhibitor removal kit, were not significantly different (p value = 0.098, 274 Kruskall-Wallis test). For SARS-CoV-2, the mean recovery rates of the RdRp_IP4 gene reached 275 64.1 ± 50.2% and 32.4% ± 20.2% using ultrafiltration and protein precipitation, respectively. 276 Additional purification of the RNA extracts led to mean recovery rates of 55.8 ± 46.9% and 277 23.5 ± 15.0% for ultrafiltration and protein precipitation, respectively. Thus, the mean recovery 278 rate obtained for SARS-CoV-2 (RdRp_IP4 gene) using ultrafiltration were twice as high as those 279 obtained using protein precipitation. Moreover, the highest recovery rate was obtained using samples, collected between 2 April and 28 May 2020, were analysed in one replicate using both 333 the ultrafiltration procedure on 100 mL and the direct analysis of 5 mL to quantify the SARS-CoV-2 334 and FRNAPH GGII genomes. 335 The FRNAPH GGII genome could be quantified in both 5 mL and 100 mL of wastewater in all the 336 samples ( Figure 1 ). The mean concentrations reached 2.1 × 10 7 ± 1.1 × 10 7 gc/L and 337 1.6 × 10 7 ± 1.4 × 10 7 gc/L in unconcentrated and concentrated samples, respectively. From these 338 concentration values, the recovery rates of FRNAPH GGII ranged from 14.1% to 133.8%. 339 We propose a recovery rate of over 10% as a quality control, to validate the results. This goes The aim of this study was to define the dynamics of viral concentration in wastewater during the 98 first lockdown of the French population. This objective required a simple, specific, and sensitive 99 approach to quantifying the SARS-CoV-2 genome in wastewater. In the present study, we took 100 advantage of the wide circulation of SARS-CoV-2 in north-eastern France to compare two methods wastewater were both filtered by centrifugation at 1,500 × g for 15 min. After each centrifugation 135 step, the concentrate was recovered by inverting the system and applying centrifugation (1,000 × 136 g for 2 min). The resulting concentrate's volume was around 1.5 mL. The ultrafilter was then 137 washed with 3.5 mL of deionised water. The washing solution was added to the 1.5 mL 138 concentrate to produce the final concentrate sample (5 mL). In order to recover the maximum 139 amount of virus genome from the ultrafilter, two further washing steps were undertaken, each 140 using 5 mL NucliSENS® lysis buffer (bioMérieux) for an incubation time of 5 min. The entire volume 141 (15 mL) was then used for nucleic acid extraction. Twelve water samples were subjected to this 142 procedure. 143 PEG 6000 precipitation was performed in a 250-mL centrifuge bottle containing 3 g beef extract 144 powder, 3 g NaCl, and 0.37 g glycine for 100 mL of wastewater. After the dissolution of the beef 145 extract powder, 20 g of PEG 6000 were added. The sample was gently stirred at 4°C for 2 h and 146 then maintained at 4°C overnight. The pellet obtained after centrifugation at 4,500 × g and 4°C for 147 45 min was resuspended in deionised water to obtain a concentrated 5 mL sample. Ten millilitres 148 of NucliSENS® lysis buffer were then added to the concentrate. After incubation for 10 min at 149 room temperature, the entire volume (15 mL) was used for nucleic acid extraction. This 150 concentration method was tested on the first four water samples of our study. 151 In tandem with each concentration procedure, 5 mL samples of unconcentrated water were used 152 for viral genome extraction. They were submitted to the same procedure as the concentrated 153 samples. Ten millilitres of NucliSENS® lysis buffer was added to the water, which was incubated at 154 room temperature for 10 min, prior to the nucleic acid extraction. 241 The epidemiological surveillance of SARS-CoV-2 by quantifying its genome in wastewater requires 242 reliable methods of concentration and detection. Beginning our study in a region that was highly 243 impacted by COVID-19 allowed us to estimate recovery rates for SARS-CoV-2. 244 FRNAPH are usually present in wastewater at concentrations that are relatively stable over time 245 and around the world (Lucena et al., 2003) . We obtained just under 2.5 × 10 7 gc/L which was high 246 enough to allow us to evaluate both recovery rates and PCR inhibition problems. Using these 247 bacteriophages, the presence of PCR inhibitors was detected at even a low volume (5 mL) of 248 unconcentrated wastewater. Indeed, the PCR inhibition varied between 88% and 100% in 249 undiluted RNA extracts (n=5) and between 14% and 42% in 1/10 diluted RNA extracts (n=5). 250 Following phenol-chloroform purification, the PCR inhibitors had been completely removed from 5 251 mL of wastewater. In these five wastewater samples, the level of contamination by SARS-CoV-2 252 also enabled the quantification of the virus in 5 mL unconcentrated samples and showed that, 253 following phenol-chloroform purification, the concentrations of SARS-CoV-2 genome detected in 257 2011; Hartard et al., 2015) . The phenol-chloroform purification method was systematically applied 258 in the following experiments. A complementary method for the removal of PCR inhibitors in RNA 259 extracts was tested during the comparison of the concentration methods (as described below). 260 Two concentration procedures, based on protein precipitation using PEG 6 000 and on 261 ultrafiltration using the Centricon® 70-Plus 100 kD device, respectively, were compared. Recovery 262 rates for both FRNAPH GGII and SARS-CoV-2 could be determined (Table 1) precipitation. It also led to a decrease in the standard deviation in both concentration methods. 272 The concentration values obtained for FRNAPH GGII using the two concentration methods, with or 273 without the use of the PCR inhibitor removal kit, were not significantly different (p value = 0.098, 274 Kruskall-Wallis test). For SARS-CoV-2, the mean recovery rates of the RdRp_IP4 gene reached 275 64.1 ± 50.2% and 32.4% ± 20.2% using ultrafiltration and protein precipitation, respectively. 276 Additional purification of the RNA extracts led to mean recovery rates of 55.8 ± 46.9% and 277 23.5 ± 15.0% for ultrafiltration and protein precipitation, respectively. Thus, the mean recovery 278 rate obtained for SARS-CoV-2 (RdRp_IP4 gene) using ultrafiltration were twice as high as those 279 obtained using protein precipitation. Moreover, the highest recovery rate was obtained using (Table 1 ). The statistical analysis of the data obtained for both the RdRp_IP4 and E 286 genes showed significantly higher recovery rates for ultrafiltration than for protein precipitation (p 287 value = 0.009, Wilcoxon signed-rank test). 288 Recovery rates and the inhibition of molecular detection methods applied for SARS-CoV-2 in 289 wastewater have been poorly described. Nevertheless, our SARS-CoV-2 recovery rate was higher The FRNAPH GGII genome could be quantified in both 5 mL and 100 mL of wastewater in all the 336 samples ( Figure 1 ). The mean concentrations reached 2.1 × 10 7 ± 1.1 × 10 7 gc/L and 337 1.6 × 10 7 ± 1.4 × 10 7 gc/L in unconcentrated and concentrated samples, respectively. From these 338 concentration values, the recovery rates of FRNAPH GGII ranged from 14.1% to 133.8%. 339 We propose a recovery rate of over 10% as a quality control, to validate the results. This goes 383 We developed a method of concentrating SARS-CoV-2 from wastewater with one of the highest La Rosa, G., Iaconelli, M., Mancini, P., Bonanno Ferraro, G., Veneri, C., Bonadonna, L., Lucentini, L., 

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Mining population exposure and 434 community health via wastewater-based epidemiology

Detection of 2019 novel coronavirus

nCoV) by real-time RT-PCR

Performance 444

Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories

E1871

Novel coronavirus infection and gastrointestinal tract

Occurrence of and sequence variation among F-449 specific RNA bacteriophage subgroups in feces and wastewater of urban and animal origins

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Protocol: Real-Time RT-PCR Assays for the Detection of SARS-CoV-2

First 468 proof of the capability of wastewater surveillance for COVID-19 in India through

Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of Montpellier, France. One 530 Health 10

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Survivability, partitioning, and 556 recovery of enveloped viruses in untreated municipal wastewater

Fecal specimen diagnosis 2019 novel coronavirus-infected 559 pneumonia