key: cord-0719493-ws7lp560
authors: Langeveld, J.; Schilperoort, R.; Heijnen, L.; elsinga, g.; schapendonk, c.; fanoy, e.; de schepper, e.; Koopmans, M.; de Graaf, M.; Medema, G.
title: Normalisation of SARS-CoV-2 concentrations in wastewater: the use of flow, conductivity and CrAssphages
date: 2021-11-30
journal: nan
DOI: 10.1101/2021.11.30.21266889
sha: 071e32780599e85e428d6714fcd995c50c5d0843
doc_id: 719493
cord_uid: ws7lp560

Over the course of the COVID-19 pandemic in 2020-2021, monitoring of SARS-CoV-2 RNA in wastewater has rapidly evolved into a supplementary surveillance instrument for public health. Short term trends (2 weeks) are used as a basis for policy and decision making on measures for dealing with the pandemic. Normalization is required to account for the varying dilution rates of the domestic wastewater, that contains the shedded virus RNA. The dilution rate varies due to runoff, industrial discharges and extraneous waters. Three normalization methods using flow, conductivity and CrAssphage, have been investigated on 9 monitoring locations between Sep 2020 and Aug 2021, rendering 1071 24-hour flow-proportional samples. In addition, 221 stool samples have been analyzed to determine the daily CrAssphage load per person. Results show that flow normalization supported by a quality check using conductivity monitoring is the advocated normalization method in case flow monitoring is or can be made available. Although Crassphage shedding rates per person vary greatly, the CrAssphage loads were very consistent over time and space and direct CrAssphage based normalization can be applied reliably for populations of 5600 and above.

e . They may be attributed to short term changes in population size due to commuters or 99 tourism, or to sewer system dynamics, such as spills of SSOs (sanitary sewer overflows) due 100 available. Launay et al. (2016) propose the use of electric conductivity (EC) as a proxy of the 111 dilution of wastewater due to storm water or ground water dilution. They showed that the 112 dilution calculated based on EC gives similar results as the use of inert human wastewater 113 tracers such as ibuprofen, naproxen and diclofenac. Many other human wastewater tracers 114 are less applicable as tracer due to e.g. a higher back ground variation or degradation in the 115 sewer (Gao et al., 2017) . EC was selected as a cheap parameter which can be reliably The aim of this study was to develop a procedure for quality control and normalisation of 125 wastewater samples, in order to allow quantitative trend analysis of SARS-CoV-2 in support 126 of public health decision-making. We compared and evaluated three methods for 127 normalisation: the 'standard' daily wastewater flow rate and population normalisation, electric 128 conductivity (EC) as a proxy for dilution and the human fecal biomarker crAssphage. In 129 addition, we analysed whether a combination of two normalisation methods can be used as a 130 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint and ASP2000 stations) were used to collect flow-proportional 24h composite wastewater 155 samples. The autosamplers were programmed to collect 50 mL aliquots per fixed volume of 156 wastewater (as measured by the flow sensors at the WWTPs/pumping stations). This fixed 157 volume varied between 12 m 3 (Katendrecht) and 170 m 3 (Dokhaven INF3) and was selected 158 to ensure a minimum of 100 aliquots (5 L of sample) for each 24h sample throughout the 159 year (NEN6600). All locations followed the same sampling schedule: three 24h samples per 160

week (Sun 08h00 -Mon 08h00, Tue 08h00 -Wed 08h00 and Thu 08h00 -Fri 08h00) from 161

September 2020 (Bergschenhoek: January 2021) to August 2021. Samples were stored 162 inside the autosampler at a temperature between 1°C and 5°C until sample collection (Mon, 163

Wed and Fri between 08h00 and 16h00). preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. .

The N2 and E_Sarbecco gene fragments of SARS-CoV-2 were used as qRT-PCR targets, 193 N2 was used for quantification, E_Sarbecco for confirmation (both needed to be present for a 194 positive result). Reagents and reaction conditions were as previously described (Medema et 195 al. 2020; Heijnen et al. 2021 ). All RT-PCR's were run as technical duplicates on 5 µl 196 extracted nucleic acid. Reactions were considered positive if the cycle threshold was below 197 40 cycles. Spiked MHV-A59 RNA was detected by performing a MHV-A59 specific targeting the N-gene using the primers and reaction profile described by (Raabenet al. 2007 In the study, three normalisation procedures have been developed to be able to assess flow, 215 EC and crAssphage as normalisation parameters. In each procedure, the percentage of 216 

where ddwf = average daily domestic wastewater production per person, V 24h = measured 227 wastewater volume over 24 h sampling time 228

The EC based normalisation procedure requires more additional data to enable direct 229 comparison with the flow base normalisation: 230 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint person per day and the average sewage dry weather flow, which is used as a 232 reference to account for the dilution with extraneous water during dry weather. This 233 average dry weather flow can be extracted as the 40 th percentile from a time series of 234 flow data at the sampling site (Mulder et al., 2020) . For most monitoring locations, the 235 difference between the 30 th and 50 th percentile value is relatively small. For 236 catchments with a strong seasonal variation of the amount of extraneous waters, it 237 may be necessary to derive a reference for the wet and dry season. This assessment 238 is easy to make using the methods as described in Weiß et al, 2002. 239 2. Determine the reference EC value during dry weather flow by taking the average of all 240 days with a daily flow volumes ranging between the 10 th and 50 th percentile derived 241 from a 1 year time series. Days with flows smaller than the 10 th percentile could be 242 affected by operational irregularities, such pump failures or drinking water outage, 243 while flows above the 50 th percentile could be affected by (small) storm events. 244 3. Determine the proportion of domestic sewage in a wastewater sample using EC data: 245 The crAssphage based normalisation follows the same approach as the EC based 255 normalisation and only differs in the parameter used: CrAssphage instead of EC: 256 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint calculating the proportion of domestic sewage during wet weather using eq 3). 258 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. .

All rights reserved. No reuse allowed without permission.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint rate of the domestic part of the wastewater. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint provides a good basis for quality control of SARS-CoV-2 samples. Of our 1207 SARS-CoV-2 347 samples, 32 (2.8%) yielded a low CrAssphage concentration (<10 4 GC/ml). 20 of those were 348 from Ommoord, the site with the most variable proportion of extraneous waters, and these 349 samples also had a high EC (>2000 µS/cm). In these low CrAssphage samples, SARS-CoV-350 2 N2 concentration was also low or absent. The recovery efficiency of the detection methods 351 for both viruses was low in these samples, possibly due to the presence of humic acids in 352 brackish groundwater coming via clay and peat layers entering the sewer network at this site. 353

Recovery efficiency did not decrease due to high EC due to road de-icing. The data from 354 these samples were therefore excluded from further analysis. Similarly, CrAssphage 355 provided a basis for quality control of detection methods, In August 2020, supply shortages 356 forced us to use other ultrafilters than Centricon. Even though an initial comparison between 357

Centricon and the alternative filters indicated comparable SARS-CoV-2 recoveries, the 358 CrAssphage concentrations obtained in successive wastewater samples processed with 359 these alternative filters were more than 10-fold lower than in the previous sample series. 360 Therefore, data obtained with these alternative filters were not used (data not shown). preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint developed a procedure to perform the flow and EC based normalisations in parallel. If these 375 two normalisations result in domestic wastewater proportions that differ less than 10%, the 376 flow based normalisation is considered successful and reliable. If the two methods differ 377 more than 10%, it is necessary to analyse the data in more depth and identify the cause of 378 the difference. Figure 9 gives an example for the catchment Katendrecht. Overall, there is a 379 good agreement between the flow and EC based normalisation with a few exceptions. Two 380 of the exceptions occurred close after each other in January 2021. On these days, the 381 pumping station was non-operational between January 19 th 14h00 and January 21 st 11h00. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. CrAssphage concentrations in these stool samples showed very large variation, spanning 411 more than 10 log 10 units (Figure 11 ). A small percentage (4.5%) had no detectable (<140 412 GC/ml faeces) CrAssphage in their stool sample. In the positive stool samples, a bimodal 413 distribution was observed, with peaks at 10 3.5 and at 10 8.5 GC/ml faeces. 414 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This study showed that without normalisation SARS-CoV-2 sewage data may misrepresent 448 the actual short-term trends of COVID-19 circulation in the population due to the impact of 449 rain and snowmelt in a combined sewer network. Unnormalised data do show similar long-450 term trends as normalized data, but it is the short-term (1-2 weeks) trends are the most 451 relevant to support public health actions to limit the transmission of the virus and where 452 discrepancies were considerable. Also, for comparison of SARS-CoV-2 circulation in city 453 areas (or cities), normalisation is needed to compensate for the different proportions of 454 extraneous water in different (parts of the) sewer networks. 455 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint (2/1207; 0.17%) flow was not appropriate, due to sewer blocking. Since EC captured these 457 errors, we propose flow-based normalisation in combination with an EC check. In our study, 458 EC had the added value of picking up brackish groundwater intrusion and road de-icing, the 459 latter being associated with reduced recoveries of the CrAssphage and SARS-CoV-2 460 detection methods. 461

The CrAssphage loads per population per day was very comparable between city areas and 462 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Eurosurveillance-editorial-team (2021). "Updated rapid risk assessment from ECDC on the 561 risk related to the spread of new SARS-CoV-2 variants of concern in the EU/EEA -first 562 update." Eurosurveillance 26(3): 2101211. 563 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. shows a high correlation between normalized and unnormalised data, but this is lost in the wet weather data. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint Figure 8 . Correlation between daily SARS-CoV-2 load per 100.000 inhabitants and flow based, EC based and CrAss based normalisation for INF3 catchment Figure 9 . Normalisation of SARS-CoV-2 at the Katendrecht catchment All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint Figure 11 . Distribution of CrAssphage GC/ml in human stool samples (n=221) from Rotterdam. Frequency of data below the detection limit (140 GC/ml) is shown in light blue.

Normal distribution fitted through the log-transformed concentrations in stool samples of the low shedder (<10 6.5 GC/ml; mean: 3.474; standard deviation 0.843; in grey) and the high shedder (≥10 6.5 GC/ml; mean: 8.413; standard deviation 0.950; in orange). All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.30.21266889 doi: medRxiv preprint

Genomics and epidemiology of a novel SARS

CoV-2 lineage in Manaus, Brazil

Evaluation of Sampling, Analysis, and Normalisation Methods for

SARS-CoV 2 Concentrations in Wastewater to Assess COVID-19 Burdens in Wisconsin

Evaluation of in-581 sewer transformation of selected illicit drugs and pharmaceutical biomarkers

Circulating SARS-CoV-2 variants escape 592 neutralization by vaccine-induced humoral immunity

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load 596 in wastewater using an allele-specific primer extension PCR strategy

Droplet Digital RT-PCR to detect SARS-CoV-2 variants of concern in wastewater

Monitoring SARS-CoV-2 in municipal wastewater to evaluate the success of lockdown 604 measures for controlling COVID-19 in the UK

SARS-CoV-2 606 wastewater surveillance in Germany: long-term PCR monitoring, suitability of primer/probe 607 combinations and biomarker stability

Biogeographic study of human gut-associated crAssphage suggests impacts from 610 industrialization and recent expansion

Monitoring SARS-CoV-2 circulation and diversity through 619 community wastewater sequencing

Detection and surveillance of SARS

CoV-2 genomic variants in wastewater

Shedding of SARS-CoV-2 in feces and urine and its potential role in person-to-person 628 transmission and the environment-based spread of COVID-19

Challenges in

Measuring the Recovery of SARS-CoV-2 from Wastewater

The Architecture 633 of SARS-CoV-2 Transcriptome

Using Wastewater Surveillance Data to Support the

COVID-19 Response -United States

CoV-2 prevalence by wastewater-based epidemiology

The basis of a more contagious 501Y.V1 variant of SARS-COV-2

SARS-CoV-2 in wastewater: potential 649 health risk, but also data source

Implementation of 651 environmental surveillance for SARS-CoV-2 virus to support public health decisions: 652 Opportunities and challenges

Presence of

SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence 655 in the Early Stage of the Epidemic in The Netherlands

Verwijdering van organische 658 microverontreinigingen

NEN 6600 2019

Sampling for PPCPs in 662 wastewater systems: comparison of different sampling modes and optimization strategies

Wastewater-based epidemiology as a useful tool to track SARS-CoV-2 671 and support public health policies at municipal level in Brazil

Cyclooxygenase 675 activity is important for efficient replication of mouse hepatitis virus at an early stage of 676 infection

Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore 680 sequencing. Lancet Reg Health Eur

The Characterization of Feces and 684

Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical reviews 685 in environmental science and technology

RNA viromics of

Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants

characteristic time and space scales of in-sewer processes by analysis of one year of 697 continuous in-sewer monitoring data

Passive Sampling of SARS-CoV-2 for Wastewater Surveillance

The Human Gut Virome Is Highly Diverse, Stable, and Individual 706

Acquisition, transmission and strain 708 diversity of human gut-colonizing crAss-like phages

Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement

Building knowledge of university campus population dynamics to enhance near-to-715 source sewage surveillance for SARS-CoV-2 detection

721 mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses 723 during wastewater treatment

Emergence 732 of a SARS-CoV-2 variant of concern with mutations in spike glycoprotein

2019-Novel Coronavirus (2019-nCoV) Real-Time rRT-PCR Panel Primers 734 and Probes

Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking 742 epidemiological and genetic data

Rioolvreemd water rioolwaterzuiveringsinrichting Dokhaven

Clinical Characteristics of 138 Hospitalized Graham

1.351 and B.1.1.7 to Antibody Neutralization

Infiltration and inflow in combined sewer 754 systems: long-term analysis

Co-quantification of crAssphage increases 757 confidence in wastewater-based epidemiology for SARS-CoV-2

Quantification of SARS-CoV-2 RNA in Wastewater Settled Solids at Eight Publicly Owned 762

Treatment Works in Northern California Shows Strong Association with COVID-19 Incidence

Virological assessment of 768 hospitalized patients with COVID-2019

Wastewater Surveillance of 1 SARS-CoV-2 across 40 U.S. states

Evidence for Gastrointestinal 775 Infection of SARS-CoV-2

City-level

SARS-CoV-2 sewage surveillance

All rights reserved. No reuse allowed without permission

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted