key: cord-0268143-69287rvh
authors: Monteiro, S.; Rente, D.; Cunha, M. V.; Marques, T.; Cardoso, E.; Alvaro, P.; Vilaca, J.; Ribeiro, J.; Silva, M.; Coelho, N.; Broco, N.; Carvalho, M.; Santos, R.
title: Concentration of SARS-CoV-2 from large volumes of raw wastewater is enhanced with the inuvai R180 system
date: 2021-07-22
journal: nan
DOI: 10.1101/2021.07.21.21260907
sha: 16fb3f95098a9f6a46ef3607fd12e402f85c9fac
doc_id: 268143
cord_uid: 69287rvh

Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a powerful tool to complement syndromic surveillance: first, as an early-warning system for the spread of the virus in the community, second, to find hotspots of infection, and third, to aid in the early detection and follow-up of circulating virus variants. Although detection of SARS-CoV-2 in raw wastewater may be prompted with good recoveries during periods of high community prevalence, in the early stages of population outbreaks concentration procedures are required to overcome low viral concentrations. Several methods have become available for the recovery of SARS-CoV-2 from raw wastewater, generally involving filtration. However, these methods are limited to small sample volumes, possibly missing the early stages of virus circulation, and restrained applicability across different water matrices. The aim of this study was thus to evaluate the performance of three methods enabling the concentration of SARS-CoV-2 from large volumes of wastewater: i) hollow fiber filtration using the inuvai R180, with an enhanced elution protocol and polyethylene glycol (PEG) precipitation; ii) PEG precipitation; and iii) skimmed milk flocculation. The performance of the three approaches was evaluated in wastewater from multiple wastewater treatment plants (WWTP) with distinct singularities, according to: i) effective volume; ii) percentage of recovery; iii) extraction efficiency; iv) inhibitory effect; and v) the limits of detection and quantification (The inuvai R180 system had the best performance, with detection of spiked controls across all samples, average recovery percentages of 64% for SARS-CoV-2 control and 68% for porcine epidemic diarrhea virus (PEDV), with low variability. The inuvai R180 enables the scalability of volumes without negative impact on the costs, time for analysis, and recovery/inhibition. Moreover, hollow fiber filters favor the concentration of different microbial taxonomic groups. Such combined features make this technology attractive for usage in environmental waters monitoring.

Surveillance of wastewater for epidemiological purposes has been previously used in 55 public health, with the most important and successful example being the polio 56 eradication program (GPEI, 2021) . Given the ongoing Coronavirus disease 2019 57 7 cells were grown in T25 flasks and inoculated with 100 μL of viral stock. At 2h post 129 infection, DMEM supplemented with 0.3% tryptose phosphate broth, 100 units/mL of 130 penicillin (Lonza), 100 units/mL of streptomycin (Lonza), and 10 μg/μL trypsin, was 131 added to the flasks. Flasks were then incubated at 37 (± 1) ºC in 5% CO2 for 4 days. 132 PEDV were recovered following three cycles of freeze/thawing and centrifugation at 133 1,100 xg for 10 min. Quantification was performed by RT-dPCR as described on 134 section 2.5 using the primers and probes from Supplementary Table S1 (Zhou et al., 135 2017) , following nucleic acid extraction as described on section 2.4. After absolute 136 quantification by RT-dPCR (as described below), a stock solution was prepared in 137

DNase/RNase free water to obtain a PEDV final concentration of 1.21 x 10 4 GC/L in 138 wastewater. The same stock was used in all experiments described below. 139 140

Twenty-four-hour composite samples were collected, on two separate rounds, from 142 five wastewater treatment plants (WWTP) in Portugal (Serzedelo, Gaia, Alcântara, 143 Beirolas and Guia). The first round comprised samples collected between April 27 and 144

May 8, 2020 (n = 8; n = 2 for Serzedelo, Gaia and Beirolas; n = 1 for Alcântara and 145

Guia) and the second round comprised samples collected between July 6-10, 2020 (n 146 = 8; n = 2 for Serzedelo, Gaia and Guia; n = 1 for Alcântara and Beirolas). In each 147 round, the samples were transported to the laboratory, refrigerated and within eight 148 hours of collection. Samples collected in April-May were seeded with SARS-CoV-2 149 control whereas samples collected in July were seeded with PEDV. Raw wastewater 150 samples were kept at 37 (± 1) ºC for seven days to ensure that the levels of SARS-151

CoV-2 RNA, if and where existing, decreased substantially prior to analysis. SARS-152 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CoV-2 control and PEDV were seeded at concentrations of 2.27 x 10 4 GC/L and 1.21 153

x 10 4 GC/L, respectively (quantified as described previously). 154

Seeded raw wastewater samples were aliquoted and concentrated using three 155 methods: (i) hollow fiber with the newly developed inuvai R180 filters (inuvai, a division 156 of Fresenius Medical Care, Germany) followed by PEG precipitation (method 1); (ii) 157 direct PEG precipitation (method 2); and (iii) skimmed-milk flocculation (method 3). All 158 methods were tested using the same initial volume of wastewater (1-L) for a more 159 accurate comparison. 160

Method 1 employed the use of hollow fiber filters: 1-L of raw wastewater was filtered 161 through inuvai R180 filters using a peristaltic pump with a flow rate of 250 mL/min. The 162 elution was performed in three steps: (i) air forward push using 60 mL of air; (ii) 163 backflush with 250 mL of elution buffer (1´ PBS with 0.01% NaPP and 0.01% Tween 164 80/0.001% antifoam) at a flow rate of 140-280 mL/min; and (iii) forward flush using 50 165 mL of elution buffer. The final elution volume was 300 mL. Samples were further 166 concentrated by precipitation with 20% (w/v) PEG 8000 overnight (Blanco et al., 2019) . 167

Samples were centrifuged at 10,000 ´g for 30 min, the supernatant discarded, and the 168 pellet resuspended in 5 mL 1´ PBS, pH 7.4. 169

Method 2 used PEG precipitation: 20% PEG 8000 was added directly to 1-L of raw 170 wastewater, with overnight precipitation followed by centrifugation as described above 171 for method 1. Method 3 employed skimmed milk flocculation, performed in accordance 172

with Calgua et al. (2008) . Briefly, a pre-flocculated solution of 1% (w/v) skimmed milk 173 pH 3.5 was prepared in artificial seawater. The solution of skimmed milk was then 174 added to a final concentration of 0.01% (w/v) to 1-L of previously acidified raw 175 wastewater (pH 3.5). Samples were stirred for 8h at room temperature and flocs were 176 allowed to sediment for another 8h. Supernatant was carefully removed without 177 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 disturbing the sediment. The final volume (approximately 500 mL) was centrifuged at 178 7,000 ´g for 30 min at 12 ºC. The supernatant was carefully discarded, and the pellet 179 resuspended in 0.2 M phosphate buffer at pH 7.5 to a final volume of 5 mL. All 180 concentrates were stored at -80 (± 10) ºC until further analysis. 

RT-dPCR was used to determine the exact concentration of SARS-CoV-2 and PEDV 198 spiked controls. Controls were amplified using the AgPath-ID One-Step RT-PCR kit 199 (Thermo Fischer Scientific) with the set of primers and probe described on 200

Supplementary Table S1 (PEDV; E_Sarbecco and RdRP assays). The 15 μL reaction 201 mixture consisted of 7.5 μL of 2´ RT-PCR buffer, 0.6 μL of 25´ RT-PCR enzyme mix, 202 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. 10-fold dilutions in RNase/DNase-free water. Positive and NTC controls were also 224 added to each PCR assay. Limits of detection (LoD) and quantification (LoQ) were 225 determined in RNase/DNase-free water. The LoD was considered the lowest 226 concentration of target that could be consistently detected (in more than 95% 227 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. The mean recovery efficiency of SARS-CoV-2 control and PEDV for each method was 233 calculated using the copies quantified by RT-qPCR as follows (Eq. The mean and standard deviation for each method were also calculated. 238 239

To minimize nucleic acid carry-over and cross-contamination, sampling concentration, 241 extraction procedures and RT-qPCR/RT-dPCR were performed in separate rooms of 242 the laboratory. A process blank and extraction blank were included for each 243 concentration method and each nucleic acid extraction, respectively. As described 244 above, and before spiking, all wastewater samples were aged to decay potentially 245 present SARS-CoV-2 RNA; following aging, all spiked samples were tested in parallel 246

with the corresponding unseeded samples to rule out or estimate the contribution of 247 potentially native SARS-CoV-2 and PEDV. 248 249

All data analyses were performed with SPSS Statistics 26 (IBM). Repeated 251 measurement ANOVA was conducted to compare the differences between the 252 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint parameters estimated for the three methods. In all cases, p-values < 0.05 were 253 considered statistically significant. 254 255 3. Results and discussion 256

Appropriate quantification of the controls used in spiking experiments and in standard 258 curve for RT-qPCR is extremely important, as it will influence downstream data 259 interpretation. That is why we opted for RT-dPCR, with high precision and sensitivity, 

All unseeded wastewater samples were negative for the presence of SARS-CoV-2 273 and PEDV. Samples were chosen in periods with low number of daily COVID-19 cases 274 (mean for entire country, 287 from April 27 to May 8, and 374, between July 6 and 10, 275 2020) (DGS, 2020). All process and extraction blanks were negative. 276 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint

The effective volume tested within each method was the same (2.2 mL): all methods 277 started with the same initial volume (1-L) of wastewater, followed by concentration 278 steps prior to extraction and sediment resuspension in 5 mL of elution buffer; samples 279 tested across the three methods were extracted using the same extraction protocol, 280 and the same volumes and dilutions were analyzed by RT-qPCR. Nonetheless, the 281 inuvai R180 filters (method 1) enabled the filtration of 2.5 -5-L of raw wastewater. 282

Increasing the initial volume of sample with the inuvai R180 filters would conduct to an 283 increment of the effective volume assayed from 2.2 mL to 5.5 -11 mL without further 284 increases in the concentration time, the concentrate volume, costs for analysis, and 285

RT-qPCR inhibition. On the other hand, increasing the volume of filtration in the 286 skimmed milk flocculation method (and therefore, theoretically, increasing the effective 287 volume assayed; method 3) would imply an increase of skimmed milk and artificial 288 seawater, as well as of HCl to adjust the pH; the volume of concentrated matter and, 289 therefore, of the concentrate would also increase, leading to a decrease in the 290 efficiency of extraction and an increase of inhibitory effects on RT-qPCR. Additionally, 291

increasing the processing volume would require the acquisition of larger volume 292 sample containers, which would also take up more space in the laboratory. 293

Concomitantly, increasing the processing volume when using solely PEG precipitation 294 (method 2) implicates increasing substantially the volume to be centrifuged, which 295 increases the time spent in the concentration step and the costs due to the usage of 296 larger amounts of PEG. 297 SARS-CoV-2 control and PEDV were used to compare concentration recoveries. The 298 highest average percentage of recovery was obtained with the inuvai R180 system at 299 64% (± 6%) for SARS-CoV-2 control and 68% (± 7%) for PEDV, with global recoveries 300 varying between 50 and 82% (Fig. 1A) . 301 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint 14 PEG precipitation had the lowest percentage of recovery for PEDV (9% (± 5%)). 302

Recovery with skimmed milk performed only slightly better (14% (± 8%)) (Fig. 1A) . 303

Recovery using SARS-CoV-2 control was similar for PEG and skimmed milk (4% (± 304 2%)). There were statistically significant differences in the lower recovery percentage 305 of PEG and skimmed milk compared to inuvai R180 (F(1, 3) = 14.94, p = 0.03 for 306 PEDV and F(1, 3) = 171.7, p = 0.006 for SARS-CoV-2 control). 307 308

The inuvai R180 was the single method that consistently led to nucleic acid detection 309 in all samples. Concentration using PEG and skimmed milk led to the detection of 310 PEDV in 50% of the samples, while detection of SARS-CoV-2 control was attained in 311 38% and 63% of the samples, respectively. 312

The method using the inuvai R180 system led to detection by RT-qPCR of the highest 313 mean concentration of genome copies, for both targets: 8.98 and 4.25 GC/reaction for 314 SARS-CoV-2 and PEDV, respectively. Concentration with PEG (1.19 and 0.21 315 GC/reaction for SARS-CoV-2 and PEDV, respectively) and skimmed milk (1.74 316 GC/reaction for SARS-CoV-2 and 0.28 GC/reaction for PEDV) showed similar results 317 (Fig. 1B) . 318

Our recovery values using the inuvai R180 system were similar to those reported for 319 MHV, while enabling an increase in the filtration volume (Ahmed et al., 2020) . For PEG 320 precipitation and skimmed milk flocculation the recoveries were slightly higher than 321 those reported by Philo et al. (2021) . The authors used a concentration of 14% (w/v) 322

of PEG compared to 20% (w/v) PEG in our study. The use of higher concentrations of 323 PEG, although implying increased costs, has been shown to increase the recovery of 324 enveloped viruses from 31% to 51% (Blanco et al., 2019) . In our study, recovery values 325 for PEG precipitation were higher than those reported by Pérez-Cataluña et al. (2021) 326 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 when using similar nucleic acid extraction method (spin column). McMinn et al. (2021) 327 developed a method for the recovery of coronavirus from raw wastewater also using 328 hollow fibers as a primary concentration approach, followed by Concentrating Pipette 329

Select TM (CP Select TM ), reporting overall recovery values for human coronavirus OC43 330 of 22%. Differences in recovery between our study and that of McMinn et al. (2021) 331 may be attributed to the filter that used in our study (inuvai R180 vs Rexeed), coupled 332

with an enhanced elution strategy with three steps that we adopted, and/or to the 333 secondary concentration protocol. The inuvai R180 filter has a reduced nominal pore 334 size (≤ 5.5 nm with a correspondent cut-off ≤ 18.8 Kda) compared to the Rexeed 15S, 335 which has a more open pore structure. Additionally, the filter used in our study has a 336 larger membrane area (1.8 m 2 for inuvai R180 vs 1.5 m 2 for Rexeed S15) and larger 337 fiber inner diameters (220 μm for inuvai R180 vs 185 μm for Rexeed S15). In addition 338 to the optimized elution and secondary concentration protocols, such features might 339 help justify the differences registered in the recovery efficiencies of our study and 340

McMinn et al. (2021) . 341

The extraction efficiency using MNV as proxy averaged 70% (±19%) for inuvai R180 342 protocol. Extraction efficiencies for PEG precipitation and skimmed milk flocculation 343 averaged 50% (±15%) and 36 (±13%), respectively. 344

Detection of SARS-CoV-2 control and PEDV using the inuvai R180 system was 345 consistently achieved with the 1/4-fold dilution, while for undiluted spiked samples, 346 only 38% could be detected without inhibition. PEG precipitation was the single 347 method that detected both targets from undiluted samples, although inhibition still 348 occurred (as evidenced subsequently by testing the 4-and 10-fold dilution). As for the 349 skimmed milk concentration method, detection in undiluted concentrates was found 350 for 75% of the samples, although inhibition still occurred (as measured by the 351 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint dilutions). These results indicate that inhibitory effects exerted upon RT-qPCR could 352 be confirmed for the three methods under comparison. 353

Overall, our results showed that the inuvai R180 system coupled with an improved 354 elution protocol is highly suitable for the detection of SARS-CoV-2 and PEDV, 355 exhibiting the highest percentage of detection and mean recovery value. Additionally, 356 this method also showed greater extraction efficiency and larger volume processing 357 without increased cost or time for downstream analyses. Furthermore, the 358 performance of the inuvai system showed consistency across raw wastewater 359 samples from different catchments / WWTP, including the Serzedelo WWTP, which is 360 highly impacted by industrial effluents (tannery industry) and therefore an extremely 361 complicated matrix to work with altogether, a result corroborated by the Pan-European 362

Umbrella study (Gawik et al., 2021) . In the Umbrella study, raw wastewater samples 363 from different European countries were collected and sent for analysis in a centralized 364 laboratory. In parallel, the same samples were also analyzed in each country for 365 comparison of results. The centralized European laboratory was unable to recover 366 SARS-CoV-2 RNA from Serzedelo raw wastewater presenting low recovery 367 percentages (0.1%) and lower concentrations of crAssphage compared to the other 368 samples analyzed. The same sample, analyzed by our group and using the inuvai 369 R180 system, was positive for SARS-CoV-2 and the concentration of crAssphage was 370 3-log above that detected by the centralized laboratory. These results demonstrate the 371 difficulty of working with this raw wastewater, highlighting the need to test method 372 performance in raw wastewater from different origins. 373 374

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The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low 574 prevalence area

Clinical characteristics of 138 577 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan

CoV-2 in different types of clinical specimens

Timeline of WHO's response to COVID-19 -30

Evaluation of lockdown effect on SARS-CoV-2 586 dynamics through viral genome quantification in waste water

Evidence for 590 gastrointestinal infection of SARS-CoV-2

pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral 594

After establishing the inuvai R180 system as gold-standard for primary concentration, 376 the efficiency of the relative quantification method (RT-qPCR) was assessed by 377 calculating the LoD and LoQ for the E_Sarbecco and RdRp assays using SARS-CoV-378 2 control. Fig. 2 displays the subset of points from the standard curve to determine the 379 LoD and LoQ. 380The LoD was 3.99 GC and 5.52 GC per reaction for the E_Sarbecco and RdRp 381 assays, respectively. This corresponded to a method LoD of 2.73 x 10 3 GC/L for 382E_Sarbecco and 3.79 x 10 3 GC/L for RdRp using the inuvai R180 system. 383As for the LoQ, the results were 66 GC and 178 GC per reaction for the E_Sarbecco 384and RdRp assays, respectively. This corresponded to a method LoQ of 4.56 x 10 4 385 GC/L for E_Sarbecco and 1.22 x 10 5 GC/L for RdRp assay. 386The LoD obtained in our study were inferior to those obtained by Philo et al. (2021) . 387 Pérez-Cataluña et al. (2021) reported similar LoD for E_Sarbecco assay, while also 388 presenting method-dependence LoD. Gonzalez et al. (2020) , testing the CDC assay 389 (N1, N2, and N3), reported different theoretical limits of detection depending on the 390 RT-qPCR assay used but the LoD were similar to those obtained in our study. A 391 comparison between the performance of our method (evaluated through LoD and 392LoQ) and the method reported by McMinn et al. (2021) would have been useful, given 393 that the authors have also used hollow-fiber filters for primary concentration, but such 394 parameter information is missing on the former report. In fact, information on LoQ is 395 missing from most publications with very few exceptions, such as LaTurner et al. 396 (2021) who, while testing five distinct concentration methods, reported LoQ ranging 397 from 2.76 x 10 5 to 8.39 x 10 6 GC/L. Philo et al. (2021) calculated their LoQ in nuclease-398 free water to be 100 gene copies per reaction for all CDC assays. 399 400 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 18

Data from our study demonstrates the importance of validating concentration 402 procedures using seeded controls. Although other studies have tested the efficiency 403 of concentration and extraction methods, this study showed the stability of the inuvai 404 R180 system for the recovery of seeded controls in raw wastewater from WWTP with 405 different composition particularities, including effluents from the tannery industry. A 406 single concentration method may not necessarily be ideal to be used in waters from 407 different backgrounds. In this study, the inuvai R180 system with improved three-step 408 elution protocol was selected for monitoring SARS-CoV-2 in raw wastewaters. Such 409 system is attractive as it enables the concentration of large volumes of raw 410 wastewater, while also being useful to concentrate larger volumes of samples from 411 other origins, such as treated wastewater, environmental waters and drinking water. 412This feature enables handling a single concentration method across different water 413 types without sensitivity loss, increasing costs or time for analysis, while also allowing 414 a less challenging result comparison. 415For an effective environmental surveillance to be put in place, not only for SARS-CoV-416 2 but also for potential future pandemics involving enveloped virus, it is paramount to 417 have validated methods. Nonetheless, comparisons between published methods are 418 difficult as they differ in many aspects including: i) seeding controls; ii) concentration 419 methods; iii) extraction methods; iv) diagnostic and quantification molecular assays 420 and genome targets; v) and mostly, the accepted performance levels. Some 421 publications only mention the recovery efficiency (Ahmed et al., 2020; McMinn et al., 422 2021) , others mention the recovery efficiency and the LoD but not LoQ (Gonzalez et 423 al., 2020; Randazzo et al., 2020; Pérez-Cataluña et al., 2021) , some mention LoQ but 424 not LOD (LaTurner et al., 2021) , while other studies show all data performance, 425 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. all' approach, that is having a single standardized method worldwide for the 429 concentration of SARS-CoV-2, may not be the best approach. This was demonstrated 430 with the Umbrella study (Gawik et al., 2021) , due to several issues, including: (i) 431 laboratories already have their own preferred methods with performances studied; (ii) 432 the methods may not be useful for application in less economically developed 433 countries; (iii) or simply because it is difficult to get a hold of laboratory 434 materials/equipment (as it was the case of ultrafiltration filters or ultracentrifuges). 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 24 LaTurner, Z., Zong, D., Kalvapalle, P., Gamas, K., Terwilliger, A., Crosby, T., Ali, P., 549 Avadhanula, V., Santos, H., Weesner, K., Hopkins, L., Piedra, P., Maresso, A.W., 550 Stadler, L.B., 2021. Evaluating recovery, cost, and throughput of different 551 concentration methods for SARS-CoV-2 wastewater-based epidemiology. Water Res. 552 197, 117043. doi: 10.1016 /j.watres.2021 .117043 553 McMinn, B.R., Korajkic, A., Kelleher, J., Herrmann, M.P, Pemberton, A.C., Ahmed, W., 554 Villegas, E.N., Oshima, K., 2021 Norovirus for water quality assessment. Plos One 12(7), e0179985. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint 595Zhou, X., Zhang, T., Song, D., Huang, T., Peng, Q., Chen, Y., Li, A., Zhang, F., Wu, 596 Q., Ye, Y., Tang, Y., 2017. Comparison and evaluation of conventional RT-PCR SYBR 597 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.21.21260907 doi: medRxiv preprint 28 610 Fig. 2 . LoD for SARS-CoV-2 relative quantification assays. Subset of standard curve points used to determine the 611 smallest concentration of SARS-CoV-2 detected by E_Sarbecco assay at a 95% confidence level (A). Curve to 612 determine the smallest concentration of SARS-CoV-2 detected by RdRp assay at a 95% confidence level (B).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021