key: cord-0325342-uxh09kny
authors: Barua, V. B.; Juel, M. A. I.; Blackwood, A. D.; Clerkin, T.; Ciesielski, M.; Sorinolu, A. J.; Holcomb, D. A.; Young, I.; Kimble, G.; Sypolt, S.; Engel, L. S.; Noble, R. T.; Munir, M.
title: Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: a six-month long study in Charlotte, North Carolina
date: 2021-09-24
journal: nan
DOI: 10.1101/2021.09.23.21258047
sha: 224b749c381a8ca1b5ed5f6864506b30325c2f86
doc_id: 325342
cord_uid: uxh09kny

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of the wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant moderate to a strong positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases using a lag that ranged from 7 to 12 days. A major finding of this study is that despite small differences, both RT-qPCR and RT-ddPCR performed well for tracking the SARS-CoV-2 virus across WWTP of a range of sizes and metropolitan service functions.

diagnosed, and asymptomatic individuals. This area of active research will yield beneficial 70 information for guiding public health decisions. 71 WBE is a potential approach for understanding the proliferation of SARS-CoV-2 within a 72 community as the viral RNA is shed by infected individuals into wastewater (Hasan et al., 2021; 73 Hemalatha et al., 2021) . Aoust et al. (2021) reported that the surges in SARS-CoV-2 RNA in 74 wastewater were observed 48 h prior to clinical testing and 96 h prior to hospitalization. 75 Wastewater sampling captures the community signal comprising both symptomatic and 76 asymptomatic individuals (Bivins et al., 2020; Peccia et al., 2020) , suggesting the value of WBE 77 as an impartial surveillance system at a community level when making public health decisions. To date, SARS-CoV-2 wastewater surveillance studies have mostly employed RT-qPCR for viral 99 quantification (Ahmed, Angel, et al., 2020; Chik et al., 2021; Gerrity et al., 2021; Haramoto et 100 al., 2020; Medema et al., 2020; Nemudryi et al., 2020; Peccia et al., 2020; Randazzo et al., 2020; 101 Sherchan et al., 2020; Westhaus et al., 2021; Wurtzer et al., 2020; Zhao et al., 2021) rather than 102 RT-ddPCR (Gonzalez et al., 2020; Gonzalez et al., 2021) . Only a few research groups have used 103 both RT-qPCR and RT-ddPCR quantification (Aoust, Graber, et al., 2021; Ciesielski et al., 2021; 104 Dumke et al., 2021; Graham et al., 2021) . The study conducted by both Graham et al. (2021) and 105 1 L Nalgene sample collection bottle was used as a field blank. The field blank was exposed to 123 the same environment and transported to the laboratory in coolers packed with ice along with the 124 wastewater samples. The collected samples were processed immediately after reaching the 125 laboratory. A recent study conducted by Pecson et al. (2021) observed that SARS-CoV-2 126 quantitation was slightly higher in pasteurized samples after recovery correction. Sample Asuragen, Austin, TX). Samples were then vortexed and incubated at room temperature for 10 159 minutes to facilitate viral recovery from the filter surface (Gibas et al., 2021; Juel et al., 2021) . 

164 . CC-BY-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) preprint (Table S1 ). The reaction mixture comprised a total volume of 20 μL containing 5 µL 168 extracted RNA template, 10 µL iTaq universal probes reaction mix (Bio-Rad), 0.5 µL iScript 169 reverse transcriptase (Bio-Rad), 1.5 µL (500 nM) primers along with a (125 nM) probe and 3 µL 170 of nuclease-free water. The thermocycling conditions employed were 25°C for 2 min, 50°C for 

Precise QC metrics were considered to assess the detection sensitivity of CDC recommended N1 182 and N2 assays for both workflow 1 (RT-qPCR) and workflow 2 (RT-ddPCR). QC was taken into 183 consideration throughout the whole study to avoid ambiguous interpretation of the obtained 184 results. The positive and negative controls used during each of the steps for both the workflows 185 (1 and 2) were in accordance with MIQE (Bustin et al., 2009) and the digital MIQE (dMIQE 186 . CC-BY-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) preprint 

BCoV was spiked into wastewater samples as a proxy for SARS CoV-2, which could be 190 measured throughout the extraction and RT-qPCR process. 6300 copies of BCoV vaccine was 191 spiked per mL of wastewater. The initial titer of BCoV vaccine was quantified by prior to spiking. The average BCoV recovery for each of the WWTP was observed to be 21-193 31%. 

Single-stranded RNA from Twist Bioscience was extracted in the same manner as wastewater 202 influent samples. The RNA standard was quantified using RT-ddPCR prior to extraction. 10-203 fold serial dilution was performed with the extracted RNA over four orders of magnitude for 204 generating N1 and N2 standard curves. Detailed information has been provided in the 205 supplementary file (Fig. S1 ). The amplification efficiency was 90% for both N1 and N2 assay 206 with an R 2 value of 0.998 and 0.997, respectively which was within the acceptable range as 207 specified in MIQE guidelines (Bustin et al., 2009 ). 

. CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint assay was determined by running an extended series of dilutions of the RNA based SARS-CoV-2 211 positive control (Twist Bioscience) in six replicates with as few as 1 copy/reaction (three-fold 212 dilution series towards the lower end). The threshold cycle at which signals were observed for all 213 the three replicates with a standard deviation less than 1 was considered to be the Cq of LoD 214 (CqLoD). Cq values of 37.07 and 37.78 for N1 and N2 assays, respectively were converted to 215 copies per reaction using the equation (1) to get the LoD for the assay.

216

Where, EAMP represents exponential amplification value of RT-qPCR assay, evaluated as EAMP= 218 10 -1/m , b represents the intercept and m represents the slope. The LoD for workflow 1 was 219 determined as 3000 copies/L of wastewater for both targets. 

The dilution method was used for the determination of the RT-qPCR inhibition (Graham et al., 222 2021). A dilution series of 1:1, 1:2, 1:5 and 1:10 was performed on a subset of samples (n=10) 223 for assessing inhibition. If the diluted sample showed a more than 1 Cq difference between the 224 actual and theoretically expected change in Cq, then the undiluted samples were considered 225 inhibited. There was no inhibition observed for the N1 target but there was with N2. A dilution 226 1:2 was selected to continue inhibition testing as further dilution resulted in Cq values beyond 227 LoD or as non-detectable and the quantification data was updated accordingly. 

• RNA extraction and master mix preparation for molecular quantification was conducted 230 in two different biosafety cabinets in two separate laboratories next to each other to 231 reduce contamination potential.

. CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint recovery (23%) were re-extracted and re-quantified.

• Samples were considered positive when a minimum of two out of three replicates showed 235 amplification above LoD for N1 and N2 assay. (Table S7a) . 248 RT-ddPCR was utilized to quantify SARS-CoV-2 RNA copies targeting N1 and N2, described 249 previously (Table S1) , and utilizing a two-step reverse transcription and RT-ddPCR. Purified 250 RNA was reverse transcribed using Superscript VILO IV MM (ThermoFisher Waltham, MA.).

Briefly, 50μL of the eluate was combined with 20μL 5X VILO IV MM, 1μL (160 copies) mouse 252 lung RNA (p/n R1334152-50 BioChain Newark, CA) and 29μL of DEPC water for a total 253 reaction volume of 100 μL (Table S7a) . Reverse transcription was performed on a C1000 deep 254 block thermal cycler (BioRad) with the following conditions: 25℃ for 10 minutes, 50℃ for 10 255 . CC-BY-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. All assay conditions were previously optimized and established by the Noble Laboratory.

Droplet generation was performed in accordance with manufacturer's instructions, and then 269 droplets were amplified in a C1000 thermal cycler with the following temperature profile: 10 270 min at 95°C for initial denaturation, 40 cycles of 94°C for 30 s, and 55°C for 60 s, followed by 271 98°C for 10 min, with a ramp rate of 2℃ per sec, then an indefinite hold at 12℃. After RT-272 ddPCR cycling was complete, the plate was placed in a QX200 instrument (Bio-Rad) and (Table S6) . 307 162 copies of mouse lung total RNA were spiked into the reverse transcription master mix and 308 the recovery was measured using a mouse ACTB assay (Life Technologies). Recovery was 309 measured by dividing the concentration of the unknown sample by the negative extraction 310 control and multiplying by 100 (Table S7b) . 

For the determination of LoD using RT-ddPCR, the Limit of Blank (LoB) was elucidated from 318 eight replicates of negative matrix samples derived from influent collected at multiple WWTP 319 throughout eastern NC. The LOB was calculated as the mean concentration of all sixty-four 320 replicates and the LOD was then calculated as two standard deviations beyond the defined LOB 321 (Hayden et al., 2013) . The LOQ was determined to be never less than 3 positive droplets no 322 matter the number of merged wells, which for this study was two, resulting in 10 μL or 10% of 323 . CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint the RNA eluate and is equal to a concentration of 10 copies. The detailed LOB, LOD, and LOQ 

The following formula was utilized for both workflow 1 and 2 to determine the recovery is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint (https://covid19.ncdhhs.gov/dashboard/data-behind-dashboards). We calculated daily incident 344 cases as the difference between the current and previous day's reported cumulative cases, 345 carrying the last non-missing value forward as necessary.

Zip code and sewershed boundaries do not typically align (Fig.3) . Daily case counts for each CC-BY-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 this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint The detection frequency and trend of SARS-CoV-2 RNA in the municipal influent 375 wastewater of Charlotte was observed by both RT-qPCR and RT-ddPCR using N1 and N2 376 . CC-BY-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 this version posted September 24, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 to N2 target (Table 3) . About 27.83% of samples detected positive with the N1 target did not 380 show any signal with the N2 target. In addition, the N2 assay showed inhibition while N1 did not 381 (Table S4 and S5). On the other hand, RT-ddPCR performed well in detecting SARS-CoV-2 382 using both N1 and N2 targets, though the N2 target was quantified in a higher percentage (36-383 48%) of samples (Table 3) . When comparing the molecular platform, RT-ddPCR showed more 384 sensitivity than RT-qPCR in quantifying SARS-CoV-2 RNA in wastewater samples. However, 385 SARS-CoV-2 RNA was quantified more readily using the N1 target across all samples using 386 both platforms. As such, downstream analysis was conducted only using N1 data. SARS-CoV-2 387 positivity agreement between the two molecular platforms was 74.4% while the negative 388 agreement was 52.6%. The overall percent agreement was 71% with the Cohen's Kappa 396 . CC-BY-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 overall quantitative data generated using both RT-qPCR and RT-ddPCR for the WWTP A, 404 B, C was positively correlated (ρ=0.569, p<0.0001) with statistical significance. The agreement 405 between the platforms is shown using a range of colors corresponding to concentrations between 406 3.00E+03 copies/L and 2.05E+05 (Fig.4) . RT-qPCR and RT-ddPCR generated similar SARS-

CoV-2 RNA concentration data across the duration of the study which is indicated by the 408 consistency between colors for both platforms on any given collection date. However, the 409 . CC-BY-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 concentration of SARS-CoV-2 in the municipal influent wastewater was correlated with the 427 clinically reported COVID-19 case numbers for Mecklenburg County, Charlotte, NC. The 428 SARS-CoV-2 concentration quantified by both RT-qPCR and RT-ddPCR in the influent 429 municipal wastewater of Charlotte for all the WWTP were plotted against the clinically reported 430 7-day average COVID-19 cases for zip codes served by each plant (Fig.5) . From Fig. 5a, 5b and   431 5c it is evident that the trends of reported COVID-19 cases match with the influent wastewater 432 . CC-BY-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. (Bibby et al., 2021; Olesen et al., 2021) . Even 449 if the influent wastewater concentration data provided a predictive lead to the reported COVID-450 19 cases, it is interesting to note that the trend of the raw SARS-CoV-2 concentration data 451 generated from the influent wastewater is similar to the reported COVID-19 cases. Additionally, 452 SARS-CoV-2 concentration upsurge as quantified by both RT-qPCR and RT-ddPCR at a certain 453 WWTP and decrease in another WWTP suggested that WBE provided us with the specific 454 location where individuals are most or least infected than just the copies/L. Hasan et al. (2021) 455 . CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 has also reported a similar observation where they have suggested the significant potential of CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.23.21258047 doi: medRxiv preprint approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications 556 . CC-BY-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-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-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) preprint CC-BY-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) preprint CC-BY-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) preprint CC-BY-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) preprint

The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10. 1101 

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