key: cord-0301288-cbx4qmgn
authors: Hewitt, J.; Trowsdale, S.; Armstrong, B.; Chapman, J. R.; Carter, K.; Croucher, D.; Billiau, C.; Sim, R.; Gilpin, B.
title: Sensitivity of wastewater-based epidemiology for detection of SARS-CoV-2 RNA in a low prevalence setting
date: 2021-08-26
journal: nan
DOI: 10.1101/2021.08.24.21258577
sha: 0447e67465b3a4eeebad79cb9584d3bd30085929
doc_id: 301288
cord_uid: cbx4qmgn

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005 % and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a more realistic proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread around 64 the world since first detected in late 2019 (World Health Organisation 2020). For many 6 exacerbated when the number of new cases per day, rather than total cases (i.e., incidence 88 rather than prevalence) is used, given that individuals may continue to shed SARS-CoV-2 89 RNA for weeks following diagnosis (Cevik et al. 2021 ). While not ideal, daily incidence is 90 the best proxy for total case load in many countries. 91 Aotearoa New Zealand adopted an COVID-19 elimination strategy with the intent of 92 reducing the incidence of cases in the community to zero (e.g., Baker (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint (Hewitt et al. 2009 ). Working stocks of 10 6 TCID 50 /mL 138 FIPV and 10 6 PFU/mL MNV were prepared from the initial virus preparations by diluting in 139 2% Eagle's Minimal Essential Media (Gibco, Waltham, MA). Aliquots were stored at -80 °C. 140 Prior to virus concentration (Section 2.3), 2 µL of FIPV and MNV working stocks were 141 added per 100 mL wastewater (e.g., 10 µL each of FIPV and MNV added to 500 mL). For 142 each batch of samples processed, 10 µL each of FIPV and MNV working stocks were also 143 added to 2.5 mL phosphate bu ered saline (PBS), pH 7.2 and stored at 4 °C (for up to 18 hr).

This was referred to as the FIPV/MNV in PBS process control.

A SARS-CoV-2 positive extraction quality control was prepared from a clinical specimen 146 submitted for diagnostic testing (ESR, Porirua, NZ). The virus suspension was heat 147 inactivated at 56 °C for 30 min, diluted 1/1000 in viral transport media, 200 µL aliquots 148 prepared and stored at -80 °C until required for RNA extraction. The SARS-CoV-2 extraction 149 control was quantified (as described below) and determined as approx. 30,000 genome copies 150 (GC)/mL. Following seeding of FIPV and MNV into wastewater (Section 2.2), viruses from both liquid 153 and solid fractions were recovered and concentrated using PEG precipitation. Wastewater 154 (500 mL) was centrifuged at 10,000 x g for 20 min at 4 °C. The supernatant was transferred 155 to a clean bottle and temporarily stored at 4 °C (maximum 2 hr). The solids were resuspended 156 in a 1:8 (w/v) ratio of glycine buffer (0.05 M glycine, 3% beef extract), and pH adjusted to 157 9.0. The bottles were sonicated in an ultrasonic water bath for 2 min, and then placed on a 158 horizontal shaker at 200 rpm for 30 min at room temperature. Subsequently, the glycine 159 buffer mixture was centrifuged at 10,000 x g for 20 min at 4 °C and the supernatant 160 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Mannheim, Germany) was used to extract viral nucleic acid from a 200 µL aliquot of each 174 wastewater concentrate and the FIPV/MNV in PBS process control. One modification was 175 made to the standard protocol; samples were centrifuged at 10,000 x g for 30 sec prior to 176 addition to the filter. To monitor for RT-qPCR inhibition and/or poor RNA extraction, a 1:4 177 dilution of the concentrate and the FIPV/MNV in PBS process control was also prepared 178 prior to RNA extraction. For each batch of RNA extractions, a positive (200 µL pre-aliquoted 179 heat inactivated SARS-CoV-2 suspension) and negative (water) extraction control were used.

Viral RNA was stored at 4 °C (maximum 1 hr) or at -80 °C until cDNA synthesis. Table S1 . For all targets, viral cDNA (5 184 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint converted to GC/reaction using the standard curve, and then expressed as GC/L wastewater 232 using the formula:

where V final was the final volume of concentrate (mL), V processed was the initial volume of 234 wastewater processed (L), V eluted was the volume following RNA extraction (µL), V extracted 235 was the volume of concentrate extracted (µL), and V PCR was the volume of RNA used in the 236 RT-qPCR reaction (µL). GC/L was then converted to log 10 GC/L wastewater.

When three or four replicates were positive, but the calculated mean GC/reaction value was 

FIPV and MNV process control recoveries were calculated using the formula, (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint Epidemiological data on notified COVID-19 cases (symptomatic or asymptomatic) were 253 obtained from the EpiSurv Surveillance database (https://surv.esr.cri.nz/episurv). These (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint collected. We therefore used this sampling date as a proxy "onset date" and that the shedding 277 profile was the same as symptomatic cases. Dates relating to occupation and shedding density contribution have been adjusted to reflect 285 the date that wastewater was collected. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Mean (± bootstrap 95% confidence intervals (CI)) process control recovery efficiencies were 

From 9 July to 6 November 2020, there were a total of 335 COVID-19 cases at the MIQF. Of 305 the 335 total cases, 212 were symptomatic and 123 were asymptomatic. Symptomatic cases 306 had an illness onset which ranged from 30 days before arriving at the MIQF to 3 days after 307 arrival, with an average onset of illness 3.3 days before arrival at the MIQF. Overall, 308 COVID-19 cases stayed at the MIQF from 5 -52 (mean of 13) days (Fig. 2) . During the four-309 month study, one cluster of 179 community cases, mostly residing in the WWTP catchment 310 being studied, was reported. All cases were rapidly identified (onset of illness 31 July -11 311 September 2020), with 155 cases being transferred to the MIQF between 11 August and 11 312 September (Fig 2) . On 29 August 2020, the total number of cases at the MIQF peaked at 107.

This was equivalent to 40 infectious cases (model 2) or 16 relative infectious cases (model 3), 314 and 17 new daily cases (model 4). There were no other known COVID-19 cases outside of 315 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Fig. S1a) . In samples diluted 1:4 prior to RNA extraction, FIPV and MNV recovery 342 data showed that, overall, there was no significant RT-qPCR inhibition detected in the MIQF 343 or WWTP wastewater samples (Fig. S1b) . Mean (± SD) PCR Cq values for samples ranged from 26.2 (± 0.1) to 37.9 (± 0.4) (Fig. 3a) . replicates. For these ten samples, mean (± SD) PCR Cq values ranged from 33.9 (± 0.7) to 354 38.1 (± 1.2) (Fig. 3b) , with SARS-CoV-2 RNA concentrations that ranged from 3.3 to 3.8 355 log 10 GC/L wastewater from the WWTP influent. For SARS-CoV-2 RNA detections with 1 356 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint CoV-2 RNA (Fig. 4a and 4b) . In addition to simply counting COVID-19 total cases, shedding 376 was modelled on the more restricted shedding assumptions described above. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. When SARS-CoV-2 RNA was detected in the MIQF wastewater (n = 108), there were 6 -389 107 total cases (i.e., 5 -89 per 100,000 population) (model 1). This is equivalent to 3 -60 390 (i.e., 2.5 -50 per 100,000 population) infectious cases (model 2) and 0.2 -9.1(i.e., 0.2 -7.6 391 per 100,000 population) relative infectious cases (model 3). Non-detections of SARS-CoV-2 392 RNA in the MIQF wastewater were associated with 5 -11 total MIQF cases (i.e., 4 -9 per 393 100,000 population) compared to at least 6 total cases when SARS-CoV-2 RNA was 394 detected. The MIFQ wastewater samples with SARS-CoV-2 RNA concentrations more than 395 4.0 log 10 GC/L were collected on days when there were more cases (i.e., 7 -107 cases (i.e., (Table 1) . 398 It is clear from the data that detecting SARS-CoV-2 RNA at the WWTP was more likely 399 when there were at least 8 total cases (i.e., using model 1) (Table 2 ). This threshold reduced 400 to 4 cases using model 2 and one cases using model 3. SARS-CoV-2 RNA detections at the 401 WWTP, particularly those with three or four RT-qPCR replicates, were associated with an 402 increase in the number of total (model 1), infectious (model 2), relative infectious (model 3), 403 or new daily (model 4) cases in the catchment (Table 2) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Logistic regression models were fitted to data excluding and including the period of the 414 community COVID-19 cluster for each shedding model (Fig. 5) . When the data during the 415 community cluster were excluded, increases in relative infectious cases and new daily cases, 416 was not significantly associated with SARS-CoV-2 RNA detection at the WWTP (df = 67, (Table S2) . (Table S2) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The proportion of symptomatic to asymptomatic cases at the MIQF varied with SARS-CoV-2 436 RNA detections at the WWTP (Fig. 6) . The interquartile range (IQR) of ratios was higher in (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint amount of viral RNA within the sample is below the limit of detection. Alternatively, the 465 sample could be a true negative (i.e., no SARS-CoV-2 RNA is present in the sample) but be a 466 false negative at the community scale because the sample collected did not capture the 467 intermittent and low levels of SARS-CoV-2 RNA in the wastewater system. Even if sampling 468 could somehow test all the wastewater in a community, shedding from infectious individuals 469 whose residence is served by a septic tank or who are otherwise not connected to the 10 GC/mL (i.e., 10 4 GC/L) wastewater. Our data tested these estimates and found that 480 detection with 0.00005% prevalence would be highly unlikely, whereas detection with a 1% 481 prevalence would be highly likely. Indeed, detection with prevalence of 0.005% was possible, 482 but was more reliable (90-100%) at a 0.03% or higher prevalence (i.e., 30 total cases in 483 100,000 population). The sensitivity reported in our study is similar to that reported by Hata (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint 5 cases per 100,000 population (0.005%) (Table 3) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. cases to estimate a positive detection of SARS-CoV-2 RNA therefore had little support 518 beyond 10 new daily cases due to the low case prevalence during the study period (Fig. 5c) .

Shedding by asymptomatic cases 520 Our data suggest that symptomatic individuals may shed a higher amount of SARS-CoV-2 521 RNA into wastewater than asymptomatic individuals (Fig. 6) . Wastewater testing is 522 potentially one of the best ways to detect asymptomatic cases in a community, as they are 523 otherwise unlikely to be tested unless they are close contacts of symptomatic cases. During 524 our study, asymptomatic case load was not significantly associated with increased SARS-525 CoV-2 RNA detection in wastewater for any shedding models. It is possible that 526 asymptomatic cases in our study may have been tested longer after infection than 527 symptomatic cases, many of whom were tested immediately upon developing symptoms. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. shedding assumption model) of detecting SARS-CoV-2 RNA in wastewater. As such, 560 wastewater can be used as both an early warning system of community outbreaks and for 561 longer term surveillance. However, it is important to carefully consider how cases are 562 quantified/modelled, and the relative contributions of symptomatic and asymptomatic 563 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. collection, analysis, and interpretation of data; manuscript writing or the submission process. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.21258577 doi: medRxiv preprint

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