key: cord-0428934-n2t7nc5o
authors: Daigle, J.; Racher, K.; Hazenberg, J.; Yeoman, A.; Hannah, H.; Duong, D.; Mohammed, U.; Spreitzer, D.; Gregorchuk, B. S. J.; Head, B. M.; Meyers, A. F. A.; Sandstrom, P. A.; Nichani, A.; Brooks, J. I.; Mulvey, M. R.; Mangat, C. S.; Becker, M. G.
title: A sensitive and rapid wastewater test for SARS-COV-2 and its use for the early detection of a cluster of cases in a remote community
date: 2021-08-18
journal: nan
DOI: 10.1101/2021.08.13.21262039
sha: 95cd0878e347901e2b6c8206e2c30823e8f7c685
doc_id: 428934
cord_uid: n2t7nc5o

Throughout the COVID-19 pandemic, wastewater surveillance has been used to monitor trends in SARS-CoV-2 prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert System, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest the GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories.

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, public health officials 68 worldwide have investigated methods to detect community transmission of the severe acute respiratory 69 syndrome virus 2 (SARS-CoV-2). One such method includes environmental surveillance of wastewater, or 70 wastewater-based epidemiology (WBE), that has been used for poliovirus monitoring ( Despite these advantages, WBE testing typically requires a large laboratory due to the need for 86 multiple types of instruments and equipment. Samples are often processed using lengthy and complicated 87 protocols for concentration, extraction, and molecular testing that require specially trained individuals. 88

Sample transport and shipping to centralized laboratories also causes delays, adding additional hours to 89 days of sample processing time, negating its benefit as an early warning system (Larsen and Wigginton, 90 2020) . 91

To date, no field-deployable and rapid test for SARS-CoV-2 in wastewater has been developed. settings these assays display a low sensitivity when compared to the gold standard nucleic acid 97 . CC-BY-NC 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) Table 1 ). The RT-qPCR reaction was prepared as per 183 the manufacturer's instructions with a primer concentration of 500 nM and probe concentration of 125 184 nM. The following cycling conditions were used: 25°C for 2 minutes; 50°C for 15 minutes; 95°C for 2 185 minutes; followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. RT-qPCR was performed 186 with two technical replicates. Quantification of the SARS-CoV-2 N1 and N2 CT values was performed using 187 . CC-BY-NC 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. 

To determine if the GeneXpert SARS-CoV-2 test was compatible with wastewater, gamma-199 irradiated SARS-CoV-2 was serially diluted in wastewater that was negative for SARS-CoV-2 as determined Canadian communities (Method A, Materials and Methods). In total, 30 samples were collected from 208 communities with active COVID-19 cases, and 15 samples from were collected from communities with no 209 known COVID-19 activity ( Figure 1 ). The identity of the specimens was censored until test results were 210 collated. One sample produced a loading error on the GeneXpert system, possibly due to clogging of the 211 test cartridge; however, a repeat test was performed successfully. All samples were tested concurrently 212 with our laboratory-developed test for COVID-19 targeting the solids fraction of wastewater, and for a 213 subset, the supernatant fraction was also tested. Our laboratory-developed tests detected SARS-CoV-2 in 214 all 30 wastewater samples taken from communities with active cases of COVID-19 ( Figure 1 ). The viral 215 concentration ranging from 9.2 to 216 cp/mL (solids fraction). Of these positives, 22 were reported as 216 positive on the GeneXpert, seven were endpoint positive (weak positive), and one was negative for an 217 . CC-BY-NC 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 August 18, 2021. ; https://doi.org/10.1101/2021.08.13.21262039 doi: medRxiv preprint agreement of 96.6%. As expected, our laboratory-developed test did not detect SARS-CoV-2 in any of the 218 wastewater samples collected from communities with no active cases of COVID-19 (n = 15). The 219

GeneXpert assay also reported all of these samples as negative for SARS-CoV-2 (negative agreement of 220 100%). 221 concentration was then compared to estimates from the PHAC-NML laboratory-developed solids assay 238 ( Fig. 2B ). There was a moderate/strong correlation (r = 0.724) between the predicted SARS-CoV-2 239 concentrations of the GeneXpert and the laboratory-developed solids assay (Fig. 2B) . 240

communities without known cases 242

The PHAC National Wastewater Surveillance Program has been testing wastewater in the 243 Northwest Territories since August 2020. Wastewater samples were shipped from the Northwest 244

Territories to PHAC-NML in Winnipeg for testing with a transit time of 1-4 days, with a further two days 245 required for testing and reporting. In March 2021, a GeneXpert system was deployed to Yellowknife to 246 . CC-BY-NC 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 August 18, 2021. ; support an on-site wastewater surveillance program and expedite test reporting. GeneXpert surveillance 247 in Yellowknife formally began on March 26 with wastewater tested multiple times weekly (Fig 3) . 248

Preceding the GeneXpert pilot study initiated on March 26, wastewater samples from Yellowknife 249 were shipped to and tested exclusively at the PHAC-NML laboratory in Winnipeg, Canada, a distance of 250 approximately 1745 kilometres (Fig 4A) . These wastewater samples were collected from two major lift 251 stations that capture wastewater from >85% of the Yellowknife population ( Fig 4B) . Currently, no options exist for mobile and rapid testing of wastewater. This study explored the 278 GeneXpert system as a candidate technology for this purpose, and performed a pilot study in Yellowknife, 279

Canada. The data presented here demonstrates that the GeneXpert SARS-CoV-2/Flu/RSV assay can 280 reliably detect SARS-CoV-2 in wastewater at concentrations above 32 cp/mL ( Figure S1; Figure 1) . 281

Sensitivity of the assay can be improved further through concentration methods such as centrifugal 282 filtration, which facilitated detection of SARS-CocV-2 below 10 cp/mL (Fig 2B) . Although effective, 283 concentration by centrifugal filters is not ideal, as it requires use of a centrifuge, expensive specialized 284 filters, and additional processing time by the operator. Future work should investigate more rapid and 285 deployable methods for concentration, such as filter syringes or concentrating pipettes (Gonzalez et al., 286 2020). Alternatively, concentration of the sample may not be required if the GeneXpert system is used to 287 monitor wastewater from a smaller system (small neighborhood or institutional samples). 288

At the observed level of sensitivity, the GeneXpert is capable of serving as an early detection 289 system in remote communities. This is supported by data from the Yellowknife pilot project, where the 290 GeneXpert system detected SARS-CoV-2 in wastewater before public health officials were aware of its 291 presence in the community. Previously, Yellowknife relied exclusively on wastewater testing at a 292

Winnipeg-based testing facility (PHAC-NML), which delayed results by approximately 4-7 days due to 293 shipping and sample processing time. In the time between sample collection in Yellowknife and 294 completion of testing in Winnipeg, seven positives wastewater samples were recorded on the GeneXpert 295 system and five COVID-19 cases were already identified in the community. Since this pilot, the 296 Government of Northwest Territories has further expanded GeneXpert testing to include seven additional 297 remote communities. Together, this clearly demonstrates the utility of rapid, deployable wastewater 298 testing for SARS-CoV-2. 299

In terms of efficient resource allocation, a single GeneXpert cartridge can effectively screen an 300 entire community with much broader coverage than a single clinical test, and is likely a more sustainable 301 and cost-effective option for community surveillance in post-vaccination scenarios. Additionally, because 302 of the widespread distribution GeneXpert systems for SARS-CoV-2, HIV, tuberculosis, and influenza 303 testing, this technology provides a means to improve global access to wastewater surveillance. This 304 includes remote Canadian communities as well as remote areas of South America, Africa, and Asia. 305 . CC-BY-NC 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 August 18, 2021. ; https://doi.org/10.1101/2021.08.13.21262039 doi: medRxiv preprint There are some limitations to the GeneXpert technology. Notably, the targets are determined by 306 the manufacturer and the system itself is optimized for clinical testing. The system currently does not 307 monitor for SARS-CoV-2 variant detection, nor include fecal indicators such as pepper mild mottle virus 308 that may be useful for data normalization (D'Aoust et al., 2021b). As the cartridge uses an internal filter 309 there is also a risk of clogging; however, this was only observed once in this study and the sample was 310 successfully processed in a repeat test. The input volume of the GeneXpert SARS-CoV-2/Flu/RSV test is 311 also quite low at 300 µL, which may introduce sampling biases when testing large volumes of 312 heterogeneous wastewater. This sampling bias makes any quantitative data from the GeneXpert less 313 reliable unless a concentration step is performed. Ideally, wastewater-specific test cartridges for the 314 GeneXpert could be developed with optimized targets and larger input volumes; or a similar large-volume 315 assay could be developed using a competing technology. 316

The Xpert Xpress SARS-CoV-2/Flu/RSV assay used in this study is also used clinically for the 317 detection of Influenza A, Influenza B, and Respiratory Syncytial Virus. Therefore, it is theoretically possible 318 that this assay will be able to detect these other pathogens in wastewater in addition to SARS-CoV-2. In 319 this study, we only observed one positive detection of Influenza A in a wastewater sample from a major 320 surveillance for remote and isolated communities; however, these locations are often separated by large 333 distances creating logistical complications. As the second largest country by land mass, wastewater tests 334 will need to be deployable for an effective and equitable Canadian national wastewater surveillance 335 program for COVID-19. Additionally, WBE has not been substantially applied to low-resource communities 336 . CC-BY-NC 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 August 18, 2021. ; https://doi.org/10.1101/2021.08.13.21262039 doi: medRxiv preprint (Naughton et al., 2021) . Vaccinations and effective clinical surveillance has not reached many parts of the 337 developing world, and these factors increase the risk and impact from the introduction of COVID-19 into 338 these communities (Ritchie et al., 2020) . 339 340 5. Conclusions: 341

In conclusion, the GeneXpert can be used for wastewater testing for SARS-CoV-2, improving access 342 to wastewater based epidemiology, and providing rapid results allowing for an immediate public health 343 response. In particular, this technology could be applied in support wastewater surveillance in remote 344 communities where it may provide early warning of SARS-CoV-2 outbreaks. It is likely wastewater 345 surveillance will become increasingly important in the post-vaccination pandemic landscape as individuals 346 with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be clinically 347 tested. Although not specifically designed for this purpose, the GeneXpert system provides a stopgap 348 measure until a purposely-designed wastewater rapid test for SARS-CoV-2 becomes available for remote . CC-BY-NC 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 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 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 August 18, 2021. CC-BY-NC 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 August 18, 2021. ; https://doi.org/10.1101/2021.08.13.21262039 doi: medRxiv preprint 609

Supplementary Figure 1 : Detection of SARS-CoV-2 in wastewater using the GeneXpert system. Gamma irradiated SARS-CoV-2 culture fluid was diluted in wastewater to final concentrations between 16 -128 copies (cp/mL). Tests were performed in duplicate. The GeneXpert detected SARS-CoV-2 in all replicates with an input equal to or greater than 32 cp/mL.

. CC-BY-NC 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 August 18, 2021. ; https://doi.org/10.1101/2021.08.13.21262039 doi: medRxiv preprint

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