Wastewater-based surveillance (WBS) has shown to be an effective tool in monitoring the spread of SARS-CoV-2 and has helped guide public health actions. Consequently, WBS has expanded to now include the monitoring of mpox virus (MPXV) to contribute to its mitigation efforts. In this study, we demonstrate a unique sample processing and a molecular diagnostic strategy for MPXV detection that can inform on the epidemiological situation of mpox outbreaks through WBS. We conducted WBS for MPXV in 22 Canadian wastewater treatment plants (WWTPs) for 14 weeks. Three MPXV qPCR assays were assessed in this study for the detection of MPXV which include the G2R assays (G2R_WA and G2R_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, and an in-house-developed assay that we have termed G2R_NML. The G2R_NML assay was designed using reference genomes from the 2022 MPXV outbreak and provides a larger qPCR amplicon size to facilitate Sanger sequencing. Results show that all three assays have similar limits of detection and are able to detect the presence of MPXV in wastewater. The G2R_NML assay produced a significantly greater number of Sanger sequence-confirmed MPXV results compared to the CDC G2R assays. Detection of MPXV was possible where provincial surveillance indicated overall low caseloads, and in some sites forewarning of up to several weeks was observed. Overall, this study proposes that WBS of MPXV provides additional information to help fill knowledge gaps in clinical case-surveillance and is potentially an essential component to the management of mpox.

The sudden emergence and spread of Monkeypox in non-endemic parts of the world is currently not well understood. Infections are often mis-diagnosed and surveillance strategies are scarce. Wastewater-based surveillance (WBS) of human Monkeypox virus (MPXV) can help supplement our current clinical surveillance and mitigation efforts. WBS has shown to be an effective tool in monitoring the spread of other infectious pathogens, such as SARS-CoV-2 and its variants, and has helped guide public health actions. In this study, we describe how WBS can be used to detect MPXV in wastewater. We conducted WBS for MPXV in 22 wastewater treatment plants (WWTPs) over a period of 14 weeks. Nucleic acids were extracted using the MagAttract PowerMicrobiome DNA/RNA extraction kit. Three real-time qPCR assays were assessed for the detection of MPXV in wastewater. These included the G2R assays (G2R\_WA and G2R\_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, as well as an in-house-developed assay (G2R\_NML). The G2R\_G (generic) assay was designed to detect both the Congo and West African clades (re-named to Clades one and two, respectively) of viruses while the G2R\_WA assay was designed to detect the West African clade (Clade one). The G2R\_NML assay was designed using reference genomes of the 2022 MPXV outbreak. Our results show that all three assays have similar limits of detection and are all able to detect the presence of MPXV in wastewater. Following detection through real time qPCR, Sanger sequencing was performed on the resulting amplicon products, with the assembled contigs then undergoing analysis using nucleotide Basic Local Alignment Search Tool (BLAST). Due in part to the longer amplicon size of the G2R_NML assay, a significantly greater number of positive detections were identified as originating from MPXV compared to the CDC G2R assays. The ability to detect trace amounts of MPXV in wastewater as well as obtain Sanger sequence confirmation, has allowed for the successful surveillance of this virus in wastewater.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study was funded by internal funds (Government of Canada)### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (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 SARSCoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.