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Background WW surveillance enables real time monitoring of SARS-CoV-2 burden in defined sewer catchment areas. Here, we assessed the occurrence of total, Delta and Omicron SARS-CoV-2 RNA in sewage from three tertiary-care hospitals in Calgary, Canada. Methods Nucleic acid was extracted from hospital (H) WW using the 4S-silica column method. H-1 and H-2 were assessed via a single autosampler whereas H-3 required three separate monitoring devices (a-c). SARS-CoV-2 RNA was quantified using two RT-qPCR approaches targeting the nucleocapsid gene; N1 and N200 assays, and the R203K/G204R and R203M mutations. Assays were positive if Cq< 40. Cross-correlation function analyses (CCF) was performed to determine the time-lagged relationships between WW signal and clinical cases. SARS-CoV-2 RNA abundance was compared to total hospitalized cases, nosocomial-acquired cases, and outbreaks. Statistical analyses were conducted using R. Results Ninety-six percent (188/196) of WW samples collected between Aug/21-Jan/22 were positive for SARS-CoV-2. Omicron rapidly supplanted Delta by mid-December and this correlated with lack of Delta-associated H-transmissions during a period of frequent outbreaks. The CCF analysis showed a positive autocorrelation between the RNA concentration and total cases, where the most dominant cross correlations occurred between -3 and 0 lags (weeks) (Cross-correlation values: 0.75, 0.579, 0.608, 0.528 and 0.746 for H-1, H-2, H-3a, H-3b and H-3c; respectively). VOC-specific assessments showed this positive association only to hold true for Omicron across all hospitals (cross-correlation occurred at lags -2 and 0, CFF value range between 0.648 -0.984). We observed a significant difference in median copies/ml SARS-CoV-2 N-1 between outbreak-free periods vs outbreaks for H-1 (46 [IQR: 11-150] vs 742 [IQR: 162-1176], P< 0.0001), H-2 (24 [IQR: 6-167] vs 214 [IQR: 57-560], P=0.009) and H-3c (2.32 [IQR: 0-19] vs 129 [IQR: 14-274], P=0.001). Conclusion WW surveillance is a powerful tool for early detection and monitoring of circulating SARS-CoV-2 VOCs. Total SARS-CoV-2 and VOC-specific WW signal correlated with hospitalized prevalent cases of COVID-19 and outbreak occurrence. Disclosures All Authors: No reported disclosures.

Background New tools capable of dynamic assessment of the varying burden of Clostridioides difficile infections are required to mitigate increased patient morbidity, mortality, and health costs. Wastewater (WW)-based epidemiology (WBE) is an emerging science, enabling comprehensive, inclusive, and unbiased assessment of populations, spatially and temporally. We sought to detect, quantify and track C. difficile across a range of scales using WBE. Methods WW collected from two hospitals; the Rockyview General Hospital (RGH; 600 beds) and Peter Lougheed Centre (PLC; 550 beds) both based in Calgary, were compared to that from a municipal WW Treatment Plant (WWTP) in Calgary, Canada. DNA was extracted from pellets collected after WW centrifugation. A multiplexed quantitative PCR assay was used to quantify the abundance of C. difficile 16S rRNA and toxin A (tcdA) genes. These were then assessed as raw values or as normalized ratios to three fecal biomarker genes: total bacterial 16S rRNA, human 18S rRNA, and Bacteroides HF183 16S rRNA. Kruskal-Wallis and Mann-Whitney tests were performed using RStudio and GraphPad Prism (version 9.3.1). Results Eight weekly samples collected from the RGH demonstrated significant changes in the levels of total C. difficile 16S rRNA gene and tcdA over time (P=0.0004 and P=0.0005, respectively, Kruskal-Wallis). Similar trends in total C. difficile and tcdA burden over time were observed when gene copies were normalized with the three fecal biomarker genes. Over a separate 13-week comparison, C. difficile and tcdA gene target abundance was greater in hospital WW (RGH and PLC) than in community-based samples from the WWTP (P=0.048 and P=0.012, respectively, Mann-Whitney). There was no significant difference in C. difficile and tcdA gene target abundance between RGH and PLC (P=0.896 and P=0.343, respectively, Mann-Whitney). Clostridioides difficile genes in wastewater measured by quantitative PCR. C. difficile 16S rRNA and tcdA gene abundance normalized as a ratio against total bacterial load (16S rRNA) varies over time and is markedly increased in hospitals relative to a municipal wastewater treatment plant in Calgary, Canada. Conclusion WW surveillance is a powerful tool that can monitor the burden the C. difficile across a range of scales in real-time. This tool could augment infection prevention and control and antimicrobial stewardship programs to better understand factors that contribute to colonization and infection. Disclosures Thomas J. Louie, MD, Artugen: Advisor/Consultant|Artugen: Grant/Research Support|Crestone: Advisor/Consultant|Crestone: Grant/Research Support|Finch Therapeutics: Advisor/Consultant|Finch Therapeutics: Grant/Research Support|Rebiotix: Advisor/Consultant|Rebiotix: Grant/Research Support|Seres Therapeutics: Advisor/Consultant|Seres Therapeutics: Grant/Research Support|summit plc: Advisor/Consultant|summit plc: Grant/Research Support|Vedanta Biosciences: Advisor/Consultant|Vedanta Biosciences: Grant/Research Support.

In order to detect the SARS-CoV-2 variants of concern (VOCs), five real-time reverse transcriptase PCR (rRT-PCR) assays were designed to target the critical discriminatory mutations responsible for the following amino acid changes in the spike protein: two Δ69-70 + N501Y + E gene triplexes (one optimized for Alpha [B.1.1.7] and one optimized for Omicron [B.1.1.529]), a K417N + 242-244 wild-type duplex, a K417T + E484K duplex, and a L452R + P681 + E484Q triplex. Depending on the assay, sensitivity was 98.97-100% for the detection of known VOC-positive samples, specificity was 97.2-100%, limit of detection was 2-116 copies/reaction, intra- and interassay variability was less than 5%, and no cross-reactivity with common respiratory pathogens was observed with any assay. A subset of rRT-PCR- positive VOC samples were further characterized by genome sequencing. A comparison of the lineage designation by the VOC rRT-PCR assays and genome sequencing for the detection of the Alpha, Beta, Gamma, Delta and Omicron variants showed clinical sensitivities of 99.97-100 %, clinical specificities of 99.6-100 %, positive predictive values of 99.8-100%, and negative predictive values of 99.98-100 %. We have implemented these rRT-PCR assays targeting discriminatory single nucleotide polymorphisms for ongoing VOC screening of SARS-CoV-2 positive samples for surveillance purposes. This has proven extremely useful in providing close to real-time molecular surveillance to monitor the emergence of Alpha, the replacement of Alpha by Delta, and the replacement of Delta by Omicron. While the design, validation and implementation of the variant specific PCR targets is an ever-evolving approach, we find the turn-around-time, high throughput and sensitivity to be a useful complementary approach for SARS-CoV-2 genome sequencing for surveillance purposes in the province of Alberta, Canada.