Understanding the cellular responses to low-dose ionizing radiation exposure (≤ 0.1 Gy) is essential for developing evidence-based radiation protection policies. Unlike high-dose radiation exposure, which is known to alter gene expression and protein synthesis, the cellular impact of low-dose exposure remains less defined. To address this gap, ribosome profiling was performed on normal human lung fibroblast cells exposed to low (0.1 Gy) and high (1 Gy) doses of 60Cobalt gamma (γ) rays. At 1 and 6 h postirradiation, global protein synthesis remained unchanged at low dose; however, specific mRNAs showed altered translation efficiency. These translational shifts occurred despite minimal changes at the transcriptional level, indicating that translation offers a more sensitive readout of early low-dose γ-radiation effects. The small GTP-binding protein RAB33B was identified as a translationally upregulated target. This study emphasizes that conventional transcriptome analyses do not fully capture gene expression dynamics and suggests that selective translation could contribute to a distinct cellular response to low-dose irradiation.

Since the pandemic, interest in Wastewater and environmental surveillance (WES) has expanded rapidly, alongside advances in PCR-based detection and the need for reliable normalization strategies. One widely adopted approach to quantification in WES involves normalizing pathogen measurements using human-associated fecal biomarkers such as tobamoviruses. While pepper mild mottle virus and tomato brown rugose fruit virus have been extensively studied and utilized in hundreds of WES studies, the broader tobamovirus genus remains relatively underexplored. We reviewed over 270 published studies, assessed the suitability of 43 tobamoviruses for application as indicators of human fecal contamination or for normalization biomarkers for WES applications. Tobamoviruses were systematically evaluated based on host range, likelihood of human dietary exposure and gastrointestinal passage, environmental persistence, and evidence of detection in wastewater. This analysis identifies 11 tobamoviruses with significant potential as current or emerging biomarkers across diverse geographic and dietary contexts. Our analyses identify 11 tobamoviruses with significant potential as biomarkers across global wastewater contexts. Tobamoviruses have proven suitable for wastewater and environmental surveillance normalization due to their frequent and high-volume introduction into the human food chain through staple crops, in which they induce only mild or asymptomatic infections. This results in reliable, high-abundance measurements in municipal wastewater.

Wastewater-based surveillance (WBS) has emerged as an important complementary tool for infectious disease monitoring during COVID-19 pandemic, especially in high-income countries. In contrast, adoption of WBS in low-and middle-income countries (LMICs) remains constrained by resource limitations, inadequate infrastructure, and technical challenges, while methodological practices are insufficiently documented to guide future preparedness. This systematic review (January 2020-January 2025) synthesizes WHO-guided WBS methodologies applied to six priority pathogens: SARS-CoV-2, Influenza, RSV, MPXV, Salmonella, and Poliovirus across LMICs. Following PRISMA guidelines, 2370 records were screened, and 180 studies from 37 LMICs were included. Our analysis indicates that WBS of infectious diseases has been implemented across centralized and decentralized settings using diverse sampling and analytical methods. Despite methodological variability, WBS confirmed the presence of viral signals (i.e., SARS-CoV-2) days to weeks before clinical surges, underscoring its early warning potential. Future research in LMICs should prioritize pathogen-specific protocol development, investment in WBS, and integration with national surveillance frameworks to strengthen preparedness, global health equity, and pandemic response.

Nucleic acid amplification tests (NAATs) are exquisitely sensitive and specific, able to accurately and quickly monitor vanishingly small amounts of RNA or DNA in wastewater-based public health surveillance applications. Yet, multiplexing samples and target assays is difficult and the dependence of NAATs on enzymes increases false negative rates with inhibitor-rich environmental samples. The Nanostring nCounter system (NNS) is a nucleic acid hybridization platform with a capability for high multiplexing (upwards of 800 probes) that directly counts RNA or DNA biomarkers without reverse transcription or amplification. This study determines the feasibility of direct detection and quantification of genetic markers of public health significance in wastewater samples using NNS and compares the performance with gold-standard NAAT assays targeting antimicrobial resistance (AMR) DNA loci, 16S DNA, SARS-CoV-2 RNA, and the fecal content markers, Pepper mild mottle virus (PMMoV) RNA and crAssphage DNA. We demonstrate that the direct detection and quantification of high- and medium-copy markers, including PMMoV RNA, beta-lactamase, and carbapenemase DNA, in wastewater extracts is both feasible and accurate when benchmarked against gold-standard NAATs. Low-copy detection and monitoring daily trends of SARS-CoV-2 N RNA was achievable but was not as robust as NAATs. Due to the lower analytical sensitivity of NNS compared to NAATs and the requirement of a 18 h hybridization step, NNS may not be suitable to provide early warning of incident cases to public health. However, as the scope of wastewater surveillance expands to monitor a broad range of nucleic acid-based biomarkers, the target and sample multiplexing capability of NNS, combined with reduced hands-on time and ease-of-analysis, are distinct advantages. Thus, NNS has a role to play in wastewater and environmental monitoring applications, especially for AMR surveillance.

Clinical genomic surveillance is regarded as the gold standard for monitoring SARS-CoV-2 variants globally. However, as the pandemic wanes, reduced testing poses a risk to effectively tracking the trajectory of these variants within populations. Wastewater-based genomic surveillance that estimates variant frequency based on its defining set of alleles derived from clinical genomic surveillance has been successfully implemented. This method has its challenges, and allele-specific (AS) RT-qPCR or RT-dPCR may instead be used as a complementary method for estimating variant prevalence. Demonstrating equivalent performance of these methods is a prerequisite for their continued application in current and future pandemics. Here, we compared single-allele frequency using AS-RT-qPCR, to single-allele or haplotype frequency estimations derived from amplicon-based sequencing to estimate variant prevalence in wastewater during emergent and prevalent periods of Delta, Omicron, and two sub-lineages of Omicron. We found that all three methods of frequency estimation were concordant and contained sufficient information to describe the trajectory of variant prevalence. We further confirmed the accuracy of these methods by quantifying the diagnostic performance through Youden's index. The Youden's index of AS-RT-qPCR was reduced during the low prevalence period of a particular variant while the same allele in sequencing was negatively influenced due to insufficient read depth. Youden's index of haplotype-based calls was negatively influenced when alleles were common between variants. Coupling AS-RT-qPCR with sequencing can overcome the shortcomings of either platform and provide a comprehensive picture to the stakeholders for public health responses.

Passive sampling provides a cost-effective alternative to conventional auto-sampling and serves as a valuable approach for wastewater surveillance in resource-limited settings. However, the feasibility of passive sampling for SARS-CoV-2 wastewater genomic surveillance (WWGS) remains underexplored in wastewater resource recovery facility (WWRF). In this study, we collected influent wastewater samples using an autosampler, COSCa-ball and Torpedo passive samplers from inlet of WWRF serving the city of Ottawa, Canada. We enriched, extracted, quantified, and sequenced samples targeting SARS-CoV-2 genome using a widely used ARTIC tiled amplicon approach. Our findings show that daily SARS-CoV-2 RNA levels were similar (p > 0.05) across the sampling methods. Although similar viral RNA was captured from passive samples, we found genomic recovery of SARS-CoV-2 from passive samplers was influenced by targeted sequencing read length, with shorter (300 bp) reads resulting in lower recovery than longer (600 bp) reads. Our study confirmed near-complete recovery of SARS-CoV-2 genomes (≥ 90 %) from the autosampler, COSCa-ball, and Torpedo samplers using longer reads. Genome sequencing parameters such as the number of raw reads, trimmed reads, mapped reads, depth of coverage and % of genome coverage was identical (p > 0.05) among the sampling methods. Genomic analyses showed similar (p > 0.05) single nucleotide variant profiles (SNV) and lineage prevalence across sampling methods, and concordance with the available clinical surveillance. Overall, the findings suggest that passive sampling of wastewater is a viable, cost-effective alternative for population-scale genomic surveillance of SARS-CoV-2 and may allow for surveillance of other pathogens, supporting future pandemic preparedness efforts.

Passive sampling has proven to be a reliable and cost-effective method in wastewater and environmental surveillance (WES) during the COVID-19 pandemic. In passive sampling, wastewater solids were collected from the wastewater networks, homogenized in solutes and analyzed the supernatant to measure the SARS-CoV-2 RNA concentrations in wastewater. However, the direct impact of wastewater solids content collected via passive sampling on SARS-CoV-2 RNA measurements has not been previously evaluated. In this study, we analyzed wastewater solids collected using Auto, Torpedo, COSCa-ball samplers, and primary sludge samples from a wastewater treatment plant to measure SARS-CoV-2 RNA concentrations in wastewater. Results showed significant variation (p < 0.05) in wastewater solids content (i.e., TS and VS) across Auto, Torpedo, COSCa-ball samplers, and primary sludge samples. Despite differences in solids content, SARS-CoV-2 RNA concentrations in wastewater solids from passive samplers can be effectively compared (p > 0.05) to autosampler and primary sludge samples. To evaluate the influences of wastewater solids content on SARS-CoV-2 RNA measurement, we used a linear mixed-effects model. The model demonstrated that wastewater solids content had no direct effect on SARS-CoV-2 RNA measurements across the sampling methods and primary sludge samples. Overall, this study established a standardized experimental approach for implementing passive samplers as a viable alternative to conventional autosampler in WES for emerging pathogens.

Wastewater genomic surveillance (WWGS) of SARS-CoV-2 is typically performed using influent wastewater, but the approach is challenging due to degradation as well as low target concentrations in wastewater. This could be alleviated by utilizing primary sludge; however, this matrix is prone to sequencing library failures. Our study focuses on developing a robust primary sludge-based SARS-CoV-2 genome sequencing method. The study was conducted using 30 parallel influent wastewater and primary sludge samples collected during three different time periods, under three clinically predominant SARS-CoV-2 Omicron lineages in Ottawa, Canada. Results showed that our approach consistently recovered near-complete (≥90%) SARS-CoV-2 genomes from both influent wastewater and primary sludge samples. Prevalent lineage and single nucleotide variant (SNV) profiles were identical (p > 0.05) between influent wastewater and primary sludge. Further analysis indicated that a similar (p > 0.05) number of rare SNVs were detected between influent wastewater and primary sludge. Overall, our approach enables the sequencing of the most concentrated sources of genetic material within the wastewater matrix, providing valuable insights for public health forecasting of infectious disease prevalence beyond the COVID-19 pandemic.

ADVERTISEMENT RETURN TO ISSUEPREVViewpointNEXTA Call to Wastewater Researchers to Support Phage Therapy in the Global Fight against Antibiotic ResistanceOcean Thakali*Ocean ThakaliDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaChildren's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario K1H 8L1, Canada*[email protected]More by Ocean ThakaliView Biographyhttps://orcid.org/0000-0002-6649-2322, Shen WanShen WanDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaMore by Shen Wan, Md. Pervez KabirMd. Pervez KabirDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaMore by Md. Pervez Kabir, Xin TianXin TianDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaMore by Xin Tian, Patrick D'AoustPatrick D'AoustDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaMore by Patrick D'Aoust, Anand TiwariAnand TiwariExpert Microbiology Unit, Finnish Institute for Health and Welfare, 70701 Kuopio, FinlandMore by Anand Tiwari, Kyle BibbyKyle BibbyDepartment of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United StatesMore by Kyle Bibbyhttps://orcid.org/0000-0003-3142-6090, Samendra SherchanSamendra SherchanCenter of Research Excellence in Wastewater-based Epidemiology, Morgan State University, Baltimore, Maryland 21251, United StatesMore by Samendra Sherchan, Charles GerbaCharles GerbaWater & Energy Sustainable Technology Center, University of Arizona, Tucson, Arizona 85745, United StatesMore by Charles Gerba, Anthony William MaressoAnthony William MaressoTAILΦR Laboratories, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, United StatesMore by Anthony William Maresso, Tyson GraberTyson GraberChildren's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario K1H 8L1, CanadaMore by Tyson Graber, and Robert DelatollaRobert DelatollaDepartment of Civil Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, CanadaMore by Robert DelatollaCite this: ACS EST Water 2024, 4, 4, 1177–1179Publication Date (Web):February 7, 2024Publication History Received29 December 2023Accepted23 January 2024Revised20 January 2024Published online7 February 2024Published inissue 12 April 2024https://doi.org/10.1021/acsestwater.3c00844Copyright © 2024 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views766Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (2 MB) Get e-AlertscloseSUBJECTS:Antibiotic resistance,Bacteria,Therapeutics,Viruses,Wastewater Get e-Alerts

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Wastewater surveillance (WWS) of SARS-CoV-2 has become a crucial tool for monitoring COVID-19 cases and outbreaks. Previous studies have indicated that SARS-CoV-2 RNA measurement from testing solid-rich primary sludge yields better sensitivity compared to testing wastewater influent. Furthermore, measurement of pepper mild mottle virus (PMMoV) signal in wastewater allows for precise normalization of SARS-CoV-2 viral signal based on solid content, enhancing disease prevalence tracking. However, despite the widespread adoption of WWS, a knowledge gap remains regarding the impact of ferric sulfate coagulation, commonly used in enhanced primary clarification, the initial stage of wastewater treatment where solids are sedimented and removed, on SARS-CoV-2 and PMMoV quantification in wastewater-based epidemiology. This study examines the effects of ferric sulfate addition, along with the associated pH reduction, on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary clarified sludge through jar testing. Results show that the addition of Fe 3+ concentrations in the conventional 0 to 60 mg/L range caused no effect on SARS-CoV-2 N1 and N2 gene region measurements in wastewater solids. However, elevated Fe 3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV-normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). The observed pH reduction from coagulant addition did not contribute to the increased PMMoV measurements, suggesting that this phenomenon arises from the partitioning of PMMoV viral particles into wastewater solids.

Wastewater-based surveillance of human disease offers timely insights to public health, helping to mitigate infectious disease outbreaks and decrease downstream morbidity and mortality. These systems rely on nucleic acid amplification tests for monitoring disease trends, while antibody-based seroprevalence surveys gauge community immunity. However, serological surveys are resource-intensive and subject to potentially long lead times and sampling bias. We identified and characterized a human antibody repertoire, predominantly secretory IgA, isolated from a central wastewater treatment plant and building-scale wastewater collection points. These antibodies partition to the solids fraction and retain immunoaffinity for SARS-CoV-2 and Influenza A virus antigens. This stable pool could enable real-time tracking for correlates of vaccination, infection, and immunity, aiding in establishing population-level thresholds for immune protection and assessing the efficacy of future vaccine campaigns.

This study presents a comprehensive analysis of the decay patterns of endogenous SARS-CoV-2 and Pepper mild mottle virus (PMMoV) within wastewaters spiked with stool from infected patients expressing COVID-19 symptoms, and hence explores the decay of endogenous SARS-CoV-2 and PMMoV targets in wastewaters from source to collection of the sample. Stool samples from infected patients were used as endogenous viral material to more accurately mirror real-world decay processes compared to more traditionally used lab-propagated spike-ins. As such, this study includes data on early decay stages of endogenous viral targets in wastewaters that are typically overlooked when performing decay studies on wastewaters harvested from wastewater treatment plants that contain already-degraded endogenous material. The two distinct sewer transport conditions of dynamic suspended sewer transport and bed and near-bed sewer transport were simulated in this study at temperatures of 4 °C, 12 °C and 20 °C to elucidate decay under these two dominant transport conditions within wastewater infrastructure. The dynamic suspended sewer transport was simulated over 35 h, representing typical flow conditions, whereas bed and near-bed transport extended to 60 days to reflect the prolonged settling of solids in sewer systems during reduced flow periods. In dynamic suspended sewer transport, no decay was observed for SARS-CoV-2, PMMoV, or total RNA over the 35-h period, and temperature ranging from 4 °C to 20 °C had no noticeable effect. Conversely, experiments simulating bed and near-bed transport conditions revealed significant decreases in SARS-CoV-2 and total RNA concentrations by day 2, and PMMoV concentrations by day 3. Only PMMoV exhibited a clear trend of increasing decay constant with higher temperatures, suggesting that while temperature influences decay dynamics, its impact may be less significant than previously assumed, particularly for endogenous RNA that is bound to dissolved organic matter in wastewater. First order decay models were inadequate for accurately fitting decay curves of SARS-CoV-2, PMMoV, and total RNA in bed and near-bed transport conditions. F-tests confirmed the superior fit of the two-phase decay model compared to first order decay models across temperatures of 4 °C-20 °C. Finally, and most importantly, total RNA normalization emerged as an appropriate approach for correcting the time decay of SARS-CoV-2 exposed to bed and near-bed transport conditions. These findings highlight the importance of considering decay from the point of entry in the sewers, sewer transport conditions, and normalization strategies when assessing and modelling the impact of viral decay rates in wastewater systems. This study also emphasizes the need for ongoing research into the diverse and multifaceted factors that influence these decay rates, which is crucial for accurate public health monitoring and response strategies.

Leukemia inhibitory factor, a member of the interleukin-6 cytokine family, plays a central role in homeostasis and disease. Interestingly, some of the pleiotropic effects of leukemia inhibitory factor have been attributed to the modulation of macrophage functions although the molecular underpinnings have not been explored at a genome-wide scale. Herein, we investigated leukemia inhibitory factor-driven transcriptional changes in murine bone marrow-derived macrophages by RNA sequencing. In silico analyses revealed a selective and time-dependent remodeling of macrophage gene expression programs associated with lipid metabolism and cell activation. Accordingly, a subset of leukemia inhibitory factor-upregulated transcripts related to cholesterol metabolism and lipid internalization was validated by real-time quantitative polymerase chain reaction. This was accompanied by a leukemia inhibitory factor-enhanced capacity for lipid accumulation in macrophages upon incubation with oxidized low-density lipoprotein. Mechanistically, leukemia inhibitory factor triggered the phosphorylation (Y705 and S727) and nuclear translocation of the transcription factor STAT3 in bone marrow-derived macrophages. Consistent with this, ingenuity pathway analysis identified STAT3 as an upstream regulator of a subset of transcripts, including Il4ra, in leukemia inhibitory factor-treated macrophages. Notably, leukemia inhibitory factor priming enhanced bone marrow-derived macrophage responses to interleukin-4-mediated M2 polarization (i.e. increased arginase activity and accumulation of transcripts encoding for M2 markers). Conversely, leukemia inhibitory factor stimulation had no significant effect in bone marrow-derived macrophage responses to M1-polarizing stimuli (interferon-gamma and lipopolysaccharide). Thus, our study provides insight into the transcriptional landscape of leukemia inhibitory factor-treated macrophages, shedding light on its role in lipid metabolism and M2 polarization responses. A better understanding of the regulatory mechanisms governing leukemia inhibitory factor-driven changes might help informing novel therapeutic approaches aiming to reprogram macrophage phenotypes in diseased states (e.g. cancer, atherosclerosis, and infection). Leukemia inhibitory factor (LIF) promotes a transcriptional signature that enhances macrophage alternative activation and lipid accumulation.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Wastewater-based surveillance is a valuable approach for monitoring COVID-19 at community level. Monitoring SARS-CoV-2 variants of concern (VOC) in wastewater has become increasingly relevant when clinical testing capacity and case-based surveillance are limited. In this study, we ascertained the turnover of six VOC in Alberta wastewater from May 2020 to May 2022. Wastewater samples from nine wastewater treatment plants across Alberta were analysed using VOC-specific RT-qPCR assays. The performance of the RT-qPCR assays in identifying VOC in wastewater was evaluated against next generation sequencing. The relative abundance of each VOC in wastewater was compared to positivity rate in COVID-19 testing. VOC-specific RT-qPCR assays performed comparatively well against next generation sequencing; concordance rates ranged from 89% to 98% for detection of Alpha, Beta, Gamma, Omicron BA.1 and Omicron BA.2, with a slightly lower rate of 85% for Delta (p < 0.01). Elevated relative abundance of Alpha, Delta, Omicron BA.1 and BA.2 were each associated with increased COVID-19 positivity rate. Alpha, Delta and Omicron BA.2 reached 90% relative abundance in wastewater within 80, 111 and 62 days after their initial detection, respectively. Omicron BA.1 increased more rapidly, reaching a 90% relative abundance in wastewater after 35 days. Our results from VOC surveillance in wastewater correspond with clinical observations that Omicron is the VOC with highest disease burden over the shortest period in Alberta to date. The findings suggest that changes in relative abundance of a VOC in wastewater can be used as a supplementary indicator to track and perhaps predict COVID-19 burden in a population.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Extracellular vesicles (EVs) are entering the clinical arena as novel biologics for infectious diseases, potentially serving as the immunogenic components of next generation vaccines. However, relevant human assays to evaluate the immunogenicity of EVs carrying viral antigens are lacking, contributing to challenges in translating rodent studies to human clinical trials. Here, we engineered EVs to carry SARS-CoV-2 Spike to evaluate the immunogenicity of antigen-carrying EVs using human peripheral blood mononuclear cells (PBMCs). Delivery of Spike EVs to PBMCs resulted in specific immune cell activation as assessed through T cell activation marker expression. Further, Spike EVs were taken up largely by antigen-presenting cells (monocytes, dendritic cells and B cells). Taken together, this human PBMC-based system models physiologically relevant pathways of antigen delivery, uptake and presentation. In summary, the current study highlights the suitability of using human PBMCs for evaluating the immunogenicity of EVs engineered to carry antigens for infectious disease therapeutics.