Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada.

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

Tracking Emergence and Spread of SARS-CoV-2 Omicron Variant in Large and Small Communities by Wastewater Monitoring in Alberta, Canada.

Wastewater monitoring of SARS-CoV-2 enables early detection and monitoring of the COVID-19 disease burden in communities and can track specific variants of concern. We determined proportions of the Omicron and Delta variants across 30 municipalities covering >75% of the province of Alberta (population 4.5 million), Canada, during November 2021-January 2022. Larger cities Calgary and Edmonton exhibited more rapid emergence of Omicron than did smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a medium-sized northern community that has many workers who fly in and out regularly. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden by late December, before the peak in newly diagnosed clinical cases throughout Alberta in mid-January. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of SARS-CoV-2 variants.

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada.

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Regenerative NanoOctopus Based on Multivalent-Aptamer-Functionalized Magnetic Microparticles for Effective Cell Capture in Whole Blood.

Isolation of specific rare cell subtypes from whole blood is critical in cellular analysis and important in basic and clinical research. Traditional immunomagnetic cell capture suffers from suboptimal sensitivity, specificity, and time- and cost-effectiveness. Mimicking the features of octopuses, a device termed a "NanoOctopus" was developed for cancer cell isolation in whole blood. The device consists of long multimerized aptamer DNA strands, or tentacle DNA, immobilized on magnetic microparticle surfaces. Their ultrahigh sensitivity and specificity are attributed to multivalent binding of the tentacle DNA to cell receptors without steric hindrance. The simple, quick, and noninvasive capture and release of the target cells allows for extensive downstream cellular and molecular analysis, and the time- and cost-effectiveness of fabrication and regeneration of the devices makes them attractive for industrial manufacture.

How Does Reference Site Selection Influence Interpretation of Omics Data?: Evaluating Liver Transcriptome Responses in Male Rainbow Darter (Etheostoma caeruleum) across an Urban Environment.

Studies quantifying the influence of reference site selection on transcriptomic profiles in aquatic organisms exposed to complex mixtures are lacking in the literature, despite the significant implications of such research for the interpretation of omics data sets. We measured hepatic transcriptomic responses in fish across an urban environment in the central Grand River watershed (Ontario, Canada). Adult male rainbow darter (RBD) (Etheostoma caeruleum) were collected from nine sites at varying distances from two major municipal wastewater treatment plants (MWWTPs) (Waterloo, Kitchener), including three upstream reference sites. The transcriptomic response in RBD was independently compared with that of fish from each of the three reference sites. Data collected in fish downstream of the Waterloo MWWTP (poorest effluent quality) suggested that ∼15.5% of the transcriptome response was influenced by reference site selection. In contrast, at sites where the impact of MWWTPs was less-pronounced and fish showed less of a transcriptome response, reference site selection had a greater influence (i.e., ∼56.9% of transcripts were different depending on the site used). This study highlights the importance of conducting transcriptomics studies that leverage more than one reference site, and it broadens our understanding of the molecular responses in fish in dynamic natural environments.

Reduction of Intersex in a Wild Fish Population in Response to Major Municipal Wastewater Treatment Plant Upgrades.

Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.

Distribution, Partitioning and Bioaccumulation of Substituted Diphenylamine Antioxidants and Benzotriazole UV Stabilizers in an Urban Creek in Canada.

Substituted diphenylamine antioxidants (SDPAs) and benzotriazole UV stabilizers (BZT-UVs), previously under reported classes of organic contaminants, were determined in sediment, water, and freshwater biota in an urban creek in Canada. SDPAs and BZT-UVs were frequently detected in all matrices including upstream of the urban area in a rural agricultural/woodlot region, suggesting a ubiquitous presence and bioaccumulation of these emerging contaminants. Spatial comparisons were characterized by higher levels of SDPAs downstream compared with the upstream, implying a possible influence of the urban activities on the antioxidant contamination in the sampling area. In sediment, 4,4'-bis(α,α-dimethylbenzyl)-diphenylamine (diAMS), dioctyl-diphenylamine (C8C8), and dinonyl-diphenylamine (C9C9) were the most dominant congeners of SDPAs, with concentrations up to 191 ng/g (dry weight, d.w.). Benthic invertebrates Crayfish (Orcoescties spp.) had larger body burdens of SDPAs and BZT-UVs compared to pelagic fish (hornyhead chub (Nocomis biguttatus) and common shiner (Luxilus cornutus)) in the creek and partitioning coefficients demonstrated that sediment was the major reservoir of these contaminants. This is the first report of bioaccumulation and partitioning behaviors of SDPAs and BZT-UVs in freshwater environments.

Assessing the bioremediation potential of algal species indigenous to oil sands process-affected waters on mixtures of oil sands acid extractable organics.

Surface mining extraction of bitumen from oil sand in Alberta, Canada results in the accumulation of oil sands process-affected water (OSPW). In attempts to maximize water recycling, and because its constituents are recognized as being toxic, OSPW is retained in settling basins. Consequently, research efforts are currently focused on developing remediation strategies capable of detoxifying OSPW to allow for eventual release. One potential bioremediation strategy proposes to utilize phytoplankton native to the Alberta oil sand region to sequester, break down, or modify the complex oil sands acid extractable organic (AEO) mixtures in OSPW. Preliminary attempts to quantify changes in total oil sands AEO concentration in test solutions by ESI-MS following a 14-day algal remediation period revealed the presence of unknown organic acids in control samples, likely released by the phytoplankton strains and often of the same atomic mass range as the oil sands AEO under investigation. To address the presence of these "biogenic" organic acids in test samples, ESI-MS in MRM mode was utilized to identify oil sands AEO "marker ions" that were a) present within the tested oil sands AEO extract and b) unique to the oil sands AEO extract only (e.g. atomic masses different from biogenic organic acids). Using this approach, one of the 21 tested algal strains, Stichococcus sp. 1, proved capable of significantly reducing the AEO marker ion concentration at test concentrations of 10, 30, and 100mgL(-1). This result, along with the accelerated growth rate and recalcitrance of this algal strain with exposure to oil sands AEO, suggests the strong potential for the use of the isolated Stichococcus sp. 1 as a candidate for bioremediation strategies.

Adsorption of DNA oligonucleotides by titanium dioxide nanoparticles.

Titanium dioxide (TiO2) or titania shows great promise in detoxification and drug delivery. To reach its full potential, it is important to interface TiO2 with biomolecules to harness their molecular recognition function. To this end, DNA attachment is an important topic. Previous work has mainly focused on long double-stranded DNA or single nucleotides. For biosensor development and targeted drug delivery, it is more important to use single-stranded oligonucleotides. Herein, the interaction between fluorescently labeled oligonucleotides and TiO2 nanoparticles is reported. The point of zero charge (PZC) of TiO2 is around 6 in water or acetate buffer; therefore, the particles are positively charged at lower pH. However, if in phosphate or citrate buffer, the particles are negatively charged, even at pH ∼2, suggesting strong adsorption of buffer anions. DNA adsorption takes place mainly via the phosphate backbone, although the bases might also have moderate contributions. Peptide nucleic acids (PNAs) with an amide backbone cannot be adsorbed. DNA adsorption is strongly affected by inorganic anions, where phosphate and citrate can strongly inhibit DNA adsorption. DNA adsorption is promoted by adding salt or lowering pH. DNA adsorption is accompanied with fluorescence quenching, and double-stranded DNA showed reduced quenching, allowing for the detection of DNA using TiO2 nanoparticles.

Venlafaxine exposure alters mitochondrial respiration and mitomiR abundance in zebrafish brains.

Wastewater treatment plant (WWTP) effluent often releases pharmaceuticals like venlafaxine (a serotonin-norephinephrine reuptake inhibitor antidepressant) to freshwater ecosystems at levels causing adverse metabolic effects on fish. Changes to fish metabolism can be regulated by epigenetic mechanisms like microRNA (small RNA molecules that regulate mRNA translation), including regulating mitochondrial mRNAs. Nuclear-encoded microRNAs regulate mitochondrial gene expression in mammals, and have predicted effects in fish. We aimed to identify whether venlafaxine exposure changed mitochondrial respiration and resulted in differentially abundant mitochondrial microRNA (mitomiRs) in zebrafish brains. In vitro exposure of brain homogenate to below environmentally relevant concentrations of venlafaxine (<1 µg/L) caused a decrease in mitochondrial respiration, although this was not driven by changes to mitochondrial Complex I or II function. To identify whether these effects occur in vivo, zebrafish were exposed to 1 µg/L venlafaxine for 0, 1, 6, 12, 24, and 96 h. In vivo, venlafaxine exposure had no significant effects on brain mitochondrial respiration; however, select mitomiRs (dre-miR-301a-5p, dre-miR-301b-3p, and dre-miR-301c-3p) were also measured, because they were bioinformatically predicted to regulate mitochondrial cytochrome c oxidase subunit I (COI) abundance. These mitomiRs were differentially regulated based on venlafaxine exposure (with miR-301c-3p abundance differing during the day and miR-301b-3p being lower in exposed fish at night), and with respect to sex and time sampled. Overall, the results demonstrated that in vitro venlafaxine exposure to zebrafish brain caused a decrease in mitochondrial respiration, but these effects were not seen after acute in vivo exposure. Results may have differed because in vivo exposure allows for fish to mitigate effects through mechanisms that could include mitomiR regulation, and because fish were only acutely exposed. Environ Toxicol Chem 2024;43:1569-1582. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Impacts of caffeine on fathead minnow behaviour and physiology.

Pollution from regularly used substances such as pharmaceuticals, cleaning agents, and even food and beverages is an increasing problem in the environment. Caffeine, a commonly ingested stimulant, is one such contaminant that has been detected in aquatic environments worldwide. Yet, little is known about how ecologically relevant concentrations of caffeine influence the morphology, behaviour, and physiology of exposed organisms. To address this knowledge gap, we exposed fathead minnow (Pimephales promelas) to three caffeine treatments: a freshwater control (nominal: 0 ng/L), a low (nominal: 1,000 ng/L) and high environmentally relevant dose (nominal: 10,000 ng/L), for 35 days. We tested the learning abilities, anxiety, metabolic rates, and morphological features of exposed vs. control fish. Caffeine exposure did not affect the ability of fish to learn but did influence anxiety levels. Over the course of repeated anxiety testing, unexposed control fish visited a black square more often while fish exposed to low levels of caffeine did not, potentially indicating that these fish remained in a more anxious state. While caffeine did not impact metabolism, fish growth, or body size, it was associated with lower liver investment-although this response was only observed in our low caffeine treatment. Overall, our results suggest that even relatively low concentrations of caffeine may impact the liver size and anxiety of exposed fish, but further research is needed to assess how extended exposure to caffeine impacts fitness. Given the increase in anthropogenic contaminants in aquatic environments, it is important that we continue to investigate their effects on the organisms exposed to them.

Identifying transcriptomic indicators of tertiary treated municipal effluent in longnose dace (Rhinichthys cataractae) caged under semi-controlled conditions in experimental raceways.

To evaluate effects of tertiary treated wastewater treatment plant effluent (MWWE) on transcriptomic responses in longnose dace (Rhinichthys cataractae; LND) we conducted a semi-controlled study in experimental raceways (Advancing Canadian Water Assets facility) imbedded in the Pine Creek treatment plant (Calgary, AB). LND collected from a reference site in the Bow River (REF) were caged in raceways containing either 5 % Pine Creek effluent (PC) or Bow River water (BR; control) over 28 d. Liver transcriptomes were analyzed in males and females sampled on days 7, 14 and 28 from BR and PC, and compared to REF fish on day 0. Concurrent with the caging, selected environmental substances of concern were analyzed in the BR and PC. Significantly different unigenes (SDUs) in females (vs males) within both BR and PC raceways increased over time and compared to REF fish. Moreover, SDUs in females and males within the same treatment (i.e., BR, PC) showed a temporal increase as well as compared to REF fish. Time was the dominant factor affecting SDUs, whereas sex and treatment had less of an impact on the transcriptome profiling. Gene Set Enrichment Analysis of BR vs PC over time revealed effects on genes involved in growth, metabolism of carbohydrates and lipids, and immune system on day 7; however, by day 28, 80-100 % of the transcripts localized to enriched biomarkers were associated with tissue immune responses in both sexes. Exposure to 5 % effluent had significant effects on female liver somatic index but no effects were observed on other phenotypic health indices in either sex. BR was used as the source of reference water, but analyses showed trace amounts of ESOCs. Analyses did not point towards definitive response patterns that could be used in field-based ecotoxicogenomic studies on the impacts of well-treated MWWE but suggested compromised adaptive immune responses.

Implementing an adaptive, two-tiered SARS-CoV-2 wastewater surveillance program on a university campus using passive sampling.

Building-level wastewater-based surveillance (WBS) has been increasingly applied upstream from wastewater treatment plants to conduct targeted monitoring for SARS-CoV-2. In this study, a two-tiered, trigger-based wastewater surveillance program was developed on a university campus to monitor dormitory wastewater. The objective was to determine if passive sampling with cotton gauze as a sampling medium could be used to support institution-level public health action. Two nucleocapsid gene targets (N1 and N2) of SARS-CoV-2 as well as the endogenous fecal indicator pepper mild mottle virus (PMMoV) were quantified using RT-qPCR. >500 samples were analyzed during two contrasting surveillance periods. In the Fall of 2021 community viral burden was low and a tiered sampling network was able to isolate individual clinical cases at the building-scale. In the Winter of 2022 wastewater signals were quickly elevated by the emergence of the highly transmissible SARS-CoV-2 Omicron (B.1.1.529) variant. Prevalence of SARS-CoV-2 shifted surveillance objectives from isolating cases to monitoring trends, revealing both the benefits and limitations of a tiered surveillance design under different public health situations. Normalization of SARS-CoV-2 by PMMoV was not reflective of upstream population differences, suggesting saturation of the material occurred during the exposure period. The passive sampling method detected nearly all known clinical cases and in one instance was able to identify one pre-symptomatic individual days prior to confirmation by clinical test. Comparisons between campus samplers and municipal wastewater influent suggests that the spread of COVID-19 on the campus was similar to that of the broader community. The results demonstrate that passive sampling is an effective tool that can produce semi-quantitative data capable of tracking temporal trends to guide targeted public health decision-making at an institutional level. Practitioners of WBS can utilize these results to inform surveillance program designs that prioritize efficient resource use and rapid reporting.

Microparticles in Wild and Caged Biota, Sediments, and Water Relative to Large Municipal Wastewater Treatment Plant Discharges.

Anthropogenically modified microparticles including microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether biotic exposures are elevated downstream of these outfalls. In the fall of 2019, the present study examined whether microparticle levels in resident fish, environmental samples, and caged organisms were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River, Ontario, Canada. Wild rainbow darters (Etheostoma caeruleum) were collected from a total of 10 sites upstream and downstream of both WWTPs, along with surface water and sediment samples to assess spatial patterns over an approximately 70-km river stretch. Amphipods (Hyalella azteca), fluted-shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were also caged upstream and downstream of one WWTP for 14 or 28 days. Whole amphipods, fish digestive tracts, and mussel tissues (hemolymph, digestive glands, gills) were digested with potassium hydroxide, whereas environmental samples were processed using filtration and density separation. Visual identification, measurement, and chemical confirmation (subset only) of microparticles were completed. Elevated abiotic microparticles were found at several upstream reference sites as well as at one or both wastewater-impacted sites. Microparticles in amphipods, all mussel tissues, and wild fish did not show patterns indicative of increased exposures downstream of effluent discharges. In contrast, elevated microparticle counts were found in trout caged directly downstream of the outfall. Across all samples, cellulose fibers (mainly blue and clear colors) were the most common. Overall, results suggest little influence of WWTP effluents on microparticles in biota but rather a ubiquitous presence across most sites that indicates the importance of other point and nonpoint sources to this system. Environ Toxicol Chem 2024;43:1047-1061. © 2024 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Accelerated cascade melanoma therapy using enzyme-nanozyme-integrated dissolvable polymeric microneedles.

Dissolvable polymeric microneedles (DPMNs) have emerged as a powerful technology for the localized treatment of diseases, such as melanoma. Herein, we fabricated a DPMN patch containing a potent enzyme-nanozyme composite that transforms the upregulated glucose consumption of cancerous cells into lethal reactive oxygen species via a cascade reaction accelerated by endogenous chloride ions and external near-infrared (NIR) irradiation. This was accomplished by combining glucose oxidase (Gox) with a NIR-responsive chloroperoxidase-like copper sulfide (CuS) nanozyme. In contrast with subcutaneous injection, the microneedle system highly localizes the treatment, enhancing nanomedicine uptake by the tumor and reducing its systemic exposure to the kidneys and spleen. NIR irradiation further controls the potency and toxicity of the formulation by thermally disabling Gox. In a mouse melanoma model, this unique combination of photothermal, starvation, and chemodynamic therapies resulted in complete tumor eradication (99.2 ± 0.8 % reduction in tumor volume within 10 d) without producing signs of systemic toxicity. By comparison, other treatment combinations only resulted in a 42-76.5 % reduction in tumor growth. The microneedle patch design is therefore not only highly potent but also with regulated toxicity and improved safety.

Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant.

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid-liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2. •Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect.

SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

Polarity control for nonthiolated DNA adsorption onto gold nanoparticles.

Gold nanoparticles (AuNPs) functionalized with thiolated DNA have enabled many studies in nanoscience. The strong thiol/gold affinity and the nanoscale curvature of AuNPs allow the attached DNA to adapt an upright conformation favorable for hybridization. Recently, it has been shown that nonthiolated DNA can also be attached via DNA base adsorption. Without a thiol label, both ends of the DNA and even internal bases could be adsorbed, decreasing the specificity of subsequent molecular recognition reactions. In this work, we employed a modular sequence design approach to systematically study the effect of DNA sequence on adsorption polarity. A block of poly adenine (poly-A) could be used to achieve a high density of DNA attachment. When the poly-A block length is short (e.g., below 5-7), the loading was independent of the block length, and the conjugate cannot hybridize to its cDNA effectively, suggesting a random attachment controlled by adsorption kinetics. Increasing the block length leads to reduced capacity but improved hybridization, suggesting that more DNA with the desired conformation was adsorbed due to the thermodynamic effects of poly-A binding. The design can be further improved by including capping sequences rich in T or G. Finally, a more general double-stranded DNA approach was described to be suitable for DNA that cannot satisfy the above-mentioned design requirements.