Wastewater surveillance monitoring of SARS-CoV-2 variants of concern and dynamics of transmission and community burden of COVID-19.

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.

Number of COVID-19 cases required in a population to detect SARS-CoV-2 RNA in wastewater in the province of Alberta, Canada: Sensitivity assessment.

With a unique and large size of testing results of 1,842 samples collected from 12 wastewater treatment plants (WWTP) for 14 months through from low to high prevalence of COVID-19, the sensitivity of RT-qPCR detection of SARS-CoV-2 RNA in wastewater that correspond to the communities was computed by using Probit analysis. This study determined the number of new COVID-19 cases per 100,000 population required to detect SARS-CoV-2 RNA in wastewater at defined probabilities and provided an evidence-based framework of wastewater-based epidemiology surveillance (WBE). Input data were positive and negative test results of SARS-CoV-2 RNA in wastewater samples and the corresponding new COVID-19 case rates per 100,000 population served by each WWTP. The analyses determined that RT-qPCR-based SARS-CoV-2 RNA detection threshold at 50%, 80% and 99% probability required a median of 8 (range: 4-19), 18 (9-43), and 38 (17-97) of new COVID-19 cases /100,000, respectively. Namely, the positive detection rate at 50%, 80% and 99% probability were 0.01%, 0.02%, and 0.04% averagely for new cases in the population. This study improves understanding of the performance of WBE SARS-CoV-2 RNA detection using the large datasets and prolonged study period. Estimated COVID-19 burden at a community level that would result in a positive detection of SARS-CoV-2 in wastewater is critical to support WBE application as a supplementary warning/monitoring system for COVID-19 prevention and control.

Does normalization of SARS-CoV-2 concentrations by Pepper Mild Mottle Virus improve correlations and lead time between wastewater surveillance and clinical data in Alberta (Canada): comparing twelve SARS-CoV-2 normalization approaches.

Wastewater-based surveillance (WBS) data normalization is an analyte measurement correction that addresses variations resulting from dilution of fecal discharge by non-sanitary sewage, stormwater or groundwater infiltration. No consensus exists on what WBS normalization parameters result in the strongest correlations and lead time between SARS-CoV-2 WBS data and COVID-19 cases. This study compared flow, population size and biomarker normalization impacts on the correlations and lead times for ten communities in twelve sewersheds in Alberta (Canada) between September 2020 and October 2021 (n = 1024) to determine if normalization by Pepper Mild Mottle Virus (PMMoV) provides any advantages compared to other normalization parameters (e.g., flow, reported and dynamic population sizes, BOD, TSS, NH3, TP). PMMoV concentrations (GC/mL) corresponded with plant influent flows and were highest in the urban centres. SARS-CoV-2 target genes E, N1 and N2 were all negatively associated with wastewater influent pH, while PMMoV was positively associated with temperature. Pooled data analysis showed that normalization increased ρ-values by almost 0.1 and was highest for ammonia, TKN and TP followed by PMMoV. Normalization by other parameters weakened associations. None of the differences were statistically significant. Site-specific correlations showed that normalization of SARS-CoV-2 data by PMMoV only improved correlations significantly in two of the twelve systems; neither were large sewersheds or combined sewer systems. In five systems, normalization by traditional wastewater strength parameters and dynamic population estimates improved correlations. Lead time ranged between 1 and 4 days in both pooled and site-specific comparisons. We recommend that WBS researchers and health departments: a) Investigate WWTP influent properties (e.g., pH) in the WBS planning phase and use at least two parallel approaches for normalization only if shown to provide value; b) Explore normalization by wastewater strength parameters and dynamic population size estimates further; and c) Evaluate purchasing an influent flow meter in small communities to support long-term WBS efforts and WWTP management.

Wastewater-Based Surveillance Is an Effective Tool for Trending COVID-19 Prevalence in Communities: A Study of 10 Major Communities for 17 Months in Alberta.

The correlations between SARS-CoV-2 RNA levels in wastewater from 12 wastewater treatment plants and new COVID-19 cases in the corresponding sewersheds of 10 communities were studied over 17 months. The analysis from the longest continuous surveillance reported to date revealed that SARS-CoV-2 RNA levels correlated well with temporal changes of COVID-19 cases in each community. The strongest correlation was found during the third wave (r = 0.97) based on the population-weighted SARS-CoV-2 RNA levels in wastewater. Different correlations were observed (r from 0.51 to 0.86) in various sizes of communities. The population in the sewershed had no observed effects on the strength of the correlation. Fluctuation of SARS-CoV-2 RNA levels in wastewater mirrored increases and decreases of COVID-19 cases in the corresponding community. Since the viral shedding to sewers from all infected individuals is included, wastewater-based surveillance provides an unbiased and no-discriminate estimation of the prevalence of COVID-19 compared with clinical testing that was subject to testing-seeking behaviors and policy changes. Wastewater-based surveillance on SARS-CoV-2 represents a temporal trend of COVID-19 disease burden and is an effective and supplementary monitoring when the number of COVID-19 cases reaches detectable thresholds of SARS-CoV-2 RNA in wastewater of treatment facilities serving various sizes of populations.

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.

Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater.

Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology. In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E). Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%). Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

Effect of a Compound Energy Field with Temperature and Ultrasonic Vibration on the Material Properties and Bending Process of TC2 Titanium Alloy.

Due to the low formability and forming quality of titanium alloy, the forming process of a compound energy field (CEF) with temperature and ultrasonic vibration was proposed. Tensile tests were carried out to investigate the effect of the CEF on the true stress-strain curve, yield strength, elastic modulus, and other mechanical properties of the TC2 titanium alloy. Bending tests assisted by CEF were also performed to investigate the effect of different parameters of the CEF on bending force, spring-back, bending fillet radius, and microstructure of TC2 titanium. The results demonstrate that compared to the process under a single-temperature field, the CEF can reduce yield strength, elastic modulus, bending force, bending fillet, and the spring-back angle, which shows that the CEF can further increase the high-temperature softening effect of TC2 titanium. Furthermore, this effect becomes more remarkable when ultrasonic vibration energy increases. As a result, the formability of titanium alloy can be improved.

Early warning and rapid public health response to prevent COVID-19 outbreaks in long-term care facilities (LTCF) by monitoring SARS-CoV-2 RNA in LTCF site-specific sewage samples and assessment of antibodies response in this population: prospective study protocol.

INTRODUCTION: The COVID-19 pandemic has an excessive impact on residents in long-term care facilities (LTCF), causing high morbidity and mortality. Early detection of presymptomatic and asymptomatic COVID-19 cases supports the timely implementation of effective outbreak control measures but repetitive screening of residents and staff incurs costs and discomfort. Administration of vaccines is key to controlling the pandemic but the robustness and longevity of the antibody response, correlation of neutralising antibodies with commercial antibody assays, and the efficacy of current vaccines for emerging COVID-19 variants require further study. We propose to monitor SARS-CoV-2 in site-specific sewage as an early warning system for COVID-19 in LTCF and to study the immune response of the staff and residents in LTCF to COVID-19 vaccines. METHODS AND ANALYSIS: The study includes two parts: (1) detection and quantification of SARS-CoV-2 in LTCF site-specific sewage samples using a molecular assay followed by notification of Public Health within 24 hours as an early warning system for appropriate outbreak investigation and control measures and cost-benefit analyses of the system and (2) testing for SARS-CoV-2 antibodies among staff and residents in LTCF at various time points before and after COVID-19 vaccination using commercial assays and neutralising antibody testing performed at a reference laboratory. ETHICS AND DISSEMINATION: Ethics approval was obtained from the University of Alberta Health Research Ethics Board with considerations to minimise risk and discomforts for the participants. Early recognition of a COVID-19 case in an LTCF might prevent further transmission in residents and staff. There was no direct benefit identified to the participants of the immunity study. Anticipated dissemination of information includes a summary report to the immunity study participants, sharing of study data with the scientific community through the Canadian COVID-19 Immunity Task Force, and prompt dissemination of study results in meeting abstracts and manuscripts in peer-reviewed journals.

The prevalence and levels of enteric viruses in groundwater of private wells in rural Alberta, Canada.

The prevalence and levels of enteric viruses in untreated groundwater of private wells used for drinking and/or agricultural practices in rural Alberta were studied using the qPCR panel assay, integrated cell culture with qPCR and cell culture in the volume of 500 liters per sample through serial sampling. Seven viruses were assessed including adenovirus, rotavirus, norovirus, astrovirus, sapovirus, reovirus and JC virus. Five viruses were detected with an overall positive detection rate of 6.33 % (45 of 711 samples). The most frequently detected virus was adenovirus (48.9%, 22/45) followed by rotavirus (44.4%, 20/45), reovirus (20%, 9/45), JC virus (6.7%, 3/45) and norovirus (6.7%, 3/45). There was no significant difference in the positive detection rates, ranging from 1.1% to 3.4% by various well settings used for broiler farms, cow/calf farms, feedlots and rural acreages. Effects of well characteristics (aquifer type, well depth, static level of water, well seal) and well completion lithology on potential viral contamination of groundwater of private wells were also analyzed upon available data. The findings demonstrate that occurrence of enteric viruses is low and viral contamination is sporadic in groundwater of private wells in rural Alberta. Conventional fecal bacterial indicators (coliform and/or E. coli) were not a representative marker for viral contamination in groundwater wells in rural Alberta.

Prevalence, levels and seasonal variations of human enteric viruses in six major rivers in Alberta, Canada.

The prevalence and seasonal variation of 7 viruses in 6 major rivers in Alberta were assessed using a combination of qPCR, cell culture and integrated cell culture with qPCR (ICC-qPCR). Water samples were collected monthly from rivers at different sites upstream and downstream of major urban centers. Seven viruses including rotavirus, adenovirus, astrovirus, norovirus, sapovirus, JC virus and enterovirus, were detected in at least one of the water samples at each site using qPCR. Rotavirus was most common with concentration ranging from 2.3 to 4.5 log10 genomic equivalent (GE) copies/L. Norovirus, sapovirus, astrovirus, adenoviruses and JC virus peaked during the winter (November to March). Viruses were most prevalent at the Bow River sampling site downstream of the City of Calgary, followed by the North Saskatchewan River site downstream of the City of Edmonton and the Red Deer River site downstream of the City of Red Deer. The detection rates and quantity of viruses had significant difference in the sampling sites between upstream and downstream of major urban centers (p < 0.001). 14% of the samples tested positive using viral culture indicating the presence of infectious viruses in river. Sequencing analysis identified human rotavirus in 75% of the samples collected from downstream versus 37% of the samples collected from upstream sites (p < 0.02). Multivariate binary regression showed that human activity in watersheds is a significant determinant of viruses in Alberta's Rivers. The discharge from wastewater treatment plants may be the possible sources of viral contamination. Seasonal coincidence of acute viral gastroenteritis outbreaks and monthly peak occurrence of enteric viruses in river water implies potential impact of waterborne viruses on human health.

The role of the sexual partner in managing erectile dysfunction.

Erectile dysfunction (ED) has detrimental social and psychological effects on the quality of life of affected individuals and their sexual partners. When medical intervention is introduced to treat ED, physicians, nurses, and clinical educators should consider this disorder as a shared health problem for the men with ED and their sexual partners. New therapeutics such as phosphodiesterase-5 inhibitors improve erectile function in affected men, and the ultimate goal of medical intervention for ED should be achievement of a satisfactory sex life for couples engaged sexual relationships. Sexual partners of men with ED have an important role in its management and improvement in quality of sex life; therefore, they should be involved in assessment of, diagnosis, education, counselling, and choice of therapy. This sexual- partner-engaged approach might assist treatment and rehabilitation, helping the couples affected by ED to achieve a high-quality sex life.

Evaluation of the inactivation of human Coxsackievirus by thermophilic and mesophilic anaerobic digestion using integrated cell culture and reverse transcription real-time quantitative PCR.

The virucidal effects of anaerobic digestion were evaluated using human Coxsackievirus as a model for the Enterovirus family. Coxsackievirus was inactivated completely by thermophilic anaerobic digestion (TAD). By 4 h no living and infectious virus remained and no detectable viral RNA was present after 2 days in TAD (7.0 log reduction). Compared to TAD, 2.6 log reduction of viral RNA was achieved by 14 days in mesophilic anaerobic digestion (MAD) (p < 0.0001). Although cytopathogenic effect was not observed in the cultured cells, low levels of intracellular viral RNA were detected after one day of MAD treatment indicating that Coxsackievirus had infected the cells but could not replicate. The combination of thermal and biochemical effects in TAD plays a critical role for viral disinfection. The results of this study indicate that selection of the right configuration of anaerobic digestion for treatment of biowaste may reduce the risk of viral contamination to the environment and water source.

Evaluation of the matrix effect of thermophilic anaerobic digestion on inactivation of infectious laryngotracheitis virus using real-time PCR and viral cell culture.

The matrix effect of the thermophilic anaerobic digestion (TAD) process on inactivation of infectious laryngotracheitis virus (ILTV) was evaluated. Viral cell culture and real-time PCR were used for assessing removal of the viral infectivity and degradation of viral DNA, respectively. Results showed that the TAD-derived matrix alone can inactivate the virus and destroy the nucleic acid helix core of ILTV in a time-and- dose-dependent manner. No cytopathogenic effect (CPE) was observed in the cells exposed to ILTV pre-treated with TAD matrix for 1.5h in experiment 1 and for 16h in experiment 2. There was a significant statistical difference between TAD matrix treated and non-treated cultures (p<0.001, Chi-test). Amplifiable ILT viral DNA was reduced 2.27 log by 1.5h-treatment and was not present by 16h-treatment with TAD matrix, indicating complete viral DNA fragmentation. The TAD process is an environmentally friendly way for disposing of poultry biowaste and carcasses.

Using thermophilic anaerobic digestate effluent to replace freshwater for bioethanol production.

The possibility of using anaerobic digestate effluent (ADE) to replace freshwater and nutrients for bioethanol production was explored. The ethanol concentration yielded from ADE and post-centrifuged ADE supernatant was 79.60±1.75 g/L and 78.33±1.66 g/L, respectively, with a 24% dry mass (DM) of soft wheat. Ethanol production was enhanced in ADE by as much as 18% in comparison to the production in freshwater (66.61±0.28 g/L, p<0.01). Without yeast nutrients, ADE fermentation yielded an ethanol concentration of 81.10±2.87 g/L, which was significantly higher than that in freshwater fermentation (59.67±1.79 g/L). Analysis showed that ADE contained rich nitrogen, proteins and minerals. After one-step distillation, the ethanol concentration attained was 700.05±46.20 g/L in ADE as compared to 622.79±32.22 g/L in freshwater (p<0.05).

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti-hepatitis C virus activity in vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus.

UNLABELLED: Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC(50)) values of 0.01 to 0.14 microM for genotype 1, with half maximal effective concentration (EC(50)s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 +/- 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. CONCLUSION: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment.

Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application.

Compounds with in vitro anti-hepatitis C virus (HCV) activity are often advanced directly into clinical trials with limited or no in vivo efficacy data. This limits prediction of clinical efficacy of compounds in the HCV drug pipeline, and may expose human subjects to unnecessary treatment effects. The scid-Alb-uPA mouse supports proliferation of transplanted human hepatocytes and subsequent HCV infection. Cohorts of genotype 1a HCV-infected mice were treated with interferon alpha-2b(IFN-alpha), BILN-2061 (anti-NS3 protease), or HCV371 (anti-NS5B polymerase). Mice treated with 1350 IU/g/day IFN-alpha intramuscularly for 10 to 28 days demonstrated reduced viral titers compared with controls in all five experiments (P < .05, t test); viral titers rebounded after treatment withdrawal. A more pronounced antiviral effect with IFN-alpha was seen in genotype 3a-infected mice. Pilot studies with BILN2061 confirmed exposure to 10X replicon EC50 at trough and reduced viral titer over 2 log at 4 days. In a second 7-day study, mean HCV RNA titers dropped 1.1 log in BILN2061-treated animals, 0.6 log in IFN-treated mice, and rose 0.2 log in controls (P = .013, ANOVA). Pre-existing mutants with partial resistance to BILN2061 were identified by sequencing both the human inoculum and sera from treated mice. The polymerase inhibitor HCV371 yielded a decline in HCV titers of 0.3 log relative to vehicle-treated controls (P = NS). Performance of all three antiviral regimens in the chimeric mouse model paralleled responses in humans. In conclusion, this system may help selection of lead compounds for advancement into human trials with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection.

Application of the trak-C HCV core assay for monitoring antiviral activity in HCV replication systems.

The Ortho trak-C immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5pg, and exhibits linear detection up to 100pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-alpha using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C assay has several advantages over RNA-based methods of viral monitoring.

Synthetic thermoreversible polymers are compatible with osteoinductive activity of recombinant human bone morphogenetic protein 2.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is currently in clinical studies as part of an implantable device that contains a biomaterial carrier. Implant retention of rhBMP-2 by the biomaterial carrier is important for the osteoinductive activity. To control in situ retention of rhBMP-2, thermoreversible polymers were synthesized and characterized, and their compatibility with rhBMP-2-induced osteoinduction was investigated. The results indicated that polymers with a controlled "solubility <--> insolubility" transition temperature could be prepared from N-isopropylacrylamide, ethylmethacrylate, and N-acryloxysuccinimide (NASI). NASI-containing polymers were able to conjugate to rhBMP-2 without additional cross-linkers. Implantation in the rat ectopic model, where alkaline phosphatase and calcium deposition were utilized as markers of osteoinductive activity, indicated that rhBMP-2 mixed with the polymers were effective for osteoinduction. Moreover, rhBMP-2 conjugated to the chosen polymers was as effective as native rhBMP-2 in inducing ALP activity and calcium deposition. We conclude that thermoreversible polymers are compatible with rhBMP-2-induced osteogenesis and can serve as novel biomaterials for rhBMP-2 delivery.