Understanding common population markers for SARS-CoV-2 RNA normalization in wastewater - A review.

Wastewater monitoring and epidemiology have seen renewed interest during the recent COVID-19 pandemic. As a result, there is an increasing need to normalize wastewater-derived viral loads in local populations. Chemical tracers, both exogenous and endogenous compounds, have proven to be more stable and reliable for normalization than biological indicators. However, differing instrumentation and extraction methods can make it difficult to compare results. This review examines current extraction and quantification methods for ten common population indicators: creatinine, coprostanol, nicotine, cotinine, sucralose, acesulfame, androstenedione 5-hydroindoleacetic acid (5-HIAA), caffeine, and 1,7-dimethyluric acid. Some wastewater parameters such as ammonia, total nitrogen, total phosphorus, and daily flowrate were also evaluated. The analytical methods included direct injection, dilute and shoot, liquid/liquid, and solid phase extraction (SPE). Creatine, acesulfame, nicotine, 5-HIAA and androstenedione have been analysed by direct injection into LC-MS; however, most authors prefer to include SPE steps to avoid matrix effects. Both LC-MS and GC-MS have been successfully used to quantify coprostanol in wastewater, and the other selected indicators have been quantified successfully with LC-MS. Acidification to stabilize the sample before freezing to maintain the integrity of samples has been reported to be beneficial. However, there are arguments both for and against working at acidic pHs. Wastewater parameters mentioned earlier are quick and easy to quantify, but the data does not always represent the human population effectively. A preference for population indicators originating solely from humans is apparent. This review summarises methods employed for chemical indicators in wastewater, provides a basis for choosing an appropriate extraction and analysis method, and highlights the utility of accurate chemical tracer data for wastewater-based epidemiology.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays.

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.

A wastewater-based risk index for SARS-CoV-2 infections among three cities on the Canadian Prairie.

Wastewater surveillance (WWS) is useful to better understand the spreading of coronavirus disease 2019 (COVID-19) in communities, which can help design and implement suitable mitigation measures. The main objective of this study was to develop the Wastewater Viral Load Risk Index (WWVLRI) for three Saskatchewan cities to offer a simple metric to interpret WWS. The index was developed by considering relationships between reproduction number, clinical data, daily per capita concentrations of virus particles in wastewater, and weekly viral load change rate. Trends of daily per capita concentrations of SARS-CoV-2 in wastewater for Saskatoon, Prince Albert, and North Battleford were similar during the pandemic, suggesting that per capita viral load can be useful to quantitatively compare wastewater signals among cities and develop an effective and comprehensible WWVLRI. The effective reproduction number (Rt) and the daily per capita efficiency adjusted viral load thresholds of 85 × 106 and 200 × 106 N2 gene counts (gc)/population day (pd) were determined. These values with rates of change were used to categorize the potential for COVID-19 outbreaks and subsequent declines. The weekly average was considered 'low risk' when the per capita viral load was 85 × 106 N2 gc/pd. A 'medium risk' occurs when the per capita copies were between 85 × 106 and 200 × 106 N2 gc/pd. with a rate of change <100 %. The start of an outbreak is indicated by a 'medium-high' risk classification when the week-over-week rate of change was >100 %, and the absolute magnitude of concentrations of viral particles was >85 × 106 N2 gc/pd. Lastly, a 'high risk' occurs when the viral load exceeds 200 × 106 N2 gc/pd. This methodology provides a valuable resource for decision-makers and health authorities, specifically given the limitation of COVID-19 surveillance based on clinical data.

RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern

: There are no standardized protocols for quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater to date, especially for population normalization. Here, a pipeline was developed, applied, and assessed to quantify SARS-CoV2 and key variants of concern (VOCs) RNA in wastewater at Saskatoon, Canada. Normalization approaches using recovery ratio and extraction efficiency, wastewater parameters, or population indicators were assessed by comparing to daily numbers of new cases. Viral load was positively correlated with daily new cases reported in the sewershed. Wastewater surveillance (WS) had a lead time of approximately 7 days, which indicated surges in the number of new cases. WS revealed the variant alpha and delta driving the third and fourth wave, respectively. The adjustment with the recovery ratio and extraction efficiency improved the correlation between viral load and daily new cases. Normalization of viral concentration to concentrations of the artificial sweetener acesulfame K improved the trend of viral load during the Christmas and New Year holidays when populations were dynamic and variable. Acesulfame K performed better than pepper mild mottle virus, creatinine, and ammonia for population normalization. Hence, quality controls to characterize recovery ratios and extraction efficiencies and population normalization with acesulfame are promising for precise WS programs supporting decision-making in public health.

Rapid transition between SARS-CoV-2 variants of concern Delta and Omicron detected by monitoring municipal wastewater from three Canadian cities.

Monitoring the communal incidence of COVID-19 is important for both government and residents of an area to make informed decisions. However, continuous reliance on one means of monitoring might not be accurate because of biases introduced by government policies or behaviours of residents. Wastewater surveillance was employed to monitor concentrations of SARS-CoV-2 RNA in raw influent wastewater from wastewater treatment plants serving three Canadian Prairie cities with different population sizes. Data obtained from wastewater are not directly influenced by government regulations or behaviours of individuals. The means of three weekly samples collected using 24 h composite auto-samplers were determined. Viral loads were determined by RT-qPCR, and whole-genome sequencing was used to charaterize variants of concern (VOC). The dominant VOCs in the three cities were the same but with different proportions of sub-lineages. Sub-lineages of Delta were AY.12, AY.25, AY.27 and AY.93 in 2021, while the major sub-lineage of Omicron was BA.1 in January 2022, and BA.2 subsequently became a trace-level sub-variant then the predominant VOC. When each VOC was first detected varied among cities; However, Saskatoon, with the largest population, was always the first to present new VOCs. Viral loads varied among cities, but there was no direct correlation with population size, possibly because of differences in flow regimes. Population is one of the factors that affects trends in onset and development of local outbreaks during the pandemic. This might be due to demography or the fact that larger populations had greater potential for inter- and intra-country migration. Hence, wastewater surveillance data from larger cities can typically be used to indicate what to expect in smaller communities.

Transmission of SARS-CoV-2 virus and ambient temperature: a critical review.

The coronavirus disease 2019 (COVID-19) pandemic has brought unprecedented public health, and social and economic challenges. It remains unclear whether seasonal changes in ambient temperature will alter spreading trajectory of the COVID-19 epidemic. The probable mechanism on this is still lacking. This review summarizes the most recent research data on the effect of ambient temperature on the COVID-19 epidemic characteristic. The available data suggest that (i) mesophilic traits of viruses are different due to their molecular composition; (ii) increasing ambient temperature decreases the persistence of some viruses in aquatic media; (iii) a 1°C increase in the average monthly minimum ambient temperatures (AMMAT) was related to a 0.72% fewer mammalian individuals that would be infected by coronavirus; (iv) proportion of zoonotic viruses of mammals including humans is probably related to their body temperature difference; (v) seasonal divergence between the northern and southern hemispheres may be a significant driver in determining a waved trajectory in the next 2 years. Further research is needed to understand its effects and mechanisms of global temperature change so that effective strategies can be adopted to curb its natural effects. This paper mainly explores possible scientific hypothesis and evidences that local communities and authorities should consider to find optimal solutions that can limit the transmission of SARS-CoV-2 virus.

Enlightenment from the COVID-19 Pandemic: The Roles of Environmental Factors in Future Public Health Emergency Response.

The coronavirus disease 2019 (COVID-19) pandemic is challenging the current public health emergency response systems (PHERSs) of many countries. Although environmental factors, such as those influencing the survival of viruses and their transmission between species including humans, play important roles in PHERSs, little attention has been given to these factors. This study describes and elucidates the roles of environmental factors in future PHERSs. To improve countries' capability to respond to public health emergencies associated with viral infections such as the COVID-19 pandemic, a number of environmental factors should be considered before, during, and after the responses to such emergencies. More specifically, to prevent pandemic outbreaks, we should strengthen environmental and wildlife protection, conduct detailed viral surveillance in animals and hotspots, and improve early-warning systems. During the pandemic, we must study the impacts of environmental factors on viral behaviors, develop control measures to minimize secondary environmental risks, and conduct timely assessments of viral risks and secondary environmental effects with a view to reducing the impacts of the pandemic on human health and on ecosystems. After the pandemic, we should further strengthen surveillance for viruses and the prevention of viral spread, maintain control measures for minimizing secondary environmental risks, develop our capability to scientifically predict pandemics and resurgences, and prepare for the next unexpected resurgence. Meanwhile, we should restore the normal life and production of the public based on the "One Health" concept, that views global human and environmental health as inextricably linked. Our recommendations are essential for improving nations' capability to respond to global public health emergencies.

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.

Fighting against the second wave of COVID-19: Can honeybee products help protect against the pandemic?

Coronavirus Disease (COVID-19) has infected people in 210 nations and has been declared a pandemic on March 12, 2020 by the World Health Organization (WHO). In the absence of effective treatment and/or vaccines for COVID-19, natural products of known therapeutic and antiviral activity could offer an inexpensive, effective option for managing the disease. Benefits of products of honey bees such as honey, propolis, and bee venom, against various types of diseases have been observed. Honey bees products are well known for their nutritional and medicinal values, they have been employed for ages for various therapeutic purposes. In this review, promising effects of various bee products against the emerging pandemic COVID-19 are discussed. Products of honey bees that contain mixtures of potentially active chemicals, possess unique properties that might help to protect, fight, and alleviate symptoms of COVID-19 infection.