Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids.

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 Fe3+ 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 Fe3+ 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.

Understanding the dynamic relation between wastewater SARS-CoV-2 signal and clinical metrics throughout the pandemic.

Wastewater surveillance (WWS) of SARS-CoV-2 was proven to be a reliable and complementary tool for population-wide monitoring of COVID-19 disease incidence but was not as rigorously explored as an indicator for disease burden throughout the pandemic. Prior to global mass immunization campaigns and during the spread of the wildtype COVID-19 and the Alpha variant of concern (VOC), viral measurement of SARS-CoV-2 in wastewater was a leading indicator for both COVID-19 incidence and disease burden in communities. As the two-dose vaccination rates escalated during the spread of the Delta VOC in Jul. 2021 through Dec. 2021, relations weakened between wastewater signal and community COVID-19 disease incidence and maintained a strong relationship with clinical metrics indicative of disease burden (new hospital admissions, ICU admissions, and deaths). Further, with the onset of the vaccine-resistant Omicron BA.1 VOC in Dec. 2021 through Mar. 2022, wastewater again became a strong indicator of both disease incidence and burden during a period of limited natural immunization (no recent infection), vaccine escape, and waned vaccine effectiveness. Lastly, with the populations regaining enhanced natural and vaccination immunization shortly prior to the onset of the Omicron BA.2 VOC in mid-Mar 2022, wastewater is shown to be a strong indicator for both disease incidence and burden. Hospitalization-to-wastewater ratio is further shown to be a good indicator of VOC virulence when widespread clinical testing is limited. In the future, WWS is expected to show moderate indication of incidence and strong indication of disease burden in the community during future potential seasonal vaccination campaigns.

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities.

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) will have greater value as an important diagnostic tool. An in-depth analysis and understanding of the metrics derived from WWS is required to interpret and utilize WWS-acquired data effectively (McClary-Gutierrez et al., 2021; O'Keeffe, 2021). In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven cities in Canada over periods ranging from 8 to 21 months. This work demonstrates that significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing (resulting in a reduction to testing access and a reduction in the number of daily tests) in these communities, despite increases in the wastewater signal. Furthermore, the WC ratio decreased significantly in 6 of the 7 studied locations, serving as a potential signal of the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community (40-60 % allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community (40-60 % allelic proportion). Finally, a significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant's greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when community immunity was high. The WC ratio, used as an additional monitoring metric, could complement clinical case counts and wastewater signals as individual metrics in its potential ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity.

COVID-19 wastewater surveillance in rural communities: Comparison of lagoon and pumping station samples.

Wastewater-based epidemiology/wastewater surveillance has been a topic of significant interest over the last year due to its application in SARS-CoV-2 surveillance to track prevalence of COVID-19 in communities. Although SARS-CoV-2 surveillance has been applied in more than 50 countries to date, the application of this surveillance has been largely focused on relatively affluent urban and peri-urban communities. As such, there is a knowledge gap regarding the implementation of reliable wastewater surveillance in small and rural communities for the purpose of tracking rates of incidence of COVID-19 and other pathogens or biomarkers. This study examines the relationships existing between SARS-CoV-2 viral signal from wastewater samples harvested from an upstream pumping station and from an access port at a downstream wastewater treatment lagoon with the community's COVID-19 rate of incidence (measured as percent test positivity) in a small, rural community in Canada. Real-time quantitative polymerase chain reaction (RT-qPCR) targeting the N1 and N2 genes of SARS-CoV-2 demonstrate that all 24-h composite samples harvested from the pumping station over a period of 5.5 weeks had strong viral signal, while all samples 24-h composite samples harvested from the lagoon over the same period were below the limit of quantification. RNA concentrations and integrity of samples harvested from the lagoon were both lower and more variable than from samples from the upstream pumping station collected on the same date, indicating a higher overall stability of SARS-CoV-2 RNA upstream of the lagoon. Additionally, measurements of PMMoV signal in wastewater allowed normalizing SARS-CoV-2 viral signal for fecal matter content, permitting the detection of actual changes in community prevalence with a high level of granularity. As a result, in sewered small and rural communities or low-income regions operating wastewater lagoons, samples for wastewater surveillance should be harvested from pumping stations or the sewershed as opposed to lagoons.

Adhesion of annexin 7 deficient erythrocytes to endothelial cells.

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca(2+)](i) by Ca(2+) ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7(-/-)) than in erythrocytes from wild type mice (anxA7(+/+)). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7(-/-) and anx7(+/+)-mice following increase of [Ca(2+)](i) by Ca(2+) ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7(-/-) than in anx7(+/+)-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7(-/-) than in anx7(+/+)-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.

Thymoquinone-induced platelet apoptosis.

Thymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca(2+) activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca(2+) activity utilizing Fluo 3-AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase-3 and inactive procaspase-3, mitochondrial potential utilizing DiOC(6) fluorescence and ceramide by FACS analysis of ceramide-binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca(2+) activity, mitochondrial depolarization, and ceramide formation. P-selectin exposure and integrin α(IIb) ß(3) activation did not increase in response to TQ. Nominal absence of extracellular Ca(2+) blunted but did not fully abolish the TQ-induced activation of caspase-3. The effects of TQ on platelets are significantly abolished with phosphoinositide-3 kinase (PI3K) inhibitor wortmannin and G-protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K-dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization, and caspase-3 activation.