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 corona- virus 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"National Microbiology Laboratory||Water Services at the Cities of Ottawa and Barrie||Ottawa Public Health||Simcoe Muskoka District Health Unit|| Public Health Ontario||Ontario Wastewater Surveillance Initiativ

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 extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community’s wastewater (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’s wastewater (40-60% allelic proportion). Finally, a rapid and 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 vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.Ontario's Ministry of Environment, Conservation and Parks||Alberta Healt

Neue FaKtoren in Thrombozytenaktivierung und Apoptose

The non-nucleated platelets are the key players in thrombosis and haemostasis and versatile mediators of inflammation and immunity. Their life, death and actions define illness and health of men. This dissertation attempted to find what events lead to the apoptotic death in platelets by nutraceutical thymoquinone, pathogen-associated peptidoglycan, the antibiotic vancomycin and finally how platelet activation and death by a recently reported chemokine CXCL16 influence platelet function. Thymoquinone triggers suicidal death of blood platelets in a Phosphoinositide 3Kinase-dependent manner, possibly through a G-protein Coupled Receptor family opioid receptor; an effect paralleled by increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization, and caspase-3 activation. HPLC-purified fractions of peptidoglycan from Staphylococcus aureus 113 triggers apoptosis of platelets, characterized by annexin-V binding, increase of [Ca2+]i, mitochondrial depolarization, caspase-3 activation and integrin alphaIIbbeta3 upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in the absence of extracellular Ca2+ and by pancaspase inhibitor zVAD-FMK (1 µM). Vancomycin decreased cell volume, triggered annexin V-binding, stimulated ceramide formation and activated caspase 3. The annexin V-binding was significantly blunted by removal of extracellular Ca2+ but not by pan-caspase inhibition with zVAD-FMK (1µM). Vancomycin also triggers thromboxane B2 release from platelets in cyclooxygenase dependent way. The chemokine CXCL16 significantly up-regulated expression of P-selectin and activated integrin alphaIIbbeta3 at the platelet surface. Consistent with these findings the effects of CXCL16 on platelet activation were inhibited by PI3K inhibitors wortmannin and LY294002 as well as by Akt inhibitor SH-6 (20 µM). The stimulation of adhesion by CXCL16 was again prevented by wortmannin, LY294002 and SH-6. Apyrase and inhibition of the platelet purinergic receptors P2Y1 (MRS2179, 100 µM) and especially P2Y12 (Cangrelor, 10 µM) blunted CXCL16-triggered platelet activation as well as CXCL-16-induced platelet adhesion under high arterial shear stress in vitro indicating that CXCL16 activates platelets via ADP release-dependent paracrine activation. In conclusion the inflammatory chemokine CXCL16 triggers platelet activation and adhesion via PI3K/Akt signaling pathway and paracrine activation suggesting a decisive role for CXCL16 in the pathogenesis of atherosclerosis and thrombo-occlusive diseases.Thrombozyten spielen sowohl in thrombotischen und hämostatischen Prozessen eine wesentliche Rolle als auch in entzündlichen sowie Immunreaktionen. Für die Gesundheit des Menschen sind sie von essentieller Bedeutung. Im Rahmen dieser Doktorarbeit konnte herausgefunden werden, welche Ereignisse in Thrombozyten bei Kontakt mit dem Nahrungsergänzungsmittel Thymoquinone, dem Pathogen-assoziierten Peptidoglycan und dem Antibiotikum Vancomycin zu programmiertem Zellungergang führen und wodurch das Chemokin CXCL-16 Einfluss auf die Plättchenfunktion hat. Thymoquinone löst in Thrombozyten PI3-Kinase-abhängig und downstream eines G-Proteins Apoptose aus, wodurch es zu einer gesteigerten intrazellulären Ca2+-Menge, zu erhöhter Ceramidbildung, zu mitochondrialen Depolarization sowie zur Aktivierung von Caspase-3 kommt. HPLC-gereinigte Fraktionen von Peptidoglycan aus Staphylococcus aureus 113 führt in Blutplättchen zur Annexin-V-Bindung, dadurch zu einer Erhöhung der [Ca2+]i, zu mitochondriale Depolarisation sowie zu Caspase-3-Aktivierung und Integrin alphaIIbbeta33-Hochregulation. Die Annexin-V-Bindung wurde durch anti-TLR-2-Antikörper, die Abwesenheit von extrazellulärem Ca2+ sowie durch den Pan-Caspase-Inhibitor zVAD-FMK signifikant reduziert. Ebenso verringert Vancomycin das Zellvolumen, triggert die Annexin V-Bindung, stimuliert die Bildung von Cerabid sowie von aktiver Caspase-3. Die Bindung von Annexin V konnte mittels des Pan-Caspase-Inhibitors zVAD-FMK sowie durch die Entfernung von [Ca2+]e erfolgreich verhindert werden. Zusätzlich triggert Vancomycin abhängig von p38 MAPK und der Cyclooxygenase die Thromboxan B2-Freisetzung aus Thrombozyten. Das Chemokin CXCL16 reguliert die Expression von P-Selektin hoch und aktiviert Integrin alphaIIbbeta3 an der Plättchenoberfläche. Mit diesen Ergebnissen kohärent ist die Inhibition der beschriebenen Auswirkungen von CXCL16 sowie dessen Adhäsion durch die PI3K-Inhibitoren Wortmannin und LY294002 sowie durch den Akt Inhibitor SH-6. Die Inhibition des P2Y1 sowie des P2Y12-Rezeptors führte zu einer deutlichen Abnahme der CXCL16-induzierten Plättchenadhäsion bei hoher Flussrate in vitro, wodurch die Schlussfolgerung zulässig ist, dass CXCL16 Plättchen ADP-abhängig parakrin aktiviert. Zusammenfassend lässt sich sagen, dass CXCL16 Plättchen mittels PI3K-Akt sowie parakriner Prozesse aktiviert und dadurch eine maßgebliche Rolle in der Pathogenese von Atherosklerose sowie thombo-okklusiven Erkrankungen spielt

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 Ca2+ concentration ([Ca2+]i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca2+]i by Ca2+ 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 [Ca2+]i by Ca2+ 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

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2+]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca2+]i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation

Enhanced ionomycin induced eryptosis and adhesion of erythrocytes from annexin7-deficient mice. A.

<p>Histogram of annexin V-binding reflecting phosphatidylserine exposure in a representative experiment of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>) and their wild type control mice (<i>anx7^+/+</i>) exposed for 30 min to Ca²⁺ ionophore ionomycin (1 µM). <b>B.</b> Arithmetic means ± SEM (n = 8−9) of the percentage of annexin V-binding erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) exposed for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant (p<0.001) difference from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7^+/+</i> erythrocytes (ANOVA). <b>C.</b> Arithmetic means ± SEM (n = 8−9) of the forward scatter of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) exposed for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant (p<0.001) difference from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7^+/+</i> erythrocytes (ANOVA). <b>D.</b> Arithmetic means ± SEM (n = 6) of the number of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) adhering to the human umbilical vein endothelial cells (HUVEC) following exposure for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant difference (p<0.001) from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7^+/+</i> erythrocytes (ANOVA).</p

Role of CXCL16 in dynamic adhesion erythrocytes to anti-CXCl16-treated endothelial cells under arterial shear stress. A.

<p>Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythorcytes were pretreated for 30 minutes with Ringer solution without (control) or with 1 µM ionomycin. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of ionomycin (1 µM), ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA). <b>B.</b> Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythrocytes were pretreated for 12 hours with Ringer with (control), or without (-Glucose) glucose. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of glucose, ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA). C. Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7^−/−</i>, black bars) and their wild type control mice (<i>anx7^+/+</i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythrocytes were pretreated for 2 hours with isotonic (control) or hypertonic (550 mM sucrose) Ringer. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of hyperosmotic shock, ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA).</p