Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium.

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID-19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT-qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS-CoV-2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS-CoV-2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS-CoV-2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS-CoV-2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS-CoV-2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Two Separate Clusters of SARS-CoV-2 Delta Variant Infections in a Group of 41 Students Travelling from India: An Illustration of the Need for Rigorous Testing and Quarantine.

We report two clusters of SARS-CoV-2 B.1.617.2 (Delta variant) infections in a group of 41 Indian nursing students who travelled from New Delhi, India, to Belgium via Paris, France. All students tested negative before departure and had a second negative antigen test upon arrival in Paris. Upon arrival in Belgium, the students were quarantined in eight different houses. Four houses remained COVID-free during the 24 days of follow-up, while all 27 residents of the other four houses developed an infection during quarantine, including the four residents who were fully vaccinated and the two residents who were partially vaccinated. Genome sequencing revealed two distinct clusters affecting one and three houses, respectively. In this group of students, vaccination status did not seem to prevent infection nor decrease the viral load. No severe symptoms were reported. Extensive contact tracing and 3 months of nationwide genomic surveillance confirmed that these outbreaks were successfully contained and did not contribute to secondary community transmission in Belgium. These clusters highlight the importance of repeated testing and quarantine measures among travelers coming from countries experiencing a surge of infections, as all infections were detected 6 days or more after arrival.

Regulator of G-protein signaling 18 controls megakaryopoiesis and the cilia-mediated vertebrate mechanosensory system.

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.

Validation of a food-frequency questionnaire for Flemish and Italian-native subjects in Belgium: The IMMIDIET study.

OBJECTIVE: To validate an integrated food-frequency questionnaire (FFQ) developed to assess habitual food intake of Flemish and Italian-native subjects in Belgium as part of the European Collaborative Dietary Habit Profile in European Communities With Different Risk of Myocardial Infarction: the Impact of Migration as a Model of Gene/Environment Interaction (IMMIDIET Project). METHODS: The semiquantitative FFQ contained 322 items on food and food preparation. FFQs filled by a sample (n = 70) of the Flemish-Flemish and Flemish-Italian IMMIDIET subpopulations were randomly selected. Five 24-h recalls, administered over a period of 1 y by the same sample, served for validation. Energy and macronutrients were calculated using the Dutch NEVO and the Belgian NUBEL food composition tables. Intakes of energy and macronutrients estimated by the FFQ and repeated 24-h recall, respectively, were compared by means of correlation coefficients, classification into quartiles, and Bland-Altman plotting. RESULTS: The FFQ overestimated intake of energy and most macronutrients by 40-70%. This overestimation largely disappeared when values were expressed as energy percentage. Correlations ranked from 0.40 to 0.60 for energy and most macronutrients (median 0.53); correlations were lower (null to 0.41) for fat and higher (up to 0.90) for alcohol. Classification in quartiles of intake showed good agreement: 83% were classified in the same or adjacent quartile of energy, and 66-90% for macronutrients. Correlations and classification of macronutrient intake into quartiles remained similar when macronutrients were expressed as energy percentage. Stratification according to ethnic subgroup, age, body mass index, or social status showed no differences. CONCLUSION: The IMMIDIET FFQ is a valuable tool for studies of the role of energy and macronutrients in disease etiology or outcome, but less suitable for estimating absolute intake levels.

Laboratory detection of the antiphospholipid syndrome via calibrated automated thrombography.

Lupus anticoagulants (LAC) consist of antiphospholipid antibodies, detected via their anticoagulant properties in vitro. Strong LAC relate to thromboembolic events, a hallmark of the antiphospholipid syndrome. We have analyzed whether detection of this syndrome would benefit from thrombin generation measurements. Therefore, calibrated automated thrombography was done in normal plasma (n = 30) and LAC patient plasma (n = 48 non-anticoagulated, n = 12 on oral anticoagulants), diluted 1:1 with a normal plasma pool. The anti-beta2-glycoprotein I monoclonal antibody 23H9, with known LAC properties, delayed the lag time and reduced the peak height during thrombin generation induction in normal plasma dose-dependently (0-150 microg/ml). At variance, LAC patient 1:1 plasma mixtures manifested variable lag time prolongations and/or peak height reductions. Coupling these two most informative thrombin generation parameters in a peak height/lag time ratio, and upon normalization versus the normal plasma pool, this ratio distributed normally and was reduced in the plasma mixtures, for 59/60 known LAC plasmas. The normalized peak height/lag time ratio correlated well with the normalized dilute prothrombin time, diluted Russell's viper venom time and silica clotting time, measured in 1:1 plasma mixtures (correlation coefficients 0.59-0.72). The anticoagulant effects of activated protein C (0-7.5 nM) or 23H9 (0-150 microg/ml), spiked in the 1:1 LAC plasma mixtures were reduced for the majority of patients, compatible with functional competition between patient LAC and activated protein C and LAC and 23H9, respectively. Hence, the normalized thrombin generation-derived peak height/lag time ratio identifies LAC in plasma with high sensitivity in a single assay, irrespective of the patient's treatment with oral anticoagulants.

Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes.

The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

Angiogenesis enhances factor IX delivery and persistence from retrievable human bioengineered muscle implants.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies.

Understanding the antiphospholipid syndrome and its treatment.

The antiphospholipid syndrome (APS) is a clinicopathologic disorder characterized by the persistent presence of anticardiolipin antibodies, lupus anticoagulants, or both in the plasma of patients with arterial or venous thrombosis or obstetric complications. The antiphospholipid antibodies involved in this syndrome have a relatively weak affinity for phospholipid-binding proteins such as b(2) glycoprotein I and prothrombin. In certain conditions they form bivalent antibody-protein complexes that bind with a relatively high affinity to negatively charged phospholipid surfaces. Evidence is accumulating for a pathogenetic role of antiphospholipid antibodies via interference with surface-mediated anticoagulant and procoagulant processes. The syndrome is characterized by the recurrence of thrombotic and obstetric complications. Retrospective studies have suggested that patients with APS and thrombosis need a high-intensity anticoagulant treatment. A few small prospective studies support treatment with a targeted INR of 2 to 3.

Quality assessment of oral anticoagulation in Belgium, as practiced by a group of general practitioners.

OBJECTIVE: In Belgium oral anticoagulation therapy is mainly supervised by general practitioners (GPs). This study aims to evaluate the quality of management of oral anticoagulation by Belgian GPs and to verify the relation between time in range and a set of potentially influencing co-variables. METHODS: In a retrospective cross-sectional study, involving 66 GP-practices, the INR-values obtained over a 6-month period were analysed. All INR-values were determined by a single clinical laboratory and additional medical information was provided by the GPs. Linear mixed models have been used to model the patient-specific percentage INR in target as a function of different co-variables. RESULTS: 737 patients were included in the study. Patients who underwent a surgical intervention with an interruption of the anticoagulation during the study were excluded. Patients were only included after the initial starting-up period. 5890 INR-values were obtained. A total of 92,566 days of therapy was evaluated. 50% of the day values were within 0.5 INR-units from target (and 66% within 0.75 INR-units from target). In a multiple regression model, a significant relation between the percentage of time in range and the target INR (2.5 or 3.5) and the gender of the patient was shown. The incidence rate for major bleeding was 5.5/100 patient years (and 3.5/100 patient years for thrombo-embolic events). CONCLUSION: The quality of management of oral anticoagulation by the GPs in Belgium is suboptimal. It is unknown whether interventions such as guidelines, feedback, point-of-care monitoring and computer-assisted anticoagulation monitoring could improve the results.

Thrombophilia in mice expressing a tissue factor variant lacking its transmembrane and cytosolic domain.

Mice with a targeted truncation in the gene encoding tissue factor of blood coagulation (TF) to eliminate the cytosolic domain and carrying a neo(R) cassette in intron 5 unexpectedly displayed severe spontaneous thrombosis in various vascular beds. Thrombosis was observed in heterozygous TF(+/neo) mice, causing death of over 50% of adults within 36 weeks of birth, and fulminantly exacerbating in pregnant females. Homozygous TF(neo/neo) mice were more severely affected and died within 7 weeks after birth. These TF(neo) mice primarily synthesized a mutant mRNA aberrantly spliced from exon 5 to neo(R), encoding an apparently non-vesicle-binding soluble TF lacking both the transmembrane and cytosolic domain, but still capable of blood coagulation induction. This severe thrombotic phenotype associated with the presence of a non-anchored soluble TF variant underscores the recently recognized significance of circulating TF for thrombus formation and development.

Heparin influences human platelet behavior in cardiac surgery with or without cardiopulmonary bypass.

The objective was to investigate whether the platelet dysfunction in cardiac surgery is caused by hemodilution or by shear stress due to cardiopulmonary bypass (CPB). Platelet count and function were prospectively analyzed in two groups of patients undergoing cardiac surgery either with or without CPB (n = 40). In the first study (n = 20; 10 patients with and 10 without CPB), platelet counts were assessed at seven time points. In the second study (n = 20; 10 patients with and 10 without CPB), platelet function was studied with platelet aggregometry at different points during surgery: (a) after induction of anesthesia; (b) after sternotomy; and (c) 1 h after heparin. In the first study, the CPB group showed a significant decrease in platelet count starting after sternotomy (230 +/- 34 vs. 182 +/- 25, P < 0.05) and a maximum decrease at day 1 postoperative (96 +/- 34, P < 0.05). A similar observation was made in the non-CBP group. In the second study, a significant decrease of ADP (54 +/- 13% vs. 38 +/- 9%, P < 0.05), AA (76 +/- 16% vs. 22 +/- 14%, P < 0.05), and Collagen (66 +/- 13% vs. 37 +/- 11%, P < 0.05) induced platelet aggregation was observed at MOMENT d compared to the beginning of surgery in the CPB group. In the non-CBP group a significant decrease was observed in AA-induced platelet aggregation at MOMENT d (83% +/- 4 vs. 44% +/- 14, P < 0.05). The reduction in platelet count is similar with or without cardiopulmonary bypass and is due to pure hemodilution. Platelet function reduces significantly after heparin administration. Hemodilution and predominantly heparin are the causes of platelet dysfunction after cardiac surgery.

The Belgian Improvement Study on Oral Anticoagulation Therapy: a randomized clinical trial.

AIMS: In Belgium, general practitioners (GPs) mainly manage oral anticoagulation therapy. To improve the quality of oral anticoagulation management by GPs and to compare different models and interventions, a randomized clinical trial was performed. METHODS AND RESULTS: Stratified randomization divided 66 GP-practices into four groups. A 6-month retrospective analysis assessed the baseline quality. In the prospective study, each group received education on oral anticoagulation, anticoagulation files, and patient information booklets (groups A, B, C, and D). Group B additionally received feedback every 2 months on their anticoagulation performance; group C determined the international normalized ratio (INR) with a CoaguChek device in the doctor's office or at the patient's home; and group D received Dawn AC computer assisted advice for adapting oral anticoagulation. For the different groups, the time spent in target INR range (Rosendaal's method) and adverse events related to anticoagulation were determined and compared with the same quality indicators at baseline. There was a significant increase in per cent of time within 0.5 INR from target, from 49.5% at baseline to 60% after implementing the different interventions. However, neither the per cent in target range nor the event rates differed among the four groups. CONCLUSION: The interventions significantly improved the quality of management of oral anticoagulation by Belgian GPs, mainly as a result of an education and support programme.

Plasma glycocalicin as a source of GPIbalpha in the von Willebrand factor ristocetin cofactor ELISA.

We have previously demonstrated that the von Willebrand factor ristocetin cofactor activity (VWF:RCo), used in the diagnosis of vonWillebrand disease (VWD), can be accurately determined via ELISA by measuring the ristocetin-induced binding of VWF to a captured recombinant fragment of GPIbalpha (rfGPIbalpha, AA 1-289) (Vanhoorelbeke et al., Thromb Haemost 2000; 83: 107-13). This ELISA is more reliable than the currently used platelet agglutination test. Normal plasma contains relatively high concentrations of glycocalicin, a proteolytic fragment of GPIbalpha. We therefore studied whether non-purified plasma glycocalicin can replace rfGPIbalpha in our ELISA. Of 42 anti-GPIbalpha monoclonal antibodies (MAbs) capable of binding plasma glycocalicin, only one MAb captured glycocalicin in a spatial orientation exposing the VWF-binding site in glycocalicin, allowing a specific and dose-dependent ristocetin-mediated VWF-binding. Intra- and interassay variability were comparable with those for the rfGPIbalpha based VWF:RCo ELISA. The VWF:RCo activity of plasma from 33 normal individuals, 19 type 1, 16 type 2A, 9 type 2B, 8 type 2M and 7 type 3VWD patients was determined with this ELISA and allowed a clear identification of VWD patients. Furthermore, determination of the VWF:RCo/VWF:Ag ratio resulted in the discrimination between type 1 and type 2 VWD patients. Results for the glycocalicin based and the rfGPIbalpha based VWF:RCo ELISAs were in good agreement (r=0.943). There was also a good correlation between the glycocalicin based ELISA and the standard platelet agglutination test (r=0.963). In conclusion, to diagnose VWD, a VWF:RCo ELISA based on antibody immobilized plasma glycocalicin can be performed reliably.

Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.

Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-beta2-glycoprotein I (beta 2GPI) antibodies. Antibody-induced dimerization of beta 2GPI increases the affinity of beta 2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of beta 2GPI that mimic effects of beta 2GPI-anti-beta 2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric beta 2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-beta 2GPI or some, but not all, monoclonal anti-beta 2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-beta 2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by beta 2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric beta 2GPI can interact with apolipoprotein E receptor 2 (apoER2'), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric beta 2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2'.

Thrombogenicity of beta 2-glycoprotein I-dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster.

We previously showed that beta(2)-glycoprotein I (beta(2)GPI)-dependent lupus anticoagulants (LAs) form bivalent antigen-antibody complexes with high affinity for phospholipids; these complexes are responsible for their in vitro anticoagulant effect. We now studied the role of these bivalent complexes in arterial thrombosis in the hamster. Three monoclonal antibodies (mAbs) raised against human beta(2)GPI were selected on the basis of their cross-reactivity with hamster beta(2)GPI. Two of these, one with LA activity, 5H2, and one with only anticardiolipin properties, 11E8, were infused at 0 to 10 mg/kg prior to photochemically induced vessel damage. 5H2 promoted thrombus formation dose dependently, raising the thrombus size from 6.0 arbitrary units (AU) in controls (n = 9) to 65.0 AU in the high-dose group (10 mg/kg, n = 6, P =.007). The LA(-) mAb 11E8 and mAb 27A8, reactive with human beta(2)GPI exclusively, did not significantly promote thrombus formation. In a second set of experiments, intact mAb 5H2 was compared to its fragments. Intact mAb 5H2 at 3.3 mg/kg and the equimolar dose of F(ab')(2) fragments (2.2 mg/kg) promoted thrombus formation equally well (55.8 AU, n = 8 and 62.5 AU, n = 7, respectively); mAb 5H2-derived Fab' fragments were inactive. Immunohistochemical analysis showed platelet-rich thrombi, with 5H2 or its F(ab')(2) fragments mainly bound to individual platelets. Our results indicate that bivalent immune complex formation plays an important role in the genesis of arterial thrombosis by certain antiphospholipid antibodies. Cellular activation via the Fc portion of these immune complexes, however, is not essential, because F(ab')(2) fragments of 5H2 still promote thrombus formation.