In the context of the current global pandemic and the limitations of the RT-PCR test, we propose a novel deep learning architecture, DFCN (Denoising Fully Connected Network). Since medical facilities around the world differ enormously in what laboratory tests or chest imaging may be available, DFCN is designed to be robust to missing input data. An ablation study extensively evaluates the performance benefits of the DFCN as well as its robustness to missing inputs. Data from 1088 patients with confirmed RT-PCR results are obtained from two independent medical facilities. The data includes results from 27 laboratory tests and a chest x-ray scored by a deep learning model. Training and test datasets are taken from different medical facilities. Data is made publicly available. The performance of DFCN in predicting the RT-PCR result is compared with 3 related architectures as well as a Random Forest baseline. All models are trained with varying levels of masked input data to encourage robustness to missing inputs. Missing data is simulated at test time by masking inputs randomly. DFCN outperforms all other models with statistical significance using random subsets of input data with 2-27 available inputs. When all 28 inputs are available DFCN obtains an AUC of 0.924, higher than any other model. Furthermore, with clinically meaningful subsets of parameters consisting of just 6 and 7 inputs respectively, DFCN achieves higher AUCs than any other model, with values of 0.909 and 0.919.

Objectives:The novel coronavirus disease 19 (COVID-19), caused by SARS-CoV-2, spreads rapidly across the world. The exponential increase in the number of cases has resulted in overcrowding of emergency departments (ED). Detection of SARS-CoV-2 is based on an RT-PCR of nasopharyngeal swab material. However, RT-PCR testing is time-consuming and many hospitals deal with a shortage of testing materials. Therefore, we aimed to develop an algorithm to rapidly evaluate an individual's risk of SARS-CoV-2 infection at the ED. Methods:In this multicenter retrospective study, routine laboratory parameters (C-reactive protein, lactate dehydrogenase, ferritin, absolute neutrophil and lymphocyte counts), demographic data and the chest X-ray/CT result from 967 patients entering the ED with respiratory symptoms were collected. Using these parameters, an easy-to-use point-based algorithm, called the corona-score, was developed to discriminate between patients that tested positive for SARS-CoV-2 by RT-PCR and those testing negative. Computational sampling was used to optimize the corona-score. Validation of the model was performed using data from 592 patients. Results:The corona-score model yielded an area under the receiver operating characteristic curve of 0.91 in the validation population. Patients testing negative for SARS-CoV-2 showed a median corona-score of 3 vs. 11 (scale 0-14) in patients testing positive for SARS-CoV-2 (p<0.001). Using cut-off values of 4 and 11 the model has a sensitivity and specificity of 96 and 95%, respectively. Conclusions:The corona-score effectively predicts SARS-CoV-2 RT-PCR outcome based on routine parameters. This algorithm provides the means for medical professionals to rapidly evaluate SARS-CoV-2 infection status of patients presenting at the ED with respiratory symptoms.

SummaryBackgroundDue to the large number of patients with coronavirus disease 19 (COVID-19), rapid diagnosis at the emergency department (ED) is of critical importance. In this study we have developed a flowchart based on two well-known diagnostic methods: the ‘corona-score’ and the ‘CO-RADS’. This flowchart can be used in hospitals that use chest-CT, instead of chest X-ray, for COVID-19 suspected patients at the ED.MethodsED patients (n=1904) from the Jeroen Bosch Hospital, Amphia Hospital, HagaHospital, Elisabeth TweeSteden Hospital, Bernhoven Hospital and Slingeland Hospital were included. A laboratory-based ‘corona-score’, without radiology, called the ‘lab-corona-score’ was combined with a chest-CT based radiology scoring system (CO-RADS), to develop a flowchart. The performance was assessed by sensitivity/specificity analyses using the RT-PCR outcome or the physician’s final diagnosis as golden standard.ResultsOut of the 1904 patients, 611 (32.1%) patients tested positive for the SARS-CoV-2 virus. The lab-corona-score alone had an AUC of 0.86, a sensitivity of 87% and a specificity of 88% using cut-off values of 0-2 (negative) and 8-10 (positive). Of 255 patients, from the Amphia and Slingeland Hospitals, a CO-RADS score was determined. The flowchart, which combined the ‘CO-RADS’ with the ‘lab-corona-score’, was developed based on data from Slingeland Hospital (sensitivity 97%, specificity 96%). Hereafter, the performance of the flowchart was validated using an independent dataset from Amphia hospital, and reached a sensitivity of 98% and specificity of 93%. A decision could be made in 79% of the patients, which was correct in 95% of the cases.ConclusionThis flowchart, based on radiology (CO-RADS) and clinical chemistry parameters (lab-corona-score), results in a rapid and accurate diagnosis of COVID-19 at the ED.

Article Blood sampling after COVID-19 − How to organize large scale phlebotomy services in the post SARS CoV-2 era was published on September 1, 2020 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 58, issue 9).

BACKGROUND:Prothrombin complex concentrate (PCC) is used to reverse vitamin K antagonist (VKA)-induced anticoagulation. Prothrombin time-derived international normalized ratio (INR) measurements are widely used in determining the required PCC dose, but this approach requires reappraisal. The aim of the present study was to determine the added value of the thrombin generation assay (TGA) compared with the INR in guidance of VKA reversal by PCC. METHODS:In an open, observational study, INR and TGA measurements were carried out on plasma samples from phenprocoumon-treated patients receiving VKA reversal. Following both analytical methods, PCC dosing correlates were calculated and compared retrospectively. Alternatively, in vitro PCC spiking experiments were performed. RESULTS:As expected, an exponential relationship between PCC dose and INR was found. For the TGA parameters peak thrombin and endogenous thrombin potential (ETP), however, this relationship was found to be linear throughout the full therapeutic range. Additional computational analysis showed a positive correlation (r²=0.7) between the initial INR and PCC dose required for a target INR of 2.1, which was completely lost at a lower target INR. In contrast, a positive correlation (r²=0.8) between initial ETP as well as peak height and PCC dose required to obtain parameter normalization was found. These correlates appeared useful for calculating PCC dose. CONCLUSIONS:Our results support the current debate questioning the rationale for the use of the INR in the management of anticoagulation by VKA. Compared with INR, TGA-based calculations may enable a more accurate PCC dosing regimen for patients requiring VKA reversal.

Background— New vessel formation contributes to organ development during embryogenesis and tissue repair in response to mechanical damage, inflammation, and ischemia in adult organisms. Early angiogenesis includes formation of an excessive primitive network that needs to be reorganized into a secondary vascular network with higher hierarchical structure. Vascular pruning, the removal of aberrant neovessels by apoptosis, is a vital step in this process. Although multiple molecular pathways for early angiogenesis have been identified, little is known about the genetic regulators of secondary network development. Methods and Results— Using a transcriptomics approach, we identified a new endothelial specific gene named FYVE, RhoGEF, and PH domain–containing 5 (FGD5) that plays a crucial role in vascular pruning. Loss- and gain-of-function studies demonstrate that FGD5 inhibits neovascularization, indicated by in vitro tube-formation, aortic-ring, and coated-bead assays and by in vivo coated-bead plug assays and studies in the murine retina model. FGD5 promotes apoptosis-induced vaso-obliteration via induction of the hey1-p53 pathway by direct binding and activation of cdc42. Indeed, FGD5 correlates with apoptosis in endothelial cells during vascular remodeling and was linked to rising p21 CIP1 levels in aging mice. Conclusion— We have identified FGD5 as a novel genetic regulator of vascular pruning by activation of endothelial cell–targeted apoptosis.

Ilse Geudens, Robert Herpers, Karlien Hermans, Inmaculada Segura, Carmen Ruiz de Almodovar, Jeroen Bussmann, Frederik De Smet, Wouter Vandevelde, Benjamin M. Hogan, Arndt Siekmann, Filip Claes, John C. Moore, Anna Silvia Pistocchi, Sonja Loges, Massimiliano Mazzone, Giovanni Mariggi, Francoise Bruyere, Franco Cotelli, Dontscho Kerjaschki, Agnes Noel, Jean-Michel Foidart, Holger Gerhardt, Annelii Ny, Tobias Langenberg, Nathan D. Lawson, Henricus J. Duckers, Stefan Schulte-Merker, Peter Carmeliet, Mieke Dewerchin

Objective—To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. Methods and Results—In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. Conclusion—These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

textabstractThe extensive networks of blood and lymphatic vessels within the vertebrate body are essential for the transport and delivery of fluids, gases, macromolecules and cells, and play important roles in facilitating immune responses. The development of the vascular tree requires a highly coordinated interplay of hierarchical genetic and environmental factors, ultimately leading to the formation of a functional network of interconnected tubules that efficiently perfuses tissues. Through the use of various in vitro and in vivo model systems many major interactors in vascular development have been identified in the past decade. The zebrafish (Danio rerio) system in particular offers several advantages for in vivo studies, and has played a pivotal role in new discoveries within the angiogenesis field (Lawson and Weinstein, 2002). Further understanding of the mechanisms whereby blood and lymphatic vessels form is an important question that could yield therapeutic options to alleviate vascular disorders, which are leading causes of mortality.

Ilse Geudens, Robert Herpers, Karlien Hermans, Inmaculada Segura, Carmen Ruiz de Almodovar, Jeroen Bussmann, Frederik De Smet, Wouter Vandevelde, Benjamin M. Hogan, Arndt Siekmann, Filip Claes, John C. Moore, Anna Silvia Pistocchi, Sonja Loges, Massimiliano Mazzone, Giovanni Mariggi, Françoise Bruyère, Franco Cotelli, Dontscho Kerjaschki, Agnes Noël, Jean-Michel Foidart, Holger Gerhardt, Annelii Ny, Tobias Langenberg, Nathan D. Lawson, Henricus J. Duckers, Stefan Schulte-Merker, Peter Carmeliet, Mieke Dewerchin

The development of arteries, veins and lymphatics from pre-existing vessels are intimately linked processes controlled by a number of well-studied reiteratively acting signalling pathways. To delineate the mechanisms governing vessel formation in vivo, we performed a forward genetic screen in zebrafish and isolated the mutant expando. Molecular characterisation revealed a loss-of-function mutation in the highly conserved kinase insert region of flt4. Consistent with previous reports, flt4 mutants were deficient in lymphatic vascular development. Recent studies have demonstrated a role for Flt4 in blood vessels and showed that Dll4 limits angiogenic potential by limiting Flt4 function in developing blood vessels. We found that arterial angiogenesis proceeded normally, yet the dll4 loss-of-function arterial hyperbranching phenotype was rescued, in flt4 signalling mutants. Furthermore, we found that the Flt4 ligand Vegfc drives arterial hyperbranching in the absence of dll4. Upon knockdown of dll4, intersegmental arteries were sensitised to increased vegfc levels and the overexpression of dll4 inhibited Vegfc/Flt4-dependent angiogenesis events. Taken together, these data demonstrate that dll4 functions to suppress the ability of developing intersegmental arteries to respond to Vegfc-driven Flt4 signalling in zebrafish. We propose that this mechanism contributes to the differential response of developing arteries and veins to a constant source of Vegfc present in the embryo during angiogenesis.

Neovascularization is a requirement for embryonic development and plays an important role in the pathogenesis of various diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. Previously, we conducted a genome-wide microarray analysis for vasculogenesis during murine embryonic development. The E 26 T ransformation-specific S equence 2 (ETS2) transcription factor was upregulated in Flk1+ angioblasts and vascular expression of ETS2 was validated by whole mount in situ hybridization in the developing zebrafish. We sought to assess the role of ETS2 on neovascularization during atherosclerotic lesion progression toward plaque instability. We conducted experiments in ApoE−/− mice using a vulnerable plaque (VP) model that creates vulnerable and stable plaques in the common carotid artery by flow alteration. QPCR analysis showed that ETS2 expression is augmented in vulnerable versus stable lesions (P<0,05, N=3). Stimulation with the pro-atherogenic cytokine TNF α increased ETS2 expression and induced ETS2 translocation from the golgi area to the nucleus. Western Diet increased ETS2 expression on the endothelium of the aorta of ApoE−/− animals compared with chow-fed controls, indicated by whole mount staining (P<0,05, N=4). In vitro knockdown of ETS2 by SiRNA in HUVECs diminished the expression of the cytokines MCP1, IL6, and the adhesion molecule VCAM1, under basal conditions and in response to TNF α (P<0,05, N=4). ETS2 knockdown by SiRNA impeded new vessel formation in a matrigel tube-formation assay (P<0,05, N=8). In vivo , adenoviral transduction of ETS2 in the adventitia of the VP region significantly augmented the vascular network of the adventitia compared with shamvirus treated controls. ETS2 overexpression increased both lesion size and necrotic core area, and promoted plaque destabilization by increasing intraplaque lipid and macrophage accumulation, while vascular smooth muscle cell accumulation was diminished. In conclusion, ETS2 expression correlates with plaque instability and responds to pro-atherogenic stimuli. Subsequently, ETS2 function is crucial for induction of the pro-atherogenic EC phenotype and promotes destabilization in advanced atherosclerotic lesions.

The molecular mechanisms that govern the formation of new blood vessels following ischemia remain largely unknown. In order to identify new genes involved in blood vessel formation, we performed a genome-wide microarray screen using the affymetrix platform in mice and zebrafish. Gene expression profiles of isolated Flk1+ angioblasts during murine and piscine embryonic development were compared to those of Flk1− cells. Angiotensin receptor 2 like 1 (Agtrl-1) is one of the genes that was identified. In adult C57bl/6 mice Agtrl-1 is highly expressed in mature endothelial cells and was also detected in cKit+/Flk1+/Lin- cells, but not in Sca+/Flk1+/Lin- cells. Remarkably, the number of cKit+/Flk1+/Agtrl-1 cells was increased in the ischemic area after myocardial ischemia (P<0.001), but not after hind limb ischemia. Apelin protein expression, the endogenous ligand of Agtrl-1, was also augmented by 3-fold in the infarct region of the myocardium (P<0.01) and systemic infusion of Apelin increased the recruitment cKit+/Flk1+ EPCs to the circulation by two-fold (P<0.03). Injection of Apelin into the ischemic myocardium resulted in increased homing to the injection site of this specific population and a two-fold increase in neovascularization of the ischemic tissue (P<0.01), consequently improving cardiac function, left ventricular dimensions and diminished scar formation. These findings indicate for the first time, that apelin in response to myocardial ischemia could function as a specific chemoattractant for Agtrl-1+ EPCs, stimulating migration into the infarcted area and subsequently promoting neovascularisation and myocardial repair.

The specification of arteries and veins is an essential process in establishing and maintaining a functional blood vessel system. Incorrect arteriovenous specification disrupts embryonic development but has also been diagnosed in human syndromes such as hypotrichosis-lymphedema-telangiectasia, characterized by defects in blood and lymphatic vessels and associated with mutations in SOX18. Here we characterize the role of sox7 and sox18 during zebrafish vasculogenesis. Sox7 and sox18 are specifically expressed in the developing vasculature, and simultaneous loss of their function results in a severe loss of the arterial identity of the presumptive aorta which instead expresses venous markers, followed by dramatic arteriovenous shunt formations. Our study identifies members of the Sox family as key factors in specifying arteriovenous identity and will help to better understand hypotrichosis-lymphedema-telangiectasia and other diseases.