MAIN BLOOD SERUM METABOLITES RESPONSIBLE FOR THE DISCRIMINATION OF HOSPITALIZED PATIENTS WITH MODERATE SYMPTOMS WITH OR WITHOUT COVID-19

Introduction: The variation in human blood serum metabolites resulting from an infection can assist in understanding mechanisms of pathogen action and body response and improve diagnosis. Aim: To map serum signatures of hospitalized symptomatic patients, positive or negative to SARS-CoV-2. Methods: Patients (n = 64) admitted to Anhembi Field Municipal Hospital, a hospital set up for initial care to patients with moderate symptoms, were analyzed being discriminated in positive (n = 32) or negative patients. Age and gender were matched to ensure homogeneity in the basal metabolic rates. Three Nuclear Magnetic Resonance (NMR) data set were recorded on Bruker AVANCE III 600 MHz spectrometer for serum samples analyzed in MetaboAnalyst 5.0 software platform. Results and discussion: The mean age of groups was 54.92 ±12.41 and 54.30 ±12.15, for positive and negative patients, divided in 16 female and 16 male. The ethnicity was 56.2% vs 46.8% caucasian, 34.3% mixed race in both groups, and 9.3% 12.5% vs black in positive and negative groups, respectively. BMI was 24 ±6.93 vs 33.5 ±7.85 in comparison to positive and negative patients, respectively. In both groups 50% of patients presented alveolar infiltrate. Although the groups were not paired by comorbidities, they were homogeneous ensuring that the metabolic variation is due to COVID-19 as similar percentage of patients with arterial hypertension, diabetes and dyslipidemia. Clinical symptoms were also remarkably similar between the groups in relation to: fever, dry cough, dyspnea and myalgia. The Partial Least Squares - Discriminant Analysis (PLS-DA) performed onto noesy1d data discriminated positively from negative patients. Also, it covered lower variance. Combining NMR techniques, it was possible to depict the main metabolites that distinguished the COVID-19 signatures. Alanine, glucose, cholesterol, and glutamine were increased, and lactate decreased in COVID-19. Conclusion: These results suggest NMR as an excellent tool to differentiate hospitalized patients with moderate symptoms as COVID-19 positive or negative. The Ethics Research Committee of the University of Campinas approved all of the experimental procedures, and all individuals signed the informed consent form.

HEMOSTASTATIC ALTERATIONS WITH DISEASE SEVERITY IN THE EARLY SYMPTOMATIC PHASE OF COVID-19

Introduction: The main factors associated with disease severity in Covid-19 are age, sex, body weight, hypertension, and diabetes. Biomarkers of hemostatic activation have been shown to be independent predictors of disease severity in different populations. Aim: To evaluate whether biomarkers of hemostatic activation were associated with clinical outcomes in patients admitted to a field hospital set up to provide initial care to patients in the early symptomatic phase of Covid-19. Methods: Data and samples were obtained from June to September 2020. Laboratory evaluation included complete blood counts, PT, aPTT, fibrinogen, D-dimer, factor VIII activity, Von Willebrand Factor (VWF) (activity and antigen), C reactive protein (CRP) and P-selectin (ELISA). Patients were segregated by outcome, with clinical worsening defined as need for ICU, mechanical ventilation, pulmonary embolism, deep vein thrombosis or death. Results and discussion: In total 209 were enrolled in the study, of which 24 presented clinical deterioration (11.5%). In both groups there was more male patients. In the group of clinical worsening the mean age was 58.1 and improvement was 53.6 years old. Concerning smoking, 3.2% of patients that improved smoke. Regarding pulmonar infiltrate, it was verified in 50% in the group that worsening versus 41% in clinical improvement. No differences could be observed between patient subgroups regarding the presence of fever (63.2% vs. 62.5%), dry cough (75.1% vs. 87.5%) and dyspnea (65.9% vs. 54.2%) at admission. As main comorbidities, the groups presented chronic obstructive pulmonary disease (2.2% vs 8.3%), asthma (3.2% vs 4.2%), chronic heart failure (1.1% vs 8.3%), arterial hypertension (46% vs 41.7%) and diabetes (28.1% vs 33.3%) in comparing improved with clinical deterioration patients. In general, it was verified a significant decrease in platelet number (p = 0.0426), and an increase in the parameters of aPTT (0.0084), CRP (p = 0.0450), vWF antigen (p = 0.0022) and ristocetin cofactor (p = 0.0032). Conclusion: Our results demonstrate that hemostasis activation is associated with clinical deterioration even at the early phases of Covid-19. The Ethics Research Committee of the University of Campinas approved all of the experimental procedures, and all individuals signed the informed consent form.

AVALIAÇÃO DOS NÍVEIS CIRCULANTES DE MEDIADORES DA INTEGRIDADE DA BARREIRA ENDOTELIAL NA COVID-19 E SUA RELAÇÃO COM A ATIVAÇÃO DA HEMOSTASIA

Objetivos: a imunotrombose consiste no processo que envolve a ativação concomitante da imunidade inata, hemostasia e endotélio como parte da resposta a patógenos, e vem sendo colocada no centro da fisiopatologia da Covid-19. Um elemento menos explorado da imunotrombose é a ruptura da barreira endotelial (BE), que permite o acesso dos leucócitos aos tecidos inflamados. Entre os reguladores da integridade da BE destacam-se as vias que envolvem a angiopoietina (Ang) 1 e 2 e seu receptor Tie2, e a via do VEGF-A/VE-caderina (VEC). Além deste papel, foi recentemente demonstrado que a ativação da via Ang/Tie2 inibe a ativação endotelial e a expressão de fator tecidual, estabilizando o endotélio no estado quiescente. Neste estudo determinamos os níveis circulantes de mediadores da integridade da BE na Covid-19, e exploramos sua associação com a gravidade da doença, assim como com a ativação da hemostasia através de um painel abrangente de biomarcadores. Materiais e métodos: as amostras foram obtidas de 30 pacientes internados por Covid-19 devido à hipoxemia e achados tomográficos típicos, e recrutados para um estudo clínico (REBEC: U1111-1250-1843). As amostras foram coletadas em até 24h do diagnóstico, antes de qualquer intervenção terapêutica. Os níveis de reguladores da BE foram medidos por métodos imunológicos (Elisa ou multiplex), e o de biomarcadores da hemostasia por kits comerciais específicos. Um grupo de 30 indivíduos saudáveis pareados por idade e sexo foram utilizados como controle. Dados clínicos e laboratoriais foram obtidos dos prontuários digitais. Resultados: o tempo médio de internação foi de 12,9 ± 9,8 dias, e 12 pacientes (40%) necessitaram de UTI. O dímero D médio foi de 3.609 ± 14.440 ng/mL. Os níveis circulantes de todos reguladores da integridade da BE encontraram-se aumentados em pacientes, quando comparado com controles (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p < 0.0001;Ang2: 1.926 (1.275-3.134) vs 1.215 (9-1.444) pg/mL, p < 0.0001;Tie2: 10.753 ± 2.377 vs 8.603 ± 1.851 pg/mL, p < 0.0001 e VEGF-A: 94.7 (73.4-116.0) vs 45.9 (39.7-57.0), p < 0.0001. Além disso, os níveis de alguns destes reguladores se associaram significativamente a desfechos de relevância clínica, a saber: (i) extensão da lesão pulmonar na tomografia: Ang2 e VEGF-A;(ii) tempo de internação em UTI: VEGF-A. Interessantemente, observamos correlações consistentes e significativas entre os níveis de reguladores da BE a proteínas envolvidas na ativação da hemostasia (fibrinogênio, VWF: Ag, uPAR, PAI-1 e P-selectina). Discussão: o interesse no estudo de reguladores da integridade da BE na Covid-19 já se justifica pelo fato de a doença envolver tanto o comprometimento da barreira alvéolo-capilar quanto a ativação da angiogênese, como demonstrado por outros autores. Nossos resultados reforçam a relevância destas vias através da associação observada com desfechos clínicos. Além disso, os resultados mostram pela primeira vez uma associação entre mediadores da integridade da BE e um painel amplo de biomarcadores da ativação da hemostasia, sugerindo um crosstalk entre estas vias na Covid-19, como demonstrado recentemente no contexto da sepse. Conclusões: nossos resultados apontam que a via Ang/Tie2 deve ser considerada um alvo terapêutico atrativo na Covid-19, por representar um elemento central da imunotrombose nestes pacientes.

AVALIAÇÃO DA INTEGRIDADE DA BARREIRA ENDOTELIAL E SUA RELAÇÃO COM AS MANIFESTAÇÕES CLÍNICAS E LABORATORIAIS NA COVID-19

Objetivos: Os mecanismos fisiopatológicos que determinam a gravidade da Covid-19 estão associados a ativação da hemostasia e da imunidade inata, em um processo coletivamente referido como imunotrombose, e que envolve ativação plaquetária, geração de NETs (do inglês, Nucleo extracelular traps), expressão de fator tecidual, ativação do complemento e ativação endotelial. Um elemento importante da ativação endotelial é a quebra da barreira endotelial (BE), que ocorre para facilitar o acesso de leucócitos aos tecidos, onde contribuem para erradicação dos patógenos. No entanto, a avaliação da integridade da BE é desafiadora, exigindo o uso de modelos celulares. O objetivo desse estudo foi avaliar o efeito do soro de pacientes com Covid-19 sobre a integridade da BE em monocamadas de células endoteliais, e sua correlação com características clínicas da doença. Materiais e métodos: A população do estudo consistiu em 30 pacientes com Covid-19 que apresentavam comprometimento pulmonar confirmado por tomografia de tórax, e necessidade de internação hospitalar por hipoxemia e 30 controles saudáveis pareados por sexo e idade. Os pacientes recrutados fizeram parte de um estudo clínico (REBEC: U1111-1250-1843), e as amostras utilizadas nesta avaliação foram obtidas no momento da internação, antes de qualquer intervenção. Monocamadas de células endoteliais de duas fontes (HUVECs: células de cordão umbilical;HULECs: células endoteliais pulmonares) foram estimuladas com soro de pacientes e indivíduos saudáveis (diluição 15% em meio de cultura) e a integridade da BE foi avaliada por um sensor de impedância celular (ECIS;Eletric Cell-substrate Impedance Sensing System) continuamente por 36 horas. Biomarcadores de gravidade e relacionados à ativação da hemostasia foram avaliados por kits comerciais. Dados clínicos foram obtidos a partir dos prontuários digitais. Resultados: O soro de pacientes com Covid-19 induziu quebra de BE significativamente mais acentuada que o de indivíduos saudáveis em HUVECs nos tempos 15 min (p < 0,01);30 min (p ≤ 0,001);1h (p ≤ 0,0001);2h (p ≤ 0,0001);3h (p ≤ 0,0001);4h (p ≤ 0,01) e 5h (p ≤ 0,05). Estes resultados foram confirmados no modelo de células endoteliais pulmonares (HULECs). A magnitude da quebra apresentou correlação significativa com desfechos clínicos relevantes como tempo de internação total (RS até 0.57) e tempo de UTI (RS = 0,47). Em relação a biomarcadores de interesse na Covid-19, a quebra da BE apresentou correlação significativa com neutrofilia, relação neutrófilo/linfócito, fator de Von Willebrand, fatores IX e XI, fibrinogênio, D-dímero e uPAR (Receptor de Uroquinase). Discussão: Através de um método considerado padrão-ouro para avaliação in vitro da integridade da BE nós demonstramos que componentes presentes no soro de pacientes com Covid-19 são capazes de promover a quebra da BE, e que a magnitude deste processo está relacionada à gravidade desta doença. A correlação com outros marcadores inflamatórios corrobora a conexão entre os mecanismos envolvidos na imunotrombose em pacientes com Covid-19. Conclusão: nossos resultados apontam a quebra da BE como um alvo terapêutico atrativo nestes pacientes.

COVID-19 Diagnosis and Its Impact on Thrombotic Risk in a Cohort of Patients Simultaneously Hospitalized Due to Acute Respiratory Distress Syndrome

Previous evidence suggests that the thromboembolic risk is greater among patients with COVID-19 than among those affected by other types of acute respiratory distress syndrome (ARDS). However, such comparison has been primarily based on historical cohorts. In order to reduce the possible influence of such selection bias, the main goal of this study was to evaluate thromboembolic events in patients with COVID-19 and other ARDS hospitalized in the same time period. For this reason, we have selected patients admitted from March to June, 2020 at the UNICAMP Clinical Hospital who met the ARDS clinical criteria established by the Brazilian Ministry of Health and the Berlin Definition by presenting two or more flu-like symptoms and at least one ARDS-specific manifestation (dyspnea, persistent chest pressure, oxygen saturation lower than 95% at hospital admission, or lip/face cyanosis). Symptom onset or worsening occurred 30 days before hospital admission at the latest, and COVID-19 diagnosis was confirmed or excluded by at least 2 real time polymerase chain reactions or enzyme-linked immunosorbent assays. Descriptive analysis, chi-square and t-tests, as well as binary logistic regression, were used to compare COVID-19 and non-COVID-19 patients. Of the 253 patients hospitalized due to ARDS during this period, 101 COVID-19 and 102 non-COVID-19 patients were included in this study. The remaining patients were excluded due to incomplete medical records (n=16) or absence of COVID-19 testing results (n=34). Table 1 demonstrates the included patients' demographic and clinical baseline features. Both COVID-19 and non-COVID-19 groups showed similar baseline risk of hospital-associated thrombosis (assessed by reduced mobility within the past 3 days or more, previous thromboembolism event, recognized “thrombophilia”, and infarction, stroke, trauma or surgery within the past 4 weeks) and oxygen saturation at admission (COVID-19: 92% IQR 90% to 96%;non-COVID-19: 94% IQR 91% to 97%, P=0.44). However, the need for invasive oxygenation support (37.6% vs. 14.7%, P=0.0002) and vasoactive drugs (44.6% vs. 21.6%, P=0.0006) was greater in COVID-19 than in non-COVID-19 patients. Accordingly, those infected by SARS-CoV-2 were more frequently admitted in ICU (55.4% vs. 40.2%, P=0.04) and for a longer period of time (13 days IQR 6 to 22 vs. 3 days IQR 2 to 8.3, P=0.02) than those affected by other types of ARDS. In comparison to the non-COVID-19 group, the COVID-19 group's median total hospital stay was more lasting (15 days IQR 6 to 30.5 vs. 7 days IQR 3 to 16.3, P<0.0001), and its death rate, higher (27.7% vs. 14.7%, P=0.03), as shown in Table 2. With respect to coagulation markers (Table 3), activated partial thromboplastin time and C-reactive protein levels were greater in COVID-19 than in non-COVID-19 patients, while the latter presented higher median platelet counts. There was no statistically significant difference between both study groups in regards to prothrombin time, fibrinogen, and D-dimer levels (COVID-19: 1488 ng/mL IQR 726.5 to 3476;non-COVID-19: 1773 ng/mL IQR 807.5 to 4153.8, P=0.57). Although thromboprophylaxis was more commonly administered to COVID-19 (76.2%) than non-COVID-19 patients (41.2%, P<0.0001), the incidence of thromboembolic events confirmed by imaging examination was similar between groups even after adjusting for multiple factors (age, sex, thromboprophylaxis use, arterial hypertension, and cancer): there were 7 confirmed events in 7 non-COVID-19 patients, and 13 confirmed events in 9 COVID-19 patients (adjusted OR 0.74, 95% CI 0.24-2.25, P=0.59). Table 4 demonstrates the characteristics of such thrombotic manifestations. By analyzing patients hospitalized in the same time period, we have found that although high, the thromboembolic risk in COVID-19 is similar to that in other types of ARDS, indicating that a hypercoagulable state is inherent to ARDS in general. Additionally, the obtained results show that the use of thromboprophylaxis was significantly higher among COVID-19 patients, and that there was no tatistically relevant difference between COVID-19 and non-COVID-19 patients' D-dimer levels, a commonly used coagulation marker. Such findings provide a better understanding of the thromboembolic risk associated with SARS-CoV-2 infection, and suggest that previous evidence of higher thrombosis rates in COVID-19 suffered bias from the use of historical cohorts. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

Venous thromboembolism in critically-ill patients with pneumonia in the pre-COVID-19 era: Data from a large public database

Background: The high risk of venous thromboembolism (VTE) is a hallmark of COVID-19, particularly in intensive care units (ICU) patients. However, the magnitude of this risk is a matter of debate due to studies heterogeneity, significant changes on VTE management in COVID-19 era and scarce evidence of VTE risk in ICU patients with pneumonia in the pre-COVID-19 era. Aims: To evaluate the VTE risk in the pre-COVID-19 era in a large ICU database. Methods: Data of consecutive pneumonia patients admitted to ICU were retrieved from the Medical Information Mart for Intensive Care III (MIMIC-III) . VTE incidence during ICU stay was described. The association of thromboprophylaxis and VTE risk was determined by logistic regression, adjusted for age, sex, SOFA score, pneumonia diagnosis and type of ICU. Results: Among 6,842 pneumonia patients admitted to ICU, the median ICU stay was 11 (IQR 6-20) days. Tables 1 and 2 summarizes patients' characteristics and outcomes. 486 patients were diagnosed with VTE after a median of 3 (IQR 1-11) days in ICU. The overall cumulative incidence of VTE was 7% (95%CI 6.4-7.6), corresponding to a daily VTE incidence of 0.51% (95%CI 0.47-0.56). 1788 patients received thromboprophylaxis (out of 2958 for whom that data was available). The cumulative incidence of VTE was 10.7% (95%CI 8.9-12.6) among patients without thromboprophylaxis and 6.5% (95%CI 5.4-7.8) among those with thromboprophylaxis. Overall mortality was 19.3%, that was similar among patients with and without VTE (20.6% and 19.2%, respectively). (Table Presented) Conclusions: In pre-COVID-19 era, VTE rates in ICU patients with pneumonia was not substantially different from those reported in COVID-19 when VTE diagnosis is based on clinical suspicion. The risk of VTE was reduced by 46% with thromboprophylaxis. These findings can serve as comparator for future studies aiming at evaluating the impact of VTE on COVID-19.

Circulating levels of platelet, endothelial and red blood cell microvesicles in COVID-19: Association with biomarkers of coagulation, fibrinolysis and angiogenesis

Background: Hemostasis activation is a hallmark of COVID-19, and an integral part of a broader host response known as immunothrombosis. Microvesicles (MVs) are lipid-bilayer particles released from cells, and thought to be relevant mediators of immunothrombosis, due to their capacity to transfer proteins such as tissue factor (TF) and fibrinolytic mediators between different cells. Aims: To quantify platelet, endothelial cell (EC) and red blood cell (RBC) MVs in plasma from patients with COVID-19 and to explore their association with immunothrombosis mediators. Methods: Samples were obtained from patients admitted to a COVID-19 ward as part of a clinical trial. Samples were collected at admission, before any study intervention. MVs levels were measured in double centrifuged platelet poor plasma by flow cytometry. Coagulation, fibrinolysis and endothelial activation markers were measured by functional or immunological methods. The study was approved by the IRB and all participants provided written informed consent. Results: Data were obtained from 30 patients and 30 age and sexmatched healthy individuals. Mean time from symptoms onset and length of hospital stay (LOS) were 8.1 ± 2.3 days and 12.9 ± 9.8 days respectively. Twelve patients (40%) required intensive care (ICU), and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Platelet, EC and RBC MV counts are shown in Table 1. No association was observed between MV counts and clinical markers of disease severity such as LOS (total or in ICU), oximetry, and lung CT extension score. Associations between MV counts and biomarkers of coagulation, fibrinolysis and EC activation are shown in Table 2. Conclusions: Increased levels of MV from platelets and EC were observed in COVID-19. The association of these MVs with markers of coagulation and fibrinolytic activation, and with elements of the Tie2/Ang1 pathway warrant additional studies on the contribution of these pathways to the pathogenesis of COVID-19.

Association of the angiopoietin/TIE2 and VEGF-a pathways with clinical and laboratory markers of disease severity incovid-19

Background : Endothelial barrier (EB) disruption is an important part of immunothrombosis, allowing the access of leukocytes to inflamed tissues. Pathways involving angiopoietin (Ang) 1 and 2 and their receptor Tie2, and VEGF-A/VE-cadherin (VEC) are key regulators of EB integrity. While the association of these mediators with sepsis severity have been known for more than 15 years, it was only recently that their role in coagulation activation was described. Moreover, these proteins also mediate angiogenesis, which has been shown to be upregulated in COVID-19. Aims : To measure circulating levels of key mediators of Ang/Tie2 and VEGF-A pathways in COVID-19 patients, and to explore their association with disease severity and hemostatic activation. Methods : Samples were obtained from patients admitted to a COVID-19 ward, within 24 h from COVID-19 confirmation. EB mediator levels were measured by immunological methods (Elisa or multiplex assays). The study was approved by the IRB and all participants provided written informed consent. Results : Data were obtained from 30 patients and 30 age and sexmatched healthy individuals. Mean length of hospital stay (LOS) was 12.9 ± 9.8 days respectively, twelve patients (40%) required intensive care (ICU), and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Levels of EB mediators are shown in Table 1. Associations between these parameters with relevant clinical and laboratory markers of disease severity are shown in Table 2. Conclusions : All mediators of EB disruption were significantly elevated in COVID-19 patients. In addition, these mediators were consistently associated with proteins involved in immunothrombosis, in particular with fibrinogen, VWF:Ag, uPAR, PAI-1 and P-selectin. Clinically significant associations were observed between Ang-2 and VEGF-A and extension of lung disease (for both) and ICU stay (for VEGF-A). Additional studies are warranted to explore the crosstalk between Ang/Tie2 and VEGF-A pathways with hemostasis in COVID-19.

Artificial neural networks to predict COVID-19 progression of moderate hospitalized patients using early clinicalparameters and blood tests

Background : The early prediction of Covid-19 progression could improve patient's treatment. It is important to develop mathematical models to perform this task using simple blood tests. Aims : To obtain a neural network (ANN) to predict the progression (death vs discharge and intubation vs discharge) of Covid-19 in patients with confirmed diagnosis. Methods : The patients included in this work were diagnosed with Covid-19 by RT-PCR. All data were collected from hospitalized patients admitted to Anhembi Field Municipal Hospital (São Paulo-Brazil), a hospital set up for initial care to patients with moderate symptoms during the pandemic, between June/2020 and October/2020. Blood was collected at the patient's admission. The inputs considered were: sex, age, ethinicity, body mass index, tabagism, ex-tabagism, alveolar infiltrate, arterial hypertension, diabetes, heart rate, respiration rate, body temperature, oxygen saturation, D-dimer, activated partial thromboplastin time, prothrombin time, levels of: hemoglobin, platelet, leukocytes, lymphocytes, monocytes, neutrophils, lactate dehydrogenase, C-reactive protein, and creatinine. Two ANNs were proposed, as shown at Table 1. The best ANN was defined by a 5-fold cross-validation scheme. Finally, a test step was performed to verify the ANN performance. ANNs with one and two hidden layers were tested. The number of neurons ranged from 5 to 35. Results : The main results are shown at Table 2. The best models were obtained with different ANN's structures, which show the influence of the different outcome. The models presented high ACC, AUC, PPV, NPV, and TNR. The ANN 2 presented better performance than ANN 1. We believe that this may be due the data homogeneity that rises from the inclusion criteria adopted in the study. Conclusions : The results showed that the ANNs could be used to predict the progression of Covid-19 based on simple blood tests. The models could be used in the future after an external validation with high number of patients.

Combining artificial neural networks and blood tests to diagnose COVID-19 infection

Background : Fast and accurate diagnosis of COVID-19 is important to prevent dissemination and disease progression. Artificial Intelligence is known as a universal fitting tool and can be used on the formulation of predictive models for the disease's diagnosis. Aims : Obtain a neural network (ANN) to diagnose patients as positive or negative COVID-19 based on patient data and blood tests. Methods : Data from 678 patients with moderate symptoms from the Anhembi Field Municipal Hospital (São Paulo-Brazil), followed between June/2020 and October/2020 were used. Covid-19 by RTPCR was confirmed in 460 patients. The inputs considered were: sex, age, ethinicity, body mass index, tabagism, ex-tabagism, alveolar infiltrate, arterial hypertension, diabetes, heart rate, respiration rate, body temperature, oxygen saturation, D-dimer, activated partial thromboplastin time, prothrombin time, levels of: hemoglobin, platelet, leukocytes, lymphocytes, monocytes, neutrophils, lactate dehydrogenase, C-reactive protein, and creatinine. Blood was collected at the patient's admission. The ANNs had 25 inputs and the output was the Covid-19 diagnosis. The best ANN was defined by a 5-fold cross-validation scheme. Then, a test step was performed to assess the model's performance. ANNs with one and two hidden layers were proposed. The number of neurons ranged from 5 to 35. Results : The best result was obtained with an ANN containing 25 and 30 neurons in the first and second hidden layers, respectively. All the statistical parameters found for the best model are shown at Table 1. The model presented accuracy of 83.3 %, high capacity for the prediction of true positives (PPV = 0.917 and LR+ = 5.188), and moderate probability to indicate false negatives (LR-= 0.202). Conclusions : The results showed that the ANNs are promising to diagnose Covid-19 based on clinical parameters and blood tests. After future refinements and proper validation, this model could be used to diagnose Covid-19 on daily basis.

Contact system and intrinsic pathway are activated and associated with clinical and laboratory outcomes in COVID-19patients

Background : Hemostasis activation is considered a key pathogenic element of COVID-19, as evidenced by the frequency of lung microvascular thrombosis and of venous thromboembolism. As shown in other conditions, pathways that mediate hemostasis activation during inflammation are not necessarily the same as those that drive hemostasis in non-inflammatory states. In particular, contact system and intrinsic pathway activation have been increasingly explored as potential mediators of hemostasis activation and prothrombotic states observed in both sterile and infectious inflammatory conditions. Aims : To assess the activation of contact system and intrinsic pathway activation in COVID-19. Methods : Protease:serpin complexes were measured in plasma from inpatients with COVID-19 and healthy individuals (HI). All samples were collected within 24 h of COVID-19 diagnosis. Associations with laboratory and clinical markers of hemostasis activation and disease severity were explored. The study was approved by the IRB and all participants provided written informed consent. Results : In total, 30 patients with COVID-19 and 30 age and sex-matched HI were enrolled. Main characteristics of the study population are shown in Table 1. As expected, higher levels of classical coagulation activation parameters were observed in COVID-19 patients. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased levels of several protease:serpin complexes (Table 2). Interestingly, a consistent and significant association was observed between FIXa:antithrombin complexes with both clinical endpoints (need of ICU admission, length of ICU stay, total length of stay and extent of lung CT alterations), and with laboratory markers of immunothrombosis (neutrophil:lymphocyte ratio, C-reactive protein, D-dimer, PAP, VWF:Ag), as well as with several other activation markers of contact system and intrinsic pathway Conclusions : Contact system and intrinsic pathway activation contribute to hemostasis activation in COVID-19. The association of FIXa:antithrombin complexes with disease severity suggests that these pathways contribute to the pathogenesis of the prothrombotic state of COVID-19.