Markers of blood-brain barrier disruption increase early and persistently in COVID-19 patients with neurological manifestations (preprint)

Background: Coronavirus disease 2019 (COVID-19) leads to peripheral and central disorders, frequently with neurological implications. Blood-brain barrier disruption (BBBd) has been hypothesized as a mechanisms in the acute phase. We tested whether markers of BBBd, brain injury and inflammation could help identify a blood signature for disease severity and neurological complications. Methods: Biomarkers of BBBd (MMP-9, GFAP), neuronal damage (NFL) and inflammation (PPIA, IL-10, TNF) were measured by SIMOA, AlphaLISA and ELISA, in two COVID-19 patient cohorts with high disease severity (ICU Covid; n=79) and neurological complications (NeuroCovid; n=78), and in two control groups with no COVID-19 history: healthy subjects (n=20) and patients with amyotrophic lateral sclerosis (ALS; n=51). Results: Biomarkers of BBBd and neuronal damage were high in COVID-19 patients, with levels similar to or higher than in ALS. NeuroCovid patients had lower levels of PPIA but higher levels of MMP-9 than ICU Covid patients. There was evidence of different temporal dynamics in ICU Covid compared to NeuroCovid patients with PPIA and IL-10 levels highest in ICU Covid patients in the acute phase. In contrast, MMP-9 was higher in the acute phase in NeuroCovid patients, with severity-dependency in the long term. We also found clear severity-dependency of NFL and GFAP. Conclusions: The overall picture points to an increased risk of neurological complications in patients with high levels of biomarkers of BBBd. Our observations may provide hints for therapeutic approaches mitigating BBBd to reduce the neurological damage in the acute phase and potential dysfunction in the long term.

The usefulness of D-dimer as a predictive marker for mortality in patients with COVID-19 hospitalized during the first wave in Italy. (preprint)

Background The coronavirus disease 2019 (COVID-19) presents an urgent threat to global health. Identification of predictors of poor outcomes will assist medical staff in treatment and allocating limited healthcare resources. Aims The primary aim was to study the value of D-dimer as a predictive marker for in-hospital mortality. Methods This was a cohort study. The study population consisted of hospitalized patients (age >18 years), who were diagnosed with COVID-19 based on real-time PCR at 9 hospitals during the first COVID-19 wave in Lombardy, Italy (Feb-May 2020). The primary endpoint was in-hospital mortality. Information was obtained from patient records. Statistical analyses were performed using a Fine-Gray competing risk survival model. Model discrimination was assessed using Harrells C-index and model calibration was assessed using a calibration plot. Results Out of 1049 patients, 501 patients had evaluable data. Of these 501 patients, 96 died. The cumulative incidence of in-hospital mortality within 30 days was 20% (95CI: 16%-23%), and the majority of deaths occurred within the first 10 days. A prediction model containing D-dimer as the only predictor had a C-index of 0.66 (95%CI: 0.61-0.71). Overall calibration of the model was very poor. The addition of D-dimer to a model containing age, sex and co-morbidities as predictors did not lead to any meaningful improvement in either the C-index or the calibration plot. Conclusion The predictive value of D-dimer alone was moderate, and the addition of D-dimer to a simple model containing basic clinical characteristics did not lead to any improvement in model performance.

New susceptibility loci for severe COVID-19 by detailed GWAS analysis in European populations (preprint)

Due to the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), deepening the host genetic contribution to severe COVID-19 may further improve our understanding about underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany, as well as hypothesis-driven targeted analysis of the human leukocyte antigen (HLA) region and chromosome Y haplotypes. We include detailed stratified analyses based on age, sex and disease severity. In addition to already established risk loci, our data identify and replicate two genome-wide significant loci at 17q21.31 and 19q13.33 associated with severe COVID-19 with respiratory failure. These associations implicate a highly pleiotropic ~0.9-Mb 17q21.31 inversion polymorphism, which affects lung function and immune and blood cell counts, and the NAPSA gene, involved in lung surfactant protein production, in COVID-19 pathogenesis.

No changes of parameters nor coagulation activation in healthy subjects vaccinated for SARS-Cov-2

Background Recent reports of thrombotic events after SARS-Cov-2 vaccination raised concern. However, modifications of the most common coagulation parameters after vaccination are unknown. Aims We measured parameters of coagulation including (i) basic coagulation tests, (ii) procedures aimed to assess the ex-vivo potential capacity to generate thrombin and (iii) in vivo thrombin activity. We also assessed anti-platelet factor 4 (aPF4) with two methods. Design Laboratory measurements were performed for a cohort of subjects (n = 30) before (baseline) and after (7 and 21days after first dose, and 14days after second dose) SARS-Cov-2 vaccination. Results All subjects received the Pfizer-BioNTech vaccine, and none developed symptomatic thrombotic events during the study period. None of the parameters showed clinically relevant variations at different time-points before and after vaccination. Only platelet count showed a slight increase, and F1.2 and the thrombin generation parameters ETP and ETP-TM ratio, showed a small decline, at the last time-point after vaccination when compared to baseline. aPF4 was negative in all the subjects, except two, who were positive (one with the chemiluminescent and the other with the ELISA assay). Conclusions The study shows no modifications of the coagulation parameters nor the presence of biochemical signs of coagulation activation following the administration of the Pfizer-BioNTech vaccine.

The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis (preprint)

Background. Respiratory failure is a key feature of severe Covid-19 and a critical driver of mortality, but for reasons poorly defined affects less than 10% of SARS-CoV-2 infected patients. Methods. We included 1,980 patients with Covid-19 respiratory failure at seven centers in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe (Milan, Monza, Madrid, San Sebastian and Barcelona) for a genome-wide association analysis. After quality control and exclusion of population outliers, 835 patients and 1,255 population-derived controls from Italy, and 775 patients and 950 controls from Spain were included in the final analysis. In total we analyzed 8,582,968 single-nucleotide polymorphisms (SNPs) and conducted a meta-analysis of both case-control panels. Results. We detected cross-replicating associations with rs11385942 at chromosome 3p21.31 and rs657152 at 9q34, which were genome-wide significant (P<5x10-8) in the meta-analysis of both study panels, odds ratio [OR], 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.14x10-10 and OR 1.32 (95% CI, 1.20 to 1.47; P=4.95x10-8), respectively. Among six genes at 3p21.31, SLC6A20 encodes a known interaction partner with angiotensin converting enzyme 2 (ACE2). The association signal at 9q34 was located at the ABO blood group locus and a blood-group-specific analysis showed higher risk for A-positive individuals (OR=1.45, 95% CI, 1.20 to 1.75, P=1.48x10-4) and a protective effect for blood group O (OR=0.65, 95% CI, 0.53 to 0.79, P=1.06x10-5). Conclusions. We herein report the first robust genetic susceptibility loci for the development of respiratory failure in Covid-19. Identified variants may help guide targeted exploration of severe Covid-19 pathophysiology.

Early phases of COVID-19 are characterized by a reduction of lymphocyte populations and the presence of atypical monocytes (preprint)

Background Severe acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown. Methods We evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis and the presence of association with inflammatory biomarkers and 28-days mortality. Results Lymphocytopenia was present in 51 of 63 (80.9%) patients. This reduction was mirrored also on CD8+ lymphocytes (128 cells/uL), natural killer cells (67 cells/uL) and natural killer T cells (31 cells/uL). Monocytes were preserved in total number but displayed a subpopulation composed mainly of cells with a reduced expression of both CD14 and HLA-DR. A direct correlation was found between serum values of IL-6 and the frequency of Th2 lymphocytes (R=0.17; p=0.04) but not with the monocytes count (R=0.01; p=0.60). Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (p=0.028) and CD4+ cells (p=0.042) and higher mean percentages of CD8+/CD38+/HLA-DR+ lymphocytes (p=0.026). Conclusions The early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2 lymphocytes and less immunocompetent monocytes, which include atypical mononuclear cells.