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

BackgroundCoronavirus 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. MethodsBiomarkers 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). ResultsBiomarkers 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. ConclusionsThe 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.

External validation of risk scores to predict in-hospital mortality in patients hospitalized due to coronavirus disease 2019.

BackgroundThe coronavirus disease 2019 (COVID-19) presents an urgent threat to global health. Prediction models that accurately estimate mortality risk in hospitalized patients could assist medical staff in treatment and allocating limited resources. AimsTo externally validate two promising previously published risk scores that predict in-hospital mortality among hospitalized COVID-19 patients. MethodsTwo cohorts were available; a cohort of 1028 patients admitted to one of nine hospitals in Lombardy, Italy (the Lombardy cohort) and a cohort of 432 patients admitted to a hospital in Leiden, the Netherlands (the Leiden cohort). The primary endpoint was in-hospital mortality. All patients were adult and tested COVID-19 PCR-positive. Model discrimination and calibration were assessed. ResultsThe C-statistic of the 4C mortality score was good in the Lombardy cohort (0.85, 95CI: 0.82-0.89) and in the Leiden cohort (0.87, 95CI: 0.80-0.94). Model calibration was acceptable in the Lombardy cohort but poor in the Leiden cohort due to the model systematically overpredicting the mortality risk for all patients. The C-statistic of the CURB-65 score was good in the Lombardy cohort (0.80, 95CI: 0.75-0.85) and in the Leiden cohort (0.82, 95CI: 0.76-0.88). The mortality rate in the CURB-65 development cohort was much lower than the mortality rate in the Lombardy cohort. A similar but less pronounced trend was found for patients in the Leiden cohort. ConclusionAlthough performances did not differ greatly, the 4C mortality score showed the best performance. However, because of quickly changing circumstances, model recalibration may be necessary before using the 4C mortality score.

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

BackgroundThe 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. AimsThe primary aim was to study the value of D-dimer as a predictive marker for in-hospital mortality. MethodsThis 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. ResultsOut 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. ConclusionThe 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.

SARS-CoV-2 spike conformation determines plasma neutralizing activity

Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.

The production of anti-PF4 antibodies in anti-phospholipid antibody-positive patients is not affected by COVID-19 vaccination

Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4) have been described in heparin-induced thrombocytopenia (HIT) but also in patients positive for anti-phospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based COVID-19 vaccines. We investigated whether COVID-19 vaccination affects the production of anti-PF4 immunoglobulins detectable by solid phase assay in aPL-positive patients and their ability to induce in vitro platelet activation. Anti-PF4 were found in 9/126 aPL-positive patients, 4/50 COVID-19, 9/49 other infections and 1/50 aPL-negative systemic lupus erythematosus patients. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose sera failed to cause platelet aggregations. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination. In conclusion, heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titer as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination.

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

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic [~]0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

Effect of anakinra on mortality in COVID-19: a patient level meta-analysis

BackgroundAnakinra may represent an important therapy to improve the prognosis of COVID-19 patients. This meta-analysis using individual patient data was designed to assess the efficacy and safety of anakinra treatment in patients with COVID-19. MethodsBased on a pre-specified protocol (PROSPERO: CRD42020221491), a systematic literature search was performed in MEDLINE (PubMed), Cochrane, medRxiv.org, bioRxiv.org and clinicaltrials.gov databases for trials in COVID-19 comparing administration of anakinra with standard-of-care and/or placebo. Individual patient data from eligible trials were requested. The primary endpoint was the mortality rate and the secondary endpoint was safety. FindingsLiterature search yielded 209 articles, of which 178 articles fulfilled screening criteria and were full-text assessed. Aggregate data on 1185 patients from 9 studies were analyzed and individual patient data on 895 patients from 6 studies were collected. Most studies used historical controls. Mortality was significantly lower in anakinra-treated patients (38/342 [11{middle dot}1%]) as compared with 137/553 (24{middle dot}8%) observed in patients receiving standard-of-care and/or placebo on top of standard-of-care (137/553 [24{middle dot}8%]); adjusted odds ratio (OR), 0{middle dot}32; 95% CI, 0{middle dot}20 to 0{middle dot}51; p <0{middle dot}001. The mortality benefit was similar across subgroups regardless of diabetes mellitus, ferritin concentrations, or baseline P/F ratio. The effect was more profound in patients exhibiting CRP levels >100 mg/L (OR 0{middle dot}28,95%CI 0{middle dot}27-1{middle dot}47). Safety issues, such as increase of secondary infections, did not emerge. InterpretationAnakinra may be a safe anti-inflammatory treatment option in patients hospitalized with moderate-to-severe COVID-19 pneumonia to reduce mortality, especially in the presence of hyperinflammation signs such as CRP>100mg /L. FundingSobi. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSince the emergence of the COVID-19 pandemic, numerous drugs have been tried in an effort to prevent major detrimental consequences, such as respiratory and multiorgan failure and death. Early during the pandemic, it was realized that drugs aiming to regulate the immune host reaction may play an important role in the treatment of COVID-19. Evidence from a small number of patients with moderate or severe COVID-19 treated with anakinra, and interleukin-1 receptor antagonist, has suggested therapeutic efficacy. We systematically searched all available literature and aimed to present cumulative evidence of anakinra treatment in COVID-19 and the related effect on mortality. Added value of this studyThis is the first patient-level analysis on the effect of anakinra treatment in COVID-19 patients, which, on the one hand, suggests a significant benefit in the reduction of mortality and on the other hand, reassures safety of the treatment. Most importantly, the current study identifies a subgroup of patients with CRP>100mg/L, that may benefit most from treatment with anakinra. Confirmation of these effects in larger randomized clinical trials (RCTs) is urgently needed. Implications of all the available evidenceAnakinra may be an effective and safe immunomodulatory treatment in moderate-to-severe cases of pneumonia due to COVID-19 to prevent unfavorable outcomes. Anakinra may be helpful to avoid adverse events, such as breakthrough infections observed often with dexamethasone use, and may be considered an alternative in specific subgroups of patients e.g. diabetics. Larger trials, summarized in the Table, are ongoing and their results are urgently needed to investigate anakinras best place in the treatment of COVID-19. O_TBL View this table: org.highwire.dtl.DTLVardef@37134borg.highwire.dtl.DTLVardef@1d3ca11org.highwire.dtl.DTLVardef@1774b08org.highwire.dtl.DTLVardef@df281borg.highwire.dtl.DTLVardef@c2188d_HPS_FORMAT_FIGEXP M_TBL C_TBL

A distinct metabolic profile associated with a fatal outcome in COVID-19 patients during early epidemic in Italy

Leveraging the unique biological resource based upon the initial COVID-19 patients in Policlinico di Milano (Italy), our study provides the first metabolic profile associated with a fatal outcome. The identification of potential predictive biomarkers offers a vital opportunity to employ metabolomics in a clinical setting as diagnostic tool of disease prognosis upon hospital admission.

Inflammatory biomarkers in pregnant women with COVID-19: a retrospective cohort study

Coronavirns disease 2019 is a pandemic viral disease affecting also obstetric patients and uncertainties exist about the prognostic role of inflammatory biomarkers and hemocytometry values in patients with this infection. To clarify that, we assessed the values of several inflammatory biomarkers and hemocytometry variables in a cohort of obstetric patients hospitalized with coronavirus disease 2019 and we correlated the values at admission with the need of oxygen supplementation during the hospitalization. Overall, among 27 (61%) pregnant women and 17 (39%) post-partum women, 6 (14%) patients received oxygen supplementation and 2 (4%) required admission to intensive care unit but none died. During hospitalization neutrophils (p=0.002), neutrophils to lymphocytes ratio (p=0.037) and C reactive protein (p<0.001) decreased significantly, whereas lymphocytes (p<0.001) and platelets (p<0.001) increased. Leukocytes and lymphocytes values at admission were correlated with oxygen need, with respectively a 1% and 5% higher risk of oxygen supplementation for each 1,000 cells decrease. Overall, in obstetric patients hospitalized with coronavirus disease 2019, C reactive protein is the inflammatory biomarker that better mirrors the course of the disease whereas D-dimer or ferritin are not reliable predictors of poor outcome. Care to the need of oxygen supplementation should be reserved to patients with reduced leukocytes or lymphocytes values at admission.

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

BackgroundRespiratory 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. MethodsWe 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. ResultsWe 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). ConclusionsWe 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.

Characteristics of 1,573 healthcare workers who underwent nasopharyngeal swab for SARS-CoV-2 in Milano, Lombardy, Italy

BackgroundThe management of healthcare workers (HCWs) exposed to confirmed cases of COVID-19 is still a matter of debate. It is unclear whether these subjects should be tested in the absence of symptoms and if those can guide diagnosis. MethodsOccupational and clinical characteristics of all the consecutive HCWs who performed a nasopharyngeal swab for the detection of SARS-CoV-2 in a University Hospital from February 24, 2020, to March 31, 2020, were collected. Frequencies of positive tests were compared according to selected variables. Multivariable logistic regression analyses were then applied. FindingsPositive tests were 138 among 1,573 HCWs (8.8%, 95% confidence interval [CI]: 7.4-10.3), with a marked difference between symptomatic (20.2%, 95% CI: 16.7-24.1) and asymptomatic (3.7%, 95% CI: 2.7-5.1) subjects (p<0.001). Physicians were the group with the highest frequency of positive tests (10.6%, 95% CI: 8.3-13.4) whereas clerical workers and technicians displayed the lowest frequency (2.9%, 95% CI: 0.8-7.3). The likelihood of being positive increased with the number of reported symptoms and the strongest predictors of a positive test were taste and smell alterations (odds ratio [OR] = 29.7) and fever (OR = 7.21). The median time from first positive test to a negative test was 23 days (95% CI: 19-24). InterpretationIn this Italian group of HCWs exposed to confirmed cases of COVID-19 the presence of symptoms, especially taste and smell alterations and fever, was associated with SARS-CoV-2 infection. The median time to clear the virus from nasopharynx was 23 days. Fundingnone related to the content of this manuscript. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for articles published in English up to April 25, 2020, using the keywords "SARS-CoV-2", "COVID-19", "2019-nCoV", AND "healthcare workers","HCW", AND "testing", "nasopharyngeal swab". We found one article: Roll-out of SARS-CoV-2 testing for healthcare workers at a large NHS Foundation Trust in the United Kingdom, March 2020 published in Euro Surveillance. Reviewing the pre-print website medRxiv with the same keywords we identified two additional studies: SARS-CoV-2 infection in Health Care Workers in a large public hospital in Madrid, Spain, during March 2020, and SARS-CoV-2 infection in 86 healthcare workers in two Dutch hospitals in March. Added value of this studyWe showed that, even if symptomatic healthcare workers had a much higher probability of positive test, almost one third of those infected were asymptomatic. Specific symptoms, namely taste and smell alterations and fever, were strongly associated with the infection. Finally, the median time to clear the virus from nasopharynx was 23 days. Implications of all the available evidenceScreening strategies for healthcare workers exposed to COVID-19 patients should take in account the significant proportion of asymptomatic carriers and the predictive role of specific symptoms. Moreover, healthcare workers coming back to work after a positive test should be aware of the long-time of viral shedding from nasopharynx.

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

BackgroundSevere 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. MethodsWe 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. ResultsLymphocytopenia was present in 51 of 63 (80.9%) patients. This reduction was mirrored also on CD8+ lymphocytes (128 cells/L), natural killer cells (67 cells/L) and natural killer T cells (31 cells/L). 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). ConclusionsThe early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2 lymphocytes and less immunocompetent monocytes, which include atypical mononuclear cells. eTOC-At diagnosis patients with COVID-19 have lymphocytopenia -Monocytes with both normal or altered scatter properties display a reduced expression of CD14 and HLA-DR in most of COVID-19 patients -Patients who die in the 28 days from admission have lower values of CD3+ and CD4+ cells and higher percentages of activated CTL cells compared to those who survive