Abstract
Background
Studies quantifying SARS-CoV-2 have focused on upper respiratory tract or plasma viral RNA with inconsistent association with clinical outcomes. The association between plasma ...viral antigen levels and clinical outcomes has not been previously studied. Our aim was to investigate the relationship between plasma SARS-CoV-2 nucleocapsid antigen (N-antigen) concentration and both markers of host response and clinical outcomes.
Methods
SARS-CoV-2 N-antigen concentrations were measured in the first study plasma sample (D0), collected within 72 h of hospital admission, from 256 subjects admitted between March 2020 and August 2021 in a prospective observational cohort of hospitalized patients with COVID-19. The rank correlations between plasma N-antigen and plasma biomarkers of tissue damage, coagulation, and inflammation were assessed. Multiple ordinal regression was used to test the association between enrollment N-antigen plasma concentration and the primary outcome of clinical deterioration at one week as measured by a modified World Health Organization (WHO) ordinal scale. Multiple logistic regression was used to test the association between enrollment plasma N-antigen concentration and the secondary outcomes of ICU admission, mechanical ventilation at 28 days, and death at 28 days. The prognostic discrimination of an externally derived “high antigen” cutoff of N-antigen ≥ 1000 pg/mL was also tested.
Results
N-antigen on D0 was detectable in 84% of study participants. Plasma N-antigen levels significantly correlated with RAGE (
r
= 0.61), IL-10 (
r
= 0.59), and IP-10 (
r
= 0.59, adjusted
p
= 0.01 for all correlations). For the primary outcome of clinical status at one week, each 500 pg/mL increase in plasma N-antigen level was associated with an adjusted OR of 1.05 (95% CI 1.03–1.08) for worse WHO ordinal status. D0 plasma N-antigen ≥ 1000 pg/mL was 77% sensitive and 59% specific (AUROC 0.68) with a positive predictive value of 23% and a negative predictive value of 93% for a worse WHO ordinal scale at day 7 compared to baseline. D0 N-antigen concentration was independently associated with ICU admission and 28-day mechanical ventilation, but not with death at 28 days.
Conclusions
Plasma N-antigen levels are readily measured and provide important insight into the pathogenesis and prognosis of COVID-19. The measurement of N-antigen levels early in-hospital course may improve risk stratification, especially for identifying patients who are unlikely to progress to severe disease.
There remains a need to better identify patients at highest risk for developing severe Coronavirus Disease 2019 (COVID-19) as additional waves of the pandemic continue to impact hospital systems. We ...sought to characterize the association of receptor for advanced glycation end products (RAGE), SARS-CoV-2 nucleocapsid viral antigen, and a panel of thromboinflammatory biomarkers with development of severe disease in patients presenting to the emergency department with symptomatic COVID-19.
Blood samples were collected on arrival from 77 patients with symptomatic COVID-19, and plasma levels of thromboinflammatory biomarkers were measured.
Differences in biomarkers between those who did and did not develop severe disease or death 7 days after presentation were analyzed. After adjustment for multiple comparisons, RAGE, SARS-CoV-2 nucleocapsid viral antigen, interleukin (IL)-6, IL-10 and tumor necrosis factor receptor (TNFR)-1 were significantly elevated in the group who developed severe disease (all
<0.05). In a multivariable regression model, RAGE and SARS-CoV-2 nucleocapsid viral antigen remained significant risk factors for development of severe disease (both
<0.05), and each had sensitivity and specificity >80% on cut-point analysis.
Elevated RAGE and SARS-CoV-2 nucleocapsid viral antigen on emergency department presentation are strongly associated with development of severe disease at 7 days. These findings are of clinical relevance for patient prognostication and triage as hospital systems continue to be overwhelmed. Further studies are warranted to determine the feasibility and utility of point-of care measurements of these biomarkers in the emergency department setting to improve patient prognostication and triage.
Thromboinflammatory complications are well described sequalae of Coronavirus Disease 2019 (COVID-19), and there is evidence of both hyperreactive platelet and inflammatory neutrophil biology that ...contributes to the thromoinflammatory milieu. It has been demonstrated in other thromboinflammatory diseases that the circulating environment may affect cellular behavior, but what role this environment exerts on platelets and neutrophils in COVID-19 remains unknown. We tested the hypotheses that 1) plasma from COVID-19 patients can induce a prothrombotic platelet functional phenotype, and 2) contents released from platelets (platelet releasate) from COVID-19 patients can induce a proinflammatory neutrophil phenotype.
We treated platelets with COVID-19 patient and disease control plasma, and measured their aggregation response to collagen and adhesion in a microfluidic parallel plate flow chamber coated with collagen and thromboplastin. We exposed healthy neutrophils to platelet releasate from COVID-19 patients and disease controls and measured neutrophil extracellular trap formation and performed RNA sequencing.
We found that COVID-19 patient plasma promoted auto-aggregation, thereby reducing response to further stimulation
. Neither disease condition increased the number of platelets adhered to a collagen and thromboplastin coated parallel plate flow chamber, but both markedly reduced platelet size. COVID-19 patient platelet releasate increased myeloperoxidasedeoxyribonucleic acid complexes and induced changes to neutrophil gene expression.
Together these results suggest aspects of the soluble environment circulating platelets, and that the contents released from those neutrophil behavior independent of direct cellular contact.
Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals
, many others experience milder symptoms. Here, to gain a ...more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)
across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.
Full text
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of ...comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS.
Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung ...endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells.
Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera.
Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness.
SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.
The soluble receptor for advanced glycation end-products (sRAGE) is a marker of alveolar type I cell injury associated with outcomes COVID-19 pneumonia. How plasma sRAGE changes over time and whether ...it remains associated with long-term clinical outcomes beyond a single measurement in COVID-19 has not been well-studied. We studied two cohorts in randomized clinical trials of monoclonal antibody treatment for COVID-19 (bamlanivimab and tixagevimab/cilgavimab). We first studied the association between baseline plasma sRAGE and 90-day clinical outcomes, which had been previously demonstrated in the bamlanivimab cohort, among hospitalized patients with COVID-19 supported with high flow nasal oxygen (HFNO) or non-invasive ventilation (NIV) in the tixagevimab/cilgavimab study. Next, we investigated the relationship between day 3 sRAGE and 90-day outcomes and how plasma sRAGE changes over the first 3 days of hospitalization in both clinical trial cohorts. We found that plasma sRAGE in the highest quartile in the HFNO/NIV participants in the tixagevimab/cilgavimab trial was associated with a significantly lower rate of 90-day sustained recovery (recovery rate ratio 0.31, 95% CI 0.14-0.71, p=0.005) and with a significantly higher rate of 90-day mortality (HR 2.49, 95% CI 1.15-5.43, p = 0.021) compared with the lower three quartiles. Day 3 plasma sRAGE in both clinical trial cohorts remained associated with 90-day clinical outcomes. The trajectory of sRAGE was not influenced by treatment assignment. Our results indicate that plasma sRAGE is a valuable prognostic marker in COVID-19 up to three days after initial hospital presentation.
Two molecular phenotypes have been identified in acute respiratory distress syndrome (ARDS). In the ROSE (Reevaluation of Systemic Early Neuromuscular Blockade) trial of cisatracurium in moderate to ...severe ARDS, we addressed three unanswered questions:
) Do the same phenotypes emerge in a more severe ARDS cohort with earlier recruitment;
) Do phenotypes respond differently to neuromuscular blockade? and
) What biological pathways most differentiate inflammatory phenotypes?
We performed latent class analysis in ROSE using preenrollment clinical and protein biomarkers. In a subset of patients (
= 134), we sequenced whole-blood RNA using enrollment and Day 2 samples and performed differential gene expression and pathway analyses. Informed by the differential gene expression analysis, we measured additional plasma proteins and evaluated their abundance relative to gene expression amounts.
In ROSE, we identified the hypoinflammatory (60.4%) and hyperinflammatory (39.6%) phenotypes with similar biological and clinical characteristics as prior studies, including higher mortality at Day 90 for the hyperinflammatory phenotype (30.3% vs. 61.6%;
< 0.0001). We observed no treatment interaction between the phenotypes and randomized groups for mortality. The hyperinflammatory phenotype was enriched for genes associated with innate immune response, tissue remodeling, and zinc metabolism at Day 0 and collagen synthesis and neutrophil degranulation at Day 2. Longitudinal changes in gene expression patterns differed dependent on survivorship. For most highly expressed genes, we observed correlations with their corresponding plasma proteins' abundance. However, for the class-defining plasma proteins in the latent class analysis, no correlation was observed with their corresponding genes' expression.
The hyperinflammatory and hypoinflammatory phenotypes have different clinical, protein, and dynamic transcriptional characteristics. These findings support the clinical and biological potential of molecular phenotypes to advance precision care in ARDS.
In sepsis and acute respiratory distress syndrome (ARDS), heterogeneity has contributed to difficulty identifying effective pharmacotherapies. In ARDS, two molecular phenotypes (hypoinflammatory and ...hyperinflammatory) have consistently been identified, with divergent outcomes and treatment responses. In this study, we sought to derive molecular phenotypes in critically ill adults with sepsis, determine their overlap with previous ARDS phenotypes, and evaluate whether they respond differently to treatment in completed sepsis trials.
We used clinical data and plasma biomarkers from two prospective sepsis cohorts, the Validating Acute Lung Injury biomarkers for Diagnosis (VALID) study (N=1140) and the Early Assessment of Renal and Lung Injury (EARLI) study (N=818), in latent class analysis (LCA) to identify the optimal number of classes in each cohort independently. We used validated models trained to classify ARDS phenotypes to evaluate concordance of sepsis and ARDS phenotypes. We applied these models retrospectively to the previously published Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial and Vasopressin and Septic Shock Trial (VASST) to assign phenotypes and evaluate heterogeneity of treatment effect.
A two-class model best fit both VALID and EARLI (p<0·0001). In VALID, 804 (70·5%) of the 1140 patients were classified as hypoinflammatory and 336 (29·5%) as hyperinflammatory; in EARLI, 530 (64·8%) of 818 were hypoinflammatory and 288 (35·2%) hyperinflammatory. We observed higher plasma pro-inflammatory cytokines, more vasopressor use, more bacteraemia, lower protein C, and higher mortality in the hyperinflammatory than in the hypoinflammatory phenotype (p<0·0001 for all). Classifier models indicated strong concordance between sepsis phenotypes and previously identified ARDS phenotypes (area under the curve 0·87-0·96, depending on the model). Findings were similar excluding participants with both sepsis and ARDS. In PROWESS-SHOCK, 1142 (68·0%) of 1680 patients had the hypoinflammatory phenotype and 538 (32·0%) had the hyperinflammatory phenotype, and response to activated protein C differed by phenotype (p=0·0043). In VASST, phenotype proportions were similar to other cohorts; however, no treatment interaction with the type of vasopressor was observed (p=0·72).
Molecular phenotypes previously identified in ARDS are also identifiable in multiple sepsis cohorts and respond differently to activated protein C. Molecular phenotypes could represent a treatable trait in critical illness beyond the patient's syndromic diagnosis.
US National Institutes of Health.
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