Distinct pulmonary and systemic effects of dexamethasone in severe COVID-19.

Dexamethasone is the standard of care for critically ill patients with COVID-19, but the mechanisms by which it decreases mortality and its immunological effects in this setting are not understood. Here we perform bulk and single-cell RNA sequencing of samples from the lower respiratory tract and blood, and assess plasma cytokine profiling to study the effects of dexamethasone on both systemic and pulmonary immune cell compartments. In blood samples, dexamethasone is associated with decreased expression of genes associated with T cell activation, including TNFSFR4 and IL21R. We also identify decreased expression of several immune pathways, including major histocompatibility complex-II signaling, selectin P ligand signaling, and T cell recruitment by intercellular adhesion molecule and integrin activation, suggesting these are potential mechanisms of the therapeutic benefit of steroids in COVID-19. We identify additional compartment- and cell- specific differences in the effect of dexamethasone that are reproducible in publicly available datasets, including steroid-resistant interferon pathway expression in the respiratory tract, which may be additional therapeutic targets. In summary, we demonstrate compartment-specific effects of dexamethasone in critically ill COVID-19 patients, providing mechanistic insights with potential therapeutic relevance. Our results highlight the importance of studying compartmentalized inflammation in critically ill patients.

Causes and attributable fraction of death from ARDS in inflammatory phenotypes of sepsis.

BACKGROUND: Hypoinflammatory and hyperinflammatory phenotypes have been identified in both Acute Respiratory Distress Syndrome (ARDS) and sepsis. Attributable mortality of ARDS in each phenotype of sepsis is yet to be determined. We aimed to estimate the population attributable fraction of death from ARDS (PAFARDS) in hypoinflammatory and hyperinflammatory sepsis, and to determine the primary cause of death within each phenotype. METHODS: We studied 1737 patients with sepsis from two prospective cohorts. Patients were previously assigned to the hyperinflammatory or hypoinflammatory phenotype using latent class analysis. The PAFARDS in patients with sepsis was estimated separately in the hypo and hyperinflammatory phenotypes. Organ dysfunction, severe comorbidities, and withdrawal of life support were abstracted from the medical record in a subset of patients from the EARLI cohort who died (n = 130/179). Primary cause of death was defined as the organ system that most directly contributed to death or withdrawal of life support. RESULTS: The PAFARDS was 19% (95%CI 10,28%) in hypoinflammatory sepsis and, 14% (95%CI 6,20%) in hyperinflammatory sepsis. Cause of death differed between the two phenotypes (p < 0.001). Respiratory failure was the most common cause of death in hypoinflammatory sepsis, whereas circulatory shock was the most common cause in hyperinflammatory sepsis. Death with severe underlying comorbidities was more frequent in hypoinflammatory sepsis (81% vs. 67%, p = 0.004). CONCLUSIONS: The PAFARDS is modest in both phenotypes whereas primary cause of death among patients with sepsis differed substantially by phenotype. This study identifies challenges in powering future clinical trials to detect changes in mortality outcomes among patients with sepsis and ARDS.

Rapidly improving ARDS differs clinically and biologically from persistent ARDS.

BACKGROUND: Rapidly improving acute respiratory distress syndrome (RIARDS) is an increasingly appreciated subgroup of ARDS in which hypoxemia improves within 24 h after initiation of mechanical ventilation. Detailed clinical and biological features of RIARDS have not been clearly defined, and it is unknown whether RIARDS is associated with the hypoinflammatory or hyperinflammatory phenotype of ARDS. The purpose of this study was to define the clinical and biological features of RIARDS and its association with inflammatory subphenotypes. METHODS: We analyzed data from 215 patients who met Berlin criteria for ARDS (endotracheally intubated) and were enrolled in a prospective observational cohort conducted at two sites, one tertiary care center and one urban safety net hospital. RIARDS was defined according to previous studies as improvement of hypoxemia defined as (i) PaO2:FiO2 > 300 or (ii) SpO2: FiO2 > 315 on the day following diagnosis of ARDS (day 2) or (iii) unassisted breathing by day 2 and for the next 48 h (defined as absence of endotracheal intubation on day 2 through day 4). Plasma biomarkers were measured on samples collected on the day of study enrollment, and ARDS phenotypes were allocated as previously described. RESULTS: RIARDS accounted for 21% of all ARDS participants. Patients with RIARDS had better clinical outcomes compared to those with persistent ARDS, with lower hospital mortality (13% vs. 57%; p value < 0.001) and more ICU-free days (median 24 vs. 0; p value < 0.001). Plasma levels of interleukin-6, interleukin-8, and plasminogen activator inhibitor-1 were significantly lower among patients with RIARDS. The hypoinflammatory phenotype of ARDS was more common among patients with RIARDS (78% vs. 51% in persistent ARDS; p value = 0.001). CONCLUSIONS: This study identifies a high prevalence of RIARDS in a multicenter observational cohort and confirms the more benign clinical course of these patients. We report the novel finding that RIARDS is characterized by lower concentrations of plasma biomarkers of inflammation compared to persistent ARDS, and that hypoinflammatory ARDS is more prevalent among patients with RIARDS. Identification and exclusion of RIARDS could potentially improve prognostic and predictive enrichment in clinical trials.

Host and Microbe Blood Metagenomics Reveals Key Pathways Characterizing Critical Illness Phenotypes.

Rationale: Two molecular phenotypes of sepsis and acute respiratory distress syndrome, termed hyperinflammatory and hypoinflammatory, have been consistently identified by latent class analysis in numerous cohorts, with widely divergent clinical outcomes and differential responses to some treatments; however, the key biological differences between these phenotypes remain poorly understood.Objectives: We used host and microbe metagenomic sequencing data from blood to deepen our understanding of biological differences between latent class analysis-derived phenotypes and to assess concordance between the latent class analysis-derived phenotypes and phenotypes reported by other investigative groups (e.g., Sepsis Response Signature [SRS1-2], molecular diagnosis and risk stratification of sepsis [MARS1-4], reactive and uninflamed).Methods: We analyzed data from 113 patients with hypoinflammatory sepsis and 76 patients with hyperinflammatory sepsis enrolled in a two-hospital prospective cohort study. Molecular phenotypes had been previously assigned using latent class analysis.Measurements and Main Results: The hyperinflammatory and hypoinflammatory phenotypes of sepsis had distinct gene expression signatures, with 5,755 genes (31%) differentially expressed. The hyperinflammatory phenotype was associated with elevated expression of innate immune response genes, whereas the hypoinflammatory phenotype was associated with elevated expression of adaptive immune response genes and, notably, T cell response genes. Plasma metagenomic analysis identified differences in prevalence of bacteremia, bacterial DNA abundance, and composition between the phenotypes, with an increased presence and abundance of Enterobacteriaceae in the hyperinflammatory phenotype. Significant overlap was observed between these phenotypes and previously identified transcriptional subtypes of acute respiratory distress syndrome (reactive and uninflamed) and sepsis (SRS1-2). Analysis of data from the VANISH trial indicated that corticosteroids might have a detrimental effect in patients with the hypoinflammatory phenotype.Conclusions: The hyperinflammatory and hypoinflammatory phenotypes have distinct transcriptional and metagenomic features that could be leveraged for precision treatment strategies.

Low Perfusion and Missed Diagnosis of Hypoxemia by Pulse Oximetry in Darkly Pigmented Skin: A Prospective Study.

BACKGROUND: Retrospective clinical trials of pulse oximeter accuracy report more frequent missed diagnoses of hypoxemia in hospitalized Black patients than White patients, differences that may contribute to racial disparities in health and health care. Retrospective studies have limitations including mistiming of blood samples and oximeter readings, inconsistent use of functional versus fractional saturation, and self-reported race used as a surrogate for skin color. Our objective was to prospectively measure the contributions of skin pigmentation, perfusion index (PI), sex, and age on pulse oximeter errors in a laboratory setting. METHODS: We enrolled 146 healthy subjects, including 25 with light skin (Fitzpatrick class I and II), 78 with medium (class III and IV), and 43 with dark (class V and VI) skin. We studied 2 pulse oximeters (Nellcor N-595 and Masimo Radical 7) in prevalent clinical use. We analyzed 9763 matched pulse oximeter readings (pulse oximeter measured functional saturation [Sp o2 ]) and arterial oxygen saturation (hemoximetry arterial functional oxygen saturation [Sa o2 ]) during stable hypoxemia (Sa o2 68%-100%). PI was measured as percent infrared light modulation by the pulse detected by the pulse oximeter probe, with low perfusion categorized as PI < 1%. The primary analysis was to assess the relationship between pulse oximeter bias (difference between Sa o2 and Sp o2 ) by skin pigment category in a multivariable mixed-effects model incorporating repeated-measures and different levels of Sa o2 and perfusion. RESULTS: Skin pigment, PI, and degree of hypoxemia significantly contributed to errors (bias) in both pulse oximeters. For PI values of 1.0% to 1.5%, 0.5% to 1.0%, and <0.5%, the P value of the relationship to mean bias or median absolute bias was <.00001. In lightly pigmented subjects, only PI was associated with positive bias, whereas in medium and dark subjects bias increased with both low perfusion and degree of hypoxemia. Sex and age was not related to pulse oximeter bias. The combined frequency of missed diagnosis of hypoxemia (pulse oximeter readings 92%-96% when arterial oxygen saturation was <88%) in low perfusion conditions was 1.1% for light, 8.2% for medium, and 21.1% for dark skin. CONCLUSIONS: Low peripheral perfusion combined with darker skin pigmentation leads to clinically significant high-reading pulse oximeter errors and missed diagnoses of hypoxemia. Darkly pigmented skin and low perfusion states are likely the cause of racial differences in pulse oximeter performance in retrospective studies.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Identifying molecular phenotypes in sepsis: an analysis of two prospective observational cohorts and secondary analysis of two randomised controlled trials.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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). INTERPRETATION: 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. FUNDING: US National Institutes of Health.

Distinct pulmonary and systemic effects of dexamethasone in severe COVID-19.

Dexamethasone is the standard of care for critically ill patients with COVID-19, but the mechanisms by which it decreases mortality and its immunological effects in this setting are not understood. We performed bulk and single-cell RNA sequencing of the lower respiratory tract and blood, and plasma cytokine profiling to study the effect of dexamethasone on systemic and pulmonary immune cells. We find decreased signatures of antigen presentation, T cell recruitment, and viral injury in patients treated with dexamethasone. We identify compartment- and cell- specific differences in the effect of dexamethasone in patients with severe COVID-19 that are reproducible in publicly available datasets. Our results highlight the importance of studying compartmentalized inflammation in critically ill patients.

Time to Extubation Among ARDS Subjects With and Without COVID-19 Pneumonia.

BACKGROUND: Pneumonia from COVID-19 that results in ARDS may require invasive mechanical ventilation. This retrospective study assessed the characteristics and outcomes of subjects with COVID-19-associated ARDS versus ARDS (non-COVID) during the first 6 months of the COVID-19 pandemic in 2020. The primary objective was to determine whether mechanical ventilation duration differed between these cohorts and identify other potential contributory factors. METHODS: We retrospectively identified 73 subjects admitted between March 1 and August 12, 2020, with either COVID-19-associated ARDS (37) or ARDS (36) who were managed with the lung protective ventilator protocol and required >48 h of mechanical ventilation. Exclusion criteria were the following: <18 years old or the patient required tracheostomy or interfacility transfer. Demographic and baseline clinical data were collected at ARDS onset (ARDS day 0), with subsequent data collected on ARDS days 1-3, 5, 7, 10, 14, and 21. Comparisons were made by using the Wilcoxon rank-sum test (continuous variables) and chi-square test (categorical variables) stratified by COVID-19 status. A Cox proportional hazards model assessed the cause-specific hazard ratio for extubation. RESULTS: The median (interquartile range) mechanical ventilation duration among the subjects who survived to extubation was longer in those with COVID-19-ARDS versus the subjects with non-COVID ARDS: 10 (6-20) d versus 4 (2-8) d; P < .001. Hospital mortality was not different between the two groups (22% vs 39%; P = .11). The competing risks Cox proportional hazard analysis (fit among the total sample, including non-survivors) revealed that improved compliance of the respiratory system and oxygenation were associated with the probability of extubation. Oxygenation improved at a lower rate in the subjects with COVID-19-associated ARDS than in the subjects with non-COVID ARDS. CONCLUSIONS: Mechanical ventilation duration was longer in subjects with COVID-19-associated ARDS compared with the subjects with non-COVID ARDS, which may be explained by a lower rate of improvement in oxygenation status.

Early plasma angiopoietin-2 is prognostic for ARDS and mortality among critically ill patients with sepsis.

Angiopoietin-2 (Ang-2) is associated with vascular endothelial injury and permeability in the acute respiratory distress syndrome (ARDS) and sepsis. Elevated circulating Ang-2 levels may identify critically ill patients with distinct pathobiology amenable to targeted therapy. We hypothesized that plasma Ang-2 measured shortly after hospitalization among patients with sepsis would be associated with the development of ARDS and poor clinical outcomes. To test this hypothesis, we measured plasma Ang-2 in a cohort of 757 patients with sepsis, including 267 with ARDS, enrolled in the emergency department or early in their ICU course before the COVID-19 pandemic. Multivariable models were used to test the association of Ang-2 with the development of ARDS and 30-day morality. We found that early plasma Ang-2 in sepsis was associated with higher baseline severity of illness, the development of ARDS, and mortality risk. The association between Ang-2 and mortality was strongest among patients with ARDS and sepsis as compared to those with sepsis alone (OR 1.81 vs. 1.52 per log Ang-2 increase). These findings might inform models testing patient risk prediction and strengthen the evidence for Ang-2 as an appealing biomarker for patient selection for novel therapeutic agents to target vascular injury in sepsis and ARDS.

Association of SARS-CoV-2 nucleocapsid viral antigen and the receptor for advanced glycation end products with development of severe disease in patients presenting to the emergency department with COVID-19.

Introduction: 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. Methods: Blood samples were collected on arrival from 77 patients with symptomatic COVID-19, and plasma levels of thromboinflammatory biomarkers were measured. Results: 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 p<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 p<0.05), and each had sensitivity and specificity >80% on cut-point analysis. Discussion: 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.

Effects of the circulating environment of COVID-19 on platelet and neutrophil behavior.

Introduction: 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. Methods: 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. Results: We found that COVID-19 patient plasma promoted auto-aggregation, thereby reducing response to further stimulation ex-vivo. 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. Discussion: 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.

Plasma metabolic profiling implicates dysregulated lipid metabolism and glycolytic shift in hyperinflammatory ARDS.

Using latent class analysis (LCA) of clinical and protein biomarkers, researchers have identified two phenotypes of the acute respiratory distress syndrome (ARDS) with divergent clinical trajectories and treatment responses. We investigated whether plasma metabolites differed among patients with LCA-derived hyperinflammatory and hypoinflammatory ARDS, and we tested the prognostic utility of adding metabolic clusters to LCA phenotypes. We analyzed data from 93 patients with ARDS and sepsis enrolled in a multicenter prospective cohort of critically ill patients, comparing 970 metabolites between the two LCA-derived phenotypes. In all, 188 metabolites were differentially abundant between the two LCA-derived phenotypes. After adjusting for age, sex, confounding medications, and comorbid liver and kidney disease, 82 metabolites remained significantly different. Patients with hyperinflammatory ARDS had reduced circulating lipids but high levels of pyruvate, lactate, and malate. Metabolic cluster and LCA-derived phenotypes were each significantly and independently associated with survival. Patients with hyperinflammatory ARDS may be experiencing a glycolytic shift leading to dysregulated lipid metabolism. Metabolic profiling offers prognostic information beyond what is captured by LCA phenotypes alone. Deeper biological profiling may identify key differences in pathogenesis among patients with ARDS and may lead to novel targeted therapies.

Integrated host-microbe plasma metagenomics for sepsis diagnosis in a prospective cohort of critically ill adults.

We carried out integrated host and pathogen metagenomic RNA and DNA next generation sequencing (mNGS) of whole blood (n = 221) and plasma (n = 138) from critically ill patients following hospital admission. We assigned patients into sepsis groups on the basis of clinical and microbiological criteria. From whole-blood gene expression data, we distinguished patients with sepsis from patients with non-infectious systemic inflammatory conditions using a trained bagged support vector machine (bSVM) classifier (area under the receiver operating characteristic curve (AUC) = 0.81 in the training set; AUC = 0.82 in a held-out validation set). Plasma RNA also yielded a transcriptional signature of sepsis with several genes previously reported as sepsis biomarkers, and a bSVM sepsis diagnostic classifier (AUC = 0.97 training set; AUC = 0.77 validation set). Pathogen detection performance of plasma mNGS varied on the basis of pathogen and site of infection. To improve detection of virus, we developed a secondary transcriptomic classifier (AUC = 0.94 training set; AUC = 0.96 validation set). We combined host and microbial features to develop an integrated sepsis diagnostic model that identified 99% of microbiologically confirmed sepsis cases, and predicted sepsis in 74% of suspected and 89% of indeterminate sepsis cases. In summary, we suggest that integrating host transcriptional profiling and broad-range metagenomic pathogen detection from nucleic acid is a promising tool for sepsis diagnosis.

Plasma SARS-CoV-2 nucleocapsid antigen levels are associated with progression to severe disease in hospitalized COVID-19.

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.

Mass cytometry reveals a conserved immune trajectory of recovery in hospitalized COVID-19 patients.

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection.

COVID-19-associated Lung Microvascular Endotheliopathy: A "From the Bench" Perspective.

Rationale: 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). Objectives: 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. Methods: 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. Measurements and Main Results: 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. Conclusions: 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.

Immediate myeloid depot for SARS-CoV-2 in the human lung.

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic1, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.

Immediate myeloid depot for SARS-CoV-2 in the human lung.

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.