Differential Association of Cytomegalovirus with Acute and Post-Acute Covid-19

Background: Asymptomatic Cytomegalovirus (CMV) infection reshapes systemic immune responses and its replication can be both a consequence and cause of inflammation. As CMV resides in the same tissues affected by SARSCoV- 2, we hypothesized that asymptomatic CMV co-infection might modify the pathogenesis of both acute and post-acute COVID-19. Method(s): Participants had current or prior nucleic acid-confirmed SARS-CoV-2 infection in the COVID-19 Multi-Phenotyping for Effective Therapies (COMET, n=219), Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC, n=244) or the Long-term Impact of Infection with Novel Coronavirus (LIINC, n=327) cohorts. We assessed the relationship between CMV serostatus and odds of hospitalization and plasma SARS-CoV-2 N antigen levels during acute COVID-19 as well as post-acute "Long COVID" symptoms, defined as >=1 of 32 COVID-19-attributed symptoms present at least 60 days following initial symptom onset. Result(s): Among 758 participants, 518 were hospitalized for their acute COVID-19 episode. CMV seropositivity was independently associated with a 1.9-fold increased odds of hospitalization for acute COVID-19, after adjustment for age, sex, race, ethnicity, HIV status, prior autoimmune disease, diabetes, and obesity (p=0.01, A). Among those hospitalized, CMV seropositivity was also associated with higher plasma SARS-CoV-2 N antigen levels (median 936 vs. 323 pg/ml, P=0.03, B), which remained significant after adjustment for potential confounders, but not with ICU admission (n=209), death (n=58), or thrombotic events (n=31). In contrast to its relationship to acute COVID-19 disease severity, CMV seropositivity was independently associated with a 48% decreased odds of having neurocognitive Long COVID symptoms such has headache and brain fog 4 months after initial COVID-19 diagnosis (P=0.036). Conversely, serologic evidence of Epstein-Barr Virus (EBV) reactivation and HIV both increased the odds of these symptoms (C). Conclusion(s): CMV seropositivity is associated with a 1.9-fold higher odds of hospitalization in people with acute COVID-19 and a nearly 3-fold higher SARS-CoV-2 antigen burden in hospitalized patients. In contrast, CMV seropositivity is associated with a decreased odds of neurocognitive Long COVID symptoms, while other chronic viral co-infections like EBV reactivation and HIV are associated with an increased odds of this complication. The biologic mechanisms mediating these relationships are unknown, but warrant further investigation. (Figure Presented).

A living WHO guideline on drugs for covid-19

Clinical question What is the role of drug interventions in the treatment and prevention of covid-19? Recommendations The first version on this living guidance focuses on corticosteroids. It contains a strong recommendation for systemic corticosteroids in patients with severe and critical covid-19, and a weak or conditional recommendation against systemic corticosteroids in patients with non-severe covid-19. Corticosteroids are inexpensive and are on the World Health Organisation list of essential medicines. How this guideline was created This guideline reflects an innovative collaboration between the WHO and the MAGIC Evidence Ecosystem Foundation, driven by an urgent need for global collaboration to provide trustworthy and living covid-19 guidance. A standing international panel of content experts, patients, clinicians, and methodologists, free from relevant conflicts of interest, produce recommendations for clinical practice. The panel follows standards, methods, processes, and platforms for trustworthy guideline development using the GRADE approach. We apply an individual patient perspective while considering contextual factors (that is, resources, feasibility, acceptability, equity) for countries and healthcare systems. The evidence A living systematic review and network meta-analysis, supported by a prospective meta-analysis, with data from eight randomised trials (7184 participants) found that systemic corticosteroids probably reduce 28 day mortality in patients with critical covid-19 (moderate certainty evidence;87 fewer deaths per 1000 patients (95% confidence interval 124 fewer to 41 fewer)), and also in those with severe disease (moderate certainty evidence;67 fewer deaths per 1000 patients (100 fewer to 27 fewer)). In contrast, systemic corticosteroids may increase the risk of death in patients without severe covid-19 (low certainty evidence;absolute effect estimate 39 more per 1000 patients, (12 fewer to 107 more)). Systemic corticosteroids probably reduce the need for invasive mechanical ventilation, and harms are likely to be minor (indirect evidence). Understanding the recommendations The panel made a strong recommendation for use of corticosteroids in severe and critical covid-19 because there is a lower risk of death among people treated with systemic corticosteroids (moderate certainty evidence), and they believe that all or almost all fully informed patients with severe and critical covid-19 would choose this treatment. In contrast, the panel concluded that patients with non-severe covid-19 would decline this treatment because they would be unlikely to benefit and may be harmed. Moreover, taking both a public health and a patient perspective, the panel warned that indiscriminate use of any therapy for covid-19 would potentially rapidly deplete global resources and deprive patients who may benefit from it most as potentially lifesaving therapy. Updates This is a living guideline. Work is under way to evaluate other interventions. New recommendations will be published as updates to this guideline. Readers note This is version 1 of the living guideline, published on 4 September (BMJ 2020;370:m3379) version 1. Updates will be labelled as version 2, 3 etc. When citing this article, please cite the version number. Submitted August 28 Accepted August 31Copyright © Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to.

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.

Dexamethasone Decreases Respiratory Tract and Systemic Pro-Inflammatory Signaling in Patients with Covid-19 Acute Respiratory Distress Syndrome

Introduction: Dexamethasone decreases mortality in patients with severe COVID-19. The effects of dexamethasone on inflammation and repair in patients with severe COVID-19 are not well understood. We integrated tracheal aspirate (TA) and peripheral blood bulk/single-cell RNA sequencing to study the effect of dexamethasone on patients with COVID-19 ARDS. Methods: We studied selected patients from a cohort of adults with COVID-19 admitted to three hospitals in San Francisco, California from April 2020 to February 2021. Immunosuppression was not used to treat COVID-19 ARDS at these hospitals prior to July 2020, but was routinely used in these patients after this date. For this analysis, we included patients who were mechanically ventilated for COVID-19 ARDS for whom sequencing samples were available within four days of intubation. We excluded patients who received steroids prior to July 2020, subjects who received immunosuppression other than dexamethasone (e.g., tocilizumab) prior to sample collection, and chronically immunosuppressed subjects. We compared bulk RNASeq from TA and single cell RNASeq from TA and whole blood from subjects who received dexamethasone to subjects who did not receive dexamethasone. In addition, we studied the effect of dexamethasone on peripheral blood cytokine concentrations to confirm the effects of observed changes in gene expression. Results: TA bulk RNASeq was available from 20 subjects (six dexamethasone, 14 non-dexamethasone). There was no significant difference in age, sex, smoking, or BMI between groups. After correcting for multiple comparisons, 947 genes were differentially expressed in TA from subjects who received dexamethasone. Ingenuity Pathway Analysis predicted decreased activation of interferon, JAK/STAT, and NLRP12 signaling in subjects who received dexamethasone (Figure 1A). TA scRNASeq samples were available from ten dexamethasone-treated subjects and nine non-dexamethasone subjects. Whole blood scRNAseq samples were available for seven dexamethasone and eight non-dexamethasone subjects (Figure 1B). Eight subjects (three treated with dexamethasone) had both TA and whole blood scRNAseq samples available for analysis. Dexamethasone had distinct effects on the proportions of immune cells in tracheal aspirates and whole blood (Figure 1C). In 36 dexamethasone vs 42 non-dexamethasone subjects, treatment with dexamethasone was associated with significantly increased concentrations of IL-10 and decreased concentrations of IL-6 (Figure 1D). Conclusions: Dexamethasone decreases pro-inflammatory gene expression in the respiratory tract and peripheral blood of patients with COVID-19 ARDS. The effect of dexamethasone on specific cell populations may be distinct in the respiratory tract and peripheral blood.

Novel EHR-Derived SARS-CoV-2 Pneumonia Subphenotypes Show Divergent Clinical Outcomes with Differential Responses to Corticosteroids

Introduction: In COVID-19-related acute respiratory distress syndrome (ARDS), two distinct subphenotypes have been identified with differential outcomes and responses to corticosteroid therapy. We aimed to evaluate (1) whether clinical data can identify subgroups in a broader group of patients with SARS-CoV-2 pneumonia and (2) the extent to which corticosteroids demonstrate heterogeneity of treatment effect across such subgroups. Methods: We retrospectively studied all SARS-CoV-2 patients hospitalized for >24 hours and requiring oxygen support across 11 BJC HealthCare hospitals from June-December 2020. We excluded the initial surge (March-May 2020), as clinical care was heterogeneous and corticosteroid use low during this period. Using prespecified routinely-collected vital sign and laboratory indicator variables, we sought distinct clinical subphenotypes of SARS-CoV-2 pneumonia through latent class analysis (LCA). Across LCA subphenotypes, we evaluated the relationship between corticosteroid treatment and patient outcomes. We used multivariable logistic regression (dependent variable = composite of death/hospice) to explore treatment interaction between corticosteroid exposure and LCA subphenotype, adjusting for age and maximal SOFA score within 24 hours of admission as surrogates for indication. Results: The 3-class LCA model best fit the 1845-patient cohort (p=0.007). Class-1 (n=1456) had mean standardized values of all indicator variables near zero;Class-2 (n=235) manifested profound isolated hypoxemia;and Class-3 (n=154) displayed multiorgan failure, shock, and neutrophilia (Figure-1A). Despite representing <25% of the cohort, Classes 2 (n=109, 46%) and 3 (n=70, 46%) comprised >50% of the primary outcome (vs Class-1: n=151, 10%;p<0.001). Corticosteroids were more frequently administered in Class-2 (n=215, 91%) than in Class-1 (n=1071, 74%) or Class-3 (n=110, 71%, p<0.001;Figure-1B). Adjusted analyses demonstrated interaction between LCA class and corticosteroid treatment for the primary outcome (Class-1, p=0.003;Class-3, p=0.002). Corticosteroids were associated with increased adjusted odds for the primary outcome in Class-1 (aOR 2.11, 95% CI 1.33-3.50, p=0.002) and decreased adjusted odds for the primary outcome in Class-3 (aOR 0.44, 95% CI 0.19-0.98, p=0.048). Class-2 showed no outcome differences between the corticosteroid and noncorticosteroid groups (aOR 1.03, 95% CI 0.38-2.81, p=0.95). Conclusions: Three distinct subphenotypes of SARS-CoV-2 pneumonia demonstrate different clinical outcomes and corticosteroid response. No clear effect was seen among patients with isolated hypoxemia, whereas those with multiorgan failure appeared to benefit. Our findings suggest that among hospitalised patients in our healthcare system, corticosteroid therapy was associated with increased risk of harm. Prospective studies are needed to evaluate the efficacy of corticosteroids in SARS-CoV-2 pneumonia in predictively-enriched trials.

Baseline Plasma Viral Antigen Levels Are Associated with Clinical Deterioration in Hospitalized Covid-19: Potential Value for Stratifying Patients for Therapeutic Interventions

RATIONALE: Some biomarkers of host response to viral infection are associated with COVID-19 outcomes, but these biomarkers do not directly measure viral burden. 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 viral antigen concentration and proximal clinical deterioration in hospitalized patients. METHODS: SARS-CoV-2 nucleocapsid antigen concentrations were measured using a validated microbead immunoassay (Quanterix, NIH/NIAID laboratory) in plasma collected at enrollment from 256 subjects in a prospective observational cohort of hospitalized patients with COVID-19 from 3 hospitals, admitted between March 2020 and August 2021. Relationships between viral antigen concentration and clinical status at 1 week as measured by the World Health Organization (WHO) ordinal scale as well as ICU admission were assessed. Models were adjusted for age and sex, baseline comorbidities including immunosuppression, endogenous neutralizing antibodies, baseline COVID-19 severity, smoking status, remdesivir therapy, steroid therapy, and vaccine status. Missing covariate data were imputed using multiple imputation by chained equations. RESULTS: The median viral antigen concentration for the 35 subjects who deteriorated by 1 week was 4507 (IQR 1225-9665) pg/mL compared to 494 (IQR 18-3882) pg/mL in the 212 subjects who did not (p = 0.0004 Figure a). Using ordinal regression, each doubling in viral antigen concentration was significantly associated with a worse WHO ordinal scale at 1 week (unadjusted OR 1.07, 95% CI 1.02-1.13;adjusted OR 1.10, 95% CI 1.02-1.18). Among 168 patients not in the ICU at baseline, the median viral antigen concentration for the 40 patients who progressed to the ICU was 4697 (IQR 482- 10410) pg/mL vs. 459 (IQR 15-3062) pg/mL in the 128 patients who did not progress to require ICU care (p = 0.0001 Figure b). Using logistic regression, each doubling in viral antigen concentration was significantly associated with ICU admission (unadjusted OR 1.18, 95% CI 1.06-1.32, adjusted OR 1.40, 95% CI 1.11-1.76). CONCLUSIONS: Higher plasma viral antigen concentration at hospital admission is independently associated with a significantly worse clinical status at 1 week and a higher odds of ICU admission among hospitalized patients with COVID-19. This novel finding indicates that plasma viral antigen concentration may identify hospitalized COVID-19 patients at highest risk of short-term clinical deterioration in both clinical practice and research. Results of plasma antigen tests are available within 2-3 hours and could be integrated for identifying hospitalized COVID-19 patients who might benefit from early intervention.

Hyperinflammatory ARDS Is Characterized by Interferon-High, Neutrophil-Predominant Inflammation in the Lower Respiratory Tract

Background: Latent class analyses in patients with acute respiratory distress syndrome (ARDS) have identified “hyper-inflammatory” and “hypo-inflammatory” phenotypes with divergent clinical outcomes and treatment responses. ARDS phenotypes are defined using plasma biomarkers and clinical variables. It is currently unknown if these phenotypes have distinct pulmonary biology and if pre-clinical models of disease replicate the biology of either phenotype. Methods: 45 subjects with ARDS (Berlin Definition) and 5 mechanically ventilated controls were selected from cohorts of mechanically ventilated patients at UCSF and ZSFG. Patients with COVID-19 were excluded from this analysis. A 3-variable classifier model (plasma IL-8, protein C, and bicarbonate;Sinha 2020) was used to assign ARDS phenotypes. Tracheal aspirate (TA) RNA was analyzed using established bulk and single-cell sequencing pipelines (Langelier 2018, Sarma 2021). Differentially expressed (DE) genes were analyzed using Ingenuity Pathway Analysis (IPA). Microbial community composition was analyzed with vegan. Fgsea was used to test for enrichment of gene sets from experimental ARDS models in genes that were differentially expressed between each phenotype and mechanically ventilated controls. Results: Bulk RNA sequencing (RNAseq) was available from 29 subjects with hypoinflammatory ARDS and 10 subjects with hyperinflammatory ARDS. 2,777 genes were differentially expressed between ARDS phenotypes. IPA identified several candidate upstream regulators of gene expression in hyperinflammatory ARDS including IL6, TNF, IL17C, and interferons (Figure 1A). 2,953 genes were differentially expressed between hyperinflammatory ARDS and 5 ventilated controls;in contrast, only 243 genes were differentially expressed between hypoinflammatory ARDS and controls, suggesting gene expression in the hypoinflammatory phenotype was more heterogeneous. Gene sets from experimental models of acute lung injury were enriched in hyperinflammatory ARDS but not in hypoinflammatory ARDS (Figure 1B). Single cell RNA sequencing (scRNAseq) was available from 6 additional subjects with ARDS, of whom 3 had hyperinflammatory ARDS. 14,843 cells passed quality control filters. Hyperinflammatory ARDS subjects had a markedly higher burden of neutrophils (Figure 1C), including a cluster of stressed neutrophils expressing heat shock protein RNA that was not present in hypoinflammatory ARDS. Expression of a Th1 signature was higher in T cells from hyperinflammatory ARDS. Differential expression analysis in macrophages identified increased expression of genes associated with mortality in a previous study of ARDS patients (Morell 2019). Conclusions: The respiratory tract biology of ARDS phenotypes is distinct. Hyperinflammatory ARDS is characterized by neutrophilic inflammation with distinct immune cell polarization. Transcriptomic profiling identifies candidate preclinical disease models that replicate gene expression observed in hyperinflammatory ARDS.

Revisiting the ROSE Trial - Why Did Participants with ARDS Identified as Hispanic/Latino Have Lower Mortality When Randomized to Receive Early Neuromuscular Blockade?

Rationale: The ROSE trial was a multicenter unblinded randomized clinical trial comparing early neuromuscular blockade (NMB) to usual care in patients with moderate to severe ARDS (NEJM 2019). This trial (n=1006) was stopped early for futility yet a subgroup analysis found that among Hispanic/Latino participants the NMB intervention group had a significantly lower mortality (32%) compared to those in the control group (53.7% p=0.02 for interaction). To evaluate potential contributors to these differences we compared baseline clinical and biological characteristics among Hispanic/Latino participants in the intervention vs control group. Methods: We compared demographics primary ARDS risk factor illness severity ventilatory parameters comorbidities and plasma biomarkers at baseline between the NMB intervention and control group for all 118 Hispanic/Latino patients recruited to the ROSE trial (11.6% of the trial population). We used multiple logistic regression to examine whether the mortality difference by treatment group would persist after controlling for the factors that differed significantly between groups. Results: At baseline Hispanic/Latino participants randomized to the control group had greater disease severity scores (APACHE III SOFA;p<0.05 for both) and a higher prevalence of shock (p=0.01) compared to those randomized to the intervention. There were no significant differences between groups in causes of lung injury or baseline ventilatory parameters. In an unadjusted logistic regression model the NMB intervention was significantly associated with mortality (OR 0.42;95%CI 0.20-0.89 p=0.02). The NMB intervention was no longer significantly associated with mortality when adjusting for severity of by illness by either SOFA score (OR 0.53;95%CI 0.24-1.20 p=0.13), APACHE III (OR 0.51, 95%CI 0.20- 1.30 p=0.16) or shock as defined by the need for vasopressors (OR 0.48, 95%CI 0.22-1.03, p=0.06). Hispanic/Latino participants in the control group had significantly higher interleukin-8 (p=0.02) and lower bicarbonate (p=0.045) than those in the intervention group. Conclusion: Together these clinical and biomarker data support the conclusion that the lower mortality associated with NMB in the Hispanic/Latino subgroup may have been partially due to baseline imbalances in systemic severity of illness. This finding underscores the need to cautiously interpret apparent treatment benefits within small subgroups. The COVID-19 pandemic has highlighted ethnic and racial disparities in ARDS. Future trials will benefit from increased representation of populations that are disproportionately affected to minimize the impact of spurious findings related to small sample sizes while creating more statistical power to prospectively address disparities.

Tracheal aspirate sequencing identifies unique features of dysregulated host response in SARS-CoV 2 associated acute respiratory distress syndrome

Background: The coronavirus disease 2019 (COVID-19) pandemic has led to a rapid increase in the incidence of acute respiratory distress syndrome (ARDS). The distinct features of pulmonary biology in COVID-19 ARDS compared to other causes of ARDS, including other lower respiratory tract infections (LRTIs), are not well understood. Methods: Tracheal aspirates (TA) and plasma were collected within five days of intubation from mechanically ventilated adults admitted to one of two academic medical centers. ARDS and LRTI diagnoses and were verified by study physicians. Subjects were excluded if they received immunosuppression. TA from subjects with COVID-ARDS was compared to gene expression in TA from subjects with other causes of ARDS (OtherARDS) or mechanically ventilated control subjects without evidence of pulmonary pathology (NoARDS). Plasma concentrations of IL-6, IL-8, and protein C also were compared between these groups. Upstream regulator and pathway analysis was performed on significantly differentially expressed genes with Ingenuity Pathway Analysis (IPA). Subgroup analyses were performed to compare gene expression in COVID to ARDS associated with other viral LRTIs and bacterial LRTIs. The association of interferon-stimulated gene expression with SARS-CoV2 viral load was compared to the same association in nasopharyngeal swabs in a cohort of subjects with mild SARS-CoV2. Results: TA sequencing was available from 15 subjects with COVID, 32 subjects with other causes of ARDS (OtherARDS), and 5 mechanically ventilated subjects without evidence of pulmonary pathology (NoARDS). 696 genes were differentially expressed between COVID and OtherARDS (Figure 1A). IL-6, IL-8, B-cell receptor, and hypoxia inducible factor-1a signaling were attenuated in COVID compared to OtherARDS. Peroxisome proliferator-activated receptor (PPAR) and PTEN signaling were higher in COVID compared to OtherARDS (Figure 1B). Plasma levels of IL-6, IL-8, and protein C were not significantly different between COVID and OtherARDS. In subgroup analyses, IL-8 signaling was higher in COVID compared to viral LRTI, but lower than bacterial LRTI. Type I/III interferon was higher in COVID compared to bacterial ARDS, but lower compared to viral ARDS (Figure 1C). Compared to nasopharyngeal swabs from subjects with mild COVID-19, expression of several interferon stimulated genes was less strongly correlated with SARS-CoV2 viral load in TA (Figure 1D). IPA identified several candidate medications to treat COVID-19, including dexamethasone, G-CSF, and etanercept. Conclusions: TA sequencing identifies unique features of the host response in COVID-19. These differentially expressed pathways may represent potential therapeutic targets. An impaired interferon response in the lung may increase susceptibility to severe SARS-COV2.