Subphenotypes of Self-Reported Symptoms and Outcomes in Long COVID: a prospective cohort study with latent class analysis. (preprint)

Objective: To characterize subphenotypes of self-reported symptoms and outcomes(SRSOs) in Post-acute sequelae of COVID-19(PASC). Design: Prospective, observational cohort study of PASC subjects. Setting: Academic tertiary center from five clinical referral sources. Participants: Adults with COVID-19 [≥] 20 days before enrollment and presence of any new self-reported symptoms following COVID-19. Exposures: We collected data on clinical variables and SRSOs via structured telephone interviews and performed standardized assessments with validated clinical numerical scales to capture psychological symptoms, neurocognitive functioning, and cardiopulmonary function. We collected saliva and stool samples for quantification of SARS-CoV-2 RNA via qPCR. Primary and Secondary outcomes of measure: Description of PASC SRSOs burden and duration, derivation of distinct PASC subphenotypes via latent class analysis (LCA), and relationship between viral load with SRSOs and PASC subphenotypes. Results: Baseline data for 214 individuals were analyzed. The study visit took place at a median of 197.5 days after COVID-19 diagnosis, and participants reported ever having a median of 9/16 symptoms (interquartile range 6-11) after acute COVID, with muscle-aches, dyspnea, and headache being the most common. Fatigue, cognitive impairment, and dyspnea were experienced for a longer time. Participants had a lower burden of active symptoms (median 3, interquartile range 1-6) than those ever experienced (p<0.001). Unsupervised LCA of symptoms revealed three clinically-active PASC subphenotypes: a high burden constitutional symptoms (21.9%) , a persistent loss/change of smell and taste (20.6%) , and a minimal residual symptoms subphenotype (57.5%). Subphenotype assignments were strongly associated with self-assessments of global health, recovery and PASC impact on employment (p<0.001). Viral persistence (5.6% saliva and 1% stool samples positive) did not explain SRSOs or subphenotypes. Conclusions: We identified distinct PASC subphenotypes and highlight that although most symptoms progressively resolve, specific PASC subpopulations are impacted by either high burden of constitutional symptoms or persistent olfactory/gustatory dysfunction, requiring prospective identification and targeted preventive or therapeutic interventions.

Trajectories of host-response biomarkers and inflammatory subphenotypes in COVID-19 patients across the spectrum of respiratory support. (preprint)

Purpose: Enhanced understanding of the dynamic changes in the dysregulated inflammatory response in COVID-19 may help improve patient selection and timing for immunomodulatory therapies. Methods: We enrolled 323 COVID-19 inpatients on different levels of baseline respiratory support: i) Low Flow Oxygen (37%), ii) Non-Invasive Ventilation or High Flow Oxygen (NIV_HFO, 29%), iii) Invasive Mechanical Ventilation (IMV, 27%), and iv) Extracorporeal Membrane Oxygenation (ECMO, 7%). We collected plasma samples upon enrollment and days 5 and 10 to measure host-response biomarkers. We classified subjects into inflammatory subphenotypes using two validated predictive models. We examined clinical, biomarker and subphenotype trajectories and outcomes during hospitalization. Results: IL-6, procalcitonin, and Angiopoietin-2 were persistently elevated in patients at higher levels of respiratory support, whereas sRAGE displayed the inverse pattern. Patients on NIV_HFO at baseline had the most dynamic clinical trajectory, with 26% eventually requiring intubation and exhibiting worse 60-day mortality than IMV patients at baseline (67% vs. 35%, p<0.0001). sRAGE levels predicted NIV failure and worse 60-day mortality for NIV_HFO patients, whereas IL-6 levels were predictive in IMV or ECMO patients. Hyper-inflammatory subjects at baseline (<10% by both models) had worse 60-day survival (p<0.0001) and 50% of them remained classified as hyper-inflammatory on follow-up sampling at 5 days post-enrollment. Receipt of combined immunomodulatory therapies (steroids and anti-IL6 agents) was associated with markedly increased IL-6 and lower Angiopoietin-2 levels (p<0.05). Conclusions: Longitudinal study of systemic host responses in COVID-19 revealed substantial and predictive inter-individual variability, influenced by baseline levels of respiratory support and concurrent immunomodulatory therapies.

Noninvasive diagnosis of secondary infections in COVID-19 by sequencing of plasma microbial cell-free DNA. (preprint)

Background: Secondary infection (SI) diagnosis in COVID-19 is challenging, due to overlapping clinical presentations, practical limitations in obtaining samples from the lower respiratory tract (LRT), and low sensitivity of microbiologic cultures. Research Question: Can metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) help diagnose SIs complicating COVID-19? Study Design and Methods: We enrolled 42 inpatients with COVID-19 classified as microbiologically-confirmed SI (Micro-SI, n=8), clinically-diagnosed SI (Clinical-SI, n=13, i.e. empiric antimicrobials), or no clinical suspicion for SI (No-Suspected-SI, n=21) at time of enrollment. From baseline and follow-up plasma samples (days 5 and 10 post-enrollment), we quantified mcfDNA for all detected microbes by mcfDNA sequencing and measured nine host-response biomarkers. From LRT samples among intubated subjects, we quantified bacterial burden with 16S rRNA gene quantitative PCR. Results: We performed mcfDNA-Seq in 82 plasma samples. Sequencing was successful in 60/82 (73.2%) samples, which had significantly lower levels of human cfDNA than failed samples (p<0.0001). McfDNA detection was significantly higher in Micro-SI (15/16 [94%]) compared to Clinical-SI samples (8/14 [57%], p=0.03), and unexpectedly common in No-Suspected-SI samples (25/30 [83%]), similar to detection rate in Micro-SI. We detected culture-concordant mcfDNA species in 13/16 Micro-SI samples (81%) and mcfDNA levels tracked with SI outcome (resolution or persistence) under antibiotic therapy. McfDNA levels correlated significantly with LRT bacterial burden (r=0.74, p=0.02) as well as plasma biomarkers of host response (white blood cell count, IL-6, IL-8, and SPD, all p<0.05). Baseline mcfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log10 mcfDNA, p=0.03). Interpretation: High circulating levels of mcfDNA in a substantial proportion of patients with COVID-19 without clinical suspicion for SI suggest that SIs may often remain undiagnosed. McfDNA-Seq, when clinically available, can offer a non-invasive diagnostic tool for pathogen identification, with prognostic value on host inflammatory response and clinical outcomes.

Radiographic Assessment of Lung Edema (RALE) Scores are Highly Reproducible and Prognostic of Clinical Outcomes for Inpatients with COVID-19 (preprint)

INTRODUCTION: Chest imaging is necessary for diagnosis of COVID-19 pneumonia, but current risk stratification tools do not consider radiographic severity. We quantified radiographic heterogeneity among inpatients with COVID-19 with the Radiographic Assessment of Lung Edema (RALE) score on Chest X-rays (CXRs). METHODS: We performed independent RALE scoring by [≥]2 reviewers on baseline CXRs from 425 inpatients with COVID-19 (discovery dataset), we recorded clinical variables and outcomes, and measured plasma host-response biomarkers and SARS-CoV-2 RNA load from subjects with available biospecimens. RESULTS: We found excellent inter-rater agreement for RALE scores (intraclass correlation co-efficient=0.93). The required level of respiratory support at the time of baseline CXRs (supplemental oxygen or non-invasive ventilation [n=178]; invasive-mechanical ventilation [n=234], extracorporeal membrane oxygenation [n=13]) was significantly associated with RALE scores (median [interquartile range]: 20.0[14.1-26.7], 26.0[20.5-34.0] and 44.5[34.5-48.0], respectively, p<0.0001). Among invasively-ventilated patients, RALE scores were significantly associated with worse respiratory mechanics (plateau and driving pressure) and gas exchange metrics (PaO2/FiO2 and ventilatory ratio), as well as higher plasma levels of IL-6, sRAGE and TNFR1 levels (p<0.05). RALE scores were independently associated with 90-day survival in a multivariate Cox proportional hazards model (adjusted hazard ratio 1.04[1.02-1.07], p=0.002). We validated significant associations of RALE scores with baseline severity and mortality in an independent dataset of 415 COVID-19 inpatients. CONCLUSION: Reproducible assessment of radiographic severity revealed significant associations with clinical and physiologic severity, host-response biomarkers and clinical outcome in COVID-19 pneumonia. Incorporation of radiographic severity assessments may provide prognostic and treatment allocation guidance in patients hospitalized with COVID-19.

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19 (preprint)

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19. (preprint)

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in simultaneously collected longitudinal samples from mechanically-ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (r=0.83, p<10-8) and then declined in parallel except in non-survivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early in severe disease.

Longitudinal changes in age and race of patients with SARS-CoV-2 in a multi-hospital health system (preprint)

BackgroundThe COVID-19 pandemic continues to affect the United States and the world. Media reports have suggested that the wave of the alpha variant in the Spring of 2021 in the US caused more cases among younger patients and racial and ethnic subgroups. ApproachWe analyzed electronic health record data from a multihospital health system to test whether younger patients accounted for more cases and more severe disease, and whether racial disparities are widening. We compared demographics, patient characteristics, and hospitalization variables for patients admitted from November 2020 through January 2021 to those admitted in March and April 2021. ResultsWe analyzed data for 37, 502 unique inpatients and outpatients at 21 hospitals from November 1, 2020 to April 30, 2021. Compared to patients from November through January, those with positive tests in March and April were younger and less likely to die. Among patients under age 50, those with positive tests in March and April were three times as likely to be hospitalized and twice as likely to require ICU admission or mechanical ventilation. Individuals identified as Black represented a greater proportion of cases and hospitalizations in March and April as compared to November through January. ConclusionsWe found that relative COVID-19 hospitalization rates for younger individuals and individuals identified as Black were rising over time. These findings have important implications for ongoing public health measures to mitigate the impact of the pandemic.

CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease is autocrine TNF-α/TNFRI-dependent (preprint)

Lymphopenia is common in severe COVID-19 disease, yet the mechanisms are poorly understood. In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared to recovered, mild disease (p<0.0001). In severe disease, immunodominant CD4+ T cell responses to Spike-1(S1) produced increased in vitro TNF-, but impaired proliferation and increased susceptibility to activation-induced cell death (AICD). CD4+TNF-+ T cell responses inversely correlated with absolute CD4+ counts from severe COVID-19 patients (n=76; R=-0.744, P<0.0001). TNF- blockade including infliximab or anti-TNFRI antibodies strikingly rescued S1-specific CD4+ proliferation and abrogated S1-AICD in severe COVID-19 patients (P<0.001). Single-cell RNAseq demonstrated downregulation of Type-1 cytokines and NF{kappa}B signaling in S1-stimulated CD4+ cells with infliximab treatment. Lung CD4+ T cells in severe COVID-19 were reduced and produced higher TNF- versus PBMC. Together, our findings show COVID-19-associated CD4+ lymphopenia and dysfunction is autocrine TNF-/TNFRI-dependent and therapies targeting TNF- may be beneficial in severe COVID-19.

Bifurcated monocyte states are predictive of mortality in severe COVID-19 (preprint)

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1{beta}) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1{beta} and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.

Lower respiratory tract myeloid cells harbor SARS-CoV-2 and display an inflammatory phenotype (preprint)

SARS-CoV-2 pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the lower respiratory tract, which may contribute to morbidity and mortality. We describe endotracheal aspirate samples from seven patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. We note SARS-CoV-2 virions within lower respiratory tract myeloid cells shown by electron tomography, immunofluorescence confocal imaging, and immuno-electron microscopy. Endotracheal aspirates are primarily composed of mononuclear and polymorphonuclear leukocytes. These myeloid cells that harbor virus are frequently positive for CD14 and/or CD16 and most display an inflammatory phenotype marked by expression of IL-6 and tissue factor mRNA transcript and protein expression.