Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, standard PBMC isolation from blood requires technical expertise and over 2 hours of on-site processing using Ficoll density gradient separation (“Ficoll”) for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize on-site processing, we developed cryopreservation with PBMC recovery offsite (Cryo-PRO), a method of immediate on-site whole-blood cryopreservation and subsequent batched PBMC isolation in a central laboratory prior to sequencing. We compared multimodal single-cell immune profiling results from samples processed using Cryo-PRO versus standard on-site Ficoll separation in 23 patients with sepsis. Key outputs, including cell substate fractions, marker genes, and surface protein expression were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis. Capture of T cell receptor transcripts was also comparable across both methods. Cryo-PRO reduced on-site sample processing time from more than 2 hours to less than 15 minutes and was reproducible across 2 enrollment sites, thus demonstrating potential for expanding multimodal single-cell analyses in multicenter studies of sepsis and other diseases.

OBJECTIVES:. Understanding the mechanistic impact of fostamatinib, a spleen tyrosine kinase inhibitor, in severe COVID-19 using biomarkers associated with disease severity is crucial for the development of host-directed therapeutics. We analyzed samples from a randomized clinical trial to investigate the impact of fostamatinib on multiple inflammatory biomarkers associated with COVID-19 disease severity. DESIGN:. Secondary analyses of biomarkers from a randomized clinical trial. SETTING:. Multicenter randomized clinical trial. PATIENTS:. A total of 400 adults hospitalized with COVID-19 were enrolled in a phase 3 randomized clinical trial. Absolute neutrophil counts (ANCs) were analyzed across 392 patients and biomarkers were measured in 190 patients with available plasma samples. INTERVENTIONS:. Adults hospitalized with COVID-19 were randomized to receive either fostamatinib (150 mg bid) or placebo. ANCs and 24 biomarkers were assessed at day 0 and over time using a multiplexed Meso Scale Discovery assay (Meso Scale Diagnostics LLC, Rockville, MD). MEASUREMENTS AND MAIN RESULTS:. At day 0, participants with World Health Organization ordinal scale 5–7 had elevated ANC counts, compared with ordinal scale 4. In addition, the levels of neutrophil-associated biomarkers, inflammatory cytokines, and mediators of endothelial dysfunction at day 0 were increased in the participants who were ordinal scale 5–7 vs. ordinal scale 4. Randomization to fostamatinib compared with placebo resulted in a decrease in ANC and several neutrophil-associated biomarkers, pro-inflammatory cytokines, and mediators of endothelial dysfunction/tissue damage. This differential finding was also demonstrated in a subgroup of patients (n = 85) with a hypoinflammatory phenotype. LIMITATIONS:. Missing plasma samples and neutral phase 3 trial results. CONCLUSIONS:. Randomization to fostamatinib resulted in lower neutrophil counts and levels of circulating biomarkers in hospitalized patients with COVID-19; however, the observed impact of fostamatinib was modest compared with prior studies.

BACKGROUND:The Surviving Sepsis Campaign guidelines stratify antibiotic urgency in suspected sepsis by shock status, recommending treatment within 1 h for patients with shock and within 3 h for possible sepsis without shock. UK National Institute for Health and Care Excellence (NICE) guidelines use National Early Warning Score 2 (NEWS2) risk categories, with a window of 1 h for the group at highest risk of mortality (aggregate score ≥7). We aimed to compare these strategies for identifying patients in whom antibiotic delays are associated with mortality. METHODS:We conducted a retrospective cohort study between May 31, 2015, and Feb 28, 2024, of adults (age ≥18 years) admitted via the emergency departments of nine hospitals within the Mass General Brigham health-care system (MA, USA) and treated for suspected infection, defined as blood culture sampling and intravenous antibiotic administration within 6 h of emergency department arrival. Exclusion criteria included transition to palliative care or death within 6 h of arrival to emergency department, transfer from acute care hospitals, psychiatric or obstetric admissions, missing vital signs or key laboratory test results within 12 h of arrival, oral or intravenous antibiotic receipt before arrival, insufficient data to calculate NEWS2 scores, and positive SARS-CoV-2 PCR test results within 2 days of arrival. We estimated hourly associations between time to antibiotic administration and hospital mortality using multivariable logistic regression with generalised estimating equations, stratified by shock (defined as persistent hypotension and lactate >2 mmol/L) and by laboratory-upgraded NEWS2 scores (NEWS2+), which were calculated per UK NICE guidance by shifting patients up one category if they had neutropenia, lactate concentration higher than 2 mmol/L, or laboratory evidence of new organ dysfunction within 3 h of arrival. FINDINGS:115 464 encounters with suspected infection were identified, of which 15 797 were ineligible based on exclusion criteria. The final analysis cohort included 71 593 patients who contributed 99 667 encounters (52 835 [53%] male, 46 828 [47%] female). 4867 (4·9%) encounters had shock. NEWS2+ scores were 0 in 9672 (9·7%) encounters, 1-4 in 37 310 (37·4%) encounters, 5-6 in 26 134 (26·2%) encounters, and ≥7 in 26 551 (26·6%) encounters. Each additional hour to antibiotic administration was associated with higher mortality in encounters with shock (adjusted odds ratio [OR] 1·15, 95% CI 1·07-1·25; 1·5% absolute increase per hour, 95% CI 0·5-2·4), but not in those without shock (1·03, 1·00-1·05). Delays were also associated with higher mortality in encounters with NEWS2+ ≥7 (1·07, 1·04-1·11; 0·5% absolute increase per hour, 95% CI 0·3-0·8), including 22 902 (86·3%) of 26 551 encounters without shock (1·05, 1·01-1·09; 0·4% absolute increase per hour, 95% CI 0·1-0·6%). No association was observed in encounters with NEWS2+ scores less than 7. INTERPRETATION:The NICE-defined high-risk NEWS2 category (scores ≥7) identified patients in whom each additional hour to antibiotic administration was associated with increased mortality, including many without shock, whereas no association was observed for lower NEWS2 scores. These findings suggest that NEWS2 might better target urgent antibiotic therapy beyond shock-based assessment alone. FUNDING:US Agency for Healthcare Research and Quality and US Centers for Disease Control and Prevention.

Rationale: There is heterogeneity in the manifestations of patients with coronavirus disease 2019 (COVID-19). The T cell receptor (TCR) repertoire is responsible for pathogen recognition, and selection of suboptimal TCRs is implicated in an impaired response. We leveraged a deep learning pipeline to characterize the epitope affinities of TCRs in patients with COVID-19 to elucidate the relationship between TCR-epitope affinity and disease severity. Methods: We enrolled 351 patients presenting to the emergency room at a tertiary care hospital with febrile respiratory illness of which 302 patients had confirmed SARS-CoV-2 infection by nasal PCR. Clinical features and peripheral blood were collected at enrollment and on days 3, 7, 14, and 28. Single-cell RNA sequencing and TCR profiling were performed for each sample. Utilizing TCRconv, a previously reported deep learning pipeline combining an embedding model and convolutional neural network (CNN), we created embeddings from the CDR3 and TCRβ chain sequences in the VDJ database, and trained the CNN to predict affinity for 71 available epitopes across 14 species. We generated embeddings for each sequence in our samples and applied the CNN to predict epitope affinities. We modeled the association between TCRβ V/J expression with severity on a modified WHO ordinal scale using linear models adjusting for age, sex, and COVID-19 status and examined epitope specificity of severity-associated TCRβ V/J combinations. Results: Clonality was associated with age(p=2e-9) and age-adjusted severity(p=4e-4). Across 85,355 TCR sequences, epitopes from SARS-CoV-2 were predicted for 15,341(18.0%) sequences. Limiting the analysis to the 2,227 sequences with high-confidence predictions(ŷ>0.5) revealed 548(24.6%) sequences associated with SARS-CoV-2 epitopes. Across these sequences we identified 78 unique V/J gene combinations associated with severity (FDRBenjamini-Hochberg<0.05, FC>2) of which 30(38.5%) were associated with SARS-CoV-2 epitopes (TFE [nsequences=75, nV/J =12], YLQ [nsequences =72, nV/J=10], NYN [nsequences =17, nV/J =7], QYI [nsequences =1, nV/J=1]). Specificity within this set of genes was defined by the J gene with TRBJ1-2, TBRJ2-2, TRBJ2-7, TRBJ2-1 corresponding with TFE(10/12), YLQ(7/7), NYN(10/10), and QYI(1/1), respectively. However, expression of TRBV5 family genes corresponded with the strongest acuity-associated combination for YLQ(p=1.0E-3), NYN(p=8.26E-4), and QYI(p=1.0E-3). Conclusion: The diversity and composition of the TCR repertoire are associated with disease severity in COVID-19. However, the majority of severity-associated TCRs have affinity for non-SARS-CoV-2 epitopes. Although the TRBJ genes drive epitope specificity, expression of genes from the TRBV5 family yielded the strongest acuity association, suggesting that TRBV5 family expression may yield TCRs with suboptimal specificity across multiple SARS-CoV-2 epitopes.

Sepsis, defined as life-threatening acute organ dysfunction due to infection, is generally considered a hospital-based issue. However, sepsis usually begins in the community, where knowledge of sepsis is scarce, diagnosis is difficult, and resources vary. Community-based interventions might offer the best opportunity for prevention, prompt diagnosis, and improved outcomes. In this Viewpoint, we address current gaps and limitations in understanding of sepsis in the community and outline research priorities, clinical priorities, and existing initiatives across four domains: mitigation (ie, reduction of population-based sepsis risk), monitoring (ie, screening for sepsis in individuals at high risk of sepsis), measurement (ie, identification of sepsis in the community), and management (ie, treatment of sepsis in the community). We propose a pathway to improve the care of individuals with, or at risk of, sepsis in the community and delineate the next steps to advance the field.

BACKGROUND:Treatment guidelines recommend rapidly treating all patients with suspected sepsis with broad-spectrum antibiotics. This may contribute to antibiotic overuse. We quantified the incidence of antibiotic overtreatment and possible antibiotic-associated harms among patients with suspected sepsis. METHODS:We reviewed the medical records of 600 adults treated for suspected sepsis with anti-methicillin-resistant Staphylococcus aureus and/or antipseudomonal β-lactam antibiotics in the emergency departments of 7 hospitals, 2019-2022, to assess their post hoc likelihood of infection, whether narrower antibiotics would have sufficed in retrospect, and possible antibiotic-associated complications. We used generalized estimating equations to assess associations between likelihood of infection and hospital mortality. RESULTS:Of 600 patients, 411 (68.5%) had definite (48.0%) or probable (20.5%) bacterial infection and 189 (31.5%) had possible but less likely (18.3%) or definitely no (13.2%) bacterial infection. Among patients with definite/probable bacterial infection, 325 of 411 (79.1%) received antibiotics that were overly broad in retrospect. Potential antibiotic-associated complications developed in 104 of 600 (17.3%) patients within 90 days, most commonly new infection or colonization with organisms resistant to first-line agents (48/600 [8.0%]). Mortality was higher for patients with less likely/definitely no bacterial infection versus definite/probable bacterial infections (9.0% vs 4.9%; adjusted odds ratio [aOR], 2.25 [95% confidence interval{CI}, 1.70-2.98]), but antibiotic-associated complication rates were similar (14.8% vs 18.5%; aOR, 0.79 [95% CI, .60-1.05]). CONCLUSIONS:Among 600 patients treated with broad-spectrum antibiotics for possible sepsis, 1 in 3 most likely did not have a bacterial infection, 4 in 5 of those with bacterial infections were treated with regimens that were broader than necessary in retrospect, and 1 in 6 developed antibiotic-associated complications.

Importance:The Centers for Medicare & Medicaid Services Severe Sepsis and Septic Shock Management Bundle (SEP-1) is supported by observational studies that report SEP-1 compliance is associated with lower mortality. Most studies, however, adjusted for limited confounders and provided little insight into why bundle-compliant care was not provided. Objectives:To identify the clinical factors that complicate the diagnosis and management of sepsis and assess their association with SEP-1 compliance and mortality. Design, Setting, and Participants:This retrospective cohort study was conducted among 590 adults with sepsis in the emergency department of 4 academic hospitals from January 1, 2019, to December 31, 2022. Patients' medical records were reviewed between September 2022 and December 2023. Main Outcomes and Measures:Study outcomes were (1) characteristics of patients who received SEP-1-compliant care vs characteristics of patients who received noncompliant care and (2) association between SEP-1 compliance and hospital mortality using multivariable models to adjust for successively more potential confounders (first demographics and comorbidities, then infection source, then severity of illness, and then clinical markers of complexity). Results:Of 590 patients with sepsis (median age, 65 years [IQR, 53-77 years]; 329 men [55.8%]), 335 (56.8%) received SEP-1-compliant care, and 225 (43.2%) received noncompliant care. Compared with patients in the compliant group, patients in the noncompliant group were more likely to be 65 years or older (142 [55.7%] vs 158 [47.2%]; odds ratio [OR], 1.41 [95% CI, 1.01-1.95]), to have multiple comorbidities (Elixhauser score >20: 99 [38.8%] vs 99 [29.6%]; OR, 1.51 [95% CI, 1.07-2.13]), and to have a higher incidence of septic shock (107 [42.0%] vs 107 [31.9%]; OR, 1.54 [95% CI, 1.10-2.16]), kidney dysfunction (87 [34.1%] vs 80 [23.9%]; OR, 1.65 [95% CI, 1.15-2.37]), and thrombocytopenia (43 [16.9%] vs 37 [11.0%]; OR, 1.16 [95% CI, 1.02-2.62]) on presentation. Compared with patients in the compliant group, those in the noncompliant group also had more nonfebrile presentations (136 [53.3%] vs 121 [36.1%]; OR, 2.02 [95% CI, 1.45-2.82]), impaired mental status (92 [36.1%] vs 94 [28.1%]; OR, 1.45 [95% CI, 1.02-2.05]), need for bedside procedures (57 [22.4%] vs 41 [12.2%]; OR, 2.06 [95% CI, 1.33-3.21]), acute concurrent noninfectious illnesses (140 [54.9%] vs 151 [45.1%]; OR, 1.48 [95% CI, 1.07-2.06]), and noninfectious illness as the primary factor associated with their presentation (84 [32.9%] vs 71 [21.2%]; OR, 1.82 [95% CI, 1.08-3.08]). SEP-1 compliance was associated with lower crude mortality rates compared with noncompliance (40 [11.9%] vs 41 [16.1%]; unadjusted OR, 0.60 [95% CI, 0.37-0.98]), but there was no statistically significant difference between groups after successively adjusting for demographics and comorbidities (adjusted OR [AOR], 0.71 [95% CI, 0.42-1.18]), infection source (AOR, 0.71 [95% CI, 0.43-1.20]), severity of illness (AOR, 0.86 [95% CI, 0.50-1.49]), and clinical markers of complexity (AOR, 1.08 [95% CI, 0.61-1.91]). Conclusions and Relevance:In this cohort study of adults with sepsis, complex clinical presentations were more common among patients whose treatment was noncompliant with SEP-1. These nuances are poorly captured in most observational studies but confound the association between SEP-1 compliance and mortality.

Abstract Background Immune response variation among sepsis patients leads to substantial clinical heterogeneity that makes treatment challenging. Bulk RNA-sequencing of circulating immune cells has been used in an attempt to resolve sepsis heterogeneity, and has facilitated classification of patients into different subtypes, or “endotypes”. The Sepsis Response Signature (SRS) classification comprises 3 endotypes: SRS1 represents an immunosuppressed transcriptional profile with high mortality; SRS2 a relatively immunocompetent response with low mortality, and SRS3 is a profile close to a normal immune response. However, because bulk RNA-seq does not distinguish the transcriptional contributions of different cell types, mechanistic insights from these classifications are lacking, thus limiting clinical utility. Single-cell RNA-sequencing (scRNA-seq) enables cell-specific transcriptomics and has the potential to demonstrate the cellular basis of sepsis endotypes. In this study, we performed scRNA-seq on blood from sepsis patients to investigate the differences in immune cell transcriptional states among SRS endotypes.Figure 1.Fractional abundance of monocyte substate 1 (MS1) is highest in the Sepsis Response Signature 1 (SRS1) endotype Fraction of MS1 cells relative to all peripheral blood mononuclear cells (PBMCs) in SRS1 (n= 10), SRS2 (n= 33), and SRS3 (n= 11) patients. Boxes show the median and inter-quartile range (IQR) for each endotype, with whiskers extending to 1.5x the IQR in either direction from the top or bottom quartile. Two-sided Wilcoxon rank-sum test with Benjamini-Hochberg correction used for comparisons; ***p<0.001, ****p<0.0001. Methods Mononuclear immune cells were isolated from the blood of sepsis patients (n=54) at hospital presentation and the transcriptomes of ∼1500 single cells per sample were analyzed. By aggregating gene counts for each patient, we generated pseudobulked transcriptional profiles to approximate whole blood gene expression. We then leveraged the SepstratifieR machine learning framework to stratify patients into SRS endotypes and compared the proportions of immune cell transcriptional states across the endotypes. Results A monocyte transcriptional state – monocyte substate 1 (MS1), was differentially expanded across SRS endotypes (Fig.1). MS1 abundance was significantly higher for patients in the SRS1 endotype compared to SRS2 (p< 0.001) or SRS3 (p< 0.0001). Conclusion Identifying cellular signals underlying endotypes may improve understanding of sepsis immunobiology and open paths to precision therapy. This study demonstrates the expansion of a monocyte transcriptional state in the SRS1 endotype. MS1 cells inhibit T cell activation and proliferation, suggesting a potential mechanistic explanation for the immunosuppressive state of SRS1 patients. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support

Sepsis is a dynamic syndrome of immune dysregulation and a leading cause of global mortality. Although immune suppression is recognized as a hallmark of sepsis, the temporal dynamics and mechanistic drivers of immune dysfunction remain incompletely understood. Here, we performed longitudinal single-cell transcriptomic and proteomic profiling of peripheral blood from 98 adults with sepsis or sterile inflammation, alongside 12 healthy controls, capturing immune trajectories from initial clinical presentation through recovery. Integration of RNA and surface protein data revealed an early expansion of a transcriptionally reprogrammed CD14+ monocyte population (MS1) exhibiting features of monocytic myeloid-derived suppressor cells (M-MDSCs), including downregulation of HLA-DR and upregulation of alarmins ( S100A8 , S100A9 ), resistin, and clusterin. M-MDSCs arise from emergency myelopoiesis and contribute to adaptive immune suppression through impaired antigen presentation and T cell inhibition. MS1 abundance peaked at initial presentation and declined progressively during the first week of clinical management in most patients. In contrast, CD8+ naive and CD4+ memory T cells exhibited sustained depletion, with recovery occurring at convalescence in only a subset of patients. Plasma proteomic profiling identified cytokines and growth factors, including IL-6 and resistin, that may contribute to MS1 induction and suppressive activity. IL-6 exhibited a kinetic trajectory that closely paralleled MS1 abundance, peaking early and declining during recovery. Resistin levels were positively correlated with MS1 abundance across all timepoints from acute sepsis through convalescence. These dynamics suggest temporally distinct cytokine roles in initiating and sustaining immunosuppressive myeloid responses in sepsis. Together, these findings define a high-resolution, time-resolved immune atlas of human sepsis, linking emergency myelopoiesis to downstream adaptive immune suppression. Our results suggest that coordinated dynamics between myeloid and lymphoid compartments shape early immune trajectories in sepsis and highlight MDSC-targeting pathways as potential therapeutic avenues. ### Competing Interest Statement M.R is an employee of Genentech, Inc. and owns equity in Roche. P.C.B. serves as a consultant to or equity holder in several companies including 10X Technologies/10X Genomics, GALT/Isolation Bio, Next Gen Diagnostics, Cache DNA, Concerto Biosciences, Stately Bio, Ramona Optics, Bifrost Biosystems, and Amber Bio. P.C.B.'s lab has received funding from Calico Life Sciences, Merck, and Genentech for unrelated research. N.H. holds equity in BioNTech and is an advisor for Related Sciences/Danger Bio. ### Funding Statement This work was supported in part by the National Institutes of Health (NIH R01AI153142). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board of the Massachusetts General Hospital gave ethical approval for this work (IRB number 2017P001681) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Background Timely management of sepsis with early targeted antimicrobial therapy improves patient outcomes. Rapid molecular assays (RMAs) have emerged, enabling the detection of bloodstream infection (BSI) with a shorter turnaround time than blood cultures (BCs). The accuracy of several RMAs has not been comprehensively reviewed. We aimed to identify commercial RMAs reported in the literature and evaluate their diagnostic performance compared to BC. Methods A systematic review and meta-analysis was conducted, covering MEDLINE, Cochrane Library, Embase, and Web of Science from inception to September 23, 2024. Eligible studies included patients with suspected or documented BSI, tested with both an RMA (turnaround time of <= 12 h, targeting >= 20 pathogens) and BC. Non- original research articles and animal studies were excluded. The primary outcomes were pooled sensitivity and specificity of RMAs for pathogen detection compared to BC. Bivariate analysis was used to produce summary receiver operating characteristic plots and diagnostic metric measures stratified by different units of analysis (sample versus patient), RMA types, and patient populations. Risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) and Quality Assessment of Diagnostic Accuracy Studies-Comparative (QUADAS-C) tools. The study was registered with PROSPERO, CRD42022377280. Findings A total of 63,916 articles were identified, of which 104 were included in the qualitative synthesis and 75 in the quantitative synthesis, covering 17,952 samples and 11,393 patients analyzed separately. Eleven RMAs were identified, with four included in the RMA-based subgroup analysis (LightCycler SeptiFast Test MGRADE (R), IRIDICA BAC BSI assay, SepsiTest, MagicPlex Sepsis Test) and fi ve additional ones in the pooled analysis (UMDSelectNA, VYOO (R), MicrobScan assay, MicrobScan-Kairos24/7, REBA Sepsis-ID test). Two RMAs were included in the qualitative synthesis only (InfectID-BSI, Pilot Gene Technology droplet digital polymerase chain reaction). Pooled specificity of RMAs was higher (0.858, 95% confidence interval (CI) 0.830-0.883) than sensitivity (0.659, 95% CI 0.594-0.719) by patient. Sensitivities varied by RMA type from 0.492 (95% CI 0.390-0.594, MagicPlex Sepsis Test) to 0.783 (95% CI 0.662-0.870, IRIDICA BAC BSI assay) by patient. Specificities varied more by patient population, ranging from 0.811 (95% CI 0.716-0.879) in the intensive care population to 0.892 (95% CI 0.838-0.930) in the emergency department population, by patient. Similar metrics were observed when the analysis was done by sample. Risk of bias was judged to be high in all included articles. Interpretation Despite their shorter turnaround time, low sensitivity means RMAs cannot replace BCs. However, our data indicate that RMAs may have value as an add-on test by increasing pathogen detection rates. Higher-sensitivity RMAs are needed which could possibly be achieved by expanding pathogen coverage and increasing blood sample volumes. High-quality implementation studies and standardized reporting are required to assess the clinical advantages of RMAs. Funding Centre for Translational Medicine, Semmelweis University. Copyright (c) 2024 The Authors. Published by Elsevier Ltd.

The alpha-variant wave of the COVID-19 pandemic provided a unique opportunity to study, at single-cell resolution, how near-universal exposure to the same pathogen can lead to either effective or dysfunctional immune responses in humans. Although single-cell RNA-sequencing studies have characterized immune cellular features of COVID-19, they have not shown how tocilizumab treatment changes these features at single-cell resolution, or which features might persist into convalescence. In this study, we analyzed 2.5 million circulating immune cells from 428 patients across time points (840 PBMC samples), encompassing three contemporaneous SARS-CoV-2 cohorts: acutely infected patients across five WHO disease severity levels and three time points, patients from the first randomized control trial to study the efficacy of tocilizumab in the management of COVID-19, and convalescent patients three months after infection. We used linear modeling to integrate multiple data types - including single-cell RNA-seq, CITE-seq, TCR and BCR sequencing, viral load measurements, viral neutralization assays, detection of 75 autoantibodies, HLA genotype data, and serum proteomics covering 1,463 targets - to derive the most comprehensive view to-date of the biological features of COVID-19 disease severity. Our findings show that myeloid-derived suppressor cells (MDSCs) act as a key immunologic pivot point in severe COVID-19. Myeloid dysfunction, which is marked by impaired antigen presentation, drives a non-productive adaptive immune response, as reflected by reduced expression of B and T cell gene programs involved in antigen recognition, immune synapse formation, and cytotoxicity. Severe disease is also linked to autoantibodies targeting type I interferons, influenced by specific HLA-DQB1 allelic variants, and strongly correlated with serum IL-6 levels. Tocilizumab treatment eliminates CLU -expressing MDSCs and ISG-positive myeloid subsets, restores antigen presentation, and reactivates productive adaptive immunity. These changes align with improved clinical outcomes and better clinical laboratory measures, including reduced CRP. While many immunologic abnormalities in acute severe COVID-19 resolve during convalescence 3-months post-infection, we observed persistently high ICOS expression in regulatory T cells, potentially linking acute infection to chronic post-COVID syndromes. Overall, we define distinct innate and adaptive host immune responses associated with acute, IL-6-responsive, and convalescent SARS-CoV-2 infection. Our multimodal and high-dimensional dataset with curated clinical metadata provides a foundational and clinically relevant resource for modeling host immune response biology in health and disease.

Abstract Background Early pathogen ID and targeted treatment are key to reducing bloodstream infection (BSI) morbidity and mortality. Current diagnostics rely on culture which takes 1-2 days for ID and longer for antimicrobial susceptibility testing (AST) results, or molecular assays with limited panels, few resistance markers, and high false positive rates. We report interim results of a first-in-kind comprehensive ID and predictive AST assay directly from patient blood samples. The system can deliver results in ∼8 hrs, uses ultra-high enrichment of microbial DNA directly from blood, whole-genome sequencing, and a predictive AST machine-learning algorithm to identify a broad range of species and pathogen/drug combinations. Methods We enrolled subjects with suspicion of BSI from 3 EDs and 1 ICU/inpatient in 4 Boston area hospitals in 2 IRB approved observational studies. We collected whole blood in SPS vacutainers and 10mL were processed at Day Zero Diagnostics (DZD) and sequenced on an Oxford Nanopore platform. Sequencing data were analyzed by Keynome® algorithms to determine pathogen ID and predict AST profiles. Tables 1 & 2 list current on-panel pathogens and drug models tested. Performance was compared to hospital microbiology lab phenotypic ID/AST results from blood cultures collected within 0-24 hours of the research draw. Results Species-level ID results in 225 subjects (6525 calls) demonstrated 80.0% sensitivity, 99.9% specificity, 64% PPV, and 99.9% agreement with clinical culture. Eight distinct species were identified among the 20 (8.9%) clinical culture positive samples with on-panel organisms. Thirteen samples had sufficient genome coverage for on-panel AST predictions demonstrating 92.3% categoric agreement with phenotypic AST. Conclusion Our data suggests the DZD diagnostic system can provide accurate ID and AST results directly from blood in patients with suspected BSI. To our knowledge these results are the first demonstration of whole genome recovery and comprehensive ID & AST directly from patient blood samples. This assay has the potential to revolutionize speed to diagnosis of BSI, thus facilitating targeted therapy, improved outcomes, and reduced development of antimicrobial resistance. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support Peter Hou, MD, Center for Disease Control: Grant/Research Support|Day Zero Diagnostics: Grant/Research Support|iDoc Telehealth Solutions: Ownership Interest Michael Donnino, MD, Day Zero Diagnostics: Grant/Research Support Archana Asundi, MD, Day Zero Diagnostics: Grant/Research Support|Gilead Sciences: Grant/Research Support|GSK/ViiV Healthcare: Grant/Research Support|Theratechnologies: Grant/Research Support Zoe H. Rogers, MPH, Day Zero Diagnostics: employment Emma Briars, PhD, Day Zero Diagnostics: employment Alison Gassett, MPH, Day Zero Diagnostics: employment Alexander Reidel, BS, Day Zero Diagnostics: employment Alexis Campbell, MA, Day Zero Diagnostics: Grant/Research Support Jason Wittenbach, PhD, Day Zero Diagnostics: employment Nicole Billings, PhD, Day Zero Diagnostics: employment

Abstract Background Though immune dysregulation often plays a key role in disease, characterizing circulating immune cells in critical illnesses is challenging. Studies using single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) reveals the type and function of patients' immune cells (cell substates) that could advance diagnostics and therapeutics, particularly in diseases defined by immune response heterogeneity such as sepsis. However, PBMC isolation from blood is required for scRNA-seq, and the gold-standard method (density gradient separation, or ‘Ficoll’) requires two hours of onsite processing, precluding large-scale sample collection at most clinical sites. Simplifying sample collection could enable participation of more health care centers, as well as the over-enrollment of patients for later retrospective adjudication, better reflecting the true patient heterogeneity in diseases.Figure 1.Workflow depicting the process from patient presentation in a health care center to sequencing results. The WB cryo method expedites sample collection in clinical settings by eliminating the need for Ficoll PBMC separation, a time-intensive process requiring laboratory equipment and trained operators. Differences in methods are highlighted in yellow. Methods We developed an alternative PBMC isolation method from cryopreserved whole blood (WB-cryo) using magnetic- and fluorescence- activated cell sorting (Fig. 1). We isolated PBMCs using the WB-cryo and Ficoll methods from patients with sepsis (n = 5) who sought evaluation at an Emergency Department and one healthy control. We performed scRNA-seq to identify and compare cell substate proportions and marker genes for key substates.Figure 2.Results of scRNA-seq on PBMCs isolated using Ficoll and WB cryo methods (n = 5 patients with sepsis, 1 healthy control). a) Uniform Manifold Approximation and Projection (UMAP) of separately clustered and annotated scRNA-seq data from each method; Bm denotes memory B cells, Bn: naive B cells, NK: natural killer cells, Mono: monocytes, MS1: monocyte substate 1, DC: dendritic cells, Tn: naive T cells, Tm: memory T cells, gdT: gamma delta T cells, Treg: regulatory T cells. b) correlation plot of resulting cell substate proportions (R = 0.87, p < 0.0001). Results Analysis included 9,996 cells isolated by Ficoll and 15,482 cells from WB-cryo. Sequencing quality was comparable between methods, as were cell substate fractions (Pearson correlation 0.87, p < 0.0001) (Fig. 2), and marker genes for a key monocyte substate enriched in patients with sepsis (19 of top 20 marker genes shared). WB-cryo reduced onsite sample processing time from 2 hours to < 10 minutes. Conclusion PBMCs isolated by WB-cryo retained similar transcriptional states when compared to PBMCs isolated using Ficoll, with > 10-fold less onsite hands-on time. With further validation, WB-cryo may expand scRNA-seq study enrollment in sepsis and other diseases. Ongoing work in a larger cohort (n = 23 patients with sepsis) processed across two clinical sites is underway to validate our isolation method as a way to extend the utility of scRNA-seq in multisite studies of complex and heterogeneous conditions like sepsis. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.