Multivariate protein landscape of host response in hospitalised patients with suspected infection in the emergency department

Abstract

Suspected infection requiring hospitalisation has highly heterogenous presentation. Yet, variances in host response and its implications are largely unknown. In this multicentre cohort of 3802 individual patients presenting to the Emergency Department (ED) with suspected infection requiring hospitalisation, we apply uniform manifold approximation and projections and K-means clustering to 29 plasma proteins to identify biologically discrete host response clusters. In this work, we first describe two large clusters, called "Dysregulated" and "Undifferentiated", with abnormal protein concentrations and adverse outcomes in the former. Through further clustering, we identify 4 sub-clusters in the Dysregulated cluster, each with discrete biological signatures, clinical correlates, and outcomes. Clusters 3 and 4 are characterised by renal impairment and viral infections respectively. Clusters 5 and 6 are associated with bacterial culture positivity, with the former consistent with an immunosuppressed signature and worse outcomes, and the latter with gram-negative bacteria, higher IL-6 and IL-8, and better outcomes despite higher vasopressor use. These clusters are a biologically driven approach to characterising acute suspected infection and may lead to more targeted therapeutics.

Fig. 1. UMAP of 29 plasma proteins in the population.

Panel (A): 3-dimensional representation of the UMAP of all 3802 patients included in the analysis. Panel (B): Distribution of frequently encountered comorbidities overlayed on a 2-dimensional representation of the UMAP. Panel (C): Highlighted area localised to past medical history of liver disease, alcohol abuse, and coagulopathy on a 2-dimensional representation of the UMAP (left panel; in red). Mean standardised values of protein biomarkers used in UMAP among patients in the area highlighted in the UMAP with a history of liver disease, alcohol abuse, and coagulopathy (centre panel). Mean standardised values of continuous clinical variables extracted from the electronic health record among patients in the area highlighted in the UMAP with a history of liver disease, alcohol abuse, and coagulopathy (right panel). Panel (D): Highlighted area localised to past medical history of solid tumour and or metastatic disease on a 2-dimensional representation of the UMAP (left panel; in red). Mean standardised values of protein biomarkers among patients in the area highlighted in the UMAP (centre panel). Mean standardised values of continuous clinical variables extracted from the electronic health record among patients in the area highlighted in the UMAP (right panel). Values were standardised to the mean values of the entire cohort for each variable in Panel (C and D). ALT Alanine Aminotransferase, AST Aspartate Aminotransferase, BUN Blood Urea Nitrogen, CRP C-reactive protein, DBP Diastolic blood pressure, FLT-3 ligand Fms-related tyrosine kinase 3 ligand, GCSF Granulocyte Colony-Stimulating Factor, GMCSF Granulocyte-Macrophage Colony-Stimulating Factor, IL Interleukin, IP-10 Interferon Gamma-Induced Protein 10, MCP Monocyte Chemoattractant Protein, MIP Macrophage Inflammatory Protein, NGAL Neutrophil Gelatinase-Associated Lipocalin, NLR Neutrophil:Lymphocyte, PDL-1 Programmed Death-Ligand 1, RDW Red Cell Distribution Width, SBP Systolic Blood Pressure, SF Ratio SpO₂/FiO₂, sTREM-1 Soluble Triggering Receptor Expressed on Myeloid Cells, TNF Tumour Necrosis Factor, TRAIL TNF-related apoptosis-inducing ligand, UMAPL Uniform Manifold Approximation and Projection, VCAM-1 Vascular Cell Adhesion Molecule-1, VEGF Vascular Endothelial Growth Factor, WBC White Blood Cell Count.

Fig. 2. Phase 1 K-means clustering of the UMAP.

Panel (A): The two clusters identified using K-means, the Dysregulated and Undifferentiated Clusters. Panel (B): Differences in mean standardised values of protein biomarkers used to create UMAP between patients in the Dysregulated and Undifferentiated Clusters. Panel (C): Differences in Mean standardised values of continuous clinical variables extracted from the electronic health record between patients in the Dysregulated and Undifferentiated Clusters. Values were standardised to the mean values of the entire cohort for each variable in Panels (B, C). Panel (D): Differences in primary and secondary outcomes between the Dysregulated and Undifferentiated Clusters. Panel (E): Odd ratio of developing the primary outcome (death or discharge > 7 days after admission) if antibiotics were delayed by more than three hours after order of blood cultures in the subset of patients who received antibiotics within 12 h after the time of the first vital sign (n = 2879). P-value is from the likelihood ratio test assessing the addition of the interaction term for cluster assignment and antibiotic delay status (yes/no) in a multivariable model with the primary outcome as the dependent variable. The odds ratios and corresponding 95% confidence intervals are shown with the point and error bars, respectively. ALT Alanine Aminotransferase; AST Aspartate Aminotransferase, BUN Blood Urea Nitrogen, CRP C-reactive protein, DBP: Diastolic blood pressure, FLT-3 ligand Fms-related tyrosine kinase 3 ligand, GCSF Granulocyte Colony-Stimulating Factor, GMCSF Granulocyte-Macrophage Colony-Stimulating Factor, IL Interleukin, IP-10 Interferon Gamma-Induced Protein 10, MCP Monocyte Chemoattractant Protein, MIP Macrophage Inflammatory Protein, NGAL Neutrophil Gelatinase-Associated Lipocalin, NLR Neutrophil:Lymphocyte, PDL-1 Programmed Death-Ligand 1, RDW ed Cell Distribution Width, SBP Systolic Blood Pressure, SF Ratio SpO₂/FiO₂, sTREM-1 Soluble Triggering Receptor Expressed on Myeloid Cells, TNF Tumour Necrosis Factor, TRAIL TNF-related apoptosis-inducing ligand, UMAPL Uniform Manifold Approximation and Projection, VCAM-1 Vascular Cell Adhesion Molecule-1, VEGF Vascular Endothelial Growth Factor, WBC White Blood Cell Count.

Fig. 3. Phase 2 K-means re-clustering of the Undifferentiated cluster.

Panel (A): Depiction of the two subclusters identified in the Undifferentiated cluster, named Cluster 1 (light blue) and Cluster 2 (purple), on the 3-dimensional UMAP. Panel (B): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 1. Panel (C): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 2. Values were standardised to the mean values of the entire cohort for each variable in Panels (B and C). Panel (D): Differences in outcomes between Clusters 1 and 2. ALT Alanine Aminotransferase, AST Aspartate Aminotransferase, BUN Blood Urea Nitrogen, CRP C-reactive protein, DBP Diastolic blood pressure, FLT-3 ligand Fms-related tyrosine kinase 3 ligand; GCSF Granulocyte Colony-Stimulating Factor; GMCSF Granulocyte-Macrophage Colony-Stimulating Factor; IL Interleukin; IP-10 Interferon Gamma-Induced Protein 10; MCP Monocyte Chemoattractant Protein; MIP Macrophage Inflammatory Protein; NGAL Neutrophil Gelatinase-Associated Lipocalin; NLR Neutrophil:Lymphocyte; PDL-1 Programmed Death-Ligand 1; RDW Red Cell Distribution Width; SBP Systolic Blood Pressure; SF Ratio SpO₂/FiO₂; sTREM-1 Soluble Triggering Receptor Expressed on Myeloid Cells; TNF Tumour Necrosis Factor; TRAIL TNF-related apoptosis-inducing ligand; UMAPL Uniform Manifold Approximation and Projection; VCAM-1 Vascular Cell Adhesion Molecule-1; VEGF Vascular Endothelial Growth Factor; WBC White Blood Cell Count.

Fig. 4. Phase 2 K-means re-clustering of the Dysregulated cluster.

Panel (A): Depiction of the four subclusters identified in the Dysregulated cluster named Cluster 3 (orange), Cluster 4 (brown), Cluster 5 (green), and Cluster 6 (yellow) on the 3-dimensional UMAP. Panel (B): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 3. Panel (C): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 4. Panel (D): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 5. Panel (E): Mean standardised values of protein biomarkers (left panel) and continuous clinical variables (right panel) in Cluster 4. Values were standardised to the mean values of the entire cohort for each variable in Panels B–E. ALT Alanine Aminotransferase, AST Aspartate Aminotransferase, BUN Blood Urea Nitrogen; CRP C-reactive protein; DBP Diastolic blood pressure; FLT-3 ligand Fms-related tyrosine kinase 3 ligand; GCSF Granulocyte Colony-Stimulating Factor; GMCSF Granulocyte-Macrophage Colony-Stimulating Factor; IL Interleukin; IP-10 Interferon Gamma-Induced Protein 10; MCP Monocyte Chemoattractant Protein; MIP Macrophage Inflammatory Protein; NGAL Neutrophil Gelatinase-Associated Lipocalin; NLR Neutrophil:Lymphocyte; PDL-1 Programmed Death-Ligand 1; RDW Red Cell Distribution Width; SBP Systolic Blood Pressure; SF Ratio SpO₂/FiO₂; sTREM-1 Soluble Triggering Receptor Expressed on Myeloid Cells; TNF Tumour Necrosis Factor; TRAIL TNF-related apoptosis-inducing ligand; UMAPL Uniform Manifold Approximation and Projection; VCAM-1 Vascular Cell Adhesion Molecule-1; VEGF Vascular Endothelial Growth Factor; WBC White Blood Cell Count.

Fig. 5. Clinical characteristics of Clusters 3–6.

Panel (A): Differences in outcomes between Cluster 3–6. Panel (B): Distribution of positive cultures stratified by Gram staining projected onto the 2-dimensional UMAP, with subcluster areas depicted by encircled areas. Panel (C): Distribution of viral pathogens identified projected onto the 2-dimensional UMAP. Panel (D): Odds ratio of clinical features associated with each cluster generated in logistic regression models looking at all patients in the dysregulated group (n = 1973). The odds ratios and corresponding 95% confidence intervals are shown with the point and surrounding error bars. Panel (E): Differences in selected protein biomarkers between patients with positive blood cultures with Gram-negative species in Clusters 5 and 6. The middle line in each violin plot represents the median. Statistical significance was determined by Wilcoxon rank sum tests. Exact p-values: CRP (9.86e-16), GCSF (2.99e-18), IL-10 (5.84e-20), IL-15 (1.94e-10), IL-6 (1.11e-17), IL-7 (5.30e-11), MCP-1(1.55e-26), MIP3 Alpha (3.79e-10). CRP C-reactive protein, GCSF Granulocyte Colony-Stimulating Factor, IL Interleukin, MCP-1 Monocyte Chemoattractant Protein-1, MIP-3 Macrophage Inflammatory Protein-3, SOFA Sequential organ failure assessment score. *p < 0.05; **p < 0.01; ***p < 0.001.