BAL Hierarchical clustering and Principal Component Analysis
13 subjects had complete BAL and plasma immune markers and available Ct SARS-CoV-2 RT-PCR. Immune markers IL-1β IL-10, IL-6, IL-12, MCP-1, IFN-γ and IP-10 were the most relevant variables in separating subjects into 2 clusters: cluster 1 comprised the majority of the subjects (11 patients 84.6%) while cluster 2 was characterized by higher inflammatory markers (2 patients, 15.4%). The model with 3 clusters further divided cluster 1, i.e. the hypoinflammatory cluster, into low and high viral load as measured by the Ct SARS-CoV-2 RT-PCR: the new cluster 3 (3 of 13 patients, 23.1%) had higher semi-quantitative SARS-CoV-2 RT-PCR compared to the new cluster 1 (8 of 13, 61.5%) (Fig. 1 Panels A and B).
Overall, 88% of the variance was accounted for by the first 3 principal components (PC) (Figs.1 and 2 and supplemental Table E2). PC1 explains the highest variance at 63% with IFN-γ, IL-10, IL-6, IL-1β, IL-12, IP-10 and MCP-1, having the highest loadings (negatively correlated). The largest group of subjects (84.6%), clusters 1 and 3 (i.e. the hypoinflammatory clusters), are located on the positive side of PC1. PC2 explains 15% of the variance and it is mainly based on the Ct values (positively correlated). Clusters 2 and 3 are located on opposite sides of PC2. PC3 explained 10% of the variance with IL-10, Ct and MCP-1 being the highest contributors (positively correlated). Overall, we can label cluster 1 as hypoinflammatory low viral load, cluster 2 as hyperinflammatory and cluster 3 as hypoinflammatory high viral load.
Tables 1 and E1 illustrate the distribution of clinical variables, laboratory results and outcomes between the entire cohort and the BAL clusters. The frequency of immunocompromising conditions, prior administration of remdesivir and dexamethasone were similar in all groups. Cytokine concentrations were higher in the hyperinflammatory cluster 2. This was also characterized by higher use of vasopressors, lower PaO2/FiO2 ratio and neutrophilic predominance of the BAL. Subjects in the hypoinflammatory high viral load cluster (Cluster 3) had bronchoscopy performed more rapidly following intubation, had a predominantly lymphocytic BAL and had higher mortality.