The overview of the study and analyses is detailed in Supplementary Fig. 1. A total of 1,395 patients met the inclusion criteria for the study of whom we excluded 324 patients who did not receive any vasoactive support. The median age of the patients included in the study (n = 1,071) was 5.3 years (quartile 1: 1.7; quartile 3: 11.0 years). The derivation set was comprised of 646 patients and the validation set included 425 patients.
Latent profile analyses in the derivation set revealed two phenotypes. Differences in standardized variables between the two phenotypes are shown in Fig. 1. One of the phenotypes (n = 126, 19.5%) was characterized by high Angiopoietin-2/Tie-2 ratio, Angiopoietint-2, soluble thrombomodulin (sTM), interleukin 8 (IL-8), and intercellular adhesion molecule 1 (ICAM-1) and low Tie-2 and Angiopoietin-1, which we designated as the ‘inflamed’ phenotype. This group was characterized by a high serum creatinine, blood urea nitrogen (BUN), lactate, a high international normalized ratio (INR), and low platelet counts. We labeled the remaining patients (n = 520, 80.5%), characterized by the absence of such features, as the ‘uninflamed’ phenotype.
Table 1 shows the comparisons between phenotypes in the derivation and validation sets – the latter based on the assignments of our SVM classifier. There were no differences in age and sex comparing phenotypes. Although patients who were ‘inflamed’ were more likely to have had a history of oncologic disease or bone marrow transplantation than those ‘uninflamed’ in the derivation set, there were no statistically significant differences in the validation set. Patients with an ‘inflamed’ phenotype had a trend toward higher rates of positive blood cultures in the derivation set (26.2% vs. 19.2%, p = 0.08), which reached statistical significance in the validation set (33.8% vs. 20.6%, p = 0.016), relative to those ‘uninflamed’. There were no significant differences in the type of pathogen. Patients with an ‘inflamed’ phenotype had higher baseline illness severity and significantly worse clinical outcomes in the derivation and validation sets. Finally, patients with an ‘inflamed’ phenotype were more likely to have been prescribed adjunctive corticosteroids by treating physicians, relative to those ‘uninflamed’.
Patients with an ‘inflamed’ phenotype had over 5-fold higher odds of 7-day mortality (adj. OR 5.6, 95% CI: 3.6–8.6, p < 0.001), over 4-fold higher odds of 28-day mortality (adj. OR 4.4, 95% CI: 3.0-6.4, p < 0.001), and nearly 4-fold higher odds of complicated course (adj. OR 3.9, 95% CI: 2.8–5.5, p < 0.001) relative to those ‘uninflamed’. Results of interactions between phenotypes and common sepsis therapies on patient outcomes are detailed in Table 2. Patients with an ‘inflamed’ phenotype were more likely to have received ≥ 100 ml/kg of fluid on day 1 of PICU admission, ≥ 2 vasoactive agents, corticosteroids, required intubation and continuous renal replacement therapy (CRRT) support with commensurately worse outcomes, relative to those who ‘uninflamed’. We did not identify any significant interaction between phenotype and sepsis therapies on outcomes with one exception. Patients with an ‘inflamed’ phenotype who received ≥ 2 antimicrobial therapies had a significantly higher rate of complicated course in comparison with those ‘uninflamed’ who received ≥ 2 antimicrobial therapies (65.5% vs 26.6%, interaction p-value 0.021).
Transcriptomic data was available in 144 patients. We identified 44 differentially expressed genes (DEGs) when comparing patients with ‘inflamed’ (n = 17) vs. ‘uninflamed’ phenotype (n = 127), of which 25 genes were overexpressed and 19 were underexpressed. Biological pathways enriched among patients with an ‘inflamed’ phenotype relative to those ‘uninflamed’ corresponded to activation of the immune system, cytokine signaling, neutrophil degranulation, and antimicrobial peptides. CIBERSORT analyses identified that the proportion of neutrophils was lower among patients with an ‘inflamed’ phenotype relative to those ‘uninflamed’. Expression data was available for 14 overexpressed and 5 underexpressed genes, identified through DEG analyses, in the integrated single-cell dataset. After correction for multiple comparisons, genes overexpressed among those with an ‘inflamed’ phenotype corresponded to those expressed by developing neutrophils, proliferating T lymphocytes/Natural Killer (NK) cells, and NK cells. In contrast, genes underexpressed among those with an ‘inflamed’ phenotype corresponded to those expressed by mature neutrophils. These data are shown in Fig. 2; with additional details presented in the Online Supplement.
A total of 233 patients in the study had data on established gene-expression endotype and latent profile phenotype assignments. There was no statistically significant association between endotypes and phenotypes in the cohort (Pearson χ2 test, p-value of 0.08). Figure 3 shows the Kaplan Meier survival curves based on gene-expression endotype (A vs. B), latent profile phenotype (‘inflamed’ vs. ‘uninflamed’), and an integrated scheme where we considered all four possible combinations of endotype and phenotype assignment. Patients classified as endotype B & ‘uninflamed’ had the lowest mortality risk. Relative to this group, those classified as endotype A & ‘inflamed’ had an over 12-fold (RR: 12.5, 95% CI: 3.8, 41.2, p < 0.001) higher relative risk of mortality; those with endotype B & ‘inflamed’ had a nearly 5-fold increase in mortality (RR; 4.8, 95% CI: 1.1, 20.1, p = 0.032); those with endotype A & ‘uninflamed’ had an over 3-fold increase in mortality (RR: 3.6, 95%CI: 1.2, 11.1, p = 0.024). There were no statistically significant differences in mortality between the latter two subclasses.