Figures
Overview of the study findings. TNF-R1: tumour necrosis factor receptor 1; TREM-1: triggering receptor expressed on myeloid cells 1. Figure partially created with BioRender.com.
- FIGURE 1
Overview of the three COVID-19 cohorts in which plasma biomarkers were measured. The external validation cohort was derived from a publicly available dataset of nonintubated COVID-19 patients in whom plasma proteins were measured by the Olink proximity extension assay [21]. ICU: intensive care unit. Figure partially created with BioRender.com.
- FIGURE 2
Mortality analysis of COVID-19 patients admitted to the general ward stratified by age decade. a) Kaplan–Meier plot of patients stratified by age decade. b) Risk of 30-day mortality with age modelled as a continuous variable. Given the nonlinear relationship between age and mortality, a restricted cubic spline function with three inner knots at default quantile locations was used. To calculate the odds ratio, the reference was set to 60 years of age. c) The same method as b), but now the 30-day mortality odds ratio is adjusted for demographics (inclusion hospital, sex and inclusion wave), age-related comorbidities (hypertension, diabetes, malignancies, immunosuppression, and chronic cardiac, neurological, respiratory and kidney disease), age-related chronic medication (antiplatelet and anticoagulant drugs), and COVID-19-related treatments both before and after sampling (corticosteroids including dexamethasone, anti-interleukin-6, imatinib, remdesivir and antibiotics).
- FIGURE 3
Principal component analysis (PCA) of host response domain differences between age groups: a) endothelial and coagulation response, b) systemic inflammation and organ damage, c) cytokines and d) chemokines. Principal components (PCs) 1 and 2 are plotted per domain. For each domain, the x- and y-axes are labelled with the percentage of the total variance within that domain that is explained by PC1 and PC2, respectively. The complete contribution of each biomarker to a PC score is depicted in supplementary table S6. The ellipse indicates the central 10% of each age group, colour coded as indicated in the key at the bottom of the figure. The arrows indicate the direction (arrow orientation) and strength (arrow length) of the correlation between each biomarker and the PCs. Next to each PCA plot are box plots with 1.5 interquartile range whiskers of PC1 and PC2. Herein, upper p-values were obtained by ANOVA between age groups: ρ-values with accompanying p-values were generated using a Spearman's correlation with ageing on a continuous scale. Note that a negative association of a PC with ageing may still reflect a positive association with biomarker concentration, as reflected by the direction of the arrows. Post-hoc testing was done with a Tukey test. *: p<0.05; **: p<0.01; ***: p<0.001. ANG: angiopoietin; sTie-2: soluble Tie-2; sE-selectin: soluble E-selectin; sThrombomodulin: soluble thrombomodulin; sVCAM-1: soluble vascular cellular adhesion molecule 1; PAI-1: plasminogen activator inhibitor 1; sCD31: soluble cluster of differentiation 31; sRAGE: soluble receptor for advanced glycation end-products; sTNF-R1: soluble tumour necrosis factor receptor 1; sTREM-1: soluble triggering receptor expressed on myeloid cells 1; SP-D: surfactant protein D; CD40L: CD40 ligand; PD-L1: programmed death ligand 1; CCL:C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; IL: interleukin; TNF: tumour necrosis factor; GM-CSF: granulocyte–macrophage colony-stimulating factor; IFN: interferon.
- FIGURE 4
Association of host response biomarkers with ageing. a) Heatmap depicting the magnitude of biomarker differences (Hedges’ g) between patients ≥70 years compared with other age groups. p-values were obtained from a linear (if linear) or cubic spline regression analysis (if nonlinear) in which age was modelled as a continuous variable. The adjusted model included demographics, age-related comorbidities, age and biomarker-related chronic medication, and COVID-19-related immunomodulating treatments before sampling (see Methods for details). Red indicates higher levels in patients ≥70 years and blue indicates lower levels in patients ≥70 years. #: biomarkers with a nonlinear relationship with ageing on a continuous scale. b) Volcano plot depicting the strength of the correlation between a biomarker and ageing. Red dots represent a significant positive correlation, blue dots represent a significant negative correlation and grey dots represent a nonsignificant correlation. Both the adjusted and unadjusted p-values are multiple testing corrected using the Benjamini–Hochberg (BH) procedure for testing 43 biomarkers. *: p<0.05; **: p<0.01; ***: p<0.001. ANG: angiopoietin; sTie-2: soluble Tie-2; sE-selectin: soluble E-selectin; sThrombomodulin: soluble thrombomodulin; sVCAM-1: soluble vascular cellular adhesion molecule 1; PAI-1: plasminogen activator inhibitor 1; sCD31: soluble cluster of differentiation 31; sRAGE: soluble receptor for advanced glycation end-products; sTNF-R1: soluble tumour necrosis factor receptor 1; sTREM-1: soluble triggering receptor expressed on myeloid cells 1; SP-D: surfactant protein D; CD40L: CD40 ligand; PD-L1: programmed death ligand 1; CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; IL: interleukin; TNF: tumour necrosis factor; GM-CSF: granulocyte–macrophage colony-stimulating factor; IFN: interferon.
- FIGURE 5
Mediation analysis for ageing-associated mortality and host response biomarkers upon admission to the general ward. a) Quadrant plot. The x-axis depicts the increase in the 30-day mortality odds ratio per 25% increase of the biomarker derived from an unadjusted logistic regression with the log-transformed biomarker as the explanatory variable and 30-day mortality as the response variable. The y-axis shows the percentage change in the biomarker concentration per 1-year increase in age which was derived from an unadjusted linear regression analysis with the log-transformed biomarker as the response variable. Biomarkers in both the top-right and bottom-left corners are most likely associated with an age-dependent increase in 30-day mortality. The significance of the coefficient was multiple testing corrected using the Benjamini–Hochberg procedure for testing 43 biomarkers. b) Quadrant plot. The same method as in a), but both coefficients are now adjusted for demographics (inclusion hospital, sex and inclusion wave), age-related comorbidities (hypertension, diabetes, malignancies, immunosuppression, and chronic cardiac, neurological, respiratory and kidney disease), age- and biomarker-related chronic medication (antiplatelet and anticoagulant drugs), and COVID-19-related treatments both before and after sampling (corticosteroids including dexamethasone, anti-interleukin (IL)-6, imatinib, remdesivir and antibiotics). c) Diagram of mediation analysis. The adjusted model contained the same covariates as in b). d) Unadjusted and e) adjusted mediation analysis results. Only biomarkers and principal components (PCs) significantly associated with ageing and 30-day mortality were analysed. Confidence intervals were obtained from 1000 times bootstrapping. The higher the proportion of mediation, the stronger the association of the age-dependent differences in that biomarker and the age-dependent increase in 30-day mortality. The PCs and their contributing biomarkers are depicted in figure 3. The complete contribution of each biomarker to a PC score is depicted in supplementary table S6. Endocoag: endothelial and coagulation; ANG: angiopoietin; sTie-2: soluble Tie-2; sE-selectin: soluble E-selectin; sThrombomodulin: soluble thrombomodulin; sVCAM-1: soluble vascular cellular adhesion molecule 1; PAI-1: plasminogen activator inhibitor 1; sCD31: soluble cluster of differentiation 31; sRAGE: soluble receptor for advanced glycation end-products; sTNF-R1: soluble tumour necrosis factor receptor 1; sTREM-1: soluble triggering receptor expressed on myeloid cells 1; SP-D: surfactant protein D; CD40L: CD40 ligand; PD-L1: programmed death ligand 1; CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; IL: interleukin; TNF: tumour necrosis factor; GM-CSF: granulocyte–macrophage colony-stimulating factor; IFN: interferon.
Tables
- TABLE 1
Baseline characteristics of COVID-19 patients on admission to the general ward
Age decade p-value <50 years (n=89) ≥50– <60 years (n=111) ≥60– <70 years (n=135) ≥70 years (n=129) Demographics Age, years 44 (37–47) 55 (53–57) 64 (62–67) 76 (72–82) <0.001 Male 49 (55.1) 68 (61.3) 83 (61.5) 78 (60.5) 0.784 BMI, kg·m−2 30.0 (27.4–33.2) 27.8 (25.2–32.1) 28.7 (25.6–32.8) 27.5 (24.3–30.1) 0.002 Duration of symptoms#, days 8 (7–10) 9 (7–11) 8 (6–11) 8 (5–10) 0.109 Smoking status 0.009 Current smoker 4 (4.5) 4 (3.6) 8 (5.9) 8 (6.2) Ex-smoker 17 (19.1) 27 (24.3) 48 (35.6) 53 (41.1) Never-smoker 60 (67.4) 70 (63.1) 73 (54.1) 55 (42.6) Unknown 8 (9.0) 10 (9.0) 6 (4.4) 13 (10.1) COVID-19 vaccination status 0.262 Yes 2 (2.2) 2 (1.8) 8 (5.9) 7 (5.4) No 86 (96.6) 106 (95.5) 120 (88.9) 119 (92.2) Unknown 1 (1.1) 3 (2.7) 7 (5.2) 3 (2.3) Comorbidities and (selected) chronic medication Charlson score¶ 0.0 (0–1.0) 1.0 (1.0–2.0) 3.0 (2.0–4.0) 4.0 (4.0–6.0) <0.001 Hypertension 17 (19.1) 32 (28.8) 64 (47.4) 68 (52.7) <0.001 Cardiac disease 13 (14.6) 16 (14.4) 44 (32.6) 51 (39.5) <0.001 Respiratory disease 14 (15.7) 20 (18.0) 25 (18.5) 33 (25.6) 0.261 Diabetes 19 (21.3) 13 (11.7) 35 (25.9) 41 (31.8) 0.003 Kidney disease 5 (5.6) 4 (3.6) 8 (5.9) 19 (14.7) 0.005 Neurological disease 5 (5.6) 3 (2.7) 9 (6.7) 21 (16.3) 0.001 Prior malignancy 3 (3.4) 1 (0.9) 13 (9.6) 14 (10.9) 0.005 Immunosuppression+ 10 (11.2) 4 (3.6) 16 (11.9) 5 (3.9) 0.016 Antiplatelet drugs 1 (1.1) 5 (4.5) 28 (20.7) 19 (14.7) <0.001 Anticoagulant drugs 5 (5.6) 4 (3.6) 7 (5.2) 24 (18.6) <0.001 Disease severity on admission 4C Mortality§ 4 (3–5) 3 (2–5) 4 (3–6) 6 (4–7) <0.001 qSOFA 1 (0–1) 1 (0–1) 1 (0–1) 1 (0–1) 0.002 MEWS 3 (2–4) 3 (2–4) 3 (2–4) 3 (2–4) 0.102 CURBƒ 0 (0–1) 0 (0–1) 0 (0–1) 1 (0–1) <0.001 Treatment at day of admission Supplementary oxygen therapy 80 (89.9) 103 (92.8) 121 (89.6) 115 (89.1) 0.784 High-flow nasal cannula 0 (0.0) 0 (0.0) 2 (1.5) 2 (1.6) 0.382 Noninvasive ventilation 1 (1.1) 0 (0.0) 2 (1.5) 1 (0.8) 0.647 Routine laboratory markers Leukocyte count, ×109 L−1 6.1 (4.6–8.3) 6.0 (4.8–8.6) 6.4 (4.9–7.9) 6.9 (5.2–8.6) 0.306 Neutrophil count, ×109 L−1 4.8 (3.3–5.9) 4.4 (3.2–6.6) 5.0 (3.4–6.3) 5.2 (3.9–6.9) 0.074 Lymphocyte count, ×109 L−1 1.00 (0.70–1.20) 0.97 (0.70–1.20) 0.93 (0.70–1.20) 0.75 (0.50–1.05) 0.001 Neutrophil/lymphocyte ratio 4.8 (2.8–7.2) 4.5 (2.9–8.6) 4.8 (3.3–7.5) 6.4 (4.1–10.5) 0.002 C-reactive protein, mg·L−1 98 (49–126) 76 (43–136) 83 (51–141) 100 (57–147) 0.369 Platelet count, ×109 L−1 220 (184–255) 225 (181–276) 215 (149–281) 207 (151–266) 0.434 Creatinine, µmol·L−1 74 (63–92) 79 (65–90) 82 (67–101) 87 (70–113) 0.001 Data are presented as median (interquartile range) or n (%), unless otherwise stated. BMI: body mass index; qSOFA: quick Sequential Organ Failure Assessment; MEWS: Modified Early Warning Score; CURB: confusion, urea >7 mmol·L−1, respiratory rate ≥30 breaths·min−1, blood pressure <90 mmHg (systolic) or ≤60 mmHg (diastolic). #: prior to admission; ¶: Charlson score was calculated without the age component; +: defined as a history of an organ transplant, immune deficiency or chronic use of immunosuppressants (see supplementary table S7 for details); §: 4C Mortality, a validated COVID-19 score [26], was calculated without the age and obesity component; ƒ: CURB score was calculated without the age component.
- TABLE 2
Treatments, disease course and outcome of COVID-19 patients after admission to the general ward
Age decade p-value <50 years (n=89) ≥50– <60 years (n=111) ≥60– <70 years (n=135) ≥70 years (n=129) Treatment Supplementary oxygen therapy 84 (94.4) 107 (96.4) 131 (97.0) 125 (96.9) 0.737 High-flow nasal cannula 2 (2.2) 8 (7.2) 19 (14.1) 17 (13.2) 0.012 Noninvasive ventilation 1 (1.1) 0 (0.0) 3 (2.2) 8 (6.2) 0.015 Invasive ventilation 8 (9.0) 10 (9.0) 22 (16.3) 9 (7.0) 0.073 Remdesivir 12 (13.5) 10 (9.0) 14 (10.4) 17 (13.2) 0.670 Chloroquine 0 (0.0) 2 (1.8) 0 (0.0) 1 (0.8) 0.284 Monoclonal antibodies against SARS-CoV-2 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.8) 0.457 Antibiotics in the first 7 days of admission 31 (34.8) 40 (36.0) 70 (51.9) 65 (50.4) 0.010 Immunomodulating therapies Dexamethasone 6 mg 72 (80.9) 89 (80.2) 102 (75.6) 103 (79.8) 0.730 Of which before sampling 62 (69.7) 79 (71.2) 96 (71.1) 93 (72.1) 0.985 Other corticosteroids# 4 (4.5) 3 (2.7) 6 (4.4) 3 (2.3) 0.713 Of which before sampling 3 (3.4) 2 (1.8) 5 (3.7) 1 (0.8) 0.392 Interleukin-6 inhibitors 10 (11.2) 11 (9.9) 13 (9.6) 12 (9.3) 0.971 Of which before sampling 4 (4.5) 1 (0.9) 3 (2.2) 3 (2.3) 0.426 Anti-C5a antibody 0 (0.0) 0 (0.0) 1 (0.7) 1 (0.8) 0.677 Of which before sampling 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) >1.000 Imatinib 1 (1.1) 5 (4.5) 3 (2.2) 11 (8.5) 0.027 Of which before sampling 0 (0.0) 1 (0.9) 2 (1.5) 4 (3.1) 0.279 Clinical course Thrombosis 5 (5.6) 12 (10.8) 9 (6.7) 18 (14.0) 0.110 Of which pulmonary embolism¶ 5 (5.6) 10 (9.0) 9 (6.7) 17 (13.2) 0.175 Of which deep venous thrombosis¶ 0 (0.0) 4 (3.6) 0 (0.0) 2 (1.6) 0.054 Length of hospital stay, days 4 (2–7) 4 (3–8) 6 (3–11) 7 (4–11) <0.001 ICU admission+ 9 (10.1) 15 (13.5) 26 (19.3) 13 (10.1) 0.113 ICU stay, days 15 (8–29) 11 (9–13) 8 (5–14) 8 (1–17) 0.450 Readmission§ 4 (4.5) 5 (4.5) 6 (4.4) 10 (7.8) 0.581 Mortalityƒ 30-day 1 (1.1) 2 (1.8) 16 (11.9) 39 (30.2) <0.001 90-day 2 (2.2) 2 (1.8) 16 (11.9) 43 (33.3) <0.001 Data are presented as median (interquartile range) or n (%), unless otherwise stated. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; ICU: intensive care unit. #: prednisolone and hydrocortisone; ¶: numbers do not add up to 100% as some patients suffered from both pulmonary embolism and deep venous thrombosis; +: ICU admission after sampling; §: for any cause within 28 days of the initial admission; ƒ: for 98.2% of patients, worsening of COVID-19 was reported as a causal element for mortality by the treating physician.
Supplementary Material
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