86 Chapter 5 was approved by the institutional review boards or ethical committees in each country and/or site, and all participants or their legally authorized representative provided written, informed consent for additional data and sample collection. The protocol definition of an ICU-acquired pneumonia was described in detail elsewhere [11] and in Additional file 1. Briefly, four clinical criteria were assessed daily: new antibiotic use, new blood cultures performed, new chest radiograph or computed tomography done, or another new reason to suspect pneumonia. In cases with one positive answer, a combination of major and minor criteria was assessed to categorize patients as having protocol-defined pneumonia, or not [11]. The diagnosis of pneumonia, based on criteria assessed daily, triggered the collection of an “event” blood sample; there were no cases in whom the diagnosis pneumonia was later refuted. The current project was designed as a nested case-control study within the ASPIREICU population. A case was defined as a patient who developed a (protocol-defined) ICUacquired pneumonia at least 48 hours after ICU admission. A control was defined as a patient who did not develop a protocol-defined ICU-acquired pneumonia. Comorbidities and causative pathogens were defined as described in Additional file 1. Sample collection and assays Ethylenediaminetetraacetic acid (EDTA) anticoagulated blood was obtained upon enrolment into ASPIRE-ICU (baseline). In cases, a follow up blood sample was obtained on the day pneumonia was diagnosed and on day 7 after inclusion (Additional file 1: Figure S1); in controls, a follow-up sample was drawn on day 7 after inclusion. Biomarkers were categorized into four pathophysiological domains: interleukin (IL)-6, IL-8, IL-10, and IL-1 receptor antagonist (IL-1RA)(reflecting cytokine release); matrix metalloproteinase (MMP)-8, soluble triggering receptor expressed on myeloid cells (sTREM)-1, soluble cluster of differentiation (sCD)163, soluble receptor for advanced glycation endproducts (sRAGE), tenascin-C and procalcitonin (reflecting systemic inflammation); sE-selectin, soluble vascular cell adhesion protein (sVCAM)-1, fractalkine, syndecan-1, soluble thrombomodulin, angiopoietin-1, and angiopoietin-2 (reflecting endothelial activation and function); soluble tissue factor and D-dimer (reflecting coagulation activation). For further details, see Additional file 1. Statistical analysis All biomarker values were logarithmically transformed and analyzed using linear mixed model analyses. Calculation of principal component analysis (PCA) plots was done by a singular value decomposition of the centered and scaled data matrix including the (logged) protein plasma biomarkers for key pathophysiological pathways. The mixed model was fitted taking the group (cases versus controls), the discrete timepoint (i.e. baseline, event - only for cases - and/or day 7), and their interaction as fixed effects, and patient-specific intercepts as random effects unless otherwise stated. The interaction term (group x timepoints) revealed whether there was a difference in biomarker trajectory over time between cases and controls, where the patient-specific intercept accounted for repeated measures within each patient. In additional analyses, we adjusted for the following predefined variables that may confound the relationship between ICU-acquired pneumonia and plasma biomarker levels: site of enrolment, age, sex, body mass index, Charlson comorbidity index, reason for admission (medical,
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