Kallirroi Kefala

C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients

RATIONALE Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood. OBJECTIVES To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment. METHODS Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions. MEASUREMENTS AND MAIN RESULTS Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody. CONCLUSIONS Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.

Diagnostic importance of pulmonary interleukin-1β and interleukin-8 in ventilator-associated pneumonia

Background Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. Methods A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >104 colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from “non-VAP”. Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. Results Seventy-two patients had recoverable lavage—24% had VAP. BALF interleukin-1β (IL-1β), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1β generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1β <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <104 cfu/ml. Conclusions BALF IL-1β and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.

Evaluation of the effect of diagnostic methodology on the reported incidence of ventilator-associated pneumonia

Background: The optimal method for diagnosing ventilator-associated pneumonia (VAP) is controversial and its effect on reported incidence uncertain. This study aimed to model the impact of using either endotracheal aspirate or bronchoalveolar lavage on the reported incidence of pneumonia and then to test effects suggested from theoretical modelling in clinical practice. Methods: A three-part single-centre study was undertaken. First, diagnostic performance of aspirate and lavage were compared using paired samples from 53 patients with suspected VAP. Secondly, infection surveillance data were used to model the potential effect on pneumonia incidence and antibiotic use of using exclusively aspirate or lavage to investigate suspected pneumonia (643 patients; 110 clinically suspected pneumonia episodes). Thirdly, a practice change initiative was undertaken to increase lavage use; pneumonia incidence and antibiotic use were compared for the 12 months before and after the change. Results: Aspirate overdiagnosed VAP compared with lavage (89% vs 21% of clinically suspected cases, p<0.0001). Modelling suggested that changing from exclusive aspirate to lavage diagnosis would decrease reported pneumonia incidence by 76% (95% CI 67% to 87%) and antibiotic use by 30% (95% CI 20% to 42%). After the practice change initiative, lavage use increased from 37% to 58%. Although clinically suspected pneumonia incidence was unchanged, microbiologically confirmed VAP decreased from 18 to 9 cases per 1000 ventilator days (p = 0.001; relative risk reduction 0.61 (95% CI 0.46 to 0.82)), and mean antibiotic use fell from 9.1 to 7.2 antibiotic days (21% decrease, p = 0.08). Conclusions: Diagnostic technique impacts significantly on reported VAP incidence and potentially on antibiotic use.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia

Background Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. Objectives We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. Methods A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >104 colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. Results Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). Conclusions Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

The cyclin-dependent kinase inhibitor AT7519 accelerates neutrophil apoptosis in sepsis-related acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20 hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.

S79 Potential diagnostic significance of neutrophil proteases in ventilator-associated pneumonia

Introduction and Objectives The clinical diagnosis of ventilator-associated pneumonia (VAP) remains notoriously difficult, as several non-infective conditions mimic VAP. Microbiological confirmation of the diagnosis using conventional cultures typically takes 48–72 h. Identification of molecules measurable within a short time frame and closely associated with microbiologically confirmed VAP is therefore highly desirable. VAP is associated with significant influx of activated neutrophils into the alveolar space. We postulated that extracellular neutrophil proteases in bronchoalveolar lavage fluid (BALF) may reliably identify VAP in suspected cases. Methods Fifty-four intubated and mechanically ventilated patients in the intensive care unit developed clinically suspected VAP and were recruited. Bronchoalveolar lavage (BAL) was performed using a standardised protocol. An aliquot of BALF was sent to the diagnostic microbiology laboratory for quantitative culture, with confirmation of VAP defined as growth of a pathogen(s) at >104 colony forming units/ml. Remaining BALF was centrifuged. The following neutrophil-specific proteases were assayed in cell-free BALF supernatant—matrix metalloproteinase (MMP)-8 and MMP-9 by Luminex assay, and human neutrophil elastase (HNE) by enzyme-linked immunosorbent assay. Urea was simultaneously measured in serum and BALF, and used to correct for the dilution of epithelial lining induced by BAL. Receiver operating characteristic (ROC) curves were constructed and optimal specificity and sensitivity for each marker calculated. Results Eleven patients (20%) had confirmed VAP. For HNE (cut off 670ng/ml) the ROC area under curve (AUC) was 0.87 (p<0.0001), sensitivity 93%, specificity 79%. For MMP-8 (13 ng/ml), ROC AUC was 0.81 (p<0.005), sensitivity 91%, specificity 63%. For MMP-9 (22 ng/ml), ROC AUC was 0.79 (p<0.005), sensitivity 82%, specificity 63%. Conclusions Neutrophil proteases are strongly associated with confirmed infection in cases of suspected VAP. The values for HNE, in particular, compare extremely favourably with any previously published equivalent values. These data suggest that neutrophil protease concentrations in BALF deserve further attention as potentially diagnostic markers for VAP. They further suggest that neutrophil proteases, inappropriately released into the extracellular space, may contribute to the pathophysiology of VAP.