BreathDx - molecular analysis of exhaled breath as a diagnostic test for ventilator-associated pneumonia: protocol for a European multicentre observational study.

BACKGROUND: The diagnosis of ventilator-associated pneumonia (VAP) remains time-consuming and costly, the clinical tools lack specificity and a bedside test to exclude infection in suspected patients is unavailable. Breath contains hundreds to thousands of volatile organic compounds (VOCs) that result from host and microbial metabolism as well as the environment. The present study aims to use breath VOC analysis to develop a model that can discriminate between patients who have positive cultures and who have negative cultures with a high sensitivity. METHODS/DESIGN: The Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia (BreathDx) study is a multicentre observational study. Breath and bronchial lavage samples will be collected from 100 and 53 intubated and ventilated patients suspected of VAP. Breath will be analysed using Thermal Desorption - Gas Chromatography - Mass Spectrometry (TD-GC-MS). The primary endpoint is the accuracy of cross-validated prediction for positive respiratory cultures in patients that are suspected of VAP, with a sensitivity of at least 99% (high negative predictive value). DISCUSSION: To our knowledge, BreathDx is the first study powered to investigate whether molecular analysis of breath can be used to classify suspected VAP patients with and without positive microbiological cultures with 99% sensitivity. TRIAL REGISTRATION: UKCRN ID number 19086, registered May 2015; as well as registration at www.trialregister.nl under the acronym 'BreathDx' with trial ID number NTR 6114 (retrospectively registered on 28 October 2016).

Synergistic effects of chemical insult and toll-like receptor ligands on dendritic cell activation.

There is considerable interest in the development of in vitro methods for the identification of contact sensitizers, including those that use cultured dendritic cells (DC), key players in cutaneous immune responses. Chemical allergens, such as dinitrobenzene sulfonic acid (DNBS), or skin irritants, such as benzene sulfonic acid (BS), induce modest changes in DC phenotype. In an attempt to increase the sensitivity of DC responses, DC have been co-cultured with chemical and DC activators (toll-like receptor [TLR] ligands). Cells were cultured with DNBS or BS at doses of equivalent cytotoxicity, together with sub-optimal doses of selected TLR ligands (Pam(3)Cys-Ser-(Lys)(4) [PAM], TLR1-2; macrophage-activating lipopeptide-2 [MALP-2], TLR6-2; or flagellin; TLR5). Both chemicals caused a decline in cell viability. DNBS induced a higher proportion of late apoptotic/necrotic cells whereas BS was associated with early apoptotic cells, suggesting different mechanisms of cell death. Some synergy was observed for interleukin (IL)-6 and tumor necrosis factor alpha production for DC co-cultured with BS and MALP-2/PAM. In contrast, there were marked synergistic effects on IL-6 secretion when DC were cultured with DNBS and flagellin. It may be possible to exploit this enhanced sensitivity of flagellin-activated DC for chemical allergen for the development of in vitro skin sensitization assays.