The dynamics of the pulmonary microbiome during mechanical ventilation in the intensive care unit and the association with occurrence of pneumonia.

RATIONALE: Ventilator-associated pneumonia (VAP) is the most common nosocomial infections in patients admitted to the ICU. The adapted island model predicts several changes in the respiratory microbiome during intubation and mechanical ventilation. OBJECTIVES: We hypothesised that mechanical ventilation and antibiotic administration decrease the diversity of the respiratory microbiome and that these changes are more profound in patients who develop VAP. METHODS: Intubated and mechanically ventilated ICU-patients were included. Tracheal aspirates were obtained three times a week. 16S rRNA gene sequencing with the Roche 454 platform was used to measure the composition of the respiratory microbiome. Associations were tested with linear mixed model analysis and principal coordinate analysis. MEASUREMENTS AND MAIN RESULTS: 111 tracheal aspirates were obtained from 35 patients; 11 had VAP, 18 did not have VAP. Six additional patients developed pneumonia within the first 48 hours after intubation. Duration of mechanical ventilation was associated with a decrease in α diversity (Shannon index; fixed-effect regression coefficient (ß): -0.03 (95% CI -0.05 to -0.005)), but the administration of antibiotic therapy was not (fixed-effect ß: 0.06; 95% CI -0.17 to 0.30). There was a significant difference in change of ß diversity between patients who developed VAP and control patients for Bray-Curtis distances (p=0.03) and for Manhattan distances (p=0.04). Burkholderia, Bacillales and, to a lesser extent, Pseudomonadales positively correlated with the change in ß diversity. CONCLUSION: Mechanical ventilation, but not antibiotic administration, was associated with changes in the respiratory microbiome. Dysbiosis of microbial communities in the respiratory tract was most profound in patients who developed VAP.

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).

Levels of cytokines in broncho-alveolar lavage fluid, but not in plasma, are associated with levels of markers of lipid peroxidation in breath of ventilated ICU patients.

Alkanes and alkenes in the breath are produced through fatty acid peroxidation, which is initialized by reactive oxygen species. Inflammation is an important cause and effect of reactive oxygen species. We aimed to evaluate the association between fatty acid peroxidation products and inflammation of the alveolar and systemic compartment in ventilated intensive care unit (ICU) patients.Volatile organic compounds were measured by gas chromatography and mass spectrometry in the breath of newly ventilated ICU patients within 24 h after ICU admission. Cytokines were measured in non-directed bronchial lavage fluid (NBL) and plasma by cytometric bead array. Correlation coefficients were calculated and presented in heatmaps.93 patients were included. Peroxidation products in exhaled breath were not associated with markers of inflammation in plasma, but were correlated with those in NBL. IL-6, IL-8, IL-1ß and TNF-α concentration in NBL showed inverse correlation coefficients with the peroxidation products of fatty acids. Furthermore, NBL IL-10, IL-13, GM-CSF and IFNγ demonstrated positive associations with breath alkanes and alkenes. Correlation coefficients for NBL cytokines were high regarding peroxidation products of n-6, n-7 and particularly in n-9 fatty acids.Levels of lipid peroxidation products in the breath of ventilated ICU patients are associated with levels of inflammatory markers in NBL, but not in plasma. Alkanes and alkenes in breath seems to be associated with an anti-inflammatory, rather than a pro-inflammatory state in the alveoli.

Next-generation sequencing of the BRCA1 and BRCA2 genes for the genetic diagnostics of hereditary breast and/or ovarian cancer.

Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 hereditary breast and/or ovarian cancer patients. We first validated this approach in a cohort of 115 samples with previously known BRCA1 and BRCA2 mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq BRCA1 and BRCA2 panel. The DNA Libraries were pooled, barcoded, and sequenced using an Ion Torrent Personal Genome Machine sequencer. The combination of different robust bioinformatics tools allowed detection of all previously known pathogenic mutations and polymorphisms in the 115 samples, without detecting spurious pathogenic calls. We then used the same assay in a discovery cohort of 95 uncharacterized hereditary breast and/or ovarian cancer patients for BRCA1 and BRCA2. In addition, we describe the allelic frequencies across 210 hereditary breast and/or ovarian cancer patients of 74 unique definitely and likely pathogenic and uncertain BRCA1 and BRCA2 variants, some of which have not been previously annotated in the public databases. Targeted next-generation sequencing is ready to substitute classic molecular methods to perform genetic testing on the BRCA1 and BRCA2 genes and provides a greater opportunity for more comprehensive testing of at-risk patients.

Analysis of the airway microbiota of healthy individuals and patients with chronic obstructive pulmonary disease by T-RFLP and clone sequencing.

UNLABELLED: Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. OBJECTIVE: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.

Detection of microorganisms in exhaled breath condensate during acute exacerbations of COPD.

BACKGROUND AND OBJECTIVE: One hallmark of COPD is colonization and infection of the lung. Acute exacerbations of COPD (AECOPD) are acute deteriorations of the chronic disease and are associated with a change of the pulmonary microbial balance. The collection of exhaled breath condensate (EBC) can be used to non-invasively determine markers of lung disease. The aim of the present study was to compare the results of assays based on the detection of microbial nucleic acids from EBC and from spontaneous sputum in patients with AECOPD. METHODS: EBC and sputa of 29 adults with AECOPD were obtained. Isolated DNA or RNA were used as starting material for the PCR assays to detect Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza viruses (AH 1, AH 3) and respiratory syncytial virus. RESULTS: Bacterial or viral nucleic acids were identified in 14 EBC and 21 sputa from 29 patients. Results from EBC did not correlate well with those from sputum. Viral and S. pneumoniae nucleic acids were detected only in sputum, whereas L. pneumophila DNA was only found in EBC. In three EBC and 10 sputa nucleic acids of more than one microorganism was detected. CONCLUSIONS: Bacterial nucleic acids can be identified in EBC of COPD patients with exacerbations. The results obtained from EBC and sputum did not correlate well.

The role of vitamin D in pulmonary disease: COPD, asthma, infection, and cancer.

The role of vitamin D (VitD) in calcium and bone homeostasis is well described. In the last years, it has been recognized that in addition to this classical function, VitD modulates a variety of processes and regulatory systems including host defense, inflammation, immunity, and repair. VitD deficiency appears to be frequent in industrialized countries. Especially patients with lung diseases have often low VitD serum levels. Epidemiological data indicate that low levels of serum VitD is associated with impaired pulmonary function, increased incidence of inflammatory, infectious or neoplastic diseases. Several lung diseases, all inflammatory in nature, may be related to activities of VitD including asthma, COPD and cancer. The exact mechanisms underlying these data are unknown, however, VitD appears to impact on the function of inflammatory and structural cells, including dendritic cells, lymphocytes, monocytes, and epithelial cells. This review summarizes the knowledge on the classical and newly discovered functions of VitD, the molecular and cellular mechanism of action and the available data on the relationship between lung disease and VitD status.