Laboratory contamination in airway microbiome studies.

BACKGROUND: The low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. We have examined the impact of laboratory contamination on samples collected from the lower airways by protected (through a sterile catheter) bronchoscopy and explored various in silico approaches to dealing with the contamination post-sequencing. Our analyses included quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene. RESULTS: The mean bacterial load varied by sample type for the 23 study subjects (oral wash>1st fraction of protected bronchoalveolar lavage>protected specimen brush>2nd fraction of protected bronchoalveolar lavage; p < 0.001). By comparison to a dilution series of know bacterial composition and load, an estimated 10-50% of the bacterial community profiles for lower airway samples could be traced back to contaminating bacterial DNA introduced from the laboratory. We determined the main source of laboratory contaminants to be the DNA extraction kit (FastDNA Spin Kit). The removal of contaminants identified using tools within the Decontam R package appeared to provide a balance between keeping and removing taxa found in both negative controls and study samples. CONCLUSIONS: The influence of laboratory contamination will vary across airway microbiome studies. By reporting estimates of contaminant levels and taking use of contaminant identification tools (e.g. the Decontam R package) based on statistical models that limit the subjectivity of the researcher, the accuracy of inter-study comparisons can be improved.

Factors associated with coronary heart disease in COPD patients and controls.

BACKGROUND: COPD and coronary heart disease (CHD) frequently co-occur, yet which COPD phenotypes are most prone to CHD is poorly understood. The aim of this study was to see whether COPD patients did have a true higher risk for CHD than subjects without COPD, and to examine a range of potential factors associated with CHD in COPD patients and controls. METHODS: 347 COPD patients and 428 non-COPD controls, were invited for coronary computed tomography angiography (CCTA) and pulmonary CT. Arterial blood gas, bioelectrical impedance and lung function was measured, and a detailed medical history taken. The CCTA was evaluated for significant coronary stenosis and calcium score (CaSc), and emphysema defined as >10% of total area <-950 Hounsfield units. RESULTS: 12.6% of the COPD patients and 5.7% of the controls had coronary stenosis (p<0.01), whereas 55.9% of the COPD patients had a CaSc>100 compared to 31.6% of the controls (p<0.01). In a multivariable model adjusting for sex, age, body composition, pack-years, CRP, cholesterol/blood pressure lowering medication use and diabetes mellitus, the OR (95% CI) for having significant stenosis was 1.80 (0.86-3.78) in COPD patients compared with controls. In a similar model, the OR (95% CI) for having CaSc>100 was 1.68 (1.12-2.53) in COPD patients compared with controls. Examining the risk of significant stenosis and CaSc>100 among COPD patients, no variable was associated with significant stenosis, whereas male sex [OR 2.85 (1.56-5.21)], age [OR 3.74 (2.42-5.77)], statin use [OR 2.23 (1.23-4.50)] were associated with CaSc>100, after adjusting for body composition, pack-years, C-reactive protein, use of angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), diabetes, emphysema score, GOLD category, exacerbation frequency, eosinophilia, and hypoxemia. CONCLUSION: COPD patients were more likely to have CHD, but neither emphysema score, lung function, exacerbation frequency, nor hypoxemia predicted presence of either coronary stenosis or CaSc>100.

Complications and discomfort after research bronchoscopy in the MicroCOPD study.

BACKGROUND: Data on discomfort and complications from research bronchoscopy in chronic obstructive pulmonary disease (COPD) and asthma is limited. We present complications and discomfort occurring within a week after bronchoscopy, and investigate personal and procedural risk factors. METHODS: 239 subjects with COPD, asthma or without lung disease underwent research bronchoscopies as part of a microbiome study of the lower airways (the MicroCOPD study). Bronchoscopy was done in the supine position with oral scope insertion with the option of light conscious alfentanil sedation. Sampling consisted of protected specimen brushes, bronchoalveolar lavage, small volume lavage and for some, endobronchial biopsies. Bleeding, desaturation, cough, haemodynamic changes, dyspnoea and other events that required an unplanned intervention or early termination of bronchoscopy were prospectively recorded. Follow-up consisted of a telephone interview where subjects rated discomfort and answered questions about fever sensation and respiratory symptoms in the week following bronchoscopy. RESULTS: An unplanned intervention or early termination of bronchoscopy was required in 25.9% of bronchoscopies. Three subjects (1.3%) experienced potentially severe complications, of which all recovered without sequelae. COPD subjects experienced more dyspnoea than controls. Sedation and lower age was associated with less unplanned intervention or premature termination. About half of the subjects (47.7%) reported fever. Discomfort was associated with postprocedural fever, dread of bronchoscopy, higher score on the COPD Assessment Test and never-smoking. In subjects undergoing more than one bronchoscopy, the first bronchoscopy was often predictive for complications and postprocedural fever in the repeated bronchoscopy. CONCLUSION: Research bronchoscopies were not associated with more complications or discomfort in COPD subjects. 47.7% experienced postbronchoscopy fever sensation, which was associated with discomfort.

Motivation and response rates in bronchoscopy studies.

BACKGROUND: Bronchoscopy is frequently used to sample the lower airways in lung microbiome studies. Despite being a safe procedure, it is associated with discomfort that may result in reservations regarding participation in research bronchoscopy studies. Information on participation in research bronchoscopy studies is limited. We report response rates, reasons for non-response, motivation for participation, and predictors of participation in a large-scale single-centre bronchoscopy study ("MicroCOPD"). METHODS: Two hundred forty-nine participants underwent at least one bronchoscopy in addition to being examined by a physician, having lung function tested, and being offered a CT scan of the heart and lungs (subjects > 40 years). Each participant was asked an open question regarding motivation. Non-response reasons were gathered, and response rates were calculated. RESULTS: The study had a response rate just above 50%, and men had a significantly higher response rate than women (56.5% vs. 44.8%, p = 0.01). Procedural fear was the most common non-response reason. Most participants participated due to perceived personal benefit, but a large proportion did also participate to help others and contribute to science. Men were less likely to give exclusive altruistic motives, whereas subjects with asthma were more likely to report exclusive personal benefit as main motive. CONCLUSION: Response rates of about 50% in bronchoscopy studies make large bronchoscopy studies feasible, but the fact that participants are motivated by their own health status places a large responsibility on the investigators regarding the accuracy of the provided study information.

Protected sampling is preferable in bronchoscopic studies of the airway microbiome.

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.