The airway microbiota of stable COPD : Association with exacerbation frequency and the risks associated with bronchoscopic data collection

Background: Acute exacerbations of COPD are an important cause of mortality and morbidity in patients with COPD. It is incompletely understood why some COPD patients experience frequent exacerbations, while others rarely or never exacerbate. Studies have suggested that the microbiome of the lungs is different in patients with different exacerbation frequencies. Most studies use sputum samples prone to contamination from the upper airway. Bronchoscopic sampling could improve the quality of the samples, but is a more invasive approach. Aims: The overall aim of the PhD project is to investigate if the airway microbiota in subjects with stable COPD is associated with exacerbation frequency and to assess the complications and discomforts (including rates and predictors) associated with bronchoscopic data collection in participants with and without COPD. Materials and Methods: For the first paper, we performed a systematic literature search on complications and discomfort of non-therapeutic bronchoscopy in PubMed. Titles and abstracts of retrieved search hits were sorted according to inclusion and exclusion criteria. The second and third paper uses data collected in the Bergen COPD Microbiome Study (MicroCOPD). Individuals with and without COPD underwent bronchoscopy including protected bronchoalveolar lavage (BAL) (in participants with FEV1 >30% of predicted), protected specimen brushes (PSB), small volume lavage, and in 1/3 of bronchoscopies, endobronchial biopsies. In addition to bronchoscopic sampling, participants provided oral wash samples. For each bronchoscopic procedure, there was one negative control sample of the phosphate-buffered-saline used for the microbial samples. Some participants underwent more than one bronchoscopy. Light sedation with alfentanil was offered to participants. Immediate complications, defined as any event requiring an unplanned intervention or early termination of the procedure, were recorded. Participants were interviewed after a week regarding discomfort, respiratory symptoms and fever sensation. Participants with COPD were followed with telephone interviews every three months for one year regarding exacerbations. Microbial samples and negative controls went through laboratory processing including DNA extraction, PCR and sequencing of the 16S rRNA gene. Extensive bioinformatic processing of sequencing data and microbiota analyses were performed using QIIME2 and R. Pre-processing included bioinformatic identification and removal of contaminant sequences. We them compared bacterial taxonomy and alpha and beta diversity in individuals with and without COPD exacerbations in the follow-up. Results: Bronchoscopy is generally a safe procedure with low mortality and few severe complications, but the literature shows a wide range of specific complication rates, and it was not possible to conclude on discomfort or predictors. In MicroCOPD, 239 participants underwent bronchoscopy once, 61 underwent more than one bronchoscopy. Complications occurred in 25.9% of first bronchoscopies. The rate of potentially severe complications was 1.3%. Participants with COPD experienced more dyspnoea than participants without lung disease. Sedation and lower age were associated with less complications. 47.7% reported fever. Discomfort was associated with fever, dread of bronchoscopy, high COPD Assessment Test score, and never-smoking. Complications and fever in a first bronchoscopy were often predictive for complications and fever in a second bronchoscopy. We found no difference in alpha and beta diversity between participants with and without COPD exacerbations, and no ASV or genus was found to be consistently differentially abundant or distributed between the groups. Conclusions: Bronchoscopy is a generally safe procedure, even in research into COPD, but is not free of risk. Bronchoscopy was associated with frequent need for unplanned interventions, discomfort and fever sensation in MicroCOPD. We found no association between the lung microbiota at stable state and exacerbations of COPD.Doktorgradsavhandlin

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.publishedVersio

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.publishedVersio

The airway microbiota of stable COPD : Association with exacerbation frequency and the risks associated with bronchoscopic data collection

Background: Acute exacerbations of COPD are an important cause of mortality and morbidity in patients with COPD. It is incompletely understood why some COPD patients experience frequent exacerbations, while others rarely or never exacerbate. Studies have suggested that the microbiome of the lungs is different in patients with different exacerbation frequencies. Most studies use sputum samples prone to contamination from the upper airway. Bronchoscopic sampling could improve the quality of the samples, but is a more invasive approach. Aims: The overall aim of the PhD project is to investigate if the airway microbiota in subjects with stable COPD is associated with exacerbation frequency and to assess the complications and discomforts (including rates and predictors) associated with bronchoscopic data collection in participants with and without COPD. Materials and Methods: For the first paper, we performed a systematic literature search on complications and discomfort of non-therapeutic bronchoscopy in PubMed. Titles and abstracts of retrieved search hits were sorted according to inclusion and exclusion criteria. The second and third paper uses data collected in the Bergen COPD Microbiome Study (MicroCOPD). Individuals with and without COPD underwent bronchoscopy including protected bronchoalveolar lavage (BAL) (in participants with FEV1 >30% of predicted), protected specimen brushes (PSB), small volume lavage, and in 1/3 of bronchoscopies, endobronchial biopsies. In addition to bronchoscopic sampling, participants provided oral wash samples. For each bronchoscopic procedure, there was one negative control sample of the phosphate-buffered-saline used for the microbial samples. Some participants underwent more than one bronchoscopy. Light sedation with alfentanil was offered to participants. Immediate complications, defined as any event requiring an unplanned intervention or early termination of the procedure, were recorded. Participants were interviewed after a week regarding discomfort, respiratory symptoms and fever sensation. Participants with COPD were followed with telephone interviews every three months for one year regarding exacerbations. Microbial samples and negative controls went through laboratory processing including DNA extraction, PCR and sequencing of the 16S rRNA gene. Extensive bioinformatic processing of sequencing data and microbiota analyses were performed using QIIME2 and R. Pre-processing included bioinformatic identification and removal of contaminant sequences. We them compared bacterial taxonomy and alpha and beta diversity in individuals with and without COPD exacerbations in the follow-up. Results: Bronchoscopy is generally a safe procedure with low mortality and few severe complications, but the literature shows a wide range of specific complication rates, and it was not possible to conclude on discomfort or predictors. In MicroCOPD, 239 participants underwent bronchoscopy once, 61 underwent more than one bronchoscopy. Complications occurred in 25.9% of first bronchoscopies. The rate of potentially severe complications was 1.3%. Participants with COPD experienced more dyspnoea than participants without lung disease. Sedation and lower age were associated with less complications. 47.7% reported fever. Discomfort was associated with fever, dread of bronchoscopy, high COPD Assessment Test score, and never-smoking. Complications and fever in a first bronchoscopy were often predictive for complications and fever in a second bronchoscopy. We found no difference in alpha and beta diversity between participants with and without COPD exacerbations, and no ASV or genus was found to be consistently differentially abundant or distributed between the groups. Conclusions: Bronchoscopy is a generally safe procedure, even in research into COPD, but is not free of risk. Bronchoscopy was associated with frequent need for unplanned interventions, discomfort and fever sensation in MicroCOPD. We found no association between the lung microbiota at stable state and exacerbations of COPD

Participation in research bronchoscopy: a literature review

Bronchoscopy is the preferred method for collecting biological samples from the lower airways of subjects in clinical research. However, ensuring participation in clinical research can be challenging when the research includes an invasive procedure. For this report we reviewed the literature to look for information on participation in research bronchoscopy studies to better design our own study, the Bergen COPD Microbiome study (MicroCOPD). We performed a systematic literature search on participation in research bronchoscopy studies in February 2014 using the search engines of PubMed and EMBASE. The literature search resulted in seven relevant papers. Motivation was an end point in six of the seven papers, but reasons for declining participation and recruitment strategies also seemed important. Human subjects participate in research bronchoscopy studies for personal benefit and altruistic reasons. Inconvenience associated with research, in addition to fear of procedures, is considered a barrier. Radio, especially news stations, generated the most inquiries for a clinical study involving bronchoscopy. There is a lack of information on participation in research bronchoscopy studies in the literature. A bronchoscopy study has been initiated at Haukeland University Hospital, Bergen, Norway, to examine the role of the microbiome in COPD, and participation will be explored as a substudy

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

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

The pulmonary mycobiome—A study of subjects with and without chronic obstructive pulmonary disease

Background: The fungal part of the pulmonary microbiome (mycobiome) is understudied. We report the composition of the oral and pulmonary mycobiome in participants with COPD compared to controls in a large-scale single-centre bronchoscopy study (MicroCOPD). Methods: Oral wash and bronchoalveolar lavage (BAL) was collected from 93 participants with COPD and 100 controls. Fungal DNA was extracted before sequencing of the internal transcribed spacer 1 (ITS1) region of the fungal ribosomal RNA gene cluster. Taxonomic barplots were generated, and we compared taxonomic composition, Shannon index, and beta diversity between study groups, and by use of inhaled steroids. Results: The oral and pulmonary mycobiomes from controls and participants with COPD were dominated by Candida, and there were more Candida in oral samples compared to BAL for both study groups. Malassezia and Sarocladium were also frequently found in pulmonary samples. No consistent differences were found between study groups in terms of differential abundance/distribution. Alpha and beta diversity did not differ between study groups in pulmonary samples, but beta diversity varied with sample type. The mycobiomes did not seem to be affected by use of inhaled steroids. Conclusion: Oral and pulmonary samples differed in taxonomic composition and diversity, possibly indicating the existence of a pulmonary mycobiome