Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

RATIONALE: Bronchiectasis is characterised by acute exacerbations but the biological mechanisms underlying these events is poorly characterised. Objectives To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. METHODS: 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation prior to receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral or both. Sputum inflammatory assessments included label free Liquid chromography/mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16s rRNA sequencing was used to characterise the microbiome. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacteria was identified in 103 samples (86%) and a high bacterial load (total bacterial load >10(7) copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients with rhinovirus being the most common virus (31%). PCR was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, Il-1beta and CXCL8. There markers were particularly associated with bacterial and bacterial+viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral and eosinophilic events in both hypothesis led, and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating 4 subtypes of exacerbation. CONCLUSION: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses and inflammatory dysregulation.

Objective sputum colour assessment and clinical outcomes in bronchiectasis: data from the European Bronchiectasis Registry (EMBARC).

BACKGROUND: A validated 4-point sputum colour chart can be used to objectively evaluate the levels of airway inflammation in bronchiectasis patients. In the European Bronchiectasis Registry (EMBARC), we tested whether sputum colour would be associated with disease severity and clinical outcomes. METHODS: We used a prospective, observational registry of adults with bronchiectasis conducted in 31 countries. Patients who did not produce spontaneous sputum were excluded from the analysis. The Murray sputum colour chart was used at baseline and at follow-up visits. Key outcomes were frequency of exacerbations, hospitalisations for severe exacerbations and mortality during up to 5-year follow-up. RESULTS: 13 484 patients were included in the analysis. More purulent sputum was associated with lower forced expiratory volume in 1 s (FEV1), worse quality of life, greater bacterial infection and a higher bronchiectasis severity index. Sputum colour was strongly associated with the risk of future exacerbations during follow-up. Compared to patients with mucoid sputum (reference group), patients with mucopurulent sputum experienced significantly more exacerbations (incident rate ratio (IRR) 1.29, 95% CI 1.22-1.38; p<0.0001), while the rates were even higher for patients with purulent (IRR 1.55, 95% CI 1.44-1.67; p<0.0001) and severely purulent sputum (IRR 1.91, 95% CI 1.52-2.39; p<0.0001). Hospitalisations for severe exacerbations were also associated with increasing sputum colour with rate ratios, compared to patients with mucoid sputum, of 1.41 (95% CI 1.29-1.56; p<0.0001), 1.98 (95% CI 1.77-2.21; p<0.0001) and 3.05 (95% CI 2.25-4.14; p<0.0001) for mucopurulent, purulent and severely purulent sputum, respectively. Mortality was significantly increased with increasing sputum purulence, hazard ratio 1.12 (95% CI 1.01-1.24; p=0.027), for each increment in sputum purulence. CONCLUSION: Sputum colour is a simple marker of disease severity and future risk of exacerbations, severe exacerbations and mortality in patients with bronchiectasis.

Structural Lung Disease and Clinical Phenotype in Bronchiectasis Patients: The EMBARC CT Study.

RATIONALE: Chest computed tomography -scans (CTs) are essential to diagnose and monitor bronchiectasis (BE). To date, little quantitative data is available about the nature and extent of structural lung abnormalities (SLA) on CTs of BE patients. OBJECTIVES: to investigate SLA on CTs of patients with bronchiectasis and the relationship of SLAs to clinical features using the European Bronchiectasis Registry (EMBARC) Methods: CTs from BE patients included in the EMBARC registry were analyzed using the validated Bronchiectasis Scoring Technique for CT (BEST-CT). BEST-CT subscores are expressed as % of total lung volume. Scored items are: atelectasis/consolidation (%ATCON), bronchiectasis with and without mucus plugging (%BEMP, %BEwMP), airway wall thickening (%AWT), mucus plugging (%MP), ground-glass opacities (%GGO), bullae (%BUL), airways and parenchyma (%A,%P). Four composite scores were calculated: Total BE (%TBE=%BEMP+%BEwMP), total MP (%TMP=%BEMP+%MP), total inflammatory changes (%TinF=%ATCON+%BEMP+%MP+%GGO) and total disease (%DIS= all but %A & %P).¬ Measurments and Main Results: CTs of 524 BE patients were analyzed. Mean (range) of subscores were: %TBE 4.6 (2.3-7.7), %TMP 4.2 (1.2-8.1), %TinF 8.3 (3.5-16.7) and %DIS 14.9 (9.1-25.9). BE associated with primary ciliary dyskinesia was associated with more SLA, while COPD was associated with less SLA. Lower FEV1, longer disease duration, Pseudomonas aeruginosa and NTM infection, and severe exacerbations were all independently associated with worse SLA. CONCLUSION: Patients with bronchiectasis have highly heterogeneous type and extent of structural lung abnormalities. Strong relationships between radiological disease and clinical features suggest CT analysis may be a useful tool for clinical phenotyping.

Combined approaches, including long-read sequencing, address the diagnostic challenge of HYDIN in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD), a disorder of the motile cilia, is now recognised as an underdiagnosed cause of bronchiectasis. Accurate PCD diagnosis comprises clinical assessment, analysis of cilia and the identification of biallelic variants in one of 50 known PCD-related genes, including HYDIN. HYDIN-related PCD is underdiagnosed due to the presence of a pseudogene, HYDIN2, with 98% sequence homology to HYDIN. This presents a significant challenge for Short-Read Next Generation Sequencing (SR-NGS) and analysis, and many diagnostic PCD gene panels do not include HYDIN. We have used a combined approach of SR-NGS with bioinformatic masking of HYDIN2, and state-of-the-art long-read Nanopore sequencing (LR_NGS), together with analysis of respiratory cilia including transmission electron microscopy and immunofluorescence to address the underdiagnosis of HYDIN as a cause of PCD. Bioinformatic masking of HYDIN2 after SR-NGS facilitated the detection of biallelic HYDIN variants in 15 of 437 families, but compromised the detection of copy number variants. Supplementing testing with LR-NGS detected HYDIN deletions in 2 families, where SR-NGS had detected a single heterozygous HYDIN variant. LR-NGS was also able to confirm true homozygosity in 2 families when parental testing was not possible. Utilising a combined genomic diagnostic approach, biallelic HYDIN variants were detected in 17 families from 242 genetically confirmed PCD cases, comprising 7% of our PCD cohort. This represents the largest reported HYDIN cohort to date and highlights previous underdiagnosis of HYDIN-associated PCD. Moreover this provides further evidence for the utility of LR-NGS in diagnostic testing, particularly for regions of high genomic complexity.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Sputum metagenomics of people with bronchiectasis.

Background: The microbiota in the sputum of people with bronchiectasis has repeatedly been investigated in cohorts of different geographic origin, but so far has not been studied to the species level in comparison to control populations including healthy adults and smokers without lung disease. Methods: The microbial metagenome from sputa of 101 European Bronchiectasis Registry (EMBARC) study participants was examined by using whole-genome shotgun sequencing. Results: Our analysis of the metagenome of people with bronchiectasis revealed four clusters characterised by a predominance of Haemophilus influenzae, Pseudomonas aeruginosa or polymicrobial communities with varying compositions of nonpathogenic commensals and opportunistic pathogens. The metagenomes of the severely affected patients showed individual profiles characterised by low alpha diversity. Importantly, nearly 50% of patients with severe disease were grouped in a cluster characterised by commensals. Comparisons with the sputum metagenomes of healthy smokers and healthy nonsmokers revealed a gradient of depletion of taxa in bronchiectasis, most often Neisseria subflava, Fusobacterium periodonticum and Eubacterium sulci. Conclusion: The gradient of depletion of commensal taxa found in healthy airways is a key feature of bronchiectasis associated with disease severity.

Bronchiectasis and asthma: Data from the European Bronchiectasis Registry (EMBARC).

BACKGROUND: Asthma is commonly reported in patients with a diagnosis of bronchiectasis. OBJECTIVE: The aim of this study was to evaluate whether patients with bronchiectasis and asthma (BE+A) had a different clinical phenotype and different outcomes compared with patients with bronchiectasis without concomitant asthma. METHODS: A prospective observational pan-European registry (European Multicentre Bronchiectasis Audit and Research Collaboration) enrolled patients across 28 countries. Adult patients with computed tomography-confirmed bronchiectasis were reviewed at baseline and annual follow-up visits using an electronic case report form. Asthma was diagnosed by the local investigator. Follow-up data were used to explore differences in exacerbation frequency between groups using a negative binomial regression model. Survival analysis used Cox proportional hazards regression. RESULTS: Of 16,963 patients with bronchiectasis included for analysis, 5,267 (31.0%) had investigator-reported asthma. Patients with BE+A were younger, were more likely to be female and never smokers, and had a higher body mass index than patients with bronchiectasis without asthma. BE+A was associated with a higher prevalence of rhinosinusitis and nasal polyps as well as eosinophilia and Aspergillus sensitization. BE+A had similar microbiology but significantly lower severity of disease using the bronchiectasis severity index. Patients with BE+A were at increased risk of exacerbation after adjustment for disease severity and multiple confounders. Inhaled corticosteroid (ICS) use was associated with reduced mortality in patients with BE+A (adjusted hazard ratio 0.78, 95% CI 0.63-0.95) and reduced risk of hospitalization (rate ratio 0.67, 95% CI 0.67-0.86) compared with control subjects without asthma and not receiving ICSs. CONCLUSIONS: BE+A was common and was associated with an increased risk of exacerbations and improved outcomes with ICS use. Unexpectedly we identified significantly lower mortality in patients with BE+A.

The Association Between Bronchiectasis and Chronic Obstructive Pulmonary Disease: Data from the European Bronchiectasis Registry (EMBARC).

RATIONALE AND OBJECTIVE: Bronchiectasis and COPD are associated conditions but misdiagnosis is believed to be common. A recently published international consensus definition of bronchiectasis (BE) and COPD association: The ROSE criteria (radiological bronchiectasis(R), obstruction: FEV1/FVC ratio<0.7 (O), symptoms (S) and exposure:≥10 pack year smoking (E) allows objective diagnosis of the BE-COPD association. METHODS: Analysis of the EMBARC registry, a prospective observational study of patients with CT confirmed bronchiectasis from 28 countries. The ROSE criteria were used to objectively defined BE-COPD association. Key outcomes during up to 5-years follow-up were exacerbations, hospitalization and mortality. MEASUREMENT AND MAIN RESULTS: 16730 patients with bronchiectasis were included. 4336 had a co-diagnosis of COPD and these patients had more exacerbations, worse quality of life and higher severity scores. We observed marked overdiagnosis of COPD using the ROSE criteria: 22.2% of patients with a diagnosis of COPD did not have airflow obstruction and 31.9% did not have a history of ≥10 pack years smoking. Therefore the proportion meeting the ROSE criteria for COPD was 2157 (55.4%). Compared to patients without COPD, patients meeting ROSE criteria had increased risk of exacerbations and exacerbations resulting in hospitalisation during follow-up (IRR 1.25 95%CI 1.15-1.35 and 1.69 95%CI 1.51-1.90 respectively) but patients with a diagnosis of COPD who did not meet ROSE criteria also had increased risk of exacerbations. CONCLUSIONS: The label of COPD is often applied to bronchiectasis patients without objective evidence of airflow obstruction and smoking history. Patients with a clinical label of COPD have worse clinical outcomes.

A BEAT-PCD consensus statement: a core outcome set for pulmonary disease interventions in primary ciliary dyskinesia.

Background: Consistent use of reliable and clinically appropriate outcome measures is a priority for clinical trials, with clear definitions to allow comparability. We aimed to develop a core outcome set (COS) for pulmonary disease interventions in primary ciliary dyskinesia (PCD). Methods: A multidisciplinary international PCD expert panel was set up. A list of outcomes was created based on published literature. Using a modified three-round e-Delphi technique, the panel was asked to decide on relevant end-points related to pulmonary disease interventions and how they should be reported. First, inclusion of an outcome in the COS was determined. Second, the minimum information that should be reported per outcome. The third round finalised statements. Consensus was defined as ≥80% agreement among experts. Results: During the first round, experts reached consensus on four out of 24 outcomes to be included in the COS. Five additional outcomes were discussed in subsequent rounds for their use in different subsettings. Consensus on standardised methods of reporting for the COS was reached. Spirometry, health-related quality-of-life scores, microbiology and exacerbations were included in the final COS. Conclusion: This expert consensus resulted in a COS for clinical trials on pulmonary health among people with PCD.

Airway 'Resistotypes' and Clinical Outcomes in Bronchiectasis.

INTRODUCTION: Application of whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis highlights a diverse pool of antimicrobial resistance genes: the 'resistome', the clinical significance of which remains unclear. METHODS: Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n=280) including the international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 study (CAMEB 2; n=251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing P. aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing and the bronchiectasis resistome evaluated in association with clinical outcomes and underlying host microbiomes. RESULTS: The bronchiectasis resistome features a unique resistance gene profile and elevated counts of aminoglycoside, bicyclomycin, phenicol, triclosan and multi-drug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles including increased macrolide and multi-drug resistance genes associate with shorter intervals to next exacerbation, while distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant 'resistotypes' RT1 and RT2, the latter characterized by poor clinical outcomes, increased multi-drug resistance and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favourable resistome profile demonstrating reduced resistance gene diversity. CONCLUSION: The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis 'resistotypes' link to clinical disease and are modifiable through targeted antimicrobial therapy. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Extensive acute and sustained changes to neutrophil proteomes post-SARS-CoV-2 infection.

BACKGROUND: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery. METHODS: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale. RESULTS: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors. CONCLUSIONS: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.

Inflammatory Molecular Endotypes in Bronchiectasis: A European Multicenter Cohort Study.

Rationale: Although inflammation and infection are key disease drivers in bronchiectasis, few studies have integrated host inflammatory and microbiome data to guide precision medicine. Objectives: To identify clusters among patients with bronchiectasis on the basis of inflammatory markers and to assess the association between inflammatory endotypes, microbiome characteristics, and exacerbation risk. Methods: Patients with stable bronchiectasis were enrolled at three European centers, and cluster analysis was used to stratify the patients according to the levels of 33 sputum and serum inflammatory markers. Clusters were compared in terms of microbiome composition (16S ribosomal RNA sequencing) and exacerbation risk over a 12-month follow-up. Measurements and Main Results: A total of 199 patients were enrolled (109 [54.8%] female; median age, 69 yr). Four clusters of patients were defined according to their inflammatory profiles: cluster 1, milder neutrophilic inflammation; cluster 2, mixed-neutrophilic and type 2; cluster 3, most severe neutrophilic; and cluster 4, mixed-epithelial and type 2. Lower microbiome diversity was associated with more severe inflammatory clusters (P < 0.001), and ß-diversity analysis demonstrated distinct microbiome profiles associated with each inflammatory cluster (P = 0.001). Proteobacteria and Pseudomonas at phylum and genus levels, respectively, were more enriched in clusters 2 and 3 than in clusters 1 and 4. Furthermore, patients in cluster 2 (rate ratio [RR], 1.49; 95% confidence interval [CI], 1.16-1.92) and cluster 3 (RR, 1.61; 95% CI, 1.12-2.32) were at higher risk of exacerbation over a 12-month follow-up compared with cluster 1, even after adjustment for prior exacerbation history. Conclusions: Bronchiectasis inflammatory endotypes are associated with distinct microbiome profiles and future exacerbation risk.

Restoring Ciliary Function: Gene Therapeutics for Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by defects in motile cilia, which play an important role in several organ systems. Lung disease is a hallmark of PCD, given the essential role of cilia in airway surface defense. Diagnosis of PCD is complicated due to its reliance on complex tests that are not utilized by every clinic and also its phenotypic overlap with several other respiratory diseases. Nonetheless, PCD is increasingly being recognized as more common than once thought. The disease is genetically complex, with several genes reported to be associated with PCD. There is no cure for PCD, but gene therapy remains a promising therapeutic strategy. In this review, we provide an overview of the clinical symptoms, diagnosis, genetics, and current treatment regimens for PCD. We also describe PCD model systems and discuss the therapeutic potential of different gene therapeutics for targeting the intended cellular target, the ciliated cells of the airway.

Clinical outcomes of bronchiectasis in India: data from the EMBARC/Respiratory Research Network of India registry.

BACKGROUND: Identifying risk factors for poor outcomes can help with risk stratification and targeting of treatment. Risk factors for mortality and exacerbations have been identified in bronchiectasis but have been almost exclusively studied in European and North American populations. This study investigated the risk factors for poor outcome in a large population of bronchiectasis patients enrolled in India. METHODS: The European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India (EMBARC-India) registry is a prospective observational study of adults with computed tomography-confirmed bronchiectasis enrolled at 31 sites across India. Baseline characteristics of patients were used to investigate associations with key clinical outcomes: mortality, severe exacerbations requiring hospital admission, overall exacerbation frequency and decline in forced expiratory volume in 1 s. RESULTS: 1018 patients with at least 12-month follow-up data were enrolled in the follow-up study. Frequent exacerbations (≥3 per year) at baseline were associated with an increased risk of mortality (hazard ratio (HR) 3.23, 95% CI 1.39-7.50), severe exacerbations (HR 2.71, 95% CI 1.92-3.83), future exacerbations (incidence rate ratio (IRR) 3.08, 95% CI 2.36-4.01) and lung function decline. Coexisting COPD, dyspnoea and current cigarette smoking were similarly associated with a worse outcome across all end-points studied. Additional predictors of mortality and severe exacerbations were increasing age and cardiovascular comorbidity. Infection with Gram-negative pathogens (predominantly Klebsiella pneumoniae) was independently associated with increased mortality (HR 3.13, 95% CI 1.62-6.06), while Pseudomonas aeruginosa infection was associated with severe exacerbations (HR 1.41, 95% CI 1.01-1.97) and overall exacerbation rate (IRR 1.47, 95% CI 1.13-1.91). CONCLUSIONS: This study identifies risk factors for morbidity and mortality among bronchiectasis patients in India. Identification of these risk factors may support treatment approaches optimised to an Asian setting.

A Hybrid Architecture (CO-CONNECT) to Facilitate Rapid Discovery and Access to Data Across the United Kingdom in Response to the COVID-19 Pandemic: Development Study.

BACKGROUND: COVID-19 data have been generated across the United Kingdom as a by-product of clinical care and public health provision, as well as numerous bespoke and repurposed research endeavors. Analysis of these data has underpinned the United Kingdom's response to the pandemic, and informed public health policies and clinical guidelines. However, these data are held by different organizations, and this fragmented landscape has presented challenges for public health agencies and researchers as they struggle to find relevant data to access and interrogate the data they need to inform the pandemic response at pace. OBJECTIVE: We aimed to transform UK COVID-19 diagnostic data sets to be findable, accessible, interoperable, and reusable (FAIR). METHODS: A federated infrastructure model (COVID - Curated and Open Analysis and Research Platform [CO-CONNECT]) was rapidly built to enable the automated and reproducible mapping of health data partners' pseudonymized data to the Observational Medical Outcomes Partnership Common Data Model without the need for any data to leave the data controllers' secure environments, and to support federated cohort discovery queries and meta-analysis. RESULTS: A total of 56 data sets from 19 organizations are being connected to the federated network. The data include research cohorts and COVID-19 data collected through routine health care provision linked to longitudinal health care records and demographics. The infrastructure is live, supporting aggregate-level querying of data across the United Kingdom. CONCLUSIONS: CO-CONNECT was developed by a multidisciplinary team. It enables rapid COVID-19 data discovery and instantaneous meta-analysis across data sources, and it is researching streamlined data extraction for use in a Trusted Research Environment for research and public health analysis. CO-CONNECT has the potential to make UK health data more interconnected and better able to answer national-level research questions while maintaining patient confidentiality and local governance procedures.