Targeting respiratory microbiomes in COPD and bronchiectasis.

INTRODUCTION: This review summarizes our current understanding of the respiratory microbiome in COPD and Bronchiectasis. We explore the interplay between microbial communities, host immune responses, disease pathology and treatment outcomes. AREAS COVERED: We detail the dynamics of the airway microbiome, its influence in chronic respiratory diseases, and analytical challenges. Relevant articles from PubMed and Medline searches between Jan 2010 and March 2024 were retrieved and summarized. The review examines clinical correlations of the microbiome in COPD and bronchiectasis, assessing how current therapies impact upon it. The potential of emerging immunotherapies, anti-inflammatories and antimicrobial strategies are discussed, with focus on the pivotal role of commensal taxa in maintaining respiratory health and the promising avenue of microbiome remodeling for disease management. EXPERT OPINION: Given the heterogeneity in microbiome composition and its pivotal role in disease development and progression, a shift toward microbiome-directed therapeutics is appealing. This transition, from traditional 'pathogen-centric' diagnostic and treatment modalities to those acknowledging the microbiome, can be enabled by evolving cross-disciplinary platforms which have the potential to accelerate microbiome-based interventions into routine clinical practice. Bridging the gap between comprehensive microbiome analysis and clinical application, however, remains challenging, necessitating continued innovation in research, diagnostics, trials and therapeutic development pipelines.

Predictors of persistent poor control and validation of ASSESS score: Longitudinal 5-year follow-up of severe asthma cohort.

Background: Longitudinal predictors of persistent poor asthma control in severe asthma (SA) cohort remain scarce. The predictive value of the asthma severity scoring system (ASSESS) in the SA cohort outside the original study and in the Asian population is unknown. Objective: We sought to determine the 5-year longitudinal outcome of patients with SA and validate the use of ASSESS score in predicting future outcomes in SA. Methods: A prospective longitudinal observational study of patients with SA attending the multidisciplinary specialist SA clinic of the Singapore General Hospital from 2011 to 2021 was conducted. The number of exacerbations and asthma control test results were recorded yearly for 5 consecutive years. The ASSESS score was computed at baseline, and the area under the receiver-operating characteristic curve for predicting persistent poor asthma control was generated. Results: Of the 489 patients recruited into the study, 306 patients with 5-year follow-up data were analyzed. Seventy-three percent had type 2 inflammation with increased overall exacerbations over 5 years (rate ratio, 2.55; 95% CI, 1.31-4.96; P = .006) relative to non-type 2 SA. In the multivariate model, bronchiectasis, gastroesophageal reflux disease, and an asthma control test score of less than 20 were significantly associated with persistent poor asthma control over 5 years. ASSESS scores were good at predicting persistent poor asthma control with an area under the receiver-operating characteristic curve of 0.71 (95% CI, 0.57-0.84). Conclusions: Bronchiectasis and gastroesophageal reflux disease are predictors for persistent poor asthma control and targeted traits for precision medicine in SA. The ASSESS score has a good prediction for persistent poor asthma control over 5 years.

RNA is a key component of extracellular DNA networks in Pseudomonas aeruginosa biofilms.

The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. Extracellular RNA (eRNA) can also be present, contributing to the structural integrity of biofilms. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. Here, we show that eRNA associates with extracellular DNA (eDNA) to form matrix fibres in Pseudomonas aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA led to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix was enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalised with eDNA fibres in the biofilm matrix, as shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA was also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. Thus, our results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.

Applying Next-Generation Sequencing and Multi-Omics in Chronic Obstructive Pulmonary Disease.

Microbiomics have significantly advanced over the last decade, driven by the widespread availability of next-generation sequencing (NGS) and multi-omic technologies. Integration of NGS and multi-omic datasets allow for a holistic assessment of endophenotypes across a range of chronic respiratory disease states, including chronic obstructive pulmonary disease (COPD). Valuable insight has been attained into the nature, function, and significance of microbial communities in disease onset, progression, prognosis, and response to treatment in COPD. Moving beyond single-biome assessment, there now exists a growing literature on functional assessment and host-microbe interaction and, in particular, their contribution to disease progression, severity, and outcome. Identifying specific microbes and/or metabolic signatures associated with COPD can open novel avenues for therapeutic intervention and prognosis-related biomarkers. Despite the promise and potential of these approaches, the large amount of data generated by such technologies can be challenging to analyze and interpret, and currently, there remains a lack of standardized methods to address this. This review outlines the current use and proposes future avenues for the application of NGS and multi-omic technologies in the endophenotyping, prognostication, and treatment of COPD.

Sensitisation to recombinant Aspergillus fumigatus allergens and clinical outcomes in COPD.

BACKGROUND: Variable clinical outcomes are reported with fungal sensitisation in chronic obstructive pulmonary disease (COPD), and it remains unclear which fungi and what allergens associate with the poorest outcomes. The use of recombinant as opposed to crude allergens for such assessment is unknown. METHODS: A prospective multicentre assessment of stable COPD (n=614) was undertaken in five hospitals across three countries: Singapore, Malaysia and Hong Kong. Clinical and serological assessment was performed against a panel of 35 fungal allergens including crude and recombinant Aspergillus and non-Aspergillus allergens. Unsupervised clustering and topological data analysis (TDA) approaches were employed using the measured sensitisation responses to elucidate if sensitisation subgroups exist and their related clinical outcomes. RESULTS: Aspergillus fumigatus sensitisation was associated with increased exacerbations in COPD. Unsupervised cluster analyses revealed two "fungal sensitisation" groups. The first was characterised by Aspergillus sensitisation and increased exacerbations, poorer lung function and worse prognosis. Polysensitisation in this group conferred even poorer outcome. The second group, characterised by Cladosporium sensitisation, was more symptomatic. Significant numbers of individuals demonstrated sensitisation responses to only recombinant (as opposed to crude) A. fumigatus allergens f 1, 3, 5 and 6, and exhibited increased exacerbations, poorer lung function and an overall worse prognosis. TDA validated these findings and additionally identified a subgroup within Aspergillus-sensitised COPD of patients with frequent exacerbations. CONCLUSION: Aspergillus sensitisation is a treatable trait in COPD. Measuring sensitisation responses to recombinant Aspergillus allergens identifies an important patient subgroup with poor COPD outcomes that remains overlooked by assessment of only crude Aspergillus allergens.

Healthcare utilization and health-related quality of life of severe asthma patients in Singapore.

BACKGROUND: Notwithstanding unequivocal consensus on the disproportionate effect of severe asthma (SA) on asthma morbidity, healthcare utilization, quality of life, work impairment and socioeconomic burden, the burden of SA patients in Singapore has not been appraised. OBJECTIVES: To determine the burden of disease and extent of quality of life impairment in SA patients in Singapore. METHODS: A cross-sectional analysis of SA patients seen in Singapore General Hospital (2020-2021) to investigate emergency healthcare utilization, oral corticosteroid (OCS) burden and health-related quality of life (HRQoL) with primary endpoint EuroQoL-5 Dimension three-level (EQ-5D-3L) scores. The empirical measurement properties of the EQ-5D utility index in SA were comprehensively assessed through multivariate regression analyses. RESULTS: A total of 336 SA patients were recruited, 51.2% of SA patients had at least one acute healthcare resource utilization during the previous year, with 25.6% of patients having an emergency healthcare visit to the hospital. Overall mean (SD) EQ-5D-3L and EQ-5D-3L utility scores in SA patients were 6.22 (1.51) and 0.77 (0.30), respectively. EQ-5D utility scores were 0.14 lower in uncontrolled vs controlled asthma and 0.09 lower in the presence of severe exacerbation, whereas barely changed by maintenance OCS dose and airflow limitation. CONCLUSION: SA patients were found to have high disease burden, high healthcare resource utilization and OCS use, low biologics usage, poor HRQoL and utility in comparison with other chronic diseases.

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Managing adult asthma during the COVID-19 pandemic: A 2022 review and current recommendations.

INTRODUCTION: This review aims to examine asthma management during the COVID-19 pandemic. METHOD: Relevant recommendations and articles were identified by respiratory professional societies and PubMed search using the terms "asthma" and "COVID-19", and examined for relevance and inclusion in this study. RESULTS: Recommendations for the management of asthma have remained similar but are now supported by new evidence between the years 2020 and 2022. Patients with well-controlled, mild-to-moderate asthma are unlikely to be at increased risk of acquiring COVID-19 or having worse outcomes from COVID-19. All asthma patients should receive COVID-19 vaccination. Spirometry can be performed with the usual strict infection control procedures unless there is a suspicion of COVID-19. Mask-wearing and other health measures remain important for asthma patients. CONCLUSION: While previous recommendations were largely based on expert opinion, the tremendous amount of literature published since the pandemic first emerged 2 years ago has helped guide respiratory professional bodies to update their recommendations. This study provides a timely review of the various recommendations and can be used to guide healthcare professionals in managing asthma patients, as the world prepares for a future with COVID-19 becoming endemic. The long-term consequences of COVID-19 infection in asthma patients and the ripple effects of COVID-19 remain uncertain and deserve ongoing study.

Cluster phenotypes in a non-idiopathic pulmonary fibrosis fibrotic interstitial lung diseases cohort in Singapore.

Background: Non-idiopathic pulmonary fibrosis fibrosing interstitial lung diseases (F-ILDs) may demonstrate a progressive disease trajectory similar to idiopathic pulmonary fibrosis (IPF). We aimed to identify novel F-ILD phenotypes in a multi-ethnic South-East Asian population. Methods: F-ILD subjects (n=201) were analysed using unsupervised hierarchical cluster analysis and their outcomes compared against IPF (n=86). Results: Four clusters were identified. Cluster 1 (n=53, 26.4%) comprised older Chinese males with high body mass index (BMI) and comorbidity burden, higher baseline forced vital capacity (FVC) percentage predicted and lower diffusing capacity of the lung for carbon monoxide (DLCO) percentage predicted. They had similar mortality to IPF. Cluster 2 (n=67, 33.3%) had younger female non-smokers with low comorbidity burden, groundglass changes on high-resolution chest computed tomography (HRCT) and a positive anti-nuclear antibody (ANA) titre ≥1:160. They had lower baseline FVC and higher DLCO, low mortality and slower lung function decline. Cluster 3 (n=42, 20.9%) consisted male smokers with low comorbidity burden, emphysema on HRCT and high baseline lung function. They had low mortality and slow lung function decline. Cluster 4 (n=39, 19.4%) was the highest risk and comprised of mainly Indians with high BMI. They had the highest proportion of ischemic heart disease (IHD) and previous pulmonary tuberculosis. Subjects had the lowest baseline lung function, highest mortality, and fastest lung function decline. Survival differences across clusters remained significant following adjustment for treatment. Conclusions: We identified four distinct F-ILD clinical phenotypes with varying disease trajectories. This demonstrates heterogeneity in F-ILD and the need for complementary approaches for classification and prognostication beyond ATS/ERS guideline diagnosis.

Clinical Aspergillus Signatures in COPD and Bronchiectasis.

Pulmonary mycoses remain a global threat, causing significant morbidity and mortality. Patients with airways disease, including COPD and bronchiectasis, are at increased risks of pulmonary mycoses and its associated complications. Frequent use of antibiotics and corticosteroids coupled with impaired host defenses predispose patients to fungal colonization and airway persistence, which are associated with negative clinical consequences. Notably, Aspergillus species remain the best-studied fungal pathogen and induce a broad spectrum of clinical manifestations in COPD and bronchiectasis ranging from colonization and sensitization to more invasive disease. Next-generation sequencing (NGS) has gained prominence in the field of respiratory infection, and in some cases is beginning to act as a viable alternative to traditional culture. NGS has revolutionized our understanding of airway microbiota and in particular fungi. In this context, it permits the identification of the previously unculturable, fungal composition, and dynamic change within microbial communities of the airway, including potential roles in chronic respiratory disease. Furthermore, inter-kingdom microbial interactions, including fungi, in conjunction with host immunity have recently been shown to have important clinical roles in COPD and bronchiectasis. In this review, we provide an overview of clinical Aspergillus signatures in COPD and bronchiectasis and cover the current advances in the understanding of the mycobiome in these disease states. The challenges and limitations of NGS will be addressed.

High Frequency of Allergic Bronchopulmonary Aspergillosis in Bronchiectasis-COPD Overlap.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is associated with frequent exacerbations and poor outcomes in chronic respiratory disease, but remains underdiagnosed. The role of fungal sensitization in bronchiectasis-COPD overlap (BCO) is unknown. RESEARCH QUESTION: What is the occurrence and clinical relevance of Aspergillus sensitization and ABPA in BCO when compared with individuals with COPD or bronchiectasis without overlap? STUDY DESIGN: Prospective, observational, cross-sectional study. METHODS: We prospectively recruited 280 patients during periods of clinical stability with bronchiectasis (n = 183), COPD (n = 50), and BCO (n = 47) from six hospitals across three countries (Singapore, Malaysia, and Scotland). We assessed sensitization responses (as specific IgE) to a panel of recombinant Aspergillus fumigatus allergens and the occurrence of ABPA in relationship to clinical outcomes. RESULTS: Individuals with BCO show an increased frequency and clinical severity of ABPA compared with those with COPD and bronchiectasis without overlap. BCO-associated ABPA is associated with more severe disease, higher exacerbation rates, and lower lung function when compared with ABPA occurring in the absence of overlap. BCO with a severe bronchiectasis severity index (BSI; > 9) is associated significantly with the occurrence of ABPA that is unrelated to underlying COPD severity. CONCLUSIONS: BCO demonstrates a high frequency of ABPA that is associated with a severe BSI (> 9) and poor clinical outcomes. Clinicians should maintain a high index of suspicion for the potential development of ABPA in patients with BCO with high BSI.

Spillover Effects of COVID-19 on Essential Chronic Care and Ways to Foster Health System Resilience to Support Vulnerable Non-COVID Patients: A Multistakeholder Study.

OBJECTIVES: Little empirical research exists on how key stakeholders involved in the provision of care for chronic conditions and policy planning perceive the indirect or "spillover" effects of the COVID-19 on non-COVID patients. This study aims to explore stakeholder experiences and perspectives of the impact of COVID-19 on the provision of care for chronic conditions, evolving modalities of care, and stakeholder suggestions for improving health system resilience to prepare for future pandemics. DESIGN: Qualitative study design. SETTING AND PARTICIPANTS: This study was conducted during and after the COVID-19 lockdown period in Singapore. We recruited a purposive sample of 51 stakeholders involved in care of non-COVID patients and/or policy planning for chronic disease management. They included health care professionals (micro-level), hospital management officers (meso-level), and government officials (macro-level). METHODS: In-depth semi-structured interviews were conducted. All interviews were digitally recorded, transcribed verbatim, and thematically analyzed. RESULTS: Optimal provision of care for chronic diseases may be compromised through the following processes: lack of "direct" communication between colleagues on clinical cases resulting in rescheduling of patient visits; uncertainty in diagnostic decisions due to protocol revision and lab closure; and limited preparedness to handle non-COVID patients' emotional reactions. Although various digital innovations enhanced access to care, a digital divide exists due to uneven digital literacy and perceived data security risks, thereby hampering wider implementation. To build health system resilience, stakeholders suggested the need to integrate digital care into the information technology ecosystem, develop strategic public-private partnerships for chronic disease management, and give equal attention to the provision of holistic psychosocial and community support for vulnerable non-COVID patients. CONCLUSIONS AND IMPLICATIONS: Findings highlight that strategies to deliver quality chronic care for non-COVID patients in times of public health crisis should include innovative care practices and institutional reconfiguration within the broader health system context.

The current understanding and future directions for sputum microbiome profiling in chronic obstructive pulmonary disease.

PURPOSE OF REVIEW: Next-generation sequencing (NGS) has deepened our understanding of the respiratory microbiome in health and disease. The number of microbiome studies employing sputum as an airway surrogate has continued to increase over the past decade to include multiple large multicentre and longitudinal studies of the microbiome in chronic obstructive pulmonary disease (COPD). In this review, we summarize the recent advances to our understanding of the bacteriome, virome and mycobiome in COPD. RECENT FINDINGS: Diverse microbiome profiles are reported in COPD. The neutrophilic Haemophilus-predominant bacteriome remains a prominent COPD phenotype, relatively stable over time and during exacerbations. Studies of the virome remain limited but reveal a potential involvement of viruses and bacteriophages particularly during COPD exacerbations and advancing disease severity. Mycobiome signatures, even in stable COPD are associated with poorer clinical outcomes including mortality. SUMMARY: The sputum microbiome in COPD is being increasingly recognized for its clinical relevance, even in the stable state. Future studies integrating microbial kingdoms holistically (i.e. bacterial, viral and fungal) will provide deeper insight into its functionality including the relevance of microbial interactions and effect of treatment on microbiome-associated clinical outcomes.

Heterogeneity of non-cystic-fibrosis bronchiectasis in multiethnic Singapore: A prospective cohort study at a tertiary pulmonology centre.

INTRODUCTION: Non-cystic fibrosis bronchiectasis (NCFB) is a highly heterogenous disease. We describe the clinical characteristics of NCFB patients and evaluate the performance of Bronchiectasis Severity Index (BSI) in predicting mortality. METHODS: Patients attending the bronchiectasis clinic between August 2015 and April 2020 with radiologically proven bronchiectasis on computed tomography were recruited. Clinical characteristics, spirometry, radiology, microbiology and clinical course over a median period of 2.4 years is presented. RESULTS: A total of 168 patients were enrolled in this prospective cohort study. They were predominantly women (67.8%), Chinese (87.5%) and never-smokers (76.9%). Median age of diagnosis was 64 years (interquartile range 56-71) and the most common aetiology was "idiopathic" bronchiectasis (44.6%). Thirty-nine percent had normal spirometries. Compared to female patients, there were more smokers among the male patients (53.8% versus 8.5%, P<0.001) and a significantly larger proportion with post-tuberculous bronchiectasis (37.0% vs 15.8%, P=0.002). Fifty-five percent of our cohort had a history of haemoptysis. Lower body mass index, presence of chronic obstructive pulmonary disease, ever-smoker status, modified Reiff score, radiological severity and history of exacerbations were risk factors for mortality. Survival was significantly shorter in patients with severe bronchiectasis (BSI>9) compared to those with mild or moderate disease (BSI<9). The hazard ratio for severe disease (BSI>9) compared to mild disease (BSI 0-4) was 14.8 (confidence interval 1.929-114.235, P=0.01). CONCLUSION: The NCFB cohort in Singapore has unique characteristics with sex differences. Over half the patients had a history of haemoptysis. The BSI score is a useful predictor of mortality in our population.

Integrative microbiomics in bronchiectasis exacerbations.

Bronchiectasis, a progressive chronic airway disease, is characterized by microbial colonization and infection. We present an approach to the multi-biome that integrates bacterial, viral and fungal communities in bronchiectasis through weighted similarity network fusion ( https://integrative-microbiomics.ntu.edu.sg ). Patients at greatest risk of exacerbation have less complex microbial co-occurrence networks, reduced diversity and a higher degree of antagonistic interactions in their airway microbiome. Furthermore, longitudinal interactome dynamics reveals microbial antagonism during exacerbation, which resolves following treatment in an otherwise stable multi-biome. Assessment of the Pseudomonas interactome shows that interaction networks, rather than abundance alone, are associated with exacerbation risk, and that incorporation of microbial interaction data improves clinical prediction models. Shotgun metagenomic sequencing of an independent cohort validated the multi-biome interactions detected in targeted analysis and confirmed the association with exacerbation. Integrative microbiomics captures microbial interactions to determine exacerbation risk, which cannot be appreciated by the study of a single microbial group. Antibiotic strategies probably target the interaction networks rather than individual microbes, providing a fresh approach to the understanding of respiratory infection.

Respiratory Mycoses in COPD and Bronchiectasis.

Chronic obstructive pulmonary disease (COPD) and bronchiectasis represent chronic airway diseases associated with significant morbidity and mortality. Bacteria and viruses are commonly implicated in acute exacerbations; however the significance of fungi in these airways remains poorly defined. While COPD and bronchiectasis remain recognized risk factors for the occurrence of Aspergillus-associated disease including chronic and invasive aspergillosis, underlying mechanisms that lead to the progression from colonization to invasive disease remain uncertain. Nonetheless, advances in molecular technologies have improved our detection, identification and understanding of resident fungi characterizing these airways. Mycobiome sequencing has revealed the complex varied and myriad profile of airway fungi in COPD and bronchiectasis, including their association with disease presentation, progression, and mortality. In this review, we outline the emerging evidence for the clinical importance of fungi in COPD and bronchiectasis, available diagnostic modalities, mycobiome sequencing approaches and association with clinical outcomes.

The airway microbiome in COPD, bronchiectasis and bronchiectasis-COPD overlap.

OBJECTIVE: To review the airway microbiome in chronic obstructive pulmonary disease (COPD), bronchiectasis and bronchiectasis-COPD overlap (BCO). DATA SOURCE AND STUDY SELECTION: Relevant studies were selected from PubMed, Google scholar, EMBASE and Web of Science. All studies involving human microbiomes, published in the English language, and using the search terms "COPD", "Chronic Obstructive Pulmonary Disease", "Bronchiectasis", "BCO" or "Bronchiectasis and COPD overlap", AND "microbiome", "mycobiome" or "metagenomics" were included. RESULTS: Despite variability in sampling methods and specimen types used, microbiome composition remains relatively comparable in COPD and bronchiectasis with prominence of Proteobacteria, Firmicutes and Bacteroidetes. Alterations to airway microbiomes occur in association to disease severity and/or exacerbations in COPD and bronchiectasis. Decreased alpha diversity and Haemophilus-predominant microbiomes are associated with poorer survival in COPD, while, in bronchiectasis, Pseudomonas-predominant microbiomes demonstrate high exacerbation frequency and greater symptom burden while Aspergillus-dominant mycobiome profiles associate with exacerbations. The role of the microbiome in BCO remains understudied. CONCLUSION: Use of next-generation sequencing has revolutionised our detection and understanding of the airway microbiome in chronic respiratory diseases such as COPD and bronchiectasis. Targeted amplicon sequencing reveals important associations between the respiratory microbiome and disease outcome while metagenomics may elucidate functional pathways. How best to apply this information into patient care, monitoring and treatment, however, remains challenging and necessitates further study.

A high-risk airway mycobiome is associated with frequent exacerbation and mortality in COPD.

INTRODUCTION: The chronic obstructive pulmonary disease (COPD) bacteriome associates with disease severity, exacerbations and mortality. While COPD patients are susceptible to fungal sensitisation, the role of the fungal mycobiome remains uncertain. METHODS: We report the largest multicentre evaluation of the COPD airway mycobiome to date, including participants from Asia (Singapore and Malaysia) and the UK (Scotland) when stable (n=337) and during exacerbations (n=66) as well as nondiseased (healthy) controls (n=47). Longitudinal mycobiome analysis was performed during and following COPD exacerbations (n=34), and examined in terms of exacerbation frequency, 2-year mortality and occurrence of serum specific IgE (sIgE) against selected fungi. RESULTS: A distinct mycobiome profile is observed in COPD compared with controls as evidenced by increased α-diversity (Shannon index; p<0.001). Significant airway mycobiome differences, including greater interfungal interaction (by co-occurrence), characterise very frequent COPD exacerbators (three or more exacerbations per year) (permutational multivariate ANOVA; adjusted p<0.001). Longitudinal analyses during exacerbations and following treatment with antibiotics and corticosteroids did not reveal any significant change in airway mycobiome profile. Unsupervised clustering resulted in two clinically distinct COPD groups: one with increased symptoms (COPD Assessment Test score) and Saccharomyces dominance, and another with very frequent exacerbations and higher mortality characterised by Aspergillus, Curvularia and Penicillium with a concomitant increase in serum sIgE levels against the same fungi. During acute exacerbations of COPD, lower fungal diversity associates with higher 2-year mortality. CONCLUSION: The airway mycobiome in COPD is characterised by specific fungal genera associated with exacerbations and increased mortality.

Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of Aspergillus species in the Human Airway.

BACKGROUND: Prior studies illustrate the presence and clinical importance of detecting Aspergillus species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of Aspergillus in specimens from the human airway. Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway Aspergillus when compared to standard qPCR. METHODS: The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in n = 20 sputum specimens obtained from non-diseased (n = 4), chronic obstructive pulmonary disease (COPD; n = 8) and non-cystic fibrosis bronchiectasis (n = 8) patients where Aspergillus status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed. RESULTS: Standard qPCR and ddPCR were both able to detect, even at low abundance, Aspergillus species (Aspergillus fumigatus - A. fumigatus and Aspergillus terreus - A. terreus) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of A. terreus particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR. CONCLUSION: ddPCR has greater sensitivity for A. terreus detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of A. terreus species in chronic respiratory disease states such as bronchiectasis.

Metagenomics Reveals a Core Macrolide Resistome Related to Microbiota in Chronic Respiratory Disease.

Rationale: Long-term antibiotic use for managing chronic respiratory disease is increasing; however, the role of the airway resistome and its relationship to host microbiomes remains unknown.Objectives: To evaluate airway resistomes and relate them to host and environmental microbiomes using ultradeep metagenomic shotgun sequencing.Methods: Airway specimens from 85 individuals with and without chronic respiratory disease (severe asthma, chronic obstructive pulmonary disease, and bronchiectasis) were subjected to metagenomic sequencing to an average depth exceeding 20 million reads. Respiratory and device-associated microbiomes were evaluated on the basis of taxonomical classification and functional annotation including the Comprehensive Antibiotic Resistance Database to determine airway resistomes. Co-occurrence networks of gene-microbe association were constructed to determine potential microbial sources of the airway resistome. Paired patient-inhaler metagenomes were compared (n = 31) to assess for the presence of airway-environment overlap in microbiomes and/or resistomes.Measurements and Main Results: Airway metagenomes exhibit taxonomic and metabolic diversity and distinct antimicrobial resistance patterns. A "core" airway resistome dominated by macrolide but with high prevalence of ß-lactam, fluoroquinolone, and tetracycline resistance genes exists and is independent of disease status or antibiotic exposure. Streptococcus and Actinomyces are key potential microbial reservoirs of macrolide resistance including the ermX, ermF, and msrD genes. Significant patient-inhaler overlap in airway microbiomes and their resistomes is identified where the latter may be a proxy for airway microbiome assessment in chronic respiratory disease.Conclusions: Metagenomic analysis of the airway reveals a core macrolide resistome harbored by the host microbiome.