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The wider availability of information on genomes has become essential for future advances in fungal biology, pathogenesis and epidemiology, and for the discovery of new drugs and diagnostics. MycopathologiaGENOMES is designed for the rapid publication of new genomes of human and animal pathogenic fungi using a checklist-based, standardized format.

Rationale: Allergic sensitization is associated with poor clinical outcomes in asthma, chronic obstructive pulmonary disease, and cystic fibrosis; however, its presence, frequency, and clinical significance in non–cystic fibrosis bronchiectasis remain unclear. Objectives: To determine the frequency and geographic variability that exists in a sensitization pattern to common and specific allergens, including house dust mite and fungi, and to correlate such patterns to airway immune-inflammatory status and clinical outcomes in bronchiectasis. Methods: Patients with bronchiectasis were recruited in Asia (Singapore and Malaysia) and the United Kingdom (Scotland) (n = 238), forming the Cohort of Asian and Matched European Bronchiectasis, which matched recruited patients on age, sex, and bronchiectasis severity. Specific IgE response against a range of common allergens was determined, combined with airway immune-inflammatory status and correlated to clinical outcomes. Clinically relevant patient clusters, based on sensitization pattern and airway immune profiles (“immunoallertypes”), were determined. Measurements and Main Results: A high frequency of sensitization to multiple allergens was detected in bronchiectasis, exceeding that in a comparator cohort with allergic rhinitis (n = 149). Sensitization was associated with poor clinical outcomes, including decreased pulmonary function and more severe disease. “Sensitized bronchiectasis” was classified into two immunoallertypes: one fungal driven and proinflammatory, the other house dust mite driven and chemokine dominant, with the former demonstrating poorer clinical outcome. Conclusions: Allergic sensitization occurs at high frequency in patients with bronchiectasis recruited from different global centers. Improving endophenotyping of sensitized bronchiectasis, a clinically significant state, and a “treatable trait” permits therapeutic intervention in appropriate patients, and may allow improved stratification in future bronchiectasis research and clinical trials. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) Published version Supported by the Singapore Ministry of Health’s National Medical Research Council under its Transition Award NMRC/TA/0048/2016 (S.H.C.) and Changi General Hospital Research grant CHF2016.03-P (T.B.L.). The work performed at NUS was supported by the Singapore Ministry of Education Academic Research Fund, SIgN, and National Medical Research Council grants N-154-000-038-001, R-154-000-404-112, R-154-000-553-112, R-154-000-565-112, R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597, SIgN-06-006, SIgN-08-020, and NMRC/1150/2008 (F.T.C.); J.D.C. is supported by the GSK/British Lung Foundation Chair of Respiratory Research.

Inhaled antibiotic nanoparticles have emerged as an effective strategy to control infection in bronchiectasis lung owed to their mucus-penetrating ability. Using ciprofloxacin (CIP) as the model antibiotic, we evaluated dry powder inhaler (DPI) formulations of two classes of antibiotic nanoparticles (i.e. liposome and nanoplex) in their (1) physical characteristics (i.e. size, zeta potential, CIP payload, preparation efficiency), (2) dissolution in artificial sputum medium, (3) ex vivo mucus permeability, (4) antimicrobial activity against Pseudomonas aeruginosa in mucus, (5) cytotoxicity towards human lung epithelium cells, and (6) in vitro aerosolization efficiency. The results showed that the CIP nanoplex exhibited fast dissolution with CIP supersaturation generation, in contrast to the slower release of the liposome (80 versus 30% dissolution after 1 h). Both nanoparticles readily overcame the mucus barrier attributed to their nanosize and mucus-inert surface (50% permeation after 1 h), leading to their similarly high antipseudomonal activity. The CIP liposome, however, possessed much lower CIP payload than the nanoplex (84% versus 3.5%), resulting in high lipid contents in its DPI formulation that led to higher cytotoxicity and lower aerosolization efficiency. The CIP nanoplex thus represented a superior formulation owed to its simpler preparation, higher CIP payload hence lower dosage, better aerosolization, and lower cytotoxicity. Ministry of Health (MOH) National Medical Research Council (NMRC) Accepted version This research is supported by (1) LKCMedicine-SCBE Collaborative Grant (CG-04/16) (S.H.C and K.H.O), (2) LKCMedicine Start-Up Grant (S.H.C) both from Nanyang Technological University, and (3) the Singapore Ministry of Health’s National Medical Research Council under its Transition Award (NMRC/TA/0048/2016) (S.H.C). The authors would like to acknowledge The Academic Respiratory Initiative for Pulmonary Health (TARIPH) for the collaboration supports.

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. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) Published version This research is supported by the Singapore General Hospital Research Grant (SRG-OPN-06-2021) (P.Y. Tiew) and the Singapore Ministry of Health's National Medical Research Council under its Clinician-Scientist Individual Research Grant (MOH-000141) (S.H. Chotirmall) and Clinician-Scientist Award (MOH-000710) (S.H. Chotirmall). K. Tsaneva-Atanasova gratefully acknowledges the financial support of the EPSRC via grant EP/T017856/1. F.T. Chew (Singapore) received grants from the National University of Singapore (N-154-000-038-001), Singapore Ministry of Education Academic Research Fund (R-154-000-191-112, R-154-000-404-112, R-154-000-553-112, R-154-000-565-112, R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597, R-154-000-A91-592, R-154-000-A95-592, R154-000-B99-114), Biomedical Research Council (Singapore) (BMRC/01/1/21/18/077, BMRC/04/1/21/19/315, BMRC/APG2013/108), Singapore Immunology Network (SIgN-06-006, SIgN-08-020), National Medical Research Council (Singapore) (NMRC/1150/2008), and the Agency for Science Technology and Research (Singapore) (H17/01/a0/008 and APG2013/108). Funding information for this article has been deposited with the Crossref Funder Registry.

The respiratory tract harbors a stable and diverse microbial population within an extracellular mucus layer. Mucus provides a formidable defense against infection and maintaining healthy mucus is essential to normal pulmonary physiology, promoting immune tolerance and facilitating a healthy, commensal lung microbiome that can be altered in association with chronic respiratory disease. How one maintains a specialized (healthy) microbiome that resists significant fluctuation remains unknown, although smoking, diet, antimicrobial therapy, and infection have all been observed to influence microbial lung homeostasis. In this review, we outline the specific role of polymerizing mucin, a key functional component of the mucus layer that changes during pulmonary disease. We discuss strategies by which mucin feed and spatial orientation directly influence microbial behavior and highlight how a compromised mucus layer gives rise to inflammation and microbial dysbiosis. This emerging field of respiratory research provides fresh opportunities to examine mucus, and its function as predictors of infection risk or disease progression and severity across a range of chronic pulmonary disease states and consider new perspectives in the development of mucolytic treatments.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections. Published version This study was partially supported by an unrestricted education grant from Gilead Sciences through their investigator sponsored research programme. J. Rhodes, T.S., A.P.B., P.S.D., D.A.J. and M.C.F. were supported by grants from the NERC (nos. NE/P001165/1 and NE/P000916/1), the UK Medical Research Council (MRC) (no. MR/R015600/1) and Wellcome Trust (no. 219551/Z/19/Z). D.A.J. is also funded by the Medical Research Council (grant no. MR/V037315/1) and Cystic Fibrosis Trust (grant no. SRC015). D.A.J. is funded by the Department of Health and Social Care (DHSC) Centre for Antimicrobial Optimisation (CAMO), Imperial College London. The views expressed in this publication are those of the authors and not necessarily those of the DHSC, National Health Service or National Institute for Health Research (NIHR). M.C.F. is a CIFAR Fellow in the Fungal Kingdom programme. K.D. was supported by a PhD studentship awarded by the School of Medicine, Trinity College Dublin. P.G.M. and J. Renwick (Dublin) received a project grant from the National Children’s Hospital, Tallaght University Hospital, which in part supported this work. A.W. and E.B. are supported by the Wellcome Trust Strategic Award (grant no. 097377), MRC Centre for Medical Mycology (grant no. MR/N006364/2) at the University of Exeter and a Biotechnology and Biological Sciences Research Council EASTBIO grant (no. BB/M010996/1). The authors also acknowledge the Imperial College London Cystic Fibrosis Strategic Research Centre and NIHR CAMO.

Infections caused by opportunistic fungal pathogens are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about the extent to which susceptible patients acquire infection from drug resistant genotypes in the environment. Here, we present a population genomic analysis of the mouldAspergillus fumigatusfrom across the United Kingdom and Republic of Ireland. First, we show occurrences where azole resistant isolates of near identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Second, we find that the fungus is structured into two clades (‘A’ and ‘B’) with little interclade recombination and the majority of environmental azole resistance genetically clustered inside Clade A. Genome-scans show the impact of selective sweeps across multiple regions of the genome. These signatures of positive selection are seen in regions containing canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, whilst other regions under selection have no defined function. Phenotyping identified genes in these regions that could act as modifiers of resistance showing the utility of reverse genetic approaches to dissect the complex genomic architecture of fungal drug resistance. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

International audience; The 2020 COVID-19 pandemic had a profound impact on the publishing landscape. The 'pre-peer-review' publication model is likely to become common as a lag in publishing is not acceptable in a pandemic or other time! Mycopathologia is well placed to adopt such changes with its improved editorial processes, article formats, author engagements, and published articles' access and citation. Mycopathologia had an improved journal impact factor and article downloads in 2018-2019. A limited sampling suggested a slight decrease in the total submissions in 2019 (352 articles) compared to 2018 (371 articles). However, the acceptance rate improved to 30% in 2019 from 19% in 2018. Nearly half of all submissions in 2019 were rejected before peer-review or transferred to other Springer Nature journals. The published articles were contributed from 34 different countries, with authors from China, the USA, and Brazil among the top three contributors. An enhanced editorial oversight allowed peer-reviewers to focus on fewer articles that were well-matched to their expertise, which led to lower rejection rates post-peer-review. The introduction of MycopathologiaGENOME and MycopathologiaIMAGE article types received a good reception with notable downloads and citations.

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. Ministry of Health (MOH) Nanyang Technological University This research is supported by the Singapore Ministry of Health's National Medical Research Council under its Transition Award (NMRC/TA/0048/2016 to S.H.C.); the Clinician-Scientist Individual Research Grant (MOH-000141 to S.H.C.); NTU Integrated Medical, Biological and Environmental Life Sciences (NIMBELS) (NIM/03/2018 to S.H.C.); the British Lung Foundation through the GSK/British Lung Foundation Chair of Respiratory Research (to J.D.C.) and the Scottish Government Chief Scientist Office (SCAF/17/03). K.T.-A. gratefully acknowledges the financial support of the EPSRC via grant EP/N014391/1.

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease. Published version

Respiratory infections are an important and frequent cause of morbidity and mortality globally. Sex and gender-based differences in lung infection are recognized and gradually gaining importance due to the potential for gender-tailored therapeutics. While sex and gender differences are widely acknowledged in the evaluation of chronic respiratory disease states such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF), acute and chronic respiratory infection complicate all of these and, in themselves, depending on age and organism, demonstrate sex differences. Males are disadvantaged in the occurrence and severity of lower respiratory tract infections such as pneumonia, while females suffer more commonly with upper respiratory tract ailments including tonsillitis and sinusitis. Differences in genetics and immunity have been forwarded as explanations for such differences; however, it is likely that a complex interplay of sex steroid hormones, host immunity, genetics, anatomical variation, and lung physiology, in addition to sociocultural and behavioral factors, influences the observed sex differences in respiratory infection. This chapter aims to assess the current state of the literature in this field and expound the range of its contributory factors.

Allergic asthma, driven by T helper 2 cell-mediated immune responses to common environmental antigens, remains the most common respiratory disease in children. Perfluorinated chemicals (PFCs) are environmental contaminants of great concern, because of their wide application, persistence in the environment, and bioaccumulation. PFCs associate with immunological disorders including asthma and attenuate immune responses to vaccines. The influence of PFCs on the immunological response to allergens during childhood is unknown. We report here that a major PFC, perfluorooctane sulfonate (PFOS), inactivates house dust mite (HDM) to dampen 5-wk-old, early weaned mice from developing HDM-induced allergic asthma. PFOS further attenuates the asthma protective effect of the microbial product lipopolysaccharide (LPS). We demonstrate that PFOS prevents desensitization of lung epithelia by LPS, thus abolishing the latter's protective effect. A close mechanistic study reveals that PFOS specifically binds the major HDM allergen Der p1 with high affinity as well as the lipid A moiety of LPS, leading to the inactivation of both antigens. Moreover, PFOS at physiological human (nanomolar) concentrations inactivates Der p1 from HDM and LPS in vitro, although higher doses did not cause further inactivation because of possible formation of PFOS aggregates. This PFOS-induced neutralization of LPS has been further validated in primary human cell models and extended to an in vivo bacterial infection mouse model. This study demonstrates that early life exposure of mice to a PFC blunts airway antigen bioactivity to modulate pulmonary inflammatory responses, which may adversely affect early pulmonary health. Ministry of Education (MOE) Ministry of Health (MOH) Nanyang Technological University National Environmental Agency (NEA) This work is supported by the Singapore Ministry of Education Academic Research Fund Tier 1 (04MNP000567C120), Nanyang Technological University Harvard School of Public Health Initiative for Sustainable Nanotechnology (M4082370.030), the Singapore National Environment Agency (M4061617), and Singapore Ministry of Health’s National Medical Research Council under its Clinician–Scientist Individual Research Grant (MOH-000141), the Open Fund Individual Research Grant (OFIRG/0076/2018), the Lee Kong Chian School of Medicine under the Dean’s Postdoctoral Fellowship, and the Wong Peng Onn Fellowship (002823-00001).

Graphical abstract, Traditionally, human microbiology has been strongly built on the laboratory focused culture of microbes isolated from human specimens in patients with acute or chronic infection. These approaches primarily view human disease through the lens of a single species and its relevant clinical setting however such approaches fail to account for the surrounding environment and wide microbial diversity that exists in vivo. Given the emergence of next generation sequencing technologies and advancing bioinformatic pipelines, researchers now have unprecedented capabilities to characterise the human microbiome in terms of its taxonomy, function, antibiotic resistance and even bacteriophages. Despite this, an analysis of microbial communities has largely been restricted to ordination, ecological measures, and discriminant taxa analysis. This is predominantly due to a lack of suitable computational tools to facilitate microbiome analytics. In this review, we first evaluate the key concerns related to the inherent structure of microbiome datasets which include its compositionality and batch effects. We describe the available and emerging analytical techniques including integrative analysis, machine learning, microbial association networks, topological data analysis (TDA) and mathematical modelling. We also present how these methods may translate to clinical settings including tools for implementation. Mathematical based analytics for microbiome analysis represents a promising avenue for clinical translation across a range of acute and chronic disease states.

Fungal infection in association with lung disease has emerged as a significant clinical problem. Owing to a ubiquitous environmental abundance, fungal spores, inhaled daily, can reach even the smallest airways. Although healthy individuals have effective immune mechanisms to clear this, individuals with anatomically abnormal airways and chronic respiratory disease (CRD) such as bronchiectasis are at higher risk of colonization and complications. Use of high-throughput sequencing has allowed insight into the pulmonary microbiome. This is well characterized for bacteria, in both healthy individuals and those with CRD; however, analysis of the fungal microbiome (the mycobiome) has lagged because of technical challenges. Despite the existence of fungi in healthy and diseased states, most published work to date has focused on CRD, in which judgments on fungal identity and burden may be confounded by use of inhaled corticosteroids and the underlying disease. This highlights a critical need to understand the airway mycobiome in healthy (nondiseased) individuals. Although the effect of aging on lung microbiomes remains to be established, our recent work illustrates that aging may potentially associate with specific microbes. Here, for the first time, we characterize the airway mycobiome in healthy subject pairs (first-degree relatives) of Asian descent. Nanyang Technological University This research is supported by the Ageing Research Institute for Society and Education (ARISE), Nanyang Technological University, Singapore [ARISE/2017/6; S.H.C].

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.

Volume 11, no. 5, e01774-20, 2020, [https://doi.org/10.1128/mBio.01774-20][1]. Professor Birgitta Henriques-Normark’s last name was missing a hyphen between Henriques and Normark in the original article. The byline should appear as shown above. Also, the affiliations and affiliation letters are

Background: A lack of study on the COPD mycobiome exists. Methods: We performed the largest multicenter evaluation of the COPD mycobiome including patients from Asia (Singapore and Malaysia) and the UK (Scotland) (n=380). Non-diseased controls (n=47) were assessed and longitudinal analyses performed during and following exacerbations (n=34). COPD mycobiomes were examined based on mortality and occurrence of serum specific-IgE against selected fungi. Results: A distinct mycobiome profile in COPD evidenced by increased α-diversity (p Conclusion: Frequent COPD exacerbators are characterized by Aspergillus, Penicillium and Curvularia and illustrate poorer survival and systemic specific-IgE responses against these fungi allowing COPD risk stratification based on airway mycobiomes. Funding: Singapore Ministry of Health’s National Medical Research Council under its Research Training Fellowship (NMRC/Fellowship/0049/2017) (P.Y.T) and Clinician-Scientist Individual Research Grant (MOH-000141) (S.H.C). The TARDIS study is funded by Glaxosmithkline & British Lung Foundation (Fellowship to JDC).

Background: To date, no study has integrated bacterial, viral and fungal microbiomes to provide a holistic assessment of the true in-vivo state. Methods: We report the first integrated ‘multi-biome’ analysis integrating bacteriome, virome and mycobiomes in bronchiectasis (n=217) employing weighted Similarity Network Fusion coupled to co-occurrence network analysis to evaluate the ‘interactome’. Interactome dynamics at baseline, during exacerbations and following recovery was assessed in n=17 patients with bronchiectasis. Results: “Integrative microbiomics” identifies patients at high-risk of exacerbation. Frequent exacerbators exhibit reduced α-diversity and elevated antagonistic interactions. Assessment of the Pseudomonas-interactome reveals that its network rather than abundance is key in determining exacerbation risk. Longitudinal ‘interactome’ analysis reveals increased antagonistic interactions during exacerbations which resolve following treatment within an otherwise stable microbiome. The interactome has ‘core’ and ‘ancillary’ components, the latter altered during exacerbation. Conclusion: “Integrative microbiomics” captures interactions determining exacerbation risk otherwise unaccounted by specific microbes. Antibiotic strategies likely target interaction networks rather than individual microbes providing fresh approaches to understanding respiratory infection. Funding: Singapore Ministry of Health’s National Medical Research Council under its Transition Award (NMRC/TA/0048/2016) (S.H.C); Clinician-Scientist Individual Research Grant (MOH-000141) (S.H.C) and the NTU Integrated Medical, Biological and Environmental Life Sciences (NIMBELS) [NIM/03/2018] (S.H.C).

Background: Macrolide antibiotics are increasingly used in frequent exacerbators with chronic respiratory disease (CRD) however the role of the airway resistome and its relationship to the host microbiome remains unknown. Methods: We derived airway microbiomes and corresponding resistomes using deep sequencing metagenomic approaches from airway specimens of individuals with and without CRD (severe asthma, COPD and bronchiectasis) (n=85). Gene-Microbe association networks were built using compositionality corrected general boosted linear models to determine possible microbial sources of the airway resistome. Results: Antibiotic resistance profiles exhibit variability across healthy and diseased states with greatest resistance observed in COPD and bronchiectasis. A ‘core’ airway resistome dominated by macrolide resistance but with high prevalence of β-lactam, fluoroquinolone and tetracycline resistance genes exists, and, is independent of disease status or prior antibiotic exposure. Dysbiosis of the respiratory microbiome is evident across CRDs and gene-microbe association networks of the ‘core’ resistome revealed Streptococcus and Actinomyces as potential microbial reservoirs of macrolide resistance including the ermX, ermF and msrD genes. Conclusion: Functional metagenomics of the airway reveals a core macrolide resistome harboured by the host microbiome with clinical implications for macrolide use across CRD. Funding: Singapore Ministry of Health’s National Medical Research Council under its Transition Award (NMRC/TA/0048/2016) (S.H.C); Clinician-Scientist Individual Research Grant (MOH-000141) (S.H.C) and the NTU Integrated Medical, Biological and Environmental Life Sciences (NIMBELS) [NIM/03/2018] (S.H.C).

Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection., Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.

Highlights • Platypnea-orthodeoxia syndrome (POS) is observed in COVID-19 acute respiratory distress syndrome (ARDS) survivors. • POS is associated with older age, lower body mass index and varying degrees of dyspnea. • Arterial to end-tidal carbon dioxide and alveolar to arterial oxygen partial pressure differences were persistently elevated. • POS is likely a gravitational exacerbation of intrapulmonary shunt in ARDS due to COVID-19 specific changes. • POS may cause alarm and requires adjustment in the rehabilitation approach during the recovery period., Platypnea-orthodeoxia syndrome (POS) is a rare clinical syndrome characterized by orthostatic oxygen desaturation and positional dyspnea from supine to an upright position. We observed POS in 5 of 20 cases of severe 2019 novel coronavirus (COVID-19) pneumonia, which demonstrated persistently elevated shunt fraction even after liberation from mechanical ventilation. POS was first observed during physiotherapy sessions; median oxygen desaturation was 8 % (range: 8–12 %). Affected individuals were older (median 64 vs 53 years old, p = 0.05) and had lower body mass index (median 24.7 vs 27.6 kg/m2, p = 0.03) compared to those without POS. While POS caused alarm and reduced tolerance to therapy, this phenomenon resolved over a median of 17 days with improvement of parenchymal disease. The mechanisms of POS are likely due to gravitational redistribution of pulmonary blood flow resulting in increased basal physiological shunting and upper zone dead space ventilation due to the predominantly basal distribution of consolidative change and reported vasculoplegia and microthrombi in severe COVID-19 disease.

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.

BACKGROUND: COPD is a heterogeneous disease demonstrating inter-individual variation. A high COPD prevalence in Chinese populations is described, but little is known about disease clusters and prognostic outcomes in the Chinese population across Southeast Asia. We aim to determine if clusters of Chinese patients with COPD exist and their association with systemic inflammation and clinical outcomes.RESEARCH QUESTION: We aim to determine if clusters of Chinese patients with COPD exist and their association with clinical outcomes and inflammation.STUDY DESIGN AND METHODS: Chinese patients with stable COPD were prospectively recruited into two cohorts (derivation and validation) from six hospitals across three Southeast Asian countries (Singapore, Malaysia, and Hong Kong; n = 1,480). Each patient was followed more than 2 years. Clinical data (including co-morbidities) were employed in unsupervised hierarchical clustering (followed by validation) to determine the existence of patient clusters and their prognostic outcome. Accompanying systemic cytokine assessments were performed in a subset (n = 336) of patients with COPD to determine if inflammatory patterns and associated networks characterized the derived clusters.RESULTS: Five patient clusters were identified including: (1) ex-TB, (2) diabetic, (3) low comorbidity: low-risk, (4) low comorbidity: high-risk, and (5) cardiovascular. The cardiovascular and ex-TB clusters demonstrate highest mortality (independent of Global Initiative for Chronic Obstructive Lung Disease assessment) and illustrate diverse cytokine patterns with complex inflammatory networks.INTERPRETATION: We describe clusters of Chinese patients with COPD, two of which represent high-risk clusters. The cardiovascular and ex-TB patient clusters exhibit high mortality, significant inflammation, and complex cytokine networks. Clinical and inflammatory risk stratification of Chinese patients with COPD should be considered for targeted intervention to improve disease outcomes.

Laser printers emit high levels of nanoparticles (PM0.1) during operation. Although it is well established that toners contain multiple engineered nanomaterials (ENMs), little is known about inhalation exposures to these nanoparticles and work practices in printing centers. In this report, we present a comprehensive inhalation exposure assessment of indoor microenvironments at six commercial printing centers in Singapore, the first such assessment outside of the United States, using real-time personal and stationary monitors, time-integrated instrumentation, and multiple analytical methods. Extensive presence of ENMs, including titanium dioxide, iron oxide, and silica, was detected in toners and in airborne particles collected from all six centers studied. We document high transient exposures to emitted nanoparticles (peaks of ∼500 000 particles/cm3, lung-deposited surface area of up to 220 μm2/cm3, and PM0.1 up to 16 μg/m3) with complex PM0.1 chemistry that included 40-60 wt % organic carbon, 10-15 wt % elemental carbon, and 14 wt % trace elements. We also record 271.6-474.9 pmol/mg of Environmental Protection Agency-priority polycyclic aromatic hydrocarbons. These findings highlight the potentially high occupational inhalation exposures to nanoparticles with complex compositions resulting from widespread usage of nano-enabled toners in the printing industry, as well as inadequate ENM-specific exposure control measures in these settings. Nanyang Technological University Accepted version Nanyang Technological University—Harvard School of Public Health Initiative for Sustainable Nanotechnology (NTU‐Harvard SusNano; NTU-HSPH-17001)

An increased fungal presence in the cystic fibrosis (CF) airway is associated with immune and clinical consequences including sensitisation to fungal allergens, reduced lung function and allergic bronchopulmonary aspergillosis (ABPA). Key drivers of fungal colonisation in CF remain unclear and are related to impaired mucociliary clearance, aberrant innate immunity and a skewed Th2 airway hyperresponsiveness. The commonest fungal pathogen, Aspergillus fumigatus, represents the predominant species that chronically colonises the CF lung, perpetuating a spectrum of varied disease states that each pose scientific, diagnostic and therapeutic challenges to CF scientists and clinicians. Recent progress in this field has explored the emergence of other fungal genera including Candida, Scedosporium, Exophiala, Rasamsonia and Malassezia, in the setting of CF, while culture-independent studies including next-generation sequencing provide a new perspective on the diversity, complexity and resistance profile of the CF mycobiome. System-based analyses of microbial-host interaction have further uncovered novel paradigms in CF pathogenesis including inter-kingdom signalling and the gut-lung axis, where fungi play an emerging and critical role. Current treatment modalities against CF-associated fungal pathogens aim at targeting host inflammation (with steroids) and fungal presence (with antifungals), and the approaches are accompanied by a weak evidence base coupled to a dearth of randomised controlled clinical trials proving efficacy. More specifically, emerging immunotherapeutic approaches targeting Th2-mediated hyperresponsiveness, multi-omics technologies and fungal sequencing platforms represent promising areas for future work that require strong and sustained investment to improve our understanding of the complex host-microbe-immunological interaction that underpins CF-associated fungal diseases.

Pseudomonas aeruginosa is a major pathogen responsible for both acute and chronic infection. Known as a colonising pathogen of the cystic fibrosis (CF) lung, it is implicated in other settings such as bronchiectasis. It has the ability to cause acute disseminated or localised infection particularly in the immunocompromised. Human hormones have been highlighted as potential regulators of bacterial virulence through crosstalk between analogous "quorum sensing" (QS) systems present in the bacteria that respond to mammalian hormones. Pseudomonas aeruginosa is known to utilise interconnected QS systems to coordinate its virulence and evade various aspects of the host immune system activated in response to infection. Several human hormones demonstrate an influence on P. aeruginosa growth and virulence. This inter-kingdom signalling, termed "microbial endocrinology" has important implications for host-microbe interaction during infection and, potentially opens up novel avenues for therapeutic intervention. This phenomenon, supported by the existence of sexual dichotomies in both microbial infection and chronic lung diseases such as CF is potentially explained by sex hormones and their influence on the infective process. This review summarises our current understanding of the microbial endocrinology of P. aeruginosa, including its endogenous QS systems and their intersection with human endocrinology, pathogenesis of infection and the host immune system.

Advances in platform technologies facilitate the design of largescale “multi-omic” studies that encompass genomic, transcriptomic, proteomic, epigenomic, metabolomic and microbiomic components, each representing different views of a single biological specimen [1]. While useful, this is analogous to the ‘Flatland’ jeu d'esprit where the same reality (i.e., a sphere of constant diameter) is subject to different interpretations (i.e., circles of varying diameter) depending on one’s point of view (from various 2-D cross sections). Although each -omics approach has value, they can be even more useful if holistically modelled through appropriate integration. While ‘mono-omic’ analysis has been extremely beneficial, from a systems medicine perspective, this may fail to capture the emergent properties of an individual system and hence may yield limited understanding of non-linear and dynamic features, all of which are increasingly evident in the pathogenesis of respiratory disease [1]. There is clearly a growing need for a more holistic ‘all in’ integration methodology that leverages each distinct -omic dataset derived from multi-omic studies (Figure 1). Although several integrative methodologies are available (e.g. mixOmics, Anvi'o and integrOmics), Similarity Network Fusion (SNF) has emerged as an appropriate, applicable, and robust method in respiratory disease [2-4]. Ministry of Health (MOH) Nanyang Technological University This research is supported by the Singapore Ministry of Health’s National Medical Research Council under its Clinician-Scientist Individual Research Grant (MOH-000141) (S.H.C) and the NTU Integrated Medical, Biological and Environmental Life Sciences (NIMBELS) [NIM/03/2018] (S.H.C). KTA gratefully acknowledges the financial support of the EPSRC via grant EP/T017856/1.

Background Severe asthma is a largely heterogeneous disease with varying phenotypic profiles. The relationship between specific allergen sensitization and asthma severity, particularly in Asia, remains unclear. We aim to study the prevalence of specific allergen sensitization patterns and investigate their association with outcomes in a severe asthma cohort in an Asian setting. Methods We conducted a cross-sectional study of patients receiving step 4 or 5 Global Initiative for Asthma treatment. Univariate and multivariate analyses were performed to assess the association between sensitization to a specific identifiable allergen by skin prick test (SPT) and uncontrolled asthma (defined in our study as the use of ≥2 steroid bursts or hospitalization in the past year, a history of near-fatal asthma or evidence of airflow obstruction on spirometry). Results Two hundred and six severe asthma patients (mean age 45±17 years, 99 [48.1%] male) were evaluated. Of them, 78.2% had a positive SPT to one or more allergens. The most common allergen to which patients were sensitized was house dust mites (Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae). Also, 11.7% were sensitized to Aspergillus species. On multivariate analysis, Aspergillus sensitization was associated with uncontrolled asthma (odds ratio 6.07, 95% confidence interval 1.80-20.51). In particular, Aspergillus sensitization was independently associated with the use of ≥2 steroid bursts in the past year (odds ratio 3.05, 95% confidence interval 1.04-8.95). No similar associations of uncontrolled asthma with sensitization to any other allergens were found. Conclusion High allergen, specifically Aspergillus sensitization was observed in the Asian population with severe asthma by SPT. Aspergillus sensitization was specifically associated with frequent exacerbations and a greater corticosteroid requirement. An improved understanding of the severe asthma with Aspergillus sensitization phenotype is warranted, which is likely a subgroup of severe asthma with fungal sensitization.

COPD is a major global concern, increasingly so in the context of ageing populations. The role of infections in disease pathogenesis and progression is known to be important, yet the mechanisms involved remain to be fully elucidated. While COPD pathogens such as Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are strongly associated with acute exacerbations of COPD (AECOPD), the clinical relevance of these pathogens in stable COPD patients remains unclear. Immune responses in stable and colonized COPD patients are comparable to those detected in AECOPD, supporting a role for chronic colonization in COPD pathogenesis through perpetuation of deleterious immune responses. Advances in molecular diagnostics and metagenomics now allow the assessment of microbe–COPD interactions with unprecedented personalization and precision, revealing changes in microbiota associated with the COPD disease state. As microbial changes associated with AECOPD, disease severity and therapeutic intervention become apparent, a renewed focus has been placed on the microbiology of COPD and the characterization of the lung microbiome in both its acute and chronic states. Characterization of bacterial, viral and fungal microbiota as part of the lung microbiome has the potential to reveal previously unrecognized prognostic markers of COPD that predict disease outcome or infection susceptibility. Addressing such knowledge gaps will ultimately lead to a more complete understanding of the microbe–host interplay in COPD. This will permit clearer distinctions between acute and chronic infections and more granular patient stratification that will enable better management of these features and of COPD.

Introduction: Chronic obstructive pulmonary disease accounts for a large burden of lung disease. It can 'overlap' with other respiratory diseases including bronchiectasis, fibrosis and obstructive sleep apnea (OSA). While COPD alone confers morbidity and mortality, common features with contrasting clinical outcomes can occur in COPD 'overlap syndromes'. Areas covered: Given the large degree of heterogeneity in COPD, individual variation to treatment is adopted based on its observed phenotype, which in turn overlaps with features of other respiratory disease states such as asthma. This is coined asthma-COPD overlap syndrome ('ACOS'). Other examples of such overlapping clinical states include bronchiectasis-COPD ('BCOS'), fibrosis-COPD ('FCOS') and OSA-COPD ('OCOS'). The objective of this review is to highlight similarities and differences between the COPD-overlap syndromes in terms of risk factors, pathophysiology, diagnosis and potential treatment differences. Expert commentary: As a consequence of COPD overlap syndromes, a transition from the traditional 'one size fits all' treatment approach is necessary. Greater treatment stratification according to clinical phenotype using a precision medicine approach is now required. In this light, it is important to recognize and differentiate COPD overlap syndromes as distinct disease states compared to individual diseases such as asthma, COPD, fibrosis or bronchiectasis. Accepted version

There is currently enormous interest in studying the role of the microbiome in health and disease. Microbiome's role is increasingly being applied to respiratory diseases, in particular COPD, asthma, cystic fibrosis and bronchiectasis. The changes in respiratory microbiomes that occur in these diseases and how they are modified by environmental challenges such as cigarette smoke, air pollution and infection are being elucidated. There is also emerging evidence that gut microbiomes play a role in lung diseases through the modulation of systemic immune responses and can be modified by diet and antibiotic treatment. There are issues that are particular to the Asia-Pacific region involving diet and prevalence of specific respiratory diseases. Each of these issues is further complicated by the effects of ageing. The challenges now are to elucidate the cause and effect relationships between changes in microbiomes and respiratory diseases and how to translate these into new treatments and clinical care. Here we review the current understanding and progression in these areas.