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Fungal disease is an increasingly recognised global clinical challenge associated with high mortality. Early diagnosis of fungal infection remains problematic due to the poor sensitivity and specificity of current diagnostic modalities. Advances in sequencing technologies hold promise in addressing these shortcomings and for improved fungal detection and identification. To translate such emerging approaches into mainstream clinical care will require refinement of current sequencing and analytical platforms, ensuring standardisation and consistency through robust clinical benchmarking and its validation across a range of patient populations. In this stateof-the-art review, we discuss current diagnostic and therapeutic challenges associated with fungal disease and provide key examples where the application of sequencing technologies has potential diagnostic application in assessing the human 'mycobiome'. We assess how ready access to fungal sequencing may be exploited in broadening our insight into hostfungal interaction, providing scope for clinical diagnostics and the translation of emerging mycobiome research into clinical practice.

COPD (11, 12). RARB is expressed in human lung tissue, bronchial epithelial cells, and airway smooth muscle cells (10), and knockout of this gene could lead to premature alveolar septation in mice (13), suggesting its fundamental function in lung development, normal physiology, and capacity. In this study, we observed the positive association between PAH exposure and hypermethylation in RARB promoter; a putative effect may exist on decreasing expression of RARB caused by PAHs. There was also a suggestive association of rs1529672-A allele with reduced RARB expression. Considering the important biological roles of RARB in lung development, we speculated that the decreased expression of RARB may act as the common target for PAH exposure and rs1529672, suggesting a possible mechanism underlying the statistical gene-environment interaction. Strengths of this study were the prospective design and results validation by two independent cohorts of populations with a wide range of PAH exposure. The limitations of this study included not investigating the SNPs reported after March 2017, along with variants in genes involved in PAH metabolism and related biological pathways. Future longitudinal studies in other ethnic populations and the well-designed functional studies are warranted to validate these associations and elucidate putative mechanisms for the PAH-RARB interaction in lung function impairment. In conclusion, the GWAS reported RARB rs1529672 may modify the effect of PAH exposure on annual FEV 1 /FVC decline. The results emphasize the urgency of reducing environmental PAH levels and the importance of lung function monitoring in the routine physical examination, especially for high-risk populations. n Author disclosures are available with the text of this letter at www.atsjournals.org. Acknowledgment: The authors thank all participants in this study as well as all volunteers for collecting the samples and questionnaires.

Our development and usage of engineered nanomaterials has grown exponentially despite concerns about their unfavourable cardiorespiratory consequence, one that parallels ambient ultrafine particle exposure from vehicle emissions. Most research in the field has so far focused on airway inflammation in response to nanoparticle inhalation, however, little is known about nanoparticle-microbiome interaction in the human airway and the environment. Emerging evidence illustrates that the airway, even in its healthy state, is not sterile. The resident human airway microbiome is further altered in chronic inflammatory respiratory disease however little is known about the impact of nanoparticle inhalation on this airway microbiome. The composition of the airway microbiome, which is involved in the development and progression of respiratory disease is dynamic, adding further complexity to understanding microbiota-host interaction in the lung, particularly in the context of nanoparticle exposure. This article reviews the size-dependent properties of nanomaterials, their body deposition after inhalation and factors that influence their fate. We evaluate what is currently known about nanoparticle-microbiome interactions in the human airway and summarise the known clinical, immunological and toxicological consequences of this relationship. While associations between inhaled ambient ultrafine particles and host immune-inflammatory response are known, the airway and environmental microbiomes likely act as intermediaries and facilitate individual susceptibility to inhaled nanoparticles and toxicants. Characterising the precise interaction between the environment and airway microbiomes, inhaled nanoparticles and the host immune system is therefore critical and will provide insight into mechanisms promoting nanoparticle induced airway damage.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.