Residential exposure to Aspergillus spp. is associated with exacerbations in COPD

Abstract

Background

Sensitisation to Aspergillus fumigatus is linked to worse outcomes in patients with COPD; however, its prevalence and clinical implications in domestic (residential) settings remains unknown.

Methods

Individuals with COPD (n=43) recruited in Singapore had their residences prospectively sampled and assessed by shotgun metagenomic sequencing including indoor air, outdoor air and touch surfaces (a total of 126 specimens). The abundance of environmental A. fumigatus and the occurrence of A. fumigatus (Asp f) allergens in the environment were determined and immunological responses to A. fumigatus allergens determined in association with clinical outcomes including exacerbation frequency. Findings were validated in 12 individuals (31 specimens) with COPD in Vancouver, Canada, a climatically different region.

Results

157 metagenomes from 43 homes were assessed. 11 and nine separate Aspergillus spp. were identified in Singapore and Vancouver, respectively. Despite climatic, temperature and humidity variation, A. fumigatus was detectable in the environment from both locations. The relative abundance of environmental A. fumigatus was significantly associated with exacerbation frequency in both Singapore (r=0.27, p=0.003) and Vancouver (r=0.49, p=0.01) and individuals with higher Asp f 3 sensitisation responses lived in homes with a greater abundance of environmental Asp f 3 allergens (p=0.037). Patients exposed and sensitised to Asp f 3 allergens demonstrated a higher rate of COPD exacerbations at 1-year follow-up (p=0.021).

Conclusion

Environmental A. fumigatus exposure in the home environment including air and surfaces with resulting sensitisation carries pathogenic potential in individuals with COPD. Targeting domestic A. fumigatus abundance may reduce COPD exacerbations.

Shareable abstract

High abundance of inhalable Aspergillus in the indoor home environment associates with COPD exacerbations https://bit.ly/48UmZol

Introduction

Aspergillus is a ubiquitous filamentous fungal pathogen associated with negative clinical consequences in individuals with chronic respiratory diseases including asthma, COPD, bronchiectasis and cystic fibrosis [1–5]. A. fumigatus possesses a myriad pathogenic characteristics and virulence factors including thermotolerance, hypoxic adaptation and potent secondary metabolites and efflux pumps that allow persistence and survival, even in the harshest environmental conditions [6, 7].

Important recent work from our group and others demonstrates the relevance of host mycobiomes, sensitisation responses to Aspergillus spp. in the environment, and the relevance of crude versus specific allergens in COPD [3, 4, 8]. Emerging evidence clearly links clinical Aspergillus signatures, disease progression, heightened severity and greater mortality in COPD [3, 4, 8–10]. Notably, sensitisation to Aspergillus-related allergens is associated with increased exacerbation frequency, poorer lung function and worse overall prognosis in COPD, while allergic bronchopulmonary aspergillosis in bronchiectasis–COPD overlap confers greater disease severity and poorer clinical outcomes [3, 8, 9]. A high-risk mycobiome profile characterised by an increased abundance of Aspergillus is related to sensitisation and higher mortality in COPD [4]. However, contrasting results have been reported, with some studies illustrating no relationship between Aspergillus sensitisation and lung function decline, attributed to the differing study cohorts and geographic locations [10, 11].

While Aspergillus spp. appear to have negative impacts on individuals with COPD, little is known about their abundance within the immediate domestic (residential) environment and whether any such direct exposure has effects on clinical outcomes. Here, in this two-country multicentre study, we sought to evaluate the presence of environmental Aspergillus in outdoor air (balcony), indoor air (bedroom) and across surfaces (fans and air-conditioning filters) in the homes of individuals with COPD, using a state-of-the-art metagenomics approach, and relate these data to important clinical outcomes in COPD.

Methods

Patient recruitment

Individuals aged ≥40 years with stable COPD and attending specialist respiratory outpatient clinics at four participating tertiary hospitals across two countries were included: Singapore General Hospital, Tan Tock Seng Hospital and Changi General Hospital (Singapore) and St Paul's Hospital (Vancouver, Canada). Participants were recruited prospectively between 2018 and 2020, with COPD being defined according to the 2018 Global Initiative for Chronic Obstructive Lung Disease guidelines [12]. All participants provided their smoking history, COPD Assessment Test (CAT) scores, exacerbation history and COPD treatments. Stable COPD was defined as remaining exacerbation-free in the 4 weeks preceding study recruitment [13]. Exacerbations were defined as a worsening of underlying symptoms, requiring systemic corticosteroids and/or antibiotics and/or hospitalisation [12]. Frequent exacerbators were defined as having two or more moderate exacerbations and/or one or more hospitalised exacerbations annually [12]. Patients with active mycobacterial infection, those on oral corticosteroids 4 weeks preceding the recruitment, receiving immunosuppression of any kind and/or antifungal therapy were excluded. Patients recruited from Singapore were prospectively followed for a year following study sampling and exacerbation frequency recorded.

Ethics

This study was approved by the institutional review boards of all participating hospitals under CIRB 2017/2109 and NTU IRB-2017-05-035 (Singapore) and Vancouver (H18-01836). Written informed consent was obtained from all participants.

Environmental sampling

Home visits were scheduled for each participant within 1 month of clinical recruitment. Home sampling was performed between January 2018 and February 2020 in Singapore and between September 2019 and March 2020 in Vancouver, Canada. During these visits, filter-based SASS3100 air samplers (Research International, Monroe, WA, USA) were placed in participant bedrooms and balconies (or other outdoor air sources adjacent to the home) to sample indoor and outdoor air sources, respectively. Samplers were set to run for eight consecutive hours (overnight) at a flow rate of 100 L·min⁻¹ according to previously described protocols for metagenomic assessment of indoor and outdoor air [14, 15]. Surface swabs of fans and air-conditioning filters were performed using 4N6Floq (Copan, Murrieta, CA, USA) swabs pre-moistened with 0.1% PBS-Triton-X100 [16–18]. Biomass was removed from filters by washing in 0.1% PBS-Triton-X100 with sonication at room temperature for 1 min, after which samples were concentrated using a 0.02 µm Anodisc filter with vacuum manifold (Whatman, Little Chalfont, UK) [15].

DNA extraction and shotgun metagenomic sequencing

To ensure consistency, all library preparation and metagenomic sequencing were performed at a single site (Nanyang Technological University, Singapore). The DNeasy PowerWater kit (Qiagen, Germantown, MD, USA) was employed for DNA extraction from concentrated Anodisc filters in parallel with filter and reagent blanks [14, 15]. Library preparation and sequencing was performed using established low-biomass sequencing protocols optimised as described previously on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) [8, 14, 15]. Filter, reagent and sequencing blanks versus samples are illustrated in supplementary figure S1. Sequencing depth and read counts are illustrated in supplementary figure S2.

Blood sampling and immunological assays

Plasma samples from all participants in Singapore were used to measure the specific immunoglobulin-E response to recombinant Aspergillus fumigatus (rAsp f) allergens: rAsp f 1, 3, 5 and 6 using immune-dot-blot assays, as described previously [8].

Additional methods on recording temperature, humidity, particulate matter with aerodynamic diameter <10 µm (PM₁₀) and <2.5 μm (PM_2.5), metagenomic sequencing and data processing, immunological assays, sputum sampling and targeted internal transcribed spacer (ITS) sequencing are provided in the supplementary methods.

Statistical analysis

Data distribution was evaluated by the Shapiro–Wilk test and presented as median (interquartile range (IQR)) for non-normally distributed data. Comparisons between groups was performed with a Mann–Whitney U-test or Kruskal–Wallis test for continuous variables and Chi-squared or Fisher-exact tests, as appropriate, for categorical variables. Comparisons between multiple groups was corrected for false discovery rate using the Benjamini–Hochberg correction. The risk ratio for exacerbation analyses was adjusted for age, body mass index (BMI), smoking status and lung function (as forced expiratory volume in 1 s (FEV₁) % predicted). p<0.05 was considered statistically significant. The relative abundance of A. fumigatus was calculated using the total fungal read count as the denominator. All statistical analyses were performed using R (version 4.2.2; R Foundation for Statistical Computing, Vienna, Austria). A repository with all processed data and codes to reproduce the results in this manuscript is accessible at the following site: https://github.com/Jereu12/Asp/blob/main/Code/Figures%20R%20script.

Data availability

All the sequencing data are available at the National Center for Biotechnology Information sequence read archives under project accession numbers PRJNA609892, PRJNA608611 and PRJNA1100783.

Results

Home visits complete with shotgun metagenomic sequencing of indoor air (n=55), outdoor air (n=55) and swabs of surfaces including air-conditioner filters and/or fans (n=47) were performed at residences of individuals with COPD (n=55) in two countries with contrasting geography and climate: Singapore (n=43) and Canada (Vancouver) (n=12). A summary of patient demographics and clinical characteristics is provided in table 1.

TABLE 1.

Demographic and clinical data summarising the study population of individuals with COPD recruited from Singapore and Vancouver, Canada

	Overall	Singapore	Vancouver	p-value
Participants	55	43	12
Age years	72.0 (67.0–75.0)	72.0 (68.5–75.5)	70.0 (65.0–72.0)	ns
Sex				0.024
Female	2 (3.6)	0 (0)	2 (16.7)
Male	53 (96.4)	43 (100)	10 (83.3)
BMI kg·m−2	23.6 (20.2–27.4)	22.7 (19.1–26.7)	28.2 (23.0–31.9)	0.021
Smoking status				ns
Current	21 (38.2)	18 (41.9)	3 (25.0)
Never-smoker	1 (1.8)	1 (2.3)	0 (0.0)
Ex-smoker	33 (60.0)	24 (55.8)	9 (75.0)
Smoking pack-years	50.0 (32.0–60.0)	50.0 (36.0–60.0)	39.5 (17.6–65.0)	ns
CAT score	10.0 (7.0–18.0)	10.0 (5.0–18.0)	12.5 (7.8–20.8)	ns
FEV1 % predicted	59.0 (46.5–73.5)	53.0 (41.5–66.0)	83.0 (63.8–85.8)	0.001
FEV1/FVC %	56.0 (46.4–63.0)	56.0 (44.5–64.0)	59.3 (54.6–61.6)	ns
Frequent exacerbators (≥2 moderate or ≥1 hospitalisation) in the past year	17 (30.9)	14 (32.6)	3 (25.0)	ns
Treatment				0.004
LAMA	3 (5.5)	2 (4.7)	1 (8.3)
LABA/LAMA	23 (41.8)	22 (51.1)	1 (8.3)
LABA/ICS	3 (5.5)	1 (2.3)	2 (16.7)
LABA/LAMA/ICS	24 (43.6)	18 (41.9)	6 (50.0)
Indoor temperature °C	28.4 (24.8–30.0)	29.4 (27.8–30.1)	21.7 (20.5–22.7)	<0.001
Indoor relative humidity %	72.5 (55.2–75.8)	73.8 (70.7–77.1)	41.7 (37.2–47.2)	<0.001
Home type				<0.001
Landed	5 (9.1)	0 (0)	5 (41.7)
Apartment/townhouse	50 (90.1)	43 (100)	7 (58.3)

Data are presented as n, median (interquartile range) or n (%), unless otherwise stated. BMI: body mass index; CAT: COPD Assessment Test; FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity; LAMA: long-acting muscarinic antagonist; LABA: long-acting β-agonist; ICS: inhaled corticosteroid; ns: nonsignificant.

A. fumigatus predominates in the Singaporean home environment

11 different Aspergillus spp. were identified in the home environment in Singapore. Notably, A. fumigatus was detected in all three types of environmental samples: indoor and outdoor air and surfaces (figure 1). Conversely, the remaining Aspergillus spp. were predominantly isolated from indoor air and surfaces. A. steynii, A. glaucus and A. nidulans were present in the indoor air and all 11 Aspergillus spp. were detected on the residential surfaces (figure 1). Having identified the high occurrence of A. fumigatus within the home environment, we next evaluated its association with other environmental parameters. The relative abundance of environmental A. fumigatus positively correlated with several indoor environmental measures including temperature (r=0.2, p=0.029) and particulate matter: PM_2.5 (r=0.32, p<0.001) and PM₁₀ (r=0.32, p<0.001), but importantly not with relative humidity (r=−0.14, p=0.13) (figure 2a–d).

FIGURE 1.

Diversity of Aspergillus spp. detected in the home environment of patients with COPD. UpSet plot illustrating the frequency of Aspergillus spp. isolated in outdoor (balcony) air, indoor (bedroom) air and surfaces including air-conditioner filters and/or fan swabs. Bar charts corresponding to the number of homes with detectable Aspergillus spp. in the outdoor air, indoor air and on surfaces.

FIGURE 2.

Environmental detection of Aspergillus fumigatus correlates with increased temperature and particulate matter. Scatterplots illustrating correlations between the relative abundance of detectable A. fumigatus within the home environment and a) indoor temperature, b) particulate matter with aerodynamic diameter <2.5 µm (PM_2.5), c) particulate matter with aerodynamic diameter <10 µm (PM₁₀) and d) relative humidity. Black dotted lines correspond to Spearman's regression and grey shaded areas represent 95% confidence intervals. ns: nonsignificant.

Environmental A. fumigatus is significantly associated with exacerbation risk in COPD

We evaluated the relationship between environmental detection of A. fumigatus in the home environment and key clinical COPD outcomes. By assessing exacerbation frequency at baseline (in the year preceding recruitment to the study) and at 1-year follow-up, we detected a significantly positive correlation between relative abundance of environmental A. fumigatus and exacerbation frequency at baseline (r=0.27, p=0.003; figure 3a) with a trend towards significance at 1-year follow-up (r=0.14, p=0.19, figure 3b). This relationship was most evident in homes with higher A. fumigatus abundance (i.e. ≥1% within the respective environmental sample) with significantly higher exacerbation frequency at both baseline (median 2, IQR 0–4; p=0.023) and at 1-year follow-up (median 1, IQR 1–2; p=0.003) relative to homes with moderate A. fumigatus abundance (<1% within the respective environmental sample) (figure 3c and d). The relationship between environmental A. fumigatus abundance and exacerbation frequency remains statistically significant following multivariate analysis adjusted for age, BMI, smoking status, and FEV₁ % pred for both baseline (risk ratio 4.1, 95% CI 1.5–11.0; p<0.001) and 1-year follow-up exacerbations (risk ratio 4.83, 95% CI 1.70–14.0; p<0.001), which further adjusted for baseline exacerbator status (figure 3e and f). Importantly, no association between environmental A. fumigatus and either FEV₁ % pred or CAT scores was detected (supplementary figure S3). While A. fumigatus positively correlated with indoor temperature, PM_2.5 and PM₁₀, these parameters individually showed no significant correlation with exacerbation frequency (supplementary figure S4), demonstrating that the association between environmental A. fumigatus and exacerbations was independent of these environmental parameters.

FIGURE 3.

Environmental Aspergillus fumigatus associates with increased exacerbation frequency in individuals with COPD. Correlations between the relative abundance of A. fumigatus detected in environmental samples (air, surfaces) and number of exacerbations a) in the year preceding study recruitment and b) at 1-year follow-up. Black dotted lines correspond to Spearman's regression and grey shaded areas represent 95% confidence intervals. Scatter boxplots illustrating number of exacerbations in c) the year preceding study recruitment and d) at 1-year follow-up in homes with moderate (i.e. <1% relative abundance) and high (≥1% relative abundance) of A. fumigatus in environmental samples (air, surfaces). Forest plots for exacerbation risk ratio in e) the year preceding study recruitment and f) at 1-year follow-up adjusted for age, body mass index (BMI), smoking status, lung function (forced expiratory volume in 1 s (FEV₁) % predicted) and baseline exacerbation rate (between patients with moderate and high exposure to environmental A. fumigatus). Error bars indicate 95% confidence intervals with red dots denoting significance and grey dots representing nonsignificance. FE: frequent exacerbator status at baseline. *: p<0.05, **: p<0.01.

Having detected a significant association between levels of environmental Aspergillus and COPD exacerbation frequency, we next explored host mycobiome–environment relationships by performing targeted amplicon ITS sequencing using sputum prospectively obtained during home visits in a subset of patents (n=20). Sputum mycobiome analysis revealed no significant associations between sputum and environmental Aspergillus, suggesting that any effect on exacerbation frequency was not a consequence of increased sputum Aspergillus in our patients (supplementary figure S5).

A. fumigatus allergens are present in the home environment and associated with exacerbation frequency

Prior studies have shown associations between A. fumigatus sensitisation, particularly to specific A. fumigatus allergens: Asp f 1, 3, 5 and 6 with COPD exacerbations [3, 8]; therefore, we next sought to assess whether such allergens were present in the home environment and any association their presence may have with exposure and sensitisation in our COPD cohort. To address this, metagenomic reads (abundance) from indoor and outdoor air and surfaces were aligned to the World Health Organization/International Union of Immunological Societies allergen nomenclature. To date, 23 Asp f allergens have been identified, and all were detectable in the home environment of patients with COPD at varying frequency (figure 4a). All Asp f allergens were detected on surfaces, with higher abundance of Asp f 3 (p<0.001), 22 (p=0.0116), 27 (p=0.0135) and 34 (p=0.007) compared to indoor and outdoor air. Asp f 1,7, 15 and 17 were absent in the indoor air and Asp f 29 was not detected in outdoor air. Next, we evaluated the potential for direct relationships between A. fumigatus environmental exposure and host sensitisation responses by screening plasma from our cohort for Asp f 1, 3, 5 and 6, all established responses linked to exacerbations [3]. Patients demonstrating significant sensitisation responses to Asp f 3 were found to have an increased relative abundance of Asp f 3 allergen within their home environment, conferring higher exposure relative to non-sensitised patients (median 1.3%, IQR 1.0–1.6% versus median 1.0%, IQR 0.6–1.3%; p=0.043) (figure 4b). Interestingly, no such association between exposure and sensitisation response to Asp f 1, 5 or 6 allergen was found; however, all these allergens were present in very low abundance in the studied homes with only two patients having any detectable Asp f 1 allergen in their home environment (figure 4c and d). As Asp f 3 exposure appeared related to a measurable sensitisation response in our cohort, we next assessed its association with clinical outcomes. Patients with both exposure and a sensitisation response to Asp f 3 allergen had poorer FEV₁ % pred (median 40.0% pred, IQR 35.8–47.0% pred versus median 58.0% pred, IQR 47.5–67.0% pred; p=0.028, figure 4e) and significantly more exacerbations at 1-year follow-up (median 2, IQR 1–2 versus median 0, IQR 0–1; p=0.044, figure 4f) relative to patients with exposure but without any sensitisation response to Asp f 3 allergen. We did not detect similar relationships with either CAT scores or baseline exacerbations (figure 4g and h). Collectively, these data support the notion that environmental exposure to Asp f 3 allergen may promote sensitisation in some COPD patients, which in turn leads to poorer clinical outcomes.

FIGURE 4.

Specific Aspergillus fumigatus allergens in the home environment of individuals with COPD. a) Heatmap demonstrating metagenome reads of specific A. fumigatus allergens in the home environment of patients with COPD: outdoor air, indoor air and surfaces. Scatter boxplots illustrating the relative abundance of environmental b) Asp f 3, c) Asp f 5 and d) Asp f 6 allergens in the home environment of patients with COPD assessed by the presence or absence of sensitisation responses to the respective allergens. e) Lung function (as forced expiratory volume in 1 s (FEV₁) % predicted), f) number of exacerbations at 1-year follow-up, g) symptoms (by COPD Assessment Test (CAT) score) and h) number of exacerbations in the year preceding study recruitment between COPD patients exposed (but not sensitised) and both exposed and sensitised to Asp f 3. E: exposed (detectable significant presence of Asp f 3 in the home environment) but not sensitised; E+S: exposed and sensitised to Asp f 3. *: p<0.05, ns: nonsignificant.

The relationship between environmental A. fumigatus and exacerbation frequency in COPD validates in Vancouver, Canada

Having established the high abundance and negative impact of A. fumigatus in COPD within the tropical climate of Singapore, next we evaluated whether similar findings could be validated in an alternative geographic region, one with contrasting seasonal variation and a temperate climate. We thus performed equivalent home sampling and metagenomic sequencing of outdoor and indoor air and swabs of surfaces in the homes of 12 patients with COPD residing in Vancouver, Canada, using identical protocols as described. Compared to participants in Singapore, participants in Vancouver had milder disease with higher median FEV₁ % pred, were more likely to be on triple inhaler therapy, and were more likely to live in landed homes (table 1).

The indoor air temperature (median 21.7°C in Vancouver versus 29.4°C in Singapore, p<0.001) and relative humidity (median 41.7% in Vancouver versus 73.8% in Singapore, p<0.001) differed significantly between the two locations (table 1). Variation in the environmental fungi were observed between geographic locations (supplementary figure S6a and b). Only nine Aspergillus spp. were detectable within the home environment of COPD patients residing in Vancouver (figure 5a). Notably, A. terreus, A. mulundensis and A. novofumigatus were only detected in Vancouver, while A. nidulans, A. aculeatinus, A. welwitschiae, A. awamori and A. pseudoglaucus were only present in Singapore (figure 5a and supplementary figure S7). The prevalence of A. fumigatus was significantly higher in Singapore (98.4% versus 77.4%, p<0.001) (supplementary figure S7). Despite the variable abundance of A. fumigatus across both locations, its effect on exacerbation frequency remained and appeared independent of geographic location or patient origin with a positive correlation identified (r=0.4, p=0.027) between exacerbation frequency and environmental A. fumigatus abundance in the home environment of COPD patients from Vancouver (figure 5b). No associations with CAT scores or FEV₁ % pred were identified (figure 5c and d; supplementary figure S3).

FIGURE 5.

Aspergillus spp. in the home environment associates with exacerbation frequency in COPD independent of geographic origin. a) Bubble plot illustrating the metagenome reads of various Aspergillus spp. detected in indoor air and surface swabs within the homes of patients with COPD from Vancouver and Singapore, respectively. Scatterplots illustrating the correlation between the relative abundance of environmental A. fumigatus and b) number of COPD exacerbations in the year preceding study recruitment, c) symptoms (by COPD Assessment Test (CAT) score) and d) lung function (as forced expiratory volume in 1 s (FEV₁) % predicted) in COPD patients from Vancouver, Canada. Black dotted lines correspond to Spearman's regression and the grey shaded area represents the 95% confidence intervals. ns: nonsignificant.

Finally, we assessed the presence of specific A. fumigatus (Asp f) allergens in the home environment across our two geographically distinct locations. Patients with COPD residing in Singapore have exposure to a significantly higher number of environmental Asp f allergens in their homes relative to Vancouver (median 8, IQR 7–8 versus median 6, IQR 5–7; p=0.0005) (figure 6a and supplementary figure S8), and the number of Asp f allergens in the home environment significantly associates with frequent exacerbator status (median 8, IQR 7–9 versus median 7, IQR 6–8; p=0.028) (figure 6b), but not with symptoms or lung function (figure 6c and d).

FIGURE 6.

Number of specific Aspergillus fumigatus allergens detected in the home environment associates with frequent exacerbations in COPD. Scatter boxplots illustrating the number of specific A. fumigatus allergens detected in the home environment in a) Vancouver and Singapore, b) frequent and nonfrequent COPD exacerbators, c) COPD with low and high symptomatic burden (by COPD Assessment Test (CAT) score), and d) between Global Initiative for Chronic Obstructive Lung Disease (GOLD) group (forced expiratory volume in 1 s (FEV₁) % predicted) groups. GOLD 1: FEV₁ ≥80% pred; GOLD 2: FEV₁ 50–79% pred; GOLD 3: FEV₁ 30–49% pred; GOLD 4: FEV₁ <30% pred. *: p<0.05, ***: p<0.001, ns: nonsignificant.

Discussion

Despite the global burden of COPD and the repeated warnings to our healthcare systems of the imminent crisis that this disease will cause as our population ages and climate heats up, therapies for COPD remain inadequate, failing to meaningfully reverse outcomes [19]. In the face of slow drug development, our study presents evidence in support of a novel strategy towards COPD therapeutics by considering the pathogenic potential of the home environment. Our findings support that 1) A. fumigatus and its associated allergens are abundant in the air of homes in two distinct geographic and climatic settings and the surface environment in Singapore and 2) that this abundance is significantly associated with COPD exacerbations.

Our study adds to the growing body of literature on the pathogenic implications of Aspergillus in COPD, but for the first time identifies the home environment as a major exposure source [4, 8]. Importantly, exacerbation frequency is not significantly associated with sputum A. fumigatus (although it should be noted that this was tested in a subset of the cohort). Instead, environmental A. fumigatus allergens and allergic sensitisation to A. fumigatus appears to play a key role in mediating the relationship between environment and disease. While indoor A. fumigatus exposure has been assessed using floor dust or electrostatic dust collectors through culture or PCR methods in COPD patients, no association was found between colonisation and the environmental Aspergillus levels, similar to our findings [20–22]. The role A. fumigatus colonisation and COPD exacerbations remain unclear, with contrasting results reported in relation to exacerbation frequency [23, 24]. Other factors such as the use of inhaled corticosteroids and host genetic factors remain key considerations that may influence the persistence of A. fumigatus and consequences in COPD.

Patients with higher Asp f 3 abundance in their home were more likely to be sensitised; these patients demonstrated both worse FEV₁ % pred and worse exacerbation frequency at 1 year. Asp f 3 is a peroxisomal membrane protein, one of high abundance and a major Aspergillus allergen [25]. It is present in conidia and hyphae, and exhibits peroxidase activity, protecting Aspergillus from reactive oxygen species. Asp f 3 represents a virulence factor contributing to pulmonary aspergillosis in mouse models [26, 27]. Importantly, proteomic analysis did not identify it as an important protein in patients with invasive aspergillosis [28]. Nonetheless, rAsp f 3 is shown to be useful in diagnosing allergic bronchopulmonary aspergillosis in individuals with asthma and cystic fibrosis [29]. Cross-reactivity is common across fungal allergens, for instance, A. fumigatus contains various homologue allergens that cross-react with other species, including A. nidulans and Candida boidinii; however, the clinical significance of these observations remain unclear [30]. Previously, our group has demonstrated the importance of specific A. fumigatus 1, 3, 5 and 6 sensitisation in worsening COPD exacerbations, lung function and BODEx (BMI, airway obstruction, dyspnoea and exacerbations) scores [3]. Here, we extend these findings by demonstrating that a key source for such sensitisation, specifically Asp f 3, may be coming from the home (residential) environment.

Several environmental factors are associated with the presence of A. fumigatus spores in indoor environments. These include the burden of outdoor fungal spores, high moisture levels, temperature fluctuations, pollutant levels, poor ventilation, lack of proper insulation (leading to moisture accumulation) and indoor condensation, all of which are established to promote fungal growth [31]. In addition, activities such as occupational exposure to agricultural resources, frequent vacuuming and not removing shoes on entering homes are also associated with fungal exposure and healthcare visits for COPD [32]. In line with earlier work, we showed that high temperature, PM_2.5 and PM₁₀ were positively correlated with environmental A. fumigatus. No associations have been observed between air humidity and environmental A. fumigatus. Rather, surface water holds most influence over the growth of fungi as opposed to air humidity [33].

These findings raise the possibility that depletion and/or eradication of A. fumigatus in the home may be one approach in reducing exacerbations and improving COPD outcomes. Measures such as removing visible indoor mould, maintaining good indoor ventilation and cleanliness and the potential use of high-efficiency particulate arresting-filtered vacuum cleaners and air filtration devices in the home setting may potentially reduce a patient's overall mycotic exposure. However, these interventions require prospective study in COPD before true efficacy can be determined [34]. This strategy may be particularly useful for patients living in regions of high temperature, PM_2.5 and PM₁₀, all factors demonstrated here to be associated with higher environmental A. fumigatus. These results confirm the increasingly recognised relationship between pollution, A. fumigatus and poor lung outcomes, recently described by Lin et al. [35], who found that the detrimental impact of PM_2.5 on lung function was primarily mediated through airway Aspergillus abundance. Although worldwide PM_2.5 and PM₁₀ levels have decreased since 2011 [36], rising global temperatures, climate change [37] and spikes in fine particulate matter due to increasingly more frequent weather events including wildfires [38] raise concerns that A. fumigatus exposure will increase with time. This is particularly alarming given the evidence that mixtures of combustion-derived particulates with biological components in aerosols have potent immunological effects [39].

Although our study has notable strengths, including validation in a distinctly different geographic location, the use of state-of-the-art environmental metagenomic sequencing and the prospective capture of exacerbation events, it is not without limitations. Our cohort is predominantly male in both Singapore and Vancouver and whether the relationship between environmental A. fumigatus and exacerbations holds in females remains unanswered. Second, we are unable to infer causation, and the mechanism through which environmental A. fumigatus leads to exacerbation events is not fully established. While we postulate that sensitisation is part of the causal pathway, a greater understanding of host responses to environmental A. fumigatus would be gained by deeper airway sampling in a longitudinal study design. Third, while air and surface swab (fan and air-condition filter) sampling were performed in each individual patient's bedroom, the precise individual exposures (i.e. time spent in bedroom) and other bedroom sources of exposure including the bed, mattress, pillow and cloth coverings remain unclear. Moreover, other factors such as work and other environmental exposures beyond the bedroom were not assessed. Furthermore, the lack of an intervention to reduce fungal exposures in this study and thus improve exacerbation frequency limits the interpretation of our findings. Fourth, taxonomic classification was performed using the Kaiju classifier, which uses a protein-level classification approach. Using this method, direct information about the depth and genome coverage of each species cannot be provided. Finally, the residential fungal mycobiome is far richer than simply A. fumigatus and understanding the role that other fungal species (and their interactomes) may play in the domestic patient environment has yet to be determined. The interplay between bacterial and fungal species in the home environment would similarly be of unique interest to explore. Notwithstanding these limitations, we demonstrate that the air and surfaces of a patient's domestic setting carry a mycotic burden with important clinical implications in COPD. For these patients, the home mycobiome represents an exciting new therapeutic target to improve clinical outcomes for COPD.

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