Aspergillus-Associated Endophenotypes in Bronchiectasis

Abstract

Bronchiectasis is a chronic condition of global relevance resulting in permanent and irreversible structural airway damage. Bacterial infection in bronchiectasis is well studied; however, recent molecular studies identify fungi as important pathogens, either independently or in association with bacteria. Aspergillus species are established fungal pathogens in cystic fibrosis and their role is now increasingly being recognized in noncystic fibrosis bronchiectasis. While the healthy airway is constantly exposed to ubiquitously present Aspergillus conidia in the environment, anatomically damaged airways appear more prone to colonization and subsequent infection by this fungal group. Aspergilli possess diverse immunopathological mechanistic capabilities and when coupled with innate immune defects in a susceptible host, such as that observed in bronchiectasis, it may promote a range of clinical manifestations including sensitization, allergic bronchopulmonary aspergillosis, Aspergillus bronchitis, and/or invasive aspergillosis. How such clinical states influence “endophenotypes” in bronchiectasis is therefore of importance, as each Aspergillus-associated disease state has overlapping features with bronchiectasis itself, and can evolve, depending on underlying host immunity from one type into another. Concurrent Aspergillus infection complicates the clinical course and exacerbations in bronchiectasis and therefore dedicated research to better understand the Aspergillus-host interaction in the bronchiectasis airway is now warranted.

Keywords

noncystic fibrosis bronchiectasis - Aspergillus - endophenotypes - fungi - mycobiome

Bronchiectasis is a chronic, progressive, and irreversible airways disease characterized by bronchial dilatation and copious sputum production, and it is often complicated by recurrent chronic pulmonary infections and exacerbations.[1] Globally, the disease is increasing in prevalence despite continued under recognition, and it remains an important cause of respiratory morbidity and poorer quality of life.[2] While approximately 50% of cases are idiopathic, one of the commonest causes of bronchiectasis is postinfection, predominantly after severe pneumonia or tuberculosis. Other important causes include cystic fibrosis (CF), primary ciliary dyskinesia, immunodeficiencies, and allergic bronchopulmonary aspergillosis (ABPA). However, importantly bronchiectasis is often seen in relation to other primary lung diseases including severe asthma and/or chronic obstructive pulmonary disease (COPD). In this article, we use the term “bronchiectasis” in relation to etiologies unrelated to CF.

While bacteria are traditionally recognized as the predominant infection-driving pathogens in bronchiectasis, recent advances in next generation microbiome sequencing approaches reveal an important role for other kingdoms including fungi. Fungi, such as Aspergillus may act in isolation as respiratory pathogens, or alternately in complex interplay with established bronchiectasis pathogens such as Pseudomonas.[3] [4] Disease progression is facilitated through the vicious cycle, or the more recently described vicious vortex of infection, inflammation, epithelial dysfunction, and impaired mucociliary clearance.[5] Considering this model of pathogenesis in relation to fungal exposure and disease, a healthy immunocompetent host, armed with effective mucociliary clearance mechanisms and robust immunity, can successfully clear inhaled fungal conidia and avoid disease.[6] [7] [8] [9] [10] Hosts such as those with established bronchiectasis, however, lack such protective mechanisms, making them inherently susceptible to fungal colonization and/or infection. Aspergillus fumigatus is therefore recognized as an important colonizer of the bronchiectasis airway and remains the most widely recognized fungus in relation to bronchiectasis. Despite high frequencies of airway Aspergillus in bronchiectasis, much of our current understanding in regard to its pathogenic potential is extrapolated from other respiratory disease states such as CF and COPD. However, this filamentous fungal pathogen has been associated with increased mucus production, purulence, and exacerbation frequency in bronchiectasis.[11]

Aspergillus-associated lung disease states and their related clinical consequences in bronchiectasis remain understudied and warrant attention in view of their increasing recognition, frequency, and clinical importance.[4] [12] [13] Traditional and challenging methods of fungal detection including culture and/or microscopy are now complemented by more sophisticated and sensitive molecular detection methods including quantitative polymerase chain reaction (qPCR) and, increasingly, next-generation sequencing (NGS) approaches, which have allowed earlier detection and therefore renewed attention to the deleterious consequences of Aspergillus-associated pathologies in chronic lung diseases such as bronchiectasis.[3] Here, we review the key Aspergillus-associated pathologies as relevant to bronchiectasis, with a focus on NGS approaches in the context of endophenotyping this complex and heterogenous respiratory disease.

The Clinical Significance and Spectrum of Aspergillus-Associated Disease in Bronchiectasis

While the role of Aspergillus in CF, asthma and COPD is recognized, Aspergillus-associated disease in the setting of bronchiectasis remains understudied and significant gaps exist regarding its epidemiology, pathogenesis, diagnosis, and management.[12] [14] Fungal cultures are not routinely performed and remain limited by their lack of sensitivity to detect fungi in comparison with molecular approaches including NGS, which thus far have only been employed in research settings.[15] [16] Variable recovery rates of Aspergillus from sputum culture, ranging from 6.9 to 24.0%, are routinely reported between centers, demonstrating the significant diagnostic challenge this organism presents in routine clinical practice ([Fig. 1]).[11] [17] Chronic and invasive forms of Aspergillus-associated disease are more readily detectable in those immunocompromised; however, less consensus on diagnostic approaches in stable immunocompetent bronchiectasis exists. The role and relevance of serum galactomannan, a widely used biomarker in chronic and invasive aspergillosis (IA), is not fully understood in the context of bronchiectasis, particularly in patients with features of early pulmonary aspergillosis. An additional diagnostic challenge in bronchiectasis are the largely indistinguishable radiological features that differentiate underlying bronchiectatic change from ongoing pulmonary aspergillosis, leading to under-recognition of the latter.[14] Dependent on underlying host immunity, Aspergillus can act independently to cause direct pulmonary damage leading to bronchiectasis or alternately trigger a spectrum of syndromes that complicate preexisting bronchiectasis[13] [18] [19] [20]([Fig. 2]). The ubiquitous nature of Aspergillus spores in the surrounding environment coupled to their small size (2–5 µm) favor dispersal to the most distal airways, and the inhaled fungal burden may be especially high in hot and humid environments potentially explaining the geographic variation in Aspergillus-associated disease.[16] The clinical gamut of Aspergillus lung disease primarily depends on fungal–host interaction, a key area of ongoing research.[7] [12] [21] [22] The balance between host defenses and Aspergillus proliferation broadly determines the clinical outcome, which can range from asymptomatic colonization to sensitization and ABPA in the immunocompetent and allergic to chronic and invasive disease in the immunocompromised[7] [10] [18] [23] ([Fig. 2]). The type and severity of Aspergillus disease therefore directly relates to the host immune response and underlying anatomical abnormalities, including bronchiectasis, that demonstrate immunodeficiency states and high frequencies of sensitization in a significant number of patients.[24] It is also common to observe Aspergillus-related pathology evolve from one defined clinical state into another, in the same patient and over time, under the influence of the immune system and/or treatment the patient may be receiving such as steroids. Such dynamic ongoing change to host immunity, particularly in the setting of bronchiectasis, increase the difficulties in diagnosing and managing such infections.[14]

Fig. 1 Diagnostic challenges in Aspergillus-associated disease in bronchiectasis. NCFB, noncystic fibrosis bronchiectasis; CF, cystic fibrosis; NGS, next generation sequencing.

Fig. 2 The clinical spectrum of pulmonary Aspergillus-associated disease. ABPA, allergic bronchopulmonary aspergillosis; CPA, chronic pulmonary aspergillosis; IA, invasive aspergillosis.

Broadly, clinical syndromes related to pulmonary aspergillosis in the setting of bronchiectasis may be classified as allergic disease, saprophytic infection, or invasive disease[20] ([Fig. 2]). ABPA represents one of the commonest Aspergillus-related endophenotypes in bronchiectasis and represents a hypersensitivity response to Aspergillus antigens, located in the airway in a sensitized host. Involving an unrestrained immune-inflammatory airway response by macrophages, neutrophils, and fungal proteases, excessive damage to the airways can result.[13] A complex association between ABPA and bronchiectasis exists, and its underlying pathogenic mechanisms are of increasing research interest. ABPA is an established cause of bronchiectasis but more commonly is a consequence of preexisting disease. An ABPA prevalence of up to 10% is observed in idiopathic bronchiectasis, while causal links between ABPA and bronchiectasis are described due to the persistent and exaggerated immune response characteristic of ABPA.[25] [26] Current data suggest that ABPA leads to poorer clinical outcomes and a higher risk of exacerbations in bronchiectasis.[27] [28] [29] Disproportionate immune responses and variable Aspergillus virulence can predispose to fungal bronchitis and chronic pulmonary aspergillosis (CPA) in bronchiectasis, comparable to that observed in other chronic lung diseases such as COPD.[12] [30] Interestingly, tuberculous and nontuberculous mycobacterial (NTM) infection established bronchiectasis etiologies closely associated with CPA occurrence.[19] [31] Direct correlations between NTM and A. fumigatus sensitization are observed, and both Aspergillus lung disease and NTM remain important independent predictors of mortality in patients with bronchiectasis.[19] [31] Cavitary lesions in individuals with post-tuberculosis (TB) bronchiectasis can result in saprophytic noninvasive Aspergillomas, while coexisting bronchiectasis in patients with asthma or COPD increase the risk of Aspergillus-associated disease while conversely, chronic Aspergillus infection in any setting can increase the chance of developing subsequent bronchiectasis.[32] [33] [34] Invasive aspergillosis represents the most serious and lethal form of Aspergillus-associated disease, most commonly seen in the immunocompromised, but importantly described in association with structural airway damage such as that seen in bronchiectasis which increases risk.[13]

Aspergillus-Associated Disease and Its Related Endophenotypes in Bronchiectasis

Increasing molecular evidence including NGS studies reveal important Aspergillus-associated endophenotypes in bronchiectasis which merit recognition owing to their potential increased risk of exacerbations and poorer clinical outcomes coupled to the inherent complexity in their diagnosis and management.[12] The etiopathogenesis of aspergillosis in bronchiectasis is wide ranging and leads to both the development of further bronchiectasis and decompensation of existing disease.[13] Studies in patients with bronchiectasis and aspergillosis illustrate that they experience a higher frequency of hospitalization, exacerbation, and overall poorer clinical outcomes, especially in older individuals and those receiving chronic antibiotic therapy.[13] [14]

Allergic Bronchopulmonary Aspergillosis

Most studies on aspergillosis in bronchiectasis have focused on the development of bronchiectasis because of ABPA, and the accompanying clinical consequences of coinfection. Bronchiectasis develops over time following acute ABPA, and pathogenesis directly relates to the host immune response including type I, III, and IV hypersensitivity reactions to Aspergillus antigens, immunoglobulin mediation, and the degranulation of mast cells and eosinophils in airways during ABPA exacerbations. Taken together, like the vicious cycle of bronchiectasis itself, these events lead to a self-perpetuating cycle of inflammation and airway dilatation.[35] [36] This causes mucus impaction, atelectasis, and eventually permanent structural damage to the airway resulting in bronchiectasis.[37] Mucus plugging itself, through selective induction of MUC5AC by A. fumigatus is described as a possible mechanism toward developing ABPA.[38] A recent multicenter study demonstrated trends toward higher ABPA occurrence in patients with higher Bronchiectasis Severity Index, suggesting a potential link between existing disease severity, occurrence of ABPA, and further disease progression.[2] A major challenge however remains the difficultly in determining true ABPA incidence in bronchiectasis, due to overlapping symptoms with infective exacerbations and variability in the applied diagnostic criteria across geographic regions.[39] [40] [41] Central bronchiectasis was traditionally viewed as an important feature characteristic of ABPA-related bronchiectasis. However, there are other causes of central bronchiectasis and some ABPA patients display solely peripheral bronchiectasis.[34] [42] The characterization of ABPA in bronchiectasis is also further complicated as the presence of bronchiectasis itself is considered an independent criteria to establish a diagnosis of ABPA.[43] Immunodeficiency remains another important etiology of bronchiectasis and primary immunodeficiency disorders including defects in the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase complex in chronic granulomatous disease (CGD) or STAT3 mutations can predispose to Aspergillus colonization and Aspergillus-associated disease. Notably, secondary immunosuppression through necessary steroid use in ABPA treatment confers the risk of transitioning to other Aspergillus-associated disease states including CPA.[41] [44] [45]

Chronic Pulmonary Aspergillosis

CPA, also referred to as semi-invasive or subacute aspergillosis, is most commonly observed as a complication of established and severe bronchiectasis.[13] [34] [46] Unlike IA, that remains characterized by vascular invasion, CPA is limited to slowly progressing cavitary lesions of the lung parenchyma that follows Aspergillus infection ([Table 1]).[47] Denning et al propose a CPA classification into three distinct categories based on radiological pattern as clinical features overlap.[48] Chronic cavitary pulmonary aspergillosis (CCPA) is characterized by multiple progressive cavitary lung lesions while the presence of fibrosis, as sequelae of these expanding cavities lead to chronic fibrosing pulmonary aspergillosis (CFPA). The final category, chronic necrotizing pulmonary fibrosis (CNPA), or subacute invasive pulmonary aspergillosis (subacute IPA) is distinguished by its slowly progressive and invasive disease due to the enlargement of a single cavity ([Table 1]). This latter condition occurs most frequently in individuals with significant immune compromise including diabetes and those receiving long-term corticosteroid therapy. Long-term follow-up of clinical progression in CPA demonstrates that all affected individuals had some preexisting lung disease, while approximately just over one quarter demonstrate bronchiectasis-related change on either radiology and/or histology.[48] A South Korean-based evaluation of pulmonary aspergillosis observed similar proportions of patients with underlying bronchiectasis and further documented strong associations with NTM infection.[49] Of note, coinfection with NTM in bronchiectasis also independently associates with higher mortality.[31]

Table 1 Summary of degree of tissue invasion based on type of Aspergillus-associated disease

Noninvasive no tissue invasion by hyphae	Invasive hyphae invade tissue
	Superficial		Deep
Colonization/Sensitization	CCPA	CPA	IPA
ABPA	CFPA		IA
Aspergilloma	CNPA/Subacute IPA
	ATB

Abbreviations: ABPA, allergic bronchopulmonary aspergillosis; ATB, Aspergillus tracheo-bronchitis; CCPA, chronic cavitary pulmonary aspergillosis; CFPA, chronic fibrosing pulmonary aspergillosis; CNPA, chronic necrotizing pulmonary aspergillosis; CPA, chronic pulmonary aspergillosis; IA, invasive aspergillosis; IPA, invasive pulmonary aspergillosis.

Invasive Pulmonary Aspergillosis

Tissue invasion, either by angioinvasion or invasion of the airway, by septate fungal hyphae indicates IA ([Table 1]). This is most prevalent in the severely immunosuppressed or those with preexisting chronic respiratory pathology such as bronchiectasis.[18] [34] This is a serious fungal consequence that associates with high mortality. A. fumigatus is the most reported species causing IPA although infections with others such as Aspergillus niger, Aspergillus flavus, and Aspergillus terreus are reported.[13] [34] Prolonged neutropenia is an important risk factor; however, IPA is documented in immunocompetent hosts including advanced COPD and/or bronchiectasis.[34] [50] It can be particularly challenging to detect IPA, especially in non-neutropenic states such as that observed in bronchiectasis, leaving such patients undiagnosed for long periods as the disease progresses. Chronic airway damage such as that characteristic of bronchiectasis predisposes to Aspergillus colonization, and it remains highly probable that such structural change, and its accompanying immune-related effects are an under-recognized risk factor for IPA. Immunodeficiency syndromes including CGD occur with coexisting bronchiectasis and represent additional risks for the development of IPA.[51] [52] [53] Whether routine screening for IPA is warranted in bronchiectasis care remains to be determined; however, it is advised, especially in high-risk patients with bronchiectasis and a significant immunodeficiency.

Aspergillus Tracheo-Bronchitis

ATB is considered a subgroup of IPA, but unlike IPA, tissue invasion is confined to the superficial mucosal layers of the tracheo-bronchial tree ([Table 1]).[18] [34] This condition has lacked overall study but is identified in small groups of patients (∼3%) among large cohorts, where aspergillosis was assessed.[54] In the context of bronchiectasis, it predominantly occurs in patients with minor immune deficits and is characterized by mucoid impaction of the airways and bronchial plugging, with or without accompanying ulceration.[54] [55] Other forms of ATB are described and include obstructive, pseudomembranous and ulcerative subtypes.[34] [56] Most interestingly, unlike IA, obstructive ATB may present without evidence of mucosal invasion and is characterized by an absence of airway inflammation; however, this form is yet to be described explicitly in association to bronchiectasis and warrants further study.[56]

Aspergilloma

Aspergillomas are a saprophytic manifestation of pulmonary aspergillosis. They represent a localized mass of hyphae and cellular debris, often developing in areas of lung with preexisting structural damage, including lung cavities in patients with post-tuberculosis bronchiectasis.[47] [57] Most remain asymptomatic and are detected incidentally; however, life-threatening hemoptysis is a dreaded complication if fungal hyphae invade the bronchial vasculature. In this setting, surgical resection of the mass or embolization of the involved vasculature may be necessary, especially with a large or otherwise intermittent frequent bleeds. The precise occurrence of aspergillomas in bronchiectasis remains unknown and is likely underestimated.

Bronchiectasis Overlap Syndromes and Aspergillus-Associated Disease

Bronchiectasis may overlap with the presence of other chronic respiratory diseases including asthma and COPD. These present inherent diagnostic challenges due to common symptoms and the relative lack of clinical guidelines for identification and management.[58] [59] [60] Patients with severe asthma and concurrent fungal sensitization are more prone to colonization with A. fumigatus, and studies have shown an approximate twofold increase in the risk for developing bronchiectasis, which is then complicated by poor lung function.[61] [62] It remains unclear what specifically predisposes these patients to Aspergillus sensitization in the first place, but what is clearly evident is that once this occurs, it represents an important risk for the subsequent development and progression of bronchiectasis, likely due to the chronically inflamed airways and the consequent remodeling process. Bronchiectasis COPD overlap is diagnosed when patients fulfill physiological and structural diagnostic criteria for both COPD and bronchiectasis.[63] Bronchiectasis has been commonly associated with COPD and when present is identified as an independent risk for mortality.[64] [65] [66] [67] [68] In a study of COPD patients, bronchiectasis was interestingly most frequently observed in patients with demonstrable sensitization to Aspergillus antigens. Furthermore, these “sensitized” patients exhibited a higher frequency of coinfection with bacterial pathogens such as P. aeruginosa, which in itself complicates bronchiectasis.[69] Such work further emphasizes the existence of Aspergillus endophenotypes in bronchiectasis, which first must be recognized before we can consider the various approaches necessary for accurate diagnosis, an understanding of their clinical course and required therapeutic interventions. While Aspergillus-associated disease endophenotypes in relation to bronchiectasis are clearly influenced by the presence of an overlap syndrome, the spectrum of Aspergillus-associated disease itself means that some disease states can coexist in the same patient or evolve from one entity to another dependent on the underlying host immune system. General risks for the evolution of disease include multiple respiratory pathologies, prolonged corticosteroid therapy, high fungal load, and/or host genetic susceptibility.[34] Aspergillomas and ABPA are frequently codiagnosed and likely due to an expanding bronchiectasis developing cavitation and subsequent Aspergillus colonization.[70] [71] Alternately, hyperimmune responses cause the evolution of an aspergilloma into ABPA or ABPA treated with prolonged steroids can result in CPA.[72] ABPA with an element of invasion is rare but is linked to prolonged corticosteroid use where immune suppression promotes a displacement of hyphae from the airways into the lung parenchyma.[73] Concurrent Aspergillus pathologies while presenting an even more significant diagnostic challenge need to be recognized as they can lead to more severe bronchiectasis and a higher exacerbation risk.

The Contribution of Nonfumigatus and Other Fungi in Bronchiectasis

While A. fumigatus remains the most common fungi implicated in bronchiectasis-related aspergillosis, other Aspergillus species including A. niger, A. versicolor, and A. flavus have been identified in relation to bronchiectasis.[30] A. niger, A. flavus, and A. terreus are reported to induce ABPA while the less frequent A. nidulans does exhibit an association with CGD and an aggressive course of disease.[30] [52] [74] [75] [76] [77] [78] [79] Importantly, coinfection by more than a single Aspergillus species has also been reported.[80] [81] Geographic variation in nonfumigatus species have been described: A. niger and A. terreus are more prevalent in Japan while A. flavus predominates in India and China.[30] [77] [82] [83] A key study in bronchiectasis, the Cohort of Asian and Matched European Bronchiectasis (CAMEB) study, demonstrates that A. fumigatus profiles dominate patients of Asian origin while in an age- and sex-matched cohort of European origin, A. terreus was more frequent. Correlation with airway conidial burden reiterated this regional variation, even when both species coexisted, and interestingly, higher conidial burdens were associated with a greater number of exacerbations.[84] Other fungi that commonly associate with bronchiectasis include Fusarium, Mucor, Rhizopus, and Scedosporium.[14] [36] Yeasts, including Candida albicans and Exophiala dermatitidis have also been isolated in bronchiectasis.[11] [36] C. albicans, for instance, was isolated in over 40% of patients with bronchiectasis in a Spanish study while E. dermatitidis albeit rarely does cause a significant deterioration in pulmonary function.[11] A global review on allergic bronchopulmonary mycosis caused by fungi other than Aspergillus reports that up to 60% of the identified cases can be caused by C. albicans, an important consideration in bronchiectasis.[85]

The Pulmonary Mycobiome in Bronchiectasis

Many fungi have been proposed as contributors to airway infection in chronic lung diseases including bronchiectasis.[36] With increasing urbanization, climate change, and the ubiquitous nature of fungal presence in the surrounding environment, the relationship between fungi and human lung disease has received renewed attention.[86] [87] Early culture-based assessment coupled to deep metagenomic sequencing now illustrates the complexity of fungal consortia that exist in outdoor air and demonstrate cyclical variability in abundance, whose consequences for human respiratory health remain to be fully appreciated.[88] [89] [90] Fungal conidia, owing to their small size, can reach the smallest airways, but are then removed by innate immune mechanisms including mucociliary clearance and macrophage engulfment.[91] Daily fungal exposure, although plentiful, is of little consequence in healthy immunocompetent individuals due to their successful elimination by the tightly regulated host mucosal defenses.[91] [92] In bronchiectasis, however, chronic infection and associated immunopathogenic dysfunction lead to dysregulated host responses and fungal colonization.[11] [93] Inflammatory cytokines, elastases, and matrix metalloproteinases (MMPs) then damage the structural integrity of the airway and lead to anatomic distortion and subsequent fungal sensitization, an increasingly recognized contributor to the pathology of bronchiectasis.[94] [95] [96] Several studies now underscore the importance of fungi in bronchiectasis, in particular the increased colonization by Aspergillus and Candida species, the increased expression of antifungal chitinase enzymes, and the heightened sensitization response to fungal antigens.[11] [24] [84] [97] Given these developments, the role of the mycobiome, a collective assessment of fungal consortia present in the lung has become a key the subject of focused for NGS-based analyses in bronchiectasis.[98] [99]

Bronchiectasis is a markedly heterogenous disease which differs further based on geographic boundaries, ethnicities, etiologies, and response to therapy.[100] Most clinical intervention to date focuses on prevention of exacerbations and airway clearance although targeting microbes such as P. aeruginosa in the airway confers clinical benefit. Recent NGS sequencing studies uncover a milieu of complex multikingdom organisms including bacteria, viruses, and fungi that potentially interact within the bronchiectasis airway, which explains the inherent heterogeneity and vastly contrasting clinical course observed between patients.[3] [99] The European Multicenter Bronchiectasis Audit and Research Collaboration consensus statement for bronchiectasis attempts to address these important gaps in our understanding of this disease and identified exploration of the pulmonary mycobiome as a key research priority.[101] The recent CAMEB study is notable for its first report on the pulmonary mycobiome in bronchiectasis across continents and in age- and sex-matched populations from distinct geographical regions. It provided key insights into Aspergillus-associated disease in bronchiectasis.[84] By using high throughput 18S-28S ITS sequencing, this work first reaffirms the importance of Aspergillus and Aspergillus-associated disease in bronchiectasis and identifies distinct mycobiome profiles dominated by A. terreus in patients from Dundee, Scotland and A. fumigatus in patients from Singapore and Kuala Lumpur, Malaysia. These findings exemplify the existence and relevance of considering geographic differences in the clinical assessment of the bronchiectasis mycobiome. Quantification of conidial burden by quantitative polymerase chain reaction (q-PCR) reveals significant associations between higher conidial burden and occurrence of exacerbations. Further clinical correlations were elucidated by grouping patients based on meeting criteria for the various Aspergillus-associated disease states. Patients with serological ABPA (sABPA) had more severe disease, greater exacerbations, and poorer lung function when compared with those Aspergillus colonized and/or sensitized. This serves to demonstrate the clinical significance of Aspergillus in the etiopathogenesis and progression of bronchiectasis and, screening for this fungus, even in clinically stable states may offer important clinical insight.

While the Aspergilli remain the best characterized fungi in the bronchiectasis airway, Candida species are the most widely detected.[4] [14] [98] Other important fungi to emerge from culture-based studies include Saccharomyces cerevisiae, Trichosporon spp., Scedosporium, and Penicillium.[14] Given the inherent challenges and lack of standardization in fungal culture protocols, it is possible that these data are skewed and underestimate the true in vivo composition of the bronchiectasis mycobiome.[102] [103] Emerging NGS-based mycobiome research in bronchiectasis performed by our group, and others offers fresh insight into the mycobiome in this setting and remain less susceptible to the biases of culture-based assessments.[84] [99] The CAMEB study, as previously described also highlighted an overrepresentation of fungal taxa that go beyond Aspergillus and include Penicillium and Cryptococcus while concurrently identifying, in view of its study design, several taxa that vary geographically. This latter group includes Simplicillium and Trichosporon which predominate in Asians, and Wickerhamomyces, Clavispora, and Cryptococcus that demonstrate a higher abundance in Europeans. High Basidiomycota loads within a bronchiectasis mycobiome was generally associated with a more favorable prognosis; however, some key Basidiomycota fungi including Trichosporon, Cryptococcus, Clavispora, Alternaria, Botrytis, Wickerhamomyces, and Cladosporium were relevant in Aspergillus sensitization and sABPA demonstrating the complexity of the bronchiectasis mycobiome and need for future ongoing research to better understand its clinical correlates and usefulness in patient stratification.[16] [84]

Immunoallertypes and the Role of Aspergillus Sensitization in Endophenotyping Bronchiectasis

Heightened Th2 responses are associated with fungal sensitization and allergy and remain a component of several chronic respiratory disease states.[36] Early suggestions of a role in bronchiectasis came from studies in CF, where sensitization and allergy correlate with the development of CF-ABPA and lung function declines.[104] [105] [106] Further work revealed that even outside the CF setting, that a significantly elevated atopic response was detectable in bronchiectasis and, that the association between sensitization and lung function decline, as identified in CF, remained consistent.[107] Interestingly, sensitization to A. fumigatus was identified as a risk factor for the development of bronchiectasis in COPD cohorts, with most of the heightened risk attributed to the recombinant A. fumigatus allergens rAsp f1 or f3.[69] Building on these existing works, and employing the CAMEB cohort, our group next assessed the frequency and clinical relevance of fungal sensitization in stable bronchiectasis. We identified a high prevalence of both the Aspergillus fungi and related sensitization response including significant (and largely clinically undetected) proportions of sABPA.[12] [24] [36] [108] [109] Significant levels of polysensitization, going beyond fungi and including common environmental allergens were identified independent of patient origin. These include crude allergens of the house dust mites Dermatophagoides pteronyssinus and Blomia tropicalis and the fungi Alternaria alternata. Importantly, a comprehensive panel of A. fumigatus recombinant allergens was explored and includes rAsp f 1, f 2, f 6, f 8, f 15, and f 17. Comprehensive immune-inflammatory profiling was concurrently performed and when assessed in combination to airway sensitization responses in bronchiectasis revealed two distinct “immuno-allertypes” including a predominantly house dust mite sensitized patient group, characterized by a chemokine-dominant airway profile including growth-regulated oncogene (CXCL1), monocyte chemoattractant protein-1 (CCL2), and eotaxin-1 (CCL11) in addition to the anti-inflammatory cytokines interleukin 1RA, interleukin 10, and granulocyte colony–stimulating factor. In contrast, a second group of patients with a predominantly fungal-driven sensitization response, and poorer clinical outcomes was also identified. This group, marked by a proinflammatory airway cytokine signature including tumor necrosis factor α (TNF-α), IL-1α, and IL-1β demonstrated significant associations with poorer lung function and increased disease severity.[24] [110] A marked geographic variation in allergic profiles was also evident, suggestive of perhaps distinct endophenotypes that warrant further study. Asians with bronchiectasis in the CAMEB cohort exhibit a higher sensitization to house dust mite allergens and the A. fumigatus major allergen, rAsp 1, in contrast to Europeans who had higher levels of sensitization to Alternaria and the A. fumigatus allergens rAsp f 6, f 8, f 15, and f 17. A further dissection of these patterns within each “immuno-allertype” revealed specific endophenotypic subgroups relating to a patient's country of origin, reflective of the clinical heterogeneity in sensitization responses that likely exist. While the overall picture is complex, the observed increases in sensitization responses to the major Aspergillus allergen rAsp1 in Asian patients remains consistent with the higher detected A. fumigatus conidial burden in this region suggesting that a combination of geographic origin, host response, and fungal exposure levels all have importance. Endophenotypic variability, based on sensitization pattern and other features, is therefore an important consideration for patient stratification and the design of clinical trials in bronchiectasis, particularly when multiple centers across wide ranging geographic regions are included. Careful consideration is required to the presence of underlying sensitization in bronchiectasis, and while complex, it likely contributes to disease heterogeneity.[111] [112] This complexity extends further to the underlying etiology of bronchiectasis at the individual level, and recent work substantiates this, illustrating the critical importance of fungal sensitization in bronchiectasis while identifying TB-related bronchiectasis as an independent risk factor for Aspergillus sensitization.[113] Taken together, collective data does suggest that sensitization in bronchiectasis, particularly to fungi is significant and clinically relevant. This provides scope for improved patient stratification and potentially the development of targeted and personalized interventions based on geographic origin.

Going Beyond Fungi: Interkingdom Interaction in the Bronchiectasis Airway

While clearly relevant, the mycobiome cannot be considered in isolation, given its existence within an integrated microbial ecosystem in the airway that encompasses fungi, bacteria, and viruses, all of which contribute to bronchiectasis.[16] While bacteriomes have been investigated, the role of viruses and the “virome” including bacteriophages remains poorly understood in bronchiectasis and necessitates research.[114] [115] [116] As studies continue to emerge that improve our understanding of the airway microbiome and its relevance to human health and disease, it is apparent that holistic “multibiome” analyses, which encompasses complex microbial interaction networks, represents a logical progression in bronchiectasis microbiome research efforts. Efforts to model host microbiomes as an integrated microbial network have already been advanced in CF to understand exacerbations.[117] Such models seek to assess the microbiome as a network, integrating coexisting, commensal, and/or ‘pathobiont’ microbes in addition to classical “culprit” organisms. Network-based analysis can therefore account more accurately for observed clinical differences seen in patient cohorts and represent a promising platform for further explication of microbiome-driven endophenotypes of respiratory disease including bronchiectasis.[118] The plausibility of potentially variable interactions is evident in existing clinical and co-culture analyses of P. aeruginosa and A. fumigatus, the most well-studied interkingdom interaction of relevance to respiratory disease.[119] [120] [121] Interkingdom communication between fungi and bacteria remains an active research area with relevance to bronchiectasis, where the application of “multibiome; approaches may yield insight into complex and currently poorly understood endophenotypes.[3] [16] [84] [122] While each individual microbial kingdom has its own relevance and is examined independently, an integrated and holistic interkingdom approach remains an important and likely rich avenue for future microbiome studies in bronchiectasis.

Conclusion

The emerging role of fungi and in particular Aspergillus in bronchiectasis has been the subject of numerous studies and remains a key research priority.[101] Although significant progress has already been made, further studies focusing on epidemiology, strain variation, and clinical relevance in relation to bronchiectasis endophenotypes are required. Our current understanding is largely shaped by the increasing applied systems-level analysis that employs data-rich microbial profiling of host airways and their associated systemic response. Recent exploratory work underscores the potential of data-driven molecular approaches to identify and stratify patients according to their underlying fungal endophenotypes. While current work on the bronchiectasis mycobiome provides a platform for future research, it also highlights several challenges for the field including the development and optimization of ITS protocols to ensure adequate fungal coverage as compared with those robustly established for the bacterial microbiome.[99] [123] Metagenomics offers an alternate approach, but here challenges also exist, most notably in the development and availability of public reference databases that lag significantly behind those available for bacteria, which potentially leads to classification errors.[124] [125] Notwithstanding this, the exploratory microbiome studies have been steadily increasing in bronchiectasis, which provides scope for improved patient stratification and resolving the inherent disease heterogeneity that exists.[3] [117] Applying intrakingdom analytical approaches may be the next logical step to address key knowledge gaps in bronchiectasis mycology. Longitudinal measures of Aspergillus sensitization and its associated immune response may allow the identification of “at risk” groups at an early stage and prior to the onset of overt fungal disease, permitting the identification of fungal endophenotypes at the molecular level that can be individually targeted for appropriate intervention. An improved understanding of the role for antifungal therapy in bronchiectasis is also required, therefore addressing fungal endophenotypes and their accompanying clinical outcomes remain a priority. The role of bacterial taxa such as P. aeruginosa and members of the NTM family, both of which can interact with Aspergillus may contribute to fungal endophenotypes and disease progression in bronchiectasis and warrant dedicated investigation. In addition to leveraging key technologies, an increased international effort to address the geographic variation through the establishment and maintenance of large bronchiectasis cohorts will be important to drive this field forward and recognize the true relevance of Aspergillus and other fungi in bronchiectasis.

Conflict of Interest

None declared.

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Publication History

Article published online:
14 July 2021