Table of Contents

m3 Aspergillus fumigatus Scientific Information Allergen Epidemiology Clinical Relevance Prevention and Therapy Molecular Aspects Explained Results

Whole Allergen

m3 Aspergillus fumigatus

m3 Aspergillus fumigatus Scientific Information

Type:

Whole Allergen

Allergen code:

Family:

Trichocomaceae

Route of Exposure:

Inhalation

Source Material:

Conidia (Asexual Spores) and mycelium

Latin Name:

Aspergillus fumigatus

Other Names:

Neosartorya fumigata, Sartorya fumigata (obsolete)

Allergen

Summary

Aspergillus fumigatus (A. fumigatus) is a saprotroph and opportunistic filamentous fungus widely distributed in the environment. A. fumigatus is a spore-forming, thermotolerant fungus that reproduces asexually through spores in the form of conidia. It is a cosmopolitan pathogen distributed all over the five continents (Europe, Asia, America, Africa and Australia). Inhalation is the main route of exposure to Aspergillus spores. A. fumigatus is the causal agent of allergic and infectious diseases affecting dozens of millions of people globally. Allergic bronchopulmonary aspergillosis (ABPA) is the most severe Aspergillus-related allergic disease, affecting 1-2.5% of asthmatic patients and up to 10% of cystic fibrosis patients during their lifetime. Other significant allergic Aspergillus-related diseases are allergic fungal rhinosinusitis (AFRS) and severe asthma with fungal sensitization (SAFS). Up to now, 23 allergens of A. fumigatus have been characterized. Among them, Asp f 1 and Asp f 2 are major allergens, Asp f 3 has extensive cross-reactivity, and Asp f 4 and Asp f 6 are associated with ABPA diagnosis. Moreover, Asp f 1 and Asp f 2 are markers of genuine sensitization to A. fumigatus. Cross-reactivity has been mainly observed between A. fumigatus and other fungal species.

Nature

Aspergillus fumigatus (A. fumigatus) is a saprotroph and opportunistic filamentous fungus widely distributed in the environment. It sporulates abundantly with airborne conidia (asexual spores). It can grow and survive at a wide range of temperatures (25°C to 55°C) (1, 2). However, seasonal variation is seen in its growth influenced by geographical location (1). Elevated spore counts are reported to occur mostly during the summer, monsoon and autumn seasons due to effect of temperature and humidity in certain regions like India (1, 3). In contrast, there are studies, which reported weak or no correlation between A. fumigatus spore counts and seasonal variation with the persistent occurrence of low level of A. fumigatus throughout the year (1).

The conidia of A. fumigatus are small (2-3 µm), much more hydrophobic than those produced by other molds and can disperse easily. A. fumigatus shares remarkable stress tolerance features, such as thermotolerance (A. fumigatus conidia survive at 70°C), xerotolerance, resistance to acid pH, tolerance to low O₂ levels, and nutritional versatility with other Aspergillus species (2, 4, 5).

Habitat

The main ecological niche of A. fumigatus is environmental decaying organic matter, where it contributes to carbon and nitrogen recycling. Consequently, A. fumigatus is abundant in soil, decaying plants, stored grain and fruit, silage, compost, stagnant water, sewage. The optimal conditions for A. fumigatus growth and conidiogenesis are a temperature of 37°C and an oxygen-rich gaseous environment. Under these conditions, A. fumigatus growth occurs almost independently of the degree of moisture and water activity (i.e., proportion of water which is unbound and thus available for microorganismal growth) (2, 5-7).

Taxonomy

There are more than 300 species belonging to the genus Aspergillus. Among them A. fumigatus is the most frequently occurring species in the environment (8) and also the most relevant for human disease. Below is the taxonomic classification of A. fumigatus.

Taxonomic tree of Aspergillus fumigatus (8)

Domain	Eukaryota
Kingdom	Fungi
Phylum	Ascomycota
Class	Eurotiomycetes
Order	Eurotiales
Family	Trichocomaceae
*Genus*	Aspergillus
*Species*	A. fumigatus

Domain	Eukaryota

Tissue

A. fumigatus is a spore-forming filamentous fungus that reproduces asexually by producing spores in the form of conidia. Conidial heads are typically columnar (up to 400 x 50 µm) and uniseriate. Conidiophore stipes are short, smooth-walled, and have conical-shaped terminal vesicles, which support a single row of phialides on the upper two thirds of the vesicle. Conidia grow into hyphae whose cell wall is basically composed of fibrillar β-1, 3-glucan and chitin (8).

Epidemiology

Worldwide Distribution

In the vast majority of humans, daily inhalation of A. fumigatus spores does not result in disease, thanks to effective mucociliary clearance and innate immune defenses involving neutrophils and macrophages (9). However, A. fumigatus is the causal agent of allergic and infectious diseases affecting dozens of millions of people worldwide (10).

Aspergillus-related allergic (type I hypersensitivity, IgE) diseases mainly affect asthmatic and cystic fibrosis (CF) patients, although rhino-sinusal forms may develop in otherwise nonaffected subjects (11). Aspergillus sensitization, evidenced by skin or blood specific IgE tests, is associated with poorer lung function in adults and children with asthma, CF and chronic obstructive bronchopulmonary disease (COPD) (12-14).

Hypersensitivity pneumonitis (type III hypersensitivity, IgG) due to A. fumigatus may develop in the context of occupational exposure, for example in farmers and in rural, malt, or stucco workers (15, 16).

Allergic bronchopulmonary aspergillosis (ABPA), the most severe Aspergillus-related allergic disease, has an estimated global prevalence of almost 5,000,000, affecting mostly asthmatic and CF patients (10, 17).

ABPA is estimated to affect 1-2.5% of asthmatic patients and up to 10% of CF patients (18-20). These figures may vary with the severity of asthma, comorbidities, fungal ecology, diagnostic criteria, and country of residence (21, 22). Considering the subgroup of A. fumigatus-sensitized asthma, a systematic review and meta-analysis found that ABPA diagnostic criteria were met by 40% of patients (95% CI 27–53, range 6% - 68%). A. fumigatus sensitization (AFS) itself is a frequent finding in bronchial asthma, demonstrated in 28% of patients (95% CI 24–34, range 15% - 48%). The prevalence of ABPA was found to be high (70%) in severe asthmatic patients as shown in a study among 50 severe asthmatic patients (23). Finally, the prevalence of ABPA appeared as significantly higher in industrialized than in developing countries (25.6% vs. 12.7%, p = 0.0001) (21). Also, it was reported significantly higher in adults than in children (10.1% vs. 8.9%, p<0.0001) (20).

Other allergic Aspergillus-related diseases are allergic fungal rhinosinusitis (AFRS) and severe asthma with fungal sensitization (SAFS) (24, 25).The prevalence of AFRS is reported to range from 2.7 to 19% (24). AFRS is a unique form of immune-mediated non-invasive fungal rhinosinusitis (26). It is definitely higher in arid and tropical climates, such as in the Asia-Pacific region, Australia, Thailand, Malaysia, India, the Middle East, Saudi Arabia, North Africa, and southeastern and southwestern parts of the US, especially the Mississippi basin (26, 27). Reported prevalence in general population may be as high as 100 to 500 per 100,000 in India and Israel. Climate influence is prominent, as demonstrated by a prevalence of 0.4% in Northern US states compared to over 10% in Southern ones. AFRS can be caused by A. fumigatus, but also by other Aspergillus species, e.g. A. flavus, and by dematiaceous fungi (26 - 28).

SAFS is another phenotype of severe asthma associated with fungal sensitization in adults having overlapping characteristics with ABPA. However, it is distinct from ABPA as it presents without serious bronchopulmonary impairment (bronchiectasis and mucus plugging) and is diagnosed only on exclusion of ABPA (14, 29). As per a multi-national prevalence estimate analysis of 43 reports, the average global incidence of SAFS was observed to be 154 cases/100,000 cases. Further, the incidence in South and Central America was found to be significantly higher than other continents like Asia, Europe, Africa or North America (10). Prevalence rate of SAFS due to Aspergillus species (spp.) sensitization was found to be 18.3% among the 109 uncontrolled severe asthmatic patients in a cross-sectional study in Singapore (30). Similarly a Japanese study on 124 severe asthma patients reported sensitization prevalence of Aspergillus spp.to be 11% (31). Aspergillus-related infections can be divided into invasive and non-invasive forms, with the former preferentially affecting immunocompromised patients. Invasive aspergillosis (IA) has an estimated global annual incidence of 4.10 cases/100,000 cases, and chronic pulmonary aspergillosis (CPA) is estimated at a global annual incidence of 22 cases/1,000,000 cases as per a global and multi-national prevalence study. A high mortality rate is associated with such infections (10).

Pediatric issues

Allergic A. fumigatus-related diseases such as ABPA, SAFS, and AFRS are slightly less frequent in children than in adults. ABPA occurrence in children younger than 4 years is infrequent (10).

Environmental Characteristics

Living environment

Soil is the main habitat of Aspergillus spp. which has adapted to grow in adverse environmental conditions (29). The most prominent outdoor places where it grows are plants and dead and decayed organic matter. Freshwater and marine habitats are also favorable niches. In indoor environments it is mostly found on the surfaces of buildings, household appliances, in drinking water, and on dust (2). A. fumigatus can grow at 37°C, explaining its ability to colonize the airways and induce potentially severe infections in immunocompromised hosts (32).

Worldwide distribution

A. fumigatus is a cosmopolitan, coping with virtually all environments: deserts, forests, wetland, and cultivated soils around the world (33). Further, it is a ubiquitous pathogen distributed all over the globe covering all the five continents (Europe, Asia, America, Africa and Australia) (32, 34).

Route of Exposure

Main

Large amounts of conidia are released from the mycelial mass in the atmosphere. Humans are estimated to inhale 100 to 1,000 conidia each day from the atmosphere. Due to their small size, in the absence of efficient muco-ciliary clearance, Aspergillus conidia easily reach lung alveoli and may cause colonization even in normal subjects, and respiratory tract infections in susceptible hosts (8, 35).

Secondary

Other secondary route of A. fumigatus exposure may include transmission through contaminated water, also referred to as ‘wet route of transmission’. This may include drinking of contaminated water, accidental inhalation of fluid into the lungs or even through contact with exposed wounds (36). The widespread presence of A. fumigatus in stagnant water and water supply systems in hospitals poses considerable infectious risks for immunocompromised patients (37, 38). This source of contamination from potable water supply of hospitals still poses a hazard risk for immunocompromised patients as per a recent study (39).

Clinical Relevance

Aspergillus spp. is known to cause a spectrum of lung disorders including allergic (AFRS, ABPA, IgE-mediated asthma, SAFS) and infectious diseases (invasive aspergillosis, chronic pulmonary aspergillosis) (17, 25, 40).

Allergic rhinitis and rhinosinusitis

AFRS is classified as a form of non-invasive, eosinophilic, chronic fungal rhinosinusitis (27, 28). From a pathophysiologic viewpoint, AFRS is closely related to ABPA, exhibiting inflammation, Th2-skewed anti-A. fumigatus lymphocyte responses with eosinophil involvement, while IgE production is inconsistently demonstrated. AFRS is rarely found alone, as it has been demonstrated that 66% of AFRS patients may have co-existing allergic rhinitis (AR), 90% of them are fungi-sensitized (increased IgE levels) and approximately 50% may have co-existing asthma (26, 27). Sensitization is more frequent in patients with atopic diseases; conversely, apparent monosensitization to molds is infrequent. In atopic patients, fungal antigenic material is released and induces a chain of immunologic reactions favoring the development of AFRS. In a study on 131 AR patients, 32 (24.4%) displayed Aspergillus sensitization, of whom 28 had associated sinusitis; however, only 2 patients met all the criteria for allergic Aspergillus rhinosinusitis (27, 41). AFRS can be complicated by concomitant fungal presence and persistence in non-invasive forms, e.g. mycetoma (aspergilloma), a fungal ball in the sinonasal cavities (29, 42), or even invasive forms with local or systemic infection. The latter group is beyond the scope of the allergy referral, as their treatment relies on antifungal medication and surgery (24, 42).

Asthma

The two most important conditions associated with sensitization to Aspergillus spp. in asthmatic patients are ABPA and SAFS (25, 43).

Allergic bronchopulmonary aspergillosis (ABPA)

ABPA is an immune-mediated lung disease due to hypersensitivity to Aspergillus that mostly occurs in individuals suffering from asthma and CF (17, 43), however it can also occur in non-asthmatics (40). Sensitized individuals may develop allergic reactions on recurrent inhalation of Aspergillus spores, that is mainly a type I (IgE-mediated) allergic reaction. Additionally, type-III (IgG-mediated immune complex) and type IV (cell-mediated, mainly eosinophils) reactions also play a role (25, 40, 43).

Typically, ABPA follows a flare and relapse pattern. Asthma exacerbations, poor asthma control, recurrent pulmonary infiltrate resulting in irreversible bronchial remodeling with bronchiectasis form the natural history of ABPA. Another distinctive feature of ABPA is expectoration of brownish mucous plugs (mucus along with fungal hyphae and eosinophils), observed in 5-69% of the patients and representing the clinical counterpart of the computed tomography scan “mucoid impactions” (17, 40). Asthma severity in ABPA patients can range from mild intermittent asthma to severe persistent asthma that may be corticosteroid dependent (19). In CF patients, ABPA-related exacerbation with reduced lung function can be accompanied by hemoptysis, increased microbial colonization, pneumothorax, and worsened nutritional status (23). In both asthmatic and CF patients, untreated ABPA results in severe lung damage with central bronchiectasis, fibrosis and respiratory failure (17, 40).

The diagnosis of ABPA in asthma or CF patients is complicated due to overlapping clinical features and involves clinical history, immunological findings (such as increased levels of both total serum IgE and A. fumigatus–sIgE as well as Aspergillus-specific IgG), radiological observations (17, 23). The ISHAM (International Society for Human and Animal Mycology) working group has proposed a classification of ABPA in seven stages, ranging from asymptomatic (serological) expression to respiratory failure (17, 40).

A prospective study on 35 severe asthmatic ABPA patients reported the prevalence of serological ABPA at 68.5%, compared 31.4% for that of the full-blown form of ABPA with bronchiectasis (23).

Severe asthma with fungal sensitization (SAFS)

Aspergillus species sensitization has been reported to enhance asthma severity, resulting in worsening of asthma control. A subset of patients diagnosed with severe asthma and fungal sensitization was defined as SAFS (30, 31, 44). In contrast to ABPA, SAFS does not result in mucoid impactions or bronchiectasis. Additionally, it is associated only with severe asthma, unlike ABPA that can also occur in mild and moderate asthmatic patients (25).

A retrospective study conducted in United Kingdom (UK) among 135 patients with severe asthma reported a prevalence of 60% for SAFS due to A. fumigatus (44). In Singapore and Japan, the reported prevalence of Aspergillus spp. sensitization among severe asthmatic patients was 11.7% (n=206) and 11% (n=124) respectively (30, 31). The occurrence of SAFS was reported at a younger age as compared to patients without fungal sensitization (31). In accordance to this study, another study conducted in UK on 82 children (4-17 years) reported presence of SAFS in 46% children with onset of asthma at early age (45).

Atopic Dermatitis (AD)

A cross-sectional study in China conducted among 59 AD patients without respiratory disease found Aspergillus spp. sensitization in 81.4% of patients along with other fungal sensitizations like Penicillium spp. or Candida spp. (46). The contribution of cross-reactions is probably important as suggested by a retrospective study conducted in the Czech Republic, which found only 8% of 60 AD patients were sensitized to Asp f 1, a marker of genuine sensitization to A. fumigatus (47).

Other Diseases

Due to its ability to interact with multiple players of the host immune response, A. fumigatus induces a continuous spectrum of allergic and infectious diseases (10). (see above, “Epidemiology” section).

Prevention and Therapy

Prevention Strategies

Avoidance

Aspergillus spp. is an environmental pathogen (health-care-acquired linked with humid and dusty environment), infection may occur due to contamination of the health-care facility. The implicated environmental vehicles are mainly air filters, air conditioners, damaged ventilation system, air filter frames etc.(48). To prevent the spread of A. fumigatus spores, HEPA filtration limits the conidial air counts (36). A protected environment with high efficiency HEPA filtration along with laminar flow, least air leakage in the room, increased number of hourly air changes, as well as regular maintenance of HEPA filtration systems are recommended in hospital rooms (particularly for immunocompromised patients) (48-50).

Molecular Aspects

Allergenic molecules

Fungal species are characterized by a large array of allergenic molecules, and A. fumigatus is an outstanding example, with 23 characterized allergens featured in the WHO/IUIS database accessed on February 8^th, 2021 (51). The allergens of A. fumigatus are difficult to classify as major and minor allergens owing to high variation in the sensitization pattern among different allergic manifestations of A. fumigatus (52). (pg 131). The table below provides the information of the allergenic molecules of A. fumigatus identified by WHO/IUIS (51).

Allergens	Molecular weight	Biochemical name	Allergenicity
Asp f 1	18 kDa	Mitogilin family	· Major secreted allergen (53, 54) · Species specific and marker of genuine sensitization (54, 55). · Not present in spores, produced after gemination process (55) · Significant in asthmatic and ABPA patients (54). · Recognised by 83% of ABPA patients and 45% of A. fumigatus allergic patients (52) (pg 133)
Asp f 2	37 kDa		· Major allergen (54). · Species specific and marker of genuine sensitization (54, 55). · Intracellular allergen solely identified by ABPA patients (asthma and CF) (52). (pg 366) · Recognised by 100% of ABPA patients while none of the A. fumigatus allergic patients (52). (pg 133)
Asp f 3	19 kDa	Peroxysomal protein	· Species non-specific (54). · ABPA-related intracellular protein (54). · Recognised by 88% of ABPA patients and 52% of A. fumigatus allergic patients (52) (pg 133). · May be helpful in differentiating between ABPA and non-ABPA patients (54).
Asp f 4	30 kDa	unknown	· Species specific (55, 56) · Specific for ABPA diagnosis (53) · ABPA-related intracellular protein solely recognized by ABPA patients (asthma and CF) (52) (pg 132 and 134). · Recognised by 80% of ABPA patients while none of the A. fumigatus allergic patients (52) (pg 133).
Asp f 5	40 kDa	Metalloprotease	· Recognised by 93% of ABPA patients and 74% of A. fumigatus allergic patients (52) (pg 133).
Asp f 6	26.5 kDa	Mn superoxide dismutase	· Species non-specific (54). · Specific for ABPA diagnosis (53) · ABPA-related intracellular protein solely recognized by ABPA patients (asthma and CF) (52) (pg 132 and 134). · High IgE levels observed in patients with comorbid atopic dermatitis in A. fumigatus sensitized asthmatic patietns (57) · Recognised by 55% of ABPA patients while none of the A. fumigatus allergic patients (52) (pg 133).
Asp f 7	12 kDa		· Recognised by 46% of ABPA patients and 29% of A. fumigatus allergic patients (52) (pg 133).
Asp f 8	11 kDa	Ribosomal protein P2	· Recognised by 15% of ABPA patients and 8% of A. fumigatus allergic patients (52) (pg 133).
Asp f 9	34 kDa		· Recognised by 89% of ABPA patients and 31% of A. fumigatus allergic patients (52) (pg 133).
Asp f 10	34 kDa	Aspartate protease	· Recognised by 28% of ABPA patients and 3% of A. fumigatus allergic patients (52) (pg 133).
Asp f 11	24 kDa	Peptidyl-prolyl isomerase	· Recognised by 90% of A. fumigatus allergic patients (52) (pg 133).
Asp f 12	90 kDa	Heat shock protein P90	· Recognized by serum IgE and IgG from ABPA patients (58, 59)
Asp f 13	34 kDa	Alkaline serine protease	· Detected in the airway of AIA patients and may co-relate with asthma severity (60).
Asp f 15	16 kDa	Unknown	· NA
Asp f 16	43 kDa	Unknown	· Recognised by 70% of ABPA patients (52) (pg 133).
Asp f 17	27 kDa		· NA
Asp f 18	34 kDa	Vacuolar serine protease	· Recognised by 79% of A. fumigatus allergic patients (52) (pg 133).
Asp f 22	46 kDa	Enolase	· Recognised by 86% of A. fumigatus allergic patients (52) (pg 133).
Asp f 23	44 kDa	L3 ribosomal protein	· Identified following IgG and IgE screening of cDNA library with sera from ABPA patients (61)
Asp f 27	18 kDa	Cyclophilin	· Recognised by 75% of ABPA patients (52) (pg 133).
Asp f 28	13 kDa	Thioredoxin	· Recognised by 30% of ABPA patients (52) (pg 133).
Asp f 29	13 kDa	Thioredoxin	· Recognised by 50% of ABPA patients (52) (pg 133).
Asp f 34	20 kDa	PhiA cell wall protein	· Recognised by 93% of ABPA patients and 46% of A. fumigatus allergic patients (52) (pg 133).

Allergens	Molecular weight	Biochemical name	Allergenicity

kDa: kilodaltons, IgE: Immunoglobulin E, AIA: A. fumigatus sensitized asthma ABPA: Allergic bronchopulmonary aspergillosis, CF: Cystic fibrosis, NA: Not available.

Biomarkers of severity

Major allergens Asp f 1 and Asp f 2 are considered as markers of genuine sensitization to A. fumigatus and could help differentiate primary sensitization from cross-reactive sensitization (54 - 56). However, sensitization to Asp f 1 and Asp f 2 is reported in both ABPA and asthmatic patients (54). IgE reactivity to Asp f 3, Asp f 4 and Asp f 6 is considered specific for ABPA in the context of genuine sensitization (IgE to Asp f 1 or Asp f 2), but Asp f 3 and Asp f 6 are cross-reactive and not specific for A. fumigatus sensitization, while Asp f 4 is said to be specific for A. fumigatus (54, 55). Thus, combined detection of IgE reactivity to all 5 allergens of A. fumigatus can help to identify A. fumigatus sensitized ABPA patients (54).

Furthermore, IgE reactivity to Asp f 1, Asp f 4, and Asp f 6 was found to be higher in ABPA patients as compared to asthmatic patients. Thus, the levels of IgE antibodies towards all 5 allergens could help to differentiate between ABPA and A. fumigatus sensitized asthma patients (53). In addition, mono-sensitization to Asp f 6 was reported in association to AD, possibly as a result of cross-reaction with fungi other than A. fumigatus, thus possessing a very low risk of ABPA (55).

Cross-reactivity

Extensive cross-reactivity is linked to the homology between allergens found in A. fumigatus and other allergenic molds, mainly Cladosporium, Penicillium, Alternaria and Malassezia.

The table below provides the list of most common cross-reactive A. fumigatus allergens with other fungal allergens (55):

A. fumigatus allergen	Fungal Homologs IUIS allergens	Cross-reactive species
Asp f 3	Pen c 3 Cand b 2 Mala f 3, 4	Penicillium citrinum Candida boidinii Malassezia furfur
Asp f 5	-	Aspergillus oryzae (52) (pg 134).
Asp f 6	Alt a 14 Mala s 11	Alternaria alternata Malassezia sympodialis
Asp f 8	Alt a 5 Cla h 5	Alternaria alternata Cladosporium herbarum
Asp f 12	Alt a 3 Mala s 10	Alternaria alternata Malassezia sympodialis
Asp f 13	Pen ch 13	Penicillium chrysogenum (53)
Asp f 18	Pen ch 18	Penicillium chrysogenum (53)
Asp f 22	Pen c 22 Alt a 6 Cla h 6	Penicillium citrinum Alternaria alternata Cladosporium herbarum
Asp f 23	Pen b 26 Alt a 12 Cla h 12	Penicillium brevicompactum Alternaria alternata Cladosporium herbarum
Asp f 27	Mala s 6	Malassezia sympodialis
Asp 28, 29	Mala s 13	Malassezia sympodialis

A. fumigatus allergen	Fungal Homologs IUIS allergens	Cross-reactive species

Explained Results

Allergen information

A. fumigatus is a common aeroallergen of the indoor and outdoor environments. It is a saprotroph and opportunistic filamentous fungus (1). Major allergens Asp f 1 and Asp f 2 are markers of genuine sensitization to A. fumigatus (54).

Clinical information

A. fumigatus is the causal agent of potentially severe allergic (allergic rhino-sinusitis, ABPA, IgE-mediated asthma, SAFS) and infectious diseases with high rates of morbidity and mortality (17, 25, 40).

Cross-reactivity

Aspergillus fumigatus shows important cross-reactivity, mainly with other fungi and yeast species (52, 53, 55).

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