mold-sensitive asthma

CLIN REV ALLERGY 183 3:183-196, 1985

Mold-Sensitive Asthma

Manuel Lopez and John E. Salvaggio

It has long been established that fungal spores are present in the atmosphere in high concentration, considerably in excess of pollen grains. For example, Cladosporium spores can outnumber pollen grains by a ratio of 1000 : 1. The sources of atmospheric fungal spores include, dying vegetation, leaves, stems, foliage, and branches of living plants, as well as large macroscopic fungi, such as the mushroom family. Throughout the years, pollen has been widely studied as an aeroallergen, but less is known about fungal spores. Many fungal spores are known to induce respiratory reactions in sensitized individuals, as demonstrated by the presence of wheal and flare skin reactivity and provocative inhalation challenge testing. Most studies of fungi as aeroallergens have been limited to the Fungi Imperfecti class, with little information available concerning the possible allergenicity of members of other fungal classes. For example, the Basidiomycete class, which numbers between 20,000 and 25,000 species, and has a reported high atmosphere spore concentration in many areas of the world, 1~ has only recently begun to be studied. 4 The literature is also still inadequate with regard to the production of allergic asthma by inhaled fungal spores, and there is little information available on characterization of antigenic fractions of common fungal aeroallergens, even though extracts of many species of the Fungi Imperfecti are used routinely for skin testing and immunotherapy. 5-9 Thus, the clinical impact of airborne fungi on asthma is not accurately known, and the characterization and stand- ardizafion of allergens from a wide range of fungal species will be necessary before this type of information can be obtained.

C la s s i f i ca t ion a n d T a x o n o m y

The Eumycetes, or true fungi, are members of the phylum Thallophyta i.e., plants that lack definite roots, stems, or leaf structures. Some taxonomists, however, place them in a kingdom distinct from both plants and animals, although the presence of cell walls reflects a closer association with plants. Unlike algae, they do not contain chlorophyll. They may function as either obligate or facultative parasites, but are unable to s);nthesize their own food and depend on other organisms for nutrients. In spite of their tremendous

From the Clinical Immunology Laboratory (M.L.) and the Department of Medicine (J.E.S.), Tulane University School of Medicine, New Orleans, Louisiana.

Send correspondence and requests for reprints to Manuel Lopez, MD, Clinical Immunology Laboratory, Tulane University School of Medicine, New Orleans, LA 70112.

© 1985 Elsevier Science Publishing Co., Inc. 52 V~mderbilt Ave., New York, NY 10017

0731-8235/85/$03.30

184 M. Lopez and J. E. Salvaggio

variation in morphology, only two primary structural units exist: 1) common fungi, composed of branching hyphae often divided by septa (aggregates of these hyphae are collectively referred to as mycelia), and 2) yeasts, which represent a minority of species, grow as single cells, and reproduce by an eccentric process of budding to form daughter cells or by central division.

The major classes of fungi that predominant ly contribute to the airborne spore load are discussed below (Table 1).

Table 1. Classification of Eumycetes

Phylum Thallophyta Major classes that contribute to airborne spore load:

1. Phycoinycetes Rhizopus Mucor

2. Ascornycetes Saccharomycetacea

Saccharomyces Torulaceae

Rhodotorula Chaetorniaceae

Chaetomium

3. Basidiomycetes Arnanitaceae Tricholomataceae Rhodophyllaceae Agaricaceae Coprinaceae

4. Fungi Imperfecti orders 1. Dematiaceae

Alternaria Curvilaria Spondilocladium Helminthosporium Cladosporium Sternphylium Nigrospora Pullularia

2. Moniliaceae AspergiUus Botrytis Glioicladiurn Monilia Mycogone Penicillium Paecilomyces

3. Sphaeriodiaceae Phoma

4. Tuberculariaceae Fusarium

Mold-Sensitive Asthma 185

Class Phycomycetes Of this class, the subclass oomycetes is of little allergenic importance. A second subclass, the zygomycetes, which includes the common sugar and bread molds and contains the important genera Rhizopus and Mucor, is clinically important. Many species of this class are prominent in soil, composts, and food residues.

Class Ascomycetes Ascomycetes reproduce sexually by cytoplasmic fusion, followed by formation of an ascus or sack in which sexual spores (ascospores) develop without any permanent wall attachment. They reproduce asexually, forming conidia, either by budding, fragmentation, or formation of special hyphae (conidiophores). The black molds, yeasts, blue molds, and cup fungi are important groups of this class. Saccharomyces, a yeast, and Chaetomium are members of this class that are known to produce allergic respiratory symptoms in humans.

Class Basidiomycetes These are the club fungi, so-called because after mycelial growth, a fruiting body is formed and club-shaped structures called basidia develop where basidiospores are produced. The Basidiomycetes reproduce asexually by means of conidia and fragmentation of hyphae. Sexual reproduction occurs by means of cytoplasmic fusion, formation of the fruiting bodies, and development of the basidia. This class comprises many diverse forms, including puffballs, common rusts and smuts, mushrooms, and mirror yeasts. The great majority of Basidiomycetes, like most Ascomycetes, do not grow on common laboratory media. They feed on decaying organic matter and wood or exist as parasites of cereal grains, other plants, and trees. Abundant small, spiny smut telio- spores and rust urediospores are often quite prominent in the atmosphere and may be discharged in bursts during times of high humidity. Although little is known about the relationship between basidiospores and asthma at present, they have recently been shown to be allergenic in humans and im- munogenic in the rabbit.* Atopic asthmatics may demonstrate IgE-mediated hypersensitivity to Basidiomycete antigens, as evidenced by positive immediate wheal and flare skin reactivity and/or radioaUergosorbent test (PAST). Thus, awareness of the potential importance of basidiospores as aeroaUergens should become more apparent in the future, especially in view of their high atmospheric concentrations during the summer and fall months.

Class Deuteromycetes, or Fungi Imperfecti This class is the largest and most diverse and contains most of the fungi allergenic for humans. In this class, sexual reproduction is extremely rare, and the means of common asexual reproduction is by conidia. Their nutrition


is either by parasitic means on plants and occasionally animals or saprobic on decaying organic matter. Four orders are commonly distinguished: sphae- riopsidales, of which the genus Phoma is a common allergenic mold; melan- oconiales, which contains important plant pathogens but no known human aeroallergens; and moniales, which contains three families accounting for a large number of the fungi causing allergic respiratory disease in humans. "Mycelia Sterilia" is a descriptive term for all mycelia that grow on culture plates without spores or fruiting body production. A few of these fungi are derived from basidiospores and ascospores, but most lack known affinities with perfect forms. Many additional organisms fail to sporulate on culture media in spite of excellent growth.

Atmospheric Concentration The air spore content comes mainly from species highly adapted to using wind energy for their dispersal. Wind dispersal of spores has three prindpal stages: 1) spore liberation, 2) dispersion, 3) deposition. The particular species and concentration represented in the air at a given time depends on temperature, degree of precipitation, prevailing winds, seasonal climatologic factors, circadian patterns of sunlight and darkness, availability of substrates, and degree of both substrate and atmospheric moisture. For example, the vegetative hyphae of most fungi grow best between 18 ° and 32°C, whereas other thermotolerant fungi, such as several of the Aspergillus species, grow over a wide temperature range, and certain thermophilic forms grow best between 65 ° and 75°C. The growth and dispersal of some spores, such as basidiospores, is also markedly affected by atmospheric moisture. These spores are propelled into the atmosphere by a process dependent on the presence of free water, and they increase markedly in concentration during periods of rainfall and dampness. Conversely, spores of other fungi, such as Clados- porium, Alternaria, and Helminthosporium, are blown free by wind, and this type of "dry spore" increases in concentration with diminishing humidity and increased air flow. These species are often abundant during the mid-day period of maximal sunlight. Fungi also readily invade and propagate inside homes, and spores from these sources are often a source of perennial allergic symptoms. Areas of mold growth in the interior of homes are frequently noted, particularly growth of certain Deuteromycete species such as Penicil- lium and Aspergillus. Many specific interior areas are known to be prime sites for domestic mold growth. Among these are garbage containers, food storage areas, upholstery, wallpaper, areas of increased moisture such as shower curtains, window moldings, portable window units, and damp base- ments. Emissions from cold mist vaporizers, in which high levels of fungi particles are present, have been implicated in production of asthma in mold- sensitive patients. 1°

Sampling for airborne fungal spores is important for several reasons. It can establish airborne spore loads for different environments and it can aid in disease diagnosis, as spore prevalence and composition may suggest the appropriate fungal extracts for skin testing and/or provocative inhalation challenge testing. Sampling may indirectly aid in the evaluation of the effective-


ness of eliminating allergen sources, particularly with regard to indoor spore concentrations. The availability of data on spore counts may also allow correlation between airborne spore loads, climatologic variables, and sudden increases in asthma in particular geographic, occupational, or environmental settings. Such comparisons have been particularly useful in studying certain forms of epidemic asthma, such as New Orleans asthma. 1H3

Many methods are available to identify and quantitate airborne spore loads. The simplest, but least accurate, method employs the Durham gravitational sampler, which allows the gravitational deposition of spores on a sticky slide. This methods tends to obscure the prevalence of small spores in favor of larger heavier ones. Simple gravitation onto Petri plates followed by culture has also been widely employed in the past. This method limits the spores identified, depending on their growth characteristics. For example, species of the Fungi Imperfecti class grow very well in standard culture media, in contrast to Basidiomycete spores, many of which do not grow in artificial media. A variety of other devices are available for more accurate semiquan- titation of spores, including rotating impactors (rotorod, rotobar, and rotoslide samplers) and suction impactors (Hirst spore trap and its modification, the Burkard sampler) (Fig. 1). A particularly useful device for spore studies is the Anderson sampler, which combines suction and impaction methods with a nutrient Agar Petri Dish culture method. In this sampler, a series of Petri dishes are placed atop one another in close proximity. Air passes through a series of sieves, with pores of decreasing diameter located between the plates, so that larger spores are deposited on the first plate and smaller size spores on the other plates, depending on the size of the sieve. Details of these and other sampling methods have been well described elsewhere.~4

Airborne surveys have been performed in many areas throughout the world. In a study by Gregory and Hirst, 2 the mean spore concentrations between June and October was 125,000 spores/m 3. The most common spore type was Cladosporium, which accounted for 47% of the total; basidiospores were the second most common, with 31% and Alternaria amounted to 2% of the catch. In contrast, pollen made up 1% of the total count. Atmospheric mold counts in Canada by Collins-Williams and Beit ~s reported Alternaria, Cladosporium, and yeasts as the most numerous molds, with a peak season between the middle of July and October. Basidiomycetes were quite prominent in July and August. In the United States, several studies have shown that Cladosporium Altemaria, and basidiospores are the most common atmospheric spores.~6"17 The indoor pattern of fungal spores, which is important to mold-sensitive patients, reflects the outdoor spore composition plus an independent intrinsic fungal flora dominated by small spored Deuteromycetes, including Penicil- lium, Aspergillus, Rhizopus, Mucor, and yeasts. ~s

S e a s o n a l P a t t e r n s

A defined "season" for molds often does not exist because of their universal presence in the atmosphere and their marked adaptability to environmental conditions. Outdoor levels range from peaks in late summer and fall to very low concentrations during periods of snow cover. In the Gulf South area of


Figure 1. Common air samplers. (A) Durham Gravitational; (B) rotorod samplers; (C) Bur- kard sampler.

the United States, many ascospores and spores of the Fungi Imperfecti appear during the initial warming periods in early spring. 19 After this initial peak, they persist throughout the summer months, often with a second peak during the fall months, followed by slight diminution during the cooler months of December and January. Basidiospores are also present throughout the year, with peaks during the humid, moist, late summe~" and early fall months. In the Great Lakes areas, various ascospores appear during early spring and disappear by midsummer, whereas others persist throughout the growing season. Mold-sensitive patients have perennial symptoms, often with exac-


erbations during the midsummer and fall season. In areas with cold winters, a significant relief of symptoms occurs after the first snowfall.

Cl in ical R e l e v a n c e o f M o l d s in A s t h m a

The role of fungi in asthma has long been considered to be established, based on anecdotal evidence. Although positive immediate and delayed skin reactivity is obtained with crude extracts of many fungal species, the clinical relevance of the reactivity is often uncertain. Because most atopic individuals demonstrate broad patterns of wheal and flare skin reactivity to multiple pollen, fungal, animal dander, and house dust allergens, the contribution of fungal allergens to production of symptoms in any given patient is often difficult to assess. This is due to many factors, among which are the following: 1) The choice and method of preparing fungal extracts for skin testing and provocative challenge has varied markedly among investigators. 2) The quality and potency of mold allergenic extracts has often been poor and variable. 3) Relatively few organisms have been studied in detail, and many common fungi still need initial clinical evaluation and testing. 4) A single, well-defined, "mold season" usually does not occur, making it especially difficult to select a study population with unique seasonal mold allergy.

Although there is an agreement among allergists that inhaled fungi are responsible for production of upper and lower respiratory tract symptoms, there is a paucity of data on the actual incidence of asthma induced by inhaled fungal spores. Efforts to correlate symptom patterns with sensitivity to fungi have been difficult due to the complexity of the airborne spore load as well as the lack of a clear-cut mold season with prolonged exposure to a variety of fungi. Therefore, clinical impressions are based primarily on patterns of skin reactivity to a wide range of poorly characterized and standardized fungal extracts, plus anecdotal evidence relating to the development of symptoms in areas where heavy atmospheric concentrations of fungal spores are known to be present. The respiratory symptoms produced by fungi are thought by some to be delayed in onset and chronic, rather than acute, as induced by pollen. By inhalation challenge, three types of airway response to inhaled fungi extracts may be demonstrated, particularly with Aspergillus and Can- dida allergens. 26"27 These consist of: 1) immediate bronchospasm, which re- solves within 1-2 hr; 2) immediate bronchospasm, followed after 4--6 hr by recurrence; and 3) late asthmatic reaction, appearing after 4-6 hr of challenge without a preceding immediate reaction. In such challenge studies, both the immediate and subsequent late reactions can be blocked with cromolyn sodium and adrenergic agonists, whereas only the late responses can be ab- rogated with corticosteroids.

The following are the most commonly incriminated allergenic fungal species:

1. Cladosporium The most widely distributed fungi, often reaching atmosphere concentrations up to 5000 spores/m.

2. Alternaria--Known to be highly allergenic based on wheal and flare skin reactivity. A specific allergen (Alternaria 1) has been isolated and characterized. 33


3. Aspergillus--Widely distributed in outdoor as well as indoor environments. Aspergillus fumigatus is the etiologic agent of allergic broncho- pulmonary aspergillosis.

4. Helminthosporium--Often gives rates of wheal and flare skin reactivity equal to those elicited by Alternaria.

5. Epicoccum--Prevalent in temperate regions, especially grassland and agricultural areas. In certain surveys it has been described as the most important source of spores isolated from the atmosphere, along with Ctadosporium and Alternaria.

6. Fusarium, Penicillium, and Geotrichum. 7. Several of the zygomycetes, such as Rhizopus and Mucor; these are

often abundant in damp basement areas. 8. Several members of the ascomycetes, airborne concentrations of which

often reach several thousand per meter of air. 9. Many of the Basidiomycetes, which are being found with increasing

prevalence in the atmosphere in worldwide studies.

In general, because there is no clear mold season, the diagnosis of mold allergy is based on the identification of sensitivity by skin test, RAST, and inhalation challenge.

D i a g n o s i s of F u n g a l A l l e r g y a n d D e t e c t i o n of F u n g a l A l l e r g e n s

Skin Reactivity Wheal and flare skin reactions to crude allergenic extracts of fungi have long been documented to occur in asthmatic patients, and skin testing with fungal allergens is routinely employed in most allergy clinics. However, because of the lack of characterization, purification, and standardization of fungal allergens, skin test results continue to be highly variable. It is known that the same strains of an organism grown under identical culture conditions and for the same time period can produce extracts that vary markedly in potency, s,2° A recent study by Aas et al. 21 of patients with suspected mold allergy using skin testing with various commercial extracts showed that the incidence of positive tests to Cladosporium herbarium varied from 12% to 65%, depending on the extract employed. Cultures of Aspergillus fumigatus grown under controlled conditions may show differences in gross appearance and antigenic composition. 22 Because of this variability in allergenic extracts, it has been suggested that standardized pools of a wide variety of strains be employed for skin testing to ensure the presence of a sufficient number of determinants from a representative group of strains of a particular species.

There is a great variation in the reported frequency of positive skin reactivity to fungal extracts in patients with asthma from different countries, varying between 3% and 4% in studies from Sweden and Switzerland to above 80% in certain studies in the United States. 2~-z5 There are many potential reasons for these varying results, including the general lack of standardization and characterization of extracts, plus the fact that neither wheal and flare skin reactivity to fungal antigens nor perennial symptoms alone provide definite


proof that clinically relevant fungal allergy is present. In such cases, inhalation provocative testing can be of great benefit.

Inhalation Challenge

Provocation testing by inhalation can establish a definitive cause and effect relationship and make a more objective evaluation of the significance of positive skin reactions possible. However, there have been relatively few studies of provocation challenge tests with mold extracts, and the results have often been ambiguous because of the lack of standardized antigens and inhalation challenge techniques. Pepys and Hutchcroft 26 have described bronchial reactivity following inhalation provocative challenge testing of patients with asthma who exhibited immediate wheal and flare reactivity to Candida albicans. Some patients had immediate asthmatic symptoms, a second group demonstrated immediate symptoms followed by further asthmatic reactions after 8-16 hr, and others demonstrated only late asthmatic responses. Similar results were obtained by Itkin and Dennis, 27 who noted a rough correlation between positive bronchial reactivity and skin reactivity to Candida antigens. The potential diagnostic value of provocation tests in asthma associated with suspected fungal hypersensitivity has also been emphasized in other areas of the world. ~-~°

Serologic Studies RAST and ELISA. The RAST technique, although employed routinely to

detect IgE antibodies against various pollens, mites, danders, and other in- halants, is still not used with great confidence to identify fungal IgE antibodies. With this technique, allergens are coupled to a solid phase by means of (cyanogen bromide) activation of polysaccharide polymers on cellulose discs with the formation of a reactive amino group. Soluble protein allergens may then be covalently bound to the reactivated polymer through an amino group on the allergens. Although results of RAST with Alternaria and related fungal species suggest that these allergens contain free amino groups, many fungal allergens may also contain polysaccharides that cannot be readily coupled to cellulose discs. The use of better characterized antigens have given better RAST values. For example, studies by Yunginger et al. 31~ with crude Alter- naria extracts have indicated that a major allergenic fraction, with a molecular weight in excess of 5000 daltons, elutes in the void volume of Sephadex G25. Using this protein- and carbohydrate-containing peak, these investigators were able to produce satisfactory and strongly positive RAST results with the sera of Alternaria-sensitive subjects. Potency of these Alternaria extracts, as measured both by RAST inhibition and direct RAST, was also strongly cor- related to potency as measured by skin testing. In contrast, however, neither weight per volume nor protein nitrogen content of crude extracts were related to allergenic potency as measured by skin testing or by RAST, and many crude commercial extracts of Alternaria were shown to differ in allergen content by as much as 3000-fold. In a similar manner, Aukrust and Aas 34 have shown that better allergens can be obtained using a partially purified prep- aration of Cladosporium herbarium than when using a crude extract. The overall


results using Alternaria and Cladosporium antigens show that RAST can be used to detect IgE antifungal antibody, particularly when partially characterized and purified allergen preparations are employed.

Other techniques, such as enzyme-linked immunoabsorbent assay (ELISA), have been successfully employed for detecting IgE antibody, as well as antibodies of other immunoglobulin classes, against fungal antigens o!f a pre- dominantly polysaccharide nature. 35"36 This assay depends on the fact that antigens can be covalently attached to a solid-phase support, such as cellulose or polyacrylamide, and that satisfactory passive adsorption can also be obtained to tubes, beads, or microplates made of nylon, polystyrene, polyvinyl, or polypropylene. The antibodies are linked to an enzyme, with the complex retaining both immunologic and enzymatic activity. Antigen-antibody reactions can be detected by adding the specific enzyme substrate and measuring the color change. This technique is less expensive, uses reagents of longer shelf life, and can detect nonprotein antigens.

CIE and CRIE. These techniques are particularly useful for the identification of individual allergens in crude extracts. 37 Initially, standard two-dimensional crossed-immunoelectrophoresis (CIE) is performed by migrating crude fungal extract horizontally in 1% agarose gel, followed by a second vertical migration in gel sections containing the appropriate rabbit anfifungal serum. The different antigens in the mixture are precipitated by the antiserum and can be seen as arcs or cones (Fig. 2). In cross-radioimmunoelectrophoresis (CRIE), the plates from CIE are incubated with patient serum, and specific IgE antibodies bind to the allergen containing precipifin cones. These allergen-IgE precipitates are visualized by a second incubation with 125I--anfi-IgE and subsequent autoradiography on x-ray films (Fig. 3). From patterns of IgE binding in CRIE, it is possible to draw conclusions regarding the importance of individual allergens in mold extracts as contrasted with the many nonallergenic antigens. It is also possible to gain information on the frequency of patients having specific IgE antibodies against a particular fungal allergen, and by this method, define the major allergens for each fungal extract. Through the use of CRIE, a tracer system can be developed to monitor each important fungal allergen during purification and to quanfitate them when comparing different raw materials. This facilitates both the comparison of different raw materials in a meaningful manner and the controlled purification of extracts without loss of important allergens.

T h e r a p e u t i c C o n s i d e r a t i o n s

The elimination of fungal spores from the environment is a virtually impos- sible task due to the ubiquitous occurrence of fungal spores in both indoor and outdoor environments. However, exposure can be decreased in certain industrial, environmental, occupational, or peculiar geographic situations where particularly high concentrations of fungal spores are present. Pharmaco- therapy for mold-Lnduced asthma is the same as that for asthma caused by other inhaled allergens, such as pollen. These include the use of conventional bronchodilators, the prophylactic use of cromolyn sodium, and corticosteroids applied topically to the respiratory mucosa or used systemically. The role of


Figure 2. Examples of Fungi Imperfecti spores. (A) Alternaria, (B) Curvularia, (C) Helminthosporium, (D) Cladosporium, (E) Stemphylium.


Figure 3. Examples of Basidiomycete spores. (A) Rust spores, (B) Ganoderma ( x 50), (C) Ustilago (smut) (x 50), (D) Rhodophyllus (x 20).


i m m u n o t h e r a p y in m o l d - s e n s i t i v e a s t h m a is h ighly con t rovers ia l , a n d the re is a v i r tua l ab sence of p u b l i s h e d da ta to pe rmi t f i rm conc lus ions in this regard. 38 T h e ques t i on of i m m u n o t h e r a p y for f u n g a M n d u c e d a s t h m a clear ly requ i res s tudy , as it is l ikely tha t funga l spo res are a m a j o r cause of b ronch ia l a s t h m a in cer ta in c l imates . The re are severa l ma jo r p r o b l e m s in cons ide r ing s tud ies of i m m u n o t h e r a p y w i t h m o l d extracts . The i r var iabi l i ty a n d low quality m a k e it difficult to eva lua t e resul ts , a n d it is p r o b a b l e tha t i m m u n o t h e r a p y wi th the cu r ren t ly avai lab le funga l ex t rac ts will no t inc lude a suff ic ient ly b r o a d s p e c t r u m of a p p r o p r i a t e a l lergens . T h e complex i ty of funga l ex t rac ts will f u r the r c o m p o u n d the se p r o b l e m s , as c o m p a r e d to o the r a l lergenic extracts , funga l extracts are c o n s i d e r a b l y m o r e c o m p l e x and var iable . Thus , the n u m b e r of d i f fe ren t an t igen ic d e t e r m i n a n t s t ha t the pa t i en t ' s i m m u n e s y s t e m has to h a n d l e s i m u l t a n e o u s l y is v e r y h igh. This m a y lead to a lesser i m m u n e re- s p o n s e t h a n w o u l d h a v e b e e n ob t a ined if a s ingle a l lergenic f rac t ion h a d b e e n injected. In spi te of t he se l imi ta t ions , clinical a n d anecdo ta l e v i d e n c e does a t tes t to the fact t ha t m a n y pa t i en t s w i t h b r o a d p a t t e r n s of w h e a l a n d flare skin reac t iv i ty to c r u d e funga l ex t rac ts a n d bronchia l a s t h m a i m p r o v e af ter i m m u n o t h e r a p y w i t h c rude funga l allergefiic extracts . Al lergis ts t h r o u g h o u t the c o u n t r y t rea t m o l d - i n d u c e d a s t h m a w i t h i m m u n o t h e r a p y o n a regu la r basis , a l t h o u g h aga in , it m u s t be e m p h a s i z e d tha t a p p r o p r i a t e l y cont ro l led i m m u n o t h e r a p y s tud i e s w i th m o l d ex t rac t s have s i m p l y no t b e e n car r ied out .

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mold-sensitive asthma

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