Sensitization to Airborne Ascospores, Basidiospores, and Fungal Fragments in Allergic Rhinitis and Asthmatic Subjects in San Juan, Puerto Rico

Background: Fungal spores are the predominant biological particulate in the atmosphere of Puerto Rico, yet their potential as allergens has not been studied in subjects with respiratory allergies. The purpose of this study was to determine the level of sensitization of subjects with respiratory allergies to these particles. Methods: Serum samples were drawn from 33 subjects with asthma, allergic rhinitis, or nonallergic rhinitis and 2 controls with different skin prick test reactivity. An MK-3 sampler was used to collect air samples and the reactivity of the sera to fungal particles was detected with a halogen immunoassay. Results: All subjects reacted to at least 1 fungal particle. Thirty-one subjects reacted to ascospores, 29 to basidiospores, 19 to hyphae/fungal fragments, and 12 to mitospores. The median percentage of haloes in allergic rhinitis subjects was 4.82% while asthma or nonallergic rhinitis subjects had values of 1.09 and 0.39%, respectively. Subjects with skin prick tests positive to 3, 2, 1, or no extract had 5.24, 1.09, 1.61, and, 0.57% of haloed particles, Received: June 22, 2010 Accepted after revision: September 30, 2010 Published online: February 22, 2011 Correspondence to: Dr. Benjamín Bolaños-Rosero Department of Microbiology, School of Medicine, UPR-MSC PO Box 365067 San Juan 00936-5067 (Puerto Rico) Tel. +1 787 759 8228, Fax +1 787 756 8475, E-Mail benjamin.bolanos @ upr.edu © 2011 S. Karger AG, Basel Accessible online at: www.karger.com/iaa D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 5: 23 :4 8 A M Sensitization to Airborne Fungal Particulates Int Arch Allergy Immunol 2011;155:322–334 323 tant factor [2, 11, 12] , others propose a significant role of environmental variables including outdoor airborne allergens [9, 10, 13] . For this reason, it is important to directly study the role of aeroallergens in inducing episodes of rhinitis and asthma. The predominant biological airborne particulates in many temperate and tropical regions are pollen and fungal spores [14–16] , and some studies have revealed that fungal fragments are also prevalent [17–21] . Furthermore, a significant number of species of pollen and fungi have been described as allergens or as having the potential to be allergenic [22–24] . Nevertheless, in many geographic locations fungal spores are the principal biological component in the atmosphere and various studies have identified their importance in episodes of respiratory allergic diseases [22, 23, 25–28] . Therefore, it is reasonable to expect that approximately 10% of the population could be allergic to airborne molds. In a recently published study, we characterized the biological airborne particulates in the atmosphere of San Juan, Puerto Rico (SJ) [14] , in which basidiospores and ascospores represented over 90% of the outdoor biological particulates and fungal spores throughout the year. In addition, there was an intradiurnal variation directly affected by the high humidity and dew point but inversely affected by wind and wind gusts. This is not the case in temperate regions, in which mitospores (deuteromycetes or Fungi Imperfecti) such as Cladosporium and Alternaria are the most common. Numerous studies have described the allergenic potential of these spores in temperate areas [16, 29–31] , but very few have demonstrated the same potential with basidiospores and ascospores, or directly with airborne fungal spores, due to the difficulty of preparing extracts [24–26, 32, 33] . Sensitization to a specific allergen can be detected in vivo via the skin prick test (SPT) or in vitro (e.g. radioallergosorbent test) by determining the presence and levels of immunoglobulin E (IgE) when the serum of a patient is challenged with an extract [1, 34] . One important limitation of these tests is that they are dependent on the extract’s quality which is known to be affected by many variables including intraspecies mutations, batch-tobatch variation, and culture conditions [24, 34, 35] . In addition, extracts are not available for many fungi or are very difficult to prepare, which could lead to a subdiagnosis of fungal allergies. Testing a whole-air sample extract would not be specific because of the heterogeneity of biological airborne particulates, and the presence of endotoxin may lead to secondary reactions. The halogen immunoassay, initially developed by O’Meara et al. [36] to detect inhaled cat allergen, described in detail by Tovey et al. [37] , and later refined for fungal allergens by Mitakakis et al. [38] and Green et al. [19–21, 35, 39, 40] is an immunoblotting method with some similarities to the enzyme-link immunosorbent assay and Western blot techniques. Advantages of this technique are that it is not dependent on any extract and the particle to be tested is induced to elute allergen. In addition, the assay is performed on a sealed protein binding membrane that captures the allergen-IgE complexes that can be observed under the microscope. It has been used to test various mitosporic fungi and nonfungal allergens (e.g. cat) and, more importantly, it can also be used to test against air samples collected with a volumetric air sampler. Given the importance of airborne fungal spores in allergic respiratory diseases and their predominance in many geographical areas, the purpose of this study was to determine the sensitization of sera from subjects with asthma, allergic rhinitis, or nonallergic rhinitis to airborne fungal particulates present in the atmosphere. Because of our previous aerobiological study in SJ, our hypothesis was that the predominant airborne spores in our atmosphere (basidiospores and ascospores) have higher percentages of reactivity compared to mitosporic fungi. Materials and Methods Recruitment of Volunteers With the approval of the Institutional Review Board (IRB) of the University of Puerto Rico, Medical Sciences Campus (UPRMSC), participants were selected from among staff and students of the UPR-MSC and patients seen by UPR-MSC faculty-physicians at external allergy clinics. Inclusion criteria for the study were a self-reported history of suffering from nasal allergies or asthma, having had a stable medical regimen for the previous 30 days, being noninstitutionalized, and being 1 15 years of age. Exclusion criteria were the current use of oral steroids for the treatment of asthma or being in an immunosuppresor regimen, any type of cancer within the last 6 months, infection of the upper respiratory tract within the last 2 weeks, being a smoker, having dementia or other conditions that would not allow the accurate self-reporting of data, having self-reported poor compliance with any medical therapy, being institutionalized, and being pregnant. The purpose of the study was explained to each individual in understandable terms and subjects were presented with an informed-consent form. Thirty-three participants consented to being part of the study. All of them were 1 22 years of age, with female volunteers being predominantly represented (30 females and 3 males). Allergic rhinitis was diagnosed based on the history of the current use of medications for rhinitis or nasal symptoms as determined by the nasal symptoms score and/or physician-diagnosed allergic rhinitis in addition to a positive skin test to relevant antigens based on the subject’s history. Nonallergic rhinitis was diagnosed in the presence of current nasal symptoms, medication D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 5: 23 :4 8 A M Rivera-Mariani /Nazario-Jiménez / López-Malpica /Bolaños-Rosero Int Arch Allergy Immunol 2011;155:322–334 324 use, and a negative skin test. Negative controls were asymptomatic subjects who did not use any medications for rhinitis and who had negative skin test results. Asthma was diagnosed based on pulmonary symptoms of wheeze, cough, and shortness of breath, as well as on medication use and physician diagnosis. Chronic obstructive pulmonary disease (COPD) was diagnosed based on pulmonary symptoms and smoking history. When arranging subjects into groups based on the diagnosed disease, those who had allergic rhinitis and asthma were placed in the asthma group. The only subject with COPD was classified with asthmatics as this subject was also suffering from asthma. Human Sera Serum samples were drawn by trained medical technicians from 33 subjects ( table 1 ) with no respiratory disease, asthma, or allergic or nonallergic rhinitis. SPT were performed using commercial and crude basidiospore extracts and the samples were stored at –80 ° C until analysis. In the SPT, a wheal 3 mm larger than that of the negative control was considered positive. In addition, the IgE levels were determined at a reference laboratory using an Advia Centaur  CP Immunoassay System (Bayer Diagnostics, Greenburgh, N.Y., USA). In 4 of the subjects (with allergic rhinitis) the volume of sera was insufficient to determine both the IgE titer and the reactivity with a halogen immunoassay; for these participants, only the halogen immunoassay was used. The battery of SPT extracts included commercial extracts (Greer Laboratories, Lenoir, N.C., USA) of mitosporic fungi ( Penicillium spp. , Aerobasidium spp., Alternaria spp., Cladosporium spp., Acremonium spp., Fusarium spp., and Aspergillus spp.), 1 ascomycete ( Chaetomium spp.), animals (dog, cat, mite, and cockroach), pollen (Bermuda, Johnson, Bahia, and trees), and crude extracts from basidiomycetes’ spore prints (Ganoderma applanatum, Chlorophyllum molybdites, and Pleurotus ostreatus) which were prepared as previously described by Horner et al. [32 ] . Collection of Air Samples An 80 ! 20 mm strip of ABgene PCR sealing film (Fisher Scientific, New Hampton, N.H., USA) was attached, with the adhesive surface facing upwards, to a microscope slide. The prepared slide was placed in an Allergenco MK-3 sampler (Environmental Monitoring Systems, Inc., Charleston, S.C., USA) situated on the roof top of the main building of the UPR-RCM ( 100 m above ground level) located in SJ. Outdoor air samples were collected at a volume of 15 liters/m 3 of air in March, May, June, and December of 2009 and in January of 2010. Air sampling was done only on days during these months when participants were recruited and sera were available for testing. The samples were collected from 4 p.m. to 8 a.m., which are the hours with the highest fungal concentration in the SJ atmosphere, at intervals of 10 min of sampling every 110 min of relapse. Although these hours may not reflect actual periods of individual exposure, we wanted to test as many fungal spores and particulates as possible. Sampling during daytime hours would have yielded very few spores or other nonbiological particulates. At 8 a.m. the slide was removed from the sampler and processed with the halogen immunoassay. In the afternoon (4 p.m.) of the same day another slide with adhesive tape was placed in the sampler. The same procedure was repeated on the days on which sera was drawn until enough air samples had been collected for all of the sera because on 1 adhesive film a limited number of mixed-cellulose ester protein binding membrane squares could be attached. Halogen Immunoassay The patients’ sera were blindly analyzed with the halogen immunoassay ( fig. 1 a) before the clinical, IgE level, and SPT data were provided. The assay was performed as described by Green et al. [39] but instead of the air sample being collected on the membrane it was collected on the adhesive tape as mentioned above. Table 1. C linical data, IgE levels, and SPT results of the patients included in the study Patient Condition Disease code IgE (IU/ml) SPT (+) 1 NAR 472 ND Gano, Chlor, grass 2 AR, COPD 477.8 107.1 Mite, grass 3 AR 477.8 ND Mite, trees 4 AR, asthma 493 117.8 Mite, grass 5 AR 477.8 16.6 Mite 6 AR 477.8 ND Gano, mite, cat 7 AR 477.8 ND Pen, mite, cockr, cat 8 AR, asthma, conjunct 493 49.6 Mite, Gano 9 Neg ctrl control 5.7 None 10 NAR 472 4.9 None 11 Neg ctrl control 8.7 Fusarium 12 AR, asthma 493 6.5 Pleu, cockr 13 NAR 472 1 None 14 Chronic rhinitis 472 29 Unable to be determined 15 NAR 472 43.5 Gano 16 NAR 472 1.5 None 17 AR 477.8 338.8 Pen 18 AR, asthma 493 27.5 Mite 19 NAR 472 648.1 None 20 NAR, AD 472 461.9 None 21 AR, asthma 493 64.6 Cockr 22 NAR 472 14.4 None 23 AR, asthma 493 210.4 Pleu, Gano 24 AR, asthma 493 491.6 Gano 25 AR 477.8 242.8 Pen, mite 26 NAR 472 79.4 None 27 NAR 472 12.3 Gano, Chlor, Pleu, mite 28 AR 477.8 755.3 Grass 29 Asthma 493 1,442 Mite, grass, Asp, Alt, Gano, Chlor, mite 30 AR 477.8 307.7 Grass 31 AR 477.8 100.5 Grass 32 AR 477.8 2,017.1 Asp, Alt, Gano, mite, grass, Chlor, Pleu 33 Asthma 493 1,101.4 Grass A D = Atopic dermatitis; AR = allergic rhinitis; conjunct = conjunctivitis; COPD = chronic obstructive pulmonary disease; NAR = nonallergic rhinitis; neg ctrl = negative control; Alt = Alternaria spp.; Asp = Aspergillus spp; Chlor = Chlorophyllum molybdites; cockr = cockroach; Gano = Ganoderma applanatum; Pleu = Pleurotus ostreatus. D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 5: 23 :4 8 A M Sensitization to Airborne Fungal Particulates Int Arch Allergy Immunol 2011;155:322–334 325 1 2 3