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Acta Médica Costarricense

versión On-line ISSN 0001-6002versión impresa ISSN 0001-6012

Acta méd. costarric vol.54 no.4 San José oct./dic. 2012

 

Original

Analysis of the reactions of type I hypersensitivity to the Basidiomycetes in a Costa Rican allergic population during 2009

Daniela Jaikel-Víquez1,2 y Olman Riggioni-Cordero2

1. Center for Research in Tropical Diseases, Faculty of Microbiology, University of Costa Rica.
2. Center for Diagnosis and Treatment of Allergy. Sources of support: The work was conducted with support from the Center for Diagnosis and Treatment of Allergy.
Correspondence: rivilab@hotmail.com


Abstract

Aim: Costa Rica is a tropical country in which a high concentration of aerial basidiospores has been found, which measure between 4-13 μm and are capable of arriving to the lower respiratory system, surpassing the nasal barrier. This study aims to find possible sources of environmental contamination by basidiomycetes and to determine if it is necessary to routinely analyze this allergen in the Costa Rican population that suffer from rhino bronchial allergy.

Methods: The percentage of patients allergic to the extracts of basidiomycetes, specifically  smuts like Sporisoriumcruentumand Ustilagocynodontisand rusts Pucciniagraminissubsp. graminiswas determined. Also, we collected panicles of Panicum maximum to assess their contamination by basidiomycetes.

Results: We found that 59.23% of patients were allergic to mushrooms and that the fungi type for which more patients had allergy is the basidiomicetes. At the same time, a 100% of the collected panicles were contaminated with a fungus of the genus Ustilago.

Conclusion: Most of the patients studied were allergic to basidiomycetes, therefore, we strongly recommend that Costa Rican allergists constantly test for these extracts.

Keywords: Basidiomycetes, allergy, fungi, Ustilago.


In our country there have been several studies on the identification of the different species of basidiomycetes, based on the search for basidiocarps producers, in areas like Monteverde and Volcano Poás.1-3 However, in the Central Valley it is not usually observe a large concentration of basidiocarps that justifies the high rate of basidiospores (23% of total air fungal spores) (; Jaikel, et al., thesis to opt for a Bachelor’s degree in Microbiology and Clinical Chemistry at UCR) in the environment which suggests that the major basidiomycetes in this area are the rust type fungi or the coal type fungi, which parasitize various plants, but do not produce basidiocarp.5-8 An example of this is found in grasses such as Panicum maximum (Guinea), which is found in large areas of the country, both in the Central Valley and beyond,9 and shows that its panicle is colonized by a fungus of pigmented spores.

In 1951, there were the first reportsof the relationship between basidiospores and respiratory  symptoms,10 as they are known as contact allergens and inhalation allergens causing bronchial  asthma, chronic cough, hypersensitivity pneumonitis, contact dermatitis11-13 and even anafilaxia.15

But, not only are spores cause allergy: studies with the Pleurotusostreatus and the Coprinusquadrifidus showed that extracts from the pile usand stipe contain allergens in approximately equal amounts or in excess of the amounts present in extracts of spores.10

Hypersensitivity to the Basiomycets/ Jaikel-Víquez y Riggioni-Cordero

The use of farms within buildings, specialized for  growing edible mushrooms, has become popular in Japan, North America and Europe, due to its great success.

Unfortunately, during harvest, millions of spores are released into the environment and they cause the “fog of spores”, which can obstruct the visibility in these rooms. This is the main reason that in a study of workers who grow Hypsizigusmarmoreus (Bunashimeji) one third presented chronic cough, after a few months of starting to work, and three years later, up to 90% of the workers showed positive precipitins in their serum to fungal spores, 40% left the farm due to severe respiratory allergic disease and 5% developed hipersensibilitypneumonitis.15

Regarding the immune response, macrophages / monocytes, NK cells and NK T cells are crucial in the innate immune response mounted against these pathogens and host T lymphocytes are important during the adaptive response. Because the spores of Basidiomycetes are composed of protein and lipid complexes it is believed that CD1 is involved in their recognition. The CD1b is an important marker of monocytes and activated dendritic cells ant it also presents glycolipids from the outer cell wall of Mycobacterium tuberculosis to the non conventional T cells, the double negative as αβT and / or γδT. Furthermore, the CD1d recognizes and binds to internal and external glycolipids stimulating NK cells. Finally, it has been shown that in these patients there is an increase in IL-13, a decrease in IFN-γ, an increase of Th2 lymphocytes and Th2/Th1 ratio.15

IL-13 plays an important role in the development of airway hyperresponsiveness and in isotype switching to IgE production by B lymphocytes, like IL-4. Furthermore, it induces an independent asthmatic phenotype of lymphocytes, suggesting that it acts, at least in mice, directly on non-immune cells in the bronchial tissues. This allows the inference that asthma in workers of mushroom farms, can be caused by inflammation mediated by Th2-type cytokines, especially IL-13.15 Basidiomycetes are capable of inducing, increased IL- 13, stimulation of the cascade of allergic inflammation and low levels of IFN-γ, which by itself is poor in atopic population, by a defect not well understood until now.16-18

Despite the importance of basiodiomicetes in public  health, there are few or almost no health centers, both in  Costa Rica and the rest of Central America, which have  incorporated extracts from basidiomycetes in their kits for  the diagnosis of immediate hypersensitivity diseases, and  only ZygomycetesAscomycetes are tested. Thanks to the  above, this study aims to analyze the type I hypersensitivity reactions to basidiomycetes in an allergic population in  Costa Rican, from January to December 2009.

The first aerobiological study conducted in Costa Rica,  demonstrated the importance of basidiomycetes in air  samples from schools in the province of Heredia, and it was made in 2008 (Jaikel, et al., Thesis to qualify for the title of degree in Microbiology and Clinical Chemistry at UCR),  and it warned about the lack of national or central-american research on such allergens.

This is the first study conducted in the country, which aims to demonstrate the potential sources of pollution responsible for high concentration of aerial basidiospores and pretends to assess routine analysis with extracts from different basidiomycetes in the evaluation of the allergic patient.

Materials and methods

Study population: We conducted a retrospective study  based on the records of the Allergy Section in the Heredian  Medical Clinic, between January and December 2009. We  studied 130 atopic patients from different regions of the country: 81 female and 49 male, with an age range of 6-86  years (mean 35.5 years).

First, we determined the percentage of patients that where allergic to extracts of basidiomycetes: coal type Sporisorium cruentumand rust type Pucciniagraminissubsp. graminis, as a whole, by linking data from positive prick tests for these fungi, in 2009, with the total population of patients of the Heredian Medical Clinic during that same year. Then, the percentage of patients allergic to rust type and type coal fungi was obtained separately in order to assess which is the most important.

As part of the Clincs protocol, four mixtures are used for testing fungi Ascomycetes, Zygomycetes and imperfect fungi, namely:

a.  Blend A or mixture of Aspergillus spp.: Aspergillusclavatus, Aspergillusfumigatus, Aspergillusterreus, Aspergillusniger and Aspergillus spp.

b.  Blend B or mixture of hyaline fungi other than Aspergillus: Chaetomium sp. Fusarium sp. Geotrichum sp. Mucor sp. Paecilomyces sp. Penicillium sp. Rhizopus sp. and Trichophytonrubrum.

c.  Blend C or mixture of sooty fungus: Alternaria sp. Cladophialophora (Cladosporium) carrioni, Cladosporiumcladosporioides, Cladosporium sp. and Curvulariasp.

d.  Blend D or mixture of yeast: Candida albicans and Rhodoturolarubra.

Data were collected concerning the percentage of  patients allergic to each of these fungal mixtures and the  associated clinical manifestations of such patients. Data on  sex, age and place of residence, were analyzed for possible demographic relationships regarding patients allergic to Basidiomycetes. Then, we determined the percentages of each disease in the total of allergic patients to these extracts.

However, because the Heredian Medical Clinic patients are,  mostly, atopic patients, the information obtained on the  various manifestations was analyzed comprehensively with main environmental allergens: dust mites  (Dermatophagoidespteronyssinus, Tyrophagusputrescentiae

and B. tropicalis) and grass pollens, as in Costa Rica  polysensitization is the rule, not the exception, in the allergic population.

We used the chi-square test to determine the association  of variables between different types of fungi and the clinical  manifestations.

The Bioethics Committee of the University of Costa Rica approved the research protocol (Session No. 206, VI-Doc 8897-2010), as it was within the ethical and humane principles of research. The Center for Diagnosis and Treatment of Allergy approved the revision of the clinical records of these tests.

Collection of panicle Panicum maximum contaminated with Basidiomycetes: we conducted a field study to find basidiomycetes pollution in the province of Heredia, Costa Rica.

The plant chosen was the grass Panicum maximum, which is distributed as wild foliage at the edge of the road, up to 1100 meters above sea level, especially between May  and November.19 The collection took place in the following cantons: Central Canton (Mercedes district), Barva and San Pablo. The plants were cutted at the level of stem and they were placed in individual plastic bags and they were labeled.

There were a total of 23 panicles, which were analyzed under the light stereoscope. The basidiomycetes spores that were found underwent a wet montage with blue lactophenol,  which helped with the identification of the genre it  belonged.

The contaminated pollens were sown in glass bottles with 25 ml of dextrose agar Sabouraud, and stored at room temperature for one month.

Basidiomycetes extract preparation isolated from Panicum maximum for skin testing: The material obtained from the culture in Dextrose Agar Sabouraud was washed three times with acetone to remove the lipids, and then fungal structures were preserved in liquid nitrogen.

Allergenic material was extracted in phenolated saline buffer 0.4% (Evans solution) with continued agitation for 48 hours at 4 ° C at a concentration weight / volume (W / V) of 1/10. It was centrifuged at 3500 rpm for 30 minutes. The supernatant was removed and filtered through a sterile milliporomembrane of 0.22 micrometers.

Standardization of extract for skin testing: an in vitrostandardization was performed, in which the amount of total proteins was specified by the Dumas method, for the crude extract obtained from the basidiomycete isolate panicle of Panicum maximum and for the three extracts of commercial basidiomycetes GREER (Sporisoriumcruentum, Ustilagocynodontis and Pucciniagraminis subsp. graminis). All four extracts are at a concentration of 1/10 P / V. Based on the results, we determined the concentration at which the extract can be used.

Results

As seen in Table 1, from the patients studied, 59.23% are allergic to fungi, being basidiomycetes 40.77%, yeasts 30.77%, hyaline fungi other than Aspergillus spp. 26.92%, and Aspergillus spp. and sooty fungus, both 13.08%. Similarly, in the most important group of fungi, which is the Basidiomycetes, 79.25% corresponds to the allergy against rust fungus Pucciniagraminis subsp. graminis, and 50.94% against the coal type.

According to the analysis of demographic data (Tables 2, 3 and 4), it should be noted that there are no significant differences found between the percentage of female patients allergic to Basidiomycetes as compared to males, or between age groups, but there was a decrease of positive prick tests in the population over 40 years, with respect to the one under that age. It was not possible to perform the analysis by province, as the number of patients was not sufficient to assign them any statistical value.

Table 5 shows the distribution of these allergic patients by diagnosis. The main clinical features associated with patients Heredian Medical Clinic, that were allergic to basidiomycetes, were: rhinitis and conjunctivitis and rhinitis, conjunctivitis and asthma. We found no patient whose diagnosis was exclusively bronchial asthma or atopic dermatitis. At the same time, 88.68% of patients allergic to any basidiomycete are allergic to house dust mites and storage mites, and 81.13%, to grass pollens, as befits the rule of the polysensitizationin the costarican atopic patient.

When performing the chi-square test for allergy variables Aspergillus spp. against a diagnosis of asthma, we found that these two variables are dependent, but not with the other types of fungi and this disease (Table 6).

Finally, 100% of panicles had fungal contamination (characterized by black spots) in at least 25 of its inflorescences (Figure 1) identifying the isolation as a basidiomycete coal type belonging to the genus Ustilago (Table 7).

Discussion

The results demonstrate that the Basidiomycetes corresponds to an important group as allergens, since they are causing atopic reactions in 40.77% of patients analyzed, and that 59.23% of allergic patients to fungus are allergic to them. In turn, what is most important is that 19.23% of patients are allergic to these fungi only, so it can be under diagnosed in their allergic disease, if it is not routinely tested with the Basidiomycetes allergens. In view of the results, we recommend using these fungi as part of the routine in symptomatic patients with negative skin tests. It should be emphasized that the main types of these fungi that cause allergies are the rust type, so the source of this sensibilization should be determined. Furthermore, a higher allergenic potential is reported, because it has about 6 times more protein than coal type.

There was no variation in the percentage of positivity obtained to the studued allergens by sex. Similarly, there is no link between the age groups of 0-12 years and 13 to 40 years, but there is a link between them and the group older than 40 years, which may be due to lower type I reactivity in this population, as it is described that the most allergic activity occurs in children and young people. This phenomenon is also reported for newborns and infants, however, in our study population the youngest patient was 6 years.20

With regard to the symptoms caused by basidiomycetes and fungi in general, literature reports that they are a cause of asthma, 14 but in this study population it was not the case, since there was no relationship of dependence between variables. Association was found only with this diagnosis in patients allergic to Aspergillus spp., which corresponds to spores that have a size of 2 micrometers or less, so they can reach the pulmonary alveoli. Not so with the Basidiomycetes, as their spores, although small, are larger than the 4 μm.11, 21

The majority of allergic patients were diagnosed with acute basidiomycetes of allergic rhinitis and allergic conjunctivitis and allergic rhinitis, bronchial asthma and allergic conjunctivitis, but no significant association with any particular pathology. This is because the allergic population of tropical countries is a population with sensitivity to many different allergens, including allergy to house dust mites and storage mites, to grass pollen, so it is not surprising that 88 68% of allergic patients allergic to mites are also allergic tobasidiomycetes, and 81.13% also allergic to pollens.

The Panicum maximumpanicles harvested were entirely colonized by the fungus Ustilago sp. identified as coal type, showing that these plants are an important source of fungal perennial allergens in the environment, and when they disperse at air level,7 they easily come in contact with the allergic patient. As mentioned above, almost 80% of allergic patients are allergic to basidiomycetes rust type fungi, so it is proposed as a continuation of this study, trying to locate sources of fungal contamination by rust, taking as a starting point the coffee plantations, which are often colonized with coffee rust: Hemilieiavastatrix.

As medicinal chemistry evolves, active materials has been prepared and synthesize exactly with a known composition, which can be administered in precise amounts and with confidence in the reproducibility of the effects of the different drugs used. However, the allergenic extracts are still the exception to this trend. Therefore, the purpose of standardization of allergens is to offer the allergists, extracts with known concentrations of allergens for diagnosis and immunotherapy of allergic diseases.22

The protein concentration for the two extracts obtained from Ustilago sp. (commercial and homemade) are equal, which shows that nationally allergens can be produced based on indigenous raw materials, with the same quality as in the transnational laboratories. It should be noted that one of the reasons for the variability in the mold studies for type I hypersensitivity, is the amount of the own enzymes of the fungus, which cause fungal allergens to be unstable while stored, such as allergens of domestic cockroach, so it is recommended to change them often with short due dates, of about a year, in the aqueous extracts, making the diagnosis difficult and expensive. The importance of environmental analysis should highlighted to determine the biodiversity characteristics of each region of the country and to offer each patient a more accurate diagnosis and an effective immunotherapy.

Conflict of interest: The authors report no conflict of  interest.

Acknowledgments: The authors thank Mr. Bernal Burgos, who helped with electronic microscopy photographs, and Mrs. Mercedes Sanchez, in charge of maintenance of the micoteca and who helped prepare Ustilago extracts.


References

1.Gómez L. Los basidiomicetes de Costa Rica. V. Paxillaceae (Agaricales, Boletineae). Brenesia. 1992; 38: 105-113.         [ Links ]

2.Gómez L. Los basidiomicetes de Costa Rica. VI. Bondarzewiaceae (Agaricales, Russulineae). Brenesia. 1993; 39-40: 1-3.         [ Links ]

3.Gómez L, Kisimova- Horovitz L. Basidiomicetes de Costa Rica. Nuevas especies de Exobasidium (Exobasidiaceae) y registros de Cryptobasidiales. Rev Biol Trop. 1998; 46: 4.         [ Links ]

4.Jaikel D, Hernández S, Riggioni O, Gross N, Salas I. Contaminación fúngica ambiental en tres centros de enseñanza primaria del Cantón Central de Heredia. Trabajo final de graduación para optar por el grado de licenciatura en Microbiología y Química Clínica. Universidad de Costa Rica. 2008.         [ Links ]

5.Deacon J. Modern Micology. Ter. Ed. England: Blackwell Science, 1997: 26-27.         [ Links ]

6.Munkacsi A, Stoxen S, May G. Ustilago maydis populations tracked maize through domestication and cultivation in the Americas. Proc R Soc B. 2008; 275: 1037–1046.         [ Links ]

7.Stolze-Rybczynski J, Cui Y, Stevens M, Davis D, Fischer M, Money N. Adaptation of the Spore Discharge Mechanism in the Basidiomycota. PLoS ONE. 2009; 4.         [ Links ]

8.Germain H, Bergeron M, Bernier L, La flamme G, Hamelin R. Patterns of colonization and spread in the fungal spruce pathogen Onnia tomentosa. Mol Ecol. 2009.         [ Links ]

9.Riggioni O, Montiel M, Fonseca J, Jaramillo O, Carvajal E, Rosencwaig P, Colmenares A. Hipersensibilidad tipo I a pólenes de gramíneas (por especie) en pacientes con rinitis alérgica. Rev Biol Trop. 1994; 42: 71-6.         [ Links ]

10.Davis W, Horner W, Salvaggio J, Lehrer S. Basidiospore allergens: analysis of Coprinus quadrifidus spore, cap, and stalk extracts. Clin Exp Allergy. 1988; 18: 261-267.         [ Links ]

11.Tanaka H, Saikai T, Sugawara H, Takeya I; Tsunematsu K, Matsuura A, Abe S. Workplace-Related Chronic Cough on a Mushroom Farm. Chest. 2002; 122:1080-1085.         [ Links ]

12.Ogawa H, Fujimura M, Takeuchi Y, Makimura K. The importance of basidiomycetous fungi cultured from the sputum of chronic idiopathic cough: a study to determine the existence of recognizable clinical patterns to distinguish CIC from non-CIC. Respir Med. 2009; 103: 1492-7.         [ Links ]

13.Ahmed M, Ishino T, Takeno S, Hurakawa K. Bilateral allergic fungal rhinosinusitis caused by Schizophillum commune and Aspergillus niger. A case report. Rhinology. 2009; 47: 217-221.         [ Links ]

14.Ichikawa K, Ito R, Kobayashi Y, Aihara M, Osuna H, Aihara Y. A pediatric case of anaphylaxis caused by Matsutake Mushroom (Tricholoma Matsutake) ingestion. Allergol Int. 2006; 55: 85-88.         [ Links ]

15.Saikai T, Tanaka H, Sato N, Matsuura A. Mushroom plant workers experience a shift towards a T helper type 2 dominant state: contribution of innate immunity to spore antigen. Clin Exp Immunol. 2004; 135:119–124.         [ Links ]

16.Bullens D, Decreane A, Dilssen E, Meyts I, De Bock K, Dupont J, Ceuppens J. Type III IFN-γ mRNA expression in sputum of adult and school-aged asthmatics. Clin Exp Allergy. 2008; 38: 1459-1467.         [ Links ]

17.Germ G. Interferon γ and infections in the allergic population. World Allergy Organiz J. 2007: 3.         [ Links ]

18.Jiménez A, López-Gonzalez M, Solano F, Candí A, Delgado F, Pintado E, Lucas M. Hypomethylation of DNA and resistance to apoptosis in tonsilar hypertrophy in children. Pediatr Allergy & Immunol. 2006; 17: 218-220.         [ Links ]

19.Burger W. Flora costarricensis. Prim. ed. USA: Fieldiana Botany, 1980: 371-374.         [ Links ]

20.Roitt I, Brostoff J, Male D, Roth D. Immunology. Set. ed. USA: Mosby Elsevier, 2007.         [ Links ]

21.American College of Allergy and Immunology. Aeroallergen identification.USA: Allergy Research Laboratory, University of Michigan, 1989.         [ Links ]

22.Anderson M, Baer H. In vitro methods for standardization of allergenic extracts. Clin Rev Allergy. 1986; 4:363-370.         [ Links ]


Received: June 20, 2011 Accepted: August 6, 2012

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