Print version ISSN 0036-4665
Rev. Inst. Med. trop. S. Paulo vol.44 no.5 São Paulo Oct. 2002
Knowledge of anemophilous fungi in a given city or region is important for the ecological diagnosis and specific treatment of allergic manifestations induced by inhaled allergens. In order to diagnose the presence of anemophilous fungi, several qualitative and quantitative techniques are used depending on the study place. This study of fungal air spores was performed with a Rotorod Sampler®, an equipment which samples the air through a plastic rod attached to an electric engine that makes it spin fast enough to collect the particles in the air. The samples were collected once a week during 24 hours using the standard cycle of the manufacturers. A total of 52 samples were obtained from April 2000 through March 2001. The results revealed prevalence of ascosporos (50.49%), Cladosporium (17.86%), Aspergillus/Penicillium (15.03%), basidiosporos (3.84%), rusts (3.82%), and Helminthosporium (2.49%), and a lesser frequency of Botrytis (1.22%), Alternaria (1.19%), smuts (0.90%), Curvularia (0.87%), Nigrospora (0.61%), and Fusarium (0.08%). Also, 1.59% of the spores detected here could not be identified by the systematic key used. More fungal spores were observed during the summer than during the autumn.
KEYWORDS: Aeroallergens; Airborne; Anemophilous fungi; Spores.
Fungi disseminate their spores in the environment through the atmospheric air, water, insects, man, and animals. Anemophilous fungi are those whose spores are spread by the atmospheric air. Qualitative and quantitative knowledge of these fungi in a given region is of great importance and concern because they can cause several respiratory diseases in man such as asthma and rhinitis when inhaled2.
Among the fungi groups that spread air spores and are important aeroallergens are Zygomycota, Ascomycota, Basidiomycota and Deuteromycota18.
Zygomycota are represented by Rhyzopus and Mucor. Ascomycota deliver asci carrying ascospores and are represented by Leptosphaeria, Chaetomium and Venturia. Basidiomycota deliver basidiospores and are represented by fungi that are pathogenic for plants, i.e, rusts and smuts. Deuteromycota have the greatest number of fungi in their asexual reproduction phase, such as the Aspergillus, Penicillium, Cladosporium, Alternaria among others. Ascomycota, Basidiomycota, Zigomycota and Deuteromycota have the greatest number of well-known causative agents of allergic symptoms5.
Spore formation by fungi go through a number of processes, and different types of spores are produced by the same fungus at the same time in response to varying environmental conditions. Spores are developed by sexual reproduction (teleomorphic or perfect phase) either through nuclear fission or without nuclear fissure, resulting in the anamorphic or imperfect phase. Thus, some spores of different fungi are morphologically similar, making them impossible to identify by gender (e.g., Aspergillus and Penicillium). Other spores are so small, transparent, or devoid of distinctive characteristics that they cannot be identified (Phoma, Neurospora and Candida). Many other spores, especially ascospores and basidiospores, are not identified as specific particles1.
Qualitative and quantitative techniques are used to diagnose the presence of anemophilous fungi. Qualitative techniques include Petri dishes exposures or cover glass research. These are exposed for some time in the environment. This method is based on the sporulation characteristics of fungi, and thus cannot be used to identify them when fungi fail to sporulate on the medium, neither to quantitate them8,11. Quantitative techniques include the use of equipment such as Burkard®, Rotorod Sampler®, and others that can collect fungi spores on a continuous basis. Subsequently these can be identified through light microscopy according to their morphological characteristics and quantitated by cubic meter of air measured for 24 hours.
Research on fungi conducted in Brazil so far is mostly qualitative, and there are no data from surveys using intermittent sampling equipment like Rotorod Sampler®, Burkard® and others. Few are the publications about fungi prevalence in the atmospheric air of Brazilian cities. The published studies show a higher incidence of the following genera:
Cladosporium, Penicillium, Aspergillus, Rhodotorula, Aureobasidium, Candida, Fusarium, Curvularia, Rhizopus, Helminthosporum, Trichoderma, and others at a lower incidence9,10,12,13,14,15,16,18.
In Porto Alegre, HOMRICH (1961) investigated indoor environments and surroundings for the presence of fungi and found Aspergillus to be the most prevalent genus, followed by Penicillium, Rhizopus and Mucor10. It is important to emphasize that there is a difference between fungi present in indoor environments and in the atmospheric air. Of course, dwelling conditions determine a greater or lesser probability of fungal growth. About 300 species of fungi have already been described as allergenic. In the world, species belonging to Alternaria, Cladosporium, Aspergillus, and Penicillium are the most frequent ones10.
Knowledge of anemophilous fungi of a given city or region is important for the ecological diagnosis and specific treatment of allergic manifestations induced by inhaled allergens12.
The composition of the atmospheric fungal population in Porto Alegre was unknown. Since the spores of these fungi have clear antigenic properties, it is possible that such elements have played a sensitization role among atopic individuals in Porto Alegre. The identification of the fungal microbiota of our city was essential to obtain this knowledge.
The main purpose of this study was to investigate the prevalence, quantity, and seasonal variation of anemophilous fungi in the atmospheric air of Porto Alegre.
MATERIALS AND METHODS
The sampling of fungal air spores was performed with a Rotorod Sampler® placed at 23 meters of height on the top of the building of the Federal Foundation of Medical Sciences of Porto Alegre, located downtown.
The samples were collected once a week using the standard cycle of the manufacturer (i.e., one-minute collection + 9-minute pause, for 24 hours). The equipment's collector, made of a plastic rod measuring 1.52 X 1.52 X 32 mm, was previously greased with silicone before starting the cycle. Only the external surface of the collector rods were used for the procedure, according to manufacturer's advice.
After the end of cycle, the collector rod was dyed with Calberla's dye, put under a cover glass (22 X 22 mm), and examined under a light microscope at a magnification of 40X or 100X. Counts were performed throughout the cover glass extension. The particle counts on this surface are related to the samples quantity of air per cubic meter in 24 hours sampling. The final concentration is expressed as number of particles per cubic meter of air.
In this study 52 samples were collected and 3,773 fungal air spores were detected and identified in the period of April 2000 through March 2001.
The identification of fungal air spores was performed using the systematic key recommended by the American Academy of Allergy, Asthma & Immunology (AAAAI), 19973.
Our data on fungal air spores refer to the period of April 2000 through March 2001.
Wherever possible, fungi were identified as far as their genera, in genera groups such as Aspergillus/Penicillium, or only the spore form, such as ascospores and basidiospores, and their frequency was calculated in relation to the total count of spores.
Table 1 shows the results about the identification and number of anemophilous fungal spores found in this study. Note that the number of spores varied widely across the months.
As can be seen in Fig. 1, three peaks were observed in the months of August, November, and March within the studied period.
Table 2 shows the anemophilous spore frequency in the four seasons of the year within the studied period.
The standardization of aeroallergens, according to CHAPMANN6, generates comparative data from year to year and season to season. These data can allow to establish the beginning and the end of a period, to identify sporulation peaks, and to quantitate the number of spores in a given season of the year.
Our study was performed in only one site of Porto Alegre because of the impossibility of using the equipment in other sites in the same period.
The results shown in Table 1 encompass all seasons of the year. The detected prevalence of spores was as follows (in descending order): ascosporos (50.48%), Cladosporium (17.86%), Aspergillus/Penicillium (15.03%), basidiosporos (3.84%), rusts (3.82%), and Helminthosporium (2.49%). Less prevalent were Botrytis (1.22%), Alternaria (1.19%), smuts (0.90%), Curvularia (0.87%), Nigrospora (0.61%), and Fusarium (0.08%). Also, 1.59% of the spores detected here could not be identified by the systematic key used. This difficulty is due to spores of different genera presenting the same morphologic aspect and no distinctive characteristics to allow their classification, a problem faced by everyone doing research on these fungi.
Our findings failed to correlate with the degree of environmental pollution or with winds speed.
In Rio of Janeiro, PASSARELLI et al.16 detected Rhodotorula and Cladosporium with marked seasonal periodicity from May to October, Penicillium with the same incidence throughout the year except for December and January, when incidence rates were decreased. The distribution of Aspergillus was uniform with no seasonal variation, and Fusarium had no defined seasonal periodicity; the other fungi showed no seasonal variation.
In this study, 3,773 fungal spores were detected and identified. All genera were found in every season of the year (Table 2), except for Fusarium, Botrytis, Nigrospora and smuts. Aspergillus/Penicillium, ascosporos, basidiosporos, Nigrospora, rusts and smuts showed a higher incidence in summer, while Cladosporium had a higher incidence in winter and Botrytis, Curvularia and Helminthosporium in autumn; Alternaria was found at the same number in spring and summer. Fusarium was not found in winter, spring, and summer, Botrytis was not found in spring and summer, and smuts were not detected in autumn.
Results in Table 2 show a higher incidence of fungal spores in the summer, and a lower incidence during the fall months. According to BURGE et al.5 the higher prevalence of fungal spores was observed during the dry and hot seasons. In our study, however, we did not measure the air humidity to verify this observation.
In a study performed in a few Brazilian cities, OLIVEIRA LIMA et al.14 concluded that the most frequent fungi in the atmosphere of these cities were Aspergillus, Penicillium, Cladosporium, Fusarium, Rhodotorula, Rhizopus, Aureobasidium Curvularia, Helminthosporium, Candida, Trichoderma, and Phoma, as well as others with a lesser incidence. The genera Alternaria showed an irregular pattern of incidence. According to the authors, these findings match those of other American and European countries.
CHAPMANN6 pointed out that the prevalence of air spores does not always identify clinically important inhalant allergens. Indeed, in spite of the high number of Cladosporium spores found here, it presented low sensibility.
Our overall data indicate the presence of a large number of fungal air-spores in Porto Alegre, with a higher incidence of ascospores, Cladosporium, Aspergillus, and Penicillium. In general, those fungi with a higher incidence in our study are the same as those most frequently found in other Brazilian cities through qualitative studies10,12,13,15,18.
The occurrence of a great number of fungal spores emphasizes the importance of studying anemophilous fungi in Porto Alegre. In addition, the anemophilous fungi found here reinforce Bernd's finding4 that 13.8% of patients with respiratory allergies showed sensibility to air fungi.
The continuous presence of spores of genera Cladosporium, Aspergillus, and Penicillium alerts health professionals to the importance of continuously monitoring patients with allergies for these microorganisms.
Fungos anemófilos na cidade de Porto Alegre, Rio Grande do Sul, Brasil
O conhecimento dos fungos anemófilos em determinada cidade ou região é importante para o diagnóstico etiológico e o tratamento específico de manifestações alérgicas provocadas por estes alérgenos inalantes. Várias técnicas são preconizadas para coleta e identificação de fungos anemófilos na dependência do local estudado. Nesta pesquisa foi utilizado o equipamento Rotorod Sampler® que retira a amostra do ar através de um bastão preso a um motor elétrico que o faz girar rapidamente e as partículas suspensas no ar são recolhidas pelo bastão. A coleta foi realizada uma vez por semana, durante 24 horas, correspondendo a um ciclo de coleta. Totalizando 52 coletas entre abril 2000 a março de 2001. Os resultados apresentaram-se com prevalência de ascosporos (50,49%), Cladosporium (17,86%), Aspergillus/Penicillium (15,03%), basidiosporos (3,84%), rusts (3,82%) e Helminthosporum (2,49%), com menor freqüência Botrytis (1,22%), Alternaria (1,19%), smuts (0,90%), Curvularia (0,87%), Nigrospora (0,61%) e Fusarium (0,08%). Não foram possíveis de serem identificados 1,59% dos esporos de fungos anemófilos observados neste estudo. O maior número de esporos foi observado no verão e o menor no outono.
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Received: 19 December 2001
Accepted: 19 July 2002
(1) Fundação Faculdade Federal de Ciências Médicas de Porto Alegre, Rio Grande do Sul, Brazil
(2) Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
(3) Doutoranda no Curso de Pós Graduação em Ciências Veterinárias da Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Correspondence to: Adelina Mezzari, Rua Sarmento Leite 245, 90050-170 Porto Alegre, RS, Brazil. e-mail: email@example.com