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Acta Médica Costarricense
versão On-line ISSN 0001-6002versão impressa ISSN 0001-6012
Acta méd. costarric vol.54 no.4 San José Out./Dez. 2012
Original
Analysis
of the reactions of type I hypersensitivity to the Basidiomycetes
in a Costa Rican allergic population during 2009
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:
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.
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
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-
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
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
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
Collection
of panicle Panicum
maximum contaminated with Basidiomycetes:
we conducted
a field study to find basidiomycetes
pollution in the
The plant
chosen was the grass Panicum
maximum, which is distributed as wild foliage at the edge of the road,
up to
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.
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