Grass pollen sensitization in dogs in Paraná, Brazil: comparison between percutaneous and intradermal testing

Abstract

Canine atopic dermatitis is an inflammatory, genetic, pruritic and chronic dermatosis that affects between 10 and 30% of dogs and one of the most important allergens is grass pollen. The objective of this study was to evaluate the sensitization to grass pollen allergens in dogs with canine atopic dermatitis and to compare intradermal skin test (IDT) with percutaneous test (PT). For this study, ten healthy dogs and 39 dogs with atopic dermatitis were tested. Dogs were submitted to IDT and PT for Lolium multiflorum, Cynodon dactylon and Paspalum notatum. The IDT and PT tests were compared using the Proportion Test. All healthy dogs were negative to both tests. Ten atopic dogs (25.6%) responded positively to the PT and none were positive in IDT. C. dactylon, L. multiflorum and P. notatum were responsible for positive reactions in 70%, 70% and 30% of positive dogs, respectively. The number of positive reactions in PT were statistically higher than IDT (P<0.05). In conclusion, grass pollen can be important source of allergens for dogs in Paraná state (Brazil) and the PT showed higher sensitization to grass pollen in dogs with atopic dermatitis than IDT.
Keywords:
dogs; grass pollen; atopic dermatitis; prick test

Full text

Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021
Grass pollen sensitization in dogs in Paraná, Brazil: comparison between
percutaneous and intradermal testing
[Sensibilização a polens em cães no Paraná, Brasil: comparação entre o teste percutâneo
e o teste intradérmico]
A.P.S. Cerdeiro1, M.R. Farias2*, V.E.S. Cunha3, G.V. Secchi1, B.D. Pacheco1,
C.O.R. Alcalá1, R.G.R. Ganho4
1Aluna de pós-graduação ˗ Pontifícia Universidade Católica do Paraná ˗ Curitiba, PR
2 Pontifícia Universidade Católica do Paraná ˗ Curitiba, PR
3FDA Allergenic ˗ Rio de Janeiro, RJ
4Aluno de pós-graduação ˗ Pontifícia Universidade Católica do Paraná ˗ Curitiba, PR
ABSTRACT
Canine atopic dermatitis is an inflammatory, genetic, pruritic and chronic dermatosis that affects between
10 and 30% of dogs and one of the most important allergens is grass pollen. The objective of this study was
to evaluate the sensitization to grass pollen allergens in dogs with canine atopic dermatitis and to compare
intradermal skin test (IDT) with percutaneous test (PT). For this study, ten healthy dogs and 39 dogs with
atopic dermatitis were tested. Dogs were submitted to IDT and PT for Lolium multiflorum, Cynodon
dactylon and Paspalum notatum. The IDT and PT tests were compared using the Proportion Test. All
healthy dogs were negative to both tests. Ten atopic dogs (25.6%) responded positively to the PT and none
were positive in IDT. C. dactylon, L. multiflorum and P. notatum were responsible for positive reactions in
70%, 70% and 30% of positive dogs, respectively. The number of positive reactions in PT were statistically
higher than IDT (P<0.05). In conclusion, grass pollen can be important source of allergens for dogs in
Paraná state (Brazil) and the PT showed higher sensitization to grass pollen in dogs with atopic dermatitis
than IDT.
Keywords: dogs, grass pollen, atopic dermatitis, prick test
RESUMO
A dermatite atópica canina é uma dermatose inflamatória, genética, prurítica e crônica que afeta entre 10% e
30% dos cães, e um dos alérgenos mais importantes são os polens de gramíneas. O objetivo deste estudo é
avaliar a sensibilização a alérgenos de polens de gramíneas em cães com dermatite atópica e comparar o teste
intradérmico (TID) com o teste percutâneo (TP). Para o estudo, 10 cães hígidos e 39 cães com dermatite atópica
foram testados. Estes foram submetidos ao TID e ao TP para Lolium multiflorum, Cynodon dactylon e Paspalum
notatum. TID e TP foram comparados usando-se o teste de proporção. Todos os cães hígidos foram negativos
em ambos os testes. Dez cães atópicos (25,6%) responderam positivamente ao TP e nenhum ao TID. C. dactylon,
L. multiflorum e P. notatum foram responsáveis por reações positivas de 70%, 70% e 30% dos cães positivos,
respectivamente. O número de reações positivas no TP foi estatisticamente maior que no TID (P<0,05). Foi
concluído que os polens de gramíneas podem ser importantes fontes de alérgenos para cães no estado do Paraná
(Brasil) e que o TP mostrou maior sensibilização a polens em cães com dermatite atópica que o TID.

Palavras-chave: cães, polens de gramíneas, dermatite atópica, teste percutâneo
INTRODUCTION
Canine atopic dermatitis (CAD) is a pruritic,
inflammatory, and chronic dermatopathy, with a
genetic predisposition, affecting between 10 and
Recebido em 29 de junho de 2020
Aceito em 6 de abril de 2021
*Autor para correspondência (corresponding author)
E-mail: marconi.puc@terra.com.br
30% of the canine population (Carlotti, 2012).
CAD results in the loss of the integumentary
physical barrier function and increased
immunoreactivity. It is estimated that
approximately 80% of dogs with CAD have
http://dx.doi.org/10.1590/1678-4162-12111
A.P.S. Cerdeiro
http://orcid.org/0000-0003-1466-322X
M.R. Farias
http://orcid.org/0000-0003-4428-1767
V.E.S. Cunha
http://orcid.org/0000-0002-1910-098X
G.V. Secchi
http://orcid.org/0000-0002-2046-633X
B.D. Pacheco
http://orcid.org/0000-0002-1546-6685
C.O.R. Alcalá
http://orcid.org/0000-0001-9755-8366
R.G.R. Ganho
https://orcid.org/0000-0002-0353-8813

Cerdeiro et al.
822 Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021
increased levels of the specific antibody
immunoglobulin E (IgE), mainly as a response to
environmental, microbial and food allergens
(Pucheu-Haston et al. 2015). The environmental
major allergens inducing CAD come from house
dust mite allergens, weeds, shrubs, and grasses
(Prélaud, 2014). In people with atopic dermatitis
IgE mediated by extrinsic factors, it was found
that exposure to grass pollens induces a
significant worsening of cutaneous symptoms
(Werfel et al., 2016).
The major allergenic grasses in south Brazil are
Lolium multiflorum (LM) or Ryegrass, Cynodon
dactylon (CD) or Bermuda grass, and Paspalum
notatum (PN) or Bahia Grass. The subfamily
Pooideae, to which belongs the LM, has the major
allergens in groups 1 and 5 and may confer cross-
reactivity with other species of the same
subfamily and with species of other subfamilies
(Vieira, 2012). Among them, Lolium multiflorum
is considered to be the most important allergen in
humans in south Brazil (Rosário Filho, 2012).
Veterinary studies to verify the incidence of
pollinosis in Brazil are rare. A recent study that
used intradermal skin test (IDT) in 58 dogs with
atopic dermatitis in Rio Grande do Sul state,
demonstrated a positive response to CD (24.13%),
and PN (12%) (Pereira et al., 2015). Sensitization
to LM was not evaluated.
Percutaneos test (PT) has been used in medicine
since its first description by Jack Pepys in 1975 as
an alternative with lower risk of systemic
reactions compared to IDT. Studies performed in
cats and dogs showed papule reactions easier to
interpret, no cutaneous and systemic reactions and
good specificity (Rocha, 2012; Rossi et al., 2013,
Gentry e Messinger, 2016). The objective of this
study was to evaluate the sensitization to grass
pollen allergens in dogs with CAD and to compare
IDT with PT.
MATERIAL AND METHODS
The present study was approved by the Ethics
Committee for the Use of Animals at the
Pontifical Catholic University of Paraná (registry
number: 1027/2016). It was a cross-sectional,
non-randomized and controlled study. Ten
healthy dogs, 5 females and 5 males, average age
of 5 years, and 39 dogs with atopic dermatitis, 26
females, average age of 5 years, irrespective of
breed, from the Pontifical Catholic University of
Paraná Veterinary Clinic School were included.
There were not included in this study dogs that
received antihistamines (1 week), oral and topical
glucocorticoids (2 weeks) or long-term injectable
glucocorticoids (4 weeks), according to Olivry’s
study of anti-allergic drugs optimal and minimal
withdrawal times before allergic tests in dogs
(2013). Females in estrus, gestation or lactation
and dogs with chronic systemic diseases or with
any other dermatopathy were also not included.
All dogs in this study were submitted to the IDT
and PT sequentially in the same day by the same
examinator, who previously did a proficiency test
as described by Cunha et al. (2015).
For IDT, the skin was shaved using a grooming
machine and blade 40 in the lateral region of the
thorax. After careful cleaning of the skin with a
physiological solution, 10 application points were
marked at an interval of 3cm with a dermographic
pen. For both groups, 0.9% phenolated saline and
a histamine solution (0.05 mg/ml) were used as a
negative and positive controls, respectively.
Sensitization to pollen was evaluated using
standardized extracts of LM, CD and PN at the
concentration of 100 UBE /ml. All extracts were
supplied by FDA Allergenic (Rio de Janeiro,
Brazil). The intradermal applications were
performed using 0.5-ml insulin syringes, attached
to hypodermic needles (13 x 4,5mm), containing
0.05ml of each grass pollen extract. Fifteen
minutes after application, the papules were
marked with a dermographic pen to facilitate the
reading. The cut-off value was established by
calculating the mean for the negative control (CN)
and positive control (CP) values. The responses to
the tested antigens were considered positive when
the mean diameter of the papules was ≥ the
calculated cut-off value.
In the PT, histamine hydrochloride (10 mg/ml)
and saline (containing 0.45% phenol) were used
as the positive and negative control, respectively.
Glycerinated allergenic extracts of LM, CD and
PN were used at the concentration of 10.000
UBE/mL. All the extracts were supplied by FDA
Allergenic (Rio de Janeiro, Brazil). After
applying a drop of each extract or control
solutions on the skin, the device (Duotip-test II®,
Multi-test Brazil) was passed through the drop at
a 45° to 60° angle to the skin. The skin was then
gently lifted, creating a small break in the
epidermis through which the suspected allergen
solution penetrates. Then an absorbent paper was

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Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021 823
used to remove the excess of solutions. After 15
minutes, the test sites were observed, and the
papules were evaluated. Papules that were at least
3mm larger than the negative control were
considered positive (Van der Valk et al., 2016).
The results of the PT and IDT were compared in
a descriptive way and then by the proportion test
using the Minitab16 software (Minitab Inc., State
College, Pennsylvania), with significance level
set at α=0.05 (P≤0.05).
RESULTS
All dogs in Group 1 were negative to the allergen
extracts in the IDT and PT. The mean histamine
papule diameter in the IDT and PT was 15 and
7.45mm, respectively. The positive reactions in
PT ranged from 3 to 6mm, with formation of
pseudopods in some cases. The comparison
between papular histamine reaction in IDT and
positive reaction to grass pollen in PT is shown in
Figure 1. In DAC group, none of the dogs were
positive for the pollen tested in IDT. Ten dogs
(25.6%) reacted to grass pollen with PT. Seven
dogs (18%) were positive to CD and LM, while
three dogs (7.7%) were positive to PN. Two dogs
(5.1%) reacted to three species tested and three
dogs (7.7%) to two species. Monosensitization
was observed in five dogs (12.8%) as shown in
Table 1. The statistical analysis with Proportion
Test showed that the PT was more effective in
identifying sensitization to CD and LM
(p=0,002), and PN (p=0,036) compared to IDT.
Figure 1. Comparison of papular histamine reaction in the intradermal test (left) and positive for grass
pollen in the prick test (right) in millimeters. Note the formation of a pseudopod (arrow).
Table 1. Results of sensitization among ten reactive dogs in to grass pollen through the Percutaneous Test
Pollen/Dog
1
2
3
4
5
6
7
8
9
10
CD
+
-
+
+
-
+
+
+
-
+
LM
-
+
+
-
+
+
+
+
+
-
PN
-
-
-
-
-
+
+
-
-
+
CD – Cynodon dactylon; LM – Lolium multiflorum; PN – Paspalum notatum.
DISCUSSION
In the present study, the PT and IDT showed high
specificity, justified by the lack of positive
animals in the control group; these solutions were
tested previously for irritative threshold in healthy
dogs (unpublished data) and were tested again in
this study to prove these findings. It is believed
that IDT based on the irritation threshold have
high specificity in the diagnosis of allergic
sensitization (Hensel et al., 2004). It is estimated
that the pollen sensitization in allergic people in
Brazil varies from 16% to 34%, being more
prevalent in the South and Center-West of the
country (Vieira et al., 2011; Vieira, 2014;
Sopelete et al., 2014). In dogs, the prevalence of
grass pollen sensitization ranges from 10 to 27.7%
(Masuda et al., 2000; Roussel et al., 2013). In
Brazil, one study using IDT identified 10.3% of
sensitization in atopic dogs using grass polens
extracts (Pereira et al., 2015). In the present study
using PT, the prevalence of sensitization to grass
pollen was (25.6%) and CD and LM were the
most important species.

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824 Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021
This could be explained by the wide distribution
of these species in the South region of Brazil,
which can be found along roads, backyards, and
parks (Viera, 2012). LM may provoke rhino
conjunctivitis and allergic asthma in people
(Rosário Filho, 2012) due to its wide distribution
and an increase in its cultivation over the years. It
has high allergenic potential due to its large-scale
production and rapid spread in the wind
(Bonissoni and Neto, 2014). In Curitiba, South of
Brazil, a study done in 1999 found positive results
using PT for LM in 4.7% of children aged 13 to
14 years and 15.4% of adults over 20 years of age
(Esteves et al., 1999). A study conducted in 2002
found positivity for Lolium perene of 16.5% in
skin allergic tests (Rosário Filho, 2002). In
veterinary medicine, the incidence of sensitization
to LM has not been evaluated so far and the
present study revealed a positive response to this
grass among atopic dogs (12.8%) similar to
previous findings.
Additionally, other species such as CD are
allergenic and produce allergy symptoms beyond
September and December (Rosário Filho, 2012).
Sensitization to CD was detected in 16.8 and 80%
of patients with rhinitis in Curitiba (Araújo et al.,
2015) and Rio Grande do Sul (Vieira et al..,
2011), respectively. A previous study using IDT
in Brazil found that 24.13% of atopic dogs were
sensitized to CD (Pereira et al., 2015). In the
present study, the positive response to this grass
was similar to the other studies cited. The lowest
incidence of positive response was observed with
PN. Positive reactions to PN were accompanied
by positive reactions to CD, suggesting the
possibility of cross-reaction between these
species. In dogs, the major allergens of grasses are
yet to be identified; hence we could not test the
hypothesis of cross-reaction between and PN in
this study.
Among the ten animals with positive pollen
responses through PT, 2 were positive only to CD,
2 to CD and LM, 2 to CD, LM and PN, 3 only LM
and 1 to CD and PN. Therefore, there was no
pattern of response suggestive of cross reactivity.
The exception was the 3 PN positive dogs that
were also CD positive. However, 4 dogs were CD
positive but not PN positive. These results may
indicate true sensitization to both species and
cross reactivity between species, however
suggesting that CD extracts may contain their own
antigens. Due to the small number of positive tests
(n=10) and the heterogeneous pattern of
responses, it was not possible to perform an
adequate statistical analysis for evaluation of
cross-reactivity among the species tested. For
cross-reactivity studies, the ideal would be the use
of immunochemical methods such as Cross-
Inhibition ELISA, which is beyond the scope of
this study.
The negative results in the present study can be
explained by the sensitization to other
environmental allergens, mainly from house dust
mites species, species, as Dermatophagoides
farinae, D. pteronyssinus and Blomia tropicalis, as
shown by Cunha et al. in studies done in Brazil
(Cunha et al., 2007; Cunha, 2012). Besides that, it
is estimated that about 30% of patients with
clinical diagnosis of atopic dermatitis have
negative skin tests (Bauer et al., 2010). This
suggests an innate, cellular inflammatory
component that mediates pruritus development
and symptoms related to atopic dermatitis in some
patients, with no clear relation with sensitization
and IgE formation (Halliwell, 2006). These
findings could also explain some negative results
observed. No systemic or local adverse reactions
were observed in the PT and IDT, suggesting the
safety of these tests in dogs. Some dogs presented
only local effects characterized by pruritus and
erythema, which disappeared in a few hours. In
humans, the PT generates fewer adverse reactions
than IDT, and fatal reactions can occur in the latter
(Bernstein et al., 2008).
CONCLUSION
The prick test was more effective in identifying
grass pollen sensitization in dogs with atopic
dermatitis than the intradermal skin test.
Sensitization to grass pollen occurred in about a
quarter of the subjects and was more common to
CD and LM through the prick test. Future studies
are recommended to evaluate the efficacy of
allergen-specific immunotherapy in dogs
sensitized to these grass species.
REFERENCES
ARAÚJO, L.M.L.; ROSARIO, N.A.; MARI, A.
Molecular-based diagnosis of respiratory allergic
diseases in children from Curitiba, a city in
Southern Brazil. Allergol. Immunopathol., v.44,
p.18-22, 2015.

Grass pollen sensitization…
Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021 825
BAUER, C.L.; HENSEL, P.; AUSTEL, M.;
KEYS, D. Determination of irritant threshold
concentrations to weeds, trees and grasses through
serial dilutions in intradermal testing on healthy
clinically nonallergic dogs. Vet. Dermatol., v.21,
p.192-197, 2010.
BERNSTEIN, I.L.; LI, J.T.; BERNSTEIN, D.I. et
al. Allergy diagnostic testing: an updated practice
parameter. Ann. Allergy Asthma Immunol., v.100,
p.S1-S148, 2008.
BONISSONI, E.; PASSOS NETO, J. O Lolium
multiflorum associado ao aumento do consumo de
anti-histamínicos. Visão Acad., v.15, p.81-99,
2014.
CARLOTTI, D.N. Clinical signs of canine atopic
dermatitis and concurrent Diseases. In: WORLD
CONGRESS OF VETERINARY
DERMATOLOGY AND THE WORLD
ASSOCIATION FOR VETERINARY
DERMATOLOGY, 7., 2012, Bordeaux.
Proceedings… Bordeaux: WAVD, 2012. p.58-66.
CUNHA, V.E.S. Serological identification of
house dust mite allergens in dogs with atopic
dermatitis. Pesqui. Vet. Bras., v.32, p.917-921,
2012.
CUNHA, V.E.S.; HAHNSTADT, R.L.;
SOARES, A.M.B.; FACCINI, J.L.H. Evaluation
of skin sensitivity in dogs bearing allergic
dermatitis to standardized allergenic extract of
house dust and storage mites. Pesqui. Vet. Bras.,
v.27, p.341-344, 2007.
CUNHA, V.E.S.; PEREIRA, D.T.; FARIAS,
M.R. et al. Intradermal Test - Proficiency testing
in dogs. In: CONGRESO
LATINOAMERICANO DE DERMATOLOGÍA
VETERINARIA, 3., 2015, Buenos Aires.
Anales... Buenos Aires: [ASIS], 2015.
ESTEVES, P.C.; ROSÁRIO FILHO, N.A.;
TRIPPIA, S.G.; CALEFFE, L.G. Sensibilização
atópica em escolares e adultos de Curitiba,
Paraná. Rev. Bras. Alergia Imunopatol., v.22,
p.156-160, 1999.
FERNÁNDEZ-CALDAS, E. On mite allergy in
dogs and humans. Int. Arch. Allergy Immunol.,
v.160, p.329-330, 2013.
GENTRY, C.M.; MESSINGER, L. Comparison
of intradermal and percutaneous testing to
histamine, saline and nine allergens in healthy
adult cats. Vet. Dermatol., v.27, p.370-e92, 2016.
HALLIWELL, R. Revised nomenclature for
veterinary allergy. Vet. Immunol. Immunopathol.,
v.114, p.207-208, 2006.
HENSEL, P.; AUSTEL, M.; MEDLEAU, L. et al.
Determination of threshold concentration of
allergens and evaluation of two different
histamine concentrations in canine intradermal
testing. Vet Dermatol., v.15, p.304-308, 2004.
MASUDA, K.; SAKAGUCHI, M.; FUJIWARA,
S.; KURATA, K. et al. Positive reactions to
common allergens in 42 atopic dogs in Japan. Vet.
Immunol. Immunopathol., v.73, p.193-204, 2000.
OLIVRY, T. Evidence-bsed guidelines for anti-
allergic drug withdrawal times before allergen-
specific intradermal and IgE serological tests in
dogs. Vet. Dermatol., v.24, p.225-e49, 2013.
PEREIRA, D.T.; CUNHA, V.E.S.; SCHMIDT,
C. et al. Sensitization study of dogs with atopic
dermatitis in the central region of Rio Grande do
Sul. Arq. Bras. Med. Vet. Zootec., v.67, p.1533-
1538, 2015.
PRÉLAUD, P. Allergens and environmental
influence. In: NOLI, C.; FOSTER, A.;
ROSENKRANTZ, W. Veterinary allergy.
Oxford: Wileey Blackwell, 2014. p.24-31.
PUCHEU-HASTON, C.M.; BIZIKOVA, P.;
EISENSCHENK, M.N.C. et al. Review: the role
of antibodies, autoantigens and food alleregens in
canine atopic dermatitis. Vet. Dermatol., v.26,
p.115-e30, 2015.
RANDALL, M.; HILLIER, A.; COLE, L.K. et al.
Quantitation of house dust mites and house dust
mite allergens in the microenvironment of dogs.
Am. J. Vet. Res., v.64, p.1580-1588, 2003.
ROCHA, M.I.L. Skin prick tests – preliminary
evaluation of this technique for the diagnosis of
canine atopic dermatitis sensitization. 68f. 2012.
68f. Dissertation (Masters in Veterinary
Medicine) - Technical University of Lisboa, POR.
ROSÁRIO FILHO, N.A. Alergia ao pólen de
gramíneas: “back to the future”. Rev. Bras.
Alergia Imunopatol., v.35, p.82-84, 2012.
ROSÁRIO FILHO, N.A. Sensibilização atópica a
aeroalérgenos em crianças asmáticas em Curitiba.
J. Paranaense Ped., v.3, p.80-82, 2002.

Cerdeiro et al.
826 Arq. Bras. Med. Vet. Zootec., v.73, n.4, p.821-826, 2021
ROSSI, M.A.; MESSINGER, L.; OLIVRY, T.;
HOONTRAKOON, R. A pilot study of the
validation of percutaneous testing in cats. Vet.
Dermatol., v.24, p.488-493, 2013.
ROUSSEL, A.J.; BRUET, V.; BORDEAU, P.J.
Characterisation of dog sensitization to grass
pollen in western France from 1999 to 2010. Vet.
Rec., v.172, p.686, 2013.
SOPELETE, M.C.; JOSÉ VAZ, L.A.;
TAKETOMI, E.A. Sensitization do Cynodon
dactylon and Paspalum notatum grass pollens in
pollinosis patients in Uberlândia, Minas Gerais.
INTERNATIONAL SCIENTIFIC
CONFERENCE, 2014, Rio de Janeiro.
Proceedings... Rio de Janeiro: WAO, 2014.
(Abstracts).
VAN DER VALK, J.P.M.; GERTH R.W.;
HOORN, E. et al. Measurement and interpretation
of skin prick test results. Clin. Transl. Allergy, v.6,
n.8, 2016.
VIEIRA, F.A.M. Gramíneas tropicais
alergênicas: Cynodon dactylon e Paspalum
notatum – Uma visão para o trópico brasileiro.
Rev. Bras. Alergia Imunol., v.35, n.1, 2012.
VIEIRA, F.A.M. Sensibilização polínica
associada à polinose no trópico brasileiro: um
desafio atual para o diagnóstico clínico. Braz. J.
Allergy Immunol., v.2, p.81-82, 2014.
VIEIRA, F.M.; BRAGA, G.L.; FERREIRA, E.M.
Cynodon dactylon e Paspalum notatum: revisar
conceitos de sensibilização polínica por
gramíneas no Sul do Brasil? Rev. Bras. Alergia
Imunol., v.34, p.218-9, 2011.
WERFEL, T.; HERATIZADEH, A.; NIEBUHR,
M. et al. Exacerbation of atopic dermatitis on
grass pollen exposure in an environmental
challenge chamber. J. Allergy Clin. Immunol.,
v.136, p.96-103, 2016.