ISAC multiplex testing – results of examination in 100 patients suffering from atopic dermatitis

ABSTRACT

To find the molecular components with the highest underlying probability of sensitization in patients suffering from atopic dermatitis and in subgroup of patients with allergic rhinitis, asthma bronchiale and to determine whether there is some difference of sensitization in mild, moderate and severe form of AD, in patients suffering from asthma bronchiale and allergic rhinitis. The complete dermatological and allergological examination including the examination of the sensitization to molecular allergens with Multiplex ISAC testing was performed. Altogether 100 atopic dermatitis patients were examined – 48 men, 52 women, the average age 40.9 years; the sensitization to molecular components was confirmed in 93% of patients. The highest observed sensitization rate was 61.0% to grass specific molecule Phl p 1, the second most frequent sensitization was 57.0% to Betulaceae-specific molecule Bet v 1. Frequently observed sensitizations were those to PR-10 proteins, NPC2 proteins family, Uteroglobin and Lipocalin. In severe form of atopic dermatitis sensitization to molecular components of NPC2 proteins family, Uteroglobin, Lipocalin and Aspergillus were recorded with a significant higher occurrence.

KEYWORDS:

• Atopic dermatitis
• molecular components
• severity of atopic dermatitis
• asthma bronchiale
• allergic rhinitis
• underlying probability of sensitization

Introduction

Atopic dermatitis (AD) is a common, chronic inflammatory skin disease characterized by immune abnormalities and a disturbed epidermal barrier, resulting in increased transepidermal water loss (TEWL) and permeation of allergens, irritants and microbes. The key role of filaggrin (FLG), a protein contained in the granular layer of the epidermis regulating the aggregation of keratin filaments, was evidenced in atopic dermatitis as several loss-of-function mutations in FLG gene or FLG deficiency contribute to epidermal barrier dysfunction and was strongly associated with AD (Czarnowicki et al., 2017; Paller et al., 2019). The progression of atopic dermatitis to asthma and allergic rhinitis during the first several years of life is a phenomenon known as “atopic march”. A majority of patients with AD developed sensitizations to common inhalant allergens and the sensitization level was associated with the severity of AD as well as the risk of developing asthma (Czarnowicki et al., 2017; Paller et al., 2019). Although the mechanism explaining the atopic march remains to be fully elucidated, it is evident that the direct contact of skin with allergens could trigger signals to initiate Th2 allergic response (Czarnowicki et al., 2017; Paller et al., 2019). Emerging data now suggest that epithelial cell-derived cytokines such as thymic stromal lymphopoietin (TSLP), IL-33, and IL-25 may drive the progression from atopic dermatitis to asthma and food allergy (Czarnowicki et al., 2017; Paller et al., 2019).

Atopic dermatitis can be stratified according to different criteria, such as age, severity, complications and other factors. During the last decade, great advances have been made in the clinical characterization of atopic dermatitis phenotypes. However, the identification of different biomarkers that characterize each phenotype is essential for the development of individualized atopic dermatitis therapies. The differentiation of atopic dermatitis in extrinsic or intrinsic is based on total and specific serum IgE levels and a history of family or personal atopy, factors that are characteristic for extrinsic atopic dermatitis. Previous studies suggested that extrinsic atopic dermatitis had a predominant T helper 2 polarization compared with intrinsic atopic dermatitis phenotype. However, the concept has been recently challenged by the observation that mRNA levels of the T helper 2 cytokines IL-4, IL-5, IL-13 and IL-31 were augmented in the skin lesions of both atopic dermatitis phenotypes. In addition, T-cell subsets such as T helper 17 and T helper 22 were found to be augmented in intrinsic atopic dermatitis, with a higher immune activation in intrinsic atopic dermatitis compared with extrinsic atopic dermatitis (Suárez-Fariñas et al., 2013).

Various allergens play a role in the elicitation or exacerbation of eczematous skin lesions in atopic dermatitis, and much research effort has been focused on improving diagnostic tests to identify causative allergens. The main allergenic sources are foods, fungi, trees, weeds, grasses, mites and finally animals; with the largest number of allergenic proteins being found in foods and the smallest in animals (Czarnowicki et al., 2017; Paller et al., 2019). Currently, allergens could be defined as proteins, glycoproteins, lipoproteins, or protein-conjugated haptens, which have unique molecular and structural properties (Czarnowicki et al., 2017; Paller et al., 2019). In today’s clinical practice patients’ skin is used as screening organ for diagnosing type 1 allergy. According to European guidelines skin prick testing with a panel of 18 allergen extracts is recommended, in the US between 10–50 allergens are used. The specificity and sensitivity of skin testing is individually highly variable depending on age, body mass and skin barrier status (Jensen-Jarolim et al., 2017). Molecular allergy diagnosis using singleplex allergens or multiplex allergen microarrays are typical methods of precision medicine (Bousquet et al., 2016; Jensen-Jarolim et al., 2017) and they enhance the specificity of IgE-diagnosis in polysensitized respiratory allergies (Passalacqua et al., 2013), can be applied in food allergies (Foong et al., 2016; Wuthrich, 2014) and atopic dermatitis (Foong et al., 2016; Melioli et al., 2011), and may even reveal unexplained anaphylaxis (Jensen-Jarolim et al., 2017). Immunocap ISAC is an in vitro assay for the measurement of allergen specific IgE antibodies in human plasma (Jensen-Jarolim et al., 2017; Suárez-Fariñas et al., 2013). It is intended to aid in the diagnosis of IgE-mediated allergic disorders (Boyce et al., 2011; Hatzler et al., 2012; Jung et al., 2015; Muraro et al., 2014; Prosperi et al., 2014; Spergel et al., 2015).

In our previous studies, we evaluated also the occurrence of sensitization to food and inhalant allergens in patients suffering from AD, but the sensitization was determined according to extract specific IgE, atopy patch test and skin prick tests without using molecular allergy diagnosis (Čelakovská & Bukač, 2017). Although it is difficult to establish directly the frequency to which sensitization occurs through the skin, the observation that atopic dermatitis tends to precede atopic disease at other sites has led to the proposal that the skin may be an important site in the initiation of the atopic march. In fact, even in the absence of atopic dermatitis, children with skin barrier defects are still at a higher risk for asthma than healthy children, suggesting that the skin may serve as a site for sensitization to allergens even when allergic skin inflammation is absent (Lack et al., 2003). Only few reports demonstrate the comorbidity of allergy to food and inhalant allergens, the severity of atopic dermatitis evaluated with SCORAD index, the relation to the occurrence of asthma bronchiale and allergic rhinitis (Bousquet & Van Cauwenberge, 2001; Hanifin & Rajka, 1980; Röckmann et al., 2014). Asthma is a heterogeneous, multifactorial disease with variable and mostly reversible respiratory pathway obstruction based on a chronic bronchial inflammatory reaction. The symptoms (cough, rhonchus, wheezing, chest tightness, or shortness of breath) are variable and correlated with expiratory flow limitation. Although bronchial hyperresponsiveness (BHR) is often present, the current Global Initiative for Asthma (GINA) Guidelines no longer include it as a necessary or sufficient criterion for diagnosis (GINA. Global Strategy for asthma management and prevention – Update 2015. www.ginasthma.com). Allergic rhinitis was defined as a process which included three cardinal symptoms: sneezing, nasal obstruction and mucus discharge. Symptoms occur with allergen exposure in the allergic patient (Bousquet & Van Cauwenberge, 2001).

Aim of the study

The aim of this study is to find the molecular components with the highest underlying probability of sensitization in patients suffering from atopic dermatitis and in subgroup of patients with allergic rhinitis, asthma bronchiale according to the ISAC Multiplex examination. These molecular components may play an important role in the atopic march. Our other aim is to determine whether there is some difference of sensitization to examined molecular components in mild, moderate and severe form of AD, in patients suffering from asthma bronchiale and allergic rhinitis.

Patients and methods In the period 2018–19, 100 patients suffering from atopic dermatitis at the age of 14 years and older were examined. All these patients were examined in the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech republic.

Inclusion criteria: (1) age 14 years and over (2) atopic dermatitis as defined by the criteria of Hanifin and Rajka (Hanifin & Rajka, 1980). The severity of atopic dermatitis evaluated with SCORAD index (Röckmann et al., 2014).

Exclusion criteria: sytemic immunosupresive therapy – sytemic corticosteroids, cyclosporin, methotrexat, azathioprin, biological therapy, pregnancy, breastfeeding. Patients suffering from oncological disease. Patients with atopic dermatitis having other systemic diseases were excluded from the study as well.

Dermatological examination

Complete dermatological examination was performed in patients included in the study. Severity of atopic dermatitis was scored in agreement with SCORAD, with the assessment of topography items (affected skin area), intensity criteria and subjective parameters. To measure the extent of atopic dermatitis, the rule of nines was applied on a front/back drawing of the patient’s inflammatory lesions. The extent was graded 0–100 points. The intensity part of the SCORAD index consists of six items: erythema, oedema/papules, excoriations, lichenification, crusts and dryness. Each item was graded on a scale 0–3. The subjective items included daily pruritus and sleeplessness. Both subjective items were graded on a 10-cm visual analog scale and the maximum subjective score was 20 points. All items were filled out in the SCORAD evaluation form. The SCORAD index formula was: A/5 + 7B/2 + C. In this formula A is defined as the extent (0–100 points), B is defined as the intensity (0–18 points) and C is defined as the subjective symptoms (0–20 points). The severity of atopic dermatitis is evaluated with SCORAD as a mild form to 20 points, as moderate over 20–50 points, as a severe form over 50 points (Röckmann et al., 2014). The evaluation of SCORAD score was performed every two months during the study (Röckmann et al., 2014).

Bronchial asthma

The diagnosis of bronchial asthma (AB), was determined according to the guidelines of the GINA at allergy outpatients clinic of the Institute of Clinical Immunology and Allergology, Faculty Hospital Hradec Kralove, Czech Republic (GINA. Global Strategy for asthma management and prevention – Update 2015. www.ginasthma.com).

Allergic rhinitis

The evaluation of allergic rhinitis (AR), was made according to the allergy testing and personal history of the Institute of Clinical Immunology and Allergology, Faculty Hospital Hradec Kralove, Czech Republic (Bousquet & Van Cauwenberge, 2001). AR was defined as a process which included three cardinal symptoms: sneezing, nasal obstruction and mucus discharge. Symptoms occur with allergen exposure in the allergic patient (Bousquet & Van Cauwenberge, 2001).

This study is approved by the Ethics committee of Faculty Hospital Hradec Králové, Charles University, Czech republic.

Examination of specificIgE to molecular components

The serum level of the sIgE was measured by the component-resolved diagnosis microarray-based sIgE detection assay ImmunoCAP ISAC (Phadia, Thermo Fisher Scientific, Uppsala, Sweden). ImmunoCAP ISAC is a solid-phase multiple immunoassay which enables to determine 112 different components from 51 allergen sources (Jakob et al., 2015; Melioli et al., 2011). The allergens are applied in triplicates to ensure the test reproducibility. The ImmunoCAP ISAC (Phadia AB, Uppsala, Sweden) system consists of a glass slide with immobilized allergens, a scanner and software. Thirty microlitres of serum sample is required to test a predefined allergen panel of 112 allergen components derived from 51 common allergen sources including food, inhalation allergens, latex and venoms. The assay is manual and takes about 3.5 h. A description of the technology is found in the study by Melioli et al. (2011). Samples were assayed in 2011 at Phadia, Uppsala, using a research version of the ISAC 112 system. Results were analyzed in independent standard units (ISU). The ISU correlate with the kU_A/l of ImmunoCAP units although they are not interchangeable (Melioli et al., 2011). Both microarray methods are regulatory classified as semiquantitative methods; however, they both provide quantitative IgE data. The specific IgE values are presented in arbitrary units called ISAC Standardized Units (measuring range of 0.3–00 ISU-E). The level of specific IgE higher than 0.3 ISU-E was assessed as positive. The level of molecular components in ISU –E was evaluated: <0.3 – negative, 0.3–0.9 ISU –E low positivity, 0.9–15 ISU –E moderate positivity, above 15 ISU –E very high positivity (Jakob et al., 2015; Melioli et al., 2011).

Statistical analysis

We analysed the data to determine whether there is some difference in sensitization to examine molecular components in mild, moderate and severe form of AD, in patients suffering from asthma bronchiale and allergic rhinitis. Allergens were ordered in the decreasing order to find out the one with the highest probability. We continued by testing the difference between the highest underlying probability and the following one and we found out that the null hypothesis of equality of those underlying probabilities was confirmed. It follows that we have to consider a set of allergens with the highest probability.

The question of calculating which allergens fall in the set of those with the highest probability is found in the study of change-point problem. We consider events with the highest probabilities, denote their probabilities as p(1), p(2), …, p(k) and ask if we may include one more event with probability p(k + 1). The null hypothesis may be written as H0: p(i) = p for i = 1, …, k + 1 for some probability p common to all the k + 1 events, wheras the alternative hypothesis is Ha: p(i) = p for i = 1, …, k but p(k+1) is not equal to p. This says that the event with this distinct probability p(k + 1) is not in the set of events with the highest probability. Since the probabilities p(1), …, p(k) are the same, we can add all the numbers of successes (positive results of tests) and all the numbers of trials in binomial distributions with the same parameter p and, provided the trials are independent, the cumulative sum of the numbers of successes out of the cumulative sum of trials have a binomial distribution with the same parameter p. We compare this probability p with the probability p(k + 1) of the next k + 1 -st event.

As Worsley would put it, essentially, we are dealing with the association in a 2 × 2 contingency table. Our conclusion is that such an association may be tested by implementing the well-known chi-squared test (Worsley, 1983). Essentially the method of maximum likelihood estimates K by choosing the value of k that gives the strongest association in a 2 × 2 contingency table formed by combining all periods up to and including k and all periods after k, against success and failure (Worsley, 1983).

We tested if there are significant differences in relative frequencies of molecular components in mild, moderate and severe forms of AD, in patients suffering from asthma bronchiale, and allergic rhinitis. We formed a 2 by 3 (in the case of severity of AD) or 2 by 2 (in the case of asthma bronchiale and allergic rhinitis) tables. Such a table was evaluated by the usual Chi-squared test. The p-value is displayed in the right hand column. Unfortunately, zeroes appeared in many of the tables and one has to be careful in making conclusions.

Results

Patients

Altogether 112 different components from 51 allergen sources were examined in 100 atopic dermatitis patients included in the study (48 men and 52 women with the average age 40.9 years and with the average SCORAD 39, s.d.13.1 points). The mild form of AD was recorded in 14 patients (14%), moderate form in 58 patients (58%), severe form in 28 patients (28%). In the included patients, 55 patients (55%) suffer from asthma bronchiale and 78 patients (78%) suffer from rhinitis. The positive results in sensitization to molecular components were recorded in 93 patients (93%). The characteristics of patients are shown in Table 1. We evaluated the order of molecular components with the underlying probability of the sensitization in all included patients in general (Table 2) and then in patients with mild, moderate and severe form of AD, in subgroup of patients with asthma bronchiale and allergic rhinitis (Tables 3–7). In all tables, we distinguish between mainly species-specific components and mainly cross-reactive components. We show the sensitization only to the most frequent molecular components in our results, because the tables with 112 molecular components seem to be too big. The list of molecular components in mild, moderate and severe form of AD is shown in Table 3. There is no component with the highest underlying probability in patients suffering from mild form. On the other hand, the first 19 molecular components with the highest underlying probability were found in patients suffering from moderate form and the first 29 molecular components in patients with severe form (Table 3). The comparison of the relative frequency of sensitization to molecular components in patients suffering from the moderate form of AD (58 patients = 100%) and severe form (28 patients = 100%) of AD in comparison to patients with mild form of AD (14 patients = 100%) is shown in Complement to Table 3. Typically in patients suffering from a severe form the sensitization to molecular components such as Phl p1, Der f 2, Der p 2, Can f 1, Fel d 1, Fel d 4, Pen m2, Equ c 1, Asp f 6, Mus m 1 and Api g 1 has a higher occurrence.

Table 1. The characteristic of patients with atopic dermatitis.

– 100 patients examined – 48 men, 52 women, the average age 40.9 years

– The average SCORAD 39 s.d. 13.1 points

– The positive results in sensitization to molecular components – 93 patients (93 %)

– Mild form of AD patients 14 (14 %)

– Moderate form of AD patients 58 (58 %)

– Severe form of Ad patients 28 (28 %)

– Number of patients with asthma bronchiale – 55 (55 %)

– Number of patients with allergic rhinitis – 78 (78 %)

Table 2. The list of molecular components in all included patients (100 patients = 100 %). The order of first 18 molecular components is recorded with the higher underlying probability of the occurrence – marked as extra bold. The underlying probability of the occurrence is calculated according to Worsley (22).

Molecular components	Allergen	Biochemical name Protein family	Number of patients	Frequency in %
Phlp1	Timothy	β-expansin	61	61.0
Betv 1*	Birch	PR-10 protein	57	57.0
Phlp4	Timothy	berberine – bridge enzym	52	52.0
Cynd1	Bermuda grass	Glycoprotein	49	49.0
Mald1*	Apple	PR-10 protein	47	47.0
Derp2	House dust mite	NPC2 proteins family	46	46.0
Prup1*	Peach	PR-10 protein	46	46.0
Cora1.0101*	Hazelnut	PR-10 protein	45	45.0
Derf2	House dust mite	NPC2 proteins family	45	45.0
Phlp5	Timothy	Grass group 5, ribonuclease	43	43.0
Alng1*	Alder	PR-10 protein	43	43.0
Phlp6	Timothy	Grass group 6	42	42.0
Feld1	Cat	Uteroglobin	42	42.0
Cora1.0401*	Hazelnut	PR-10 protein	42	42.0
Phlp2	Timothy	Grass group 2	39	39.0
Canf1	Dog	Lipocalin	39	39.0
Arah8*	Peanut	PR-10 protein	38	38.0
Derp1	House dust mite	Cystein protease	36	36.0
Derf1	House dust mite	Cystein protease	34	34.0
Glym4*	Soy	PR-10 protein	32	32.0
Artv 1	Mugwort	Defensin like protein	29	29.0
Alta1	Alternaria	Alternaria	29	29.0
Feld4	Cat	Lipocalin	29	29.0
Equc1	Horse	Lipocalin	27	27.0
Canf5	Dog	Kallikrein	26	26.0
Plaa2	Plane tree	Polygalactouronase	24	24.0
Actd8	Kiwi	PR-10 protein	24	24.0
Penm2	Shrimp	Arginin kinase	22	22.0
Apig1*	Celery	PR-10 protein	22	22.0
MUXF3	Sugar epitope from bromelaine		22	22.0

*mainly cross-reactive components.

Table 3. The list of molecular components in mild, moderate and severe form of AD in 100 patients. There is no component with the highest underlying probability in patients suffering from mild form. On the other hand, we found a set of molecular components with the highest underlying probability in moderate and severe form – marked extra bold, *mainly cross-reactive components. The underlying probability of the occurrence is calculated according to Worsley (22).

Severity of atopic dermatitis – number of patients (relative frequency, given severity)
Mild form in 14 patients = 100 %			Moderate form in 58 patients = 100 %			Severe form in 28 patients = 100 %
Molecular component	No. of patients	Relative frequency %	Molecular component	No. of patients	Relative frequency %	Molecular component	No. of patients	Relative frequency %
Betv1*	8	57.1	Phlp1	37	63.8	Phlp1	20	71.4
Cynd1	6	42.9	Betv1*	33	56.9	Cynd1	19	67.8
Alng1*	6	42.9	Phlp4	30	51.7	Phlp4	18	64.2
Cora1.0401*	6	42.9	Mald1*	28	48.3	Derf2	18	64.2
Phlp1	4	28.6	Phlp5	27	46.6	Derp2	18	64.2
Phlp2	4	28.6	Phlp6	26	44.8	Cora1.0101*	16	57.1
Phlp4	4	28.6	Derp2	26	44.8	Betv1*	16	57.1
Cora1.0101*	4	28.6	Canf1	26	44.8	Feld1	16	57.1
Alta1	4	28.6	Prup1*	26	44.8	Mald1*	16	57.1
Prup1*	4	28.6	Cora1.0101*	25	43.1	Prup1*	16	57.1
Arah8*	4	28.6	Derf2	25	43.1	Phlp2	15	53.5
Derp1	3	21.4	Cora1.0401*	25	43.1	Alng1*	15	53.5
Mald1*	3	21.4	Cynd1	24	41.4	Feld4	15	53.5
Phlp5	2	14.3	Feld1	24	41.4	Arah8*	15	53.5
Phlp6	2	14.3	Alng1*	22	37.9	Phlp5	14	50.0
Olee1	2	14.3	Phlp2	20	34.5	Phlp6	14	50.0
Derf1	2	14.3	Derf1	19	32.8	Derp1	14	50.0
Derf2	2	14.3	Derp1	19	32.8	Equc1	14	50.0
Derp2	2	14.3	Arah8*	19	32.8	Penm2	14	50.0
Feld1	2	14.3	Artv1	17	29.3	Derf1	13	46.4
Glym4*	2	14.3	Glym4*	17	29.3	Canf1	13	46.4
Phlp11	1	7.1	Alta1	16	27.6	Glym4*	13	46.4
Phlp7*	1	7.1	Phlp11	15	25.9	Artv1	11	39.2
Cupa1	1	7.1	Feld4	14	24.1	Aspf6	11	39.2
Cryj1	1	7.1	Canf5	14	24.1	Canf5	11	39.2
Plaa2	1	7.1	Actd8	14	24.1	Cora1.0401*	11	39.2
Betv4*	1	7.1	MUXF3	14	24.1	Plaa2	10	35.7
Artv1	1	7.1	Plaa2	13	22.4	Musm1	10	35.7
Blot5	1	7.1	Equc1	13	22.4	Apig1*	10	35.7
Derp10*	1	7.1	Actd2*	13	22.4	Alta1	9	32.1
Blag7*	1	7.1	Apig1*	12	20.7	Alta6	9	32.1
Lepd2	1	7.1	Cupa1	10	17.2	Actd8	9	32.1
Canf5	1	7.1	Plal1	10	17.2	Olee9	8	28.5
Actd2*	1	7.1	Aspf6	10	17.2	Canf2	8	28.5
Actd8	1	7.1	Musm1	10	17.2	Lepd2	7	25.0
Anis3*	1	7.1	Olee9	9	15.5	Vesv5	7	25.0
Penm1*	1	7.1	Canf2	9	15.5	Actd2*	7	25.0
Penm2	1	7.1	Cryj1	8	13.8	MUXF3	7	25.0
Penm4	1	7.1	Mera1*	7	12.1	Cryj1	6	21.2
MUXF3	1	7.1	Alta6	7	12.1	Olee1	6	21.2

Complement to Table 3. The comparison of the relative frequency of sensitization to molecular components in patients suffering from the moderate form of AD (58 patients = 100 %) and severe form (28 patients = 100 %) of AD in comparison to patients with mild form of AD (14 patients = 100 %). Significant differences are marked extra bold. Typically in patients suffering from a severe form the sensitization to molecular components such as Phl p1, Der f 2, Der p 2, Can f 1, Fel d 1, Fel d 4, Pen m2, Equ c 1, Asp f 6, Mus m 1 and Api g 1 has a higher occurrence.

   

Molecular component	Allergen	Biochemical function	Mild form of AD 14 patients (=100 %)	Moderate form of AD 58 patients (=100 %)	Severe form of AD 28 patients (=100 %)	p– value
Phl p 1	Timothy	β-expansin	4 (28.6 %)	37 (63.8 %)	20 (71.4 %)	0.022
Bet v1*	Birch	PR-10 protein	8 (57.1 %)	33 (56.9 %)	16 (57.1 %)	1.000
Phl p 4	Timothy	Berberine –bridge enzym	4 (28.6 %)	30 (51.7 %)	18 (64.3 %)	0.092
Cyn d 1	Bermuda grass	Glycoprotein	6 (42.9 %)	24 (41.4 %)	19 (67.8 %)	0.063
Mal d 1*	Apple	PR-10 protein	3 (21.4 %)	28 (48.3 %)	16 (57.1 %)	0.088
Cor a 1.0101*	Hazelnut	PR-10 protein	4 (28.6 %)	25 (43.1 %)	16 (57.1 %)	0.194
Pru p 1	Peach	PR-10 protein	4 (28.6 %)	26 (44.8 %)	16 (57.1 %)	0.208
Der f 2	House dust mite	NPC2 proteins family	2 (14.3 %)	25 (43.1 %)	18 (64.3 %)	0.008
Der p 2		NPC2 proteins family	2 (14.3 %)	26 (44.8 %)	18 (64.3 %)	0.009
Can f 1	Dog	Lipocalin	0 %	26 (44.8 %)	13 (46.4 %)	0.005
Fel d 1	Cat	Uteroglobin	2 (14.3 %)	24 (41.4 %)	16 (57.1 %)	0.029
Cor 1.0401*	Hazelnut	PR-10 protein	6 (42.9 %)	25 (43.1 %)	11 (39.2 %)	0.943
Der f 1	House dust mite	Cystein protease	2 (14.3 %)	19 (32.8 %)	13(46.4 %)	0.111
Der p 1		cystein protease	3 (21.4 %)	19 (32.8 %)	14 (50.0 %)	0.140
Ara h 8*	Peanut	PR-10 protein	4 (28.6 %)	19 (32.8 %)	15 (53.5 %)	0.130
Aln g 1*	Alder	PR-10 protein	6 (42.9 %)	22 (37.9 %)	15 (53.5 %)	0.390
Phl p 2	Timothy	Grass group 2	4 (28.6 %)	20 (34.5 %)	15 (53.5 %)	0.162
Fel d 4	Cat	Lipocalin	0 (0.0 %)	14 (24.1 %)	15 (53.5 %)	0.001
Phl p 5	Timothy	Grass group 5, ribonuclease	2 (14.3 %)	27 (46.6 %)	14 (50.0 %)	0.062
Phl p 6	Timothy	Grass group 6	2 (14.3 %)	26 (44.8 % )	14 (50.0 %)	0.069
Equ c 1	Horse	Lipocalin	0 (0.0 %)	13 (22.4 %)	14 (50.0 %)	0.001
Pen m 2	Shrimp	Arginin kinase	1 (7.1 %)	7 (12.1 %)	14 (50.0 %)	0.000
Gly m 4*	Soy	PR-10 protein	2 (14.3 %)	17 (29. 3% )	13 (46.4 %)	0.087
Asp f 6	Aspergillus	MnSOD highly conserved allergen	0 (0.0 %)	10 (17.2 %)	11 (39.2 %)	0.007
Can f 5	Dog	Kallikrein	1 (7.1 %)	14 (24.1 %)	11 (39.2 %)	0.072
Pla a 2	Plane tree	Polygalactouronase	1 (7.1 %)	13 (22.4 %)	10 (35.7 %)	0.113
Mus m1	Mouse	Lipocalin	0 (0.0 %)	10 (17.2 %)	10 (35.7 %)	0.017
Api g 1*	Celery	PR-10 protein	0 (0.0 %)	12 (20.7 %)	10 (35.7 %)	0.029

*mainly cross-reactive components.

Table 4. The order of molecular components in subgroups of patients suffering from asthma bronchiale and in patients without asthma bronchiale. The first 19 molecular components with the highest underlying probability (marked extra bold) in patients suffering from asthma bronchiale were found to be the same as those molecular components in patients not suffering from asthma bronchiale. We cannot say that one single component has a highest probability. The underlying probability of the occurrence is calculated according to Worsley (22).

Positive results of sensitization to molecular components in 100 patients according to the occurrence of asthma bronchiale
Asthma bronchiale yes, 55 patients = 100 %			Asthma bronchiale no, 45 patients = 100 %
Molecular components	Number of patients	Frequency %	Molecular components	Number of patients	Frequency %
Phlp1	37	67.2	rBetv1*	27	60.0
Cynd1	30	54.5	Phlp1*	24	53.3
Phlp4	30	54.5	Phlp4	22	48.8
Betv1*	30	54.5	Alng1*	21	46.6
Derf2	29	52.7	Cynd1*	19	42.2
Derp2	29	52.7	Cora1.0101*	19	42.2
Mald1*	28	50.9	Mald1*	19	42.2
Phlp6	27	49.1	Prup1*	19	42.2
Prup1*	27	49.1	Cora1.0401*	18	40.0
Phlp2	26	47.2	Phlp5	17	37.7
Phlp5	26	47.2	Derp2	17	37.7
Cora1.0101*	26	47.2	Derf2	16	35.5
Feld1	26	47.2	Feld1	16	35.5
Canf1	24	43.6	Phlp6	15	33.3
Cora1.0401*	24	43.6	Canf1	15	33.3
Derf1	23	41.8	Arah8*	15	33.3
Derp1	23	41.8	Phlp2	13	28.8
Arah8*	23	41.8	Derp1*	13	28.8
Alng1*	22	40.0	Canf5	13	28.8
Glym4*	20	36.3	Artv1	12	26.6
Feld4	19	34.5	Glym4*	12	26.6
Alta1	18	32.7	Alta1	11	24.4
Equc1	18	32.7	Derf1	11	24.4
Artv1	17	30.9	Feld4	10	22.2
MUXF3	16	29.1	Actd8	10	22.2

Complement to Table 4. The comparison of the relative frequency of sensitization to molecular components in patients suffering from asthma bronchiale (55 patients = 100 %) and in patients without asthma bronchiale (45 patients = 100 %). No significant difference between frequencies of sensitization to molecular components was found, although we can observe the increasing frequency of sensitization almost in all molecular components in patients suffering from asthma bronchiale.

Table 4. Continued

Molecular component	Biochemical function	Asthma bronchiale yes, 55 patients = 100 %		Asthma bronchiale no, 45 patients = 100 %		p – value
		Number of patients	Frequency %	Number of patients	Frequency %
Phlp1	β-expansin	37	67.2	24	53.3	0.155
Cynd1	glycoprotein	30	54.5	19	42.2	0.220
Phlp4	berberine-bridge enzym	30	54.5	22	48.9	0.573
Betv 1*	PR-10 protein	30	54.5	27	60.0	0.584
Derf2	NPC2 proteis family	29	52.7	16	35.6	0.086
Derp2	NPC2 proteis family	29	52.7	17	37.8	0.136
Mald1*	PR-10 protein	28	50.9	19	42.2	0.387
Phlp6	grass group 6	27	49.1	15	33.3	0.112
Prup1*	PR-10 protein	27	49.1	19	42.2	0.493
Phlp2	grass group 2	26	47.3	13	28.9	0.061
Phlp5	grass group 5	26	47.3	17	37.8	0.340
Cora1.0101*	PR-10 protein	26	47.3	19	42.2	0.614
Feld1	Uteroglobin	26	47.3	16	35.6	0.238
Canf1	Lipocalin	24	43.6	15	33.3	0.293
Cora1.0401*	PR-10 protein	24	43.6	18	40.0	0.714
Derf1	Cystein protease	23	41.8	11	24.4	0.068
Derp1	Cystein protease	23	41.8	13	28.9	0.180
Arah8*	PR-10 protein	23	41.8	15	33.3	0.384
Alng1*	PR-10 protein	22	40.0	21	46.7	0.503
Glym4*	PR-10 protein	20	36.3	12	26.7	0.301
Feld4	Uteroglobin	19	34.5	10	22.2	0.177
Alta1	Alternaria	18	32.7	11	24.4	0.364
Equc1	Lipocalin	18	32.7	9	20.0	0.154
Artv 1	Defensine like protein	17	30.9	12	26.6	0.642

*mainly cross-reactive components.

Table 5. The order of molecular components in patients suffering from allergic rhinitis and in patients without allergic rhinitis. The first 9 molecular components with the highest underlying probability (marked extra bold) in patients suffering from allergic rhinitis were found to be different from those not suffering from allergic rhinitis. The underlying probability of the occurrence is calculated according to Worsley (22).

Positive results of molecular components in 100 patients according to the occurrence of allergic rhinits
Allergic rhinitis yes – 78 patients = 100 %			Allergic rhinitis no – 22 patients = 100%
Molecular components	Number of patients	Frequency %	Molecular components	Number of patients	Frequency %
Phlp1	51	65.4	Phlp1	10	45.5
Betv 1*	49	62.8	Betv 1*	8	36.3
Phlp4	46	58.9	Cora1.0401*	7	31.8
Cynd1	44	56.4	Phlp4	6	27.3
Mald1*	42	53.8	Alng1*	6	27.3
Cora1.0101*	41	52.6	Derp2	6	27.3
Prup1*	41	52.6	Cynd1	5	22.7
Derf2	40	51.3	Phlp5	5	22.7
Derp2	40	51.3	Phlp6	5	22.7
Phlp5	38	48.7	Artv 1	5	22.7
Feld1	38	48.7	Derf2	5	22.7
Phlp6	37	47.4	Mald1*	5	22.7
Alng1*	37	47.4	Prup1*	5	22.7
Canf1	37	47.4	Arah8*	5	22.7
Phlp2	35	44.9	Phlp2	4	18.2
Cora1.0401*	35	44.9	Cora1.0101*	4	18.2
Arah8*	33	42.3	Derf1	4	18.2
Derp1	32	41.0	Derp1	4	18.2
Derf1	30	38.5	Feld1	4	18.2
*mainly cross-reactive components.
Complement to Table 5. The comparison of the relative frequency of sensitization to molecular components in patients suffering from allergic rhinitis (78 patients = 100 %) and in patients without allergic rhinitis (22 patients = 100 %). The significant difference is marked extra bold. Typically in patients suffering allergic rhinitis the sensitization to molecular components such as Bet v 1, Phl p 4, Phl p 6, Cyn d 1, Mal d 1, Cora 1.0101, Pru p 1, Der f 2, Der p 2, Phl p 5, Fel d 1, Can f 1, Phl p 2, Der p 1 has a higher occurrence.
		Allergic rhinitis yes 78 patients = 100 %		Allergic rhinitis no 22 patients = 100%
Molecular components	Protein family	Number of patients	Frequency %	Number of patients	Frequency %	p-value
Phlp 1	Beta expansin	51	65.4	10	45.5	0.091
Betv 1*	PR-10 Protein	49	62.8	8	36.3	0.027
Phlp 4	Berberine – bridge enzym	46	58.9	6	27.3	0.009
Cynd 1	Glycoprotein	44	56.4	5	22.7	0.005
Mald 1*	PR-10 Protein	42	53.8	5	22.7	0.010
Cora 1.0101*	PR-10 Protein	41	52.6	4	18.2	0.004
Prup 1*	PR-10 Protein	41	52.6	5	22.7	0.013
Derf 2	NPC2 protein family	40	51.3	5	22.7	0.017
Derp 2	NPC2 protein family	40	51.3	6	27.3	0.046
Phlp 5	Grass group 5	38	48.7	5	22.7	0.030
Feld 1	Uteroglobin	38	48.7	4	18.2	0.010
Phlp 6	Grass group 6	37	47.4	5	22.7	0.038
Alng 1*	PR-10 Protein	37	47.4	6	27.3	0.092
Canf 1	Lipocalin	37	47.4	2	9.1	0.001
Phlp 2	Grass group 2	35	44.9	4	18.2	0.023
Cora1.0401*	PR-10 Protein	35	44.9	7	31.8	0.273
Arah 8*	PR-10 Protein	33	42.3	5	22.7	0.095
Derp 1	Cystein protease	32	41.0	4	18.2	0.049
Derf 1	Cystein protease	30	38.5	4	18.2	0.076

*mainly cross-reactive components.

Table 6. The molecular components with the simultaneously confirmed highest underlying probability of occurrence in patients suffering from allergic rhinitis, asthma bronchiale and severe form of AD – the relative frequency of sensitization to these molecular components in comparison to patients without asthma bronchiale, without allergic rhinitis and to patients with mild form of AD. The significant difference in the relative frequency of sensitization is marked extra bold. The underlaying probability of the occurrence is calculated according to Worsley (22).

Molecular components	The relative frequency of sensitization
	Asthma bronchiale		p – value	Allergic rhinitis		p – value	Severity of AD		p – value
	Yes, 55 patients (=100 %)	No, 45 patients (=100 %)		Yes, 78 patients (=100 %)	No, 22 patients (=100 %)		Severe form 28 patients (=100 %)	Mild form 14 patients (=100 %)
Phl p 1	67.3 %	53.3 %	0.155	65.4 %	45.5 %	0.091	71.4 %	28.6 %	0.022
Bet v 1*	54.5 %	60.0 %	0.584	62.8 %	36.6 %	0.027	57.1 %	57.1 %	1.000
Phl p 4	54.5 %	48.8 %	0.573	58.9 %	27.3 %	0.009	64.3 %	28.6 %	0.092
Cyn d 1	54.5 %	42.2 %	0.220	56.4 %	22.7 %	0.005	67.8 %	42.9 %	0.063
Mal d 1*	50.9 %	42.2 %	0.387	53.8 %	22.7 %	0.010	57.1 %	21.4 %	0.088
Cor a 1.0101*	47.3 %	42.2 %	0.614	52.6 %	18.2 %	0.004	57.1 %	28.6 %	0.194
Pru p 1*	49.1 %	42.2 %	0.493	52.6 %	22.7 %	0.013	57.1 %	28.6 %	0.208
Der f 2	52.7 %	35.5 %	0.086	51.3 %	22.7 %	0.017	64.3 %	14.3 %	0.008
Der p 2	52.7 %	37.7 %	0.136	51.3 %	27.3 %	0.046	64.3 %	14.3 %	0.009
Can f 1	43.6 %	33.3 %	0.293	47.4 %	9.1 %	0.001	46.4 %	0 %	0.005
Fel d 1	47.2 %	35.5 %	0.238	48.7 %	18.2 %	0.010	57.1 %	14.3 %	0.029
Cor 1.0401*	43.6 %	40.0 %	0.714	44.9 %	31.8 %	0.273	39.2 %	42.9 %	0.943
Der f 1	41.8 %	24.4 %	0.068	38.4 %	14.3 %	0.076	46.4 %	14.3 %	0.111
Der p 1	41.8 %	28.9 %	0.180	41.0 %	21.4 %	0.049	50.0 %	21.4 %	0.140
Ara h 8*	41.8 %	33.3 %	0.384	42.3 %	28.6 %	0.095	53.6 %	28.6 %	0.130
Aln g 1*	40.0 %	46.7 %	0.503	47.4 %	42.9 %	0.092	53.6 %	42.9 %	0.390

*mainly cross – reactive components.

Table 7. The molecular components with the confirmed significant difference of occurrence in patients suffering from allergic rhinitis and severe form of AD. Typically in patients suffering from a severe form of AD and from allergic rhinitis the sensitization to molecular components such as Der f 2, Der p 2, Can f 1 and Fel d 1 has a higher occurrence simultaneously.

Allergic rhinitis – 78 patients (=100 %)			Severe form of AD – 28 patients (=100 %)
Molecular component	Allergen	Frequency %	Molecular component	Allergen	Frequency %
Betv 1*	Birch PR-10 protein	62.8	Phl p 1	Timothy CCD bearing protein	71.4
Phlp 4	Timothy CCD bearing protein, berberine – bridge enzym	58.9	Der f 2	House dust mite NPC2 proteins family	64.3
Cyn d 1	Bermuda grass Glycoprotein	56.4	Der p 2	House dust mite NPC2 proteins family	64.3
Mal d 1*	Apple PR-10 protein	53.8	Can f 1	Dog Lipocalin	46.4
Cora1.0101*	Hazel nut PR-10 protein	52.6	Fel d 1	Cat Uteroglobin	57.1
Pru p 1*	Peach PR-10 protein	52.6	Fel d 4		53.5
Der f 2	House dust mite NPC2 proteins family	51.3	Equ c 1	Horse Lipocalcin	50.0
Der p 2	House dust mite NPC2 proteins family	51.3	Pen m 2	Shrimp Arginin kinase	50.0
Phl p 5	Timothy Grass group 5	48.7	Asp f 6	Aspergillus	39.2
Fel d 1	Cat Uteroglobin	48.7	Mus m 1	Mouse Lipocalcin	35.7
Phl p 6	Timothy Grass group 6	47.4	Api g 1*	Celery PR – 10 protein	35.7
Can f 1	Dog Lipocalin	47.4
Phl p 2	Timothy Grass group 2	44.9
Der p 1	House dust mite Cystein protease	41.0

*mainly cross – reactive components.

The order of molecular components in subgroup of patients suffering from asthma bronchiale and in patients without asthma bronchiale is shown in Table 4. The first 19 molecular components with the highest underlying probability in patients suffering from asthma bronchiale were found to be the same as those molecular components in patients not suffering from asthma bronchiale. We cannot say that one single component has a highest probability. The comparison of the relative frequency of sensitization to molecular components in patients suffering from asthma bronchiale (55 patients = 100%) and in patients without asthma bronchiale (45 patients = 100%) is shown in Complement to Table 4. No significant difference between frequencies of sensitization to molecular components was found in these subgroups of patients, although we can observe the increasing frequency almost in all molecular components in patients suffering from asthma bronchiale.

We evaluated the order of molecular components in patients suffering from allergic rhinitis and in patients without allergic rhinitis. The order of molecular components in patients suffering from allergic rhinitis and in patients without allergic rhinitis is shown in Table 5. The first 9 molecular components with the highest underlying probability in patients suffering from allergic rhinitis were found to be different from those not suffering from allergic rhinitis. The comparison of the relative frequency of sensitization to molecular components in patients suffering from allergic rhinitis (78 patients = 100%) and in patients without allergic rhinitis (22 patients = 100%) is shown in Complement to Table 5. Typically in patients suffering from allergic rhinitis, the sensitization to molecular components such as Bet v 1, Phl p 4, Phl p 6, Cyn d 1, Mal d 1, Cora 1.0101, Pru p 1, Der f 2, Der p 2, Phl p 5, Fel d 1, Can f 1, Phl p 2, Der p 1 has a higher occurrence.

The molecular components with the simultaneously confirmed highest underlying probability of occurrence in patients suffering from allergic rhinitis, asthma bronchiale and severe form of AD are recorded in Table 6. The molecular components such as Phl p 1, Phl p 4, Bet v 1 (and PR −10 proteins), Der f 2, Der p 2 (House dust mite, NPC2 proteins family), Can f 1 (Dog, Lipocalin), Fel d 1 (Cat, Uteroglobin), Der f 1, Der p1 (House dust mite, cystein protease) were recorded with the simultaneously confirmed highest underlying probability of sensitization in patients suffering from allergic rhinitis, asthma bronchiale, moderate and severe form of AD. These molecular components may play an important role in the atopic march. We show the relative frequency of sensitization to these molecular components in comparison to patients without asthma bronchiale, without allergic rhinitis and to patients with mild form of AD. The significant difference in frequency (p-value <0.05) is marked extra bold (Table 6). As Bermuda grass pollen is not present in our region, possible cross- reactivity with β-expansins from other grasses could be the explanation for the results with high sensitization to Cyn d 1 in in our study. The molecular components with the confirmed significant difference of occurrence in patients suffering from allergic rhinitis and severe form of AD are recorded in Table 7. Typically in patients suffering from a severe form of AD and from allergic rhinitis the sensitization to molecular components such as Der f 2, Der p 2, Can f 1 and Fel d 1 has a higher occurrence simultaneously.

Discussion

The sense of our study was to evaluate in AD patients the sensitization to molecular components according to Multiplex ISAC testing and to find some of molecular components which may play the most important role in AD patients and in atopic march. There are few studies dealing with this question in adolescents and adults suffering from atopic dermatitis according to the databases in Medline, Pubmed and Web of Science. It needs to be emphasized that in this paper we focus only on sensitization rates and not their clinical relevance without using specific provocation tests.

There is currently no advice on the use of ISAC testing worldwide (Boyce et al., 2011; Hatzler et al., 2012; Jung et al., 2015; Muraro et al., 2014; Prosperi et al., 2014; Spergel et al., 2015). Hatzler et al. (2012) looked at the IgE response to grass pollen antigens and demonstrated that sensitization can occur years before the onset of clinical disease through a process called “molecular spreading”. Some studies show a strong correlation between results with the ISAC112 microarray test, and SPT and other specific IgE tests (Huss-Marp et al., 2015; Williams et al., 2016) with a particularly good correlation in allergies to pollen (Ahlgrim et al., 2015) and to house dust mites (Jung et al., 2015). It is accepted that molecular allergy diagnosis improves the risk evaluation, sorts out genuine from cross-reactive sensitizations, improves the overall predictive value of the diagnostic results, as well as the accuracy of the resulting allergen immunotherapy. In daily routine maximally 112 allergens can be tested at a time, but in experimental approaches more than 170 molecules have proven possible (Lupinek et al., 2014). The WAO-ARIA-GA2LEN consensus document (Canonica et al., 2013) states that molecular-based allergy diagnostics, may be used by the expert in the second-line diagnostic workup, thus equivalent with extract-based skin prick- and IgE-testing. The first “Molecular Allergology User’s Guide” was urgently needed as recently published by the European Academy of Allergy and Clinical Immunology (EAACI) (Matricardi et al., 2016). Allergy screening with the ISAC multiplex allergen array not only with a similar fidelity leads to allergy diagnosis, but is favourable in polysensitized patients, in small children with limited skin area, but higher strain, in elderly when skin tests get less reliable (Jensen-Jarolim & Jensen, 2016; Kondo et al., 1998; Yagami et al., 2015), in all settings of inflamed or atopic skin and when medications interfering with skin prick testing cannot be discontinued. ISAC testing has high sensitivity and specificity (Panzner et al., 2014), and showed a strong correlation with singleplex tests including IgE and skin prick testing with extracts, specifically for respiratory allergens with slight alterations from allergen to allergen (Ahlgrim et al., 2015; Huss-Marp et al., 2015; Jung et al., 2015; Williams et al., 2016).

Our study evaluates the sensitization to molecular components with the use of Multiplex ISAC testing in the group of 100 atopic dermatitis patients. The sensitization to molecular components was confirmed in 93 patients (93%). The highest observed sensitization rate was 61.0% to grass specific molecule Phl p 1, the second most frequent sensitization was 57. 0% to Betulaceae-specific molecule Bet v 1. Frequently observed sensitizations were those to PR-10 proteins, NPC2 proteins family, Uteroglobin and Lipocalin. We found that the order of molecular components in mild form of AD is not statistically significant, on the other hand, we found a set of molecular components with the highest underlying probability in moderate and severe form. So far, we cannot decide which allergen has the highest probability in the mild form of AD; an approach like this would require a much larger number of patients. Patients suffering from severe form of AD differ from patients with moderate and mild form in several characteristics. Severe AD patients were characterized by the fact that they reacted to a larger panel of environmental allergens than patients with moderate AD and with mild AD. In patients suffering from severe form of AD, the sensitization to molecular components such as Can f 1 (Dog, lipocalin), Fel d 4 (Cat, lipocalin), Equ c 1 (Horse, lipocalin), Asp f 6 (Aspergillus, MnSOD highly conserved allergen), Mus m 1 (Mouse, lipocalin) and Api g 1 (Celery, PR – 10 protein) was confirmed with the higher probability, but in patient suffering from mild form of AD, the sensitization to these components is negative. We cannot say that one single component has a highest probability in patients with asthma bronchiale and in patients without asthma bronchiale. Regarding the allergic rhinitis, the first 9 molecular components with the highest underlying probability were found to be different from those not suffering from allergic rhinitis. After calculation of the relative frequency, we can observe in the majority of molecular components the increase in relative frequency from mild to moderate and to severe form of AD and in patients with asthma bronchiale and in patients with allergic rhinitis. But the statistically significant difference was confirmed only in some of these molecular components as it is shown in Tables. No significant difference between frequencies of sensitization to molecular components was found in the subgroup of patients suffering from asthma bronchiale, although we can observe the increasing frequency almost in all molecular components in patients suffering from asthma bronchiale.

According to our results, Phl p 1 is the leading molecular component in patients suffering from severe form of AD and in subgroup of patients suffering from asthma bronchiale and allergic rhinitis. Phl p 1 is in most patients the “initiator” molecule. Moreover, even in the few grass pollen-allergic patients who start their sensitization process with other molecules, IgE against Phl p 1 are produced in early childhood. Therefore, IgE to Phl p 1 is an essential marker in grass pollen-allergic patients to establish “true sensitization”. The absence of IgE to Phl p 1 does not exclude “true” sensitization to grass pollen, which might be due to isolated IgE sensitization to other major allergenic proteins (e.g. Phl p 5) but makes it rather unlikely. (Matricardi et al., 2016). Phl p 4 is a major allergenic protein of grass pollen and contains extremely highly cross-reactive carbohydrate determinants (CCD). This explains why in several epidemiological studies IgE positivity to Phl p 4 scores over 90% of the grass pollen-allergic patients (Matricardi et al., 2016), as it can be observed in our study. The second most frequent sensitization was 57. 0% to Betulaceae-specific molecule Bet v 1. Birch, followed by alder and hazel, represents the most potent cause of tree pollen allergy. Interestingly, there is now a difference in the relative frequency in mild (57.1%), moderate (56.9%) and severe form (57.1%) in the sensitization to Bet v 1. There is no difference in this sensitization regarding the occurrence of asthma bronchiale in our study, but the sensitization is significantly higher in subgroup of patients suffering from allergic rhinitis (62.8% versus 36.3%). Frequently observed sensitizations were those to PR-10 proteins, as expected (Mal d 1 in 47.0%, Pru p 1 in 46.0%, Cora 1.0101 in 45.0%, Aln g 1 in 43.0%, Cora 1.0401 in 42.0%, Ara h 8 in 38.0%, Gly m 4 in 32.0, Act d 8 in 24.0 and Api g 1 in 22.0%). From PR-10 Proteins, the significantly higher occurrence was observed to Api g1 in patients suffering from severe form of AD and to Mal d 1, Pru p 1 and Cor 1.0.01 in subgroup of patients with allergic rhinitis.

Regarding the sensitization to House dust mite, molecular components Der p 1 and Der r p 2 are present in faecal particles and are strongly associated with asthma (Matricardi et al., 2016). For these allergens there is good evidence that they have an important role in the symptoms of rhinitis and asthma as it is documented in our study also. In 2014, it was reported that IgE antibodies to Der p 11 are more common in sera from patients with atopic dermatitis (Banerjee et al., 2015). Thus, sensitization to this allergen may reflect the fact that the eczematous skin allows easy penetration of allergens even with molecular weight as high as 100,000. In ISAC testing, the molecular component Der p 11 is not present, so we cannot compare it with our results. Although the House dust mites (HDM) allergens are present in the mite bodies, the main allergenic sources are the mite faeces which, with a diameter higher than 10 μm (Douwes et al., 2000), can be easily inhaled into the airways and consequently be entered deep into the lungs (Valerio et al., 2005). Whereas the full composition of mite faeces remains to be determined, we can easily speculate that mite faeces contain not only the identified HDM allergens but also numerous non allergenic mite proteins and macromolecules. In the context of innate immunity activation, all these protein and non-protein compounds could putatively participate in those stimulations. Consequently, we cannot consider HDM strictly as an allergen carrier but also as an important transporter of microbial PAMPs able to trigger innate immunity. At least three different microbial PAMPs can be detected routinely in the mite faeces and/or in the mite environment: LPS, β-glucan and chitin. House dust, the mites’ natural home environment contains large amounts of LPS and/or bacteria as well as β-glucans and/or fungi which can be associated with HDM (Andersen, 1991) Moreover, chitin, a glucosamine-based polymer not only present in the fungi cell wall but also in the mite exoskeleton, has been found in the faeces (Post et al., 2012). Our results show, that the sensitization to HDM in severe form of AD is connected with the sensitization to molecular components from animals and fungi (Can f 1, Fel d 1, Fel d 4, Equ c 1, Asp f 6, Mus m 1).

A number of cat, dog and horse allergens have been described. Lipocalins constitute the most important allergen protein family (Matricardi et al., 2016). Most of them are major allergens: Equ c 1, Can f 1, Can f 6 and Fel d 4. Lipocalins are characterized by a common three-dimensional structure and a low sequence identity. They are synthesized in salivary glands and are dispersed into the environment by saliva and dander. Serum albumins are highly cross-reactive molecules generally considered as minor allergens. They are abundant in saliva and dander. Fel d 1, the major cat allergen, is an uteroglobin expressed in salivary glands and skin. The production of Fel d 1 is related to sexual hormones (Matricardi et al., 2016).

The sensitization to Asp f 6 (Aspergillus fumigatus) was recorded with a significantly higher occurrence in patients suffering from severe form of AD. The incidence of sensitization for individual Aspergillus fumigatus allergens strongly depends on the status of the patient. Interestingly, the allergens like Asp f 2, 4 and 6 seem to be exclusively recognized by patients with allergic broncho – pulmonary aspergillosis both, in asthma and cystic fibrosis although large confirmatory studies are still lacking. In our study, the sensitization to Asp f 6 was recorded in no patients suffering from mild form of AD, but in 17.2% of patients with moderate form and in 39.2% in patients suffering from severe form of AD. Regarding the sensitization to Asp f 1 and Asp f 3, no sensitization to these molecular components was recorded in mild form of AD, but the sensitization is 3.4% in moderate form and 14.0% in severe form of AD in both of these components.

In our previous studies, we evaluated the occurrence of sensitization to food and inhalant allergens in patients suffering from AD. The challenge test was performed according to the results of examinations sIgE and APT atopy patch tests with suspected foods. The diagnostic workup should comprise not only the laboratory methods, but also the diagnostic hypoallergenic diet and the challenge test in patients with suspected food allergy (Čelakovská et al., 2015a; Čelakovská et al., 2015b). Our previous study demonstrates that there is a significant association between the severity of AD and the incidence of perennial rhinitis, asthma bronchiale and the worsening of atopic dermatitis in relation to food (Čelakovská & Bukač, 2011).

In another previous publication we evaluated the results of ISAC in 81 patients suffering from AD, average age 41.7 years. The positivity was recorded most frequently to molecular components of timothy, birch, house dust mite, animal’s dander, bermuda grass, peach, apple, hazel pollen, kiwi, peanut and hazelnut, Alternaria and Aspergillus. Molecular components such as Equ c 1 (Horse), Alt a 6 (Alternaria), Fel d 1, Fel d 4 (Cat) and Can f 1, Can f 5 (Dog) were recorded with a significant higher occurrence simultaneously in patients suffering from severe form of AD, asthma bronchiale and allergic rhinitis (PUBL s 81 pac). At this study with 100 patients, we confirmed, that in patients suffering from a severe form of AD and from allergic rhinitis the sensitization to molecular components such as Der f 2, Der p 2, Can f1 and Fel d 1 was recorded with has a higher occurrence simultaneously (in print, Food and Agricultural Immunology).

Little is known about pollen-food allergy syndrome (PFS) in China. Ma Shikun et al. investigated the clinical characteristics, as well as sensitization patterns, of PFS in China. Clinical parameters and serum Immunoglobulin E (IgE) responses to prevalent pollens, plant foods and corresponding allergen components were evaluated. The top three most common pollen-associated allergenic foods were peach, apple and pear. Peach was the most common allergenic food in PFS patients. Patients with PFS in China showed an LTP-dominant sensitization profile and usually presented systemic reactions upon consumption of the allergenic foods (Ma et al., 2018).

Regarding the effect of probiotics in patients suffering from allergy, probiotics exhibit a variety of biological activities: they potentiate the immune response in humans and animals and suppress inflammatory and allergic responses. Kim et al. (Kim et al., 2019) examined the effects of Bifidobacterium longum IM55, Lactobacillus plantarum IM76 and their (1:1 and 1:9) mixtures (PMs) on house dust mite allergen extract (HDMA)-induced allergic rhinitis (AR) in mice. Oral administration of IM55, IM76, or PM significantly suppressed HDMA-induced allergic nasal symptoms, IL-4 and IL-5 expression in the nasal mucosa, bronchoalveolar lavage fluid (BALF), and blood, and IgE level in blood. They suppressed HDMA-induced eosinophil, mast cell and Th2 populations in BALF while the regulatory T-cell population and IL-10 expression were increased. Treatment with IM55, IM76, or PM significantly reduced HDMA-induced IL-4, IL-5, IL-13 and eosinophil peroxidase expression and increased IL-10 expression in the colon. Furthermore, their treatments suppressed HDMA-induced Proteobacteria population and Proteobacteria to Bacteroidetes ratio in the gut microbiota. In conclusion, IM55 and IM76 may mitigate AR by suppressing IL-4, IL-5 and IL-13 expression and inducing IL-10 expression through the inhibition of gut Proteobacteria population (Kim et al., 2019).

Our results demonstrate, that the molecular components such as Phl p 1 (Timothy, Beta expansin), Phl p 4 (Timothy, berberine-bridge enzyme), Bet v 1 (Birch, PR – 10 protein), Mal d 1 (Apple, PR – 10 protein), Cor a 1.0101 (Hazelnut, PR – 10 protein), Pru p 1 (Peach, PR – 10 protein), Der f 2 (House dust mite, NPC2 proteins family), Der p 2 (House dust mite, NPC2 proteins family), Can f 1 (Dog, Lipocalin), Fel d 1 (Cat, Uteroglobin), Cor a 1.0401 (Hazelnut, PR – 10 protein), Ara h 8 (Peanut, PR – 10 protein), Aln g 1 (Alder, PR – 10 protein), Der f 1 (House dust mite, cystein protease), Der p 1 (House dust mite, cystein protease) were recorded with the simultaneously confirmed highest underlying probability of sensitization in patients suffering from allergic rhinitis, asthma bronchiale, moderate and severe form of AD. In severe form of AD sensitization to molecular components of NPC2 proteins family, Uteroglobin, Lipocalin and Aspergillus were recorded with a significant higher occurrence. These molecular components may play an important role in the atopic march.

Conclusion

According to the ISAC Multiplex testing, the sensitization to molecular components was confirmed in 93% of patients suffering from AD. The highest observed sensitization rate was 61.0% to grass specific molecule Phl p 1, the second most frequent sensitization was 57. 0% to Betulaceae-specific molecule Bet v 1. Frequently observed sensitizations were those to PR-10 proteins, NPC2 proteins family, Uteroglobin and Lipocalin. The order of molecular components in mild form of AD is not statistically significant, but a set of molecular components with the highest underlying probability in moderate and severe form AD and in a subgroup of patients suffering from allergic rhinitis was recorded. In severe form of AD mainly sensitization to NPC2 proteins family, Uteroglobin, Lipocalin and Aspergillus were recorded with a significant higher occurrence. The increasing frequency of sensitization to molecular components was observed in subgroup of patients suffering from allergic rhinitis and asthma bronchiale, althoug in patients suffering from asthma bronchiale without significant difference.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Charles University of Prague [grant number Progress Charles University in Prague].