Sensitisation to molecular components of fungi in atopic dermatitis patients, the relation to the occurrence of food hypersensitivity reactions

ABSTRACT

The aim of our study is the evaluation of the relation between the sensitisation to molecular components of moulds and yeast and the occurrence of food hypersensitivity reactions in atopic dermatitis patients. The food reactions were confirmed in an open exposure test (history); the sensitisation to allergen reagents (molecular components) of moulds and yeast was confirmed with ALEX2 Allergy Explorer Multiplex examination. The relation between the sensitisation to molecular components of fungi and the food hypersensitivity reactions was evaluated with the use of Fisher’s exact test. One hundred atopic dermatitis patients were included in the study (48 men and 52 women with an average age of 40.9 years). In patients with reaction to hazelnuts, walnuts, peanuts, fish and egg, the significantly higher level of specific IgE to Asp f 3, Cla h 8, Alt a 6, Alt a 1, Mala s 6 and Asp f 6 was confirmed.

KEYWORDS:

• Atopic dermatitis
• Sensitisation to molecular components of moulds and yeast
• Food hypersensitivity reactions
• ALEX2 Allergy Explorer test

Introduction

Fungi are potent sources of allergenic molecules covering a vast variety of molecular structures including enzymes, toxins, cell wall components and phylogenetically highly conserved cross-reactive proteins (Crameri et al., 2014; Simon-Nobbe et al., 2008). In contrast to other allergenic sources, fungi are very common in the environment, and exposure to airborne spores is almost constant throughout the year. Fungi may colonise the human body, and they may damage airways by the production of toxins, proteases, enzymes and volatile organic compounds and they develop rapidly on different kinds of foods (Bush et al., 2006; Kauffman et al., 2000). Thus, moulds have a far greater impact on the patients’ immune system than pollen or other allergenic sources. The immunological mechanisms underlying mould allergies are hypersensitivity reactions of types I, II, III and IV (Crameri et al., 2006; Gaitanis et al., 2012; Green et al., 2006). The spectrum of allergic symptoms caused by these hypersensitivity reactions to fungi is very broad, including rhinitis, asthma, atopic dermatitis. Atopic dermatitis (AD) with a worldwide prevalence of 10–20% in children and of 1–3% in adults, is a chronic dermatosis characterised with a disrupted skin barrier resulting in cutaneous hyperreactivity to various environmental stimuli. Fungi with other allergenic sources such as foods, trees, weeds, grasses, mites and animals may contribute to the elicitation or exacerbation of eczematous skin lesions (Novak & Simon, 2011; Simon & Kernland Lang, 2011 Wolf & Wolf, 2012).

In addition to aeroallergens, food allergens are the most important factors causing allergic inflammation in the pathogenesis of atopic dermatitis. Identifying the causal triggers of food hypersensitivity reactions is crucial in the diagnosis of allergic conditions as well as in terms of dietary measures (Bindslev-Jensen et al., 2004; DunnGalvin et al., 2015; Jansson et al., 2013; Johansson et al., 2001; Maintz & Novak, 2007; Nwaru et al., 2014). Reactions to food in disposable individuals can be divided into two basic groups, which most often manifest themselves as food allergy (FA) and food intolerance, which have completely different pathophysiology. Food allergy is an inappropriate reaction of the immune system to a specific component of food (usually a glycoprotein). Non-allergic (non-immunological) hypersensitivity, so-called food intolerance is not mediated by the immune mechanism. Food allergy and non-allergic food hypersensitivity are included under the term food hypersensitivity reactions (Bindslev-Jensen et al., 2004; DunnGalvin et al., 2015; Jansson et al., 2013; Johansson et al., 2001; Maintz & Novak, 2007; Nwaru et al., 2014). Molecular allergology uses pure, mainly recombinant and structurally defined allergen molecules and allergen-derived epitopes to study mechanisms of IgE-associated allergy, to diagnose, and even predict the development of allergic manifestations and to treat and prevent IgE-associated allergies (Bousquet et al., 2016; Heffler et al., 2018; Jensen-Jarolim et al., 2017; Patelis et al., 2016; van Hage et al., 2017). The aim of our work is to evaluate the relation between the sensitisation to molecular components of moulds and yeast (Mala s 5, Mala s 6, Mala s 11, Sac c, Alt a 1, Alt a 6, Asp f 1, Asp f 3, Asp f 4, Asp f 6, Cla h 8 and Pen ch) and the occurrence of food hypersensitivity reactions in atopic dermatitis patients aged 14 years and older.

Methods

Patients and methods

In the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech republic we examined 100 patients suffering from atopic dermatitis in the period 2018–2020. Hanifin–Rajka criteria were used for the diagnosis of atopic dermatitis (Hanifin & Rajka, 1980). From the study were excluded patients with the systemic therapy (cyclosporin, systemic corticoids, biological therapy), pregnancy, breastfeeding and also patients having other systemic diseases. All patients underwent a complete dermatological and allergological examination. This study was approved by Ethics committee of Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic.

Examination of specific IgE to allergen reagents

The sensitisation to molecular components of mould and yeast was evaluated with Multiplex method ALEX2 Allergy Explorer. This ALEX system (Allergy Explorer) from Macro-Array Diagnostics uses molecular components bound via nanoparticles to a solid phase and contains 295 allergen reagents (117 allergenic extracts and 178 molecular components). The ALEX measuring range for specific IgE is 0.3-50 kU_A/L (quantitative) and for total IgE is 1–2500 kU/L (semiquantitative). The results are expressed as Class 0 (< 0.3 kU_A/L) – negative, Class 1 (0.3 > 1 kU_A/L) – low positivity, Class 2 (1 > 5 kU_A/L) – moderate positivity, Class 3 (5 > 15 kUA/L) – high positivity, and Class 4 (≥ 15 kU_A/L) – very high positivity. There is no significant interference from high total IgE, bilirubin, haemoglobin or triglycerides. Sensitisation to mould and yeast was confirmed in patients with positive level of specific IgE in classes 1, 2, 3, 4.

Confirmation of food hypersensitivity reactions

The reaction to examined food was confirmed in these cases:

1. In patients with the positive result in the open exposure test (early and/or late reactions) with examined food allergens.

2. In patients with the repeated reactions after the ingestion of examined food in their history; the open exposure test was not performed in these patients because of anaphylactic reaction danger.

Diagnostic elimination and diagnostic hypoallergenic diet

In all patients with a mild form of AD, an elimination diet was recommended according to the anamnestic data or the results of the specific IgE to molecular components and allergen reagents in ALEX2 Allergy Explorer test. An elimination diet was recommended with the exclusion of the suspect food from the diet for at least 14 days.

Patients with moderate to severe form of AD usually underwent a diagnostic hypoallergenic diet, usually 4–6 weeks. Foods with significant allergenicity, such as cow's milk, wheat flour, soybeans, eggs, tree nuts, peanuts, as well as foods suspected of having anamnestic data and positive laboratory results of specific IgE (ALEX2 Allergy Explorer), were excluded from the diet. Furthermore, foods with a histaminoliberating effect (eg: cheese, fish, smoked meat), foods with dyes, stabilisers and preservatives were excluded from the diet. Raw fruits and vegetables, spices and food containing additives, so-called food additives, were also excluded. The diet consisted mainly of rice, corn, potatoes, meat (excluding fish and seafood) and cooked vegetables (excluding celery, parsley and carrots) and cooked fruit. Only drinking water, mineral water and black tea were permitted. During the elimination event diagnostic hypoallergenic diets, the patient recorded the intensity of the skin lesions associated with atopic dermatitis (pruritus, erythema, sowing of new lesions, etc.). Before and after the elimination and diagnostic hypoallergenic diet, the intensity of AD was objectively evaluated using the SCORAD index.

Open exposure test (OET)

In the case of patients who underwent an elimination or diagnostic hypoallergenic diet, the symptoms of atopic dermatitis improved, their own exposure test was performed with suspicious food. Prior to the exposure test, drugs that could affect the test results, such as antihistamines, were discontinued for at least five days, and further topical skin treatment remained unrestricted. If the patient had not an acute systemic anaphylactic reaction in the past and the test food was part of a normal diet, the test was performed at home. Patients with more severe bronchial asthma and/or a systemic allergic reaction were admitted to short-term hospitalisation for OET with suspect food. OET has not been performed in patients who have reported an early allergic reaction to the ingestion of peanuts, fish or nuts due to the risk of an anaphylactic reaction.

The actual implementation of OET followed the diet mentioned above, in the period without symptoms or with regard to the course of AD disease (eg: in patients with pollen allergy, the test was not performed in the pollen season). Each test was performed according to the general scheme, with three doses of test food over two days. One dose of food was administered in divided portions at 10 minutes intervals for one hour.

Exposure test results were considered as positive if one or more of the following objective and subjective clinical reactions were observed: cutaneous (rash, urticaria, angioedema, pruritus, worsening of atopic dermatitis), gastrointestinal (oral allergy syndrome (OAS), nausea, abdominal pain, diarrhoea) and respiratory (nasal congestion, difficulty breathing). Early reactions were defined as clinical symptoms that occurred within 2 h of ingestion, late reactions were evident after at least 6 h. If worsening of AD or other clinical reactions were noted during the exposure test, the patient continued to eliminate the suspect food and subsequently the severity of AD was assessed every 3 months for 1 year to exclude the food. If the exposure test was negative, the patient could reintroduce the food and the severity of AD was assessed every 3 months for 1 year while consuming the tolerated food. If a food allergy to more than one food is suspected, another OET was performed 2–3 weeks after the first OET (Čelakovská, Ettlerová, Ettler, & Bukač 2015; Čelakovská, Ettlerová, Ettler, Vaněčková, et al., 2015).

Statistical analysis

The results of specific IgE to molecular components of moulds and yeast (Mala s 5, Mala s 6, Mala s 11, Sac c, Alt a 1, Alt a 6, Asp f 1, Asp f 3, Asp f 4, Asp f 6, Cla h 8 and Pen ch) and clinical reactions after ingestion of examined foods with the use of Fisher’s exact test of independence were evaluated in contingency tables. The correlation between the results of specific IgE to moulds and yeast and specific IgE to molecular components of examined food allergens with Fisher's exact test was also tested. The statistical software: NCSS 2019 LLC, Kaysville, Utah, USA, css.com/software/ncss was used.

Results

We included 100 atopic dermatitis patients in the study (48 men and 52 women with an average age of 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 61 patients (61%), severe form in 25 patients (25%); 55 patients (55%) suffer from asthma bronchiale and 74 patients (74%) suffer from allergic rhinitis. In all these patients we analysed the sensitisation to molecular components of moulds and yeast with ALEX2 Allergy Explorer test.

In Table 1, we show how many patients suffer from food hypersensitivity reactions to examined foods. Food hypersensitivity reactions to peanuts were recorded in 46 patients (46%), to hazelnuts in 43 patients (43%), to walnuts in 44 patients (44%), to soy in 12 patients (12%), to egg in 20 patients (20%), to fish in 11 patients (11%), to wheat flour in 12 patients (12%), to cow milk in 13 patients (13%), to celery in 21 patients (21%), to apple in 23 patients (23%), to kiwi in 15 patients (15%), to carrot in 17 patients (17%), to spices in 19 patients (19%), to peach in 18 patients (18%), to tomato in 15 patients (15%), to oranges in 26 patients (26%).

Table 1. Number of patients with clinical symptoms of food hypersensitivity reactions to examined food allergens.

	Number of patients with clinical symptoms of food hypersensitivity reactions, 100 patients included (=100 %)
	The whole number of patients with clinical symptoms	OAS	GIT	Pruritus	Worsening of AD	Worsening of breath	Urticaria
Peanuts	46 (46 %)	35 (35 %)	12 (12 %)	7 (7 %)	8 (8 %)	–	–
Walnuts	44 (44 %)	26 (26 %)	8 (8 %)	8 (8 %)	7 (7 %)	–	–
Hazelnuts	43 (43 %)	22 (22 %)	8 (8 %)	6 (6 %)	7 (7 %)	–	–
Fish	11 (11 %)	0 (%)	6 (6 %)	3 (3 %)	4 (4 %)	–	–
Apple	23 (23 %)	21 (21 %)	13 (13 %)	10 (10 %)	8 (8 %)	–	4 (4 %)
Peach	18 (18 %)	18 (18 %)	12 (12 %)	5 (5 %)	8 (8 %)	–	3 (3 %)
Celery	21 (21 %)	20 (20 %)	15 (15 %)	5 (5 %)	5 (5 %)	1 (1 %)	3 (3 %)
Egg	20 (20 %)	0	2 (2 %)	13 (13 %)	20 (20 %)	1 (1 %)	–
Soy	12 (%)	2 (2 %)	9 (9 %)	8 (8 %)	6 (6 %)	–	–
Wheat	12 (12 %)	0 (%)	3 (3 %)	12 (12 %)	10 (10 %)	0	0
Cow milk	13 (13 %)	0 (%)	10 (10 %)	2 (2 %)	3 (3 %)	1 (%)	0
Kiwi	15 (15 %)	10 (10 %)	2 (2 %)	7 (7 %)	9 (9 %)	2 (2 %)	(%)
Carrot	17 (17 %)	5 (5%)	10 (10%)	5 (5%)	5 (5 %)	0 (%)	(%)
Spices	19 (19 %)	10 (10 %)	6 (6 %)	6 (6 %)	8 (8 %)	0 (%)	2 (2 %)
Tomato	15 (15%)	10 (10 %)	4 (4 %)	6 (6 %)	7 (7 %)	0 (%)	0 (%)
Oranges	26 (26 %)	4 (4 %)	3 (3 %)	20 (20 %)	15 (15 %)	0 (%)	0 (%)

OAS, oral allergy syndrome; GIT, gastrointestinal symptoms; AD, atopic dermatitis.

The overview of allergen reagents (allergenic extracts and molecular components) of moulds and yeast according to the frequency of sensitisation in classes 0, 1, 2, 3, 4 in 100 atopic dermatitis patients ( =  100%) is shown in Table 2. Mould sensitisation was most commonly reported to Alt a 1 in 26 (26%) with a very high level of specific IgE in 22% patients and high level in 4% patients. The next most common sensitisation was noted to Mala s 11 in 24 (24%) patients, to Asp f 6 in 20 (20%) patients, to Asp f 3 and Cla h 8 in 15 (15%) patients, to Mala s 6 and Sac c in 14 (14%) patients and to Alt a 6 in 12 (12%) patients. Lower sensitisation was recorded to Mala s 5 in 10 (10%) patients, to Cla h in 6 (6%) patients, to Asp f 1 in 4 (4%) patients, to Asp f 4 and Pen ch in 3 (3%) patients. The relation between the sensitisation to molecular components of moulds and yeast and the occurrence of food hypersensitivity reaction to examined foods is recorded in Table 3. Patients suffering from sensitisation to molecular components Alt a 6, Asp f 3 suffer significantly more from food hypersensitivity reactions to hazelnuts, patients with sensitisation to Alt a 1, Alt a 6 and Asp f 3 suffer significantly more from reactions to walnuts, patients with sensitisation to Asp f 6 and Mala s 6 suffer from reaction to fish, patients with sensitisation to Cla h 8 suffer from reactions to egg, patients with sensitisation to Asp f 3 and Cla h 8 suffer from reaction to peanuts. No significant relation was confirmed between the sensitisation to fungi and moulds and the occurrence of food hypersensitivity reactions to peach, kiwi, apples, spices, oranges, celery, carrot, soy, wheat, and cow milk. In Complement to Table 3, we show an overview of foods with hypersensitivity reactions for which significantly higher sensitisation to moulds and yeast is recorded.

Table 2. The overview of allergen reagents (allergenic extracts and molecular components) of moulds and yeast according to the frequency of sensitisation in classes 0, 1, 2, 3, 4 in 100 atopic dermatitis patients (=100 %).

Allergen reagents		The number of patients in % according to the level of specific IgE in classes 0–4 (included 100 patients = 100 %)
		Class 0	Class 1	Class 2	Class 3	Class 4
Alt a 1	Alternaria alternata	74%	0%	0%	4%	22%
Alt a 6	Enolase, Alternaria alternata	88%	2%	4%	4%	2%
Asp f 1	Aspergilus fumigatus Mitogillin family	96%	2%	1%	1%	0%
Asp f 3	Aspergilus fumigatus Peroxysomal protein	85%	8%	5%	1%	1%
Asp f 4	Aspergilus fumigatus	97%	0%	1%	1%	1%
Asp f 6	Aspergilus fumigatus, Mn superoxide dismutase	80%	2%	4%	9%	5%
Cla h	Cladosporium herbarum	94%	5%	1%	0%	0%
Cla h 8	Mannitol dehydrogenase, Cladosporium herbarum	85%	14%	1%	0%	0%
Mala s 11	Manganese superoxide dismutase, Malassezia sympodialis	76%	0%	7%	1%	16%
Mala s 5	Malassezia sympodialis	90%	1%	3%	1%	5%
Mala s 6	Malassezia sympodialis, Cyclophilin	86%	5%	6%	3%	0%
Pen ch	Penicillium chrysogenum	97%	3%	0%	0%	0%
Sacc	Saccharomyces cerevisiae	86%	7%	7%	0%	0%

Class 0 (< 0.3 kU_A/L) – negative; Class 1 (0.3 > 1 kU_A/L) – low positivity; Class 2 (1 >5 kU_A/L) – moderate positivity; Class 3 (5 >15 kUA/L) – high positivity; Class 4 (≥ 15 kU_A/L) – very high positivity.

Table 3. The relation between the sensitisation to molecular components of moulds and yeast and the food hypersensitivity reactions.

Molecular components, number of patients with positive and negative specific IgE	Number of patients with (+) and without (−) clinical reactions to examined food
	Peanuts+	Peanuts−	Hazelnut+	Hazelnuts−	Walnuts+	Walnuts−	Soy+	Soy−
Mala s 5+	4	6	5	5	5	5	2	8
Mala s 5−	42	48	38	52	39	51	10	80
p-value	0.749		0.741		0.745		0.343
Mala s 6+	7	7	6	8	6	8	2	12
Mala s 6−	39	47	37	49	38	48	10	76
p-value	0.779		1.000		1.000		0.673
Mala s 11+	13	11	11	13	13	11	4	20
Mala s 11−	33	43	32	44	31	45	8	68
p-value	0.481		0.815		0.345		0.474
Alt a 1+	16	10	13	13	16	10	3	23
Alt a 1−	30	44	30	44	28	46	9	65
p-value	0.072		0.491		0.042*		1.000
Alt a 6+	9	3	9	3	10	2	1	11
Alt a 6−	37	51	34	54	34	54	11	77
p-value	0.060		0.027*		0.005*		1.000
Asp f 3+	12	3	12	3	12	3	3	12
Asp f 3−	34	51	31	54	32	53	9	76
p-value	0.005*		0.003*		0.004*		0.382
Asp f 6+	11	9	10	10	12	8	3	17
Asp f 6−	35	45	33	47	32	48	9	71
p-value	0.454		0.614		0.133		0.702
Cla h 8+	11	4	10	5	10	5	2	13
Cla h 8−	35	50	33	52	34	51	10	75
p-value	0.026*		0.052		0.088		1.000

Molecular components, number of patients with positive and negative specific IgE	Number of patients with (+) and without (−) clinical reactions to examined food
	Egg+	Egg−	Fish+	Fish−	Wheat flour+	Wheat flour−	Cow milk+	Cow milk−
Mala s 5+	4	6	0	10	1	9	0	10
Mala s 5 -	16	74	11	79	11	79	13	77
p-value	0.109		0.596		1.000		0.351
Mala s 6+	5	9	5	9	1	13	1	13
Mala s 6−	15	71	6	80	11	75	12	74
p-value	0.147		0.007*		1.000		0.686
Mala s 11+	6	18	5	19	1	23	3	21
Mala s 11−	14	62	6	70	11	65	10	66
p-value	0.560		0.127		0.283		1.000
Alt a 1+	5	21	3	23	4	22	2	24
Alt a 1−	15	59	8	66	8	66	11	63
p-value	1.000		1.000		0.504		0.504
Alt a 6+	5	7	3	9	2	10	1	11
Alt a 6−	15	73	8	80	10	78	12	76
p-value	0.060		0.125		0.634		1.000
Asp f 3+	6	9	2	13	2	13	1	14
Asp f 3−	14	71	9	76	10	75	12	73
p-value	0.072		0.668		1.000		0.684
Asp f 6+	6	14	5	15	1	19	3	17
Asp f 6−	14	66	6	74	11	69	10	70
p-value	0.222		0.040*		0.450		0.720
Cla h 8+	7	8	3	12	2	13	1	14
Cla h 8−	13	72	8	77	10	75	12	73
p-value	0.012*		0.363		1.000		0.684

Molecular components, number of patients with positive and negative specific IgE	Number of patients with (+) and without (−) clinical reactions to examined food
	Celery+	Celery−	Apple+	Apple−	Kiwi+	Kiwi−	Carrot+	Carrot−
Mala s 5+	2	8	4	6	2	8	2	8
Mala s 5−	19	71	19	71	13	77	15	75
p-value	1.000		0.232		0.643		0.677
Mala s 6+	5	9	6	8	3	11	4	10
Mala s 6−	16	70	17	69	12	74	13	73
p-value	0.164		0.083		0.436		0.250
Mala s 11+	8	16	4	20	5	19	5	19
Mala s 11−	13	63	19	57	10	66	12	64
p-value	0.147		0.578		0.345		0.546
Alt a 1+	5	21	7	19	7	19	4	22
Alt a 1−	16	58	16	58	8	66	13	61
p-value	1.000		0.595		0.060		1.000
Alt a 6+	3	9	1	11	2	10	2	10
Alt a 6−	18	70	22	66	13	75	15	73
p-value	0.712		0.286		1.000		1.000
Asp f 3+	4	11	4	11	3	12	4	11
Asp f 3−	17	68	19	66	12	73	13	72
p-value	0.512		0.743		0.693		0.278
Asp f 6+	7	13	3	17	3	17	3	17
Asp f 6−	14	66	20	60	12	68	14	66
p-value	0.123		0.552		1.000		1.000
Cla h 8+	4	11	4	11	4	11	3	12
Cla h 8−	17	68	19	66	11	74	14	71
p-value	0.512		0.743		0.232		0.716

Molecular components, number of patients with positive and negative specific IgE	Number of patients with (+) and without (−) clinical reactions to examined food
	Spices+	Spices−	Peach+	Peach−	Tomato+	Tomato−	Orange+	Orange−
Mala s 5+	4	6	2	8	2	8	2	8
Mala s 5−	15	75	16	74	13	77	24	66
p-value	0.092		1.000		0.643		1.000
Mala s 6+	4	10	4	10	3	11	5	9
Mala s 6−	15	71	14	72	12	74	21	65
p-value	0.460		0.272		0.436		0.511
Mala s 11+	6	18	5	19	4	20	7	17
Mala s 11−	13	63	13	63	11	65	19	57
p-value	0.385		0.761		0.752		0.790
Alt a 1+	4	22	7	19	3	23	9	17
Alt a 1−	15	59	11	63	12	62	17	57
p-value	0.773		0.233		0.753		0.300
Alt a 6+	1	11	3	9	1	11	5	7
Alt a 6−	18	70	15	73	14	74	21	67
p-value	0.453		0.448		0.687		0.289
Asp f 3+	3	12	3	12	2	13	7	8
Asp f 3−	16	69	15	70	13	72	19	66
p-value	1.000		0.730		1.000		0.060
Asp f 6+	5	15	4	16	2	18	5	15
Asp f 6−	14	66	14	66	13	67	21	59
p-value	0.524		0.753		0.728		1.000
Cla h 8+	2	13	4	11	2	13	5	10
Cla h 8−	17	68	14	71	13	72	21	64
p-value	0.729		0.463		1.000		0.528

Complement to Table 3. Foods with hypersensitivity reactions for which significantly higher sensitization to moulds and yeast is recorded.
Food allergen	Molecular components
Peanuts	Asp f 3 (Peroxysomal protein), Cla h 8 (Mannitol dehydrogenase)
Hazelnuts	Asp f 3 (Peroxysomal protein), Alt a 6 (Enolase).
Walnuts	Asp f 3 (Peroxysomal protein), Alt a 1, Alt a 6 (Enolase)
Fish	Asp f 6 (Mn superoxide dismutase), Mala s 6 (Cyclophilin)
Egg	Cla h 8 (Mannitol dehydrogenase)

The p-value is recorded; the significant relation is marked with p-value ≤ 0.05 extra bold*. peanuts+: food hypersensitivity reaction to peanuts confirmed; peanuts−: food hypersensitivity reactions to peanuts not confirmed.

In Table 4 and in Complement to Table 4, we show the relation between the results of specific IgE to molecular components of peanuts, walnuts, hazelnuts, egg, fish and shrimps and the results of specific IgE to molecular components of moulds and yeast. Sensitisation to molecular components of moulds and yeast was in significant relation to sensitisation to molecular components of Beta – parvalbumin, arginin kinase, 7 S globulin, 2 S albumin, PR-10 proteins and non-specific lipid transfer protein. Moreover, there is a relationship between the levels of specific IgE to Alt a 6, Cla h and the level of Cra c 6 (Troponin C). Sensitisation to Mala s 5, Mala s 6, Mala s 11, Sacc, Alt a 6, Asp f 6, Cla h and Cla h 8 is in significant relation with specific IgE results to molecular components of egg.

Table 4. Number of patients with positive results of specific IgE to molecular components of peanuts, walnuts, hazelnuts, fish, shrimps end egg and number of patients with positive results of specific IgE to molecular components of moulds and yeast.

		Molecular components of moulds and yeast, number of patients with positive sIgE
		Mala s 5	Mala s 6	Mala s 11	Sac c	Alt a 1	Alt a 6	Asp f 3	Asp f 6	Cla h 8	Cla h
		10	14	24	14	26	12	15	20	15	6
Molecular components of peanuts, walnuts, hazelnuts, fish, shrimps, egg	Number of patients with positive sIgE	The significant relation is marked with p-value ≤ 0.05
Ana o Cashew	7						0.0356
Car i Pecan	9		0.0207				0.0112	0.0035		<0.001
Cor a 1.0401 Hazelnut, PR-10 protein	44		0.0009		0.0076		0.0295	0.0004		0.0004	0.0059
Cor a 11 Hazelnut, 7 S globulin	6				0.0345		0.0016	0.0043		0.0002	0.0407
Cor a 14 Hazelnut, 2 S albumin	6			0.0282	0.0032	0.0043	0.0016	0.0043		0.0043
Jug r 2 Walnut, 7S globulin	8		0.0126		0.0126	0.0265	0.0067	0.0001		0.0001
Jug r 6 Walnut, 7S globulin	6		0.0345		0.0345		0.0219	0.0043		0.0002
Ara h 1 Peanut, 7S globulin	9			0.0055	0.0024		0.001	0.027	0.0147	0.0003	0.009
Ara h 15 Peanut, Oleosin	8						0.0067	0.0017
Ara h 6 Peanut, 2S albumin	7
Ara h 8 Peanut, PR-10 protein	33		0.0125	0.0052	0.0002			0.0007	0.0317	0.0007	0.0143
Arah 9 Peanut, nsLTP	9			0.0342	0.0207	0.0486	0.001	0.027		0.0003	0.441
Cra c 6 North Sea shrimp, troponin C	7						0.0036				0.0039
Cyp c 1 Carp, Beta-parvalbumin	6		0.0345	0.0282	0.0345				0.0139
Hom g Lobster	15			0.0446			0.0164	0.0466			0.042
Chi spp. Crab	9							0.0035		0.027
Pen m 2 Shrimp, Arginine kinase	18		0.0031	0.0116	0.0031		0.0071	0.0267	0.0463	0.0007	0.0006
Rud spp. Clam	8							0.0165
Sal s 1 Salmon, Beta-parvalbumin	7		0.0069				0.0036		0.0281
Sco s Mackerel	6			0.0282	0.0345			0.042	0.0139
Sco s 1 Mackerel, Beta-parvalbumin	6		0.0032	0.0282					0.0139
Thu a 1 Tuna, Beta-parvalbumin	7		0.0069	0.0082					0.0033
Xip g 1 Swordfish, Beta-parvalbumin	6		0.0345	0.0282	0.0345				0.0011
Gal d white Egg white	16	0.0087	0.0013	0.0208	0.0001		0.0219		0.0032		0.0022
Gal d yolk Egg – yolk	6						0.0219			0.0407
Gal d 1 Egg, ovomucoid	6		0.0345		0.0345		0.0036			0.0023	0.042
Gal d 2 Egg, ovoalbumin	7				0.0069		0.0356			0.0001	0.0091
Gal d 3 Egg, ovotransferin	7
Gal d 4 Egg, lysozyme C	6		0.0345		0.0032					0.0407
Complement to Table 4. Summary Table: The significant relation between the level of specific IgE to moulds and yeast and the level of specific IgE to molecular components of peanuts, hazelnuts, walnuts, fish, shrimps and egg. Positive results of specific IgE to molecular components of moulds and yeast are in significant relation to molecular components of Beta – parvalbumin, arginin kinase, 7S globulin, 2S albumin, PR-10 proteins and Non-specific lipid transfer protein. Moreover, there is a relationship between the levels of specific IgE to Alt a 6, Cla h and the level of Cra c 6 – Troponin C. Sensitisation to Mala 5, Mala 6, Mala 11, Sacc, Alt a 6, Asp f 6, Cla h and Cla h 8 is in significant relation with specific IgE results to molecular components of egg.
Significant relation between the positive results of specific IgE to molecular components of moulds and yeast and positive results of specific IgE to molecular components of peanuts, hazelnuts, walnuts, fish, shrimps and egg
Molecular components of moulds and yeast	Molecular components of peanuts, hazelnuts, walnuts, fish, shrimps and egg with the significant relation to specific IgE to moulds and yeast
Mala s 5	Gal d white
Mala s 11 Mn superoxide dismutase	Cor a 14 – 2S albumin Ara h 1 – 7S globulin Ara h 8 – PR 10 protein Ara h 9- Non-specific lipid transfer protein Pen m 2 – Arginin kinase Cyp c 1, Sco s 1, Thu a 1, Xip g 1-Beta-parvalbumin Hom g Gal d white
Mala s 6 Cyclophilin	Car i – pecan nut Cor a 1.0401, Ara h 8 – PR 10 protein Jug r 2, Jug r 6 – 7S globulin Pen m 2 - Arginin kinase Cyp c 1, Sal s 1, Sco s 1, Thu a 1, Xip g 1-Beta-parvalbumin Gal d white Gal d 1 - ovomucoid, Gal d 4 – Lysozyme C
Sacc	Cor a 1.0401, Ara h 8 – PR 10 protein Cor a 11. Jug r 2. Jug r 6. Ara h 1 – 7S globulin Cor a 14 – 2 S albumin Ara h 9- Non-specific lipid transfer protein Pen m 2 –Arginin kinase Cyp c 1, Sco s, Xip g 1-Beta-parvalbumin Gal d white Gal d 1-ovomucoid, Gal d 2 – ovalbumin, Gal d 4 – Lysozyme C
Alt a 1 Alt a 1 family	Cor a 14 – 2S albumin Jug r 2 – 7S globulin Ara h 9-Non-specific lipid transfer proteinCor a 14 – 2S albumin Jug r 2 – 7S globulin Ara h 9 - Non-specific lipid transfer protein
Alt a 6 Enolase	Jug r 2, Jug r 6, Ara h 1, Cor a 11 -7 S globulin Ana o – cashew nut Car i – pecan nut Cor a 1.0401 – PR 10 protein Cor a 14 – 2 S albumin Ara h 15 – oleosin Ara h 9 - Non-specific lipid transfer protein Cra c 6 – Troponin C Hom g Pen m 2 – Arginin kinase Sal s 1 – Beta-parvalbumin Gal d yolk Gal d 1-ovomucoid, Gal d 2 – ovalbumin, Gal d 3 - ovotransferrin

The hypothesis of independence from the alternative of addiction was tested. Fisher's exact test was used. The significant relation between the level of specific IgE to molecular components of moulds and yeast and specific IgE to molecular components of peanuts, hazelnuts, walnuts, fish and shrimps is recorded (the significant relation is marked with p-value ≤ 0.05). The relation was not calculated for the components of moulds and yeast for which the frequency of positive results of specific IgE is less than 6.

Discussion

The question we asked in our study has not yet been addressed in patients with atopic dermatitis. Only in our previous studies, we addressed the relationship between the results of extracting specific IgE for some fungal allergens and the incidence of food hypersensitivity reactions (Čelakovská, Bukač, Ettler, Vaneckova, Krcmova, et al. 2018; Čelakovská, Bukač, Ettler, Vaněčková, Ettlerová, et al., 2018). However, our current study goes much more in-depth as we assess sensitisation to individual molecular allergens.

Our study revealed that in patients with reaction to hazelnuts, walnuts and peanuts is significantly higher sensitisation to Asp f 3 (Peroxysomal protein), in patients with reaction to peanuts and egg is significantly higher the sensitisation to Cla h 8 (Mannitol dehydrogenase), in patients with reaction to hazelnuts and walnuts is significantly higher the sensitisation to Alt a 6 (Enolase), in patients with reaction to walnuts the sensitisation to Alt a 1, in patients with reaction to fish the sensitisation to Asp f 6 (Mn superoxide dismutase) and Mala s 6 (Malassezia sympodialis, Cyclophilin). On the other hand, no significant relation between the sensitisation to moulds and yeast and the occurrence of food hypersensitivity reactions to other food allergens was confirmed. We also compared the level of specific IgE to moulds and yeast with the level of specific IgE to hazelnuts, peanuts, walnuts, fish and egg. Sensitisation to molecular components of moulds and yeast is in significant relation to sensitisation to molecular components of Beta – parvalbumin, arginin kinase, 7 S globulin, 2 S albumin, PR-10 proteins and non-specific lipid transfer protein. Moreover, there is a relationship between the levels of specific IgE to Alt a 6, Cla h and the level of Cra c 6 (Troponin C). Sensitisation to Mala s 5, Mala s 6, Mala s 11, Sacc, Alt a 6, Asp f 6, Cla h and Cla h 8 is in significant relation with specific IgE results to molecular components of egg. In our previous study (Čelakovská et al., 2019) we evaluated the relation between the results of specific IgE to Alt a 1, Alt a 6, Cla h 8, Asp f 1, Asp f 3, Asp f 6 and the results of specific IgE to other molecular food and inhalant allergens with the use of ISAC Multiplex testing (Čelakovská et al., 2019); 60 patients with atopic dermatitis were examined (Čelakovská et al., 2019). We confirmed the significant relation between the level of specific IgE to to Alt a 1, Alt a 6, Cla h 8, Asp f 1, Asp f 3, Asp f 6 and the level of specific IgE to molecular allergens from Peanut, Walnut, Hazelnut, Atlantic Cod, Black Tiger Shrimp, Apple, Kiwi, Peach, Celery, Timothy, London Plane tree, Cedar pollen allergen, Mugwort, Cat, and Horse (Čelakovská et al., 2019).

Sensitisation to Alternaria alternata spores is considered a well-known biological contaminant and a very common potent aeroallergen source that is found in environmental samples. The most intense exposure to Alternaria alternata allergens is likely to occur outdoors; however, Alternaria and other allergenic fungi can colonise in indoor environments and thereby increase the fungal aeroallergen exposure levels. Among allergenic proteins described in this fungal species, the major allergen, Alt a 1, has been reported as the main elicitor of airborne allergies in patients affected by a mould allergy and considered a marker of primary sensitisation to Alternaria alternata (Gabriel et al., 2016 April–May).

In addition, Alternaria alternata sensitisation appears to be a trigger in the development of polysensitisation, most likely due to Alternaria alternata's ability to produce a wide and complex range of cross-reactive allergens in addition to Alt a 1, which are homologues in several other allergen sources (Gabriel et al., 2016, April–May). Studying and understanding information about the Alternaria alternata allergen may be key to explaining why sensitisation to Alternaria alternata is a risk factor for asthma and also why the severity of asthma is associated with this fungus. Recent research into the identification and characterisation of Alternaria alternata allergens has made it possible to consider new perspectives in the categorisation of allergenic fungi, exposure assessment and the diagnosis of fungal allergies (Gabriel et al., 2016, April–May). Alternaria spp. Mycotoxins have been isolated from fruits (apples, pears, melons, apricots, grapes, raisins, strawberries, olives, citrus fruits and dried figs), vegetables (tomatoes, peppers and carrots) and potatoes, as well as several processed foods made from damaged raw materials (juices, preserves, sauces). Studies have also shown that aflatoxin contaminates certain types of food, such as nuts, dried fruits and tea, as these groups are often exposed to fungi (Pavon et al., 2012).

We may ask why patients with a food hypersensitivity reaction to peanuts, walnuts, hazelnuts, fish and eggs are significantly more likely to have higher levels of specific IgE against moulds and yeasts. Due to clinical signs after ingestion of peanuts, walnuts, hazelnuts, fish and eggs, patients with atopic dermatitis are trying to eliminate these foods from their diet. One reason may be the fact that reducing the consumption of omega-3 polyunsaturated fatty acids has been proposed as a contribution to the increased prevalence of allergic diseases. Walnuts (vegetable n-3 fatty acid) and fatty fish (marine n-3 fatty acid) may have similar effects because n-3 (omega-3) polyunsaturated fatty acids (PUFAs) can regulate inflammatory processes and response (Kremmyda et al., 2011). Thus, n-3 PUFAs are expected to protect against atopic sensitisation and clinical manifestations of atopy. It has been suggested that reducing the consumption of omega-3 polyunsaturated fatty acids, which is prevalent especially in fatty fish and walnuts, contributes to an increased prevalence of allergic diseases (Kremmyda et al., 2011). Another reason for the significant direct link between fungal and yeast sensitisation and food hypersensitivity reactions to peanuts, hazelnuts, walnuts, fish and eggs may be the fact that fungal and yeast sensitisation is mostly observed in patients with multiple sensitisation to various allergens (Nolles et al., 2001; Reijula et al., 2003).

On the other hand, treenuts and peanuts can have some protective antifungal effects; this effect cannot be applied in patients eliminating these foods. Plants express large amounts of antifungal proteins for protection against fungal diseases, such as pathogen-related proteins (PR), thaumatin-like proteins, thionins, lipid transfer proteins (LTPs), plant defensins, hevein and knottin proteins, Ib-AMP and snakins. Some members of these families have been classified into PR groups, such as defensins (PR-12), thionins (PR-13) and LTP (PR-14), (Loon & Strien, 1999; Theis & Stahl, 2004; van der Weerden et al., 2013); various functions have been attributed to plant defensins. Petersson et al. discovered that 2 new peanut allergens were plant defensins with antifungal activity. In this study, a clear inhibition of the growth of the fungal species Alternaria and Cladosporium was demonstrated (Petersen et al., 2015). The lipophilic extraction strategy was successful in identifying 2 new allergens and 1 other isoform in peanuts. It belongs to a family of 12 proteins associated with pathogenesis, plant defensins (Petersen et al., 2015). Plant defensins with antifungal activity have shown promise for use in both agricultural and therapeutic conditions (Bloch & Richardson, 1991; Colilla et al., 1990; Lay & Anderson, 2005; Loon & Strien, 1999; Petersen et al., 2015; Theis & Stahl, 2004). The results of other research groups describe the antifungal activity of plant defensins on Alternaria and Cladosporium species (Kaur et al., 2011; Ntui et al., 2010; Osborn et al., 1995; Thomma et al., 2002; van der Weerden & Anderson, 2013). Garcia-Olmedo et al. and Stotz et al. conclude that plant defensins show predominantly antifungal activity, while lipid transfer proteins are directed against bacteria. Together, these 2 groups of defense proteins form a general barrier against pathogens (García-Olmedo et al., 1995; Stotz et al., 2009).

Conclusion

The significantly higher level of specific IgE to Asp f 3, Cla h 8, Alt a 6, Alt a 1, Mala s 6 and Asp f 6 was confirmed in atopic dermatitis patients with reaction to hazelnuts, walnuts, peanuts, fish and egg. No significant relation was confirmed between the sensitisation to fungi and moulds and the occurrence of food hypersensitivity reactions to peach, kiwi, apples, spices, oranges, celery, soy, carrot, wheat flour and cow milk. Positive results of specific IgE to molecular components of moulds and yeast are in significant relation to positive results of specific IgE to molecular components of Beta – parvalbumin, arginin kinase, 7 S globulin, 2 S albumin, PR-10 proteins and Non-specific lipid transfer protein. Moreover, there is a relationship between the levels of specific IgE to Alt a 6, Cla h and the level of Cra c 6 (Troponin C).

Acknowledgements

Jarmila Čelakovská is the main investigator and has contributed to selection of the patients from the out- and in-patients dermatological departments, dermatological examination and recommendations for the examination, processing of all results and publication of results. Radka Vaňková made his contribution in laboratory examination. Eva Čermáková helped in statistical analysis. Ctirad Andrýs involved in the control of laboratory examination. Jan Krejsek provided professional supervision.

Disclosure statement

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

Additional information

Funding

This work was supported by Charles University [grant number Q 40/10]; Charles University, Medical Faculty, Hradec Kralove.