Kiwi allergy in atopic dermatitis patients – analysis of specific IgE results in ALEX2 multiplex examination. Latex fruit syndrome

ABSTRACT

Our study analyses the sensitisation profile to kiwi allergens in patients suffering from atopic dermatitis with the use of ALEX2 Allergy Explorer test. The sensitisation to molecular components of latex, banana, avocado, pollen, seeds within latex fruit syndrome was also evaluated. Altogether 100 atopic dermatitis patients were examined. The incidence of clinical reaction to kiwi was observed in 15% of patients; a combination of the kiwi allergens Act d 1, Act d 2 and Act d 10 gave a diagnostic sensitivity of 33.3%. The latex fruit syndrome was recorded in one patient (1%) with the positive result to Hev b 6.02, to (Pers a) and oral allergy syndrome after kiwi ingestion. The sensitisation both to molecular components of kiwi and latex was recorded in 7% of patients. Further research on kiwi allergy should focus on the importance of chitinase and 2S albumins.

KEYWORDS:

• Kiwi allergy
• latex fruit syndrome
• molecular components
• ALEX2 Allergy Explorer

Introduction

Kiwifruit is a nutrient-dense fruit and extensive research over the last decade on the health benefits of kiwifruit has linked their regular consumption to improvements not only in nutritional status, but also benefits to digestive, immune and metabolic health. On the other hand, allergy to kiwi fruit is increasing (Lucas et al., 2004). Kiwi fruit (Actinidia) is a plant native to the Yangtze Valley of China. The seed was taken to New Zealand in 1904, and almost all kiwi cultivars outside China are descended from the two female and one male plant grown from this single introduction of seed (Lucas et al., 2004). Allergy to kiwi fruit was first described in 1981 and is increasingly reported across Europe (Lucas et al., 2004). Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying (Lucas et al., 2004).

To date, thirteen allergens have been identified in kiwifruit. Act d 1, actinidin, major allergen, represents about 50% of the total soluble protein content. The relevance of Act d 2, a thaumatin-like protein and Act d 3, a 45 kDa glycoallergen has yet to be elucidated. Act d 4 acts as an inhibitor of cysteine proteinases, Act d 5, Kiwellin, is a cystein-rich protein that may undergo in vivo and in vitro proteolytic processing by kiwifruit actinidin, Act d 6, a pectin methylesterase inhibitor that may be involved in the regulation of the fruit ripening, and Act d 7, a pectin methylesterase, seem to be recognised by a minority of allergic patients; Act d 8, corresponds to a pathogenesis-related protein class 10 (PR-10) homologous to the major allergen of birch pollen Bet v 1. Other minor allergens from kiwifruit are Act d 9, a 14 kDa profilin, Act d 10, a non-specific lipid transfer proteins (LTP) and Act d 11, a major latex protein/ripening-related protein that cross-reacts with members from the PR-10 family. Recently, two novel allergens contained in kiwifruit seeds have been described and characterised: Act d 12, a 51 kDa 11S globulin that represents a major allergen, and Act d 13, a 12 kDa 2S albumin, which is a minor allergen. Both proteins share common epitopes from peanut and tree nuts, suggesting that both allergens might be involved in cross-reactivity with those allergenic sources. While actinidin is abundantly expressed in green kiwifruits, its expression level and allergenic activity are much lower in golden kiwifruits and in certain kiwifruit cultivars (Lucas et al., 2004; Bublin et al., 2010; Bublin et al., 2011; D'Avino et al., 2011; Bublin et al., 2004). Major and relevant minor kiwi allergenic molecules are recorded in Table 1.

Table 1. Kiwi allergenic molecules (4). Molecular components examined in ALEX2 Allergy Explorer are shown extra bold*.

Molecular component	Biochemical name
	Protein family
Act d 1	Cysteine protease (actinidin)
Act d 2	Thaumatin-like protein
Act d 3	45 kDa glycoallergen, [has yet to be elucidated]
Act d 4	Phytocystatin
Act d 5	Kiwellin
Act d 8	Pathogenesis-related protein, PR-10, Bet v 1 family member
Act d 9	Profilin
Act d 10	Non-specific lipid transfer protein – nsLTP1
Act d 11	Major latex protein/ripening-related protein (MLP/RRP), Bet v 1 family member
Act d 12	Cupin, 11 S globulin
Act d 13	2S albumin

Various allergens play a role in the elicitation or exacerbation of eczematous skin lesions in atopic dermatitis patients, and much research effort has been focused on improving diagnostic tests to identify the causative allergens. The most common symptom of allergy to kiwi fruit is oral allergy syndrome, but late eczematous reactions may be also observed. Organ symptoms with severe life-threatening anaphylactic reactions appear more often in patients suffering simultaneously from other plant allergies. Diagnosing of kiwi allergy is difficult due to lack of standardised allergen extracts to perform skin prick test. The diagnosis of the allergy is mainly based on prick-by-prick tests with native kiwi peel and pulp or with water extracts from the pulp (Lucas et al., 2004; Bublin et al., 2010; Bublin et al., 2011). Component-resolved diagnostics (CRD) may enable significantly improved detection of sensitisation to kiwifruit (Lucas et al., 2004; Bublin et al., 2010; Bublin et al., 2011; D'Avino et al., 2011). Molecular allergy diagnosis (MD) using singleplex allergens or multiplex allergen microarrays are typical methods of precision medicine; MD 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 (Patelis et al., 2016; Jensen-Jarolim et al., 2017; Campana et al., 2017; Matricardi et al., 2016; van Hage et al., 2017; Heffler et al., 2018). Research into the structural similarity between allergens and the amino acid sequence homology between food allergens also helps to explain cross-reactivity between allergens, which may be clinically relevant or irrelevant (Patelis et al., 2016; Jensen-Jarolim et al., 2017; Campana et al., 2017; Matricardi et al., 2016; van Hage et al., 2017; Heffler et al., 2018). Kiwi allergens cross-react with pollens of plants, fruit and latex (Blanco, 2003) the so-called latex-fruit syndrome. Although the foods most frequently involved in latex-fruit syndrome are banana, avocado, kiwi, and chestnut, several others are also implicated. Investigations point to a group of defense-related plant proteins, class I chitinases, which cross-react with a major latex allergen, hevein, as the panallergens responsible for the syndrome (Blanco, 2003).

Aim of the study

To analyse the sensitisation profile to kiwi allergens in patients suffering from atopic dermatitis with the use of ALEX2 Allergy Explorer test and to determine the sensitivity, specificity and positive predictive value of this multiplex examination in comparison with the clinical reaction to kiwi (in the open exposure test or in the history). Our other aim is to evaluate the occurrence of latex fruit syndrome in atopic dermatitis patients with the clinical reactions to kiwi and to evaluate the sensitisation to allergen reagents (molecular components) of banana, avocado, latex, seeds, tomatoes, potatoes and pollen in patients with clinical reaction to kiwi. We analyse the relation between the results of specific IgE to molecular components of kiwi and latex.

Methods

Patients and methods

In the period 2018–20, 100 patients suffering from atopic dermatitis were examined. All these patients were examined in the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech republic. The diagnosis of atopic dermatitis was made with the Hanifin-Rajka criteria (Hanifin and Rajka, 1980). Exclusion criteria were systemic therapy (cyclosporin, systemic corticoids, biological therapy), pregnancy, and breastfeeding. Patients with atopic dermatitis having other systemic diseases were excluded from the study as well. The complete dermatological and allergological examination was performed in patients included in the study. This study was approved by Ethics committee of Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic.

Dermatological examination

Complete dermatological examination was performed in patients included in the study. Severity of atopic dermatitis was scored in agreement with SCORAD, with assessment of topography items (affected skin area), intensity criteria and subjective parameters (Wollenberg et al., 2020).

Examination of specific IgE to allergen reagents

The serum level of the specific IgE (sIgE) was measured by the components resolved diagnostic microarray-based sIgE detection assay ImmunoCAP ALEX2 Allergy Explorer test (van Hage et al., 2017; Heffler et al., 2018). It is based on a state-of-the-art proprietary nano-bead technology. The ALEX2 Allergy Explorer measuring range for specific IgE is 0.3–50 kU_A/L (quantitative) and for total IgE is 1–2500 kU/L (semiquantitative). The sample requirement is 100 µL serum or plasma. 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 kU_A/L) – high positivity, and Class 4 (≥ 15 kU_A/L) – very high positivity. The ALEX2 Allergy Explorer is commercially available, having attained CE certification, which assures that the quality of the assay, regarding LoD, precision and repeatability as well as specificity and linearity, is in line with in vitro diagnostic features. There is no significant interference from high total IgE, haemoglobin, bilirubin or triglycerides (van Hage et al., 2017; Heffler et al., 2018).

Confirmation of clinical reaction to kiwi

The kiwi allergy was confirmed in these cases: (1) In patients with the clinical reaction in the open exposure test or in the history (early and/or late reactions). (2) Sensitisation to kiwi was confirmed in patients with positive results of specific IgE to molecular components of kiwi in ALEX2 Allergy Explorer and with the negative results in the open exposure test/history.

Open exposure test

In patients without previous history about food reactions to kiwi, we recommended the elimination diet with the elimination of kiwi for the period of 5 weeks (Čelakovská et al., 2015).

Open exposure test was performed after this diet in intervals without symptoms or during a consistent period with regard to atopic dermatitis (not in pollen season in patients with pollen allergy). In generally, the open exposure test was recommended to perform with three doses of kiwi given during two days. One dose of examined food was given in incremental dosages in 10-minute intervals during one hour. The food challenge results were scored as positive if one or more of the following objective and subjective clinical reactions were noted: itching, rash, urticaria, angioedema, vomiting, wheezing, abdominal pain, diarrhoea, pruritus, or worsening of atopic dermatitis. Early reactions were defined as clinical symptoms within 2 h after the ingestion of food and late symptoms if occurring after more than 2 h. In the case the physician or the patient recorded worsening of the atopic dermatitis or some other reactions during the open exposure test, the patient continued in the elimination of suspected food and the severity of atopic dermatitis was evaluated every 3 month during 1 year. If the open exposure test was negative, the patient introduced this food in the diet regimen. The severity of atopic dermatitis was evaluated during the ingestion of food over a period of 3 months during 1 year (Čelakovská et al., 2015).

Statistical analysis

We compared the results of specific IgE to allergen reagents (molecular components) in the examination of ALEX2 Allergy Explorer testing and clinical reactions to kiwi in the open exposure test (patient’s history) test; according to these results, the sensitivity, specificity and positive predictive value were calculated (we show Rates and Confidence Intervals 95% C.I., value, lower, upper). Sensitivity was calculated as proportion of positive IgE antibody tests in patients with clinical reaction to kiwi.

We evaluated with Fisher's Exact test the relation between the occurrence of clinical reactions to kiwi and sensitisation to allergen extracts (molecular components) such as Hev b 1 (Rubber elongation factor), Hev b 3 (Small rubber particle protein), Hev b 5 (unknown function), Hev b 6. 02 (Hevein precursor), Hev b 8 (Latex, profilin), Hev b 11 (Class 1, chitinase), Mus a (Banana), Pers a (Avocado), Bet v 1 (PR-10 protein, Birch), Aln g 1 (PR-10 protein, Alder), Phl p 1 (Beta-expansin, Timothy), Cor a 1.0103 (PR-10 protein, hazel pollen), Fag s 1 (PR-10 protein, European beech), Ara h 8 (PR-10 protein,peanuts), Sol t (Potatoes), Sola l (Tomatoes), Sola l 6 (Non-specific lipid transfer protein, tomatoes) and Pru p 3 (Non-specific lipid transfer protein, peach) The significance level is chosen at 5%.

We also evaluated the relation between the results of specific IgE to molecular components of kiwi (Act d 1, Act d 2, Act d 5, Act d 10) and the results of specific IgE to molecular components of latex (Hev b 8, Bev b 6.02, Hev b 11, Hev b 3) with Fisher's Exact test. The significance level is chosen at 5%. We used statistical software: NCSS 2021 Statistical Software (2021). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.

Results

Characteristic of patients

100 atopic dermatitis patients were included in the study (49 men and 51 women with the average age 40.6 years: min. age 14 years, max. age 67 years and with the average SCORAD 39 points, SD 13.5 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 bronchial asthma and 74 patients (74%) suffer from allergic rhinitis.

Results of specific IgE to molecular components and allergen extracts of kiwi, avocado, banana, latex, timothy, birch, sesame, peanuts, European beech, hazelnuts, hazelpollen, peach, potatoes and tomatoes according to the level of specific IgE in Classes 0, 1, 2, 3, 4, Table 2.

Table 2. The detailed overview of allergen reagents (allergenic extracts and molecular components) of kiwi, banana, avocado, latex, birch, alder, timothy, European beech, peanuts, peach, hazelpollen and hazelnuts, sesame, tomatoes, potatoes 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 (%) according to the level of specific IgE in classes 0–4
	100 patients include in the study = 100%
	Class 0	Class 1	Class 2	Class 3	Class 4
Act d 1 Cystein protease	91%	4%	3%	2%	0%
Act d 10 nsLTP	96%	1%	2%	0%	1%
Act d 2 Thaumatin-like protein	86%	11%	1%	2%	0%
Act d 5 Kiwelin	99%	1%	0%	0%	0%
Hev b 1 Rubber elongation factor	99%	0%	1%	0%	0%
Hev b 11 Class I chitinase	99%	1%	0%	0%	0%
Hev b 3 Small rubber particle protein (SRPP)	95%	3%	1%	1%	0%
Hev b 5 Acidic protein, unknown function	98%	0%	2%	0%	0%
Hev b 6_02 Para rubber tree (latex)	95%	2%	2%	1%	0%
Hev b 8 Profilin	92%	2%	3%	2%	1%
Pers a Avocado	90%	5%	5%	0%	0%
Mus a Banana	93%	6%	1%	0%	0%
Bet v 1 Birch, PR 10 protein	47%	2%	6%	5%	40%
Aln g 1 Alder, PR 10 protein	57%	0%	12%	14%	17%
Ara h 8 Peanuts, PR 10 protein	67%	8%	10%	8%	7%
Cor a 1.0103 Hazel pollen, PR 10 protein	52%	2%	7%	7%	32%
Cor a 1.0401 Hazelnuts, PR 10 protein	56%	0%	14%	10%	20%
Cor a 14 Hazelnuts, 2 S albumin	94%	3%	2%	1%	0%
Ses i 1 Sesame, 2 S albumin	89%	4%	3%	1%	3%
Phlp 1 Timothy, Beta expansin	43%	5%	9%	4%	39%
Fag s 1 European beech, PR 10 protein	51%	3%	6%	15%	25%
Pru p 3 Peach, Non-specific lipid transfer protein 1 (nsLTP1)	94%	1%	2%	1%	2%
Sola l, Tomato	90%	6%	3%	1%	0%
Sola l 6, Tomatoe (nsLTP2)	95%	1%	3%	1%	0%
Sol t, Potatoes	86%	7%	3%	1%	3%

Explanation: 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.

In Table 2 we show the sensitisation to molecular components of kiwi and examined molecular components and allergen extracts, which may play the role in latex fruit syndrome. In majority of patients, we recorded a low level of positive results of specific IgE (Class 1) to molecular components of kiwi, latex, banana, avocado, potatoes, tomatoes and sesame. The very high level of specific IgE to Act d 10 and to Hev b 8 was recorded only in 1 patient (1%). On the other hand, very high level of specific IgE was recorded to molecular components of PR 10 proteins (Bet v 1, Aln g 1, Cor a 1.0103, Cor a 1.0401, Fag s 1) and to Beta expansin (Phl p 1).

Clinical reactions to kiwi

The reaction to kiwi with clinical symptoms was confirmed in 15 patients (15%) according to the convincing history and/or open exposure test. We recorded in all 15 patients (15%) early reaction such as oral allergy syndrome and pruritus of the skin.

Sensitivity, specificity and positive predictive value of allergen reagents in ALEX2 Allergy Explorer test in patients with clinical reaction to kiwi, Table 3.

Table 3. Results of specific IgE to molecular components in atopic dermatitis patients with and without the clinical reaction to kiwi. Results of specific IgE to molecular components of kiwi are evaluated as positive (classes of specific IgE 1–4) and negative (Class 0). The sensitivity, specificity and positive predictive value was calculated (we show Rates and Confidence Intervals 95% C.I., value, lower, upper). The significant relation between the results of specific IgE to molecular componnets of kiwi and the occurrence of clinical reactions was not confirmed.

	Number of patients - clinical reaction to kiwi			Sensitivity rates and confidence intervals 95% C.I.
Specific IgE to allergen reagents (molecular components)	yes 15 (15%)	no 85 (85%)	p-value		Value	Lower	Upper
Act d 1 cystein protease
Act d 1 +	3	6	0.132	Sensitivity	0.200	0.043	0.481
Act d 1 -	12	79		Specificity	0.929	0.853	0.974
				Positive predictive value	0.333	0.075	0.701
Act d 2 Thaumatin like protein
Act d 2 +	3	11	0.437	Sensitivity	0.200	0.0433	0.481
Act d 2 -	12	74		Specificity	0.871	0.7802	0.934
				Positive predictive value	0.214	0.0466	0.214

For molecular component Act d 5 and Act d 10 the sensitivity, specificity and positive predictive predictive value can not be calculated, because of low positivity.

The results of specific IgE to molecular components of kiwi and the occurrence of clinical reactions to kiwi are recorded in Table 3. For molecular component Act d 1 the sensitivity is 20.0% (4.3–48.1%), the specificity 92.9% (85.3–97.4%), positive predictive value 33.3% (7.5–70.1%). For molecular component Act d 2 the sensitivity is 20.0% (4.3–48.1%), the specificity 87.1% (78.0–93.3%), positive predictive value 21.4% (4.7–21.4%).

For molecular component Act d 5 and Act d 10 the sensitivity, specificity and positive predictive predictive value can not be calculated, because of low positivity (Table 3).

The review of patients suffering from clinical reactions to kiwi and the results of specific IgE to molecular components of kiwi (Act d 1, Act d 2, Act d 5, Act d 10), latex (Hev b 6.02), banana (Mus a), avocado (Pers a), hazelnuts (Cor a 14, 2 S albumin), tomatoes (Sola l, Sola l 6 – nsLTP), potatoes (Sol t) and sesame (Ses i 1), Table 4 (Complement to Table 4).

Table 4. The review of patients suffering from clinical reactions to kiwi. The results of specific IgE to molecular components of kiwi (Act d 1, Act d 2, Act d 5, Act d 10), latex (Hev b 6.02), banana (Mus a), avocado (Pers a), hazelnuts (Cor a 14, 2S albumin), tomatoes (Sola l, Sola l 6 − nsLTP), potatoes (Sol t) and sesame (Ses i 1), w-woman, m-man. We show the positive value of specific IgE in kUA/L.

	Allergen reagent, molecular components – result of specific IgE in kUA/L
Patient Number	Act d 1	Act d 2	Act d 5	Act d 10	Hev b 6.02	Mus a	Pers a	Cor a 14	Sola l	Sola l 6	Sol t	Ses i 1
1, w, 49 years	–	–	–	–	–	–	–	–	–	–	15.52	18.41
2, w, 45 yeasr	12.61	–	–	–	–	–	–	–	–	–	–	1.17
3, m, 22 yeasr	–	–	–	–	–	–	1.86	0.93	–	–	29.81	0.34
4, w, 49 years	–	–	–	–	–	–	–	–	–	–	26.2	–
5, m, 45 years	–	–	–	–	9.79	–	0.51	–	–	–	–	–
6, m, 16 years	–	–	–	–	–	–	–	–	–	–	–	–
7, w, 67 years	–	–	–	–	–	–	–	–	0.36	–	–	–
8, m, 35 years	0.64	6.01	–	2.96	–	0.61	1.35	2.53	3.43	10.17	2.99	0.85
9, w, 15 years	–	–	–	–	–	–	–	–	–	–	–	–
10, w, 46 years	–	–	–	–	–	–	–	–	–	3.42	–	–
11, w, 17 years	–	–	–	–	–	–	–	–	–	–	–	–
12, m, 28 years	3.32	–	–		–	–	–	–	0.70	–	–	–
13, m, 19 years,	–	0.45	–	–	–	–	–	0.85	0.76	–	0.4	–
14, w, 48 years	–	–	–	–	–	–	–	–	–	–	–	–
15, m, 38 years	–	0.34	–	–	–	–	–	–	–	–	–	–
Complement to Table 4. Cross tabulation report. The relation between the clinical reactions to kiwi and the results of specific IgE to molecular components of latex (Hev b 1, Hev b 3, Hev b 5, Hev b 6.02, Hev b 8, Hev b 11), banana (Mus a), avocado (Pers a), Hazelnuts (Cor a 14 – 2S albumin), Sesame (Ses i 1, 2S albumin), tomatoes (Sola l, Sola l 6 – nsLTP), potatoes (Sol t). The significant relation is confirmed to Sola l, Sol t, Cor a 14, the borderline relation to Ses i 1. The significant relation is shown extra bold *
	Number of patients with positive (+) and negative (−) specific IgE to molecular components
	Hev b 8+	Hev b 8−
Clinical reaction to kiwi +	0 (0%)	15 (100%)
Clinical reaction to kiwi –	8 (9.4%)	77 (90.6%)
Total	8 (8.0%)	92 (92.0%)
p-value	0.602
	Hev b 1+	Hev b 1−
Clinical reaction to kiwi +	0 (0%)	15 (100%)
Clinical reaction to kiwi –	1 (1.2%)	84 (98.8%)
total	1 (1.0%)	99 (99.0%)
p-value	0.602
	Hev b 3+	Hev b 3−
Clinical reaction to kiwi +	0 (0%)	15 (100%)
Clinical reaction to kiwi –	5 (5.9%)	80 (94.1%)
total	5 (5.0%)	95 (95.0%)
p-value	0.100
	Hev b 5+	Hev b 5−
Clinical reaction to kiwi +	0 (0%)	15 (100%)
Clinical reaction to kiwi –	2 (2.3%)	83 (97.7%)
total	2 (2.0%)	98 (98,0%)
p-value	0.100
	Hev b 6 02+	Hev b 6 02−
Clinical reaction to kiwi +	1 (6.7%)	14 (93.3%)
Clinical reaction to kiwi –	4 (4.7%)	81 (95.3%)
total	5 (5.00%)	95 (95.00%)
p-value	0.564
	Hev b 11+	Hev b 11−
Clinical reaction to kiwi +	0 (0%)	15 (100.00%)
Clinical reaction to kiwi –	1 (1.2%)	84 (98.8%)
total	1 (1.00%)	99 (99.00%)
p-value	1.000
	Mus a +	Mus a –
Clinical reaction to kiwi +	1 (6.7%)	14 (93.3%)
Clinical reaction to kiwi –	6 (7.1%)	79 (92.9%)
total	7 (7.0%)	93 (93.0%)
p-value	1.000
	Pers a +	Pers a –
Clinical reaction to kiwi +	3 (20.0%)	12 (80.0%)
Clinical reaction to kiwi –	7 (8.2%)	78 (91.8%)
total	10 (10.0%)	90 (90.0%)
p-value	0.1705
	Cor a 14 +	Cor a 14 –
Clinical reaction to kiwi +	3 (20.0%)	12 (80.0%)
Clinical reaction to kiwi –	3 (3.5%)	82 (96.4%)
total	6 (6.0%)	94 (94.0%)
p-value	0.0420*
	Ses i 1 +	Ses i 1 –
Clinical reaction to kiwi +	4 (26.7%)	11 (73.3%)
Clinical reaction to kiwi –	7 (8.2%)	78 (91.8%)
total	11 (11.0%)	89 (89 .0%)
p-value	0.058
	Sola l
Clinical reaction to kiwi +	4 (26.7%)	11 (73.3%)
Clinical reaction to kiwi –	6 (7.1%)	79 (92.9%)
total	10 (10.0%)	90 (90.0%)
p-value	0.041*
	Sola l 6
Clinical reaction to kiwi +	2 (13.3%)	13 (86.7%)
Clinical reaction to kiwi –	3 (3.5%)	82 (96.5%)
total	5(5.0%)	95 (95.0%)
p-value	0.161
	Sol t
Clinical reaction to kiwi +	5 (33.3%)	10 (66.7%)
Clinical reaction to kiwi –	9 (10.6%)	76 (89.4%)
total	14 (5.0%)	86 (95.0%)
p-value	0.034*

From 15 patients (15%) suffering from clinical reactions to kiwi, we confirmed in one patient the positive result to Hev b 6.02 and positive result to Pers a (avocado), (patient No. 5); in this patient we suspect the latex-fruit syndrome. In 9 patients (9%) with reactions to kiwi, we confirmed the positive results to molecular components of avocado and/or banana and/or hazelnuts and/or sesame and/or potatoes and/or tomatoes. In Complement to Table 4, we show the Cross tabulation report: the relation between the clinical reactions to kiwi and the results of specific IgE to molecular components of latex (Hev b 1, Hev b 3, Hev b 5, Hev b 6.02, Hev b 8, Hev b 11), banana (Mus a), avocado (Pers a), Hazelnuts (Cor a 1.0401 – PR-10 protein, Cor a 14 – 2S albumin), Sesame (Ses i 1, 2S albumin), tomatoes (Sola l, Sola l 6 – nsLTP), potatoes (Sol t). The relation was confirmed between the occurrence of clinical reaction to kiwi and sensitisation to molecular component Cor a 14 (2 S albumin, hazelnuts), to allergen extracts Sola l (tomatoes), Sol t (potatoes), borderline relation was confirmed to the sensitisation to Ses i 1 (Sesame), (p-value = 0.0581).

The review of patients suffering from clinical reactions to kiwi and the results of specific IgE to molecular components and allergen reagents of PR 10 proteins – birch (Bet v 1), alder (Aln g 1), European Beech (Fag s 1), Hazel pollen (Cor a 1.0103), peanuts (Ara h 8), timothy (Beta Expansin Phl p 1) and peach (Pru p 3, nsLTP), Table 5, Complement to Table 5.

Table 5. The review of patients suffering from clinical reactions to kiwi. The results of specific IgE to molecular components and allergen reagents of PR 10 proteins – birch (Bet v 1), alder (Aln g 1), European Beech (Fag s 1), Hazel pollen (Cor a 1.0103), peanuts (Ara h 8), timothy (Beta Expansin Phl p 1) and peach (Pru p 3, nsLTP) w-woman, m-man. We show the positive value of specific IgE in kU_A/L.

	Allergen reagent, molecular components – result of specific IgE in kUA/L
Patient number	Phl p 1	Bet v 1	Aln g 1	Cor a 1.0103	Fag s 1	Arar h 8	Pru p 3
1, w, 49 years	20.86
14.53	36.15	6.17	26.34	19.54	–	–
2, w, 45 years	–	46.86	24.74	44.77	44.81	26.67	–
3, m, 22 years	48.10	45.46	26.86	44.41	40.67	20.36	–
4, w, 49 years	22.39	–	–	–	–	–	–
5, m, 45 years	3.52
31.85	32.09	2.55	22.95	5.92	0.75	–
6, m, 16 years	40.32	–	–	–	–	–	–
7, w, 67 years	–	–	–	–	–	–	–
8, m, 35 years	46.22	35.81	–	0.59	8.19	8.78	28.28
9, w, 15 years	–	1.72	–	–	–	–	–
10, w, 46 years	10.24	4.20	–	–	1.29	–	–
11, w, 17 years*	–	–	–	–	–	–	–
12, m, 28 years	32.84	35.53	9.19	11.25	10.33	–	–
13, m, 19 years,	46.32	38.44	7.06	23.62	32.03	2.00	–
14, w, 48 years	28.64	45.02	20.24	40.76	28.9	0.73	–
15, m, 38 years	33.61	–	–	–	–	–	–
Cross tabulation report. The relation between the clinical reactions to kiwi and the results of specific IgE to molecular components of birch (Bet v 1, PR 10 protein), alder (Aln g 1, PRR 10 protein), Timothy (Phl p 1, Beta expansin), European beech (Fag s, PR-10 protein), peanuts (Ara h 8, PR 10 protein), Hazelpollen (Cor a 1.0103, PR 10 protein), peach (Pru p 3 – nsLTP).
	Number of patients with positive (+) and negative (−) specific IgE to molecular components
	Bet v 1 +	Bet v 1−
Clinical reaction to kiwi +	10 (66.7%)	5 (33.3%)
Clinical reaction to kiwi −	43 (50.6%)	42 (49.4%)
Total	53 (53.0%)	47 (47.0%)
p-value	0.277
	Aln g 1 +	Aln g 1 −
Clinical reaction to kiwi +	7 (46.7%)	8 (53.3%)
Clinical reaction to kiwi −	36 (42.3%)	49 (57.7%)
Total	43 (43.0%)	57 (57.0%)
p-value	0.783
	Phl p 1 +	Phl p 1 −
Clinical reaction to kiwi +	11 (73.3%)	4 (26.7%)
Clinical reaction to kiwi −	46 (54.1%)	39 (45.9%)
Total	57 (57.0%)	43 (43.0%)
p-value	0.258
	Cor a 1.0103 +	Cor a 1.0103 −
Clinical reaction to kiwi +	8 (53.3%)	7 (46.7%)
Clinical reaction to kiwi −	40 (47.1%)	45 (52.9%)
Total	48 (48.0%)	52 (52.0%)
p-value	0.781
	Fag s 1 +	Fag s 1 −
Clinical reaction to kiwi +	9 (60.0%)	6 (40.0%)
Clinical reaction to kiwi −	40 (47.1%)	45 (52.9%)
Total	49 (49.0%)	51 (51. 0%)
p-value	0.410
	Ara h 8 +	Ara h 8 −
Clinical reaction to kiwi +	6 (40.0%)	9 (60.0%)
Clinical reaction to kiwi −	27 (31.8%)	58 (68.2%)
Total	33 (33.0%)	67 (67. 0%)
p-value	0.560
	Pru p 3
Clinical reaction to kiwi +	1 (6.7%)	14 (93.3%)
Clinical reaction to kiwi −	5 (8.9%)	80 (94.1%)
Total	6 (6.0%)	94 (94.0%)
p-value	1.000

In patients suffering from clinical reaction to kiwi we can observe in majority of them (in 13/15 patients) sensitisation to molecular components of PR 10 proteins and in 11/15 patients sensitisation to Phl p 1. In Complement to Table 5, we show the Cross tabulation report – the relation between the clinical reactions to kiwi and the results of specific IgE to molecular components of birch (Bet v 1, PR 10 protein), alder (Aln g 1, PRR 10 protein), Timothy (Phl p 1, Beta expansin), European beech (Fag s, PR-10 protein), peanuts (Ara h 8, PR 10 protein), Hazelpollen (Cor a 1.0103, PR 10 protein), peach (Pru p 3 – nsLTP). The significant relation between the patients suffering from clinical reaction to kiwi and sensitisation to PR-10 proteins, Phl p 1 (Beta expansin – Timothy) and Pru p 3 (nsLTP – peach) was not confirmed.

The review of patients without the clinical reaction to kiwi, but with the positive results of specific IgE to molecular components of kiwi. The results of specific IgE to molecular components of latex in these patients (to Hev b 8, Hev b 6.02, Hev b 11, Hev b 3), Table 6.

Table 6. The review of patients without clinical reactions to kiwi, but with positive results of specific IgE to to Act d 1 and/or Act d 2 and/or Act d 5 and/or Act d 10 ( =  sensitisation). In these patients we show the results of specific IgE to molecular components of latex (Hev b 3, Hev b 6.02, Hev b 8, Hev b 11). Specific IgE in kU_A/L. No clinical reaction, but positive results of specific IgE to Act d 1 and/or Act d 2 and/or Act d 5 and/or Act d 10 ( =  sensitisation) were confirmed 17 patients (17%), in 7 patients (7%) we confirmed the positive results of specific IgE to molecular components of latex (to Hev b 8 and/or Hev b 6.02 and/or Hev b 11 and/or Hev b 3).

Patients with positive results of specific IgE to kiwi allergens	Allergen reagent, molecular components – result of specific IgE in kUA/L
	Act d 1	Act d 2	Act d 5	Act d 10	Hev b 8	Hev b 6.02	Hev b 11	Hev b 3
1. man, 56 years	3.08	0.86
2. woman, 32 years		0.42
3. woman, 37 years	1.4			0.64				2.29
4. man, 64 years	0.79					0.61
5. man, 42 years			0.86
6.man,60 years		0.38
7.man, 30 years		12.93
8.man, 50 years	5.56			1.32
9. woman, 15 years	0.41					0.66
10.man, 20 years		0.44						0.31
1man, 30 years		1.03		37.00			0.63
12. man, 68 years	0.32
13 man, 25 years		0.31			24.38
14.man, 43 years		0.34
15.man, 30 years		0.59
16.woman, 20 years		0.34				2.97
17.woman, 42 years		0.35

No clinical reaction to kiwi, but positive results of specific IgE to Act d 1 and/or Act d 2 and/or Act d 5 and/or Act d 10 ( =  sensitisation) were confirmed in 17 patients (17%); in 7 patients (7%) we confirmed at the same time the positive results of specific IgE to molecular components of latex (to Hev b 8 and/or Hev b 6.02 and/or Hev b 11 and/or Hev b 3). In these patients we can suppose the latex fruit syndrome (Table 6).

The relation between the results of specific IgE to molecular components of kiwi and latex, Table 7.

Table 7. The relation between the sensitisation to molecular components of kiwi and latex. In contingency tables we evaluated, if in patients with positive results of specific IgE to molecular components of kiwi (Act d 1, Act d 2, Act d 5, Act d 10) is recorded the significant relation to the sensitisation to molecular components of latex (Hev b 8, Hev b 6.02, Hev b 11 and Hev b 3). The significant relation was confirmed between Actd d 10 and Hev b 11 (p-value = 0.04); the border line relation was confirmed between Act d 1 and Hev b 6.02 (p-value = 0.0628).

Sensitisation to molecular components of latex – number of patients	Sensitisation to molecular components of kiwi – number of patients
			p-value
	Act d + 1	Act d 1-
Hev b 8 +	1	7	0.5433
Hev b 8−	8	84
Hev b 3+	1	4	0.3823
Hev b 3−	8	87
Hev b 6.02+	2	3	0.0628
Hev b 6.02−	7	88
Hev b 11+	0	1	1.000
Hev b 11-	9	90
	Act d 2 +	Act d 2-
Hev b 8 +	2	6	0.3106
Hev b 8−	12	80
Hev b 3+	1	4	0.5374
Hev b 3−	13	82
Hev b 6.02 +	1	4	0.8374
Hev b 6.02−	13	82
Hev b 11+	1	0	0.14
Hev b 11−	13	86
	Act d 5+	Act d 5-
Hev b 8+	0	8	1.000
Hev b 8−	1	91
Hev b 3+	0	5	1.000
Hev b 3−	1	94
Hev b 6.02 +	0	5	1.000
Hev b 6.02−	1	94
Hev b 11+	0	1	1.000
Hev b 11−	1	98
	Act d 10+	Act d 10 -
Hev b 8+	0	8	1.000
Hev b 8−	4	88
Hev b 3+	1	4	0.1881
Hev b 3−	3	92
Hev b 6.02+	0	5	1.000
Hev b 6.02−	4	91
Hev b 11+	1	0	0.04*
Hev b 11−	3	96

In contingency tables we evaluated, if in patients with positive results of specific IgE to molecular components of kiwi (Act d 1, Act d 2, Act d 5, Act d 10) is recorded the significant relation to the sensitisation to molecular components of latex (Hev b 8, Hev b 6.02, Hev b 11 and Hev b 3). The significant relation was confirmed between Act d 10 (ns LTP) and Hev b 11 (Class I Chitinase), (p-value = 0.04); the border line relation was confirmed between Act d 1 (Cystein protease) and Hev b 6.02 (Hevein precursor), (p-value = 0.0628).

Discussion

The purpose of our study was to evaluate the clinical reactions to kiwi in atopic dermatitis patients and to analyse the results of specific IgE to kiwi with the use of ALEX2 Allergy Explorer test. We evaluated the sensitivity, specificity and positive predictive value of this test. Our other aim was to evaluate the occurrence of a latex-fruit syndrome in atopic dermatitis patients suffering from clinical reactions to kiwi.

There has been an increase in the incidence of kiwifruit allergy in the last few years with new cases more often affecting younger infants (Lucas et al., 2004). Kiwifruit is becoming one of the more common causes of food allergy in Mediterranean countries such as France, where it is the third most common food allergen in children after milk and egg, affecting 9% of children (Rance et al., 2005). In Portugal, Vieira et al., found that kiwifruit is the most allergenic fruit, eliciting allergic symptoms in 60% of a paediatric cohort, followed by peach (50%) (Vieira et al., 2014). Spanish allergic children develop systemic reactions following kiwifruit ingestion more frequently than adults. Act d 1 and Act d 2 were major allergens in the Spanish paediatric age group (Moreno Álvarez et al., 2015).

So far, no work has been done on comparing laboratory results in ALEX2 Allergy explorer test with respect to clinical symptoms with reactions to kiwi in adolescent and adult patients suffering from atopic dermatitis.

Our study confirms, that most common symptom of kiwi fruit allergy is the oral allergy syndrome; it was observed in 15 (15%) of patients with atopic dermatitis. All these patients eliminate kiwi for a long time from their diet; after a casual ingestion, they observe mouth and throat itching, which starts rapidly after a food is placed in the mouth. Some patients described also pruritus of the skin and gastrointestinal symptoms, such as abdominal pain.

Regarding the results of specific IgE to molecular components of kiwi, a combination of the kiwi allergens Act d 1, Act d 2 and Act d 10 gave a diagnostic sensitivity of 33.3%. Some factors that might influence the lower sensitivity of ALEX2 Allergy Explorer test compared with in vivo tests, may be the smaller number of allergens tested in ALEX2 Allergy Explorer test – from 13 allergens identified in kiwifruit, only four allergens are included in ALEX2 Allergy Explorer test. Another reason for the lower sensitivity of specific IgE to molecular components of kiwi may be the fact, that patients in our study eliminate kiwi for a long time from their diet. On the other hand, we recorded high specificity of Act d 1 and Act d 2.

Bublin evaluated the use of individual allergens for component-resolved in vitro diagnosis of kiwifruit allergy. The diagnostic sensitivities of the commercial extract and of the sum of single allergens were 17% and 77%, respectively, whereas diagnostic specificities were 100% and 30%. A combination of the kiwi allergens Act d 1, Act d 2, Act d 4, and Act d 5 gave a diagnostic sensitivity of 40%, whereas diagnostic specificity remained high (90%). According to their results Bet v 1 homologue and profilin are important allergens in pollen-related kiwifruit allergy, whereas actinidin is important in monoallergy to kiwifruit, in which symptoms are often more severe (Bublin et al., 2010). In another study, Bublin measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Act d 1 can be considered a marker allergen for genuine sensitisation to kiwifruit. Our study with the use of multiplex examination shows lower sensitivity and lower positive predictive value, but higher specificity given in comparison to Bublinś studies. Lucas (Lucas et al., 2004) in his study confirmed allergy to kiwi fruit in 53% of the subjects tested with double-blind placebo-controlled food challenge test (DBPCFC), who had a previous history suggestive of kiwi allergy. Skin testing with fresh fruit has good sensitivity (93%), but poor specificity (45%) in this population; CAP sIgE and a commercially available skin test solution were both much less sensitive (54%; 75%) but had better specificity (90%; 67%) (Lucas et al., 2004). Alemán sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitised patients from a birch-free area (Alemán et al., 2004). Forty-three patients with allergy symptoms who were sensitised to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The most frequent clinical manifestation was oral allergy syndrome. The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi (Alemán et al., 2004).

D’Avino identified and characterised kirola, the 17-kDa protein of green kiwifruit (Act d 11). Act d 11 is the first member of the MLP/RRP protein family to be described as an allergen. It displays IgE co-recognition with allergens belonging to the PR-10 family, including Bet v 1 (D'Avino et al., 2011).

Garrido-Arandia et al. in their study show the importance of the N-glycan fraction in the allergic sensitisation to Act d 2 (Bublin et al., 2010). According to their results, the sugar moiety plays a significant role in sensitisation, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions (Garrido-Arandia et al., 2014).

About 30–50% of latex-allergic patients show allergic symptoms to plant-derived foods, especially fresh fruits. This association was named latex-fruit syndrome (Blanco, 2003). The fruits most commonly involved are banana, avocado, chestnut, and kiwi. Several latex allergens were discussed as mediators of the latex-fruit cross-reactivity, such as Hev b 2 (endo-β1,3-glucanase), Hev b 6.02 (hevein), Hev b 7 (patatin-like protein), Hev b 8 (profilin), and Hev b 12 (non-specific lipid-transfer protein) (Blanco, 2003).

In our study we evaluated, if in patients suffering from clinical reactions to kiwi are recorded the positive results of specific IgE to molecular components of latex, banana, avocado, pollen and seeds. From 15 patients (15%) with clinical reaction to kiwi, in one patient we confirmed the positive result to molecular component Hev b 6.02 (hevein precursor) and to Pers a (Avocado), in this patient we expect the occurrence of latex fruit syndrome. In patients with clinical reaction to kiwi we did not confirme the significantly higher sensitisation rate to PR-10 proteins; on the other hand, we confirmed significantly higher sensitisation rate to molecular component Cor a 14 (2S albumin, hazelnuts), to allergen extracts Sola l (tomatoes), Sol t (potatoes), borderline relation was confirmed to Ses i 1 (2S albumin, Sesame), (p-value = 0.0581). We can suppose, that in patients suffering from clinical reactions to kiwi may be positive results of specific IgE to Act d 13 (2S albumin), but this molecular component is not included in ALEX2 Allergy Explorer. Act d 13, a 12 kDa 2S albumin, is a minor allergen, which shares common epitopes from peanut and tree nuts, suggesting that this allergen might be involved in cross-reactivity with those allergenic sources.

No clinical reaction, but positive results of specific IgE to molecular components of kiwi were confirmed in 17 patients (17%), in 7 patients (7%) we confirmed at the same time the positive results of specific IgE to molecular components of latex – in these patients we can suppose the occurrence of latex fruit syndrome. These patients are informed about possible reactions with latex and some of them describe skin problems after contact with latex.

Evaluating the results of specific IgE to molecular components of kiwi and latex (regardless of the presence of a clinical reaction to kiwi), the significant relation was confirmed between Act d d 10 (ns LTP) and Hev b 11 (Class I Chitinase), (p-value = 0.04); the border line relation was confirmed between Act d 1 (Cystein protease) and Hev b 6.02 (Hevein precursor), (p-value = 0.0628). Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin-binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. According to O'Riordain et al., Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin-binding domain and displays a different IgE binding capacity compared with hevein (O’Riordain et al., 2002).

Radauer et al. aimed to assess the extent of cross-reactivity between hevein and hevein-like domains using sera from latex-allergic patients with and without plant food allergy. Hevein and hevein-like domains of latex, banana, and avocado chitinases were expressed in Escherichia coli as fusion proteins with the maltose-binding protein and purified by affinity chromatography. Despite considerable cross-reactivity between these allergens, no correlation between latex-associated plant food allergy and sensitisation to hevein or hevein-like domains was found (Radauer et al., 2011). Our results show, that specific IgE to molecular component Act d 10 significantly correlate with the results of specific IgE to Hev b 11, Class I Chitinase. However, low positivity must be taken into account because the positivity of Hev b 11 was recorded only in one patient (1%).

In addition to the fact that kiwi is an important alergen, it is necessary to note the beneficial effects of kiwifruit in gastrointestinal function in healthy individuals as well as in individuals with constipation and other gastrointestinal disorders (Stonehouse et al., 2013; Chang and Liu, 2009; Jung et al., 2005; Aune et al., 2017).

Kiwifruit is exceptionally high in vitamin C and contains an array of other nutrients, notably nutritionally relevant levels of dietary fibre, potassium, vitamin E and folate, as well as various bioactive components, including a wide range of antioxidants, phytonutrients and enzymes, that act to provide functional and metabolic benefits. The contribution of kiwifruit to digestive health is attracting particular attention owing to a growing body of evidence from human intervention studies. There are several plausible mechanisms of action that are likely to act together including the fibre content and type, the presence of actinidin (a natural proteolytic enzyme unique to kiwifruit) which breaks down protein and facilitates gastric and ileal digestion and other phytochemicals which may stimulate motility (Boeing et al., 2012; Kaur et al., 2010a; 2010b; Ciardiello et al., 2008).

Conclusion

The incidence of clinical reaction to kiwi in our study was observed in 15% of patients, majority of these patients suffer from oral allergy syndrome. In examination with ALEX2 Allergy explorer test, a combination of the kiwi allergens Act d 1, Act d 2 and Act d 10 gave a diagnostic sensitivity of 33.3%. The significant relation between the clinical reaction to kiwi and sensitisation to molecular component Cor a 14 (2S albumin, hazelnuts), to allergen extracts Sola l (tomatoes) and Sol t (potatoes) was confirmed. We suspect the latex fruit syndrome in one patient with the positive result to Hev b 6.02 (latex, Hevein precursor), to avocado (Pers a) and oral allergy syndrome after kiwi ingestion. The positive results of specific IgE both to molecular components of kiwi and molecular components of latex were recorded in 7% of patients. The significant relation was confirmed between results of specific IgE to Act d 10 and Hev b 11; the border line relation was confirmed between Act d 1 and Hev b 6.02. Further research on kiwi allergy should focus on the importance of chitinase and 2S albumins.

Disclosure statement

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

Additional information

Funding

This work was supported by Charles University (Cooperatio), Medical Faculty Hradec Králové.