Molecular IgE Reactivity Profiling With Micro‐Arrayed Allergens Reveals Distinct Interregional Patterns of Sensitization and a Hypoallergenic Region in Türkiye

ABSTRACT

Background

Molecular immunoglobulin E (IgE) reactivity profiling with multiple micro‐arrayed allergens is a valuable tool in allergy diagnosis and has provided fundamental insights into the evolution of allergic sensitization in longitudinal birth cohort studies. However, there is a need for systematic cross‐sectional population‐based studies.

Methods

A prospective, systematic and cross‐sectional population study was performed involving 1000 adult subjects from five centers (İstanbul, Ankara, İzmir, Kayseri, Samsun) representing different climatic areas of Türkiye (Turkey). In each center adult subjects with (n = 100) and without (n = 100) allergic symptoms who were born and grew up in the region were recruited. Subjects underwent skin prick testing with a panel of common aeroallergen extracts and were tested for IgE reactivity to 108 micro‐arrayed allergen molecules.

Results

IgE reactivity profiles of respiratory allergens differed in the five centers according to the regional allergen exposome and climate. Interestingly, Kayseri with a high‐altitude, continental climate was identified as hypoallergenic region with low frequency of allergic symptoms and strongly reduced molecular sensitization rates. Between 31%–55% of subjects without allergic symptoms showed IgE sensitization to at least one of the allergens tested but allergen‐specific IgE levels, numbers of recognized allergens and rates of poly‐sensitization were more frequent in symptomatic individuals.

Conclusions

This is the first comprehensive, systematic, prospective and cross‐sectional study investigating molecular IgE sensitization profiles in a region at the crossroads of Europe, Asia, and Africa with different climatic conditions. It reveals clinically relevant differences in regional molecular IgE sensitization profiles depending on the allergen exposome and identifies a hypoallergenic region with distinct climatic features.

A prospective, systematic and cross‐sectional population study was performed involving 1000 adult subjects from five centers representing different climatic areas of Türkiye (Turkey). Molecular IgE profiling demonstrated substantial interregional variation and identified a hypoallergenic region in the easternmost area with a continental climate. These findings emphasize the critical role of climatic and environmental factors in allergy development and provide valuable insights for targeted prevention strategies and improved allergy management. AMS, allergen molecule sensitization; CCD, cross‐reactive carbohydrate determinant; HDM, house dust mite; IgE, immunoglobulin E.

Abbreviations

AD

atopic dermatitis

AIT

allergen‐specific immunotherapy

AM

allergen molecule

AMS

AM sensitization

AR

allergic rhinitis

CCD

cross‐reactive carbohydrate determinant

FA

food allergy

HDM

house dust mite

HRP

horseradish peroxidase

IgE

immunoglobulin E

IQR

interquartile range

ISAAC

International Study of Asthma and Allergies in Childhood

nsLTP

non‐specific lipid transfer protein

OAS

oral allergy syndrome

SPT

skin prick testing

1. Introduction

The prevalence and spectrum of immunoglobulin E (IgE)‐mediated sensitizations vary significantly across countries and geographic regions [1, 2, 3, 4]. These variations are attributed to differences in allergen exposure patterns and timing, as well as the influence of protective and promoting co‐factors [5, 6]. IgE‐mediated sensitization is now recognized as a dynamic process that begins shortly after birth, diversifies throughout childhood, and eventually plateaus in adulthood [7, 8]. Consequently, examining allergic adults from various geographic regions with a shared cultural background has the potential to enhance our understanding of divergent exposomes and the factors influencing sensitization development.

Traditionally, allergy diagnosis has relied on whole‐extract‐based testing; however, the field is progressing to broad‐panel allergen molecule (AM) testing [9, 10]. The multiplexing of hundreds of AMs now enables comprehensive evaluation of a patient's sensitizations, in contrast to the extract‐based diagnosis that allergists were restricted to when components were not available. Documenting sensitization to a broad range of AMs enables the distinction between genuine sensitizations, co‐sensitizations, cross‐sensitizations, and clinically irrelevant sensitizations [11]. Shifting the focus to documenting sensitization to AMs, rather than whole extracts, has the potential to revolutionize our understanding of allergen sensitizations.

Situated at the crossroads of Asia, Europe, and Africa in the Eastern Mediterranean, Türkiye is influenced by a unique blend of cultural and environmental factors originating from Europe, the Middle East, and parts of Western Asia. This unique geographic position renders the country particularly relevant for research on diversity and disparities within the region.

The primary objective of this study was to investigate molecular allergen sensitizations in adults residing in five distinct geographic regions and to elucidate the variability of molecular allergen sensitizations within these areas. The design of our cross‐sectional study is unique because we investigated subjects who were born in the different regions of the country and grew up there. Furthermore, we investigated IgE reactivity profiles in subjects with and without allergic symptoms. Our results reveal the presence of a hypoallergenic region within the country most likely due to unique environmental and climate factors.

2. Methods

2.1. Study Design

The study was designed as a multicenter cross‐sectional study and conducted between October 1, 2022, and January 15, 2023. It aimed to establish two cohorts—symptomatic and asymptomatic for allergic diseases—across five different cities in Türkiye, with 100 participants in each group per center. In total, 1000 individuals were enrolled, with 200 participants recruited from each center.

The study sample was drawn from five cities—İstanbul, Ankara, İzmir, Kayseri, and Samsun—representing distinct geographical regions of Türkiye (Figure 1). These cities were selected to reflect the country's diversity in geography and climate. For instance, İzmir lies on the Aegean coast and has a Mediterranean climate, whereas Kayseri and Ankara are inland cities located at altitudes between 800 and 1000 m, characterized by a continental climate [12].

FIGURE 1.

Map of Türkiye and the study centers. Türkiye, with an approximate population of 80 million people, is the second most populated country in Europe. The climatic and geographical facets of Türkiye are quite variable, given its partial location in the Balkans, its lengthy coastline along both the Mediterranean and the Black Sea, and its location on the Middle East.

2.2. Target Population

Inclusion criteria were individuals 18 years of age or older who were born in the respective city, had grown up there, and were currently residing in the same city. Exclusion criteria were a history of allergen‐specific immunotherapy (AIT), residence outside the city for more than 5 years, or any educational, physical, or mental condition preventing completion of the questionnaire. The 5‐year benchmark was chosen to account for relocation during university (≈4 years) and compulsory military service (≈1 year). Furthermore, it is known from birth cohort studies that most IgE sensitizations occur during the first years of life [13].

We used the modified International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire for adults to assess the presence of allergic symptoms [14]. Symptomatic individuals were selected from the allergy department of the relevant hospital, and an additional 100 asymptomatic individuals without allergy symptoms were recruited from the general outpatient clinic. Participants were enrolled on a first‐come, first‐served basis, and enrollment was stopped when the target number of participants was reached. Each subject underwent the questionnaire after the written informed consent was obtained. Age and gender distributions were comparable between symptomatic and asymptomatic groups within each center. Median age and female proportion ranged from 29.0 to 37.5 years and 54% to 76% in the symptomatic group, and from 30.0 to 38.0 years and 52% to 64% in the asymptomatic group, across centers.

2.3. Assessment of the Allergic Symptoms

The ISAAC questionnaire [14], a widely employed tool for assessing IgE‐mediated allergic symptoms, has been translated into Turkish and had been evaluated for its validity and reliability earlier [15]. While the survey was adapted for adult participants, the fundamental question patterns remained unchanged. In order to gauge its comprehensibility, in‐depth interviews were conducted with 30 individuals. The questionnaire was subsequently utilized to classify participants into two groups: symptomatic and asymptomatic. Within the asymptomatic group, negative responses were anticipated for all questions. In contrast, for the symptomatic group, questions that received affirmative responses were used to identify specific allergic conditions.

Evidence of asthma was defined as a positive response to one of the following questions: (1) “Have you had wheezing or whistling in your chest in the past 12 months?” or (2) “Have you had a dry cough at night, not associated with a cold or chest infection, in the past 12 months?” or (3) “Have you ever had asthma?” Allergic rhinitis (AR) was determined as a positive response to one of the following questions: (1) “Have you ever experienced episodes of sneezing, runny nose, nasal congestion, or conjunctivitis with red, itchy eyes not associated with a cold or flu?” or (2) “Have you ever had hay fever?” Atopic dermatitis (AD) was assessed by the following questions: (1) “Have you ever had an itchy rash affecting specific areas of your body—such as the inner elbows, backs of the knees, fronts of the ankles, neck, or around the ears or eyes—that lasted at least 6 months?” or (2) “Have you ever had AD?” Food allergy (FA) was assessed through items inquiring about symptoms occurring after food ingestion, including oral allergy syndrome (OAS) (e.g., swelling or itching of the lips, tongue, mouth, or throat), skin manifestations (e.g., generalized urticaria, angioedema), respiratory symptoms (e.g., stridor, dyspnea, cough, wheezing), cardiovascular symptoms (e.g., hypotension), or persistent gastrointestinal symptoms (e.g., crampy abdominal pain, vomiting). The pollen‐related OAS was diagnosed by using a questionnaire which was validated previously [16].

2.4. Skin Prick Testing

All symptomatic participants underwent whole extract skin prick testing (SPT) for common aeroallergens with commercial aeroallergen extracts (Lofarma S.p.A., Milan, Italy) as performed by the relevant department prior to enrollment in the study. The panel included the following allergens: Alternaria alternata , Artemisia vulgaris , Olea europaea , Corylus avellana , Parietaria judaica , Cupressus arizonica , Ambrosia artemisiifolia , Blattella germanica , cat and dog dander, and house dust mite (HDM) (Dermatophagoides pteronyssinus and D. farinae). The tree pollen mix consisted of Alnus incana and other Betulaceae species; the weed mix included Artemisia vulgaris , Solidago virgaurea, Helianthus annuus , Xanthium strumarium , and Iva axillaris ; and the grass mix contained Dactylis glomerata , Festuca elatior , Lolium multiflorum , Phleum pratense , and Poa pratensis . To ensure reliable testing, participants discontinued antihistamines for at least 1 week before the test. A positive result was determined when the mean of the longest diameter and perpendicular diameter was ≥ 3 mm compared to the negative control [17]. The asymptomatic group did not undergo SPT.

2.5. Molecular Allergen‐Specific IgE Measurement

The allergen chips contained 108 AMs including respiratory and food allergens (Table S1). Most of the AMs included in the micro‐array were recombinant allergens expressed in Escherichia coli , insect cells or mammalian cells. Allergens were characterized regarding physicochemical properties and tested for IgE reactivity with reference sera. In Tables S1–S6, recombinant allergens were defined by “r” and allergens purified from natural allergen sources were denoted by “n”. The cross‐reactive carbohydrate determinant (CCD) marker, horseradish peroxidase (HRP), was obtained from Sigma‐Aldrich Handels Gmbh, Vienna, Austria [18].

Allergen micro‐arrays were produced at the Medical University of Vienna, Austria. For this purpose, molecules were immobilized on glass slides in triplicates (Paul Marienfeld GmbH & Co. KG, Lauda‐Königshofen, Germany) with a SciFlex array spotter (Scienion, Berlin, Germany) [19]. After spotting, the allergens were immobilized in a 75% humidity chamber at room temperature overnight on the slides and blocking buffer (30 mmol/L ethanolamine in PBS containing 0.1% Tween‐20) was added. Then micro‐arrays were stabilized with liquid plate sealer (CANDOR Bioscience GmbH, Wangen, Germany), dried by centrifugation (200 g, 1 min) in a Sigma 2–7 centrifuge using a MTP‐11113 rotor (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) vacuum‐sealed and stored at 4°C until use.

Microarrays underwent a process of washing while being stirred for 1 min in buffer B (PBS containing 0.1% Tween‐20). Upon drying of the chips via centrifugation at 600–800g for 3 min, aliquots of 30 μL of undiluted serum samples were added to each of the arrays for 2 h. In addition, control slides were incubated with a serum pool containing defined IgE levels as determined by ImmunoCAP measurements to several allergens included in the array for calibration or sample diluent was used as a negative control for 2 h at 22°C (Thermo Fisher Scientific/Phadia, Uppsala, Sweden). The arrays were spray washed, followed by a further washing step as described above, and then dried. Subsequently they were incubated with a mouse monoclonal anti‐human IgE antibody (1 μg/mL) (Roche, Basel, Switzerland) conjugated with DyLight 550‐2xPEG NHS Ester (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in the dark for 30 min at room temperature. After this, slides were washed with buffer B followed by distilled H₂O, and finally dried by centrifugation. Fluorescence signals were measured with a TECAN Power Scanner (Thermo Fisher Scientific, Grödig, Austria). Fluorescence signals were analyzed using Mapix software (Version 8.5.0) (Innopsys, Carbonne, France). A calibrator serum and sample diluent were included in each serum analysis sequence for calibration and detection of background signals. Results are expressed in ISU‐IgE (International Standardized IgE Units). Positivity was defined as an allergen‐specific level of 0.3 ISU‐IgE or greater.

2.6. Statistical Analysis

Statistical analyses were performed using the Python programming language version 3.8. As the data were not normally distributed, demographic characteristics and allergen‐specific IgE levels are reported as medians with interquartile ranges (IQR). Differences in nominal variables were assessed using the Chi‐square test or Fisher's exact test, as appropriate. The non‐parametric Mann–Whitney U test was used to differentiate allergen‐specific IgE levels between groups. p values < 0.05 were considered statistically significant.

3. Results

3.1. Demographic Characteristics of Participants and Features of Study Centers

The goal of enrolling at least 100 symptomatic and 100 asymptomatic participants was successfully met for each of the five participating centers. Comprehensive demographic data for different cities and study subgroups are presented in Table 1. Importantly, symptomatic versus asymptomatic subjects and among centers were well balanced regarding age and gender. Table S7 provides further data regarding the subjects from the different study centers. Centers were in general comparable regarding fertility rates/births per woman, mortality rates and life expectancy. Mean family sizes and housing statuses were also comparable among centers. Fewer subjects from Kayseri and Samsun had a higher educational status as compared with the subjects from İstanbul, Ankara and İzmir (Table S7). Accordingly, income and numbers of cars were lower in Kayseri and Samsun compared with the other three cities. No relevant differences were noted regarding migration statuses and health facilities among the centers.

TABLE 1.

Demographic and clinical characterization of individuals with symptoms of allergy from different regions of Türkiye.

	Istanbul	Ankara	Izmir	Kayseri	Samsun
Age, years, median (IQR)
Symptomatic, n = 100	29.0 (24.0–43.0)	34.5 (25.0–44.0)	34 (26.0–42.0)	33.5 (25.0–45.7)	37.5 (29.0–47.0)
Asymptomatic, n = 100	30.0 (20.2–46.0)	36.6 (30.0–45.0)	32.5 (22.2–40.7)	32.5 (26.0–40.0)	38.0 (27.2–52.0)
Gender (F), %
Symptomatic, n = 100	54	63	68	76	67
Asymptomatic, n = 100	52	54	63	64	63
Patients with symptoms of allergy each center, n = 100
ISAAC questionnaire data, n, (%)
Asthma	78 (78)	65 (65)	53 (53)	36 (36)	51 (51)
Allergic rhinitis/conjunctivitis	92 (92)	90 (90)	91 (91)	73 (73)	100 (100)
Atopic dermatitis	37 (37)	16 (16)	18 (18)	12 (12)	3 (3)
Food allergy	25 (25)	13 (13)	27 (27)	1 (1)	4 (4)
Urticaria/Angioedema	13 (52)	4 (31)	16 (59)	1 (100)	1 (25)
Respiratory symptoms (rhinitis, stridor, dyspnea, cough, wheezing)	11 (44)	5 (38)	6 (22)	1 (100)	2 (50)
Gastrointestinal symptoms (abdominal cramps and/or vomiting)	6 (24)	1 (8)	10 (37)	0 (0)	0 (0)
Cardiovascular symptoms (hypotension)	1 (4)	0 (0)	1 (4)	0 (0)	0 (0)
Oral allergy syndrome	11 (44)	6 (46)	10 (37)	0 (0)	1 (25)
Anaphylaxis	0 (0)	0 (0)	2 (7)	0 (0)	1 (25)
Eczama	3 (12)	1 (8)	2 (7)	0 (0)	0 (0)
Skin prick tests with allergen extracts, positive results, n (%)
Alternaria	0 (0)	3 (3)	9 (9)	3 (3)	2 (2)
Grass mix	25 (25)	31 (31)	42 (42)	22 (22)	26 (26)
Artemisia	7 (7)	8 (8)	13 (13)	2 (2)	0 (0)
Olea	24 (24)	11 (11)	19 (19)	6 (6)	14 (14)
Cupressus	0 (0)	0 (0)	3 (3)	0 (0)	16 (16)
Tree mix	5 (5)	4 (4)	2 (2)	12 (12)	7 (7)
Cat	20 (20)	25 (25)	24 (24)	13 (13)	12 (12)
Dog	17 (17)	14 (14)	8 (8)	3 (3)	7 (7)
House dust mite	58 (58)	33 (33)	36 (36)	22 (22)	70 (70)
Ragweed	11 (11)	1 (1)	0 (0)	1 (1)	22 (22)
Weed mix	8 (8)	1 (1)	27 (27)	8 (8)	24 (24)
Corylus	0 (0)	2 (2)	0 (0)	0 (0)	7 (7)
Parietaria	0 (0)	2 (2)	0 (0)	0 (0)	0 (0)
Blatella	0 (0)	7 (7)	0 (0)	5 (5)	0 (0)

However, regarding climate we noted fundamental differences in the study centers: Kayseri and Ankara had higher altitudes, much lower minimal temperatures and much less total rainfall per year than İstanbul, İzmir and Samsun (Table S7).

3.2. Respiratory and Food Allergy Is Much Less Frequent in Kayseri Than in the Other Centers

Within the symptomatic group, AR emerged as the most prevalent diagnosis, followed by asthma, AD, and FA (Table 1 and Figure 2). Notably, in Samsun, all symptomatic individuals were diagnosed with AR. İstanbul displayed the highest prevalence of asthma and AD, accounting for 78% and 37%, respectively, while Kayseri exhibited the lowest rates of asthma, AR, and FA, with 36%, 73%, and 1%, respectively. Thus Kayseri stood out among the five cities with the lowest prevalence of respiratory and FA symptoms (Table 1, Figure 2).

FIGURE 2.

Regional distribution of clinical presentation types across different parts of Türkiye.

3.3. Patients From Kayseri Show the Lowest Frequency of SPT Positivity to Most of the Respiratory Allergen Sources

In all centers, the highest sensitization rates among symptomatic patients, as determined by SPT, were observed for HDM and grass pollen allergen extracts (Table 1). The most frequent HDM sensitization rate was reported in Samsun (70%), followed by İstanbul (58%), İzmir (36%), Ankara (33%), and Kayseri (22%). Sensitization to grass pollen allergens varied from 42% in İzmir to 22% in Kayseri. Additional sensitization profiles based on SPT results are presented in Table 1. We noted that sensitization to certain pollen allergens varied: sensitization to mugwort pollen was most frequent in İzmir whereas ragweed sensitization was most common in İstanbul and Samsun (Table 1). By contrast, grass pollen‐specific sensitization was found in all five study centers.

3.4. Sensitizations to Defined Allergen Molecules

3.4.1. Sensitization Patterns in Symptomatic Versus Asymptomatic Individuals: Higher Prevalence, Levels, and Complexity of Sensitizations in Symptomatic Patients

The rate of symptomatic patients with any extract positivity on SPT was comparable to the rate of those sensitized to any AM (Table S8). When considering the differences between the symptomatic and asymptomatic groups in terms of any AM sensitizations (AMSs), including any outdoor or indoor AMs, the entire group of symptomatic individuals exhibited a higher prevalence compared with the entire group of asymptomatic individuals (69% vs. 42%, respectively) (Figure S1, Table S8, Figures 3 and 4). Furthermore, median specific‐IgE levels for pollen, cat and dog allergens but not for HDM allergens were higher in the symptomatic patients versus the asymptomatic subjects in all of the study centers (Table S1). Similar results were found when the IgE levels within the different centers were analyzed separately (Tables S2–S6). When we analyzed subjects with very high levels of sensitization (> 15 ISU) we found more sensitized patients in the symptomatic group and this difference was significant for all study centers except Kayseri (Figure S2). Furthermore, when comparing individuals with monosensitization, oligosensitization (2–4 non‐cross‐reactive AMs), or polysensitization (> 4 non‐cross‐reactive AMs) between the symptomatic and asymptomatic groups, higher rates of oligo‐ and polysensitization were consistently found for symptomatic individuals (Figure S3).

FIGURE 3.

Recognition frequency of outdoor aeroallergen molecules in individuals with and without symptoms of allergy based on regions (A: İstanbul, B: Ankara, C: İzmir, D: Kayseri, E: Samsun).

FIGURE 4.

Recognition frequency of indoor aeroallergen molecules in individuals with and without symptoms of allergy based on regions (A: İstanbul, B: Ankara, C: İzmir, D: Kayseri, E: Samsun).

Sensitization profiles and IgE levels of symptomatic individuals are presented in Table S9 according to comorbidities. The prevalence of HDM sensitization was strikingly higher than that of other allergen sensitizations among those with AD. Grass pollen, HDM, and cat emerged as the predominant sensitizations across all comorbidities (Table S9).

3.4.2. Comparative Analysis of Sensitization Rates to Allergen Molecules Across Centers: Lowest Prevalence Observed in Kayseri

AMS rates and specific IgE levels among individuals with and without allergic symptoms across the five cities are presented in Tables S2–S6. Among symptomatic individuals, sensitization to the grass pollen allergen Phl p 1 was most prevalent in İzmir, Kayseri, and Ankara, whereas sensitization to the HDM AM Der p 23 predominated in İstanbul and Samsun. Regarding overall AMSs, Kayseri had the lowest prevalence (42.5%) compared with İstanbul (63.5%), Samsun (66.5%), İzmir (53%), and Ankara (51%) (Table S8). Kayseri also exhibited the lowest proportion of symptomatic individuals with very high allergen‐specific levels (> 15 ISU) and the lowest frequency of polymolecular sensitization (> 4) (Figure S2−S3).

3.4.3. Outdoor Aeroallergens: Phl p 1 Was the Most Frequently Sensitizing Allergen in All Cities, Except Samsun, Where Amb a 1 Was the Leading Allergen

Timothy grass pollen, represented by its major AM Phl p 1, was the most commonly detected AMS in all cities except Samsun (Figure 3). The sensitization rates of Phl p 1 among symptomatic versus asymptomatic individuals were as follows: İzmir 40% versus 10%, Ankara 22% versus 13%, İstanbul 21% versus 17%, and Kayseri 16% versus 5%. Other Phleum pratense molecules—Phl p 3, 5, 2, and 6—followed Phl p 1 in frequency (Table S1). Phl p 1 and Phl p 5 are important respiratory allergens with strong clinical relevance among grass pollens. Phl p 1 sensitization levels were significantly higher in symptomatic than in asymptomatic individuals, whereas no significant difference was observed for Phl p 5, which might be attributable to the infrequent sensitization to this molecule in both groups (Table S1).

In Samsun, Amb a 1, the major ragweed allergen, was the most frequently recognized pollen allergen (21% symptomatic, 4% asymptomatic), with Phl p 1 being the second most prevalent. Notably, Amb a 1 was also the second most common outdoor aeroallergen in İstanbul (symptomatic: 15%; asymptomatic: 9%), similar to Samsun, which is partly located on the Black Sea coast. In contrast, Amb a 1 sensitization was very low in İzmir (2%) and undetectable among symptomatic individuals in Kayseri. Other weed allergens, such as the major mugwort allergen Art v 1, showed low IgE reactivity, being detected in 7% and 6% of symptomatic individuals in Kayseri and Ankara, respectively. The Parietaria major allergen Par j 2 was detected in 8% of symptomatic individuals in İzmir, but was rare (1%) in Ankara, Kayseri, and Samsun (Figure 3).

The highest sensitization rate to the major olive pollen allergen Ole e 1 was observed in İzmir, which is situated in an area with a Mediterranean climate, with 18% of symptomatic and 2% of asymptomatic individuals testing positive. The lowest rate was recorded in Kayseri (1% vs. 0%). Sensitization rates to the major birch pollen allergen Bet v 1 were very low across all study regions. Similarly, sensitization to the Alternaria, based on IgE recognition of Alt a 1, was rare, as was sensitization to mold allergens from Cladosporium (Figure 3).

3.4.4. Indoor Aeroallergens: HDM Sensitization Driven by Der p 23 Was Prevalent in Humid Regions, While Sensitization to the Major Cat Allergen Fel d 1 Frequent Across All Regions

Fel d 1, the major cat allergen, was the most frequently detected pet AM across all regions. Among symptomatic individuals, the highest positivity rate of Fel d 1 was observed in İzmir (25%), followed by Ankara (20%), İstanbul (17%), Samsun (14%), and Kayseri (13%). In contrast, sensitization to the dog allergen Can f 1 was consistently low, with rates of 5% among symptomatic individuals in İstanbul and Ankara, 4% in İzmir and Samsun, and 1% in Kayseri (Figure 4).

HDM sensitization rates tended to be higher in cities with greater annual average relative humidity, with Samsun, İstanbul, and İzmir exhibiting the highest rates, respectively (Table S7). Interestingly, although the inland cities of Ankara and Kayseri had similar relative humidity levels, Ankara exhibited a sensitization rate of 10%, which was twice as high as that of Kayseri (Figure 4). However, Kayseri had a higher altitude than Ankara and the minimal temperature was almost 10°C lower in Kayseri than in Ankara (Table S7). Sensitization to the major HDM molecules Der p 23, Der p 2, and Der p 1 was consistently observed across all centers, with Der p 23 positivity in the symptomatic group ranging from 57% to 2%. Cockroach sensitization, primarily associated with Bla g 7, was most frequently observed in İstanbul (6%). Mold allergen sensitization was generally low in all centers, with Asp f 3 detected only in İzmir and Samsun, each at 2% (Figure 4).

3.4.5. Food Allergens: Sensitizations and Genuine Reactivity Markers Were Infrequent

Food sensitization was consistently low in the whole study population (Figure 5, Table S1). IgE sensitizations to most food allergens, except for Ara h 1 (1.8%) and Ara h 9 (1%) were below 1% in the symptomatic subjects. IgE sensitization to tropomyosins, albumins and arginine kinases were likely due to cross‐reactivity with respiratory homologous allergens because IgE levels to the latter were higher than to the corresponding food allergens.

FIGURE 5.

Recognition frequency of food allergen molecules in individuals with and without symptoms of allergy based on regions (A: İstanbul, B: Ankara, C: İzmir, D: Kayseri, E: Samsun).

Sensitization to non‐specific lipid transfer proteins (nsLTPs), such as Pru p 3, and Ara h 9, showed a variable distribution across centers, with either no cases or a prevalence ranging from 1% to 3%. No sensitization to bovine serum albumin (Bos d 6) was detected among patients with FA. Positivity to Bos d 6 was identified in up to 3% of individuals in Ankara, İzmir, and Kayseri but seemed to be due to sensitization to albumin from respiratory allergen sources (e.g., Fel d 2, Can f 3). Sensitization to peanut allergens was low among patients from the five centers with Ara h 1 being the most frequently detected allergen (1.8% of patients) but specific IgE levels were low (i.e., 0.3 ISU). In contrast, no sensitization to Ara h 2 or Ara h 6 was detected in any patient with FA, suggesting a lack of clinical reactivity to peanut. However, IgE sensitization of the nsLTP from peanut, Ara h 9 was found in 1% of the symptomatic patients and 2.9% of food sensitized patients (Tables S1 and S9).

3.4.6. Ves v 5 Was the Most Frequently Detected Venom Allergen and IgE Sensitization to CCDs Was Frequent

The sensitization rates for other AMs are presented in Figure S4. Wasp (vespula vulgaris) sensitization was common in both symptomatic and asymptomatic individuals, primarily through reactivity to Ves v 5. The highest Ves v 5 positivity was observed in Ankara and Kayseri, followed by İzmir, Samsun, and İstanbul (symptomatic vs. asymptomatic: Kayseri = 2% vs. 12%; Ankara = 8% vs. 4%; İzmir = 6% vs. 2%; Samsun = 4% vs. 3%; İstanbul = 3% vs. 3%). Bee allergen sensitization was rare. Api m 1 positivity was detected in 2% of symptomatic individuals in İstanbul, while Api m 2 was observed in 1% of symptomatic individuals in İzmir and 1% of asymptomatic individuals in both İzmir and Kayseri. Sensitization to the latex allergen Hev b 8 was detected predominantly in symptomatic individuals (İzmir = 6%; Ankara = 4%; Kayseri = 4%; Samsun = 4%; İstanbul = 3%). No IgE sensitization to Ves v 1, Ves v 2, or Hev b 3 was detected in any participants. CCD positivity was detected in 14.4% of the entire symptomatic group and in 4.8% of the entire asymptomatic group.

4. Discussion

To the best of our knowledge our study is the first comprehensive, systematic, prospective and cross‐sectional study investigating molecular IgE sensitization profiles in five different regions of a country with varying climatic conditions. It is another special feature of our study that molecular IgE sensitization profiles were investigated in subjects with and without allergic symptoms who were born and grew up in the respective regions. We found that individuals with a documented history of allergic symptoms, identified through the ISAAC survey, exhibited a significantly higher prevalence, levels, and complexity of AMSs. Furthermore, notable disparities in both indoor and outdoor AMSs were observed among these individuals with a shared cultural background across the surveyed centers. Particularly noteworthy were the findings from Kayseri, the easternmost center in our study, characterized by a dry continental climate, cold winter temperatures, and high altitude. It exhibited the lowest rates, levels, and complexity of AMSs, setting it clearly apart from the other surveyed regions. Additionally, food sensitizations were rare and predominantly attributed to cross‐sensitization in Kayseri.

In this study, we focused on AMSs. Traditional and widely used allergy diagnosis methods are typically extract‐based, relying on allergen extracts that contain a mixture of various proteins/glycoproteins from the allergen source of interest. This approach has notable limitations. For example, non‐relevant allergenic proteins and CCDs may be included in the extract, resulting in false‐positive results [20, 21]. Additionally, certain AMs may be missed due to the inherent diversity within allergens, leading to false‐negative results. In contrast, allergen molecule‐based testing employs purified allergenic proteins from the allergen source, enabling a more precise and specific evaluation of a patient's sensitizations. This approach offers several key advantages: it delivers a heightened level of precision by identifying the specific AMs responsible for IgE sensitization; it enables the distinction between genuine sensitization and cross‐reactivity; and it may offer insights into the potential severity of the allergic response. The advent of allergen microarray technology and multi‐allergen testing with other platforms provides a groundbreaking opportunity to comprehensively document sensitizations to a wide panel of AMs in a single analysis, affording a comprehensive view of a patient's sensitization profile [7].

Furthermore, our study diverges from many prior studies in its study population, which consisted of a cohort of adults residing in five distinct regions with different climate and allergen exposome within a particular country. The objective was to select individuals who shared a common cultural background but were exposed to diverse geographical and climatic influences. To mitigate the potential impact of migration movements, stringent criteria were imposed, requiring that participants not only be born in the respective region but also continue to reside there without any significant periods of residence outside the region exceeding five years. The choice of adults with a comparable age as study subjects served two purposes. First, it allowed for the observation of the cumulative effects of various exposures on AMSs. Second, this approach enabled the examination of individuals who had reached a relatively stable phase in the allergic sensitization process, characterized by a plateau in their sensitization trajectory [22].

We assessed the presence of allergic disease symptoms using the well‐established questions of the ISAAC survey, which has been used in numerous previous studies [23]. Our study showed that individuals with symptoms had a higher prevalence, greater extent, and heightened complexity of molecular IgE sensitization. Additionally, 31% of symptomatic patients were non‐sensitized, likely reflecting false‐positive classification by the ISAAC questionnaire, which may be due to the fact that the ISAAC questionnaire also identifies subjects with allergy‐like symptoms without IgE sensitization. Notably, sensitizations identified in asymptomatic individuals may be attributed to several factors, including potential recall bias, the questionnaire's inherent limitations in discriminatory capabilities, distinctions between sensitization and clinical allergy, individual particularities, or possibly inconsequential sensitizations. For instance, there was a 14.4% prevalence of CCD positivity in the symptomatic group and a 4.8% prevalence in the asymptomatic group.

The most striking finding of this study was that AMSs exhibited lower frequency, less severity, and complexity in Kayseri than in Ankara another study center that shares geographical and climatic similarities. Kayseri's distinctiveness may stem from its easternmost location, higher altitude and lower winter temperature. As reported for other regions with similar climate conditions [24] and especially high altitude [25] we found a very low prevalence of HDM allergen‐specific IgE sensitizations while the sensitization profiles in these two centers were similar regarding grass and cat AMSs ranking as the top two, indicating the presence of similar encounters contributing to the development of sensitizations. Nevertheless, Kayseri consistently displayed markedly lower rates across a range of indoor, outdoor, food, and other AMS metrics, both in symptomatic and asymptomatic groups. The unique characteristics of Kayseri, such as its arid climate and high elevation, may be responsible for a dampening effect on pollination [26] in contrast to Ankara. Furthermore, being situated in the easternmost spectrum of the study centers, Kayseri may have experienced lesser influence from the allergy‐inducing factors associated with a Westernized lifestyle, which is mainly characterized by urbanization, reduced microbial exposure, and dietary shifts with increased consumption of processed and fast foods, decreased intake of fresh fruits, vegetables, and omega‐3 [27, 28, 29]. The differentiation in allergy prevalence between the Western and Eastern world is recognized as a complex matter influenced by numerous factors [30]. While caution is necessary in drawing broad conclusions, it is hypothesized that various key factors, including urbanization, stringent hygiene practices, dietary habits, lifestyle choices, air quality, and healthcare practices, may contribute to the higher prevalence of allergies in Western regions [1, 31, 32]. Ultimately, the relatively low prevalence of allergies in Kayseri may be attributed to its continental arid climate, elevated altitude, and less westernized lifestyle, although the latter is not completely documented within the scope of this study.

Besides the key finding that Kayseri is a hypoallergenic area in Türkiye additional remarkable findings were made. Ambrosia artemisiifolia (ragweed) is a major cause of seasonal asthma and AR in areas where it thrives [32, 33]. Recent reports have shown that northern Italy, certain parts of Türkiye, France, Romania, Ukraine, and parts of Russia have the highest pollen integrals [34, 35, 36]. In Türkiye, ragweed invasions have been detected along the Black Sea coast and in provinces neighboring Ankara, such as Bolu [34, 37]. Our study is consistent with these findings, as sensitization to the major ragweed allergen Amb a 1 was detected in two provinces bordering the Black Sea, Samsun, and İstanbul, as well as in Ankara, which is near Bolu. Previously, ragweed was not included in the country's allergen panel, but our study suggests that it is an emerging aeroallergen [38]. Moreover, in Samsun, located on the Black Sea coast, Amb a 1 was the dominant outdoor aeroallergen, surpassing Phl p 1, which was the most common AMS in the other study centers. Driven by climate change, ragweed is projected to expand its range in Europe [39]. Our results highlight the increasing importance of ragweed in public health planning and allergy management.

Timothy grass pollen ( Phleum pratense ) is widely recognized as the leading allergen source in both Europe and Türkiye [40, 41], and it holds a prominent position as the primary allergen in temperate climates [42]. Phl p 1, the hallmark of primary grass sensitization, and the other major allergens Phl p 2, 5, and 6 [43], emerged as the predominant AMs across all of the study centers. Notably, Phl p 1 exhibits substantial homology and cross‐reactivity with other group 1 grass pollen allergens, suggesting potential sensitization to various grass species [44]. Within the scope of our study, the lowest rates of Phleum sensitivity were observed in Kayseri and Samsun. The low prevalence in Kayseri can be attributed to its arid continental climate. Samsun, in contrast, was characterized by the highest relative humidity and, correspondingly, the highest levels of Dermatophagoides pteronyssinus sensitization. It is noteworthy that prior research has noted an inverse correlation between the prevalence of Dermatophagoides pteronyssinus and Phleum pratense allergies [45]. A possible explanation for this intriguing observation is the likelihood that individuals residing in regions with elevated humidity tend to spend more time indoors, potentially influencing their allergen sensitization patterns.

Discrepancies in olive pollen sensitization rates were also observed across the surveyed provinces. Olive trees are predominantly cultivated in the western and southern regions of Türkiye, which are characterized by a Mediterranean climate. Consistent with this geographical distribution, İzmir emerged as the center with the highest and most robust sensitization to the olive pollen AM, Ole e 1. The rate observed in our study was comparable to the Ole e 1 sensitization previously reported for İzmir in a molecular study of seasonal AR patients from Southern European countries (18% vs. 23%) [46]. Olive pollen is well‐documented for its heigh allergenicity and its strong association with asthma exacerbation [47]. Previous reports have indicated that Ole e 1 is the most frequently sensitized AM in olive pollen, followed by Ole e 2 [48]. In our study, individuals selectively sensitized to Ole e 2 represented a minority, accounting for 3.8% of symptomatic patients. The prevalence of olive sensitization in İstanbul can be attributed to the presence of limited olive tree growth in this region. Whereas Ole e 1 sensitizations in Ankara and Samsun may be associated with sensitization to Fraxinus pollen, specifically Fra e 1, which exhibits significant cross‐reactivity with Ole e1 [49].

HDMs are ubiquitous in regions with high humidity, which is a recognized pivotal factor in the development of HDM sensitization [50]. Correspondingly, our study revealed that provinces with high humidity levels, such as Samsun and İstanbul, exhibited the highest rates of HDM sensitization. Conversely, HDM sensitization was notably infrequent in Ankara and nearly absent in Kayseri. Furthermore, our findings highlight the prominence of Der p 23 as the primary HDM AM, alongside Der p 2 and Der p 1. Recent research has shown that the rate of Der p 23 monosensitization in HDM‐allergic patients ranges from 2% to 8% [51], and that Der p 23 has been confirmed as a clinically relevant major allergen [52, 53, 54]. Our data not only reaffirm the importance of Der p 23 in adults but also support recommendations advocating its inclusion in molecular analysis panels and immunotherapy protocols [55].

Cat sensitization, led by the major cat allergen Fel d 1, was widespread in all centers. Cat allergens are ubiquitous because of their small molecular size and ease of transfer [56]. A detection rate of 95% was observed for Fel d 1 in residences where cats were not present [57]. In addition, Heinrich et al. indicated that non‐cat owners are also at risk for high cat allergen exposure, in regions with a large population of cat owners [58]. Recent studies showed that cat ownership is increasing in Türkiye, especially in metropolises where rapid urbanization is experienced [59]. In Türkiye the urban population is composed of 77% of the country's population [60]. This increase in pet ownership in urban environments may reflect the evolving needs of urban residents. They may be increasingly seeking companionship, emotional comfort, and a higher quality of life to meet the challenges of urbanization. The fact that the rate of cat sensitization was higher, especially in the country's three largest metropolises, namely İstanbul, Ankara, and İzmir, compared with the other two provinces, may reflect that people living in these centers feel the above‐mentioned needs more and prefer cat companionship.

Numerous research findings have advanced our understanding of FAs. A recent review highlighted that adults in North‐central Europe have limited IgE recognition of food allergens, with sensitizations predominantly directed toward cross‐reactive PR‐10 proteins [61]. Bet v 1, a major trigger of PR‐10–related pollen–food allergy syndrome in Northern Europe [62], exhibited a low sensitization rate in our population. Given the highest sensitization rate and IgE level, Cor a 1 may represent a potential trigger of PR‐10 sensitization in our cohort, reflecting Türkiye's leading role in global hazelnut production and its high per capita consumption [63]. Conversely, Warren et al. reported a high prevalence of FAs among North American adults of 10.8% [64, 65]. A recent European study revealed varying rates of FAs, with the highest prevalence in Switzerland and Spain, and the lowest incidence in Eastern Europe and Greece [66].

In our study, IgE sensitization to peanut allergens in Türkiye was very rare like in certain other parts of Europe (e.g., Italy, Spain) [3]. Tropomyosin, serum albumin, and nsLTP emerged as prominent AMs potentially associated with food sensitizations. It is important to note that the documented sensitizations are likely not genuine, but cross‐sensitizations. Notably, the anticipated high nsLTP sensitization typically associated with Mediterranean regions was relatively low in our study. Sensitizations to serum albumin could potentially be linked to pet sensitizations [67, 68]. Although comprehensive national data are lacking, regional studies in Türkiye suggest that hen's egg, seafood, tree nuts, seeds, fruits, and vegetables are the most common food allergens in adults [69, 70], while hen's egg, milk, tree nuts, sesame, and peanut are most common in children [71, 72]. Despite hen's egg being the leading allergen in both groups, egg allergy was rarely observed in our cohort. A multicenter study on pollen–food allergy syndrome reported kiwi as the predominant allergen in İzmir and almond in İstanbul among adults [73]. According to the inclusion of the major fish allergen and the major shrimp allergen we assume that we did not miss subjects with sensitization to seafood. The inclusion of a complete panel of peanut allergens in our micro‐array ensures that true peanut sensitization is detected. However, previous studies using plant food allergen extracts containing CCDs may have overestimated sensitization to plant food. In fact, we found that 14.4% of the whole study population of symptomatic subjects showed IgE sensitization to the CCD marker. However, our panel of plant food allergens was not complete and we may therefore have missed patients with IgE sensitization to plant food allergens derived from nuts, fruits, vegetables and seeds. As previously hypothesized, the prevalence of FAs appears to be influenced by culinary traditions and exposure to cross‐reactive AMs, such as those originating from birch pollen [66]. Additionally, consistent with our findings, the rates of sensitization to Bet v 1 and PR‐10 food allergens were very low.

Our study found that the sensitization rate of Ves v 5, the primary allergen associated with wasp venom [74], was approximately 5% on average. Another molecular analysis study reported a combined detection rate of 10% for Api m 1 and Ves v 5 sensitization in the tested cohort of patients [75]. Molecular analysis plays a pivotal role in both diagnosis and quantification of the prevalence of venom allergy. Sturm et al. reported a noteworthy finding, indicating that 65% of patients with wasp allergy who did not have detectable extract‐specific IgE, displayed sensitization to Ves v 5 [76]. An independent study conducted in Germany revealed a significant association between the presence of Ves v 1 and/or Ves v 5 positivity and the manifestation of large local reactions in adults who had not experienced stings‐related anaphylaxis [77]. The variability in Ves v 5 sensitization prevalence among diverse populations underscores the need to acknowledge and carefully consider detected Ves v 5 positivity. It is paramount to recognize the significance of this finding, as it can cause unnecessary anxiety in individuals; however, it should be evaluated within the scope of clinical findings.

Considering the findings of previous reports from Türkiye, evaluating additional allergen sources not included in our panel can provide a better understanding of sensitization profiles in future studies. In a study conducted in Ankara among allergic adult patients, sensitization to storage mites, including Acarus siro, Lepidoglyphus destructor, and Tyrophagus putrescentiae, was reported in 21%–23% of cases [78]. Low sensitization rates to the storage mite Blo t allergens observed in our cohort may reflect limited cross‐sensitization between Blo t allergens and other allergens from storage mite species or house dust mites [79, 80]. Another study from Ankara reported sensitization to Blattella germanica in 11.9% and to Periplaneta americana in 7.4% of allergic children, with only 30% of cockroach‐sensitized subjects co‐sensitized to both cockroach species [81]. Due to the IgE cross‐reactivity of certain allergens from cockroach and house dust mite [82], the molecular allergen micro‐arrays including cockroach and mite allergen molecules might be of interest. Additionally, Amaranthaceae pollen, particularly from Amaranthus and Chenopodium species, may constitute an important allergenic source in Türkiye, with central regions showing the highest sensitization rates [83], presenting a possible allergen sources for future studies on regional sensitization patterns.

Our study's strengths lie in its comprehensive characterization of AMS profiles and regional disparities through a cross‐sectional assessment of a large and comparable cohort, marking the first such standardized endeavor. Subsequent investigations that employ regional analyses through molecular dissection could map susceptibility patterns on a global scale. Despite its strengths, our study has certain limitations. We did not assess various factors that could influence the severity and variety of sensitizations, such as housing conditions, dietary habits, exposure to air pollution, and smoking. As a result, our findings are based on descriptive statistics, making it difficult to establish relationships between sensitization patterns and these unexamined factors. However, it is important to note that the primary focus of our study was to identify regional disparities among individuals with and without allergic symptoms. Another limitation is the lack of contemporaneous data on regional pollen calendars and counts across the five participating cities, which could have provided important context regarding variations in pollen exposure. However, data from previous studies are available for three of the centers (İstanbul and İzmir [84]; Ankara [85]), but not for Samsun or Kayseri. In these three centers, the reported pollen profiles are consistent with the observed sensitization profiles, with Poaceae, Ambrosia, Olea, and Artemisia being the most frequently detected aeroallergens. The array used in the study may potentially lack some molecular allergens and exhibit low analytic sensitivity in individuals with low total IgE values. This potential limitation could account for the absence of AMS in a small subset of symptomatic individuals. The exclusive inclusion of adults may have affected the assessment of some important FAs such as milk and hen's egg, which are clinically relevant early in life and for most patients natural remission is frequently achieved before adulthood. Lastly, there may be concerns about our recruitment approach, as participants were recruited from a single center within each city, potentially reflecting the population of that specific center. However, it is essential to clarify that these centers were the recognized referral centers in their respective regions, and the sensitization profiles observed in the study aligned with the expected regional distinctions of these centers.

In conclusion, our findings highlight substantial disparities in sensitization profiles and the prevalence of AMSs in individuals with the same cultural background, even within regions with similar geographical and climatic characteristics. A comprehensive understanding of these discrepancies and the resultant exposomal influences is of significant importance in the development of preventive and therapeutic strategies for allergic diseases.

Author Contributions

The Allergochip working group: Maria‐Roxana Buzan, Kuan‐Wei Chen, Monica Daniela Cotarcă, Alexandra Dubovets, Pia Gattinger, Alexander Karaulov, Evgenii Kozlov, Carmen Panaitescu, Thomas Schlederer, Daria Trifonova, Mikhail Tulaev, Susanne Vrtala, Milena Weber, Lauriana‐Eunice Zbîrcea expressed, purified and characterized weed pollen, pet and house dust mite allergens and prepared the micro‐array and detection reagents used in this study. The Allergochip working group was involved in revising the manuscript critically. Rudolf Valenta and Huey‐Jy Huang were involved in data interpretation, design of the study, writing, reviewing and revising the manuscript. Bülent Enis Şekerel contributed to the study design, led and supervised the fieldwork, and was involved in drafting, visualizing, critically reviewing, writing, and revising the manuscript. Alp Kazancioglu coordinated data collection, performed molecular IgE testing and formal analysis, prepared the original draft, and handled figure preparation and data visualization. Ali Fuat Kalyoncu, İsmet Bulut, Okan Gulbahar, Murat Turk, and Sengul Aksakal contributed to data collection and revision of the manuscript. All authors approved the final version of the manuscript.

Ethics Statement

Ethical approval for this multicenter study was obtained from the Institutional Review Board of Hacettepe University Faculty of Medicine (GO22/794). The analysis of allergen micro‐arrays was performed at the Medical University of Vienna with the permission of the Ethics Committee of the Medical University of Vienna (EK1641/2014).

Conflicts of Interest

Rudolf Valenta received research grants from HVD Biotech, Vienna, Austria; Worg Pharmaceuticals, Hangzhou, China; HVD Biotech, Vienna, Austria; and Viravaxx, Vienna, Austria. Rudolf Valenta serves as a consultant for HVD. The other authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The authors with a Russian affiliation declare that they have prepared the article in their “personal capacity” and/or that they are employed at an academic/research institution where research or education is the primary function of the entity.

Supporting information

Appendix S1: all70145‐sup‐0001‐AppendixS1.pdf.

Kazancioglu A., Huang H.‐J., Aksakal S., et al., “Molecular IgE Reactivity Profiling With Micro‐Arrayed Allergens Reveals Distinct Interregional Patterns of Sensitization and a Hypoallergenic Region in Türkiye,” Allergy 81, no. 4 (2026): 1247–1264, 10.1111/all.70145.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.