Multiple Allergen Simultaneous Test for Food Allergens Cannot Screen Wheat-Dependent, Exercise-Induced Anaphylaxis and α-Gal Syndrome

Abstract

Screening tests for specific immunoglobulin E (sIgE) to food allergens, such as the multiple allergen simultaneous test (MAST), are widely used in patients with suspected food allergies in South Korea. We evaluated whether MAST could effectively screen wheat-dependent exercise-induced anaphylaxis (WDEIA) and α-gal syndrome (AGS). We retrospectively reviewed patients with WDEIA and AGS diagnosed with unequivocal history and positive sIgE results for omega-5 gliadin and α-gal using ImmunoCAP, respectively. The clinical manifestations and results of MAST and ImmunoCAP (sIgE to wheat for WDEIA and beef/pork for AGS) were reviewed. In the MAST and ImmunoCAP results, class 0 (<0.35 units in each test) was considered negative. Medical records of 45 patients with WDEIA and 39 patients with AGS were reviewed. For WDEIA, 37 (82.2%) of patients had a history of anaphylaxis. Among those positive for omega-5 gliadin sIgE, 39 (87.7%) and 25 (55.6%) tested positive for gluten- and wheat-sIgE using ImmunoCAP, respectively. MAST performed on 15 patients yielded positive results for wheat-sIgE in 5 (33.3%). For AGS, 23 (59.0%) of patients had a history of anaphylaxis. Among those positive for α-gal sIgE, 32 (85.7%) and 37 (96.4%) tested positive for pork- and beef-sIgE using ImmunoCAP, respectively, whereas MAST could not detect sIgE for pork and beef (0%, 0/17). MAST for sIgE to food allergens cannot screen WDEIA and AGS. The tests for sIgE to a specific component of food allergen, such as omega-5 gliadin for WDEIA and α-gal for AGS, should be used to screen WDEIA and AGS.

Graphical Abstract

Anaphylaxis is a potentially life-threatening allergic emergency. Its incidence has been on the rise, and the anaphylaxis induced by new triggers has emerged.¹ The increase in peanut allergies was first recognized in the 1990s.¹,² The incidence of anaphylaxis by galactose-alpha-1,3-galactose (α-gal), a novel antigen widely distributed in non-primate mammalian tissues, referred to as α-gal syndrome (AGS) or red meat allergy, has been steadily increasing³ after its initial report as cetuximab anaphylaxis in 2008.⁴ Identifying and avoiding the causative agent through a thorough review of medical history based on the temporal relationship that anaphylactic symptoms start shortly after exposure to the triggers and the repeatability of symptoms following exposure to the specific antigen is crucial to prevent anaphylaxis.⁵ In addition, testing for specific immunoglobulin E (IgE) for suspected antigens is also useful. Skin prick tests and multiple allergen simultaneous tests (MAST) have been used to screen specific IgE to multiple allergens. MAST has been widely used in several countries, including South Korea, since it is a simple blood test that is not affected by current medications.

Food is one of the most common causes of anaphylaxis,⁶ and some food allergies exhibit atypical presentations in temporal relationships and repeatability due to specific triggers. Wheat-dependent exercise-induced anaphylaxis (WDEIA) and AGS are some examples. WDEIA typically requires triggering stimuli, such as physical exertion, after the consumption of wheat flour. Without such stimuli, symptoms may not manifest, even after wheat consumption. AGS is characterized by a delayed onset of 6–8 hours after exposure, and individuals may react to various foods having α-gal, including pork, beef, and meat broth. To diagnose WDEIA and AGS, tests for the IgE against specific allergen components are used: omega-5 gliadin of wheat for WDEIA⁷ and α-gal of meat for AGS.⁸ However, data on whether MAST effectively screens for WDEIA and AGS is lacking. For AGS, data regarding IgE test results for meat-related allergies is limited, and for WDEIA, the detection rate of specific IgE for wheat has been inconsistent.⁷,⁹

Therefore, it is necessary to investigate whether MAST can effectively screen for WDEIA and AGS, especially given the recent increase in food allergies and anaphylaxis. This study evaluated the detection rates for food-specific IgE using the MAST and ImmunoCAP assay for those with WDEIA and AGS.

We reviewed the medical records of patients with WDEIA and AGS who visited the Kangwon National University Hospital allergy clinic between January 2014 and September 2023. WDEIA and AGS were diagnosed by an allergist with an unequivocal clinical history and positive results for specific IgE against omega-5 gliadin and α-gal, respectively, using the ImmunoCAP assay.

Demographic data, symptom duration, and anaphylaxis history were reviewed. In evaluating the unequivocal clinical history for WDEIA and AGS, along with adequate temporal relationships and reproducibility by specific triggers, cases with only skin symptoms were included considering anaphylaxis risk if skin symptoms were judged to be typical type I reactions.⁵,¹⁰ However, cases without typical skin manifestations or any possibility of shock from other diseases and anaphylaxis by other triggers were excluded. A history of anaphylaxis was determined when there were typical skin symptoms and severe dyspnea or symptoms suggesting low blood pressure that required emergency room visits or similar levels.⁵ For MAST, the AdvanSure AlloScreen (LG Chem, Seoul, Korea) was used to test for 66 common allergens. The results of the ImmunoCAP (Thermo Fisher Scientific, Waltham, MA, USA) assay on wheat and gluten for patients with WDEIA, and pork and beef for patients with AGS were reviewed. In both tests, the results were divided into seven classes (0–6) according to the concentration range: class 0 (<0.35 IU/mL in the MAST and <0.35 kU/L in the ImmunoCAP assay) was considered negative. The titer of specific IgE measured by ImmunoCAP, >100 and <0.10, were regarded as 100 and 0, respectively. For disease duration, cases <1 month were regarded as 1 month. Three patients with AGS and one with WDEIA had insufficient data on disease onset and duration. Mann–Whitney U test was used to compare the serum levels of specific IgE according to anaphylaxis history. Statistical analyses (Mann–Whitney U, Spearman, or chi-square tests) were performed using SPSS version 26.0 software (IBM Corp., Armonk, NY, USA). p-values <0.05 were considered statistically significant. The study protocol was approved by the hospital’s Institutional Review Board (IRB number: KNUH-2023-01-013).

The medical records of 45 patients with WDEIA were reviewed retrospectively. Table 1 shows the demographic and clinical characteristics along with the results of each assay. Most of the patients were male 29 (64.4%). The median age (range) and disease duration were 44 (17–79) years and 16.5 (1–240) months, respectively. A total of 37 (82.2%) of patients had a history of anaphylaxis. In the ImmunoCAP assay (Table 1), 25 (55.6%) of patients had positive results for wheat-specific IgE and 39 (87.7%) for gluten-specific IgE. Regarding symptom severity, the serum levels of specific IgE for omega-5 gliadin, gluten, and wheat were significantly higher in patients with WDEIA with a history of anaphylaxis than in those with only urticaria (Fig. 1A), and the difference was still significant, excluding one outlier (91.60 kU/L) in specific IgE for omega-5 gliadin (p=0.006). Analysis after transforming the crude values into log values provided the same results. MAST was performed in only 15 patients, and 5 (33.3%) had positive results with a median titer of 0.80 (0.35–4.68) IU/mL, which corresponded to classes 1 (0.35–0.69 IU/mL) to 3 (3.50–17.49 IU/mL).

Table 1. Demographic and Clinical Characteristics of Patients with WDEIA and AGS.

Characteristics			Patients with WDEIA (n=45)	Patients with AGS (n=39)
Age, yr			44.5±18.0	65.5±9.5
Male			29 (64.4)	19 (48.7)
History of anaphylaxis			37 (82.2)	23 (59.0)
Duration			(n=44)	(n=36)
	1 month		9 (20.5)	8 (22.2)
	1–12 months		11 (25.0)	10 (27.8)
	1–5 years		14 (31.8)	7 (19.4)
	>5 years		10 (22.7)	11 (30.6)
Positivity of specific IgE tests
	ImmunoCAP assay		(n=45)	(n=39)
		Omega-5 gliadin specific IgE	45 (100)*	-
		Gluten-specific IgE	39 (87.7)	-
		Wheat-specific IgE	25 (55.6)	-
		α-gal specific IgE	-	39 (100)
		Pork-specific IgE	-	32 (85.7)
		Beef-specific IgE	-	37 (96.4)
	MAST		(n=15)	(n=17)
		Wheat-specific IgE	5 (33.3)	-
		Pork-specific IgE	-	0 (0)
		Beef-specific IgE	-	0 (0)

WDEIA, wheat-dependent exercise-induced anaphylaxis; AGS, α-gal syndrome; MAST, multiple allergen simultaneous test; IgE, immunoglobulin E.

Data are presented as mean±standard deviation or n (%).

^*Positive results were defined as ≥class 1 in each tests.

Fig. 1. Comparison of the serum level of specific IgE among patients with anaphylaxis and those with urticaria. (A) Specific IgE antibodies for omega-5 gliadin, wheat, and gluten among patients with WDEIA. (B) Specific IgE antibodies for α-gal among patients with α-gal syndrome. IgE, immunoglobulin E; WDEIA, wheat-dependent exercise-induced anaphylaxis.

A total of 39 patients with AGS were enrolled in this study (Table 1). The patients’ median (range) age was 66 (36–84) years, and 19 (48.7%) were male. A total of 23 (59.0%) of patients with AGS had anaphylaxis history. The serum levels of specific IgE for α-gal, pork, and beef did not differ between patients with AGS with and without anaphylaxis history (Fig. 1B). Although specific IgE for pork 32 (85.7%) and beef 37 (96.4%), as measured by ImmunoCAP, showed high positive rates, MAST showed negative results (0%, 0/17) for specific IgE for pork and beef (Table 1).

Several foods trigger food-dependent exercise-induced anaphylaxis (FDEIA). Detection of food allergen-specific IgE has been recommended as a screening test for FDEIA, and is also included in the diagnostic criteria.¹¹,¹²,¹³ Although it is the most common FDEIA, evidence to support screening tests for wheat-specific IgE for WDEIA has been limited, and there have been conflicting data. A previous study reported that the challenge-confirmedsensitivity of skin-prick tests with gluten was 100% (n=16) in patients with WDEIA, 94% for skin-prick tests with wheat flour, and 50% for skin-prick tests with wheat extract.⁹ A low detection rate of wheat allergen-specific IgE in patients with WDEIA was observed in a study of 50 Japanese patients with positive wheat/exercise challenges; the sensitivities of wheat- and gluten-specific IgE testing using the ImmunoCAP assay were 48% and 56%, respectively.⁷ The current study clearly showed that wheat-specific IgE is not well-detected in MAST and ImmunoCAP among patients with WDEIA.

Recent reports indicate that AGS is increasing in the US, and more may be undiagnosed.³ α-gal is not a typical peptide antigen but a carbohydrate antigen that arises through post-transcriptional changes involving carboxylation of proteins.¹⁴ Since it is widely distributed in non-primate mammals, including beef, pork, and lamb, these foods could elicit allergic reactions among patients with AGS. In addition, since the distribution of α-gal may vary across organs, symptom occurrence may depend on the consumption of different parts—meat, intestines, or appendages—and the quantity ingested. Consequently, diagnosing AGS solely based on medical history can be challenging. Moreover, AGS is characterized by a delayed onset after exposure, hypothesized to be attributed to the delayed absorption of fatty tissues where a significant distribution of α-gal is observed.¹⁴,¹⁵ These difficulties underscore the need for a reliable screening test for these patients. AGS diagnosis is based on clinical history and the presence of specific IgE for α-gal. If the antibodies are ≥2 IU/mL or >2% of the total IgE, diagnosis is very likely.⁸ Reports about the results of specific IgE for food allergens, such as pork and beef, in AGS are limited. Skin prick testing with commercial whole-meat extracts often does not provide reliable results.¹⁶ This study showed that MAST is not useful for screening for AGS, and that specific IgE for α-gal is needed for AGS diagnosis and differential diagnosis for cat-pork syndrome.¹⁷

Since this study included patients from a single institution’s allergy clinic, it may not represent the epidemiology of their respective conditions. In this study, the patients with AGS were older than those with WDEIA. Both conditions showed a high rate of anaphylaxis (82.2% of patients with WDEIA and 59% of patients with AGS); therefore, considerable attention and patient education regarding anaphylaxis are needed in clinical practice. The serum level of specific IgE for omega-5-gliadin was significantly high in patients with WDEIA who had a history of anaphylaxis compared to those without anaphylaxis. This finding may serve as a reference for assessing symptom severity. Conversely, the current study demonstrated that serum levels of α-gal specific IgE are unrelated to the severity of AGS symptoms, consistent with previous findings.⁸ Some ticks, including lone star ticks (Amblyomma americanum) in the US, have been confirmed as culprits for AGS.¹⁸ In South Korea, there is a similar species called ‘Ixodes nipponensis.’¹⁹ However, although not extensively confirmed in this study, there were more individuals who reported no tick bites than those with a history of tick bites.

Component-resolved diagnostics (CRD) in food allergies is used to characterize the molecular components of food allergen involved in a specific IgE-mediated response. CRD can improve diagnostic accuracy and predict the causal relationship between specific IgE and symptoms.²⁰ This study highlighted the necessity of CRD to screen for WDEIA and AGS. However, detailed history taking remains crucial, as only approximately 80% of WDEIA cases tested positive for specific IgE for omega-5-gliadin.⁷

One limitation of this study was its small sample size. However, as WDEIA and AGS are uncommon, the number of enrolled patients with each disease in this study was the highest among studies related to WDEIA or AGS in South Korea. Moreover, the current study is sufficient to demonstrate that MAST for specific IgE to foods cannot be used to screen for WDEIA or AGS. Additionally, provocation tests were not performed to confirm the diagnosis of AGS or WDEIA. In this retrospective study, we included only patients with clear histories and positive results for each CRD test, applying strict exclusion and enrollment criteria. Provocation tests for AGS and WDEIA are seldom necessary in clinical practice, as most patients are diagnosed based on their medical history and CRD test results. Diagnosis is confirmed by observing the patient’s clinical response to a diet that excludes red meat or wheat.

Given the increased prevalence of food allergies and atypical manifestations with high anaphylaxis rates, the establishment of accurate screening tests is crucial. This study showed that MAST is inadequate for WDEIA and AGS screening, underscoring the necessity of CRD for each condition. The lack of awareness regarding the potential false negatives associated with MAST for AGS and WDEIA could lead to confusion in identifying the causes of food allergies. It is crucial to prevent overuse and overconfidence in MAST and to highlight the importance of clinical history and CRD as screening tests for patients with a suspected history of AGS, WDEIA, or anaphylaxis of unknown cause.