Diagnostics and monitoring in Hymenoptera venom allergy: current and future perspectives.

Low sensitivity of commercially available rApi m 1 for diagnosis of honeybee venom allergy

Detection of IgE to recombinant Api m 1 and rVes v 5 is valuable but not sufficient to distinguish bee from wasp venom allergy

BackgroundA longstanding debate in allergy is whether or not specific immunoglobulin-E antibodies (sIgE), recognizing cross-reactive carbohydrate determinants (CCD), are able to elicit clinical symptoms. In pollen and food allergy, ≥20% of patients display in-vitro CCD reactivity based on presence of α1,3-fucose and/or β1,2-xylose residues on N-glycans of plant (xylose/fucose) and insect (fucose) glycoproteins. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE).Methodology/Principal FindingsTomato and/or potato plants with stable reduction of Lyc e 2 (tomato) or CCD formation in general were obtained via RNA interference, and gene-silencing was confirmed by immunoblot analyses. Two different CCD-positive patient groups were compared: one with tomato and/or potato food allergy and another with hymenoptera-venom allergy (the latter to distinguish between CCD- and peptide-specific reactions in the food-allergic group). Non-allergic and CCD-negative food-allergic patients served as controls for immunoblot, basophil activation, and ImmunoCAP analyses. Basophil activation tests (BAT) revealed that Lyc e 2 is no key player among other tomato (glyco)allergens. CCD-positive patients showed decreased (re)activity with CCD-reduced foodstuff, most obvious in the hymenoptera venom-allergic but less in the food-allergic group, suggesting that in-vivo reactivity is primarily based on peptide- and not CCD-sIgE. Peptide epitopes remained unaffected in CCD-reduced plants, because CCD-negative patient sera showed reactivity similar to wild-type. In-house-made ImmunoCAPs, applied to investigate feasibility in routine diagnosis, confirmed BAT results at the sIgE level.Conclusions/SignificanceCCD-positive hymenoptera venom-allergic patients (control group) showed basophil activation despite no allergic symptoms towards tomato and potato. Therefore, this proof-of-principle study demonstrates feasibility of CCD-reduced foodstuff to minimize ‘false-positive results’ in routine serum tests. Despite confirming low clinical relevance of CCD antibodies, we identified one patient with ambiguous in-vitro results, indicating need for further component-resolved diagnosis.

The composition of venom extracts, cross-reactive carbohydrate determinants (CCD) and the component-resolved diagnostics (CRD) are important fields of investigation. IgE-reactivity to CCD complicates the interpretation of IgE to Hymenoptera venoms, especially in patients with multiple-positivity. We analyzed the clinical importance of CRD and CCD-inhibition for selection of allergens for venom immunotherapy (VIT). In 71 patients, we measured specific IgE (sIgE) to honeybee venom (HBV), wasp venom (WV), hornet venom (HV), CCD, and recombinant allergens: phospholipase A2 (rApi m 1), hyaluronidase (rApi m 2), icarapin (rApi m 10), antigen 5 (rVes v 5), and phospholipase A1 (Immunoblot). In 29/71 HBV/WV/HV/CCD-positive patients CCD-inhibition was performed. According to CRD and CCD-inhibition, we identified true sensitization and defined groups of multiple-positive patients who needed CCD-inhibition before starting VIT. sIgE-rApi m 1, sIgE-rApi m 2, and sIgE-rApi m 10 were detected in 65.7%, 68.4%, and 58%, respectively. In HBV allergic patients, CRD sensitivity was 86.8%. In WV allergic patients, sensitivity of sIgE-rVes v 5 was 94%. True multiple-sensitization was found in 44.8% of HBV/WV/HV/CCD-positive patients after CCD-inhibition. Patients with multiple venom- and CCD-positivity had more frequent severe allergic reactions (p<0.001). CCD-inhibition was helpful in HBV/WV/HV/CCD-positive patients who were negative to all tested recombinant honeybee allergens. Persistence of HBV-positivity after CCD-inhibition requires CRD to other honeybee recombinant allergens. CRD, using a profile of five most important recombinant allergens and CCD, has a high sensitivity for the diagnosis of venom allergy, especially in patients positive to several venom extracts. CRD and CCD-inhibition are helpful to reveal the clinically relevant, true sensitization and improve the selection of venoms for long-lasting VIT.

Safety of COVID-19 vaccination in patients with clonal mast cell disorders

Background: In hymenoptera venom allergy, about 75% of detected in vitro double positivity to yellow jacket and honeybee venom is ascribed to specific IgE (sIgE) directed against cross-reactive carbohydrate determinants (CCDs). To date, for the detection of CCD-sIgE, different carbohydrate antigens and methods are used. The most suitable one still has to be identified. Methods: Eighty-seven patients with confirmed hymenoptera venom allergy and venom sIgE values of ≧0.7 kU/l were investigated. Sixty-five patients showed sIgE reactivity to both yellow jacket and honeybee venom, 22 were venom mono positive and served as controls. Occurrence of CCD-sIgE was determined using bromelain, horseradish peroxidase (HRP) and MUXF³ on system A, and ascorbic acid oxidase (AAO), bromelain and HRP on system B. Further, a reference standard for CCD-sIgE evaluation was created: CCD positivity was assumed when at least 4 of the 6 test results were positive. Results: According to the defined reference standard, 45/65 venom double positive patients exhibited CCD-sIgE. Using system A, comparison with the reference standard revealed sensitivity and specificity values of 96 and 97%, respectively, for MUXF³, 100 and 100%, respectively, for bromelain, and 96 and 97%, respectively, for HRP. Using system B, sensitivity and specificity was 98 and 97%, respectively, for AAO, 62 and 95%, respectively, for bromelain, and 96 and 69%, respectively, for HRP. Results of the 3 test allergens obtained with system A showed strong correlations (r = 0.932–0.976), whereas results with system B showed lower correlations (r = 0.714–0.898). Conclusions: All 3 test allergens used with system A are suitable for CCD-sIgE detection in hymenoptera venom allergy. With system B, only AAO seems to be a reliable tool.

Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylation

BackgroundIt is hypothesized that because of higher mast cell numbers and mediator release, mastocytosis predisposes patients for systemic immediate-type hypersensitivity reactions to certain drugs including non-steroidal anti-inflammatory drugs (NSAID).ObjectiveTo clarify whether patients with NSAID hypersensitivity show increased basal serum tryptase levels as sign for underlying mast cell disease.MethodsAs part of our allergy work-up, basal serum tryptase levels were determined in all patients with a diagnosis of NSAID hypersensitivity and the severity of the reaction was graded. Patients with confirmed IgE-mediated hymenoptera venom allergy served as a comparison group.ResultsOut of 284 patients with NSAID hypersensitivity, 26 were identified with basal serum tryptase > 10.0 ng/mL (9.2%). In contrast, significantly (P = .004) more hymenoptera venom allergic patients had elevated tryptase > 10.0 ng/mL (83 out of 484; 17.1%). Basal tryptase > 20.0 ng/mL was indicative for severe anaphylaxis only in venom allergic subjects (29 patients; 4x grade 2 and 25x grade 3 anaphylaxis), but not in NSAID hypersensitive patients (6 patients; 4x grade 1, 2x grade 2).ConclusionsIn contrast to hymenoptera venom allergy, NSAID hypersensitivity do not seem to be associated with elevated basal serum tryptase levels and levels > 20 ng/mL were not related to increased severity of the clinical reaction. This suggests that mastocytosis patients may be treated with NSAID without special precautions.

Anaphylactic Reactions After Discontinuation of Hymenoptera Venom Immunotherapy: A Clonal Mast Cell Disorder Should Be Suspected

Mastocytosis and/or elevated basal serum tryptase may be associated with severe anaphylaxis. To analyse Hymenoptera venom-allergic patients with regard to basal tryptase in relation to the severity of sting reactions and the safety and efficacy of venom immunotherapy. Basal serum tryptase was measured in 259 Hymenoptera venom-allergic patients (158 honey bee, 101 Vespula). In 161 of these (104 honey bee, 57 Vespula), a sting challenge was performed during venom immunotherapy. Nineteen of the 259 patients had an elevated basal serum tryptase. Evidence of cutaneous mastocytosis as documented by skin biopsy was present in 3 of 16 patients (18.8%). There was a clear correlation of basal serum tryptase to the grade of the initial allergic reaction (P<0.0005). Forty-one of the 161 sting challenged patients reacted to the challenge, 34 to a bee sting and 7 to a Vespula sting. Thereof, 10 had an elevated basal serum tryptase, i.e. 1 (2.9%) of the reacting and 2 (2.9%) of the non-reacting bee venom (BV) allergic individuals, as compared to 3 (42.9%) of the reacting and 4 (8%) of the non-reacting Vespula venom-allergic patients. Thus, there was a significant association between a reaction to the sting challenge and an elevated basal serum tryptase in Vespula (chi2=6.926, P<0.01), but not in BV-allergic patients. Systemic allergic side-effects to venom immunotherapy were observed in 13.9% of patients with normal and in 10% of those with elevated basal serum tryptase. An elevated basal serum tryptase as well as mastocytosis are risk factors for severe or even fatal shock reactions to Hymenoptera stings. Although the efficacy of venom immunotherapy in these patients is slightly reduced, most of them can be treated successfully. Based on currently available data, lifelong treatment has to be discussed in this situation.

Component resolution reveals additional major allergens in patients with honeybee venom allergy

Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy.

Spiking venom with rVes v 5 improves sensitivity of IgE detection in patients with allergy to Vespula venom

Hymenoptera venom allergy is the most common cause of anaphylaxis in adults and the second-most frequent in children. The proper diagnosis of this life-threatening allergy remains a challenge. This review focuses on the current knowledge regarding diagnostics of Hymenoptera venom allergy. The paper includes a brief description of the representatives of Hymenoptera order and the composition of their venoms. Then, diagnostic tests for allergy to Hymenoptera venom are described. Common diagnostic problems, especially double positivity in tests for IgE antibodies specific to honeybee and wasp venom, are also discussed. Special attention is paid to the search for new diagnostic capabilities using modern methodologies. Multidimensional molecular analysis offers an opportunity to characterize changes in body fluids associated with Hymenoptera venom allergy and yields a unique insight into the cell status. Despite recent developments in the diagnostics of Hymenoptera venom allergy, new testing methodologies are still needed to answer questions and doubts we have.

Introduction and objectives The diagnosis of baker’s asthma as part of health surveillance schemes in some UK supermarkets relies on determining sensitisation to wheat flour and/or alpha amylase. Recently, data have emerged suggesting that serum IgE analysis in bakers may be complicated by the presence of clinically irrelevant specific IgE to cross-reactive carbohydrate determinants (CCDs), which are complex-type Asn (N)-linked glycan structures commonly formed in plants. Potentially this might lead to false positive flour specific IgE assays, which would have an impact on bakers undergoing surveillance. The aim of this study was to identify the prevalence of CCD sensitisation in UK bakers and investigate the impact of CCD specific IgE within a health surveillance setting. Methods Serum samples from UK bakers attending our occupational asthma clinic (n = 209) were analysed for specific IgE to CCD (MUXF 3 ) using ImmunoCAP assay (Phadia). Any positive samples were further tested for specific IgE to grass pollen, and competitive inhibition assays were used to determine cross-reactivity between CCD, flour and grass pollen. Results Sensitisation rates to CCD in our population of UK bakers were low (7%) despite high sensitisation rates to grass pollen (48%) and flour (60%). Sensitisation to CCD was more prevalent in those sensitised to either flour or grass than in those not sensitised to flour (11.5% vs 0%, p Conclusions Our study demonstrated that a minority of bakers were sensitised to CCD and, interestingly, this was associated with being co-sensitised with both flour and grass. It is unlikely that CCDs have major implications for the health surveillance for UK bakeries. In the minority of bakers with CCD specific IgE, there was some suggestion that CCDs may play a role in the cross-reactivity between flour and grass pollen, although in others it was less likely. Within the clinical setting, it may be prudent to measure CCD specific IgE in bakers who are co-sensitised to both flour and grass pollen.