Diagnostic therapeutic care pathway for pediatric food allergies and intolerances in Italy: a joint position paper by the Italian Society for Pediatric Gastroenterology Hepatology and Nutrition (SIGENP) and the Italian Society for Pediatric Allergy and Immunology (SIAIP).

Epidemiologic data suggest an increased prevalence of pediatric food allergies and intolerances (FAIs) during the last decades. This changing scenario has led to an increase in the overall healthcare costs, due to a growing demand for diagnostic and treatment services. There is the need to establish Evidence-based practices for diagnostic and therapeutic intervention that could  be adopted in the context of public health policies for FAIs are needed.This joint position paper has been prepared by a group of experts in pediatric gastroenterology, allergy and nutrition from the Italian Society for Pediatric Gastroenterology Hepatology and Nutrition (SIGENP) and the Italian Society for Pediatric Allergy and Immunology (SIAIP). The paper is focused on the Diagnostic Therapeutic Care Pathway (DTCP) for pediatric FAIs in Italy.

A comprehensive analysis of the components of common weed pollen and related allergens in patients with allergic diseases in southern China.

BACKGROUND: Pollen allergens are vital contributors to allergic diseases. The frequency and coreactivity pattern of allergens are closely related to geographical distribution. OBJECTIVE: In this study, we aimed to characterize the prevalence of the molecular components of the common weed pollen allergens, birch pollen, walnut, and cross-reactive carbohydrate determinant (CCD), as well as investigate the relationship between the allergens and CCD in Chinese pollen-sensitized patients with allergic diseases. METHODS: Based on previous vegetation surveys, serum samples from 165 pollen-sensitized patients with allergic diseases in Guangdong Province in southern China were used to test 19 crude allergen extracts, their components, and CCD using component-resolved diagnosis (CRD). Moreover, the potential associations among CCD, allergens, and their components were described. RESULTS: In the 165 samples, the most common sensitized allergens were goosefoot (43.0%), ragweed (40.6%), walnut (37.6%), walnut tree (37.6%), and mugwort (37.0%), followed by platane (35.2%), cocklebur (27.9%), and birch (24.2%). The positivity rate of CCD was 39.4%. Among the samples positive for mugwort, 11 (18.0%), 15 (24.6%), and 15 (24.6%) were positive for Art v 1, Art v 2, and Art v 3, respectively. Among the 67 patients sensitized to ragweed, only five (7.5%) were positive for Amb a 1. In the 40 patients sensitized to birch, Bet v 2 had the highest positivity rate (40.0%). There were 62 patients who were sensitized to walnut. Their components had a lower positivity rate (less than 15%). The hierarchical clustering and optimal scale analysis showed that Art v 4 and Bet v 2 were closely related, and 91.9% of CCD-positive samples were polysensitized. Meanwhile, Spearman's rank correlation method showed that CCD was closely correlated with the sensitization of crude allergen extracts, and there was a low correlation between CCD and allergen components. CCD was highly correlated with goosefoot, ragweed, and walnut trees (r＞0.8). Moreover, there was a strong relationship between the levels of Jug r 3 and Art v 3 (r = 0.78; P < 0.001). CONCLUSIONS: In southern China, the weed pollens (ragweed, cocklebur, and goosefoot) exhibited higher positivity rates in adults and had a stronger relationship with CCD but not with mugwort. The positivity rate of allergen components was not high. CCD-positive samples were always polysensitized.

Unraveling the Diagnosis of Kiwifruit Allergy: Usefulness of Current Diagnostic Tests.

OBJECTIVES: To determine the usefulness of the in vitro and in vivo methods used in the diagnosis of kiwifruit allergy and to specifically assess the impact of seed proteins on sensitivity. METHODS: We performed skin prick tests (SPTs) using various commercial extracts, homemade pulp, and seed extracts and prick-prick tests with kiwifruit on 36 allergic patients. The presence of specific IgE (sIgE) was assessed using the ImmunoCAP (kiwifruit extract), ELISA (Act d 1, Act d 2), ISAC, and FABER assays. Immunoblotting of seed extract was carried out, and a single-blind oral food challenge was performed with whole seeds in seed-sensitized individuals. RESULTS: The prick prick test with kiwifruit demonstrated the highest diagnostic capacity (81.8% sensitivity and 94.1% specificity) among the in vivo tests. The sIgE levels measured using ImmunoCAP (kiwifruit extract) showed a similar sensitivity to that of global ISAC and FABER (63.9%, 59.5%, and 58.3%, respectively). Act d 1 was the major allergen. Sensitization to Act d 1 was associated with positive sIgE results to whole kiwifruit extract detected by ImmunoCAP (P<.000). A positive SPT result to kiwifruit seeds was associated with severe symptoms induced by kiwifruit (P=.019) as a marker of advanced disease, but not with clinically relevant sensitization. Challenge testing with kiwifruit seeds performed on 8 seed-sensitized patients yielded negative results. CONCLUSION: Sensitization to Act d 1 is associated with a positive result in conventional diagnostic techniques, whereas kiwifruit seed sensitization does not increase the sensitivity of the diagnostic techniques evaluated.

Development of a sensitive and stable chemiluminescent immunoassay for detection of birch pollen allergic specific IgE based on recombinant Bet v1 protein.

Background Quantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein. Methods rBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system. Results The developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results. Conclusions The proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity.

Hazelnut Allergy.

Background and Objectives: Hazelnuts are frequently involved in IgE-mediated reactions and represent the main culprit of nut allergy in Europe. The clinical presentation varies from mild symptoms limited to the oropharynx [oral allergy syndrome (OAS)], due to the cross-reaction with homologues in pollen allergens and more severe events caused by the primary sensitization to highly stable molecules contained in hazelnuts. The aim of this review is to summarize the most relevant concepts in the field of hazelnut allergy and to provide a practical approach useful in the clinical practice Materials and Methods: References were identified by PubMed searches dating from January 2000 up to November 2020 using the search terms: "component resolved diagnosis" and "Hazelnut allergy. Results: The storage proteins Cor a 9 and Cor a 14 resulted highly specific for primary hazelnut allergy and strongly associated with severe reactions, while the cross reactive Cor a 1, an homolog of the birch Bet v1, were related to OAS. Any cut-off has shown a specificity and sensitivity pattern as high as to replace the oral food challenge (OFC), which still remains the gold standard in the diagnosis of hazelnut allergy. To date there is still no definitive treatment. Hazelnut free-diet and treatment of symptoms with emergency management, including the prescription of auto-injective epinephrine, still represent the main approach. Oral allergen immunotherapy (AIT) appears a promising therapeutic strategy and the definition of individual clinical threshold would be useful for sensitized individuals, caregivers, and physicians to reduce social limitation, anxiety, and better manage food allergy. Conclusions: An accurate diagnostic work-up including clinical history, in vivo and in vitro test including component resolved diagnosis and OFC are essential to confirm the diagnosis, to assess the risk of a severe reaction, and to prescribe an adequate diet and treatment.

Component resolved diagnosis by recombinant allergens in patients with allergies to inhalants.

Molecular characterization of IgE reactivity of specific individual components of allergenic extracts is now possible due to the technology of recombinant allergens derived from studies of molecular biology of allergic pathology. The identification of the immunoreactivity to single allergenic components in allergic subjects allows to specifically define her/his allergic profile and obtain the so-termed Component Resolved Diagnosis (CRD). Molecular allergens can be classified into those that induce the respiratory allergic reactivity and those that identify the food-related allergic pathology. It is also essential to identify those molecular allergens whose immunoreactivity is able to connect the two clinical conditions: respiratory symptoms and food allergy symptoms. The present study was conducted on 50 patients with a clinical history of hypersensitivity to pollen and/or allergy and positivity to Skin Prick Test. The sera were analyzed in our laboratories and the panel of recombinant allergens was applied in the case of positivity of the specific IgE. Of the 50 patients enrolled, 31 were selected as positive to 4 main pan-allergen Bet v1, Par j2, Art v1 and Phl p1; among these, 14 subjects showed one allergen-specific IgE towards natural extracts of tested foods even in absence of clinical history. CRD allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in: a) resolving genuine vs cross-reactive sensitization in poly-sensitized patients, b) assessing, in selected cases, the risk of severe, systemic vs mild, local reactions in food allergy, and c) identifying patients and triggering allergens for specific immunotherapy (ITS). In light of our results, we believe that the transition from a diagnostic based on the use of allergenic extracts to another one based on the use of single allergenic molecules that is able to define the specific allergenic profile of each patient, seems to be able to revolutionize the allergy diagnosis.

Component-resolved diagnostics can be useful for identifying hazelnut allergy in Japanese children.

BACKGROUND: Cor a 9 and Cor a 14 are effective markers for predicting hazelnut allergy. However, there have been no reports on the component-resolved diagnostics (CRD) of hazelnut allergy using an oral food challenge (OFC) for diagnosis in Asia. We hypothesized that CRD would improve the accuracy of diagnosing hazelnut allergies in Japanese children. METHODS: We recruited 91 subjects (median age: 7.3 years) who were sensitized to hazelnuts and had performed a hazelnut OFC at the National Hospital Organization Sagamihara National Hospital between 2006 and 2017. All subjects were classified as allergic or asymptomatic to 3 g of hazelnuts. The sIgE levels (hazelnut/Cor a 1/Cor a 8/Cor a 9/Cor a 14/alder pollen) were measured using ImmunoCAP. We aimed to determine the predictive factors of hazelnut allergy. RESULTS: Nine subjects (10%) were allergic to ≤3 g of hazelnuts. Levels of sIgE for Cor a 9 in hazelnut-allergic subjects were significantly higher than those in asymptomatic subjects (4.47 vs. 0.76 kUA/L, p = 0.039). Levels of sIgE to alder pollen and Cor a 1 in hazelnut-allergic subjects were significantly lower than those in asymptomatic subjects (<0.10 vs 13.0 kUA/L, p = 0.004; <0.10 vs 5.03 kUA/L, p = 0.025). The area under the receiver operating characteristics curve for hazelnut/alder/Cor a 1/Cor a 9 was 0.55/0.78/0.72/0.71, respectively, with p = 0.651/0.006/0.029/0.040, respectively. CONCLUSIONS: The findings of a high sIgE level for Cor a 9 and a low sIgE level for Cor a 1 can improve the diagnostic accuracy to better identify Japanese children sensitized to hazelnuts.

Allergen Recognition Patterns in Walnut Allergy Are Age Dependent and Correlate with the Severity of Allergic Reactions.

BACKGROUND: Walnut is an important elicitor of food allergy in children and adults with a high rate of severe reactions. Multicenter studies using a common clinical protocol and a comprehensive allergen are lacking. OBJECTIVE: To investigate potential correlations between molecular sensitization patterns and clinical characteristics of walnut-allergic patients. METHODS: A total of 91 walnut-allergic subjects and 24 tolerant controls from Switzerland, Germany, and Spain were included. Walnut allergy was established by food challenge in all but anaphylactic subjects. Specific IgE (sIgE) to walnut extract, rJug r 1 (2S albumin), rJug r 3 (nonspecific lipid transfer protein 1), nJug r 4 (11S globulin), rJug r 5 (PR-10 protein), 2 vicilin fractions, profiling, and cross-reactive carbohydrate determinant was determined by ImmunoCAP. A threshold of 0.10 kUA/L was used for positivity. RESULTS: Sensitivity of sIgE to walnut extract was 87% and increased to 96% for the sum of all walnut components. sIgE to walnut extract and all walnut components, except rJug r 5, was significantly higher in patients younger than 14 years at inclusion. Stratification by age at onset of walnut allergy led to similar results. All patients younger than 14 years had severe reactions, whereas 38% of patients 14 years or older were mild reactors. Severe reactors (n = 70) had higher sIgE levels than did mild reactors (n = 21) to walnut extract (P < .0001), rJug r 1 (P < .0001), nJug r 4 (P = .0003), and both vicilin fractions (P < .0001), but not to Jug r 3 and Jug r 5. CONCLUSIONS: Sensitization to walnut storage proteins is acquired in childhood and correlates with severe reactions. sIgE levels to storage proteins Jug r 1 and Jug r 4 and vicilin fractions, but not to nonspecific lipid transfer protein and PR-10 proteins, correlate with systemic reactions to walnut.

Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

BACKGROUND: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity. OBJECTIVES: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE. METHODS: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes. RESULTS: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2. CONCLUSION: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Artemisia pollen allergy in China: Component-resolved diagnosis reveals allergic asthma patients have significant multiple allergen sensitization.

BACKGROUND: Artemisia pollen allergy is a major cause of asthma in Northern China. Possible associations between IgE responses to Artemisia allergen components and clinical phenotypes have not yet been evaluated. This study was to establish sensitization patterns of four Artemisia allergens and possible associations with demographic characteristics and clinical phenotypes in three areas of China. METHODS: Two hundred and forty patients allergic to Artemisia pollen were examined, 178 from Shanxi and 30 from Shandong Provinces in Northern China, and 32 from Yunnan Province in Southwestern China. Allergic asthma, rhinitis, conjunctivitis, and eczema symptoms were diagnosed. All patients' sera were tested by ImmunoCAP with mugwort pollen extract and the natural components nArt v 1, nArt ar 2, nArt v 3, and nArt an 7. RESULTS: The frequency of sensitization and the IgE levels of the four components in Artemisia allergic patients from Southwestern China were significantly lower than in those from the North. Art v 1 and Art an 7 were the most frequently recognized allergens (84% and 87%, respectively), followed by Art v 3 (66%) and Art ar 2 (48%). Patients from Northern China were more likely to have allergic asthma (50%) than patients from Southwestern China (3%), and being sensitized to more than two allergens increased the risk of allergic asthma, in which co-sensitization to three major allergens Art v 1, Art v 3, and Art an 7 is prominent. CONCLUSIONS: Component-resolved diagnosis of Chinese Artemisia pollen-allergic patients helps assess the potential risk of mugwort-associated allergic asthma.

Food-dependent, exercise-induced anaphylaxis in a patient allergic to peach.

Determining the single factor that triggered anaphylactic shock can be challenging. We present an interesting case of a 25-year-old female patient with recurrent anaphylactic reactions developing after eating various foods, particularly in presence of co-factors of allergic reactions. Symptoms occurred after consumption of various kinds of foods - peach, pancakes with cottage cheese and fruit, a meal from a Chinese restaurant - all eaten on other occasions without symptoms. During diagnosis, skin prick tests were negative for all tested allergen extracts (both inhalatory and food) from Allergopharma. Prick by prick tests were positive for the peach - wheal diameter - 6 mm, nectarine - 4 mm (histamine 4 mm, negative control 0 mm). Increased levels of asIgE were found for allergens of peach (0.55 kU/L).Open challenge test with one mid-size peach combined with the physical exercise challenge test was positive. ImmunoCAP ISAC test indicated increased levels of IgE specific for the lipid transfer protein (LTP) for walnut (nJug r 3), peach (Pru p 3), wheat (rTri a 14) and plane tree (rPla a 3). The patient was diagnosed with food-dependent, exercise-induced anaphylaxis associated with an allergy to lipid transport proteins (LTPs).

How molecular diagnosis may modify immunotherapy prescription in multi-sensitized pollen-allergic children.

INTRODUCTION: Specific immunotherapy (SIT) is used to treat asthma and allergic rhinitis, and a dose-response relationship has been found for SIT efficacy, creating a need to accurately select the allergen used in therapy. This need is especially pronounced in poly-sensitized children living in areas where different pollen allergen sources coexist in the same season, as this circumstance complicates diagnostic efforts. In such cases, component-resolved diagnosis (CRD) can increase diagnostic accuracy and aid in SIT prescription. MATERIALS AND METHODS: We hypothesized that CRD results would lead to modifications in classical immunotherapy prescription based on sources such as medical history, season of symptom presentation, and skin testing. We studied a sample of children indicated for immunotherapy in whom classical methods had not pointed out the most relevant allergen due to sensitization to more than two pollens. We used a small panel of recombinant allergens, analyzing the percentage of changes to prescription considering the findings of molecular studies. RESULTS: Of the 70 children included, CRD led to modified immunotherapy prescription in 54.3%. Indications of single-allergen therapy increased from 18% to 51% when CRD was included. The decision to prescribe immunotherapy was reversed following CRD in 9.3% of cases. DISCUSSION: CRD use alters the choice of specific immunotherapy in poly-sensitized children. A wide panel of recombinant allergens may not be necessary to improve immunotherapy indication using molecular techniques; rather, a smaller panel adapted to include those allergens prevalent in the geographical area in question appears to be sufficient for more effective immunotherapy, also leading to an improved cost-benefit ratio.

Allergen component analysis as a tool in the diagnosis and management of occupational allergy.

We are now in the epoch of "molecular allergology" and numerous clinically relevant allergenic molecules are available improving the performance of in vitro allergen tests and allergen detection methods. This review is focusing on characterized occupational allergens and their implementation into the in vitro diagnosis for occupational allergy and in allergen detection methods. More than 400 occupational agents are identified and documented as being 'respiratory sensitizers', but currently only a limited number of them are characterized on the molecular level and available for routine diagnosis as native or recombinant allergens. One exception, however, is natural rubber latex (NRL) from Hevea brasiliensis still remaining an important occupational allergen source. Characterization of 15 NRL allergens led to the development of assays for the determination of allergen content of NRL materials and the implementation of component-resolved diagnosis (CRD) for specific IgE antibody measurement. Microarray or singleplex using recombinant or native allergens are reliable tools for NRL allergy diagnosis. In addition, NRL allergy is an excellent model for improving extract-based specific IgE measurement by amplification of NRL extract preparation with stable recombinant major allergen rHev b 5. Despite the many efforts to characterize the occupationally relevant wheat allergens for baker's asthma, the most frequently occurring forms of occupational asthma, the results are highly diverse. Wheat sensitization profiles of bakers showed great interindividual variability and no wheat allergen could be classified as the major allergen. For diagnosis of baker's asthma, a whole wheat extract is still the best option for specific IgE determination. But single wheat allergens might help to discriminate between wheat-induced food allergy, grass pollen allergy and baker's asthma. For workplace-related allergens like coffee, wood, soybean, seafood and moulds allergens are characterized and few of them are available, but their relevance for occupational sensitization routes should be verified in the further studies.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Is eosinophilic esophagitis an equivalent of pollen allergic asthma? Analysis of biopsies and therapy guided by component resolved diagnosis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by esophageal dysfunction and, histologically, by eosinophilic inflammation. There is not a clear etiologic treatment. Biopsies analysis using plant histology methods may show callose and pollen tubes in the esophageal mucosa. Component-resolved diagnosis (CRD) with microarrays could detect possible allergens involved and indicate an elimination diet and allergen immunotherapy (AIT). METHODS: One hundred and twenty-nine patients with EoE were tested for environmental and food allergens. CRD, histological and botanical analysis were performed. Clinical scores and endoscopic biopsy were performed every six months for three years. Fifty healthy patients, 50 asthmatics due to pollen, and 53 celiac disease patients were included as comparison groups. CRD-directed AIT was administered in 91 EoE patients and elimination diet in 140 patients (87 EoE and all 53 CD patients). RESULTS: CRD detected allergen hypersensitivity in 87.6% of patients with EoE. The predominant allergens were grass group 1 (55%), lipid transfer proteins (LTP) of peach and mugwort, hazelnuts and walnuts. Callose from pollen tubes was found in 65.6% of biopsies. After CRD-guided elimination diet and/or AIT, 101 (78.3%) EoE patients showed significant clinical improvement (p<0.017) and 97 (75.2%) were discharged (negative biopsy, no symptoms, no medication) without relapse. AIT-treated patients had better outcomes (odds ratio 177.3, 95% CI 16.2-1939.0). CONCLUSION: CRD-directed AIT and/or elimination diet was efficient in treating EoE patients and was well tolerated.

Concomitant sensitization to legumin, Fag e 2 and Fag e 5 predicts buckwheat allergy.

BACKGROUND: Buckwheat (Fagopyrum esculentum) has become increasingly popular as a healthy food in Europe. However, for sensitized individuals, consumption can cause anaphylactic reactions. The aim of this study was to identify individual well-characterized buckwheat allergens for component-resolved diagnosis. METHODS: Patients were selected by positive skin prick test to buckwheat and divided into two groups: (1) sensitized to buckwheat without clinical symptoms and (2) buckwheat allergy. Buckwheat proteins were extracted from raw buckwheat seeds, purified applying a combination of protein precipitation and chromatographic methods, and analyzed by IgE immunoblotting and ELISA. RESULTS: Buckwheat-allergic patients had a significantly larger median skin prick test weal diameter for buckwheat than the sensitized group and the positive control. Also, IgE immunoblotting clearly showed a distinct pattern in sera from allergic patients when compared to sensitized individuals. Several IgE-reactive proteins were purified from crude buckwheat extract, namely legumin (Fag e 1 plus its large subunit), Fag e 2 (2S albumin), and newly identified Fag e 5 (vicilin-like) as well as hevein-like antimicrobial peptides, designated Fag e 4. All four allergens showed superior diagnostic precision compared to extract-based ImmunoCAP with high sensitivity as well as high specificity. CONCLUSIONS: Patients with clinical symptoms clearly show a distinct allergen recognition pattern. We characterized a buckwheat vicilin-like protein as a new relevant marker allergen, designated Fag e 5. Additionally, another new allergen, Fag e 4, potentially important for cross-reactivity to latex was added to the allergen panel of buckwheat. Further, our data show that the full-length legumin comprising both, large and small subunit should be applied for component-resolved diagnosis. Our data indicate that concomitant sensitization to legumin, Fag e 2 and Fag e 5, predicts buckwheat allergy.

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during in vitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. OBJECTIVE: We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. METHODS: IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). RESULTS: Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r = 0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n = 15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r = 0.61) and to all blank ImmunoCAPs (r > 0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. CONCLUSIONS: Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

Greater Real-Life Diagnostic Efficacy of Allergen Molecule-Based Diagnosis for Prescription of Immunotherapy in an Area with Multiple Pollen Exposure.

BACKGROUND: Allergen molecule-based diagnosis has been suggested to facilitate the identification of disease-causing allergen sources and the prescription of allergen-specific immunotherapy (AIT). The aim of the current study was to compare allergen molecule-based IgE serology with allergen extract-based skin testing for the identification of the disease-causing allergen sources. The study was conducted in an area where patients are exposed to pollen from multiple sources (trees, grasses, and weeds) at the same time to compare the diagnostic efficiency of the 2 forms of diagnosis. METHODS: Patients from Astana, Kazakhstan, who suffered from pollen-induced allergy (n = 95) were subjected to skin prick testing (SPT) with a local panel of tree pollen, grass pollen, and weed pollen allergen extracts and IgE antibodies specific for marker allergen molecules (nArt v 1, nArt v 3, rAmb a 1, rPhl p 1, rPhl p 5, rBet v 1) were measured by ImmunoCAP. Direct and indirect costs for diagnosis based on SPT and marker allergen-based IgE serology as well as direct costs for immunotherapy depending on SPT and serological test results were calculated. RESULTS: The costs for SPT-based diagnosis per patient were lower than the costs for allergen molecule-based IgE serology. However, allergen molecule-based serology was more precise in detecting the disease-causing allergen sources. A lower number of immunotherapy treatments (n = 119) was needed according to molecular diagnosis as compared to extract-based diagnosis (n = 275), which considerably reduced the total costs for diagnosis and for a 3-year treatment from EUR 1,112.30 to 521.77 per patient. CONCLUSIONS: The results from this real-life study show that SPT is less expensive than allergen molecule-based diagnostic testing, but molecular diagnosis allowed more precise prescription of immunotherapy which substantially reduced treatment costs and combined costs for diagnosis and treatment.

Possible effect of landscape design on IgE recognition profiles of two generations revealed with micro-arrayed allergens.

The aim of this study was to investigate possible effects of landscape design on the IgE sensitization profile toward inhalant allergens in patients with respiratory allergy from Uzbekistan where green areas have been changed during the last two decades by a State program. Sera from two different generations of Uzbek (n=58) and, for control purposes, from two generations of Austrian (n=58) patients were analyzed for IgE reactivity to 112 different micro-arrayed allergen molecules by ImmunoCAP ISAC technology. Changes in molecular IgE sensitization profiles to pollen allergens in the young vs the middle-aged Uzbek population were associated with replanting, whereas those in the Vienna populations reflected natural changes in plant growth. Our data indicate that anthropologic as well as natural changes in the biome may have effects on IgE sensitization profiles already from one to another generation.