Original Article
The Value of Specific IgE to Peanut and Its Component Ara h 2 in the Diagnosis of Peanut Allergy Lucila C. Lopes de Oliveira, MDa,b, Martina Aderholda, Marianne Brill, PhDc, Gabriele Schulza, Claudia Rolinck-Werninghaus, MDa, E.N. Clare Mills, PhDd, Bodo Niggemann, MDa,e, Charles Kirov Naspitz, MDb, Ulrich Wahn, MDa, and Kirsten Beyer, MDa Berlin, Germany; São Paulo, Brazil; Vienna, Austria; and Norwich, United Kingdom
What is already known about this topic? Studies have suggested peanut-specific IgE above defined cutoff levels or sensitization to Ara h 2 can predict clinical relevant peanut allergy and is helpful in avoidance of unnecessary oral food challenges. Allergen-specific IgG and IgG4 are also detectable in healthy individuals and do not play a role in peanut allergy diagnostic. What does this article add to our knowledge? Analysis of our data emphasizes that patients with high levels of peanutspecific or Ara h 2especific IgE can still be clinical tolerant. Our data confirm that peanut-specific IgG and IgG4 levels are comparable in individual who are allergic and in tolerant individuals. How does this study impact current management guidelines? Interpretation of in vitro test results should be done with care; neither proposed cutoff levels for peanut-specific IgE nor component-resolved diagnostics can replace an oral food challenge for all patients with suspected peanut allergy. Measurement of peanut-specific IgG or IgG4 should not be done in the diagnostic workup for suspected peanut allergy.
BACKGROUND: To avoid unnecessary oral food challenges, which are time consuming, stressful, and risky, improved in vitro diagnostic methods for food allergy such as component resolved diagnostics are still under investigation. OBJECTIVE: To investigate the role of whole peanut- and peanut-component (Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 8)especific IgE levels in the diagnostic procedure of peanut allergy as well as the diagnostic properties of peanut-specific IgG and IgG4. METHODS: Sixty-one children underwent oral peanut challenge tests for diagnostic purposes irrespective of their peanut-specific IgE levels. Peanut-specific serum IgE, IgG, and
IgG4 levels were determined by ImmunoCAP FEIA and specific IgE against individual peanut proteins by Immuno Solid-phase Allergen Chip. RESULTS: Thirty-four of 61 patients (56%) had a peanut allergy. No significant difference was observed for peanutspecific IgG or peanut-specific IgG4 levels between patients who were allergic and tolerant patients, whereas peanut-specific IgE was significant higher in patients who were allergic than in tolerant patients (P < .005). Twenty-five of 61 children had peanut-specific IgE above a previously proposed cutoff level of 15 kUA/L; however, 7 of these 25 children (28%) were clinically tolerant. Ara h 2-specific IgE was significantly lower in tolerant
a
Institute of Food Research; has received lecture fees from Imperial College; owns stock in Standard Life; and has received travel support from ILSI, EAACI, the University of Bologna, and the British High Commission. K. Beyer has received research support from the EU, the German Research Foundation, the Danone Foundation for the Treatment of Peanut Allergy; has received consultancy fees from Danone, Novartis, ALK, Meda Pharma, and Unilever; and has received lecture fees from Danone, Phadia, Infectopharm, CSL Behring, Novartis, UCB, Meda Pharma, Med Update, Allergopharma, and Thermo Fischer. The rest of the authors declare that they have no relevant conflicts. Received for publication March 31, 2013; revised May 10, 2013; accepted for publication May 11, 2013. Cite this article as: Lopes de Oliveira LC, Aderholz M, Brill M, Schulz G, RolinckWerninghaus C, Mills ENC, et al. The value of specific IgE to peanut and its component Ara h 2 in the diagnosis of peanut allergy. J Allergy Clin Immunol: In Practice 2013;1:394-8. http://dx.doi.org/10.1016/j.jaip.2013.05.010. Corresponding author: Kirsten Beyer, MD, Department of Paediatric Pneumology and Immunology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail: [email protected]. 2213-2198/$36.00 Ó 2013 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaip.2013.05.010
Department of Pediatric Pneumology and Immunology, Charité Universitätsmedizin Berlin, Berlin, Germany b Division of Allergy and Clinical Immunology, Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil c VBC Genomics, Vienna, Austria d Institute of Food Research, Norwich, United Kingdom e Pediatric Allergology and Pneumology, German Red Cross Hospital Westend, Berlin, Germany This work was funded in part within the collaborative research initiative EuroPrevall, an integrated project funded by the European Commission under the 6th Framework Programme (FOOD-CT-2005-514000), and in part by the German Research Foundation DFGeBE3991/1-1. Conflicts of interest: M. Aderhold has received grants from the European Union and the German Research Foundation. M. Brill has received research and travel support from EuroPrevall. C. Mills has received research support from the European Commission, the Biological and Biotechnological Sciences Research Council, the UK Food Standards Agency, DBV Technologies, the UK Technology Strategy Board, and the European Food Safety Authority; is on the Novartis Board; is a member of the UK Food Standards Agency Advisory Committee on Novel Foods and Processes; has received consultancy fees from Pepsico International; is employed by the University of Manchester and the
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Abbreviations used Ig- Immunoglobulin ISAC- Immuno Solid-phase Allergen Chip
than in patients with allergies (P < .0001). Interestingly, 94% of the patients with peanut allergies showed IgE-binding to Ara h 2. Unfortunately, 26% of the sensitized but tolerant patients have shown IgE binding to Ara h 2 too. CONCLUSIONS: Neither the level of specific IgE to peanut nor to Ara h 2 was able to clearly distinguish patients with clinical relevant peanut allergy from those who were clinical tolerant in our population. As expected, peanut-specific IgG and IgG4 did not improve the diagnostic procedure. Ó 2013 American Academy of Allergy, Asthma & Immunology (J Allergy Clin Immunol: In Practice 2013;1:394-8) Key words: IgE; IgG; IgG4; Peanut allergy; Immuno Solid-phase Allergen Chip; Component-resolved diagnostics; Ara h 1; Ara h 2; Ara h 3; Ara h 6; Ara h 8
Peanut allergy is a common disease, and its prevalence appears to be increasing in many countries.1-3 Resolution of peanut allergy is uncommon, and children with a peanut allergy tend to have their disease life-long.2 Importantly, accidental ingestion often results in anaphylactic reactions, and fatal cases have been reported.4,5 The diagnosis of peanut allergy results in a tremendous change in life. Strict dietary restrictions are necessary, and the patients need to avoid a large variety of foods that contain peanuts, including products with precautionary labeling. Because patients with a peanut allergy tend to have severe reactions after accidental ingestion,2 the prescription of epinephrine self-injectors is common. These aspects associated with the fear of a possible anaphylactic reaction are related to impairment on quality of life.6-8 Therefore, the diagnosis of peanut allergy results in serious consequences for the patients and their families. Immunologic parameters, which correlate completely to the clinical manifestation of peanut allergy have not been identified yet. It has been shown that patients with allergies (symptomatic) have higher levels of peanut-specific IgE when compared with asymptomatic ones.6 Because oral food challenges for diagnostic purposes are time consuming, stressful, and not exempt of risks, other diagnostic tools have been studied. Sampson6 observed that a peanut-specific IgE higher than 15 kUA/L was able to predict 95% of the patients with allergy and suggested this peanutspecific IgE cutoff level as an alternative diagnostic procedure. Later on, the same results were observed by Clark and Ewan.7 It has been speculated that food allergenespecific IgG and IgG4 may also be helpful to define the clinical reactivity. It has been observed in oral immunotherapy studies that peanutspecific IgG4 increased in parallel with the development of oral tolerance.8,9 However, in hen’s egg allergy, the level of hen’s eggespecific IgG4 did not correlate with the clinical reactivity10 and the usage of food allergenespecific IgG4 is not recommended as a diagnostic test in food allergy.11,12 More recently, the usage of a component-resolved diagnostic has been evaluated. Especially, measurement of specific IgE to Ara h 2 appears to be of great value. Codreanu et al13 were able to show that 79% of the patients who were pollen sensitized showed IgE binding to peanut, despite their tolerance to
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peanut.13 In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/L.13 IgE to Ara h 2 seems to improve specificity in detecting patients with allergy among peanut-sensitized ones.14,15 Similarly, Nicolaou et al16 showed that the usage of Ara h 2especific IgE for the correct diagnosis of peanut allergy was very helpful. In this population, a sensitivity of 100% and a specificity of 96% were found when using a cutoff value of 0.35 kU/L for Ara h 2especific IgE.16 The aim of the present study was to investigate the role of peanut-specific IgE, IgG, and IgG4 levels in the diagnostic procedure of peanut allergy and determine the accuracy of a proposed diagnostic decision point of 15kUA/L in our population. In parallel, the usefulness of specific IgE antibody determination to individual peanut proteins was investigated in a well-defined population.
METHODS Study population Sixty-one infants and children referred to the Charité Allergy Center, a tertiary level clinic, for evaluation of suspected IgEmediated peanut allergy were included into the study. Ages ranged from 0.9 to 13.6 years, with a median age of 4.1 years. Sixty-two percent of the patients were boys and 46% had a diagnosis of atopic eczema. The study was approved by the local ethical committee of the University Children’s Hospital of Charité, Universitätsmedizin Berlin. Oral food challenges Oral food challenges were performed with homogenized roasted peanut kernels. In detail, 30 mg, 60 mg, 130 mg, 250 mg, 500 mg, 1 g, and 2 g of peanut protein were given orally every 30 minutes. After the challenge was completed, the patient remained under clinical supervision for at least 2 hours. In case of clinical tolerance, the patient received a subsequent dose of roasted peanuts in a total amount of 4 g of peanut protein another day. Full emergency equipment and medications were at hand during all the procedures. The use of antihistamine was suspended 72 hours before the challenge. The food challenges were scored as positive if objective clinical reactions were noted, such as urticaria, angioedema, vomiting, diarrhea, wheezing, stridor, rhino conjunctivitis, or a drop in blood pressure. Blood sampling Venous blood samples of all patients were collected before oral provocation tests and obtained serum probes were aliquoted and stored at 20 C until further analysis. Fluorescence enzyme immunoassays Determination of peanut-specific IgE, IgG, and IgG4 was done by fluorescence enzyme immunoassay by using ImmunoCAP 250 System (Phadia, Uppsala, Sweden). Specific IgE titers were quantified in protein units designated as kilounits of antibody per liter (KUA/L) with a lower detection limit of 0.35 KUA/L and an upper limit of 100 KUA/L as proposed by the manufacturer. Detection limits for specific IgG and IgG4 were 15 to >100 kU/L)
6/7 (86) 7/8 (88) 17/17 (100)
4/7 (57) 8/8 (100) 17/17 (100)
2/7 (29) 6/8 (75) 17/17 (100)
2/12 (17) 1/8 (13) 4/7 (57)
4/12 (33) 6/8 (75) 6/7 (86)
5/12 (42) 5/8 (63) 5/7 (71)
perform, because precautionary labeling of prepacked food is common and results in a restricted diet and the permanent fear of accidental reactions. To be prepared for the treatment of a potential anaphylactic reaction, patients with a peanut allergy are advised to carry self-injectable adrenaline at all times. Therefore, the diagnosis peanut allergy has a great impact on the quality of life of patients and their caregivers. Double-blind, placebo-controlled oral food challenges are considered the criterion standard in the diagnostic approach of food allergy.20 Because they are expensive and bear the risk of severe reactions, in vitro tests are wanted to give an accurate diagnosis without the necessity of an oral food challenge. For this purpose, the level of peanut-specific IgE has been correlated with the likelihood of having a clinical relevant peanut allergy and a cutoff level of 15 kUA/L has been proposed.6 Children above this level would be considered as peanut allergic with an accuracy of 95%. When applying this diagnosis cutoff level of peanutspecific IgE level more than 15 kUA/L in our study population, 25 patients would have been diagnosed with peanut allergy. Among them, there are 7 children who were asymptomatic at the food challenge, which resulted in a rate of a false-positive diagnosis of 28%. These patient would all probably be considered, unnecessarily, as patients who were allergic and receive therapy, including dietary advice and the prescription of rescue medication, including adrenaline self-injectors, which could possibly result in the impairment of quality of life. The difference in our results and the results previously reported is probably due to population characteristics. Although in the population studied by Sampson,6 not only the median value but also the peanut-specific range between patients who were allergic and tolerant patients were very distinct, in our population we noticed overlapping of peanut-specific IgE values. One of the main strengths of the current study is that all patients underwent an oral peanut challenge. This is different from previous studies in which a substantial number of peanut allergic patients (43% and 85%, respectively) were based on convincing histories for ethical reasons.6 A convincing history was defined as an immediate allergic reaction to food, which developed after the isolated ingestion of the suspected food and required emergency management by a physician within the previous 2 years.6 Tolerance development to peanut is rare but should not be neglected. Moreover, it is well known that the diagnosis of food allergy based on patient history results in an overestimation of food allergy, whereas oral food challenges express the actual clinical response to the provided allergen.3 Therefore, the studies that generated and validated the specific IgE cutoff level of 15 kUA/L for the diagnosis of peanut allergy6,7 were not based on 100% diagnostic accuracy confirmed by the food challenge.
Furthermore, cutoff points calculated as 95% positive predictive value are a parameter greatly influenced by the prevalence of the disease in the studied population. In this present study, peanut allergy prevalence was 56%, smaller than the 69% and 81%, respectively, presented by Sampson’s retrospective and prospective analysis,6 so it would be theoretically expected that a cutoff established with a higher prevalence would provide a safety range when applied to a less allergic population.6 Despite this, our results could not confirm the previously established decision points. Importantly, peanut-specific IgG and IgG4 were proven not to be helpful for the diagnosis of peanut allergy. This is in line with observations from other studies in regard to hen’s egg allergy10 and confirms the statement from the European Academy of Allergy and Clinical Immunology and AAAAI that food-specific IgG and IgG4 should not be used in the diagnostic approach of food allergy.11,12 More recently, it has been shown that the measurement of specific IgE to individual peanut components seems to improve the diagnostic procedure; especially, the measurement of specific IgE against the seed storage protein Ara h 2 seems to be highly correlated with the clinical relevance of an observed sensitization to peanuts.13-16 Codreanu et al13 were able to show that 79% of the patients who are pollen sensitized showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/L. Use of a threshold of 0.23 kU/L for Ara h 2 specificity was 96% and sensitivity was 93%.13 These results are quite different from the finding in our study when using the ISAC system. In our patients, a diagnosis of peanut allergy based on specific IgE to Ara h 2 would have resulted in a wrong diagnosis in one out of four patients as similarly found by Keet et al.21 The main difference between the study by Codreanu et al13 and our study is that the tolerant groups from Codreanu et al13 were all patients with pollen allergy who had elevated peanut-specific IgE due to cross-reactivity between PR10 proteins. In our study, all tolerant patients had suspected peanut allergy. Many of them were primarily sensitized to peanuts but clinically tolerant. Some might have outgrown their disease. DunnGalvin et al22 published an interesting diagnostic prediction model for peanut allergy in children by using 6 predictors (sex, age, history, skin prick test, peanut-specific Ig E, and total IgE minus peanut specific IgE), but, unfortunately, we could not apply it within our setting because some of the parameters used were not available. However, a most recent article, by Klemans et al,23 validated this model and updated it by adding allergic rhinitis, atopic dermatitis, and sIgE to peanut
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components Ara h 1, 2, 3, and 8 as candidate predictors (measured by ImmunoCAP). Moreover, they developed a new model based only on a specific IgE to peanut components. The discriminative ability of specific IgE to Ara h 2 alone was almost comparable with that of the updated model.23 In another study, from the United Kingdom, sensitization profiles between children with peanut allergy and peanut tolerant children by using a microarray with peanut components similar to our study were compared.14 The group detected marked differences in the pattern of component recognition between children with peanut allergy and peanut tolerant children. They found that the peanut component Ara h 2 was the most important predictor of clinical allergy.14 In a subsequent study that used the Phadia ImmunoCAP, the group was able to show that a cutoff for Ara h 2especific IgE of 0.03 kU/L resulted in a correct diagnosis in 98% of the children.16 This is in marked difference to the results in our study. The major difference to our study, however, was that threefourths of the patients with peanut allergy in the study by Nicolaou14 were not challenged, but the diagnosis of peanut allergy was based on patient history and peanut-specific IgE >15 kU/L, whereas, in our study, all children who were allergic underwent oral food challenge tests. Some discrepancies with prior studies may also be due to using different assay systems. In summary, we were able to show, in a group of wellcharacterized children who underwent oral food challenge test irrespective of their peanut-specific IgE levels, that the diagnosis of peanut allergy solely based on the measurement of specific IgE to peanut or Ara h 2 can be misleading. In our article, we would like to highlight that every second patient who were peanut tolerant and with high levels of peanut-specific IgE (>15 kU/L) recognizes Ara h 2 and that, especially these patients, might be wrongly classified as peanut allergic. Therefore, despite in vitro test advances, physician-supervised oral food challenge still remains the criterion standard in the diagnostic approach of peanut allergy.24 REFERENCES 1. Sicherer SH, Munoz-Furlong A, Godbold JH, Sampson HA. US prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year follow-up. J Allergy Clin Immunol 2010;125:1322-6. 2. Sicherer SH. Epidemiology of food allergy. J Allergy Clin Immunol 2011;127: 594-602. 3. Rona RJ, Keil T, Summers C, Gislason D, Zuidmeer L, Sodergren E, et al. The prevalence of food allergy: a meta-analysis. J Allergy Clin Immunol 2007;120: 638-46. 4. Hompes S, Kohli A, Nemat K, Scherer K, Lange L, Rueff F, et al. Provoking allergens and treatment of anaphylaxis in children and adolescents: data from the anaphylaxis registry of German-speaking countries. Pediatr Allergy Immunol 2011;22:568-74.
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5. Jarvinen KM. Food-induced anaphylaxis. Curr Opin Allergy Clin Immunol 2011;11:255-61. 6. Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-6. 7. Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-5. 8. Blumchen K, Ulbricht H, Staden U, Dobberstein K, Beschorner J, de Oliveira LC, et al. Oral peanut immunotherapy in children with peanut anaphylaxis. J Allergy Clin Immunol 2010;126:83-91. 9. Varshney P, Jones SM, Scurlock AM, Perry TT, Kemper A, Steele P, et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol 2011; 127:654-60. 10. Ahrens B, Lopes de Oliveira LC, Schulz G, Borres MP, Niggemann B, Wahn U, et al. The role of hen’s egg-specific IgE, IgG and IgG4 in the diagnostic procedure of hen’s egg allergy. Allergy 2010;65:1554-7. 11. Stapel SO, Asero R, Ballmer-Weber BK, Knol EF, Strobel S, Vieths S, et al. Testing for IgG4 against foods is not recommended as a diagnostic tool: EAACI Task Force Report. Allergy 2008;63:793-6. 12. Bock SA. AAAAI support of the EAACI Position Paper on IgG4. J Allergy Clin Immunol 2010;125:1410. 13. Codreanu F, Collignon O, Roitel O, Thouvenot B, Sauvage C, Vilain AC, et al. A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis. Int Arch Allergy Immunol 2011;154:216-26. 14. Nicolaou N, Poorafshar M, Murray C, Simpson A, Winell H, Kerry G, et al. Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnosis. J Allergy Clin Immunol 2010;125: 191-7. 15. Lieberman JA, Glaumann S, Batelson S, Borres MP, Sampson HA, Nilsson C. The utility of peanut components in the diagnosis of IgE-mediated peanut allergy among distinct populations. J Allergy Clin Immunol 2013;1: 75-82. 16. Nicolaou N, Murray C, Belgrave D, Poorafshar M, Simpson A, Custovic A. Quantification of specific IgE to whole peanut extract and peanut components in prediction of peanut allergy. J Allergy Clin Immunol 2011;127:684-5. 17. Marsh J, Rigby N, Wellner K, Reese G, Knulst A, Akkerdaas J, et al. Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis. Mol Nutr Food Res 2008;52(Suppl 2):S272-85. 18. Deinhofer K, Sevcik H, Balic N, Harwanegg C, Hiller R, Rumpold H, et al. Microarrayed allergens for IgE profiling. Methods 2004;32:249-54. 19. Harwanegg C, Hutter S, Hiller R. Allergen microarrays for the diagnosis of specific IgE against components of cow’s milk and hen’s egg in a multiplex biochip-based immunoassay. Methods Mol Biol 2007;385:145-57. 20. Eigenmann PA, Oh JW, Beyer K. Diagnostic testing in the evaluation of food allergy. Pediatr Clin North Am 2011;58:351-62. ix. 21. Keet CA, Johnson K, Savage JH, Hamilton RG, Wood RA. Evaluation of Ara h 2 IgE thresholds in the diagnosis of peanut allergy in a clinical population. J Allergy Clin Immunol 2013;1:101-3. 22. DunnGalvin A, Galy D, Cullinane C, Stenke E, Keeton D, ErlewynLajeunesse M, et al. Highly accurate prediction of food challenge outcome using routinely available clinical data. J Allergy Clin Immunol 2011;127:633-9. 23. Klemans RJB, Otte D, Knol M, Knol EF, Meijer Y, Gmelig-Meyling FHJ, et al. The diagnostic value of specific IgE to Ara h 2 to predict peanut allergy in children is comparable to validated and updated diagnostic prediction model. J Allergy Clin Immunol 2013;131:157-63. 24. Sicherer SH, Wood RA. Advances in diagnosing peanut allergy. J Allergy Clin Immunol 2013;1:1-13.
The Value of Specific IgE to Peanut and Its Component Ara h 2 in the Diagnosis of Peanut Allergy Lucila C. Lopes de Oliveira, MDa,b, Martina Aderholda, Marianne Brill, PhDc, Gabriele Schulza, Claudia Rolinck-Werninghaus, MDa, E.N. Clare Mills, PhDd, Bodo Niggemann, MDa,e, Charles Kirov Naspitz, MDb, Ulrich Wahn, MDa, and Kirsten Beyer, MDa Berlin, Germany; São Paulo, Brazil; Vienna, Austria; and Norwich, United Kingdom
What is already known about this topic? Studies have suggested peanut-specific IgE above defined cutoff levels or sensitization to Ara h 2 can predict clinical relevant peanut allergy and is helpful in avoidance of unnecessary oral food challenges. Allergen-specific IgG and IgG4 are also detectable in healthy individuals and do not play a role in peanut allergy diagnostic. What does this article add to our knowledge? Analysis of our data emphasizes that patients with high levels of peanutspecific or Ara h 2especific IgE can still be clinical tolerant. Our data confirm that peanut-specific IgG and IgG4 levels are comparable in individual who are allergic and in tolerant individuals. How does this study impact current management guidelines? Interpretation of in vitro test results should be done with care; neither proposed cutoff levels for peanut-specific IgE nor component-resolved diagnostics can replace an oral food challenge for all patients with suspected peanut allergy. Measurement of peanut-specific IgG or IgG4 should not be done in the diagnostic workup for suspected peanut allergy.
BACKGROUND: To avoid unnecessary oral food challenges, which are time consuming, stressful, and risky, improved in vitro diagnostic methods for food allergy such as component resolved diagnostics are still under investigation. OBJECTIVE: To investigate the role of whole peanut- and peanut-component (Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 8)especific IgE levels in the diagnostic procedure of peanut allergy as well as the diagnostic properties of peanut-specific IgG and IgG4. METHODS: Sixty-one children underwent oral peanut challenge tests for diagnostic purposes irrespective of their peanut-specific IgE levels. Peanut-specific serum IgE, IgG, and
IgG4 levels were determined by ImmunoCAP FEIA and specific IgE against individual peanut proteins by Immuno Solid-phase Allergen Chip. RESULTS: Thirty-four of 61 patients (56%) had a peanut allergy. No significant difference was observed for peanutspecific IgG or peanut-specific IgG4 levels between patients who were allergic and tolerant patients, whereas peanut-specific IgE was significant higher in patients who were allergic than in tolerant patients (P < .005). Twenty-five of 61 children had peanut-specific IgE above a previously proposed cutoff level of 15 kUA/L; however, 7 of these 25 children (28%) were clinically tolerant. Ara h 2-specific IgE was significantly lower in tolerant
a
Institute of Food Research; has received lecture fees from Imperial College; owns stock in Standard Life; and has received travel support from ILSI, EAACI, the University of Bologna, and the British High Commission. K. Beyer has received research support from the EU, the German Research Foundation, the Danone Foundation for the Treatment of Peanut Allergy; has received consultancy fees from Danone, Novartis, ALK, Meda Pharma, and Unilever; and has received lecture fees from Danone, Phadia, Infectopharm, CSL Behring, Novartis, UCB, Meda Pharma, Med Update, Allergopharma, and Thermo Fischer. The rest of the authors declare that they have no relevant conflicts. Received for publication March 31, 2013; revised May 10, 2013; accepted for publication May 11, 2013. Cite this article as: Lopes de Oliveira LC, Aderholz M, Brill M, Schulz G, RolinckWerninghaus C, Mills ENC, et al. The value of specific IgE to peanut and its component Ara h 2 in the diagnosis of peanut allergy. J Allergy Clin Immunol: In Practice 2013;1:394-8. http://dx.doi.org/10.1016/j.jaip.2013.05.010. Corresponding author: Kirsten Beyer, MD, Department of Paediatric Pneumology and Immunology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail: [email protected]. 2213-2198/$36.00 Ó 2013 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaip.2013.05.010
Department of Pediatric Pneumology and Immunology, Charité Universitätsmedizin Berlin, Berlin, Germany b Division of Allergy and Clinical Immunology, Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil c VBC Genomics, Vienna, Austria d Institute of Food Research, Norwich, United Kingdom e Pediatric Allergology and Pneumology, German Red Cross Hospital Westend, Berlin, Germany This work was funded in part within the collaborative research initiative EuroPrevall, an integrated project funded by the European Commission under the 6th Framework Programme (FOOD-CT-2005-514000), and in part by the German Research Foundation DFGeBE3991/1-1. Conflicts of interest: M. Aderhold has received grants from the European Union and the German Research Foundation. M. Brill has received research and travel support from EuroPrevall. C. Mills has received research support from the European Commission, the Biological and Biotechnological Sciences Research Council, the UK Food Standards Agency, DBV Technologies, the UK Technology Strategy Board, and the European Food Safety Authority; is on the Novartis Board; is a member of the UK Food Standards Agency Advisory Committee on Novel Foods and Processes; has received consultancy fees from Pepsico International; is employed by the University of Manchester and the
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Abbreviations used Ig- Immunoglobulin ISAC- Immuno Solid-phase Allergen Chip
than in patients with allergies (P < .0001). Interestingly, 94% of the patients with peanut allergies showed IgE-binding to Ara h 2. Unfortunately, 26% of the sensitized but tolerant patients have shown IgE binding to Ara h 2 too. CONCLUSIONS: Neither the level of specific IgE to peanut nor to Ara h 2 was able to clearly distinguish patients with clinical relevant peanut allergy from those who were clinical tolerant in our population. As expected, peanut-specific IgG and IgG4 did not improve the diagnostic procedure. Ó 2013 American Academy of Allergy, Asthma & Immunology (J Allergy Clin Immunol: In Practice 2013;1:394-8) Key words: IgE; IgG; IgG4; Peanut allergy; Immuno Solid-phase Allergen Chip; Component-resolved diagnostics; Ara h 1; Ara h 2; Ara h 3; Ara h 6; Ara h 8
Peanut allergy is a common disease, and its prevalence appears to be increasing in many countries.1-3 Resolution of peanut allergy is uncommon, and children with a peanut allergy tend to have their disease life-long.2 Importantly, accidental ingestion often results in anaphylactic reactions, and fatal cases have been reported.4,5 The diagnosis of peanut allergy results in a tremendous change in life. Strict dietary restrictions are necessary, and the patients need to avoid a large variety of foods that contain peanuts, including products with precautionary labeling. Because patients with a peanut allergy tend to have severe reactions after accidental ingestion,2 the prescription of epinephrine self-injectors is common. These aspects associated with the fear of a possible anaphylactic reaction are related to impairment on quality of life.6-8 Therefore, the diagnosis of peanut allergy results in serious consequences for the patients and their families. Immunologic parameters, which correlate completely to the clinical manifestation of peanut allergy have not been identified yet. It has been shown that patients with allergies (symptomatic) have higher levels of peanut-specific IgE when compared with asymptomatic ones.6 Because oral food challenges for diagnostic purposes are time consuming, stressful, and not exempt of risks, other diagnostic tools have been studied. Sampson6 observed that a peanut-specific IgE higher than 15 kUA/L was able to predict 95% of the patients with allergy and suggested this peanutspecific IgE cutoff level as an alternative diagnostic procedure. Later on, the same results were observed by Clark and Ewan.7 It has been speculated that food allergenespecific IgG and IgG4 may also be helpful to define the clinical reactivity. It has been observed in oral immunotherapy studies that peanutspecific IgG4 increased in parallel with the development of oral tolerance.8,9 However, in hen’s egg allergy, the level of hen’s eggespecific IgG4 did not correlate with the clinical reactivity10 and the usage of food allergenespecific IgG4 is not recommended as a diagnostic test in food allergy.11,12 More recently, the usage of a component-resolved diagnostic has been evaluated. Especially, measurement of specific IgE to Ara h 2 appears to be of great value. Codreanu et al13 were able to show that 79% of the patients who were pollen sensitized showed IgE binding to peanut, despite their tolerance to
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peanut.13 In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/L.13 IgE to Ara h 2 seems to improve specificity in detecting patients with allergy among peanut-sensitized ones.14,15 Similarly, Nicolaou et al16 showed that the usage of Ara h 2especific IgE for the correct diagnosis of peanut allergy was very helpful. In this population, a sensitivity of 100% and a specificity of 96% were found when using a cutoff value of 0.35 kU/L for Ara h 2especific IgE.16 The aim of the present study was to investigate the role of peanut-specific IgE, IgG, and IgG4 levels in the diagnostic procedure of peanut allergy and determine the accuracy of a proposed diagnostic decision point of 15kUA/L in our population. In parallel, the usefulness of specific IgE antibody determination to individual peanut proteins was investigated in a well-defined population.
METHODS Study population Sixty-one infants and children referred to the Charité Allergy Center, a tertiary level clinic, for evaluation of suspected IgEmediated peanut allergy were included into the study. Ages ranged from 0.9 to 13.6 years, with a median age of 4.1 years. Sixty-two percent of the patients were boys and 46% had a diagnosis of atopic eczema. The study was approved by the local ethical committee of the University Children’s Hospital of Charité, Universitätsmedizin Berlin. Oral food challenges Oral food challenges were performed with homogenized roasted peanut kernels. In detail, 30 mg, 60 mg, 130 mg, 250 mg, 500 mg, 1 g, and 2 g of peanut protein were given orally every 30 minutes. After the challenge was completed, the patient remained under clinical supervision for at least 2 hours. In case of clinical tolerance, the patient received a subsequent dose of roasted peanuts in a total amount of 4 g of peanut protein another day. Full emergency equipment and medications were at hand during all the procedures. The use of antihistamine was suspended 72 hours before the challenge. The food challenges were scored as positive if objective clinical reactions were noted, such as urticaria, angioedema, vomiting, diarrhea, wheezing, stridor, rhino conjunctivitis, or a drop in blood pressure. Blood sampling Venous blood samples of all patients were collected before oral provocation tests and obtained serum probes were aliquoted and stored at 20 C until further analysis. Fluorescence enzyme immunoassays Determination of peanut-specific IgE, IgG, and IgG4 was done by fluorescence enzyme immunoassay by using ImmunoCAP 250 System (Phadia, Uppsala, Sweden). Specific IgE titers were quantified in protein units designated as kilounits of antibody per liter (KUA/L) with a lower detection limit of 0.35 KUA/L and an upper limit of 100 KUA/L as proposed by the manufacturer. Detection limits for specific IgG and IgG4 were 15 to >100 kU/L)
6/7 (86) 7/8 (88) 17/17 (100)
4/7 (57) 8/8 (100) 17/17 (100)
2/7 (29) 6/8 (75) 17/17 (100)
2/12 (17) 1/8 (13) 4/7 (57)
4/12 (33) 6/8 (75) 6/7 (86)
5/12 (42) 5/8 (63) 5/7 (71)
perform, because precautionary labeling of prepacked food is common and results in a restricted diet and the permanent fear of accidental reactions. To be prepared for the treatment of a potential anaphylactic reaction, patients with a peanut allergy are advised to carry self-injectable adrenaline at all times. Therefore, the diagnosis peanut allergy has a great impact on the quality of life of patients and their caregivers. Double-blind, placebo-controlled oral food challenges are considered the criterion standard in the diagnostic approach of food allergy.20 Because they are expensive and bear the risk of severe reactions, in vitro tests are wanted to give an accurate diagnosis without the necessity of an oral food challenge. For this purpose, the level of peanut-specific IgE has been correlated with the likelihood of having a clinical relevant peanut allergy and a cutoff level of 15 kUA/L has been proposed.6 Children above this level would be considered as peanut allergic with an accuracy of 95%. When applying this diagnosis cutoff level of peanutspecific IgE level more than 15 kUA/L in our study population, 25 patients would have been diagnosed with peanut allergy. Among them, there are 7 children who were asymptomatic at the food challenge, which resulted in a rate of a false-positive diagnosis of 28%. These patient would all probably be considered, unnecessarily, as patients who were allergic and receive therapy, including dietary advice and the prescription of rescue medication, including adrenaline self-injectors, which could possibly result in the impairment of quality of life. The difference in our results and the results previously reported is probably due to population characteristics. Although in the population studied by Sampson,6 not only the median value but also the peanut-specific range between patients who were allergic and tolerant patients were very distinct, in our population we noticed overlapping of peanut-specific IgE values. One of the main strengths of the current study is that all patients underwent an oral peanut challenge. This is different from previous studies in which a substantial number of peanut allergic patients (43% and 85%, respectively) were based on convincing histories for ethical reasons.6 A convincing history was defined as an immediate allergic reaction to food, which developed after the isolated ingestion of the suspected food and required emergency management by a physician within the previous 2 years.6 Tolerance development to peanut is rare but should not be neglected. Moreover, it is well known that the diagnosis of food allergy based on patient history results in an overestimation of food allergy, whereas oral food challenges express the actual clinical response to the provided allergen.3 Therefore, the studies that generated and validated the specific IgE cutoff level of 15 kUA/L for the diagnosis of peanut allergy6,7 were not based on 100% diagnostic accuracy confirmed by the food challenge.
Furthermore, cutoff points calculated as 95% positive predictive value are a parameter greatly influenced by the prevalence of the disease in the studied population. In this present study, peanut allergy prevalence was 56%, smaller than the 69% and 81%, respectively, presented by Sampson’s retrospective and prospective analysis,6 so it would be theoretically expected that a cutoff established with a higher prevalence would provide a safety range when applied to a less allergic population.6 Despite this, our results could not confirm the previously established decision points. Importantly, peanut-specific IgG and IgG4 were proven not to be helpful for the diagnosis of peanut allergy. This is in line with observations from other studies in regard to hen’s egg allergy10 and confirms the statement from the European Academy of Allergy and Clinical Immunology and AAAAI that food-specific IgG and IgG4 should not be used in the diagnostic approach of food allergy.11,12 More recently, it has been shown that the measurement of specific IgE to individual peanut components seems to improve the diagnostic procedure; especially, the measurement of specific IgE against the seed storage protein Ara h 2 seems to be highly correlated with the clinical relevance of an observed sensitization to peanuts.13-16 Codreanu et al13 were able to show that 79% of the patients who are pollen sensitized showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/L. Use of a threshold of 0.23 kU/L for Ara h 2 specificity was 96% and sensitivity was 93%.13 These results are quite different from the finding in our study when using the ISAC system. In our patients, a diagnosis of peanut allergy based on specific IgE to Ara h 2 would have resulted in a wrong diagnosis in one out of four patients as similarly found by Keet et al.21 The main difference between the study by Codreanu et al13 and our study is that the tolerant groups from Codreanu et al13 were all patients with pollen allergy who had elevated peanut-specific IgE due to cross-reactivity between PR10 proteins. In our study, all tolerant patients had suspected peanut allergy. Many of them were primarily sensitized to peanuts but clinically tolerant. Some might have outgrown their disease. DunnGalvin et al22 published an interesting diagnostic prediction model for peanut allergy in children by using 6 predictors (sex, age, history, skin prick test, peanut-specific Ig E, and total IgE minus peanut specific IgE), but, unfortunately, we could not apply it within our setting because some of the parameters used were not available. However, a most recent article, by Klemans et al,23 validated this model and updated it by adding allergic rhinitis, atopic dermatitis, and sIgE to peanut
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components Ara h 1, 2, 3, and 8 as candidate predictors (measured by ImmunoCAP). Moreover, they developed a new model based only on a specific IgE to peanut components. The discriminative ability of specific IgE to Ara h 2 alone was almost comparable with that of the updated model.23 In another study, from the United Kingdom, sensitization profiles between children with peanut allergy and peanut tolerant children by using a microarray with peanut components similar to our study were compared.14 The group detected marked differences in the pattern of component recognition between children with peanut allergy and peanut tolerant children. They found that the peanut component Ara h 2 was the most important predictor of clinical allergy.14 In a subsequent study that used the Phadia ImmunoCAP, the group was able to show that a cutoff for Ara h 2especific IgE of 0.03 kU/L resulted in a correct diagnosis in 98% of the children.16 This is in marked difference to the results in our study. The major difference to our study, however, was that threefourths of the patients with peanut allergy in the study by Nicolaou14 were not challenged, but the diagnosis of peanut allergy was based on patient history and peanut-specific IgE >15 kU/L, whereas, in our study, all children who were allergic underwent oral food challenge tests. Some discrepancies with prior studies may also be due to using different assay systems. In summary, we were able to show, in a group of wellcharacterized children who underwent oral food challenge test irrespective of their peanut-specific IgE levels, that the diagnosis of peanut allergy solely based on the measurement of specific IgE to peanut or Ara h 2 can be misleading. In our article, we would like to highlight that every second patient who were peanut tolerant and with high levels of peanut-specific IgE (>15 kU/L) recognizes Ara h 2 and that, especially these patients, might be wrongly classified as peanut allergic. Therefore, despite in vitro test advances, physician-supervised oral food challenge still remains the criterion standard in the diagnostic approach of peanut allergy.24 REFERENCES 1. Sicherer SH, Munoz-Furlong A, Godbold JH, Sampson HA. US prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year follow-up. J Allergy Clin Immunol 2010;125:1322-6. 2. Sicherer SH. Epidemiology of food allergy. J Allergy Clin Immunol 2011;127: 594-602. 3. Rona RJ, Keil T, Summers C, Gislason D, Zuidmeer L, Sodergren E, et al. The prevalence of food allergy: a meta-analysis. J Allergy Clin Immunol 2007;120: 638-46. 4. Hompes S, Kohli A, Nemat K, Scherer K, Lange L, Rueff F, et al. Provoking allergens and treatment of anaphylaxis in children and adolescents: data from the anaphylaxis registry of German-speaking countries. Pediatr Allergy Immunol 2011;22:568-74.
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5. Jarvinen KM. Food-induced anaphylaxis. Curr Opin Allergy Clin Immunol 2011;11:255-61. 6. Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-6. 7. Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-5. 8. Blumchen K, Ulbricht H, Staden U, Dobberstein K, Beschorner J, de Oliveira LC, et al. Oral peanut immunotherapy in children with peanut anaphylaxis. J Allergy Clin Immunol 2010;126:83-91. 9. Varshney P, Jones SM, Scurlock AM, Perry TT, Kemper A, Steele P, et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol 2011; 127:654-60. 10. Ahrens B, Lopes de Oliveira LC, Schulz G, Borres MP, Niggemann B, Wahn U, et al. The role of hen’s egg-specific IgE, IgG and IgG4 in the diagnostic procedure of hen’s egg allergy. Allergy 2010;65:1554-7. 11. Stapel SO, Asero R, Ballmer-Weber BK, Knol EF, Strobel S, Vieths S, et al. Testing for IgG4 against foods is not recommended as a diagnostic tool: EAACI Task Force Report. Allergy 2008;63:793-6. 12. Bock SA. AAAAI support of the EAACI Position Paper on IgG4. J Allergy Clin Immunol 2010;125:1410. 13. Codreanu F, Collignon O, Roitel O, Thouvenot B, Sauvage C, Vilain AC, et al. A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis. Int Arch Allergy Immunol 2011;154:216-26. 14. Nicolaou N, Poorafshar M, Murray C, Simpson A, Winell H, Kerry G, et al. Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnosis. J Allergy Clin Immunol 2010;125: 191-7. 15. Lieberman JA, Glaumann S, Batelson S, Borres MP, Sampson HA, Nilsson C. The utility of peanut components in the diagnosis of IgE-mediated peanut allergy among distinct populations. J Allergy Clin Immunol 2013;1: 75-82. 16. Nicolaou N, Murray C, Belgrave D, Poorafshar M, Simpson A, Custovic A. Quantification of specific IgE to whole peanut extract and peanut components in prediction of peanut allergy. J Allergy Clin Immunol 2011;127:684-5. 17. Marsh J, Rigby N, Wellner K, Reese G, Knulst A, Akkerdaas J, et al. Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis. Mol Nutr Food Res 2008;52(Suppl 2):S272-85. 18. Deinhofer K, Sevcik H, Balic N, Harwanegg C, Hiller R, Rumpold H, et al. Microarrayed allergens for IgE profiling. Methods 2004;32:249-54. 19. Harwanegg C, Hutter S, Hiller R. Allergen microarrays for the diagnosis of specific IgE against components of cow’s milk and hen’s egg in a multiplex biochip-based immunoassay. Methods Mol Biol 2007;385:145-57. 20. Eigenmann PA, Oh JW, Beyer K. Diagnostic testing in the evaluation of food allergy. Pediatr Clin North Am 2011;58:351-62. ix. 21. Keet CA, Johnson K, Savage JH, Hamilton RG, Wood RA. Evaluation of Ara h 2 IgE thresholds in the diagnosis of peanut allergy in a clinical population. J Allergy Clin Immunol 2013;1:101-3. 22. DunnGalvin A, Galy D, Cullinane C, Stenke E, Keeton D, ErlewynLajeunesse M, et al. Highly accurate prediction of food challenge outcome using routinely available clinical data. J Allergy Clin Immunol 2011;127:633-9. 23. Klemans RJB, Otte D, Knol M, Knol EF, Meijer Y, Gmelig-Meyling FHJ, et al. The diagnostic value of specific IgE to Ara h 2 to predict peanut allergy in children is comparable to validated and updated diagnostic prediction model. J Allergy Clin Immunol 2013;131:157-63. 24. Sicherer SH, Wood RA. Advances in diagnosing peanut allergy. J Allergy Clin Immunol 2013;1:1-13.
