Cross-reactivity among cacao and tree nuts in a paediatric allergic patientTo the Editor:Chocolate is produced from cacao beans from the Theobroma cacao tree, through a multistep process involving fermentation, drying, roasting, nib grinding and refining, and tempering, to ensure its stability and flavour [1].Most studies have reported beneficial effects of polyphenols from cacao consumption on health, specially on the cardiovascular system [2] and cognitive functions [3], among others. According the International Cocoa Organization (ICCO), the total estimated world production of cacao beans in 2023–24 was 4.368 tons, principally from Africa [4], being Europe the largest regional market, accounting for a significant share of cocoa consumption.Even though chocolate is widely consumed in the world, food allergy to cacao is unfrequent and reports on true allergy are scarce.We present the case of a 9-year old boy who for the last year, presented with oral pruritus every time he ingested almonds, cashew nuts, walnuts and hazelnuts. He also developed oral itchiness as well as vomiting and lethargy, minutes after ingesting cacao containing foods. He tolerated peanuts and sunflower and sesame seeds.Regarding his atopic background, he suffered from exercise induced asthma and mild rhinitis, being sensitised to dust mites.Skin prick test with tree nuts, peanut and cacao extracts (Diater and Leti Laboratories, Madrid, Spain) yielded positive for almond (15x8mm), hazelnut (17x10mm), walnut (7x8mm), pecan nut (20x10mm), macadamia nut (10x5mm), pistachio nut (25x10mm), cashew nut (20x15mm), pine nut (5x2mm), peanut (5x2,5mm), sesame seeds (3x5mm).Prick by prick with pure cacao powder 100% (Valor Cacao®) was positive (5x5mm). Serum specific IgE determination to cacao by ImmunoCAP (ThermoFisher Scientific, Madrid, Spain) was 0.36kU/L. Total serum IgE was 702kU/L.ALEX® assay showed patient’s specific IgE recognition of Ana o 2 (11S globuline) and Ana o 3 (2S albumin), from cashew nut; Cor a 9 (11S globuline) and Cor a 11 (7/8 globuline), from hazelnut; Jug r 1 (2S albumin), Jug r 2 (7/8 globulin), Jug r 4 (11S globulin) and Jug r 6 (7/8 globuline), from walnut; Pis v 1 (2S albumin), Pis v 2 (11S globuline), and Pis v 3 (7/8 globuline), from pistachio nut; Pru du from almond; Ses I from sesame seeds; Hel a from sunflower seeds; Pap a 2S albumin from poppy seeds.SDS-PAGE-Immunoblotting was performed according to the Laemmli method [5] to estimate de molecular mass of the IgE-reactive proteins from samples from two different and popular cacao brands in Spain: Valor® Cacao (pure cacao bean powder with no tree nut traces as reported by manufacturer) and Cola Cao® (sugar, defatted cocoa, and a cola-malted cereal cream, made from wheat flour, barley malt extract, and kola nut extract, with no tree nut traces as indicated in label), under reducing and non-reducing conditions. Cacao proteins were previously isolated: protein extraction from Cola-Cao® yielded a concentration of 453mcg/ml and from Valor® Cacao, 426 mcg/ml. A diffuse protein profile was obtained by SDS-PAGE without clearly defined bands from both extracts (Figure 1). This result is likely due to the complex composition of cacao, which contains a relatively low number of soluble proteins mixed with polyphenols, lipids, and carbohydrates. Even so, a ~16 kDa band from the Valor Cacao® extract was recognised by patient’s specific IgE (Figure 2).For Western Blot inhibition, pure cacao extract (Valor Cacao®) was used. Total inhibition of the 16 kDa band was shown after incubating patient’s serum with cashew nut and walnut extracts, respectively. Hazelnut caused only partial inhibition (Figure 2).Perfetti et al [6], reported the case of a confectionery worker suffering from occupational asthma due to cacao. They identified six different bands from cacao powder by SDS-PAGE: three between 10 and 20kDa, two of 30kDa and one of 50kDa. Although this patient showed IgE-mediated sensitisation to cacao by SPT and RAST, there was no IgE binding to any of those bands detected most probably due to the low levels of specific IgE, as suggested by them.In another recent case report, Nin Valencia et al [7] presented a cacao allergic patient, previously sensitised to walnut and almond, who recognised a 17, 5kDa and a 17,5-21kDa bands from cacao by SDS-immunoblotting. Partial cross-reactivity among those tree nuts and cacao was demonstrated by blotting inhibiton.We hypothesize that the 16kDa band found by us and the 17,5kDa band described by Nin Valencia et al [7] could correspond to same protein, being specifically a fragment of a vicilin-like globulin storage protein. This vicilin consists of three subunits with apparent molecular masses of 47 kDa, 31 kDa, and 15 kDa, respectively, which are derived from fragmentation of a 66- kDa precursor. These subunits have been shown to retain IgE-binding epitopes [8], which could explain their recognition by both patients’ specific IgE.In the case we present, total inhibition of IgE binding to cacao by cashew nut and walnut suggested primary sensitisation to these nuts. Moreover, partial inhibition by hazelnut sugests cross-reactivity between hazelnut and cacao. On the other hand, we did not find cross-reactivity between cacao and almond, contrary to that reported by Nin Valencia et al [7].Although rare, few clinical cases of food allergy to cacao [7, 9] as well as cacao as a cause of occupational asthma in confectionery workers, have been reported [7, 10].Lopes et al [9] reported three paediatric patients sensitised to cacao as demonstrated by SPT and serum cacao specific IgE who developed anaphylactic reactions during food challenge. Two of them were allergic to tree nuts as well. In the case we present, we did not perform food challenge with cacao because the patient’s mother had tried several brands of pure cacao at home, that the patient did not tolerate, developing oral pruritus and vomiting after its ingestion. At the present time they agreed to start oral tolerance induction with cacao.In summary, although chocolate allergy is commonly attributed to the presence of other allergens included in its composition, as nuts, peanuts or milk, true allergy to cacao should be considered.In the present report, we could demonstrate cross-reactivity among cacao and tree nuts as cashew, walnut and hazelnut, suggesting that primary tree nut allergy might predispose to the development of cacao allergy.Written consent for permission to publish was obtained from patient’s parents, following recommendation of the Ethics Committee of A Coruña-Ferrol (Spain).Key words: chocolate, cacao, allergy, tree nuts, cross-reactivity, SDS-Page, immunoblotting.Word count: 1031.
