Seafood allergy: crossreactivity of shrimp specific IgE with ascaridoids allergens
(Ascaris lumbricoides and Anisakis simplex)
L Delgado, RM Murta, L Cunha, M Sequeira, J Ferraz de Oliveira, MG Castel-Branco Immunoallergology Unit and Immunology Department, H. S. João, Faculty of Medicine. Porto, Portugal.
IgE-mediated reactions are the most severe of food hypersensitive disorders, and are potentially life threatening (anaphylaxis, laryngeal edema, asthma). Fish and shellfish are common causes of food allergy, in both children and adults, and the detection of seafood-specific IgE by reliable tests is particularly relevant to diagnosis of these at-risk patients. There is now evidence of extensive cross-reactivity of IgE to major fish allergens (parvalbumins) (1) and major shellfish (crustaceans and molluks) allergens (tropmyosins) (2,3). Fish nematodes, like Anisakis simplex, have been recently recognized as an important cause of seafood-related allergy in adults (4,5), but accurate in vitro diagnostic tests are still lacking (6,7). In the Mediterranean diet several mixtures of seafood (fish and shellfish) are very common in different dishes (e.g. caldeirada, paella, bouillabaisse) raising difficulties in the identification of the culprit allergen in seafood related allergy.
Aims: to evaluate the sensitization to Anisakis simplex in patients with shellfish allergy, and to study a possible crossreactivity of shrimp specific IgE with ascaridoids (Anisakis and Ascaris lumbricoides) allergens.
Methods: 10 patients with typical symptoms (urticaria/ angioedema, dermatitis, diarrhea or anaphylaxis) after ingestion, inhalation or skin contact with shellfish, positive skin prick tests with commercial allergens and/or the natural food and positive specific IgE (UniCAPä) to shrimp. Sera specific IgE (UniCAPä), to fish/shellfish mixture (FSmx) and individual allergens (codfish, shrimp, Ascaris lumbricoides and Anisakis simplex) - were positive if >0.35 kUA//L (0.7 kUA/L for Ascaris and Anisakis). Specific IgE Imunoblotting assays with shrimp (Penaeus aztecus) and Al (Ascaris lumbricoides) and As (Anisakis simplex) allergen strips were performed using the AlaSTAT-AlaBlot System (DPC). The frequency and intensity of the antibody binders to each protein band was detected by the Plus Steck Scanner and evaluated with the Quantisoftware program (Biosoft). In order to study the crossreactivity between the shrimp and Anisakis simplex and Ascaris lumbricoides, imunoblot and CAP inhibition tests with both fluid phase and solid phase allergens were performed with a representative serum. In the specific IgE inhibition with shrimp liquid extract, the pre-diluted serum (1:2) was pre-incubated with 10 ml shrimp extract allergen (Greer Laboratories, Inc.), during 2 hours at room temperature, and the determination of the shrimp, Anisakis s. and Ascaris l. specific IgE was performed by the UniCAPä and AlaBLOT methods. In the specific IgE inhibition with solid-phase shrimp, Anisakis simplex and Ascaris lumbricoides, the serum was pre-incubated with the relevant allergen coupled to the solid phase of the UniCAP during 1 h at room temperature. The eluted sample was tested in the imunoblotting assay and UniCAPä for shrimp specific IgE.
Results: Results of the skin prick tests (commercial and/or natural food) and specific IgE to shrimp, codfish, Anisakis simplex (As), Ascaris lumbricoides (Al), and the multiallergen panel of fish/shellfish mixture (fx2) determined by UniCAPä are shown in the Table 1- skin prick tests (commercial and/or natural food) and specific IgE to shrimp, codfish, Anisakis simplex (As), Ascaris lumbricoides (Al), and the multiallergen panel of fish/shellfish mixture (fx2) (UniCAPä).
All patients had positive results to Anisakis simplex and/or to Ascaris lumbricoides; Shrimp specific-IgE levels (27.8 0.87-141.0 kUA/l) were significantly correlated with Anisakis simplex-
Fig.1-specific IgE to shrimp vs Anisakis simplex - (6.9 kUA/l, 0.35 -34.3 kUA/l, rs= 0.97 p=0.001) and Ascaris lumbricoides-
Fig.2- specific IgE to shrimp vs Ascaris lumbricoides- (5.5 kUA/l, 0.6 -57.2 kUA/l, rs= 0.94, p=0.05) positivity. Total inhibition of Ascaris, Anisakis and shrimp specific IgE was obtained after pre-incubation of a highly positive sample with shrimp liquid extract, (CAP test)-
Table 2- Shrimp, Anisakis simplex, Ascaris lumbricoides specific IgE inhibition with shrimp liquid extract (UniCAPä). The intensity of the bands of the pre-incubated serum with shrimp liquid extract and the native serum was compared by imunoblotting, and total inhibition was confirmed for shrimp and Ascaris lumbricoides specific IgE.
Fig.3- AlaBLOT shrimp specific IgE Fig.4 - AlaBLOT Ascaris lumbricoides specific IgE. Conversely, pre-incubation of this sample with solid-phase, Ascaris and Anisakis allergen partially inhibited shrimp reactivity (40-50% in UniCAPä).
Fig.4 - AlaBLOT Ascaris lumbricoides specific IgE. Conversely, pre-incubation of this sample with solid-phase, Ascaris and Anisakis allergen partially inhibited shrimp reactivity (40-50% in UniCAPä).
Table3- Shrimp specific IgE inhibition with shrimp, Anisakis simplex, Ascaris lumbricoides solid phase in the UniCAPä and AlaBLOT.
Conclusions: in patients with shellfish allergy, the frequent co-existence of positive specific IgE to Anisakis simplex may be related with shrimp and ascaridoids allergen crossreactivity. Based on these in vitro methods, the clinical significance of ascaridoids sensitization is difficult to establish in patients with shellfish-related allergy. Better characterized and purified Anisakis simplex allergens are clearly needed to overcome this problem.
Alergia aos mariscos: reacções cruzadas entre a IgE específica para o camarão e os ascaridoides (Ascaris lumbricoides e Anisakis simplex)
L Delgado, RM Murta, L Cunha, M Sequeira, J Ferraz de Oliveira, MG Castel-Branco Unidade de Imunoalergologia e Serviço de Imunologia, H. de S. João, Faculdade de Medicina. Porto, Portugal.
As reacções IgE-mediadas são as formas mais graves de hipersensilidade alimentar e são potencialmente fatais (anafilaxia, edema laríngeo, asma). O peixe e mariscos são causas muito comuns de alergia alimentare, não só em crianças, mas também em adultos, pelo que a detecção de IgE específica para peixes e mariscos por métodos in vitro, é particularmente importante no diagnóstico deste doentes de risco. Está hoje bem demonstrada a existência de reacções cruzadas entre as proteinas alergénicas do peixe- parvalbuminas (1)- e também entre os alergénios do marisco (crustáceos e moluculos) - tropomiosina (2,3). Os nemátodes que infectam animais marinhos, como o Anisakis simplex, foram recentemente reconhecidos como uma importante e frequente causa de alergia desencadeada pela ingestão de peixe e/ou marisco em adultos (4,5). Contudo, não estão ainda definidos os melhores testes no diagnóstico in vitro. Na dieta Mediterranica várias combinações de peixes e mariscos são frequentemente utilizadas em diferentes pratos (p.ex caldeirada, paella, bouillabaisse), levantando também dificuldades na identificação dos alergénios responsáveis em caso de alergia após a ingestão de peixes e mariscos.
Objectivo: avaliar a sensibilização ao Anisakis simplex de doentes com alergia a mariscos e estudar a possível existência de reacções cruzadas da IgE específica ao camarão com os alergénios ascaridoides (Anisakis simplex, Ascaris lumbricoides).
Métodos: 10 doentes com sintomas típicos (urticária, angioedema, dermatite, diarreia ou anafilaxia) após ingestão, inalação ou contacto com mariscos, testes “prick” positivos a alergénios comerciais e/ou alimentos naturais e IgE específica ao camarão positiva (UniCAPä). Determinaram-se as IgE específicas (UniCAPä) à mistura peixe/marisco (FSmx) e aos alergénios individuais (bacalhau, camarão, Ascaris lumbricoides e Anisakis simplex) - tendo sido considerados positivos se >0.35 kUA/L (0.7 kUA/L para Ascaris e Anisakis). Efectuaram-se também testes de IgE específica por imunoblotting para o camarão (Penaeus aztecus) e Ascaris lumbricoides (Al) e Anisakis simplex (As), utilizando o sistema AlaBLOT (DPC). A frequência e intensidade das bandas de proteinas alergénicas reconhecidas, foram lidas no scanner Plus Steck e analisadas com o auxílio do programa Quantisoftware (Biosoft). Posteriormente efectuaram-se testes de inibição com extractos em fase líquida e em fase sólida, por ambos os métodos (imunoblot e CAP), utilizando um soro representativo, com o objectivo de estudar as reacções cruzadas entre o camarão, Anisakis simplex e Ascaris lumbricoides. Nos testes de inibição com extracto de camarão líquido, o soro pre-diluído (1:2) foi pre-incubado com 10 l de extracto líquido de camarão (Greer Laboratories, Inc.) durante duas horas à temperatura ambiente, após o que se determinaram as IgEs específicas do camarão, Anisakis s. and Ascaris l., pelos métodos UniCAPä and AlaBLOT. Para os testes de inibição com extractos de camarão, Anisakis simplex e Ascaris lumbricoides em fase sólida, o soro foi pré-incubado com o alergénio relevante ligado à fase sólida do UniCAP durante 1 h, à temperatura ambiente. A amostra eluída foi testada por imunoblotting e por RAST para o camarão.
Resultados: Os resultados dos skin prick tests (comerciais e/ou alimentos naturais) e das IgEs específicas para o camarão, bacalhau, Anisakis simplex (As), Ascaris lumbricoides (Al), e para o painel multialergénico de mistura de peixe/marisco (fx2), determinados pelo UniCAPä são apresentados na tabela 1. Table 1- skin prick tests (commercial and/or natural food) and specific IgE to shrimp, codfish, Anisakis simplex (As), Ascaris lumbricoides (Al), and the multiallergen panel of fish/shellfish mixture (fx2) (UniCAPä). Todos os doentes foram positivos ao Anisakis simplex e/ou ao Ascaris lumbricoides; Os níveis de positividade obtidos para a IgE específica ao camarão (27.8 kUA/l, 0.87-141.0 kUA/l) correlacionam-se significativamente com os do Anisakis simplex- Fig.1-specific IgE to shrimp vs Anisakis simplex - (6.9 kUA/l, 0.35 -34.3 kUA/l, rs= 0.97 p=0.001) e os do Ascaris lumbricoides- Fig.2- specific IgE to shrimp vs Ascaris lumbricoides- (5.5 kUA/l, 0.6 -57.2 kUA/l, rs= 0.94, p=0.05). Após a pré-incubação de um soro fortemente positivo com extracto de camarão líquido, obteve-se a inibição total da IgE específica ao Ascaris l., Anisakis s. (CAP)-Table 2- Shrimp, Anisakis simplex, Ascaris lumbricoides specific IgE inhibition with shrimp liquid extract (UniCAPä). Compararam-se as intensidades das bandas do soro pré-incubado com extracto líquido de camarão e o soro nativo, por imunoblotting, confirmando-se a inibição total da IgE específica para o camrarão e para o Ascaris lumbricoides. Fig.3- AlaBLOT shrimp specific IgE Fig.4 - AlaBLOT Ascaris lumbricoides specific IgE. Contrariamente, ao pré-incubar esta mesma amostra com os alergénios Ascaris e Anisakis em fase sólida, apenas se conseguiu inibir parcialmente a reactividade ao camarão (40-50% no UniCAPä).Table3- Shrimp specific IgE inhibition with shrimp, Anisakis simplex, Ascaris lumbricoides solid phase in the UniCAPä and AlaBLOT.
Conclusões: em doentes com alergias ao marisco, a frequente coexistência de IgE específica ao Anisakis simplex pode estar relacionada com reacções cruzadas entre os alergénios camarão e ascaridoides. Com base nestes métodos in vitro, a importância clínica da sensibilização aos ascaridoides é díficil de estabelecer nos doentes com alergia a mariscos. Melhor caracterização e purificação dos principais alergénios do Anisakis simplex é imperativa para responder a esta questão.
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