Anaphylactic Reactions to Novel Foods: Case Report of a Child with Severe Crocodile Meat Allergy

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Anaphylactic Reactions to Novel Foods: Case Report of a Child With Severe Crocodile Meat Allergy Natalia Ballardini, MD, PhD,a, b, c Anna Nopp, PhD,d Carl Hamsten, MSc, PhD,d Mirja Vetander, MD, PhD,a, b, e Erik Melén, MD, PhD,a, b Caroline Nilsson, MD, PhD, b,f Markus Ollert, MD, PhD,g, h Carsten Flohr, MD, PhD,i Annette Kuehn, MD, PhD,g Marianne van Hage, MD, PhDd

Availability of “exotic” foods is steadily increasing. In this report, we abstract describe the first case of anaphylaxis to crocodile meat. The patient was a 13-year-old boy with severe immunoglobulin E–mediated allergy to chicken meat. When tasting crocodile meat for the first time, he developed an anaphylactic reaction. Cross-reactivity between chicken and crocodile meat was suspected to have triggered this reaction. Basophil activation and immunoglobulin E testing confirmed the boy’s allergic reaction to crocodile a e meat proteins. Molecular analysis identified a crocodile α-parvalbumin, Institute of Environmental Medicine, Centre for Allergy Research, and fDepartment of Clinical Science with extensive sequence homology to chicken α-parvalbumin, as the and Education, Södersjukhuset, Karolinska Institutet, main cross-reactive allergen. We conclude that crocodile meat can be a Stockholm, Sweden; dClinical Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, and potent food allergen and patients with allergy to chicken meat should be Karolinska University Hospital, Stockholm, Sweden bSachs’ advised to avoid intake of meat from crocodile species. Both foods and Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden; cSt John’s Institute of Dermatology, King’s College people travel around the world and accessibility to exotic foods is steadily London, London, United Kingdom; gDepartment of Infection growing. As a result, novel allergic cross-reactivities are likely to become a and Immunity, Luxembourg Institute of Health, Esch-sur- Alzette, Luxembourg; hDepartment of Dermatology and challenge in the management of food allergy and, as our report illustrates, Allergy Center, Odense Research Center for Anaphylaxis, cross-reactivity has to be considered even between foods that might not University of Southern Denmark, Odense, Denmark; and i intuitively be perceived as related. St John’s Institute of Dermatology, Guy’s and St Thomas’ Hospital NHS Foundation Trust and King’s College London, London, United Kingdom

Dr Ballardini conceptualized and designed the study, was responsible for care of the patient, and drafted the ﬁ rst manuscript; Dr van Hage designed Food allergy is an increasing PRESENTATION the study and was responsible for the initial problem in the pediatric population immunoglobulin E (IgE) analyses, immunoblots, and is estimated to affect 8% of A 13-year-old boy presented to the and cross-inhibition experiments, and critically reviewed and revised the manuscript; Drs Kuehn children with a high negative pediatric emergency department and Ollert planned and carried out the molecular impact on quality of life. 1 – 3 Allergy at Sachs’ Children and Youth experiments, subsequent IgE analysis, immunoblots, to chicken meat has been reported Hospital in Stockholm, Sweden, and cross-inhibition tests, and critically reviewed both in children and adults and is due to an anaphylactic reaction and revised the manuscript; Dr Nopp conceptualized estimated to be rare, but reliable to crocodile meat. He had been a and designed the study, performed basophil allergen threshold sensitivity analysis, and reviewed 4–8 prevalence data are missing. regular outpatient at the hospital’s and revised the manuscript; Dr Hamsten carried Cross reactivity between chicken pediatric allergy department since 5 out the initial IgE analyses, immunoblots, and cross- and duck, turkey, and goose has years of age when he was diagnosed inhibition experiments and critically reviewed and been described.4 Little is known with chicken meat allergy (specific revised the manuscript; Dr Vetander performed about the causative allergens for immunoglobulin E [IgE], 19 kU /L). the literature review and reviewed and revised the A manuscript; Dr Nilsson participated in patient care chicken meat allergy. There are He was otherwise healthy and ate no reports of allergic reactions to all foods except poultry. At 7 years crocodile meat. Although crocodiles of age, an adrenaline autoinjector To cite: Ballardini N, Nopp A, Hamsten C, et al. belong to the reptile group, they was prescribed after an anaphylactic Anaphylactic Reactions to Novel Foods: Case are also the closest living relatives reaction due to accidental Report of a Child With Severe Crocodile Meat Allergy. Pediatrics. 2017;139(4):e20161404 of birds.9 consumption of turkey. Although strict

Downloaded from www.aappublications.org/news by guest on September 24, 2021 PEDIATRICS Volume 139 , number 4 , April 2017 :e 20161404 CASE REPORT avoidance of chicken and turkey meat for content of DNA and protein from crocodile extract at a concentration was recommended, he continued to chicken and turkey. In addition, the of 1 mg inhibitor/mL for immunoblot experience anaphylactic reactions crocodile filet was used to prepare a and 200 μg inhibitor/mL for ELISA. due to accidental consumption of protein extract. 11 Briefly, the muscle Parvalbumins were detected by using foods contaminated by chicken or tissue was grinded in liquid nitrogen an antiparvalbumin IgG antibody mix turkey. As a consequence, he was followed by extraction in lysis buffer (Swant, Marly, Switzerland; Abcam, reluctant to eat foods outside of (50 mM Tris-HCl, pH 8, 150 mM Cambridge, MA). his home, which had a significant NaCl, and 1% Triton X-100). Extract impact on his social life. More supernatants were dialyzed against Biomolecular Allergen recently, his father, a professional phosphate-buffered saline (pH 7.2) Characterization chef, bought and prepared crocodile before basophil activation assay and Chicken and crocodile parvalbumins meat to create a novel meal for his specific IgE–binding analysis. For were isolated by ion exchange and son. The boy reacted at first bite the basophil activation assay and gel filtration chromatography as with itch in the mouth and throat, measurements of allergen-specific previously reported. 7, 14 Protein facial urticaria, conjunctivitis, IgE, muscle tissue was homogenized purity of chicken and crocodile angioedema, chest tightness, and followed by extraction in phosphate- parvalbumins was confirmed by breathing difficulties. Intramuscular buffered saline (pH 7.4). silver-stained, two-dimensional adrenaline and β-2 agonist inhalation SDS-PAGE. 11 Crocodile parvalbumin was administered at home by the Measurement of Allergen-Speciﬁ c isoforms were additionally separated parents. At the pediatric emergency IgE by isoelectric focusing (Offgel, department, he presented with IgE antibodies against chicken Agilent, Diegem, Belgium) before facial urticaria, angioedema around (f83), turkey (f284), and crocodile an intact mass measurement by the eyes, bilateral redness of the meat proteins (5 μg of biotinylated mass spectrometry (MS) analysis. sclera, and heavy breathing but no crocodile extract coupled to Parvalbumins were trypsinized for bronchoconstriction. After 4 hours of Streptavidin ImmunoCAP) and total MS peptide mass fingerprint analysis, 11 observation, he was free of symptoms IgE were measured by ImmunoCAP which revealed the complete and discharged. (Phadia AB/Thermo Fisher Scientific, sequence of 1 crocodile parvalbumin Uppsala, Sweden). isoform. Unidentified peptides of Reviewing the literature, we found the other isoform were sequenced no reports of allergic reactions Basophil Activation Assay de novo on a second MALDI TOF/ to crocodile. We had previously TOF instrument (AB Sciex 5800, identified α-parvalbumin as a Basophil allergen threshold Framingham, MA), identifying 92% relevant chicken meat allergen 7 and sensitivity (CD-sens) was performed 12,13 of the protein sequence. Protein because another report showed that as previously described. Briefly, structures were calculated using parvalbumin is expressed in the tail cells were stimulated with increasing Modeller 9v2 software, 15, 16 and muscle of Alligator mississippiensis, 10 concentrations of crocodile or chicken parvalbumin (P43305; 2KYF) we hypothesized that the reaction chicken extracts (0.5–500 ng was used to establish the surface to crocodile was due to IgE cross- protein/mL) or negative controls model. reactivity between α-parvalbumins in (peanut, birch, casein) and then chicken and crocodile meat. analyzed by flow cytometry. Allergen Analysis by IgE Immunoblot RESULTS and Enzyme-Linked Immunosorbent METHODS No contamination of the crocodile Assay meat by chicken or turkey residues Written informed consent was Chicken and crocodile meat was demonstrated. The boy’s obtained from the patient and his extracts were separated by serum immunoglobulin E (sIgE) parents. SDS-PAGE (sodium dodecyl levels were 40 kUA/L to chicken, sulfate polyacrylamide gel 14 kU /L to turkey, and 6 kU /L Allergen Preparation A A electrophoresis).11 Patient serum to crocodile meat extract, whereas Crocodile filet was purchased was diluted 5× in 3% bovine total serum-IgE was 190 kU/L. from the same store that sold the serum albumin (Sigma-Alderich, CD-sens was clearly positive to both crocodile meat that caused the Bornem, Belgium) for immunoblot crocodile and chicken meat extracts allergic reaction. It was analyzed and 5–10× for IgE enzyme-linked (Fig 1A). In immunoblot analysis, by the certified laboratory of the immunosorbent assay (ELISA). 11 For the patient’s IgE bound to chicken Swedish National Food Agency IgE cross-inhibition, patient serum (14 kDa, 19/22 kDa, 30 kDa) and (Livsmedelsverket; Uppsala, Sweden) was preincubated with chicken or crocodile meat proteins (18–50 kDa)

Downloaded from www.aappublications.org/news by guest on September 24, 2021 e2 BALLARDINI et al (Fig 1B). IgE-binding to chicken phylogenetic relationship. In another allergens was not inhibited by recent publication, we reported on preincubation with crocodile extract, 36 patients (including 17 children whereas IgE-binding was inhibited <14 years old) with chicken meat by preincubation with chicken allergy and showed that 61% had extract ( Fig 1B). Because chicken specific IgE against the allergen Gal d is never consumed raw, an extract 8, chicken parvalbumin. 17 Therefore, from heated chicken meat was also most patients with chicken meat included in the immunoblot analysis. allergy might also be at risk for The patient’s sIgE recognized a single allergic reactions to crocodile meat. 14-kDa chicken protein only, which We believe that chicken meat allergic was confirmed to be parvalbumin by individuals should be informed about using an antiparvalbumin antibody. the potential risk of cross-reactivity Homologous crocodile parvalbumins and advised to avoid meat from were detected as 2 bands at 6 and 14 crocodile species. kDa (potentially 2 isoforms, denoted Both foods and people travel around “6-kDa” and “14-kDa parvalbumin”). the world and accessibility to “exotic” The patient’s sIgE levels were foods is steadily growing. Food safety 108 kU /L to purified chicken A FIGURE 1 is an important aspect to be addressed α-parvalbumin and 25 kU /L to A Basophil reactivity and immunoblot with chicken before the introduction of these new crocodile 14-kDa parvalbumin. The and crocodile meat extracts. A, Basophil reactivity products to the market. 18 Assessment selective IgE reactivity to crocodile (CD-sens) against crocodile (○) and chicken (▪) of potential allergenicity of novel foods parvalbumins was confirmed by IgE extracts. CD-sens was also performed against peanut, birch, and casein as negative controls is highly challenging because it is not immunoblot, where the patient’s IgE and basophils from a nonallergic blood donor well understood what makes a food antibodies only detected the 14-kDa were tested against crocodile and chicken protein a potent allergen. However, as but not the 6-kDa isoform ( Fig 1B). extracts with negative results (not shown in this case illustrates, additional research After preincubation of serum with ﬁ gure). B, Detection of IgE-reactive chicken and crocodile proteins. Patient serum (P) was concerning allergic cross-reactivities the respective homolog, IgE reactivity analyzed for IgE reactivity to raw/heated meat is needed so that clinicians will be able to chicken α-parvalbumin and extracts and puriﬁ ed crocodile parvalbumin to give accurate advice to food-allergic 14-kDa crocodile parvalbumin was by immunoblot. IgE cross-inhibition (Pinh) was tested by serum preincubation with chicken patients intending to broaden their efficiently inhibited (94% and 100%, and crocodile extracts before immunoblot. +, diet. respectively). Amino acid sequencing antiparvalbumin antibody (arrows); ★, IgE- revealed the complete sequence of reactive proteins; M, molecular weight marker. the 14-kDa (108/108 amino acids) CONCLUSIONS and most of the 6-kDa crocodile parvalbumin (100/108 amino acids). and crocodile meat. Our hypothesis Crocodile meat can be a potent The protein identity of the 14-kDa of clinical cross-reactivity via highly food allergen. Because patients crocodile parvalbumin compared conserved B cell epitopes on these with allergy to chicken meat are with the chicken homolog was 94%, parvalbumins could be confirmed at risk for reacting to crocodile and a molecular model comparison by IgE cross-inhibition assays meat, clinical testing with crocodile visualized extensive surface identity using the purified chicken and meat may need to be included in of both allergens (Fig 2). crocodile allergens. Furthermore, the diagnostic workup of these we showed extensive sequence individuals. Novel allergic cross- identity representing putative reactivities are likely to become a challenge in the management DISCUSSION linear epitopes and high structural homology representing putative of food allergy and, as our report To the best of our knowledge, this conformational epitopes. Although illustrates, cross-reactivity has is the first report of a food-allergic chicken was assumed to be the to be considered even between reaction to crocodile meat. We primary sensitization source, the foods that might not intuitively be demonstrate that this anaphylactic patient had experienced anaphylactic perceived as related. reaction, confirmed by the positive reactions to turkey as well. These basophil challenge (CD-sens) with reactions can be explained by our ACKNOWLEDGMENTS crocodile meat, was due to cross- previous finding that chicken and reactivity of a highly homologous turkey α-parvalbumin are 100% We thank M. Sandberg and Y. Sjögren α-parvalbumin found in both chicken identical, 7 which reflects their close Bolin at the Swedish National Food

Downloaded from www.aappublications.org/news by guest on September 24, 2021 PEDIATRICS Volume 139 , number 4 , April 2017 e3 ABBREVIATIONS CD-sens: basophil allergen threshold sensitivity ELISA: enzyme-linked immunosorbent assay IgE: immunoglobulin E MS: mass spectrometry sIgE: serum immunoglobulin E

FIGURE 2 Comparison of chicken and crocodile α-parvalbumin. A, Three-dimensional model of chicken parvalbumin, protein surface coloring by identity to crocodile parvalbumin. Red, identical residues; gray, variable residues. B, Amino acid sequence alignment of chicken and crocodile parvalbumins as allergens involved in the clinical cross-reaction to both meats.

Agency (Uppsala, Sweden) for and turkey DNA by real time PCR. We also thank Dr K. Arumugam analysis of crocodile meat for (1) We thank D. Revets (Luxembourg (Luxembourg Institute of Health) for detection of poultry protein by use Institute of Health) for excellent establishing the structural model of of ELISA and (2) detection of chicken technical support with MS analysis. chicken and crocodile parvalbumin. and reviewed and revised the manuscript; Drs Melén and Flohr provided intellectual input and reviewed and revised the manuscript; and all authors approved the fi nal manuscript as submitted. DOI: 10.1542/peds.2016-1404 Accepted for publication Sep 22, 2016 Address correspondence to Natalia Ballardini, MD, PhD, Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, SE-171 77 Stockholm, Sweden. E-mail: [email protected] PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright © 2017 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no fi nancial relationships relevant to this article to disclose. FUNDING: This work was supported by research grants from the Swedish Research Council, the Stockholm County Council, the Swedish Asthma and Allergy Association’s Research Foundation, the Swedish Cancer and Allergy Foundation, the Hesselman Foundation, Karolinska Institutet, the Karin and Sten Mörtstedt Initiative on Anaphylaxis, and the Ministry of Higher Education and Research in Luxembourg. POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential confl icts of interest to disclose.

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Updated Information & including high resolution figures, can be found at: Services http://pediatrics.aappublications.org/content/early/2017/03/06/peds.2 016-1404 References This article cites 18 articles, 2 of which you can access for free at: http://pediatrics.aappublications.org/content/early/2017/03/06/peds.2 016-1404#BIBL Subspecialty Collections This article, along with others on similar topics, appears in the following collection(s): Allergy/Immunology http://www.aappublications.org/cgi/collection/allergy:immunology_s ub Permissions & Licensing Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://www.aappublications.org/site/misc/Permissions.xhtml Reprints Information about ordering reprints can be found online: http://www.aappublications.org/site/misc/reprints.xhtml

Downloaded from www.aappublications.org/news by guest on September 24, 2021 Anaphylactic Reactions to Novel Foods: Case Report of a Child With Severe Crocodile Meat Allergy Natalia Ballardini, Anna Nopp, Carl Hamsten, Mirja Vetander, Erik Melén, Caroline Nilsson, Markus Ollert, Carsten Flohr, Annette Kuehn and Marianne van Hage Pediatrics originally published online March 8, 2017;

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