IgE Mediated Shellfish Allergy in Children—A Review

Shellfish is a leading cause of food allergy and anaphylaxis worldwide. Recent advances in molecular characterization have led to a better understanding of the allergen profile. High sequence homology between shellfish species and between shellfish and house dust mites leads to a high serological cross-reactivity, which does not accurately correlate with clinical cross-reactions. Clinical manifestations are immediate and the predominance of perioral symptoms is a typical feature of shellfish allergy. Diagnosis, as for other food allergies, is based on SPTs and specific IgE, while the gold standard is DBPCFC. Cross-reactivity between shellfish is common and therefore, it is mandatory to avoid all shellfish. New immunotherapeutic strategies based on hypoallergens and other innovative approaches represent the new frontiers for desensitization.


Introduction
Shellfish allergy is one of the main food allergies worldwide [1] and a leading cause of food-induced anaphylaxis [2]. This review aims to summarize the epidemiological aspects, clinical manifestations and management of shellfish allergy.

Taxonomy
Shellfish is defined as any edible marine invertebrate. Crustaceans are a subphylum of the phylum Arthropoda. Commonly consumed crustaceans, such as shrimps (or prawns, a synonym of shrimp for those of larger size and less curved), crabs, lobsters and crayfish, belong to the order Decapoda, literally "ten-footed". Decapods have five pairs of legs on the main thoracic body along with five pairs of swimming legs on the abdomen and share a close evolutionary relationship to arachnids (dust mites, spiders, etc.) and insects (cockroaches, edible insects) [3].
Mollusks belong to the phylum Mollusca. Mollusks commonly consumed by humans belong to the classes Bivalvia, Gastropoda and Cephalopoda. Bivalves (clams, mussels) have a shell made of two systems called valves, which are generally symmetrical. Gastropods (sea slug, sea snails) are characterized by a shell without bilateral symmetry. Cephalopods (cuttlefish, squid, octopus) have a shell that is internally reduced or completely absent and a voluminous head [4].

Epidemiology
The prevalence of shellfish allergy in the Western world is approximately 0.5% [5][6][7]. Shellfish allergy often occurs in late childhood or adolescence. Therefore, the prevalence of Table 1. Epidemiological data about shellfish allergy in children in different countries from 2012 to 2022. SR: self/parent report, PC: physician confirmation through skin prick tests, sIgE or oral food challenge.

Country
Year
The threshold triggering the allergic reaction depends on the patient; in fact, high thresholds of reactivity have been reported, as well as severe reactions after the intake of traces of shellfish. It has been reported that a woman with shrimp allergy developed anaphylaxis 1 min after kissing her boyfriend, who had eaten shrimp <1 h before [44,45,47]. Furthermore, shellfish has been shown to be a trigger of exercise-induced food-dependent anaphylaxis in children [48,49].
Sensitization to shellfish occurs not only by oral intake but also by skin contact and inhalation of aerosolized particles during processing or cooking. Inhalation sensitization typically occurs in workers exposed to shellfish particulates resulting from several processing activities [50][51][52]. Occupational inhalation exposure leads to rhinoconjunctivitis, nasal pruritus, asthma, coughing, urticaria, rash and hardly systemic reactions [53][54][55][56][57]. The presence of shellfish-induced occupational respiratory symptoms increases the risk of developing allergic reactions to ingested shellfish [58]. Occupational exposure may also induce contact urticaria or contact dermatitis [54,55,59].
Contrasting data on rates of tolerance development in children with shellfish allergy have been provided. Natural resolution after 5-10 years has been described in a percent-age of patients ranging from 3.9% in a questionnaire study including both adults and children [60] up to 46% in a study conducted with an oral challenge in children with non-anaphylactic reactions [61]. It is unclear whether the latter findings are restricted to a subgroup of children.

Allergens
Many allergens have been characterized in several species of crustaceans and mollusks and are registered with the International Union of Immunological Societies (WHO/IUIS Allergen Nomenclature Database). The main shellfish allergens are reviewed in Table 2.
The homology of the amino acid sequence of tropomyosin between crustaceans is 88-100%, between crustaceans and insects or mites is about 80%, between crustaceans and mollusks is 55-65% and between crustaceans and fish is 55%. The homology between mollusks ranges from 70% to 98% and between mollusks and fish is about 55%. A homology greater than 80% should be regarded as potentially cross-reactive [6].
Microarray techniques have enabled describing linear peptides involved in sensitization to allergens. To date, eight epitopes of tropomyosin have been identified [71].

Arginine Kinase
Arginine kinase (AK) is an enzyme identified in several crustaceans and a mollusk and it is involved in the regulation of cellular ATP levels [70]. Shrimp AK (Pen m 2) is the second most clinically relevant allergen following tropomyosin, as 10-20% of shellfishallergic patients are sensitized to arginine kinase [45,77]. AK is less resistant to heat than tropomyosin [78,79]. The volatility could be responsible for symptoms induced by the inhalation of vapors [80,81].
Arginine kinase is involved in cross-reactivity between shellfish and edible insects [82].

Myosin Light Chain
Myosin light chain (MLC) is a part of the myosin macromolecular complex in muscle proteins [83]. The frequency of sensitization to this enzyme in shellfish-allergic patients varies between 19 and 55% [68,84]. Shellfish MLC is heat-and pH-stable [85]. Generally, a sensitization to MLC is found together with a sensitization to tropomyosin. However, some reports describe cases of shrimp allergy in which the shrimp MLC (Pen m 3) was the only responsible allergen [77,83,86].

Sarcoplasmic Calcium-Binding Protein
Sarcoplasmic calcium-binding protein (SCP) is a highly heat-resistant and stable protein [87,88]. Sensitization to SCP (Pen m 4) often accompanies sensitization to tropomyosin and is common in children, having been observed in up to 85% of pediatric patients [70,89].

Cross-Reactivity
There is a major risk of cross-sensitization and clinical cross-reactivity between crustaceans, mollusks, house dust mites and insects [79]. Tropomyosin is the major allergen implicated in cross-reactivity [52,71,76,102,103]. The high frequency of cross-reactivity complicates the identification of the primary culprit allergen both with skin allergy testing (SPTs) and molecular testing (sIgE) [104][105][106].
Tropomyosin of shellfish also shares homologies in sequence with tropomyosin of other invertebrates, including house dust mites (Der p 10). Sensitization to dust mites via inhalation has been hypothesized to secondarily trigger cross-reactivity to shellfish. This route of sensitization may also explain the late age of onset and the predominance of oral symptoms typical of shellfish allergy [72,76]. The data are supported by the high sensitization to both dust mites and shellfish in atopic populations [112][113][114][115]. On the contrary, sensitization to dust mites as cross-reaction after shellfish ingestion has also been proposed but less frequently [76,97].
Insects are largely consumed in China. In Europe, it was recently allowed to use some insects such as mealworms, crickets and locusts as foods. Patients with allergy to shrimp or house dust mites are at risk of developing clinical allergy to edible insects [116,117]. Most shrimp-allergic patients are sensitized to mealworm, although they did not previously eat it [118]. Co-sensitization to tropomyosin, arginine kinase, myosin light chain and triosephosphate isomerase probably explain cross-reactivity among insects, shrimp and mites.
There is limited evidence of cross-reactions between shellfish and fish, even though up to about 20% of adults with mollusk allergy have self-reported to be allergic to fin fish [30,119]. This coexistence may be explained by the homology between fish and shellfish tropomyosin. Overall, patients with shellfish allergy should not follow a fish-free diet [119,120].

Diagnosis
A thorough medical history, including ingested shellfish species, interval time after ingestion, symptoms, timing of resolution and treatment, is essential to properly target allergy testing. Diagnostic workup is made difficult by the wide variety of shellfish species and by cross-reactivity between shellfish. Furthermore, sensitization may be primary or secondary to cross-reactivity among homologous proteins of other invertebrates such as cockroaches and house dust mites.
The determination of serum-specific IgE could also prove useful to demonstrate sensitization. Specific IgEs are highly influenced by cross-reactivity in vitro, without clinical correlates. A negative predictive value of IgE to shrimps (91.3%) is comparable to that of SPTs (90%), while a positive predictive value seems higher (41-71% for IgE, 33.3% for SPTs) but still inaccurate [69,123].
Molecular diagnosis may be a promising approach to increase in vitro diagnostic accuracy [68,84,109,124]. Tropomyosin sensitization is critical in the diagnosis of shellfish allergy although other allergens can be involved [71]. Two shrimp tropomyosin are commercialized, Pen a 1 and Pen m 1. Shrimp tropomyosin (Pen m 1) is the major allergen in shrimp-sensitized patients. However, the clinical significance of positive IgE to Pen m 1 is unclear since the frequency of sensitization to Pen m 1 ranged from 34% to 63% in populations with shrimp allergy from Hong Kong, Thailand, Japan, Brazil, Spain and Italy [84,109,[125][126][127].
In Hong Kong, in patients with shrimp allergy, the area under the curve of shrimp SPTs was 0.74, that of specific IgE to shrimp was 0.75, that of Pen m 1-sIgE was 0.70, that of Pen m 4-sIgE was 0.77, that of Pen m 6-sIgE was 0.78, that of Pen m 13-sIgE (fatty acid-binding protein) was 0.77 and that of Pen m 14-sIgE (glycogen phosphorylase) was 0.59; the areas under the curve of the same IgEs in Thailand were 0.7, 0.7, 0.89, 0.96, 0.86, 0.81 and 0.54, respectively [128]. While for other food allergies, a link between specific epitopes and allergic reactivity (for example persistent or more severe allergy) has been found, in shellfish allergy, the clinical significance of different allergens is not yet clear. Also, the recognition of epitopes differs between children and adults [70].
Basophil activation test for shrimp reached an area under the curve of 0.88 in a Chinese population with shrimp allergy [125].
A recent study first assessed the diagnostic value of the nasal allergen provocation test (NAPT) in shellfish allergy diagnosis, showing that it differentiated between shrimp-allergic and -tolerant subjects with high sensitivity (90%) and specificity (89%) [86].
The use of the ExiLe technology is also under study. ExiLe is based on a rat basophilic leukemia cell line transfected with the a/b/g subunits of the human IgE receptor FceRI and the luciferase reporter gene (RS-ATL8). The test is based on the idea that the measurement of luciferase's signal reflects the degree of IgE crosslinking [129]. ExiLe technology has been promisingly demonstrated to have better diagnostic accuracy than SPTs and sIgE [125,130].
Oral food challenge remains the gold standard for confirming the diagnosis of food allergies, although it is time-consuming and expensive and carries the risk of severe allergic reactions. An initial dose of 3 mg of shrimp proteins has been proposed. The dose is then increased every 15 to 30 min [131][132][133]. The protocol should be individualized to achieve the recommended daily dose based on the age of the patient. On average, about 0.1-1.0 g of shellfish pulp must be ingested to trigger an allergic response [111].
The gold standard is the double-blind placebo-controlled food test (DBPCFC). In clinical practice, tests are usually performed open unless there are diagnostic doubts.

Differential Diagnosis
Non-IgE-mediated forms of shellfish allergy, mainly represented by food proteininduced enterocolitis syndrome (FPIES), are also described [134]. Clinical manifestations of FPIES are different from IgE-mediated symptoms and consist of profuse vomiting, typically 1-3 h after ingestion, diarrhea, pallor, hypothermia and hypotension or flaccidity.
Adverse reactions to shellfish may also be caused by non-immunologic mechanisms. As filter feeders, these shellfish may accumulate bacteria (e.g., Vibrio, Klebsiella), viruses (e.g., Hepatitis A) and toxins produced by algae (shellfish poisoning syndromes). Shellfish may also be infested by Anisakis, a parasitic nematode mainly found in fish, but also in large crustaceans and cephalopods. The parasite is ingested while eating raw or undercooked seafood, then fails to reproduce in the human host and dies in about 3 weeks. The acute gastric form, occurring 2-8 h after the ingestion, consists of diffuse epigastric pain, fever, nausea and vomiting. Chronic intestinal anisakiasis occurs 5-7 days after ingestion, following the attachment of the larvae to the intestinal mucosa with the formation of granulomas and abscesses in the intestinal wall. Patients previously exposed to Anisakis may also develop hypersensitivity to worm antigens with the development of allergic reactions or anaphylaxis upon re-exposure to live or dead larvae [111,[135][136][137][138][139][140][141][142][143][144]. Different diagnoses of shellfish allergy are reviewed in Table 3.

Management
Management of shellfish allergy is based on avoidance of shellfish and treatment with rescue medication in case of an allergic episode. Some patients with shellfish allergy can tolerate particular crustaceans or mollusks. However, cross-reactivity is frequent, so patients with crustacean or mollusk allergy should avoid all shellfish species to which they are sensitized also because of contamination risk, unless tolerance is demonstrated by food challenge. Contact with shellfish cooking vapors should also be avoided. In case of severe reactions, the prescription of adrenaline autoinjectors with a personalized emergency action plan is recommended.
The goal of novel therapies for food allergy is to desensitize patients and restore food tolerance in order to improve patients' quality of life. Allergen-specific immunotherapy (AIT) involves the administration of gradually increasing amounts of allergen extracts to induce desensitization. AIT for shrimp has not yet been introduced in clinical practice. Preliminary data suggest that it is safe and well tolerated [145]. The main problem with this approach is that shrimp extracts are heterogeneous in allergen content, leading to diverse responses. A recent study reported the safety and efficacy of omalizumab-facilitated oral immunotherapy for shrimp allergy [146]. Allergen-specific AIT with tropomyosin has been studied in a murine model, showing successful desensitization, but with potentially serious side effects [147].
Since tropomyosin is highly allergenic, research in shellfish immunotherapy has also focused on the development of hypoallergenic variants of tropomyosin to improve the safety of AIT. Hypoallergenic tropomyosin is obtained through methods including enzymatic crosslinking, polypeptide fragmentation and epitope manipulation [130,148,149].
AIT with linear peptides corresponding to T-cell epitopes is also under study [150][151][152]. A recently proposed therapeutic approach is based on T-cell epitopes and CpG-ODN agonist of Toll-like receptor 9 (TLR9) in nanoparticles. CpG-ODN is a TLR9 ligand known to downregulate the established Th2 response and induce Th1 immunity [153,154].
The correlation between shellfish and dust mites suggests the possibility that AIT for dust mites may lead to the improvement of shellfish allergy [155,156]. On the other hand, cases have been reported of shrimp allergy following mite-specific immunotherapy [157][158][159].
Anti-IgE therapies such as omalizumab and anticytokine drugs are traditional nonspecific treatments that can be used alone or in combination with AIT for desensitization.

Conclusions
Shrimp allergy is an increasing worldwide problem affecting not only adults but also children, and is a frequent cause of anaphylaxis. Molecular characterization may potentially allow a better description of shellfish allergenic profiles. The clinical relevance of the different allergens and epitopes remains to be determined and could lead to improvements in diagnostic and therapeutic approaches. More reliable and specific diagnostic tools that correlate with clinical reactivity are needed. New immunotherapeutic strategies based on hypoallergens and other innovative approaches represent the new frontiers for desensitization.