# B09 – Edible insects

Kitty C. M. Verhoeckx, André C. Knulst

## Highlights

* Clinically relevant cross-reactivity between mealworm and shrimp has been found.

* Primary sensitization to insects (mealworms) is possible.

* Important allergenic proteins are tropomyosin and arginine kinase (cross-reactivity).

* Currently good diagnostic tools for insect food allergy are missing.

* In case of unclear clinical history and serology, food challenges are necessary to confirm the diagnosis.

## 1 – The allergen sources

With an increasing world population and demand for sustainable food sources, insects are a promising alternative source of protein.⁹⁰⁷ Almost 2000 insect species are consumed globally by approximately two billion people.⁹⁰⁸ Insects are consumed in Asia, Latin America, and Africa. Entomophagy is not yet common practice in Europe and North America (FAO, 2013). Nevertheless, people already unknowingly ingest approximately 500g of insect traces per year.⁹¹⁰ The top eight most frequently consumed insect orders are Coleoptera (beetles), Lepidoptera (caterpillars), Hymenoptera (ants, wasps, and bees), Orthoptera (locusts, grasshoppers, and crickets), Hemiptera (leafhoppers, plant hoppers, cicadas, scale insects, and true bugs), Odonata (dragonflies), Isoptera (termites), and Dyptera (flies).

Because insects were not frequently eaten in Europe before May 1997, they are classified as novel food by the EU Commission.⁹¹¹ Recently the EU Commission approved the introduction of the yellow mealworm (*Tenebrio molitor*) on the food market.⁹¹² Insects such as the yellow mealworm consist mainly of protein, fat and fibre and are proposed to be consumed as a whole, dried insect or in the form of powder, added to various products such as energy bars, pasta, and biscuits. Information on adverse reactions after eating insects is scarce, they are sporadically reported in case reports. The prevalence of food allergy to insects was only described in three population studies. In Laos a prevalence of 7.6% was found under entomophagists consuming insects,⁹¹³ in China 18% of reported cases of anaphylaxis to food was related to the ingestion of insects⁹¹⁴ and in Korea 3.1% of food allergic patients were allergic to silkworm.⁹¹⁵ More information can be found on allergic reactions caused by insect sting bites or inhalant allergies due to insect exposure, e.g., to cockroach. For information on these allergies we refer to other chapters (B05, B20, and B21).

## 2 – Allergen families

According to the simplified phylogenic tree (Figure 76) insects are part of the Arthropoda phylum and closely related to Crustacea (shrimp and lobster) and to the subclass Acari (house dust mite), which are (food) allergenic sources. They contain the well-known invertebrate pan-allergens tropomyosin and arginine kinase.⁹¹⁶

Tropomyosin belongs to the tropomyosin family and has been identified as a major allergen in house dust mite (Der p 10), crustaceans (Pen m 1), moths (Bomb m 3), and cockroach (Bla g 7) but also in herring worm *Anisakis simplex* (Ani s 3) and common roundworm *Ascaris lumbricoides* (Asc I 3) (see also chapters B04, B05, B12, B13, C05). Until now 39 tropomyosins have been registered, 27 as food allergen, 11 as airway allergen and one as injection allergen, according to the WHO/IUIS allergen nomenclature subcommittee. Tropomyosin typically consists of two parallel alpha-helical tropomyosin molecules that are wound around each other forming a coiled-coil dimer and are characterized by high amino acid (AA)-sequence identity (Figure 77).³³⁴ Arginine kinase, belongs to the ATP guanido phosphotransferase family, and is an enzyme present in insects and crustaceans. According to the WHO-IUIS allergen nomenclature committee until now 13 arginine kinases have been registered as allergens, 7 food allergens (crab (Cal b 2, Scy p 2), shrimp (Pen m 2, Cra c 2, Lit v 2), crayfish (Pro c 2), silk moth (Bomb m 1), 6 airway allergens (cockroach (Bla g 9, Per a 9), house dust mite (Der p 20, Der f 20), Indian meal moth (Plo I 1), and storage mite (Tyr p 20). Arginine kinases have a highly conserved amino acid sequence among various invertebrate species that are characterized by a β-sheet domain surrounded by α-helices.⁹¹⁷ No 3D structure of insect arginine kinase is currently available.

## 3 – Allergenic molecules (epidemiology including geography and function)

In UniProt many isoforms of tropomyosin (7566 entries) and arginine kinase (8754 entries) can be found. Of these, officially only silkworm (Bombyx mori) arginine kinase (Bomb m 1) and tropomyosin (Bomb m 3) are recognized by the WHO-IUIS (www.allergen.org) database as an insect food allergen.

Mealworms (*Tenebrio molitor*) were recently authorized by the European Commission as (novel) food for humans. IgE binding to many different proteins from mealworm was described, namely: tropomyosin, arginine kinase, paramyosin, chitinase, troponin C, myosin light and heavy chain, hexamerin, α-amylase, trypsin-like proteinase, cockroach-like allergen and larval cuticle protein. These proteins were identified using immunoprecipitation with serum from shrimp and mealworm allergic patients and LC-MS⁹¹⁸,⁹¹⁹ (Figure 78).
# B09 – Edible insects

Kitty C. M. Verhoeckx, André C. Knulst

## Highlights

* Clinically relevant cross-reactivity between mealworm and shrimp has been found.

* Primary sensitization to insects (mealworms) is possible.

* Important allergenic proteins are tropomyosin and arginine kinase (cross-reactivity).

* Currently good diagnostic tools for insect food allergy are missing.

* In case of unclear clinical history and serology, food challenges are necessary to confirm the diagnosis.

## 1 – The allergen sources

With an increasing world population and demand for sustainable food sources, insects are a promising alternative source of protein.⁹⁰⁷ Almost 2000 insect species are consumed globally by approximately two billion people.⁹⁰⁸ Insects are consumed in Asia, Latin America, and Africa. Entomophagy is not yet common practice in Europe and North America (FAO, 2013). Nevertheless, people already unknowingly ingest approximately 500g of insect traces per year.⁹¹⁰ The top eight most frequently consumed insect orders are Coleoptera (beetles), Lepidoptera (caterpillars), Hymenoptera (ants, wasps, and bees), Orthoptera (locusts, grasshoppers, and crickets), Hemiptera (leafhoppers, plant hoppers, cicadas, scale insects, and true bugs), Odonata (dragonflies), Isoptera (termites), and Dyptera (flies).

Because insects were not frequently eaten in Europe before May 1997, they are classified as novel food by the EU Commission.⁹¹¹ Recently the EU Commission approved the introduction of the yellow mealworm (*Tenebrio molitor*) on the food market.⁹¹² Insects such as the yellow mealworm consist mainly of protein, fat and fibre and are proposed to be consumed as a whole, dried insect or in the form of powder, added to various products such as energy bars, pasta, and biscuits. Information on adverse reactions after eating insects is scarce, they are sporadically reported in case reports. The prevalence of food allergy to insects was only described in three population studies. In Laos a prevalence of 7.6% was found under entomophagists consuming insects,⁹¹³ in China 18% of reported cases of anaphylaxis to food was related to the ingestion of insects⁹¹⁴ and in Korea 3.1% of food allergic patients were allergic to silkworm.⁹¹⁵ More information can be found on allergic reactions caused by insect sting bites or inhalant allergies due to insect exposure, e.g., to cockroach. For information on these allergies we refer to other chapters (B05, B20, and B21).

## 2 – Allergen families

According to the simplified phylogenetic tree (Figure 76) insects are part of the Arthropoda phylum and closely related to Crustacea (shrimp and lobster) and to the subclass Acari (house dust mite), which are (food) allergenic sources. They contain the well-known invertebrate pan-allergens tropomyosin and arginine kinase.⁹¹⁶

Tropomyosin belongs to the tropomyosin family and has been identified as a major allergen in house dust mite (Der p 10), crustaceans (Pen m 1), moths (Bomb m 3), and cockroach (Bla g 7) but also in herring worm *Anisakis simplex* (Ani s 3) and common roundworm *Ascaris lumbricoides* (Asc I 3) (see also chapters B04, B05, B12, B13, C05). Until now 39 tropomyosins have been registered, 27 as food allergen, 11 as airway allergen and one as injection allergen, according to the WHO/IUIS allergen nomenclature subcommittee. Tropomyosin typically consists of two parallel alpha-helical tropomyosin molecules that are wound around each other forming a coiled-coil dimer and are characterized by high amino acid (AA)-sequence identity (Figure 77).³³⁴ Arginine kinase, belongs to the ATP guanido phosphotransferase family, and is an enzyme present in insects and crustaceans. According to the WHO-IUIS allergen nomenclature committee until now 13 arginine kinases have been registered as allergens, 7 food allergens (crab (Cal b 2, Scy p 2), shrimp (Pen m 2, Cra c 2, Lit v 2), crayfish (Pro c 2), silk moth (Bomb m 1), 6 airway allergens (cockroach (Bla g 9, Per a 9), house dust mite (Der p 20, Der f 20), Indian meal moth (Plo I 1), and storage mite (Tyr p 20). Arginine kinases have a highly conserved amino acid sequence among various invertebrate species that are characterized by a β-sheet domain surrounded by α-helices.⁹¹⁷ No 3D structure of insect arginine kinase is currently available.

## 3 – Allergenic molecules (epidemiology including geography and function)

In UniProt many isoforms of tropomyosin (7566 entries) and arginine kinase (8754 entries) can be found. Of these, officially only silkworm (Bombyx mori) arginine kinase (Bomb m 1) and tropomyosin (Bomb m 3) are recognized by the WHO-IUIS (www.allergen.org) database as an insect food allergen.

Mealworms (*Tenebrio molitor*) were recently authorized by the European Commission as (novel) food for humans. IgE binding to many different proteins from mealworm was described, namely: tropomyosin, arginine kinase, paramyosin, chitinase, troponin C, myosin light and heavy chain, hexamerin, α-amylase, trypsin-like proteinase, cockroach-like allergen and larval cuticle protein. These proteins were identified using immunoprecipitation with serum from shrimp and mealworm allergic patients and LC-MS⁹¹⁸,⁹¹⁹ (Figure 78).
<figure><img src="image_175.png" /><figcaption>FIGURE 76 Simplified representation of the phylogenetic relationship between insects, mites, and crustaceans</figcaption></figure>

Allergenic proteins where also reported for locust (Patanga succincta): arginine kinase, enolase, GAPDH, hexamerin and pyruvate kinase⁹²⁰; for cricket: arginine kinase, hexamerin-like protein 2, tropomyosin⁹²¹⁻⁹²⁴ and for Indian meal moth: arginine kinase.⁷⁵⁵ Table 31 only lists allergenic proteins where information on prevalence of IgE binding was previously reported.

Below the different putative allergens are briefly described. Tropomyosin is a muscle protein that, together with myosin and actin, is involved in muscle contraction.

Arginine kinase is an enzyme present in insects and crustaceans and contributes to cellular homeostasis by catalysing the transfer of phosphate between ATP and arginine.⁹²⁵

Other putative allergenic proteins in silkworms are paramyosin, chitinase⁹²⁶ and a 27-kDa glycoprotein.⁹²⁷ Paramyosin is a myosin filament-related protein found in the striated muscle of invertebrates, which plays an important role in the process of myosin filament assembly and mainly acts as a major muscle component in invertebrates. Paramyosin belongs to the paramyosin family. Paramyosin is known as an inhalation allergen in mites (BLOT 11, Der f 11, and Der p 11) and as a food allergen in Veined rapa whelk, *Rapana venosa*, a sea snail (Rap v 2) and *Anisakis simplex* (Anis s 2).

Chitinase belongs to the chitinase family (family 18 of glycoside hydrolases (GH18) and is a component of the exoskeletal of
<figure><img src="image_175.png" /><figcaption>FIGURE 76 Simplified representation of the phylogenetic relationship between insects, mites, and crustaceans</figcaption></figure>

Allergenic proteins where also reported for locust (*Patanga succincta*): arginine kinase, enolase, GAPDH, hexamerin and pyruvate kinase⁹²⁰; for cricket: arginine kinase, hexamerin-like protein 2, tropomyosin⁹²¹⁻⁹²⁴ and for Indian meal moth: arginine kinase.⁷⁵⁵ Table 31 only lists allergenic proteins where information on prevalence of IgE binding was previously reported.

Below the different putative allergens are briefly described. Tropomyosin is a muscle protein that, together with myosin and actin, is involved in muscle contraction.

Arginine kinase is an enzyme present in insects and crustaceans and contributes to cellular homeostasis by catalysing the transfer of phosphate between ATP and arginine.⁹²⁵

Other putative allergenic proteins in silkworms are paramyosin, chitinase⁹²⁶ and a 27-kDa glycoprotein.⁹²⁷ Paramyosin is a myosin filament-related protein found in the striated muscle of invertebrates, which plays an important role in the process of myosin filament assembly and mainly acts as a major muscle component in invertebrates. Paramyosin belongs to the paramyosin family. Paramyosin is known as an inhalation allergen in mites (BLOT 11, Der f 11, and Der p 11) and as a food allergen in Veined rapa whelk, *Rapana venosa*, a sea snail (Rap v 2) and *Anisakis simplex* (Anis s 2).

Chitinase belongs to the chitinase family (family 18 of glycoside hydrolases (GH18) and is a component of the exoskeletal of
arthropods. Chitinase is a hydrolytic enzyme that breaks down glycosidic bonds in chitin.⁹²⁸ Allergenic chitinase (inhalant and food) can be found in mites (e.g., Der p 15) and in cockroach (Bla g 12 and Per a 12), fruits such as bananas (Mus a 2), pomegranate (Pun g 14) and avocado (Pers a 1) (see chapter B15).

Troponin C belongs to the troponin C family and is the calcium-sensing component of the troponin complex that can attach to and detach from tropomyosin. Troponin C is involved in muscle

<figure><img src="image_176.png" /><figcaption>FIGURE 77 3D structure of tropomyosin from *Bombyx mori* (Q1HPU0 (TPM1_BOMMO), Q1HPU0 | SWISS-MODEL Repository (expasy.org)</figcaption></figure>

contraction.⁹²⁹ Troponin C is also recognized as a food allergen in shrimp (Cra c 6 and Pen m 6). Myosin light and heavy chain belonging to the myosin family, are part of the sarcomeric units in muscle tissue. Myosin is involved in muscle contraction.⁹²⁹ Myosin light chain is also recognized as a food allergen in various shrimp species (Art fr 5, Cra c 5, Lit v 3, and Pen m 3).

Hexamerin belongs to the tyrosinase family, closely related to arthropod hemocyanins, is present in insect haemolymph as a storage protein and sometimes has a transporter function. In addition, hexamerin is incorporated in the cuticle and possibly involved in humoral immune defence.⁹³⁰ Alpha-amylase belongs to the glycoside hydrolase 13 (GH13) family and serves as a key digestive enzyme in most insects. Alpha-amylase hydrolyses starch into simple sugar units, which provides energy for survival and the development of insects.⁹³¹ Allergenic α-amylases can be found in mites (Der p 4, Der f 4), cockroach (Bla g 1, Per a 11), all inhalation allergens and as food allergens in barley (Hor v 16).

Trypsin like proteinase belongs to the proteases of the mixed nucleophile, superfamily A and is involved in protein digestion. Allergenic trypsin is mostly found in mites (Blo t 3, Der f 3, and Der p 3) as an inhalant allergen. Cockroach like allergen is a nitrile-specifier protein with a detoxifying function localized in the midgut

<figure><img src="image_177.png" /><figcaption>FIGURE 78 Allergen components from silkworm and mealworm. The same colour is used for related proteins.</figcaption></figure>

<table><caption>TABLE 31 Relevant insect allergens *unpublished data</caption><thead><tr><th>Species</th><th>Allergenic molecule</th><th>Protein family</th><th>Frequency of IgE</th><th>MW (kDa)</th></tr></thead><tbody><tr><td rowspan="2">Silkworm</td><td>Bomb m 1</td><td>Arginine Kinase</td><td>100% (n=10)<sup>1705</sup> [doi:10.1159/000210375]</td><td>42</td></tr><tr><td>Bomb m 3</td><td>Tropomyosin</td><td>53.3% (n=15)<sup>1706</sup> [doi:10.3892/mmcr.2017.6373]</td><td>38</td></tr><tr><td rowspan="2">Mealworm</td><td>NA</td><td>Arginine Kinase</td><td>23.1% (n=13)*</td><td>27</td></tr><tr><td>NA</td><td>Tropomyosin</td><td>76.9% (n=13)*</td><td>34</td></tr><tr><td>Tenebrio melitor</td><td>NA</td><td>LCP AIA</td><td>100% (n=2)<sup>915</sup></td><td>18</td></tr></tbody></table>
arthropods. Chitinase is a hydrolytic enzyme that breaks down glycosidic bonds in chitin.⁹²⁸ Allergenic chitinase (inhalant and food) can be found in mites (e.g., Der p 15) and in cockroach (Bla g 12 and Per a 12), fruits such as bananas (Mus a 2), pomegranate (Pun g 14) and avocado (Pers a 1) (see chapter B15).

Troponin C belongs to the troponin C family and is the calcium-sensing component of the troponin complex that can attach to and detach from tropomyosin. Troponin C is involved in muscle

contraction.⁹²⁹ Troponin C is also recognized as a food allergen in shrimp (Cra c 6 and Pen m 6). Myosin light and heavy chain belonging to the myosin family, are part of the sarcomeric units in muscle tissue. Myosin is involved in muscle contraction.⁹²⁹ Myosin light chain is also recognized as a food allergen in various shrimp species (Art fr 5, Cra c 5, Lit v 3, and Pen m 3).

Hexamerin belongs to the tyrosinase family, closely related to arthropod hemocyanins, is present in insect haemolymph as a storage protein and sometimes has a transporter function. In addition, hexamerin is incorporated in the cuticle and possibly involved in humoral immune defence.⁹³⁰ Alpha-amylase belongs to the glycoside hydrolase 13 (GH13) family and serves as a key digestive enzyme in most insects. Alpha-amylase hydrolyses starch into simple sugar units, which provides energy for survival and the development of insects.⁹³¹ Allergenic α-amylases can be found in mites (Der p 4, Der f 4), cockroach (Bla g 1, Per a 11), all inhalation allergens and as food allergens in barley (Hor v 16).

Trypsin like proteinase belongs to the proteases of the mixed nucleophile, superfamily A and is involved in protein digestion. Allergenic trypsin is mostly found in mites (Blo t 3, Der f 3, and Der p 3) as an inhalant allergen. Cockroach like allergen is a nitrile-specifier protein with a detoxifying function localized in the midgut

<figure><img src="image_176.png" /><figcaption>FIGURE 77 3D structure of tropomyosin from *Bombyx mori* (Q1HPU0 (TPM1_BOMMO), Q1HPU0 | SWISS-MODEL Repository (expasy.org)</figcaption></figure>

<figure><img src="image_177.png" /><figcaption>FIGURE 78 Allergen components from silkworm and mealworm. The same colour is used for related proteins.</figcaption></figure>

<table><caption>TABLE 31 Relevant insect allergens *unpublished data</caption><thead><tr><th>Species</th><th>Allergenic molecule</th><th>Protein family</th><th>Frequency of IgE</th><th>MW (kDa)</th></tr></thead><tbody><tr><td rowspan="2">Silkworm</td><td>Bomb m 1</td><td>Arginine Kinase</td><td>100% (n=10)<sup>1705</sup> [doi:10.1159/000210375]</td><td>42</td></tr><tr><td>Bomb m 3</td><td>Tropomyosin</td><td>53.3% (n=15)<sup>1706</sup> [doi:10.3892/mmr.2017.6373]</td><td>38</td></tr><tr><td rowspan="2">Mealworm</td><td>NA</td><td>Arginine Kinase</td><td>23.1% (n=13)*</td><td>27</td></tr><tr><td>NA</td><td>Tropomyosin</td><td>76.9% (n=13)*</td><td>34</td></tr><tr><td>Tenebrio melitor</td><td>NA</td><td>LCP AIA</td><td>100% (n=2)<sup>915</sup></td><td>18</td></tr></tbody></table>
microvillar part of the insect. The cockroach allergen-like protein can only be found in the yellow mealworm (Tenebrio molitor). Larval cuticle protein (family unknown) is a mealworm specific protein, having a conserved domain in arthropod cuticles known as R&R consensus and binds chitin. The chitin-binding complex links the soft internal tissue to the exoskeleton of the larvae.⁹¹⁸

## 4 – Sensitization to individual molecules and their clinical relevance

Cross-reactivity/co-sensitization was assessed for mealworm, cricket, grasshopper, black soldier fly, moth, locust, termite, and cockroach with serum obtained from crustacean and/or house dust mite (HDM) allergic patients. The pan-allergens arginine kinase and tropomyosin were frequently involved in insect cross-reactivity between different insect species and crustaceans (e.g., shrimp) because tropomyosins and arginine kinases from different species are highly homologous (> 70% sequence identity) (Figure 79). The sequence identity of other allergenic proteins from insects (e.g., paramyosin, chitinase) ranges from 35 to 90%.⁹³³ Unfortunately, the clinical relevance of this cross-reactivity has not been investigated.⁹³³

Only one study with 15 shrimp allergic patients showed clinically relevant co-sensitization of mealworm in 13 patients in a double-blind placebo-controlled food challenge (DBPCFC). All 15 patients were sensitized to mealworm extract (basophil activation test (BAT), the ImmunoCAP Specific IgE test and Western blot).⁹³⁴

Ten mealworm allergic patients had IgE against tropomyosin and 3 against arginine kinase (Table 31). These 15 patients had also slgE

against House cricket, Giant mealworm, Lesser mealworm, African grasshopper, Large wax moth and Black soldier fly and this slgE was able to activate basophils. Tropomyosin and arginine kinase were the most dominant allergens responsible for cross-reactivity between shrimp and the tested insects, but cross-reactivity to other allergens could not be excluded. It is most likely that shrimp allergic patients will react to various edible insects, but the clinical relevance needs to be further assessed.⁹³⁵ Primary sensitization to edible insects (mealworm) was demonstrated in two studies, within a total of 6 subjects, that all worked in a facility that reared or processed mealworms, which suggests exposure different from ingestion (e.g., inhalation or skin contact) and might have played a role in the onset of primary mealworm allergy as well.

Two employees, who worked in the production of yellow mealworm flour, complained after repetitive exposure to mealworm (rhinocconjunctivitis, itching and contact erythema) when entering the rearing room. Both subjects were used to eat edible insects such as wax moth, crickets and black soldier fly without any complaints. They experienced oral allergy syndrome (OAS) the first time eating a hamburger containing mealworms. Both patients refused to undergo a provocation test. The skin prick test for inhalant and food allergens were negative except for grass in subject 1 and Alternaria in subject 2. Specific IgE was found for mealworm extract but was negative for HDM and shrimp. The results indicate a primary food allergy to mealworm. The cockroach allergen like protein, early-staged encapsulation protein and troponin C were identified as responsible proteins.⁹³⁶

Another study showed that two mealworm breeders became food allergic to mealworm after repeated ingestion and exposure to mealworm (DBPCFC proven mealworm allergy) while two other

<figure><img src="image_178.png" /><figcaption>FIGURE 79 Cross-reactivity of tropomyosin (TM) and arginine kinase (AK) between different allergenic sources (Solid line=tropomyosin; dashed line=arginine kinase)</figcaption></figure>
microvillar part of the insect. The cockroach allergen-like protein can only be found in the yellow mealworm (Tenebrio molitor). Larval cuticle protein (family unknown) is a mealworm specific protein, having a conserved domain in arthropod cuticles known as R&R consensus and binds chitin. The chitin-binding complex links the soft internal tissue to the exoskeleton of the larvae.⁹¹⁸

## 4 – Sensitization to individual molecules and their clinical relevance

Cross-reactivity/co-sensitization was assessed for mealworm, cricket, grasshopper, black soldier fly, moth, locust, termite, and cockroach with serum obtained from crustacean and/or house dust mite (HDM) allergic patients. The pan-allergens arginine kinase and tropomyosin were frequently involved in insect cross-reactivity between different insect species and crustaceans (e.g., shrimp) because tropomyosins and arginine kinases from different species are highly homologous (> 70% sequence identity) (Figure 79). The sequence identity of other allergenic proteins from insects (e.g., paramyosin, chitinase) ranges from 35 to 90%.⁹³³ Unfortunately, the clinical relevance of this cross-reactivity has not been investigated.⁹³³

Only one study with 15 shrimp allergic patients showed clinically relevant co-sensitization of mealworm in 13 patients in a double-blind placebo-controlled food challenge (DBPCFC). All 15 patients were sensitized to mealworm extract (basophil activation test (BAT), the ImmunoCAP Specific IgE test and Western blot).⁹³⁴

Ten mealworm allergic patients had IgE against tropomyosin and 3 against arginine kinase (Table 31). These 15 patients had also slgE

against House cricket, Giant mealworm, Lesser mealworm, African grasshopper, Large wax moth and Black soldier fly and this slgE was able to activate basophils. Tropomyosin and arginine kinase were the most dominant allergens responsible for cross-reactivity between shrimp and the tested insects, but cross-reactivity to other allergens could not be excluded. It is most likely that shrimp allergic patients will react to various edible insects, but the clinical relevance needs to be further assessed.⁹³⁵ Primary sensitization to edible insects (mealworm) was demonstrated in two studies, within a total of 6 subjects, that all worked in a facility that reared or processed mealworms, which suggests exposure different from ingestion (e.g., inhalation or skin contact) and might have played a role in the onset of primary mealworm allergy as well.

Two employees, who worked in the production of yellow mealworm flour, complained after repetitive exposure to mealworm (rhinocconjunctivitis, itching and contact erythema) when entering the rearing room. Both subjects were used to eat edible insects such as wax moth, crickets and black soldier fly without any complaints. They experienced oral allergy syndrome (OAS) the first time eating a hamburger containing mealworms. Both patients refused to undergo a provocation test. The skin prick test for inhalant and food allergens were negative except for grass in subject 1 and Alternaria in subject 2. Specific IgE was found for mealworm extract but was negative for HDM and shrimp. The results indicate a primary food allergy to mealworm. The cockroach allergen like protein, early-staged encapsulation protein and troponin C were identified as responsible proteins.⁹³⁶

Another study showed that two mealworm breeders became food allergic to mealworm after repeated ingestion and exposure to mealworm (DBPCFC proven mealworm allergy) while two other

<figure><img src="image_178.png" /><figcaption>FIGURE 79 Cross-reactivity of tropomyosin (TM) and arginine kinase (AK) between different allergenic sources (Solid line=tropomyosin; dashed line=arginine kinase)</figcaption></figure>
mealworm breeders only experienced complaints when working in the rearing facility. The mealworm food allergic breeders had higher slgE to mealworm, consumed larger amounts of mealworm (~50 g), and were exposed for a longer period (7-9 years) than the two with respiratory allergy (~1 g mealworm and 2-5 years of exposure). This might suggest that occupational exposure for a longer period of time and/or oral exposure with high doses are required to develop a food allergy to mealworm. The mealworm allergic breeders were not allergic to shrimp (the open challenge was negative) or any other food, which suggested a primary food allergy to mealworm. The culprit allergens were the larval cuticle proteins A1A, A2B, and A3A.⁹¹⁸ The serum of the 4 mealworm allergic breeders had only slgE against some of the tested insects (House cricket, Giant mealworm, Lesser mealworm, African grasshopper, Large wax moth, and Black soldier fly) and basophils' activation was not seen for all insects. This might suggest that primary mealworm allergy is not indicative of insect allergy and suggests the possibility of species-specific insect allergy when primarily sensitized to insect-specific proteins.⁹³⁵ This is confirmed by the fact that the mealworm allergic workers were able to eat wax moth, crickets and black soldier fly without any complaints.⁹³⁶

Insects intended for food formulations are necessarily subjected to postharvest processing, e.g., blanching, pasteurization, and sterilization to ensure their microbiological safety. It is well known that heat processing could affect the allergenic potency of proteins. Unfortunately, there is limited information concerning the effects of processing on the allergenicity of insects. Most studies investigated the effect on IgE binding, and the results are contradictory, possibly due to solubility issues.⁹³³,⁹³⁷ Furthermore, the impact of treatment on the IgE-binding capacity does not necessarily correlate with clinical symptoms. One study investigated the effect of thermal processing of mealworms on IgE-binding capacity and IgE cross-linking (basophil activation and skin prick test) and showed that processing did not lower IgE binding and functionality of mealworm allergens.⁹³⁸ Another study showed that both, thermal processing and hydrolysis using food grade enzymes of locust proteins abrogated the functionality of locust allergens in a skin prick test (n=5).⁹³⁹

## 5 – Clinical management

Diagnosis of insect food allergy is not routinely performed, because insects are seldom eaten in Europe and North America and diagnostic tests are not available yet.

The diagnosis of food allergy to insects has to start with a careful clinical history, followed by a prick-to-prick test or skin tests with commercial extracts (not yet available) and/or slgE tests. Many insects can cross-react with shrimp/shellfish, so that it is advisable to include these in the evaluation (Figure 80).

In addition, sera can be tested for the presence of IgE to tropomyosin of the insect or to other (more or less related) species (e.g., shrimp, HDM, *anisakis*), because tropomyosins are very homologous amongst different species, a positive test could indicate cross-reactive insect food allergy to shrimp.

Availability of other individual allergens from insects is limited and can therefore not be used for the diagnosis of primary or secondary insect food allergy. Extracts of the whole insect can also be used to measure slgE (ELISA, BLOT). A positive test could either indicate a cross-reactive or primary food allergy.

Ideally, a food challenge (open or DBPCFC) is performed with the suspected insect(s) to confirm or exclude food allergy. A food challenge with shrimp/shellfish should be considered to determine if insect allergy is cross-reactivity or a primary food allergy. In case of confirmation of food allergy to insect(s), elimination from the diet has to be advised and cross-reactive allergies need to be discussed (Figure 80).

## 6 – Clinical cases

Various case reports were described, reporting on food allergic reactions to different insects, e.g., (larvae of) beetles: mealworm, sago worm, lentil weevil, larvae of moths: silkworm, mopane worm, pine processionary caterpillar, woolly bear caterpillar, *Clanis bilineata*, and other insects such as locusts, crickets, grasshoppers, cicadas, and bees. Allergy was also reported following ingestion of carmine (E120), a colour additive, which is not a protein but a chemical pigment, obtained from female *Dactylopius coccus var. Costa*.⁹³³ Of all insects, allergy to silkworm (7 cases) and the food additive carmine (9 cases) were most frequently described. It is highly likely that not all clinical cases are described, so the prevalence of insect allergy is underestimated. Moreover, it can be expected that the prevalence of allergy to insects will increase, as the global consumption is increasing, due to the recent acceptance of mealworms to the European market. Below 3 cases of insect food allergy are highlighted.

### Case 1⁹⁴⁰

#### Clinical History

A 47-year-old man experienced a severe allergic reaction, within 30 min anaphylaxis after the first consumption of approx. 5 grams of cricket (*Acheta domestica*) and mealworm (*Tenebrio molitor*) mixture. He developed nausea, erythema of the neck, cough and difficulty of breathing, requiring the use of antihistamines, corticosteroids and epinephrine followed by a 24-hour hospitalization. No co-factors, such as alcohol, NSAIDs, or exercise were reported by the patient. The patient reported, anaphylactic reactions occurring at the age of 20 and 24 years following consumption of crab, mussels, and ground snails. These reactions manifested as hives, gastrointestinal symptoms and breathing difficulties which led to practicing strict exclusion of all crustaceans, molluscs and gastropods from his diet.

#### Test with extracts

The patient had positive skin prick tests (SPT, not commercially available) to native cricket, mealworm, crab, mussel, and snail. Skin prick tests were also positive for shrimp despite the absence of any clinical reaction and negative for house dust mite (HDM). Specific IgE was positive to shrimp extract, and HDM. Reactivity was further investigated with basophil activation tests (BAT) with cricket,
mealworm breeders only experienced complaints when working in the rearing facility. The mealworm food allergic breeders had higher slgE to mealworm, consumed larger amounts of mealworm (~50 g), and were exposed for a longer period (7-9 years) than the two with respiratory allergy (~1 g mealworm and 2-5 years of exposure). This might suggest that occupational exposure for a longer period of time and/or oral exposure with high doses are required to develop a food allergy to mealworm. The mealworm allergic breeders were not allergic to shrimp (the open challenge was negative) or any other food, which suggested a primary food allergy to mealworm. The culprit allergens were the larval cuticle proteins A1A, A2B, and A3A.⁹¹⁸ The serum of the 4 mealworm allergic breeders had only slgE against some of the tested insects (House cricket, Giant mealworm, Lesser mealworm, African grasshopper, Large wax moth, and Black soldier fly) and basophils' activation was not seen for all insects. This might suggest that primary mealworm allergy is not indicative of insect allergy and suggests the possibility of species-specific insect allergy when primarily sensitized to insect-specific proteins.⁹³⁵ This is confirmed by the fact that the mealworm allergic workers were able to eat wax moth, crickets and black soldier fly without any complaints.⁹³⁶

Insects intended for food formulations are necessarily subjected to postharvest processing, e.g., blanching, pasteurization, and sterilization to ensure their microbiological safety. It is well known that heat processing could affect the allergenic potency of proteins. Unfortunately, there is limited information concerning the effects of processing on the allergenicity of insects. Most studies investigated the effect on IgE binding, and the results are contradictory, possibly due to solubility issues.⁹³³,⁹³⁷ Furthermore, the impact of treatment on the IgE-binding capacity does not necessarily correlate with clinical symptoms. One study investigated the effect of thermal processing of mealworms on IgE-binding capacity and IgE cross-linking (basophil activation and skin prick test) and showed that processing did not lower IgE binding and functionality of mealworm allergens.⁹³⁸ Another study showed that both, thermal processing and hydrolysis using food grade enzymes of locust proteins abrogated the functionality of locust allergens in a skin prick test (n=5).⁹³⁹

## 5 – Clinical management

Diagnosis of insect food allergy is not routinely performed, because insects are seldom eaten in Europe and North America and diagnostic tests are not available yet.

The diagnosis of food allergy to insects has to start with a careful clinical history, followed by a prick-to-prick test or skin tests with commercial extracts (not yet available) and/or slgE tests. Many insects can cross-react with shrimp/shellfish, so that it is advisable to include these in the evaluation (Figure 80).

In addition, sera can be tested for the presence of IgE to tropomyosin of the insect or to other (more or less related) species (e.g., shrimp, HDM, *anisakis*), because tropomyosins are very homologous amongst different species, a positive test could indicate cross-reactive insect food allergy to shrimp.

Availability of other individual allergens from insects is limited and can therefore not be used for the diagnosis of primary or secondary insect food allergy. Extracts of the whole insect can also be used to measure slgE (ELISA, BLOT). A positive test could either indicate a cross-reactive or primary food allergy.

Ideally, a food challenge (open or DBPCFC) is performed with the suspected insect(s) to confirm or exclude food allergy. A food challenge with shrimp/shellfish should be considered to determine if insect allergy is cross-reactivity or a primary food allergy. In case of confirmation of food allergy to insect(s), elimination from the diet has to be advised and cross-reactive allergies need to be discussed (Figure 80).

## 6 – Clinical cases

Various case reports were described, reporting on food allergic reactions to different insects, e.g., (larvae of) beetles: mealworm, sago worm, lentil weevil, larvae of moths: silkworm, mopane worm, pine processionary caterpillar, woolly bear caterpillar, *Clanis bilineata*, and other insects such as locusts, crickets, grasshoppers, cicadas, and bees. Allergy was also reported following ingestion of carmine (E120), a colour additive, which is not a protein but a chemical pigment, obtained from female *Dactylopius coccus var. Costa*.⁹³³ Of all insects, allergy to silkworm (7 cases) and the food additive carmine (9 cases) were most frequently described. It is highly likely that not all clinical cases are described, so the prevalence of insect allergy is underestimated. Moreover, it can be expected that the prevalence of allergy to insects will increase, as the global consumption is increasing, due to the recent acceptance of mealworms to the European market. Below 3 cases of insect food allergy are highlighted.

### Case 1⁹⁴⁰

#### Clinical History

A 47-year-old man experienced a severe allergic reaction, within 30 min anaphylaxis after the first consumption of approx. 5 grams of cricket (*Acheta domestica*) and mealworm (*Tenebrio molitor*) mixture. He developed nausea, erythema of the neck, cough and difficulty of breathing, requiring the use of antihistamines, corticosteroids and epinephrine followed by a 24-hour hospitalization. No co-factors, such as alcohol, NSAIDs, or exercise were reported by the patient. The patient reported, anaphylactic reactions occurring at the age of 20 and 24 years following consumption of crab, mussels, and ground snails. These reactions manifested as hives, gastrointestinal symptoms and breathing difficulties which led to practicing strict exclusion of all crustaceans, molluscs and gastropods from his diet.

#### Test with extracts

The patient had positive skin prick tests (SPT, not commercially available) to native cricket, mealworm, crab, mussel, and snail. Skin prick tests were also positive for shrimp despite the absence of any clinical reaction and negative for house dust mite (HDM). Specific IgE was positive to shrimp extract, and HDM. Reactivity was further investigated with basophil activation tests (BAT) with cricket,
<figure><img src="image_179.png" /><figcaption>FIGURE 80 Diagnostic algorithm for insect food allergy</figcaption></figure>

mealworm, shrimp, and snail extracts. All but shrimp extract induced basophil activation.

### Food challenge

An open oral food challenge test was carried out with shrimp and was negative up to 100 g, excluding shrimp allergy.

### Test with molecules

No sensitization to shrimp allergens Pen m 1 (tropomyosin), Pen m 2 (arginine kinase) or Pen m 4 (sarcoplasmic calcium-binding protein) was found with the ImmunoCAP ISAC allergen microarray. In addition, no IgE(s) to HDM, cockroaches or *Anisakis simplex* allergens was detected with ImmunoCAP ISAC.

### Conclusion

Taken together, the data suggested that the occurrence of an anaphylactic reaction upon the first consumption of insect was explained by cross-allergy between crickets and mealworm, with mussel, crab, and snail, however, without the involvement of shrimp or HDM. In addition, the culprit allergen did not appear to be either tropomyosin, arginine kinase or the sarcoplasmic calcium-binding protein of crustaceans.

### Case 2⁹²²

#### Clinical History

A 50-year-old woman experienced oral pruritus, oral itching, and oropharyngeal and lingual oedema after cricket ingestion. She could,

however, handle them in the kitchen without any problem. She also showed similar symptoms with the grasshopper. She ate scorpions, worms, and tarantulas without any symptoms.

#### Test with extracts

The patient had sIgE-positive for shrimp and against four cricket extracts: 0.6 kU/L for G. assimilis, *G. bimaculatus*, and A. domesticus, and 0.8 kU/L for G. sigillatus, A. domesticus, and G. bimaculatus being the species most frequently used in human food.

A skin prick test for *D. pteronyssinus* and *D. farinae* was positive, and so was a prick-to-prick test with shrimp head; however, a prick-by-prick test with shrimp body was negative. Prick-to-prick with four cricket species, including *Gryllus assimilis*, *Gryllus bimaculatus*, *Grylloides sigillatus*, and *Acheta domesticus* were also positive.

#### Food challenge

An oral provocation test with shrimp showed that she could safely eat them peeled.

#### Test with molecules

The patient's serum recognized a 75 kDa protein on the immunoblot, which was identified as hexamerin-like protein 2 with LC-MS.

#### Conclusion

The patient was food allergic to cricket and probably grasshopper but not to peeled shrimp. This is most likely due to sIgE against hexamerin-like protein 2, which is present in the shrimp head.
<figure><img src="image_179.png" /><figcaption>FIGURE 80 Diagnostic algorithm for insect food allergy</figcaption></figure>

mealworm, shrimp, and snail extracts. All but shrimp extract induced basophil activation.

### Food challenge

An open oral food challenge test was carried out with shrimp and was negative up to 100 g, excluding shrimp allergy.

### Test with molecules

No sensitization to shrimp allergens Pen m 1 (tropomyosin), Pen m 2 (arginine kinase) or Pen m 4 (sarcoplasmic calcium-binding protein) was found with the ImmunoCAP ISAC allergen microarray. In addition, no IgE(s) to HDM, cockroaches or *Anisakis simplex* allergens was detected with ImmunoCAP ISAC.

### Conclusion

Taken together, the data suggested that the occurrence of an anaphylactic reaction upon the first consumption of insect was explained by cross-allergy between crickets and mealworm, with mussel, crab, and snail, however, without the involvement of shrimp or HDM. In addition, the culprit allergen did not appear to be either tropomyosin, arginine kinase or the sarcoplasmic calcium-binding protein of crustaceans.

### Case 2⁹²²

#### Clinical History

A 50-year-old woman experienced oral pruritus, oral itching, and oropharyngeal and lingual oedema after cricket ingestion. She could,

however, handle them in the kitchen without any problem. She also showed similar symptoms with the grasshopper. She ate scorpions, worms, and tarantulas without any symptoms.

#### Test with extracts

The patient had sIgE-positive for shrimp and against four cricket extracts: 0.6 kU/L for G. assimilis, *G. bimaculatus*, and A. domesticus, and 0.8 kU/L for G. sigillatus, A. domesticus, and G. bimaculatus being the species most frequently used in human food.

A skin prick test for *D. pteronyssinus* and *D. farinae* was positive, and so was a prick-to-prick test with shrimp head; however, a prick-by-prick test with shrimp body was negative. Prick-to-prick with four cricket species, including *Gryllus assimilis*, *Gryllus bimaculatus*, *Grylloides sigillatus*, and *Acheta domesticus* were also positive.

#### Food challenge

An oral provocation test with shrimp showed that she could safely eat them peeled.

#### Test with molecules

The patient's serum recognized a 75 kDa protein on the immunoblot, which was identified as hexamerin-like protein 2 with LC-MS.

#### Conclusion

The patient was food allergic to cricket and probably grasshopper but not to peeled shrimp. This is most likely due to sIgE against hexamerin-like protein 2, which is present in the shrimp head.
Case 3⁹⁴¹

Clinical History

A 15-year-old Zimbabwean boy experienced within 10 minutes headache, dyspnea, cough, wheeze, palatal pruritus, urticaria, tongue, and lip swelling after eating mopane worm, the larva of the emperor moth (Imbrasia belina). Symptoms gradually resolved after antihistamine use, but drowsiness persisted for 48 hours. The boy had eczema, which resolved after the age of 2 years. Since the age of 8 years, he had mouth and ear itching to mopane worm ingestion. Summer seasonal rhinitis started at the age of 3 years. He was neither asthmatic nor drug or latex allergic. His mother has allergic rhinitis, asthma, and oral allergy syndrome. Two siblings had a food allergy and allergic rhinitis, respectively. No long-term medications are used.

Test with extracts

Skin prick testing was positive for tree pollen (oak, Acacia), grass pollen (Bermuda, Timothy, maize), English plantain, dust mite mix (Dermatophagoides farinae, Dermatophagoides pteronyssinus), and cockroach (Blattella germanica). SPT results were negative to moulds, cats, and dogs. SPT with mopane worm was strongly positive (7 mm wheal and 15 mm flare).

A total IgE level of 622 kU/L was found. Positive the ImmunoCAP Specific IgE test results were as follows: D. pteronyssinus, 5.07 kU/L; Anisakis, 0.06 kU/L; tree mix, 0.06 kU/L; weed mix, 0.55 kU/L; grass mix, 4.98 kU/L; mealworm, 3.39 kU/L; Schistosoma, 0.59 kU/L; and Ascaris, 0.18 kU/L.

Food challenge

Not available.

Test with molecules

Allergen microarray test results (ImmunoCAP ISAC, Thermo Fisher Scientific, Uppsala, Sweden) were positive for the following: Bermuda grass (nCyn d 1): 23 ISU - Timothy grass (rPhl p 1): 4.6 ISU - Japanese cedar (nCry j 1): 1.7 ISU - cypress (Cup a 1): 14 ISU - D. pteronyssinus (nDer p 1): 2.3 ISU - D. pteronyssinus (nDer p 2): 2.8 ISU - D. farinae (rDer f 2): 3 ISU - olive (nOle e 2): 0.9 ISU - and latex (rHev b 8): 0.7 ISU.

Microarray test results were negative to foods (fruits, nuts, peanut, milk, egg, soybean, wheat, shrimp, codfish, carp), pollens (plane, ragweed, mugwort, saltwort), tropomyosin in shrimp (rPen a 1, nPen i 1, nPen m 1), D. pteronyssinus (rDer p 10), cockroach (nBla g 7), and Anisakis simplex (rAni s 3). Western blot with mopane worm extract showed a positive band at 50 kDa, which was not identified.

Conclusion

The results of both SPT and Western blot to MW were positive, confirming sensitization. sensitization to dust mites (SPT, ImmunoCAP Specific IgE and ImmunoCAP ISAC tests), cockroach (SPT), Ascaris (the ImmunoCAP Specific IgE), and Anisakis (ImmunoCap RAST and Western blot) suggest a cross-sensitization. This may result from glutathione transferases or tropomyosin, which have been implicated in cross-reactivity among Ascaris, dust mites, cockroaches, crustaceans, and molluscs.

7 – Research and future perspectives

Currently only allergens (tropomyosin and arginine kinase) from silkworms are recognized by the WHO-IUIS (www.allergen.org) database as a food allergen, while more and more evidence is available that also other insects contain putative food allergens. In addition to tropomyosin and arginine kinase, other proteins might be involved.

Clinically relevant IgE binding and cross-reactivity was only found for mealworm in shrimp allergic patients. Unfortunately for other insects, no data are available yet.

None of the case reports found confirmed the food allergy to insects with a food challenge.

Mealworms were recently approved by the EU Commission as a novel food. Allergic patients and their health care providers should be informed about the possible risk the introduction of insect proteins might pose and labelling of mealworm allergens should be required by authorities. Not only cross-reactive risk may be expected but also primary food allergy caused by insects may occur.

Most studies investigated shrimp allergic and HDM allergic patients with IgE to tropomyosin but also 22% of the HDM allergic patients without tropomyosin, arginine kinase or shrimp sensitization and 16% of a seasonal rhinitis population showed mealworm-protein reactive IgE. Although a higher prevalence of sensitization to mealworm was found in the population of shrimp allergic patients (88%), HDM allergy and seasonal rhinitis are much more prevalent.⁹⁴² The clinical relevance of this IgE binding should therefore be investigated. It should be noted that sensitization to mealworm in the seasonal rhinitis and HDM allergic populations could be caused by cross-reactivity to other insects. Such primary sensitization may result from historical exposure to insect proteins. We are all exposed to insect allergens, both aerosolized and as food contamination. Primary allergy to mealworms was also demonstrated. In theory, besides the development of such primary insect allergy, cross-reactive allergies might develop leading to broader insect-protein sensitization and allergy. Reasoning the other way around, theoretically, primary insect allergy could lead to the development of cross-reactive allergy to shrimp or HDM. This should be investigated in the future for instance by post-launch monitoring. Post-launch monitoring would help to identify new allergies when novel (insect) foods will enter the food market and to increase awareness in patients at risk and their caregivers.

Advices for use of molecular diagnostics for insects

1. Specific IgE to tropomyosins of different species (Shrimp, HDM and Anisakis) is a marker for potential sensitization and (cross)allergy to insects.

2. Different extraction buffers should be used to extract as many proteins from insects as possible for a good diagnosis of insect sensitization.
Case 3⁹⁴¹

Clinical History

A 15-year-old Zimbabwean boy experienced within 10 minutes headache, dyspnea, cough, wheeze, palatal pruritus, urticaria, tongue, and lip swelling after eating mopane worm, the larva of the emperor moth (Imbrasia belina). Symptoms gradually resolved after antihistamine use, but drowsiness persisted for 48 hours. The boy had eczema, which resolved after the age of 2 years. Since the age of 8 years, he had mouth and ear itching to mopane worm ingestion. Summer seasonal rhinitis started at the age of 3 years. He was neither asthmatic nor drug or latex allergic. His mother has allergic rhinitis, asthma, and oral allergy syndrome. Two siblings had a food allergy and allergic rhinitis, respectively. No long-term medications are used.

Test with extracts

Skin prick testing was positive for tree pollen (oak, Acacia), grass pollen (Bermuda, Timothy, maize), English plantain, dust mite mix (Dermatophagoides farinae, Dermatophagoides pteronyssinus), and cockroach (Blattella germanica). SPT results were negative to moulds, cats, and dogs. SPT with mopane worm was strongly positive (7 mm wheal and 15 mm flare).

A total IgE level of 622 kU/L was found. Positive the ImmunoCAP Specific IgE test results were as follows: D. pteronyssinus, 5.07 kU/L; Anisakis, 0.06 kU/L; tree mix, 0.06 kU/L; weed mix, 0.55 kU/L; grass mix, 4.98 kU/L; mealworm, 3.39 kU/L; Schistosoma, 0.59 kU/L; and Ascaris, 0.18 kU/L.

Food challenge

Not available.

Test with molecules

Allergen microarray test results (ImmunoCAP ISAC, Thermo Fisher Scientific, Uppsala, Sweden) were positive for the following: Bermuda grass (nCyn d 1): 23 ISU - Timothy grass (rPhl p 1): 4.6 ISU - Japanese cedar (nCry j 1): 1.7 ISU - cypress (Cup a 1): 14 ISU - D. pteronyssinus (nDer p 1): 2.3 ISU - D. pteronyssinus (nDer p 2): 2.8 ISU - D. farinae (rDer f 2): 3 ISU - olive (nOle e 2): 0.9 ISU - and latex (rHev b 8): 0.7 ISU.

Microarray test results were negative to foods (fruits, nuts, peanut, milk, egg, soybean, wheat, shrimp, codfish, carp), pollens (plane, ragweed, mugwort, saltwort), tropomyosin in shrimp (rPen a 1, nPen i 1, nPen m 1), D. pteronyssinus (rDer p 10), cockroach (nBla g 7), and Anisakis simplex (rAni s 3). Western blot with mopane worm extract showed a positive band at 50 kDa, which was not identified.

Conclusion

The results of both SPT and Western blot to MW were positive, confirming sensitization. sensitization to dust mites (SPT, ImmunoCAP Specific IgE and ImmunoCAP ISAC tests), cockroach (SPT), Ascaris (the ImmunoCAP Specific IgE), and Anisakis (ImmunoCap RAST and Western blot) suggest a cross-sensitization. This may result from glutathione transferases or tropomyosin, which have been implicated in cross-reactivity among Ascaris, dust mites, cockroaches, crustaceans, and molluscs.

7 – Research and future perspectives

Currently only allergens (tropomyosin and arginine kinase) from silkworms are recognized by the WHO-IUIS (www.allergen.org) database as a food allergen, while more and more evidence is available that also other insects contain putative food allergens. In addition to tropomyosin and arginine kinase, other proteins might be involved.

Clinically relevant IgE binding and cross-reactivity was only found for mealworm in shrimp allergic patients. Unfortunately for other insects, no data are available yet.

None of the case reports found confirmed the food allergy to insects with a food challenge.

Mealworms were recently approved by the EU Commission as a novel food. Allergic patients and their health care providers should be informed about the possible risk the introduction of insect proteins might pose and labelling of mealworm allergens should be required by authorities. Not only cross-reactive risk may be expected but also primary food allergy caused by insects may occur.

Most studies investigated shrimp allergic and HDM allergic patients with IgE to tropomyosin but also 22% of the HDM allergic patients without tropomyosin, arginine kinase or shrimp sensitization and 16% of a seasonal rhinitis population showed mealworm-protein reactive IgE. Although a higher prevalence of sensitization to mealworm was found in the population of shrimp allergic patients (88%), HDM allergy and seasonal rhinitis are much more prevalent.⁹⁴² The clinical relevance of this IgE binding should therefore be investigated. It should be noted that sensitization to mealworm in the seasonal rhinitis and HDM allergic populations could be caused by cross-reactivity to other insects. Such primary sensitization may result from historical exposure to insect proteins. We are all exposed to insect allergens, both aerosolized and as food contamination. Primary allergy to mealworms was also demonstrated. In theory, besides the development of such primary insect allergy, cross-reactive allergies might develop leading to broader insect-protein sensitization and allergy. Reasoning the other way around, theoretically, primary insect allergy could lead to the development of cross-reactive allergy to shrimp or HDM. This should be investigated in the future for instance by post-launch monitoring. Post-launch monitoring would help to identify new allergies when novel (insect) foods will enter the food market and to increase awareness in patients at risk and their caregivers.

Advices for use of molecular diagnostics for insects

1. Specific IgE to tropomyosins of different species (Shrimp, HDM and Anisakis) is a marker for potential sensitization and (cross)allergy to insects.

2. Different extraction buffers should be used to extract as many proteins from insects as possible for a good diagnosis of insect sensitization.
# B10 - Cow's milk allergy

Anna Nowak-Wegrzyn, Rebecca Grohman, Antonella Muraro

## Highlights

* Cow's milk allergy is the most common food allergy universally and is often outgrown.

* The utility of IgE to allergen components does not exceed that of IgE to cow's milk extract to support the diagnosis of cow's milk allergy.

* Sensitization to heat-resistant proteins (e.g., casein) and sequential epitopes have been associated with reactivity to baked milk and persistent milk allergy.

## 1 - The allergen sources

Cow's milk (CM) is a liquid product of the mammary glands of cow's (Bos domesticus). It is commonly consumed in large quantities by children and adults in a liquid form, as well as in a form of various dairy products, such as cheeses, butter, yogurt, and cream. CM is a base source for the majority of infant formulas, including hypoallergenic hydrolyzed and amino acid-based formulas. CM is commonly the first foreign protein introduced into the diet of infants who are not exclusively breast-fed. CM and dairy products are the major source of protein, calories, and calcium in a diet of infants and young children under the age of 2 years, and elimination of CM poses a risk for nutritional deficiencies. CM proteins are among the most common food allergens in infant and children with IgE and non-IgE-mediated food allergy, and among adults with eosinophilic esophagitis. The frequency of CM allergy has been estimated to range from 0.5 to 7.5% in westernized countries.⁷³,⁷⁴,⁹⁴,⁹⁴¹ Nevertheless, the perceived prevalence of allergic reactions to CM milk is much higher than the actual number of true cases of CM allergy. CM proteins are classified as class I food allergens, due to their resistance to digestion and heating. They do induce sensitization via gastrointestinal tract. Proteins in CM have a high sequence homology (>80%) with proteins from goat and sheep and are highly clinically cross-reactive (>90%) with these species. By contrast, the laboratory and clinical cross-reactivity is very low (<5%) with milks from donkey, mare, buf-falo, or camel.⁹⁴²

## 2 - Major and relevant minor allergenic molecules

CM contains approximately 30 to 35 g of proteins per liter. Under the influence of rennin or upon acidification of the milk to pH 4.6, proteins segregate into 2 fractions: casein which contains approximately 80% of the CM proteins and whey (lactoserum) which contains approximately 20% of the CM proteins (Table 32). All of the proteins present in cow's milk are also present in human breast milk, with an exception of beta-lactoglobulin. Caseins, beta-lactoglobulin

and alpha-lactalbumin are considered major allergens, i.e., more than 50% of the individuals with CMA are sensitized to these proteins. Most of the patients are polysensitized to several proteins.⁹⁴³,⁹⁴⁴

Casein supplies amino acids, carbohydrates, and the two inorganic elements calcium and phosphorus. Casein fraction is very resistant to high temperatures, retaining strong IgE binding after 90 minutes of boiling at >90°C.⁹⁴⁵ Except for short alpha-helical regions, caseins have little secondary or tertiary structure.⁹⁴⁶,⁹⁴⁷ The caseins of cow's milk exist in the form of colloidal complexes called micelles. The micelles contain an amorphous micellar calcium phosphate core, surrounded by a casein shell.⁹⁴⁶,⁹⁴⁷ (Figure 81, from ref 10).⁹⁴⁷

Alpha₁s₁-casein is the most abundant protein of bovine milk. It exists as a major and minor form and is highly phosphorylated. Alpha2-casein is also highly phosphorylated and has four isoforms. Beta-casein has one isoform. Limited hydrolysis of beta-casein by endogenous peptides (e.g., plasmin) present in milk produces gamma-caseins 1, 2, and 3. Kappa-casein is the only casein soluble in the presence of calcium ions. It also has the smallest amount of phosphate, with phosphorylation sites being present only in the C-terminal region. Kappa-casein is the only casein to contain carbohydrate moieties.

Whey is a mixture of beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), bovine serum albumin (~8%) and immunoglobulins.⁹⁴⁸ These are soluble in their native forms, independent of pH. Whey proteins are more sensitive to heating than caseins and lose IgE binding following 15-20 minutes of boiling at >90°C.⁹⁴⁵

Alpha-lactalbumin is a protein present in the milk of almost all mammals. In primates, alpha-lactalbumin expression is upregulated in response to the hormone prolactin and increases the production of lactose.⁹⁴² Alpha-lactalbumin forms the regulatory subunit of the lactose synthase (LS) heterodimer and beta-1,4-galactosyltransferase forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring galactose moieties to glucose. As a multimer, alpha-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal and/or antitumor activity.

Beta-lactoglobulin under physiological conditions forms dimers but dissociates to a monomer below pH 3. Beta-lactoglobulin solutions form gels in various conditions, when the native structure is sufficiently destabilized to allow aggregation.⁹⁴³ No clear function has been identified for beta-lactoglobulin, although it binds to several hydrophobic molecules, suggesting potential role in their transport. Beta-lactoglobulin is the only CM protein that is not present in the human breast milk.

Bovine serum albumin (BSA) is a globular, water-soluble, unglycosylated serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood and plays a major role in stabilizing extracellular fluid volume by contributing to oncotic pressure of plasma. BSA is highly homologous with human serum albumin and albumins of other species, e.g., cow (beef), cat, and dog. BSA has been identified as one of the major beef allergens and is responsible for clinical cross-reactivity between CM and raw beef.⁹⁴⁸⁻⁹⁵¹ Immunoglobulins present in CM are predominantly of the G class. Immunoglobulins may play a role in cross-reactivity with beef.⁹⁴⁹
# B10 - Cow's milk allergy

Anna Nowak-Wegrzyn, Rebecca Grohman, Antonella Muraro

## Highlights

* Cow's milk allergy is the most common food allergy universally and is often outgrown.

* The utility of IgE to allergen components does not exceed that of IgE to cow's milk extract to support the diagnosis of cow's milk allergy.

* Sensitization to heat-resistant proteins (e.g., casein) and sequential epitopes have been associated with reactivity to baked milk and persistent milk allergy.

## 1 - The allergen sources

Cow's milk (CM) is a liquid product of the mammary glands of cow's (*Bos domesticus*). It is commonly consumed in large quantities by children and adults in a liquid form, as well as in a form of various dairy products, such as cheeses, butter, yogurt, and cream. CM is a base source for the majority of infant formulas, including hypoallergenic hydrolyzed and amino acid-based formulas. CM is commonly the first foreign protein introduced into the diet of infants who are not exclusively breast-fed. CM and dairy products are the major source of protein, calories, and calcium in a diet of infants and young children under the age of 2 years, and elimination of CM poses a risk for nutritional deficiencies. CM proteins are among the most common food allergens in infant and children with IgE and non-IgE-mediated food allergy, and among adults with eosinophilic esophagitis. The frequency of CM allergy has been estimated to range from 0.5 to 7.5% in westernized countries.⁷³,⁷⁴,⁹⁴,⁹⁴¹ Nevertheless, the perceived prevalence of allergic reactions to CM milk is much higher than the actual number of true cases of CM allergy. CM proteins are classified as class I food allergens, due to their resistance to digestion and heating. They do induce sensitization via gastrointestinal tract. Proteins in CM have a high sequence homology (>80%) with proteins from goat and sheep and are highly clinically cross-reactive (>90%) with these species. By contrast, the laboratory and clinical cross-reactivity is very low (<5%) with milks from donkey, mare, buf-falo, or camel.⁹⁴²

## 2 - Major and relevant minor allergenic molecules

CM contains approximately 30 to 35 g of proteins per liter. Under the influence of rennin or upon acidification of the milk to pH 4.6, proteins segregate into 2 fractions: casein which contains approximately 80% of the CM proteins and whey (lactoserum) which contains approximately 20% of the CM proteins (Table 32). All of the proteins present in cow's milk are also present in human breast milk, with an exception of beta-lactoglobulin. Caseins, beta-lactoglobulin

and alpha-lactalbumin are considered major allergens, i.e., more than 50% of the individuals with CMA are sensitized to these proteins. Most of the patients are polysensitized to several proteins.⁹⁴³,⁹⁴⁴

Casein supplies amino acids, carbohydrates, and the two inorganic elements calcium and phosphorus. Casein fraction is very resistant to high temperatures, retaining strong IgE binding after 90 minutes of boiling at >90°C.⁹⁴⁵ Except for short alpha-helical regions, caseins have little secondary or tertiary structure.⁹⁴⁶,⁹⁴⁷ The caseins of cow's milk exist in the form of colloidal complexes called micelles. The micelles contain an amorphous micellar calcium phosphate core, surrounded by a casein shell.⁹⁴⁶,⁹⁴⁷ (Figure 81, from ref 10).⁹⁴⁷

Alpha₁s₁-casein is the most abundant protein of bovine milk. It exists as a major and minor form and is highly phosphorylated. Alpha2-casein is also highly phosphorylated and has four isoforms. Beta-casein has one isoform. Limited hydrolysis of beta-casein by endogenous peptides (e.g., plasmin) present in milk produces gamma-caseins 1, 2, and 3. Kappa-casein is the only casein soluble in the presence of calcium ions. It also has the smallest amount of phosphate, with phosphorylation sites being present only in the C-terminal region. Kappa-casein is the only casein to contain carbohydrate moieties.

Whey is a mixture of beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), bovine serum albumin (~8%) and immunoglobulins.⁹⁴⁸ These are soluble in their native forms, independent of pH. Whey proteins are more sensitive to heating than caseins and lose IgE binding following 15-20 minutes of boiling at >90°C.⁹⁴⁵

Alpha-lactalbumin is a protein present in the milk of almost all mammals. In primates, alpha-lactalbumin expression is upregulated in response to the hormone prolactin and increases the production of lactose.⁹⁴² Alpha-lactalbumin forms the regulatory subunit of the lactose synthase (LS) heterodimer and beta-1,4-galactosyltransferase forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring galactose moieties to glucose. As a multimer, alpha-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal and/or antitumor activity.

Beta-lactoglobulin under physiological conditions forms dimers but dissociates to a monomer below pH 3. Beta-lactoglobulin solutions form gels in various conditions, when the native structure is sufficiently destabilized to allow aggregation.⁹⁴³ No clear function has been identified for beta-lactoglobulin, although it binds to several hydrophobic molecules, suggesting potential role in their transport. Beta-lactoglobulin is the only CM protein that is not present in the human breast milk.

Bovine serum albumin (BSA) is a globular, water-soluble, unglycosylated serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood and plays a major role in stabilizing extracellular fluid volume by contributing to oncotic pressure of plasma. BSA is highly homologous with human serum albumin and albumins of other species, e.g., cow (beef), cat, and dog. BSA has been identified as one of the major beef allergens and is responsible for clinical cross-reactivity between CM and raw beef.⁹⁴⁸⁻⁹⁵¹ Immunoglobulins present in CM are predominantly of the G class. Immunoglobulins may play a role in cross-reactivity with beef.⁹⁴⁹
<table><caption>TABLE 32 Allergens in Cow's Milk (source: IUIS Allergen Database, July 2015)</caption><thead><tr><th>Protein name</th><th>Allergen name</th><th>Molecular mass (kDa)</th><th>AA #</th><th>Tertiary structure</th></tr></thead><tbody><tr><td colspan="5">Curd (coagulum) - Casein family</td></tr><tr><td>Caseins</td><td>Bos d 8</td><td>20-30</td><td></td><td>Caseins don't have a rigid tertiary structure but develop a random coil conformation</td></tr><tr><td>Alpha s1-casein</td><td>Bos d 9</td><td>23.6</td><td>199</td><td></td></tr><tr><td>Alpha s2-casein</td><td>Bos d 10</td><td>25.2 2</td><td>207</td><td></td></tr><tr><td>Beta-casein</td><td>Bos d 11</td><td>4</td><td>209</td><td>stabilized by hydrophobic interactions</td></tr><tr><td>Kappa-casein</td><td>Bos d 12</td><td>19</td><td>169</td><td></td></tr><tr><td colspan="5">Whey (lactoserum)</td></tr><tr><td>Alpha-lactalbumin</td><td>Bos d 4</td><td>14.2</td><td>123; 4 disulphide bridge, 70% homology with human alpha-lactalbumin</td><td></td></tr><tr><td>Beta-lactoglobulin<br>Protein family:<br>lipocalins</td><td>Bos d 5</td><td>18.3; exists as a dimer</td><td>162; 2 disulphide bridges, one free cysteine; exists as isoforms A and B; binds and carries hydrophobic molecules</td><td></td></tr><tr><td>Bovine serum albumin;<br>Serum albumins</td><td>Bos d 6</td><td>67</td><td>583</td><td></td></tr><tr><td>Immunoglobulins (mostly IgG)<br>Family: Immunoglobulins</td><td>Bos d 7</td><td>160</td><td></td><td></td></tr><tr><td>Lactoferrin<br>Family: Transferrins</td><td></td><td>80</td><td>703; forms two homologous globular domains named N-and C-lobes Lactoferrin exists in various polymeric forms: monomers to tetramers xac</td><td></td></tr></tbody></table>

<figure><img src="image_180.png" /></figure>

<figure><img src="image_181.png" /></figure>

<figure><img src="image_182.png" /></figure>

<figure><img src="image_183.png" /></figure>

<figure><img src="image_184.png" /></figure>

Lactoferrin is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions. Lactoferrin is one of the transferrin proteins that transfer iron to the cells and control the level of free iron in the blood and external secretions. Lactoferrin is one of the components of the immune system of the body; it has antimicrobial activity (bacteriocide, fungicide) and is part of the innate immune defense, mainly at mucosal surfaces. In particular, lactoferrin provides antibacterial activity to human infants. Lactoferrin interacts with DNA and RNA, polysaccharides and heparin. Lactoferrin is a minor allergen in CM.⁹⁴⁴

## 3 – Sensitization to individual molecules and its clinical relevance

The patterns of sensitization to the individual CM proteins vary significantly by study population and age of the affected individuals. In general, most of the affected subjects are polysensitized to

several casein and whey proteins. Caseins, beta-lactoglobulin and alpha-lactalbumin are the major allergens, with over 50% of CM-allergic subjects having evidence of IgE antibodies directed at these proteins. IgE sensitization to caseins, beta-lactoglobulin and alpha-lactalbumin is closely related, whereas IgE sensitization to BSA is independent of other CM proteins, and may reflect cross-reactivity with beef.⁹⁴⁹ (Table 33).

### Effect of heating on CM protein allergenicity

CM proteins contain both conformational and sequential IgE-binding epitopes. Children with persistent milk allergy have been shown to predominantly generate IgE antibodies directed against sequential casein epitopes.⁹⁵²,⁹⁵³ Extensive heating e.g., baking, affects the allergenicity of CM protein, with caseins being more resistant to heating compared with whey proteins that are susceptible to heating. Heating of beta-lactoglobulin results in formation of the intermolecular disulphide bonds and binding to other food proteins that result in a reduced allergenicity of beta-lactoglobulin.⁹⁵⁴ The majority (70-80%) of the CM-allergic children tolerate CM as an ingredient in the baked products.³⁸⁵,⁹⁵⁴ Reactivity to baked milk is a marker of a more severe and more persistent CM allergy. Inclusion
<table><caption>TABLE 32 Allergens in Cow's Milk (source: IUIS Allergen Database, July 2015)</caption><thead><tr><th>Protein name</th><th>Allergen name</th><th>Molecular mass (kDa)</th><th>AA #</th><th>Tertiary structure</th></tr></thead><tbody><tr><td colspan="5">Curd (coagulum) - Casein family</td></tr><tr><td>Caseins</td><td>Bos d 8</td><td>20-30</td><td></td><td>Caseins don't have a rigid tertiary structure but develop a random coil conformation</td></tr><tr><td>Alpha s1-casein</td><td>Bos d 9</td><td>23.6</td><td>199</td><td></td></tr><tr><td>Alpha s2-casein</td><td>Bos d 10</td><td>25.2 2</td><td>207</td><td></td></tr><tr><td>Beta-casein</td><td>Bos d 11</td><td>4</td><td>209</td><td>stabilized by hydrophobic interactions</td></tr><tr><td>Kappa-casein</td><td>Bos d 12</td><td>19</td><td>169</td><td></td></tr><tr><td colspan="5">Whey (lactoserum)</td></tr><tr><td>Alpha-lactalbumin</td><td>Bos d 4</td><td>14.2</td><td>123; 4 disulphide bridge, 70% homology with human alpha-lactalbumin</td><td></td></tr><tr><td>Beta-lactoglobulin<br>Protein family:<br>lipocalins</td><td>Bos d 5</td><td>18.3; exists as a dimer</td><td>162; 2 disulphide bridges, one free cysteine; exists as isoforms A and B; binds and carries hydrophobic molecules</td><td></td></tr><tr><td>Bovine serum albumin;<br>Serum albumins</td><td>Bos d 6</td><td>67</td><td>583</td><td></td></tr><tr><td>Immunoglobulins (mostly IgG)<br>Family: Immunoglobulins</td><td>Bos d 7</td><td>160</td><td></td><td></td></tr><tr><td>Lactoferrin<br>Family: Transferrins</td><td></td><td>80</td><td>703; forms two homologous globular domains named N-and C-lobes Lactoferrin exists in various polymeric forms: monomers to tetramers xac</td><td></td></tr></tbody></table>

<figure><img src="image_180.png" /></figure>

<figure><img src="image_181.png" /></figure>

<figure><img src="image_182.png" /></figure>

<figure><img src="image_183.png" /></figure>

<figure><img src="image_184.png" /></figure>

Lactoferrin is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions. Lactoferrin is one of the transferrin proteins that transfer iron to the cells and control the level of free iron in the blood and external secretions. Lactoferrin is one of the components of the immune system of the body; it has antimicrobial activity (bacteriocide, fungicide) and is part of the innate immune defense, mainly at mucosal surfaces. In particular, lactoferrin provides antibacterial activity to human infants. Lactoferrin interacts with DNA and RNA, polysaccharides and heparin. Lactoferrin is a minor allergen in CM.⁹⁴⁴

## 3 – Sensitization to individual molecules and its clinical relevance

The patterns of sensitization to the individual CM proteins vary significantly by study population and age of the affected individuals. In general, most of the affected subjects are polysensitized to

several casein and whey proteins. Caseins, beta-lactoglobulin and alpha-lactalbumin are the major allergens, with over 50% of CM-allergic subjects having evidence of IgE antibodies directed at these proteins. IgE sensitization to caseins, beta-lactoglobulin and alpha-lactalbumin is closely related, whereas IgE sensitization to BSA is independent of other CM proteins, and may reflect cross-reactivity with beef.⁹⁴⁹ (Table 33).

### Effect of heating on CM protein allergenicity

CM proteins contain both conformational and sequential IgE-binding epitopes. Children with persistent milk allergy have been shown to predominantly generate IgE antibodies directed against sequential casein epitopes.⁹⁵²,⁹⁵³ Extensive heating e.g., baking, affects the allergenicity of CM protein, with caseins being more resistant to heating compared with whey proteins that are susceptible to heating. Heating of beta-lactoglobulin results in formation of the intermolecular disulphide bonds and binding to other food proteins that result in a reduced allergenicity of beta-lactoglobulin.⁹⁵⁴ The majority (70-80%) of the CM-allergic children tolerate CM as an ingredient in the baked products.³⁸⁵,⁹⁵⁴ Reactivity to baked milk is a marker of a more severe and more persistent CM allergy. Inclusion
of the baked products containing CM into the diet of children with CM allergy is associated with more rapid advancement to inclusion of liquid milk, but there is no conclusive evidence that it accelerates development of tolerance to unheated CM.⁹⁵⁵ High levels of specific IgE antibodies directed against casein are predictive of clinical reactivity to baked milk.⁹⁵⁶,⁹⁵⁷ In a peptide microarray assay, subjects with persistent milk allergy had increased epitope diversity to caseins and beta-lactoglobulin compared with those who outgrew their CM allergy.⁹⁵⁸ Baked milk-tolerant subjects had IgE-binding

patterns similar to those who had outgrown CM allergy, but IgG4-binding patterns that were more similar to those of the allergic group. Binding to higher numbers of IgE peptides was associated with more severe allergic reactions during an oral CM challenge. There was no association between IgG4 peptides and clinical features of milk allergy. Using a competitive peptide microarray assay, CM-allergic patients had a combination of high- and low-affinity IgE binding, whereas baked milk-tolerant subjects and those who had outgrown their CM allergy had primarily low-affinity binding.

<figure><img src="image_185.png" /><figcaption>FIGURE 81 Casein supplies amino acids, carbohydrates, and the two inorganic elements calcium and phosphorus. Casein fraction is very resistant to high temperatures, retaining strong IgE binding after 90 minutes of boiling at >90°C⁹⁵⁰. Except for short alpha-helical regions, caseins have little secondary or tertiary structure⁹⁵¹, ⁹⁵². The caseins of cow's milk exist in the form of colloidal complexes called micelles. The micelles contain an amorphous micellar calcium phosphate core, surrounded by a casein shell⁹⁵¹, ⁹⁵². (Figure from ref⁹⁴⁷).</figcaption></figure>

## 4 - Clinical management

Diagnosis of CM allergy begins with an assessment of clinical history and an assessment of the potential immunologic mechanism involved in the reactions.

### Suspected IgE-mediated CM allergy

**Diagnostic testing:** Routine testing involves skin prick (SPT) and/or serologic testing with complete CM extract. Molecular diagnosis is not recommended for standard evaluation of suspected CM allergy. Diagnostic decision points have been proposed; they vary by population studied and age. Negative SPT and undetectable serum level of CM-specific IgE antibodies have a very high negative predictive value >90% for IgE-mediated CM allergy. The positive predictive value of the test increases with an increased size of the wheal of the SPT and serum level of the specific CM-IgE antibody (Table 34).

Molecular diagnosis may be helpful for evaluation of reactivity to baked milk, based on the differential resistance to heating among the CM protein. As caseins are more resistant to extensive heating, higher levels of casein-specific IgE are associated with increased likelihood of reactivity to baked milk. Basophil activation test with CM proteins has been utilized in a research setting, but it is not yet recommended for a routine diagnosis of CM allergy.⁹⁵⁶,⁹⁵⁹

<table><caption>TABLE 33 Sensitisation and cross-reactivity patterns of the CM proteins</caption><thead><tr><th>Allergen name</th><th>Allergenicity</th><th>Sensitisation rate % among those reactive to CM</th><th>Laboratory cross-reactivity</th><th>Clinical cross-reactivity</th></tr></thead><tbody><tr><td colspan="5"><strong>Curd (coagulum) - Casein family</strong></td></tr><tr><td>Caseins (Bos d 8)</td><td>Major</td><td>63</td><td rowspan="5">>85% with sheep and goat milk caseins</td><td rowspan="5">>90%</td></tr><tr><td>Alpha s1-casein (Bos d 9)</td><td>Major</td><td>98 *</td></tr><tr><td>Alpha s2-casein (Bos d 10)</td><td>Major</td><td>94 *</td></tr><tr><td>Beta-casein (Bos d 11)</td><td>Major</td><td>91 *</td></tr><tr><td>Kappa-casein (Bos d 12)</td><td>Major</td><td>91 *</td></tr><tr><td colspan="5"><strong>Whey (lactoserum)</strong></td></tr><tr><td>Alpha-lactalbumin (Bos d 4)</td><td>Major</td><td>51</td><td rowspan="5">80% with beef</td><td rowspan="5">15-20% with raw beef</td></tr><tr><td>Beta-lactoglobulin (Bos d 5)</td><td>Major</td><td>61</td></tr><tr><td>Bovine serum albumin (Bos d 6)</td><td>Minor</td><td>43</td></tr><tr><td>Immunoglobulins (Bos d 7)</td><td>Minor</td><td>36</td></tr><tr><td>Lactoferrin</td><td>Minor</td><td>35</td></tr></tbody></table>
of the baked products containing CM into the diet of children with CM allergy is associated with more rapid advancement to inclusion of liquid milk, but there is no conclusive evidence that it accelerates development of tolerance to unheated CM.⁹⁵⁵ High levels of specific IgE antibodies directed against casein are predictive of clinical reactivity to baked milk.⁹⁵⁶,⁹⁵⁷ In a peptide microarray assay, subjects with persistent milk allergy had increased epitope diversity to caseins and beta-lactoglobulin compared with those who outgrew their CM allergy.⁹⁵⁸ Baked milk-tolerant subjects had IgE-binding

patterns similar to those who had outgrown CM allergy, but IgG4-binding patterns that were more similar to those of the allergic group. Binding to higher numbers of IgE peptides was associated with more severe allergic reactions during an oral CM challenge. There was no association between IgG4 peptides and clinical features of milk allergy. Using a competitive peptide microarray assay, CM-allergic patients had a combination of high- and low-affinity IgE binding, whereas baked milk-tolerant subjects and those who had outgrown their CM allergy had primarily low-affinity binding.

<figure><img src="image_185.png" /><figcaption>FIGURE 81 Casein supplies amino acids, carbohydrates, and the two inorganic elements calcium and phosphorus. Casein fraction is very resistant to high temperatures, retaining strong IgE binding after 90 minutes of boiling at >90°C⁹⁵⁰. Except for short alpha-helical regions, caseins have little secondary or tertiary structure⁹⁵¹, ⁹⁵². The caseins of cow's milk exist in the form of colloidal complexes called micelles. The micelles contain an amorphous micellar calcium phosphate core, surrounded by a casein shell⁹⁵¹, ⁹⁵². (Figure from ref⁹⁴⁷).</figcaption></figure>

## 4 – Clinical management

Diagnosis of CM allergy begins with an assessment of clinical history and an assessment of the potential immunologic mechanism involved in the reactions.

### Suspected IgE-mediated CM allergy

**Diagnostic testing:** Routine testing involves skin prick (SPT) and/or serologic testing with complete CM extract. Molecular diagnosis is not recommended for standard evaluation of suspected CM allergy. Diagnostic decision points have been proposed; they vary by population studied and age. Negative SPT and undetectable serum level of CM-specific IgE antibodies have a very high negative predictive value >90% for IgE-mediated CM allergy. The positive predictive value of the test increases with an increased size of the wheal of the SPT and serum level of the specific CM-IgE antibody (Table 34).

Molecular diagnosis may be helpful for evaluation of reactivity to baked milk, based on the differential resistance to heating among the CM protein. As caseins are more resistant to extensive heating, higher levels of casein-specific IgE are associated with increased likelihood of reactivity to baked milk. Basophil activation test with CM proteins has been utilized in a research setting, but it is not yet recommended for a routine diagnosis of CM allergy.⁹⁵⁶,⁹⁵⁹

<table><caption>TABLE 33 Sensitisation and cross-reactivity patterns of the CM proteins</caption><thead><tr><th>Allergen name</th><th>Allergenicity</th><th>Sensitisation rate % among those reactive to CM</th><th>Laboratory cross-reactivity</th><th>Clinical cross-reactivity</th></tr></thead><tbody><tr><td colspan="5"><strong>Curd (coagulum) - Casein family</strong></td></tr><tr><td>Caseins (Bos d 8)</td><td>Major</td><td>63</td><td rowspan="5">>85% with sheep and goat milk caseins</td><td rowspan="5">>90%</td></tr><tr><td>Alpha s1-casein (Bos d 9)</td><td>Major</td><td>98 *</td></tr><tr><td>Alpha s2-casein (Bos d 10)</td><td>Major</td><td>94 *</td></tr><tr><td>Beta-casein (Bos d 11)</td><td>Major</td><td>91 *</td></tr><tr><td>Kappa-casein (Bos d 12)</td><td>Major</td><td>91 *</td></tr><tr><td colspan="5"><strong>Whey (lactoserum)</strong></td></tr><tr><td>Alpha-lactalbumin (Bos d 4)</td><td>Major</td><td>51</td><td rowspan="5">80% with beef</td><td rowspan="5">15-20% with raw beef</td></tr><tr><td>Beta-lactoglobulin (Bos d 5)</td><td>Major</td><td>61</td></tr><tr><td>Bovine serum albumin (Bos d 6)</td><td>Minor</td><td>43</td></tr><tr><td>Immunoglobulins (Bos d 7)</td><td>Minor</td><td>36</td></tr><tr><td>Lactoferrin</td><td>Minor</td><td>35</td></tr></tbody></table>
<table><caption>TABLE 34 Proposed specific IgE diagnostic decision points for CM allergy diagnosis derived from studies in children, majority of whom had atopic dermatitis. This might be a practical guidance for the clinical ambulatory setting, aiming to identify the optimal candidates for OFC and to limit unnecessary OFC. It would however be recommended that specific values are developed for the local population. It is important to recognize that OFC can always be performed at the discretion of the treating physician despite the elevated results of the allergy tests. In the research setting, DBPCFC OFC are recommended regardless of the CM-sIgE level.</caption><thead><tr><th></th><th>CM-sIgE [kUA/L]</th><th>OFC to unheated milk</th></tr></thead><tbody><tr><td>&gt;95% PPV</td><td>≥15;<sup>52</sup><br/>≥ 5 if less than 1 year old<sup>53</sup></td><td>Defer</td></tr><tr><td>&gt;50% to &lt;95% PPV</td><td>5-15</td><td>Consider the OFC based on the clinician and patient's/ family preference, social and nutritional importance of dairy, history of recent reactions and type of symptoms</td></tr><tr><td>&lt;50% PPV</td><td>&lt;5<br/>CM skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;95% PPV</td><td>≥8<sup>54</sup><br/>Casein-sIgE [kU/L]</td><td>Defer<br/>OFC to baked milk</td></tr><tr><td>&gt;95% PPV</td><td>≥10<sup>21</sup></td><td>Defer</td></tr><tr><td>&gt;50% to &lt;95% PPV</td><td>5-10</td><td>Consider the OFC based on the clinician and patient's/ family preference, social and nutritional importance of dairy, history of recent reactions and type of symptoms</td></tr><tr><td>&lt;50% PPV</td><td>≤5<sup>21</sup><br/>CM skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;90% NPV</td><td>&lt;12<sup>55</sup><br/>Casein skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;90% NPV</td><td>≤9<sup>55</sup></td><td>Perform</td></tr></tbody></table>

**Not recommended:** Testing for CM-specific IgG / IgG4 antibodies is not recommended in the diagnosis of CM allergy as these antibodies reflect the presence of CM in the diet, not an allergy.

**Elimination-Challenge testing:** In general, the conclusive diagnosis of CM allergy requires elimination of CM proteins from the diet followed by a supervised oral food challenge. Double-blind placebo controlled oral food challenge (DBPCFC) remains the gold standard for food allergy diagnosis and it is commonly utilized in the research setting. Open controlled challenge can replace DBPCFC in the children younger than 2 years of age and serve as a useful screening

test for patients of any age in the clinical setting. The initial assessment of reactivity to baked milk is also recommended to be conducted under the physician-supervised food challenge condition because children reactive to baked milk may experience anaphylaxis. However, such approach may be unnecessarily restrictive and not practical when access to food challenges is limited due to the paucity of allergy specialists or during the COVID-19 pandemic.⁹⁶⁰ Several reports indicated that home introduction of baked milk and egg may be safely done in carefully selected young children, e.g., under the age 3 years, without prior history of anaphylaxis or wheezing from any causes and skin prick test wheal diameter less than 8 mm for cow's milk.⁹⁶¹,⁹⁶² Home introduction starts from a significantly lower dose of baked milk and progresses slowly over the course of days as compared to a single supervised feeding over hours to a higher dose of baked food. However, severe and even delayed reactions are possible and caution is needed.⁹⁶³,⁹⁶⁴ The implementation of this approach must be adapted to the local context including quick access to emergency facilities, if required.

### Suspected non IgE-mediated CM allergy

**Laboratory testing:** There is no reliable laboratory diagnostic testing for non-IgE-mediated CM allergy.⁷⁴,⁹⁴⁴ Atopy patch testing may be considered in selected cases of EoE but not as a routine diagnostic test.⁷⁴ Lymphocyte transformation test, serum CM-specific IgG / IgG4 antibodies, or stool measurements of pro-inflammatory mediators (e.g., calprotectin, eosinophilic cationic protein, eosinophil derived neurotoxin) are not recommended [1]. As some non-IgE-mediated disorders may be associated with a concomitant IgE-mediated food allergy, testing for CM-specific IgE antibodies may be utilized in such cases, e.g., eosinophilic esophagitis (EoE) and food protein-induced enterocolitis syndrome (FPIES) to diagnose IgE-mediated CM allergy. The ultimate confirmation of diagnosis in non-IgE-mediated CM allergy requires an elimination of CM proteins from the diet and followed by an oral CM challenge. With an exception of FPIES, reintroduction of CM can be done at home. When FPIES is suspected, reintroduction during a supervised food challenge should be considered, due to the risk of severe reactions (hypotension). Tolerance to baked milk among patients with non-IgE-mediated CM allergy has not been systematically characterized. Based on anecdotal reports, a subset of patients with EoE might tolerate baked milk in the diet; however, such patients likely represent a minority, unlike with IgE-mediated CM allergy.

### Management of CM allergy

Management primarily relies on dietary avoidance of CM proteins. However, emerging data emphasize a shift from the traditional passive approach of avoidance, to a proactive one that seeks to modulate the immune system.⁹⁶⁵ In infants and young children, substituting alternative sources of protein, calories, and calcium with a specialized hypoallergenic formula may be necessary. The alternative formula choices include casein-hydrolysate, whey-hydrolysate, and amino-acid based formulas, as well as soy-based and rice hydrolysate.⁹⁶⁶ The selection of the most appropriate formula depends on the age and allergic profile of the child. Soy formulas are based on an intact protein and are not hypoallergenic but can be a suitable
<table><caption>TABLE 34 Proposed specific IgE diagnostic decision points for CM allergy diagnosis derived from studies in children, majority of whom had atopic dermatitis. This might be a practical guidance for the clinical ambulatory setting, aiming to identify the optimal candidates for OFC and to limit unnecessary OFC. It would however be recommended that specific values are developed for the local population. It is important to recognize that OFC can always be performed at the discretion of the treating physician despite the elevated results of the allergy tests. In the research setting, DBPCFC OFC are recommended regardless of the CM-sIgE level.</caption><thead><tr><th></th><th>CM-sIgE [kUA/L]</th><th>OFC to unheated milk</th></tr></thead><tbody><tr><td>&gt;95% PPV</td><td>≥15;<sup>52</sup><br/>≥ 5 if less than 1 year old<sup>53</sup></td><td>Defer</td></tr><tr><td>&gt;50% to &lt;95% PPV</td><td>5-15</td><td>Consider the OFC based on the clinician and patient's/ family preference, social and nutritional importance of dairy, history of recent reactions and type of symptoms</td></tr><tr><td>&lt;50% PPV</td><td>&lt;5<br/>CM skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;95% PPV</td><td>≥8<sup>54</sup><br/>Casein-sIgE [kU/L]</td><td>Defer<br/>OFC to baked milk</td></tr><tr><td>&gt;95% PPV</td><td>≥10<sup>21</sup></td><td>Defer</td></tr><tr><td>&gt;50% to &lt;95% PPV</td><td>5-10</td><td>Consider the OFC based on the clinician and patient's/ family preference, social and nutritional importance of dairy, history of recent reactions and type of symptoms</td></tr><tr><td>&lt;50% PPV</td><td>≤5<sup>21</sup><br/>CM skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;90% NPV</td><td>&lt;12<sup>55</sup><br/>Casein skin prick test mean wheal diameter, mm</td><td>Perform</td></tr><tr><td>&gt;90% NPV</td><td>≤9<sup>55</sup></td><td>Perform</td></tr></tbody></table>

**Not recommended:** Testing for CM-specific IgG / IgG4 antibodies is not recommended in the diagnosis of CM allergy as these antibodies reflect the presence of CM in the diet, not an allergy.

**Elimination-Challenge testing:** In general, the conclusive diagnosis of CM allergy requires elimination of CM proteins from the diet followed by a supervised oral food challenge. Double-blind placebo controlled oral food challenge (DBPCFC) remains the gold standard for food allergy diagnosis and it is commonly utilized in the research setting. Open controlled challenge can replace DBPCFC in the children younger than 2 years of age and serve as a useful screening

test for patients of any age in the clinical setting. The initial assessment of reactivity to baked milk is also recommended to be conducted under the physician-supervised food challenge condition because children reactive to baked milk may experience anaphylaxis. However, such approach may be unnecessarily restrictive and not practical when access to food challenges is limited due to the paucity of allergy specialists or during the COVID-19 pandemic.⁹⁶⁰ Several reports indicated that home introduction of baked milk and egg may be safely done in carefully selected young children, e.g., under the age 3 years, without prior history of anaphylaxis or wheezing from any causes and skin prick test wheal diameter less than 8 mm for cow's milk.⁹⁶¹,⁹⁶² Home introduction starts from a significantly lower dose of baked milk and progresses slowly over the course of days as compared to a single supervised feeding over hours to a higher dose of baked food. However, severe and even delayed reactions are possible and caution is needed.⁹⁶³,⁹⁶⁴ The implementation of this approach must be adapted to the local context including quick access to emergency facilities, if required.

### Suspected non IgE-mediated CM allergy

**Laboratory testing:** There is no reliable laboratory diagnostic testing for non-IgE-mediated CM allergy.⁷⁴,⁹⁴⁴ Atopy patch testing may be considered in selected cases of EoE but not as a routine diagnostic test.⁷⁴ Lymphocyte transformation test, serum CM-specific IgG / IgG4 antibodies, or stool measurements of pro-inflammatory mediators (e.g., calprotectin, eosinophilic cationic protein, eosinophil derived neurotoxin) are not recommended [1]. As some non-IgE-mediated disorders may be associated with a concomitant IgE-mediated food allergy, testing for CM-specific IgE antibodies may be utilized in such cases, e.g., eosinophilic esophagitis (EoE) and food protein-induced enterocolitis syndrome (FPIES) to diagnose IgE-mediated CM allergy. The ultimate confirmation of diagnosis in non-IgE-mediated CM allergy requires an elimination of CM proteins from the diet and followed by an oral CM challenge. With an exception of FPIES, reintroduction of CM can be done at home. When FPIES is suspected, reintroduction during a supervised food challenge should be considered, due to the risk of severe reactions (hypotension). Tolerance to baked milk among patients with non-IgE-mediated CM allergy has not been systematically characterized. Based on anecdotal reports, a subset of patients with EoE might tolerate baked milk in the diet; however, such patients likely represent a minority, unlike with IgE-mediated CM allergy.

### Management of CM allergy

Management primarily relies on dietary avoidance of CM proteins. However, emerging data emphasize a shift from the traditional passive approach of avoidance, to a proactive one that seeks to modulate the immune system.⁹⁶⁵ In infants and young children, substituting alternative sources of protein, calories, and calcium with a specialized hypoallergenic formula may be necessary. The alternative formula choices include casein-hydrolysate, whey-hydrolysate, and amino-acid based formulas, as well as soy-based and rice hydrolysate.⁹⁶⁶ The selection of the most appropriate formula depends on the age and allergic profile of the child. Soy formulas are based on an intact protein and are not hypoallergenic but can be a suitable
alternative to cow's milk. Soya milk should be used with caution, especially in younger infants with gastrointestinal manifestations as they are at risk of reacting to soya when this is introduced in place of cow's milk via an inflamed gut. In non-IgE-mediated food allergy, co-reactivity between cow's milk and soya in the first 6 months of life is about 40%. Nutritional consultation is recommended for those with severe form of CM allergy, multiple food allergies and poor growth. Multiple studies of CM allergy have shown that avoidance of CM compared with avoidance of other allergens resulted in greater deficits in height and weight.⁹⁶⁷ Education about recognition of allergic symptoms and prompt treatment of anaphylaxis is crucial in the patients at risk for anaphylaxis. As most children outgrow CM by school age, periodic re-evaluations every 6-12 months with laboratory testing and oral food challenges are recommended. A drop in the specific CM IgE level by 50% or more over 12-24 months is a favorable prognostic indicator of developing tolerance.⁹⁶⁸ Basophil activation tests, in combination with serum specific IgE and SPT, can also help identify patients that have developed tolerance to CM.¹⁸¹ Children with peak lifetime CM-IgE >50 kU/L are more likely to retain milk allergy until teenage years and may need less frequent testing.⁹⁶⁹ Introduction of baked products with CM should be attempted under physician supervision for patients with IgE-mediated FA. Baked milk products may be tolerated by a subset of patient with EoE.⁹⁷⁰ It is unknown if children with FPIES can tolerate baked milk products and therefore strict avoidance is recommended.

### Cross-reactivity with beef and oligosaccharide galactose-α-1,3-galactose (α-gal) syndrome

The prevalence of beef allergy among those with CM allergy has been estimated as 13-20%.⁹⁷¹ Conversely, the prevalence of CM allergy among those with beef allergy is considerably higher. In a double-blind placebo-controlled food challenge study of 335 patients with atopic dermatitis (AD) and possible food hypersensitivity, 11 were found to have symptomatic beef allergy, 8 (73%) of which were also sensitive to CM. Mammalian meat allergy or α-gal syndrome is a recently identified delayed food allergy associated with tick bites, leading to the development of IgE to the α-gal. Many patients with α-gal syndrome have IgE antibodies that recognize α-gal present in CM extract and have positive SPT to CM. However, avoidance of dairy products is not routinely recommended in these patients, as most patients are able to tolerate CM or cheese.²⁰ In a study of 24 patients with IgE to α-gal, 14/24 participants showed tolerance to CM despite positive skin prick test and serum titers. While avoidance of mammalian meat is recommended in α-gal syndrome, avoidance of cow's milk is not always required, in patients with clinical tolerance of CM.⁹⁷²

### Natural history of CM allergy

CM allergy is the most common childhood allergy with a prevalence estimated of 2.5% including both IgE and non-IgE-mediated reactions.⁹⁶⁹,⁹⁷³,⁹⁷⁴ Sensitization typically occurs within 1 year of age. Multiple prospective and retrospective observational cohort studies in the US, UK and Israel have estimated the rates of resolution to be approximately 50% by age 10.⁹⁷⁵ A study of 244 with CM allergy by

Wood et al found resolution at 52.6% at median age of 63 months, with CM-IgE, milk SPT wheal size and AD severity as important predictors of prognosis.⁹⁷⁶ Skripak et al reviewed 807 patients with CM allergy and estimated rates of resolution at 4, 8, 12, and 16 years to be 19%, 42%, 64% and 79% respectively. Those with persistent CM allergy had higher CM-IgE levels up until 16 years of age and those with concurrent asthma and allergic rhinitis were associated with worse outcomes.⁹⁶⁹ Other studies have shown faster rates of tolerance. A Danish birth cohort of 1749 children with CM allergy showed tolerance in 56% at 1 year and 77% at 2 years.⁹⁷⁷ Resolution in non-IgE-mediated CM allergy have been shown to occur more rapidly compared with IgE-mediated CM allergy. The EuroPrevall birth cohort study found that tolerance at 1 year occurred in 100% of those with non-IgE CM allergy, compared with 57% in those with IgE-mediated CM allergy. In contrast to childhood CM allergy, adult onset of IgE-mediated CM allergy is rare but characterized by more severe reactions including anaphylaxis that occur with low eliciting doses starting at 0.3 mg CM protein.⁹⁷⁸

### Prevention of CM allergy

Early introduction of peanut and egg has been now established in the prevention of peanut and egg allergies, especially in population with high prevalence of the disease.⁹⁷⁹ However, the evidence has been less clear with regards to CM ingestion. A recent review article of very early CM introduction within the first month of life discussed three observational studies associated with decreased incidence of CM allergy.⁹⁷⁵ A randomized control of 491 infants showed that daily ingestion between 1-2 months of age of at least 10 mL of CM formula reported 2 CMA cases (0.8%) among the 242 members of the ingestion group and 17 CMA cases (6.8%) among the 249 participants in the avoidance group (risk ratio = 0.12; 95% CI = 0.01-0.50; P < .001). The risk difference was 6.0% (95% CI = 2.7-9.3%).⁹⁸⁰ This is an area ongoing area of research that may impact future consensus guidelines on timing to introduce CM.

### Novel therapies for CM allergy

Oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy routes have been evaluated for CM allergy with promising results in clinical trials.⁹⁸¹,⁹⁸² OIT, SLIT, and EPIT utilize native CM proteins in a form of a CM powder. In a trial comparing CM OIT and SLIT, 10% receiving SLIT (maintenance daily dose 7 mg CM) were desensitized, 60% receiving SLIT/low dose OIT (maintenance daily dose 1000 mg CM) were desensitized, and 80% receiving SLIT/high dose OIT (maintenance daily dose 2000 mg CM) were desensitized.⁴⁰⁵ In general, CM SLIT was associated with very mild side effects mostly oro-pharyngeal pruritus, whereas CM OIT was associated with more systemic side effects, involving gastrointestinal tract (nausea, vomiting, abdominal pain, diarrhea), or respiratory system (rhinorrhea, sneezing, congestion, cough, wheezing).³⁸³ CM OIT has been associated with cases of EoE. More studies are needed to determine the potential of inducing permanent oral tolerance to milk with CM OIT. In a small pilot study, after 90 days, CM EPIT treatment tended to increase the cumulative tolerated dose, from a mean ± SD of 1.77 ± 2.98 mL at day 0 to 23.61 ± 28.61 mL at day 90.⁹⁶⁷
alternative to cow's milk. Soya milk should be used with caution, especially in younger infants with gastrointestinal manifestations as they are at risk of reacting to soya when this is introduced in place of cow's milk via an inflamed gut. In non-IgE-mediated food allergy, co-reactivity between cow's milk and soya in the first 6 months of life is about 40%. Nutritional consultation is recommended for those with severe form of CM allergy, multiple food allergies and poor growth. Multiple studies of CM allergy have shown that avoidance of CM compared with avoidance of other allergens resulted in greater deficits in height and weight.⁹⁶⁷ Education about recognition of allergic symptoms and prompt treatment of anaphylaxis is crucial in the patients at risk for anaphylaxis. As most children outgrow CM by school age, periodic re-evaluations every 6-12 months with laboratory testing and oral food challenges are recommended. A drop in the specific CM IgE level by 50% or more over 12-24 months is a favorable prognostic indicator of developing tolerance.⁹⁶⁸ Basophil activation tests, in combination with serum specific IgE and SPT, can also help identify patients that have developed tolerance to CM.¹⁸¹ Children with peak lifetime CM-IgE >50 kU/L are more likely to retain milk allergy until teenage years and may need less frequent testing.⁹⁶⁹ Introduction of baked products with CM should be attempted under physician supervision for patients with IgE-mediated FA. Baked milk products may be tolerated by a subset of patient with EoE.⁹⁷⁰ It is unknown if children with FPIES can tolerate baked milk products and therefore strict avoidance is recommended.

### Cross-reactivity with beef and oligosaccharide galactose-α-1,3-galactose (α-gal) syndrome

The prevalence of beef allergy among those with CM allergy has been estimated as 13-20%.⁹⁷¹ Conversely, the prevalence of CM allergy among those with beef allergy is considerably higher. In a double-blind placebo-controlled food challenge study of 335 patients with atopic dermatitis (AD) and possible food hypersensitivity, 11 were found to have symptomatic beef allergy, 8 (73%) of which were also sensitive to CM. Mammalian meat allergy or α-gal syndrome is a recently identified delayed food allergy associated with tick bites, leading to the development of IgE to the α-gal. Many patients with α-gal syndrome have IgE antibodies that recognize α-gal present in CM extract and have positive SPT to CM. However, avoidance of dairy products is not routinely recommended in these patients, as most patients are able to tolerate CM or cheese.²⁰ In a study of 24 patients with IgE to α-gal, 14/24 participants showed tolerance to CM despite positive skin prick test and serum titers. While avoidance of mammalian meat is recommended in α-gal syndrome, avoidance of cow's milk is not always required, in patients with clinical tolerance of CM.⁹⁷²

### Natural history of CM allergy

CM allergy is the most common childhood allergy with a prevalence estimated of 2.5% including both IgE and non-IgE-mediated reactions.⁹⁶⁹,⁹⁷³,⁹⁷⁴ Sensitization typically occurs within 1 year of age. Multiple prospective and retrospective observational cohort studies in the US, UK and Israel have estimated the rates of resolution to be approximately 50% by age 10.⁹⁷⁵ A study of 244 with CM allergy by

Wood et al found resolution at 52.6% at median age of 63 months, with CM-IgE, milk SPT wheal size and AD severity as important predictors of prognosis.⁹⁷⁶ Skripak et al reviewed 807 patients with CM allergy and estimated rates of resolution at 4, 8, 12, and 16 years to be 19%, 42%, 64% and 79% respectively. Those with persistent CM allergy had higher CM-IgE levels up until 16 years of age and those with concurrent asthma and allergic rhinitis were associated with worse outcomes.⁹⁶⁹ Other studies have shown faster rates of tolerance. A Danish birth cohort of 1749 children with CM allergy showed tolerance in 56% at 1 year and 77% at 2 years.⁹⁷⁷ Resolution in non-IgE-mediated CM allergy have been shown to occur more rapidly compared with IgE-mediated CM allergy. The EuroPrevall birth cohort study found that tolerance at 1 year occurred in 100% of those with non-IgE CM allergy, compared with 57% in those with IgE-mediated CM allergy. In contrast to childhood CM allergy, adult onset of IgE-mediated CM allergy is rare but characterized by more severe reactions including anaphylaxis that occur with low eliciting doses starting at 0.3 mg CM protein.⁹⁷⁸

### Prevention of CM allergy

Early introduction of peanut and egg has been now established in the prevention of peanut and egg allergies, especially in population with high prevalence of the disease.⁹⁷⁹ However, the evidence has been less clear with regards to CM ingestion. A recent review article of very early CM introduction within the first month of life discussed three observational studies associated with decreased incidence of CM allergy.⁹⁷⁵ A randomized control of 491 infants showed that daily ingestion between 1-2 months of age of at least 10 mL of CM formula reported 2 CMA cases (0.8%) among the 242 members of the ingestion group and 17 CMA cases (6.8%) among the 249 participants in the avoidance group (risk ratio = 0.12; 95% CI = 0.01-0.50; P < .001). The risk difference was 6.0% (95% CI = 2.7-9.3).⁹⁸⁰ This is an area ongoing area of research that may impact future consensus guidelines on timing to introduce CM.

### Novel therapies for CM allergy

Oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy routes have been evaluated for CM allergy with promising results in clinical trials.⁹⁸¹,⁹⁸² OIT, SLIT, and EPIT utilize native CM proteins in a form of a CM powder. In a trial comparing CM OIT and SLIT, 10% receiving SLIT (maintenance daily dose 7 mg CM) were desensitized, 60% receiving SLIT/low dose OIT (maintenance daily dose 1000 mg CM) were desensitized, and 80% receiving SLIT/high dose OIT (maintenance daily dose 2000 mg CM) were desensitized.⁴⁰⁵ In general, CM SLIT was associated with very mild side effects mostly oro-pharyngeal pruritus, whereas CM OIT was associated with more systemic side effects, involving gastrointestinal tract (nausea, vomiting, abdominal pain, diarrhea), or respiratory system (rhinorrhea, sneezing, congestion, cough, wheezing).³⁸³ CM OIT has been associated with cases of EoE. More studies are needed to determine the potential of inducing permanent oral tolerance to milk with CM OIT. In a small pilot study, after 90 days, CM EPIT treatment tended to increase the cumulative tolerated dose, from a mean ± SD of 1.77 ± 2.98 mL at day 0 to 23.61 ± 28.61 mL at day 90.⁹⁶⁷
# 5 - Clinical cases

## Case 1:

Parents of a 2-year old child with atopic dermatitis and history of milk-induced generalized urticaria at the age 6 months inquire about the likelihood of their child outgrowing milk allergy. There are no additional allergic reactions to milk or milk products. Skin prick test with a commercial cow milk extract is positive at a mean wheal diameter at 10mm. Serum CM-specific IgE antibody level is 17 kU/L. Based on these results, the child has more than 95% chances of reacting to liquid milk. However, considering that about 70-80% of milk allergic children tolerate milk in the baked products, further diagnostic testing is performed. Serum specific IgE antibodies directed against casein level is 4.5 kU/L and beta-lactoglobulin IgE is 25 kU/L. Based on the level of the casein-specific IgE, it is estimated that the

likelihood of tolerating baked milk products in form of a muffin is approximately 50%. A physician-supervised oral challenge with baked milk in a form of a muffin is performed in the office and the child tolerates it without an adverse reaction. Baked milk products are incorporated into the diet.

## Case 2:

A 10-year-old asthmatic male has history of severe anaphylaxis to trace amounts of milk in a cookie. He wants to know what his chances of outgrowing his milk allergy are. His CM-IgE is 75 kU/L, casein IgE is 90 kU/L; SPT to CM extract is 20mm diameter. Based on his past history of anaphylaxis to baked milk and the current test results highly predictive of clinical reactivity to both baked and unheated milk, it is likely that he will remain-milk allergic until his teenage years.
# 5 - Clinical cases

## Case 1:

Parents of a 2-year old child with atopic dermatitis and history of milk-induced generalized urticaria at the age 6 months inquire about the likelihood of their child outgrowing milk allergy. There are no additional allergic reactions to milk or milk products. Skin prick test with a commercial cow milk extract is positive at a mean wheal diameter at 10mm. Serum CM-specific IgE antibody level is 17 kU/L. Based on these results, the child has more than 95% chances of reacting to liquid milk. However, considering that about 70-80% of milk allergic children tolerate milk in the baked products, further diagnostic testing is performed. Serum specific IgE antibodies directed against casein level is 4.5 kU/L and beta-lactoglobulin IgE is 25 kU/L. Based on the level of the casein-specific IgE, it is estimated that the

likelihood of tolerating baked milk products in form of a muffin is approximately 50%. A physician-supervised oral challenge with baked milk in a form of a muffin is performed in the office and the child tolerates it without an adverse reaction. Baked milk products are incorporated into the diet.

## Case 2:

A 10-year-old asthmatic male has history of severe anaphylaxis to trace amounts of milk in a cookie. He wants to know what his chances of outgrowing his milk allergy are. His CM-IgE is 75 kU/L, casein IgE is 90 kU/L; SPT to CM extract is 20mm diameter. Based on his past history of anaphylaxis to baked milk and the current test results highly predictive of clinical reactivity to both baked and unheated milk, it is likely that he will remain-milk allergic until his teenage years.
# B11 - Allergy to egg

Mattia Giovannini, Philippe A. Eigenmann, Jean-Christoph Caubet, and Antonella Muraro

## Highlights

* Egg allergy is one of the most frequent food allergies in children.

* The use of egg white components can help distinguishing between allergy to baked, cooked and raw egg.

* The use of egg white components is clinically helpful for distinguishing between transient and persistent allergy to eggs.

* The result of an IgE test can confirm the diagnosis of egg allergy in the case of a clear clinical history of reaction; in equivocal cases, an oral food challenge to egg may be needed to clarify the diagnosis.

## 1 - The allergen sources

Hen's egg is a ubiquitous food eaten in most parts of the world. It is a cheap and easily accessible food source, used in many homemade dishes but also widely used by the food industry in processed foods. Allergenicity of baked egg (180°C for at least 20 minutes) might be reduced because interaction with the food matrix might block epitope access, and heating might destroy conformational epitopes. Individuals are mostly exposed to egg proteins in foods. Nevertheless, egg proteins can be found in aerosolized particles produced by cooking. Respiratory clinical manifestations to aerosolized egg proteins have been reported in bakery workers.⁹⁸³ Exposure to egg proteins via the respiratory route might also contribute to primary sensitization to eggs, similar to what has been

shown for peanut proteins⁹⁸⁴ Moreover, an increased risk of peanut allergy has been reported in infants with atopic dermatitis after a low-dose exposure to peanut proteins through the skin. It appears likely that low-dose cutaneous exposure can end in allergic sensitization to egg.⁹⁸⁶

## 2 - Major and relevant minor allergenic molecules and their clinical relevance

Five proteins most commonly involved in allergic reactions to hen's egg have been identified and characterized (Gal d 1 to 5, see Figure 82 and Table 35). Despite being present in a lower quantity in egg white than ovalbumin, ovomucoid is probably the immunodominant egg allergen.⁹⁸⁸,⁹⁸⁹ Among the various physicochemical characteristics, resistance to chemical denaturation has a direct clinical significance. Structural modification of egg allergens might allow safe consumption of cooked/baked egg-containing foods.

Clinical cross-reactivity occurs between various bird egg proteins (e.g., hen, turkey, duck and seagull).⁹⁸⁷ Thus, avoidance of other bird's eggs should be recommended when providing dietary guidance to egg-allergic patients.

## 3 - Clinical relevance, diagnosis and management

According to the IgE sensitivity of a given patient, three different clinical scenarios should be distinguished in a patient with a positive skin prick test or detectable levels of IgE to egg proteins⁹⁹⁰,⁹⁹¹:

### 1. Sensitized to eggs but clinically tolerant

Can eat all forms of eggs. Such patients will generally present a positive serum IgE test to egg white, in a low to mid-range value,

<figure><img src="image_186.png" /><figcaption>FIGURE 82 Major egg allergens</figcaption></figure>
# B11 - Allergy to egg

Mattia Giovannini, Philippe A. Eigenmann, Jean-Christoph Caubet, and Antonella Muraro

## Highlights

* Egg allergy is one of the most frequent food allergies in children.

* The use of egg white components can help distinguishing between allergy to baked, cooked and raw egg.

* The use of egg white components is clinically helpful for distinguishing between transient and persistent allergy to eggs.

* The result of an IgE test can confirm the diagnosis of egg allergy in the case of a clear clinical history of reaction; in equivocal cases, an oral food challenge to egg may be needed to clarify the diagnosis.

## 1 - The allergen sources

Hen's egg is a ubiquitous food eaten in most parts of the world. It is a cheap and easily accessible food source, used in many homemade dishes but also widely used by the food industry in processed foods. Allergenicity of baked egg (180°C for at least 20 minutes) might be reduced because interaction with the food matrix might block epitope access, and heating might destroy conformational epitopes. Individuals are mostly exposed to egg proteins in foods. Nevertheless, egg proteins can be found in aerosolized particles produced by cooking. Respiratory clinical manifestations to aerosolized egg proteins have been reported in bakery workers.⁹⁸³ Exposure to egg proteins via the respiratory route might also contribute to primary sensitization to eggs, similar to what has been

shown for peanut proteins⁹⁸⁴ Moreover, an increased risk of peanut allergy has been reported in infants with atopic dermatitis after a low-dose exposure to peanut proteins through the skin. It appears likely that low-dose cutaneous exposure can end in allergic sensitization to egg.⁹⁸⁶

## 2 - Major and relevant minor allergenic molecules and their clinical relevance

Five proteins most commonly involved in allergic reactions to hen's egg have been identified and characterized (Gal d 1 to 5, see Figure 82 and Table 35). Despite being present in a lower quantity in egg white than ovalbumin, ovomucoid is probably the immunodominant egg allergen.⁹⁸⁸,⁹⁸⁹ Among the various physicochemical characteristics, resistance to chemical denaturation has a direct clinical significance. Structural modification of egg allergens might allow safe consumption of cooked/baked egg-containing foods.

Clinical cross-reactivity occurs between various bird egg proteins (e.g., hen, turkey, duck and seagull).⁹⁸⁷ Thus, avoidance of other bird's eggs should be recommended when providing dietary guidance to egg-allergic patients.

## 3 - Clinical relevance, diagnosis and management

According to the IgE sensitivity of a given patient, three different clinical scenarios should be distinguished in a patient with a positive skin prick test or detectable levels of IgE to egg proteins⁹⁹⁰,⁹⁹¹:

### 1. Sensitized to eggs but clinically tolerant

Can eat all forms of eggs. Such patients will generally present a positive serum IgE test to egg white, in a low to mid-range value,

<figure><img src="image_186.png" /><figcaption>FIGURE 82 Major egg allergens</figcaption></figure>
<table><caption>TABLE 35 Allergenic molecules of hen's egg and clinical relevance of specific proteins Adapted from [989]</caption><thead><tr><th>Protein name</th><th>MW (kDa)</th><th>Protein family</th><th>Biological function(s)</th><th>Resistance to heating and chemical denaturation</th><th>Clinical relevance</th></tr></thead><tbody><tr><td colspan="6"><strong>Egg White Proteins</strong></td></tr><tr><td>Ovomucoid (Gal d1)</td><td>28</td><td>Kazal-type serine protease inhibitor</td><td>serine protease inhibition activity antibacterial activity</td><td>high</td><td>Heat-stable and highly allergenic. Risk for reaction to all forms of egg. High levels of specific IgE might indicate sustained egg allergy.</td></tr><tr><td>Ovalbumin (Gal d 2)</td><td>45</td><td>serine protease inhibitor</td><td>storage protein?</td><td>low</td><td>Heat-labile. Most abundant egg white protein. Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Ovotransferrin or ovalbumin (Gal d 3)</td><td>76-77</td><td>transferrin</td><td>iron-binding capacity with antimicrobial activity</td><td>low</td><td>Heat-labile. Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Egg lysozyme (Gal d 4)</td><td>14.3</td><td>glycoside hydrolase family 22</td><td>antibacterial activity</td><td>moderate</td><td>Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Ovomucin</td><td>165</td><td>contains trypsin inhibitor-like domains</td><td>heavily glycosylated protein with potent antiviral activities</td><td>n.a.</td><td></td></tr><tr><td colspan="6"><strong>Egg Yolk Proteins</strong></td></tr><tr><td>Phosvitin</td><td>35</td><td>transferase?</td><td>Metal-chelating agent</td><td>n.a.</td><td></td></tr><tr><td>α-livetin (Gal d 5)</td><td>65-70</td><td>serum albumin</td><td>bind ions, fatty acids, hormones in physiological conditions</td><td>n.a.</td><td></td></tr><tr><td>Apovitellenins I</td><td>9.5</td><td>very low-density lipoprotein</td><td>potent lipoprotein lipase inhibitor</td><td>n.a.</td><td></td></tr><tr><td>Apovitellenins VI (orapoprotein B)</td><td>170</td><td>unknown</td><td>lipid-binding activity</td><td>n.a.</td><td></td></tr></tbody></table>

as well as a negative or low serum IgE test to ovomucoid. Serum specific IgE to ovalbumin might be elevated in a similar range to the test to egg white.

## 2. Allergic to raw or partially raw eggs only

Tolerant to baked eggs or cooked eggs. These patients will generally present similarly to scenario one, with a positive serum IgE test to egg white, in a low to mid-range value, as well as a negative or low serum IgE test to ovomucoid. Serum specific IgE to ovalbumin might be elevated in a similar range to the test to egg white.

## 3. Allergic to all forms of egg

These patients generally have serum specific IgE to egg white in the middle to upper range. They might also have elevated serum specific IgE to ovomucoid and to ovalbumin.

### Clinical history

The case history is decisive. It needs to be assessed if the patient has a concomitant atopic disease (e.g., atopic eczema), which might predispose to a positive test to egg white. If the child has a history of an allergic reaction after eating eggs, the history needs to specify to which form of egg the child reacted (baked, cooked, or raw eggs).

### Skin prick test (SPT)

Skin prick test can be done with commercial egg white extracts or with raw eggs. Both have good accuracy for showing IgE sensitization. Extracts of major egg allergens (ovomucoid, ovalbumin or others) are not commercially available and are not used in routine diagnostic testing. A systematic review on diagnosis of egg allergy in children using cut-offs established that heated egg allergy seems
<table><caption>TABLE 35 Allergenic molecules of hen's egg and clinical relevance of specific proteins Adapted from [989]</caption><thead><tr><th>Protein name</th><th>MW (kDa)</th><th>Protein family</th><th>Biological function(s)</th><th>Resistance to heating and chemical denaturation</th><th>Clinical relevance</th></tr></thead><tbody><tr><td colspan="6"><strong>Egg White Proteins</strong></td></tr><tr><td>Ovomucoid (Gal d1)</td><td>28</td><td>Kazal-type serine protease inhibitor</td><td>serine protease inhibition activity antibacterial activity</td><td>high</td><td>Heat-stable and highly allergenic. Risk for reaction to all forms of egg. High levels of specific IgE might indicate sustained egg allergy.</td></tr><tr><td>Ovalbumin (Gal d 2)</td><td>45</td><td>serine protease inhibitor</td><td>storage protein?</td><td>low</td><td>Heat-labile. Most abundant egg white protein. Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Ovotransferrin or ovalbumin (Gal d 3)</td><td>76-77</td><td>transferrin</td><td>iron-binding capacity with antimicrobial activity</td><td>low</td><td>Heat-labile. Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Egg lysozyme (Gal d 4)</td><td>14.3</td><td>glycoside hydrolase family 22</td><td>antibacterial activity</td><td>moderate</td><td>Risk for clinical reaction to raw or slightly heated egg.</td></tr><tr><td>Ovomucin</td><td>165</td><td>contains trypsin inhibitor-like domains</td><td>heavily glycosylated protein with potent antiviral activities</td><td>n.a.</td><td></td></tr><tr><td colspan="6"><strong>Egg Yolk Proteins</strong></td></tr><tr><td>Phosvitin</td><td>35</td><td>transferase?</td><td>Metal-chelating agent</td><td>n.a.</td><td></td></tr><tr><td>α-livetin (Gal d 5)</td><td>65-70</td><td>serum albumin</td><td>bind ions, fatty acids, hormones in physiological conditions</td><td>n.a.</td><td></td></tr><tr><td>Apovitellenins I</td><td>9.5</td><td>very low-density lipoprotein</td><td>potent lipoprotein lipase inhibitor</td><td>n.a.</td><td></td></tr><tr><td>Apovitellenins VI (orapoprotein B)</td><td>170</td><td>unknown</td><td>lipid-binding activity</td><td>n.a.</td><td></td></tr></tbody></table>

as well as a negative or low serum IgE test to ovomucoid. Serum specific IgE to ovalbumin might be elevated in a similar range to the test to egg white.

## 2. Allergic to raw or partially raw eggs only

Tolerant to baked eggs or cooked eggs. These patients will generally present similarly to scenario one, with a positive serum IgE test to egg white, in a low to mid-range value, as well as a negative or low serum IgE test to ovomucoid. Serum specific IgE to ovalbumin might be elevated in a similar range to the test to egg white.

## 3. Allergic to all forms of egg

These patients generally have serum specific IgE to egg white in the middle to upper range. They might also have elevated serum specific IgE to ovomucoid and to ovalbumin.

### Clinical history

The case history is decisive. It needs to be assessed if the patient has a concomitant atopic disease (e.g., atopic eczema), which might predispose to a positive test to egg white. If the child has a history of an allergic reaction after eating eggs, the history needs to specify to which form of egg the child reacted (baked, cooked, or raw eggs).

### Skin prick test (SPT)

Skin prick test can be done with commercial egg white extracts or with raw eggs. Both have good accuracy for showing IgE sensitization. Extracts of major egg allergens (ovomucoid, ovalbumin or others) are not commercially available and are not used in routine diagnostic testing. A systematic review on diagnosis of egg allergy in children using cut-offs established that heated egg allergy seems
very likely if SPTs with egg white extract are >5mm in children <2 years and >11mm in children ≥2 years. In children <2 years, raw egg allergy appears very likely when SPTs with egg white extract are ≥4mm; in children ≥2 years, raw egg allergy appears very likely when SPTs with egg white extract are ≥10mm.⁹⁹²

### Specific IgE testing

IgE to the following allergens are commercially available for testing: egg, egg white, egg yolk, ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), egg lysozyme (Gal d 4).

Testing for specific IgE to egg white is, in general, mostly recommended for primary diagnosis of egg allergy in children. Egg white extract combines the most common major allergens recognized by egg allergic patient (ovomucoid and ovalbumin) and therefore constitutes the most accurate test for the initial diagnostic step.⁹⁹³ Moreover, levels of specific IgE to egg white might indicate the severity of egg allergy.⁹⁹⁴ In addition, egg white specific IgE levels have been studied in order to determine cut-off values indicative of true clinical egg allergy. However, diagnostic cut-offs vary with the characteristics of the patient populations, for instance age and presence or absence of eczema, and thus should be applied to populations with similar characteristics.⁹⁹⁵⁻⁹⁹⁸ A systematic review on diagnosis of egg allergy in children using cut-offs established that in children <2 years, raw egg allergy seems very likely when specific IgEs are ≥1.7 kU/l and in children ≥2 years raw egg allergy seems very likely when specific IgEs are ≥7.3 kU/l.⁹⁹²

Molecular diagnosis can be helpful to distinguish patients who are reactive to raw or partially raw eggs only from patients who are allergic to all forms of egg. Previous studies have defined a positive decision point for at least 95% clinical specificity for ovomucoid-specific IgE to diagnose allergy to cooked/baked eggs.⁹⁹⁹,¹⁰⁰⁰ Differences between studies done with different patient populations can limit the application of cut-off values to other populations.⁹⁹²,¹⁰⁰¹ However, high levels of specific IgE to ovomucoid can support the diagnosis of cooked/baked eggs allergy and persistent egg allergy as well¹⁰⁰²,¹⁰⁰³ (Table 35).

The heat-labile egg white allergen ovalbumin can contribute to distinguish between the various pattern of clinical reactivity to eggs. Sequential testing starting with IgE measurement to egg white, followed by measuring IgE to ovalbumin and ovomucoid, can significantly improve the diagnosis of raw and cooked/baked egg allergy.⁹⁹⁹,¹⁰⁰⁰ (Table 35).

It has been postulated that egg extracts modified by denaturation for mimicking the heating of eggs or egg digestion in the gut might provide more accurate proteins for clinical diagnosis. For distinguishing between egg sensitized subjects and patients allergic to all forms of eggs, native egg proteins provide reliable extracts for diagnosis as determined by receiver operating characteristic curves. For more refined diagnosis, denatured egg allergens might be helpful.¹⁰⁰⁰ Nevertheless, the clinical utility of such tests needs to be confirmed in larger patient populations.

### Oral food challenge

The oral food challenge can be performed as open, single blind or double-blind, placebo-controlled challenge. Patients with

subjective symptoms should preferably be challenged in a double-blind, placebo-controlled challenge. Food challenges are helpful to get a definite diagnosis, in cases with a discrepancy between the clinical history and specific IgE results, as well as for the follow-up of food allergy. Depending on the clinical question, eggs might be given as raw, cooked, or in baked forms. Routine testing does not include challenges with isolated egg proteins. A study regarding the natural history of egg allergy reported that it resolved in 49.3% of egg-allergic children at a median age of 72 months.¹⁰⁰⁴ Thus, due to the natural history of egg allergy, which is frequently outgrown by 5-7 years of age, natural tolerance acquisition should be tested at regular intervals in average 6-12 months in the absence of an accidental reaction.⁷³ Moreover, introduction of baked egg might accelerate tolerance acquisition to all forms of eggs, but this requires further research.³⁹²,¹⁰⁰⁵

### Clinical Management

Avoidance diet should be restricted to the form of egg not tolerated by the patient. All other forms should be regularly consumed. The diagnostic work-up, including history, SPT, specific IgE and oral food challenge as appropriate should aim to correctly identify forms of eggs to which the patient is tolerant. In addition to allergen avoidance, patients should be provided with medication, such as antihistamines, adrenaline auto-injectors and salbutamol, for treatment of acute allergic reactions due to accidental ingestions.

### Allergen-specific immunotherapy

Various studies have shown clinical efficacy for specific oral tolerance induction protocols.³⁷⁷ Nevertheless, this procedure is not yet applicable to all patients.

## 5 – Clinical cases

### Case 1

**History:** Girl, 8 months old, severe atopic eczema. Allergy testing is performed for ruling out food allergy as a triggering factor of her severe atopic eczema. She has never eaten eggs, neither isolated nor in processed foods.

**SPT:** 10 mm to egg white, negative to milk, wheat, soy, fish, peanut and hazelnut.

**In-vitro testing:** Total IgE 1825 kU/L, specific IgE to egg white 5.02 kU/L, ovoalbumin 1.64 kU/L, ovomucoid 0.82 kU/L.

**Oral challenge:** Negative with baked products, well-tolerated, without immediate reactions or flaring of atopic eczema. The egg is progressively introduced at home in baked forms and in pasta with eggs.

**Diagnosis:** Sensitization to egg white in the context of severe atopic eczema.

**Recommendation:** Continue eggs in baked forms, retesting and perform a food challenge before introducing egg in cooked or raw forms.

### Case 2

**History:** Girl, 13 months old, in good health. She has eaten cooked eggs, either isolated or processed foods, without any clinical
very likely if SPTs with egg white extract are >5mm in children <2 years and >11mm in children ≥2 years. In children <2 years, raw egg allergy appears very likely when SPTs with egg white extract are ≥4mm; in children ≥2 years, raw egg allergy appears very likely when SPTs with egg white extract are ≥10mm.⁹⁹²

### Specific IgE testing

IgE to the following allergens are commercially available for testing: egg, egg white, egg yolk, ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), egg lysozyme (Gal d 4).

Testing for specific IgE to egg white is, in general, mostly recommended for primary diagnosis of egg allergy in children. Egg white extract combines the most common major allergens recognized by egg allergic patient (ovomucoid and ovalbumin) and therefore constitutes the most accurate test for the initial diagnostic step.⁹⁹³ Moreover, levels of specific IgE to egg white might indicate the severity of egg allergy.⁹⁹⁴ In addition, egg white specific IgE levels have been studied in order to determine cut-off values indicative of true clinical egg allergy. However, diagnostic cut-offs vary with the characteristics of the patient populations, for instance age and presence or absence of eczema, and thus should be applied to populations with similar characteristics.⁹⁹⁵⁻⁹⁹⁸ A systematic review on diagnosis of egg allergy in children using cut-offs established that in children <2 years, raw egg allergy seems very likely when specific IgEs are ≥1.7 kU/l and in children ≥2 years raw egg allergy seems very likely when specific IgEs are ≥7.3 kU/l.⁹⁹²

Molecular diagnosis can be helpful to distinguish patients who are reactive to raw or partially raw eggs only from patients who are allergic to all forms of egg. Previous studies have defined a positive decision point for at least 95% clinical specificity for ovomucoid-specific IgE to diagnose allergy to cooked/baked eggs.⁹⁹⁹,¹⁰⁰⁰ Differences between studies done with different patient populations can limit the application of cut-off values to other populations.⁹⁹²,¹⁰⁰¹ However, high levels of specific IgE to ovomucoid can support the diagnosis of cooked/baked eggs allergy and persistent egg allergy as well¹⁰⁰²,¹⁰⁰³ (Table 35).

The heat-labile egg white allergen ovalbumin can contribute to distinguish between the various pattern of clinical reactivity to eggs. Sequential testing starting with IgE measurement to egg white, followed by measuring IgE to ovalbumin and ovomucoid, can significantly improve the diagnosis of raw and cooked/baked egg allergy.⁹⁹⁹,¹⁰⁰⁰ (Table 35).

It has been postulated that egg extracts modified by denaturation for mimicking the heating of eggs or egg digestion in the gut might provide more accurate proteins for clinical diagnosis. For distinguishing between egg sensitized subjects and patients allergic to all forms of eggs, native egg proteins provide reliable extracts for diagnosis as determined by receiver operating characteristic curves. For more refined diagnosis, denatured egg allergens might be helpful.¹⁰⁰⁰ Nevertheless, the clinical utility of such tests needs to be confirmed in larger patient populations.

### Oral food challenge

The oral food challenge can be performed as open, single blind or double-blind, placebo-controlled challenge. Patients with

subjective symptoms should preferably be challenged in a double-blind, placebo-controlled challenge. Food challenges are helpful to get a definite diagnosis, in cases with a discrepancy between the clinical history and specific IgE results, as well as for the follow-up of food allergy. Depending on the clinical question, eggs might be given as raw, cooked, or in baked forms. Routine testing does not include challenges with isolated egg proteins. A study regarding the natural history of egg allergy reported that it resolved in 49.3% of egg-allergic children at a median age of 72 months.¹⁰⁰⁴ Thus, due to the natural history of egg allergy, which is frequently outgrown by 5-7 years of age, natural tolerance acquisition should be tested at regular intervals in average 6-12 months in the absence of an accidental reaction.⁷³ Moreover, introduction of baked egg might accelerate tolerance acquisition to all forms of eggs, but this requires further research.³⁹²,¹⁰⁰⁵

### Clinical Management

Avoidance diet should be restricted to the form of egg not tolerated by the patient. All other forms should be regularly consumed. The diagnostic work-up, including history, SPT, specific IgE and oral food challenge as appropriate should aim to correctly identify forms of eggs to which the patient is tolerant. In addition to allergen avoidance, patients should be provided with medication, such as antihistamines, adrenaline auto-injectors and salbutamol, for treatment of acute allergic reactions due to accidental ingestions.

### Allergen-specific immunotherapy

Various studies have shown clinical efficacy for specific oral tolerance induction protocols.³⁷⁷ Nevertheless, this procedure is not yet applicable to all patients.

## 5 - Clinical cases

### Case 1

**History:** Girl, 8 months old, severe atopic eczema. Allergy testing is performed for ruling out food allergy as a triggering factor of her severe atopic eczema. She has never eaten eggs, neither isolated nor in processed foods.

**SPT:** 10 mm to egg white, negative to milk, wheat, soy, fish, peanut and hazelnut.

**In-vitro testing:** Total IgE 1825 kU/L, specific IgE to egg white 5.02 kU/L, ovoalbumin 1.64 kU/L, ovomucoid 0.82 kU/L.

**Oral challenge:** Negative with baked products, well-tolerated, without immediate reactions or flaring of atopic eczema. The egg is progressively introduced at home in baked forms and in pasta with eggs.

**Diagnosis:** Sensitization to egg white in the context of severe atopic eczema.

**Recommendation:** Continue eggs in baked forms, retesting and perform a food challenge before introducing egg in cooked or raw forms.

### Case 2

**History:** Girl, 13 months old, in good health. She has eaten cooked eggs, either isolated or processed foods, without any clinical
manifestations from 8 months of age. The girl is given for the first time a chocolate mousse made with raw beaten egg white. She presents within minutes a facial rash spreading to the upper thorax, a dry cough, and several episodes of sneezing. The signs and symptoms rapidly disappear after the administration of an oral antihistamine.

**SPT:** 5 mm to egg white.

**In-vitro testing:** Specific IgE to egg white 3.65 kU/L, ovoalbumin 1.56 kU/L, ovomucoid 0.78 kU/L. A low ovomucoid allergen-specific IgE (relatively to the specific IgE to egg white) is indicative of probable tolerance to cooked egg, which the girl tolerated.

**Diagnosis:** Allergy to raw eggs only.

**Recommendation:** Eggs well-tolerated in baked foods or cooked can be eaten. Elimination diet of incompletely cooked or raw eggs in any form. Follow-up at 25 months of age with measurement of SPT and allergen-specific IgE to egg white, ovoalbumin and ovomucoid, assess clinical reactivity with oral food challenge if there is a reasonable chance of tolerance acquisition.

### Case 3

**History:** Boy, 9 months old, history of moderate atopic eczema. He eats for the first time a hard-boiled egg. Present within minutes an urticarial rash over the thorax, followed by an episode of vomiting. The clinical manifestations rapidly disappear after the administration of an oral antihistamine.

**SPT:** 9 mm to egg white.

**In-vitro testing:** Specific IgE to egg white 18.23 kUA/L, ovoalbumin 17.12 kUA/L, ovomucoid 8.56 kU/L. **Diagnosis:** Allergy to all forms of eggs.

**Recommendation:** Eggs in all forms and foods containing eggs need to be avoided. Follow-up at 21 months of age with measurement of SPT and allergen-specific IgE to egg white, ovoalbumin and ovomucoid, assess clinical reactivity with oral food challenge at first to the baked eggs if there is a reasonable chance of tolerance acquisition. Not thoroughly cooked eggs and raw eggs will probably need to be continued to be avoided.

## 6 – Conclusion

At the present stage, the measurement of serum IgE or skin prick testing to egg white should represent the first diagnostic test when assessing a patient with suspected egg allergy. Using tests with egg white components is most helpful for fine-tuning the diagnosis to predict tolerance to baked, cooked and raw eggs and for the follow-up of egg allergy. A definite diagnosis should always be made in relation to the clinical history, and if necessary, by a standardized food challenge. For general recommendations about food allergy diagnosis, the reader might also refer to the European Academy of Allergy and Clinical Immunology Food Allergy Guidelines.
manifestations from 8 months of age. The girl is given for the first time a chocolate mousse made with raw beaten egg white. She presents within minutes a facial rash spreading to the upper thorax, a dry cough, and several episodes of sneezing. The signs and symptoms rapidly disappear after the administration of an oral antihistamine.

**SPT:** 5 mm to egg white.

**In-vitro testing:** Specific IgE to egg white 3.65 kU/L, ovoalbumin 1.56 kU/L, ovomucoid 0.78 kU/L. A low ovomucoid allergen-specific IgE (relatively to the specific IgE to egg white) is indicative of probable tolerance to cooked egg, which the girl tolerated.

**Diagnosis:** Allergy to raw eggs only.

**Recommendation:** Eggs well-tolerated in baked foods or cooked can be eaten. Elimination diet of incompletely cooked or raw eggs in any form. Follow-up at 25 months of age with measurement of SPT and allergen-specific IgE to egg white, ovoalbumin and ovomucoid, assess clinical reactivity with oral food challenge if there is a reasonable chance of tolerance acquisition.

### Case 3

**History:** Boy, 9 months old, history of moderate atopic eczema. He eats for the first time a hard-boiled egg. Present within minutes an urticarial rash over the thorax, followed by an episode of vomiting. The clinical manifestations rapidly disappear after the administration of an oral antihistamine.

**SPT:** 9 mm to egg white.

**In-vitro testing:** Specific IgE to egg white 18.23 kUA/L, ovoalbumin 17.12 kUA/L, ovomucoid 8.56 kU/L. **Diagnosis:** Allergy to all forms of eggs.

**Recommendation:** Eggs in all forms and foods containing eggs need to be avoided. Follow-up at 21 months of age with measurement of SPT and allergen-specific IgE to egg white, ovoalbumin and ovomucoid, assess clinical reactivity with oral food challenge at first to the baked eggs if there is a reasonable chance of tolerance acquisition. Not thoroughly cooked eggs and raw eggs will probably need to be continued to be avoided.

## 6 – Conclusion

At the present stage, the measurement of serum IgE or skin prick testing to egg white should represent the first diagnostic test when assessing a patient with suspected egg allergy. Using tests with egg white components is most helpful for fine-tuning the diagnosis to predict tolerance to baked, cooked and raw eggs and for the follow-up of egg allergy. A definite diagnosis should always be made in relation to the clinical history, and if necessary, by a standardized food challenge. For general recommendations about food allergy diagnosis, the reader might also refer to the European Academy of Allergy and Clinical Immunology Food Allergy Guidelines.
# B12 - Allergy to fish and *Anisakis simplex*

Leticia de las Vecillas, Lars K. Poulsen, Martine Morisset, Annette Kuehn

## Highlights

* Fish species may differ by their allergenic potency.

* Proteins present in fish muscle, roe, skin or blood can elicit fish allergy.

* Less than 1% of the general population suffer from a fish allergy.

* Allergens from fish versus from shellfish and the fish parasite *Anisakis* simplex are not the same.

* Parvalbumin is the major fish allergen, but IgE spreading to other allergens is also common.

* *Anisakis* simplex allergy is diagnosed best using a combination of extract and single molecules while excluding sensitization to other parasites.

## 1 - The allergen source

**FISH**—Fish together with egg, milk and crustaceans represent the animal kingdom in the “big eight” group of food allergens, to which the majority of food allergic patients react. As fish is both an important food component and a potent source of food allergen, fish has also become a part of the European Union regulation of food labelling (EU regulation No 1169/2011), as allergy hazard of fish-containing commercial food products.¹⁹⁵ Despite the broad biodiversity among fish (more than 30,000 individual species have been described),

commonly consumed species are members of the Osteichthyes group (bony fish) and belong to a limited number of orders, the salmon-like (Salmoniformes), cod-like (Gadiformes), perch-like (Perciformes), herring-like (Clupeiformes), carp-like (Cypriniformes), catfish-like (Siluriformes), and platfish (Pleuronectiformes) (Figure 83).

Globally, a high number of fish species is commercially available. The market share of these species varies in different countries according to regional production sites and eating habits. While cod and salmon are important food fish in Europe, other low-value freshwater species are popular in Asia (e.g., grass carp and Asian carp). Fish allergens have been described in about 40 species, but detailed analysis of the allergy-eliciting molecules was performed mainly for fish, which are commonly consumed in the European area such as carp, cod, salmon, trout and tuna.¹⁹⁵ Meanwhile the knowledge on food allergy to other species such as barramundi and catfish is growing.¹⁰⁰⁹,¹⁰¹⁰ A large and clinically relevant cross-reactivity seems to exist between parvalbumins of different fish species (see chapter C11). The following fish products can be elicitors of mild to severe allergic reactions in sensitized patients.

**Fish meat**—The largest allergenic activity resides in the muscle of the fish.¹⁹⁵ Fish is consumed as cooked, fried, pickled or even raw food product. Food processing seems not to affect the allergenic potency of the fish but rather the allergen content, which varies in different species.¹⁰⁰⁹ Parvalbumins, the major fish allergens, are highly abundant in the fish muscle. With a serving size of 200 g cod filet, the consumer might ingest up to 0.5 g of parvalbumin per meal. Other fish allergens present in the muscle are enolases, aldolases, collagen, tropomyosin and others. As a food ingredient, fish must be listed specifically on a product label regardless of the percentage of content.

**Eggs, roe, caviar**—There are case reports that caviar has elicited allergic reactions. Typically, roe is consumed in its raw form. Fish

<figure><img src="image_187.png" /><figcaption>FIGURE 83 Most important food fish are members of seven taxonomic orders.</figcaption></figure>
# B12 - Allergy to fish and *Anisakis simplex*

Leticia de las Vecillas, Lars K. Poulsen, Martine Morisset, Annette Kuehn

## Highlights

* Fish species may differ by their allergenic potency.

* Proteins present in fish muscle, roe, skin or blood can elicit fish allergy.

* Less than 1% of the general population suffer from a fish allergy.

* Allergens from fish versus from shellfish and the fish parasite *Anisakis* simplex are not the same.

* Parvalbumin is the major fish allergen, but IgE spreading to other allergens is also common.

* *Anisakis* simplex allergy is diagnosed best using a combination of extract and single molecules while excluding sensitization to other parasites.

## 1 - The allergen source

**FISH**—Fish together with egg, milk and crustaceans represent the animal kingdom in the “big eight” group of food allergens, to which the majority of food allergic patients react. As fish is both an important food component and a potent source of food allergen, fish has also become a part of the European Union regulation of food labelling (EU regulation No 1169/2011), as allergy hazard of fish-containing commercial food products.¹⁹⁵ Despite the broad biodiversity among fish (more than 30,000 individual species have been described),

commonly consumed species are members of the Osteichthyes group (bony fish) and belong to a limited number of orders, the salmon-like (Salmoniformes), cod-like (Gadiformes), perch-like (Perciformes), herring-like (Clupeiformes), carp-like (Cypriniformes), catfish-like (Siluriformes), and platfish (Pleuronectiformes) (Figure 83).

Globally, a high number of fish species is commercially available. The market share of these species varies in different countries according to regional production sites and eating habits. While cod and salmon are important food fish in Europe, other low-value freshwater species are popular in Asia (e.g., grass carp and Asian carp). Fish allergens have been described in about 40 species, but detailed analysis of the allergy-eliciting molecules was performed mainly for fish, which are commonly consumed in the European area such as carp, cod, salmon, trout and tuna.¹⁹⁵ Meanwhile the knowledge on food allergy to other species such as barramundi and catfish is growing.¹⁰⁰⁹,¹⁰¹⁰ A large and clinically relevant cross-reactivity seems to exist between parvalbumins of different fish species (see chapter C11). The following fish products can be elicitors of mild to severe allergic reactions in sensitized patients.

**Fish meat**—The largest allergenic activity resides in the muscle of the fish.¹⁹⁵ Fish is consumed as cooked, fried, pickled or even raw food product. Food processing seems not to affect the allergenic potency of the fish but rather the allergen content, which varies in different species.¹⁰⁰⁹ Parvalbumins, the major fish allergens, are highly abundant in the fish muscle. With a serving size of 200 g cod filet, the consumer might ingest up to 0.5 g of parvalbumin per meal. Other fish allergens present in the muscle are enolases, aldolases, collagen, tropomyosin and others. As a food ingredient, fish must be listed specifically on a product label regardless of the percentage of content.

**Eggs, roe, caviar**—There are case reports that caviar has elicited allergic reactions. Typically, roe is consumed in its raw form. Fish

<figure><img src="image_187.png" /><figcaption>FIGURE 83 Most important food fish are members of seven taxonomic orders.</figcaption></figure>
muscle allergens are not relevant in this context. Vitellogenin has been identified as an important fish egg allergen.¹⁹⁵ This protein and its metabolites represent nearly the total protein content in roe. The knowledge about fish egg allergens improved over the past year, including the production of the first recombinant molecules.¹⁰¹¹

**Fish gelatin, isinglass, and similar products**—Concern has been raised as to whether fish-derived products such as fish gelatin may have allergenic properties. Fish gelatin, hydrolysed collagen, is made from fish skin and bones. Isinglass is derived from fish swim bladder and largely contains collagen. Food (beverages, candy), pharmaceutical (gel capsules and coatings) or biological (vaccines, sublingual immunotherapy) products may contain these ingredients. Allergenicity appears to be inherent to collagen-like products¹⁰¹² but might also stem from contaminations by fish meat residues. Consumers are not aware of these fish-derived food ingredients as they are exempted from the food labelling regulation.

**Fish blood**—Fish hemin (fish blood) or other blood proteins have been used by the food industry as additives or processing aids, but it seems to be a relevant source of allergens only in the fish-processing environment. Occupational asthma might be linked to the aerosolization of potentially blood-derived allergens during processing of fish. Serum albumin has been suggested as a potential allergen, but this and other allergens could be not confirmed so far.¹⁰¹³,¹⁰¹⁴

The term seafood comprises both fish and shellfish (e.g., shrimps, crabs, lobster, mussels, oysters, octopus, squid¹⁰¹⁵). Considering the large phylogenetic distance between fish and these other organisms it is not surprising that little cross-reactivity occurs. Therefore, seafood other than fish as allergenic food will not be discussed further in the context of fish allergy (see chapter B13). Nevertheless, a case of cross-allergy between fish and shrimp tropomyosins has recently been reported.¹⁰¹⁶

**ANISAKIS SIMPLEX**—*Anisakis* simplex is a parasite of the Nematoda phylum, which is able to induce IgE sensitization.¹⁰¹⁷ It is increasingly recognized as a relevant allergenic component mostly in fish.¹⁰¹⁸ It is important to keep *Anisakis* simplex allergy in consideration when diagnosing patients experiencing allergic reactions after eating fish mainly when specific fish such as European hake, Atlantic horse mackerel, blue whiting or anchovies are involved. Debenedetti et al (2019); see comment in reference list ¹⁰¹⁶ Humans become an incidental host after eating raw or undercooked fish containing live larvae. Freezing fish at -20°C for at least 24 h will kill the larvae, however. When parasitized fish is consumed, larvae can penetrate the gastrointestinal mucosa and induce abdominal pain, digestive symptoms and sometimes fever (anisakiasis). Upon reexposure, sensitized individuals may also develop allergic signs. Reactions can include typical allergic and/or gastrointestinal symptoms (gastroallergic anisakiasis). It is assumed that sensitization can also occur to dead larvae material i.e., without first developing anisakiasis. In this case freezing will not ascertain full safety, albeit the parasites are killed. It is important to keep *Anisakis* simplex in consideration when diagnosing patients experiencing allergic reactions following fish ingestion. When testing for IgE against parasites it should be remembered that there is a considerable cross-reactivity between different nematode species e.g., *Anisakis* and Ascaris.¹⁰²⁰,¹⁰²¹

## 2 – Fish allergens (parvalbumin and others) and *Anisakis* allergens.

**FISH**—A search in the WHO/IUIS database currently reveals 40 entries while the database Allergen Online (www.allergenonline.org, version 21) comprises 83 fish allergens of known sequence. Sixteen and 40, respectively, of these belong to the parvalbumin family (Table 36). Further allergens are enolases (n=5), aldolases (n=4), tropomyosin (n=3), vitellogenin from salmon roe (n=1) and others (n=11). These are discussed further below.

### Fish as an allergenic source

* Fish species may differ by their allergenic potency.

* Allergy might be elicited by proteins present in fish muscle, skin or blood.

* Allergens from fish and shellfish (e.g., crustaceans, molluscs) are not the same.

* *Anisakis* simplex, a parasite residing in fish muscle, can be another source of IgE-mediated hypersensitivity after fish ingestion.

**Fish meat**—The dominating major allergen in fish muscle is parvalbumin of which the codfish molecule Gad c 1 was the first to be identified (see chapter C11).¹⁹⁵ Subsequently, studies were performed with a number of homologous proteins such as Gad m 1 from Atlantic cod, Cyp c 1 from Common carp and Sal s 1 from Atlantic salmon (Figure 84; left panel). Parvalbumins are small muscle proteins (10-12 kDa) of remarkable stability towards physicochemical effects by food processing. During fish preparation and cooking, these allergens may become aerosolized and inhaled causing respiratory symptoms.¹⁰¹⁴,¹⁰²²,¹⁰²³ Because of specific characteristics of their protein structure, these calcium-binding allergens belong to the so-called EF-hand family (Figure 85).¹⁰²⁴ The parvalbumin levels vary considerably in different fish tissues and species.¹⁰⁰⁹ Carp and herring muscle contain about 100-times more parvalbumin than mackerel and tuna. Most fish-allergic patients have specific IgE to these allergens (Table 36). Highly conserved parvalbumin epitopes have been used to explain not only IgE but also clinical cross-reactivity among various fish species. Parvalbumins cluster into two molecular subtypes, parvalbumins from the alpha- and the beta-lineage. Common fish allergens, as listed in Table 36, are beta-parvalbumins. Alpha-parvalbumins, such as parvalbumins found in ray and shark, appear to have minor cross-reactivity with beta-homologues (see chapter C11). Beyond parvalbumin, other fish allergens were identified, namely 50 kDa-enolases and 40 kDa-aldolases from cod, salmon, tuna and more recently Cyp c 2 from carp and Pan h 2 and Pan h 3 from catfish.¹⁰¹⁰,¹⁰²² These glycolytic enzymes are highly expressed in the fish muscle. Their potency as food allergens still needs to be defined as they are less stable than parvalbumins. However, a number of fish-allergic subjects seem to
muscle allergens are not relevant in this context. Vitellogenin has been identified as an important fish egg allergen.¹⁹⁵ This protein and its metabolites represent nearly the total protein content in roe. The knowledge about fish egg allergens improved over the past year, including the production of the first recombinant molecules.¹⁰¹¹

**Fish gelatin, isinglass, and similar products**—Concern has been raised as to whether fish-derived products such as fish gelatin may have allergenic properties. Fish gelatin, hydrolysed collagen, is made from fish skin and bones. Isinglass is derived from fish swim bladder and largely contains collagen. Food (beverages, candy), pharmaceutical (gel capsules and coatings) or biological (vaccines, sublingual immunotherapy) products may contain these ingredients. Allergenicity appears to be inherent to collagen-like products¹⁰¹² but might also stem from contaminations by fish meat residues. Consumers are not aware of these fish-derived food ingredients as they are exempted from the food labelling regulation.

**Fish blood**—Fish hemin (fish blood) or other blood proteins have been used by the food industry as additives or processing aids, but it seems to be a relevant source of allergens only in the fish-processing environment. Occupational asthma might be linked to the aerosolization of potentially blood-derived allergens during processing of fish. Serum albumin has been suggested as a potential allergen, but this and other allergens could be not confirmed so far.¹⁰¹³,¹⁰¹⁴

The term seafood comprises both fish and shellfish (e.g., shrimps, crabs, lobster, mussels, oysters, octopus, squid¹⁰¹⁵). Considering the large phylogenetic distance between fish and these other organisms it is not surprising that little cross-reactivity occurs. Therefore, seafood other than fish as allergenic food will not be discussed further in the context of fish allergy (see chapter B13). Nevertheless, a case of cross-allergy between fish and shrimp tropomyosins has recently been reported.¹⁰¹⁶

**ANISAKIS SIMPLEX**—*Anisakis* simplex is a parasite of the Nematoda phylum, which is able to induce IgE sensitization.¹⁰¹⁷ It is increasingly recognized as a relevant allergenic component mostly in fish.¹⁰¹⁸ It is important to keep *Anisakis* simplex allergy in consideration when diagnosing patients experiencing allergic reactions after eating fish mainly when specific fish such as European hake, Atlantic horse mackerel, blue whiting or anchovies are involved. Debenedetti et al (2019); see comment in reference list ¹⁰¹⁶ Humans become an incidental host after eating raw or undercooked fish containing live larvae. Freezing fish at -20°C for at least 24 h will kill the larvae, however. When parasitized fish is consumed, larvae can penetrate the gastrointestinal mucosa and induce abdominal pain, digestive symptoms and sometimes fever (anisakiasis). Upon reexposure, sensitized individuals may also develop allergic signs. Reactions can include typical allergic and/or gastrointestinal symptoms (gastroallergic anisakiasis). It is assumed that sensitization can also occur to dead larvae material i.e., without first developing anisakiasis. In this case freezing will not ascertain full safety, albeit the parasites are killed. It is important to keep *Anisakis* simplex in consideration when diagnosing patients experiencing allergic reactions following fish ingestion. When testing for IgE against parasites it should be remembered that there is a considerable cross-reactivity between different nematode species e.g., *Anisakis* and Ascaris.¹⁰²⁰,¹⁰²¹

## 2 – Fish allergens (parvalbumin and others) and *Anisakis* allergens.

**FISH**—A search in the WHO/IUIS database currently reveals 40 entries while the database Allergen Online (www.allergenonline.org, version 21) comprises 83 fish allergens of known sequence. Sixteen and 40, respectively, of these belong to the parvalbumin family (Table 36). Further allergens are enolases (n=5), aldolases (n=4), tropomyosin (n=3), vitellogenin from salmon roe (n=1) and others (n=11). These are discussed further below.

### Fish as an allergenic source

* Fish species may differ by their allergenic potency.

* Allergy might be elicited by proteins present in fish muscle, skin or blood.

* Allergens from fish and shellfish (e.g., crustaceans, molluscs) are not the same.

* *Anisakis* simplex, a parasite residing in fish muscle, can be another source of IgE-mediated hypersensitivity after fish ingestion.

**Fish meat**—The dominating major allergen in fish muscle is parvalbumin of which the codfish molecule Gad c 1 was the first to be identified (see chapter C11).¹⁹⁵ Subsequently, studies were performed with a number of homologous proteins such as Gad m 1 from Atlantic cod, Cyp c 1 from Common carp and Sal s 1 from Atlantic salmon (Figure 84; left panel). Parvalbumins are small muscle proteins (10-12 kDa) of remarkable stability towards physicochemical effects by food processing. During fish preparation and cooking, these allergens may become aerosolized and inhaled causing respiratory symptoms.¹⁰¹⁴,¹⁰²²,¹⁰²³ Because of specific characteristics of their protein structure, these calcium-binding allergens belong to the so-called EF-hand family (Figure 85).¹⁰²⁴ The parvalbumin levels vary considerably in different fish tissues and species.¹⁰⁰⁹ Carp and herring muscle contain about 100-times more parvalbumin than mackerel and tuna. Most fish-allergic patients have specific IgE to these allergens (Table 36). Highly conserved parvalbumin epitopes have been used to explain not only IgE but also clinical cross-reactivity among various fish species. Parvalbumins cluster into two molecular subtypes, parvalbumins from the alpha- and the beta-lineage. Common fish allergens, as listed in Table 36, are beta-parvalbumins. Alpha-parvalbumins, such as parvalbumins found in ray and shark, appear to have minor cross-reactivity with beta-homologues (see chapter C11). Beyond parvalbumin, other fish allergens were identified, namely 50 kDa-enolases and 40 kDa-aldolases from cod, salmon, tuna and more recently Cyp c 2 from carp and Pan h 2 and Pan h 3 from catfish.¹⁰¹⁰,¹⁰²² These glycolytic enzymes are highly expressed in the fish muscle. Their potency as food allergens still needs to be defined as they are less stable than parvalbumins. However, a number of fish-allergic subjects seem to
<table><caption>TABLE 36 Major and relevant minor fish allergenic molecules</caption><thead><tr><th>Order</th><th>English name (species)</th><th>Allergenic molecule<sup>A</sup></th><th>Biochemical name</th><th>Prevalence [%]<sup>B</sup></th><th>MW [kDa]</th></tr></thead><tbody><tr><td rowspan="2">Clupeiformes</td><td>Herring (Clupea harengus)</td><td>Clu h 1</td><td>parvalbumin</td><td>45</td><td>12</td></tr><tr><td>Pilchard (Sardinops sagax)</td><td>Sar sa 1</td><td>parvalbumin</td><td>80</td><td>12</td></tr><tr><td rowspan="3">Cypriniformes</td><td rowspan="2">Carp (Cyprinus carpio)</td><td>Cyp c 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td>Cyp c 2</td><td>enolase</td><td>17</td><td>47</td></tr><tr><td>Grass carp (Ctenopharyngodon idella)</td><td>Cten i 1</td><td>parvalbumin</td><td>94</td><td>9</td></tr><tr><td rowspan="3">Gadiformes</td><td>Atlantic cod ( Gadus callarias )</td><td>Gad c 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td rowspan="2">Atlantic cod ( Gadus morhua )</td><td>Gad m 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td>Gad m 2</td><td>enolase</td><td>56</td><td>50</td></tr><tr><td rowspan="6">Perciformes</td><td rowspan="3">Tuna (Thunnus albacares)</td><td>Thu a 1</td><td>parvalbumin</td><td>95</td><td>11</td></tr><tr><td>Thu a 2</td><td>enolase</td><td>19</td><td>50</td></tr><tr><td>Thu a 3</td><td>aldolase</td><td>13</td><td>40</td></tr><tr><td rowspan="2">Barramundi (Lates calcarifer)</td><td>Lat c 1</td><td>parvalbumin</td><td>77-83</td><td>11.5</td></tr><tr><td>Lat c 6</td><td>collagen</td><td>22</td><td>130, 140</td></tr><tr><td>Mozambique tilapia (Oreochromis mossambicus)</td><td>Ore m 4</td><td>tropomyosin</td><td>100</td><td>33</td></tr><tr><td>Indian mackerel (Rastrelliger kanagurta)</td><td>Ras k 1</td><td>parvalbumin</td><td>83</td><td>11.3</td></tr><tr><td>Atlantic mackerel (Scomber scombrus)</td><td>Sco s 1</td><td>parvalbumin</td><td>95</td><td>12</td></tr><tr><td>Swordfish (Xiphias gladius)</td><td>Xip g 1</td><td>parvalbumin</td><td>71</td><td>11.5</td></tr><tr><td>Pleuronectiformes</td><td>Megrim (Lepidorhombus whiffiagonis)</td><td>Lep w 1</td><td>parvalbumin</td><td>100</td><td>11.5</td></tr><tr><td rowspan="10">Salmoniformes</td><td>Pacific salmon (Oncorhynchus keta)</td><td>Onc k 5</td><td>vitellogenin</td><td>nd</td><td>18</td></tr><tr><td>Rainbow trout (Oncorhynchus mykiss)</td><td>Onc m 1</td><td>parvalbumin</td><td>95</td><td>12</td></tr><tr><td rowspan="9">Salmon (Salmo salar)</td><td>Sal s 1</td><td>parvalbumin</td><td>49-64</td><td>12</td></tr><tr><td>Sal s 2</td><td>enolase</td><td>24-34</td><td>50</td></tr><tr><td>Sal s 3</td><td>aldolase</td><td>16-26</td><td>40</td></tr><tr><td>Sal s 4</td><td>tropomyosin</td><td>13</td><td>37</td></tr><tr><td>Sal s 6</td><td>collagen</td><td>22</td><td>130, 140</td></tr><tr><td>Sal s 7</td><td>creatine kinase</td><td>14</td><td>43</td></tr><tr><td>Sal s 8</td><td>triose-P isomerase</td><td>34</td><td>25</td></tr><tr><td>Sal s 9</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>Redfish (Sebastes marinus)</td><td>Seb m 1</td><td>parvalbumin</td><td>95</td><td>11</td></tr><tr><td rowspan="10">Siluriformes</td><td rowspan="10">Striped catfish (Pangasianodon hypophthalmus)</td><td>Pan h 1</td><td>parvalbumin</td><td>42</td><td>11</td></tr><tr><td>Pan h 2</td><td>enolase</td><td>21</td><td>50</td></tr><tr><td>Pan h 3</td><td>aldolase</td><td>21</td><td>40</td></tr><tr><td>Pan h 4</td><td>tropomyosin</td><td>6-32</td><td>35</td></tr><tr><td>Pan h 7</td><td>creatine kinase</td><td>10</td><td>43</td></tr><tr><td>Pan h 8</td><td>triose-P isomerase</td><td>19</td><td>21</td></tr><tr><td>Pan h 9</td><td>pyruvate kinase</td><td>6</td><td>65</td></tr><tr><td>Pan h 10</td><td>lactate DH</td><td>13</td><td>34</td></tr><tr><td>Pan h 11</td><td>glucose-6-P DH</td><td>8</td><td>60</td></tr><tr><td>Pan h 13</td><td>glyceraldehyde-3-P DH</td><td>6</td><td>36</td></tr></tbody></table>

have IgE against these allergens (Table 36). In-vitro cross-reactivity occurs for homologues from cod, salmon and tuna. Other fish muscle allergens are relevant, e.g., tropomyosin, creatine kinase and triosephosphate isomerase described in salmon and catfish as well as pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate

dehydrogenase, and glyceraldehyde-3-dehydrogenase discovered in catfish.¹⁰¹⁰ It can be expected that further studies will confirm the clinical value of including those allergens into a diagnostic panel.

Fish gelatin and collagen—Collagen consists of three individual polypeptide chains corresponding to two alpha-subunits (a1, a2;

<footnote>A Allergens recognized by the WHO/IUIS allergen nomenclature subcommittee (for parvalbumins from other species, see chapter ‘Parvalbumins’).</footnote>

<footnote>B Prevalences are extracted from www.allergen.org/literature reference cited there. DH, dehydrogenase; P, phosphate. nd, not determined.</footnote>
<table><caption>TABLE 36 Major and relevant minor fish allergenic molecules</caption><thead><tr><th>Order</th><th>English name (species)</th><th>Allergenic molecule<sup>A</sup></th><th>Biochemical name</th><th>Prevalence [%]<sup>B</sup></th><th>MW [kDa]</th></tr></thead><tbody><tr><td rowspan="2">Clupeiformes</td><td>Herring (Clupea harengus)</td><td>Clu h 1</td><td>parvalbumin</td><td>45</td><td>12</td></tr><tr><td>Pilchard (Sardinops sagax)</td><td>Sar sa 1</td><td>parvalbumin</td><td>80</td><td>12</td></tr><tr><td rowspan="3">Cypriniformes</td><td rowspan="2">Carp (Cyprinus carpio)</td><td>Cyp c 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td>Cyp c 2</td><td>enolase</td><td>17</td><td>47</td></tr><tr><td>Grass carp (Ctenopharyngodon idella)</td><td>Cten i 1</td><td>parvalbumin</td><td>94</td><td>9</td></tr><tr><td rowspan="3">Gadiformes</td><td>Atlantic cod ( Gadus callarias )</td><td>Gad c 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td rowspan="2">Atlantic cod ( Gadus morhua )</td><td>Gad m 1</td><td>parvalbumin</td><td>100</td><td>12</td></tr><tr><td>Gad m 2</td><td>enolase</td><td>56</td><td>50</td></tr><tr><td rowspan="6">Perciformes</td><td rowspan="3">Tuna (Thunnus albacares)</td><td>Thu a 1</td><td>parvalbumin</td><td>95</td><td>11</td></tr><tr><td>Thu a 2</td><td>enolase</td><td>19</td><td>50</td></tr><tr><td>Thu a 3</td><td>aldolase</td><td>13</td><td>40</td></tr><tr><td rowspan="2">Barramundi (Lates calcarifer)</td><td>Lat c 1</td><td>parvalbumin</td><td>77-83</td><td>11.5</td></tr><tr><td>Lat c 6</td><td>collagen</td><td>22</td><td>130, 140</td></tr><tr><td>Mozambique tilapia (Oreochromis mossambicus)</td><td>Ore m 4</td><td>tropomyosin</td><td>100</td><td>33</td></tr><tr><td>Indian mackerel (Rastrelliger kanagurta)</td><td>Ras k 1</td><td>parvalbumin</td><td>83</td><td>11.3</td></tr><tr><td>Atlantic mackerel (Scomber scombrus)</td><td>Sco s 1</td><td>parvalbumin</td><td>95</td><td>12</td></tr><tr><td>Swordfish (Xiphias gladius)</td><td>Xip g 1</td><td>parvalbumin</td><td>71</td><td>11.5</td></tr><tr><td>Pleuronectiformes</td><td>Megrim (Lepidorhombus whiffiagonis)</td><td>Lep w 1</td><td>parvalbumin</td><td>100</td><td>11.5</td></tr><tr><td rowspan="10">Salmoniformes</td><td>Pacific salmon (Oncorhynchus keta)</td><td>Onc k 5</td><td>vitellogenin</td><td>nd</td><td>18</td></tr><tr><td>Rainbow trout (Oncorhynchus mykiss)</td><td>Onc m 1</td><td>parvalbumin</td><td>95</td><td>12</td></tr><tr><td rowspan="9">Salmon (Salmo salar)</td><td>Sal s 1</td><td>parvalbumin</td><td>49-64</td><td>12</td></tr><tr><td>Sal s 2</td><td>enolase</td><td>24-34</td><td>50</td></tr><tr><td>Sal s 3</td><td>aldolase</td><td>16-26</td><td>40</td></tr><tr><td>Sal s 4</td><td>tropomyosin</td><td>13</td><td>37</td></tr><tr><td>Sal s 6</td><td>collagen</td><td>22</td><td>130, 140</td></tr><tr><td>Sal s 7</td><td>creatine kinase</td><td>14</td><td>43</td></tr><tr><td>Sal s 8</td><td>triose-P isomerase</td><td>34</td><td>25</td></tr><tr><td>Sal s 9</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td>Redfish (Sebastes marinus)</td><td>Seb m 1</td><td>parvalbumin</td><td>95</td><td>11</td></tr><tr><td rowspan="10">Siluriformes</td><td rowspan="10">Striped catfish (Pangasianodon hypophthalmus)</td><td>Pan h 1</td><td>parvalbumin</td><td>42</td><td>11</td></tr><tr><td>Pan h 2</td><td>enolase</td><td>21</td><td>50</td></tr><tr><td>Pan h 3</td><td>aldolase</td><td>21</td><td>40</td></tr><tr><td>Pan h 4</td><td>tropomyosin</td><td>6-32</td><td>35</td></tr><tr><td>Pan h 7</td><td>creatine kinase</td><td>10</td><td>43</td></tr><tr><td>Pan h 8</td><td>triose-P isomerase</td><td>19</td><td>21</td></tr><tr><td>Pan h 9</td><td>pyruvate kinase</td><td>6</td><td>65</td></tr><tr><td>Pan h 10</td><td>lactate DH</td><td>13</td><td>34</td></tr><tr><td>Pan h 11</td><td>glucose-6-P DH</td><td>8</td><td>60</td></tr><tr><td>Pan h 13</td><td>glyceraldehyde-3-P DH</td><td>6</td><td>36</td></tr></tbody></table>

have IgE against these allergens (Table 36). In-vitro cross-reactivity occurs for homologues from cod, salmon and tuna. Other fish muscle allergens are relevant, e.g., tropomyosin, creatine kinase and triosephosphate isomerase described in salmon and catfish as well as pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate

dehydrogenase, and glyceraldehyde-3-dehydrogenase discovered in catfish.¹⁰¹⁰ It can be expected that further studies will confirm the clinical value of including those allergens into a diagnostic panel.

Fish gelatin and collagen—Collagen consists of three individual polypeptide chains corresponding to two alpha-subunits (a1, a2;

<footnote>A Allergens recognized by the WHO/IUIS allergen nomenclature subcommittee (for parvalbumins from other species, see chapter ‘Parvalbumins’).</footnote>

<footnote>B Prevalences are extracted from www.allergen.org/literature reference cited there. DH, dehydrogenase; P, phosphate. nd, not determined.</footnote>
<figure><img src="image_188.png" /><figcaption>FIGURE 84 Most important allergens from Atlantic salmon muscle, roe, and contamination with *Anisakis* simplex</figcaption></figure>

<figure><img src="image_189.png" /><figcaption>FIGURE 85 Protein structure of parvalbumin Cyp c 1 from carp (pdb 4CPV). Two EF-hand motifs bind each a Ca2+-molecule.</figcaption></figure>

each 110 kDa) and one beta-subunit (210 kDa). These chains forming a tight right-handed twist causing them to form a rod shape triple helix. Fish gelatin is a heterogeneous product being obtained from acidic acid extraction of collagen followed by chemical hydrolysis. According to the molecular weight of fish gelatin components, it is available at different hydrolysate grades. Anaphylaxis to fish gelatin has been documented in case reports.<sup>1025,1026</sup> Fish gelatin differs considerably by its amino acid composition from mammalian homologues. It is therefore coherent that there is no cross-reactivity among these products. More recently, the allergenic potency of fish collagen was confirmed in several fish species, salmon, barramundi and catfish, corroborating earlier reports.<sup>1012,1025</sup> It is important to be aware that fish gelatin and collagen may be used as additives or processing aids in drugs, vaccines and food products normally thought to contain fish proteins, and may therefore be more prone to act as a hidden allergen.

**Roe**—The allergens of roe, also referred as caviar or fish eggs, are different from those of fish meat and fish skin. Patients with roe allergy often tolerate fish meat and vice versa. Vitellogenins are glycolipoproteins of high molecular weight (>150 kDa) belonging to the

family of lipid transport proteins. Studies of allergens from salmonid roe have led to the identification of a 35-kDa vitellogenin fragment consisting of two partly identical subunits (18 and 16 kDa) named *Onc k 5* (Figure 84).<sup>195</sup> Cross-reactivity has been proven for roe allergens from different fish species by IgE and skin testing. However, no cross-reactivity was found to homologues from chicken yolk.

**ANISAKIS SIMPLEX**—Fourteen allergens are available via official allergen names (Table 37), allergen online contains 33 entries in total.<sup>1027</sup> These cluster according to their origin from the parasite into allergens from dead/ disintegrated (somatic, SO; cuticular, C) and from living larvae (excretory/secretory, ES) <sup>1028</sup>. There is a notion that ES allergens are more potent than SO/C allergens.<sup>1017</sup> Further allergens are known from *Anisakis* pegreffii; however, focus will be given here to the officially approved allergens.

**SO allergens**—They are present in the body of the parasite. Specific IgE reactivity to those allergens relates to sensitization but not necessarily clinical allergy.

*Ani s 2* (paramyosin) and *Ani s 3* (tropomyosin) bear high homology and strong cross-reactivity to house dust mite and crustacean homologues.<sup>1017,1028</sup> The biological function of *Ani s 10*, another SO allergen, remains to be clarified.

**C allergens**—They are released during a specific window of the larval life cycle in transition from L3 to L4 stage. It seems that these antigens are involved in a chronic stimulus inducing granulomas and other chronic lesions. *Ani s 4*, an antigen that belongs to the cysteine protease inhibitors family, it has been shown to be presented not only in the excretory gland but also in the cuticle of the parasite.

**ES allergens**—These antigens are histolytic enzymes secreted through the dorsal oesophageal gland and the excretory cells on the digestive tract of L3-stage larvae. Their function is to facilitate the parasite's infiltration through the digestive mucosa of its host. *Ani s 1*, *Ani s 4*, *Ani s 5*, *Ani s 6*, *Ani s 7*, *Ani s 8*, *Ani s 9* and *Ani s 13* can induce both sensitization and elicitation of allergic symptoms.<sup>1029-1031</sup> Three of these are considered major allergens, *Ani s 1*, an inhibitor of Kunitz-type serine proteases, *Ani s 7*, a protein of unknown biological function and *Ani s 13*, a protein of haemoglobin function. Structural homologues to *Ani s 7* and *Ani s 13* in other allergen sources are unknown so far.
<figure><img src="image_188.png" /><figcaption>FIGURE 84 Most important allergens from Atlantic salmon muscle, roe, and contamination with *Anisakis* simplex</figcaption></figure>

<figure><img src="image_189.png" /><figcaption>FIGURE 85 Protein structure of parvalbumin Cyp c 1 from carp (pdb 4CPV). Two EF-hand motifs bind each a Ca2+-molecule.</figcaption></figure>

each 110 kDa) and one beta-subunit (210 kDa). These chains forming a tight right-handed twist causing them to form a rod shape triple helix. Fish gelatin is a heterogeneous product being obtained from acidic acid extraction of collagen followed by chemical hydrolysis. According to the molecular weight of fish gelatin components, it is available at different hydrolysate grades. Anaphylaxis to fish gelatin has been documented in case reports.<sup>1025,1026</sup> Fish gelatin differs considerably by its amino acid composition from mammalian homologues. It is therefore coherent that there is no cross-reactivity among these products. More recently, the allergenic potency of fish collagen was confirmed in several fish species, salmon, barramundi and catfish, corroborating earlier reports.<sup>1012,1025</sup> It is important to be aware that fish gelatin and collagen may be used as additives or processing aids in drugs, vaccines and food products normally thought to contain fish proteins, and may therefore be more prone to act as a hidden allergen.

**Roe**—The allergens of roe, also referred as caviar or fish eggs, are different from those of fish meat and fish skin. Patients with roe allergy often tolerate fish meat and vice versa. Vitellogenins are glycolipoproteins of high molecular weight (>150 kDa) belonging to the

family of lipid transport proteins. Studies of allergens from salmonid roe have led to the identification of a 35-kDa vitellogenin fragment consisting of two partly identical subunits (18 and 16 kDa) named *Onc k 5* (Figure 84).<sup>195</sup> Cross-reactivity has been proven for roe allergens from different fish species by IgE and skin testing. However, no cross-reactivity was found to homologues from chicken yolk.

**ANISAKIS SIMPLEX**—Fourteen allergens are available via official allergen names (Table 37), allergen online contains 33 entries in total.<sup>1027</sup> These cluster according to their origin from the parasite into allergens from dead/ disintegrated (somatic, SO; cuticular, C) and from living larvae (excretory/secretory, ES)<sup>1028</sup>. There is a notion that ES allergens are more potent than SO/C allergens.<sup>1017</sup> Further allergens are known from *Anisakis* pegreffii; however, focus will be given here to the officially approved allergens.

**SO allergens**—They are present in the body of the parasite. Specific IgE reactivity to those allergens relates to sensitization but not necessarily clinical allergy.

*Ani s 2* (paramyosin) and *Ani s 3* (tropomyosin) bear high homology and strong cross-reactivity to house dust mite and crustacean homologues.<sup>1017,1028</sup> The biological function of *Ani s 10*, another SO allergen, remains to be clarified.

**C allergens**—They are released during a specific window of the larval life cycle in transition from L3 to L4 stage. It seems that these antigens are involved in a chronic stimulus inducing granulomas and other chronic lesions. *Ani s 4*, an antigen that belongs to the cysteine protease inhibitors family, it has been shown to be presented not only in the excretory gland but also in the cuticle of the parasite.

**ES allergens**—These antigens are histolytic enzymes secreted through the dorsal oesophageal gland and the excretory cells on the digestive tract of L3-stage larvae. Their function is to facilitate the parasite's infiltration through the digestive mucosa of its host. *Ani s 1*, *Ani s 4*, *Ani s 5*, *Ani s 6*, *Ani s 7*, *Ani s 8*, *Ani s 9* and *Ani s 13* can induce both sensitization and elicitation of allergic symptoms.<sup>1029-1031</sup> Three of these are considered major allergens, *Ani s 1*, an inhibitor of Kunitz-type serine proteases, *Ani s 7*, a protein of unknown biological function and *Ani s 13*, a protein of haemoglobin function. Structural homologues to *Ani s 7* and *Ani s 13* in other allergen sources are unknown so far.
<table><caption>TABLE 37 Major and relevant minor *Anisakis* simplex allergenic molecules</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence [%]<sup>a</sup></th><th>MW [kDa]</th></tr></thead><tbody><tr><td>Ani s 1</td><td>Kunitz serine protease inhibitors</td><td>85</td><td>24</td></tr><tr><td>Ani s 2</td><td>Paramyosin</td><td>88</td><td>97</td></tr><tr><td>Ani s 3</td><td>Tropomyosin</td><td>nd</td><td>41</td></tr><tr><td>Ani s 4</td><td>Cysteine protease inhibitor</td><td>27</td><td>9</td></tr><tr><td>Ani s 5</td><td>SXP/RAL-2 family protein</td><td>25</td><td>15</td></tr><tr><td>Ani s 6</td><td>Serine protease inhibitor</td><td>18</td><td>7</td></tr><tr><td>Ani s 7</td><td>Glycoprotein</td><td>83–100</td><td>139</td></tr><tr><td>Ani s 8</td><td>SXP/RAL-2 family protein</td><td>25</td><td>15</td></tr><tr><td>Ani s 9</td><td>SXP/RAL-2 family protein</td><td>nd</td><td>14</td></tr><tr><td>Ani s 10</td><td>nd</td><td>39</td><td>21</td></tr><tr><td>Ani s 11</td><td>nd</td><td>47</td><td>27</td></tr><tr><td>Ani s 12</td><td>nd</td><td>57</td><td>31</td></tr><tr><td>Ani s 13</td><td>Haemoglobin</td><td>64</td><td>37</td></tr><tr><td>Ani s 14</td><td>nd</td><td>54</td><td>24</td></tr></tbody></table>

### 3 – Sensitization to individual molecules and its clinical relevance

FISH—So far, epidemiological studies on fish allergy are missing to present consistent data of sensitization to fish and fish allergens. Prevalence rates specifically to fish have been determined in studies of variable design and methodology.¹⁰³²⁻¹⁰³⁴ Overall, it seems that <1% of the global population are affected by allergy to fish. A higher percentage is observed in paediatric cohorts and in countries with long coastlines, which have a high fish consumption, as well as in regions with fish-processing industries (up to 3%). Patients get sensitized to fish not only upon ingestion but also skin contact and inhalation of fish steam during processing of this food.¹⁹⁵,¹⁰¹⁸ Occupational asthma has been reported in 7-36% of workers in industrial fish production lines.¹⁰²³

#### Clinical relevance of fish allergens

* Less than 1% of the general population suffer from a fish allergy.

* Parvalbumin is the major fish allergen (prevalence rates 70-95%).

* Most patients have often slgE to multiple fish allergens, including enolases, aldolases, collagen and tropomyosin.

Parvalbumins have been defined as pan-allergens in fish.¹⁹⁵ In this chapter, we will focus on parvalbumins from the beta-lineage, which are known as important fish allergens. More details on alpha-parvalbumins, including their low cross-reactivity with beta-homologues can be

found in the chapter C11. Sensitization rates for beta-parvalbumins were based on studies of allergen characterization. First it was concluded that 90-95% of the patients had specific IgE to these muscle proteins. Studies of the past decade showed that the fish-allergic population can be subdivided into the following clinical clusters, i. highly sensitized patients reacting to all fish, ii. oligo-sensitized patients reacting to several, specific fish and iii. patients with “selective reactions” to individual fish species only.¹⁹⁵,¹⁰³⁵ Patients of these clinical clusters vary by their IgE-recognition profiles. It was shown that the prevalence of IgE binding to parvalbumin was lower than assumed for long time. The sensitization rate to this major allergen might be rather range at about 70 to 95%, depending on the study cohort. Beyond parvalbumin, a polyclonal immune response to multiple fish allergens correlates with clinical reactivity, as demonstrated for cod, salmon and catfish allergy.¹⁰²⁵,¹⁰³⁶ A single study demonstrated that fish-allergic patients with specific IgE to cod parvalbumin might be co-sensitized to cod enolases (81%) and aldolases (58%).¹⁰¹⁰ The clinical origin and relevance of this co-sensitization is still not yet resolved. However, specific parvalbumin-negative patients seem to develop IgE antibodies to fish enolase (47%) and aldolase (41%), which is rather linked to species-specific fish allergies.¹⁰³⁷ It is important to note that there are still limited data available to delineate how many patients can be categorized in each proposed clinical cluster. Also, it has to be taken into consideration that a geographical and/or a temporal gradient might be relevant for such a prevalence date collection.

ANISAKIS SIMPLEX—The seroprevalence appears to vary between countries, such as a pronounced occurrence reported in Mediterranean countries.¹⁰³¹,¹⁰³⁸ The vast majority of allergic cases to the parasite are after food intake; however, contact dermatitis and conjunctivitis as well as occupational bronchial asthma have been described as well.¹⁰¹⁷ In the literature, there is a lack

<footnote>ᵃAllergens recognized by the WHO/IUIS allergen nomenclature subcommittee.</footnote>

<footnote>ᵇPrevalences are extracted from www.allergen.org/literature reference cited there. nd, not determined.</footnote>
<table><caption>TABLE 37 Major and relevant minor *Anisakis* simplex allergenic molecules</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence [%]<sup>a</sup></th><th>MW [kDa]</th></tr></thead><tbody><tr><td>Ani s 1</td><td>Kunitz serine protease inhibitors</td><td>85</td><td>24</td></tr><tr><td>Ani s 2</td><td>Paramyosin</td><td>88</td><td>97</td></tr><tr><td>Ani s 3</td><td>Tropomyosin</td><td>nd</td><td>41</td></tr><tr><td>Ani s 4</td><td>Cysteine protease inhibitor</td><td>27</td><td>9</td></tr><tr><td>Ani s 5</td><td>SXP/RAL-2 family protein</td><td>25</td><td>15</td></tr><tr><td>Ani s 6</td><td>Serine protease inhibitor</td><td>18</td><td>7</td></tr><tr><td>Ani s 7</td><td>Glycoprotein</td><td>83–100</td><td>139</td></tr><tr><td>Ani s 8</td><td>SXP/RAL-2 family protein</td><td>25</td><td>15</td></tr><tr><td>Ani s 9</td><td>SXP/RAL-2 family protein</td><td>nd</td><td>14</td></tr><tr><td>Ani s 10</td><td>nd</td><td>39</td><td>21</td></tr><tr><td>Ani s 11</td><td>nd</td><td>47</td><td>27</td></tr><tr><td>Ani s 12</td><td>nd</td><td>57</td><td>31</td></tr><tr><td>Ani s 13</td><td>Haemoglobin</td><td>64</td><td>37</td></tr><tr><td>Ani s 14</td><td>nd</td><td>54</td><td>24</td></tr></tbody></table>

### 3 – Sensitization to individual molecules and its clinical relevance

FISH—So far, epidemiological studies on fish allergy are missing to present consistent data of sensitization to fish and fish allergens. Prevalence rates specifically to fish have been determined in studies of variable design and methodology.<sup>1032-1034</sup> Overall, it seems that <1% of the global population are affected by allergy to fish. A higher percentage is observed in paediatric cohorts and in countries with long coastlines, which have a high fish consumption, as well as in regions with fish-processing industries (up to 3%). Patients get sensitized to fish not only upon ingestion but also skin contact and inhalation of fish steam during processing of this food.<sup>195,1018</sup> Occupational asthma has been reported in 7-36% of workers in industrial fish production lines.<sup>1023</sup>

#### Clinical relevance of fish allergens

* Less than 1% of the general population suffer from a fish allergy.

* Parvalbumin is the major fish allergen (prevalence rates 70-95%).

* Most patients have often slgE to multiple fish allergens, including enolases, aldolases, collagen and tropomyosin.

Parvalbumins have been defined as pan-allergens in fish.<sup>195</sup> In this chapter, we will focus on parvalbumins from the beta-lineage, which are known as important fish allergens. More details on alpha-parvalbumins, including their low cross-reactivity with beta-homologues can be

found in the chapter C11. Sensitization rates for beta-parvalbumins were based on studies of allergen characterization. First it was concluded that 90-95% of the patients had specific IgE to these muscle proteins. Studies of the past decade showed that the fish-allergic population can be subdivided into the following clinical clusters, i. highly sensitized patients reacting to all fish, ii. oligo-sensitized patients reacting to several, specific fish and iii. patients with “selective reactions” to individual fish species only.<sup>195,1035</sup> Patients of these clinical clusters vary by their IgE-recognition profiles. It was shown that the prevalence of IgE binding to parvalbumin was lower than assumed for long time. The sensitization rate to this major allergen might be rather range at about 70 to 95%, depending on the study cohort. Beyond parvalbumin, a polyclonal immune response to multiple fish allergens correlates with clinical reactivity, as demonstrated for cod, salmon and catfish allergy.<sup>1025,1036</sup> A single study demonstrated that fish-allergic patients with specific IgE to cod parvalbumin might be co-sensitized to cod enolases (81%) and aldolases (58%).<sup>1010</sup> The clinical origin and relevance of this co-sensitization is still not yet resolved. However, specific parvalbumin-negative patients seem to develop IgE antibodies to fish enolase (47%) and aldolase (41%), which is rather linked to species-specific fish allergies.<sup>1037</sup> It is important to note that there are still limited data available to delineate how many patients can be categorized in each proposed clinical cluster. Also, it has to be taken into consideration that a geographical and/or a temporal gradient might be relevant for such a prevalence date collection.

ANISAKIS SIMPLEX—The seroprevalence appears to vary between countries, such as a pronounced occurrence reported in Mediterranean countries.<sup>1031,1038</sup> The vast majority of allergic cases to the parasite are after food intake; however, contact dermatitis and conjunctivitis as well as occupational bronchial asthma have been described as well.<sup>1017</sup> In the literature, there is a lack

<footnote>ᵃAllergens recognized by the WHO/IUIS allergen nomenclature subcommittee.</footnote>

<footnote>ᵇPrevalences are extracted from www.allergen.org/literature reference cited there. nd, not determined.</footnote>