of consistency to differentiate between allergy and asymptomatic sensitization. In fact, traditional test such as skin prick test (SPT) and *Anisakis* simplex-specific IgE using the whole extracts have shown low specificity.¹⁰³⁸ Various studies approached the identification of clinically relevant allergens in order to discriminate genuine allergy from molecular cross-reactivity. IgE reactivity to ES-allergens showed to correlate with clinical reactivity. Due to the lack of homology with other allergens, Ani s 1 and Ani s 7 bear a good diagnostic capacity to identify true *Anisakis* simplex allergy.¹⁰³⁹,¹⁰⁴⁰ Ani s 1-specific IgE are found predominantly on patients presenting with severe allergic reaction. Though Ani s 7 is a frequently IgE-recognized molecule, it seems less clinically relevant, which can be explained by a parallel IgG4-response resulting into a protection against adverse symptoms.¹⁰⁴¹ The ES allergen Ani s 13 has been reported as another marker for primary sensitization and clinical allergy. Ani s 2 and Ani s 3 are *Anisakis* simplex pan-allergens but with high cross-reactivity to other allergen sources such as mites, insects, shellfish, cockroach, and chronomids, thus a less specific marker.¹⁰¹⁷,¹⁰¹⁸,¹⁰²⁹

Clinical relevance of *Anisakis* simplex allergens

* The seroprevalence ranges between 0.2 and 15% in the general population.

* Specific IgE to the whole extract is not always related to symptomatic allergy.

* Ani s 1 appears to be a clinically relevant allergen from *Anisakis* simplex.

4 - Clinical management

FISH—Fish allergy diagnosis is mostly based on clinical history, skin tests (prick tests to fish extract or prick-to prick with the culprit fish -raw and cooked-) and IgE tests, followed (if needed) by an oral food challenge (ideally, double-blind placebo-controlled challenge) with the fish that has elicited the reaction.¹⁹⁵,¹⁰³¹ Level of serum IgE antibodies have been correlated with the clinical reactivity to predict allergy to fish. In a US population, an IgE level of 20 kU/L to cod extract (ImmunoCAP Specific IgE test, Thermo Fisher) allowed to predict an allergy to this fish with 95% certainty.¹⁰⁴² More recently, a large food challenge-based study reported that combining an obvious clinical history of patients with slgE to cod extract >8.2 kU/L or to salmon extract >5 kU/L should result in advising to avoid consuming all fish species.¹⁰³⁶ A specific cod IgE titer >5 kU/L has been even reported to be useful regarding an unfavorable prognosis to outgrow fish allergy.¹⁰³⁵ The availability of individual allergens for IgE testing is still limited and thus, not of much help in predicting whether the patient is allergic to other fish species. However, an outline of the future diagnosis using single

allergens is presented in the "Clinical cases" and in the chapter C11. Meanwhile, a novel multiplex platform (MadX, MacroArray Diagnostics) made a number of parvalbumins from several fish species available, in addition to the other fish allergens cod enolase and aldolase (Gad m 2, Gad m 3).

Two important questions should be addressed if the initial suspicion of fish allergy is confirmed by the challenge procedure (Figure 86). Firstly, how sensitive is the patient? This can normally be deduced from the titrated challenge procedure, and the patient should be advised for future dietary precautions based on his or her individual threshold. Of note, the scheme in (Figure 87) reflects a complete workflow, rather than a temporal sequence. Skin test results might be earlier available than IgE serology. Food challenges are advised against in case of a suspected severe reaction. As reviewed under "Parvalbumins," some fish-allergic patients can develop a cross-reactivity to chicken meat due to parvalbumin cross-reactivity.¹⁹⁵ Prick-to-prick testing with chicken meat (both, leg and breast meat) and serum IgE testing to chicken meat might be recommended in case of a positive clinical history. During the procedure of diagnosis as proposed in Figure 86, the assessment of a putative *Anisakis* simplex allergy shall be included, especially if results of fish allergy tests are unequivocal.¹⁰¹⁷ Skin prick tests and IgE serology can be useful to demonstrate sensitization. It is important to keep in mind that IgE testing with *Anisakis*-extracts can produce false-positive results due to molecular cross-reactivity, such as to shellfish or insect allergens.

The second question relates to the degree of cross-reactivity between fish species (Figure 87). If the patient reacts with IgE of similar magnitude and reacts to a parvalbumin it is likely that there is a broad cross-reactivity. If the sensitization pattern suggests a more "selective" reaction to specific fish, an open challenge may be performed to confirm this tolerance. For this, the choice of the fish can be adapted according to the clinical history of the patient. Especially fish with a low parvalbumin content, such as tuna, as well as fish distantly related to cod, such as ray, a cartilaginous fish with low cross-reactive alpha-parvalbumins, might be of importance to test for oral tolerance and alternative introduction into the diet (see chapter C11).²⁰⁸ If the patient presents primarily with digestive symptoms without cutaneous signs, non-IgE-mediated food allergy might be suspected (food protein enterocolitis syndrome/FPIES in children, eosinophilic oesophagitis (EoE) in children and adults.¹⁰⁴³ Also, histamine fish poisoning leads to the induction of allergy-like symptoms.

**History of exposure**—As for diagnosis of fish-allergic patients, it should be remembered that most people are aware of ingesting fish, and thus the patient history is often quite reliable as for the exposure (which does of course not exclude other ingredients in a meal). While probably extremely rare, there are examples of patients reacting to fish allergens that are hidden in foods.¹⁹⁵ It is obviously difficult to demonstrate a 100% safety level, but several studies have suggested that some fish-derived food additives seem to have a quite low risk of causing reactions in previously sensitized fish allergic persons.
of consistency to differentiate between allergy and asymptomatic sensitization. In fact, traditional test such as skin prick test (SPT) and *Anisakis* simplex-specific IgE using the whole extracts have shown low specificity.¹⁰³⁸ Various studies approached the identification of clinically relevant allergens in order to discriminate genuine allergy from molecular cross-reactivity. IgE reactivity to ES-allergens showed to correlate with clinical reactivity. Due to the lack of homology with other allergens, Ani s 1 and Ani s 7 bear a good diagnostic capacity to identify true *Anisakis* simplex allergy.¹⁰³⁹,¹⁰⁴⁰ Ani s 1-specific IgE are found predominantly on patients presenting with severe allergic reaction. Though Ani s 7 is a frequently IgE-recognized molecule, it seems less clinically relevant, which can be explained by a parallel IgG4-response resulting into a protection against adverse symptoms.¹⁰⁴¹ The ES allergen Ani s 13 has been reported as another marker for primary sensitization and clinical allergy. Ani s 2 and Ani s 3 are *Anisakis* simplex pan-allergens but with high cross-reactivity to other allergen sources such as mites, insects, shellfish, cockroach, and chronomids, thus a less specific marker.¹⁰¹⁷,¹⁰¹⁸,¹⁰²⁹

Clinical relevance of *Anisakis* simplex allergens

* The seroprevalence ranges between 0.2 and 15% in the general population.

* Specific IgE to the whole extract is not always related to symptomatic allergy.

* Ani s 1 appears to be a clinically relevant allergen from *Anisakis* simplex.

4 - Clinical management

FISH—Fish allergy diagnosis is mostly based on clinical history, skin tests (prick tests to fish extract or prick-to prick with the culprit fish -raw and cooked-) and IgE tests, followed (if needed) by an oral food challenge (ideally, double-blind placebo-controlled challenge) with the fish that has elicited the reaction.¹⁹⁵,¹⁰³¹ Level of serum IgE antibodies have been correlated with the clinical reactivity to predict allergy to fish. In a US population, an IgE level of 20 kU/L to cod extract (ImmunoCAP Specific IgE test, Thermo Fisher) allowed to predict an allergy to this fish with 95% certainty.¹⁰⁴² More recently, a large food challenge-based study reported that combining an obvious clinical history of patients with slgE to cod extract >8.2 kU/L or to salmon extract >5 kU/L should result in advising to avoid consuming all fish species.¹⁰³⁶ A specific cod IgE titer >5 kU/L has been even reported to be useful regarding an unfavorable prognosis to outgrow fish allergy.¹⁰³⁵ The availability of individual allergens for IgE testing is still limited and thus, not of much help in predicting whether the patient is allergic to other fish species. However, an outline of the future diagnosis using single

allergens is presented in the "Clinical cases" and in the chapter C11. Meanwhile, a novel multiplex platform (MadX, MacroArray Diagnostics) made a number of parvalbumins from several fish species available, in addition to the other fish allergens cod enolase and aldolase (Gad m 2, Gad m 3).

Two important questions should be addressed if the initial suspicion of fish allergy is confirmed by the challenge procedure (Figure 86). Firstly, how sensitive is the patient? This can normally be deduced from the titrated challenge procedure, and the patient should be advised for future dietary precautions based on his or her individual threshold. Of note, the scheme in (Figure 87) reflects a complete workflow, rather than a temporal sequence. Skin test results might be earlier available than IgE serology. Food challenges are advised against in case of a suspected severe reaction. As reviewed under "Parvalbumins," some fish-allergic patients can develop a cross-reactivity to chicken meat due to parvalbumin cross-reactivity.¹⁹⁵ Prick-to-prick testing with chicken meat (both, leg and breast meat) and serum IgE testing to chicken meat might be recommended in case of a positive clinical history. During the procedure of diagnosis as proposed in Figure 86, the assessment of a putative *Anisakis* simplex allergy shall be included, especially if results of fish allergy tests are unequivocal.¹⁰¹⁷ Skin prick tests and IgE serology can be useful to demonstrate sensitization. It is important to keep in mind that IgE testing with *Anisakis*-extracts can produce false-positive results due to molecular cross-reactivity, such as to shellfish or insect allergens.

The second question relates to the degree of cross-reactivity between fish species (Figure 87). If the patient reacts with IgE of similar magnitude and reacts to a parvalbumin it is likely that there is a broad cross-reactivity. If the sensitization pattern suggests a more "selective" reaction to specific fish, an open challenge may be performed to confirm this tolerance. For this, the choice of the fish can be adapted according to the clinical history of the patient. Especially fish with a low parvalbumin content, such as tuna, as well as fish distantly related to cod, such as ray, a cartilaginous fish with low cross-reactive alpha-parvalbumins, might be of importance to test for oral tolerance and alternative introduction into the diet (see chapter C11).²⁰⁸ If the patient presents primarily with digestive symptoms without cutaneous signs, non-IgE-mediated food allergy might be suspected (food protein enterocolitis syndrome/FPIES in children, eosinophilic oesophagitis (EoE) in children and adults.¹⁰⁴³ Also, histamine fish poisoning leads to the induction of allergy-like symptoms.

**History of exposure**—As for diagnosis of fish-allergic patients, it should be remembered that most people are aware of ingesting fish, and thus the patient history is often quite reliable as for the exposure (which does of course not exclude other ingredients in a meal). While probably extremely rare, there are examples of patients reacting to fish allergens that are hidden in foods.¹⁹⁵ It is obviously difficult to demonstrate a 100% safety level, but several studies have suggested that some fish-derived food additives seem to have a quite low risk of causing reactions in previously sensitized fish allergic persons.
Case history: Immediate reaction after potential consumption of fish (product)

<figure><img src="image_190.png" /><figcaption>FIGURE 86 Diagnostic algorithm in patients with suspected fish allergy</figcaption></figure>

**Quantitative risk assessment**—As relating to the dose of fish producing food-allergic symptoms only data for fish meat are available in the published literature. According to the literature, the lowest provoking dose is in the low milligram range. A larger population was tested in the EuroPrevall project, an EU-funded project the prevalence, costs, and basis of food allergy across Europe, and an ED10 of 27.3 mg of cod protein was found, which was confirmed later.⁵⁵⁴,⁹⁸⁸,¹⁰³⁶

**Other risk factors**—It is important to notice that parvalbumins, the major fish allergens, are highly heat-stable.¹⁹⁵,²⁰⁸ Thus, their alergenicity cannot be expected to be reduced upon food processing. However, its proteolytic resistance seems to be lower, for example, to pepsin at low pH. Accordingly, maintenance of a well-functioning digestive system with low ventricular pH may be of importance for avoiding fish allergy.

**ANISAKIS SIMPLEX**—The diagnosis of anisakiasis is mainly based on the clinical presentation and the observation of larvae during an endoscopy.

**Differential diagnosis**—How the differential diagnosis is proceeded along with a genuine fish allergy diagnosis, is represented in (Figure 86). When specific IgE to *Anisakis* extract is positive in a patient with ambiguous clinical history, sensitization to invertebrates (e.g., house dust mites, crustaceans and nematodes) should be considered.¹⁰¹⁷,¹⁰²⁸,¹⁰³⁸ Previous studies have also shown that the *Anisakis* simplex allergy evaluation should also include IgE testing to Ascaris, another potentially cross-reacting nematode. When doing this, it has been shown that diagnostic specificity increases: by forming the ratio between both (*Anisakis*/*Ascaris*-IgE ≥4.4, specificity >95%).¹⁰³⁸ Further studies will be needed to elaborate on reliable diagnostic cut-off points.¹⁰³⁹,¹⁰⁴⁴

**Risk factors**—In addition to fish consumption habits, such as raw fish intake and intake of species that are known to be more often contaminated with parasites (e.g., hake, mackerel, anchovies), certain professions related to fish handling appear to be also a risk factor for sensitization.¹⁰⁴⁴
Case history: Immediate reaction after potential consumption of fish (product)

<figure><img src="image_190.png" /><figcaption>FIGURE 86 Diagnostic algorithm in patients with suspected fish allergy</figcaption></figure>

**Quantitative risk assessment**—As relating to the dose of fish producing food-allergic symptoms only data for fish meat are available in the published literature. According to the literature, the lowest provoking dose is in the low milligram range. A larger population was tested in the EuroPrevall project, an EU-funded project the prevalence, costs, and basis of food allergy across Europe, and an ED10 of 27.3 mg of cod protein was found, which was confirmed later.⁵⁵⁴,⁹⁸⁸,¹⁰³⁶

**Other risk factors**—It is important to notice that parvalbumins, the major fish allergens, are highly heat-stable.¹⁹⁵,²⁰⁸ Thus, their alergenicity cannot be expected to be reduced upon food processing. However, its proteolytic resistance seems to be lower, for example, to pepsin at low pH. Accordingly, maintenance of a well-functioning digestive system with low ventricular pH may be of importance for avoiding fish allergy.

**ANISAKIS SIMPLEX**—The diagnosis of anisakiasis is mainly based on the clinical presentation and the observation of larvae during an endoscopy.

**Differential diagnosis**—How the differential diagnosis is proceeded along with a genuine fish allergy diagnosis, is represented in (Figure 86). When specific IgE to *Anisakis* extract is positive in a patient with ambiguous clinical history, sensitization to invertebrates (e.g., house dust mites, crustaceans and nematodes) should be considered.¹⁰¹⁷,¹⁰²⁸,¹⁰³⁸ Previous studies have also shown that the *Anisakis* simplex allergy evaluation should also include IgE testing to Ascaris, another potentially cross-reacting nematode. When doing this, it has been shown that diagnostic specificity increases: by forming the ratio between both (*Anisakis*/*Ascaris*–IgE ≥4.4, specificity >95%).¹⁰³⁸ Further studies will be needed to elaborate on reliable diagnostic cut-off points.¹⁰³⁹,¹⁰⁴⁴

**Risk factors**—In addition to fish consumption habits, such as raw fish intake and intake of species that are known to be more often contaminated with parasites (e.g., hake, mackerel, anchovies), certain professions related to fish handling appear to be also a risk factor for sensitization.¹⁰⁴⁴
Case history: Immediate reaction after potential consumption of fish (product)

<figure><img src="image_191.png" /><figcaption>FIGURE 87 Diagnostic algorithm in patients with suspected allergy to single or specific fish</figcaption></figure>

5 - Clinical cases

Most cases of fish allergy present with classical food allergic symptoms short after intake of fish. Symptoms may include oral allergy syndrome, rhinitis/conjunctivitis, asthma, urticaria, and gastrointestinal symptoms. There is an increasing number of reports on non-IgE-mediated fish allergy (FPIES and EoE), involving mostly symptoms of the digestive tract.¹⁰⁴³ For the clinical cases, we will focus on IgE-mediated fish allergy to demonstrate the value of molecular diagnosis. However, FPIES and EoE should be always considered in alignment with clinical symptoms and negative IgE tests. Like genuine fish allergy, clinical manifestations of *Anisakis* simplex allergic occur also very quickly, leading to food allergy-typical symptoms.

**Case 1 (published)**¹⁰³⁶ (Figure 88)

**Clinical History:** A young patient, 11 years, had a clinical history of fish allergy with clinical symptom development on several occasions.

**Test with extracts:** Specific IgE were positive for cod extract (117 kU/L), salmon (144 kU/L) and mackerel (52.1 kU/L). Food challenge—The patient has a positive food challenges with cod, salmon and mackerel (cumulative eliciting doses 1 g, 1 g and 2 g, respectively).

**Test with molecules:** Specific IgE were found for cod, salmon and mackerel parvalbumins (121.7 kU/L, 30.3 kU/L and 49.2 kU/L, respectively) but also for enolases (61.5 kU/L, 9.5 kU/L and 1.1 kU/L, respectively) and aldolases (63.1 kU/L, 18.3 kU/L and 0.5 kU/L, respectively).

**Conclusion:** In this case, the clinical cross-reactivity to unspecific fish was confirmed by the detection of specific IgE to a broad panel of fish allergens, including parvalbumins, enolases and aldolases.

**Case 2 (published)**¹⁰¹² (Figure 89)

**Clinical History:** Female patient, 17 years old, presenting with oral allergy syndrome and rhinitis upon consumption of fish (species unknown, tuna suspected).
Case history: Immediate reaction after potential consumption of fish (product)

<figure><img src="image_191.png" /><figcaption>FIGURE 87 Diagnostic algorithm in patients with suspected allergy to single or specific fish</figcaption></figure>

5 - Clinical cases

Most cases of fish allergy present with classical food allergic symptoms short after intake of fish. Symptoms may include oral allergy syndrome, rhinitis/conjunctivitis, asthma, urticaria, and gastrointestinal symptoms. There is an increasing number of reports on non-IgE-mediated fish allergy (FPIES and EoE), involving mostly symptoms of the digestive tract.¹⁰⁴³ For the clinical cases, we will focus on IgE-mediated fish allergy to demonstrate the value of molecular diagnosis. However, FPIES and EoE should be always considered in alignment with clinical symptoms and negative IgE tests. Like genuine fish allergy, clinical manifestations of *Anisakis* simplex allergic occur also very quickly, leading to food allergy-typical symptoms.

**Case 1 (published)**¹⁰³⁶ (Figure 88)

**Clinical History:** A young patient, 11 years, had a clinical history of fish allergy with clinical symptom development on several occasions.

**Test with extracts:** Specific IgE were positive for cod extract (117 kU/L), salmon (144 kU/L) and mackerel (52.1 kU/L). Food challenge—The patient has a positive food challenges with cod, salmon and mackerel (cumulative eliciting doses 1 g, 1 g and 2 g, respectively).

**Test with molecules:** Specific IgE were found for cod, salmon and mackerel parvalbumins (121.7 kU/L, 30.3 kU/L and 49.2 kU/L, respectively) but also for enolases (61.5 kU/L, 9.5 kU/L and 1.1 kU/L, respectively) and aldolases (63.1 kU/L, 18.3 kU/L and 0.5 kU/L, respectively).

**Conclusion:** In this case, the clinical cross-reactivity to unspecific fish was confirmed by the detection of specific IgE to a broad panel of fish allergens, including parvalbumins, enolases and aldolases.

**Case 2 (published)**¹⁰¹² (Figure 89)

**Clinical History:** Female patient, 17 years old, presenting with oral allergy syndrome and rhinitis upon consumption of fish (species unknown, tuna suspected).
**Test with extracts:** Skin testing was negative with tuna and salmon. ImmunoCAP Specific IgE test for tuna extract was 3.2 kU/L.

**Food challenge:** The patient refused a food challenge. Test with molecules—IgE-ELISA was positive for purified collagen from tuna, barramundi, and salmon. IgE ELISA was negative for tuna parvalbumin. IgE reactivity was further confirmed by immunoblot analysis using purified tuna collagen and in basophil activation assay.

**Conclusion:** The patient demonstrated IgE reactivity to tuna collagen but absence of reactivity to tuna parvalbumin. Negative SPT to tuna extract may be explained by low amount of collagen in the fish extract used for SPT.

**Case 3 (published)¹⁰⁴⁵ (Figure 90)**

**Clinical History:** Male patient, 7 years old, presenting with urticaria and vomiting upon consumption of fish as well as vomiting upon consumption of chicken meat.

**Test with extracts:** Skin testing were positive with cod and chicken. ImmunoCAP Specific IgE test for cod extract was 5.4 kU/L and with chicken meat 8.9 kU/L.

**Food challenge:** The patient reacted in a food challenge upon ingestion of a dose of 100 mg chicken meat.

**Test with molecules:** IgE-ELISA was negative with purified parvalbumins and aldolases from cod and chicken. IgE ELISA was positive for both cod and chicken meat enolase (4.3 kU/L and 4.6 kU/L, respectively).

**Conclusion:** A clinical cross-reactivity was confirmed by positive IgE reactivity for cross-reactive allergens cod Gad m 2 and chicken Gal d 9.

**Case 4 (original) (Figure 91)**

**Clinical History:** A 45-year-old woman presented left hemi-abdominal pain, urticaria and dyspnea with desaturation, 30 minutes after eating raw anchovies.

**Test with extracts:** Skin testing was positive for *Anisakis* simplex extract. Specific IgE were positive for *Anisakis* simplex (14.3 kU/L) and borderline positive for anchovy (0.3 kU/L) and *Ascaris* (0.5 kU/L). The ratio for *Anisakis/Ascaris*-IgE was 28.6.

**Food challenge:** The patient refused a food challenge with anchovy.

**Test with molecules:** Specific IgE were positive for Ani s 1 and Ani s 7 in allergen-specific ELISA.

**Conclusion:** In this case, *Anisakis* simplex allergy was confirmed by the detection of IgE to specific allergen Ani s 1.

<figure><img src="image_192.png" /><figcaption>FIGURE 88 Diagnostic procedure Case 1. Orange text box = established diagnosis</figcaption></figure>

<figure><img src="image_193.png" /><figcaption>FIGURE 89 Diagnostic procedure Case 2. Orange text box = established diagnosis</figcaption></figure>

<figure><img src="image_194.png" /><figcaption>FIGURE 90 Diagnostic procedure Case 3. Orange text box = established diagnosis</figcaption></figure>
**Test with extracts:** Skin testing was negative with tuna and salmon. ImmunoCAP Specific IgE test for tuna extract was 3.2 kU/L.

**Food challenge:** The patient refused a food challenge. Test with molecules—IgE-ELISA was positive for purified collagen from tuna, barramundi, and salmon. IgE ELISA was negative for tuna parvalbumin. IgE reactivity was further confirmed by immunoblot analysis using purified tuna collagen and in basophil activation assay.

**Conclusion:** The patient demonstrated IgE reactivity to tuna collagen but absence of reactivity to tuna parvalbumin. Negative SPT to tuna extract may be explained by low amount of collagen in the fish extract used for SPT.

**Case 3 (published)¹⁰⁴⁵ (Figure 90)**

**Clinical History:** Male patient, 7 years old, presenting with urticaria and vomiting upon consumption of fish as well as vomiting upon consumption of chicken meat.

**Test with extracts:** Skin testing were positive with cod and chicken. ImmunoCAP Specific IgE test for cod extract was 5.4 kU/L and with chicken meat 8.9 kU/L.

**Food challenge:** The patient reacted in a food challenge upon ingestion of a dose of 100 mg chicken meat.

**Test with molecules:** IgE-ELISA was negative with purified parvalbumins and aldolases from cod and chicken. IgE ELISA was positive for both cod and chicken meat enolase (4.3 kU/L and 4.6 kU/L, respectively).

**Conclusion:** A clinical cross-reactivity was confirmed by positive IgE reactivity for cross-reactive allergens cod Gad m 2 and chicken Gal d 9.

**Case 4 (original) (Figure 91)**

**Clinical History:** A 45-year-old woman presented left hemi-abdominal pain, urticaria and dyspnea with desaturation, 30 minutes after eating raw anchovies.

**Test with extracts:** Skin testing was positive for *Anisakis* simplex extract. Specific IgE were positive for *Anisakis* simplex (14.3 kU/L) and borderline positive for anchovy (0.3 kU/L) and *Ascaris* (0.5 kU/L). The ratio for *Anisakis/Ascaris*-IgE was 28.6.

**Food challenge:** The patient refused a food challenge with anchovy.

**Test with molecules:** Specific IgE were positive for Ani s 1 and Ani s 7 in allergen-specific ELISA.

**Conclusion:** In this case, *Anisakis* simplex allergy was confirmed by the detection of IgE to specific allergen Ani s 1.

<figure><img src="image_192.png" /><figcaption>FIGURE 88 Diagnostic procedure Case 1. Orange text box = established diagnosis</figcaption></figure>

<figure><img src="image_193.png" /><figcaption>FIGURE 89 Diagnostic procedure Case 2. Orange text box = established diagnosis</figcaption></figure>

<figure><img src="image_194.png" /><figcaption>FIGURE 90 Diagnostic procedure Case 3. Orange text box = established diagnosis</figcaption></figure>
<figure><img src="image_195.png" /><figcaption>FIGURE 91 Diagnostic procedure Case 3. Orange text box = established diagnosis</figcaption></figure>
<figure><img src="image_195.png" /><figcaption>FIGURE 91 Diagnostic procedure Case 3. Orange text box = established diagnosis</figcaption></figure>
# B13 - Allergy to crustacean and molluscs

Sandip D. Kamath, Roni Nugraha, Dianne E. Campbell, Andreas L. Lopata

## Highlights

* Tropomyosin and arginine kinase are highly cross-reactive allergens and are responsible for clinical cross-reactivity among crustaceans, molluscs, insects, and mites.

* Some shellfish allergens can sensitize via the oral and inhalation route (tropomyosin, arginine kinase, triosephosphate isomerase, hemocyanin).

* There is a need for the incorporation of component allergen testing for mollusc allergy.

## 1 - The allergen sources

The shellfish group is included among the “Big Eight” food groups, which are responsible for more than 90% of all food allergy cases. It is estimated that up to 3% of the population are affected by food allergy to shellfish, including the crustacean and mollusc groups, depending upon geographical region.¹⁰¹⁵,¹⁰¹⁸ Shellfish allergy, particularly to shrimps, has one of the highest rates of food-induced anaphylaxis with nearly 42% of shellfish allergic adults and 12-20% of allergic children reporting anaphylaxis.¹⁰⁴⁶,¹⁰⁴⁷

It is noteworthy that although shellfish, along with fish are commonly termed as seafood, these two groups are very distinct in evolutionary terms and contain different molecular repertoires of food allergens. All shellfish species are invertebrate animals, in contrast to fish, which are regarded as lower vertebrates. Aquatic animals of other phyla have not been investigated comprehensively regarding allergy due to limited consumption (e.g., jellyfish) and hence are not discussed in this chapter. The culinary term “Shellfish” combines two major invertebrate groups, which are taxonomically very different, the crustaceans and the molluscs. The edible crustaceans belong to the subphylum Crustacea and more specifically to the order Decapoda, which can be broadly grouped into shrimps (prawns), crabs and lobsters. While “shrimps” and “prawns” belong to two different taxonomical classifications, the terms are often used interchangeably commercially and in research publications (Figure 92). The Mollusca is the second largest phylum with over 100 edible species recorded by the Food and Agriculture Organization (FAO). The three most important subphyla in terms of consumption include the Gastropoda (e.g., abalone, snail), Bivalvia (e.g., mussel, oyster) and Cephalopoda (e.g., squid, cuttlefish). Comparing evolutionary distance, crustaceans are placed closer to insects and arachnids but not molluscs, and this seems to be the major factor for molecular cross-sensitization and clinical cross-reactivity between crustaceans, house dust mites and insects.

These classifications are important for understanding clinical and immunological cross-reactivities. The allergenic proteins present in these shellfish species have frequently different isoforms and often present a challenge in allergen detection as well as accurate allergy diagnosis and management (see below). In addition, the availability and consumption of different shellfish species vary to a high degree in different parts of the world, with two-thirds of the global seafood production consumed in Asia.

Increased awareness of the high nutritional value of crustaceans and molluscs has led to a rise in shellfish consumption, and this has been associated with more frequent reported of allergic reactions. The following types of consumption and exposure can be potent elicitors of severe allergic reactions to shellfish;

A - Raw and cooked shellfish meat—Allergenic proteins are found in high concentrations in the edible muscle parts of shellfish. In crustaceans, it is found in the abdominal, tail and pincer meat. Food processing methods such as thermal or pressure treatment do not seem to denature most of the allergens but, on the contrary, may enhance their allergenic activity.¹⁰⁴⁸,¹⁶⁷⁶ The tropomyosin family represents the major heat-stable allergen present in all shellfish species, may constitute up to 20% of the total protein content and can be considered as a shellfish pan-allergen (see also Chapter C05). For food safety, food products containing shellfish must be appropriately labelled. Moreover, the European Union has mandated separate food allergen labelling for crustaceans and molluscs; however, appropriate tests are only available for crustaceans.¹⁰⁴⁹ Determining the eliciting dose of shellfish allergic reactions threshold distributions is important for the appropriate allergen labelling on seafood-based products. Recent studies have demonstrated that approximately 0.1-1.0 g of shellfish meat has to be ingested to trigger an allergic response.⁹⁸⁸ Of note, shrimp is estimated to have one of the highest ED50's (amount of allergic protein required to trigger an allergic reaction in 50% of sensitized individuals) of the “big 8” allergen groups, with about 8 g of shrimp protein (approximately half a shrimp).⁵⁵⁴ Only 1% of shrimp allergic individuals develop allergic reactions following exposures to 26 mg or more of shrimp meat. This high ED may explain variable clinical histories of tolerance to small ingested amounts, while subsequent severe reactions can be observed.

B - Food additives containing shellfish-derived proteins—Shellfish products such as dried shrimp or shrimp paste are widely used as flavouring agents in various packaged and processed foods such as instant noodles and soups. This may be a potential source and cause of accidental consumption and exposure to shellfish allergens (see Table 38 for food products indicating the presence of shellfish groups).

C - Occupational exposure—In the seafood processing industry, workers are constantly exposed to aerosolized shellfish particulate matters arising from different processing activities leading to the inhalation of airborne allergens and/or cooking fumes.¹⁰⁵⁰ Such occupational exposure to shellfish allergens may be a primary route of sensitization to shellfish, and upon re-exposure can elicit upper and lower respiratory tract symptoms such as cough, wheeze, laryngeal
# B13 - Allergy to crustacean and molluscs

Sandip D. Kamath, Roni Nugraha, Dianne E. Campbell, Andreas L. Lopata

## Highlights

* Tropomyosin and arginine kinase are highly cross-reactive allergens and are responsible for clinical cross-reactivity among crustaceans, molluscs, insects, and mites.

* Some shellfish allergens can sensitize via the oral and inhalation route (tropomyosin, arginine kinase, triosephosphate isomerase, hemocyanin).

* There is a need for the incorporation of component allergen testing for mollusc allergy.

## 1 - The allergen sources

The shellfish group is included among the “Big Eight” food groups, which are responsible for more than 90% of all food allergy cases. It is estimated that up to 3% of the population are affected by food allergy to shellfish, including the crustacean and mollusc groups, depending upon geographical region.¹⁰¹⁵,¹⁰¹⁸ Shellfish allergy, particularly to shrimps, has one of the highest rates of food-induced anaphylaxis with nearly 42% of shellfish allergic adults and 12-20% of allergic children reporting anaphylaxis.¹⁰⁴⁶,¹⁰⁴⁷

It is noteworthy that although shellfish, along with fish are commonly termed as seafood, these two groups are very distinct in evolutionary terms and contain different molecular repertoires of food allergens. All shellfish species are invertebrate animals, in contrast to fish, which are regarded as lower vertebrates. Aquatic animals of other phyla have not been investigated comprehensively regarding allergy due to limited consumption (e.g., jellyfish) and hence are not discussed in this chapter. The culinary term “Shellfish” combines two major invertebrate groups, which are taxonomically very different, the crustaceans and the molluscs. The edible crustaceans belong to the subphylum Crustacea and more specifically to the order Decapoda, which can be broadly grouped into shrimps (prawns), crabs and lobsters. While “shrimps” and “prawns” belong to two different taxonomical classifications, the terms are often used interchangeably commercially and in research publications (Figure 92). The Mollusca is the second largest phylum with over 100 edible species recorded by the Food and Agriculture Organization (FAO). The three most important subphyla in terms of consumption include the Gastropoda (e.g., abalone, snail), Bivalvia (e.g., mussel, oyster) and Cephalopoda (e.g., squid, cuttlefish). Comparing evolutionary distance, crustaceans are placed closer to insects and arachnids but not molluscs, and this seems to be the major factor for molecular cross-sensitization and clinical cross-reactivity between crustaceans, house dust mites and insects.

These classifications are important for understanding clinical and immunological cross-reactivities. The allergenic proteins present in these shellfish species have frequently different isoforms and often present a challenge in allergen detection as well as accurate allergy diagnosis and management (see below). In addition, the availability and consumption of different shellfish species vary to a high degree in different parts of the world, with two-thirds of the global seafood production consumed in Asia.

Increased awareness of the high nutritional value of crustaceans and molluscs has led to a rise in shellfish consumption, and this has been associated with more frequent reported of allergic reactions. The following types of consumption and exposure can be potent elicitors of severe allergic reactions to shellfish;

A - Raw and cooked shellfish meat—Allergenic proteins are found in high concentrations in the edible muscle parts of shellfish. In crustaceans, it is found in the abdominal, tail and pincer meat. Food processing methods such as thermal or pressure treatment do not seem to denature most of the allergens but, on the contrary, may enhance their allergenic activity.¹⁰⁴⁸,¹⁶⁷⁶ The tropomyosin family represents the major heat-stable allergen present in all shellfish species, may constitute up to 20% of the total protein content and can be considered as a shellfish pan-allergen (see also Chapter C05). For food safety, food products containing shellfish must be appropriately labelled. Moreover, the European Union has mandated separate food allergen labelling for crustaceans and molluscs; however, appropriate tests are only available for crustaceans.¹⁰⁴⁹ Determining the eliciting dose of shellfish allergic reactions threshold distributions is important for the appropriate allergen labelling on seafood-based products. Recent studies have demonstrated that approximately 0.1-1.0 g of shellfish meat has to be ingested to trigger an allergic response.⁹⁸⁸ Of note, shrimp is estimated to have one of the highest ED50's (amount of allergic protein required to trigger an allergic reaction in 50% of sensitized individuals) of the “big 8” allergen groups, with about 8 g of shrimp protein (approximately half a shrimp).⁵⁵⁴ Only 1% of shrimp allergic individuals develop allergic reactions following exposures to 26 mg or more of shrimp meat. This high ED may explain variable clinical histories of tolerance to small ingested amounts, while subsequent severe reactions can be observed.

B - Food additives containing shellfish-derived proteins—Shellfish products such as dried shrimp or shrimp paste are widely used as flavouring agents in various packaged and processed foods such as instant noodles and soups. This may be a potential source and cause of accidental consumption and exposure to shellfish allergens (see Table 38 for food products indicating the presence of shellfish groups).

C - Occupational exposure—In the seafood processing industry, workers are constantly exposed to aerosolized shellfish particulate matters arising from different processing activities leading to the inhalation of airborne allergens and/or cooking fumes.¹⁰⁵⁰ Such occupational exposure to shellfish allergens may be a primary route of sensitization to shellfish, and upon re-exposure can elicit upper and lower respiratory tract symptoms such as cough, wheeze, laryngeal
<figure><img src="image_196.png" /><figcaption>FIGURE 92 Classification of edible crustacean and mollusc species * The term prawn and shrimp are used synonymously or interchangeably in different regions. Taxonomically, they belong to different suborders of crustaceans.</figcaption></figure>

symptoms, and rhinitis.<sup>1023</sup> Occupational exposure may also result in contact urticaria<sup>1051</sup> or contact dermatitis.<sup>1052,1053</sup> Workers with shellfish-induced occupational asthma are at risk of developing allergic reactions upon ingestion of seafood.<sup>1054</sup>

D - Fish parasites -The food-borne parasite *Anisakis* or her-
ring worm is an important food allergen source. *Anisakis* is a par-
asitic nematode that mainly infects fish, but also crustaceans and
squid, and the ingestion of contaminated fish or shellfish can result
in severe allergic reactions.<sup>1055</sup> More importantly, the allergens of
the tropomyosin family are thought to be responsible for cross-
reactivity between *Anisakis* and other invertebrates such as insects,
mites and crustaceans.

2 - Allergen families

Edible shellfish species can be broadly categorized into crustaceans and molluscs (Figure 92). Crustaceans include prawns, shrimp, crab, lobster, and several other species. Molluscs include oysters, mussels, squid, octopus, and abalone. Although a few hundred different shell-fish species are consumed worldwide, nearly 80% of all allergic incidences to shellfish are reported to shrimps or prawns. This is partly due to the high production and consumption rate in comparison to other shellfish species of commercial importance. Biologically, all edible shellfish species are invertebrates belonging to Arthropoda or Mollusca phyla. Naturally, most of the identified shellfish allergens
<figure><img src="image_196.png" /><figcaption>FIGURE 92 Classification of edible crustacean and mollusc species * The term prawn and shrimp are used synonymously or interchangeably in different regions. Taxonomically, they belong to different suborders of crustaceans.</figcaption></figure>

symptoms, and rhinitis.<sup>1023</sup> Occupational exposure may also result in contact urticaria<sup>1051</sup> or contact dermatitis.<sup>1052,1053</sup> Workers with shellfish-induced occupational asthma are at risk of developing allergic reactions upon ingestion of seafood.<sup>1054</sup>

D - Fish parasites -The food-borne parasite *Anisakis* or her-
ring worm is an important food allergen source. *Anisakis* is a par-
asitic nematode that mainly infects fish, but also crustaceans and
squid, and the ingestion of contaminated fish or shellfish can result
in severe allergic reactions.<sup>1055</sup> More importantly, the allergens of
the tropomyosin family are thought to be responsible for cross-
reactivity between *Anisakis* and other invertebrates such as insects,
mites and crustaceans.

2 - Allergen families

Edible shellfish species can be broadly categorized into crustaceans and molluscs (Figure 92). Crustaceans include prawns, shrimp, crab, lobster, and several other species. Molluscs include oysters, mussels, squid, octopus, and abalone. Although a few hundred different shell-fish species are consumed worldwide, nearly 80% of all allergic incidences to shellfish are reported to shrimps or prawns. This is partly due to the high production and consumption rate in comparison to other shellfish species of commercial importance. Biologically, all edible shellfish species are invertebrates belonging to Arthropoda or Mollusca phyla. Naturally, most of the identified shellfish allergens
<table><caption>TABLE 38 List of ingredients that may contain allergenic Crustacean or Mollusc proteins</caption><thead><tr><th>Category</th><th>Examples</th></tr></thead><tbody><tr><td>Crustaceans species that may be included as food ingredients</td><td>Barnacle, Balmain bugs, Crab, Crawfish, Crayfish, Krill, Lobster, Marron, Moreton bay bugs, Prawns, Shrimp (Crevette, Scampi), Yabbie</td></tr><tr><td>Mollusc species that may be included as food ingredients</td><td>Abalone, Barnacles, Clam, Cockle, Cuttlefish, Limpet, Mussel, Octopus, Oyster, Periwinkle, Sea cucumber, Sea urchin, Scallop, Snail, Squid (calamari), Whelk</td></tr><tr><td>Food preparations that may contain shellfish</td><td>Bouillabaisse, Cuttlefish Ink, Clam chowder, Clam broth base powder, Crab extract powder, Condiments and spices, Scallop extract powder, Fish stock, Fish Sauce, Glucosamine, Lobster extract powder, Marinara, Oyster juice powder, Paella, Pescatore sauce, Prawn crackers, Prawn chips, Shrimp powder, Seafood flavouring, Surimi, Squid ink,</td></tr></tbody></table>

belong to a common set of protein families that is shared across a diverse range of species.

Shellfish allergens currently identified belong mainly to the tropomyosin (Protein family, PF00261), EF-hand (PF00036), phosphotransferase (PF00217), triosephosphate isomerase (PF00121), fatty acid-binding protein (PF00061), and hemocyanin families (PF00372) (Figures 93 and 94). The major allergens found across all crustacean and mollusc species belong to the tropomyosin family. Multiple isoforms are found depending on function and location; for example, the fast-twitch or slow-twitch isoform is found in crab tail or pincer muscle respectively.

Allergens from the tropomyosin family have a highly conserved primary structure, and this is the main reason for immunological and clinical cross-reactivity not only among crustaceans and molluscs but also among insects, mites, and nematodes (Figure 95). Arginine kinase belongs to the phosphotransferase family while myosin light chain, sarcoplasmic calcium-binding protein, and troponin C belong to the EF-hand domain family. Interestingly, all identified shellfish allergens are proteins involved in cytoskeletal functions or metabolic enzymes (see Table 39).

Due to the complexity and heterogeneity of proteins among crustaceans and molluscs, the relationship between their structure and subsequent allergenicity has only partly been elucidated. In the past, mainly tropomyosin from many crustaceans and a few molluscs had been characterized in detail. The major allergen, tropomyosin was first identified in 1993 as the major shrimp allergen.¹⁰⁵⁶ Recently, fatty acid-binding protein, filamin C and hemocyanin have been identified and characterized as shrimp allergens. In addition, paramyosin was identified recently in whelk. In the past 10 years, five additional shellfish proteins have been identified to elicit IgE-mediated hypersensitivity through ingestion and inhalation and are now officially accepted by the IUIS Allergen Nomenclature Subcommittee (Tables 39 and 40).

## 3 – Allergenic Molecules

As of 2021, 54 allergens from 20 crustacean and 8 mollusc species have been identified and registered on the WHO IUIS Allergen Nomenclature database, despite an even higher number of species and allergens described in the current literature. All of the currently

<figure><img src="image_197.png" /><figcaption>FIGURE 93 Three-dimensional structures of shellfish allergens currently identified and registered the WHO-IUIS Allergen nomenclature. The recently IUIS-registered Paramyosin and filamin C allergens are not depicted in this figure.</figcaption></figure>

identified shellfish allergens demonstrate common properties such as low molecular weight (15-75 kDa), propensity to form a dimer or high oligomer, and good aqueous solubility (Tables 39 and 40).
<table><caption>TABLE 38 List of ingredients that may contain allergenic Crustacean or Mollusc proteins</caption><thead><tr><th>Category</th><th>Examples</th></tr></thead><tbody><tr><td>Crustaceans species that may be included as food ingredients</td><td>Barnacle, Balmain bugs, Crab, Crawfish, Crayfish, Krill, Lobster, Marron, Moreton bay bugs, Prawns, Shrimp (Crevette, Scampi), Yabbie</td></tr><tr><td>Mollusc species that may be included as food ingredients</td><td>Abalone, Barnacles, Clam, Cockle, Cuttlefish, Limpet, Mussel, Octopus, Oyster, Periwinkle, Sea cucumber, Sea urchin, Scallop, Snail, Squid (calamari), Whelk</td></tr><tr><td>Food preparations that may contain shellfish</td><td>Bouillabaisse, Cuttlefish Ink, Clam chowder, Clam broth base powder, Crab extract powder, Condiments and spices, Scallop extract powder, Fish stock, Fish Sauce, Glucosamine, Lobster extract powder, Marinara, Oyster juice powder, Paella, Pescatore sauce, Prawn crackers, Prawn chips, Shrimp powder, Seafood flavouring, Surimi, Squid ink,</td></tr></tbody></table>

belong to a common set of protein families that is shared across a diverse range of species.

Shellfish allergens currently identified belong mainly to the tropomyosin (Protein family, PF00261), EF-hand (PF00036), phosphotransferase (PF00217), triosephosphate isomerase (PF00121), fatty acid-binding protein (PF00061), and hemocyanin families (PF00372) (Figures 93 and 94). The major allergens found across all crustacean and mollusc species belong to the tropomyosin family. Multiple isoforms are found depending on function and location; for example, the fast-twitch or slow-twitch isoform is found in crab tail or pincer muscle respectively.

Allergens from the tropomyosin family have a highly conserved primary structure, and this is the main reason for immunological and clinical cross-reactivity not only among crustaceans and molluscs but also among insects, mites, and nematodes (Figure 95). Arginine kinase belongs to the phosphotransferase family while myosin light chain, sarcoplasmic calcium-binding protein, and troponin C belong to the EF-hand domain family. Interestingly, all identified shellfish allergens are proteins involved in cytoskeletal functions or metabolic enzymes (see Table 39).

Due to the complexity and heterogeneity of proteins among crustaceans and molluscs, the relationship between their structure and subsequent allergenicity has only partly been elucidated. In the past, mainly tropomyosin from many crustaceans and a few molluscs had been characterized in detail. The major allergen, tropomyosin was first identified in 1993 as the major shrimp allergen.¹⁰⁵⁶ Recently, fatty acid-binding protein, filamin C and hemocyanin have been identified and characterized as shrimp allergens. In addition, paramyosin was identified recently in whelk. In the past 10 years, five additional shellfish proteins have been identified to elicit IgE-mediated hypersensitivity through ingestion and inhalation and are now officially accepted by the IUIS Allergen Nomenclature Subcommittee (Tables 39 and 40).

### 3 – Allergenic Molecules

As of 2021, 54 allergens from 20 crustacean and 8 mollusc species have been identified and registered on the WHO IUIS Allergen Nomenclature database, despite an even higher number of species and allergens described in the current literature. All of the currently

<figure><img src="image_197.png" /><figcaption>FIGURE 93 Three-dimensional structures of shellfish allergens currently identified and registered the WHO-IUIS Allergen nomenclature. The recently IUIS-registered Paramyosin and filamin C allergens are not depicted in this figure.</figcaption></figure>

identified shellfish allergens demonstrate common properties such as low molecular weight (15-75 kDa), propensity to form a dimer or high oligomer, and good aqueous solubility (Tables 39 and 40).
<figure><img src="image_198.png" /><figcaption>FIGURE 94 A summary of the different crustacean and mollusc allergens identified and registered in the WHO/IUIS Allergen nomenclature database. Allergens belonging to a common protein family are highlighted in the same colour.</figcaption></figure>

<figure><img src="image_199.png" /><figcaption>FIGURE 95 Clinically relevant cross-reactivities between shellfish (crustacean and mollusc) and invertebrate (mite, insect, and nematode) allergens. Cross-reactivity due to different allergen families is depicted in red (tropomyosin) or blue (arginine kinase) arrows.
* indicates allergens that are not currently registered in the WHO-IUIS Allergen nomenclature database.</figcaption></figure>

**Tropomyosin** is an alpha-helical coiled-coil protein that interacts with actin filaments in muscle and nonmuscle cells. Present mainly in the edible meat, it is one of the most abundant proteins representing up to 20% of the total protein content in a shrimp. This allergen is shown to be highly heat stable and relatively resistant to proteases. Several studies have shown that tropomyosin is the prime cause for IgE cross-reactivity among shellfish, insects (including edible insects) and mite species. Interestingly, recent studies have indicated

that tropomyosin may not show T-cell cross-reactivity as a function of structural stability.¹⁰⁵⁷

**Arginine kinase** has been identified in several crustaceans and one mollusc species. Arginine kinase is a phosphotransferase that catalyzes the reversible transfer of the phosphoryl group from ATP to arginine, yielding ADP and N-phosphoarginine. In higher vertebrates, creatinine kinase catalyzes this reaction. Recent studies have shown that arginine kinase is susceptible to heat treatment or
<figure><img src="image_198.png" /><figcaption>FIGURE 94 A summary of the different crustacean and mollusc allergens identified and registered in the WHO/IUIS Allergen nomenclature database. Allergens belonging to a common protein family are highlighted in the same colour.</figcaption></figure>

<figure><img src="image_199.png" /><figcaption>FIGURE 95 Clinically relevant cross-reactivities between shellfish (crustacean and mollusc) and invertebrate (mite, insect, and nematode) allergens. Cross-reactivity due to different allergen families is depicted in red (tropomyosin) or blue (arginine kinase) arrows.
* indicates allergens that are not currently registered in the WHO-IUIS Allergen nomenclature database.</figcaption></figure>

**Tropomyosin** is an alpha-helical coiled-coil protein that interacts with actin filaments in muscle and nonmuscle cells. Present mainly in the edible meat, it is one of the most abundant proteins representing up to 20% of the total protein content in a shrimp. This allergen is shown to be highly heat stable and relatively resistant to proteases. Several studies have shown that tropomyosin is the prime cause for IgE cross-reactivity among shellfish, insects (including edible insects) and mite species. Interestingly, recent studies have indicated

that tropomyosin may not show T-cell cross-reactivity as a function of structural stability.¹⁰⁵⁷

**Arginine kinase** has been identified in several crustaceans and one mollusc species. Arginine kinase is a phosphotransferase that catalyzes the reversible transfer of the phosphoryl group from ATP to arginine, yielding ADP and N-phosphoarginine. In higher vertebrates, creatinine kinase catalyzes this reaction. Recent studies have shown that arginine kinase is susceptible to heat treatment or
<table><caption>TABLE 39 Allergenic molecules present in crustacean species and registered in the WHO/IUIS Allergen Nomenclature database</caption><thead><tr><th>Species frequently implicated</th><th>Allergenic name (IUIS)</th><th>Biochemical name</th><th>Molecular weight</th><th>Heat stability</th><th>Physiological function</th><th>Route of exposure</th></tr></thead><tbody><tr><td>1 Penaeus monodon (black tigger prawn)</td><td>Cha f 1, Cra c 1, Hom a 1, Lit v 1, Mel l 1, Met e 1,</td><td>Tropomyosin</td><td>34-38 kDa</td><td>Highly heat stable</td><td>Muscle contraction</td><td>Ingestion<br>Inhalation</td></tr><tr><td>Litopenaeus vannamei (white leg shrimp)</td><td>Pan b 1, Pen m 1, Por p 1, Scy p 1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2 Penaeus aztecus (Brown shrimp)</td><td>Cra c 2, Lit v 2, Pen m 2, Scy p 2</td><td>Arginine kinase</td><td>40-45 kDa</td><td>Stable</td><td>Energy metabolism<br>in muscles</td><td>Ingestion<br>Inhalation</td></tr><tr><td>3 Homarus americanus (American lobster)</td><td>Hom a 3, Lit v 3, Pen m 3, Cra c 5, Scy p 3</td><td>Myosin light chain</td><td>17-20 kDa</td><td>Stable</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>4 Homarus americanus (American lobster)</td><td>Cra c 4, Lit v 4, Pen m 4, Scy p 4</td><td>Sarcoplasmic calcium binding protein</td><td>20-25 kDa</td><td>Stable</td><td>Muscle contraction<br>regulation</td><td>Ingestion</td></tr><tr><td>5 Crangon crangon (Sand shrimp)</td><td>Cra c 6, Hom a 6, Pen m 6</td><td>Troponin C</td><td>20-21 kDa</td><td>Unknown</td><td>Calcium-dependent<br>activation of muscle<br>contraction</td><td>Ingestion</td></tr><tr><td>6 Charybdis feriatus (Crucifix crab)</td><td>Pen m 7</td><td>Hemocyanin</td><td>75 kDa</td><td>Stable</td><td>Copper-containing<br>Oxygen transport protein, Inhalation<br>anti-microbial property</td><td>Ingestion</td></tr><tr><td>7 Portus pelagicus (Blueswimmer crab)</td><td>Arc s 8, Cra c 8 Scy p 8</td><td>Triose-Phosphate isomerase</td><td>28 kDa</td><td>Labile</td><td>Glycolysis<br>(energy metabolism)</td><td>Ingestion<br>Inhalation</td></tr><tr><td>8 Scylla paramamosain (Mud crab)</td><td>Pen m 13</td><td>Fatty-acid binding protein</td><td>20 kDa</td><td>Unknown</td><td>Transport protein for<br>lipophilic molecules<br>(fatty acids)</td><td>Ingestion</td></tr></tbody></table>

thermal food processing. This is an important fact to consider while using *in vitro* diagnostics often based on heated shrimp or crab extract to avoid bacterial contamination and therefore include mainly heat-stable proteins; component resolved diagnostics might offer a better solution. Arginine kinase has been implicated in cross-reactivity between shellfish and edible insects.⁹³⁷

Myosin light chain (MLC), troponin C & I, and sarcoplasmic calcium-binding protein (SCP) are EF-hand domain proteins. All have been identified in several crustacean species but only SCP is identified in molluscs. Similar to tropomyosin, MLC and troponin C are involved in muscle function. SCP, similar to parvalbumin, is a calcium-binding protein regulating cytosolic calcium concentration. Hemocyanin was first identified as an allergen in the giant freshwater shrimp. **Hemocyanins** are copper-containing, oxygen transport proteins mainly found in the hemolymph of invertebrate animals. Consumption of cephalothorax is common in Asian populations and could lead to higher exposure to this allergen.¹⁰⁵⁸ The 75 kD subunits create a hexameric structure for their physiological

function. Hemocyanins-derived peptides are also known to have anti-microbial properties.¹⁰⁵⁹ Hemocyanin (Pen m 7) from Black tiger prawn was recently registered as a shrimp allergen. A recent study evidenced the presence of more than 10 different isoforms in Black tiger prawn hemolymph.¹⁰⁶⁰

Triose-phosphate isomerase is a glycolytic 28 kDa protein that, catalyzing the conversion of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. It has been identified not only in shrimps and crayfish but also in cockroaches.

Fatty acid-binding protein is a 20 kDa protein that belongs to a family of intracellular transport proteins for fatty acids, eicosanoids, retinoids, and other lipophilic molecules. This protein was recently registered as a shrimp allergen; Pen m 13. The thermal stability and presence of this protein in heated shellfish extracts are not known.

Additional IgE-binding proteins have been identified and partially characterized in crustacean and molluscs, including myosin heavy chain, pyruvate kinase, enolase, and paramyosin, but not yet registered with the WHO/IUIS.¹⁰⁶¹
<table><caption>TABLE 39 Allergenic molecules present in crustacean species and registered in the WHO/IUIS Allergen Nomenclature database</caption><thead><tr><th>Species frequently implicated</th><th>Allergenic name (IUIS)</th><th>Biochemical name</th><th>Molecular weight</th><th>Heat stability</th><th>Physiological function</th><th>Route of exposure</th></tr></thead><tbody><tr><td>1 Penaeus monodon (black tigger prawn)</td><td>Cha f 1, Cra c 1, Hom a 1, Lit v 1, Mel l 1, Met e 1,</td><td>Tropomyosin</td><td>34-38 kDa</td><td>Highly heat stable</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>Litopenaeus vannamei (white leg shrimp)</td><td>Pan b 1, Pen m 1, Por p 1, Scy p 1</td><td></td><td></td><td></td><td></td><td>Inhalation</td></tr><tr><td>2 Penaeus aztecus (Brown shrimp)</td><td>Cra c 2, Lit v 2, Pen m 2, Scy p 2</td><td>Arginine kinase</td><td>40-45 kDa</td><td>Stable</td><td>Energy metabolism in muscles</td><td>Ingestion</td></tr><tr><td>3 Homarus a 3, Lit v 3, Pen m 3, Cra c 5, Scy p 3</td><td></td><td>Myosin light chain</td><td>17-20 kDa</td><td>Stable</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>4 Homarus americanus (American lobster)</td><td>Cra c 4, Lit v 4, Pen m 4, Scy p 4</td><td>Sarcoplasmic calcium binding protein</td><td>20-25 kDa</td><td>Stable</td><td>Muscle contraction regulation</td><td>Ingestion</td></tr><tr><td>5 Crangon crangon (Sand shrimp)</td><td>Cra c 6, Hom a 6, Pen m 6</td><td>Troponin C</td><td>20-21 kDa</td><td>Unknown</td><td>Calcium-dependent activation of muscle contraction</td><td>Ingestion</td></tr><tr><td>6 Charybdis feriatus (Crucifix crab)</td><td>Pen m 7</td><td>Hemocyanin</td><td>75 kDa</td><td>Stable</td><td>Copper-containing Oxygen transport protein, Inhalation anti-microbial property</td><td>Ingestion</td></tr><tr><td>7 Portus pelagicus (Blueswimmer crab)</td><td>Arc s 8, Cra c 8 Scy p 8</td><td>Triose-Phosphate isomerase</td><td>28 kDa</td><td>Labile</td><td>Glycolysis (energy metabolism)</td><td>Ingestion</td></tr><tr><td>8 Scylla paramamosain (Mud crab)</td><td>Pen m 13</td><td>Fatty-acid binding protein</td><td>20 kDa</td><td>Unknown</td><td>Transport protein for lipophilic molecules (fatty acids)</td><td>Ingestion</td></tr></tbody></table>

thermal food processing. This is an important fact to consider while using *in vitro* diagnostics often based on heated shrimp or crab extract to avoid bacterial contamination and therefore include mainly heat-stable proteins; component resolved diagnostics might offer a better solution. Arginine kinase has been implicated in cross-reactivity between shellfish and edible insects.⁹³⁷

**Myosin light chain (MLC), troponin C & I, and sarcoplasmic calcium-binding protein (SCP)** are EF-hand domain proteins. All have been identified in several crustacean species but only SCP is identified in molluscs. Similar to tropomyosin, MLC and troponin C are involved in muscle function. SCP, similar to parvalbumin, is a calcium-binding protein regulating cytosolic calcium concentration. Hemocyanin was first identified as an allergen in the giant freshwater shrimp. **Hemocyanins** are copper-containing, oxygen transport proteins mainly found in the hemolymph of invertebrate animals. Consumption of cephalothorax is common in Asian populations and could lead to higher exposure to this allergen.¹⁰⁵⁸ The 75 kD subunits create a hexameric structure for their physiological

function. Hemocyanins-derived peptides are also known to have anti-microbial properties.¹⁰⁵⁹ Hemocyanin (Pen m 7) from Black tiger prawn was recently registered as a shrimp allergen. A recent study evidenced the presence of more than 10 different isoforms in Black tiger prawn hemolymph.¹⁰⁶⁰

**Triose-phosphate isomerase** is a glycolytic 28 kDa protein that, catalyzing the conversion of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. It has been identified not only in shrimps and crayfish but also in cockroaches.

**Fatty acid-binding protein** is a 20 kDa protein that belongs to a family of intracellular transport proteins for fatty acids, eicosanoids, retinoids, and other lipophilic molecules. This protein was recently registered as a shrimp allergen; Pen m 13. The thermal stability and presence of this protein in heated shellfish extracts are not known.

Additional IgE-binding proteins have been identified and partially characterized in crustacean and molluscs, including myosin heavy chain, pyruvate kinase, enolase, and paramyosin, but not yet registered with the WHO/IUIS.¹⁰⁶¹
<table><caption>TABLE 40 Allergenic molecules present in mollusc species and registered in the WHO/IUIS Allergen Nomenclature database</caption><thead><tr><th></th><th>Species frequently implicated</th><th>Allergenic name (IUIS)</th><th>Biochemical name</th><th>Molecular weight</th><th>Heat stability</th><th>Physiological function</th><th>Route of exposure</th></tr></thead><tbody><tr><td>1</td><td>Haliotes midae (Abalone)</td><td>Hel as 1, Hal m1, Tod p 1</td><td>Tropomyosin</td><td>34-49 kDa</td><td>Highly heat stable</td><td>Muscle contraction</td><td>Ingestion<br>Inhalation</td></tr><tr><td>2</td><td>Helix aspersa (Garden snail)</td><td>Rap v 2</td><td>Paramyosin</td><td>99 kDa</td><td>Unknown</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>3</td><td>Todarodes pacificus (Flying squid)</td><td>Unassigned</td><td>Arginine kinase</td><td>40-45 kDa</td><td>Stable</td><td>Energy metabolism<br>in muscles</td><td>Ingestion<br>Inhalation</td></tr><tr><td>4</td><td>Amphioctopus fangsi (Octopus)</td><td>Unassigned</td><td>Actin</td><td>42 kDa</td><td>Heat - Labile</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>5</td><td>Crassostrea gigas and Crassostrea angulata (Pacific oyster)</td><td>Cra a 4</td><td>Sarcoplasmic calcium<br>binding protein</td><td>20-25 kDa</td><td>Unknown</td><td>Muscle contraction<br>regulation</td><td>Ingestion</td></tr></tbody></table>

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

Previously published sensitization rates to shellfish were based predominantly on skin or IgE testing to whole shellfish extracts.335,1062 Studies have estimated the prevalence of shellfish allergy to be up to 3% in the adult population.1018,1063 In the Asia-pacific region self-reported rates of shellfish allergy in children range from 0.9%-1.19% among 7 years or below, and 5.12%-7.71% in adolescents and adults.

Allergen-specific IgE sensitization to various shellfish allergens have been demonstrated (Table 41). In general, 60% of individuals with confirmed allergy to shellfish elicit specific IgE binding to tropomyosin. More importantly, it has been demonstrated that serum-specific IgE to tropomyosin is a better predictor of shrimp allergy than shrimp SPT or IgE to whole shrimp extract.1064,1065 Tropomyosin (Pen m 1) and sarcoplasmic calcium-binding protein (Pen m 4) sensitization has been associated with clinical reactivity to shrimp allergy.15 However, the sensitization profile to specific allergens can differ geographically. For example, sensitization to tropomyosin seems to be much lower in Asian countries, and this could be due to eating habits where in addition to the shellfish muscle tissue other parts are consumed, including the cephalothorax that is rich in enzymatic proteins and hemocyanin.1067

Notably, conclusions on true sensitization rates are hampered due to the highly cross-reactive nature of some shellfish allergens. The tropomyosin allergen group among the crustaceans demonstrates very strong clinical cross-reactivity (see Figure 95 and chapter C05), likely due to the high amino acid homology, with over 95% among all currently analyzed shrimps, crabs and lobsters. For example, 75% of shrimp allergic patients elicited immunological IgE cross-reactivity to crab tropomyosin Por p 1.1068

By contrast, there is very limited information about tropomyosin among the mollusc group. This allergen from various mollusc species such as abalone, mussel, oysters, squid and cockle can have amino acid homologies as low as 70%, and even lower when compared with crustacean tropomyosin Por p 1.1068

This can result in limited clinical cross-reactivity of allergic patients as demonstrated in a study where 54% of the recruited patients anaphylactic to crustaceans were tolerant to molluscs.1069 Myosin light chain and Sarcoplasmic calcium-binding protein have a lower IgE-binding frequency; however, a higher rate of IgE sensitization is observed in children as compared to adults and may be used as diagnostic markers for shrimp allergy in children.15,1066,1677 IgE sensitization to shrimp hemocyanin has been reported to be 29% in DBPCFC-confirmed shrimp allergic patients15 and 40% in a Spanish population.1070 In a recent study, strong IgE sensitization to crab hemocyanin was observed in crab-processing workers by inhalational exposure to aerosolized matter.1071 Therefore, hemocyanin-specific IgE may be a potential diagnostic marker for occupational exposure and symptoms to shellfish allergens.

## 5 – Clinical Management

### Patterns of initial sensitization

IgE-mediated allergy is most likely the most common form of adverse reactions to shellfish; however, reliable population-based epidemiological studies are lacking. As recently reviewed by Cox et al, based upon most observational cohorts and case series, it would appear that prevalence of shellfish allergy generally follows shrimp/ prawn> lobster >crab > mollusc, although this may be confounded by first exposures and subsequent avoidance of other types of shellfish.1072 As
<table><caption>TABLE 40 Allergenic molecules present in mollusc species and registered in the WHO/IUIS Allergen Nomenclature database</caption><thead><tr><th></th><th>Species frequently implicated</th><th>Allergenic name (IUIS)</th><th>Biochemical name</th><th>Molecular weight</th><th>Heat stability</th><th>Physiological function</th><th>Route of exposure</th></tr></thead><tbody><tr><td>1</td><td>Haliotes midae (Abalone)</td><td>Hel as 1, Hal m1, Tod p 1</td><td>Tropomyosin</td><td>34-49 kDa</td><td>Highly heat stable</td><td>Muscle contraction</td><td>Ingestion<br>Inhalation</td></tr><tr><td>2</td><td>Helix aspersa (Garden snail)</td><td>Rap v 2</td><td>Paramyosin</td><td>99 kDa</td><td>Unknown</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>3</td><td>Todarodes pacificus (Flying squid)</td><td>Unassigned</td><td>Arginine kinase</td><td>40-45 kDa</td><td>Stable</td><td>Energy metabolism<br>in muscles</td><td>Ingestion<br>Inhalation</td></tr><tr><td>4</td><td>Amphioctopus fangsi (Octopus)</td><td>Unassigned</td><td>Actin</td><td>42 kDa</td><td>Heat - Labile</td><td>Muscle contraction</td><td>Ingestion</td></tr><tr><td>5</td><td>Crassostrea gigas and Crassostrea angulata (Pacific oyster)</td><td>Cra a 4</td><td>Sarcoplasmic calcium<br>binding protein</td><td>20-25 kDa</td><td>Unknown</td><td>Muscle contraction<br>regulation</td><td>Ingestion</td></tr></tbody></table>

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

Previously published sensitization rates to shellfish were based predominantly on skin or IgE testing to whole shellfish extracts.335,1062 Studies have estimated the prevalence of shellfish allergy to be up to 3% in the adult population.1018,1063 In the Asia-pacific region self-reported rates of shellfish allergy in children range from 0.9%-1.19% among 7 years or below, and 5.12%-7.71% in adolescents and adults.

Allergen-specific IgE sensitization to various shellfish allergens have been demonstrated (Table 41). In general, 60% of individuals with confirmed allergy to shellfish elicit specific IgE binding to tropomyosin. More importantly, it has been demonstrated that serum-specific IgE to tropomyosin is a better predictor of shrimp allergy than shrimp SPT or IgE to whole shrimp extract.1064,1065 Tropomyosin (Pen m 1) and sarcoplasmic calcium-binding protein (Pen m 4) sensitization has been associated with clinical reactivity to shrimp allergy.15 However, the sensitization profile to specific allergens can differ geographically. For example, sensitization to tropomyosin seems to be much lower in Asian countries, and this could be due to eating habits where in addition to the shellfish muscle tissue other parts are consumed, including the cephalothorax that is rich in enzymatic proteins and hemocyanin.1067

Notably, conclusions on true sensitization rates are hampered due to the highly cross-reactive nature of some shellfish allergens. The tropomyosin allergen group among the crustaceans demonstrates very strong clinical cross-reactivity (see Figure 95 and chapter C05), likely due to the high amino acid homology, with over 95% among all currently analyzed shrimps, crabs and lobsters. For example, 75% of shrimp allergic patients elicited immunological IgE cross-reactivity to crab tropomyosin Por p 1.1068

By contrast, there is very limited information about tropomyosin among the mollusc group. This allergen from various mollusc species such as abalone, mussel, oysters, squid and cockle can have amino acid homologies as low as 70%, and even lower when compared with crustacean tropomyosin Por p 1.1068

This can result in limited clinical cross-reactivity of allergic patients as demonstrated in a study where 54% of the recruited patients anaphylactic to crustaceans were tolerant to molluscs.1069 Myosin light chain and Sarcoplasmic calcium-binding protein have a lower IgE-binding frequency; however, a higher rate of IgE sensitization is observed in children as compared to adults and may be used as diagnostic markers for shrimp allergy in children.15,1066,1677 IgE sensitization to shrimp hemocyanin has been reported to be 29% in DBPCFC-confirmed shrimp allergic patients15 and 40% in a Spanish population.1070 In a recent study, strong IgE sensitization to crab hemocyanin was observed in crab-processing workers by inhalational exposure to aerosolized matter.1071 Therefore, hemocyanin-specific IgE may be a potential diagnostic marker for occupational exposure and symptoms to shellfish allergens.

## 5 – Clinical Management

### Patterns of initial sensitization

IgE-mediated allergy is most likely the most common form of adverse reactions to shellfish; however, reliable population-based epidemiological studies are lacking. As recently reviewed by Cox et al, based upon most observational cohorts and case series, it would appear that prevalence of shellfish allergy generally follows shrimp/ prawn> lobster >crab > mollusc, although this may be confounded by first exposures and subsequent avoidance of other types of shellfish.1072 As
<table><caption>TABLE 41 Clinical relevance and IgE sensitization to allergenic molecules in crustaceans</caption><thead><tr><th>IgE Sensitisation</th><th>Reference</th><th>Allergenicity (FC and SPT)</th><th>Risk factor</th></tr></thead><tbody><tr><td colspan="4">Tropomyosin (TM)</td></tr><tr><td>Pen a 1 51% (total 45 subjects)</td><td>[1678]</td><td>61% (24 subjects)</td><td>Ingestion</td></tr><tr><td>Lit v 1 94% (34 children)<br>61% (19 adults)</td><td>[1085]</td><td>37% (45 subjects)</td><td>Inhalation</td></tr><tr><td>Pen m 1 62% (16 subjects)</td><td>[1048]</td><td>sIgE to tropomyosin is a better<br>predictor of shrimp allergy than<br>shrimp SPT or sIgE to whole<br>shrimp</td><td>Occupational exposure</td></tr><tr><td>Cra c 1 68% (31<br>subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Arginine Kinase (AK)<br>subjects)</td></tr><tr><td>Pen m 2 50% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Lit v 2 67% (34 children)<br>21% (19 adults)</td><td>[1085]</td><td></td><td>Inhalation</td></tr><tr><td>Cra c 2 29% (31 subjects)</td><td>[1679]</td><td></td><td>Occupational exposure</td></tr><tr><td colspan="4">Myosin light chain (MLC)</td></tr><tr><td>Pen m 3 31% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Lit v 3 70% (34 children)<br>31% (19 adults)</td><td>[1085]</td><td></td><td></td></tr><tr><td>Cra c 3 19% (31 subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Sarcoplasmic calcium binding protein (SCBP)</td></tr><tr><td>Pen m 4 19% (16 subjects)</td><td>[1048]</td><td>sIgE to SCBP may be a better<br>predictor of shrimp allergy in<br>children</td><td>Ingestion</td></tr><tr><td>Lit v 4 59% (34 children)<br>31% (19 adults)</td><td>[1085]</td><td></td><td></td></tr><tr><td>Cra c 4 19% (31 subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Troponin C (TnC)</td></tr><tr><td>Cra c 6 19% (31 subjects)</td><td>[1679]</td><td>Not known</td><td>Ingestion</td></tr><tr><td colspan="4">Triose phosphate isomerase (TIM)</td></tr><tr><td>Pen m 8 19% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Cra c 8 23% (31 subjects)</td><td>[1679]</td><td></td><td>Inhalation</td></tr><tr><td colspan="4">Hemocyanin</td></tr><tr><td>29% (58 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>47% (40 subjects)</td><td>[1679]</td><td></td><td>Inhalation</td></tr></tbody></table>

previously described, initial sensitization to shellfish may arise from initial oral exposure or from occupational exposure, which may be airborne (via the respiratory tract), or cutaneous (via antigen-presenting cells resident in the dermis and epidermis) (see also Table 42). In addition, it is postulated that as cockroaches and house dust mites also have allergenic tropomyosin, sensitization and allergy to shellfish may occur via primary sensitization to an insect in some individuals.¹⁰⁷²,¹⁰⁷³

Non-IgE-mediated allergy to shellfish is also described, most commonly in the form of Food Protein-Induced Enterocolitis Syndrome

(FPIES).¹⁰⁷⁴ Shellfish is also a known trigger of IgE-mediated food-dependent exercise-induced anaphylaxis (FDEIA), whereby the combination of exercise and ingestion is required to trigger an allergic reaction. Subjects with FDEIA tolerate shellfish food in the absence of exercise. In several regions, including Japan, shellfish is the second most common trigger of FDEIA, after wheat.¹⁰⁷⁵

### Clinical manifestations

In the most common manifestations of IgE-mediated shellfish allergy, symptoms occur within minutes to 2 hours following ingestion
<table><caption>TABLE 41 Clinical relevance and IgE sensitization to allergenic molecules in crustaceans</caption><thead><tr><th>IgE Sensitisation</th><th>Reference</th><th>Allergenicity (FC and SPT)</th><th>Risk factor</th></tr></thead><tbody><tr><td colspan="4">Tropomyosin (TM)</td></tr><tr><td>Pen a 1 51% (total 45 subjects)</td><td>[1678]</td><td>61% (24 subjects)</td><td>Ingestion</td></tr><tr><td>Lit v 1 94% (34 children)<br>61% (19 adults)</td><td>[1085]</td><td>37% (45 subjects)</td><td>Inhalation</td></tr><tr><td>Pen m 1 62% (16 subjects)</td><td>[1048]</td><td>sIgE to tropomyosin is a better<br>predictor of shrimp allergy than<br>shrimp SPT or sIgE to whole<br>shrimp</td><td>Occupational exposure</td></tr><tr><td>Cra c 1 68% (31<br>subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Arginine Kinase (AK)<br>subjects)</td></tr><tr><td>Pen m 2 50% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Lit v 2 67% (34 children)<br>21% (19 adults)</td><td>[1085]</td><td></td><td>Inhalation</td></tr><tr><td>Cra c 2 29% (31 subjects)</td><td>[1679]</td><td></td><td>Occupational exposure</td></tr><tr><td colspan="4">Myosin light chain (MLC)</td></tr><tr><td>Pen m 3 31% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Lit v 3 70% (34 children)<br>31% (19 adults)</td><td>[1085]</td><td></td><td></td></tr><tr><td>Cra c 3 19% (31 subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Sarcoplasmic calcium binding protein (SCBP)</td></tr><tr><td>Pen m 4 19% (16 subjects)</td><td>[1048]</td><td>sIgE to SCBP may be a better<br>predictor of shrimp allergy in<br>children</td><td>Ingestion</td></tr><tr><td>Lit v 4 59% (34 children)<br>31% (19 adults)</td><td>[1085]</td><td></td><td></td></tr><tr><td>Cra c 4 19% (31 subjects)</td><td>[1679]</td><td></td><td></td></tr><tr><td colspan="4">Troponin C (TnC)</td></tr><tr><td>Cra c 6 19% (31 subjects)</td><td>[1679]</td><td>Not known</td><td>Ingestion</td></tr><tr><td colspan="4">Triose phosphate isomerase (TIM)</td></tr><tr><td>Pen m 8 19% (16 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>Cra c 8 23% (31 subjects)</td><td>[1679]</td><td></td><td>Inhalation</td></tr><tr><td colspan="4">Hemocyanin</td></tr><tr><td>29% (58 subjects)</td><td>[1048]</td><td>Not known</td><td>Ingestion</td></tr><tr><td>47% (40 subjects)</td><td>[1679]</td><td></td><td>Inhalation</td></tr></tbody></table>

previously described, initial sensitization to shellfish may arise from initial oral exposure or from occupational exposure, which may be airborne (via the respiratory tract), or cutaneous (via antigen-presenting cells resident in the dermis and epidermis) (see also Table 42). In addition, it is postulated that as cockroaches and house dust mites also have allergenic tropomyosin, sensitization and allergy to shellfish may occur via primary sensitization to an insect in some individuals.¹⁰⁷²,¹⁰⁷³

Non-IgE-mediated allergy to shellfish is also described, most commonly in the form of Food Protein-Induced Enterocolitis Syndrome

(FPIES).¹⁰⁷⁴ Shellfish is also a known trigger of IgE-mediated food-dependent exercise-induced anaphylaxis (FDEIA), whereby the combination of exercise and ingestion is required to trigger an allergic reaction. Subjects with FDEIA tolerate shellfish food in the absence of exercise. In several regions, including Japan, shellfish is the second most common trigger of FDEIA, after wheat.¹⁰⁷⁵

Clinical manifestations

In the most common manifestations of IgE-mediated shellfish allergy, symptoms occur within minutes to 2 hours following ingestion
<table><caption>TABLE 42 Characteristics of shellfish allergy</caption><thead><tr><th></th><th>Ingestion</th><th>Inhalation</th><th>Contact</th></tr></thead><tbody><tr><td>Routes of sensitisation (IgE specific response)</td><td>1. Gastro-intestinal uptake and subsequent IgE sensitisation to stable shellfish allergens<br>2. Gastro-intestinal uptake of shellfish allergens and subsequent IgE cross-reactivity to shellfish or fish parasite allergens</td><td>1. Inhalation of air-borne particulate shellfish matter and subsequent IgE sensitisation to allergens tropomyosin and arginine kinase<br>2. Ingestion-induced IgE sensitisation and subsequent inhalation related cross-reactivity to dust mite or insect tropomyosin</td><td>1. Primary uptake and IgE sensitisation to shellfish allergens through IgE receptors on epidermal Langerhans' (dendritic) cells<br>2. IgE sensitisation to shellfish proteins via gastro-intestinal or inhalational route and subsequent IgE reactivity on skin contact</td></tr><tr><td>Affected age group</td><td>children/adolescents/ adults</td><td>adolescents / seafood processing workers/restaurant workers</td><td>children/ adolescents/ adults/ seafood processing workers/ restaurant workers</td></tr><tr><td>Shellfish allergens involved</td><td>TM (Pen m 1), AK (Pen m 2), MLC (Pen m 3), SCP (Pen m 4), TnC (Pen m 6), TIM (Pen m 8), HC (Pen m 7), FABP (Pen m 13)</td><td>Tropomyosin, Arginine kinase</td><td>Not known</td></tr><tr><td>Allergen abundance</td><td>High in fresh and cooked meat and related products</td><td>Moderate to high in aerosol allergen content of Pen m1 and Pen m2 near cooking stations</td><td>Not known</td></tr><tr><td>Thermal stability</td><td>Very high stability</td><td>High</td><td>High</td></tr><tr><td>Prevalence</td><td>2% adults<br>0.9% children</td><td>4-36% among shellfish process workers</td><td>65% among shrimp workers (irritant or allergic origin)</td></tr><tr><td>Elicitors</td><td>Fresh or cooked shellfish meat.<br>Processed foods containing shellfish</td><td>Air-borne shellfish bio-matter and cooking vapours</td><td>Wet aerosols, splash on hands and face</td></tr><tr><td>Symptoms</td><td>Generalized reactions (anaphylaxis), cutaneous (urticaria, angioedema, atopic dermatitis), gastrointestinal (pain, nausea, diarrhoea, vomiting), oral allergy syndrome</td><td>Upper and lower respiratory tract symptoms: asthma. Ocular-nasal symptoms: rhinitis, conjunctivitis</td><td>Contact dermatitis, urticaria, eczema</td></tr><tr><td>Medical diagnosis</td><td>Mild, Moderate to severe food allergic reaction to shellfish proteins</td><td>Mild, Moderate to severe respiratory symptoms which may lead to ingestion induced food allergy to shellfish proteins</td><td>Contact dermatitis and urticaria due to primary sensitisation to shellfish proteins</td></tr><tr><td>Type of food allergy</td><td>Type I – immediate onset</td><td>Type I – immediate onset</td><td>Type IV – delayed onset</td></tr><tr><td>Avoidance/Technical solution</td><td>No oral intake of crustacean or mollusc products, even small amounts</td><td>Mild, Moderate to severe respiratory symptoms which may lead to ingestion induced food allergy to shellfish proteins</td><td>Use of hand gloves and facemask for protection</td></tr><tr><td>Product declaration</td><td>Mandatory on each shellfish containing protein. Separate for crustaceans and molluscs (EU legislation)</td><td></td><td></td></tr></tbody></table>
<table><caption>TABLE 42 Characteristics of shellfish allergy</caption><thead><tr><th></th><th>Ingestion</th><th>Inhalation</th><th>Contact</th></tr></thead><tbody><tr><th>Routes of sensitisation (IgE specific response)</th><td>1. Gastro-intestinal uptake and subsequent IgE sensitisation to stable shellfish allergens<br>2. Gastro-intestinal uptake of shellfish allergens and subsequent IgE cross-reactivity to shellfish or fish parasite allergens</td><td>1. Inhalation of air-borne particulate shellfish matter and subsequent IgE sensitisation to allergens tropomyosin and arginine kinase<br>2. Ingestion-induced IgE sensitisation and subsequent inhalation related cross-reactivity to dust mite or insect tropomyosin</td><td>1. Primary uptake and IgE sensitisation to shellfish allergens through IgE receptors on epidermal Langerhans' (dendritic) cells<br>2. IgE sensitisation to shellfish proteins via gastro-intestinal or inhalational route and subsequent IgE reactivity on skin contact</td></tr><tr><th>Affected age group</th><td>children/adolescents/ adults</td><td>adolescents / seafood processing workers/restaurant workers</td><td>children/ adolescents/ adults/ seafood processing workers/ restaurant workers</td></tr><tr><th>Shellfish allergens involved</th><td>TM (Pen m 1), AK (Pen m 2), MLC (Pen m 3), SCP (Pen m 4), TnC (Pen m 6), TIM (Pen m 8), HC (Pen m 7), FABP (Pen m 13)</td><td>Tropomyosin, Arginine kinase</td><td>Not known</td></tr><tr><th>Allergen abundance</th><td>High in fresh and cooked meat and related products</td><td>Moderate to high in aerosol allergen content of Pen m1 and Pen m2 near cooking stations</td><td>Not known</td></tr><tr><th>Thermal stability</th><td>Very high stability</td><td>High</td><td>High</td></tr><tr><th>Prevalence</th><td>2% adults<br>0.9% children</td><td>4-36% among shellfish process workers</td><td>65% among shrimp workers (irritant or allergic origin)</td></tr><tr><th>Elicitors</th><td>Fresh or cooked shellfish meat.<br>Processed foods containing shellfish</td><td>Air-borne shellfish bio-matter and cooking vapours</td><td>Wet aerosols, splash on hands and face</td></tr><tr><th>Symptoms</th><td>Generalized reactions (anaphylaxis), cutaneous (urticaria, angioedema, atopic dermatitis), gastrointestinal (pain, nausea, diarrhoea, vomiting), oral allergy syndrome</td><td>Upper and lower respiratory tract symptoms: asthma. Ocular-nasal symptoms: rhinitis, conjunctivitis</td><td>Contact dermatitis, urticaria, eczema</td></tr><tr><th>Medical diagnosis</th><td>Mild, Moderate to severe food allergic reaction to shellfish proteins</td><td>Mild, Moderate to severe respiratory symptoms which may lead to ingestion induced food allergy to shellfish proteins</td><td>Contact dermatitis and urticaria due to primary sensitisation to shellfish proteins</td></tr><tr><th>Type of food allergy</th><td>Type I – immediate onset</td><td>Type I – immediate onset</td><td>Type IV – delayed onset</td></tr><tr><th>Avoidance/Technical solution</th><td>No oral intake of crustacean or mollusc products, even small amounts</td><td>Mild, Moderate to severe respiratory symptoms which may lead to ingestion induced food allergy to shellfish proteins</td><td>Use of hand gloves and facemask for protection</td></tr><tr><th>Product declaration</th><td>Mandatory on each shellfish containing protein. Separate for crustaceans and molluscs (EU legislation)</td><td></td><td></td></tr></tbody></table>
and are well known to clinicians. Allergic individuals who are primar-
ily sensitized via oral exposure are at risk of respiratory symptoms
through inhalation of cooking fumes. Likewise, individuals who have
been sensitized in an occupational setting are at risk of both symp-
toms on further respiratory exposure, and by ingestion. Sensitizing
allergens in occupational settings comprise mostly heat-stable aller-
gens (e.g., tropomyosin and arginine kinase). In this setting, cross-
reactivity among members of the crustacean group is more common
than among molluscs.

IgE sensitization can also evolve from exposure to different
crustacean or mollusc species resulting in various degrees of cross-
reactivity, prompting severe clinical symptoms after ingestion of
unrelated shellfish products. These reactions have mainly been
described in adults but also reported in children. Skin exposure to
heated and unprocessed shellfish induce IgE-mediated sensitization
to shellfish allergens with subsequent inhalant and ingestion allergies
in exposed individuals.<sup>1054</sup> In food-dependent exercise-induced ana-
phylaxis (FDEIA) triggered by shellfish, several recent reports have
attempted to identify the culprit allergens at a molecular level. 70-
kDa and 43-kDa Tris-soluble proteins identified as P75 homologue
and fructose 1,6-bisphosphate aldolase (FBPA) were speculated to
be the sensitizing molecules.<sup>1680</sup> In FPIES, the clinical presentation is
distinct from that of typical IgE-mediated symptoms and comprises
of onset of profuse vomiting, typically between 1-3 hours after inges-
tion. Other typical features include diarrhoea, pallor, floppiness (in
infants and young children), hypothermia and hypotension. Although
typically described in young infants, shellfish and fish FPIES appears
to be the most common form of FPIES in adults.<sup>1076</sup> The specific al-
lergens responsible for shellfish FPIES are yet to be identified.

Clinical Cross-Reactivity

Clinical cross-reactivity among crustaceans, between crusta-
ceans and molluscs, as well as between crustaceans and molluscs
and mites or cockroaches, is largely considered due to the high ho-
mology of several allergens, as discussed above. Based upon several
case series, it was reported that approximately 45% of individuals
with a crustacean allergy are also mollusc allergic, and between
70-80% of individuals who report mollusc, allergy has also expe-
rienced allergic reactions to crustaceans.<sup>1062,1077</sup> Individuals who
appear allergic to only particular species of shrimp have also been
reported, and exactly what determines an individual's likelihood of
clinical cross-reactivity has not been established.<sup>1078,1079</sup> To that
point, more focus has been on the allergen tropomyosin, where sev-
eral linear and conformational epitopes have been reported, which
might help to define these patterns of cross-reactivity.<sup>1080</sup> Using a
large directory of 96 shrimp tropomyosin IgE-binding epitopes, it
was demonstrated that over 50% of the epitopes were conserved
between shrimp, cockroach and mite tropomyosins.<sup>1081</sup> By contrast,
less than 20% were conserved across different molluscs, supporting
the observation that less than 50% of individuals with crustacean
allergy cross-react to a mollusc. Based on this epitope analysis a
decision tree to diagnose molecular cross-reactivity to crustacean
and mollusc tropomyosins has been developed.<sup>1082</sup> Although incom-
pletely defined, recognition of epitopes and allergens appears to

differ between shellfish allergic children and adults.<sup>1085</sup> True rates
of clinical cross-reactivity between crustaceans and molluscs are
unknown, and to date, there are no specific biomarkers or molecular
diagnostics, which can reliably identify such individuals. It is postu-
lated that developments in epitope mapping may assist in this regard.

Natural History

The natural history of IgE-mediated allergy to shellfish is not well
described. Attainment of tolerance once allergic is not common, and
unlike most other food allergies, although well described in child-
hood, onset in adulthood appear more frequent than with other
foods.<sup>1063,1077,1084</sup> Likely due to high homology, most individuals al-
lergic to one type of crustacean (shrimp, lobster, crab) will also react
to other crustacea. Regarding FPIES to shellfish in adults, limited
data suggest that adult FPIES is characterized by a significant delay
in diagnosis and a prolonged course. Likewise, children appear less
likely to attain tolerance to shellfish.<sup>1063,1085</sup>

Diagnosis

Specific targeted clinical questions related to the history of ex-
posure, interpretation of sensitization tests (e.g., SPT, IgE) and food
challenges (open or blinded) help to establish the diagnosis of shell-
fish allergy. Following workup (Figure 96) may facilitate a correct di-
agnosis. It is also important to consider that the symptoms elicited
upon shellfish exposure may have been not directly related to the
shellfish, such as *anisakis* simplex allergy, or in the case particularly
of mussels and oysters, paralytic shellfish /diarrhoetic shellfish po-
soning, which are caused by shellfish contaminated with algae pro-
ducing toxins. Here, symptoms typically occur within 2 to 3 hours of
ingestion and include tingling of the lips, tongue and throat, nausea,
headache and fever, and are clinically quite distinct for a typical IgE-
mediated allergic reaction.

In terms of seeking a history to establish or assist in confirming
a diagnosis of shellfish allergy, the following framework can assist in
gathering the essential information required to assist with diagno-
sis, which includes history, route of exposure, any relevant cofac-
tors and/or relevant cross-sensitization (such as house dust mite/
insects).

Skin prick test (SPT)

Commercial whole shellfish extracts are available and provide
reasonable results in situations where the allergen responsible is a
highly abundant allergen such as tropomyosin but are limited value
due to false-negative reactions in case of heat-sensitive allergens
(e.g., MLC; hemocyanin) or weak or non-cross-reactive allergens
(e.g., arginine kinase). Lack of availability of commercial extracts for
specific shellfish species consumed by the patient (e.g., Asia-pacific
and Southern hemisphere).

Prick-to-prick tests using fresh shellfish (with suspected offending food) are commonly performed; however, there are no established positive and negative predictive values for determining the likelihood of clinical allergy based upon the results. In addition, fresh shellfish SPT should be performed with caution and in a setting experienced in the recognition and management of anaphylaxis, as the test itself is reported to be able to trigger anaphylaxis in sensitive individuals.
and are well known to clinicians. Allergic individuals who are primar-
ily sensitized via oral exposure are at risk of respiratory symptoms
through inhalation of cooking fumes. Likewise, individuals who have
been sensitized in an occupational setting are at risk of both symp-
toms on further respiratory exposure, and by ingestion. Sensitizing
allergens in occupational settings comprise mostly heat-stable aller-
gens (e.g., tropomyosin and arginine kinase). In this setting, cross-
reactivity among members of the crustacean group is more common
than among molluscs.

IgE sensitization can also evolve from exposure to different
crustacean or mollusc species resulting in various degrees of cross-
reactivity, prompting severe clinical symptoms after ingestion of
unrelated shellfish products. These reactions have mainly been
described in adults but also reported in children. Skin exposure to
heated and unprocessed shellfish induce IgE-mediated sensitization
to shellfish allergens with subsequent inhalant and ingestion allergies
in exposed individuals.<sup>1054</sup> In food-dependent exercise-induced ana-
phylaxis (FDEIA) triggered by shellfish, several recent reports have
attempted to identify the culprit allergens at a molecular level. 70-
kDa and 43-kDa Tris-soluble proteins identified as P75 homologue
and fructose 1,6-bisphosphate aldolase (FBPA) were speculated to
be the sensitizing molecules.<sup>1680</sup> In FPIES, the clinical presentation is
distinct from that of typical IgE-mediated symptoms and comprises
of onset of profuse vomiting, typically between 1-3 hours after inges-
tion. Other typical features include diarrhoea, pallor, floppiness (in
infants and young children), hypothermia and hypotension. Although
typically described in young infants, shellfish and fish FPIES appears
to be the most common form of FPIES in adults.<sup>1076</sup> The specific al-
lergens responsible for shellfish FPIES are yet to be identified.

Clinical Cross-Reactivity

Clinical cross-reactivity among crustaceans, between crusta-
ceans and molluscs, as well as between crustaceans and molluscs
and mites or cockroaches, is largely considered due to the high ho-
mology of several allergens, as discussed above. Based upon several
case series, it was reported that approximately 45% of individuals
with a crustacean allergy are also mollusc allergic, and between
70-80% of individuals who report mollusc, allergy has also expe-
rienced allergic reactions to crustaceans.<sup>1062,1077</sup> Individuals who
appear allergic to only particular species of shrimp have also been
reported, and exactly what determines an individual's likelihood of
clinical cross-reactivity has not been established.<sup>1078,1079</sup> To that
point, more focus has been on the allergen tropomyosin, where sev-
eral linear and conformational epitopes have been reported, which
might help to define these patterns of cross-reactivity.<sup>1080</sup> Using a
large directory of 96 shrimp tropomyosin IgE-binding epitopes, it
was demonstrated that over 50% of the epitopes were conserved
between shrimp, cockroach and mite tropomyosins.<sup>1081</sup> By contrast,
less than 20% were conserved across different molluscs, supporting
the observation that less than 50% of individuals with crustacean
allergy cross-react to a mollusc. Based on this epitope analysis a
decision tree to diagnose molecular cross-reactivity to crustacean
and mollusc tropomyosins has been developed.<sup>1082</sup> Although incom-
pletely defined, recognition of epitopes and allergens appears to

differ between shellfish allergic children and adults.<sup>1085</sup> True rates
of clinical cross-reactivity between crustaceans and molluscs are
unknown, and to date, there are no specific biomarkers or molecular
diagnostics, which can reliably identify such individuals. It is postu-
lated that developments in epitope mapping may assist in this regard.

Natural History

The natural history of IgE-mediated allergy to shellfish is not well
described. Attainment of tolerance once allergic is not common, and
unlike most other food allergies, although well described in child-
hood, onset in adulthood appear more frequent than with other
foods.<sup>1063,1077,1084</sup> Likely due to high homology, most individuals al-
lergic to one type of crustacean (shrimp, lobster, crab) will also react
to other crustacea. Regarding FPIES to shellfish in adults, limited
data suggest that adult FPIES is characterized by a significant delay
in diagnosis and a prolonged course. Likewise, children appear less
likely to attain tolerance to shellfish.<sup>1063,1085</sup>

Diagnosis

Specific targeted clinical questions related to the history of ex-
posure, interpretation of sensitization tests (e.g., SPT, IgE) and food
challenges (open or blinded) help to establish the diagnosis of shell-
fish allergy. Following workup (Figure 96) may facilitate a correct di-
agnosis. It is also important to consider that the symptoms elicited
upon shellfish exposure may have been not directly related to the
shellfish, such as *anisakis* simplex allergy, or in the case particularly
of mussels and oysters, paralytic shellfish /diarrhoetic shellfish po-
soning, which are caused by shellfish contaminated with algae pro-
ducing toxins. Here, symptoms typically occur within 2 to 3 hours of
ingestion and include tingling of the lips, tongue and throat, nausea,
headache and fever, and are clinically quite distinct for a typical IgE-
mediated allergic reaction.

In terms of seeking a history to establish or assist in confirming
a diagnosis of shellfish allergy, the following framework can assist in
gathering the essential information required to assist with diagno-
sis, which includes history, route of exposure, any relevant cofac-
tors and/or relevant cross-sensitization (such as house dust mite/
insects).

Skin prick test (SPT)

Commercial whole shellfish extracts are available and provide
reasonable results in situations where the allergen responsible is a
highly abundant allergen such as tropomyosin but are limited value
due to false-negative reactions in case of heat-sensitive allergens
(e.g., MLC; hemocyanin) or weak or non-cross-reactive allergens
(e.g., arginine kinase). Lack of availability of commercial extracts for
specific shellfish species consumed by the patient (e.g., Asia-pacific
and Southern hemisphere).

Prick-to-prick tests using fresh shellfish (with suspected offending food) are commonly performed; however, there are no established positive and negative predictive values for determining the likelihood of clinical allergy based upon the results. In addition, fresh shellfish SPT should be performed with caution and in a setting experienced in the recognition and management of anaphylaxis, as the test itself is reported to be able to trigger anaphylaxis in sensitive individuals.
<figure><img src="image_200.png" /><figcaption>FIGURE 96 Diagnostic algorithm for shellfish allergy</figcaption></figure>

* More common in fish/mollusk than crustaceans

**Consider exercise food challenge if history is suggestive of exercise related reaction and tolerance in other settings

*** Cooking at temperatures above 60°C or storage in industrial freezers for 2 days is required to kill the parasite.

Few studies have attempted to define a 95% PPV for SPT to shellfish in predicting clinical allergy based upon food challenges. A mean wheal diameter >20 mm using commercial shrimp extracts and prick-to-prick was reported to provide 95% PPV in a cohort from Thailand; however, many shrimp allergic subjects do not have SPT this large, potentially limiting the value of a PPV this high.<sup>1079</sup>

### Allergen-specific IgE

Whole Shellfish extracts are commonly used, however do not have high sensitivity or specificity, and the positive predictive value of such tests appear to vary depending upon the region. Thalayasingam et al. reported shrimp-specific IgE by ImmunoCAP ISAC had relatively poor overall test performance, with a sensitivity of 62% and a specificity of 50% for detecting shrimp allergy in a Singapore based cohort of food challenge confirmed shrimp, allergic individuals.<sup>1086</sup> In addition, with the use of whole allergen extracts, there is the potential for false-negative or low titers in individuals

sensitized to low abundant allergens (such as triosephosphate isomerase). Moreover, in some situations, allergen extract does not represent the specific shellfish species consumed by the patient and may result in false-negative results (e.g., Southern versus Northern hemisphere).

Relevant major allergens are detailed in Table 43; however, they have limited commercial availability for routine diagnostics. Some purified allergens from shrimps, house dust-mite and *anisakis* are available on the allergen microchip (ImmunoCAP ISAC (Thermo Fisher Scientific); Alex (Macro Array Diagnostics) and used to quantify allergen-specific IgE. The reported sensitivity of tropomyosin varies significantly in the literature, with some reports as high as 71-88%. In a small selected cohort, tropomyosin specific IgE was found to be more specific for the diagnosis of clinical shrimp allergy than IgE to whole extract or SPT (with similar sensitivity across all three methods).<sup>1065</sup> These results contrast with that of Thalayasingam
<figure><img src="image_200.png" /><figcaption>FIGURE 96 Diagnostic algorithm for shellfish allergy</figcaption></figure>

* More common in fish/mollusk than crustaceans

**Consider exercise food challenge if history is suggestive of exercise related reaction and tolerance in other settings

*** Cooking at temperatures above 60°C or storage in industrial freezers for 2 days is required to kill the parasite.

Few studies have attempted to define a 95% PPV for SPT to shellfish in predicting clinical allergy based upon food challenges. A mean wheal diameter >20 mm using commercial shrimp extracts and prick-to-prick was reported to provide 95% PPV in a cohort from Thailand; however, many shrimp allergic subjects do not have SPT this large, potentially limiting the value of a PPV this high.<sup>1079</sup>

### Allergen-specific IgE

Whole Shellfish extracts are commonly used, however do not have high sensitivity or specificity, and the positive predictive value of such tests appear to vary depending upon the region. Thalayasingam et al. reported shrimp-specific IgE by ImmunoCAP ISAC had relatively poor overall test performance, with a sensitivity of 62% and a specificity of 50% for detecting shrimp allergy in a Singapore based cohort of food challenge confirmed shrimp, allergic individuals.<sup>1086</sup> In addition, with the use of whole allergen extracts, there is the potential for false-negative or low titers in individuals

sensitized to low abundant allergens (such as triosephosphate isomerase). Moreover, in some situations, allergen extract does not represent the specific shellfish species consumed by the patient and may result in false-negative results (e.g., Southern versus Northern hemisphere).

Relevant major allergens are detailed in Table 43; however, they have limited commercial availability for routine diagnostics. Some purified allergens from shrimps, house dust-mite and *anisakis* are available on the allergen microchip (ImmunoCAP ISAC (Thermo Fisher Scientific); Alex (Macro Array Diagnostics) and used to quantify allergen-specific IgE. The reported sensitivity of tropomyosin varies significantly in the literature, with some reports as high as 71-88%. In a small selected cohort, tropomyosin specific IgE was found to be more specific for the diagnosis of clinical shrimp allergy than IgE to whole extract or SPT (with similar sensitivity across all three methods).<sup>1065</sup> These results contrast with that of Thalayasingam
<table><caption>TABLE 43 Currently available *in vitro* and skin prick tests for diagnosis of crustacean or mollusc allergy</caption><thead><tr><th>Sub-group</th><th>Whole extract</th><th>Component allergens</th><th>SPT (test code)</th></tr></thead><tbody><tr><td>Crustaceans</td><td></td><td></td><td></td></tr><tr><td>Prawns, Shrimp</td><td>Shrimp-<br>Black tiger prawn (Penaeus monodon),<br>Northern shrimp (Pandalus borealis),<br>Velvet prawn (Metapenaeopsis barbata),<br>Shiba shrimp (Metapenaeus joyneri)<br>(ImmunoCAP, f24),<br>Northern prawn (Pandalus borealis)<br>(MADx, f515),<br>Shrimp mix (Litopaenaeus setiferus,<br>Farfantepenaeus aztecus,<br>Farfantepenaeus dourarum) (MADx, f24)</td><td>Pen a 1 (Penaeus aztecus)<br>(ImmunoCAP, f351),<br>nPen m 1 (Tropomyosin) (ImmunoCAP ISAC)<br>nPen m 2 (Arginine kinase) (ImmunoCAP ISAC),<br>nPen m 4 (Sarcoplasmic calcium-binding protein)<br>(ISAC),<br>nPen m 1 (Tropomyosin) (MADX, f517),<br>rPen m 2 (Arginine kinase) (MADX, f545),<br>rPen m 3 (Myosin light chain) (MADX, f552),<br>rPen m 4 (Sarcoplasmic calcium-binding protein)<br>(MADX, f524),<br>rCra c 6 (Troponin C) (MADX, f529)</td><td>Shrimp (6.89) ALK-Abello,<br>Soluprick<br>(Pandalus borealis)<br>Shrimp (SHRI) ALK-Abello<br>(Penaeus spp.)<br>Shrimp (120) Stallergenes,<br>Shellfish, mix (MISH) (crab, shrimp,<br>lobster, oyster) ALK-Abello<br>Shellfish Mix 4 (SHM4) (crab, clam,<br>lobster, shrimp)</td></tr><tr><td>Crabs</td><td>Crab Edible crab (Cancer pagurus)<br>(ImmunoCAP, f23), Crab (Chionocetes<br>spp.) (MADx, f23)</td><td></td><td>Crab (6.9) ALK-Abello, Soluprick<br>(Cancer pagurus)<br>Crab (CRAB) ALK-Abello<br>(Paralithodescamtschaticus)</td></tr><tr><td>Lobsters</td><td>European Lobster<br>(Homarus gammarus) (ImmunoCAP, f80),<br>(MADx, f80),<br>Spiny Lobster<br>(Panulirus vulgaris) (ImmunoCAP, f304),<br>Crayfish<br>(Astacus astacus) (ImmunoCAP, f320)</td><td></td><td>Lobster (LOBS) ALK-Abello<br>(Panulirus spp.)<br>Spiny lobster (131) Stallergenes</td></tr><tr><td>Bivalves</td><td>Blue mussel (Mytilus edulis)<br>(ImmunoCAP, f37) (MADx, f37),<br>Oyster (Ostrea edulis) (ImmunoCAP,<br>f290), (MADx, f290),<br>Clam (Ruditapes spp.) (ImmunoCAP,<br>f207), (MADx, f207),<br>Scallop (Pecten spp.) (ImmunoCAP,<br>f338), (MADx, f338)</td><td></td><td>Oyster (OYST), ALK-Abello<br>Mussel (139), Stallergenes<br>Oyster (131), Stallergenes</td></tr><tr><td>Gastropods</td><td>Abalone (Haliotis spp.) (ImmunoCAP, f346),<br>Snail (Helix aspersa) (ImmunoCAP, f314)</td><td></td><td></td></tr><tr><td>Cephalopods</td><td>Squid (Loligo vulgaris, Loligo edulis)<br>(ImmunoCAP, f258) (MADx, f258),<br>Pacific squid (Todarodes pacificus)<br>(ImmunoCAP, f58),<br>Octopus (Octopus vulgaris)<br>(ImmunoCAP, f59),</td><td></td><td></td></tr></tbody></table>

et al, who reported a low sensitivity for tropomyosin-IgE for the challenge has proven shrimp allergy (by ImmunoCAP ISAC), at 34%.¹⁰⁸⁶ Specificity, however, was superior at 85.2%.

Overall, it has been reported that the reliability of specific IgE to the whole extract is similar to that of the SPT, whereby the identification of sensitization does not correlate with clinical reactivity
<table><caption>TABLE 43 Currently available *in vitro* and skin prick tests for diagnosis of crustacean or mollusc allergy</caption><thead><tr><th>Sub-group</th><th>Whole extract</th><th>Component allergens</th><th>SPT (test code)</th></tr></thead><tbody><tr><td colspan="4">Crustaceans</td></tr><tr><td rowspan="10">Prawns, Shrimp</td><td>Shrimp-<br>Black tiger prawn (Penaeus monodon),<br>Northern shrimp (Pandalus borealis),<br>Velvet prawn (Metapenaeopsis barbata),<br>Shiba shrimp (Metapenaeus joyneri)<br>(ImmunoCAP, f24),<br>Northern prawn (Pandalus borealis)<br>(MADx, f515),<br>Shrimp mix (Litopaenaeus setiferus,<br>Farfantepenaeus aztecus,<br>Farfantepenaeus dourarum) (MADx, f24)</td><td>Pen a 1 (Penaeus aztecus)<br>(ImmunoCAP, f351),<br>nPen m 1 (Tropomyosin) (ImmunoCAP ISAC)<br>nPen m 2 (Arginine kinase) (ImmunoCAP ISAC),<br>nPen m 4 (Sarcoplasmic calcium-binding protein)<br>(ISAC),<br>nPen m 1 (Tropomyosin) (MADX, f517),<br>rPen m 2 (Arginine kinase) (MADX, f545),<br>rPen m 3 (Myosin light chain) (MADX, f552),<br>rPen m 4 (Sarcoplasmic calcium-binding protein)<br>(MADX, f524),<br>rCra c 6 (Troponin C) (MADX, f529)</td><td>Shrimp (6.89) ALK-Abello,<br>Soluprick<br>(Pandalus borealis)<br>Shrimp (SHRI) ALK-Abello<br>(Penaeus spp.)<br>Shrimp (120) Stallergenes,<br>Shellfish, mix (MISH) (crab, shrimp,<br>lobster, oyster) ALK-Abello<br>Shellfish Mix 4 (SHM4) (crab, clam,<br>lobster, shrimp)</td></tr><tr><td>Crabs</td><td>Crab Edible crab (Cancer pagurus)<br>(ImmunoCAP, f23), Crab (Chionocetes<br>spp.) (MADx, f23)</td><td>Crab (6.9) ALK-Abello, Soluprick<br>(Cancer pagurus)<br>Crab (CRAB) ALK-Abello<br>(Paralithodescamtschaticus)</td></tr><tr><td>Lobsters</td><td>European Lobster<br>(Homarus gammarus) (ImmunoCAP, f80),<br>(MADx, f80),<br>Spiny Lobster<br>(Panulirus vulgaris) (ImmunoCAP, f304),<br>Crayfish<br>(Astacus astacus) (ImmunoCAP, f320)</td><td>Lobster (LOBS) ALK-Abello<br>(Panulirus spp.)<br>Spiny lobster (131) Stallergenes</td></tr><tr><td rowspan="4">Bivalves</td><td>Blue mussel (Mytilus edulis)<br>(ImmunoCAP, f37) (MADx, f37),<br>Oyster (Ostrea edulis) (ImmunoCAP,<br>f290), (MADx, f290),<br>Clam (Ruditapes spp.) (ImmunoCAP,<br>f207), (MADx, f207),<br>Scallop (Pecten spp.) (ImmunoCAP,<br>f338), (MADx, f338)</td><td>Oyster (OYST), ALK-Abello<br>Mussel (139), Stallergenes<br>Oyster (131), Stallergenes</td></tr><tr><td>Gastropods</td><td>Abalone (Haliotis spp.) (ImmunoCAP, f346),<br>Snail (Helix aspersa) (ImmunoCAP, f314)</td><td></td></tr><tr><td>Cephalopods</td><td>Squid (Loligo vulgaris, Loligo edulis)<br>(ImmunoCAP, f258) (MADx, f258),<br>Pacific squid (Todarodes pacificus)<br>(ImmunoCAP, f58),<br>Octopus (Octopus vulgaris)<br>(ImmunoCAP, f59),</td><td></td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

et al, who reported a low sensitivity for tropomyosin-IgE for the challenge has proven shrimp allergy (by ImmunoCAP ISAC), at 34%.¹⁰⁸⁶ Specificity, however, was superior at 85.2%.

Overall, it has been reported that the reliability of specific IgE to the whole extract is similar to that of the SPT, whereby the identification of sensitization does not correlate with clinical reactivity
nor with symptom severity.¹⁰⁷⁷ Reactivity to tropomyosin may be more specific but appears variable in sensitivity across reports and geographical regions. Furthermore, there are no purified allergens available from molluscs for diagnosis.

For further interpretation of SPT and IgE outcomes see also (Figure 96 and Table 43).

## Food Challenge

Double-blind placebo-controlled food challenges are the gold standard for confirming a diagnosis of shellfish allergy. In clinical practice, they are, however, usually performed in an open, rather than blinded manner and usually in the circumstances where there is some doubt over the relationship between shellfish ingestion and allergic reaction, or where the history appears consistent, but the SPT/sIgE are low/absent. They may also be performed (much like a fish allergy) in cases where a shellfish allergic individual is interested in consuming a potentially cross-reactive mollusc. There are a variety of proposed schedules for shellfish challenges, usually commencing at low mg quantities of food, and then proceeding at 15-20 minutely intervals with sequentially larger (often semi-logarithmic) increases in quantities until an objective allergic reaction is observed. A variety of full cumulative doses are recommended in published guidelines, and because of the ED distribution of shrimp, these tend to be significantly higher than those for cow's milk, egg or peanut (ranging up to 24 g of seafood, e.g., 1-2 large shrimps).⁵⁵⁴

## Management and avoidance

Because of the high degree of likely clinically relevant cross-reactivity, it is generally recommended to avoid all shellfish containing products, even with small amounts, regardless of the grade of shellfish processing once a diagnosis of shellfish allergy has been made. Emergency action plans in case of severe reactions and personal adrenaline auto-injectors are recommended, along with avoidance of inhalation of shellfish containing protein cooking fumes/steam and vapours and touching or handling shellfish.

A common miscomprehension surrounds iodine and shellfish allergy. Individuals who are allergic to seafood are not at an increased risk of allergic reactions to iodine (e.g., topical antiseptics such as Betadine or Povidine or intravenous x-ray radio-contrast agents). Conversely, people with iodine allergy are not at increased risk of seafood allergy.

## Allergen-specific immunotherapy

At present commercially available products for allergen-specific immunotherapy of shellfish protein allergy are not available.

# 6 - Clinical cases

## Case 1 (published¹⁰⁸⁷)

**History:** 19-year-old male, developed hives, circumoral swelling and systemic symptoms—faintness, shortness of breath, vomiting, diarrhoea, and abdominal cramp after running. He was bought into the Emergency Department (EMD). He had consumed a home-cooked

meal containing shrimp before exercise. The patient also had rashes when ingesting clams. He had previously been able to eat large amounts of shrimps without any problems but could not recall if he had ever exercised directly after eating a large shrimp meal.

**SPT:** Negative to an extensive list of foods, including wheat and shrimp but positive to Der p, Der f, Blo t. sIgE-omega-5-gliadin negative Diagnosis: Food-dependent exercise-induced anaphylaxis (FDEIA) triggered by shrimp.

**Recommendation:** Given adrenaline for anaphylaxis in ED. Must avoid shellfish 2 hours before or following strenuous exercise. In cases where the diagnosis is in doubt, an exercise challenge can be performed, both without any prior feeding (i.e., fasted) and then following consumption of a shrimp containing meal. Such challenges are complex and can potentially trigger severe anaphylaxis and should be performed carefully by staff and centres familiar with the procedures and with experience in the management of severe anaphylaxis.

## Case 2 (published¹⁰⁸⁸)

**History:** 22-months old Latin-American male with a history of cow's milk allergy, reactive airway disease, and eczema and no history of allergic rhinitis symptoms. On ingestion of one shrimp, there was immediate swelling of eyes and hives on his face without difficulty in breathing. Symptoms subsided within 24 hours on the administration of diphenhydramine.

**In-vitro testing:** Serum-specific IgE showed levels of 12.6 kU/L to shrimp, 12.3 kU/L to milk, 0.55 kU/L to egg white, 0.72 kU/L to wheat, 0.41 kU/L to soybean, 0.61 kU/L to peanut, and <0.35 kU/L to codfish.

**Recommendations:** Strict avoidance of shrimp and other shellfish in his diet. Autoinjector epinephrine was prescribed. No further episodes of angioedema or hives were observed.

# 7 - Research and future perspectives

The diagnosis and management of shellfish allergy are complicated by several homologous, cross-reactive proteins, including tropomyosin and arginine kinase. This results in patients having allergic sensitization (positive test) to several crustacean and mollusc species, however, often without demonstrating clinical reactivity, hampering the identification of true sensitization rates to specific species. Future studies need to establish sensitization rates to different shellfish allergens in different geographic environments, as this will directly impact of sensitivity and specificity of different *in vivo* and *in vitro* tests. The impact of environmental exposure should be taken into account, as high exposure and sensitization rates to cockroaches and HDM could increase allergen-specific IgE reactivity. Further investigation into the cross-sensitization to pan-allergens such as tropomyosin from inhaled HDM and cockroach will provide greater insight into the pathogenesis of the mite-shellfish oral allergy syndrome. Further clinical research is needed to analyze the relationship between sIgE specific sensitization profiles to tropomyosin from different shellfish species and other
nor with symptom severity.¹⁰⁷⁷ Reactivity to tropomyosin may be more specific but appears variable in sensitivity across reports and geographical regions. Furthermore, there are no purified allergens available from molluscs for diagnosis.

For further interpretation of SPT and IgE outcomes see also (Figure 96 and Table 43).

## Food Challenge

Double-blind placebo-controlled food challenges are the gold standard for confirming a diagnosis of shellfish allergy. In clinical practice, they are, however, usually performed in an open, rather than blinded manner and usually in the circumstances where there is some doubt over the relationship between shellfish ingestion and allergic reaction, or where the history appears consistent, but the SPT/sIgE are low/absent. They may also be performed (much like a fish allergy) in cases where a shellfish allergic individual is interested in consuming a potentially cross-reactive mollusc. There are a variety of proposed schedules for shellfish challenges, usually commencing at low mg quantities of food, and then proceeding at 15-20 minutely intervals with sequentially larger (often semi-logarithmic) increases in quantities until an objective allergic reaction is observed. A variety of full cumulative doses are recommended in published guidelines, and because of the ED distribution of shrimp, these tend to be significantly higher than those for cow's milk, egg or peanut (ranging up to 24 g of seafood, e.g., 1-2 large shrimps).⁵⁵⁴

## Management and avoidance

Because of the high degree of likely clinically relevant cross-reactivity, it is generally recommended to avoid all shellfish containing products, even with small amounts, regardless of the grade of shellfish processing once a diagnosis of shellfish allergy has been made. Emergency action plans in case of severe reactions and personal adrenaline auto-injectors are recommended, along with avoidance of inhalation of shellfish containing protein cooking fumes/steam and vapours and touching or handling shellfish.

A common miscomprehension surrounds iodine and shellfish allergy. Individuals who are allergic to seafood are not at an increased risk of allergic reactions to iodine (e.g., topical antiseptics such as Betadine or Povidine or intravenous x-ray radio-contrast agents). Conversely, people with iodine allergy are not at increased risk of seafood allergy.

## Allergen-specific immunotherapy

At present commercially available products for allergen-specific immunotherapy of shellfish protein allergy are not available.

# 6 - Clinical cases

## Case 1 (published¹⁰⁸⁷)

**History:** 19-year-old male, developed hives, circumoral swelling and systemic symptoms—faintness, shortness of breath, vomiting, diarrhoea, and abdominal cramp after running. He was bought into the Emergency Department (EMD). He had consumed a home-cooked

meal containing shrimp before exercise. The patient also had rashes when ingesting clams. He had previously been able to eat large amounts of shrimps without any problems but could not recall if he had ever exercised directly after eating a large shrimp meal.

**SPT:** Negative to an extensive list of foods, including wheat and shrimp but positive to Der p, Der f, Blo t. sIgE-omega-5-gliadin negative Diagnosis: Food-dependent exercise-induced anaphylaxis (FDEIA) triggered by shrimp.

**Recommendation:** Given adrenaline for anaphylaxis in ED. Must avoid shellfish 2 hours before or following strenuous exercise. In cases where the diagnosis is in doubt, an exercise challenge can be performed, both without any prior feeding (i.e., fasted) and then following consumption of a shrimp containing meal. Such challenges are complex and can potentially trigger severe anaphylaxis and should be performed carefully by staff and centres familiar with the procedures and with experience in the management of severe anaphylaxis.

## Case 2 (published¹⁰⁸⁸)

**History:** 22-months old Latin-American male with a history of cow's milk allergy, reactive airway disease, and eczema and no history of allergic rhinitis symptoms. On ingestion of one shrimp, there was immediate swelling of eyes and hives on his face without difficulty in breathing. Symptoms subsided within 24 hours on the administration of diphenhydramine.

**In-vitro testing:** Serum-specific IgE showed levels of 12.6 kU/L to shrimp, 12.3 kU/L to milk, 0.55 kU/L to egg white, 0.72 kU/L to wheat, 0.41 kU/L to soybean, 0.61 kU/L to peanut, and <0.35 kU/L to codfish.

**Recommendations:** Strict avoidance of shrimp and other shellfish in his diet. Autoinjector epinephrine was prescribed. No further episodes of angioedema or hives were observed.

# 7 - Research and future perspectives

The diagnosis and management of shellfish allergy are complicated by several homologous, cross-reactive proteins, including tropomyosin and arginine kinase. This results in patients having allergic sensitization (positive test) to several crustacean and mollusc species, however, often without demonstrating clinical reactivity, hampering the identification of true sensitization rates to specific species. Future studies need to establish sensitization rates to different shellfish allergens in different geographic environments, as this will directly impact of sensitivity and specificity of different *in vivo* and *in vitro* tests. The impact of environmental exposure should be taken into account, as high exposure and sensitization rates to cockroaches and HDM could increase allergen-specific IgE reactivity. Further investigation into the cross-sensitization to pan-allergens such as tropomyosin from inhaled HDM and cockroach will provide greater insight into the pathogenesis of the mite-shellfish oral allergy syndrome. Further clinical research is needed to analyze the relationship between sIgE specific sensitization profiles to tropomyosin from different shellfish species and other
invertebrates, including HDM and insects. Advice on future dietary precautions based on molecular reactivity is currently difficult to provide to patients, without oral food challenges. There is an urgent need to identify more specific marker allergens for IgE testing to discriminate between poly- or mono-sensitized patients to the crustacean,

mollusc and other invertebrates. Advice for use of molecular diagnostics for crustacean and mollusc (1) Specific IgE to shrimp tropomyosin has a positive predictive value of 0.72 in the diagnosis of shrimp allergy. (2) Tropomyosin and sarcoplasmic calcium-binding protein sensitization are associated with clinical reactivity to shrimp.
invertebrates, including HDM and insects. Advice on future dietary precautions based on molecular reactivity is currently difficult to provide to patients, without oral food challenges. There is an urgent need to identify more specific marker allergens for IgE testing to discriminate between poly- or mono-sensitized patients to the crustacean,

mollusc and other invertebrates. Advice for use of molecular diagnostics for crustacean and mollusc (1) Specific IgE to shrimp tropomyosin has a positive predictive value of 0.72 in the diagnosis of shrimp allergy. (2) Tropomyosin and sarcoplasmic calcium-binding protein sensitization are associated with clinical reactivity to shrimp.
# B14 - Allergy to mammalian meat

Marianne van Hage, Tilo Biedermann, Christiane Hilger, Thomas A.E. Platts-Mills

## Highlights

* Farm animals provide a major part of the diet in many parts of the world

* Sensitization to meat can be acquired through different routes (inhaled, oral, skin)

* New forms of allergic reactions to meat have been recognized (cat-pork and red meat)

* IgE assays including meat allergen sources and components will help identifying the patients

* The only effective treatment is avoidance of relevant meat sources

## 1 - The allergen sources

Meat derived from domesticated mammals has been an important part of the human diet for at least ten thousand years. The animal tissue sold and eaten as meat includes blood, fat, innards, and tendons, as well as muscles. There is inevitably some cross-reactivity with serum proteins and milk proteins from the same animal. Milk is relevant only for goats, sheep, and cows in the USA and Europe, but it is important to remember that camels and other animals are milked in some parts of the world.

## 2 - Allergen families

Further, although there is a limited range of animals that are sold commercially in the west, a much larger variety of wild animals may be hunted and eaten in rural communities worldwide (Table 44). Farm animals provide a major part of the diet in western societies and in many other parts of the world. These animals are prized for their meat and organs (Figure 97). In addition, there are hundreds of forms of processed meat, including sausages, salami, bacon, etc. Meat also incorporates significant quantities of protein derived from serum, which includes many proteins that are recognized as allergens in cow's milk. Other products derived from these mammals include different forms of fat, paté, and gelatin, which is derived from tendons, cartilage, or skin.

## 3 - Allergen molecules, epidemiology and geographical variation

Initially the identification of meat derived allergens focused on protein antigens recognized by patients who reported allergic reactions that occurred rapidly after exposure. Most of these cases presented in childhood and many of the allergens were species specific proteins.⁹⁵¹,¹⁰⁸⁹ However, it was already clear that some mammalian proteins showed cross-reactivity between species, and this included both immunoglobulins and albumins.¹⁰⁸³,¹⁰⁹⁰ The most important allergens from beef, *Bos domesticus*, are serum albumin (Bos d 6), actin, myoglobulin and immunoglobulin IgG (Bos d 7) (Tables 44 and 45) and of these, serum albumin and immunoglobulin are the major allergens.⁹⁵¹,⁹⁷⁴,¹⁰⁸³,¹⁰⁹⁰ Beef-allergic children have been reported to

<table><caption>TABLE 44 Animals contributing to meat consumption and reactions</caption><thead><tr><th>Animals</th><th>Source</th><th>Milk</th><th>Allergen molecules* #</th><th>Alpha-gal Reactions</th></tr></thead><tbody><tr><td><i>Bos domesticus</i> (cow)</td><td>Farm</td><td>+++</td><td>Twelve (10)<br>Bos d 2-13</td><td>Yes</td></tr><tr><td><i>Sus domesticus</i> (pig)</td><td>Farm/feral</td><td>No</td><td>Albumin (1)<br>Sus s 1</td><td>Yes</td></tr><tr><td><i>Capra aegagrus</i> (goat)</td><td>Farm/D/Feral</td><td>++</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Ovis aries</i> (sheep)</td><td>Farm</td><td>++</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Equus caballus</i> (horse)</td><td>Farm</td><td>++</td><td>Six (2)<br>Equ c 1-6</td><td>Yes</td></tr><tr><td><i>Odocoileus virginianus</i> (deer)</td><td>Wild/Farm</td><td>No</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Oryctolagus cuniculus</i> (rabbit)</td><td>Wild/D/Farm</td><td>No</td><td>Three (0)<br>Ory c 2-4</td><td>Yes</td></tr><tr><td><i>Cavia porcellus</i> (guinea pig)</td><td>D/Farm/wild</td><td>No</td><td>Five (0)<br>Cav p 1-4,6</td><td>?</td></tr></tbody></table>

D= domesticated; *IUIS Allergen Nomenclature Sub-Committee at www.allergen.org; #Number in brackets indicates the number of allergens defined as food allergen. Alpha-gal is not included as single allergen in the table as the carbohydrate is basically present in all mammalian tissues and bound to many proteins
# B14 – Allergy to mammalian meat

Marianne van Hage, Tilo Biedermann, Christiane Hilger, Thomas A.E. Platts-Mills

## Highlights

* Farm animals provide a major part of the diet in many parts of the world

* Sensitization to meat can be acquired through different routes (inhaled, oral, skin)

* New forms of allergic reactions to meat have been recognized (cat-pork and red meat)

* IgE assays including meat allergen sources and components will help identifying the patients

* The only effective treatment is avoidance of relevant meat sources

## 1 – The allergen sources

Meat derived from domesticated mammals has been an important part of the human diet for at least ten thousand years. The animal tissue sold and eaten as meat includes blood, fat, innards, and tendons, as well as muscles. There is inevitably some cross-reactivity with serum proteins and milk proteins from the same animal. Milk is relevant only for goats, sheep, and cows in the USA and Europe, but it is important to remember that camels and other animals are milked in some parts of the world.

## 2 – Allergen families

Further, although there is a limited range of animals that are sold commercially in the west, a much larger variety of wild animals may be hunted and eaten in rural communities worldwide (Table 44). Farm animals provide a major part of the diet in western societies and in many other parts of the world. These animals are prized for their meat and organs (Figure 97). In addition, there are hundreds of forms of processed meat, including sausages, salami, bacon, etc. Meat also incorporates significant quantities of protein derived from serum, which includes many proteins that are recognized as allergens in cow's milk. Other products derived from these mammals include different forms of fat, paté, and gelatin, which is derived from tendons, cartilage, or skin.

## 3 – Allergen molecules, epidemiology and geographical variation

Initially the identification of meat derived allergens focused on protein antigens recognized by patients who reported allergic reactions that occurred rapidly after exposure. Most of these cases presented in childhood and many of the allergens were species specific proteins.⁹⁵¹,¹⁰⁸⁹ However, it was already clear that some mammalian proteins showed cross-reactivity between species, and this included both immunoglobulins and albumins.¹⁰⁸³,¹⁰⁹⁰ The most important allergens from beef, *Bos domesticus*, are serum albumin (Bos d 6), actin, myoglobulin and immunoglobulin IgG (Bos d 7) (Tables 44 and 45) and of these, serum albumin and immunoglobulin are the major allergens.⁹⁵¹,⁹⁷⁴,¹⁰⁸³,¹⁰⁹⁰ Beef-allergic children have been reported to

<table><caption>TABLE 44 Animals contributing to meat consumption and reactions</caption><thead><tr><th>Animals</th><th>Source</th><th>Milk</th><th>Allergen molecules* #</th><th>Alpha-gal Reactions</th></tr></thead><tbody><tr><td><i>Bos domesticus</i> (cow)</td><td>Farm</td><td>+++</td><td>Twelve (10)<br>Bos d 2-13</td><td>Yes</td></tr><tr><td><i>Sus domesticus</i> (pig)</td><td>Farm/feral</td><td>No</td><td>Albumin (1)<br>Sus s 1</td><td>Yes</td></tr><tr><td><i>Capra aegagrus</i> (goat)</td><td>Farm/D/Feral</td><td>++</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Ovis aries</i> (sheep)</td><td>Farm</td><td>++</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Equus caballus</i> (horse)</td><td>Farm</td><td>++</td><td>Six (2)<br>Equ c 1-6</td><td>Yes</td></tr><tr><td><i>Odocoileus virginianus</i> (deer)</td><td>Wild/Farm</td><td>No</td><td>None (0)</td><td>Yes</td></tr><tr><td><i>Oryctolagus cuniculus</i> (rabbit)</td><td>Wild/D/Farm</td><td>No</td><td>Three (0)<br>Ory c 2-4</td><td>Yes</td></tr><tr><td><i>Cavia porcellus</i> (guinea pig)</td><td>D/Farm/wild</td><td>No</td><td>Five (0)<br>Cav p 1-4,6</td><td>?</td></tr></tbody></table>

D= domesticated; *IUIS Allergen Nomenclature Sub-Committee at www.allergen.org; #Number in brackets indicates the number of allergens defined as food allergen. Alpha-gal is not included as single allergen in the table as the carbohydrate is basically present in all mammalian tissues and bound to many proteins
<figure><img src="image_201.png" /><figcaption>**FIGURE 97** Any of these products can include serum proteins including proteins present in cow's milk (e.g., albumins, globulins, thyroglobulin) *Gelatin is made from tendons, cartilage, or skin and is a protein with varying quantities of glycosylation, used widely in food.</figcaption></figure>

react to bovine serum albumin on SPT, but only some of them do so during challenge.⁹⁵¹

Despite the truly enormous quantities of meat eaten worldwide there are only a limited number of allergens defined (Table 44). Indeed, despite the extensive consumption of meat from sheep, goats and deer, there are no relevant allergens included in the IUIS database. Even for rabbit and guinea-pig, where there are 4 and 5 allergens, respectively, in the database, all of the defined allergens were recognized on the basis of inhalant symptoms. Thus, the only species with a significant number of food allergens recognized is the cow where ten of the allergenic proteins are recognized as food allergens. However, even in that case, most of the allergens were initially identified as allergens in cow's milk. Indeed, the majority of reported reactions to beef in childhood have occurred in cow's milk allergic children.⁹⁵¹,⁹⁷⁴,¹⁰⁹¹ The same can occur with reactions to goat or sheep meat, with goat's milk or sheep milk as the primary sensitizer, but this is much less common. It is difficult to access the world literature on reactions to meat, because a large proportion of the populations eating goat and sheep have only primitive medical care. However, it is likely that the main meat proteins taken orally have very little allergenicity in man.¹⁰⁹²

In the last twenty years, two new forms of allergic reactions to meat have been recognized. In both of these syndromes, presentation is most often in adult life and in both cases the relevant allergens are characterized by extensive cross-reactivity between different mammals. First, it was recognized that some patients who had allergic reactions to pork, were reacting because of pre-existing IgE antibodies to cat albumin that cross-react with pork albumin (pork-cat syndrome).⁷⁹⁵,¹⁰⁹³ Secondly, an enigmatic allergic reaction to the monoclonal antibody cetuximab led to the recognition that a surprisingly large number of individuals in the South East of the United States had IgE antibodies to the disaccharide galactose alpha-1, 3-galactose (alpha-gal).¹⁰⁹⁴ This oligosaccharide is a blood group substance of the nonprimate mammals and is present on all forms of tissue including red meat,¹⁰⁹⁵,¹⁶³⁴ organs such as kidney,¹⁰⁹⁶,¹⁶³³ gelatin,¹⁰⁹⁷ and cat

<table><caption>**TABLE 45** Allergens available for diagnosis for different forms of mammalian meat allergy</caption><thead><tr><th>Clinical syndrome</th><th>Allergens involved</th><th>Available allergen components</th></tr></thead><tbody><tr><td>Pork-cat syndrome</td><td>serum albumins</td><td>Fel d 2, Can f 3, Sus s 1, Bos d 6</td></tr><tr><td>Alpha-gal syndrome (AGS)</td><td>alpha-gal</td><td>alpha-gal</td></tr><tr><td>Meat allergy related to milk</td><td>milk allergens</td><td>Bos d 4-6, Bos d 8</td></tr><tr><td>Primary meat allergy</td><td>meat allergens</td><td>Bos d 6, Sus s 1</td></tr></tbody></table>

<table><caption>**TABLE 46** Routes of sensitization for allergens related to allergic reactions to meat</caption><tr><td>Inhaled:</td><td>Cat albumins related to systemic reactions to pork</td></tr><tr><td>Oral:</td><td>Cow's milk allergens related to anaphylactic reactions to beef</td></tr><tr><td>Skin:</td><td>Tick bites leading to alpha-gal sensitisation</td></tr></table>

IgA¹⁰⁹⁸ but also in some drugs (e.g., cetuximab, anti-venom, pancreatic enzymes, gelatin-based plasma expanders) and gelatin containing vaccines (e.g., varicella, measles, mumps, rubella, Zostavax).¹⁰⁹⁹ For a detailed description of the alpha-gal epitope, please see chapter "The role of CCD." The term "alpha-gal syndrome" (AGS) is the preferred term to describe allergic reactions to mammalian meat, which are based on sIgE to alpha-ga²⁰,¹¹⁰⁰ I. AGS has been reported worldwide.¹¹⁰¹⁻¹¹⁰⁴

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

Prior to the year 2000, it was generally assumed that sensitization to food antigens was induced by oral exposure. However, we now have at least two alternative routes of exposure (Table 46).
<figure><img src="image_201.png" /><figcaption>FIGURE 97 Any of these products can include serum proteins including proteins present in cow's milk (e.g., albumins, globulins, thyroglobulin) *Gelatin is made from tendons, cartilage, or skin and is a protein with varying quantities of glycosylation, used widely in food.</figcaption></figure>

react to bovine serum albumin on SPT, but only some of them do so during challenge.⁹⁵¹

Despite the truly enormous quantities of meat eaten worldwide there are only a limited number of allergens defined (Table 44). Indeed, despite the extensive consumption of meat from sheep, goats and deer, there are no relevant allergens included in the IUIS database. Even for rabbit and guinea-pig, where there are 4 and 5 allergens, respectively, in the database, all of the defined allergens were recognized on the basis of inhalant symptoms. Thus, the only species with a significant number of food allergens recognized is the cow where ten of the allergenic proteins are recognized as food allergens. However, even in that case, most of the allergens were initially identified as allergens in cow's milk. Indeed, the majority of reported reactions to beef in childhood have occurred in cow's milk allergic children.⁹⁵¹,⁹⁷⁴,¹⁰⁹¹ The same can occur with reactions to goat or sheep meat, with goat's milk or sheep milk as the primary sensitizer, but this is much less common. It is difficult to access the world literature on reactions to meat, because a large proportion of the populations eating goat and sheep have only primitive medical care. However, it is likely that the main meat proteins taken orally have very little allergenicity in man.¹⁰⁹²

In the last twenty years, two new forms of allergic reactions to meat have been recognized. In both of these syndromes, presentation is most often in adult life and in both cases the relevant allergens are characterized by extensive cross-reactivity between different mammals. First, it was recognized that some patients who had allergic reactions to pork, were reacting because of pre-existing IgE antibodies to cat albumin that cross-react with pork albumin (pork-cat syndrome).⁷⁹⁵,¹⁰⁹³ Secondly, an enigmatic allergic reaction to the monoclonal antibody cetuximab led to the recognition that a surprisingly large number of individuals in the South East of the United States had IgE antibodies to the disaccharide galactose alpha-1, 3-galactose (alpha-gal).¹⁰⁹⁴ This oligosaccharide is a blood group substance of the nonprimate mammals and is present on all forms of tissue including red meat,¹⁰⁹⁵,¹⁶³⁴ organs such as kidney,¹⁰⁹⁶,¹⁶³³ gelatin,¹⁰⁹⁷ and cat

<table><caption>TABLE 45 Allergens available for diagnosis for different forms of mammalian meat allergy</caption><thead><tr><th>Clinical syndrome</th><th>Allergens involved</th><th>Available allergen components</th></tr></thead><tbody><tr><td>Pork-cat syndrome</td><td>serum albumins</td><td>Fel d 2, Can f 3, Sus s 1, Bos d 6</td></tr><tr><td>Alpha-gal syndrome (AGS)</td><td>alpha-gal</td><td>alpha-gal</td></tr><tr><td>Meat allergy related to milk</td><td>milk allergens</td><td>Bos d 4-6, Bos d 8</td></tr><tr><td>Primary meat allergy</td><td>meat allergens</td><td>Bos d 6, Sus s 1</td></tr></tbody></table>

<table><caption>TABLE 46 Routes of sensitization for allergens related to allergic reactions to meat</caption><tr><td>Inhaled:</td><td>Cat albumins related to systemic reactions to pork</td></tr><tr><td>Oral:</td><td>Cow's milk allergens related to anaphylactic reactions to beef</td></tr><tr><td>Skin:</td><td>Tick bites leading to alpha-gal sensitisation</td></tr></table>

IgA¹⁰⁹⁸ but also in some drugs (e.g., cetuximab, anti-venom, pancreatic enzymes, gelatin-based plasma expanders) and gelatin containing vaccines (e.g., varicella, measles, mumps, rubella, Zostavax).¹⁰⁹⁹ For a detailed description of the alpha-gal epitope, please see chapter "The role of CCD." The term "alpha-gal syndrome" (AGS) is the preferred term to describe allergic reactions to mammalian meat, which are based on sIgE to alpha-ga²⁰,¹¹⁰⁰ I. AGS has been reported worldwide.¹¹⁰¹⁻¹¹⁰⁴

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

Prior to the year 2000, it was generally assumed that sensitization to food antigens was induced by oral exposure. However, we now have at least two alternative routes of exposure (Table 46).
Sensitization to cat albumin occurs predominantly in patients who own cats and is assumed to occur by inhaling dander particles carrying this protein. Thus the route of sensitization to the cross-reacting pork albumin reflects a sensitization that was initially established by inhaled exposure. The second alternative route is through the skin. For the oligosaccharide alpha-gal, the only established route for sensitization is by tick bites, although sensitization by inhalation, e.g., animal dander, occupational allergens is conceivable. Different tick species that have been implicated: *Amblyomma americanum* in the USA; *Amblyomma sculptum* in Brazil, *Ixodes holocyclus* in Australia; *Ixodes ricinus* in Europe and *Haemophysis longicornis*, and *Amblyomma testudinarium* in Japan. The presence of alpha-gal in the gut and salivary glands of *I. ricinus* has been reported<sup>1105,1106</sup> as well as alpha-gal-carrying proteins in saliva from the ticks *I. ricinus*, *A. sculptum*, *H. longicornis*, *Hyalomma marginatum*.<sup>1107,1108,1631,1632</sup> Furthermore, the IgE-binding saliva proteins have shown to be vitellogenins.<sup>1108</sup> There is good evidence that tick bites are necessary since children or adults raised in arctic areas, where ticks are not present, can eat meat carrying this oligosaccharide without inducing IgE-mediated sensitization to alpha-gal.<sup>1109</sup> However, it is possible that also other ecto-parasites such as "chiggers" could be relevant.<sup>1110</sup>

AGS has several novel features that are relevant to diagnosis and management.²⁰ The onset is in the majority of cases in adult life after eating meat without problems for many years, the reactions start 2-6 hours after mammalian meat consumption, the patients have IgE against alpha-gal and nearly all report to have been tick bitten.⁹⁷⁵,¹⁰⁹⁶,¹¹¹¹,¹¹¹² The majority of the patients suffer from urticaria and/or gastrointestinal symptoms. The most severe form of allergic reactions, anaphylaxis, is common. Furthermore, atopy has been shown to increase the risk of anaphylaxis.¹¹¹⁴ Some patients may tolerate red meat at some occasions but have severe reactions on others. This could be due to cofactors such as exercise, nonsteroidal anti-inflammatory medications, and alcohol that may modify the allergic response.¹⁰⁹⁶ In addition, the reaction is dependent on the concentration of alpha-gal in the meal: It is highest in innards like kidney when compared to muscle meat.¹¹¹³ The presence of alpha-gal in furry animal extracts has revealed the need of molecular allergen diagnosis to successfully identified AGS patients who are primarily sensitized to, e.g., cat, dog or horse.¹¹¹⁴

As the alpha-gal epitope is structurally related to blood group B, patients belonging to this blood group have been shown to have a reduced risk of developing the disease due to self-tolerance to the B-antigen.¹¹¹²,¹¹¹⁵ Beyond allergy, Dr. Platts-Mills and colleagues have reported that alpha-Gal sensitization could be a risk factor for coronary artery disease. They noted that there was significantly worse coronary artery disease in patients who had IgE to alpha-gal.¹¹¹⁶

## 5 – Clinical management

The diagnosis is based on (i) history, (ii) skin test and/or IgE antibody assays, and (iii) challenge protocols (Figure 98).

The main parts of management are accurate diagnosis and education. The only effective form of treatment for allergic reactions to meat is to avoid the relevant source or sources. As AGS also plays a role in occupational settings, this may require a change of profession, i.e., if cooks are affected.¹¹¹⁷ Discussion of avoidance may require written protocols, as well as discussion of the many forms in which proteins derived from meat are eaten. With meat sensitization in childhood it may be necessary to go dairy-free to establish a symptom-free condition. After that it should be possible to carry out challenge tests if necessary and to progressively modify details of the diet. For pork-cat syndrome, it is usually sufficient to avoid pork and pork products. In some cases, the cross-reactivity with beef albumin is strong enough to give symptoms with beef products. As albumins are thermolabile proteins, well-cooked meat is often tolerated whereas ham and sausage are not. Challenges may be useful to assess the tolerance of well cooked beef in children.

The diagnosis of alpha-gal sensitivity may be obvious from the history, skin tests or IgE analyses. However, it may be useful to have a panel of serum IgE assays to establish the diagnosis of meat reactions. This will need to include IgE to alpha-gal, beef, and pork, as well as IgE antibodies to milk, cat and cat albumin. Chicken, turkey and cod can be used as negative controls. If the IgE antibody concentration to alpha-gal is greater than or equal to 2 kU/L or more than 2% of the total IgE, this makes the diagnosis very likely. Furthermore, if the patient responds clinically to a diet avoiding red meat, this is a good criterion for diagnosis.²⁰

Full avoidance of all products containing alpha-gal is not easy because this includes all products derived from mammals including dairy.¹¹¹⁸ However, most cases require a significant dose of red meat (i.e., ≥20 g of meat) to cause reactions, and over 80% of cases can tolerate milk and milk products. In many cases, the patients have already recognized what they can tolerate before they present to physicians. As in other IgE-mediated food allergies, mastocytosis increases the risk for severe reactions also for alpha-gal allergic individuals. Thus, measurement of serum tryptase in the investigation of red meat allergy is recommended. Drugs of porcine origin such as pancrelipase and Enzynorm f contain alpha-gal and should be administered with caution in affected patients.¹¹¹⁹ Oral challenge tests may be necessary in patients where the diagnosis remains unclear or who do not always show reactions in response to red meat exposure. In the latter, cofactors may need to be included to elucidate clinically overt reactions. In these cases, patient management may also include the advise to regularly consume small amounts of meat that were tolerated in the absence of augmentation or cofactors to allow maintenance of tolerance.

Moreover, patients should be informed that further tick bites can maintain or lead to increases in the concentration of alpha-gal IgE.¹¹²⁰,¹¹²¹ Appropriate avoidance, e.g., clothing and sprays should be recommended. Cofactors (for example, alcohol, ASS, physical exercise) can increase intestinal absorption and substitute for higher sensitivity to red meat.¹⁰⁹⁶

There are no consistent studies using immunotherapy for meat allergy, nor are there studies using anti-IgE as therapy.
Sensitization to cat albumin occurs predominantly in patients who own cats and is assumed to occur by inhaling dander particles carrying this protein. Thus the route of sensitization to the cross-reacting pork albumin reflects a sensitization that was initially established by inhaled exposure. The second alternative route is through the skin. For the oligosaccharide alpha-gal, the only established route for sensitization is by tick bites, although sensitization by inhalation, e.g., animal dander, occupational allergens is conceivable. Different tick species that have been implicated: *Amblyomma americanum* in the USA; *Amblyomma sculptum* in Brazil, *Ixodes holocyclus* in Australia; *Ixodes ricinus* in Europe and *Haemophysis longicornis*, and *Amblyomma testudinarium* in Japan. The presence of alpha-gal in the gut and salivary glands of *I. ricinus* has been reported<sup>1105,1106</sup> as well as alpha-gal-carrying proteins in saliva from the ticks *I. ricinus*, *A. sculptum*, *H. longicornis*, *Hyalomma marginatum*.<sup>1107,1108,1631,1632</sup> Furthermore, the IgE-binding saliva proteins have shown to be vitellogenins.<sup>1108</sup> There is good evidence that tick bites are necessary since children or adults raised in arctic areas, where ticks are not present, can eat meat carrying this oligosaccharide without inducing IgE-mediated sensitization to alpha-gal.<sup>1109</sup> However, it is possible that also other ecto-parasites such as "chiggers" could be relevant.<sup>1110</sup>

AGS has several novel features that are relevant to diagnosis and management.²⁰ The onset is in the majority of cases in adult life after eating meat without problems for many years, the reactions start 2-6 hours after mammalian meat consumption, the patients have IgE against alpha-gal and nearly all report to have been tick bitten.⁹⁷⁵,¹⁰⁹⁶,¹¹¹¹,¹¹¹² The majority of the patients suffer from urticaria and/or gastrointestinal symptoms. The most severe form of allergic reactions, anaphylaxis, is common. Furthermore, atopy has been shown to increase the risk of anaphylaxis.¹¹¹⁴ Some patients may tolerate red meat at some occasions but have severe reactions on others. This could be due to cofactors such as exercise, nonsteroidal anti-inflammatory medications, and alcohol that may modify the allergic response.¹⁰⁹⁶ In addition, the reaction is dependent on the concentration of alpha-gal in the meal: It is highest in innards like kidney when compared to muscle meat.¹¹¹³ The presence of alpha-gal in furry animal extracts has revealed the need of molecular allergen diagnosis to successfully identified AGS patients who are primarily sensitized to, e.g., cat, dog or horse.¹¹¹⁴

As the alpha-gal epitope is structurally related to blood group B, patients belonging to this blood group have been shown to have a reduced risk of developing the disease due to self-tolerance to the B-antigen.¹¹¹²,¹¹¹⁵ Beyond allergy, Dr. Platts-Mills and colleagues have reported that alpha-Gal sensitization could be a risk factor for coronary artery disease. They noted that there was significantly worse coronary artery disease in patients who had IgE to alpha-gal.¹¹¹⁶

## 5 – Clinical management

The diagnosis is based on (i) history, (ii) skin test and/or IgE antibody assays, and (iii) challenge protocols (Figure 98).

The main parts of management are accurate diagnosis and education. The only effective form of treatment for allergic reactions to meat is to avoid the relevant source or sources. As AGS also plays a role in occupational settings, this may require a change of profession, i.e., if cooks are affected.¹¹¹⁷ Discussion of avoidance may require written protocols, as well as discussion of the many forms in which proteins derived from meat are eaten. With meat sensitization in childhood it may be necessary to go dairy-free to establish a symptom-free condition. After that it should be possible to carry out challenge tests if necessary and to progressively modify details of the diet. For pork-cat syndrome, it is usually sufficient to avoid pork and pork products. In some cases, the cross-reactivity with beef albumin is strong enough to give symptoms with beef products. As albumins are thermolabile proteins, well-cooked meat is often tolerated whereas ham and sausage are not. Challenges may be useful to assess the tolerance of well cooked beef in children.

The diagnosis of alpha-gal sensitivity may be obvious from the history, skin tests or IgE analyses. However, it may be useful to have a panel of serum IgE assays to establish the diagnosis of meat reactions. This will need to include IgE to alpha-gal, beef, and pork, as well as IgE antibodies to milk, cat and cat albumin. Chicken, turkey and cod can be used as negative controls. If the IgE antibody concentration to alpha-gal is greater than or equal to 2 kU/L or more than 2% of the total IgE, this makes the diagnosis very likely. Furthermore, if the patient responds clinically to a diet avoiding red meat, this is a good criterion for diagnosis.²⁰

Full avoidance of all products containing alpha-gal is not easy because this includes all products derived from mammals including dairy.¹¹¹⁸ However, most cases require a significant dose of red meat (i.e., ≥20 g of meat) to cause reactions, and over 80% of cases can tolerate milk and milk products. In many cases, the patients have already recognized what they can tolerate before they present to physicians. As in other IgE-mediated food allergies, mastocytosis increases the risk for severe reactions also for alpha-gal allergic individuals. Thus, measurement of serum tryptase in the investigation of red meat allergy is recommended. Drugs of porcine origin such as pancrelipase and Enzynorm f contain alpha-gal and should be administered with caution in affected patients.¹¹¹⁹ Oral challenge tests may be necessary in patients where the diagnosis remains unclear or who do not always show reactions in response to red meat exposure. In the latter, cofactors may need to be included to elucidate clinically overt reactions. In these cases, patient management may also include the advise to regularly consume small amounts of meat that were tolerated in the absence of augmentation or cofactors to allow maintenance of tolerance.

Moreover, patients should be informed that further tick bites can maintain or lead to increases in the concentration of alpha-gal IgE.¹¹²⁰,¹¹²¹ Appropriate avoidance, e.g., clothing and sprays should be recommended. Cofactors (for example, alcohol, ASS, physical exercise) can increase intestinal absorption and substitute for higher sensitivity to red meat.¹⁰⁹⁶

There are no consistent studies using immunotherapy for meat allergy, nor are there studies using anti-IgE as therapy.
<figure><img src="image_202.png" /><figcaption>FIGURE 98 Diagnostic algorithm in patients with allergic reactions to meat</figcaption></figure>

## 6 - Clinical cases

### Case 1 (original)

**History:** Patient, 56-year-old female, who visited the emergency room (ER) in early autumn due to urticaria. She had eaten a sausage five hours earlier. Two months later she once more visited the ER due to an anaphylactic reaction six hours after eating a hamburger. She had during the summer obtained several tick bites and had noted prolonged redness and itching at the site of the tick bite.

**In vitro testing:** IgE against alpha-gal 80 kU/L, beef 14 kU/L, pork 7.6 kU/L and milk 1.7 kU/L.

**Diagnosis:** Red meat allergy.

**Recommendation:** She became symptom-free after avoiding red meat.

### Case 2 (original)

**Clinical history:** An 18-year-old male patient described five, mostly nocturnal, anaphylactic reactions with hives, nausea, abdominal pain, and dyspnea in the last ten years with unknown trigger. In four of five episodes, allergic reactions occurred 3-5 h after a meal containing pork meat, once with physical exercise (cycling) where cutaneous reactions occurred shortly (1 h) after. The patient was otherwise healthy and had no history of atopy or other (food) allergies.

No further episodes occurred as the patient had become a vegetarian. There was a history of tick bites in the patient's childhood.

**In vitro testing:** Specific IgE to alpha-gal was highly positive (29.6 kU/L) and negative to pork (<0.1 kU/L), beef (<0.1 kU/L), chicken (<0.1 kU/L) and omega-5 gliadin (<0.1 kU/L). The tryptase level was 2.13 µg/L (<11.4 µg/L).

**in vivo testing:** Skin prick test was negative to beef, lamb, pork, and cow's milk, but prick-to-prick test revealed positive reactions to raw and cooked pork kidney. Intradermal testing was positive to the gelatin colloid plasma expander Gelafundin® 4% (gelatin polysuccinate) diluted 1:100.

**Food challenge:** An oral challenge was performed with cooked pork kidney (17 g) under careful monitoring. The patient developed urticaria approx. 3 h after challenge and was treated with antihistamines and corticosteroids.

**Diagnosis:** Late-onset anaphylaxis to red meat based on IgE recognizing alpha-gal.

**Recommendation:** The patient was informed to avoid red meat especially in combination with cofactors such as alcohol, acetylsalicylic acid or exercise as well as oral ingestion of large amounts of gelatin. Furthermore, an allergy pass listing Cetuximab and gelatin colloid plasma expander Gelafundin® was handed out.
<figure><img src="image_202.png" /><figcaption>FIGURE 98 Diagnostic algorithm in patients with allergic reactions to meat</figcaption></figure>

## 6 - Clinical cases

### Case 1 (original)

**History:** Patient, 56-year-old female, who visited the emergency room (ER) in early autumn due to urticaria. She had eaten a sausage five hours earlier. Two months later she once more visited the ER due to an anaphylactic reaction six hours after eating a hamburger. She had during the summer obtained several tick bites and had noted prolonged redness and itching at the site of the tick bite.

**In vitro testing:** IgE against alpha-gal 80 kU/L, beef 14 kU/L, pork 7.6 kU/L and milk 1.7 kU/L.

**Diagnosis:** Red meat allergy.

**Recommendation:** She became symptom-free after avoiding red meat.

### Case 2 (original)

**Clinical history:** An 18-year-old male patient described five, mostly nocturnal, anaphylactic reactions with hives, nausea, abdominal pain, and dyspnea in the last ten years with unknown trigger. In four of five episodes, allergic reactions occurred 3-5 h after a meal containing pork meat, once with physical exercise (cycling) where cutaneous reactions occurred shortly (1 h) after. The patient was otherwise healthy and had no history of atopy or other (food) allergies.

No further episodes occurred as the patient had become a vegetarian. There was a history of tick bites in the patient's childhood.

**In vitro testing:** Specific IgE to alpha-gal was highly positive (29.6 kU/L) and negative to pork (<0.1 kU/L), beef (<0.1 kU/L), chicken (<0.1 kU/L) and omega-5 gliadin (<0.1 kU/L). The tryptase level was 2.13 µg/L (<11.4 µg/L).

**in vivo testing:** Skin prick test was negative to beef, lamb, pork, and cow's milk, but prick-to-prick test revealed positive reactions to raw and cooked pork kidney. Intradermal testing was positive to the gelatin colloid plasma expander Gelafundin® 4% (gelatin polysuccinate) diluted 1:100.

**Food challenge:** An oral challenge was performed with cooked pork kidney (17 g) under careful monitoring. The patient developed urticaria approx. 3 h after challenge and was treated with antihistamines and corticosteroids.

**Diagnosis:** Late-onset anaphylaxis to red meat based on IgE recognizing alpha-gal.

**Recommendation:** The patient was informed to avoid red meat especially in combination with cofactors such as alcohol, acetylsalicylic acid or exercise as well as oral ingestion of large amounts of gelatin. Furthermore, an allergy pass listing Cetuximab and gelatin colloid plasma expander Gelafundin® was handed out.
It should be emphasized that skin prick test responses to beef, lamb, pork, and cow's milk can be very small in size or negative. In adults, intradermal skin tests may give much clearer results (Figure 98).

**Case 3 (original)**

**Clinical history:** A 34-year-old female presented to the clinic with repeated episodes of oral itching, with or without systemic urticaria following eating pork. She had a long history of cat and dog exposure and was known to be clinically allergic to cats.

**In vitro testing:** Serum results showed IgE to cat 34 kU/L, pork 13.5 kU/L, cat albumin (Fel d 2) 95 kU/L and alpha-gal <0.35 kU/L. Absorption studies showed 90% reduction with cat albumin, 70% with dog albumin and 10% with pork albumin.

**Diagnosis:** The implication is that her primary sensitization was to cat albumin and the symptoms after eating pork were due to cross-reactivity between the albumins. She responded fully to a diet avoiding pork.

reactions. From a clinical perspective biomarker/s that can identify early symptomatology that over time develops in severe allergic reactions, anaphylaxis, are warranted. Moreover, as only certain individuals develop anti-alpha-gal IgE there are possible other contributing factors, adjuvants, which need to be identified in tick saliva.

**Textbox**

* IgE to pork and cat albumin are markers of pork-cat syndrome

* IgE to alpha-gal is a marker of mammalian meat allergy (AGS)

* Patients with alpha-gal syndrome (AGS) should avoid tick bites

## 7 – Research and future perspectives

As the tick population is increasing, mammalian meat allergy will become more common. This underlines that more knowledge regarding the disease is needed, e.g., which are the mechanisms of the delayed
It should be emphasized that skin prick test responses to beef, lamb, pork, and cow's milk can be very small in size or negative. In adults, intradermal skin tests may give much clearer results (Figure 98).

**Case 3 (original)**

**Clinical history:** A 34-year-old female presented to the clinic with repeated episodes of oral itching, with or without systemic urticaria following eating pork. She had a long history of cat and dog exposure and was known to be clinically allergic to cats.

**In vitro testing:** Serum results showed IgE to cat 34 kU/L, pork 13.5 kU/L, cat albumin (Fel d 2) 95 kU/L and alpha-gal <0.35 kU/L. Absorption studies showed 90% reduction with cat albumin, 70% with dog albumin and 10% with pork albumin.

**Diagnosis:** The implication is that her primary sensitization was to cat albumin and the symptoms after eating pork were due to cross-reactivity between the albumins. She responded fully to a diet avoiding pork.

reactions. From a clinical perspective biomarker/s that can identify early symptomatology that over time develops in severe allergic reactions, anaphylaxis, are warranted. Moreover, as only certain individuals develop anti-alpha-gal IgE there are possible other contributing factors, adjuvants, which need to be identified in tick saliva.

**Textbox**

* IgE to pork and cat albumin are markers of pork-cat syndrome

* IgE to alpha-gal is a marker of mammalian meat allergy (AGS)

* Patients with alpha-gal syndrome (AGS) should avoid tick bites

## 7 – Research and future perspectives

As the tick population is increasing, mammalian meat allergy will become more common. This underlines that more knowledge regarding the disease is needed, e.g., which are the mechanisms of the delayed
# B15 - Fruit and vegetable allergy

## Barbara Ballmer-Weber, Karin Hoffmann-Sommergruber

### Highlights

* Allergies to fruits and vegetables can either be due to cross-sensitization with pollen allergens or are due to "true" food allergens

* The majority of plant food allergens can be assigned to a restricted number of protein families

* Frequently observed IgE cross-reactivity does not always coincide with clinical relevance

* Prick to prick testing using raw plant food is often superior to extract based testing

* Food challenges are the method of choice to rule out clinically silent IgE cross-reactivity

## 1 - The allergen sources

Plant foods, especially fruits and vegetables are part of a healthy diet and their consumption is recommended for prevention of cardiovascular and metabolic disorders. However, in predisposed individuals, food allergic reactions are caused/induced upon uptake of a range of fruits and vegetables.

### Fruits

While the range of allergenic fruits is broad, the majority of frequent inducers of allergic reactions belong to the *Rosaceae* family. Therefore, this botanical family will be described in more detail. Among those, pyrenocarps (e.g., apple, pear) and stone fruits (e.g., peach) but also nuts (almond) are able to induce food allergic symptoms in atopic patients.

Fruits are consumed raw or processed, and peel, pulp and seeds contain allergens. For example, the nonspecific lipid transfer proteins (nsLTPs) are accumulated in the outer layer of fruits, and by removing the peel, the allergen exposure can be reduced. Also, certain apple cultivars are known to have low allergen content for the Pathogenesis-related 10 (PR-10) proteins such as Santana and Elise and nsLTPs such as Santana and Ecolette, while others are expressing higher amounts of these allergens such as Golden Delicious (high Bet v 1 and LTP content).1122,1123 Unfortunately, the reduced levels of Mal d 1 do not always coincide with low levels of Mal d 3, thus an overall hypoallergenic apple is so far not available. Also postharvest treatment processes may have impact on allergen levels, as storage under defined conditions has suggested for Mal d 1 and Mal d 3 levels.1124 Furthermore, fruits can be eaten in cakes, desserts, jams and jellies, as ingredients of dishes and as fruit juices either after pasteurization or without heat treatment, also mere cutting an apple into pieces may result in an improved tolerance due to oxidative degradation of Mal d 1. In "biological cosmetics" fruit extracts are also used and may pose an unexpected risk in highly sensitized patients.

In this chapter kiwifruit has been chosen as an example of a "novel food," entering the European market in the late 70 s of the 20th century, and thus eventually developing into a relevant allergen source, previously not anticipated as such. Furthermore, citrus fruits, banana, melon, and grapes represent plant food allergen sources, affecting mostly Southern European patients/consumers. The well-known latex fruit syndrome, comprising allergic reactions against latex products and fruits and vegetables containing cross-reactive allergens will only be briefly mentioned in this chapter, more detailed information is provided in chapter B22.

### Vegetables

Also vegetables represent a relevant source of allergens, with celery being an allergenic food to be labeled on food products according to the EU allergen legislation (European Directive 2007/68/EC). Celery and carrot, both belonging to the botanical Apiaceae family can be consumed raw as well as cooked, and several studies have investigated the impact on heat treatment on individual allergens, thus up- or downregulating their allergenic capacity. Especially in celery, the bulb (tuber; celeriac), and the green parts (stalks) are eaten raw as well as cooked. In addition, celery seeds can be used as a spice as well and are offered either as "celery salt" alone or as an ingredient in spice mixtures to be used for various dishes. In the latter case the presence of celery derived proteins may not be that evident and may lead to unexpected reactions in predisposed individuals. Also celery seed oil is sometimes used as a food ingredient or in cosmetics. In addition to celery and carrot, tomato and bell pepper are well known allergenic foods. In the recent past, additional tomato allergens belonging to the seed storage proteins have been identified from the seeds of the tomato fruit.

Finally a brief overview on Cannabis allergy is provided, reflecting the increasing number of allergic cases upon Cannabis exposure.

## 2 - Major and relevant minor allergenic molecules

### Fruits

Within the *Rosaceae* PR-10 protein family allergenic members are known from apple, peach, apricot, pear, raspberry, and strawberry. These proteins are major allergens for instance in apple and peach, and are located in the pulp and skin of the fruits. In general, PR-10 proteins are labile proteins at extreme pH conditions and their structure is affected upon heat treatment and endogenous inhibitors such as polyphenols.1126 They are constitutively expressed in plant tissues. In addition, they are upregulated upon environmental stress and pathogen attack. They are supposed to act as plant steroid carriers. PR-10 proteins in fruits are supposed to induce mild local reactions in patients. Also heat treatment of fruits (e.g., pasteurized fruit juices and jams) affects PR-10 allergenicity (see also further information on allergenic PR10-proteins in chapter C02). Nonspecific lipid transfer proteins (nsLTPs) are small proteins with a rigid tertiary structure formed by 4 disulfide bridges. Their function is to transport lipids across cell membranes. For example, allergens from this protein family are identified from
# B15 - Fruit and vegetable allergy

## Barbara Ballmer-Weber, Karin Hoffmann-Sommergruber

### Highlights

* Allergies to fruits and vegetables can either be due to cross-sensitization with pollen allergens or are due to "true" food allergens

* The majority of plant food allergens can be assigned to a restricted number of protein families

* Frequently observed IgE cross-reactivity does not always coincide with clinical relevance

* Prick to prick testing using raw plant food is often superior to extract based testing

* Food challenges are the method of choice to rule out clinically silent IgE cross-reactivity

## 1 - The allergen sources

Plant foods, especially fruits and vegetables are part of a healthy diet and their consumption is recommended for prevention of cardiovascular and metabolic disorders. However, in predisposed individuals, food allergic reactions are caused/induced upon uptake of a range of fruits and vegetables.

### Fruits

While the range of allergenic fruits is broad, the majority of frequent inducers of allergic reactions belong to the *Rosaceae* family. Therefore, this botanical family will be described in more detail. Among those, pyrenocarps (e.g., apple, pear) and stone fruits (e.g., peach) but also nuts (almond) are able to induce food allergic symptoms in atopic patients.

Fruits are consumed raw or processed, and peel, pulp and seeds contain allergens. For example, the nonspecific lipid transfer proteins (nsLTPs) are accumulated in the outer layer of fruits, and by removing the peel, the allergen exposure can be reduced. Also, certain apple cultivars are known to have low allergen content for the Pathogenesis-related 10 (PR-10) proteins such as Santana and Elise and nsLTPs such as Santana and Ecolette, while others are expressing higher amounts of these allergens such as Golden Delicious (high Bet v 1 and LTP content).1122,1123 Unfortunately, the reduced levels of Mal d 1 do not always coincide with low levels of Mal d 3, thus an overall hypoallergenic apple is so far not available. Also postharvest treatment processes may have impact on allergen levels, as storage under defined conditions has suggested for Mal d 1 and Mal d 3 levels.1124 Furthermore, fruits can be eaten in cakes, desserts, jams and jellies, as ingredients of dishes and as fruit juices either after pasteurization or without heat treatment, also mere cutting an apple into pieces may result in an improved tolerance due to oxidative degradation of Mal d 1. In "biological cosmetics" fruit extracts are also used and may pose an unexpected risk in highly sensitized patients.

In this chapter kiwifruit has been chosen as an example of a "novel food," entering the European market in the late 70 s of the 20th century, and thus eventually developing into a relevant allergen source, previously not anticipated as such. Furthermore, citrus fruits, banana, melon, and grapes represent plant food allergen sources, affecting mostly Southern European patients/consumers. The well-known latex fruit syndrome, comprising allergic reactions against latex products and fruits and vegetables containing cross-reactive allergens will only be briefly mentioned in this chapter, more detailed information is provided in chapter B22.

### Vegetables

Also vegetables represent a relevant source of allergens, with celery being an allergenic food to be labeled on food products according to the EU allergen legislation (European Directive 2007/68/EC). Celery and carrot, both belonging to the botanical Apiaceae family can be consumed raw as well as cooked, and several studies have investigated the impact on heat treatment on individual allergens, thus up- or downregulating their allergenic capacity. Especially in celery, the bulb (tuber; celeriac), and the green parts (stalks) are eaten raw as well as cooked. In addition, celery seeds can be used as a spice as well and are offered either as "celery salt" alone or as an ingredient in spice mixtures to be used for various dishes. In the latter case the presence of celery derived proteins may not be that evident and may lead to unexpected reactions in predisposed individuals. Also celery seed oil is sometimes used as a food ingredient or in cosmetics. In addition to celery and carrot, tomato and bell pepper are well known allergenic foods. In the recent past, additional tomato allergens belonging to the seed storage proteins have been identified from the seeds of the tomato fruit.

Finally a brief overview on Cannabis allergy is provided, reflecting the increasing number of allergic cases upon Cannabis exposure.

## 2 - Major and relevant minor allergenic molecules

### Fruits

Within the *Rosaceae* PR-10 protein family allergenic members are known from apple, peach, apricot, pear, raspberry, and strawberry. These proteins are major allergens for instance in apple and peach, and are located in the pulp and skin of the fruits. In general, PR-10 proteins are labile proteins at extreme pH conditions and their structure is affected upon heat treatment and endogenous inhibitors such as polyphenols.1126 They are constitutively expressed in plant tissues. In addition, they are upregulated upon environmental stress and pathogen attack. They are supposed to act as plant steroid carriers. PR-10 proteins in fruits are supposed to induce mild local reactions in patients. Also heat treatment of fruits (e.g., pasteurized fruit juices and jams) affects PR-10 allergenicity (see also further information on allergenic PR10-proteins in chapter C02). Nonspecific lipid transfer proteins (nsLTPs) are small proteins with a rigid tertiary structure formed by 4 disulfide bridges. Their function is to transport lipids across cell membranes. For example, allergens from this protein family are identified from
*Rosaceae* fruits (apple, peach, apricot, cherry, plum, pear, raspberry, strawberry, and mulberry),³⁵ citrus fruits, kiwifruit, banana, and grape. They are major allergens and primarily located in the outer tissue layers (peel) of fruits. Upon pathogen attack they are upregulated and therefore classified as PR-14. In contrast to PR-10 allergens, they are stable proteins not affected by low pH environment and heat treatment. However, at neutral pH their resistance to heat treatment is much lower as compared at acidic pH.¹¹²⁷ In general, severe, generalized allergic symptoms are correlated with nsLTPs' intake. An inverse relationship between severity of LTP induced symptoms in peach and co-sensitization to profilin and Bet v 1 has been reported.¹¹²⁸,¹¹²⁹ Further information on allergenic nsLTPs is provided in chapter C03.

Profilins are small proteins with an ubiquitous expression throughout the plant kingdom. They are functional in various important cell-signaling pathways and bind actin. These small proteins are of intermediate to low stability when subjected to heat treatment. sensitization to profilin is frequently observed in patients; however; it often lacks clinical relevance. Allergens from the profilin family have been identified in *Rosaceae* fruits (e.g., apple, peach, pear, strawberry), citrus fruits (sweet orange, litchi), banana, kiwifruit, and melon. Further information on allergenic profilins is provided in chapter C01.

Thaumatin-like proteins share a common 3 dimensional rigid structure defined by conserved cysteine residues forming 8 disulfide bridges. These proteins are expressed in ripening fruits and are upregulated upon biotic and abiotic stress (PR-5). They are regarded as minor allergens, based on data obtained from apple, peach, cherry, green kiwifruit, and banana.

The gibberellin-regulated protein (GRP), peamaclein, was identified from peach. This small protein is upregulated upon biotic stress and located in the peach peel.⁵⁵⁹ Later on, GRPs have been described in other fruits such as apricot, pomegranate, orange, and cherry⁵⁹¹,¹¹³⁰ (see also chapter C09).

In pear another allergen, an isoflavone reductase related protein was identified, which showed allergenic activity in a small group of patients allergic to pear.¹¹³¹

The green kiwifruit contains PR-10, nsLTP, profilin, and thaumatin-like proteins with allergenic activity. The cysteine protease, actinidin, enzymatically degrades seed storage proteins and is upregulated in blossoms and fruits. In kiwifruit monosensitized allergic patients it is a major allergen. In addition, a number of minor allergens have been identified such as phytocystatin, kiwellin, pectin-methylesterase and its inhibitor and a major latex-protein, which belongs to the Bet v 1 superfamily. 2S albumins and 11S globulins localized in the seeds were also characterized as allergens.¹¹³²

While actinidin is abundantly expressed in green kiwifruits, its expression level and allergenic activity is much lower in golden kiwifruits¹¹³³ and in certain kiwifruit cultivars.¹¹³⁴

Banana contains profilin, nsLTP and thaumatin-like proteins with allergenic activity. In addition, beta-1,3 glucanase (PR-2) and class I chitinase (PR-3), both degrading fungal cell walls and the exoskeleton of insects, are banana allergens and contribute to the

cross-reactivity with latex allergens. In pomegranate, a class III chitinase has been described. From citrus fruits nsLTPs type 1 were identified, germin-like proteins, and gibberellin-regulated proteins as relevant allergens. In contrast to other fruits, profilins are regarded as major allergens with clinical relevance in citrus fruits.¹¹³⁵

From melon profilin,¹¹³⁶ cucumisin, an alkaline serine protease,¹¹³⁷ and a member of the PR-1 family are identified as allergens.¹¹³⁸

Papaya contains an allergenic endopolygalacturonase, Caripol 2,¹¹⁴⁰ (Figures 99 and 100, and Table 47).

## Vegetable

In celery, the PR-10 protein is a major allergen, especially in Central Europe. Also, profilin is supposed to sensitize a relevant number of celeriac allergic patients. Less frequently, sensitization to the FAD-containing oxidase, a glycoprotein, is observed. In this case, the carbohydrate moieties of this enzyme seem to be relevant for the IgE-binding capacity.¹¹⁴¹ In the recent past, nsLTPs have been identified from celery. While the nsLTP type 1 is expressed in the stalks, the nsLTP type 2 is found in the tuber.¹¹⁴²,¹¹⁴³ Only limited IgE-cross-reactivity is observed between those two different proteins. Recently a new celery allergen, Api g 7, a defensin like protein 1, was detected in celery tuber.¹¹⁴⁴ Similarly to celery, the PR-10 protein is a major allergen in carrot. At least 2 isoforms of Dau c 1 seem to be responsible for sensitization and provide only partial cross-reactivity. In addition, profilin has been identified as a minor allergen.

Finally, the isoflavone-reductase-like protein is the most recently characterized food allergen. However, neither data on the prevalence of sensitization are available nor the clinical relevance of this allergen is known to date. From tomato, profilin was identified as a minor allergen. Additionally, beta-fructofuranosidase and cyclophilin and a PR-10 protein, are minor allergens. Recently nsLTPs, both, type 1 and type 2 were characterized; however, little is known about their prevalence in sensitization. For bell pepper, a gibberellin-regulated protein, profilin and the thaumatin-like protein—called osmotin-like protein—have been identified as allergens. However, data about their relevance for diagnosis are rather limited.

A class I chitinase was identified from avocado. Finally from potato, several allergens were identified including patatin, cathepsin D inhibitor, cysteine protease inhibitor, and a serine protease inhibitor (Figures 99 and 101; Table 48).

# 3 – Sensitization to individual molecules and their clinical relevance

## Epidemiology and sensitization/cross-reactivity rates

Prevalence data for plant food allergies are scarce, and the available data so far originate from a few studies. In a systematic review by Zuidmeer et al. the overall prevalence for fruits ranged from 0.1 to 4.3%.¹¹⁴⁵ Within a European Community Respiratory
*Rosaceae* fruits (apple, peach, apricot, cherry, plum, pear, raspberry, strawberry, and mulberry),³⁵ citrus fruits, kiwifruit, banana, and grape. They are major allergens and primarily located in the outer tissue layers (peel) of fruits. Upon pathogen attack they are upregulated and therefore classified as PR-14. In contrast to PR-10 allergens, they are stable proteins not affected by low pH environment and heat treatment. However, at neutral pH their resistance to heat treatment is much lower as compared at acidic pH.¹¹²⁷ In general, severe, generalized allergic symptoms are correlated with nsLTPs' intake. An inverse relationship between severity of LTP induced symptoms in peach and co-sensitization to profilin and Bet v 1 has been reported.¹¹²⁸,¹¹²⁹ Further information on allergenic nsLTPs is provided in chapter C03.

Profilins are small proteins with an ubiquitous expression throughout the plant kingdom. They are functional in various important cell-signaling pathways and bind actin. These small proteins are of intermediate to low stability when subjected to heat treatment. sensitization to profilin is frequently observed in patients; however; it often lacks clinical relevance. Allergens from the profilin family have been identified in *Rosaceae* fruits (e.g., apple, peach, pear, strawberry), citrus fruits (sweet orange, litchi), banana, kiwifruit, and melon. Further information on allergenic profilins is provided in chapter C01.

Thaumatin-like proteins share a common 3 dimensional rigid structure defined by conserved cysteine residues forming 8 disulfide bridges. These proteins are expressed in ripening fruits and are upregulated upon biotic and abiotic stress (PR-5). They are regarded as minor allergens, based on data obtained from apple, peach, cherry, green kiwifruit, and banana.

The gibberellin-regulated protein (GRP), peamaclein, was identified from peach. This small protein is upregulated upon biotic stress and located in the peach peel.⁵⁵⁹ Later on, GRPs have been described in other fruits such as apricot, pomegranate, orange, and cherry⁵⁹¹,¹¹³⁰ (see also chapter C09).

In pear another allergen, an isoflavone reductase related protein was identified, which showed allergenic activity in a small group of patients allergic to pear.¹¹³¹

The green kiwifruit contains PR-10, nsLTP, profilin, and thaumatin-like proteins with allergenic activity. The cysteine protease, actinidin, enzymatically degrades seed storage proteins and is upregulated in blossoms and fruits. In kiwifruit monosensitized allergic patients it is a major allergen. In addition, a number of minor allergens have been identified such as phytocystatin, kiwellin, pectin-methylesterase and its inhibitor and a major latex-protein, which belongs to the Bet v 1 superfamily. 2S albumins and 11S globulins localized in the seeds were also characterized as allergens.¹¹³²

While actinidin is abundantly expressed in green kiwifruits, its expression level and allergenic activity is much lower in golden kiwifruits¹¹³³ and in certain kiwifruit cultivars.¹¹³⁴

Banana contains profilin, nsLTP and thaumatin-like proteins with allergenic activity. In addition, beta-1,3 glucanase (PR-2) and class I chitinase (PR-3), both degrading fungal cell walls and the exoskeleton of insects, are banana allergens and contribute to the

cross-reactivity with latex allergens. In pomegranate, a class III chitinase has been described. From citrus fruits nsLTPs type 1 were identified, germin-like proteins, and gibberellin-regulated proteins as relevant allergens. In contrast to other fruits, profilins are regarded as major allergens with clinical relevance in citrus fruits.¹¹³⁵

From melon profilin,¹¹³⁶ cucumisin, an alkaline serine protease,¹¹³⁷ and a member of the PR-1 family are identified as allergens.¹¹³⁸

Papaya contains an allergenic endopolygalacturonase, Caripol 2,¹¹⁴⁰ (Figures 99 and 100, and Table 47).

## Vegetable

In celery, the PR-10 protein is a major allergen, especially in Central Europe. Also, profilin is supposed to sensitize a relevant number of celeriac allergic patients. Less frequently, sensitization to the FAD-containing oxidase, a glycoprotein, is observed. In this case, the carbohydrate moieties of this enzyme seem to be relevant for the IgE-binding capacity.¹¹⁴¹ In the recent past, nsLTPs have been identified from celery. While the nsLTP type 1 is expressed in the stalks, the nsLTP type 2 is found in the tuber.¹¹⁴²,¹¹⁴³ Only limited IgE-cross-reactivity is observed between those two different proteins. Recently a new celery allergen, Api g 7, a defensin like protein 1, was detected in celery tuber.¹¹⁴⁴ Similarly to celery, the PR-10 protein is a major allergen in carrot. At least 2 isoforms of Dau c 1 seem to be responsible for sensitization and provide only partial cross-reactivity. In addition, profilin has been identified as a minor allergen.

Finally, the isoflavone-reductase-like protein is the most recently characterized food allergen. However, neither data on the prevalence of sensitization are available nor the clinical relevance of this allergen is known to date. From tomato, profilin was identified as a minor allergen. Additionally, beta-fructofuranosidase and cyclophilin and a PR-10 protein, are minor allergens. Recently nsLTPs, both, type 1 and type 2 were characterized; however, little is known about their prevalence in sensitization. For bell pepper, a gibberellin-regulated protein, profilin and the thaumatin-like protein—called osmotin-like protein—have been identified as allergens. However, data about their relevance for diagnosis are rather limited.

A class I chitinase was identified from avocado. Finally from potato, several allergens were identified including patatin, cathepsin D inhibitor, cysteine protease inhibitor, and a serine protease inhibitor (Figures 99 and 101; Table 48).

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

### Epidemiology and sensitization/cross-reactivity rates

Prevalence data for plant food allergies are scarce, and the available data so far originate from a few studies. In a systematic review by Zuidmeer et al. the overall prevalence for fruits ranged from 0.1 to 4.3%.¹¹⁴⁵ Within a European Community Respiratory
<figure><img src="image_203.png" /><figcaption>nsLTP</figcaption></figure>

<figure><img src="image_204.png" /><figcaption>Profilin</figcaption></figure>

<figure><img src="image_205.png" /><figcaption>PR-10</figcaption></figure>

<figure><img src="image_206.png" /><figcaption>Defensin like</figcaption></figure>

<figure><img src="image_207.png" /><figcaption>TLP</figcaption></figure>

<figure><img src="image_208.png" /><figcaption>Actinidin</figcaption></figure>

<figure><img src="image_209.png" /><figcaption>GRP</figcaption></figure>

<figure><img src="image_210.png" /><figcaption>Chitinase</figcaption></figure>

<figure><img src="image_211.png" /></figure>

Health Survey (ECRHS) overall sensitization rates for fruits were assessed by Burney and colleagues in 2010 and 2014. Peach was the most frequent inducer of sensitization increasing from 5.4% to 7.9% in 2014. Apple ranked second with a sensitization rate of 4.2% and 6.5% followed by kiwifruit with 3.6% and 5.2% sensitizing capacity.¹¹⁴⁶, ¹¹⁵¹ Prevalence data on allergen-specific sensitization have been generated by a few Europe-wide studies. For apple the SAFE study provided data on Mal d 1-Mal d 4,³⁶ while the peach allergens were investigated in Spanish and Italian studies.⁹⁹⁹,¹¹²⁹,¹¹⁴⁸ reflecting the clear cut difference in the frequency of LTP-sensitization predominant in the Southern European areas as compared to the PR-10 sensitization detected in areas with Fagales pollen exposure. Several

kiwifruit studies were performed including a multicenter, within the Europrevall project and single center studies.¹¹⁴⁹,¹¹⁵⁰

Vegetables

In the systematic review the overall prevalence of food allergies caused by vegetables is around 1.4%.¹¹⁴⁵ The sensitization rates for vegetables were assessed by Burney et al. within the ECRHS study. Sensitization to carrot was determined as 3.6% and increased in 2014 to 5.0%, while celeriac sensitization was observed in 3.5% and 6,3% of the general population, respectively.¹¹⁴⁶,¹¹⁵¹ In celeriac allergy, allergen-specific sensitization prevalence was investigated within the EuroPrevall project.¹¹⁵² For carrot allergy one study investigated the sensitization rate in Switzerland, Denmark and Spain.¹¹⁵²

**FIGURE 99** Molecular structures of well-known food allergen protein families. ID numbers of the crystal structures accessible in the PDB databank https://www.rcsb.org: Non specific lipid transfer proteins (nsLTP from peach, Pru p 3; PDB:2B5S); Profilin (birch pollen; Bet v 2; PDB:1CQA); PR-10 proteins (celeriac, Apig 1; PDB:2BK0); Defensin like proteins (mugwort, Art v 1;PDB:2KPY); Thaumatin-like proteins (TLP from cherry, Pru av 2; PDB:2AHN); Actinidin (from kiwifruit; A. arguta; PDB:3P5X); Gibberellin-regulated protein (GRP; potato; snakin like; PDB:5E5Y); Chitinase (from papaya; PDB:3CQL)

<figure><img src="image_212.png" /></figure>

**FIGURE 100** Peach and kiwifruit as selected examples of fruit allergen sources. Proteins belonging to the PR-10 family are depicted in yellow, TLPs in light blue, nsLTPs in green; profilins in red; GRP in light lilac; seed storage proteins (2S albumins and cupins in grey); kiwifruit specific allergens: Act d 1, Act 3-Act d 5 in dark blue
<figure><img src="image_203.png" /><figcaption>nsLTP</figcaption></figure>

<figure><img src="image_204.png" /><figcaption>Profilin</figcaption></figure>

<figure><img src="image_205.png" /><figcaption>PR-10</figcaption></figure>

<figure><img src="image_206.png" /><figcaption>Defensin like</figcaption></figure>

<figure><img src="image_207.png" /><figcaption>TLP</figcaption></figure>

<figure><img src="image_208.png" /><figcaption>Actinidin</figcaption></figure>

<figure><img src="image_209.png" /><figcaption>GRP</figcaption></figure>

<figure><img src="image_210.png" /><figcaption>Chitinase</figcaption></figure>

<figure><img src="image_211.png" /></figure>

Health Survey (ECRHS) overall sensitization rates for fruits were assessed by Burney and colleagues in 2010 and 2014. Peach was the most frequent inducer of sensitization increasing from 5.4% to 7.9% in 2014. Apple ranked second with a sensitization rate of 4.2% and 6.5% followed by kiwifruit with 3.6% and 5.2% sensitizing capacity.¹¹⁴⁶, ¹¹⁵¹ Prevalence data on allergen-specific sensitization have been generated by a few Europe-wide studies. For apple the SAFE study provided data on Mal d 1-Mal d 4,³⁶ while the peach allergens were investigated in Spanish and Italian studies.⁹⁹⁹,¹¹²⁹,¹¹⁴⁸ reflecting the clear cut difference in the frequency of LTP-sensitization predominant in the Southern European areas as compared to the PR-10 sensitization detected in areas with Fagales pollen exposure. Several

kiwifruit studies were performed including a multicenter, within the Europrevall project and single center studies.¹¹⁴⁹,¹¹⁵⁰

Vegetables

In the systematic review the overall prevalence of food allergies caused by vegetables is around 1.4%.¹¹⁴⁵ The sensitization rates for vegetables were assessed by Burney et al. within the ECRHS study. Sensitization to carrot was determined as 3.6% and increased in 2014 to 5.0%, while celeriac sensitization was observed in 3.5% and 6,3% of the general population, respectively.¹¹⁴⁶,¹¹⁵¹ In celeriac allergy, allergen-specific sensitization prevalence was investigated within the EuroPrevall project.¹¹⁵² For carrot allergy one study investigated the sensitization rate in Switzerland, Denmark and Spain.¹¹⁵²

**FIGURE 99** Molecular structures of well-known food allergen protein families. ID numbers of the crystal structures accessible in the PDB databank https://www.rcsb.org: Non specific lipid transfer proteins (nsLTP from peach, Pru p 3; PDB:2B5S); Profilin (birch pollen; Bet v 2; PDB:1CQA); PR-10 proteins (celeriac, Apig 1; PDB:2BK0); Defensin like proteins (mugwort, Art v 1;PDB:2KPY); Thaumatin-like proteins (TLP from cherry, Pru av 2; PDB:2AHN); Actinidin (from kiwifruit; A. arguta; PDB:3P5X); Gibberellin-regulated protein (GRP; potato; snakin like; PDB:5E5Y); Chitinase (from papaya; PDB:3CQL)

<figure><img src="image_212.png" /></figure>

**FIGURE 100** Peach and kiwifruit as selected examples of fruit allergen sources. Proteins belonging to the PR-10 family are depicted in yellow, TLPs in light blue, nsLTPs in green; profilins in red; GRP in light lilac; seed storage proteins (2S albumins and cupins in grey); kiwifruit specific allergens: Act d 1, Act 3-Act d 5 in dark blue