allergens are from *B. germanica* and *P. americana*, although homologous from other species have been purified. Satinover S et al. found that *Bla* g 2 and *Bla* g 5 have the highest IgE prevalences among five cockroach allergens tested in US patients,⁷⁴⁶ but there are important differences in the profiles of IgE reactivity among individual patients and populations worldwide. Three recent studies in US cohorts, using a larger set of 8-10 cockroach allergens, highlight that other allergens (*Bla* g 3, *Bla* g 6, *Bla* g 9, *Bla* g 11) also show high prevalence among subjects highly sensitized to cockroach.⁷⁵⁴,⁷⁵⁹,⁷⁶² One of the main findings from these studies is that there are no immunodominant cockroach allergens for B and T cell reactivity.

In a study performed in Taiwan to determine whether sensitization to different cockroach allergenic components correlates with different clinical manifestations and severities, eight *P. americana* allergens (Per a 1 through Per a 7 and Per a 9) were evaluated. IgE binding to Per a 2 was more frequent in patients with persistent asthma than in patients with rhinitis only, suggesting that this allergen could be a marker for more severe airway disease. Also, IgE to Per a 9 was strongly associated with rhinitis.⁷⁶³

The availability of cloned and purified allergens is allowing further investigation of their particular effects on the immune responses and the possibilities to be used as reagents for component-resolved diagnosis (CRD) and markers of severity and response to treatment. A recent study reported unique B and T cell reactivity patterns to 10 cockroach allergens per subject, without correlation with clinical phenotype/disease severity.⁷⁵⁹ The B cell reactivity to an expanded set of 8 cockroach allergens was compared in the URECA cohort between subjects with asthma and rhinitis, and subjects without these diseases. Recognition of more cockroach allergens with higher allergen-specific IgE levels was associated with asthma and rhinitis.⁷⁶²

## Clinical relevance

* Cockroach allergens are strong inducers of sensitization and asthma

* Sensitization to cockroach allergens needs to be further investigated in asthmatic patients worldwide

* In some places co-exposure to cockroaches and mite allergens occurs

* CRD could help to detect genuine sensitization to mite and cockroach allergens

In the US cockroach allergy was first recognized as a risk factor for asthma in 1979 in Kansas City, MO.⁷⁶⁴ Subsequently, it became clear that this group of insects, but particularly, the German cockroach, *Blattella germanica*, was an important cause of sensitization in many large cities.⁷⁶⁵⁻⁷⁶⁷ However, even within the US there are large regional variations related to climate and housing conditions.

Notably, the large Inner City Asthma Study published in the New England Journal in 1997 found that among children cockroach allergy and exposure to cockroach allergens were more relevant for asthma morbidity than dust mite.⁷⁶⁸ On the other hand, it is important to realize that that study was largely carried out among children living in apartments in Chicago and New York, where the winters are very dry, which impairs growth of dust mites. By contrast, in most southern towns, including Atlanta, Wilmington DE, and Dallas, particularly in areas of single housing, the pattern of sensitization is consistent to both cockroach and dust mite allergens.⁷⁶⁵⁻⁷⁶⁷ A further element of this pattern was that cockroach infestation is not common in single homes, as distinct from large apartments, unless there are at least eight months of the year where these insects can live outdoors. Thus, it is easy to see why cockroach infestation of houses and sensitization of children to cockroach allergens are significant factors in towns from tropical or sub-tropical areas and completely absent in Scandinavia and rare in suburban areas of northern states in the USA and in most northern areas of Europe. In Southern China and Korea, sensitization to cockroach allergens has been reported, sometimes associated with co-sensitization to shrimp and moth.⁷⁶⁹ The important practical fact is that home interventions to reduce exposure is possible. Since mite and cockroach co-exposure is common, a differential CRD of sensitization might be necessary. In some populations (e.g., from Colombia), there is cross-reactivity between *Bla* g 5 and other Glutathione-transferases (GST), such as Der p 8 and or Asc I 13. In these areas, the high correlation between IgE antibodies to *Bla* g 5 and *Ascaris lumbricoides* GST (Asc I 13) suggests the presence of cross-reactivity between these molecules. However, the frequency of sensitization to Asc I 13 and *Bla* g 5 in a tropical Caribbean population is only around 23% and, in comparison to that of the mite allergen Der p 2, the strength of the IgE response to these allergens was low, which makes it difficult to assess cross-reactivity.⁷⁷⁰ By contrast, no significant cross-reactivity was found among cockroach, mite and *Ascaris* GST allergens in temperate areas of the US. This result is consistent with low amino acid sequence identity at the level of the allergen molecular surfaces, despite sharing a similar three-dimensional structure.⁷⁰⁸ The clinical importance of potential cross-sensitization between helminth and cockroach GSTs should be further investigated.

*Bla* g 7 and Per a 7, two of the cockroaches' tropomyosins, are pan-allergens and positive correlations between shrimp, cockroach, and dust mite IgE levels have been described. In this study, high exposure to cockroach in the home showed significant correlation to higher IgE levels to cockroach and shrimp but not to mite. Sensitization rates to tropomyosins, including the ones from mites and cockroaches, are low in the US and Europe and high in tropical countries⁷⁷¹,⁷⁷² more likely because of cross-reactivity with helminth tropomyosins.⁷⁷³⁻⁷⁷⁵ Arginine kinases have been described as allergens not only in seafood and other sources but also in cockroaches (Per a 9) and mites (Der p 20). Figure 63 shows predicted (thin lines) and experimentally confirmed (solid lines) cross-reactivity of *B. germanica*. Potential species-specific components are also shown (no lines).
<figure><img src="image_156.png" /><figcaption>FIGURE 63 Clinically relevant cross-reactivity of cockroach allergens</figcaption></figure>

5 – Clinical Management

Cockroach allergy should be investigated in all patients with respiratory allergy (Figure 64). Diagnosis is performed by skin testing and/or measurement of specific IgE to cockroach, using crude extracts. However, inconsistent protein and allergen contents and relative B and T cell potencies have been reported in the commercially available cockroach extracts.⁷⁵⁴,⁷⁷⁶,⁷⁷⁷ To investigate the quality of commercial diagnostic skin testing extracts, Mindaye et al quantified B. germanica allergen levels in US extracts and compared them to what patients are exposed to in the environment. They used a multiple reaction monitoring assay involving liquid chromatography combined with mass spectrometry.⁷⁷⁷ While Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 levels were similar in commercial extracts and environmental samples, of concern was the fact that Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were readily present in the environment but largely absent in commercial diagnostic extracts. The absence of select allergens in US extracts may contribute to the skewing of cockroach sensitization profiles reported in the literature.⁷⁷⁷ *In vitro* testing for sensitization to components (CRD) is commercially available for Bla g 1, Bla g 2, Bla g 5 and Bla g 7. In contrast to allergens cross-reactive between mite and cockroach (e.g., tropomyosins), Bla g 1 and Bla g 2 are useful for detecting genuine sensitization to cockroaches in patients co-exposed to mites and cockroaches. The effectiveness of recombinant Bla g 2, Bla g 4, Bla g 5, Per a 1 and Per a 7 for skin testing was evaluated in cockroach allergic patients living in Brazil.⁷⁷² In this study, sensitization to Per a 7 was dominant with a frequency of 42% (likely due to frequent Ascaris infections in this area that can cause sensitization to tropomyosin), in contrast with results from other places where a heterogeneous IgE-reactivity profile among cockroach-allergic patients has been found. For example, in the US, a panel of 5 recombinant allergens (rBla g 1, rBla g 2, rBla g 4, rBla

g 5, and rPer a 7) could identify 64% of cockroach-allergic patients, and group 7 was not the dominant one as in Brazil.⁷⁴⁶ A larger battery of recombinant allergens was tested in cockroach-allergic patients in Taiwan showing that all patients reacted to at least one allergen and discovering that vitellogenin is an important allergen of *B. germanica*.⁷⁷⁸ Together, these studies suggest that a cocktail of five cockroach allergens (Bla g 1 and/or Per a 1, Bla g 2, Bla g 4, Bla g 5, Bla g 7, and/or Per a 7) would be expected to diagnose 50–64% of cockroach allergic patients worldwide. The use of an extended set of cockroach allergens (reported up to 10) can improve the diagnostic capacity of cockroach allergy.⁷⁵⁴,⁷⁵⁹

Bla g 1 and Bla g 2 allergens are secreted in the digestive system and excreted in fecal particles, being good markers of cockroach allergen exposure. Threshold levels of exposure for sensitization and asthma symptoms in the susceptible population are 2 and 8 U/g of dust; however, sensitization by chronic exposure of very low levels (1 – 10 µg/g of dust) of Bla g 2 is associated with asthma and also a risk factor for wheezing in children.⁷⁷⁹ Reducing the environmental allergen exposure in homes of patients with cockroach-induced asthma, could lead to improvement of symptoms.⁷⁸⁰ However, cockroach allergens may persist for months following eradication of the insects. A controlled intervention including professional cleaning, bait traps, insecticides, and HEPA filters, decreased allergen levels, which correlated with decreased asthma symptoms, suggesting that allergen reduction is possible but difficult because continuous efforts and nonaccessible equipment might be necessary; also, the level of expertise that would be required to achieve significant cockroach extermination should be determined.

Immunotherapy (IT) is currently performed with crude extracts and there are reports supporting its effectiveness.⁷⁸¹ In a work including four pilot studies of IT with *B. germanica* extract,
<figure><img src="image_155.png" /><figcaption>FIGURE 63 Clinically relevant cross-reactivity of cockroach allergens</figcaption></figure>

5 – Clinical Management

Cockroach allergy should be investigated in all patients with respiratory allergy (Figure 64). Diagnosis is performed by skin testing and/or measurement of specific IgE to cockroach, using crude extracts. However, inconsistent protein and allergen contents and relative B and T cell potencies have been reported in the commercially available cockroach extracts.⁷⁵⁴,⁷⁷⁶,⁷⁷⁷ To investigate the quality of commercial diagnostic skin testing extracts, Mindaye et al quantified B. germanica allergen levels in US extracts and compared them to what patients are exposed to in the environment. They used a multiple reaction monitoring assay involving liquid chromatography combined with mass spectrometry.⁷⁷⁷ While Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 levels were similar in commercial extracts and environmental samples, of concern was the fact that Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were readily present in the environment but largely absent in commercial diagnostic extracts. The absence of select allergens in US extracts may contribute to the skewing of cockroach sensitization profiles reported in the literature.⁷⁷⁷ *In vitro* testing for sensitization to components (CRD) is commercially available for Bla g 1, Bla g 2, Bla g 5 and Bla g 7. In contrast to allergens cross-reactive between mite and cockroach (e.g., tropomyosins), Bla g 1 and Bla g 2 are useful for detecting genuine sensitization to cockroaches in patients co-exposed to mites and cockroaches. The effectiveness of recombinant Bla g 2, Bla g 4, Bla g 5, Per a 1 and Per a 7 for skin testing was evaluated in cockroach allergic patients living in Brazil.⁷⁷² In this study, sensitization to Per a 7 was dominant with a frequency of 42% (likely due to frequent Ascaris infections in this area that can cause sensitization to tropomyosin), in contrast with results from other places where a heterogeneous IgE-reactivity profile among cockroach-allergic patients has been found. For example, in the US, a panel of 5 recombinant allergens (rBla g 1, rBla g 2, rBla g 4, rBla

g 5, and rPer a 7) could identify 64% of cockroach-allergic patients, and group 7 was not the dominant one as in Brazil.⁷⁴⁶ A larger battery of recombinant allergens was tested in cockroach-allergic patients in Taiwan showing that all patients reacted to at least one allergen and discovering that vitellogenin is an important allergen of *B. germanica*.⁷⁷⁸ Together, these studies suggest that a cocktail of five cockroach allergens (Bla g 1 and/or Per a 1, Bla g 2, Bla g 4, Bla g 5, Bla g 7, and/or Per a 7) would be expected to diagnose 50–64% of cockroach allergic patients worldwide. The use of an extended set of cockroach allergens (reported up to 10) can improve the diagnostic capacity of cockroach allergy.⁷⁵⁴,⁷⁵⁹

Bla g 1 and Bla g 2 allergens are secreted in the digestive system and excreted in fecal particles, being good markers of cockroach allergen exposure. Threshold levels of exposure for sensitization and asthma symptoms in the susceptible population are 2 and 8 U/g of dust; however, sensitization by chronic exposure of very low levels (1 – 10 µg/g of dust) of Bla g 2 is associated with asthma and also a risk factor for wheezing in children.⁷⁷⁹ Reducing the environmental allergen exposure in homes of patients with cockroach-induced asthma, could lead to improvement of symptoms.⁷⁸⁰ However, cockroach allergens may persist for months following eradication of the insects. A controlled intervention including professional cleaning, bait traps, insecticides, and HEPA filters, decreased allergen levels, which correlated with decreased asthma symptoms, suggesting that allergen reduction is possible but difficult because continuous efforts and nonaccessible equipment might be necessary; also, the level of expertise that would be required to achieve significant cockroach extermination should be determined.

Immunotherapy (IT) is currently performed with crude extracts and there are reports supporting its effectiveness.⁷⁸¹ In a work including four pilot studies of IT with *B. germanica* extract,
<figure><img src="image_157.png" /><figcaption>**FIGURE 64** Decision algorithm for cockroach allergy. Please note: No evidence-based proof of efficacy or long-term safety of cockroach. Immunotherapy in patients with asthma is available.</figcaption></figure>

subcutaneous IT was more effective at modifying immune parameters than sublingual IT, although both types proved to be safe. Potential cockroach allergen immunotherapy has been tested in mouse models for prophylaxis (Bla g 2 DNA vaccine) or treatment (liposome entrapped Per a 9) of airway inflammation.⁷⁸² Currently the Inner-City Asthma Consortium is performing a subcutaneous cockroach immunotherapy trial (CRITICAL) that includes allergen B and T cell component analyses. Cockroach extracts for immunotherapy are not standardized and are highly variable in allergen content.⁷⁵⁴,⁷⁷⁶ Since both, the allergen content in German cockroach extracts and the sensitization profiles determine *in vitro* extract potency for IgE reactivity, the selection of appropriate extracts to be used for immunotherapy is important.⁷⁵⁴

# 6 - Clinical Cases

## Case 1

### Asthma in an African American Child in Atlanta

An eight-year-old boy presented to an emergency department (ED) of Grady Memorial Hospital with acute asthma. He responded well to treatment in the ED and was subsequently seen in clinic. Serum assays showed high total IgE and high specific IgE to both dust mite (94 kU/L) and German cockroach (65 kU/L). A subsequent home visit identified high levels of both dust mite (Der p 1 and Der p 2) and very high levels of Bla g 2 in the dust from the bedroom and the kitchen areas (see⁷⁶⁵ for methods).

## Case 2

### 56-year-old African American Lady with Severe Atopic Dermatitis in Central Virginia

A 56-year-old lady presented to clinic in Virginia with poorly controlled AD. In the clinic she was unable to stop scratching her legs and they were severely excoriated. Her serum showed a total IgE of 3,043 kU/L and specific IgE to cockroach of 204 kU/L and to *D. pteronyssinus* of 9.4 kU/L. Furthermore, she had class 5 IgE specific for both Bla g 1 and Bla g 2. A subsequent visit to her home found extensive evidence of cockroach infestation and high levels of Bla g 2 measured by monoclonal antibody ELISA assay, on extracts of dust obtained from both bedroom and kitchen.

# 7 - Research and future perspectives

Clinical and basic research on cockroach allergy is expanding, giving a broader perspective of its successes and limitations. One important aspect will be the definition of the allergenic activity and clinical relevance of the individual IgE-binding molecules, including the evaluation of the statistical association of the IgE-binding frequency of each molecule between cases and controls. Besides, the detailed analyses of potential non-IgE-mediated mechanisms will bring a more balanced landscape of the clinical impact of individual allergens. This, together with the epidemiological surveys on IgE sensitization in additional developing countries will help to evaluate the need of regional arrays for CRD.
<figure><img src="image_156.png" /><figcaption>**FIGURE 64** Decision algorithm for cockroach allergy. Please note: No evidence-based proof of efficacy or long-term safety of cockroach. Immunotherapy in patients with asthma is available.</figcaption></figure>

subcutaneous IT was more effective at modifying immune parameters than sublingual IT, although both types proved to be safe. Potential cockroach allergen immunotherapy has been tested in mouse models for prophylaxis (Bla g 2 DNA vaccine) or treatment (liposome entrapped Per a 9) of airway inflammation.⁷⁸² Currently the Inner-City Asthma Consortium is performing a subcutaneous cockroach immunotherapy trial (CRITICAL) that includes allergen B and T cell component analyses. Cockroach extracts for immunotherapy are not standardized and are highly variable in allergen content.⁷⁵⁴,⁷⁷⁶ Since both, the allergen content in German cockroach extracts and the sensitization profiles determine *in vitro* extract potency for IgE reactivity, the selection of appropriate extracts to be used for immunotherapy is important.⁷⁵⁴

# 6 - Clinical Cases

## Case 1

### Asthma in an African American Child in Atlanta

An eight-year-old boy presented to an emergency department (ED) of Grady Memorial Hospital with acute asthma. He responded well to treatment in the ED and was subsequently seen in clinic. Serum assays showed high total IgE and high specific IgE to both dust mite (94 kU/L) and German cockroach (65 kU/L). A subsequent home visit identified high levels of both dust mite (Der p 1 and Der p 2) and very high levels of Bla g 2 in the dust from the bedroom and the kitchen areas (see⁷⁶⁵ for methods).

## Case 2

### 56-year-old African American Lady with Severe Atopic Dermatitis in Central Virginia

A 56-year-old lady presented to clinic in Virginia with poorly controlled AD. In the clinic she was unable to stop scratching her legs and they were severely excoriated. Her serum showed a total IgE of 3,043 kU/L and specific IgE to cockroach of 204 kU/L and to *D. pteronyssinus* of 9.4 kU/L. Furthermore, she had class 5 IgE specific for both Bla g 1 and Bla g 2. A subsequent visit to her home found extensive evidence of cockroach infestation and high levels of Bla g 2 measured by monoclonal antibody ELISA assay, on extracts of dust obtained from both bedroom and kitchen.

# 7 - Research and future perspectives

Clinical and basic research on cockroach allergy is expanding, giving a broader perspective of its successes and limitations. One important aspect will be the definition of the allergenic activity and clinical relevance of the individual IgE-binding molecules, including the evaluation of the statistical association of the IgE-binding frequency of each molecule between cases and controls. Besides, the detailed analyses of potential non-IgE-mediated mechanisms will bring a more balanced landscape of the clinical impact of individual allergens. This, together with the epidemiological surveys on IgE sensitization in additional developing countries will help to evaluate the need of regional arrays for CRD.
# B06 - Furry animals

## Marianne van Hage, Jon R Konradsen, Christiane Hilger

### Highlights

* IgE to Fel d 1 is as good as IgE to cat extract for diagnosing cat allergy

* Multisensitization to dog allergen molecules is associated with dog allergy

## 1 - The allergen sources

Mammalian furry animals are an important source of indoor allergens.⁷⁸³ They are considered as risk factors for the development of allergic rhinitis and asthma in the domestic and occupational environment. Pets are present in up to 60% of European and US households, with cats and dogs being the most popular pets, followed by birds and small mammals. Horse riding is a favorite leisure activity for many people. Animal allergens are present in urine and saliva⁷⁸⁴ (Figure 65). They stick to animal hair and dander and are dispersed indoors. They also adhere to human clothes and are easily transported to public places. Exposure measurement studies have shown their presence in schools, day-care centres, public transport and households of nonpet owners.⁷⁸⁵

Allergic reactions have been described upon:

* Inhalation by direct contact with the animal

* Inhalation by indirect contact in a contaminated environment

* Animal bite

* Ingestion of raw or medium cooked meat

Most sensitized patients experience allergic symptoms like rhinitis or asthma upon direct exposure to the animal. As animal allergens are easily transported by human clothes, they are ubiquitous. There is evidence that exposure to cat allergens in schools may lead to asthma exacerbations in cat-sensitized students.⁷⁸⁶ In classrooms with a high number of cat-owners, allergen levels

measured are considered to be high enough to induce sensitization to cat.⁷⁸⁷ Animal bites are also capable of provoking anaphylactic reactions. Several cases of anaphylaxis upon rodent bites have been described in the literature.⁷⁸⁸ Anaphylaxis to cat, dog or horse bites does not seem common. However, anaphylaxis to bites from cat, horse, hamster, and laboratory animals have been reported in the literature.⁷⁸⁹⁻⁷⁹¹ There is a market for "hypoallergenic pets" that are claimed to shed less allergens into their surroundings or are supposed to have genetic mutations making the responsible molecules less allergenic. However, there is no scientific evidence proving the existence of hypoallergenic dog, cat, cattle or horse breeds.⁷⁹²⁻⁷⁹⁴

Serum albumins present in meat are easily inactivated by heat, but they can induce symptoms in sensitized patients upon ingestion of raw meat such as ham or sausages.⁷⁹⁵ Horse and donkey milk have been reported as allergen source upon ingestion or upon application on the skin as an ingredient of cosmetics.⁷⁹⁶

## 2 - The allergen families

Although a number of furry animal allergens have been described, cats and dogs are the best characterized pet animals (Table 25 and Figure 66). Lipocalins constitute the most important allergen protein family and lipocalin allergens have been isolated for each furry animal477,345 (Figure 67). Lipocalins are characterized by a common three-dimensional structure and a low sequence identity (see chapter C07). They are synthesized in salivary glands and are dispersed into the environment by saliva and dander. Serum albumins are highly cross-reactive molecules generally considered as minor allergens (see chapter C04). They are abundant in saliva and dander. Fel d 1, the major cat allergen, is a secretoglobin expressed in salivary glands and skin. Two allergens that are members of the latherin protein family are known to have surfactant properties, Equ c 4 and Fel d 8. Can f 5, a prostatic kallikrein was isolated from urine of male dogs. Fel d 3 and Can f 8 belong to the cystatin A protein family. These allergens were isolated from skin and are detected in dander. The lysozymes Equ c 6 and Equ a 6 were identified as allergens in donkey and horse milk. Recently, Niemann pick type C (NPC2) proteins have been identified in both cat and dog.257,797

<figure><img src="image_158.png" /><figcaption>FIGURE 65 Animal allergen sources</figcaption></figure>
# B06 - Furry animals

## Marianne van Hage, Jon R Konradsen, Christiane Hilger

### Highlights

* IgE to Fel d 1 is as good as IgE to cat extract for diagnosing cat allergy

* Multisensitization to dog allergen molecules is associated with dog allergy

## 1 - The allergen sources

Mammalian furry animals are an important source of indoor allergens.⁷⁸³ They are considered as risk factors for the development of allergic rhinitis and asthma in the domestic and occupational environment. Pets are present in up to 60% of European and US households, with cats and dogs being the most popular pets, followed by birds and small mammals. Horse riding is a favorite leisure activity for many people. Animal allergens are present in urine and saliva⁷⁸⁴ (Figure 65). They stick to animal hair and dander and are dispersed indoors. They also adhere to human clothes and are easily transported to public places. Exposure measurement studies have shown their presence in schools, day-care centres, public transport and households of nonpet owners.⁷⁸⁵

Allergic reactions have been described upon:

* Inhalation by direct contact with the animal

* Inhalation by indirect contact in a contaminated environment

* Animal bite

* Ingestion of raw or medium cooked meat

Most sensitized patients experience allergic symptoms like rhinitis or asthma upon direct exposure to the animal. As animal allergens are easily transported by human clothes, they are ubiquitous. There is evidence that exposure to cat allergens in schools may lead to asthma exacerbations in cat-sensitized students.⁷⁸⁶ In classrooms with a high number of cat-owners, allergen levels

measured are considered to be high enough to induce sensitization to cat.⁷⁸⁷ Animal bites are also capable of provoking anaphylactic reactions. Several cases of anaphylaxis upon rodent bites have been described in the literature.⁷⁸⁸ Anaphylaxis to cat, dog or horse bites does not seem common. However, anaphylaxis to bites from cat, horse, hamster, and laboratory animals have been reported in the literature.⁷⁸⁹⁻⁷⁹¹ There is a market for "hypoallergenic pets" that are claimed to shed less allergens into their surroundings or are supposed to have genetic mutations making the responsible molecules less allergenic. However, there is no scientific evidence proving the existence of hypoallergenic dog, cat, cattle or horse breeds.⁷⁹²⁻⁷⁹⁴

Serum albumins present in meat are easily inactivated by heat, but they can induce symptoms in sensitized patients upon ingestion of raw meat such as ham or sausages.⁷⁹⁵ Horse and donkey milk have been reported as allergen source upon ingestion or upon application on the skin as an ingredient of cosmetics.⁷⁹⁶

## 2 - The allergen families

Although a number of furry animal allergens have been described, cats and dogs are the best characterized pet animals (Table 25 and Figure 66). Lipocalins constitute the most important allergen protein family and lipocalin allergens have been isolated for each furry animal477,345 (Figure 67). Lipocalins are characterized by a common three-dimensional structure and a low sequence identity (see chapter C07). They are synthesized in salivary glands and are dispersed into the environment by saliva and dander. Serum albumins are highly cross-reactive molecules generally considered as minor allergens (see chapter C04). They are abundant in saliva and dander. Fel d 1, the major cat allergen, is a secretoglobin expressed in salivary glands and skin. Two allergens that are members of the latherin protein family are known to have surfactant properties, Equ c 4 and Fel d 8. Can f 5, a prostatic kallikrein was isolated from urine of male dogs. Fel d 3 and Can f 8 belong to the cystatin A protein family. These allergens were isolated from skin and are detected in dander. The lysozymes Equ c 6 and Equ a 6 were identified as allergens in donkey and horse milk. Recently, Niemann pick type C (NPC2) proteins have been identified in both cat and dog.257,797

<figure><img src="image_157.png" /><figcaption>FIGURE 65 Animal allergen sources</figcaption></figure>
<table><caption>TABLE 25 Allergenic molecules from furry animals</caption><thead><tr><th>Allergen source</th><th>Allergen</th><th>Biochemical name</th><th>Prevalence of allergen-specific IgE among patients (%)</th><th>MW (kDa)</th></tr></thead><tbody><tr><td rowspan="10">Domestic cattle<br>Bos taurus</td><td>Bos d 2</td><td>lipocalin</td><td>&gt;90- (of cow allergic patients)</td><td>20</td></tr><tr><td>Bos d 3</td><td>S100 calcium-binding protein A7</td><td>Single cases</td><td>11</td></tr><tr><td>Bos d 4</td><td>alpha-lactalbumin</td><td>&gt;90 (of cow milk allergic patients)</td><td>14.2</td></tr><tr><td>Bos d 5</td><td>beta-lactoglobulin</td><td>&gt;90 (of cow milk allergic patients)</td><td>18.3</td></tr><tr><td>Bos d 6</td><td>serum albumin</td><td>&gt;90 (of cow milk allergic patients)</td><td>67</td></tr><tr><td>Bos d 7</td><td>immunoglobulin</td><td>IgE reactivity not reported in allergen.org</td><td>160</td></tr><tr><td>Bos d 8-12</td><td>caseins</td><td>63 (of milk-allergic children had IgE to Bos d 8)</td><td>19-30</td></tr><tr><td>Bos d 13</td><td>myosin light chain</td><td>27 (of beef allergic patients)</td><td>21</td></tr><tr><td rowspan="10">Dog<br>Canis familiaris</td><td>Can f 1</td><td>lipocalin</td><td>50-90</td><td>23-25</td></tr><tr><td>Can f 2</td><td>lipocalin</td><td>22-35</td><td>19</td></tr><tr><td>Can f 3</td><td>serum albumin</td><td>25-60</td><td>69</td></tr><tr><td>Can f 4</td><td>lipocalin</td><td>35-59</td><td>18</td></tr><tr><td>Can f 5</td><td>kallikrein</td><td>31-76</td><td>8</td></tr><tr><td>Can f 6</td><td>lipocalin</td><td>56</td><td>27-29</td></tr><tr><td>Can f 7</td><td>Niemann Pick type C2</td><td>10-20</td><td>14</td></tr><tr><td>Can f 8</td><td>cystatin</td><td>10-14</td><td>14</td></tr><tr><td rowspan="5">Guinea-pig<br>Cavia porcellus</td><td>Cav p 1</td><td>lipocalin</td><td>83</td><td>20</td></tr><tr><td>Cav p 2</td><td>lipocalin</td><td>62</td><td>17</td></tr><tr><td>Cav p 3</td><td>lipocalin</td><td>45</td><td>18</td></tr><tr><td>Cav p 4</td><td>serum albumin</td><td>41</td><td>66</td></tr><tr><td>Cav p 6</td><td>lipocalin</td><td>59</td><td>18</td></tr><tr><td>Donkey<br>Equus asinus</td><td>Equ a 6</td><td>lysozyme</td><td>single cases</td><td>15</td></tr><tr><td rowspan="5">Domestic horse<br>Equus caballus</td><td>Equ c 1</td><td>lipocalin</td><td>76-100</td><td>25</td></tr><tr><td>Equ c 2</td><td>lipocalin</td><td>33-62</td><td>17</td></tr><tr><td>Equ c 3</td><td>serum albumin</td><td>50</td><td>67</td></tr><tr><td>Equ c 4</td><td>latherin</td><td>77-100</td><td>17, 20.5</td></tr><tr><td>Equ c 6</td><td>lysozyme</td><td>single cases</td><td>15</td></tr><tr><td rowspan="8">Domestic cat<br>Felis domesticus</td><td>Fel d 1</td><td>secretoglobin</td><td>&gt;90</td><td>18</td></tr><tr><td>Fel d 2</td><td>serum albumin</td><td>14-23</td><td>69</td></tr><tr><td>Fel d 3</td><td>cystatin-A</td><td>10</td><td>11</td></tr><tr><td>Fel d 4</td><td>lipocalin</td><td>63</td><td>22</td></tr><tr><td>Fel d 5</td><td>immunoglobulin A</td><td>20-40</td><td>400</td></tr><tr><td>Fel d 6</td><td>immunoglobulin M</td><td>not reported in allergen.org</td><td>800-1000</td></tr><tr><td>Fel d 7</td><td>lipocalin</td><td>38</td><td>17.5</td></tr><tr><td>Fel d 8</td><td>latherin</td><td>19</td><td>24</td></tr><tr><td>Golden hamster<br>Mesocricetus auratus</td><td>Mes a 1</td><td>lipocalin</td><td>single cases</td><td>20.5, 24, 30</td></tr><tr><td>House mouse<br>Mus musculus</td><td>Mus m 1</td><td>lipocalin</td><td>&gt;90</td><td>17</td></tr><tr><td rowspan="4">Rabbit<br>Oryctolagus cuniculus</td><td>Ory c 1</td><td>lipocalin</td><td>&gt;90</td><td>17-18</td></tr><tr><td>Ory c 2</td><td>lipocalin</td><td>75</td><td>22</td></tr><tr><td>Ory c 3</td><td>secretoglobin</td><td>77</td><td>19-21</td></tr><tr><td>Ory c 4</td><td>lipocalin</td><td>46</td><td>24</td></tr><tr><td>Siberian hamster<br>Phodopus sungorus</td><td>Phod s 1</td><td>lipocalin</td><td>&gt;90</td><td>23</td></tr><tr><td>Rat<br>Rattus norvegicus</td><td>Rat n 1</td><td>lipocalin</td><td>&gt;90</td><td>17</td></tr><tr><td>Domestic pig<br>Sus scrofa</td><td>Sus s 1</td><td>serum albumin</td><td>single cases of cat allergic individuals</td><td>60</td></tr></tbody></table>

Molecular weight (MW) is as stated on WHO/IUIS Allergen Nomenclature Sub-Committee Website, unless otherwise referenced. Prevalences are from www.allergen.org, reference 803 and Florin-Dan P et al. World J Methodol. 2021;11:46-60 and Torres JA et al. J Biol Chem. 2014;289(34):23382-8.
<table><caption>TABLE 25 Allergenic molecules from furry animals</caption><thead><tr><th>Allergen source</th><th>Allergen</th><th>Biochemical name</th><th>Prevalence of allergen-specific IgE among patients (%)</th><th>MW (kDa)</th></tr></thead><tbody><tr><td rowspan="7">Domestic cattle<br>Bos taurus</td><td>Bos d 2</td><td>lipocalin</td><td>&gt;90- (of cow allergic patients)</td><td>20</td></tr><tr><td>Bos d 3</td><td>S100 calcium-binding protein A7</td><td>Single cases</td><td>11</td></tr><tr><td>Bos d 4</td><td>alpha-lactalbumin</td><td>&gt;90 (of cow milk allergic patients)</td><td>14.2</td></tr><tr><td>Bos d 5</td><td>beta-lactoglobulin</td><td>&gt;90 (of cow milk allergic patients)</td><td>18.3</td></tr><tr><td>Bos d 6</td><td>serum albumin</td><td>&gt;90 (of cow milk allergic patients)</td><td>67</td></tr><tr><td>Bos d 7</td><td>immunoglobulin</td><td>IgE reactivity not reported in allergen.org</td><td>160</td></tr><tr><td>Bos d 8-12</td><td>caseins</td><td>63 (of milk-allergic children had IgE to Bos d 8)</td><td>19-30</td></tr><tr><td rowspan="8">Dog<br>Canis familiaris</td><td>Bos d 13</td><td>myosin light chain</td><td>27 (of beef allergic patients)</td><td>21</td></tr><tr><td>Can f 1</td><td>lipocalin</td><td>50-90</td><td>23-25</td></tr><tr><td>Can f 2</td><td>lipocalin</td><td>22-35</td><td>19</td></tr><tr><td>Can f 3</td><td>serum albumin</td><td>25-60</td><td>69</td></tr><tr><td>Can f 4</td><td>lipocalin</td><td>35-59</td><td>18</td></tr><tr><td>Can f 5</td><td>kallikrein</td><td>31-76</td><td>8</td></tr><tr><td>Can f 6</td><td>lipocalin</td><td>56</td><td>27-29</td></tr><tr><td>Can f 7</td><td>Niemann Pick type C2</td><td>10-20</td><td>14</td></tr><tr><td rowspan="6">Guinea-pig<br>Cavia porcellus</td><td>Can f 8</td><td>cystatin</td><td>10-14</td><td>14</td></tr><tr><td>Cav p 1</td><td>lipocalin</td><td>83</td><td>20</td></tr><tr><td>Cav p 2</td><td>lipocalin</td><td>62</td><td>17</td></tr><tr><td>Cav p 3</td><td>lipocalin</td><td>45</td><td>18</td></tr><tr><td>Cav p 4</td><td>serum albumin</td><td>41</td><td>66</td></tr><tr><td>Cav p 6</td><td>lipocalin</td><td>59</td><td>18</td></tr><tr><td>Donkey<br>Equus asinus</td><td>Equ a 6</td><td>lysozyme</td><td>single cases</td><td>15</td></tr><tr><td rowspan="5">Domestic horse<br>Equus caballus</td><td>Equ c 1</td><td>lipocalin</td><td>76-100</td><td>25</td></tr><tr><td>Equ c 2</td><td>lipocalin</td><td>33-62</td><td>17</td></tr><tr><td>Equ c 3</td><td>serum albumin</td><td>50</td><td>67</td></tr><tr><td>Equ c 4</td><td>latherin</td><td>77-100</td><td>17, 20.5</td></tr><tr><td>Equ c 6</td><td>lysozyme</td><td>single cases</td><td>15</td></tr><tr><td rowspan="8">Domestic cat<br>Felis domesticus</td><td>Fel d 1</td><td>secretoglobin</td><td>&gt;90</td><td>18</td></tr><tr><td>Fel d 2</td><td>serum albumin</td><td>14-23</td><td>69</td></tr><tr><td>Fel d 3</td><td>cystatin-A</td><td>10</td><td>11</td></tr><tr><td>Fel d 4</td><td>lipocalin</td><td>63</td><td>22</td></tr><tr><td>Fel d 5</td><td>immunoglobulin A</td><td>20-40</td><td>400</td></tr><tr><td>Fel d 6</td><td>immunoglobulin M</td><td>not reported in allergen.org</td><td>800-1000</td></tr><tr><td>Fel d 7</td><td>lipocalin</td><td>38</td><td>17.5</td></tr><tr><td>Fel d 8</td><td>latherin</td><td>19</td><td>24</td></tr><tr><td>Golden hamster<br>Mesocricetus auratus</td><td>Mes a 1</td><td>lipocalin</td><td>single cases</td><td>20.5, 24, 30</td></tr><tr><td>House mouse<br>Mus musculus</td><td>Mus m 1</td><td>lipocalin</td><td>&gt;90</td><td>17</td></tr><tr><td rowspan="4">Rabbit<br>Oryctolagus cuniculus</td><td>Ory c 1</td><td>lipocalin</td><td>&gt;90</td><td>17-18</td></tr><tr><td>Ory c 2</td><td>lipocalin</td><td>75</td><td>22</td></tr><tr><td>Ory c 3</td><td>secretoglobin</td><td>77</td><td>19-21</td></tr><tr><td>Ory c 4</td><td>lipocalin</td><td>46</td><td>24</td></tr><tr><td>Siberian hamster<br>Phodopus sungorus</td><td>Phod s 1</td><td>lipocalin</td><td>&gt;90</td><td>23</td></tr><tr><td>Rat<br>Rattus norvegicus</td><td>Rat n 1</td><td>lipocalin</td><td>&gt;90</td><td>17</td></tr><tr><td>Domestic pig<br>Sus scrofa</td><td>Sus s 1</td><td>serum albumin</td><td>single cases of cat allergic individuals</td><td>60</td></tr></tbody></table>

Molecular weight (MW) is as stated on WHO/IUIS Allergen Nomenclature Sub-Committee Website, unless otherwise referenced. Prevalences are from www.allergen.org, reference 803 and Florin-Dan P et al. World J Methodol. 2021;11:46-60 and Torres JA et al. J Biol Chem. 2014;289(34):23382-8.
<figure><img src="image_159.png" /></figure>

**FIGURE 66** Molecular structures of animal allergens. The secretoglobin family is represented by Fel d 1 (1ZKR), lipocalins by Equ c 1 (1EW3), serum albumins by Equ c 3 (4F5U), cystatins by human cystatin A (1GD3) and latherins by Equ c 4 (3ZPM). In parentheses, ID numbers of the crystal structures are accessible in the PDB databank https://www.rcsb.org

### 3 – Epidemiology and geographical variation of allergenic molecules

Large epidemiology studies are based on skin prick test results and determination of specific IgE to animal dander. These studies are hampered by the fact that cross-reactive molecules such as serum albumins are present in the extracts used and this may lead to an overestimation of sensitization rates to a particular animal. Furthermore, commercial skin prick test extracts for, e.g., dog allergy diagnosis have shown extensive variation in allergen composition.²⁶¹

A survey of almost 13,000 German children and adolescents reported sensitization rates of 9.7% to dog, 8.1% to cat and 4.4% to horse dander.⁷⁹⁸ Interestingly, animal sensitization prevalence raised from 5.7% in the age group 3-6 years to 17.2% in the 14-17 years old adolescents. The Swedish BAMSE study, which is an unselected population-based birth cohort of more than 4000 children, lately reported a similar increase from 4 to 24 years, reaching 19.6% to cat, 16.9% to dog and 9.8% to horse in young adulthood.⁷⁹⁹ Furthermore, an increase in sensitization to furry animals has also been reported among adults in two population based studies measured 15 years apart, where sensitization to cat increased from 16% to 26% and to dog from 13% to 25%.⁸⁰⁰ The GA²LEN skin test study has revealed striking geographic sensitization pattern among 14 European countries.⁸⁰¹ Among patients presenting at allergy centres with suspected allergic reaction to inhalant allergens, prevalence to cats and dogs was highest in Denmark and lowest in Austria. sensitization to animals tended to be higher in Nordic countries, which probably depends on the fact that, e.g., cats are kept indoors in a higher frequency in the Northern part of Europe. Moreover, as much as 26% of European adults coming to the clinic for suspected allergy to inhalant allergens are sensitized to cat and 27% to dog.⁸⁰²

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

A number of furry animal components are now available and their clinical value has been addressed in several studies.⁸⁰³ IgE to the major cat allergen Fel d 1 has shown to be as good as IgE to cat extract for diagnosing cat allergy.⁸⁰⁴ Moreover, children with asthma due to cat have also been reported to have higher IgE antibody levels to Fel d 1 compared to children with rhinoconjunctivitis.⁸⁰⁴

Fel d 1 has likewise been reported to be the most common sensitizing cat component (8.9%) in an adult population.⁸⁰⁵ In a study among Swedish schoolchildren current asthma and asthma symptoms following contact with cats were associated with co-sensitization to the cat lipocalin Fel d 4⁸⁰⁶. Furthermore, sensitization to Fel d 4 and Fel d 2 has shown to be independently related to type-2 inflammation among young patients with asthma.⁸⁰⁷ In a longitudinal study, adults sensitized to at least one cat component (Fel d 1, Fel d 2 or Fel d 4), in addition to cat extract, had more bronchial hyperresponsiveness, higher fractional exhaled nitric oxide (FeNO) and were more likely to develop rhinitis and asthma compared to those sensitized to cat extract only.⁸⁰⁸ Moreover, IgE to Fel d 4 and Fel d 2 have also been associated with atopic dermatitis in children with cat allergy⁸⁰⁹ [32] and a single case of a cat-induced anaphylactic reaction in a child sensitized exclusively to Fel d 1 has been reported.⁸¹⁰

Molecular allergy diagnostics has shown to refine characterization of children sensitized dog dander. So far 6 dog allergen molecules (Can f 1 – Can f 6) are available for dog allergy diagnosis. Most children sensitized to dog are sensitized to more than one component, and co-sensitization to Can f 5 and Can f 1 or Can f 2 has shown to be related with asthma.⁸⁰⁶ Käck U et al. found a
<figure><img src="image_158.png" /></figure>

**FIGURE 66** Molecular structures of animal allergens. The secretoglobin family is represented by Fel d 1 (1ZKR), lipocalins by Equ c 1 (1EW3), serum albumins by Equ c 3 (4F5U), cystatins by human cystatin A (1GD3) and latherins by Equ c 4 (3ZPM). In parentheses, ID numbers of the crystal structures are accessible in the PDB databank https://www.rcsb.org

### 3 – Epidemiology and geographical variation of allergenic molecules

Large epidemiology studies are based on skin prick test results and determination of specific IgE to animal dander. These studies are hampered by the fact that cross-reactive molecules such as serum albumins are present in the extracts used and this may lead to an overestimation of sensitization rates to a particular animal. Furthermore, commercial skin prick test extracts for, e.g., dog allergy diagnosis have shown extensive variation in allergen composition.²⁶¹

A survey of almost 13,000 German children and adolescents reported sensitization rates of 9.7% to dog, 8.1% to cat and 4.4% to horse dander.⁷⁹⁸ Interestingly, animal sensitization prevalence raised from 5.7% in the age group 3-6 years to 17.2% in the 14-17 years old adolescents. The Swedish BAMSE study, which is an unselected population-based birth cohort of more than 4000 children, lately reported a similar increase from 4 to 24 years, reaching 19.6% to cat, 16.9% to dog and 9.8% to horse in young adulthood.⁷⁹⁹ Furthermore, an increase in sensitization to furry animals has also been reported among adults in two population based studies measured 15 years apart, where sensitization to cat increased from 16% to 26% and to dog from 13% to 25%.⁸⁰⁰ The GA²LEN skin test study has revealed striking geographic sensitization pattern among 14 European countries.⁸⁰¹ Among patients presenting at allergy centres with suspected allergic reaction to inhalant allergens, prevalence to cats and dogs was highest in Denmark and lowest in Austria. sensitization to animals tended to be higher in Nordic countries, which probably depends on the fact that, e.g., cats are kept indoors in a higher frequency in the Northern part of Europe. Moreover, as much as 26% of European adults coming to the clinic for suspected allergy to inhalant allergens are sensitized to cat and 27% to dog.⁸⁰²

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

A number of furry animal components are now available and their clinical value has been addressed in several studies.⁸⁰³ IgE to the major cat allergen Fel d 1 has shown to be as good as IgE to cat extract for diagnosing cat allergy.⁸⁰⁴ Moreover, children with asthma due to cat have also been reported to have higher IgE antibody levels to Fel d 1 compared to children with rhinoconjunctivitis.⁸⁰⁴

Fel d 1 has likewise been reported to be the most common sensitizing cat component (8.9%) in an adult population.⁸⁰⁵ In a study among Swedish schoolchildren current asthma and asthma symptoms following contact with cats were associated with co-sensitization to the cat lipocalin Fel d 4⁸⁰⁶. Furthermore, sensitization to Fel d 4 and Fel d 2 has shown to be independently related to type-2 inflammation among young patients with asthma.⁸⁰⁷ In a longitudinal study, adults sensitized to at least one cat component (Fel d 1, Fel d 2 or Fel d 4), in addition to cat extract, had more bronchial hyperresponsiveness, higher fractional exhaled nitric oxide (FeNO) and were more likely to develop rhinitis and asthma compared to those sensitized to cat extract only.⁸⁰⁸ Moreover, IgE to Fel d 4 and Fel d 2 have also been associated with atopic dermatitis in children with cat allergy⁸⁰⁹ [32] and a single case of a cat-induced anaphylactic reaction in a child sensitized exclusively to Fel d 1 has been reported.⁸¹⁰

Molecular allergy diagnostics has shown to refine characterization of children sensitized dog dander. So far 6 dog allergen molecules (Can f 1 – Can f 6) are available for dog allergy diagnosis. Most children sensitized to dog are sensitized to more than one component, and co-sensitization to Can f 5 and Can f 1 or Can f 2 has shown to be related with asthma.⁸⁰⁶ Käck U et al. found a
<figure><img src="image_160.png" /><figcaption>**FIGURE 67** Known allergens of furry animals, as listed in the database of the WHO/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org). Proteins belonging to the lipocalin family are depicted in blue, serum albumins are shown in orange, latherins in dark green, immunoglobulins in light grey, cystatin in purple, secretoglobin in dark red, NPC2 in light orange, kallikrein in dark grey, and lysozyme in light green.</figcaption></figure>

significant association between sensitization to the lipocalins Can f 4 and Can f 6 as well as with an increasing number of sensitizing allergen components and clinical symptoms of dog allergy in children evaluated by nasal provocation with dog dander extract.⁸¹¹ Furthermore, multi-sensitization to allergens from furry animals and high IgE levels to dog lipocalins were associated with asthma and asthma severity.¹⁶³⁸ However, monosensitization to the male dog allergen Can f 5 has been related to a negative nasal challenge.⁸¹¹ As Can f 5 is a kallikrein, monosensitization to this allergen has shown to be highly specific for sensitization to male dogs.⁸¹² Recently Schoos et al, showed that patients allergic to dog

and monosensitized to Can f 5 tolerated a conjunctival challenge with female dog extract but not with male dog extract.⁸¹³ Can f 5 has been reported to be the most common sensitizing dog component (3.6%) in an adult population.⁸⁰⁵ Up to 76% of patients with horse allergy are sensitized to Equ c 1.⁸¹⁴ In an adult Swedish population, sensitization to Equ c 1 was present in 2% and in 12% among patients with asthma.⁸⁰⁵

Using an allergen chip (MeDALL chip) containing several individual pet allergens, sera from nearly 800 randomly collected children from the BAMSE birth cohort at 4, 8 and 16 years were analyzed in relation to symptoms to these animals up to 16 yrs. The
<figure><img src="image_159.png" /><figcaption>**FIGURE 67** Known allergens of furry animals, as listed in the database of the WHO/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org). Proteins belonging to the lipocalin family are depicted in blue, serum albumins are shown in orange, latherins in dark green, immunoglobulins in light grey, cystatin in purple, secretoglobin in dark red, NPC2 in light orange, kallikrein in dark grey, and lysozyme in light green.</figcaption></figure>

significant association between sensitization to the lipocalins Can f 4 and Can f 6 as well as with an increasing number of sensitizing allergen components and clinical symptoms of dog allergy in children evaluated by nasal provocation with dog dander extract.⁸¹¹ Furthermore, multi-sensitization to allergens from furry animals and high IgE levels to dog lipocalins were associated with asthma and asthma severity.¹⁶³⁸ However, monosensitization to the male dog allergen Can f 5 has been related to a negative nasal challenge.⁸¹¹ As Can f 5 is a kallikrein, monosensitization to this allergen has shown to be highly specific for sensitization to male dogs.⁸¹² Recently Schoos et al, showed that patients allergic to dog

and monosensitized to Can f 5 tolerated a conjunctival challenge with female dog extract but not with male dog extract.⁸¹³ Can f 5 has been reported to be the most common sensitizing dog component (3.6%) in an adult population.⁸⁰⁵ Up to 76% of patients with horse allergy are sensitized to Equ c 1.⁸¹⁴ In an adult Swedish population, sensitization to Equ c 1 was present in 2% and in 12% among patients with asthma.⁸⁰⁵

Using an allergen chip (MeDALL chip) containing several individual pet allergens, sera from nearly 800 randomly collected children from the BAMSE birth cohort at 4, 8 and 16 years were analyzed in relation to symptoms to these animals up to 16 yrs. The
authors reported that IgE to Fel d 1 and Can f 1 in childhood are predictive markers of allergy to cat or dog, respectively, at 16 years. Furthermore, IgE to Can f 1 was the most important prognostic marker of dog allergy and superior to IgE to dog allergen extract. IgE to Can f 5 is to a lower extent associated with allergy to dog than IgE to Can f 1.³¹²

Sensitization to lipocalins, which are predominantly derived from furry animals, has been associated with asthma in children⁸¹⁵ and multiple sensitization towards lipocalins, kallikrein and secretoglobin components with increased bronchial inflammation in severe asthmatics.⁸¹⁶ In addition, in children with severe asthma and allergy towards furry animals, sensitization to Can f 2 (22% vs. 0%, p = 0.009) and Equ c 1 (51% vs. 25%, p = 0.03) was shown to be more common than in children with controlled asthma.⁸¹⁷ Furthermore, in adults, sensitization to Fel d 1, Can f 1, Can f 2 and Can f 3 and polysensitization (sensitization to more than 2 components) was associated with rhinitis, asthma and asthma severity and related with increased FeNO and eosinophil levels.⁸¹⁸

Although a number of small furry animal allergens have been isolated, there are no epidemiological studies nor studies on the clinical relevance of single components.

With respect to allergen-specific IgG and IgG4, Käck et al. found no significant differences in IgG- or IgG4 levels to dog dander or to any dog allergen molecule between dog dander sensitized children with a positive and a negative provocation test with dog dander extract.²⁶² They concluded that the responses rather reflected exposure than tolerance to dog, in line with the study by Burnett and colleagues.⁸¹⁹

Epidemiological studies have shown that the presence of animal allergens in the indoor environment has been associated with an increased risk of developing allergic symptoms. Monitoring of allergen contamination allows to determine allergen levels and to assess eviction strategies. Methods of dust collection and antibody-based allergen quantification assays allow to measure allergen levels of Fel d 1, Fel d 4, Can f 1, Equ c 1, Mus m 1, Ory c 3, Cav p 1 and Rat n 1 in settled dust, but not all are commercially available.⁸²⁰

## 5 – Clinical management

Exposure to furry animals can lead to different sensitization patterns with different clinical implications. A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or allergen-specific IgE using extracts from furry animals will confirm sensitization. In this context the dose of exposure is also of importance. As furry animals contain cross-reactive molecules such as serum albumins, some of the cross-reactive lipocalins and potentially other cross-reactive molecules, it is important to define the primary allergenic source, especially if a specific immunotherapy is intended. Co-sensitization has to be distinguished from cross-sensitization. It is important to acknowledge that IgE-cross-reactivity may not always imply clinical cross-reactivity. If the cross-reactive IgE is against allergens with low to

moderate degree of sequence homology, which is the case for many of the lipocalins, the patient may not experience symptoms to these allergen sources. However, if the cross-reacting lipocalin allergens have high sequence homology, patients may experience symptoms to all these allergen sources. Moreover, there are few data on symptoms clearly related to cross-reactive molecules as monosensitization to these components seems to be rare.

Taken together: Fel d 1, Fel d 2, Fel d 4, Fel d 7 and, Can f 1 to Can f 6 are commercially available markers of cat and dog sensitization. Equ c 1 may cross-react with Fel d 4 and Can f 6.⁸²¹ The coverage is rather good for cat and dog, but only two cross-reactive molecules, Equ c 1 and Equ c 3, are available for horse and some molecules are available for component-resolved diagnosis of small furry pets. Cav p 1 and Ory c 3 are specific markers of allergy to guinea-pig and rabbit.⁸²²,⁸²³ Mes a 1 and Phod s 1 are marker allergens of the golden and dwarf hamster, respectively.⁴⁷⁷ Not all components, however, are available on all platforms.

Sensitization to major cat/dog/horse allergens (e.g., Fel d 1 / Can f 1, Can f 5/Equ c 1) are specific markers of cat/dog/ horse sensitization. Sensitized patients may experience symptoms from the upper and/or lower airways to cat/ dog/horse. IgE to Fel d 1 and Can f 1 in childhood have shown to be predictive markers of cat or dog allergy in adolescence.³¹² In patients with suspected horse allergy, only sensitization to Equ c 1 had been found to be clinically relevant.⁸¹⁶ Some lipocalins (Can f 6, Fel d 4, Equ c 1; Fel d 7 and Can f 1) share a high sequence identity and are markers of cross-sensitization (Table 26). As Can f 1 is also a primary marker of dog allergy, the decision algorithms in chapter C07 may be helpful. See also Figure 68 for clinical algorithm. The newly identified cat allergen, NPC2, has shown to have high cross-reactivity to Can f 7.²⁵⁷

Cross-reactive animal allergens with high sequence homology are, e.g., serum albumins. Serum albumins are involved in pork-cat syndrome, where sensitization to cat serum albumin represents the primary event in the development of cross-reactive IgE.⁷⁹⁵ For further information, please see chapter C04 on Serum albumins.

Other domestic animals

Bovine allergens are important inducers of occupational allergic airway diseases in cattle-exposed farmers. The European Farmers' Project Study Group has determined that the prevalence of work-related respiratory symptoms was 21.8% among cattle farmers. The

<table><caption>TABLE 26 Cross-reactive allergen from furry animals</caption><thead><tr><td>Allergen</td><td>Degree of cross-reactivity</td></tr></thead><tbody><tr><td>Can f 1, Fel d 7</td><td>moderate risk of cross-reactivity</td></tr><tr><td>Can f 3, Fel d 2, Equ c 3, Sus s 1</td><td>high risk of cross-reactivity with other serum albumins</td></tr><tr><td>Can f 6, Fel d 4, Equ c 1, Cav p 6, Mus m 1</td><td>moderate risk of cross-reactivity with some lipocalins</td></tr><tr><td>Can f 8, Fel d 3</td><td>moderate risk of cross-reactivity</td></tr><tr><td>Equ c 6, Equ a 6</td><td>high risk of cross-reactivity</td></tr></tbody></table>
authors reported that IgE to Fel d 1 and Can f 1 in childhood are predictive markers of allergy to cat or dog, respectively, at 16 years. Furthermore, IgE to Can f 1 was the most important prognostic marker of dog allergy and superior to IgE to dog allergen extract. IgE to Can f 5 is to a lower extent associated with allergy to dog than IgE to Can f 1.³¹²

Sensitization to lipocalins, which are predominantly derived from furry animals, has been associated with asthma in children⁸¹⁵ and multiple sensitization towards lipocalins, kallikrein and secretoglobin components with increased bronchial inflammation in severe asthmatics.⁸¹⁶ In addition, in children with severe asthma and allergy towards furry animals, sensitization to Can f 2 (22% vs. 0%, p = 0.009) and Equ c 1 (51% vs. 25%, p = 0.03) was shown to be more common than in children with controlled asthma.⁸¹⁷ Furthermore, in adults, sensitization to Fel d 1, Can f 1, Can f 2 and Can f 3 and polysensitization (sensitization to more than 2 components) was associated with rhinitis, asthma and asthma severity and related with increased FeNO and eosinophil levels.⁸¹⁸

Although a number of small furry animal allergens have been isolated, there are no epidemiological studies nor studies on the clinical relevance of single components.

With respect to allergen-specific IgG and IgG4, Käck et al. found no significant differences in IgG- or IgG4 levels to dog dander or to any dog allergen molecule between dog dander sensitized children with a positive and a negative provocation test with dog dander extract.²⁶² They concluded that the responses rather reflected exposure than tolerance to dog, in line with the study by Burnett and colleagues.⁸¹⁹

Epidemiological studies have shown that the presence of animal allergens in the indoor environment has been associated with an increased risk of developing allergic symptoms. Monitoring of allergen contamination allows to determine allergen levels and to assess eviction strategies. Methods of dust collection and antibody-based allergen quantification assays allow to measure allergen levels of Fel d 1, Fel d 4, Can f 1, Equ c 1, Mus m 1, Ory c 3, Cav p 1 and Rat n 1 in settled dust, but not all are commercially available.⁸²⁰

## 5 – Clinical management

Exposure to furry animals can lead to different sensitization patterns with different clinical implications. A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or allergen-specific IgE using extracts from furry animals will confirm sensitization. In this context the dose of exposure is also of importance. As furry animals contain cross-reactive molecules such as serum albumins, some of the cross-reactive lipocalins and potentially other cross-reactive molecules, it is important to define the primary allergenic source, especially if a specific immunotherapy is intended. Co-sensitization has to be distinguished from cross-sensitization. It is important to acknowledge that IgE-cross-reactivity may not always imply clinical cross-reactivity. If the cross-reactive IgE is against allergens with low to

moderate degree of sequence homology, which is the case for many of the lipocalins, the patient may not experience symptoms to these allergen sources. However, if the cross-reacting lipocalin allergens have high sequence homology, patients may experience symptoms to all these allergen sources. Moreover, there are few data on symptoms clearly related to cross-reactive molecules as monosensitization to these components seems to be rare.

Taken together: Fel d 1, Fel d 2, Fel d 4, Fel d 7 and, Can f 1 to Can f 6 are commercially available markers of cat and dog sensitization. Equ c 1 may cross-react with Fel d 4 and Can f 6.⁸²¹ The coverage is rather good for cat and dog, but only two cross-reactive molecules, Equ c 1 and Equ c 3, are available for horse and some molecules are available for component-resolved diagnosis of small furry pets. Cav p 1 and Ory c 3 are specific markers of allergy to guinea-pig and rabbit.⁸²²,⁸²³ Mes a 1 and Phod s 1 are marker allergens of the golden and dwarf hamster, respectively.⁴⁷⁷ Not all components, however, are available on all platforms.

Sensitization to major cat/dog/horse allergens (e.g., Fel d 1 / Can f 1, Can f 5/Equ c 1) are specific markers of cat/dog/ horse sensitization. Sensitized patients may experience symptoms from the upper and/or lower airways to cat/ dog/horse. IgE to Fel d 1 and Can f 1 in childhood have shown to be predictive markers of cat or dog allergy in adolescence.³¹² In patients with suspected horse allergy, only sensitization to Equ c 1 had been found to be clinically relevant.⁸¹⁶ Some lipocalins (Can f 6, Fel d 4, Equ c 1; Fel d 7 and Can f 1) share a high sequence identity and are markers of cross-sensitization (Table 26). As Can f 1 is also a primary marker of dog allergy, the decision algorithms in chapter C07 may be helpful. See also Figure 68 for clinical algorithm. The newly identified cat allergen, NPC2, has shown to have high cross-reactivity to Can f 7.²⁵⁷

Cross-reactive animal allergens with high sequence homology are, e.g., serum albumins. Serum albumins are involved in pork-cat syndrome, where sensitization to cat serum albumin represents the primary event in the development of cross-reactive IgE.⁷⁹⁵ For further information, please see chapter C04 on Serum albumins.

Other domestic animals

Bovine allergens are important inducers of occupational allergic airway diseases in cattle-exposed farmers. The European Farmers' Project Study Group has determined that the prevalence of work-related respiratory symptoms was 21.8% among cattle farmers. The

<table><caption>TABLE 26 Cross-reactive allergen from furry animals</caption><thead><tr><td>Allergen</td><td>Degree of cross-reactivity</td></tr></thead><tbody><tr><td>Can f 1, Fel d 7</td><td>moderate risk of cross-reactivity</td></tr><tr><td>Can f 3, Fel d 2, Equ c 3, Sus s 1</td><td>high risk of cross-reactivity with other serum albumins</td></tr><tr><td>Can f 6, Fel d 4, Equ c 1, Cav p 6, Mus m 1</td><td>moderate risk of cross-reactivity with some lipocalins</td></tr><tr><td>Can f 8, Fel d 3</td><td>moderate risk of cross-reactivity</td></tr><tr><td>Equ c 6, Equ a 6</td><td>high risk of cross-reactivity</td></tr></tbody></table>
<figure><img src="image_161.png" /><figcaption>2. Diagnostic algorithm for dog.</figcaption></figure>

<figure><img src="image_162.png" /><figcaption>3. Diagnostic algorithm for horse.</figcaption></figure>

<figure><img src="image_163.png" /></figure>

**FIGURE 68** Diagnostic algorithms for cat, dog, and horse allergy

main sources of bovine allergens are cow hair and dander, but allergens are also found in urine, saliva, milk, and beef. Early investigations of bovine dander extracts have identified 17 different antigenic components. Three of these, having molecular weights of 24, 22 and 20 kDa, have been characterized as major allergens. Subsequent studies have shown that the 20-kDa protein, designated as Bos d 2, is the most important allergen in cow allergen extracts and belongs to the lipocalin family of proteins (see chapter C07). ELISA are available to quantify Bos d 2 or cow hair proteins<sup>824</sup> in the air and dust samples to monitor the allergen load in occupational and home environment.

**Clinical diagnosis**

1. Skin prick test (SPT):

Commercial cat extract can be used, but dog extract has shown marked variations between companies in their content of major dog

allergens.<sup>261</sup> There are no data available on the usefulness of horse extract.

2. IgE-Testing:

Total IgE has no added value in this context. Testing of single components will allow to determine the primary sensitization source (Figure 68). Different sensitization patterns are discussed in the chapters C07 on Lipocalins and C04 Serum albumins.

3. Challenge tests

Nasal provocation testing using natural extracts is feasible in complicated cases and this option may be particularly relevant in patients sensitized to several dog allergen molecules<sup>811</sup>. Challenge tests using natural cat extracts are usually not needed but may be indicated in selected cases such as polysensitization or when discor-dances are observed between skin tests and IgE results. Challenge tests using a cat challenge chamber are only performed in clinical
<figure><img src="image_160.png" /><figcaption>2. Diagnostic algorithm for dog.</figcaption></figure>

<figure><img src="image_161.png" /><figcaption>3. Diagnostic algorithm for horse.</figcaption></figure>

<figure><img src="image_162.png" /><figcaption>FIGURE 68 Diagnostic algorithms for cat, dog, and horse allergy</figcaption></figure>

main sources of bovine allergens are cow hair and dander, but al-
lergens are also found in urine, saliva, milk, and beef. Early inves-
tigations of bovine dander extracts have identified 17 different
antigenic components. Three of these, having molecular weights
of 24, 22 and 20 kDa, have been characterized as major allergens.
Subsequent studies have shown that the 20-kDa protein, designated
as Bos d 2, is the most important allergen in cow allergen extracts
and belongs to the lipocalin family of proteins (see chapter C07).
ELISA are available to quantify Bos d 2 or cow hair proteins<sup>824</sup> in the
air and dust samples to monitor the allergen load in occupational and
home environment.

Clinical diagnosis

1. Skin prick test (SPT):

Commercial cat extract can be used, but dog extract has shown
marked variations between companies in their content of major dog

allergens.²⁶¹ There are no data available on the usefulness of horse
extract.

2. IgE-Testing:

Total IgE has no added value in this context. Testing of single
components will allow to determine the primary sensitization source
(Figure 68). Different sensitization patterns are discussed in the
chapters C07 on Lipocalins and C04 Serum albumins.

3. Challenge tests

Nasal provocation testing using natural extracts is feasible in
complicated cases and this option may be particularly relevant in
patients sensitized to several dog allergen molecules⁸¹¹. Challenge
tests using natural cat extracts are usually not needed but may be
indicated in selected cases such as polysensitization or when discor-
dances are observed between skin tests and IgE results. Challenge
tests using a cat challenge chamber are only performed in clinical
trials to evaluate efficacy of new molecules used for immunotherapy or pharmacological treatment.

### 4. Advices and avoidance

A - If the patient experiences asthma symptoms at exposure to dog or cat even after proper medication the patient should be informed that such direct and continuous exposure may have detrimental effects on health.

B - If the patient experiences asthma at indirect exposure to cat or dog despite symptomatic treatment, allergen-specific immunotherapy is recommended.

### 5. Pharmacotherapy

Symptomatic treatment as required.

### 6. Allergen-specific immunotherapy (AIT)

Allergen-specific immunotherapy with extracts from cat yield better clinical results than those from dog. The higher complexity of dog allergy sensitization patterns, the lack of preparations with an adequate balance of major allergens is likely to explain this divergence.²⁶¹,⁸²⁵,⁸²⁶ AIT for cat or dog is recommended if the patient experiences asthma at indirect exposure to cat or dog despite symptomatic treatment.

## 6 - Clinical cases

### Case 1 (original): Dog allergy

**Clinical history:** A 17 year-old-boy, diagnosed with asthma, and symptoms triggered following exposure to some but not all dogs. He had no symptoms of rhinitis.

**Test with extract:** Specific IgE to dog dander was positive (12 kU/L).

**Test with molecules:** Specific IgE was analysed against Can f 1-6 and only IgE to Can f 5 was positive (7.0 kU/L).

**Nasal challenge with dog dander extract:** The patient did not develop symptoms.

**Conclusion:** As the patients was IgE positive to the male kallikrein Can f 5, it may be possible that the patient tolerates female dogs but develop symptoms upon exposure to male dogs.

### Case 2 (original): Dog allergy

**Clinical history:** A 16 year-old-boy, diagnosed with rhinitis and asthma. He develops symptoms of rhinitis but no symptoms of asthma upon exposure to dog. His family wants to get a dog.

**Test with extract:** Specific IgE to dog dander was positive (9 kU/L).

**Test with molecules:** Specific IgE was analysed against Can f 1-6 and was positive to Can f 1 (5.3 kU/L), Can f 4 (0.5 kU/L) and Can f 6 (0.8 kU/L).

**Nasal challenge with dog dander extract:** The patient developed symptoms.

**Conclusion:** As the patient is sensitized to three dog allergens his asthma will probably worsen if the family gets a dog.

### Case 3 (theoretical): Cat allergy

**Clinical history:** A female, 25 years old, experiencing asthma symptoms to cat at indirect exposure, e.g., when travelling by public transport or visiting public places. The patient is investigated for AIT against cat.

**Test with extract:** Specific IgE to cat dander was positive (15 kU/L).

**Test with molecules:** Specific IgE was analysed against Fel d 1, Fel d 2, Fel d 4 and Fel d 7 and only IgE to Fel d 1 (12 kU/L) was positive.

**Conclusion:** As the patient is sensitized to Fel d 1, which is the major allergen in cat extract, she will most likely benefit from AIT with cat extract.

## 7 - Research and future perspectives

Molecular allergy testing in furry animal allergy is still to be considered as a complement to extract based testing. More knowledge regarding sensitization patterns associated with severe respiratory symptoms and the impact of polysensitization are needed. With respect to treatment, sensitization profiles that are likely to be associated with a positive outcome of AIT are lacking. Furthermore, studies revealing whether molecular allergy testing can be used to monitor the effect of AIT are required. This issue is further complicated by the fact that allergen extracts used for AIT vary in their content of allergens and more clinical studies are needed for evaluating AIT to dog and horse. AIT for small furry animals is still lacking.

The most important allergen molecules from furry animals are available and they may have a role in improving AIT for furry animal allergy by developing patient-tailored treatment. A vision would be to aim for mixtures of allergen molecules matching the patients' profiles. However, clinical studies testing whether AIT with furry allergen molecules will improve symptoms, efficacy, safety and quality of life are warranted.

A new approach for treating allergy to furry animals is to reduce the secretion of immunologically active allergens from the pet. This was demonstrated for Fel d 1 in a study by Satyaraj et al.⁸²⁷ where introduction of anti-Fel d 1 immunoglobulin Y in cat food reduced secretion of the immunologically active Fel d 1 and lowered Fel d 1 levels in the environment.
trials to evaluate efficacy of new molecules used for immunotherapy or pharmacological treatment.

### 4. Advices and avoidance

A - If the patient experiences asthma symptoms at exposure to dog or cat even after proper medication the patient should be informed that such direct and continuous exposure may have detrimental effects on health.

B - If the patient experiences asthma at indirect exposure to cat or dog despite symptomatic treatment, allergen-specific immunotherapy is recommended.

### 5. Pharmacotherapy

Symptomatic treatment as required.

### 6. Allergen-specific immunotherapy (AIT)

Allergen-specific immunotherapy with extracts from cat yield better clinical results than those from dog. The higher complexity of dog allergy sensitization patterns, the lack of preparations with an adequate balance of major allergens is likely to explain this divergence.²⁶¹,⁸²⁵,⁸²⁶ AIT for cat or dog is recommended if the patient experiences asthma at indirect exposure to cat or dog despite symptomatic treatment.

## 6 – Clinical cases

### Case 1 (original): Dog allergy

**Clinical history:** A 17 year-old-boy, diagnosed with asthma, and symptoms triggered following exposure to some but not all dogs. He had no symptoms of rhinitis.

**Test with extract:** Specific IgE to dog dander was positive (12 kU/L).

**Test with molecules:** Specific IgE was analysed against Can f 1-6 and only IgE to Can f 5 was positive (7.0 kU/L).

**Nasal challenge with dog dander extract:** The patient did not develop symptoms.

**Conclusion:** As the patients was IgE positive to the male kallikrein Can f 5, it may be possible that the patient tolerates female dogs but develop symptoms upon exposure to male dogs.

### Case 2 (original): Dog allergy

**Clinical history:** A 16 year-old-boy, diagnosed with rhinitis and asthma. He develops symptoms of rhinitis but no symptoms of asthma upon exposure to dog. His family wants to get a dog.

**Test with extract:** Specific IgE to dog dander was positive (9 kU/L).

**Test with molecules:** Specific IgE was analysed against Can f 1-6 and was positive to Can f 1 (5.3 kU/L), Can f 4 (0.5 kU/L) and Can f 6 (0.8 kU/L).

**Nasal challenge with dog dander extract:** The patient developed symptoms.

**Conclusion:** As the patient is sensitized to three dog allergens his asthma will probably worsen if the family gets a dog.

### Case 3 (theoretical): Cat allergy

**Clinical history:** A female, 25 years old, experiencing asthma symptoms to cat at indirect exposure, e.g., when travelling by public transport or visiting public places. The patient is investigated for AIT against cat.

**Test with extract:** Specific IgE to cat dander was positive (15 kU/L).

**Test with molecules:** Specific IgE was analysed against Fel d 1, Fel d 2, Fel d 4 and Fel d 7 and only IgE to Fel d 1 (12 kU/L) was positive.

**Conclusion:** As the patient is sensitized to Fel d 1, which is the major allergen in cat extract, she will most likely benefit from AIT with cat extract.

## 7 – Research and future perspectives

Molecular allergy testing in furry animal allergy is still to be considered as a complement to extract based testing. More knowledge regarding sensitization patterns associated with severe respiratory symptoms and the impact of polysensitization are needed. With respect to treatment, sensitization profiles that are likely to be associated with a positive outcome of AIT are lacking. Furthermore, studies revealing whether molecular allergy testing can be used to monitor the effect of AIT are required. This issue is further complicated by the fact that allergen extracts used for AIT vary in their content of allergens and more clinical studies are needed for evaluating AIT to dog and horse. AIT for small furry animals is still lacking.

The most important allergen molecules from furry animals are available and they may have a role in improving AIT for furry animal allergy by developing patient-tailored treatment. A vision would be to aim for mixtures of allergen molecules matching the patients' profiles. However, clinical studies testing whether AIT with furry allergen molecules will improve symptoms, efficacy, safety and quality of life are warranted.

A new approach for treating allergy to furry animals is to reduce the secretion of immunologically active allergens from the pet. This was demonstrated for Fel d 1 in a study by Satyaraj et al.⁸²⁷ where introduction of anti-Fel d 1 immunoglobulin Y in cat food reduced secretion of the immunologically active Fel d 1 and lowered Fel d 1 levels in the environment.
# B07 - Allergy to moulds

## Sabine Kespohl and Monika Raulf

### Highlights

* Of the huge variety of moulds worldwide, only few species can be tested.

* The mould species with greatest clinical relevance are: Alternaria alternata and Cladosporium herbarum (outdoor); Aspergillus fumigatus and Penicillium chrysogenum (indoor).

* Moulds are ubiquitous, but in contrast to spores in the air, the sensitization rate to mould is relatively low.

* Guidelines recommended diagnostic tools are skin prick tests and serological IgE measurement.

* A mould mix may be recommended as serological screening tool.

* Component-resolved mould allergen diagnosis is available for Alt a 1, Alt a 6, Cladh 8, Asp f 1 - 4 and Asp f 6.

* Component-resolved diagnosis is useful to verify allergic bronchopulmonary aspergillosis (ABPA) and also for patients sensitized to Alternaria alternata before starting specific immunotherapy (SIT).

### 1 - The source

The predicted number of fungal species varies from 0.6 - 1 million worldwide.⁸²⁸ In principle, IgE-mediated sensitization can occur against any fungal species. Fungi reproduce by spores, which are airborne spreading units, usually between 2 and 100 µm in diameter, which is small enough to infiltrate lungs and alveoli. The concentration of spores in the air depends on the temperature and humidity. The highest concentration of mould spores in outdoor air occurs in northern Europe from June to October and in southern Europe from May to August.⁸²⁹ But climate changes can increase total atmospheric mould by mean of longer spore season with possibly quantitatively higher spore releases. Furthermore, extreme weather conditions such as frequent storms, extreme rainfalls and increased flooding in many aareas of the world contribute to the increased growth and spread of moulds and spores. This applies both to outdoor mould, which often gets into indoor areas due to strong winds, and to the problem of prolonged dampness in buildings due to water damage, resulting in significantly increased occurrence of indoor mould.⁸³⁰ Besides spores, fragments of mycelial filaments (0.2 - 10 µm) are also airborne allergen carriers and can occur in even greater amounts than spores. In real life

patients are exposed to both spores and hyphae. Several studies have shown that spores and hyphal fragments are liberated from natural occurring mould cultures and became airborne.⁸³¹ It was shown that particular mould allergens, Asp f 1⁸³² and Alt a 1 were detected in spores.⁸³³,⁸³⁴ But other allergens like Alt a 8, a mannitol dehydrogenase, were exclusively localized in vacuole-like compartments of the hyphae.⁸³⁴ Fungal source material, used for preparation of diagnostic test solution, e.g., for skin prick tests, was shown to consist of a spore and hyphal material mixture.

According to medical mycology, fungi are divided into: dermatophytes, yeasts and moulds. Dermatophytes, which cause dermatophytosis on the horny layer of the skin, hair and nails. Unlike other skin fungi, they can feed on keratin (keratinase). Yeasts, on the other hand, are unicellular, spherical fungi that reproduce by budding. The most common diseases are candidiasis (Candida species) or infections / type IV allergy of the skin caused by Malassezia. By contrast, moulds often trigger respiratory problems in the form of an allergic type I (IgE-mediated) immune reaction, or an allergic type III / IV reaction as hypersensitivity pneumonitis. In immuno-compromised individuals, moulds, especially thermotolerant species such as Aspergillus fumigatus, can also grow in the lungs (mycosis / aspergillosis). The rate of sensitization to moulds in the general population is below 5%, which is significantly lower compared to those of other environmental allergens like pollen, animal allergens or mites shown in Table 27. Sensitization to fungi is more common when individuals already have allergic sensitization or suffer from asthma. In particular, Aspergillus fumigatus seemed to be important in patients with severe asthma with fungal sensitization (SAFS).⁸³⁵

### 2 - Allergen families and allergic molecules

Altogether, there are currently 113 fungal allergens listed in the official WHO/IUIS database allergens originating from 30 fungal species (www.allergen.org; 10/2021). From these 106 allergens could be found in 42 allergen families. The AllFam database of allergen families (http://www.meduniwien.ac.at/allfam/) summarizes common phylogenetic, structural and functional properties of allergens. In the following table fungal allergens are grouped according to their biological function, molecular weight, way of exposure, sensitization rates of single allergens. sensitization rates have been summarized as minor allergen (< 50% sensitization in study group) or major allergens (> 50% sensitization in study group) (Table 28) modified and updated from⁸³⁶.

<table><caption>TABLE 27 Prevalence of mould sensitization among different population groups (modified from ⁸³⁶)</caption><thead><tr><td>Moulds</td><td>General population</td><td>Patient group with allergic symptoms</td><td>Asthmatic group</td></tr></thead><tbody><tr><td>Alternaria</td><td>3-4%<sup>1231</sup>; 5%<sup>1624</sup></td><td>8-10%<sup>801</sup>; 3%<sup>1627</sup>; 9-12%<sup>1626</sup></td><td>22%<sup>835</sup>; 6%<sup>1627</sup></td></tr><tr><td>Aspergillus</td><td>2-3%<sup>1231</sup>; 3%<sup>1624</sup></td><td>4-5%<sup>801</sup>; 3%<sup>1627</sup>; 7-10%<sup>1626</sup></td><td>45%<sup>835</sup>; 11%<sup>1627</sup></td></tr><tr><td>Cladosporium</td><td>1-2%<sup>1231</sup>; 2%<sup>1624</sup></td><td>4-6%<sup>801</sup>; 2%<sup>1627</sup>; 8-10%<sup>1626</sup></td><td>24%<sup>835</sup>; 4%<sup>1627</sup></td></tr><tr><td>Penicillium</td><td>5%<sup>1624</sup>; 8%<sup>1625</sup></td><td>13-29%<sup>1626</sup></td><td>29%<sup>835</sup></td></tr></tbody></table>
# B07 – Allergy to moulds

## Sabine Kespohl and Monika Raulf

### Highlights

* Of the huge variety of moulds worldwide, only few species can be tested.

* The mould species with greatest clinical relevance are: Alternaria alternata and Cladosporium herbarum (outdoor); Aspergillus fumigatus and Penicillium chrysogenum (indoor).

* Moulds are ubiquitous, but in contrast to spores in the air, the sensitization rate to mould is relatively low.

* Guidelines recommended diagnostic tools are skin prick tests and serological IgE measurement.

* A mould mix may be recommended as serological screening tool.

* Component-resolved mould allergen diagnosis is available for Alt a 1, Alt a 6, Cla h 8, Asp f 1 - 4 and Asp f 6.

* Component-resolved diagnosis is useful to verify allergic bronchopulmonary aspergillosis (ABPA) and also for patients sensitized to Alternaria alternata before starting specific immunotherapy (SIT).

### 1 – The source

The predicted number of fungal species varies from 0.6 – 1 million worldwide.⁸²⁸ In principle, IgE-mediated sensitization can occur against any fungal species. Fungi reproduce by spores, which are airborne spreading units, usually between 2 and 100 µm in diameter, which is small enough to infiltrate lungs and alveoli. The concentration of spores in the air depends on the temperature and humidity. The highest concentration of mould spores in outdoor air occurs in northern Europe from June to October and in southern Europe from May to August.⁸²⁹ But climate changes can increase total atmospheric mould by mean of longer spore season with possibly quantitatively higher spore releases. Furthermore, extreme weather conditions such as frequent storms, extreme rainfalls and increased flooding in many aareas of the world contribute to the increased growth and spread of moulds and spores. This applies both to outdoor mould, which often gets into indoor areas due to strong winds, and to the problem of prolonged dampness in buildings due to water damage, resulting in significantly increased occurrence of indoor mould.⁸³⁰ Besides spores, fragments of mycelial filaments (0.2 - 10 µm) are also airborne allergen carriers and can occur in even greater amounts than spores. In real life

patients are exposed to both spores and hyphae. Several studies have shown that spores and hyphal fragments are liberated from natural occurring mould cultures and became airborne.⁸³¹ It was shown that particular mould allergens, Asp f 1⁸³² and Alt a 1 were detected in spores.⁸³³,⁸³⁴ But other allergens like Alt a 8, a mannitol dehydrogenase, were exclusively localized in vacuole-like compartments of the hyphae.⁸³⁴ Fungal source material, used for preparation of diagnostic test solution, e.g., for skin prick tests, was shown to consist of a spore and hyphal material mixture.

According to medical mycology, fungi are divided into: dermatophytes, yeasts and moulds. Dermatophytes, which cause dermatophytosis on the horny layer of the skin, hair and nails. Unlike other skin fungi, they can feed on keratin (keratinase). Yeasts, on the other hand, are unicellular, spherical fungi that reproduce by budding. The most common diseases are candidiasis (Candida species) or infections / type IV allergy of the skin caused by Malassezia. By contrast, moulds often trigger respiratory problems in the form of an allergic type I (IgE-mediated) immune reaction, or an allergic type III / IV reaction as hypersensitivity pneumonitis. In immuno-compromised individuals, moulds, especially thermotolerant species such as Aspergillus fumigatus, can also grow in the lungs (mycosis / aspergillosis). The rate of sensitization to moulds in the general population is below 5%, which is significantly lower compared to those of other environmental allergens like pollen, animal allergens or mites shown in Table 27. Sensitization to fungi is more common when individuals already have allergic sensitization or suffer from asthma. In particular, Aspergillus fumigatus seemed to be important in patients with severe asthma with fungal sensitization (SAFS).⁸³⁵

### 2 – Allergen families and allergic molecules

Altogether, there are currently 113 fungal allergens listed in the official WHO/IUIS database allergens originating from 30 fungal species (www.allergen.org; 10/2021). From these 106 allergens could be found in 42 allergen families. The AllFam database of allergen families (http://www.meduniwien.ac.at/allfam/) summarizes common phylogenetic, structural and functional properties of allergens. In the following table fungal allergens are grouped according to their biological function, molecular weight, way of exposure, sensitization rates of single allergens. sensitization rates have been summarized as minor allergen (< 50% sensitization in study group) or major allergens (> 50% sensitization in study group) (Table 28) modified and updated from⁸³⁶.

<table><caption>TABLE 27 Prevalence of mould sensitization among different population groups (modified from ⁸³⁶)</caption><thead><tr><td>Moulds</td><td>General population</td><td>Patient group with allergic symptoms</td><td>Asthmatic group</td></tr></thead><tbody><tr><td>Alternaria</td><td>3-4%<sup>1231</sup>; 5%<sup>1624</sup></td><td>8-10%<sup>801</sup>; 3%<sup>1627</sup>; 9-12%<sup>1626</sup></td><td>22%<sup>835</sup>; 6%<sup>1627</sup></td></tr><tr><td>Aspergillus</td><td>2-3%<sup>1231</sup>; 3%<sup>1624</sup></td><td>4-5%<sup>801</sup>; 3%<sup>1627</sup>; 7-10%<sup>1626</sup></td><td>45%<sup>835</sup>; 11%<sup>1627</sup></td></tr><tr><td>Cladosporium</td><td>1-2%<sup>1231</sup>; 2%<sup>1624</sup></td><td>4-6%<sup>801</sup>; 2%<sup>1627</sup>; 8-10%<sup>1626</sup></td><td>24%<sup>835</sup>; 4%<sup>1627</sup></td></tr><tr><td>Penicillium</td><td>5%<sup>1624</sup>; 8%<sup>1625</sup></td><td>13-29%<sup>1626</sup></td><td>29%<sup>835</sup></td></tr></tbody></table>
<table><caption>TABLE 28 Major and minor relevant allergen molecules from fungi</caption><thead><tr><th>Species</th><th>Medical mycology</th><th>Allergen</th><th>Type of protein</th><th>MW (SDS-PAGE)</th><th>Exposure</th><th>Classified as</th></tr></thead><tbody><tr><td colspan="7">Proteases (Serin proteases*) n=24</td></tr><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 15</td><td>Serine protease</td><td>58 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus flavus</td><td>Mould</td><td>Asp f 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td rowspan="5">Aspergillus fumigatus</td><td rowspan="5">Mould</td><td>Asp f 5</td><td>Metalloprotease</td><td>40 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Asp f 10</td><td>Aspartate protease</td><td>34 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td>Asp f 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Asp f 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Asp f 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus niger</td><td>Mould</td><td>Asp n 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus oryzae</td><td>Mould</td><td>Asp o 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Aspergillus versicolor</td><td>Mould</td><td>Asp v 13</td><td>Extracellular alkaline serine protease</td><td>43 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium cladosporioides</td><td>Mould</td><td>Clac 9</td><td>Vacuolar serine protease</td><td>36 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Clah 9</td><td>Vacuolar serine protease</td><td>45 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td rowspan="2">Curvularia lunata</td><td rowspan="2">Mould</td><td>Cur 11</td><td>Serine protease</td><td>31 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Cur 14</td><td>Subtilisin like serine protease</td><td>54 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Epicoccum purpurascens</td><td>Mould</td><td>Epi p 1</td><td>Serine protease</td><td>30 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Fusarium proliferatum</td><td>Mould</td><td>Fus p 9</td><td>Vacuolar serine protease</td><td>36.5 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Penicillium brevicompactum</td><td>Mould</td><td>Pen b 13</td><td>Alkaline serine protease</td><td>33 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td rowspan="2">Penicillium chrysogenum</td><td rowspan="2">Mould</td><td>Pen ch 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Pen ch 18</td><td>Vacuolar serine protease</td><td>32 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 13</td><td>Alkaline serine protease</td><td>33 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium oxalicum</td><td>Mould</td><td>Pen o 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Rhodotorula mucilaginosa</td><td>Yeast</td><td>Rho m 2</td><td>Vacuolar serine protease</td><td>31 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td rowspan="2">Trichophyton rubrum</td><td rowspan="2">Dermatophyt</td><td>Tri r 2</td><td>Putative secreted alkaline protease Alp1</td><td>29 kDa</td><td>Contact</td><td>Minor allergen</td></tr><tr><td>Tri r 4</td><td>Serine protease</td><td>85 kDa</td><td>Contact</td><td>-</td></tr><tr><td>Trichophyton tonsurans</td><td>Dermatophyt</td><td>Tri t 4</td><td>Serine protease</td><td>83 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td colspan="7">Ribosomal proteins (P1* and P2*) n=9</td></tr><tr><td rowspan="2">Alternaria alternata</td><td rowspan="2">Mould</td><td>Alt a 5</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Alt a 12</td><td>Acid ribosomal protein P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td rowspan="2">Aspergillus fumigatus</td><td rowspan="2">Mould</td><td>Asp f 8</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Asp f 23</td><td>L3 ribosomal protein</td><td>44 kDa</td><td>Airway</td><td>-</td></tr><tr><td rowspan="2">Cladosporium herbarum</td><td rowspan="2">Mould</td><td>Clah 5</td><td>Acid ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Clah 12</td><td>Acid ribosomal protein P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Fusarium culmorum</td><td>Mould</td><td>Fus c 1</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Penicillium brevicompactum</td><td>Mould</td><td>Pen b 26</td><td>Acidic ribosomal prot. P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium crustosum</td><td>Mould</td><td>Pen cr 26</td><td>60S acidic ribosomal phosphoprotein P11 kDa</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td colspan="7">Isomerases (Cyclophilins*) n=6</td></tr><tr><td rowspan="2">Aspergillus fumigatus</td><td rowspan="2">Mould</td><td>Asp f 11</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>24 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Asp f 27</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>18 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Aspergillus terreus</td><td>Mould</td><td>Asp t 36</td><td>Triosephosphate isomerase (TIM)</td><td>28 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 6</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>17 kDa</td><td>Contact</td><td>Minor allergen</td></tr><tr><td>Psilocybe cubensis</td><td>Mushroom</td><td>Psi c 2</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>16 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Rhizopus oryzae</td><td>Mould</td><td>Rhi o 2</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>18 kDa</td><td>Airway</td><td>Major allergen</td></tr></tbody><tfoot><tr><td colspan="7">ABPA: allergic bronchialpulmonary aspergillosis; *cross-reactive pan allergen families according to [1629]</td></tr></tfoot></table>
<table><caption>TABLE 28 Major and minor relevant allergen molecules from fungi</caption><thead><tr><th>Species</th><th>Medical mycology</th><th>Allergen</th><th>Type of protein</th><th>MW (SDS-PAGE)</th><th>Exposure</th><th>Classified as</th></tr></thead><tbody><tr><td colspan="7">Proteases (Serin proteases*) n=24</td></tr><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 15</td><td>Serine protease</td><td>58 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus flavus</td><td>Mould</td><td>Asp f 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td rowspan="5">Aspergillus fumigatus</td><td rowspan="5">Mould</td><td>Asp f 5</td><td>Metalloprotease</td><td>40 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Asp f 10</td><td>Aspartate protease</td><td>34 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td>Asp f 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Asp f 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Asp f 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus niger</td><td>Mould</td><td>Asp n 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus oryzae</td><td>Mould</td><td>Asp o 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Aspergillus versicolor</td><td>Mould</td><td>Asp v 13</td><td>Extracellular alkaline serine protease</td><td>43 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium cladosporioides</td><td>Mould</td><td>Clac 9</td><td>Vacuolar serine protease</td><td>36 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Clah 9</td><td>Vacuolar serine protease</td><td>45 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td rowspan="2">Curvularia lunata</td><td rowspan="2">Mould</td><td>Cur 11</td><td>Serine protease</td><td>31 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Cur 14</td><td>Subtilisin like serine protease</td><td>54 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Epicoccum purpurascens</td><td>Mould</td><td>Epi p 1</td><td>Serine protease</td><td>30 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Fusarium proliferatum</td><td>Mould</td><td>Fus p 9</td><td>Vacuolar serine protease</td><td>36.5 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Penicillium brevicompactum</td><td>Mould</td><td>Pen b 13</td><td>Alkaline serine protease</td><td>33 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td rowspan="2">Penicillium chrysogenum</td><td rowspan="2">Mould</td><td>Pen ch 13</td><td>Alkaline serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Pen ch 18</td><td>Vacuolar serine protease</td><td>32 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 13</td><td>Alkaline serine protease</td><td>33 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium oxalicum</td><td>Mould</td><td>Pen o 18</td><td>Vacuolar serine protease</td><td>34 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Rhodotorula mucilaginosa</td><td>Yeast</td><td>Rho m 2</td><td>Vacuolar serine protease</td><td>31 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td rowspan="2">Trichophyton rubrum</td><td rowspan="2">Dermatophyt</td><td>Tri r 2</td><td>Putative secreted alkaline protease Alp1</td><td>29 kDa</td><td>Contact</td><td>Minor allergen</td></tr><tr><td>Tri r 4</td><td>Serine protease</td><td>85 kDa</td><td>Contact</td><td>-</td></tr><tr><td>Trichophyton tonsurans</td><td>Dermatophyt</td><td>Tri t 4</td><td>Serine protease</td><td>83 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td colspan="7">Ribosomal proteins (P1* and P2*) n=9</td></tr><tr><td rowspan="2">Alternaria alternata</td><td rowspan="2">Mould</td><td>Alt a 5</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Alt a 12</td><td>Acid ribosomal protein P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td rowspan="2">Aspergillus fumigatus</td><td rowspan="2">Mould</td><td>Asp f 8</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Asp f 23</td><td>L3 ribosomal protein</td><td>44 kDa</td><td>Airway</td><td>-</td></tr><tr><td rowspan="2">Cladosporium herbarum</td><td rowspan="2">Mould</td><td>Clah 5</td><td>Acid ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Clah 12</td><td>Acid ribosomal protein P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Fusarium culmorum</td><td>Mould</td><td>Fus c 1</td><td>Ribosomal protein P2</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Penicillium brevicompactum</td><td>Mould</td><td>Pen b 26</td><td>Acidic ribosomal prot. P1</td><td>11 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium crustosum</td><td>Mould</td><td>Pen cr 26</td><td>60S acidic ribosomal phosphoprotein P11 kDa</td><td>11 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td colspan="7">Isomerases (Cyclophilins*) n=6</td></tr><tr><td rowspan="2">Aspergillus fumigatus</td><td rowspan="2">Mould</td><td>Asp f 11</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>24 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Asp f 27</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>18 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Aspergillus terreus</td><td>Mould</td><td>Asp t 36</td><td>Triosephosphate isomerase (TIM)</td><td>28 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 6</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>17 kDa</td><td>Contact</td><td>Minor allergen</td></tr><tr><td rowspan="2">Psilocybe cubensis</td><td rowspan="2">Mushroom</td><td>Psi c 2</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>16 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Rhi o 2</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>18 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Rhizopus oryzae</td><td>Mould</td><td>Rhi o 2</td><td>Peptidyl-prolyl cis-trans isomerase (Cyclophilin)</td><td>18 kDa</td><td>Airway</td><td>Major allergen</td></tr></tbody><tfoot><tr><td colspan="7">ABPA: allergic bronchialpulmonary aspergillosis; *cross-reactive pan allergen families according to [1629]</td></tr></tfoot></table>
Enolases* n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 6</td><td>Enolase</td><td>45 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 22</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 6</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Curvularia lunata</td><td>Mould</td><td>Cur l 2</td><td>Enolase</td><td>48 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 22</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Rhodotorula mucilaginosa</td><td>Yeast</td><td>Rho m 1</td><td>Enolase</td><td>47 kDa</td><td>Airway</td><td>Minor allergen</td></tr></table>

Dehydrogenases n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 8</td><td>Mannitol dehydrogenase</td><td>29 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td></td><td></td><td>Alt a 10</td><td>Aldehyde dehydrogenase</td><td>53 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Candida albicans</td><td>Yeast</td><td>Cand a 1</td><td>Alcohol dehydrogenase</td><td>40 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 8</td><td>Mannitol dehydrogenase</td><td>28 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td></td><td></td><td>Cl a h 10</td><td>Aldehyde dehydrogenase</td><td>53 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia furfur</td><td>Dermatophyt</td><td>Mala f 4</td><td>Mitochondrial malate dehydrogenase</td><td>35 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Peroxisomal protein (Redoxin*) n=6

<table><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 3</td><td>Peroxisomal protein (Redoxin)</td><td>19 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Candida albicans</td><td>Yeast</td><td>Cand a 3</td><td>Peroxisomal protein (DJ-1/Pfpl family)</td><td>20 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Candida boidinii</td><td>Yeast</td><td>Cand b 2</td><td>Peroxisomal membrane protein A (Redoxin)</td><td>20 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 3</td><td>Peroxisomal membrane protein (Redoxin)</td><td>18 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia furfur</td><td>Dermatophyt</td><td>Mala f 2</td><td>Peroxisomal membrane protein (Redoxin)</td><td>21 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td></td><td></td><td>Mala f 3</td><td>Peroxisomal membrane protein (Redoxin)</td><td>20 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 5</td><td>Peroxisomal membrane protein (Redoxin)</td><td>18 kDa</td><td>Contact</td><td>Minor allergen</td></tr></table>

Thioredoxins* n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 4</td><td>Disulfide isomerase (Thioredoxin)</td><td>57 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 28</td><td>Thioredoxin</td><td>13 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td></td><td></td><td>Asp f 29</td><td>Thioredoxin</td><td>13 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Fusarium culmorum</td><td>Mould</td><td>Fus c 2</td><td>Thioredoxin-like protein</td><td>13 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Coprinus comatus</td><td>Mushroom</td><td>Cop c 2</td><td>Thioredoxin</td><td>12 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 13</td><td>Thioredoxin</td><td>13 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Heat shock proteins* n=4

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 3</td><td>Heat shock protein 70</td><td>70 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 12</td><td>Heat shock protein P90</td><td>90 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 19</td><td>Heat shock protein P70</td><td>70 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 10</td><td>Heat shock protein 70</td><td>86 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Mn Superoxid dismutases* n=3

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 14</td><td>Manganese superoxide dismutase</td><td>24 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 6</td><td>Mn superoxide dismutase</td><td>26.5 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 11</td><td>manganese superoxide dismutase</td><td>23 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Transaldolases n=3

<table><tr><td>Cladosporium cladosporioides</td><td>Mould</td><td>Cl a c 14</td><td>Transaldolase</td><td>36.5 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Fusarium proliferatum</td><td>Mould</td><td>Fus p 4</td><td>Transaldolase</td><td>37.5 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Penicillium chrysogenum</td><td>Mould</td><td>Pen ch 35</td><td>Transaldolase</td><td>36.5 kDa</td><td>Airway</td><td>-</td></tr></table>

YCP (Flavodoxin) n=2

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 7</td><td>YCP4 protein</td><td>22 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 7</td><td>YCP4 protein</td><td>22 kDa</td><td>Airway</td><td>Minor allergen</td></tr></table>

Alt a 1 family n=2

<table><tr><td>Alt a 1 family n=2</td><td>Mould</td><td>Alt a 1</td><td>acidic glycoprotein</td><td>15.3/16.4 kDa</td><td>Airway</td><td>major allergen</td></tr><tr><td>Alternaria alternata</td><td>Mould</td><td>Ulo c 1</td><td>Alt a 1 homologue</td><td>17 kDa</td><td>Airway</td><td>major allergen</td></tr><tr><td>Ulocladium chartarum</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></table>
Enolases* n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 6</td><td>Enolase</td><td>45 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 22</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 6</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Curvularia lunata</td><td>Mould</td><td>Cur l 2</td><td>Enolase</td><td>48 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 22</td><td>Enolase</td><td>46 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Rhodotorula mucilaginosa</td><td>Yeast</td><td>Rho m 1</td><td>Enolase</td><td>47 kDa</td><td>Airway</td><td>Minor allergen</td></tr></table>

Dehydrogenases n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 8</td><td>Mannitol dehydrogenase</td><td>29 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td></td><td></td><td>Alt a 10</td><td>Aldehyde dehydrogenase</td><td>53 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Candida albicans</td><td>Yeast</td><td>Cand a 1</td><td>Alcohol dehydrogenase</td><td>40 kDa</td><td>Airway</td><td>Major allergen (asthma)</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 8</td><td>Mannitol dehydrogenase</td><td>28 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td></td><td></td><td>Cl a h 10</td><td>Aldehyde dehydrogenase</td><td>53 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia furfur</td><td>Dermatophyt</td><td>Mala f 4</td><td>Mitochondrial malate dehydrogenase</td><td>35 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Peroxisomal protein (Redoxin*) n=6

<table><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 3</td><td>Peroxisomal protein (Redoxin)</td><td>19 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Candida albicans</td><td>Yeast</td><td>Cand a 3</td><td>Peroxisomal protein (DJ-1/Pfpl family)</td><td>20 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Candida boidinii</td><td>Yeast</td><td>Cand b 2</td><td>Peroxisomal membrane protein A (Redoxin)</td><td>20 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 3</td><td>Peroxisomal membrane protein (Redoxin)</td><td>18 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia furfur</td><td>Dermatophyt</td><td>Mala f 2</td><td>Peroxisomal membrane protein (Redoxin)</td><td>21 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td></td><td></td><td>Mala f 3</td><td>Peroxisomal membrane protein (Redoxin)</td><td>20 kDa</td><td>Contact</td><td>Major allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 5</td><td>Peroxisomal membrane protein (Redoxin)</td><td>18 kDa</td><td>Contact</td><td>Minor allergen</td></tr></table>

Thioredoxins* n=6

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 4</td><td>Disulfide isomerase (Thioredoxin)</td><td>57 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 28</td><td>Thioredoxin</td><td>13 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td></td><td></td><td>Asp f 29</td><td>Thioredoxin</td><td>13 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Fusarium culmorum</td><td>Mould</td><td>Fus c 2</td><td>Thioredoxin-like protein</td><td>13 kDa</td><td>Airway</td><td>Major allergen</td></tr><tr><td>Coprinus comatus</td><td>Mushroom</td><td>Cop c 2</td><td>Thioredoxin</td><td>12 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 13</td><td>Thioredoxin</td><td>13 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Heat shock proteins* n=4

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 3</td><td>Heat shock protein 70</td><td>70 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 12</td><td>Heat shock protein P90</td><td>90 kDa</td><td>Airway</td><td>Minor allergen (ABPA)</td></tr><tr><td>Penicillium citrinum</td><td>Mould</td><td>Pen c 19</td><td>Heat shock protein P70</td><td>70 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 10</td><td>Heat shock protein 70</td><td>86 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Mn Superoxid dismutases* n=3

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 14</td><td>Manganese superoxide dismutase</td><td>24 kDa</td><td>Airway</td><td>-</td></tr><tr><td>Aspergillus fumigatus</td><td>Mould</td><td>Asp f 6</td><td>Mn superoxide dismutase</td><td>26.5 kDa</td><td>Airway</td><td>Major allergen (ABPA)</td></tr><tr><td>Malassezia sympodialis</td><td>Dermatophyt</td><td>Mala s 11</td><td>manganese superoxide dismutase</td><td>23 kDa</td><td>Contact</td><td>Major allergen</td></tr></table>

Transaldolases n=3

<table><tr><td>Cladosporium cladosporioides</td><td>Mould</td><td>Cl a c 14</td><td>Transaldolase</td><td>36.5 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Fusarium proliferatum</td><td>Mould</td><td>Fus p 4</td><td>Transaldolase</td><td>37.5 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Penicillium chrysogenum</td><td>Mould</td><td>Pen ch 35</td><td>Transaldolase</td><td>36.5 kDa</td><td>Airway</td><td>-</td></tr></table>

YCP (Flavodoxin) n=2

<table><tr><td>Alternaria alternata</td><td>Mould</td><td>Alt a 7</td><td>YCP4 protein</td><td>22 kDa</td><td>Airway</td><td>Minor allergen</td></tr><tr><td>Cladosporium herbarum</td><td>Mould</td><td>Cl a h 7</td><td>YCP4 protein</td><td>22 kDa</td><td>Airway</td><td>Minor allergen</td></tr></table>

Alt a 1 family n=2

<table><tr><td>Alt a 1 family n=2</td><td>Mould</td><td>Alt a 1</td><td>acidic glycoprotein</td><td>15.3/16.4 kDa</td><td>Airway</td><td>major allergen</td></tr><tr><td>Alternaria alternata</td><td>Mould</td><td>Ulo c 1</td><td>Alt a 1 homologue</td><td>17 kDa</td><td>Airway</td><td>major allergen</td></tr><tr><td>Ulocladium chartarum</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></table>
Fungal allergens can be distinguished from other allergen sources, like pollen or animal dander, by typical protein families, particularly by subtilisin-like serine proteases.⁹ These subtilisin-like serin proteases are characterized mostly as major allergens, that means more than 50% of mould sensitized subjects had specific IgE to these serine-protease allergens.⁸³⁷⁻⁸³⁹ Unfortunately, these allergens are currently not available for diagnostic purposes, therefore their clinical relevance could not be investigated in clinical trials or patient collectives. Cross-reactions were described for alkaline as well as for vacuolar serine proteases (group 13 and 18 allergens of Asp f, Asp fl, Pen b, Pen c, Pen ch and Pen o) based on positive sIgE in 20 to 80% of mould-sensitized subjects.⁸⁴⁰,⁸⁴¹

Another fungal specific cross-reactive allergen family belongs to 60S acidic ribosomal proteins. For Fuc 1 sensitization prevalence of 35% was measured in Fusarium-allergic subjects.⁸⁴⁰,⁸⁴¹ It was further shown that the mycoprotein (Quorn) produced for human consumption by continuous fermentation of Fusarium venenatum on glucose substrate could induce IgE-mediated gastrointestinal

reaction 1-4 hs after ingestion.⁸⁴² In this clinical case the 60S acidic ribosomal protein P2 of F. venantum was probably the reason.

The term fungal isomerases summarized five peptidyl-prolyl-cis-trans isomerase (cyclophilin) and one triosephosphate isomerase (TIM) as allergens. Protein family of cyclophilin were identified as allergens in mould and pollen. Mould Asp f 11 was ascertained as the major allergen (90% sensitization rates in Aspergillus sensitized subjects and Mala s 6 induced sensitization rates between 21 and 25% in patients with atopic dermatitis⁸⁴¹ [Twaroch, 2015 #1434]. The first fungal TIM was identified as allergen in Aspergillus terreus as Asp t 36 inducing allergic rhinitis and asthma in sensitized patients.⁸⁴³ TIM is highly conserved and a common allergen of sea food and mite allergy. Clinical relevance of fungal TIM has to be determined.

The Allfam enolase protein family comprised currently 12 allergens, of which six were identified in mould, four in fish, one in latex and one in chicken (Figure 69). Cross-reactions are described among enolase, Alt a 6, Cla h 6 and Hev b 9,⁸⁴⁴ as well as among Asp f 22, Pen c 22 and Alt a 6. Sensitization prevalence to fungal enolases was about 14 – 30% (minor allergen) in mould-sensitized subjects.⁸⁴⁰,⁸⁴¹

<figure><img src="image_164.png" /><figcaption>(B) Enolase AF031</figcaption></figure>

<figure><img src="image_165.png" /><figcaption>(C) Short chain dehydrogenase AF028</figcaption></figure>

<figure><img src="image_166.png" /><figcaption>(D) Redoxin AF131</figcaption></figure>

<figure><img src="image_167.png" /><figcaption>(D) Fe/Mn superoxid dismutase AF020</figcaption></figure>

<figure><img src="image_168.png" /></figure>

**FIGURE 69** Ribbon diagram of commercially available mould allergens from EMBL-EBI Protein Data Bank in Europe (www.ebi.ac.uk/pdbe) showing (A) Homo dimer of Alt a 1 from Alternaria alternata (pdbe-No: 3v0r); (B) Homo dimer of enolase of Aspergillus fumigatus (pdbe-No: 7rhv); (C) Homo tetramer of NADP-dependent mannitol dehydrogenase from Cladosporium herbarum (Cla h 8) (pdbe-No: 3gdf); (D) homo dimer of peroxiredoxin of Aspergillus fumigatus (Asp f 3) (pdbe-No: 5j9b); (E) Homo tetramer of MnSOD of Aspergillus fumigatus (Asp f 6) (pdbe-No: 1KKC). * not classified as AF031 allergen family but with biochemical function of beta-enolase according to WHO/IUIS database; ** not listed in Allfam
Fungal allergens can be distinguished from other allergen sources, like pollen or animal dander, by typical protein families, particularly by subtilisin-like serine proteases.⁹ These subtilisin-like serin proteases are characterized mostly as major allergens, that means more than 50% of mould sensitized subjects had specific IgE to these serine-protease allergens.⁸³⁷⁻⁸³⁹ Unfortunately, these allergens are currently not available for diagnostic purposes, therefore their clinical relevance could not be investigated in clinical trials or patient collectives. Cross-reactions were described for alkaline as well as for vacuolar serine proteases (group 13 and 18 allergens of Asp f, Asp fl, Pen b, Pen c, Pen ch and Pen o) based on positive sIgE in 20 to 80% of mould-sensitized subjects.⁸⁴⁰,⁸⁴¹

Another fungal specific cross-reactive allergen family belongs to 60S acidic ribosomal proteins. For Fus c 1 sensitization prevalence of 35% was measured in Fusarium-allergic subjects.⁸⁴⁰,⁸⁴¹ It was further shown that the mycoprotein (Quorn) produced for human consumption by continuous fermentation of *Fusarium venenatum* on glucose substrate could induce IgE-mediated gastrointestinal

reaction 1-4 hs after ingestion.⁸⁴² In this clinical case the 60S acidic ribosomal protein P2 of F. venantum was probably the reason.

The term fungal isomerases summarized five peptidyl-prolyl-cis-trans isomerase (cyclophilin) and one triosephosphate isomerase (TIM) as allergens. Protein family of cyclophilin were identified as allergens in mould and pollen. Mould Asp f 11 was ascertained as the major allergen (90% sensitization rates in Aspergillus sensitized subjects and Mala s 6 induced sensitization rates between 21 and 25% in patients with atopic dermatitis⁸⁴¹ [Twaroch, 2015 #1434]. The first fungal TIM was identified as allergen in *Aspergillus terreus* as Asp t 36 inducing allergic rhinitis and asthma in sensitized patients.⁸⁴³ TIM is highly conserved and a common allergen of sea food and mite allergy. Clinical relevance of fungal TIM has to be determined.

The Allfam enolase protein family comprised currently 12 allergens, of which six were identified in mould, four in fish, one in latex and one in chicken (Figure 69). Cross-reactions are described among enolase, Alt a 6, Cla h 6 and Hev b 9,⁸⁴⁴ as well as among Asp f 22, Pen c 22 and Alt a 6. Sensitization prevalence to fungal enolases was about 14 – 30% (minor allergen) in mould-sensitized subjects.⁸⁴⁰,⁸⁴¹

<figure><img src="image_163.png" /><figcaption>(B) Enolase AF031</figcaption></figure>

<figure><img src="image_164.png" /><figcaption>(C) Short chain dehydrogenase AF028</figcaption></figure>

<figure><img src="image_165.png" /><figcaption>(D) Redoxin AF131</figcaption></figure>

<figure><img src="image_166.png" /><figcaption>(D) Fe/Mn superoxid dismutase AF020</figcaption></figure>

<figure><img src="image_167.png" /></figure>

**FIGURE 69** Ribbon diagram of commercially available mould allergens from EMBL-EBI Protein Data Bank in Europe (www.ebi.ac.uk/pdbe) showing (A) Homo dimer of Alt a 1 from Alternaria alternata (pdbe-No: 3v0r); (B) Homo dimer of enolase of Aspergillus fumigatus (pdbe-No: 7rhv); (C) Homo tetramer of NADP-dependent mannitol dehydrogenase from Cladosporium herbarum (Cla h 8) (pdbe-No: 3gdf); (D) homo dimer of peroxiredoxin of Aspergillus fumigatus (Asp f 3) (pdbe-No: 5j9b); (E) Homo tetramer of MnSOD of Aspergillus fumigatus (Asp f 6) (pdbe-No: 1KKC). * not classified as AF031 allergen family but with biochemical function of beta-enolase according to WHO/IUIS database; ** not listed in Allfam
Fungal dehydrogenase were characterized as allergens covering four different allergen families and cross-reactivity was shown among mannitol dehydrogenases from Cladosporium herbarum Clah8 and Alt a 8.<sup>845,846</sup> The sensitization rate against mould dehydrogenases was between 41 and 57% in mould-sensitized subjects.<sup>841</sup>

Peroxisomal membrane proteins belong to the redoxin allergen family (Figure 69) and were exclusively found in fungi. Cross-reactive epitopes were described for Asp f 3 and peroxisomal membrane protein in *Candida boidinii*.<sup>847</sup> In particular, Asp f 3 demonstrated high slgE-binding potential of 49 - 72% in Aspergillus-sensitized subjects<sup>841</sup> and is useful for diagnostic differentiation between asthma and ABPA in Aspergillus-sensitized subjects. Five out of eight allergens characterized as thioredoxins are derived from moulds. About 50% of patients allergic to Fusarium had specific IgE against thioredoxin Fus c 2.<sup>841</sup> Another cross-reactive fungal allergen family comprised heat shock proteins (HSP 70) with Alt a 3 (5% sensitization (www.allergen.org)) and Pen c 19 (41% sensitization) in mould-sensitized subjects.<sup>840</sup>

Highly conserved protein structure is also found in manganese superoxide dismutases (MnSOD) with confirmed cross-reactivity between Asp f 6 and Alt a 1<sup>840</sup> (Figure 69). MnSOD from Aspergillus fumigatus, Asp f 6, is also useful for diagnostic differentiation between asthma and ABPA in Aspergillus-sensitized subjects.

Three fungal transaldolase allergens have been identified exclusively in mould species. IgE cross-reactivity of Cla c 14 and Pen ch 35<sup>848</sup> as well as Fus p 4 and Cla h 14<sup>849</sup> has been shown. A possible contribution of transaldolase to allergic disorders has been discussed due to its homology to human autoantigens.<sup>848,849</sup>

Mould allergens Alt a 7 and Cla h 7 belong to the flavodoxin protein family<sup>840</sup> but had only minor slgE-binding potency of 7 - 22% in mould-sensitized patients (www.allergen.org). Alt a 1, the major allergen of Alternaria alternata with a sensitization prevalence of more than 90% is one of the most clinically relevant fungal allergens.<sup>840,850</sup> Alt a 1 consists of two subunits of a unique, dimeric β-barrel structure.<sup>852</sup> and a new allergen family with unknown function, but exclusively occurrence in fungi was introduced in AllFam list, as Alt a 1 family (Figure 69).

Among clinically important A. fumigatus single allergens Asp f 1 is a major allergen in patients suffering from ABPA (80 - 85%) as well as in A. fumigatus sensitized asthmatics (50 - 84.5%).<sup>853-855</sup> Asp f 1 is

related to ribotoxins, which are known to inhibit protein translation and are highly toxic for humans and effectiveness of this allergen in diagnosis and therapy is still controversial.<sup>840</sup> The protein family was not structurally classified by Allfam until now and cross-reaction to other allergens were not published. Asp f 2 has been described as major allergen in ABPA patients (87 - 100%)<sup>840,856</sup> and was classified as metalloprotease M35 family AF211. However, there are no other allergens listed in this allergen family. Regarding Asp f 4, there was no classification to any allergen family, but in patients with cystic fibrosis or ABPA, Asp f 4 is a major allergen with about 80%.<sup>840</sup>

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

The clinical availability of both skin prick tests and serological tools for IgE-mediated mould allergy diagnosis are continuously reducing, and standardization of mould extract is still difficult.<sup>857,858</sup> The comparison of mould test solutions from different manufacturers showed a heterogeneous protein content, despite being prepared from supposedly identical allergen sources.<sup>858</sup> This might be one reason for the discrepancy between skin prick test results and serologic IgE determinations. The concordance between skin tests and serological tests can be less than 30% depending on the mould species,<sup>835,1383</sup> and skin tests were more sensitive compared with serological IgE-diagnosis. Therefore, molecular allergy diagnosis with recombinant mould allergens can offer valuable results. Using component-based serological tests rAlt a 1 and rAsp f 1, 2, 3, 4, 6 to measure serological sensitization to Aspergillus fumigatus and Alternaria alternata, 80% of extract-based serological sensitization were covered.<sup>869</sup> Even though numerous fungal allergens have been identified, there are currently only eight single mould allergens commercially available for testing as shown in (Figure 70).

IgE to rAlt a 1 (available as single allergen and on multiplex platform) was measured in patients with sensitization to *Alternaria alternata* in 47% with atopic dermatitis and up to 98% in patients with allergic asthma.<sup>841,849,859</sup> IgE-mediated sensitizations to Alternaria can be detected by testing rAlt a 1, which makes this single allergen valuable for standardization of test extracts.<sup>835,840,850,869</sup> Among

<figure><img src="image_169.png" /><figcaption>FIGURE 70 Electron-microscopic image of mould species with commercially available single allergens</figcaption></figure>
Fungal dehydrogenase were characterized as allergens covering four different allergen families and cross-reactivity was shown among mannitol dehydrogenases from Cladosporium herbarum Clah8 and Alt a 8.<sup>845,846</sup> The sensitization rate against mould dehydrogenases was between 41 and 57% in mould-sensitized subjects.<sup>841</sup>

Peroxisomal membrane proteins belong to the redoxin allergen family (Figure 69) and were exclusively found in fungi. Cross-reactive epitopes were described for Asp f 3 and peroxisomal membrane protein in *Candida boidinii*.<sup>847</sup> In particular, Asp f 3 demonstrated high slgE-binding potential of 49 - 72% in Aspergillus-sensitized subjects<sup>841</sup> and is useful for diagnostic differentiation between asthma and ABPA in Aspergillus-sensitized subjects. Five out of eight allergens characterized as thioredoxins are derived from moulds. About 50% of patients allergic to Fusarium had specific IgE against thioredoxin Fus c 2.<sup>841</sup> Another cross-reactive fungal allergen family comprised heat shock proteins (HSP 70) with Alt a 3 (5% sensitization (www.allergen.org)) and Pen c 19 (41% sensitization) in mould-sensitized subjects.<sup>840</sup>

Highly conserved protein structure is also found in manganese superoxide dismutases (MnSOD) with confirmed cross-reactivity between Asp f 6 and Alt a 1<sup>840</sup> (Figure 69). MnSOD from Aspergillus fumigatus, Asp f 6, is also useful for diagnostic differentiation between asthma and ABPA in Aspergillus-sensitized subjects.

Three fungal transaldolase allergens have been identified exclusively in mould species. IgE cross-reactivity of Cla c 14 and Pen ch 35<sup>848</sup> as well as Fus p 4 and Cla h 14<sup>849</sup> has been shown. A possible contribution of transaldolase to allergic disorders has been discussed due to its homology to human autoantigens.<sup>848,849</sup>

Mould allergens Alt a 7 and Cla h 7 belong to the flavodoxin protein family<sup>840</sup> but had only minor slgE-binding potency of 7 - 22% in mould-sensitized patients (www.allergen.org). Alt a 1, the major allergen of Alternaria alternata with a sensitization prevalence of more than 90% is one of the most clinically relevant fungal allergens.<sup>840,850</sup> Alt a 1 consists of two subunits of a unique, dimeric β-barrel structure.<sup>852</sup> and a new allergen family with unknown function, but exclusively occurrence in fungi was introduced in AllFam list, as Alt a 1 family (Figure 69).

Among clinically important A. fumigatus single allergens Asp f 1 is a major allergen in patients suffering from ABPA (80 - 85%) as well as in A. fumigatus sensitized asthmatics (50 - 84.5%).<sup>853-855</sup> Asp f 1 is

related to ribotoxins, which are known to inhibit protein translation and are highly toxic for humans and effectiveness of this allergen in diagnosis and therapy is still controversial.<sup>840</sup> The protein family was not structurally classified by Allfam until now and cross-reaction to other allergens were not published. Asp f 2 has been described as major allergen in ABPA patients (87 - 100%)<sup>840,856</sup> and was classified as metalloprotease M35 family AF211. However, there are no other allergens listed in this allergen family. Regarding Asp f 4, there was no classification to any allergen family, but in patients with cystic fibrosis or ABPA, Asp f 4 is a major allergen with about 80%.<sup>840</sup>

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

The clinical availability of both skin prick tests and serological tools for IgE-mediated mould allergy diagnosis are continuously reducing, and standardization of mould extract is still difficult.<sup>857,858</sup> The comparison of mould test solutions from different manufacturers showed a heterogeneous protein content, despite being prepared from supposedly identical allergen sources.<sup>858</sup> This might be one reason for the discrepancy between skin prick test results and serologic IgE determinations. The concordance between skin tests and serological tests can be less than 30% depending on the mould species,<sup>835,1383</sup> and skin tests were more sensitive compared with serological IgE-diagnosis. Therefore, molecular allergy diagnosis with recombinant mould allergens can offer valuable results. Using component-based serological tests rAlt a 1 and rAsp f 1, 2, 3, 4, 6 to measure serological sensitization to Aspergillus fumigatus and Alternaria alternata, 80% of extract-based serological sensitization were covered.<sup>869</sup> Even though numerous fungal allergens have been identified, there are currently only eight single mould allergens commercially available for testing as shown in (Figure 70).

IgE to rAlt a 1 (available as single allergen and on multiplex platform) was measured in patients with sensitization to *Alternaria alternata* in 47% with atopic dermatitis and up to 98% in patients with allergic asthma.<sup>841,849,859</sup> IgE-mediated sensitizations to Alternaria can be detected by testing rAlt a 1, which makes this single allergen valuable for standardization of test extracts.<sup>835,840,850,869</sup> Among

<figure><img src="image_168.png" /><figcaption>FIGURE 70 Electron-microscopic image of mould species with commercially available single allergens</figcaption></figure>
Alternaria alternata sensitized patients (92% reported allergic rhinitis and 64.2% asthma) Alt a 1 sensitization occurred significantly more frequently in children (69.8%) than in adults (30.2%) as shown in a recent Spanish study.⁸⁶⁰ Sensitization to Alt a 1 is closely associated with asthma and increased asthma medication.⁵⁵,⁸⁴¹ The protein structure of Alt a 1 (Figure 69) is formed as a dimer of acidic glycoprotein without known biochemical function, with a unique butterfly-like dimer protein structure, which was exclusively found among mould proteins.⁸⁵² Cross-reactive structures of Alt a 1-homologues were identified in other Pleosporaceae genera like Ulocladium but not in mould genera such as Aspergillus, Penicillium, or Cladosporium. A clinical trial of Alt a 1 specific immunotherapy was recently published,⁸⁶¹ showing efficacy and safety of the applied subcutaneous immunotherapy, particularly with higher dose of Alt a 1. A comparison of this newly introduced component-based Alt a 1 SIT with extract-based *Alternaria alternata* SIT has to be evaluated.

IgE to Alt a 6 (available as allergen on multiplex platform) belong to enolase family and is a minor allergen in *Alternaria alternata*-sensitized patients with sensitization rates between 20 and 30%.⁸⁶² There are currently six fungal enolases (5 mould, 1 yeast, Figure 69) sharing sequence identity of 72 – 94% and IgE-binding epitopes were shown to be highly conserved and cross-reactive.⁸⁶² Enolases were identified further as allergens in animals (food) and plants (pollen and natural rubber latex) as shown in (Figure 69). Based on the known sequence homology, and conserve IgE-binding epitopes cross-reactions between these allergens are likely.⁸⁶² To date, IgE-cross-reactions were shown by inhibition studies for rHev b 9, rAlt a 6 and rCla h 6.⁸⁴⁰ IgE to Cla h 8 (available as allergen on a multiplex platform) is a short-chain dehydrogenase (Figure 69), with sensitization prevalence of 57.1% among Cladosporium herbarum-sensitized subjects.⁸⁴¹ Cross-reactions to other members of the short chain dehydrogenase family was described for Alt a 8 from *Alternaria alternata*.⁸⁴⁰

IgE to Asp f 1, 2, 3, 4, 6 (available as single allergens and multiplex platform) are valuable tools in differential diagnosis of ABPA in asthmatics with Aspergillus fumigatus sensitization⁸⁵⁴⁻⁸⁵⁶,⁸⁶³ (see clinical case⁸⁶⁴ below). Diagnostic criteria of ABPA in patients with asthma are still based on Rosenberg-Patterson criteria,⁸⁶⁵ which are applied until today with small modifications.⁸⁵⁵ The diagnosis of ABPA can be made if six out of eight main criteria are given. One of these main ABPA diagnosis criteria is slgE-binding to Aspergillus fumigatus and previous studies showed slgE to Asp f 2 plus Asp f 4 plus Asp f 6 occurred more frequently in patients with ABPA than in asthmatic.⁸⁵⁴,⁸⁶⁶ A more recent meta-analysis⁸⁶³ investigating Asp f recombinant allergens in 26 studies (including 1694 patients) revealed that IgE to Asp f 1 or Asp f 3 had the highest sensitivity (96.7% in asthmatics and 93.3% cystic fibrosis (CF)—patients) to differentiate ABPA among these patients, but Asp f 4 or Asp f 6 had the highest specificity with 99% in asthmatics versus Asp f 6 alone with 98% in CF-patients. It is therefore not trivial to designate one specific Asp f allergen or a combination pattern of Asp f single allergens as diagnostic markers for ABPA. What has almost always been shown, however, is that slgE to recombinant Asp f allergens were

detected significantly more often and with higher concentrations in patients with ABPA.

Regarding type I allergy diagnosis a typical major allergen, comparable to Alt a 1 in Alternaria alternata, is missing in Aspergillus fumigatus, as well as in all other mould species. Recombinant Asp f allergens can be grouped into secreted allergens comprising Asp f 1 and Asp f 3 and nonsecreted allergens (Asp f 4 and Asp f 6).⁸⁶³ In a study⁸⁶⁹ investigating single Aspergillus fumigatus components in Aspergillus-sensitized patients with and without asthma most frequently slgE to rAsp f 1 with 53% and rAsp f 3 with 47% were measured, followed by rAsp f 2 and rAsp f 4 with 26% and rAsp f 6 with 16% in all sensitized subjects. There was no significant difference in slgE sensitization to single rAsp f components depending on asthma, slgE to rAsp f 1 / rAsp f 3 was measured in 46% / 46% of asthmatic and 67% / 50% of nonasthmatic Aspergillus sensitized patients. Frequency of slgE to rAsp f 2, Asp f 4 and Asp f 6 were 15 – 31% in asthmatics and 17% in nonasthmatics comparable. These results correspond with the previously described compartments of Asp f 1 and Asp f 3 as secretory proteins with high IgE-binding frequency compared with Asp f 2, Asp f 4 and Asp f 6 as intracellular proteins with lower IgE-binding frequency.

## 4 – Clinical management

For clarification of a mould-associated respiratory allergy anamnesis, prick testing or serological IgE determination are recommended according to the diagnostic allergy algorithm (Figure 71). Since more and more mould test solutions are being withdrawn from the market,⁸⁶⁷ serological IgE determination is almost the only test tool available, although it is often less sensitive than skin prick tests.¹³⁸³ In principle any mould can cause IgE sensitization and the exact determination of mould exposure is not possible in most cases. *In vitro* testing of a mould mixture (mx1) consisting of Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus and Pencillium chrysosogenum was shown to be sufficient to detect IgE reactions to all the contained individual mould species.⁸⁶⁸,¹³⁸³ If mould-associated IgE is measurable, a possible exposure should be verified anamnestically (indoor versus outdoor) and possible co-sensitizations such as grass pollen or house dust mites, which represent overlapping allergen exposures, must be investigated (Figure 71).

## 5 – Clinical Cases

The presented case report was recently published by.⁸⁶⁴

### Patient history

A 55 year old man reported rhinitis and cough with viscous secretion over two month and occasional discrete shortness of breath and mucus plug secretion associated with cough in 2010. Tree pollen allergy with seasonal rhino-conjunctivitis in March - April and oral allergy syndrome to Rosaceae fruits (e.g. apple) has been known for many years but had improved during last years. The patient had a
Alternaria alternata sensitized patients (92% reported allergic rhinitis and 64.2% asthma) Alt a 1 sensitization occurred significantly more frequently in children (69.8%) than in adults (30.2%) as shown in a recent Spanish study.⁸⁶⁰ Sensitization to Alt a 1 is closely associated with asthma and increased asthma medication.⁵⁵,⁸⁴¹ The protein structure of Alt a 1 (Figure 69) is formed as a dimer of acidic glycoprotein without known biochemical function, with a unique butterfly-like dimer protein structure, which was exclusively found among mould proteins.⁸⁵² Cross-reactive structures of Alt a 1-homologues were identified in other Pleosporaceae genera like Ulocladium but not in mould genera such as Aspergillus, Penicillium, or Cladosporium. A clinical trial of Alt a 1 specific immunotherapy was recently published,⁸⁶¹ showing efficacy and safety of the applied subcutaneous immunotherapy, particularly with higher dose of Alt a 1. A comparison of this newly introduced component-based Alt a 1 SIT with extract-based *Alternaria alternata* SIT has to be evaluated.

IgE to Alt a 6 (available as allergen on multiplex platform) belong to enolase family and is a minor allergen in *Alternaria alternata*-sensitized patients with sensitization rates between 20 and 30%.⁸⁶² There are currently six fungal enolases (5 mould, 1 yeast, Figure 69) sharing sequence identity of 72 – 94% and IgE-binding epitopes were shown to be highly conserved and cross-reactive.⁸⁶² Enolases were identified further as allergens in animals (food) and plants (pollen and natural rubber latex) as shown in (Figure 69). Based on the known sequence homology, and conserve IgE-binding epitopes cross-reactions between these allergens are likely.⁸⁶² To date, IgE-cross-reactions were shown by inhibition studies for rHev b 9, rAlt a 6 and rCla h 6.⁸⁴⁰ IgE to Cla h 8 (available as allergen on a multiplex platform) is a short-chain dehydrogenase (Figure 69), with sensitization prevalence of 57.1% among Cladosporium herbarum-sensitized subjects.⁸⁴¹ Cross-reactions to other members of the short chain dehydrogenase family was described for Alt a 8 from *Alternaria alternata*.⁸⁴⁰

IgE to Asp f 1, 2, 3, 4, 6 (available as single allergens and multiplex platform) are valuable tools in differential diagnosis of ABPA in asthmatics with Aspergillus fumigatus sensitization⁸⁵⁴⁻⁸⁵⁶,⁸⁶³ (see clinical case⁸⁶⁴ below). Diagnostic criteria of ABPA in patients with asthma are still based on Rosenberg-Patterson criteria,⁸⁶⁵ which are applied until today with small modifications.⁸⁵⁵ The diagnosis of ABPA can be made if six out of eight main criteria are given. One of these main ABPA diagnosis criteria is slgE-binding to Aspergillus fumigatus and previous studies showed slgE to Asp f 2 plus Asp f 4 plus Asp f 6 occurred more frequently in patients with ABPA than in asthmatic.⁸⁵⁴,⁸⁶⁶ A more recent meta-analysis⁸⁶³ investigating Asp f recombinant allergens in 26 studies (including 1694 patients) revealed that IgE to Asp f 1 or Asp f 3 had the highest sensitivity (96.7% in asthmatics and 93.3% cystic fibrosis (CF)—patients) to differentiate ABPA among these patients, but Asp f 4 or Asp f 6 had the highest specificity with 99% in asthmatics versus Asp f 6 alone with 98% in CF-patients. It is therefore not trivial to designate one specific Asp f allergen or a combination pattern of Asp f single allergens as diagnostic markers for ABPA. What has almost always been shown, however, is that slgE to recombinant Asp f allergens were

detected significantly more often and with higher concentrations in patients with ABPA.

Regarding type I allergy diagnosis a typical major allergen, comparable to Alt a 1 in Alternaria alternata, is missing in Aspergillus fumigatus, as well as in all other mould species. Recombinant Asp f allergens can be grouped into secreted allergens comprising Asp f 1 and Asp f 3 and nonsecreted allergens (Asp f 4 and Asp f 6).⁸⁶³ In a study⁸⁶⁹ investigating single Aspergillus fumigatus components in Aspergillus-sensitized patients with and without asthma most frequently slgE to rAsp f 1 with 53% and rAsp f 3 with 47% were measured, followed by rAsp f 2 and rAsp f 4 with 26% and rAsp f 6 with 16% in all sensitized subjects. There was no significant difference in slgE sensitization to single rAsp f components depending on asthma, slgE to rAsp f 1 / rAsp f 3 was measured in 46% / 46% of asthmatic and 67% / 50% of nonasthmatic Aspergillus sensitized patients. Frequency of slgE to rAsp f 2, Asp f 4 and Asp f 6 were 15 – 31% in asthmatics and 17% in nonasthmatics comparable. These results correspond with the previously described compartments of Asp f 1 and Asp f 3 as secretory proteins with high IgE-binding frequency compared with Asp f 2, Asp f 4 and Asp f 6 as intracellular proteins with lower IgE-binding frequency.

## 4 – Clinical management

For clarification of a mould-associated respiratory allergy anamnesis, prick testing or serological IgE determination are recommended according to the diagnostic allergy algorithm (Figure 71). Since more and more mould test solutions are being withdrawn from the market,⁸⁶⁷ serological IgE determination is almost the only test tool available, although it is often less sensitive than skin prick tests.¹³⁸³ In principle any mould can cause IgE sensitization and the exact determination of mould exposure is not possible in most cases. *In vitro* testing of a mould mixture (mx1) consisting of Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus and Pencillium chrysogenum was shown to be sufficient to detect IgE reactions to all the contained individual mould species.⁸⁶⁸,¹³⁸³ If mould-associated IgE is measurable, a possible exposure should be verified anamnestically (indoor versus outdoor) and possible co-sensitizations such as grass pollen or house dust mites, which represent overlapping allergen exposures, must be investigated (Figure 71).

## 5 – Clinical Cases

The presented case report was recently published by.⁸⁶⁴

### Patient history

A 55 year old man reported rhinitis and cough with viscous secretion over two month and occasional discrete shortness of breath and mucus plug secretion associated with cough in 2010. Tree pollen allergy with seasonal rhino-conjunctivitis in March - April and oral allergy syndrome to *Rosaceae* fruits (e.g. apple) has been known for many years but had improved during last years. The patient had a
<figure><img src="image_170.png" /><figcaption>FIGURE 71 Decision algorithm for patients with clinical history of mould associated respiratory allergy; Mould mix (mx1): Aspergillus fumigatus, Penicillium chrysogenum, Cladosporium herbarum, Alternaria alternata; D recommendation according to ¹⁴⁵; *recommendation according to ¹³⁹⁷ **optional, a cut-off IgE to Asp f 1 > 4.47 kU/L; b cut-off IgE to Asp f 2 > 1.3 kU/L; c cut-off total IgE > 417 IU/ml according to ⁸⁵⁶, SBS: sick building syndrome; MMIS: mucous-membrane irritation syndrome, ODTS: organic dust doxic syndrome (endotoxin, mycotoxins)</figcaption></figure>

good general condition and was neither obviously exposed to dust or mould nor to pets.

### Clinical examination

The clinical examination in 2010 showed apically attenuated breath sounds on auscultation in both lungs with otherwise normal findings. Lung function showed a borderline obstructive disorder with normal vital capacity, but fractionated FeNO was significantly elevated at 95 ppb, indicating a Th-2 asthma bronchiale.

### SPT

In 2010 strong skin reactions were shown in prick test to birch and ash pollen, as well as to the moulds Aspergillus fumigatus and Alternaria alternata and a weak reaction to house dust mites.

### In-vitro testing

Serologically, a massively increased total IgE (> 6000 kU/L) was measured, as well as strongly increased sIgE concentrations to Aspergillus fumigatus (78.5 kU/L, CAP class 5) and Alternaria alternata (100 kU/L, CAP class 6). Additionally, high sIgG concentration on Aspergillus fumigatus in the sense of a type III allergic reaction, as well as an eosinophilia, were indicative of a possible ABPA. Serological testing for the components rAsp f 2 (11.1 kU/L,

CAP class 3), rAsp f 4 (0.53 kU/L, CAP class 1) and rAsp f 6 (0.40 kU/L, CAP class 1) underlined the suspicion of ABPA.

### Diagnosis

According to the Rosenberg-Patterson diagnostic criteria,⁸⁶⁵ five major and two minor criteria were present and the criteria of the International Society for Human and Animal Mycology (ISHAM)⁸⁵⁵ were also fulfilled, therefore the diagnosis of ABPA was made.

### Disease progression

After a long stable course under inhaled asthma therapy and a steady decrease in total IgE and sIgE to Aspergillus fumigatus, clinical worsening occurred in 2014 and 2015 with significant increases of total IgE and sIgE to Aspergillus fumigatus and the components rAsp f 2, rAsp f 4 and rAsp f 6. The relapses were ameliorated by systemic steroid administration for several weeks, and no further attacks occurred during the last four years under inhaled asthma therapy. A chest CT performed in May 2016 did not reveal bronchiectasis.⁸⁶⁴

### Diagnostic tools

The importance of sIgE to Asp f 2, 4 and 6 in the diagnosis of ABPA in both asthma and cystic fibrosis (CF) patients, as described initially,⁸⁶⁶ and calculation of a recent meta-analysis⁸⁶³ showed
<figure><img src="image_169.png" /><figcaption>FIGURE 71 Decision algorithm for patients with clinical history of mould associated respiratory allergy; Mould mix (mx1): Aspergillus fumigatus, Penicillium chrysogenum, Cladosporium herbarum, Alternaria alternata; D recommendation according to ¹⁴⁵; *recommendation according to ¹³⁹⁷ **optional, a cut-off IgE to Asp f 1 > 4.47 kU/L; b cut-off IgE to Asp f 2 > 1.3 kU/L; c cut-off total IgE > 417 IU/ml according to ⁸⁵⁶, SBS: sick building syndrome; MMIS: mucous-membrane irritation syndrome, ODTS: organic dust doxic syndrome (endotoxin, mycotoxins)</figcaption></figure>

good general condition and was neither obviously exposed to dust or mould nor to pets.

### Clinical examination

The clinical examination in 2010 showed apically attenuated breath sounds on auscultation in both lungs with otherwise normal findings. Lung function showed a borderline obstructive disorder with normal vital capacity, but fractionated FeNO was significantly elevated at 95 ppb, indicating a Th-2 asthma bronchiale.

### SPT

In 2010 strong skin reactions were shown in prick test to birch and ash pollen, as well as to the moulds Aspergillus fumigatus and Alternaria alternata and a weak reaction to house dust mites.

### In-vitro testing

Serologically, a massively increased total IgE (> 6000 kU/L) was measured, as well as strongly increased sIgE concentrations to Aspergillus fumigatus (78.5 kU/L, CAP class 5) and Alternaria alternata (100 kU/L, CAP class 6). Additionally, high sIgG concentration on Aspergillus fumigatus in the sense of a type III allergic reaction, as well as an eosinophilia, were indicative of a possible ABPA. Serological testing for the components rAsp f 2 (11.1 kU/L,

CAP class 3), rAsp f 4 (0.53 kU/L, CAP class 1) and rAsp f 6 (0.40 kU/L, CAP class 1) underlined the suspicion of ABPA.

### Diagnosis

According to the Rosenberg-Patterson diagnostic criteria,⁸⁶⁵ five major and two minor criteria were present and the criteria of the International Society for Human and Animal Mycology (ISHAM)⁸⁵⁵ were also fulfilled, therefore the diagnosis of ABPA was made.

### Disease progression

After a long stable course under inhaled asthma therapy and a steady decrease in total IgE and sIgE to Aspergillus fumigatus, clinical worsening occurred in 2014 and 2015 with significant increases of total IgE and sIgE to Aspergillus fumigatus and the components rAsp f 2, rAsp f 4 and rAsp f 6. The relapses were ameliorated by systemic steroid administration for several weeks, and no further attacks occurred during the last four years under inhaled asthma therapy. A chest CT performed in May 2016 did not reveal bronchiectasis.⁸⁶⁴

### Diagnostic tools

The importance of sIgE to Asp f 2, 4 and 6 in the diagnosis of ABPA in both asthma and cystic fibrosis (CF) patients, as described initially,⁸⁶⁶ and calculation of a recent meta-analysis⁸⁶³ showed
that ABPA diagnose specificity based on sIgE ≥ 0.35 kU/L for Asp f 4 plus Asp f 6 was 99.2%. A further study from India⁸⁵⁶ was able to show that Asp f 1 (≥ 4.4 kU/L) and Asp f 2 (≥ 1.3 kU/L) are valuable tools to differentiate between Aspergillus fumigatus-sensitive asthmatics and asthmatic patients with ABPA with a sensitivity of 100% and a specificity of 81%. Thus, in the future, sIgE to Asp f 1 and Asp f 2 in combination with total IgE can prevail instead of sIgE to Aspergillus fumigatus extract. To confirm the ABPA diagnosis, sIgE against Asp f 4 and Asp f 6 could be measured. However, the transfer of results obtained with the serological parameters (Asp f 1 and Asp f 2) to European patient collectives still needs to be verified. As general recommendation for patients with asthma or cystic fibrosis determination of total IgE is useful to avoid overlooking ABPA.⁸⁶⁴

## 6 – Research and future perspectives

Due to the difficult production and standardization of mould allergen extracts, it is highly demanded to improve mould allergy diagnosis using single allergen components.

In the future, subtilisin-like proteases and other mould typical cross-reactive allergen families could be helpful in molecular allergy diagnosis of moulds.

Especially diagnostic evaluation of single Asp f allergens for differentiation between asthma and ABPA should be verified for more ethnic groups of patients.
that ABPA diagnose specificity based on sIgE ≥ 0.35 kU/L for Asp f 4 plus Asp f 6 was 99.2%. A further study from India⁸⁵⁶ was able to show that Asp f 1 (≥ 4.4 kU/L) and Asp f 2 (≥ 1.3 kU/L) are valuable tools to differentiate between Aspergillus fumigatus-sensitive asthmatics and asthmatic patients with ABPA with a sensitivity of 100% and a specificity of 81%. Thus, in the future, sIgE to Asp f 1 and Asp f 2 in combination with total IgE can prevail instead of sIgE to Aspergillus fumigatus extract. To confirm the ABPA diagnosis, sIgE against Asp f 4 and Asp f 6 could be measured. However, the transfer of results obtained with the serological parameters (Asp f 1 and Asp f 2) to European patient collectives still needs to be verified. As general recommendation for patients with asthma or cystic fibrosis determination of total IgE is useful to avoid overlooking ABPA.⁸⁶⁴

## 6 – Research and future perspectives

Due to the difficult production and standardization of mould allergen extracts, it is highly demanded to improve mould allergy diagnosis using single allergen components.

In the future, subtilisin-like proteases and other mould typical cross-reactive allergen families could be helpful in molecular allergy diagnosis of moulds.

Especially diagnostic evaluation of single Asp f allergens for differentiation between asthma and ABPA should be verified for more ethnic groups of patients.
# B08 - Microbial allergens /antigens

## Carole Guillet, Rebecca Czolk, Philipp Bosshard, Peter Schmid-Grendelmeier, Annette Kuehn

### Highlights

* *Malassezia* species are commensals of the normal skin flora and are part of the skin microbiome.

* Sensitization to *Malassezia* species allergens can frequently be found in head and neck type atopic dermatitis.

* Fourteen allergens from 3 *Malassezia* species have been characterized to date.

* The skin microbiome, especially *Malassezia* spp and *Staphylococcus aureus*, can be a target in AD therapy.

* The impact of commensal microbiomes on allergies and other barrier diseases is a rapidly growing research field.

## 1 - Introduction

The skin is a complex ecosystem harboring diverse and site-specific microbial communities referred to as the skin microbiome. Phylogenetic profiling of the skin microbiome of healthy individuals revealed that bacteria are predominant at most body sites.⁸⁷⁰ Additionally, fungi—also referred to as skin mycobiome—play an essential part in these microbial communities. They account for 1% to 22% of the phylogenetic composition of the skin microbiome. In healthy skin, the fungal flora almost exclusively harbors *Malassezia* species (*spp*).⁸⁷⁰

Studies suggest a significant role of the skin microbiome in the development and progression of atopic dermatitis (AD).⁸⁷¹,⁸⁷² AD is a chronic inflammatory skin disease characterized by eczematous lesions, pruritus, and a chronic/relapsing history of symptoms.⁸⁷²,⁸⁷³ Its prevalence has increased over the last decades affecting 15-30% of children and 10% of adults.⁸⁷³,⁸⁷⁴ Several factors such as an impaired barrier function of the skin, altered skin immune system, and skin microbial dysbiosis contribute to the development of AD.⁸⁷⁴,⁸⁷⁵ Cutaneous yeasts can trigger or aggravate inflammation of the skin in AD. Especially in patients with head and neck type AD, a common subtype, IgE specific to *Malassezia* antigens, can be found.⁸⁷⁵,⁸⁷⁶ This can be explained by an increased sebaceous gland activity in this area.⁸⁷⁵,⁸⁷⁶

*Malassezia* spp. specific IgE levels were identified as a marker for the severity of AD⁸⁷⁶,⁸⁷⁷ and in AD patients with elevated specific IgE to *Malassezia* allergens, antifungal treatments can be beneficial.⁸⁷⁷,⁸⁷⁸ In the following sections, we will discuss the importance of the skin microbiota in AD and review the possible interactions between microbial allergens and the immune system in atopic skin. We will focus on fungal allergens, but we will also briefly discuss the role of allergens from bacteria.

## 2 - The allergen sources

*Malassezia* is a genus of lipophilic yeasts and belonging to the phylum of Basidiomycota. Of currently 14 known species, nine can be isolated from human skin and five from animal skin (Table 29).⁸⁷⁸,⁸⁷⁹ Zoonotic transmission of *M. pachydermatis*, for example, from dogs to neonates by dog owning health care workers is possible.⁸⁷⁹,⁸⁸⁰

*Malassezia* spp. lack genes for synthesizing fatty acids and are therefore dependent on an exogenous fatty acid source, such as skin lipids, to meet their nutritional needs.⁸⁸⁰,⁸⁸¹ *M. globosa* and *M. restricta* are the two predominant species found on healthy human skin and different body sites.⁸⁸²,⁸⁸³,⁸⁸⁴ A geographical variation in distribution patterns of *Malassezia* species owing to climatic factors was found in several studies. In Japan, *M. furfur* was the most frequent species, and in Canada, Russia, and Sweden, *M. sympodialis* was the most common.⁸⁸³,⁸⁸⁴ Furthermore, a study from Switzerland and Tanzania recently showed that Swiss AD patients living in Switzerland are quite frequently sensitized to *Malassezia* spp.—whereas patients from patients with AD living in Tanzania are rarely sensitized to it. This finding suggests that there may also be significant ethnic differences between *Malassezia* sensitization.⁸⁸⁴,⁸⁸⁵ Several studies have compared the colonization of different *Malassezia* spp. in healthy and AD skin, though no consistent difference was found.⁸⁸³,⁸⁸⁴

## 3 - Allergen families

Currently, 14 different *Malassezia* allergens are characterized, and all of them are produced by three *Malassezia* species, namely *M. furfur*, *M. sympodialis*, and *M. globosa* (Figure 72). The function of some *Malassezia* spp. allergens are known.

*M. furfur* allergens Mala f 2 and Mala f 3 are peroxysomal membrane proteins, and Mala f 4 is a mitochondrial malate dehydrogenase. The functions of *M. sympodialis* allergens are known for Mala s 6, s 10, s 11, s 12, and s 13. Mala s 6 is a cyclophilin, and Mala s 13 is a thioredoxin—both being potential pan-allergens. The crystal structure of some of these allergens has been resolved (Figure 73).⁸⁸⁵,⁸⁸⁶

## 4 - Allergenic molecules (epidemiology incl. geography and function)

Thirteen *Malassezia* allergens, all of which are produced by *M. furfur* or *M. sympodialis*, are listed in the official allergen nomenclature list of the International Union of Immunological Societies (IUIS, www.allergen.org). Two *Malassezia* allergens have raised special attention, namely Mala s 11 and Mala s 13. Mala s 11 is 50% homologous in its amino acid sequence with human manganese superoxide dismutase (MnSOD)⁸⁸⁸ and 56% homologous to the MnSOD from Aspergillus fumigatus (rAsp f 6). On the Compare Database (https://comparedatabase.org) cross-reactivity to other
# B08 - Microbial allergens /antigens

## Carole Guillet, Rebecca Czolk, Philipp Bosshard, Peter Schmid-Grendelmeier, Annette Kuehn

### Highlights

* *Malassezia* species are commensals of the normal skin flora and are part of the skin microbiome.

* Sensitization to *Malassezia* species allergens can frequently be found in head and neck type atopic dermatitis.

* Fourteen allergens from 3 *Malassezia* species have been characterized to date.

* The skin microbiome, especially *Malassezia* spp and *Staphylococcus aureus*, can be a target in AD therapy.

* The impact of commensal microbiomes on allergies and other barrier diseases is a rapidly growing research field.

## 1 - Introduction

The skin is a complex ecosystem harboring diverse and site-specific microbial communities referred to as the skin microbiome. Phylogenetic profiling of the skin microbiome of healthy individuals revealed that bacteria are predominant at most body sites.⁸⁷⁰ Additionally, fungi—also referred to as skin mycobiome—play an essential part in these microbial communities. They account for 1% to 22% of the phylogenetic composition of the skin microbiome. In healthy skin, the fungal flora almost exclusively harbors *Malassezia* species (*spp*).⁸⁷⁰

Studies suggest a significant role of the skin microbiome in the development and progression of atopic dermatitis (AD).⁸⁷¹,⁸⁷² AD is a chronic inflammatory skin disease characterized by eczematous lesions, pruritus, and a chronic/relapsing history of symptoms.⁸⁷²,⁸⁷³ Its prevalence has increased over the last decades affecting 15-30% of children and 10% of adults.⁸⁷³,⁸⁷⁴ Several factors such as an impaired barrier function of the skin, altered skin immune system, and skin microbial dysbiosis contribute to the development of AD.⁸⁷⁴,⁸⁷⁵ Cutaneous yeasts can trigger or aggravate inflammation of the skin in AD. Especially in patients with head and neck type AD, a common subtype, IgE specific to *Malassezia* antigens, can be found.⁸⁷⁵,⁸⁷⁶ This can be explained by an increased sebaceous gland activity in this area.⁸⁷⁵,⁸⁷⁶

*Malassezia* spp. specific IgE levels were identified as a marker for the severity of AD⁸⁷⁶,⁸⁷⁷ and in AD patients with elevated specific IgE to *Malassezia* allergens, antifungal treatments can be beneficial.⁸⁷⁷,⁸⁷⁸ In the following sections, we will discuss the importance of the skin microbiota in AD and review the possible interactions between microbial allergens and the immune system in atopic skin. We will focus on fungal allergens, but we will also briefly discuss the role of allergens from bacteria.

## 2 - The allergen sources

*Malassezia* is a genus of lipophilic yeasts and belonging to the phylum of Basidiomycota. Of currently 14 known species, nine can be isolated from human skin and five from animal skin (Table 29).⁸⁷⁸,⁸⁷⁹ Zoonotic transmission of *M. pachydermatis*, for example, from dogs to neonates by dog owning health care workers is possible.⁸⁷⁹,⁸⁸⁰

*Malassezia* spp. lack genes for synthesizing fatty acids and are therefore dependent on an exogenous fatty acid source, such as skin lipids, to meet their nutritional needs.⁸⁸⁰,⁸⁸¹ *M. globosa* and *M. restricta* are the two predominant species found on healthy human skin and different body sites.⁸⁸²,⁸⁸³,⁸⁸⁴ A geographical variation in distribution patterns of *Malassezia* species owing to climatic factors was found in several studies. In Japan, *M. furfur* was the most frequent species, and in Canada, Russia, and Sweden, *M. sympodialis* was the most common.⁸⁸³,⁸⁸⁴ Furthermore, a study from Switzerland and Tanzania recently showed that Swiss AD patients living in Switzerland are quite frequently sensitized to *Malassezia* spp.—whereas patients from patients with AD living in Tanzania are rarely sensitized to it. This finding suggests that there may also be significant ethnic differences between *Malassezia* sensitization.⁸⁸⁴,⁸⁸⁵ Several studies have compared the colonization of different *Malassezia* spp. in healthy and AD skin, though no consistent difference was found.⁸⁸³,⁸⁸⁴

## 3 - Allergen families

Currently, 14 different *Malassezia* allergens are characterized, and all of them are produced by three *Malassezia* species, namely *M. furfur*, *M. sympodialis*, and *M. globosa* (Figure 72). The function of some *Malassezia* spp. allergens are known.

*M. furfur* allergens Mala f 2 and Mala f 3 are peroxysomal membrane proteins, and Mala f 4 is a mitochondrial malate dehydrogenase. The functions of *M. sympodialis* allergens are known for Mala s 6, s 10, s 11, s 12, and s 13. Mala s 6 is a cyclophilin, and Mala s 13 is a thioredoxin—both being potential pan-allergens. The crystal structure of some of these allergens has been resolved (Figure 73).⁸⁸⁵,⁸⁸⁶

## 4 - Allergenic molecules (epidemiology incl. geography and function)

Thirteen *Malassezia* allergens, all of which are produced by *M. furfur* or *M. sympodialis*, are listed in the official allergen nomenclature list of the International Union of Immunological Societies (IUIS, www.allergen.org). Two *Malassezia* allergens have raised special attention, namely Mala s 11 and Mala s 13. Mala s 11 is 50% homologous in its amino acid sequence with human manganese superoxide dismutase (MnSOD)⁸⁸⁸ and 56% homologous to the MnSOD from Aspergillus fumigatus (rAsp f 6). On the Compare Database (https://comparedatabase.org) cross-reactivity to other
MnSODs such as Hev b 10 and Alt b 14 is below 50% and thus cross-reactivity should not be expected. Mala s 13 is a thioredoxin and has a 45% sequence identity with human thioredoxins and can thus also lead to cross-reactivity and autoreactivity in patients suffering from AD.⁸⁷¹,⁸⁸⁷,⁸⁸⁹ Furthermore, the allergen MGL_1304 derived from M. globosa was shown to induce mast cell degranulation and trigger the release of IL-4 in basophils. Elevated levels of

IgE against this allergen in sweat were detected in AD patients and patients suffering from cholinergic urticaria.⁸⁹⁰ Crystal structures are given in (Figure 73).

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

As *Malassezia* spp. are part of the normal skin flora, specific IgG and IgM antibodies to *Malassezia* spp. can regularly be found in non-atopic human patients.⁸⁸¹ However, healthy individuals usually do not have detectable levels of *Malassezia*-specific IgE antibodies.

By contrast, 30-80% of adult AD patients are sensitized to *Malassezia* spp. as demonstrated by positive atopy patch tests, skin prick tests (SPT), or detectable serum levels of specific IgE antibodies.⁸⁹²,⁸⁹³ *Malassezia*-specific IgE is found in 5 - 27% of children and 29 - 65% of adults with AD, consistent with the rates found by SPT.⁸⁷⁶,⁸⁷⁷ The lower frequency of *Malassezia* sensitization in children compared with adults could be related to the poor growing conditions for *Malassezia* spp. in children. The lipid content of sebum, a prerequisite for skin colonization for most *Malassezia* spp., is low in children but rises during puberty.⁸⁹⁴ Accordingly, sensitization to *Malassezia* spp. seems to occur preferably in adulthood, and therefore later than the sensitization to food allergens and aeroallergens, which frequently occurs during childhood.⁸⁷⁷ The sensitization rate against particular allergens from *Malassezia* spp. is shown in the Online Repository Table 29. The currently proposed role of *Malassezia* allergens in the pathogenesis of AD is depicted in (Figure 74).

<table><caption>TABLE 29 Currently identified Malassezia species</caption><thead><tr><th>Malassezia species</th><th>Degree of cross-reactivity</th><th>Isolated from Animal</th><th>Description as species (year)</th></tr></thead><tbody><tr><td>M. caprae</td><td></td><td>x</td><td>2007</td></tr><tr><td>M. cuniculi</td><td></td><td>x</td><td>2011</td></tr><tr><td>M. dermatis</td><td>x</td><td></td><td>2002</td></tr><tr><td>M. equina</td><td></td><td>x</td><td>2007</td></tr><tr><td>M. furfur</td><td>x</td><td>x</td><td>1889</td></tr><tr><td>M. globosa</td><td>x</td><td>x</td><td>1996</td></tr><tr><td>M. japonica</td><td>x</td><td></td><td>2003</td></tr><tr><td>M. nana</td><td></td><td>x</td><td>2004</td></tr><tr><td>M. obtusa</td><td>x</td><td></td><td>1996</td></tr><tr><td>M. pachydermatis</td><td></td><td>x</td><td>1925</td></tr><tr><td>M. restricta</td><td>x</td><td></td><td>1996</td></tr><tr><td>M. slooffiae</td><td>x</td><td>x</td><td>1996</td></tr><tr><td>M. sympodialis</td><td>x</td><td>x</td><td>1990</td></tr><tr><td>M. yamatoensis</td><td>x</td><td></td><td>2004</td></tr></tbody></table>

<figure><img src="image_171.png" /><figcaption>FIGURE 72 14 known Malassezia allergens from three Malassezia species</figcaption></figure>
MnSODs such as Hev b 10 and Alt b 14 is below 50% and thus cross-reactivity should not be expected. Mala s 13 is a thioredoxin and has a 45% sequence identity with human thioredoxins and can thus also lead to cross-reactivity and autoreactivity in patients suffering from AD.<sup>871,887,889</sup> Furthermore, the allergen MGL_1304 derived from M. globosa was shown to induce mast cell degranulation and trigger the release of IL-4 in basophils. Elevated levels of

IgE against this allergen in sweat were detected in AD patients and patients suffering from cholinergic urticaria.<sup>890</sup> Crystal structures are given in (Figure 73).

5 – Sensitization to individual molecules and their clinical relevance

As *Malassezia* spp. are part of the normal skin flora, specific IgG and IgM antibodies to *Malassezia* spp. can regularly be found in non-atopic human patients.<sup>881</sup> However, healthy individuals usually do not have detectable levels of *Malassezia*-specific IgE antibodies.

By contrast, 30-80% of adult AD patients are sensitized to *Malassezia* spp. as demonstrated by positive atopy patch tests, skin prick tests (SPT), or detectable serum levels of specific IgE antibodies.<sup>892,893</sup> *Malassezia*-specific IgE is found in 5 - 27% of children and 29 - 65% of adults with AD, consistent with the rates found by SPT.<sup>876,877</sup> The lower frequency of *Malassezia* sensitization in children compared with adults could be related to the poor growing conditions for *Malassezia* spp. in children. The lipid content of sebum, a prerequisite for skin colonization for most *Malassezia* spp., is low in children but rises during puberty.<sup>894</sup> Accordingly, sensitization to *Malassezia* spp. seems to occur preferably in adulthood, and therefore later than the sensitization to food allergens and aeroallergens, which frequently occurs during childhood.<sup>877</sup> The sensitization rate against particular allergens from *Malassezia* spp. is shown in the Online Repository Table 29. The currently proposed role of *Malassezia* allergens in the pathogenesis of AD is depicted in (Figure 74).

<table><caption>TABLE 29 Currently identified Malassezia species</caption><thead><tr><th>Malassezia species</th><th>Degree of cross-reactivity</th><th>Isolated from Animal</th><th>Description as species (year)</th></tr></thead><tbody><tr><td>M. caprae</td><td></td><td>x</td><td>2007</td></tr><tr><td>M. cuniculi</td><td></td><td>x</td><td>2011</td></tr><tr><td>M. dermatis</td><td>x</td><td></td><td>2002</td></tr><tr><td>M. equina</td><td></td><td>x</td><td>2007</td></tr><tr><td>M. furfur</td><td>x</td><td>x</td><td>1889</td></tr><tr><td>M. globosa</td><td>x</td><td>x</td><td>1996</td></tr><tr><td>M. japonica</td><td>x</td><td></td><td>2003</td></tr><tr><td>M. nana</td><td></td><td>x</td><td>2004</td></tr><tr><td>M. obtusa</td><td>x</td><td></td><td>1996</td></tr><tr><td>M. pachydermatis</td><td></td><td>x</td><td>1925</td></tr><tr><td>M. restricta</td><td>x</td><td></td><td>1996</td></tr><tr><td>M. slooffiae</td><td>x</td><td>x</td><td>1996</td></tr><tr><td>M. sympodialis</td><td>x</td><td>x</td><td>1990</td></tr><tr><td>M. yamatoensis</td><td>x</td><td></td><td>2004</td></tr></tbody></table>

<figure><img src="image_170.png" /><figcaption>FIGURE 72 14 known *Malassezia* allergens from three *Malassezia* species</figcaption></figure>
<figure><img src="image_172.png" /><figcaption>**FIGURE 73** (A) Crystal structure of Mala s 13. Image from the RCSB PDB (rcsb.org) of PDB ID 2J23.<sup>886</sup> Image created with Mol*.<sup>891</sup> (B) Crystal structure of Mala s 6. Image from the RCSB PDB (rcsb.org) of PDB ID 2CFE.<sup>887</sup> Image created with Mol*.<sup>891</sup></figcaption></figure>

<figure><img src="image_173.png" /><figcaption>**FIGURE 74** Proposed mechanisms of *Malassezia* spp. allergen-induced skin inflammation in atopic dermatitis (AD)</figcaption></figure>

## 6 – Clinical relevance of sensitization

An elevated skin pH as in AD leads to an increase in release of *Malassezia* spp. allergen. These allergens can consecutively

penetrate the skin barrier, which is disturbed in atopic skin. Allergens are recognized by dendritic cells and keratinocytes via Toll-like receptor II. This stimulates the release of pro-inflammatory cytokines and induces the production of IgE antibodies. Autoreactive T cells
<figure><img src="image_171.png" /><figcaption>FIGURE 73 (A) Crystal structure of Mala s 13. Image from the RCSB PDB (rcsb.org) of PDB ID 2J23.<sup>886</sup>. Image created with Mol*.<sup>891</sup> (B) Crystal structure of Mala s 6. Image from the RCSB PDB (rcsb.org) of PDB ID 2CFE.<sup>887</sup> Image created with Mol*.<sup>891</sup></figcaption></figure>

Mala s 13

<figure><img src="image_172.png" /></figure>

Mala s 6

<figure><img src="image_173.png" /><figcaption>FIGURE 74 Proposed mechanisms of *Malassezia* spp. allergen-induced skin inflammation in atopic dermatitis (AD)</figcaption></figure>

6 – Clinical relevance of sensitization

An elevated skin pH as in AD leads to an increase in release of *Malassezia* spp. allergen. These allergens can consecutively

penetrate the skin barrier, which is disturbed in atopic skin. Allergens are recognized by dendritic cells and keratinocytes via Toll-like receptor II. This stimulates the release of pro-inflammatory cytokines and induces the production of IgE antibodies. Autoreactive T cells
cross-react between fungal and human manganese-dependent superoxide dismutase (Mala s 11) or thioredoxin (Mala s 13), hence sustaining skin inflammation.

The interaction between *Malassezia* and the skin immune system contributes to the inflammation typical of AD. *M. sympodialis* produces higher amounts of the allergen Mala s 12 when cultured under high pH conditions reflecting the higher pH of atopic dermatitis compared with normal skin. This might contribute to *Malassezia*-related skin inflammation in AD.⁸⁹⁵

## 7 – Clinical management

### Diagnosis

The diagnosis of *Malassezia*-associated AD is based on the clinical picture. It may be supported by a positive type I allergic reaction to *Malassezia* spp, measured by a positive skin prick test, or by measuring *Malassezia*-specific serum IgE with a commercially available standardized assay (the ImmunoCAP Specific IgE test m227) based upon three different *Malassezia* species.⁸⁷⁶ Furthermore, a recently developed multiplex IgE macroassay (MacroArray Diagnostics GmbH, Vienna, Austria) is available, including the allergens Mala s 5, 6, and 11. Based on its sequence homology, Asp f 6 shows high cross-reactivity with Mala s 11 and can be measured additionally. Total IgE measurements can allow to determine the sensitization attributable to the whole extract, e.g., it can be expressed as a ration or percentage; it also gives hints on the reduced sensitivity of specific gE in very low amounts of total IgE (<25 kU/l). Atopy patch testing has shown varying results. Some studies have shown no correlation between IgE and atopy patch test for *Malassezia*. By contrast, others have found a positive atopy patch test in 41% of patients with head and neck dermatitis and 30% in AD patients without head and neck involvement.⁸⁷⁶,⁸⁹²,⁸⁹⁶ Culturing *Malassezia* spp. from the skin is not commonly used in the routine clinical care of AD patients.

### Treatment

The benefit of topical or systemic antifungal treatment for clinical improvement of AD is controversial. Azole antifungals are the most commonly prescribed class of antifungals for AD patients. Azole antifungals show inhibitory effects against *Malassezia* spp. *in vitro*.⁸⁹⁴,⁸⁹⁷

Based on our anecdotal experience in routine clinical practice, topical application of ketoconazole to the face of patients with head-and-neck-type AD often improves eczema.

Several randomized, placebo-controlled trials have investigated the effect of systemic antifungal treatment on AD. Some have shown a significant difference in reduction of AD severity in patients treated with oral ketoconazole vs. placebo or oral itraconazole vs. placebo.⁸⁹⁸,⁸⁹⁹ However, the relevance of these results to routine clinical practice remains to be demonstrated.

## 8 – Clinical case⁹⁰⁰

### Clinical history

A 37-year-old patient presented with severe head and neck type atopic dermatitis (EASI score: 43). Since childhood, the patient had suffered from AD. The eczema flare-ups occurred mainly after physical exertion and heavy sweating. Other trigger factors, such as seasonal factors or the consumption of certain foods, were negative. Previous therapies included topical emollients, topical steroids, phototherapy for three months, and the use of cyclosporine for one month (intolerance due to severe headache).

### Test with extracts and molecules

Skin prick tests with the most common seasonal and year-round inhaled allergens were negative. Serum levels of total IgE (523 kU/L; norm <100 kU/L) and specific IgE against *Malassezia* spp. (m227) (53.2 kU/L; norm <0.35 kU/L) and rAsp f 6 (22.4 kU/l; norm <0.35 kU/L) were markedly elevated while, IgE against rAsp f1 and rAsp f4 were not elevated.

Patch testing showed positive reactions to *Malassezia* spp. extract (+++), as well as to the isoforms of manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus, rAsp f 6, (+++), and humans (++). (Table 30)

### Conclusion

These findings are consistent with a marked sensitization to fungal allergens (MnSOD) and their human homologues. Treatment with a peroral antifungal (itraconazole 100 mg 2x daily for two weeks) in addition to emollients and topical steroids was initiated resulting in rapid but transient improvement of his AD. Currently, the patient uses topical antifungals on his face and neck and an antifungal shampoo every four to six weeks, which has resulted in a marked improvement in eczema and a decrease in the frequency of AD flare-ups.

<table><caption>TABLE 30 Test results</caption><thead><tr><th>Serum IgE</th><th>Value [KU/L]</th><th>Interpretation</th></tr></thead><tbody><tr><td>Total IgE</td><td>523.0</td><td>Elevated</td></tr><tr><td>Inhalant allergen screening (sx1)</td><td>0.2</td><td>CAP-Class</td></tr><tr><td>Food Screening (fx5) <i>Malassezia</i></td><td>0.3</td><td>0 CAP-Class</td></tr><tr><td>spp (m227) <i>Aspergillus</i></td><td>53.2</td><td>0 CAP-Class</td></tr><tr><td>fumigatus (m3)</td><td>12.4</td><td>5 CAP-Class</td></tr><tr><td>rAsp f 6 (m222)</td><td>22.4</td><td>3 CAP-Class</td></tr><tr><td>rAsp f 1 (m218)</td><td>0.2</td><td>4 CAP-Class</td></tr><tr><td>rAsp f 4 (m221)</td><td>0.3</td><td>0 CAP-Class 0</td></tr><tr><td>Mold mix (mx2)</td><td>24.1</td><td>CAP-Class 4</td></tr></tbody></table>

<table><caption>Patch testing (assessment after 48 hours)</caption><tr><td><i>Malassezia</i> spp. Extract Fungal</td><td>+++</td><td>Highly positive</td></tr><tr><td>MnSOD (r-Asp f 6) Human</td><td>++++</td><td>Highly positive</td></tr><tr><td>MnSOD</td><td>++</td><td>Positive</td></tr></table>
cross-react between fungal and human manganese-dependent superoxide dismutase (Mala s 11) or thioredoxin (Mala s 13), hence sustaining skin inflammation.

The interaction between *Malassezia* and the skin immune system contributes to the inflammation typical of AD. *M. sympodialis* produces higher amounts of the allergen Mala s 12 when cultured under high pH conditions reflecting the higher pH of atopic dermatitis compared with normal skin. This might contribute to *Malassezia*-related skin inflammation in AD.⁸⁹⁵

## 7 - Clinical management

### Diagnosis

The diagnosis of *Malassezia*-associated AD is based on the clinical picture. It may be supported by a positive type I allergic reaction to *Malassezia* spp, measured by a positive skin prick test, or by measuring *Malassezia*-specific serum IgE with a commercially available standardized assay (the ImmunoCAP Specific IgE test m227) based upon three different *Malassezia* species.⁸⁷⁶ Furthermore, a recently developed multiplex IgE macroassay (MacroArray Diagnostics GmbH, Vienna, Austria) is available, including the allergens Mala s 5, 6, and 11. Based on its sequence homology, Asp f 6 shows high cross-reactivity with Mala s 11 and can be measured additionally. Total IgE measurements can allow to determine the sensitization attributable to the whole extract, e.g., it can be expressed as a ration or percentage; it also gives hints on the reduced sensitivity of specific gE in very low amounts of total IgE (<25 kU/l). Atopy patch testing has shown varying results. Some studies have shown no correlation between IgE and atopy patch test for *Malassezia*. By contrast, others have found a positive atopy patch test in 41% of patients with head and neck dermatitis and 30% in AD patients without head and neck involvement.⁸⁷⁶,⁸⁹²,⁸⁹⁶ Culturing *Malassezia* spp. from the skin is not commonly used in the routine clinical care of AD patients.

### Treatment

The benefit of topical or systemic antifungal treatment for clinical improvement of AD is controversial. Azole antifungals are the most commonly prescribed class of antifungals for AD patients. Azole antifungals show inhibitory effects against *Malassezia* spp. *in vitro*.⁸⁹⁴,⁸⁹⁷

Based on our anecdotal experience in routine clinical practice, topical application of ketoconazole to the face of patients with head-and-neck-type AD often improves eczema.

Several randomized, placebo-controlled trials have investigated the effect of systemic antifungal treatment on AD. Some have shown a significant difference in reduction of AD severity in patients treated with oral ketoconazole vs. placebo or oral itraconazole vs. placebo.⁸⁹⁸,⁸⁹⁹ However, the relevance of these results to routine clinical practice remains to be demonstrated.

## 8 - Clinical case⁹⁰⁰

### Clinical history

A 37-year-old patient presented with severe head and neck type atopic dermatitis (EASI score: 43). Since childhood, the patient had suffered from AD. The eczema flare-ups occurred mainly after physical exertion and heavy sweating. Other trigger factors, such as seasonal factors or the consumption of certain foods, were negative. Previous therapies included topical emollients, topical steroids, phototherapy for three months, and the use of cyclosporine for one month (intolerance due to severe headache).

### Test with extracts and molecules

Skin prick tests with the most common seasonal and year-round inhaled allergens were negative. Serum levels of total IgE (523 kU/L; norm <100 kU/L) and specific IgE against *Malassezia* spp. (m227) (53.2 kU/L; norm <0.35 kU/L) and rAsp f 6 (22.4 kU/l; norm <0.35 kU/L) were markedly elevated while, IgE against rAsp f1 and rAsp f4 were not elevated.

Patch testing showed positive reactions to *Malassezia* spp. extract (+++), as well as to the isoforms of manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus, rAsp f 6, (+++), and humans (++). (Table 30)

### Conclusion

These findings are consistent with a marked sensitization to fungal allergens (MnSOD) and their human homologues. Treatment with a peroral antifungal (itraconazole 100 mg 2x daily for two weeks) in addition to emollients and topical steroids was initiated resulting in rapid but transient improvement of his AD. Currently, the patient uses topical antifungals on his face and neck and an antifungal shampoo every four to six weeks, which has resulted in a marked improvement in eczema and a decrease in the frequency of AD flare-ups.

<table><caption>TABLE 30 Test results</caption><thead><tr><th>Serum IgE</th><th>Value [KU/L]</th><th>Interpretation</th></tr></thead><tbody><tr><td>Total IgE</td><td>523.0</td><td>Elevated</td></tr><tr><td>Inhalant allergen screening (sx1)</td><td>0.2</td><td>CAP-Class</td></tr><tr><td>Food Screening (fx5) <i>Malassezia</i></td><td>0.3</td><td>0 CAP-Class</td></tr><tr><td>spp (m227) <i>Aspergillus</i></td><td>53.2</td><td>0 CAP-Class</td></tr><tr><td>fumigatus (m3)</td><td>12.4</td><td>5 CAP-Class</td></tr><tr><td>rAsp f 6 (m222)</td><td>22.4</td><td>3 CAP-Class</td></tr><tr><td>rAsp f 1 (m218)</td><td>0.2</td><td>4 CAP-Class</td></tr><tr><td>rAsp f 4 (m221)</td><td>0.3</td><td>0 CAP-Class 0</td></tr><tr><td>Mold mix (mx2)</td><td>24.1</td><td>CAP-Class 4</td></tr></tbody></table>

<table><caption>Patch testing (assessment after 48 hours)</caption><tbody><tr><td><i>Malassezia</i> spp. Extract Fungal</td><td>+++</td><td>Highly positive</td></tr><tr><td>MnSOD (r-Asp f 6) Human</td><td>++++</td><td>Highly positive</td></tr><tr><td>MnSOD</td><td>++</td><td>Positive</td></tr></tbody></table>
Suggested serological investigations in patients with confirmed atopic eczema and suggested analyses in patients with suspicion of AD are shown in **Figure 75**.

9 – Research and future perspectives

**Atopic Dermatitis: skin microbial agents and their role** Singleplex assays currently used in the diagnosis do not provide component-resolved analysis based on *Malassezia* allergens: Complementary to the usual skin prick and extract tests, allergen-specific IgE might give an outlook on disease prognosis and severity, as said earlier. Even though molecular diagnostic tools are not yet well established in routine use, as a perspective, they might even become a helpful marker for disease endotypes. Widespread clinical use of allergen-based assays

will contribute to the elucidation of these connections. *Malassezia* spp. do not overgrow in AD patients, giving rise to the question how *Malassezia* becomes a sensitizing agent specifically in AD.⁸⁸³ From a mouse model it seems that *Malassezia* spp may have important regulatory function in AD by inducing IL-17 and related cytokines.⁸⁹⁹

Another hypothesis is that a disturbed skin barrier leads to an altered interplay between fungal and bacterial communities, triggering allergic sensitization. *Staphylococcus aureus*, one of the main bacteria in the skin microbiome, more frequently colonizes lesional (70%) and nonlesional skin (39%) of AD patients than the skin of healthy individuals (10%). *Staphylococcus aureus* enterotoxins are the primary source for sensitization. 33% of AD patients have specific IgE against enterotoxin A (SEA), and 35% against enterotoxin B (SEB), with lower sensitization numbers for other SEs. Sensitization to S. aureus allergens correlates with the severity of AD. Other bacterial strains, such

<figure><img src="image_174.png" /><figcaption>**FIGURE 75** Diagnostic algorithm in suspected atopic dermatitis and suggested serological investigations in patients with confirmed atopic eczema. Abbreviations: PFT Pulmonary Function Test. Reference: **873**</figcaption></figure>
Suggested serological investigations in patients with confirmed atopic eczema and suggested analyses in patients with suspicion of AD are shown in Figure 75.

9 - Research and future perspectives

**Atopic Dermatitis: skin microbial agents and their role** Singleplex assays currently used in the diagnosis do not provide component-resolved analysis based on *Malassezia* allergens: Complementary to the usual skin prick and extract tests, allergen-specific IgE might give an outlook on disease prognosis and severity, as said earlier. Even though molecular diagnostic tools are not yet well established in routine use, as a perspective, they might even become a helpful marker for disease endotypes. Widespread clinical use of allergen-based assays

will contribute to the elucidation of these connections. *Malassezia* spp. do not overgrow in AD patients, giving rise to the question how *Malassezia* becomes a sensitizing agent specifically in AD.⁸⁸³ From a mouse model it seems that *Malassezia* spp may have important regulatory function in AD by inducing IL-17 and related cytokines.⁸⁹⁹

Another hypothesis is that a disturbed skin barrier leads to an altered interplay between fungal and bacterial communities, triggering allergic sensitization. *Staphylococcus aureus*, one of the main bacteria in the skin microbiome, more frequently colonizes lesional (70%) and nonlesional skin (39%) of AD patients than the skin of healthy individuals (10%). *Staphylococcus aureus* enterotoxins are the primary source for sensitization. 33% of AD patients have specific IgE against enterotoxin A (SEA), and 35% against enterotoxin B (SEB), with lower sensitization numbers for other SEs. Sensitization to S. aureus allergens correlates with the severity of AD. Other bacterial strains, such

<figure><img src="image_174.png" /><figcaption>FIGURE 75 Diagnostic algorithm in suspected atopic dermatitis and suggested serological investigations in patients with confirmed atopic eczema. Abbreviations: PFT Pulmonary Function Test. Reference: 873</figcaption></figure>
as *Corynebacterium* and the *Proteobacteria*, have been associated with AD severity; however, no IgE-reactive proteins could be identified so far.⁹⁰¹ Further research is needed to understand the interplay between fungal and bacterial skin microbiome components, to illustrate different possible cause and effect mechanisms of antigen-immune interactions on the skin.

**Asthma: disrupted barriers and pathogen interactions** Other barriers between host and microbiome are also the scene of allergic reactions, such as the respiratory tract in allergic asthma or rhinitis. In the lungs, fungal and bacterial antigens appear implicated in aggravating allergy symptoms. Fungal allergens from

*Aspergillus*, *Cladosporium*, *Penicillium*, and *Alternaria* have been shown to play a significant role in asthma (see additionally chapter B07). The IgE-reactive *S. aureus* proteins involved in AD are also of importance in asthmatic lungs. Approximately 25% of asthma patients present with SEB-sIgE and 15% were sensitized to SEA. Other bacterial proteins, mostly from strains associated with lung infections, also seem to elicit an allergic response. *Chlamydia pneumoniae*'s cysteine-rich membrane protein A (CrpA), major outer membrane protein (MOMP), lectin-binding proteins (LBPs), chlamydial heat shock protein 60 (HSP60), and lipopolysaccharide (LPS) were identified to bind specific IgE. For *Haemophilus influenzae*, IgE is bound to the outer membrane proteins P4 and P6 and the surface protein C (PspC) of *Streptococcus pneumonia*. The presence of *Moraxella* in the lungs early in life has also been associated with increased asthma risk, although the mechanism and reactive structures are not known yet.⁹⁰² So far, known IgE reactive bacterial antigens are not classified as named allergens and therefore not yet available for diagnostic purposes, although the field is evolving rapidly. Measurement of IgG against bacteria may be useful in the diagnostic workup of other

immune-mediated diseases, such as hypersensitivity pneumonitis; however, this is beyond the scope of this article.

**Food allergy: intestinal microbiome and immune modulation**

The intestinal tract is one of the body's most significant barriers between the host, a complex microbial community, and foreign agents, such as food. The commensal microbiome has been associated with beneficial immune modulation, but a disturbance in the cross-talk between microbes and host may have the opposite effect. This field of research is ever-expanding and has established connections between the corruption of host barriers and allergy. Primary sensitization to peanuts through the skin, *S. aureus* colonization of the skin as a driver for food allergy, or gut microbial dysbiosis in asthmatic children have been reported.

A disrupted and dysfunctional intestinal barrier appears to be relevant in the pathogenesis of food allergy.⁹⁰³ An increase of IgE+-B-cells in patients with peanut allergy gives even credence to the thought of tissue-specific effects in allergic disease.⁹⁰⁴ This seems to be accompanied by dysbiosis, showing a pattern of over- or underrepresentation of bacterial strains to be food allergy-specific.⁹⁰³ Fecal bacteria turned out to be a target for IgE binding, leading to the question of the role of the microbiome in eliciting allergic responses.⁹⁰³ The concept of bacteria mimicking disease-related targets has recently been demonstrated in celiac disease, a chronic autoimmune disorder.⁹⁰⁵ Overall, disruptions in the delicate balance between host immune system, barrier integrity, and microbiome seem to have an influence on disease development, as well as progression and severity.⁹⁰⁷ More research will be needed to identifyagnostically relevant antigen structures on commensal bacteria and fungi to verify their IgE reactivity and biomarker capacity.
as *Corynebacterium* and the *Proteobacteria*, have been associated with AD severity; however, no IgE-reactive proteins could be identified so far.⁹⁰¹ Further research is needed to understand the interplay between fungal and bacterial skin microbiome components, to illustrate different possible cause and effect mechanisms of antigen-immune interactions on the skin.

**Asthma: disrupted barriers and pathogen interactions** Other barriers between host and microbiome are also the scene of allergic reactions, such as the respiratory tract in allergic asthma or rhinitis. In the lungs, fungal and bacterial antigens appear implicated in aggravating allergy symptoms. Fungal allergens from

*Aspergillus*, *Cladosporium*, *Penicillium*, and *Alternaria* have been shown to play a significant role in asthma (see additionally chapter B07). The IgE-reactive *S. aureus* proteins involved in AD are also of importance in asthmatic lungs. Approximately 25% of asthma patients present with SEB-sIgE and 15% were sensitized to SEA. Other bacterial proteins, mostly from strains associated with lung infections, also seem to elicit an allergic response. *Chlamydia pneumoniae*'s cysteine-rich membrane protein A (CrpA), major outer membrane protein (MOMP), lectin-binding proteins (LBPs), chlamydial heat shock protein 60 (HSP60), and lipopolysaccharide (LPS) were identified to bind specific IgE. For *Haemophilus influenzae*, IgE is bound to the outer membrane proteins P4 and P6 and the surface protein C (PspC) of *Streptococcus pneumonia*. The presence of *Moraxella* in the lungs early in life has also been associated with increased asthma risk, although the mechanism and reactive structures are not known yet.⁹⁰² So far, known IgE reactive bacterial antigens are not classified as named allergens and therefore not yet available for diagnostic purposes, although the field is evolving rapidly. Measurement of IgG against bacteria may be useful in the diagnostic workup of other

immune-mediated diseases, such as hypersensitivity pneumonitis; however, this is beyond the scope of this article.

**Food allergy: intestinal microbiome and immune modulation**

The intestinal tract is one of the body's most significant barriers between the host, a complex microbial community, and foreign agents, such as food. The commensal microbiome has been associated with beneficial immune modulation, but a disturbance in the cross-talk between microbes and host may have the opposite effect. This field of research is ever-expanding and has established connections between the corruption of host barriers and allergy. Primary sensitization to peanuts through the skin, *S. aureus* colonization of the skin as a driver for food allergy, or gut microbial dysbiosis in asthmatic children have been reported.

A disrupted and dysfunctional intestinal barrier appears to be relevant in the pathogenesis of food allergy.⁹⁰³ An increase of IgE+-B-cells in patients with peanut allergy gives even credence to the thought of tissue-specific effects in allergic disease.⁹⁰⁴ This seems to be accompanied by dysbiosis, showing a pattern of over- or underrepresentation of bacterial strains to be food allergy-specific.⁹⁰³ Fecal bacteria turned out to be a target for IgE binding, leading to the question of the role of the microbiome in eliciting allergic responses.⁹⁰³ The concept of bacteria mimicking disease-related targets has recently been demonstrated in celiac disease, a chronic autoimmune disorder.⁹⁰⁵ Overall, disruptions in the delicate balance between host immune system, barrier integrity, and microbiome seem to have an influence on disease development, as well as progression and severity.⁹⁰⁷ More research will be needed to identifyagnostically relevant antigen structures on commensal bacteria and fungi to verify their IgE reactivity and biomarker capacity.