Key Message

Allergic respiratory diseases, particularly those triggered by Alternaria alternata (A. alternata) and Olea europaea (O. europaea), represent a significant health burden in pediatric population in Southern Spain. While allergen-specific immunotherapy (AIT) is a cornerstone of treatment, data on the use of polymerized extracts containing both allergens in children remain limited. A key innovation of this study lies in addressing this gap by evaluating the effectiveness and safety of a novel immunotherapy approach by using a mixture of polymerized A. alternata and O. europaea allergen extracts in a group of pediatric patients. The polymerization ensures the stability of the allergen extracts and, critically, overcomes the challenge of mixing these two components. Native A. alternata extracts possess significant enzymatic and proteolytic activity that would degrade native O. europaea extracts, making a stable combined native formulation unfeasible. This is a key technical advantage that offers a robust and effective solution for co-sensitized patients.

Our findings demonstrate a favorable safety profile with good tolerability. We believe that these results contribute valuable insights into the potential of this combined immunotherapy approach for children suffering from allergies to A. alternata and O. europaea, offering a promising therapeutic path.

Introduction

Alternaria alternata (A. alternata) is the most common allergenic mold,¹ particularly associated with severe asthma, including emergency visits, hospitalizations, and deaths from asthma.² Sensitization is higher in children,^3–5 showing a decreasing trend with age,⁶ which could reflect the acquisition of tolerance naturally or after immunotherapy. A. alternata acts as both an indoor and outdoor allergen, especially in agricultural areas.⁷ Depending on geography, 3–10% of the general population, 12–42% of atopic individuals, and over 66% of severe asthmatics are mold-sensitized, being more prevalent in hot, dry inland regions.^8,9 Notably, most patients are sensitized to other aeroallergens,^4,8 so A. alternata is also proposed to induce co-sensitization to them.¹⁰

Olea europaea (O. europaea) (olive) pollen is, after grass, the second most significant cause of pollinosis in southern Spain, being the main cause of respiratory allergy in some Andalusian regions, with high sensitization rates, up to 80% in seasonal asthma/rhinoconjunctivitis patients, reaching 84% in areas with high exposure.^11,12

Coexisting sensitization to A. alternata and O. europaea is common in southern Spain, necessitating treatment for both allergies. Historically, mixing fungal extracts with other allergens in immunotherapy was not recommended¹³ because of mold proteases degrading pollens. The European Medicines Agency (EMA), in 2008, recommended avoiding mixtures of allergen native extracts from nonhomologous groups and extracts with high enzymatic activity.¹⁴

However, glutaraldehyde polymerization significantly reduces this enzymatic activity, allowing for stable mixtures.^15,16 For polyallergic patients, combined polymerized extracts at optimal concentrations are an option, avoiding the dilution effect by doubling the concentration of each extract before mixing. This ensures that each allergen’s concentration maintained in the final product is the same as it would be if each allergen were administered separately. The safety of mixtures containing polymerized allergen extracts of A. alternata with other polymerized allergen extracts without dilution has been reported in an observational real-world study.¹⁷

This study addresses the need for effective joint immunotherapy for A. alternata and olive pollen allergies in children, considering previous uncertainties about mixture stability and limited data on effectiveness and safety. This real-life retrospective study aims to analyze the effectiveness and safety of a combined polymerized Alternaria plus Olea allergen extract in pediatric patients.

Materials and Methods

This is a real-world, retrospective study, approved by the Ethics Committee of Andalucia, CEIM/CEI of Granada, Spain. Data from the patients’ medical records, related to the severity of RC and asthma before starting treatment with immunotherapy and at the first review (more than 6 months from the initiation of treatment) were collected by the investigators.

Methods

Patient population

After signature of parents’ inform consent, patient data were collected from medical records of pediatric subjects diagnosed with asthma and rhinitis, where both A. alternata and O. europaea sensitizations were clinically relevant.

Allergen Immunotherapy (AIT)

Subjects were treated with a mixture of two individually glutaraldehyde-polymerized extracts (A. alternata and O. europaea), adsorbed onto aluminum hydroxide (Clustoid Max^® Inmunotek SL, Alcalá de Henares. Spain). Each polymerized allergen extract in the mixture was at a concentration of 10,000 TU/mL, containing all relevant allergens.

Two administration schedules were employed: a short regimen (0.2 mL on day 1; 0.5 mL after 1 week, then monthly 0.5 mL) and a rush schedule (0.2 mL plus 0.3 mL in the contralateral arm 30 minutes later, followed by monthly 0.5 mL).

Effectiveness

Effectiveness was evaluated by comparing the severity of rhinitis and asthma before and after at least 6 months of treatment. Rhinitis was classified using ARIA guidelines:¹⁸ (1) mild intermittent; (2) moderate intermittent; (3) severe intermittent; (4) mild persistent; (5) moderate persistent; and (6) severe persistent, and asthma severity followed GEMA 5.4,¹⁹ based on medication needed to maintain control and prevent exacerbations: (1) Intermittent (step 1 of treatment); (2) mild persistent (step 2); (3) moderate persistent (Steps 3–4); and (4) severe persistent (Steps 5–6).

The medication for rhinitis was scored as described in the EAACI position paper²⁰ with slight modifications: occasional antihistamines (H1A)-1 point-; regular H1A-2 points-; and intranasal glucocorticosteroids (INGCS) with/without H1A-3 points-. In the case of asthma, the medication was scored as described in GEMA 5.4 report for the asthma treatment steps for children.²¹

Safety

Safety was assessed by recording immediate (<30 minute) and delayed (>30 minute) adverse reactions, classified as local (wheal/redness diameter) or systemic.¹⁹ Systemic reactions were graded per EAACI Position Paper (Grade I: Mild, Grade II: Moderate, Grade III: Severe, and Grade IV: Anaphylactic shock).

Statistics

Statistical analyses used Excel (Microsoft; Richmond, VA, USA) with XLSTAT AddIn (Addinsoft; New York, USA) and GraphPad Prism 9 (v 9.5.1). Shapiro–Wilk test confirmed nonnormal data distribution. Descriptive statistics used median with IQR or frequency (%). Fisher’s exact test compared rhinitis and asthma control grading scores, with Phi Coefficient for effect size. All subjects contributed data to both effectiveness and safety analyses.

Results

All 49 enrolled patients had been diagnosed with respiratory allergy to both A. alternata and O. europaea, confirmed by specific IgE and/or skin prick test (SPT) and presented with rhinitis with or without asthma. The median age at diagnosis was 9 years (IQR: 6–11), 67% were male, 24% had a personal history of atopic dermatitis, 9% had food allergy, and 63% were sensitized to other aeroallergens deemed not clinically relevant. Twelve patients had previously been treated with IT with nonpolymerized allergens (six with olive extract, one with a mixture of Olea and grass pollen, three with A. alternata, and two with other mixtures of A. alternata-Olea). Forty-nine percent of the children had a family history of allergy. Table 1 shows the demographic and epidemiological data, including other nonrelevant clinical sensitizations.

Regarding olive tree pollen allergy, 47/49 subjects had positive skin tests to native allergen extracts. The two subjects who were not tested had positive specific IgE against this pollen. The median value of specific IgE to pollen extract was 29.8 kU/L (IQR 20–100). Table 2 shows the median values of total and specific IgE.

For A. alternata, 45/49 patients had a positive skin test. The four subjects who had not been tested by SPT had specific IgE to A. alternata and to Alt a1. The median of these values of IgE to A. alternata was 10 kU/L (IQR 4.6–31.1).

The median period of treatment to evaluate the clinical response was 9 months (IQR 6–12). Ten patients followed the short administration regimen, while the rest followed the rush schedule. Before starting treatment, 84% of the subjects were in the ARIA rhinitis Steps 3–5. After the AIT period, this percentage significantly dropped to 49% (p = 0.001). At baseline, 80% were availing the rhinitis treatment classified in medication Steps 2–3. After a median of 6 months of AIT, the percentage of patients dropped to 17% (Table 3 and Figures 1A and 1B).

Figure 1 (A) pre- and post-AIT classification of rhinitis according to ARIA (1. Mild intermittent, 2. moderate intermittent, 3. severe intermittent, 4. mild persistent, and 5. moderate-severe persistent). (B) Pre- and post-AIT medication control rhinitis. Step (1) Occasional antihistamines. Step (2) Regular use of antihistamines. Step (3) Local nasal corticosteroids.

Regarding asthma, 92% of patients were in severity grades 3–4 at baseline, decreasing to 39% after AIT. The percentage of patients using drugs included in asthma treatment Steps 3–5 at baseline was 96%, decreasing to 22% after the evaluation period of AIT (Table 4 and Figures 2A and 2B).

A total of 424 injections were administered. Only two local reactions (0.47% of administrations) were reported from the start of AIT until the first follow-up visit. One reaction occurred 5 minutes post-administration and was managed with local cold application. The other was a delayed reaction, resolving with a single dose of oral antihistamine. The first reaction happened during the maintenance period, and the second during the build-up phase. Importantly, no systemic reactions were recorded.

Figure 2 (A) Pre- and post-AIT grade asthma (according to GEMA 5.4). (B) Pre- and post-AIT asthma maintenance medication.

Discussion

Allergic diseases, particularly those triggered by A. alternata and O. europaea, represent a significant health burden in pediatric populations, particularly in Southern Spain. While AIT is a cornerstone of treatment, data on the combined use of polymerized extracts of these specific allergens in children remain limited.

Our study addresses this gap by evaluating the effectiveness and safety of an immunotherapy approach using a modified (glutaraldehyde-polymerized) AIT combining A . alternata and O. europaea extracts, each at 10,000 TU/mL in a group of pediatric patients. After at least 6 months of AIT, children and adolescents demonstrated significant improvement, showing lower grades of clinical severity and reduced pharmacological treatment requirements for both rhinitis and asthma compared to pre-AIT. A key innovation of our study lies in the use of polymerized extracts for both A. alternata and O. europaea. This polymerization ensures the stability of the allergen extracts and, critically, overcomes the challenge of mixing these two components. Native A. alternata extracts possess significant enzymatic and proteolytic activity that would degrade native O. europaea extracts, making a stable combined native formulation unfeasible. This is a key technical advantage that offers a robust and effective solution for co-sensitized patients.

Fungal allergy is a well-known risk factor for severe and fatal asthma (3,8,20,21), with mold allergens often affecting the lower respiratory tract more than pollens. A. alternata is associated with greater bronchial inflammation and worse lung function,²² and a higher risk of developing asthma attacks,²³ hospitalizations,²⁴ and death from asthma.²⁵ As with pollens, the concentration of specific allergens is better associated with respiratory allergy symptoms than the concentration of spores.²⁶ Given A. alternata´s association with severe asthma, early diagnosis and treatment are crucial to prevent such adverse outcomes. Allergen Immunotherapy is unique in its ability to modify allergic disease progression, prevent asthma development in rhinitis patients, and new sensitizations.²⁷ Therefore, particularly when allergen avoidance is impossible, initiating AIT early in childhood for clinically significant asthma and/or rhinitis is vital. In spite of fungi’s importance because of their link with severe asthma, there are few efficacy studies on specific immunotherapy with these allergens.²⁸ In spite of this fact, A. alternata immunotherapy has shown efficacy in various studies.^20,29–32

To our knowledge, this is the first real-life, proof-of-concept study assessing the effectiveness and safety of subcutaneous immunotherapy with the Clustoid^® Max A. alternata plus O. europaea mixture. It demonstrates that this treatment is safe and significantly improves rhinitis and asthma severity and control. Studies on fungal immunotherapy are scarce,^33,34 and even fewer studies examine a mixture of molds with another allergen.

The proteolytic activity of molds traditionally discouraged from mixing them in native extracts with other allergens because of concerns about mixture stability. Polymerized allergoids substantially reduce this proteolytic activity, ensuring stability.^15,16 Importantly, when mixing different modified extracts, the optimal concentration of each component must be guaranteed. This study confirms that this combined treatment is highly effective for both asthma and rhinitis in children allergic to these two aeroallergens, reducing the number of injections (compared to the use of two native AIT products). Significant improvements in severity and control for both conditions were observed after a median of 9 months of treatment. This highlights the treatment’s potential to prevent progression to severe or poorly controlled asthma in children with A. alternata and O. europaea respiratory allergy. The effectiveness also suggests optimal allergen concentrations, indicating that even in a mixture of nonhomologous extracts, therapeutic doses of each component are not reduced and are adequate to reach clinical improvement.

The safety profile of this combined treatment, especially given the presence of A. alternata, could be a concern. However, its safety profile is excellent, with a very low rate (0.47%) of local reactions and no systemic reactions recorded. These safety results align with those from a large real-world observational study (738 patients, including 435 children) using polymerized A. alternata extract alone or in combination.¹⁷ This safety profile is superior to those reported in older studies with native A. alternata extracts in children. A Spanish study with native A. alternata extract reported a 1.95% reaction rate,³⁵ mostly systemic with respiratory involvement, noting higher risk in children and asthmatics, with 7 of 38 children discontinuing because of Grade III systemic reactions. A 2005 study by the Spanish pediatric allergology society showed a lower, but still higher (0.95%), incidence of adverse reactions with native A. alternata extract.³¹

Limitations of this real-world retrospective observational study include the small sample size and lack of randomization. In spite of these, a significant reduction in severity and medication need for rhinitis and asthma was observed. The strengths and novelty of this work lie in the characteristics of the studied population (polysensitized children) and the according of personalized immunotherapy composition.

In conclusion, this real-world study unequivocally demonstrates that immunotherapy using a mixture of polymerized A. alternata and O. europaea extracts is a key technical advantage that assures safety and significantly improves the severity and control of rhinitis and asthma in co-sensitized children.