Received: 20 December 2020 DOI: 10.1002/clt2.12001 RESEARCH - Accepted: 22 December 2020 Clinical and inflammatory characteristics of patients with asthma in the Spanish MEGA project cohort Manuel J. Rial1 | Maria J. Álvarez‐Puebla2 | Ebymar Arismendi3,4 | María L. Caballero5 | José A. Cañas1,3 | María J. Cruz3,6,7 | Francisco J. González‐Barcala3,8 | Juan A. Luna5 | Carlos Martínez‐Rivera3,9 | Joaquim Mullol3,10 | Xavier Muñoz3,6,7 | José M. Olaguibel2,3 | César Picado3,4 | Vicente Plaza3,11 | Santiago Quirce3,5 | Christian Romero‐Mesones7 | Francisco‐Javier Salgado12 | Beatriz Sastre1,3 | Lorena Soto‐Retes3,11 | Antonio Valero3,4 | Marcela Valverde1 | Joaquín Sastre1,3 | Victora del Pozo1,3 1 Servicio de Alergología, Departamento de Inmunología, Instituto de Investigación Sanitaria (IIS) Fundación Jiménez Díaz, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain 2 Servicio de Alergología, Complejo Hospitalario de Navarra, Pamplona, Spain 3 CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain 4 Servicio de Neumología y Alergia, Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain 5 Servicio de Alergia, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain 6 Departamento de Biología Celular, Fisiología e Inmunología, Universitat Autónoma de Barcelona, Barcelona, Spain 7 Servicio de Neumología, Hospital Vall d’Hebron, Barcelona, Spain 8 Servicio de Neumología, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain 9 Servicio de Neumología, Hospital Germans Trias i Pujol, Barcelona, Spain 10 ENT Department, Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain 11 Departamento de Medicina Respiratoria, Hospital de la Santa Creu i Sant Pau, Instituto de Investigación Biomédica Sant Pau (IIB Sant Pau), Universidad Autónoma de Barcelona. Departamento de Medicina, Barcelona, Spain 12 Department of Biochemistry and Molecular Biology, Faculty of Biology‐Biological Research Centre (CIBUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain Correspondence Manuel J. Rial, Allergy Department. IIS‐ Abstract Fundación Jiménez Díaz Av. Reyes Católicos 2 Introduction: The MEGA (MEchanism underlying the Genesis and evolution of 28040 Madrid, Spain. Email: manuterial@gmail.com Asthma) project is a multicenter cohort study carried out in eight Spanish hospitals, gathering clinical, physiological, and molecular data from patients with asthma and Funding information multimorbidities in order to gain insight into the different physiopathological Merck Company Foundation; Instituto de Salud Carlos III, Grant/Award Number: [PI15/ mechanisms involved in this disorder. 00803], [PI15/01900]; CIBERES; Sanofi, Material and Methods: We report the baseline clinical and physiological charac- Grant/Award Number: [02/055] teristics and biomarker measures of adult participants in the project with the aim of better understanding the natural history and underlying mechanisms of asthma as Joaquín Sastre and Victora del Pozo authors have codirected the work. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, pro- vided the original work is properly cited. © 2021 The Authors. Clinical and Translational Allergy published by John Wiley and Sons Ltd on behalf of European Academy of Allergy and Clinical Immunology. Clin Transl Allergy. 2021;11:e12001. wileyonlinelibrary.com/journal/clt2 1 of 10 https://doi.org/10.1002/clt2.12001 2 of 10 - RIAL ET AL. well as the associated multimorbidities across different levels of severity. We car- ried out a detailed clinical examination, pulmonary function testing, measurement of fractional exhaled nitric oxide (FeNO), blood counts, induced sputum, skin prick tests, chest computed tomography scan, asthma questionnaires, and multimorbidity assessment in 512 asthmatic patients. Results: When compared to patients with milder disease, severe asthmatic patients showed greater presence of symptoms, more exacerbations, lower asthma control, increased airflow obstruction, and higher frequency of chronic rhinosinusitis with nasal polyps, severe rhinitis, anxiety and depression, gastroesophageal reflux, and bronchiectasis. Conclusion: The MEGA project succeeded in recruiting a high number of asthma patients, especially those with severe disease, who showed lower control and higher frequency of multimorbidities. KEYWORDS asthma, biomarkers, eosinophilic asthma, inflammation, neutrophilic asthma, phenotype KEYWORDS Spanish: asma; biomarcadores; asma eosinofílica;inflamación; asma neutrofílica; fenotipos. 1 | INTRODUCTION lung function, inflammation, and bronchial hyperresponsiveness, and conduct measurements of associated multimorbidities to establish According to GEMA 5.0,1 asthma is a chronic inflammatory disease the characteristics that shape this asthma population. In addition, the of the airways in which the pathogenesis involves various cell types project will study the stability of different parameters at long term to and inflammatory mediators. In Spain, the prevalence of asthma determine changes in patient condition, exacerbations, control, bio- ranges from 1.5% to 16.7% in the adult population and is approx- markers, as well as treatments that can influence the progression of imately 10% among children.1 The current treatment approach the disease. consists of stratifying patients by phenotype (clinical, inflammatory, The aim of the present study is to describe the baseline char- and molecular)2 but also by endotype (allergic asthma, nonsteroidal acteristics of the adult asthmatic participants that make up the anti‐inflammatory drug [NSAID]‐exacerbated respiratory disease, project cohort in terms of their clinical features, frequency of mul- eosinophilic asthma, or late‐onset asthma), a strategy known as timorbidities, functional features, and inflammatory biomarkers, so as 3 “phenoendotyping.” to better understand the natural history of asthma in patients with Current medications used to treat asthma reduce inflammation different levels of disease severity. of the airways and relieve bronchospasm, but symptoms reappear with cessation of treatment. Recent studies show that over 50% of patients with asthma are not controlled,1,4 indicating a need for 2 | METHODS alternative therapies. Although novel biological treatments directed against type 2 cytokines hold promise,5,6 selection of patients most 2.1 | Design likely to respond to these treatments continues to be hindered by an inadequate understanding of the heterogeneous underlying patho- We conducted a multicenter prospective cohort study of 512 adult physiological and molecular mechanisms involved.7,8 patients with asthma. Consecutive (unselected) sampling was used to The present and future research projects should attempt to recruit patients from eight university hospitals in Spain. identify asthma phenotypes using data collected from multiple cohort studies with sufficiently large sample sizes, integrating data from several domains of the disease repeatedly measured over time. The 2.2 | Data collection MEchanism underlying the Genesis and evolution of Asthma (MEGA) project9 is an ongoing multicenter study in Spain carried out within Standard data collection methods were used in all participating the framework of the CIBER of Respiratory Diseases. This con- research centers. A electronic database and case report form (CRF) sortium will perform a number of imaging studies, determinations of were designed to collect study data.9 RIAL ET AL. - 3 of 10 2.3 | Patient selection and sampling atopy, and polyposis) to airflow obstruction (FEV1/FVC <70% post‐ BD), univariate and multivariate regression analyses were performed Asthma severity has been made according to the classification of the (Table S2). Regression analysis was performed using SPSS statistical Global Initiative for Asthma (GINA).10 Asthma diagnosis (based on software (IBM Corporate). GINA guidelines) preceded the inclusion of patients by at least 1 year. A standardized clinical history will be completed for each patient and validated Spanish versions of the following questionnaires will 3 | RESULTS be administered: Asthma Control Test (ACT),11 Test of Adherence to Inhalers (TAI),12 Asthma Quality of Life Questionnaire 3.1 | Demographic characteristics 13 14 (Mini‐AQLQ), the Sino‐Nasal Outcome Test 22 (SNOT‐22), and the Hospital Anxiety and Depression Scale (HADS).15,16 All study The demographic, functional, clinical, and inflammatory characteris- subjects will undergo a detailed clinical examination, including body tics of the MEGA cohort are summarized in Table 1. Not all data are mass index (BMI) and respiratory function tests (baseline spirometry, available for all patients, due to the absence of some data in the CRF, bronchodilator test, lung volume measurement by plethysmography, including several comorbidities such as sleep apnea, diabetes, hy- fraction of exhaled nitric oxide [FeNO] and CO transfer test [DLCO] pertension, or bronchopulmonary mycosis. using the single‐breath method), following the recommendations of A total of 512 patients were included in the study (66% women). the European Respiratory Society.17,18 Most patients were Caucasian (92%). Obesity, defined as a BMI > 30, Methacholine challenge (PC20) and induced sputum are per- was found in 25% of patients (n = 124); of these, 84% (n = 104) had formed at baseline and subsequently every 24 months. Chest severe persistent asthma and 16% (n = 20) intermittent or mild computed tomography (CT) scan and skin prick tests (SPT) with persistent asthma (p = 0.0269). Currently, 88% of the patients live in common aeroallergens were performed at the beginning of the study. an urban setting, but when these same patients were children this The panel of aeroallergens comprised the following: Dermato- percentage was lower (65.6%), and significantly higher in the most phagoides pteronyssinus, Dermatophagoides farinae, Lepidoglyphus severe asthma patients (p = 0.054). Fifty percent of patients had a destructor, Alternaria alternata, Aspergillus fumigatus, Cladosporium first‐degree relative with a history of asthma. As for smoking, 53.4% herbarum, Penicillium notatum, Cupressus arizonica, Platanus acerifolia, of patients were nonsmokers, 31% ex‐smokers, 8.2% smokers, and Olea europaea, Phleum pratense, Artemisia vulgaris, Parietaria judaica, 7% passive smokers. Asthma severity is not related with smoking Salsola kali, Blatella orientalis, and epithelia (cat and dog). SPTs will be habit. Occupational asthma was found in 7.7% of patients, and 13.4% considered positive at wheal diameters of at least 3 mm compared to had work‐related asthma. the negative control (saline); histamine (10 mg/ml) will be used as a Regarding the severity of asthma, 5% of patients had intermit- positive control. tent asthma (n = 26), 17% mild asthma (n = 90), 33.4% moderate Atopy is defined as the presence of at least one positive SPT or asthma (n = 171), and 39.6% severe asthma (n = 203) (Figure 1). A aeroallergen‐specific immunoglobulin E (IgE) in serum. total of 22 patients (4.3%) could not be correctly classified due to The DNA, serum, exhaled breath condensate, and sputum su- lack of information in the electronic registry. Bronchiectasis was pernatants have been stored at −80°C in each of the recruiting present in 7% of patients (n = 36), with 67% of them (n = 24) centers for further analysis. The detailed protocol has been published experiencing persistent severe asthma. The age of severe patients is elsewhere.9 significantly higher than those with less severity. Over the previous year, 15.5% of patients had developed more than three exacerbations, and only 1.4% did not present any exacer- 2.4 | Statistical analyses bations in this period. Of all patients who experienced exacerbations, 18.6% required hospital admission during the last year, and 9.8% Comparisons between groups were performed using the unpaired, required at least one admission in the intensive care unit (Figure 2). two‐tailed Student’s t‐test for Gaussian samples and Mann–Whitney Of the 74 patients diagnosed with NSAID hypersensitivity, 54% U‐test for non‐Gaussian samples. Normality was analyzed using the (n = 40) had severe asthma (p < 0.001). Of the 24 patients diagnosed Kolmogorov–Smirnov test. Analysis of variance with Bonferroni post with food allergy, 37.5% (n = 9) have severe asthma and 42% (n = 10) hoc test was performed for comparisons between more than two moderate persistent asthma. groups of Gaussian samples, and Kruskal–Wallis with Dunn post hoc test was applied for non‐Gaussian distributions. A p value less than 0.05 was considered significant. In addition, Spearman's correlation 3.2 | Inflammatory characteristics was used to measure the association between clinical parameters. Statistical calculations and graphs were performed with Graph- Among the studied patients, 72.3% (n = 370) had at least one positive Pad Prism 6 (GraphPad Software Inc.). SPT to the aeroallergen tested. Atopy is significantly lower in mod- In order to determine the contribution of a range of factors (sex, erate and severe asthma compared to mild asthma (p = 0.032 and age, BMI, age at onset of asthma, duration of asthma, presence of p = 0.011, respectively). One hundred eighteen patients (23%) had 4 of 10 - RIAL ET AL. TABLE 1 Demographic and clinical characteristics of patients included in the MEGA cohort N Mean ± SD Intermittent Mild Moderate Severe Demographic Age 522 47.3 ± 13 44.08 ± 13.71 42.90 ± 11.3 46.97 ± 12.1 50.50 ± 13 Female (%) 511 66.15 Ethnicity 499 Asian (%) 0.002 0.5 Black (%) 0.002 0.5 Caucasian (%) 91.99 100 82.2 92.4 94.5 Hispanic (%) 6.8 16.6 6.4 3.9 Others (%) 0.78 1.2 1.2 0.5 Type of delivery at birth 450 Dystocic (%) 7.3 13 3.5 7.6 8.2 Eutocic (%) 92.7 87 96.5 92.4 91.8 Urban residence during 461 65.6 50 58 69 65 childhood (%) Pets at home (%) 463 51 58.3 60.23 48.2 50.26 Age at onset 470 <12 years (%) 41.4 30.4 28.7 23.8 28 12–40 years 20.1 30.4 52.8 46.9 50.1 >40 years 38.5 39.2 13.5 27.3 21.2 BMI 496 27.01 ± 5 26.5 ± 5 26.15 ± 4.4 27.35 ± 5.7 27.35 ± 5.4 Smoking status 486 Never 53.2 76.9 71.9 48.2 44.8 Passive 7.5 3.4 7.6 10.4 Ex‐smoker 30.8 19.2 19.1 31.8 37.8 Smoker 8.5 3.8 5.6 12.4 7 (Mean ± SD pack‐years) 35.68 ± 92.5 12 9.75 ± 6.2 57.5 ± 128.1 15.9 ± 15.2 Alcohol consumer (%) 482 23.3 12 23.9 31.5 17.4 Athletic activity (%) 481 44.3 24 47.7 41.1 47.5 Inflammatory characteristics Atopy 481 76.9 76.9 89.9 74.6 73.1 Total IgE 458 419.1 ± 824 214.4 ± 232.4 30.4 ± 439 427.6 ± 924 492 ± 914 3 Eosinophils (cells/mm ) 489 336 ± 327 252 ± 174.3 272.5 ± 183 329.4 ± 220 390 ± 444 Eosinophils in sputum (%) 212 10.49 ± 19 4.72 ± 3.17 6.49 ± 10.7 8.8 ± 16.7 14.11 ± 22.9 FENO 340 41.82 ± 37 56.63 ± 48.17 42.48 ± 37 36.84 ± 29.6 46 ± 40 Functional parameters PC20 methacholine 198 4.63 ± 10 8.025 ± 12.14 3.073 ± 6 3.72 ± 12.9 4.66 ± 6.3 RV% 222 123 ± 44 123.2 ± 47.54 132.8 ± 42.5 113.2 ± 42.6 129.4 ± 45.18 TLC% 227 108 ± 56 95.07 ± 33.66 97.8 ± 28.45 118.2 ± 84.72 104.6 ± 20.32 FEV1 Pre‐BD (%) 493 85.71 ± 21 102.5 ± 15.8 97.09 ± 15.65 88.18 ± 17.3 75.5 ± 21.8 FEV1 Post‐BD (%) 365 86.06 ± 3 108.9 ± 16.7 100 ± 26.82 81.92 ± 37.88 81.1 ± 29.3 FVC Pre‐BD (%) 493 100.1 ± 46 108.9 ± 13.5 107 ± 16.53 101.7 ± 18.48 93.9 ± 68.4 FVC Post‐BD (%) 364 95.41 ± 59 112.3 ± 12.9 105 ± 28.28 89.5 ± 42.37 94.5 ± 80.3 FEV1/FVC Pre‐BD 474 71.81 ± 37 112.9 ± 168.6 86.75 ± 97.8 70.16 ± 14.13 67.73 ± 11.70 FEV1/FVC Post‐BD 333 76.68 ± 65 128.2 ± 198.6 95.96 ± 116.7 72.70 ± 13.3 71.33 ± 12.44 DLCO 201 95.9 ± 22 91.44 ± 14.83 97.8 ± 19.33 96.5 ± 22.35 95.22 ± 23.11 Questionaries ACT 471 20.27 ± 5 23.70 ± 1.845 21.89 ± 3.74 21.70 ± 3.68 17.97 ± 5.4 AQL 444 5.48 ± 1.38 6 ± 0.84 5.83 ± 1.33 5.87 ± 1.17 4.92 ± 1.45 SNOT‐22 416 29.5 ± 19.97 25.22 ± 1.57 24.4 ± 16.84 26 ± 18.9 35.21 ± 21 Abbreviations: ACT, Asthma Control Test; IgE, immunoglobulin E; MEGA, MEchanism underlying the Genesis and evolution of Asthma; RV, residual volume; SNOT‐22, Sino‐Nasal Outcome Test 22; TLC, total lung capacity. RIAL ET AL. - 5 of 10 F I G U R E 1 Flow chart showing visit schedule. The items appearing in the boxes are defined as follows: Sex; number of patients who completed prebronchodilator spirometry, a specific inhalation challenge with methacholine, exhaled breath condensate, the Asthma Control Test (ACT), induced sputum. Atopy is defined by at least one positive skin prick test (SPT) or detectable levels of specific IgE for any allergen tested. IgE, immunoglobulin E positive SPT to dog, 22% (n = 110) to cat, 45% (n = 230) to mites, diagnosed with food allergy. Of the 148 patients with CRSwNP, only 15% (n = 76) to pollen, 8% (n = 42) to Aspergillus, and 5.5% (n = 28) 1 patient had intermittent asthma, 10 mild asthma, 49 moderate to Alternaria (Figure 3). asthma, and 88 (59%) presented severe asthma (p < 0.001). The level of IgE was 419.1 ± 824 and tended to increase at Peripheral blood eosinophilia was measured in all patients at the greater degrees of severity. start of the study (Table 1 and Figures 1 and 4). Eighty percent of Also, 52% of patients (n = 266) had allergic rhinitis, 10% (n = 52) patients (n = 409) had a peripheral eosinophil count of ≥150 cells/ atopic dermatitis, 18% (n = 90) allergic conjunctivitis, 29% (n = 148) mm3, 53.3% (n = 273) had a count that was equal to or higher than had chronic rhinosinusitis with nasal polyps (CRSwNP), 14.5% 300 cells/mm3, 38.8% (n = 197) had a count of ≥400 cells/mm3, and (n = 74) showed hypersensitivity to NSAIDs, and 4.7 % (n = 24) were 28.3% (n = 145) had levels above 500 cells/mm3 (Figure 3). A 6 of 10 - RIAL ET AL. correlation was found between eosinophil levels above 500 and the (<3% of eosinophils and >61% of neutrophils) in 25 patients (11.8%), presence of atopy (p = 0.0375, relative risk = 0.6884, Katz's a paucigranulocytic pattern (defined as <61% neutrophils and <3% of approximation). There were no significant differences in eosinophil or eosinophils) in 52 patients (24.5%), and we observed a mixed pattern FeNO levels according to asthma severity (Figure 4), although a (>3% eosinophils and >61% neutrophils) in 64 patients (30.2%). tendency toward increased eosinophil counts was seen in severe Correlations have been made with different levels of eosinophilia asthma compared to intermittent disease (1.5‐ and 3‐fold in blood in peripheral blood (150, 300, 400, and 500 cells/mm3) and in sputum and sputum, respectively). (>2% and >3%). The results show that there are more patients with Induced sputum was available for 212 patients. An eosinophilic above 300 cells/mm3 eosinophilia in sputum, as shown in Figure 5. inflammatory profile (defined as >3% of eosinophils and <61% of The best correlation is established between more than 300 cells/mm3 neutrophils) was found in 64 patients (30.2%), a neutrophilic profile in peripheral blood and more than 2% eosinophils in sputum (Spearman r = 0.5235, p = 0.0002). 3.3 | Functional parameters Lung function test results and different asthma severity levels are shown in Figure 6. Statistically significant differences have been found between spirometry values (FEV1%, FVC%, FEV1/FVC) and the different levels of asthma severity. No statistically significant differences were found in plethysmography values (residual volume [RV], total lung capacity [TLC]%), DLCO, or in bronchial hyper- responsiveness with methacholine. F I G U R E 2 Patients with severe exacerbations according to asthma severity and hospital admissions. ER, emergency room admission; HA, hospital admissions; ICU, intensive care unit admission 3.4 | Questionnaires for disease control, quality of life, and anxiety/depression ACT scores of <20 were recorded for 34.5% of patients (n = 134). The Mini‐AQLQ questionnaire showed a mean score of 5.48 ± 1.38. These results are reflected in Figure 7 and Table 1. Correlations were made with the Fisher test to determine whether patients with ACT levels <20 had more exacerbations in the previous year, and a statistically significant association was found (p < 0.0001). Anxiety and depression were assessed using the HADS Ques- tionnaire, revealing statistically significant differences (p < 0.001) between patients with severe asthma and other severity levels. FIGURE 3 Sensitization to aeroallergens and asthma severity Patients with severe disease had scores compatible with depression levels F I G U R E 4 Blood eosinophil count and FENO levels (expressed as mean/median and standard error). No statistically significant differences (p < 0.05) were found between blood eosinophil counts and FeNO levels and the severity of asthma RIAL ET AL. - 7 of 10 F I G U R E 5 Correlation between eosinophilia in peripheral blood and sputum. The best correlation is established between more than 300 cells/mm3 in peripheral blood and more than 2% of eosinophils in sputum (correlation coefficient = 0.5235, p = 0.0002) F I G U R E 6 Spirometric, pletismography, and bronchial hyperreactivity data. There are statistically significant differences between FEV1%, FVC%, FEV1/FVC, and asthma severity levels *p < 0.05, **p < 0.01, ***p < 0.001. No statistically significant differences were found in FVC%, RV%, TLC%, and PC20 methacholine and asthma severity. RV, residual volume; TLC, total lung capacity F I G U R E 7 ACT and Mini‐AQLQ scores. Patients with severe asthma (191) had worse control with an average ACT score of 18 points (p < 0.001). Statistically significant differences *p < 0.05, **p < 0.01, ***p < 0.001 were found between different levels of asthma severity. ACT, Asthma Control Test; AQLQ, Asthma Quality of Life Questionnaire 8 of 10 - RIAL ET AL. in 17%, and scores for 39% of patients were compatible with anxiety. interesting to know whether these associations change over the Patients with moderate persistent asthma had values compatible follow‐up period and whether the predictive capacity of these asso- with depression in 8.9% of patients and with anxiety in 37.8% of ciations can be assessed. patients; among patients with intermittent and mild persistent Air trapping is a characteristic of the severely asthmatic asthma, 8% had levels compatible with depression and 24% that were population, and the RV rises with increasing severity.25 Furthermore, compatible with anxiety. air trapping has been more frequently associated with neutrophilic We performed the SNOT‐22 test, comparing the results across phenotypes and poor response to ICS with the presence of persistent different levels of asthma severity. Statistically significant differences bronchial obstruction. In our population, however, there was no were found (p < 0.001), with higher SNOT‐22 values associated with statistical difference across patients in terms of RV or TLC according more severe asthma. to severity classification. Adherence to therapy was also measured using the TAI test, A novel aspect of this study is the evaluation of rhinosinusitis by revealing somewhat poor or poor adherence in 18% of patients. means of the SNOT‐22 questionnaire. As expected, we found that higher SNOT scores (more severe rhinosinusitis) were correlated with more severe asthma. It is also important to note that NSAID intoler- 3.5 | Treatments ance was strongly associated with asthma severity in this cohort. It was not possible to calculate the average dose of ICS that During the study period, 386 patients were using an inhaled corti- patients receive due to a wide variety of devices and formulations costeroid (ICS)/long‐acting beta2‐agonist combination, 24% (n = 95) available in the Spanish market. However, all patients were classified were using a metered‐dose inhaler, and more than half of them used by severity according to the medication needed to achieve asthma a spacer. Seventy‐six percent (n = 291) of the subjects used dry control. Patients with mild asthma required low doses (200–400 μg/ powder devices: 47.07% (n = 137) Turbuhaler, 22% (n = 64) day of budesonide or equivalent), while moderate asthma required Accuhaler; 14.4% (n = 42) Ellipta, 13.4% (n = 39) Nexthaler, and 4% moderate doses (401–800 μg/day of budesonide or equivalent), and used other devices. In all patients, data concerning the inhalation severe patients required high doses (801–1600 μg/day). We were technique was obtained by direct observation, and 97% of patients also unable to measure the average dose of systemic corticosteroids rated their treatment as adequate (n = 373). due to the different corticosteroid presentations used as well as the Regarding the treatment with biological drugs, there are 95 use of depot formulations. patients treated with a biological medication, that is, 73% with When interpreting these data, it is important to note the good omalizumab (n = 69), 19% with mepolizumab (n = 18), 4% with results obtained with regard to the inhalation technique (adequate in reslizumab (n = 4), and another 4% (n = 4) with benralizumab. 97%) and adherence (adequate in 82%) in comparison to other co- horts.8,19–22 However, these results may be due to the fact that the patients were recruited in specialized centers and may not reflect 4 | DISCUSSION real life in primary care, where treatment is often inadequate.26 We found elevated peripheral blood eosinophil counts in 53.3% This is the largest cohort of Spanish asthmatic patients studied to of patients, as indicated by mean values above 300 cells/mm3, and date. Overall, the clinical characteristics of these patients are 28.3% had levels above 500 cells/mm3. An assessment of eosinophilic 19 consistent with those found in the European cohort U‐Biopred as inflammation based on differential cell count in induced sputum well as other large asthma cohorts described worldwide.8,20–24 Pa- samples revealed that 73% of the patients presented more than 2% tients included in this, the MEGA study, showed a clearly significant of eosinophils, indicating levels above other cohorts described in the association between severe asthma on the one hand and increased literature.19,22,27,28 We investigated whether an eosinophil count of symptoms and exacerbations, lower disease control, greater airflow more than 400 cells/mm3 is a risk factor for having worse asthma obstruction and higher frequency of CRSwNP, severe rhinitis, anxiety control (ACT < 20), though no statistically significant differences and depression, gastroesophageal reflux, and bronchiectasis on the were detected. Furthermore, no differences were found between other when compared to patients with milder disease. patients with eosinophil counts above or below 400 cells/mm3 and A number of findings make this cohort of asthmatic patients one early‐onset disease debuting in childhood or adolescence. Further- of great interest. Significant associations were found between airflow more, we have found a positive correlation between the levels of obstruction and various demographic and clinical features, such as eosinophilia in blood and in sputum when the cut‐off point of age, asthma duration, and the presence of CRSwNP (Table S2). These eosinophils in sputum was set at more than 2% and in peripheral data contrast with those of other cohorts, in which a strong associ- blood higher than 300 cells/mm3, a better correlation index was 24 ation was found with female sex, BMI, and presence of atopy. In our obtained than establishing the sputum cut‐off point at more than 3% cohort, these variables seem to be associated with airflow obstruc- (correlation index 0.5235 vs. 0.4819). The latter may be due to the tion, but do not have predictive ability, since the adjusted R2 is only high proportion of patients with CRSwNP (29%) and atopy included 3.4%. Random forest modeling was used to complete the regression in the sample (71%). Blood eosinophil counts above 500 cells/mm3 analysis, though this did not improve predictability. It will be correlated with the presence of atopy. It is important to note that in RIAL ET AL. - 9 of 10 this cohort, eosinophil levels are not associated with asthma control Stallergenes‐Greer, Teva, and Grants from Mundipharma outside the or age at disease onset. submitted work. Dr. Quirce reports personal fees from AstraZeneca, In asthma, sensitization to fungal, perennial, or seasonal allergens personal fees from Novartis, personal fees from Sanofi, personal fees increases the risk of uncontrolled symptoms, exacerbations, and poor from Boehringer Ingelheim, personal fees from Teva, personal fees disease outcomes.10 In severe asthma, 20% to 29% of patients typi- from ALK, personal fees from Mundipharma, personal fees from GSK, cally show sensitization to ≥1 fungal allergen, with Aspergillus being personal fees from Chiesi, personal fees from Leti, outside the sub- one of the most common.29 These patients have worse lung function, mitted work. Dr. Romero‐Mesones reports personal fees from Teva, increased risk of oral corticosteroid use and hospitalization, and a personal fees from GSK, outside the submitted work. Dr. Soto‐Retes higher degree of airflow obstruction than patients not sensitized to reports non‐financial support from CIBER de Enfermedades Respi- 30 fungal allergens. Nevertheless, our patients presented a similar rate ratorias (CIBERES), during the conduct of the study; personal fees of sensitizations to Aspergillus and Alternaria at different severities. from Stallergennes‐Greer, personal fees from Menarini, personal fees A similar finding was observed with other allergens. from Novartis, personal fees from GSK, personal fees from Hal The study has a number of limitations, such as the lack of a Allergy, personal fees from Allergy Therapeutics, personal fees from control group and the fact that patients were recruited in specialized AstraZeneca, grants from Sociedad Española de Alergologia e centers with a higher number of severe asthma patients than mild Inmunología Clinica SEAIC, grants from Sociedad Española de Neu- cases. In addition, the diagnosis of CRSwNP was based on data mologia y Cirugia Torácica SEPAR, outside the submitted work. Dr. obtained from the clinical history. Martinez Rivera reports grants and personal fees from AstraZeneca, To conclude, among the patients with asthma included in this grants and personal fees from Teva, grants and personal fees from cohort, eosinophilic asthma was the predominant phenotype, and GSK, personal fees from Novartis, personal fees from Mundipharma, many were atopic. An increase in disease severity was associated outsider the submitted work. Dr. Munoz reports personal fees from with a higher number of comorbidities, more exacerbations, worse AstraZeneca, personal fees from Boehringer Ingelheim, grants and disease control, a greater tendency to experience anxiety and personal fees from GlaxoSmithKline, grants from Menarini, personal depression, and worse lung function. The characteristics of the pa- fees from Novartis, personal fees from Teva, personal fees from tients included are consistent with those previously reported in other Mundifarma, grants and personal fees from Chiesi, personal fees cohorts. This cohort is useful for characterizing different asthma from Faes, outside the submitted work. Dr. Sastre reports grants phenotypes and in identifying associated biomarkers as well as the from Sanofi, during the conduct of the study; grants and personal fees stability of these biomarkers over time. from Sanofi, personal fees from GSK, personal fees from Novartis, Continued follow‐up of these patients will shed light on the long‐ personal fees from AstraZeneca, personal fees from Mundipharma, term factors that may influence disease severity and will likely pro- personal fees from Faes Farma, outside the submitted work. Dr. vide insight into the treatments that can influence the progression of Luna‐Porta reports other from Astra Zeneca, outside the submitted the disease, identifying the possible causes of the exacerbations and work. Dr. Olaguibel reports grants from Sanofi during the conduct of how such exacerbations affect the clinical course of the disease. the study; personal fees from AstraZeneca, personal fees from Mundipharma, outside the submitted work. Dr. Mullol reports per- ACK N O W L E D G M E N T S sonal fees and other from Sanofi‐Genzyme and Regeneron, personal This study was supported by Sanofi [02/055], Fondo de Investigación fees and other from Genentech and Novartis, grants and personal Sanitaria–FIS [PI15/00803], [PI15/01900], the Merck Health fees from Mylan Pharma, grants and personal fees from Uriach Foundation, CIBER de Enfermedades Respiratorias (CIBERES), a Group, personal fees from Mitsubishi Tanabe, personal fees from Carlos III Institute of Health initiative, and FEDER funds. The authors Menarini, personal fees from UCB Pharma, personal fees from recognize Oliver Shaw, English editor of IIS‐FJD, for his revision AstraZeneca, personal fees from GSK, personal fees from MSD, and editing of the manuscript, and Ignacio Mahillo‐Fernández for outside the submitted work. Dr. Plaza reports grants and personal statistical revision. fees from AstraZeneca, personal fees from Boehringer Ingelheim, personal fees from Merck, grants and personal fees from Chiesi, CO N F LI C T O F I N T ER E S T S personal fees from Novartis, grants from Menarini, personal fees Dr. Rial reports nonfinancial support from CIBER de Enfermedades from Sanofi, outside the submitted work. Other authors have no Respiratorias (CIBERES), during the conduct of the study; personal conflicts of interests. fees from GSK, personal fees from Allergy Therapeutics, personal fees from AstraZeneca outside the submitted work. Dr. González A UT H O R C O N TR I B UT I O N S Barcala reports personal fees from ALK, personal fees from Astra- Manuel J. Rial analyzed the patient data and was a major contributor Zeneca, personal fees from Bial, personal fees from Boehringer in writing the manuscript. Joaquín Sastre and Victora del Pozo Ingelheim, personal fees from Chiesi, personal fees from Gebro interpreted the patient data and designed the statistical study. All the Pharma, personal fees from GlaxoSmithKline, personal fees from authors have provided data to be able to carry out the study. All Laboratorios Esteve, Menarini, Mundipharma, Novartis, Rovi, Roxall, authors read and approved the final manuscript. 10 of 10 - RIAL ET AL. O RC ID 19. Shaw DE, Sousa AR, Fowler SJ, et al. Clinical and inflammatory Francisco J. González‐Barcala https://orcid.org/0000-0001-5847- characteristics of the European U‐BIOPRED adult severe asthma cohort. Eur Respir J. 2015;46(5):1308‐1321. 4784 20. Abraham B, Antó JM, Barreiro E, et al. The ENFUMOSA cross‐ Juan A. Luna https://orcid.org/0000-0003-0280-0143 sectional European multicentre study of the clinical phenotype of chronic severe asthma. Eur Respir J. 2003;22:470‐477. 21. Moore WC, Bleecker ER, Curran‐Everett D, et al. 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