Asthma and Sports: mechanisms and effects of Airway demage in elite athletes.

Academic Dissertation to be presented, with the permission of the Faculty of Medicine of the University of Porto, for public examination Dissertação de candidatura ao grau de Doutor apresentada à Faculdade de Medicina da Universidade do Porto Porto, 2016 Asthma and Sports Asma e Desporto Mechanisms and effects of airway damage in elite athletes Mecanismos e efeitos de lesão das vias aéreas em atletas de elite Mariana Couto Immunology Faculty of Medicine, University of Porto Supervised by: Professor André Moreira, MD, PhD Assistant Professor of Immunology, Faculty of Medicine, University of Porto Medical Doctor of Immunoallergology, Centro Hospitalar São João, Porto Professor Kai-Håkon Carlsen, MD, PhD Professor of Sport Medicine, Norwegian School of Sport Sciences Professor of Paediatric Allergology and Respiratory Medicine, University of Oslo Medical Consultant of Pediatrics, Oslo University Hospital, Rikshospitalet, Oslo ISBN 978-989-20-6345-4 “The deepest sin against the human mind is to believe things without evidence.” Aldous Huxley 4 Contents Abstract 6 Resumo 8 Abbreviations 12 List of original publications 14 1. INTRODUCTION 16 2. REVIEW OF THE LITERATURE 18 2.1 Asthma, physical activity and exercise 18 2.2 Asthma in athletes 18 2.3 Exercise-induced asthma: definition and heterogeneity 19 2.4 Mechanisms of asthma in athletes 21 2.4.1 The inflammatory hypothesis 22 2.4.2 The airway epithelial damage hypothesis 23 2.4.3 The neurogenic hypothesis 25 2.5 Risk factors for asthma in athletes 26 2.6 Long-term effects of sports on the airways of athletes 28 2.7 The importance of appropriate management 29 3. AIMS OF THE THESIS 32 4. MATERIAL AND METHODS 34 4.1 Participants and study design 34 4.1.1 Diagnosis of asthma and healthcare of asthmatic athletes (Study I) 35 4.1.2 Phenotypes of asthma in elite athletes (Study II) 35 4.1.3 Parasympathetic activity and bronchial hyperesponsiveness (Study III) 37 4.1.4 Anti-cholinergic drugs and BHR in elite athletes (Study IV) 38 4.1.5 Exhaled breath temperature, exercise and asthma (Study V) 38 4.1.6 Airway inflammation and long-term competitive swimming (Study VI) 39 4.2 Measurements 40 4.2.1 Lung function and reversibility (Study III, IV and V) 40 4.2.2 Bronchial hyperesponsiveness (Study III, IV and V) 41 4.2.3 Allergic sensitization (Study III, V, and VI) 41 4.2.4 Body temperature (Study V) 42 4.2.5 Autonomic nervous system activity (Study III) 42 4.2.6 Airway inflammation (Study IV, V and VI) 44 5 4.2.6.1 FeNO (Study IV and VI) 44 4.2.6.2 Induced sputum (Study V) 44 4.2.6.3 Exhaled Breath Temperature (Study V) 45 4.2.7 Physical activity (Study VI) 46 4.2.8 Assessment and diagnosis of asthma (Study I, II, III, IV, V and VI) 46 4.2.8.1 AQUA© (Study IV) 46 4.2.8.2 ISAAC questionnaire (Study VI) 46 4.3 Statistical analysis (Studies I-VI) 47 Latent Class analysis 47 Multiple factor analysis 49 Power and sample size calculations 49 4.4 Ethics 49 5. RESULTS 50 5.1 Diagnosis of asthma and healthcare of asthmatic athletes (study I) 50 5.2 Phenotypes of asthma in elite athletes (study II) 52 5.3 Dysautonomia, asthma and BHR in elite swimmers (study III) 54 5.4 Anti-cholinergic drugs for BHR in elite athletes (study IV) 57 5.5 Exhaled breath temperature, exercise and asthma (Study V) 59 5.6 Effect of long-term swimming on airways (study VI) 62 6. DISCUSSION 64 6.1 Main findings and relation to previous studies 64 6.1.1 Diagnosis of asthma and healthcare of asthmatic athletes (Study I) 64 6.1.2 Phenotypes of asthma in elite athletes (Study II) 65 6.1.3 Dysautonomia, asthma and BHR in elite swimmers (Study III) 65 6.1.4 Anti-cholinergic drugs for BHR in elite athletes (Study IV) 66 6.1.5 Exhaled breath temperature, exercise and asthma (Study V) 67 6.1.6 Effect of long-term swimming on airways (Study VI) 68 6.2 Methodological considerations – limitations and strengths 69 6.3 Implications for practice and future research 73 7. CONCLUSIONS 78 ACKNOWLEDGEMENTS 80 REFERENCES 82 ORIGINAL PUBLICATIONS 98 6 Abstract Elite athletes have an increased risk for asthma, especially those who take part in endurance sports, such as swimming or running, and in winter sports. Asthma is a significant problem for both recreational and competitive athletes and is the most common chronic condition among Olympic athletes, with obvious implications for their competing performance, health and quality of life. Some hypotheses explain how intensive physical activity may cause exercise-induced asthma. Classical postulated mechanisms include the osmotic and the cooling hypothesis, resulting in the release of inflammatory mediators that cause airway smooth muscle contraction. However, the inflammatory pathway explanation does not seem to be entirely satisfactory to justify the increased prevalence of exerciseinduced asthma in athletes. Studies are needed to better define the etiologic factors and mechanisms involved in development of asthma in elite swimmers, to ultimately propose relevant preventive and therapeutic measures. Therefore this thesis aims to investigate phenotypes and mechanisms of asthma development on elite athletes. This thesis is based on three types of studies: 1. Cross-sectional studies: a) analysis of asthmatic elite athletes to assess different phenotypes of asthma; b) analysis of asthmatic and healthy elite athletes to investigate the association between bronchial hyperresponsiveness (BHR) and parasympathetic activity measured by pupillometry; 2. Observational studies: a) prospective evaluation of swimmers to assess the effect of a swimming training session on the exhaled breath temperature in the airways of both asthmatic and non-asthmatic elite athletes; b) investigation of long-term swimming effect on airway inflammation in competitive swimmers; and c) retrospective analysis of asthma medication requests submitted by elite athletes to the Anti-Doping Authority of Portugal between 2008 and 2010 to explore the impact of asthma and anti-doping regulations in elite athletes’ health; 3) A randomized cross-over trial to compare the reversibility to inhaled ipratropium bromide with the reversibility to inhaled salbutamol and to investigate correlation between PD20 obtained with methacholine bronchial challenge and the reversibility to each of the drugs. A total of 127 swimmers participated in the observational prospective studies, 22 elite winter athletes participated in the randomized cross-over trial and 27 elite swimmers in one of the cross-sectional studies. For the other cross-sectional study, data from clinical files of elite athletes at the Portuguese database of Olympic athletes and at database of Norwegian School of Sport Sciences were analyzed. Data from asthma medication requests submitted to the Anti-Doping Authority of Portugal referring to 326 elite athletes were reviewed for the retrospective study. Since objective evidence of asthma based on the International Olympic Committee definition became mandatory for the diagnosis of asthma in athletes, the number of applications for the use of anti-asthmatic inhalers has been reduced to approximately half. By requiring additionally objective evidence to validate asthma diagnosis, guidelines improved athlete’s health care. Using latent class analysis and the IOC definition of asthma, two patterns of asthma aggregation features were identified: “atopic asthma”, 13 14 List of original publications This thesis is based on the following publications, which are referred to in the text by their roman numerals: I Couto M, Horta L, Delgado L, Capão-Filipe M, Moreira A. Impact of changes in anti-doping regulations (WADA Guidelines) on asthma care in athletes. Clin J Sport Med. 2013;23(1):74-6. doi: 10.1097/JSM.0b013e31826869ef II Couto M, Stang J, Horta L, Stensrud T, Severo M, Mowinckel P, Silva D, Delgado L, Moreira A, Carlsen KH. Two distinct phenotypes of asthma in elite athletes identified by latent class analysis. J Asthma. 2015;52(9):897-904 doi: 10.3109/02770903.2015.1067321 III Couto M, Silva D, Santos P, Queirós S, Delgado L, Moreira A. Exploratory study comparing dysautonomy between asthmatic and non-asthmatic elite swimmers. Rev Port Pneumol. 2015;21(1):22-9. doi: 10.1016/j.rppnen.2014.05.004. IV Stang J, Couto M, Carlsen KH, Stensrud T. Increased bronchial parasympathetic tone in elite cross-country and biathlon skiers: a randomised crossover study. Br J Sports Med. 2015;49(1):56-61 doi: 10.1136/bjsports-2014-094053. V Couto M, Santos P, Silva D, Delgado L, Moreira A. Exhaled breath temperature in elite swimmers: the effects of a training session in adolescents with or without asthma. Pediatr Allergy Immunol. 2015;26(6):564–570 doi: 10.1111/pai.12426 VI Couto M, Andrade P, Pereira M, Araújo J, Moreira P, Delgado L, Moreira A. Effect of competitive swimming on airway inflammation: a 3-yr longitudinal study. Pediatr Allergy Immunol. 2014;25(2):193-5. doi: 10.1111/pai.12172 The original publications are reprinted with the kind permission of the copyright holders. 15 16 1. INTRODUCTION Physical exertion is one of the many stimuli that can produce episodes of airway obstruction in asthmatic patients, so-called exercise-induced asthma (EIA). This is particularly relevant for subjects practicing competitive sports. It is well known that elite athletes have an increased risk for asthma, especially those who take part in endurance sports, such as swimming or running, and in winter sports (Carlsen, Anderson et al. 2008, Schwartz, Delgado et al. 2008, Fitch 2012). Two different clinical phenotypes of asthma in elite athletes were previously suggested (Haahtela, Malmberg et al. 2008), but these phenotypes have not been further confirmed, and the contribution of risk factors, such as environmental exposures, for the occurrence of particular phenotypes was not explored. Some hypotheses explain how intensive physical activity may cause EIA. Classical postulated mechanisms include the osmotic hypothesis (Anderson and Kippelen 2005), the disruption of the airway epithelium (Anderson and Kippelen 2008) and cooling of the airways (Carlsen, Anderson et al. 2008, Schwartz, Delgado et al. 2008), causing heat loss from the respiratory mucosa. These hypotheses consider inflammation and mast cells activation as being crucial for the development of EIA (Ali, Norsk et al. 2012). Recently, it has been debated whether the exercise-induced bronchoconstriction (EIB) that occurs in athletes during their sports career without other features of clinical asthma is identical to what is usually considered to be asthma in clinical practice, or rather has peculiar clinical and pathologic features. Some answers in the explanatory model of EIB in athletes are still lacking. It has been proposed that intensive training can have effects on autonomic regulation. In fact, autonomic nervous system activity assessed by pupillometry in endurance athletes showed increased parasympathetic activity of the pupillary light reflex (Filipe, Falcão-Reis et al. 2003). Increased parasympathetic (cholinergic) tonus could predispose to an increase in bronchomotor tone and therefore susceptibility to bronchospasm. However, the impact of dysautonomy in asthma and bronchial hyperesponsiveness (BHR) has not been fully investigated. As a proof of concept of this theory, it should be expected to observe a therapeutic effect of anticholinergic drugs in these athletes. On the other hand, evidence of the cooling hypothesis acting as mechanism of EIA would require asthmatic elite athletes to present a higher exhaled breath temperature during exercise, an increase that would also be expected in the presence of airway inflammation. Development of asthma generally occurs in young adults rather than in adolescent competitive swimmers, suggesting that airway inflammation and hyperesponsiveness develop during the training career. While the latter seems to be a transient phenomenon (Bougault, Turmel et al. 2011), whether airway inflammation persists is still debated (Helenius, Rytilä et al. 2002, Bougault, Turmel et al. 2009, Bougault, Loubaki et al. 2012). Studies are needed to better define the factors and mechanisms involved in the complex etiology and pathogenesis of asthma in athletes, ultimately leading to propose relevant therapeutic measures. Also, the long term effects of swimming need to be fully established in order to find new strategies to prevent their negative impact. 18 2. REVIEW OF THE LITERATURE 2.1 Asthma, physical activity and exercise Regular physical exercise and participation in sports are considered to be important components of a healthy life and are recommended for all individuals (Physical Activity Guidelines Advisory Committee 2009). There is unquestionable evidence that regular physical activity contributes to the primary and secondary prevention of cardiovascular diseases and several other chronic conditions (Bauman 2004). International guidelines recommend children above 2 years and youth to participate in at least 60 minutes of enjoyable, moderate-intensity physical activities every day (Physical Activity Guidelines Advisory Committee report 2009). Compared to inactive young people, physically active children and youth have higher levels of cardiorespiratory endurance (Physical Activity Guidelines Advisory Committee report 2009). Regarding asthma, evidence has shown that physical training improves cardiopulmonary fitness (Chandratilleke, Carson et al. 2012) and may even improve quality of life of asthmatics (Eichenberger, Diener et al. 2013) both in children and their caregivers (Silva, Couto et al. 2013). It has been suggested that moderate intensity physical training may decrease both total and allergen specific IgE levels (Moreira, Delgado et al. 2008). Also, it has been previously shown that intense swimming activity causes a lung growth greater than normal in children and adolescents (Silvestri, Crimi et al. 2013) and teaches airway control (Whinder 2013); also, the hot and humid conditions of swimming training have been pointed out as less asthmogenic. So, physical activity should be recommended as a supplementary therapy to medication in asthmatic subjects (Craig and Dispenza 2013, Eichenberger, Diener et al. 2013) and swimming has turned out to become a very popular sport, both among children with and without asthma (Vahlkvist and Pedersen 2009, Vahlkvist, Inman et al. 2010). But physical exertion is a powerful trigger of bronchoconstriction and symptoms in patients with asthma. Symptoms of asthma during exercise may result in avoidance of physical activity leading to detrimental consequences to physical and social well-being of patients with asthma. It is an even more relevant and important problem when considering patients practicing sports. Exercise is a frequent trigger of asthma symptoms, which impairs athlete’s performance. 2.2 Asthma in athletes Although structured exercise on a recreational level has been shown to be beneficial, repeated high-intensity exercise performed by elite athletes contributes to the development of asthma and BHR (Weiler, Anderson et al. 2010). In recent years there has been a special focus on the increased occurrence of asthma and BHR among top athletes within endurance sports (Carlsen 2009). As early as 1989, an increase in nonspecific bronchial responsiveness after heavy endurance training was found in young competitive swimmers (Carlsen, Oseid et al. 1989). Later, reports were published 19 concerning increased prevalence of asthma and BHR to methacholine among top crosscountry skiers (Larsson, Ohlsén et al. 1993, Heir and Oseid 1994). These and other studies confirmed that both BHR and airway inflammation increased through heavy endurance training (Carlsen, Oseid et al. 1989, Larsson, Ohlsén et al. 1993, Heir and Oseid 1994, Sue-Chu, Karjalainen et al. 1998, Karjalainen, Laitinen et al. 2000). In the Olympic arena, such reports were confirmed by the observed prevalence of EIA of 11% among the American 1984 summer Olympic athletes (Voy 1986); this prevalence increased to >20% among the American participants in the 1996 summer Olympic Games, and was especially high among cyclists and mountain bikers (Weiler, Layton et al. 1998). The use of asthma drugs and, in particular, inhaled 2-agonists, was shown to be highest in cross-country skiing and speed-skating followed by cycling, Nordic combined (the combination of both cross-country skiing and ski jumping) and swimming during the last three summer Olympic and the last three winter Olympic Games (Fitch 2012). So, it is nowadays well established that elite athletes have an increased risk for asthma, especially those who take part in endurance sports, such as swimming or running, and in winter sports (Carlsen, Anderson et al. 2008, Schwartz, Delgado et al. 2008, Fitch 2012). Asthma is a significant problem for both recreational and competitive athletes and is more prevalent than in the general population (Schwartz, Delgado et al. 2008). EIA is actually the most common chronic condition among Olympic athletes (Fitch 2012), with obvious implications for their health, competing performance and quality of life. There are substantial data showing that EIB occurs very commonly in athletes at all levels. Many studies have been performed in Olympic or elite-level athletes that have documented prevalence of EIB varying between 30 and 70%, depending on the population studied and methods implemented (Weiler, Bonini et al. 2007, Parsons, Hallstrand et al. 2013). 2.3 Exercise-induced asthma: definition and heterogeneity ”If from running, gymnastic exercises, or any other work, the breathing become difficult, it is called ASTHMA (αμθσα)”. The extant of Arataeus, the Cappadocian (100A.D.) It has long been recognized, even during biblical times, that physical exercise may induce asthma symptoms in susceptible individuals (Chan-Yeung, Malo et al. 2003, Weiler, Bonini et al. 2007). In fact, exercise has been implicated as the most common trigger of an acute asthma attack among elite athletes who have been clinically diagnosed with asthma and it has been estimated that up to 90% of all individuals with asthma are hyperresponsive to exercise. Nevertheless, the term exercise-induced asthma (EIA) only became popular in the 1960s and 1970s when several reports addressed the pattern of airway response to exercise and the influence of drugs on EIA, particularly in children (Jones, Buston et al. 1962, Godfrey 1974, Weiler, Bonini et al. 2007). But asthma induced by sport practicing has not always been easy to describe and recognize, and the concept that exercise may induce bronchial obstruction only in asthmatic patients has been questioned (Bonini 2008). For this reason, in 2008, a Joint Task Force was set up and 20 defined EIA as exercise-induced symptoms and signs of asthma occurring after intensive physical exercise (Carlsen, Anderson et al. 2008); exercise-induced bronchoconstriction (EIB) was defined as the reduction in lung function occurring after a standardized exercise. This has been a controversial item. The American Academy of Allergy, Asthma & Immunology Work Group Report defined EIA as the condition in which exercise induces symptoms of asthma in patients who have asthma, while the term EIB is used to describe the airway obstruction that occurs in association with exercise without regard to the presence of chronic asthma (Weiler, Bonini et al. 2007). More recently, an American Thoracic Society Clinical Practice Guideline (Parsons, Hallstrand et al. 2013) recommended to abandon the term of EIA (because exercise is not the cause, but only a trigger of asthma) and to name EIB with asthma (EIBA), the occurrence of bronchial obstruction after exercise in asthmatic patients, and EIB without asthma (EIBwA), the occurrence of bronchial obstruction in subjects without other symptoms and signs of clinical asthma. The International Olympic Committee (IOC) Independent Asthma Panel defines asthma and validates its diagnosis by the presence of either a positive bronchodilator or bronchoprovocation test. These different positions reflect the difficulties not only in defining asthma, but probably also in understanding distinct mechanisms possible occurring in relationship to asthma heterogeneity. Certainly, EIB that occurs in athletes without other features of clinical asthma has peculiar clinical and pathologic features (Parsons and Mastronarde 2005). Although some authors consider EIA as a distinct asthma phenotype (Wenzel 2006), there is considerable controversy regarding the heterogeneity of the disorder that we call asthma (Bonini, Rasi et al. 2001, Wenzel 2004, Weiler, Bonini et al. 2007). Defining phenotypes of asthma has been a major objective in recent years, as it would facilitate research into etiology and pathophysiology, targeted treatment and preventive measures, and improve prediction of long-term outcomes (Spycher, Silverman et al. 2010). For athletes with asthma, although it is generally recognized that it is highly unlikely that the asthmatic condition which develops during their sports career is identical to what is usually considered to be asthma in clinical practice (Larsson, Carlsen et al. 2005), there is no evidence until now to support clusters of grouping characteristics. Several elite athletes who are diagnosed with EIA have neither personal or family history of asthma (Rupp, Guill et al. 1992, Rupp, Brudno et al. 1993, Hammerman, Becker et al. 2002, Ali, Norsk et al. 2012), suggesting that environmental factors are more important than genetic inheritance. At rest, they seldom experience asthma symptoms (Lund, Pedersen et al. 2009), but rather they occur during high-intensity exercise. It has been claimed that athletes’ care needs further attention and that more studies are needed to further investigate if and how the asthma phenotype of elite athletes differs from that of classical asthma (Lund, Pedersen et al. 2009). The hypothesis of different phenotypes of asthma occurring in athletes was only approached once in literature, in a review article. Haahtela et al. suggested the possibility of two different clinical phenotypes of asthma occurring in elite athletes: the pattern of “classical asthma” characterized by early onset of asthma in childhood, BHR diagnosed by methacholine challenge, atopy and signs of eosinophilic airway inflammation; and another distinct phenotype with onset of symptoms during sports career, bronchial responsiveness to eucapnic hyperventilation test and a variable association with atopic markers and eosinophilic airway inflammation 21 (Haahtela, Malmberg et al. 2008). However, these phenotypes reflected the experience only with Finnish athletes, and have so far not been fully established. Furthermore, these phenotypes were defined by logistic regression analysis, which is a method centered on the variables employed. Most recent attempts to describe different disease phenotypes have been based on cluster analysis, but these methods have so far not been applied to the population of athletes. Also, no attempt was made to investigate whether the different phenotypes were related to the practice of different sports. As suggested in a recent report, the development of EIA and EIB in athletes is possibly caused by different mechanisms according to three different training environments: 1) those training in cold air; 2) swimmers training in indoor-pools; and 3) those training mainly in ambient dry air (Langdeau, Turcotte et al. 2000, Langdeau and Boulet 2001). Being able to define such distinct phenotypes would give further knowledge and understanding of the underlying mechanisms of asthma in elite athletes, and also would improve diagnosis and treatment; different therapeutic modalities could be specifically applied for the targeted phenotypes, rather than for asthmatic athletes in general, which is the current management approach. 2.4 Mechanisms of asthma in athletes EIB was initially thought to be secondary to mediator’s release from mast cells (Godfrey 1977). This hypothesis was supported by the refractory period observed after a positive exercise challenge, interpreted as the time needed for mast cell recharge, and by the preventive effect offered by mast cell stabilizing agents, such as sodium cromoglycate. Although mediator’s release does contribute to cause EIB, pathophysiologic changes induced by intense exercising are definitely more complex (Bonini and Palange 2015). Though the pathogenesis of EIA is not fully elucidated, at present it is widely accepted that it is likely multifactorial and is probably caused by exercise-induced increased ventilation and related changes in airway physiology. An increased ventilatory rate is needed to meet the higher muscular oxygen requirements during exercise. This increased ventilatory rate challenges the ability of the airways to condition the inhaled air to the correct moisture and heat levels before the air reaches the alveoli. Vigorous exercise results in the inhalation of increased volumes of relatively cold and dry air with resulting heat loss from the respiratory mucosa. One of the classical postulated mechanisms includes the osmotic, or airway-drying, hypothesis. Accordingly, the exercise-induced increased ventilation seems to be the most important factor inducing water loss and mucosal cooling and dehydration (Anderson and Kippelen 2005). At the heart rate of 140 bpm, the amount of exhaled water is approximately four times higher than during rest and equals about 60-70 mL/h. When the temperature of the inspired air and its humidity is 35°C and 75%, respectively, the water loss is 7 ml/h; whereas when these parameters are changed to -10°C and 25%, lung excretion of H2O increases up to 20 mL/h (Zielinski and Przybylski 2012). The airway surface liquid becomes hyperosmolar as water is evaporated due to the increased ventilation, providing an osmotic stimulus for water to move from any cell nearby, which results in cell shrinkage and release of inflammatory mediators that cause airway smooth 22 muscle contraction and airway obstruction (Anderson and Kippelen 2008, Carlsen, Anderson et al. 2008, Couto, Silva et al. 2013). In addition to inflammatory mediators triggered by the osmotic change, the increased ventilation during exercise cools the surface epithelium of the airways. Airway cooling stimulates cholinergic receptors in the airways, increasing airway smooth muscle tone and airway secretions. According to the cooling hypothesis, cold air inhalation results in heat loss from the respiratory mucosa and induces pulmonary vasoconstriction. During and, primarily, post-exercise, a rewarming process begins, which is a physiological consequence of the previous cooling of the airways. The rewarming process would cause secondary hyperemia, and by that increased permeability in capillaries, which contributes to a leakage of fluid from the capillaries to the submucosa. This would result in airway edema in predisposed subjects whereby mast cells are stimulated to release inflammatory mediators, leading to airway inflammation and bronchoconstriction (McFadden 1987, Makker and Holgate 1994, Anderson and Daviskas 2000, Weiler, Bonini et al. 2007, Ali, Norsk et al. 2012). 2.4.1 The inflammatory hypothesis Studies of airway pathology are scarce, but injury to the airway epithelium, over expression of cysteinyl leukotrienes, relative underprotection of prostaglandin E2 and greater airway eosinophilia have been found to be distinctive immunopathologic features of asthma with EIB, demonstrating an inflammatory basis of EIB (Hallstrand, Moody et al. 2005). Hallstrand et al. verified a relationship between columnar epithelial cells in induced sputum and severity of EIB, and an association between the concentration of columnar epithelial cells with the levels of histamine and cysteinyl leukotrienes in the airways, confirming the role of mediators’ release (Hallstrand, Moody et al. 2005). Various other investigators have documented that athletes have increased levels of chemical mediators such as histamine, cysteinyl leukotrienes and chemokines, and airway cellular inflammatory markers (Helenius, Rytilä et al. 1998, Sue-Chu, Larsson et al. 1999, Karjalainen, Laitinen et al. 2000, Lumme, Haahtela et al. 2003). The cellular markers include increased eosinophils (Helenius and Haahtela 2000), neutrophils (Sue-Chu, Karjalainen et al. 1998, Sue-Chu, Larsson et al. 1999), and/or epithelial cells (Hallstrand, Moody et al. 2005, Bougault, Turmel et al. 2009). Recently, it was demonstrated an increased presence of damage-associated molecular patterns (DAMPs) in the sputum of athletes (Seys, Hox et al. 2015), which may feature as early inducers of the proinflammatory cytokines. Interestingly, the inflammatory markers observed in athletes airways are not consistently related to lung function, BHR or disease exacerbations (Karjalainen, Laitinen et al. 2000, Helenius, Rytilä et al. 2002, Vergès, Devouassoux et al. 2005, Schwartz, Delgado et al. 2008). Studies on the effects of asthma medication, such as the antiinflammatory drugs montelukast (Helenius, Lumme et al. 2004) and inhaled corticosteroids (Sue-Chu, Karjalainen et al. 2000, Hoshino, Koya et al. 2015) in elite ice hockey players and cross-country skiers have reported no beneficial or only partial effect on asthma-like symptoms, BHR or airway cellular inflammation. Sue-Chu et al. showed 29 knowledge of remission as assessed by objective testing and natural course of the disease in former elite athletes is still missing and should be investigated (Ali, Norsk et al. 2012). 2.7 The importance of appropriate management It has been claimed that athletes’ care needs further attention (Lund, Pedersen et al. 2009). The diagnosis of asthma is this population is crucial because of potential implications not only on their general health, but also on their competing performance. Recent data indicate that asthma is often underdiagnosed and very often undertreated (Bonini, Gramiccioni et al. 2015). Asthma in elite athletes needs adequate attention and management considering that 23.1% of the 263 sudden deaths in athletes reported by Becker et al. occurred in asthmatic athletes (Becker, Rogers et al. 2004). However, setting a correct diagnosis is often challenging and poses several issues unique to this population (Couto, Moreira et al. 2012). The symptoms are often mild to moderate in severity and may cause impairment of athletic performance, but frequently are not severe enough to cause significant respiratory distress (Parsons, Hallstrand et al. 2013). Actually, symptoms have been shown to be poor predictors of asthma in athletes (Rupp, Guill et al. 1992, Rupp, Brudno et al. 1993, Rundell, Im et al. 2001, Bonini, Gramiccioni et al. 2015). The heavy training with the extremely high level of physical fitness and maximal oxygen uptake (V’O2max) makes it difficult to discriminate between physiological and pathological limitations to maximum exercise (Carlsen, Anderson et al. 2008). On the other hand, to avoid asthmatic stigma and doping concerns by fellow athletes, some athletes may be poorly motivated to complain of respiratory symptoms (Couillard, Bougault et al. 2014); in fact, it has recently been evidenced that compliance to treatment in Olympic athletes was very poor (Bonini, Gramiccioni et al. 2015). There is a lack of education in athletes and coaches regarding the recognition of exercise-induced respiratory symptoms, the high EIB/BHR prevalence in the athlete population, and the effects of asthma medication, which could contribute to inadequate symptom perception, especially in young athletes (Couillard, Bougault et al. 2014). Interestingly, a previous study underlined that the elite swimmer population is characterized by a high prevalence of symptomatic and asymptomatic BHR and/or EIB, whereas winter sport athletes often report exercise-induced cough but with a prevalence of BHR and/or EIB similar to controls (Bougault, Turmel et al. 2010). This was recently confirmed in other study on cross-country skiers (Bordeleau, Turmel et al. 2014). In a review article devoted to this issue, it was highlighted that prevalence of physician-diagnosed asthma, EIA and BHR to methacholine or other agents are homogeneous for various athletes such as sprinters, longdistance runners, football and basketball players and other track and field athletes, but for athletes training in cold air environment and for swimmers the prevalence of reported BHR is quite high; surprisingly, the prevalence of physician-diagnosed asthma is low (Langdeau and Boulet 2001). Taken together these observations raised the question: Should a systematic screening for EIB be done in athletes, especially swimmers? (Bougault, Turmel et al. 2010). 30 A number of organizations and investigators advocate screening for asthma in athletes (Holzer and Brukner 2004, Dickinson, Whyte et al. 2005, Dickinson, Whyte et al. 2006). This recommendation is pertinent and, indeed, some sporting organizations have established EIB screening programs for their internationally competitive athletes (Wilber, Rundell et al. 2000, Dickinson, Whyte et al. 2005). Yet, to date, expert working groups have not directly addressed EIB screening policy (Weiler, Bonini et al. 2007, Carlsen, Anderson et al. 2008, Schwartz, Delgado et al. 2008), and so in Portugal there aren’t any screening programmes. Since the 2002 Salt Lake City Games, the International Olympic Committee's Independent Asthma Panel required testing to validate asthma diagnosis and justify the use of inhaled 2-agonists in Olympic athletes - either a positive bronchodilator or bronchoprovocation test (Medical Commission of the International Olympic Committee 2002, Fitch, Sue-Chu et al. 2008). This strategy has provided valuable guidelines to the practicing physician. This program was educational and documented the variability in the prevalence of asthma and/or BHR and inhaled 2-agonist’s use between different sports and different countries. It provided a standard of care for the athlete with respiratory symptoms. In 2009, the World Anti-Doping Agency (WADA) followed the IOC approach, extending these guidelines to all other athletes, requiring objective evidence of asthma diagnosis to allow the use of inhaled β2-agonists (IBAs). In Portugal, changes to the WADA 2009 Prohibited List permitted rigorous screening of asthmatic athletes due to the implementation of objective criteria for inhalers use. The WADA guidelines on asthma, however, have changed in recent years: for instance, since 2012, IBAs are prohibited and require a therapeutic use exemption (TUE), except for salbutamol, formoterol and salmeterol when taken according to manufacturer’s instructions and not exceeding the published doses. Data are lacking regarding the impact of these changes in athlete’s health and medical care. 32 3. AIMS OF THE THESIS The general aim of the present study was to investigate phenotypes of asthma, and mechanisms and effects of airway damage in elite athletes. Specific questions for the study programme were:  How the definition of asthma in elite athletes impacts on its management (study I) and are there different phenotypes of asthma under this definition (study II)?  What is the relation between parasympathetic activity with asthma and bronchial hyperesponsiveness in elite swimmers? (study III), and how inhaled anticholinergic drugs perform compared with 2-agonists in elite athletes? (study IV)  Is the heat loss hypothesis supported by acute changes after vigorous exercise and does it relate to airway’s inflammation? (study V)  What are the long-term effects of competitive swimming on airway’s inflammation? (study VI) 34 4. MATERIAL AND METHODS 4.1 Participants and study design This thesis is based on three types of studies: 1) Observational studies a. Retrospective analysis of asthma medication requests submitted by elite athletes older than 16 years to the Anti-Doping Authority of Portugal (ADOP) between 2008 and 2010 to analyze the impact of asthma and antidoping regulations in elite athletes’ health (study I). b. Prospective studies: evaluation of elite swimmers to assess the effect of a swimming training session on EBT from the airways of both asthmatic and non-asthmatic elite athletes (study V); investigation if long-term swimming is associated with airway injury in swimmers (study VI). 2) Cross-sectional study of asthmatic elite athletes to assess different phenotypes of asthma (study II); examination of elite swimmers to investigate the association between asthma, bronchial hyperresponsiveness and parasympathetic activity measured by pupillometry (study III). 3) Randomized cross-over trial to compare reversibility to inhaled ipratropium bromide with reversibility to inhaled salbutamol in elite winter athletes and to explore the relation between PD20 obtained with methacholine challenge and reversibility to each drug (study IV). A total of 127 swimmers participated in observational prospective studies, 22 elite winter athletes participated in the randomized cross-over trial and 27 elite swimmers in one of the cross-sectional studies (Figure 1). Data from asthma medication requests submitted to the ADOP referring to 326 elite athletes, as well as data from clinical files of elite athletes at the Portuguese database of Olympic athletes and at database of Norwegian School of Sport Sciences were also analyzed (n=324). A summary of studies design and subjects is presented in Table 1. Figure 1 Swimmers from Futebol Clube do Porto (FCP) main swimming team, included in studies III and V. 35 Table 1. Summary of subjects and studies designs. Study Study design and subjects Asthma Gender (M/F) Age Duration Intervention I Observational, retrospective 326 requests for asthma medication 326 (100%) 254/72 27±9 2 years n.a. II Cross-sectional 324 elite athletes 150 (46) 107/43 25±7 n.a. n.a. III Cross-sectional 27 elite swimmers 11 (41) 14/13 17±3 n.a. n.a. IV Randomized, cross-over 22 elite winter athletes 16 (72) 14/8 26±5 2 weeks BD with inhaled ipratropium bromide and inhaled salbutamol V Observational 22 elite swimmers 10 (45) 10/12 17±3 1 swimming training session n.a. VI Observational, prospective 105 competitive non-elite swimmers 12 (11) 61/44 14±6 3 years n.a. Data are presented as counts (%) or mean±SD. BD: Bronchodilation; n.a.: non-applicable. 4.1.1 Diagnosis of asthma and healthcare of asthmatic athletes (Study I) A retrospective analysis of asthma medication requests submitted to the Anti-Doping Authority of Portugal between 2008 and 2010 was carried out. The study sample included all athletes older than 16 years who requested permission to use ICS and/or inhaled 2agonists for over 3 months between 2008 and 2010. Those requesting the use for shorter periods of time (e.g. less than three months) were excluded. The study protocol included collection of data on respiratory symptoms, medication requested, results from spirometry, bronchodilation tests, bronchial challenges, FeNO levels and allergic sensitization defined by the presence of at least one positive skin prick test and/or positive specific IgE. Asthma diagnosis was established according to criteria set by IOC to document asthma in athletes (Medical Commission of the International Olympic Committee 2002, Carlsen, Anderson et al. 2008). The years of 2008, 2009 and 2010 were compared to assess the impact of changes in guidelines throughout the years. 4.1.2 Phenotypes of asthma in elite athletes (Study II) An analysis of elite athletes records kept in databases files was performed. In Portugal, registries available at the Anti-doping Authority of Portugal and the Portuguese database of Olympic athletes were used; in Norway, database of medical files of Norwegian School of Sport Sciences was analyzed, including Olympic athletes participating in the 2008 summer and the 2010 winter Olympic Games. Both Portuguese and Norwegian athletes training at high competitive levels (national, international or Olympic teams) were identified through institution databases and those with available information on symptoms, 36 lung function and airway inflammation, BHR, and atopy were selected. Healthy athletes and those with other conditions rather than asthma were excluded. Study protocol included the collection of data about demographics (age, gender, height, weight and sport practiced), presence of respiratory symptoms, current use of asthma medication, presence of rhinitis or other allergic diseases (conjunctivitis, urticaria, eczema, anaphylaxis and drug, food and venom allergies) previously identified through AQUA© questionnaire (Bonini, Braido et al. 2009), lung function and reversibility, airway inflammation, BHR, and allergic sensitization. The first ever performed spirometry and the first ever performed bronchial provocation challenge, respectively, were used. From all reviewed files, 324 files had complete information available and informed consent for data use (Table 2). Table2. Features of athletes screened at the Anti-Doping Authority of Portugal and at the Norwegian School of Sports Sciences databases. Asthmatic athletes (n=150) Non-asthmatic athletes (n=174) p Male, n (%) 107 (71) 89 (51) <0.001 e Age, years 25 (14 – 40) 26 (16 - 38) 0.251 d BMI, Kg/m2 23 [23;24] 23 [22;23] 0.06 c Physician reported rhinitis, n (%) 54 (36) 33 (19) 0.003 e Other allergic disease, n (%) 20 (13) 26 (15) 0.750 e Allergic sensitization, n (%) 89 (59) 58 (33) <0.001 e Respiratory symptoms, n (%)* 138 (92) 89 (51) <0.001 e Dyspnea/ heavy breathing 48 (32) 20 (11) <0.001 e Chest tightness 12 (8) 11 (6) 0.379 e Wheezing 42 (28) 15 (9) <0.001 e Cough 44 (29) 33 (19) 0.002 e Tiredness 1 (0.7) 1 (0.6) 0.427 f Phlegm 18 (12) 15 (9) <0.001 e Asthma treatment, n (%) <0.001 f Inhaled steroids alone 9 (6) 1 (0.6) Beta-2-agonists alone 13 (9) 2 (1) Inhaled steroids + beta-2-agonists 96 (64) 13 (8) Airway obstruction a, n (%) 43 (29) 21 (12) <0.001 e FVC L 5.4 [5.1;5.7] 5.2 [5.0;5.4] 0.41 c % of predicted 114 [110;117] 112 [109;116] 0.60 c FEV1 L 4.1 [3.9;4.4] 4.3 [4.1;4.4] 0.06 c % of predicted 101 [96;106] 109 [106;111] 0.001 c FEV1/FVC 69 [65;74] 76 [72;80] 0.012 c Reversibility b, n (%) 26 (17) 1 (0.6) 0.037 f Airway hyperesponsiveness, n (%) 126 (84) 51 (29) <0.001 e FeNO, ppb 33 (6 – 213) 19 (4 – 70) 0.01 d Data presented as counts (%) and mean [95% Confidence interval] except for age and FeNO which are presented as median (min-max); BMI: Body mass index; FeNO: fraction of exhaled nitric oxide; L: litters; FVC: forced vital capacity; FEV1: forced expiratory volume in one second. a Defined as a FEV1/FVC ratio <0.70; b Defined as an increase in FEV1 200 mL and 12% from baseline; c Independent samples t-test; d Independent samples Mann-Whitney U-test; e Chi-square test; f Fisher’s exact test. *Some athletes presented more than one symptom. 37 The type of sport practiced was classified according to environmental training conditions given the previous suggestion that three different training environments could be related to different mechanisms of asthma (Langdeau, Turcotte et al. 2000, Langdeau and Boulet 2001): - water sports (swimming and water polo); - winter sports (cross-country skiing, biathlon, skeleton, alpine skiing, and ski cross); - other sports (handball, judo, triathlon, football, cycling, beach volley, rowing, athletics, sailing, badminton, canoeing, curling, equestrian, taekwondo, auto-racing, billiards, paragliding, rugby, tennis, roller hockey, kickboxing, fencing, basketball and golf). Asthma diagnosis was established by a medical doctor according to criteria set by the IOC to document asthma in athletes (Medical Commission of the International Olympic Committee 2002, Carlsen, Anderson et al. 2008), using objective evidence of either reversibility after bronchodilator administration or evidence of BHR after a bronchial provocation challenge. Of these 324 athletes, 150 athletes fulfilled asthma criteria and were included for Latent Class Analysis to define phenotypes. 4.1.3 Parasympathetic activity and bronchial hyperesponsiveness (Study III) Elite swimmers of the Futebol Clube do Porto (FCP) main swimming team were invited to participate. Recruitment was made through an invitation letter sent to all swimmers. Athletes aged above 14 year-old, who agreed to take part in the study, were enrolled. To be included, participants had to be competitive swimmers, free from any respiratory infection in the 2 weeks before testing, not to drink coffee or smoke or perform exercise on the testing day, not to wear contact lenses and withdraw their asthma medication 48 hours before (except for inhaled corticosteroids, which were asked to be suspended for at least 2 weeks prior to the study). Subjects who met any of the following criteria were excluded from the study: under any systemic medication with central nervous system effects; any topical eye treatment; systemic conditions with known ocular involvement; orbit structure damage or surrounding soft tissue with open lesion or edema at the day of testing; past history of ocular abnormalities or trauma; pregnancy; recent episode of hemoptysis; forced expiratory volume in the first second (FEV1) lower than 60% of the predicted value or 1.5 L; orally administered corticosteroids in the last month; neurological or psychiatric illness; lack of collaboration or presence of diseases that limit the patient's ability to carry out the tests; recent stroke or heart attack or malignant diseases. The study was developed in two visits. The first visit was performed between 8 to 11 am due to the circadian rhythm of pupil’s sizes (Fountas, Kapsalaki et al. 2006). Medical history and potential medication were reviewed to determine eligibility. The eligible subjects answered a structured questionnaire, and performed pupillometry, skin prick tests, spirometry and reversibility to salbutamol. The second visit took place on a different day and a bronchial challenge with methacholine was performed to assess BHR. In both visits, 38 swimmers were asked to withhold anti-asthmatic and anti-allergic medication, according to guidelines (American Thoracic Society 2000). Asthma diagnosis was established according to criteria set by IOC to document asthma in athletes (Medical Commission of the International Olympic Committee 2002, Carlsen, Anderson et al. 2008). Swimmers who fulfilled asthma diagnostic criteria where compared to healthy swimmers regarding parasympathetic activity and relation to BHR. 4.1.4 Anti-cholinergic drugs and BHR in elite athletes (Study IV) Twenty cross-country skiers and two biathlon skiers, all members of the Norwegian national teams, were included. All subjects were high-level athletes competing at a top international level. All athletes had been free from any respiratory disease for the last three weeks before the first study day, and refrained from exercise and any food or drink containing nitrate on the testing day. Subjects were asked to withhold medication, according to guidelines (American Thoracic Society 2000). The athletes attended one visit at the laboratory at the Norwegian School of Sport Sciences in Oslo for assessment of lung function, BHR to methacholine and FeNO. Prior diagnoses of asthma, EIB, allergic rhinitis and current symptoms of dyspnea, phlegm and cough during or after exercise, and use of asthma medication during the previous year were recorded with the AQUA© questionnaire (Bonini, Braido et al. 2009) and clinical interview. Later, two reversibility tests were obtained during a training camp in Val Senales, Italy, 2000 meters above sea level. The reversibility tests with inhaled ipratropium bromide and with inhaled salbutamol were performed in a randomized order on two separate days, with 24 hours between each test. Results from reversibility to inhaled ipratropium bromide and reversibility to inhaled salbutamol were compared for each athlete as well as the relation between PD20 obtained with methacholine challenge and reversibility to each drug. 4.1.5 Exhaled breath temperature, exercise and asthma (Study V) Elite swimmers of the FCP main swimming team who had been screened for asthma and atopy at the beginning of the training season (annual screening) were invited to the present study. In order to be eligible to participate in this study, a subject had to meet all the following criteria: competitive level swimmer; aged ≥14 year-old; training ≥10 hours per week; free from respiratory infection in the last 3 weeks; provided signed and dated informed consent. A potential subject who met any of the following criteria was excluded: pregnancy; recent episode of hemoptysis; forced expiratory volume in the first second (FEV1) lower than 60% of the predicted value or 1.5 L; orally administered corticosteroids in the last month; neurological or psychiatric illness; lack of collaboration or presence of diseases that limit the patient's ability to carry out the tests; recent stroke or heart attack or malignant diseases. Data from the screening visits performed at the beginning of the training season were retrospectively collected to assess eligibility and diagnosis of asthma, including 45 4.2.6.3 Exhaled Breath Temperature (Study V) EBT has been widely investigated based on the assumption that inflammation of the airways and increased vascularization of airway mucosa would influence the temperature of the air coming from the alveoli (Paredi, Kharitonov et al. 2002). Previous studies have reported increased EBT in patients with asthma compared to healthy controls (Piacentini, Peroni et al. 2007). It has been shown that, in asthmatic adults and children, EBT correlates with bronchial blood flow, FeNO levels and sputum eosinophils (Piacentini, Bodini et al. 2002, Paredi, Kharitonov et al. 2005, Piacentini, Peroni et al. 2007, Popov 2011). EBT was measured using an X-halo device (Delmedica Investments®, Singapore), five minutes before (baseline EBT) and five minutes after the swimming training session (post-exercise EBT), according to previously validated methods (Popov, Dunev et al. 2007). Briefly, the swimmers were requested to inhale freely through the nose and to exhale into the device at a rate and depth typical of their normal tidal breathing rhythm. The maneuver was continued until the built-in software of the instrument indicated that the measured value was stable (Figure 3). The decision of collecting EBT five minutes after the exercise was based upon previous studies that have shown that this was the time point in which EBT reaches the highest values, and thereafter decreases (Svensson, Nilsson et al. 2012). Measurement of EBT has been shown to be highly reproducible (Vermeulen, Barreto et al. 2014), which supports its use before and after the swimming session. Before and between measurements, the device was kept at room temperature in order to maintain a stable starting temperature. Figure 3 Collection of Exhaled Breath Temperature. 46 4.2.7 Physical activity (Study VI) Physical activity (PA) was assessed using the short seven days International Physical Activity Questionnaire (IPAQ) that provides information about frequency and duration of four domains of physical activity (sedentary activity, time spent walking and moderateand vigorousintensity physical activity) (Craig, Marshall et al. 2003). A combined total physical activity was computed as the sum of the activity domains scores (Total PA = Walking + Moderate-intensity PA + Vigorous-intensity PA) and reported as a continuous measure (Total PA score = total MET-min/week). 4.2.8 Assessment and diagnosis of asthma (Study I, II, III, IV, V and VI) For studies I, II, III, and V, asthma diagnosis was established according to criteria set by IOC to document asthma in athletes (Medical Commission of the International Olympic Committee 2002, Carlsen, Anderson et al. 2008), using objective evidence of either reversibility after bronchodilator administration or evidence of BHR after a bronchial provocation challenge. For studies IV and VI, self-administered questionnaires were used to assess previous diagnosis of asthma, as described below: 4.2.8.1 AQUA© (Study IV) Allergy Questionnaire for Athletes (AQUA©) is a validated, simple, easy-to-use, selfadministered tool that permits identification of allergy and asthma with a high positive predictive value (Bonini, Braido et al. 2009). It was developed from the European Community Respiratory Health Survey Questionnaire. On the basis of open interviews with team doctors, coaches, and athletes, questions were added about: the type, duration, and intensity of training; exercise-related allergic and infectious symptoms; social habits (smoking); drug and food supplements intake; antidoping regulations. 4.2.8.2 ISAAC questionnaire (Study VI) The International Study of Asthma and Allergies in Childhood (ISAAC) is a unique worldwide epidemiological research programme established in 1991 to investigate asthma, rhinitis and eczema in children. The ISAAC questionnaire on asthma is a validated one-page self-administered questionnaire with a specific version developed for teenagers aged 13-14 years-old (Asher, Keil et al. 1995). The questions were designed as a minimum set for inclusion in self‐completed or interview‐administered questionnaires used in population surveys of respiratory disease in children. These questions (self‐complete version) were included in a pilot study conducted among 8,000 13‐14 year olds in four centers during 1991 (Asher, Keil et al. 1995). The questionnaire has been translated into several languages using 47 standardized procedures, and the Portuguese version (Rosado-Pinto 2011) for 13-14 years respondents was used. 4.3 Statistical analysis (Studies I-VI) Data analysis was performed using Statistical Package for Social Sciences version 20.0 for Windows (SPSS, Chicago, IL, USA), considering a significance level of 0.05. In Study II, LCA models were fitted using MPlus (V.5.2; Muthen & Muthen, Los Angeles, California, USA). Besides, MedCalc Statistical System (version 10.4.6.0, Mariakerke, Belgium) was also used in study IV. Demographic data and results are given as means with 95% confidence interval (CI), or means with standard deviation (SD), or medians with interquartile range (IQR) in case of skewed distribution; categorical data are presented as counts and proportions (n, %). T-test was used for comparison of normally distributed independent continuous data, and Mann–Whitney test in case of skewed distribution. In study V, Wilcoxon test was used to compare the differences between baseline and post-exercise EBTs. In study VI, differences between groups were assessed with 1-way ANOVA for normally distributed data, or Kruskal-Wallis for non-normally distributed data. Categorical variables were compared by Chi-square or Fisher’s exact tests. Correlation analyses were performed using Pearson or Spearman’s tests as appropriate with respect to distribution of data. In study V, given the small sample size, only non-parametric tests were used. In study I, exhaled nitric oxide results were converted into personal predicted values using the FeNO Interpretation Aid tool (http://www.enovis.org), and considered increased if above 150% of predicted. Logarithmic transformation was applied to continuous data whenever a skewed distribution was observed: in studies IV and V, PD20 was log-transformed for correlation testing; in study VI, levels of FeNO and of physical activity were log-transformed. Latent Class analysis In Study II, latent class analysis (LCA) was used to uncover from a sample distinct groups of individuals homogeneous within the group (patterns), considering that the performance of an individual in a set of items is explained by a categorical latent variable with K classes, commonly called ‘latent classes’. Model interpretation was based on item profiles in each category and obtained from probabilities of endorsing each item response, conditional on class membership. The number of latent classes was defined according to Bayesian Information Criterion (BIC). Starting from one single class and increasing one class at each step, the best solution was identified when the increase of number of classes did not lead to a decrease in BIC. LCA used nine variables of importance for asthma definition or relevant for differential diagnosis (Table 4). The selection of variables was based on the assumption of their clinical relevance. The Lo-Mendell-Rubin likelihood 48 ratio test of model fit was used to quantify the likelihood that the data could be described by a model with one-less class. Table 4. Definitions of variables set for Latent Class Analysis. Variable Definition Airflow obstruction FEV1/FVC ratio lower than 0.70 Reversibility Increase of FEV1 of at least 200 mL and 12% from baseline Rhinitis a Positive answer to the question “Did any doctor diagnose you an allergic disease?” AND “Rhinitis”; OR Positive answer to the question “Do you frequently sneeze, have a running, itchy nose (apart from colds)? Any other allergic disease a Positive answer to the question “Did any doctor diagnose you an allergic disease?” (except rhinitis); OR Positive answer to the question “Have you frequently red eyes with tearing and itching?”; OR Positive answer to the question “Have you ever had severe allergic or anaphylactic reactions?”; OR Positive answer to the question “Have you ever had allergic reactions to foods?”; OR Positive answer to the question “Have you ever had allergic reactions to drugs?” Respiratory symptoms a Self-reported recurrent breathlessness, cough, wheezing, chest tightness and/or phlegm production; OR Positive answer to the question “Did any doctor diagnose you an allergic disease?” AND “Asthma”; OR Positive answer to the question “Have you ever had shortness of breath, cough and/or itching of the throat following exercise?” Asthma treatment Current or recent treatment with ICS and/or 2-agonists Airway hyperesponsiveness A fall in FEV1 ≥10% from baseline with exercise or EVH; OR a fall in FEV1 ≥15% from baseline after inhaling 22.5 ml of 4.5g% NaCl or ≤635 mg of mannitol; OR a fall in FEV1 ≥20% from baseline with methacholine: PC20 ≤ 4 mg/ml, or PD20 ≤ 400 μg (cumulative dose) or ≤ 200 μg (noncumulative dose) in those not taking ICS, and PC20 ≤ 16 mg/ml or PD20 ≤ 1600 μg (cumulative dose) or ≤ 800 μg (noncumulative dose) in those taking ICS for at least 1 month Eosinophilic inflammation FeNO levels above 25 ppb. Allergic sensitization Presence of at least one positive (mean of largest and perpendicular diameter of the wheal ≥3mm for each allergen) skin prick test or presence of positive specific IgE (≥0.35kU/L) for at least one common aeroallergen in the local geographic area a Considering AQUA©. EVH: eucapnic voluntary hyperpnea; FVC: forced vital capacity; FEV1: forced expiratory volume in one second; PC20: provocative concentration inducing a 20% decrease in FEV1; PD20: provocative dose inducing a 20% decrease in FEV1; FeNO: fraction of exhaled nitric oxide. 49 Multiple factor analysis Study of associations was performed using linear regression and general linear models. In study II, among asthmatic athletes, the risk associated with the sport training environment was estimated by using regression analysis to predict the odds of having a specific asthma pattern (phenotype), having “other sports” as reference. In study V, linear regression models were used to determine the effect of asthma and other possible explanatory variables (age, gender, height, weight, PD20, baseline EBT, intensity of the training session, axillary temperature, and the number of hours trained in the week previous to measurements) in the outcome ∆EBT. These variables were selected based on the authors’ a priori hypothesis that they may influence EBT. A univariate linear regression analysis was performed to assess the individual effect of each of the selected variables; those that were significant at the 0.25 level were included in a multiple linear regression model. A stepwise method was used to select the variables to include in the final model, taking into account their significance and effect in the adjusted r2. The effect of asthma, being a major outcome of the present study, was included in the multiple regression analysis and was kept in the final model independently of the significance level and adjusted r2 change with its inclusion. In study VI, differences in changes in FeNO after the three year follow up were assessed by general linear model adjusting on confounding factors: gender, age, allergic sensitization, physician-diagnosed asthma, and use of asthma medication. Power and sample size calculations Sample size was only calculated for study IV, the randomized cross-over trial. To achieve a high-grade correlation of ≥0.7 with power of 80%, 13 subjects were calculated to be required based upon previous measurements in top athletes. In the remaining studies, all athletes that were able to participate and were eligible were included. In study V, only 22 swimmers (of which ten with asthma) accepted to participate, retrieving a power of 49% to detect a ΔEBT of 0.43ºC (Peroni, Chinellato et al. 2012). In study III, no power calculation has been made because this was an exploratory study and the needed a priori information was not available. 4.4 Ethics All studies were conducted in accordance to the Declaration of Helsinki for Medical Research Involving Human Subjects. Protocols were approved by the Ethics Committee of Hospital São João / Faculdade de Medicina da Universidade do Porto (n_id_cic 174/12 ; n_id_ces 58/12) for studies developed in Portugal (Studies I, II, III, V, VI) and the Committee for Regional Medical Research Ethics and the Norwegian data inspectorate in Norway (Studies II and IV). All participants or their legal guardians/parents (in case of participants under 18 yearsold) signed an informed consent. 50 5. RESULTS 5.1 Diagnosis of asthma and healthcare of asthmatic athletes (study I) We analyzed requests from 326 athletes [254 males; median age 24 years (range 16-62 years)] (Figure 4). The requests were as follows: in 2008, 173 abbreviated Therapeutic use exemptions (TUEs) were submitted; in 2009 and 2010, 9 and 39 Declarations of Use (DoU) were submitted, respectively; regarding TUEs, the approval rate was 97% (74/76) in 2009 and 79% (23/29) in 2010 (p=0.005). Figure 4 Flow chart showing requests for asthma medication submitted by athletes to the AntiDoping Authority of Portugal between 2008 and 2010. * In 2008 all β2-agonists, as well as inhaled corticosteroids required only an aTUE. Approval for aTUEs was not necessary. † In 2009, salbutamol, salmeterol, terbutaline and formoterol required a TUE; use of inhaled corticosteroids only required a DoU. ‡ In 2010, salbutamol, salmeterol and inhaled steroids required DoU; for using terbutaline or formoterol use a TUE was required. aTUE: abbreviated Therapeutic use exemption; DoU: Declaration of use; ICS: inhaled corticosteroids; TUE: Therapeutic use exemption. 332 Requests 6 excluded - Those requesting the use for short periods of time (4 in 2009 and 2 in 2010) 326 Included 173 in 2008* (all aTUE) - 9 ICS - 45 β2-agonists - 119 ICS + β2agonists - 119 ICS + β2agonists 85 in 2009† 68 in 2010‡ 9 DoU (all ICS) 39 DoU - 7 ICS - 10 β2-agonists - 22 ICS + β2agonists Approval rate: 97% Approval rate: 79% 76 TUE - 9 β2-agonists - 67 ICS + β2agonists 29 TUE - 6 β2-agonists - 23 ICS + β2agonists 51 The most frequent requests were for using ICS combined with inhaled 2-agonists. Applications for inhaler use have decreased by approximately half from 2008 to 2009 (173 to 85) since objective asthma testing became mandatory. This more rigorous screening allowed withdrawal of unnecessary medication. Requests for isolated inhaled 2-agonists increased significantly from 2009 to 2010 (p=0.03), highlighting safety issues stemming from the unsupervised use of 2-agonists. The clinical and diagnostic tests performed are shown in Table 5 and the corresponding results in Table 6. No tests were reported in 2008 because all the requests submitted in that year were aTUEs. Changes to the WADA guidelines on 2-agonists in 2010 led to a dramatic decrease in the number of tests performed in Portuguese athletes with asthma (Table 5). The proportion of positive tests in 2009 and 2010 (Table 6) was similar, supporting the strategy of objective diagnosis. Table 5. Tests performed in athletes whose request to use asthma medication was approved by the Anti-doping Authority of Portugal. Tests performed 2009 (n = 83) 2010 (n = 62) P Spirometry 72 (87) 23 (37) <0.001 Bronchodilation test 37 (45) 15 (24) 0.011 Bronchoprovocation challenge 49 (59) 10 (16) <0.001 Methacholine 46 (55) 9 (15) - Mannitol 0 1 (2) - Exercise 3 (4) 0 - FeNO 15 (18) 6 (10) 0.155 SPT or sIgE 56 (67) 18 (29) <0.001 No tests were reported in 2008 as it was not necessary to provide objective evidence of asthma at that time. Data reported as n (%). SPT, skin prick tests; sIgE, specific immunoglobulin E; FeNO, fraction of exhaled nitric oxide. Table 6. Reported symptoms and positive test results in athletes with asthma who submitted a Therapeutic Use Exemption application to the Anti-Doping Authority of Portugal. 2009 n = 83 2010 n = 62 Respiratory symptoms 81 (98) 62 (100) Airflow limitation on spirometry 12/72 (17) 4/23 (17) Positive bronchodilation 20/37 (54) 8/15 (53) Positive bronchoprovocation 46/49 (94) 10/10 (100) Airway allergic inflammation a 10/15 (67) 2/6 (33) Allergic sensitization 50/56 (89) 15/18 (83) Data reported as positive/performed (%). a Defined as exhaled nitric oxide >150% of predicted value for age and height, calculated using the FeNO Interpretation Aid tool (http://www.enovis.org). 52 5.2 Phenotypes of asthma in elite athletes (study II) Among the 150 asthmatic athletes, 45 were diagnosed based on positive bronchodilation (the mean±SD of FEV1 increase was 450mL±292 and 13%±9.4), and 105 by presenting BHR after a provocation challenge: 1 positive challenge to mannitol, 3 positive challenges with exercise and the remaining 101 positive challenges with methacholine (7 reporting PC20: mean 3.9 mg/ml; 94 reporting PD20: mean 6.8 μg). LCA retrieved two clusters (Table 7): “atopic asthma” defined by allergic sensitization, increased FeNO, rhinitis and allergic co-morbidities; and “sports asthma” defined by exercise-induced respiratorysymptoms and BHR without allergic features (Figure 5 and Table 8). Table 7. Latent class analysis for asthma features in the elite athletes’ population. Number of classes1 Asthma Log L Number of parameters BIC p2 1 -503.304 9 1051 2 -472.636 19 1040 <0.001 3 -457.683 29 1060 0.0614 BIC, Bayesian information criteria; Log L, log likelihood; 1 The bold font denotes the best models according to lowest BIC; 2 Lo-Mendell-Rubin likelihood ratio test of model fit to quantify the likelihood that the data can be described by a model with one-less class. Figure 5 Percent of athletes presenting each of the features included for LCA. 53 Table 8. Characteristics of asthmatic athletes according to their asthma phenotype and variables in each assigned latent class. Total Atopic Asthma, n=104 Sports asthma, n=46 p Males, n (%) 107 81 (78) 26 (57) 0.008# Age, median±IQR in years - 23.0±12 24.5±8 0.522§ Height, mean±SD in cm - 175.4±8.7 176.5±8.7 0.530‡ Weight, mean±SD in Kg - 70.9±11.5 71.4±10.3 0.815‡ BMI, mean±SD in Kg/m2 - 23.0±2.6 22.8±1.9 0.741‡ FEV1, mean±SD in L - 4.0±0.9 4.1±0.7 0.221‡ FEV1, mean±SD in % predicted - 98.1±20.4 99.7±21.1 0.640‡ FVC, mean±SD in L - 5.1±1.0 5.3±1.1 0.413‡ FVC, mean±SD in % predicted - 108.0±15.4 113.4±15.0 0.084‡ FEV1/FVC, mean±SD - 77.9±8.9 78.7±11.1 0.649‡ Variables used in Latent Class Analysis Airflow obstruction* 0.036 No 80.5 85.3 69.4 Yes 19.5 14.7 30.6 Reversibility** 0.023 No 23.4 19.0 39.7 Yes 76.6 81.0 60.3 Rhinitis <0.001 No 64.0 51.5 90.9 Yes 36.0 48.5 9.1 Any other allergic disease <0.001 No 61.5 39.3 87.5 Yes 38.5 60.7 12.5 Respiratory symptoms 0.133 No 6.1 4.0 10.7 Yes 93.9 96.0 89.3 Asthma treatment 0.017 No 11.9 7.5 22.0 Yes 88.1 92.5 78.0 Bronchial hyperesponsiveness 0.834 No 25.7 25.0 26.9 Yes 74.3 75.0 73.1 FeNO† <0.001 Normal 62.8 44.8 84.5 Increased 37.2 55.2 15.5 Allergic sensitization <0.001 No 31.0 0 100 Yes 69.0 100 0 Data presented as percent of total, except otherwise stated; BMI: Body mass index; FeNO: fraction of exhaled nitric oxide; L: liters; FVC: forced vital capacity; FEV1: forced expiratory volume in one second; * Defined as a FEV1/FVC ratio <0.70; ** Defined as an increase in FEV1 200mL and 12% from baseline; † Defined as increased if above 25ppb; ‡ Independent samples t-test; § Independent samples Mann-Whitney U-test; # Qui-square test. Water (OR=2.87; 95%CI [1.82-4.51]) and winter (OR=8.65; 95%CI [2.67-28.03]) sport athletes had increased risk of “sports asthma” compared with other sport athletes. 54 5.3 Dysautonomia, asthma and BHR in elite swimmers (study III) Twenty-seven swimmers were included. No differences on demographic and personal characteristics were observed between groups, except for the expected significantly lower PD20 among asthmatic swimmers (Table 9). Pupillometry measurements in asthmatics compared with non-asthmatic swimmers are presented in Table 9 and Figure 6. Although lower pupil diameters and a higher percentage of constriction were observed in asthmatics, differences were not significant. Table 9. Characteristics of asthmatic and non-asthmatic swimmers included. Asthmatic swimmers (n=11) Non-asthmatic swimmers (n=16) p Males, n (%) 8 (73) 6 (38) 0.072 Age, years 17 [15-19] 18 [16-20] 0.479 BMI, kg/m2 21.5 [20.2-22.9] 21.3 [20.2-22.3] 0.725 Atopy, n (%) 5 (46) 6 (38) 0.679* Years of competition 8.9 [7.0-10.8] 9.6 [7.6-11.7] 0.602 Training hours per week 16.3 [13.1-19.4] 17.5 [16.1-18.9] 0.393 Previous diagnosis of asthma, n (%) 4 (36) 1 (6) 0.113* Previous diagnosis of rhinitis, n (%) 1 (9) 3 (18) 0.488* PD20 methacholine, μmol 0.8 [0.4-1.2] 4.6 [3.3-5.8] 0.001 Lung function FEV1/FVC 83.5 [78.3-88.8] 88.0 [84.6-91.4] 0.236 FVC Liters 5.1 [4.4-5.8] 4.9 [4.1-5.7] 0.612 % of predicted 114.5 [107.0-122.0] 114.8 [108.5-121.0] 0.957 FEV1 Liters 4.3 [3.7-4.8] 4.3 [3.7-4.9] 0.863 % of predicted 111.1 [100.7-121.5] 115.6 [109.8-121.3] 0.379 FEF25-75 Liters 4.2 [3.4-5.1] 4.7 [4.0-5.4] 0.570 % of predicted 97.3 [78.2-116.4] 109.3 [97.3-121.4] 0.230 Airway obstruction, n (%) 1 (9) 0 0.219* Increase in FEV1 after salbutamol % 5.0 [2.0-8.0] 3.4 [1.7-5.4] 0.840 Milliliters 197.0 [87.2-306.8] 149.4 [61.5-237.3] 0.922 Parasympathetic parameters Maximal diameter (in millimeters) 5.9 [4.6-7.3] 7.0 [6.4-7.6] 0.180 Minimum diameter (in millimeters) 3.9 [2.9-4.9] 4.7 [4.0-5.4] 0.708 Percent of constriction 35.1 [31.9-38.3] 33.8 [29.3-38.4] 0.295 Latency (in seconds) 0.2 [0.2-0.2] 0.2 [0.2-0.2] 0.183# ACV (in millimeters/second) 4.0 [3.5-4.6] 4.1 [3.6-4.5] 0.664 MCV (in millimeters/second) 5.7 [4.7-6.8] 5.5 [4.9-6.0] 0.592 Sympathetic parameters ADV (in millimeters/second) 0.9 [0.7-1.2] 0.9 [0.8-1.0] 0.440 T75 (in seconds) 2.4 [1.3-3.5] 3.1 [2.8-3.4] 0.154 Data reported as mean [95% Confidence interval] unless otherwise stated. * Fisher Exact test; # Mann-Whitney U test. ACV: Average constriction velocity; ADV: Average dilation velocity; BMI: body mass index; FEV1: forced expiratory volume in the first second of FVC; FVC: forced vital capacity; FEF25-75: forced expiratory flow middle portion of FVC; MCV: Maximum constriction velocity; PD20: provocative dose determining a 20% fall in FEV1; T75: the total time taken by the pupil to recover 75% of the initial resting pupil size after it reached the peak of constriction. 61 Table 14. Multiple linear regression model for predicting  EBT. Variable Standardized Coefficient B P-value 95% CI Baseline EBT -0.675 0.001 [-0.685;-0.229] Asthma -0.008 0.961 [-0.388;0.370] Axillary temperature 0.327 0.043 [0.018;1.027] R2=0.596, adjR2=0.529 p-values of the univariate analysis (including all the tested variables): sex – 0.547, age – 0.336, height – 0.025, weight – 0.196, baseline EBT – <0.001, axillary temperature – 0.089, training intensity – 0.415, asthma – 0.222, PD20 – 0.600, number of training hours in the previous week – 0.583. The following explanatory variables were initially entered into the multiple regression model, but were excluded in the final model as they did not reach significance at the 0.05 level: height, weight. Variable asthma, being a major outcome of this study, was kept in the final model independently of the significance level and adjusted r2 change with its inclusion; is coded as “0”=without asthma; “1”=with asthma. Both groups presented median values of eosinophils and epithelial cells higher and neutrophils lower than reference values for healthy populations (Belda, Leigh et al. 2000). When compared to healthy swimmers, asthmatics presented higher eosinophil and epithelial cell counts and lower neutrophils, but differences were not statistically significant (Table 12). Correlations between sputum eosinophils with baseline EBT (r=0.064, p=0.800) and EBT (r=-0.210, p=0.404) were not significant, even if considering only asthmatics (r=- 0.286, p=0.493 and r=0.048, p=0.911, for baseline EBT and EBT, respectively). Regarding neutrophils, correlations were also non-significant for the global sample for baseline EBT (r=0.194, p=0.441) and EBT (r=0.101, p=0.689) and also for the asthmatic group (r=0.500, p=0.207 and r=-0.048, p=0.911, for baseline and EBT, respectively). 62 5.6 Effect of long-term swimming on airways (study VI) From the 105 swimmers assessed at baseline visit, 86 attended the 3-years follow-up visit and were considered for final analysis. The 19 lost to follow-up subjects did not differ from the others at baseline evaluation. We observed a significant difference in changes in FeNO; those who remained active significantly increased their levels of eosinophilic airway inflammation independently of their gender, age, allergic sensitization, or asthma status (Table 15, Figure 10). After the 3-year follow-up, the prevalence of asthma, allergic rhinitis and use of asthma medication increased significantly in both groups. All subjects increased their overall physical activity levels; however, significant increases in moderate and vigorous physical activity levels were only observed in active swimmers. Table 15. Changes in airway inflammation, prevalence of asthma and rhinitis and physical activity levels in athletes according with their swimming status at baseline visit and after the 3 y follow up. Past swimmers, n=39 Active swimmers, n=47 Past vs Active swimmers Baseline Follow up Baseline Follow up FeNO†, ppb 18 (19.5) 16 (20) 14 (11) 14 (18) .008# Asthma, n (%) 6 (15) 9 (23)* 6 (13) 9 (19) * .656 Allergic rhinitis, n (%) 13 (33) 16 (41)* 11 (23) 13 (28) * .305 Asthma drugs, n (%) 5 (13) 12 (31)* 9 (19) 12 (26) * .382 Physical Activity† Walking 462 (685) 693 (2402) 396 (1188) 739 (1148) .016 Moderate 720 (2010) 1320 (1440) 120 (960) 960 (3120)** .065 Vigorous 5760 (1920) 3840 (6120) 5760 (7040) 13440 (11520)* <.001 Total 7173 (3864) 7242 (5718)* 6222 (9159) 17196 (8544)* .001 Data presented as median (IQR) unless otherwise stated; Physical activity expressed as MET-min/week; bold figures represent statistically significant differences between comparing groups; Paired samples or chi-square tests were used as appropriate and changes between groups after the follow-up were assessed by GLM adjusting on confounders for FeNO: gender, age, allergic sensitization, physician-diagnosed asthma, use of asthma medication; and on gender and age for physical activity levels, with baseline values as covariable; *p<0.001; **p=0.002; p=0.005. †FeNO and physical activity are presented as absolute values, although they were logtransformed for comparison analysis. # Comparison of ∆FeNO variation from baseline to 3 y follow-up among active and past swimmers. Figure 10 Variation of FeNO at baseline and at 3y follow-up among past and active swimmers. 63 64 6. DISCUSSION 6.1 Main findings and relation to previous studies 6.1.1 Diagnosis of asthma and healthcare of asthmatic athletes (Study I) The introduction of mandatory objective asthma diagnosis for inhaler’s use in 2009 decreased the requests submitted to the Anti-Doping Authority of Portugal by approximately half, suggesting that a large number of athletes were receiving medication based on symptoms only. The relative similarity between the proportion of positive tests in 2009 and 2010 suggests that the IOC more rigorous testing criteria strategy is reliable. This study also revealed that changes to the WADA guidelines on inhaled β2-agonists in 2010 led to a dramatic decrease in the number of tests performed in Portuguese athletes with asthma and increased the number of athletes taking inhaled β2-agonists without ICS. These findings clearly show that guidelines for asthma diagnosis have an impact on the care of athletes with asthma and influence how respiratory symptoms are managed and treated in these patients. These results are in line with a previous study that evaluated the impact of IOC-MC recommendations. Dickinson et al found that 21% of British Olympic athletes were receiving asthma medication for which there was no clinical indication (Dickinson, Whyte et al. 2005). In Portugal, changes to the WADA 2009 Prohibited List also permitted more rigorous screening of asthmatic athletes due to the implementation of objective criteria for inhaler use; the new requirements also led to the withdrawal of unnecessary medication. It improved athlete’s care by investigation of alternative diagnoses. Constant changes to WADA guidelines, however, jeopardize the achievements made to date and adversely affect the health of asthmatic athletes. Diagnosis of asthma is complex, and results of lung function, airway inflammation, and BHR provide important, complementary information that can aid asthma control. Currently, unrestricted use of inhaled salbutamol, salmeterol, or formoterol is permitted as long as specified doses are not exceeded. Such a change, however, might lead to an increased use of long-acting β2-agonists, without ICS, as recently was evidenced to occur in Olympic athletes (Bonini, Gramiccioni et al. 2015). This is a matter of concern since inhaled β2-agonists may mask worsening of airway inflammation; furthermore, airway inflammation might contribute to the downregulation of β2-agonists receptors (Bonini, Permaul et al. 2013). Therefore, although the current guidelines may seem fairer and seem to improve access to treatment among asthmatic athletes, they introduce safety issues stemming from the unsupervised use of inhaled β2-agonists. As shown by our study, in the absence of mandatory objective testing for certain asthma medications, athletes may skip lung function tests. 65 6.1.2 Phenotypes of asthma in elite athletes (Study II) This study identified two distinct phenotypes of asthma in athletes: “atopic asthma” defined by the occurrence of allergic sensitization, increased levels of FeNO, rhinitis and other allergic co-morbidities; and “sports asthma”, defined by the existence of exerciseinduced respiratory symptoms and BHR in the absence of allergic features. Moreover, specific training and environment conditions were associated with increased risk of developing “sports asthma”: athletes practicing water and winter sports present a three and a nine fold increase in their risk of “sports asthma” respectively, compared with others. The two patterns of asthma obtained in this present study are remarkably in accordance with the only previous report of phenotypes for athletes, a Finnish study relying on different study design and an a priori list of selected variables for statistical analysis (Haahtela, Malmberg et al. 2008). Our study contributes to confirm that different risk factors, such as atopy and environmental training conditions, result in different patterns of asthma. For athletes practicing water and winter sports, their “occupation” demands exposure to potentially noxious stimuli, such as sport-specific environment, in addition to frequent episodes of prolonged hyperpnoea (Price, Ansley et al. 2013). The possibility that these exposures determine different underlying mechanisms of asthma has been previously raised (Langdeau, Turcotte et al. 2000, Langdeau and Boulet 2001) and should be emphasized. The natural course of asthma in athletes has been difficult to change by antiinflammatory treatment (Helenius, Lumme et al. 2005). This highlights a possible need of a different therapeutic approach for these subjects. To define these distinct phenotypes could lead not only to further understanding underlying mechanisms of asthma in elite athletes, but, and most important from a clinical point of view, also to recognize that potentially different treatments specifically targeted for the defined phenotypic groups in relation to the specific underlying mechanism are needed. 6.1.3 Dysautonomia, asthma and BHR in elite swimmers (Study III) In this exploratory study, no significant differences were observed in parasympathetic parameters between asthmatic and non-asthmatic elite swimmers. However, in those with severe BHR, a significant difference became clear. Also, in those with clinical relevant BHR, significant correlations were observed between parasympathetic parameters and PD20 methacholine. This suggests that in these athletes the increased parasympathetic tonus is associated with BHR particularly through the contraction of the bronchial smooth muscle rather than related with other features of asthma. Previous studies supporting this hypothesis of dysautonomy associated with training are in accordance with our findings. Pichon et al. demonstrated that subjects with an increased BHR had a higher vagal tone (Pichon, de Bisschop et al. 2005), which was corroborated by Park et al. findings of a relationship between BHR and a diminished sweat secretion, tearing and salivary flow rate in healthy athletes, indicating autonomic dysfunction (Park, Stafford et al. 2008). All these studies have used BHR as an outcome measure, rather than asthma diagnosis, which is also the probable reason for the lack of 66 significant differences in parasympathetic outcomes between asthmatic and non-asthmatic swimmers in our study. In fact, while bronchoconstriction is largely dependent on airway smooth muscle cells, asthma is far more complex as a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, including airway epithelium, eosinophils, neutrophils, lymphocytes and mast cells (Holgate, Lemanske et al. 2008, Holgate 2008, Global Strategy for Asthma Management and Prevention 2012). In elite competitive adolescent swimmers, an increase in BHR correlated with the exercise intensity after 3000 meters swimming in an indoor swimming pool was demonstrated, for both asthmatic and healthy subjects (Carlsen, Oseid et al. 1989). Together with our results, this seems to point out that dysautonomia might contribute to the severity of bronchial reactivity in swimmers. 6.1.4 Anti-cholinergic drugs for BHR in elite athletes (Study IV) In this study, 23% of winter athletes had significant reversibility to inhaled ipratropium bromide and none to inhaled salbutamol. The main finding of this study was a highly significant correlation between BHR and reversibility to inhaled ipratropium bromide, but not so between BHR and reversibility to salbutamol. The significant high-grade negative correlation between a cholinergic bronchoconstrictor and an anticholinergic bronchodilator stimulus, and the lack of correlation with a β2-stimulating bronchodilator, suggest an important role of an increased parasympathetic tone and sensitivity in the development of BHR and asthma in these top endurance-trained athletes. Increased parasympathetic activity has previously been reported in endurance-trained athletes measured both by the parasympathetic activity of the eye by pupillometry in long distance runners (Filipe, Falcão-Reis et al. 2003, Kaltsatou, Kouidi et al. 2011) and of the cardiovascular system by the variation in the heart rate induced by an exercise test (Knöpfli and Bar-Or 1999). Furthermore, a significant correlation between heart rate variability indices and V’O2max was shown (Goldsmith, Bigger et al. 1997), which suggests that endurance training and increased aerobic capacity are followed by increased parasympathetic activity. These findings are supported by the present study in crosscountry skiers who, in addition to the endurance training, are also exposed to cold air, an exposure previously reported to cause parasympathetic stimulation of the airways and contribute to EIB (McFadden and Ingram 1979). Anticholinergic agents, such as ipratropium bromide, inhibit parasympathetic nerve impulses through competitive inhibition on the muscarinic acetylcholine receptors in smooth muscle and respiratory glands (de Jongste, Jongejan et al. 1991). It has been suggested that differences in the parasympathetic bronchial tone may explain why some patients are responders and others non-responders to anticholinergic treatment (Knöpfli, Bar-Or et al. 2005, Knöpfli, Luke-Zeitoun et al. 2007). As methacholine acts as a nonselective muscarinic receptor agonist, the bronchial responsiveness to methacholine may itself be considered to reflect the parasympathetic bronchial tone. In a recent study, ipratropium did not significantly influence the number and the perception of cough following exercise in cross-country skiers (Bordeleau, Turmel et al. 2014). Moreover, that study suggested that exercise-induced cough in these athletes is not mainly associated with 67 EIB, except for a subgroup of athletes which seemed to show a beneficial response to ipratropium, proposing different cough responses in this population (Bordeleau, Turmel et al. 2014). When activated, muscarinic receptors promote bronchoconstriction, glandular secretion, and blood vessel vasodilation (Lee, Jacoby et al. 2001, Bateman, Rennard et al. 2009). As there are no muscarinic receptors on airway sensory afferent nerves, it is unlikely that anticholinergic agents act through a direct effect on the cough reflex (Bateman, Rennard et al. 2009). Although respiratory symptoms are common among cross-country skiers (Larsson, Ohlsén et al. 1993, Heir and Oseid 1994), our results showed no associations between selfreported symptoms and BHR, reversibility to inhaled ipratropium bromide or inhaled salbutamol, or to FeNO. The incidence of asthma symptoms found by self-reports is higher compared with objectively measured BHR and reversibility tests. This finding confirms that objective tests are a necessity in athletes in addition to symptoms to confirm the asthma diagnosis, as previously reported (Rundell, Im et al. 2001). 6.1.5 Exhaled breath temperature, exercise and asthma (Study V) Elite swimmers presented increased EBT after a training session, supporting the hypothesis of heat loss during exercise. Interestingly, asthmatic swimmers did not experience a higher increase in EBT when compared to healthy swimmers after controlling for baseline EBT and body temperature. These findings may suggest that heat loss occurs as a physiological rather than a pathological response to exercise. Furthermore, no relation was observed between EBT and inflammatory cells in induced sputum, neither with the degree of BHR. Therefore, it is tempting to speculate that an inflammatory response to the heat loss of exercise might not be a key etiopathogenic mechanism of swimmers asthma. Our results are in accordance with the two previous studies investigating the relation of EBT to EIA (Svensson, Nilsson et al. 2012, Tufvesson, Svensson et al. 2013). Both studies showed an increase in EBT after a standardized exercise challenge at the laboratory, but no differences were observed between asthmatic and healthy controls (Svensson, Nilsson et al. 2012, Tufvesson, Svensson et al. 2013), in agreement with our results obtained with the regular exercise training of competitive swimmers. During exercise, unperfused alveoli become perfused through recruitment of pulmonary capillaries, and underperfused units receive an increased blood supply. It is therefore conceivable to expect a physiologic increase in airways temperature related to this capillary recruitment instead of a pathological mechanism related to vasodilation resulting from inflammation. That is consistent with the lack of correlation previously found between nitric oxide levels with EBT after exercise (Tufvesson, Svensson et al. 2013), as well as the lack of correlation with sputum inflammatory cell counts observed in our study. Vigorous exercise causes epithelial damage, which has likewise been linked to EIA/ EIB in elite athletes (Bougault, Turmel et al. 2009). Clara cell secretory protein (CC16) a peripheral marker for assessing the epithelial barrier disruption in the lower airways (Broeckaert and Bernard 2000, Bernard, Carbonnelle et al. 2007) was shown to correlate 68 to EBT after an exercise challenge, reflecting an overall epithelial involvement (Tufvesson, Svensson et al. 2013); no differences were found between asthmatics and healthy controls (Tufvesson, Svensson et al. 2013), which again leads to the concept of a physiological rather than a pathological response to exercise. Bronchial epithelial cells are released in higher amounts into sputum (epithelial shedding) of cold air athletes and swimmers (Bougault, Turmel et al. 2009), highlighting the role of environmental exposure impact on epithelial layer. These results are in agreement with our findings. However, we observed no significant differences between asthmatic and healthy swimmers, which support that exercise and irritant exposures contribute to a detrimental effect on the athletes’ airways but probably not the hypothesis of these two acting as etiopathogenic mechanisms of EIA. So, in the light of these results, along with previous published findings, we would like to underline that: 1) Exercise causes EBT to increase, probably due to a physiological increase in blood flow in the respiratory mucosa, rather than due to a pathological mechanism since no differences were observed between asthmatic subjects and healthy controls; 2) Mechanic noxious stimulus of repeated bouts of increased ventilation during training cause epithelial damage of the airways and susceptibility to irritative stimuli, supported by observed increased numbers of bronchial epithelial cells in athletes and high levels of CC16, regardless of their asthmatic status; 3) Due to the daily repeated training, epithelial repair is delayed and a “frustrated’ inflammatory response occurs to heal the damage of physical injury in both asthmatic and healthy swimmers. No differences were observed between asthmatic and healthy subjects with regards to the above mentioned observations, suggesting the explanatory model of EIA and bronchoconstriction in athletes will probable include the interplay between environmental training factors and athlete’s personal and genetic risk factors (Moreira, Delgado et al. 2011). 6.1.6 Effect of long-term swimming on airways (Study VI) This prospective study of competitive swimmers shows that those who remained active after 3-years significantly increased their levels of airway inflammation measured by exhaled nitric oxide compared to those who quitted swimming, independently of their gender, age, allergic sensitization, or asthma status. After the 3-year follow-up the prevalence of asthma, allergic rhinitis and use of asthma medication increased significantly in both groups. All subjects increased their overall physical activity levels; however, significant increases in moderate and vigorous physical activity levels were only observed in active swimmers. Two studies prospectively assessed BHR and airway inflammation in swimmers. Bougault et al conducted a study comparing the same swimmers during intensive training period and after at least 15 days without intense swimming (Bougault, Turmel et al. 2011), while Helenius et al followed 42 Finnish swimmers during 5 years and compared those who kept versus those who retired from swimming (Helenius, Rytilä et al. 2002). Both studies suggest that BHR is reduced when intense training is stopped. Conflicting results, however, occurred with respect to airway inflammation. In accordance with our results, Helenius et al found that after a 5-year follow-up, mild 69 eosinophilic airway inflammation was aggravated among those swimmers who remained at high-level training and tended to attenuate in swimmers who finished their sports careers. On the contrary, no significant change was observed between intense training and resting periods for swimmers’ airway inflammatory cell counts (Bougault, Turmel et al. 2011). However, in Bougault et al. study, only 7 swimmers and 2 controls were able to produce sputum in the two visits and therefore only swimmers’ data are presented (Bougault, Turmel et al. 2011). Such small numbers, together with the short time of recovery, might be responsible for the results found. During the 5 year follow-up period, Helenius et al found that the occurrence of exercise-induced bronchial symptoms and asthma tended to decrease in former swimmers, a result that differs from our findings. However, in our study, subjects remained physically active despite stopping swimming, which may responsible for keeping asthma symptoms and need of asthma medication. Also, asthma in our study was defined by doctor diagnosis, rather than by objective evidence, which might also be related to this discrepancy. The diagnosis of asthma in athletes dramatically impacts on the disease management and the definition of asthma in this population includes two specific different phenotypes. Given that both swimmers and winter sport athletes have shown a higher risk of presenting the “sports asthma” phenotype in study II, a different mechanism of asthma within these athletes may be hypothesized. Both studies III and IV support that this mechanism is most probably related to an imbalance towards an increased parasympathetic activity that predisposes to EIB. Study V further supports that other mechanism rather than the inflammation and thermal loss is associated with EIA in swimmers and study VI confirms a relative independence of the two conditions given that eosinophilic airway inflammation decreases in swimmers who finished their career while the prevalence of asthma and use of asthma medication increases significantly in both active and past adolescent swimmers. 6.2 Methodological considerations – limitations and strengths Our findings in Study I are limited by the retrospective nature of the study and the anonymity of the data collected. However, the decrease in requests observed in 2009 is not due to athletes being already covered by a previous submission, as renewal was yearly at that time. TUEs approval lasted for 4 years and we can therefore be sure that no repetitions occurred in 2010. Also, our study contributes to overcoming the paucity of data regarding asthma in Portuguese athletes. Moreover, we have evaluated how changes in WADA guidelines have impacted the clinical management of asthma in this setting. Establishing different phenotypes of asthma in elite athletes (Study II) was limited by the cross-sectional design of the study, which does not permit to identify causality; but the statistical models used to pool and characterize different clusters make this study especially useful by retrieving a clear view on asthma phenotypes in athletes. Replication 70 of results in other datasets is important when using these exploratory statistical techniques, and the two patterns of asthma obtained in this present study are remarkably in accordance with the only previously published report, a study relying on different study design and an a priori list of selected variables for statistical analysis (Haahtela, Malmberg et al. 2008). The use of different methods (both direct and indirect challenges) to assess BHR in athletes must be also pointed out as a limitation. In Study II, information was collected from medical files, so there was no possibility to homogenize tests performed by athletes in two centers. In any case, the final diagnosis was made according to IOC criteria using standardized methods. Moreover, the absence of information about age of asthma onset limits the extent of conclusion because we cannot be aware if previous presence of asthma would influence the type of sport chosen. It does not seem to be the case, based on previous literature, as the prevalence of asthma is known to increase with age both in swimmers and skiers (Stensrud, Mykland et al. 2007, Pedersen, Lund et al. 2008, Bougault and Boulet 2012). However, this study should be succeeded by new prospective studies following young athletes from adolescence until adulthood. Besides, although motivating, results provided by this exploratory analysis have to be interpreted in context of future work addressing whether the two phenotypes are relevant from a clinical perspective. Potential phenotypes require prospective validation with clinical interventional trials. Our study IV has shown that Norwegian competitive endurance winter athletes respond with a higher reversibility to ipratropium bromide than to inhaled β2-agonists, supporting that different phenotypes may reflect different mechanisms of EIA, and therefore imply different treatment modalities. The fact that only winter athletes were included in Study IV makes it difficult to generalize data for elite athletes practicing other sports. However, as shown in Study II, these athletes have a higher risk of developing a distinct asthma phenotype. Furthermore, in Study IV respiratory symptoms and asthma diagnosis were assessed only by questionnaire. However, this is unlikely to affect objective BHR and reversibility assessments. Also, the bronchodilation procedures were performed 2000 meters above the sea level, which not only does not reproduce the real-life situation of most athletes, but may induce altitude changes to some pulmonary ventilatory parameters (FVC, FEV1, and FEF25-75%) (Hashimoto, McWilliams et al. 1997). Anyway, the bronchodilator effect has not been found to increase at high altitude (Hashimoto, McWilliams et al. 1997); moreover, the high altitude is unlikely to affect differently the two bronchodilator procedures. However, in this study, eight athletes used short-acting β2agonists, ten used long-acting β2-agonists and ten athletes used ICS. A potential influence from the use of these medications on the absence of any significant bronchodilator effect after inhaled salbutamol cannot be excluded. Collection of data for this study was carried out before the competitive season, but during an intensive training period, which may have influenced the degree of BHR in the athletes. An earlier study including young competitive skiers has shown that BHR in cross-country skiers varied according to seasons and exercise training intensity (Heir and Larsen 1995). In any case, Study IV is the first randomized cross-over study comparing the bronchodilator efficacy of inhaled ipratropium bromide versus inhaled salbutamol in winter athletes, and the relationship to bronchial methacholine responsiveness. It benefits from including the Norwegian national cross-country skiing team, and thus only athletes at the elite level. Also, as self-reported 78 7. CONCLUSIONS In the present study, phenotypes and mechanisms of asthma development in elite athletes were investigated. Based on the results presented in this thesis, the following conclusions can be drawn: 1) Defining asthma in athletes implies a correct diagnosis which is crucial for management. Strict guidelines were shown to impact asthmatic athletes’ care; by starting a more rigorous screening withdrawal of unnecessary medication was possible in approximately half of the athletes. 2) Two asthma phenotypes in elite athletes were identified: “atopic asthma” and “sports asthma”. The type of sport practiced was associated with different phenotypes: water and winter sport athletes had 3 and 9-fold increased risk of “sports asthma”, respectively. 3) Differences in parasympathetic parameters between asthmatic and non-asthmatic swimmers were not significant, but among those with clinically relevant bronchial hyperesponsiveness an association was evident, supporting the relation between dysautonomia and bronchial hyperesponsiveness. 4) In elite skiers, a significantly higher reversibility was observed after inhaled ipratropium bromide compared to inhaled salbutamol. Methacholine bronchial reactivity had a highly significant inverse correlation to the cholinergic antagonism of inhaled ipratropium bromide, but not to the bronchodilating inhaled salbutamol. This markedly increased bronchial parasympathetic tone may represent an important mechanism in the development of skier’s asthma. 5) A relationship between airway’s inflammation and respiratory heat loss after vigorous exercise could not be confirmed, suggesting that the heat loss, and consequent increase in exhaled breath temperature, occur as a physiological rather than a pathological response to exercise. 6) Competitive swimmers who remained active at a 3-year follow-up significantly increased their levels of airway inflammation measured by exhaled nitric oxide levels, independently of their gender, age, allergic sensitization, or asthma status, while in those who finished swimming airway inflammation decreases. The prevalence of asthma and use of asthma medication increased significantly in both active and past adolescent swimmers, suggesting a relative independence of the two conditions. 79 80 ACKNOWLEDGEMENTS "Those who pass by us, do not go alone, and do not leave us alone; they leave a bit of themselves, and take a little of us." Antoine de Saint-Exupéry This thesis could not have been completed without guidance, support, help and cooperation of a lot of persons that passed by, leaving a bit of themselves, and to which I am deeply thankful. First and above all others, I am thankful to my supervisor Professor André Moreira. He has always encouraged me to pursue my PhD and taught me almost everything about scientific research, from designing a study to writing an article. He was the first to introduce me to the complexity of exercise-induced asthma in athletes and stimulated my curiosity regarding the open questions in this field of knowledge. His inspiring attitude, guidance, support and friendship has been invaluable and turned this thesis possible. I was very lucky to also have the support of another dedicated, knowledgeable and enthusiastic person, my co-supervisor Professor Kai-Håkon Carlsen. The work that led to this present thesis started in 2012, during a fellowship at the Norwegian School of Sports Sciences where I met him. The time spent there and the research developed allowed for joint studies, increasing the broad of the spectrum of research. His enthusiasm for respiratory research was inspiring, and I feel privileged to have had the opportunity to work with him. My gratitude goes also to all participants who volunteered to join these studies, as well as to some of our Faculty students, whose cooperation was indispensable and made this work possible. Most part of these studies was carried out at the Immunology Laboratory of Faculty of Medicine, University of Porto. My warmest appreciation goes to Professor Luís Delgado, head of the Department of Immunology, for the research facilities, encouragement, for providing inspiration and sharing his knowledge and for his excellent advices during the research work and writing process (not only of this thesis but also in several other occasions in research and life). I owe warmest thanks also to Diana Silva, my coworker and coauthor of many papers. Her help was invaluable at the laboratory work and during the writing process, and I have a profound admiration towards her extraordinary work abilities. I am most grateful to Marília Beltrão and Oksana Sokhatska for their help in many practical matters and technical assistance for blood collection as well as for induced sputum. In this context I must also deeply thank Carmo Palmares; with her experience and endless availability she turned out possible to finalize the induced sputum readings. I wish to thank to Cristina Lopes and Luís Araújo for their partnership. I have worked at the Department of Immunoallergology of Centro Hospitalar São João during my specialization. We have had a good team spirit there, and I have enjoyed working with all. Many thanks go to my colleagues for teaching me so much about 81 Immunoallergology during the beginning of my career, for understanding some of my absences and supporting me, and most of all for a friendly atmosphere at work. In particular, I wish to thank Ana Margarida Pereira for her friendship, constructive criticism and for her valuable comments and suggestions, and to Carla Martins for her invaluable help in technical aspects of methacholine bronchial challenges and her endless availability. Since I have finished my specialization, I have been working at the Allergy Unit of Hospital & Instituto CUF Porto. I owe my sincere gratitude to my supervisor, Professor João Fonseca, for his positive attitude toward my work, for interesting discussions and valuable scientific advices, availability and comprehensibility regarding my absences. I am grateful for his support throughout. I most sincerely thank Trine Stensrud and Julie Stang for including me in their study and supporting me during technical work at the Laboratory of the Norwegian School of Sport Sciences. I should also express gratitude to them, as well as Karin Carlsen for their hospitality and kindness during my stay in Oslo. Those winter months became really pleasant. I wish to thank the ORAACLE group for interesting discussions during my stay in Oslo. I am grateful to statisticians Petter Mowinckel and Milton Severo for advices on statistical methods and help with data management. Warm thanks go to all my friends for being there for me all these years – your support and friendship has been invaluable. Special thanks to my closest friends, the “sewing circle”, for your good company and invaluable help, for your continual support and for filling my life with love and happiness. Finally, I owe warmest thanks to my dear family. I am deeply grateful to my parents for their encouragement, for giving me a firm grounding for life, and for their continual love and support. My mother, with her endless affection and care has always been and will always be my role model and anchorperson. This thesis is yours! This thesis was not only about research and academic skills but also about growing as a person, facing adversities, increasing confidence and resilience and the power of perseverance and patience. With all of you that were part of this process I have learned that “Good things come to those who believe, better things come to those who are patient and the best things come to those who never give up”. Thanks! I hope that this journey also led those who passed by to take a little of it. 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Pneumonol Alergol Pol 80(4): 339-342. 98 ORIGINAL PUBLICATIONS Study I Impact of changes in antidoping regulations (WADA Guidelines) on asthma care in athletes Clin J Sport Med. 2013;23(1):74-6. BRIEF REPORT Impact of Changes in Anti-doping Regulations (WADA Guidelines) on Asthma Care in Athletes Mariana Couto, MD,*†Luís Horta, MD, PhD,‡Luís Delgado, MD, PhD,*† Miguel Capão-Filipe, MD,*§ and André Moreira, MD, PhD*† Objective: To investigate how changes to the World Anti-Doping Agency (WADA) guidelines on asthma medication requests have impacted the management of asthmatic athletes in Portugal. Design: Retrospective analysis of asthma medication requests submitted in 2008 to 2010. Setting: Portuguese Anti-Doping Authority database. Participants: Athletes requesting the use of inhaled corticosteroids and/or b 2 -agonists. Independent Variables: Demographic, therapeutic, and diagnostic test data. Main Outcome Measures: Yearly changes in number of asthma medication requests and diagnostic procedures. Results: We analyzed 326 requests: 173 abbreviated Therapeutic Use Exemptions (TUEs) in 2008 (objective tests not required), 9 Declaration of Use (DoU) and 76 TUEs in 2009, and 39 DoU and 29 TUEs in 2010. Spirometry was performed in 87% and 37% of athletes in 2009 and 2010, respectively; the corresponding figures for bronchoprovocation were 59% and 16%, almost all positive in both years. Conclusions: Applications for inhaler use have decreased by approximately half since objective asthma testing became mandatory. Our findings show that WADA guidelines have an impact on asthmatic athletes care: In 2009 a more rigorous screening was possible, leading to withdrawal of unnecessary medication. Constant changes, however, jeopardize this achievement and nowadays introduce safety issues stemming from the unsupervised use of inhaled b 2 -agonists. Key Words: asthma, airway hyperresponsiveness, anti-doping, bronchoconstriction, exercise, inhaled beta-2 agonists, sports, WADA (Clin J Sport Med 2013;23:74–76) INTRODUCTION Diagnosing asthma in athletes is challenging. Multiple phenotypes of asthma exist, and different underlying mechanisms contribute to etiopathogenesis. 1,2 Also, alternative diagnoses must be considered, 3 and in athletes, symptoms are poor predictors of this condition. 4 Objective evidence (eg, positive bronchodilator or bronchoprovocation test), thus, is needed to confirm a diagnosis in this setting. 5 The recommendation of the International Olympic Committee Medical Commission (IOC-MC) for Olympic athletes to present objective evidence of asthma before allowing the use of inhaled b 2 -agonists (IBAs), in place since 2002, has facilitated the study of how asthma impacts different sports and has benefited athletes by ensuring better care. In 2009, the World Anti-Doping Agency (WADA) followed the IOC approach, extending it to all other athletes. 6 The WADA guidelines on asthma, however, have changed in recent years. Before 2009, an abbreviated Therapeutic Use Exemption (aTUE), which did not require objective evidence of asthma, accompanied by a physician’s report of asthma, was sufficient for requesting permission to use inhaled corticosteroids (ICS) and inhaled formoterol, salbutamol, salmeterol, and terbutaline. In 2009, however, aTUEs were withdrawn and replaced by a Declaration of Use (DoU) for ICS and a full Therapeutic Use Exemption (TUE) requiring objective evidence for the same 4 IBAs. In 2010, the DoU was extended to salbutamol and salmeterol, but the other IBAs still required a TUE. The aim of this study was to assess the impact of WADA guideline changes on asthma medication requests by Portuguese athletes. METHODS We retrospectively analyzed asthma medication requests submitted to the Portuguese Anti-Doping Authority between 2008 and 2010. Athletes older than 16 years who requested permission to use ICS and/or IBAs for more than 3 months were included. Data on respiratory symptoms, medication requested, spirometry, and atopy (at least 1 positive skin prick test or positive specific IgE) were collected. A diagnosis of asthma was based on a positive bronchodilator test or bronchoprovocation test. 5 Exhaled nitric oxide results were converted to personal predicted values using the FeNO Interpretation Aid tool (http://www.enovis.org) and considered increased if above 150% of predicted. Data were expressed as median and range, and categorical variables were compared using the x 2 or Fisher Submitted for publication January 8, 2012; accepted July 5, 2012. From the *Allergy, Asthma & Sports Unit, Immunoallergology Department, Centro Hospitalar São João E.P.E., Porto, Portugal; †Immunology Laboratory, Faculty of Medicine, University of Porto, Porto, Portugal; ‡AntiDoping Authority of Portugal, Lisbon, Portugal; and §Internal Medicine Department, Baixo-Vouga Hospital Center, Aveiro, Portugal. The authors report no financial or conflicts of interest. Correspondence Author: Mariana Couto, MD, Serviço de Imunoalergologia, Centro Hospitalar São João, EPE, Alameda Prof. Hernâni Monteiro 4200-319 Porto, Portugal (m[email protected]). Copyright © 2013 by Lippincott Williams & Wilkins 74 |www.cjsportmed.com Clin J Sport Med Volume 23, Number 1, January 2013 eosinophilic airway inflammation reflected by increased exhaled nitric oxide levels (FE NO ); and another distinct phenotype with late onset of symptoms during sports career, airway responsiveness to eucapnic voluntary hyperpnoea (EVH) and a variable association with atopic markers and FE NO . These phenotypes were described only in Finnish athletes, and have not been fully established so far. Most recent efforts to describe phenotypes are based on cluster analysis. These multivariate statistical methods allow splitting the differences between patient group data into disease categories and clinically meaningful groups, therefore being less dependent on a priori assumptions. These methods have already been successfully applied within respiratory medicine [6,9–11] to identify asthma phenotypes that exhibited differences in clinical, physiological and inflammatory parameters as well as response to treatment [10,11]. However, such methods have not been applied to athletes with asthma. The objectives of the present study were to identify and characterize asthma phenotypes in elite athletes using latent class analysis (LCA) and to assess a possible association with the type of sport practiced. Methods Design and participants In the present cross-sectional study, an analysis of elite athlete records kept in database files of two different countries was performed. Portuguese and Norwegian athletes training at high competitive levels (national, international or Olympic teams) were identified through existing institution databases. In Portugal, we used registries of elite athletes available at the Portuguese Anti-doping Authority and the Portuguese database of Olympic athletes; in Norway, we analyzed medical files from the respiratory medical team of the Norwegian School of Sport Sciences, including Olympic athletes participating in the 2008 summer and 2010 winter Olympic Games. Athletes were selected according with available information on symptoms, lung function and airway inflammation, BHR, and allergic sensitization. Healthy athletes and those with other conditions rather than asthma were excluded. From all reviewed files, 324 files had complete information available and informed consent for data use. Of these 324 athletes, 150 athletes fulfilled asthma criteria and were included for LCA. The present study was conducted in accordance with Declaration of Helsinki for Medical Research Involving Human Subjects and was approved by Regional Medical Ethics Committees and Norwegian Data Inspectorate. All included subjects signed an informed consent for data usage. Definitions Asthma diagnosis was established by a medical doctor according to criteria set by the International Olympic Committee to document asthma in athletes [4,12], with objective evidence of either reversibility after bronchodilator administration or BHR after a bronchial provocation challenge. The demographic data obtained included age, gender, height, weight and sport practiced. The type of sport was classified according to environmental training conditions into water sports (swimming and water polo), winter sports (cross-country skiing, biathlon, skeleton, alpine skiing and ski cross) and other sports (speed skating, curling, handball, judo, triathlon, football, cycling, beach volley, rowing, athletics, sailing, badminton, canoeing, curling, equestrian, taekwondo, auto-racing, billiards, paragliding, rugby, tennis, roller hockey, kickboxing, fencing, basketball or golf). Medical data collected included presence of respiratory symptoms, current use of asthma medication and presence of rhinitis or other allergic diseases (conjunctivitis, urticaria, eczema, anaphylaxis and drug, food and venom allergies). These data were sampled through allergy questionnaire for athletes (AQUA) questionnaire [13] at the time of the medical consultation. For statistical purposes, variables were categorized according to the definitions presented in Table 1. Spirometry was performed in agreement with the European Respiratory Society guidelines [14] and results (forced expiratory volume in first second – FEV 1 and forced vital capacity – FVC) were presented as both absolute and predicted values, according to published reference algorithms [15]. For both airflow obstruction and BHR, the first ever performed spirometry and the first ever performed bronchial provocation challenge, respectively, were considered. Statistical analysis Results are presented as mean values [95% confidence interval (CI)], mean ± standard deviation (SD), or medians ± interquartile range (IQR) in case of skewed distribution, or counts (n, %). Independent samples t-test was used for comparison of normally distributed continuous data, and Mann–Whitney test was used on data with skewed distribution. Categorical variables were compared by Chisquare or Fisher’s exact tests. These analyses were performed using SPSS (IBM SPSS Statistics for Windows, Version 20.0, IBM Corp., Armonk, NY), considering a significance level of 0.05. LCA was used to uncover distinct groups of individuals from a sample (patterns) homogeneous within the group, considering that the performance of an individual in a set of items is explained by a categorical latent variable with K classes, commonly called ‘‘latent classes’’. Model interpretation was based on item profiles in each category and obtained from probabilities of endorsing each item response, conditional on class membership. In the present study, the number of latent classes was defined according to Bayesian Information Criterion (BIC). Starting from one single class and increasing one class at each step, the best solution was identified when the increase of number of classes did not lead to a decrease in BIC. LCA used nine variables important for asthma definition or relevant for differential diagnosis (Table 1). The selection of variables was based on the assumption of their clinical relevance for asthma definition. The Lo– Mendell–Rubin likelihood ratio test of model fit was used to quantify the likelihood that the data could be described by a model with one-less class. All LCA models were fitted using MPlus (V.5.2; Muthen & Muthen, Los Angeles, CA). Later, among asthmatic athletes, we estimated the risk associated with the sport training environment, by using regression 2M. Couto et al. J Asthma, Early Online: 1–8 Downloaded by [213.58.139.35] at 07:16 21 September 2015 analysis to predict the odds of having a specific asthma pattern (phenotype), having ‘‘other sports’’ as reference. Results Included subjects From 324 files reviewed, 150 files belonged to athletes who fulfilled asthma criteria (91 Portuguese; 59 Norwegian). Fortyfive athletes were diagnosed with asthma based on positive bronchodilation (the mean ± SD of FEV 1 increase was 450 mL ± 292 and 13% ± 9.4), and 105 by presenting airway responsiveness after a provocation challenge: 1 positive challenge to mannitol, 3 positive challenges with exercise and the remaining 101 positive challenges with methacholine (7 reporting PC 20 : mean 3.9 mg/mL; 94 reporting PD 20 : mean 6.8 mg). The remaining athletes were healthy (n¼129) or had other pathologic conditions (n¼45). Asthmatic subjects included in the present study presented airflow limitation, more reversibility to salbutamol, more BHR, atopy, rhinitis and airway inflammation assessed by FE NO (Table 2). LCA model Relying on asthma defining variables, the increase in likelihood values leveled off when increasing from one to two classes, and BIC reached its optimum value at two classes (Online Table). This result was confirmed by Lo–Mendell– Rubin likelihood ratio test. Class 1 was characterized by allergic sensitization, rhinitis and other allergic co-morbidities, and increased FE NO levels (‘‘Atopic asthma’’); while class 2 was characterized by the occurrence of respiratory symptoms and BHR, in the absence of atopic features (‘‘Sports asthma’’) (Table 3 and Figure 1). Subject’s differences between classes The athletes which were assigned to ‘‘atopic asthma’’ presented higher values of FE NO than those in ‘‘sports asthma’’ (32.2 vs. 15.7, p¼0.002). In ‘‘atopic asthma’’, 28 athletes presented increased values of FE NO , compared to only 7 among those in ‘‘sports asthma’’. Allergic diseases were evident in 60.7% of athletes in ‘‘atopic asthma’’, and in 12.5% of those assigned to ‘‘sports asthma’’, namely: conjunctivitis (48% of athletes in ‘‘atopic asthma’’ and none in ‘‘sports asthma’’), atopic eczema (12% of athletes in ‘‘atopic asthma’’ and none in ‘‘sports asthma’’), and food allergy (31% of athletes in ‘‘atopic asthma’’ and none in ‘‘sports asthma’’). Hymenoptera venom allergy, drug allergy and anaphylaxis had a similar prevalence in both Table 1. Definitions of variables set for LCA. Variable Definition Airflow obstruction FEV 1 /FVC ratio lower than 0.70 Reversibility Increase of at least 200 mL and 12% in FEV 1 Rhinitis a Positive answer to the question ‘‘Did any doctor diagnose you an allergic disease?’’ AND ‘‘Rhinitis’’ OR Positive answer to the question ‘‘Do you frequently sneeze, have a running, itchy nose (apart from colds)? Any other allergic disease a Positive answer to the question ‘‘Did any doctor diagnose you an allergic disease?’’ (except rhinitis) OR Positive answer to the question ‘‘Have you frequently red eyes with tearing and itching?’’ OR Positive answer to the question ‘‘Have you ever had severe allergic or anaphylactic reactions?’’ OR Positive answer to the question ‘‘Have you ever had allergic reactions to foods?’’ OR Positive answer to the question ‘‘Have you ever had allergic reactions to drugs?’’ Respiratory symptoms a Self-reported recurrent breathlessness, cough, wheezing, chest tightness and/or phlegm production OR Positive answer to the question ‘‘Did any doctor diagnose you an allergic disease?’’ AND ‘‘Asthma’’ OR Positive answer to the question ‘‘Have you ever had shortness of breath, cough and/or itching of the throat following exercise?’’ Asthma treatment Current or recent treatment with ICS and/or Beta 2 -agonists Airway hyperesponsiveness b A fall in FEV 1 10% from baseline with exercise or EVH OR A fall in FEV 1 15% from baseline after inhaling 22.5 ml of 4.5 g% NaCl or 635 mg of mannitol OR A fall in FEV 1 20% from baseline with methacholine: PC 20 4 mg/ml, or PD 20 400 mg (cumulative dose) or 200 mg (noncumulative dose) in those not taking ICS, and PC 20 16 mg/ml or PD 20 1600 mg (cumulative dose) or 800 mg (noncumulative dose) in those taking ICS for at least 1 month Eosinophilic inflammation The presence of FE NO levels above 25 ppb Allergic sensitization The presence of at least one positive (mean of largest and perpendicular diameter of the wheal 3 mm for each allergen and controls showing adequate reactions) skin prick test or the presence of positive specific IgE (0.35 kU/L) for at least one common aeroallergen in the local geographic area EVH, eucapnic voluntary hyperpnoea; FEV 1 , forced expiratory volume in the first second; FVC, forced vital capacity; ICS, inhaled corticosteroids; FE NO , exhaled nitric oxide; PD 20 , provocative dose of methacholine causing a 20% decrease in FEV 1 ;PC 20 , provocative concentration of methacholine causing a 20% decrease in FEV 1 . a Considering the AQUA questionnaire. b According to International Olympic Committee Medical Commission to diagnose asthma in athletes. DOI: 10.3109/02770903.2015.1067321 Phenotypes of asthma in elite athletes 3 Downloaded by [213.58.139.35] at 07:16 21 September 2015 classes (4% of athletes for both diseases). Male gender was predominant in ‘‘sports asthma’’. Regarding therapeutic, 92.5% of those athletes with ‘‘atopic asthma’’ and 78% of those with ‘‘sports asthma’’ were under anti-asthmatic drugs. Thirteen asthmatic athletes were using only short-acting b2-agonists as therapeutic – 9 (8%) among the ‘‘atopic asthma’’ and 4 (8%) among the ‘‘sports asthma’’ phenotype; the remaining athletes were on inhaled corticosteroids (ICS) alone or combined with longacting b2-agonists. Risk factors for each class A 2.87 (95%CI: 1.82–4.51) and 8.65 (95%CI: 2.67–28.03) fold increase for risk of ‘‘sports asthma’’ was observed in athletes practicing water sports and winter sports, respectively, when compared to other sports (Figure 2). Discussion Using LCA, this present study identifies two distinct phenotypes of asthma in athletes: ‘‘atopic asthma’’ defined by the occurrence of atopy, increased levels of FE NO , rhinitis and other allergic co-morbidities; and ‘‘sports asthma’’, defined by the presence of exercise-induced respiratory symptoms and BHR in the absence of allergic features. Moreover, specific training and environmental conditions are associated with an increased risk of developing ‘‘sports asthma’’, as athletes practicing water and winter sports had, respectively, a threeand ninefold increase in their risk of ‘‘sports asthma’’, when compared with others. This study allows for hypothesis generation and has several strengths. Its major strength is the new type of statistical models used to pool and characterize different clusters. This methodological approach makes this study especially useful by retrieving a clear view on asthma phenotypes in athletes. Replication of results in other datasets is important when using these exploratory statistical techniques; and the two asthma patterns obtained in this study are remarkably in accordance with the only previous report, a study relying on different study design and an a priori list of selected variables for statistical analysis [8]. Another strength of the present study is its multicentric nature, allowing the inclusion of a large sample of elite athletes, all competing at top levels, some of which are among the world’s best in their discipline with several winners of Olympic Gold medals. Athletes in this study are all competing in an elite level and, therefore, all are more prone to negative consequences of exercise ‘‘injuring’’ airways due to prolonged and repeated Table 2. Features of athletes screened at Portuguese National Anti-Doping Organization and at Norwegian School of Sports Sciences databases. Asthmatic athletes (n¼150) Non-asthmatic athletes (n¼174) p Male, n(%) 107 (71) 89 (51) 50.001 e Age, years 25 (14–40) 26 (16 – 38) 0.251 d BMI, kg/m 2 23 [23;24] 23 [22;23] 0.06 c Physician reported rhinitis, n(%) 54 (36) 33 (19) 0.003 e Other allergic disease, n(%) 20 (13) 26 (15) 0.750 e Atopy, n(%) 89 (59) 58 (33) 50.001 e Respiratory symptoms, n(%)* 138 (92) 89 (51) 50.001 e Dyspnea/heavy breathing 48 (32) 20 (11) 50.001 e Chest tightness 12 (8) 11 (6) 0.379 e Wheezing 42 (28) 15 (9) 50.001 e Cough 44 (29) 33 (19) 0.002 e Tiredness 1 (0.7) 1 (0.6) 0.427 f Phlegm 18 (12) 15 (9) 50.001 e Asthma treatment, n(%) 50.001 f Inhaled steroids alone 9 (6) 1 (0.6) Beta-2-agonists alone 13 (9) 2 (1) Inhaled steroids + b2-agonists 96 (64) 13 (8) Airway obstruction a ,n(%) 43 (29) 21 (12) 50.001 e FVC L 5.4 [5.1;5.7] 5.2 [5.0;5.4] 0.41 c % of predicted 114 [110;117] 112 [109;116] 0.60 c FEV 1 L 4.1 [3.9;4.4] 4.3 [4.1;4.4] 0.06 c % of predicted 101 [96;106] 109 [106;111] 0.001 c FEV 1 /FVC 69 [65;74] 76 [72;80] 0.012 c Reversibility b ,n(%) 26 (17) 1 (0.6) 0.037 f Airway hyperesponsiveness, n(%) 126 (84) 51 (29) 50.001 e FE NO , ppb 33 (6–213) 19 (4–70) 0.01 d Bold values indicate p50.05. Data presented as mean (95% confidence interval) except for age and FE NO which are presented as median (min–max). BMI, body mass index; FE NO , exhaled fraction of nitric oxide; L, liters; FVC, forced vital capacity; FEV 1 , forced expiratory volume in one second. a Defined as a FEV 1 /FVC ratio 50.70. b Defined as an increase in FEV 1 200 mL and 12%. c Independent samples t-test. d Independent samples Mann–Whitney Utest. e Chi-square test. f Fisher’s exact test. 4M. Couto et al. J Asthma, Early Online: 1–8 Downloaded by [213.58.139.35] at 07:16 21 September 2015 hyperpnoea. For athletes practicing water and winter sports, in addition to frequent episodes of prolonged hyperpnoea, their ‘‘occupation’’ demands exposure to potentially noxious stimuli, such as sport-specific environmental exposures [16]. Keeping in mind the close relation to environmental conditions, one could speculate whether ‘‘sports asthma’’ should be classified as a variant of occupational asthma, as recently suggested [16]. This designation could help improve the general idea of this concept of asthma dependent upon environmental factors which are part of an athlete’s occupation. The ‘‘sports asthma’’ phenotype is similar to the lateonset phenotype identified among ‘‘normal’’ asthmatics. In many cases, the late-onset phenotype appears to be more severe, less responsive to standard therapy and more related to environmental risk factors [17]. However, ‘‘sports asthma’’ tends to improve after cessation of sport participation, in what concerns airway inflammation and hyperesponsiveness [18,19]. In athletes, atopy has been long recognized to be positively associated with asthma and BHR [20,21]. Moreover, training in cold air [21] and swimming [20] were identified as risk factors for asthma. In both swimmers and cross-country skiers, the prevalence of asthma is known to increase with age [22–24], which is consistent with the hypothesis of ‘‘sports asthma’’ occurring throughout the sport career and being induced by cumulative years of exposure to environmental training conditions. The results of our study contribute to confirm that different risk factors, such as atopy and environmental training conditions, result in different patterns of asthma. The effect of these risk factors on determining different underlying mechanisms of asthma should be considered. Table 3. Characteristics of asthmatic athletes according with their asthma phenotype and variables in each assigned latent class. Total Atopic asthma, n¼104 Sports asthma, n¼46 p Male, n(%) 107 81 (78) 26 (57) 0.008 e Age, median ± IQR in years – 23.0 ± 12 24.5 ± 8 0.522 f Height, mean ± SD in cm – 175.4 ± 8.7 176.5 ± 8.7 0.530 g Weight, mean ± SD in kg – 70.9 ± 11.5 71.4 ± 10.3 0.815 g BMI, mean ± SD in kg/m 2 – 23.0 ± 2.6 22.8 ± 1.9 0.741 g FEV 1 , mean ± SD in L – 4.0 ± 0.9 4.1 ± 0.7 0.221 g FEV 1 , mean ± SD in % predicted – 98.1 ± 20.4 99.7 ± 21.1 0.640 g FVC, mean ± SD in L – 5.1 ± 1.0 5.3 ± 1.1 0.413 g FVC, mean ± SD in % predicted – 108.0 ± 15.4 113.4 ± 15.0 0.084 g FEV 1 /FVC, mean ± SD – 77.9 ± 8.9 78.7 ± 11.1 0.649 g Variables used in LCA Airflow obstruction a 0.036 No 80.5 85.3 69.4 Yes 19.5 14.7 30.6 Reversibility b 0.023 No 23.4 19.0 39.7 Yes 76.6 81.0 60.3 Rhinitis 50.001 No 64.0 51.5 90.9 Yes 36.0 48.5 9.1 Any other allergic disease c 50.001 No 61.5 39.3 87.5 Yes 38.5 60.7 12.5 Respiratory symptoms 0.133 No 6.1 4.0 10.7 Yes 93.9 96.0 89.3 Asthma treatment 0.017 No 11.9 7.5 22.0 Yes 88.1 92.5 78.0 Airway hyperesponsiveness 0.834 No 25.7 25.0 26.9 Yes 74.3 75.0 73.1 FEd NO 50.001 Normal 62.8 44.8 84.5 Increased 37.2 55.2 15.5 Atopy 50.001 No 31.0 0 100 Yes 69.0 100 0 Bold values indicate p50.05. Data presented as percentage of total, except otherwise stated. BMI, body mass index; FE NO , exhaled fraction of nitric oxide; L, liters; FVC, forced vital capacity; FEV 1 , forced expiratory volume in one second. a Defined as a FEV 1 /FVC ratio 50.70. b Defined as an increase in FEV1 200 mL and 12%. c Other allergic diseases include conjunctivitis, urticaria, eczema, anaphylaxis and drug, food and venom allergies, sampled through AQUA questionnaire. d Defined as increased if above 25 ppb. +Chi-square test. f Mann–Whitney Utest. DOI: 10.3109/02770903.2015.1067321 Phenotypes of asthma in elite athletes 5 Downloaded by [213.58.139.35] at 07:16 21 September 2015 Defining these distinct phenotypes could lead not only to further understanding the underlying mechanisms of asthma in elite athletes, but also, and most important from a practical point of view, to recognizing that potentially different treatments specifically targeted for defined phenotypic groups are needed. Optimal asthma treatment is a prerequisite for asthmatic athletes because of potential implications in performance, since airway narrowing during exercise could compromise ventilatory capacity and efficiency. However, it has been recognized that the natural course of asthma in athletes is difficult to change by ‘‘normal’’ anti-inflammatory treatment [25]. This highlights the need for a different therapeutic approach in these subjects, which leads us to the clinical implications of our study. Differences in airway response to bronchodilating drugs have been reported in the literature, and whether athletes with asthma occurring during sports career respond to anti-asthmatic drugs similarly to subjects with classic allergic or with nonallergic asthma has not been extensively studied [7] and needs further research. Most recent guidelines for treatment of exercise-induced bronchoconstriction (EIB) state a strong recommendation for using a short-acting b2-agonist before exercise in all patients with EIB [26]. However, we have recently shown that elite skiers with asthma respond better to anticholinergic treatment as compared with b2-agonists [27]. Differences in parasympathetic bronchial tone were suggested as a possible explanation to why some subjects are responders and other nonresponders to anticholinergic drugs [28,29]. It seems, therefore, that the approach of ‘‘one treatment fits all’’ is insufficient to comply with the needs of asthmatic athletes. Despite its several strengths, our study also has some limitations that must be pointed out. The first is the use of different methods (both direct and indirect challenges) to assess BHR in athletes. In the present study, information was collected from medical files, so there was no possibility to homogenize tests performed by athletes in two centers. In any case, final diagnosis was made according to IOC criteria. Another weakness to be noted is the absence of information about age of asthma onset; this limits the extent of our conclusions as we cannot be aware of whether the previous presence of asthma would influence the type of sport chosen. However, based on previous literature, it does not seem to be Figure 1. Percent of athletes presenting each of the variables included for LCA. Figure 2. Risk of presenting the ‘‘sports asthma’’ phenotype of athletes practicing water and winter sports, considering other sports as reference. 6M. Couto et al. J Asthma, Early Online: 1–8 Downloaded by [213.58.139.35] at 07:16 21 September 2015 the case as the prevalence of asthma is known to increase with age both in swimmers and skiers [22–24]. The interpretation of our results is also limited by the cross-sectional design, which is not able to identify causality; however, it is suitable for hypothesis generation. Thus, the present study should be succeeded by new prospective studies following youth athletes from adolescence until adulthood. Moreover, although motivating, results provided by this exploratory analysis have to be interpreted in context of future work, addressing whether the two phenotypes are relevant from a clinical perspective. Potential phenotypes require prospective validation with clinical interventional trials. A recent trial showed that Norwegian competitive endurance winter athletes respond with a higher reversibility to ipratropium bromide than to inhaled b2-agonists [27], helping research in this field to move forward and toward a new direction. Conclusion Using LCA on a large sample of top elite athletes from two national databases we were able to identify two patterns of asthma aggregation features based on findings routinely collected in clinical practice: ‘‘atopic asthma’’, defined by the presence of allergic sensitization, rhinitis and other allergic co-morbidities and increased FE NO ; and ‘‘sports asthma’’, defined by the presence of exercise-induced respiratory symptoms and BHR in the absence of allergic features. Moreover, exposure to particular environmental conditions of training and competition was associated with increased risk to develop ‘‘sports asthma’’ phenotype: water sports increased the risk by almost three times, whereas in winter sports the risk increased by almost nine times. Recognizing different phenotypes as a result of probable different underlying mechanisms related to environmental exposures highlights the need for distinct targeted treatments. These potential phenotypes require prospective validation by larger clinical interventional trials. If confirmed by other studies, such a model could be useful for the standardization of clinical diagnosis and future treatment of asthmatic athletes. Acknowledgements We thank to Hugo Martins, for his contribution in image designing of Figure 1. Declaration of interest The authors report no conflicts of interest. To European Academy of Allergy and Clinical Immunology for the 2011 Exchange Research Fellowship award allowing the first author to work in Oslo and therefore turned this project possible. References 1. Eichenberger PA, Diener SN, Kofmehl R, Spengler CM. Effects of exercise training on airway hyperreactivity in asthma: a systematic review and meta-analysis. Sports Med 2013;43:1157–1170. 2. Avallone KM, McLeish AC. Asthma and aerobic exercise: a review of the empirical literature. J Asthma 2013;50:109–116. 3. Fitch K. 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Increased bronchial parasympathetic tone in elite cross-country and biathlon skiers: a randomised crossover study. Br J Sports Med 2015;49:56–61. 28. Kno ¨pfli BH, Bar-Or O, Arau ´jo CGS. Effect of Ipratropium Bromide on EIB in children depends on vagal activity. Med Sci Sports Exerc 2005;37:354–359. 29. Kno ¨pfli BH, Luke-Zeitoun M, von Duvillard SP, Burki A, Bachlechner C, Keller H. High incidence of exercise-induced bronchoconstriction in triathletes of the Swiss national team. Br J Sports Med 2007;41:486–491. Supplementary material available online 8M. Couto et al. J Asthma, Early Online: 1–8 Downloaded by [213.58.139.35] at 07:16 21 September 2015 Study III Exploratory study comparing dysautonomy between asthmatic and nonasthmatic elite swimmers Rev Port Pneumol. 2015;21(1):22-9 Rev Port Pneumol. 2015;21(1):22---29 www.revportpneumol.org ORIGINAL ARTICLE Exploratory study comparing dysautonomia between asthmatic and non-asthmatic elite swimmers M. Coutoa,b,c,d,∗, D. Silvaa,b, P. Santosa, S. Queirósa, L. Delgadoa,b,c, A. Moreiraa,b,c aLaboratory of Immunology, Basic and Clinical Immunology Unit, Faculty of Medicine, University of Porto, Portugal bImmunoallergology Department, Centro Hospitalar São João EPE, Porto, Portugal cCenter for Research in Health Technologies and Information Systems (CINTESIS), Portugal dInstituto CUF & Hospital CUF Porto, Allergy Unit, Portugal Received 6 March 2014; accepted 14 May 2014 Available online 17 January 2015 KEYWORDS Airway hyperesponsiveness; Autonomic nervous system; Dysautonomia; Exercise-induced asthma; Parasympathetic activity; Swimmers Abstract Background: Dysautonomia has been independently associated with training and exerciseinduced bronchoconstriction. In addition, neurogenic airway inflammation was recently associated with swimmers-asthma. We aimed to assess the relation between autonomic nervous system and airway responsiveness of asthmatic elite swimmers. Methods: Twenty-seven elite swimmers, 11 of whom had asthma, were enrolled in this exploratory cross-sectional study. All performed spirometry with bronchodilation, skin prick tests and methacholine challenge according to the guidelines. Pupillometry was performed using PLR-200TM Pupillometer. One pupil light response curve for each eye was recorded and the mean values of pupil’s maximal and minimal diameters, percentage of constriction, average and maximum constriction velocities (parasympathetic parameters), dilation velocity, and total time to recover 75% of the initial size (sympathetic parameters) were used for analysis. Asthma was defined using IOC-MC criteria; subjects were divided into airway hyperesponsiveness (AHR) severity according to methacholine PD20 in: no AHR, borderline, mild, moderate and severe AHR. Differences for pupillary parameters between groups and after categorization by AHR severity were assessed using SPSS 20.0 (p ≤ 0.05). In individuals with clinically relevant AHR, correlation between PD20 and pupillary parameters was investigated with Spearman’s correlation test. Results: No statistically significant differences were observed between asthmatic and nonasthmatic swimmers regarding parasympathetic parameters. When stratified by AHR, maximal and minimal diameters and percentage of constriction were significantly lower among those with severe AHR. Among swimmers with clinically relevant AHR (n = 18), PD20 correlated with parasympathetic activity: maximal (r = 0.67, p = 0.002) and minimal diameters (r = 0.75, p < 0.001), percentage of constriction (r = −0.59, p = 0.011) and latency (r = 0.490, p = 0.039). ∗Corresponding author. E-mail address: [email protected] (M. Couto). http://dx.doi.org/10.1016/j.rppnen.2014.05.004 2173-5115/© 2014 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved. Document downloaded from http://www.elsevier.pt, day 19/04/2015. This copy is for personal use. Any transmission of this document by any media or format is strictly prohibited. Exploratory study comparing dysautonomia between asthmatic and non-asthmatic elite swimmers 29 of Allergy and Clinical Immunology (EAACI) in cooperation with GA2LEN. Allergy. 2008;63:387---403. 5. Capão-Filipe J, Falcão-Reis F, Castro-Correia J, Barros H. Assessment of autonomic function in high level athletes by pupillometry. Auton Neurosci. 2003;104:66---72. 6. Carlsen KH, Oseid S, Odden H, Mellbye E. The response to heavy swimming exercise in children with and without bronchial asthma. In: Oseid S, Carlsen KH, editors. Children and exercise XIII. Champaign, IL: Human Kinetics Publishers Inc.; 1989. p. 351---60. 7. Hox V, Vanoirbeek JA, Alpizar YA, Voedisch S, Callebaut I, Bobic S, et al. Crucial role of transient receptor potential ankyrin 1 and mast cells in induction of nonallergic airway hyperreactivity in mice. Am J Respir Crit Care Med. 2013;187:486---93. 8. Fountas KN, Kapsalaki EZ, Machinis TG, Boev AN, Robinson JS, Troup EC. Clinical implications of quantitative infrared pupillometry in neurosurgical patients. Neurocrit Care. 2006;5:55---60. 9. Medical Commission of the International Olympic, Committee. IOC’s medical, code. Lausanne: International Olympic Committee; 2002. 10. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. Eur Respir J. 2005;26:319---38. 11. Stanojevic S, Wade A, Stocks J, Hankinson J, Coates AL, Pan H, et al. Reference ranges for spirometry across all ages: a new approach. Am J Respir Crit Care Med. 2008;177:253---60. 12. Pellegrino R, Viegi G, Brusasco V, Crapo RO, Burgos F, Casaburi R, et al. Interpretative strategies for lung function tests. Eur Respir J. 2005;26:948---68. 13. Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, et al. Guidelines for methacholine and exercise challenge testing-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors. Am J Respir Crit Care Med. 2000;161:309---29. 14. Pichon A, Bisschop C, Diaz V, Denjean A. Parasympathetic airway response and heart rate variability before and at the end of methacholine challenge. Chest. 2005;127:23---9. 15. Park C, Stafford C, Lockette W. Exercise-induced asthma may be associated with diminished sweat secretion rates in humans. Chest. 2008;134:552---8. 16. Holgate S. Pathogenesis of asthma. Clin Exp Allergy. 2008;38:872---97. 17. Holgate S, Lemanske R Jr, O’Byrne P, Kakumanu S, Busse W. Asthma pathogenesis. In: Adkinson, editor. Middleton’s allergy: principles and practice. 7th ed. Philadelphia: Mosby; 2008. p. 893---919. 18. Haahtela T, Malmberg P, Moreira A. Mechanisms of asthma in Olympic athletes --- practical implications. Allergy. 2008;63:685---94. 19. Bougault V, Boulet LP. Airway dysfunction in swimmers. Br J Sports Med. 2012;46:402---6. 20. Bernard A. Chlorination products: emerging links with allergic diseases. Curr Med Chem. 2007;14:1771---82. 21. Voisin C, Sardella A, Marcucci F, Bernard A. Infant swimming in chlorinated pools and the risks of bronchiolitis, asthma and allergy. Eur Respir J. 2010;36:41---7. 22. Bernard A, Carbonnelle S, Michel O, Higuet S, de Burbure C, Buchet J-P, Hermans C, Dumont X, Doyle I. Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools. Occup Environ Med. 2003;60:385---94. 23. Moreira A, Palmares C, Lopes C, Delgado L. Airway vascular damage in elite swimmers. Respir Med. 2011;105:1761---5. 24. Couto M, Andrade P, Pereira M, Araújo J, Moreira P, Delgado L, Moreira A. Effect of competitive swimming on airway inflammation: a 3-yr longitudinal study. Pediatr Allergy Immunol. 2014;25:193---5. 25. Bessac B, Jordt S. Breathtaking TRP channels: TRPA1 and TRPV1 in airway chemosensation and reflex control. Physiology. 2008;23:360---70. 26. Butler C, Heaney L. Neurogenic inflammation and asthma. Inflamm Allergy Drug Targets. 2007;6:127---32. 27. Banner K, Igney F, Poll C. TRP channels: emerging targets for respiratory disease. Pharmacol Ther. 2011;130:371---84. 28. Barnes PJ. Neurogenic inflammation in airways. Int Arch Allergy Appl Immunol. 1991;94:303---9. 29. Carlsen K-H. The breathless adolescent asthmatic athlete. Eur Resp J. 2011;38:713---20. 30. Capão-Filipe M, Delgado JL, Rodrigues J, Vaz-Azevedo M. Exercise-induced respiratory symptoms in the elite athlete: evidence of a new syndrome. Allergy. 1998;53(s43):60. Document downloaded from http://www.elsevier.pt, day 19/04/2015. This copy is for personal use. Any transmission of this document by any media or format is strictly prohibited. Study IV Increased bronchial parasympathetic tone in elite cross-country and biathlon skiers: a randomised crossover study Br J Sports Med. 2015;49(1):56-61 Increased bronchial parasympathetic tone in elite cross-country and biathlon skiers: a randomised crossover study J Stang, 1 M Couto, 2,3 K-H Carlsen, 1,4,5 T Stensrud 1 For numbered affiliations see end of article. Correspondence to Julie Stang, Norwegian School of Sport Sciences, Sognsveien 220, P.O. Box 4014 Ullevål Stadion, Oslo NO-0806, Norway; julie.s[email protected] Accepted 5 November 2014 Published Online First 21 November 2014 To cite: Stang J, Couto M, Carlsen K-H, et al.Br J Sports Med 2015;49:56–61. ABSTRACT Background Increased parasympathetic activity in endurance-trained athletes has been reported by heart rate variability and pupillometry. Our primary objective was to assess parasympathetic activity and tone in the lower respiratory tract by investigating the effect of cholinergic antagonism by inhaled ipratropium bromide compared to the β 2 -receptor stimulating effect of inhaled salbutamol in elite cross-country and biathlon skiers. We also examined the medications’relationship to cholinergic sensitivity as measured by bronchial responsiveness to methacholine (PD 20met ). Methods In a randomised crossover study, 20 crosscountry and two biathlon skiers (14♂/8♀) aged 20–37 years from the Norwegian national teams measured reversibility to inhaled ipratropium bromide and inhaled salbutamol and PD 20met on three separate days. A positive reversibility test was defined as an increase in forced expiratory volume in 1 s (FEV 1 )of ≥12%. Spirometry was performed before and 45 and 15 min after inhaled ipratropium bromide and inhaled salbutamol, respectively. Bronchodilating medication was withheld according to the European Respiratory Society (ERS) guidelines. Correlations were assessed by Pearson’s correlation coefficient (r). Results Five athletes had significant reversibility after inhaled ipratropium bromide, and none after inhaled salbutamol. Twelve athletes (54.5%) had PD 20met <8 μmol (1.57 mg). PD 20met correlated negatively with ▵FEV 1 after inhaled ipratropium bromide (r=−0.85, p<0.0001), but not after inhaled salbutamol (r=−0.308, p=0.16). Conclusions In elite skiers, cholinergic sensitivity (PD 20met ) had a highly significant inverse correlation to the cholinergic antagonism of inhaled ipratropium bromide, but not at all to the bronchodilating inhaled salbutamol. This markedly increased bronchial parasympathetic tone may represent an important cholinergic role in the development of skier’s asthma. INTRODUCTION Elite endurance athletes have an increased risk of developing bronchial hyper-responsiveness (BHR) and exercise-induced asthma (EIA). 1 Reports show that cross-country and biathlon skiers, who train and compete in cold air, are among those athletes with the highest prevalence of self-reported respiratory symptoms and objectively measured BHR. 2–4 However, the mechanisms of these pathophysiological airway changes in athletes are currently not completely understood. Since the bronchoconstrictor effect of a variety of cholinergic stimulants in the airways was abolished after blocking cholinergic efferent pathways with an intravenous injection of atropine sulfate, increased cholinergic reflexes have long been proposed to contribute to BHR. 5 Cooling of the airways during exercise may cause exercise-induced bronchoconstriction (EIB) through an increase in vagal efferent tone. 6 Knöpfli and coworkers found an association between changes in heart rate variability (HRV) at the onset of exercise, thought to reflect the effect of parasympathetic stimulation on the chronotropic activity of the heart, and the bronchodilating effect of inhaled ipratropium bromide, blocking efferent cholinergic pathways in the airways, in endurance-trained cross-country runners exercising at −5°C, 7 and in children with EIB. 8 Parasympathetic activity is higher in endurancetrained athletes compared to non-athletes as assessed by HRV 7 and pupillometry. 910 Participants with BHR to methacholine (PD 20met ) had an increased HRV,reflecting a higher parasympathetic tone after a methacholine bronchial challenge, as compared to participants without BHR. 11 However, the relationship between HRV and BHR was weak, and the increased parasympathetic tone could not alone explain the increased prevalence of asthma and BHR among athletes. 12 13 Nevertheless, the heart is continuously under sympathetic and parasympathetic influence, and does not necessarily reflect a parasympathetic tone in the bronchi. Horvath et al 14 assessed airway resistance and heart rate period (inter-beat interval, milliseconds) after cholinergic blockade by atropine sulfate administration and found that vagal control of bronchial tone and heart rate were not related in resting healthy participants. 14 The primary objective of the present study was to assess a possible relationship between increased bronchial responsiveness to methacholine, reflecting the sensitivity of a cholinergic stimulation on the bronchial smooth muscle and mucous glands, and the bronchodilating effect of inhaled ipratropium bromide, blocking the effect of increased parasympathetic (cholinergic) tone on the bronchial smooth muscle in top endurance-trained elite competitive cross-country skiers. Second, we aimed to compare the bronchodilating effect of inhaled ipratropium bromide blocking parasympathetic tone with that of inhaled salbutamol as a general bronchodilating agent not affecting cholinergic receptors in this group of athletes. METHODS Subjects and design The Norwegian National team of cross-country skiing, including 20 cross-country skiers, as well as Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 1of7 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from two biathlon skiers (14♂/8♀), aged 20–37 years, who were present on a training camp in Val Senales, Italy, were included in a randomised crossover study. All participants were elite athletes competing at the top international level. All were non-smokers and did not consume snuff. Subject characteristics are described in table 1. All athletes had been free from any respiratory disease for the past 3 weeks before the first study day, and refrained from exercise and any food or drink containing nitrate on the same day of testing. Antiasthmatic medication was withheld according to the European Respiratory Society (ERS) guidelines. 15 Inhaled short-acting β 2 -agonists were withheld for 8 h before testing; inhaled long-acting β 2 -agonists, theophylline, and leukotriene antagonists were withheld for the past 72 h; antihistamines were withheld for the past 7 days; and orally administered glucocorticosteroids were withheld for the last month. Inhaled corticosteroids were not to be used on the day of testing. The athletes attended one visit at the laboratory at the Norwegian School of Sport Sciences, Oslo, Norway for assessment of lung function, BHR to methacholine (PD 20met ) and fractional exhaled nitric oxide (FE NO ). Prior diagnoses of asthma, allergic rhinitis and EIB, use of asthma medication in the last year and current symptoms of dyspnoea, phlegm and cough during or after exercise were recorded with the AQUA-questionnaire 16 and clinical interview. Two reversibility tests were obtained during a training camp in Val Senales, Italy, 2000 m above sea level. The reversibility tests were performed in a randomised order on two separate days, with 24 h between each test. All tests were performed according to current guidelines from the American Thoracic Society (ATS). 17 Data collection was conducted during September–October 2011. The study was approved by the regional medical ethics committee and carried out according to the principles stated in the Declaration of Helsinki. Signed informed consent was obtained from each participant. Procedures Measurement of lung function was performed by maximum expiratory flow-volume loops using a MasterScreen Pneumo spirometer ( Jaeger GmbH, Würzburg, Germany). The predicted values used are according to Quanjer et al. 18 The following variables were recorded: forced vital capacity (FVC), forced expiratory volume in 1 s (FEV 1 ), and forced expiratory flow at 50% of vital capacity (FEF 50 ). Methacholine bronchial challenge was performed with an inspiration-triggered nebuliser (Aerosol Provocation System, Jaeger, Würzburg, Germany). The nebuliser output was controlled and calibrated before the start of the study and weekly during the study period. After measuring baseline lung function, lung function was measured after inhaling nebulised isotonic saline (0.9%). Methacholine chloride, 32 mg/mL, was inhaled in doubling doses from a starting dose of 0.51 mmol (0.1 mg). Lung function measurements were performed 1 min after every delivered dose until FEV 1 decreased 20% from the measurement after inhaled saline. The maximum cumulative dose was 24.48 mmol (4.8 mg). A positive response to methacholine was defined as a 20% reduction in FEV 1 and the methacholine provocation dose causing a ≥20% decrease in FEV 1 was calculated by linear interpolation on the dose–response curve and recorded as PD 20met . Clinical significant BHR was defined as PD 20met ≤8mmol. After the methacholine provocation, all athletes received salbutamol inhalation (0.1 mg/mL×10 kg/body mass) by nebulisation to reverse bronchial obstruction. Measurement of FE NO was performed before lung function measurement by the Eco Medics CLD 88 sp Exhalyzer (Eco Medics AG, 8635 Duernten, Switzerland). Participants inhaled NO-free air to total lung capacity and exhaled with a standardised flow of 50 mL/s according to the ATS/ERS recommendations. 19 Mean values were used after measurements performed in triplicates. Reversibility tests for inhaled ipratropium bromide and salbutamol were performed using identical procedures on two separate days. Salbutamol (0.1 mg/mL×10 kg/body mass) and ipratropium bromide (0.500 mg/mL), respectively, were mixed in 1 mL isotonic NaCl and delivered through a Sidestream nebulising chamber (Respironics Respiratory Ltd, Chichester, UK) connected to a CR60 compressor (Medic-Aid Ltd, West Sussex, UK) at a flow rate of >6 L/min. Lung function by maximal expiratory flow volume loops was measured, as previously described, before and 15 and 45 min after inhaled salbutamol and inhaled ipratropium bromide, respectively. Clinical significant reversibility was defined as a ≥12% increase in FEV 1 from before to after inhalation. Statistical analysis Demographic data and results are expressed as mean values with 95% CIs, unless otherwise stated. Correlations were assessed by Pearson’s correlation coefficient (r p ) for normally distributed data on log 10 -transformed values of PD 20met (log 10 PD 20met ). To achieve a high-grade correlation of more than or equal to 0.7 with a power of 80%, 13 participants were calculated to be required based on previous measurements in top athletes. Differences between two measurements were analysed by Student t tests and differences in categorical data were analysed by χ 2 and Fisher’s exact tests. Statistical analyses were performed with the Statistical Package for Social Sciences (SPSS, V.21.0; Chicago, Illinois, USA) and MedCalc Statistical System (V.10.4.6.0, Mariakerke, Belgium). A p value less than or equal to 0.05 was considered statistically significant. RESULTS Subject characteristics The characteristics of the participants included in this study are presented in table 1. Sixteen athletes (73%) had a doctor’s diagnosis of asthma. Seven (32%) reported they had doctordiagnosed allergic rhinitis. Lung function was within normal range in all athletes (table 1). Reversibility and BHR Mean ▵FEV 1 after inhaled salbutamol was 4.3% (±3.8 (mean ±SD)) or 185.9 mL (±(SD)166.9), and mean ▵FEV 1 after Table 1 Subject characteristics with lung function (FEV 1 , FVC, FEF 50 ) and fractional exhaled nitric oxide (FE NO ) Mean (range, ±SD) Percentage of predicted values (range, ±SD) Height (cm) 175.5 (162.0–190.0, ±8.8) NA Weight (kg) 69.7 (52.0–85.0, ±10.5) NA Age (years) 26 (20–37, ±4.7) NA FEV 1 (L) 4.3 (3.0–5.5, ±0.7) 103 (91–130, ±9.5) FVC (L) 5.5 (3.6–7.3, ±1.2) 112 (93–131, ±11.0) FEF 50 (L/s) 4.5 (2.5–8.6, ±1.3) 86 (51–159, ±27.4) FE NO (ppb) 24.7 (9.0–113.0, ±25.6) NA FEV 1 , forced expiratory volume in 1 s; FVC, forced vital capacity; FEF 50 , forced expiratory flow at 50% of vital capacity; FE NO , fractional exhaled nitric oxide; NA, not applicable. 2 of 7 Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from inhalation of ipratropium bromide was 7.8% (±(SD)6.9) or 330.5 mL (±(SD)289.8). A significant difference between ▵FEV1 after inhaled ipratropium bromide and inhaled salbutamol of 3.4% (0.55, 6.31) was observed (p=0.002). Five athletes (4♂) had a positive reversibility to inhaled ipratropium bromide (▵FEV 1 ≥12%), and none had a positive reversibility to inhaled salbutamol (figure 1A). Mean PD 20met was 8.14 (4.86 to 13.64) (geometric mean with 95% CI). Twelve athletes (8♂) had PD 20met ≤8μmol (1.57 mg; figure 2) and six athletes (5♂) had PD 20met ≤4μmol (0.78 mg). Correlations Log 10 PD 20met correlated significantly with ▵FEV 1 after inhalation of ipratropium bromide (r=−0.85, p<0.0001; figure 1A), but not with ▵FEV 1 after inhalation of salbutamol (r=−0.308, p=0.16; figure 1B). Similarly, the mean ▵FEF 50 after inhaled ipratropium bromide of 29.2% (±(SD)20.2) was negatively associated with log 10 PD 20met (p=0.017). Mean ▵FEF 50 after inhaled salbutamol was 18.3% (±16.2), and no significant relationship with log 10 PD 20met was found. Exhaled nitric oxide Mean FE NO was increased (>20 ppb) and ranged from 9 to 113 ppb (table 1). FE NO was ≥30 ppb in five athletes (3♂), of whom four had doctor-diagnosed asthma and respiratory symptoms and used asthma medication, and one had allergic rhinitis and used antihistamines regularly. There was no difference in FE NO between athletes with allergic rhinitis (mean 26.1 ppb (3.7–48.6), n=7) and non-allergic athletes (mean 25.8 ppb (10.9–40.8), n=15). We observed no association between FE NO and PD 20met . Use of medication Seventeen athletes (77%) used inhaled corticosteroids and four (18%) used antihistamines regularly (table 2). One athlete had doctor-diagnosed allergic rhinitis and used antihistamines, inhaled corticosteroids and ipratropium bromide, but did not have doctordiagnosed asthma. All athletes with BHR (PD 20met ≤8) used inhaled corticosteroids (n=12). There were 10 athletes who had a PD 20met >8 mmol, and of these 50% used inhaled corticosteroids. Athletes with inhaled corticosteroids had PD 20met : 6.08 (3.36 to 10.99) (geometric mean with 95% CI); those without: 22.05 (12.82 to 37.92) (geometric mean with 95% CI) (p=0.025). Athletes who used inhaled corticosteroids had greater bronchodilator response to inhaled ipratropium bromide: 109.9% (106.4% to 113.3%), as compared to the athletes who did not use corticosteroids: 101.8% (98.8% to 104.7%; p=0.01). No differences were observed either in bronchodilator effects after inhaled salbutamol in athletes who used short-acting or long-acting β 2 -agonists, or in inhaled corticosteroids, respectively, as compared to athletes who did not use these medications. All athletes with positive reversibility to ipratropium bromide had PD 20met ≤8μmol (figure 1A) and doctor-diagnosed asthma and used asthma medication. Of all who used inhaled corticosteroids, four of five used short-acting β 2 -agonists and ipratropium bromide, and two of five used long-acting β 2 -agonists. Two of the athletes had doctor diagnosed allergic rhinitis, of whom one used antihistamines regularly. Presence of respiratory symptoms Nineteen athletes reported that they had respiratory symptoms during or after exercise, and three athletes were nonsymptomatic (figure 3). Cough (73%) was the most prevalent self-reported symptom, followed by phlegm (68%) and dyspnoea (41%). BHR (PD 20met ≤8μmol) was more prevalent in athletes with respiratory symptoms than in non-symptomatic athletes (figure 3). Self-reported symptoms were not associated with FE NO or ▵FEV 1 after inhalation of neither ipratropium bromide nor salbutamol. Figure 1 Relationship between the methacholine dose causing a 20% reduction in forced expiratory volume in 1 s (FEV 1 ) (PD 20met ) and (A) ▵FEV 1 (%) 45 min after inhaled ipratropium bromide and (B) ▵FEV 1 (%) 15 min after inhaled salbutamol, in elite cross-country and biathlon skiers. Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 3 of 7 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from DISCUSSION In this study, 54% of the athletes (national team cross-country and biathlon skiers) had increased BHR (PD 20met ≤8mmol), 23% had significant reversibility to inhaled ipratropium bromide (▵FEV 1 ≥12%) and none to inhaled salbutamol. The main finding of this study was a highly significant correlation between BHR (log 10 PD 20met ) and reversibility to inhaled ipratropium bromide (r=−0.85, p<0.0001), but not so between BHR and reversibility to salbutamol (r=−0.31, p=0.16). Role of the parasympathetic system in asthma symptoms BHR to inhaled methacholine may be regarded as increased sensitivity to a cholinergic stimulus, whereas increased reversibility to inhaled ipratropium bromide, which blocks acetylcholine, can be regarded as a sign of increased bronchial cholinergic (parasympathetic) tone. The high correlation between the two (increased sensitivity to cholinergic stimulation and increased cholinergic tonus), together with the bronchodilator response to the ipratropium bromide observed, suggests an important role of the parasympathetic nervous system in causing bronchial obstruction and asthma symptoms in these elite endurance trained athletes of winter sports. The significant high-grade negative correlation between a cholinergic bronchoconstrictor and an anticholinergic bronchodilator stimulus, and lack of correlation with a β 2 -stimulating bronchodilator, suggest an important role of an increased parasympathetic tone and sensitivity in the development of BHR and asthma in these top endurance-trained athletes. Increased parasympathetic activity has previously been reported in endurance-trained athletes measured both by the parasympathetic activity of the eye by pupillometry in long distance runners 910 and of the cardiovascular system by the variation in the heart rate induced by an exercise test. 7 These findings are supported by this study in cross-country skiers who, in addition to the endurance training, are also exposed to cold air during their training and competitions, an exposure previously reported to cause parasympathetic stimulation of the airways and contribute to EIB. 6 Furthermore, Goldsmith et al 20 showed a correlation of r=0.75 (p=0.0001) between HRV indices reflecting parasympathetic activity and maximal oxygen uptake (VO 2max ), which suggests that endurance training and increased aerobic capacity are followed by increased parasympathetic activity. In addition, Park et al 21 found a relationship between PD 20met and diminished sweat secretion, and between tearing rate and salivary flow rate, indicating autonomic dysfunction, in healthy athletes suspected of having EIB. These findings indicate that exercise-induced alterations of the parasympathetic branch influence bronchial tone, and may be involved in the development of BHR and asthma in susceptible elite athletes. 22 23 Figure 2 Distribution of the degree of bronchial hyper-responsiveness (BHR), measured as the methacholine dose (0.05–30 μmol) causing a 20% reduction in forced expiratory volume in 1 s (FEV 1 ) (PD 20met ), in elite Norwegian cross-country and biathlon skiers. A PD 20met less than 2 μmol is considered as severe BHR; greater than 2 μmol and less than 8 μmol is mild to moderate BHR; and greater than 8 μmol and less than 16 μmol is borderline BHR. Table 2 Prevalence of doctor-diagnosed asthma and allergic rhinitis, bronchial hyper-responsiveness (BHR), defined as a methacholine dose causing a 20% reduction in forced expiratory volume in 1 s (FEV 1 )of≤8μmol (PD 20met ), and use of medications in elite Norwegian cross-country and biathlon skiers n Total (%) Asthma 16 72.7 Allergic rhinitis 7 31.8 BHR 12 54.4 Use of medication Short-acting β 2 -agonists 8 36.4 Long-acting β 2 -agonists 10 45.5 Inhaled corticosteroids 17 77.3 Ipratropium bromide 15 68.2 Leukotriene antagonists 0 0 Antihistamines 4 18.2 4 of 7 Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from What influences the prevalence of bronchial hyper-reactivity? The prevalence of BHR and asthma is reported to be higher in endurance athletes than in strength and power athletes and nonathletes. 24 The high prevalence of asthma in endurance athletes is reportedly related to the repeated daily training activity with high ventilation rates resulting in epithelial damage of the airways, delayed repair due to the daily repetition of the training and increased airway mucosal inflammation. 22 25 In addition, the ambient training environments may contribute to the high prevalence of EIB in endurance athletes through increased exposure with high ventilation rates during exercise. This includes swimmers, triathletes, cyclists and speed skaters. 26–29 Cross-country skiing is one of the most demanding sports in regard to the aerobic metabolic system. These are highly conditioned athletes with mean peak oxygen consumption (VO 2peak ) levels of more than 6.0 L/min or approximately 80 mL/kg/min for males. 30 The athletes included in this study are among the world’s best in their discipline with several winners of Olympic Gold medals and of the World Cup in cross-country skiing. Mechanisms—Why does this occur? Cold and dry air will influence participants with EIA through increased respiratory heat and water loss during exercise, 31 and exposure to cold air combined with strenuous exercise is shown to be associated with the development of airway inflammation and epithelial damage in animal and human studies. 32–34 In this study, the prevalence of asthma diagnosis, use of asthma medication and presence of respiratory symptoms were all above 70% (table 2 and figure 3), whereas more than half of the athletes had objectively measured BHR. In regard to the high performance level of these athletes, and considering the type of sport, the asthma prevalence found in this study can be considered to be in line with previous studies. The incidence of self-reported respiratory symptoms or BHR in Swedish cross-country skiers was as high as 80%. 4 In a recent study, the evidence of asthma, defined as at least one positive objective provocation test to either methacholine or exercise, was 60% in swimmers and 29% in cold-weather athletes compared to 17% of non-athlete controls. 29 In that particular study, winter sport athletes included speed skaters in addition to cross-country and biathlon skiers, and the 25% prevalence of BHR to methacholine, defined as PD 20met ≤4μmol, was similar to that in this study. Our results are based on bronchial responsiveness assessed by a methacholine bronchial challenge and reversibility of the athletes at their current asthma treatment and asthma control during data collection. In a study by Stadelmann et al, 35 the direct assessment of BHR by PD 20met corresponded well with the indirect stimuli of an EVH-challenge in swimmers. Further studies using other bronchial challenges should be conducted in order to support our results. Testing was performed in the fall, prior to the competitive season. Heir et al 36 have found that BHR in cross-country skiers varied according to seasonal changes and exercise intensity. Their study showed that BHR, expressed as the methacholine concentration causing a fall in FEV 1 by 10% (PC 10 ), was lower at the end of winter and the competitive season and that it was highly affected by the amount of high-intensity training. The data collection of this study was carried out before the competitive season, but during an intensive training period, which may have influenced the degree of BHR in the athletes. The use of β 2 -agonists among athletes is increasing. 37 Interestingly, none of the athletes in this study showed a positive reversibility to salbutamol (▵FEV 1 ≥12%), despite the fact that 73% reported that they had asthma and 55% had clinical BHR (PD 20met ≤8μmol). Regular treatment with short-acting and long-acting β 2 -agonists may result in tolerance development to the effect of β 2 -agonists. 38 In this study, 8 athletes used shortacting β 2 -agonists and 10 used long-acting β 2 -agonists. In addition, 10 athletes used ICS. The potential influence from the use Figure 3 Presence of respiratory symptoms, and type of symptom in relation to bronchial hyper-responsiveness (BHR) (methacholine dose causing a 20% reduction in forced expiratory volume in 1 s (FEV 1 ) [PD 20met ] less than or equal to 8 μmol) in elite cross-country and biathlon skiers. Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 5 of 7 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from of these medications on the absence of any significant bronchodilator effect after inhaled salbutamol in this study cannot be excluded. Optimal asthma treatment is a pre-requisite for the asthmatic athlete, both from a competitive and health perspective. Haahtela et al 25 have described different phenotypes of asthma in athletes and individual differences in the airway response to bronchodilators are reported in the literature, especially among the athletes who develop asthma symptoms and BHR later in life and during their sport careers. 39 This is somewhat in agreement with this study where we found that athletes with a positive reversibility test to ipratropium bromide were characterised by BHR (PD 20met of <8 μmol), as well as the use of inhaled corticosteroids and self-reported exercise-induced respiratory symptoms. There was no difference observed in bronchodilator response to inhaled salbutamol between athletes using inhaled corticosteroids, short-acting or long-acting β 2 -agonists, or no asthma treatment. The high prevalence of anticholinergic drug use in this study can be explained by that previous experience with athletes of the National teams of cross-country and biathlon skiing, which has demonstrated the improved reversibility to inhaled ipratropium among this group of athletes. Therefore, reversibility tests of both salbutamol and ipratropium bromide have been performed on these elite athletes. Treatment is then based on the best effect of the reversibility tests. Another reason is that ipratropium bromide has not been listed on Wada’s World Anti-doping prohibited list of drugs. Anticholinergic agents, such as ipratropium bromide, inhibit parasympathetic nerve impulses through competitive inhibition on the muscarinic acetylcholine receptors in smooth muscle and respiratory glands. 40 It has been suggested that differences in the parasympathetic bronchial tone may explain why some patients are responders and others non-responders to anticholinergic treatment. 827 Pichon et al 11 have shown that non-athletes with BHR to methacholine also seem to have a high parasympathetic tone, measured as increased HRV. As methacholine is a synthetic choline ester that acts as a non-selective muscarinic receptor agonist, the bronchial responsiveness to methacholine may itself be considered to reflect the parasympathetic bronchial tone. Although respiratory symptoms are common among crosscountry skiers, 34 the results from this study showed no associations between self-reported symptoms and BHR, reversibility to inhaled ipratropium bromide or inhaled salbutamol, or to FE NO . The incidence of asthma symptoms found by self-reports is higher compared with objectively measured BHR to methacholine and reversibility tests. This finding extends to other studies and suggests that objective tests are a necessary addition to self-reported symptoms to ascertain the diagnosis of asthma in athletes. 41 CONCLUSION This study reported a high prevalence of BHR and asthma symptoms among top cross-country skiers. The high correlation between reversibility to ipratropium bromide and methacholine BHR in this study provides evidence that increased parasympathetic activity in the airways contributes to asthma development in elite cross-country and biathlon skiers. What are the new findings? ▸The present study demonstrated a marked increased bronchial parasympathetic tone in highly trained athletes. ▸It showed differences in responses to two different bronchodilators in elite cross-country skiers. ▸It also confirms that self-reported symptoms are not related to objective tests for asthma in athletes. How might it impact on clinical practice in the near future? ▸This highly increased bronchial parasympathetic tone may represent an important cholinergic role in the development of skier’s asthma. ▸The results from this study suggest that elite skiers with asthma respond better to anticholinergic treatment as compared with β 2 -agonists. ▸This study suggests that BHR to methacholine may be a measure of bronchial parasympathetic tone in elite cross-country and biathlon skiers. Author affiliations 1 Department of Sports Medicine, Norwegian School of Sport Sciences, Oslo, Norway 2 Immunology Lab, Faculty of Medicine, University of Porto, Porto, Portugal 3 Immunoallergology Department, Centro Hospitalar São João EPE, Porto, Portugal 4 Department of Pediatrics, Oslo University Hospital, Oslo, Norway 5 Faculty of Medicine, University of Oslo, Oslo, Norway Acknowledgements The authors would like to thank Erlend Hem at Olympiatoppen, Norway for assistance with the data collection at Val Senales, Italy, and Petter Mowinckel for statistical advice. They are grateful to all participants who took part in this study. Competing interests None. Patient consent Obtained. Ethics approval Regional Medical Ethics committee of medical and health research ethics, South East Norway, and the Norwegian data inspectorate. Provenance and peer review Not commissioned; externally peer reviewed. REFERENCES 1 Fitch KD. 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Self-reported symptoms and exercise-induced asthma in the elite athletes. Med Sci Sports Exerc 2001;33:208–13. Stang J, et al.Br J Sports Med 2015;49:56–61. doi:10.1136/bjsports-2014-094053 7 of 7 Original article group.bmj.com on January 9, 2016 - Published by http://bjsm.bmj.com/Downloaded from variables to include in the final model, taking into account their significance and effect in the adjusted r 2 . The effect of asthma, a major outcome of the present study, was included in the multiple regression analysis and kept in the final model independently of the significance level and adjusted r 2 change with its inclusion. All analyses were performed using SPSS 20.0 (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp) and considering a p <0.05 for statistical significance. Results EBT significantly increased after training (DEBT =0.32 0.57; p =0.016) (Fig 2a) without differences between asthmatic and healthy swimmers (0.15 0.39 vs. 0.45 0.68; p =0.254) (Table 2, Fig 2b). Baseline and postexercise EBT were similar between both groups (Table 2). Also, no differences were observed between those with (n =6) or without inhaled corticosteroids (p =0.853). A significant correlation was observed between baseline and post-exercise EBTs (r=0.827, p <0.001). In multiple linear regression analysis, after controlling for baseline EBT and axillary temperature, asthma was not a significant predictor of DEBT (Table 3). In univariate regression analysis, baseline EBT was the variable most strongly associated with DEBT (r 2 =0.464). Correlations between BHR (LnPD 20 ) and baseline EBT (r=0.224, p =0.533) and DEBT (r=0.044, p =0.845) for asthmatic swimmers were not significant. Both groups presented median values of eosinophils and epithelial cells higher and neutrophils lower than normal, comparatively to reference values for healthy populations (17). When compared to healthy swimmers, asthmatics presented higher eosinophil and epithelial cell counts and lower neutrophils, but with no significant differences (Table 1). Correlations between sputum eosinophils with baseline EBT (r=0.064, p =0.800) and DEBT (r=0.210, p =0.404) were not significant, even if considering only asthmatics (r=0.286, p =0.493 and r=0.048, p =0.911, for baseline EBT and DEBT, respectively). Regarding neutrophils, correlations were also non-significant for the global sample for baseline EBT (r=0.194, p =0.441) and DEBT (r=0.101, p=0.689) and also for the asthmatic group (r=0.500, p=0.207 and r=0.048, p =0.911, for baseline and DEBT, respectively). Discussion An increase in EBT after a training session was observed, supporting the hypothesis of heat loss during exercise. Interestingly, asthmatics did not experience a higher increase when compared to healthy swimmers after controlling for baseline EBT and body temperature. These results support the previous findings suggesting heat loss occurs as a physiologic rather than pathological response to exercise (10, 11). Furthermore, no relation was found between EBT and sputum inflammatory cells, neither with the degree of BHR. It is therefore tempting to speculate that an inflammatory response to the heat loss of exercise might not be a key ethiopathogenic mechanism of swimmers asthma. Our results are in accordance with previous studies exploring the relationship of EBT to EIA (10, 11). Although EBT has been widely investigated based on the assumption that airways inflammation would influence the temperature of the air coming from the alveoli (19), and has been shown to correlate (a) (b) Figure 2 Exhaled breath temperature (EBT) before and after a swimming training session considering all participants (a) and asthmatics vs others (b). Pediatric Allergy and Immunology 26 (2015) 564–570 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 567 Couto et al. Exhaled breath temperature in swimmers with bronchial blood flow, exhaled NO, and sputum eosinophils in asthmatics (3, 4, 20, 21), only two studies explored the effect of exercise (10, 11). Both studies showed an EBT increase after a laboratory standardized exercise challenge, but no differences were observed between asthmatics and controls (10, 11), in agreement with our results obtained with the regular exercise training of competitive swimmers. We have introduced several novelties. This is the first time, to our knowledge, that EBT has been evaluated in elite athletes, and also before and after a real-life training session, in order to assess whether EBT changes could support the hypothesis of heat loss due to physical exercise and inflammation occurring in the airways as mechanisms of EIA. Our study has other strengths: we have adjusted our analysis for baseline EBT, which is of particular relevance given the high dependence to ΔEBT. Also, recent studies have highlighted several factors influence EBT (22) and we have addressed that question also. We have shown that central temperature significantly influences ΔEBT in elite swimmers, and we have analyzed the possible confounding effect of several demographic variables and training characteristics. Furthermore, sputum cell counts were used to evaluate the presence, type, and degree of inflammation in the airways. During exercise, unperfused alveoli become perfused and underperfused units receive an increased blood supply. It is therefore conceivable to expect a physiologic increase in airways temperature related to capillary recruitment rather than a pathological mechanism of vasodilation resulting from inflammation, that is, consistent with the lack of correlation previously found between NO levels and EBT after exercise (11), as well as the lack of correlation with sputum inflammatory cells observed in our study. In fact, it has been proposed that airway inflammation in athletes may represent a training adaptation to injury secondary to rigorous hyperpnoea, and not necessarily related to EIA or implying detrimental effects (5, 23–25). The restorative process after injury involves plasma exudation and movement of cells into the airways, a process repeated many times during a season of training (26). This process has the potential to expose smooth muscle to a wide variety of plasmaand cell-derived substances. The exposure to these substances over time can lead to an alteration in the contractile properties of the smooth muscle, turning it more prone to bronchoconstriction. Accordingly, we have shown that although airway inflammation decreases after finishing competitive swimming, the prevalence of asthma and use of asthma medication increases significantly in both active and past adolescent swimmers, suggesting a relative independence of the two conditions (25). In the meanwhile, the IOC criteria for asthma diagnosis in athletes mostly reflect the occurrence of bronchoconstriction, while asthma in common patients is defined by a chronic course and inflammatory changes (27). Vigorous exercise causes epithelial damage, which has likewise been linked to EIA in elite athletes (28). Mechanical stress of sustained extreme breathing is believed to result in epithelial injury and turn it more prone to asthma. Clara cell secretory protein (CC16), a peripheral marker of lower airways epithelial barrier disruption, is increased after exercise in urine (29, 30) and in serum (31–33), as well as after eucapnic voluntary hyperventilation in athletes (34). It was recently shown plasmatic CC16 correlates to EBT after exercise, Table 2 Results of measurements performed in the present study, and training characteristics of asthmatic vs healthy swimmers Asthmatic swimmers (n =10) Healthy swimmers (n =12) p* Baseline EBT, °C 34.08 0.60 33.49 0.95 0.180 Post-exercise EBT, °C 34.23 0.55 33.94 0.64 0.283 Body temperature†,°C 35.97 0.42 36.02 0.31 0.771 Hours of training in previous week 9.4 2.6 9.3 3.4 0.974 Intensity of the session, n Aerobic 3 9 0.063 Mild anaerobic 5 1 Moderate/severe anaerobic 2 2 Data presented as mean SD, except otherwise stated. *Mann–Whitney U-test was used for comparisons or chi-square test in case of categorical variables. †Axillary temperature was used as a measure for body temperature. Table 3 Multiple linear regression model for predicting DEBT Variable Standardized Coefficient B p-value 95% CI Baseline EBT 0.675 0.001 [0.685; 0.229] Asthma 0.008 0.961 [0.388;0.370] Axillary temperature 0.327 0.043 [0.018;1.027] R 2 =0.596, adj R 2 =0.529. p-values of the univariate analysis (including all the tested variables): sex—0.547, age—0.336, height—0.025, weight—0.196, baseline EBT—<0.001, axillary temperature—0.089, training intensity—0.415, asthma—0.222, PD20—0.600, number of training hours in the previous week—0.583. The following explanatory variables were initially entered into the multiple regression model, but were excluded in the final model as they did not reach significance at the 0.05 level: height, weight. Variable asthma, being a major outcome of this study, was kept in the final model independently of the significance level and adjusted r 2 change with its inclusion; it is coded as ‘0’ =without asthma and ‘1’ =with asthma. 568 Pediatric Allergy and Immunology 26 (2015) 564–570 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Exhaled breath temperature in swimmers Couto et al. reflecting an overall epithelial involvement (11), but no differences were observed between asthmatic and healthy subjects (11), which again leads to the concept of a physiologic rather than a pathological response to exercise. And in fact, it seems to be only related to exercise and hyperpnea, as urinary levels of CC16 are increased after an exercise but not a mannitol challenge (30). Also the environmental conditions may increase airway epithelial damage. Bronchial epithelial cells are released in higher amounts into sputum (epithelial shedding) of cold air athletes and swimmers (28), a result also found in our study. We observed no significant differences between asthmatic and healthy swimmers, which support that exercise and irritant exposures contribute to a detrimental effect on the athletes’ airways but not as ethiopathogenic mechanisms of EIA. Age is a crucial point to bear in mind in our study. Sports asthma has been claimed to result from cumulative effects of years of training. Subjects in our sample are adolescents, so it could be argued that lack of differences could be due to their relatively young age. However, in the specific case of competitive swimmers, the environmental exposure is considered to be so strong that very frequently they have already developed BHR during their teens (7). We found a 45% prevalence of asthma diagnosis, and they are all elite swimmers with mean of 9 years of competition. There are some limitations in our study. First, the small sample size limits solid conclusions. Due to a strong effort to homogenize training characteristics and environment, only elite swimmers from the same team were included. We cannot assure that lack of differences is not related to low power; nevertheless, some results are statistically significant. Second, some swimmers were under inhaled steroids at the time of EBT collection. However, no differences were observed between those with and without therapy, a result that has also been previously reported (9). To finalize, external validation is compromised; our results are not possible to extrapolate for other sports as swimmers are a special population among elite athletes due to a potential detrimental effect of environmental exposure. So, in light of these results, along with previous published findings, the authors remark 3 points: 1) exercise increases EBT, probably due to a physiologic increase in lungs blood flow, rather than pathological mechanism, as no differences are observed between asthmatic and healthy subjects; 2) mechanic noxious stimulus of high ventilation may lead to airways epithelial damage and susceptibility to irritative stimuli, supported by increased numbers of bronchial epithelial cells in swimmers and high levels of CC16, regardless of their asthmatic status; and 3) due to daily recurrence of training, repair is delayed and a ‘frustrated’ inflammatory response occurs to heal the damage of physical injury in both asthmatic and healthy swimmers. How this relates to ethiopathogenic mechanisms of EIA is not clear and thus calls for further studies. The explanatory model of EIA in athletes will probably include the interplay between environmental training factors and athlete’s personal and genetic risk factors. Our study supports the previous hypothesis of a physiologic rather than a pathological significance of airways heat loss and inflammation in elite swimmers, but further studies are needed. Acknowledgments The authors would like to thank all subjects for their participation and the technical and administrative staff of FC Porto Swimming Section for logistical help. They would also like to thank Dr. Mar ılia Beltr~ ao, Dr. Oksana Sokhatska e Dr. Carmo Palmares (Faculdade de Medicina da Universidade do Porto—FMUP) for processing and analyzing the sputum samples; Carla Martins (Centro Hospitalar S~ ao Jo~ ao, EPE) for her availability in performing bronchial provocation challenges with methacholine; Diater  for providing methacholine for bronchial provocation challenges; Samuel Queir os (FMUP) for help in data collection; Dr. Ana Pereira (FMUP) for help in statistical analysis; and Kai-H akon Carlsen for critically reviewing the manuscript and improving it with his feedback based on his large expertise in this area. References 1. Vignola AM, Mirabella F, Costanzo G, et al. Airway remodeling in asthma. Chest 2003: 123: 417S–22S. 2. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and nonasthmatic subjects. Thorax 2001: 56: 902–6. 3. Paredi P, Kharitonov SA, Barnes PJ. Correlation of exhaled breath temperature with bronchial blood flow in asthma. Respir Res 2005: 6:1–10. 4. Piacentini GL, Peroni D, Crestani E, et al. Exhaled air temperature in asthma: methods and relationship with markers of disease. Clin Exp Allergy 2007: 37: 415–9. 5. Carlsen K-H, Anderson S, Bjermer L, et al. Exercise-induced asthma, respiratory and allergic disorders in elite athletes: epidemiology, mechanisms and diagnosis: part I of the report from the Joint Task Force of the European Respiratory Society (ERS) and the European Academy of Allergy and Clinical Immunology (EAACI) in cooperation with GA2LEN. Allergy 2008: 63: 387–403. 6. Couto M, Silva D, Delgado L, Moreira A. Exercise and airway injury in athletes. Acta Med Port 2013: 26:56–60. 7. Del Giacco SR, Carlsen K-H, Du Toit G. Allergy and sports in children. Pediatr Allergy Immunol 2012: 23:11–20. 8. Palange P, Brusasco V, Delgado L, Del Giacco S. Exercise and airway physiology: interactions with immune and allergic responses. In: Carlsen KH, Delgado L, Del Giacco S, eds. Diagnosis, Prevention and Treatment of Exercise-Related Asthma, Respiratory and Allergic Disorders in Sports European Respiratory Society Journals. Sheffield, UK: Maney Publishing, 2005: 10– 8. 9. Peroni DG, Chinellato I, Piazza M, et al. Exhaled breath temperature and exerciseinduced bronchoconstriction in asthmatic children. Pediatr Pulmonol 2012: 47: 240–4. 10. Svensson H, Nilsson D, Bjermer L, Tufvesson E. Exhaled breath temperature increases after exercise in asthmatics and controls. Respiration 2012: 84: 283–90. 11. Tufvesson E, Svensson H, Ankerst J, Bjermer L. Increase of club cell (Clara) protein (CC16) in plasma and urine after exercise challenge in asthmatics and healthy Pediatric Allergy and Immunology 26 (2015) 564–570 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 569 Couto et al. Exhaled breath temperature in swimmers controls, and correlations to exhaled breath temperature and exhaled nitric oxide. Respir Med 2013: 107: 1675–81. 12. American Thoracic Society. Guidelines for methacholine and exercise challenge testing1999. Am J Respir Crit Care Med 2000: 161: 309–29. 13. Miller MR, Hankinson J, Brusasco V, et al. Standardisation of spirometry. Eur Respir J 2005: 26: 319–38. 14. Pellegrino R, Viegi G, Brusasco V, et al. Interpretative strategies for lung function tests. Eur Respir J 2005: 26: 948–68. 15. Ara ujo L, Moreira A, Palmares C, Beltr~ ao M, Fonseca J, Delgado L. Induced sputum in children: success determinants, safety, and cell profiles. J Investig Allergol Clin Immunol 2011: 21:26–1. 16. Paggiaro PL, Chanez P, Holz O, et al. Sputum induction. Eur Respir J Suppl 2002: 37:3s–8s. 17. Belda J, Leigh R, Parameswaran K, O’Byrne PM, Sears MR, Hargreave FE. Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000: 161 (2 Pt 1): 475–8. 18. Popov TA, Dunev S, Kralimarkova TZ, Kraeva S, DuBuske LM. Evaluation of a simple, potentially individual device for exhaled breath temperature measurement. Respir Med 2007: 101: 2044–50. 19. Paredi P, Kharitonov SA, Barnes PJ. Faster rise of EBT in asthma: a novel marker of airway inflammation? Am J Respir Crit Care Med 2002: 165: 181–4. 20. Popov TA. Human exhaled breath analysis. Ann Allergy Asthma Immunol 2011: 106: 451–6. 21. Piacentini GL, Bodini A, Zerman L, et al. Relationship between exhaled air temperature and exhaled nitric oxide in childhood asthma. Eur Respir J 2002: 20: 108–11. 22. Vermeulen S, Barreto M, La Penna F, et al. Exhaled breath temperature in children: reproducibility and influencing factors. J Asthma 2014: 51: 743–50. 23. Bonsignore MR, Morici G, Riccobono L, et al. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes. Med Sci Sports Exerc 2003: 35: 1146–52. 24. Moreira A, Palmares C, Lopes C, Delgado L. Airway vascular damage in elite swimmers. Respir Med 2011: 105: 1761–5. 25. Couto M, Andrade P, Pereira M, et al. Effect of competitive swimming on airway inflammation: a 3-yr longitudinal study. Pediatr Allergy Immunol 2014: 25: 193–5. 26. Ali Z, Norsk P, Ulrik CS. Mechanisms and management of exercise-induced asthma in elite athletes. J Asthma 2012: 49: 480–6. 27. Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA) 2012 [cited 2015]. Available from: www.ginasthma.org. 28. Bougault V, Turmel J, St-Laurent J, Bertrand M, Boulet L-P. Asthma, airway inflammation and epithelial damage in swimmers and cold-air athletes. Eur Respir J 2009: 33: 740–6. 29. Bolger C, Tufvesson E, Anderson SD, et al. Effect of inspired air conditions on exerciseinduced bronchoconstriction and urinary CC16 levels in athletes. J Appl Physiol 2011: 111: 1059–65. 30. Romberg K, Bjermer L, Tufvesson E. Exercise but not mannitol provocation increases urinary Clara cell protein (CC16) in elite swimmers. Respir Med 2011: 105: 31–6. 31. Carbonnelle S, Francaux M, Doyle I, et al. Changes in serum pneumoproteins caused by short-term exposures to nitrogen trichloride in indoor chlorinated swimming pools. Biomarkers 2002: 7: 464–78. 32. Chimenti L, Morici G, Patern o A, et al. Bronchial epithelial damage after a halfmarathon in nonasthmatic amateur runners. Am J Physiol Lung Cell Mol Physiol 2010: 298: L857–62. 33. Nanson CJ, Burgess JL, Robin M, Bernard AM. Exercise alters serum pneumoprotein concentrations. Respir Physiol 2001: 127: 259–65. 34. Bolger C, Tufvesson E, Sue-Chu M, et al. Hyperpnea-induced bronchoconstriction and urinary CC16 levels in athletes. Med Sci Sports Exerc 2011: 43: 1207–13. 570 Pediatric Allergy and Immunology 26 (2015) 564–570 ª2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Exhaled breath temperature in swimmers Couto et al. Study VI Effect of competitive swimming on airway inflammation: a 3-yr longitudinal study Pediatr Allergy Immunol. 2014; 25:193-5 CORRESPONDENCE Effect of competitive swimming on airway inflammation: A 3-yr longitudinal study DOI:10.1111/pai.12172 To the Editor, In recent years, the observation that regular pool attendance, especially by young children, was associated with lung hyperpermeability and increased risk of developing asthma led to the ‘pool chlorine hypothesis’ (1). Accordingly, the increasing and largely uncontrolled exposure of young children to chlorination byproducts contaminating the air of indoor swimming pools could contribute to the childhood asthma rise in industrialized countries (1). Moreover, an increasing body of literature suggests an association between competitive swimming and asthma (2). A higher prevalence of asthma and asthma-like symptoms has been identified in elite swimmers. Environmental exposures, mechanical stress to the airways, increased prevalence of respiratory infections and dysautonomia, have been recognized as asthma contributory factors (2). Increased numbers of both eosinophils and mast cells were observed in bronchial biopsies of competitive adult swimmers, leading to believe that airway inflammation and hyper-responsiveness develop during the training career (3). While the latter seems to be a transient phenomenon (4), whether airway inflammation persists is still debated (5–7). Therefore, we aimed to assess changes in airway inflammation of swimmers during a 3-yr follow-up. Competitive non-elite young swimmers from the two main Portuguese swimming teams were invited (n =120) to participate in this cohort prospective study. Informed consent was obtained from 105, which were assessed at the baseline visit. From these, 86 attended the 3-yr follow-up visit and were included in the final analysis. No significant differences were observed between the 19 lost to follow-up subjects and the remaining (Table 1). ‘Active swimmers’ were defined as those remaining at high level of competitive swimming; those who quitted at least 6 months before the follow-up visit were considered ‘past swimmers’. None of the subjects smoked. The local hospital ethical committee approved the study. Subjects completed a self-administered questionnaire, including questions from the ISAAC questionnaire, reporting physician diagnosis of asthma and allergic rhinitis, and use of asthma medication. Physical activity (PA) was measured using the short 7 days International Physical Activity Questionnaire (IPAQ) (8). A combined total physical activity was computed as the sum of the activity domains scores (total PA =walking +moderate-intensity PA +vigorous-intensity PA) and reported as a continuous measure (total PA score =total MET-min/wk). Eosinophilic airway inflammation was assessed measuring lower airway’s exhaled nitric oxide (NO) levels according to guidelines, before a training session, using NIOX MINO (Aerocrine AB, Solna, Sweden), and expressed in parts per billion (p.p.b.). Atopy was defined by positive skin prick testing to common aeroallergens (Laboratorios LETI S.L., Spain). The study was performed at the swimming pool in which swimmers trained, except for the follow-up visit of past swimmers which occurred at the laboratory. Results were expressed as mean (SD) or, if not normally distributed, as median (interquartile range). Levels of exhaled NO and of physical activity were log-transformed because of skewed distribution. Differences between groups were assessed with one-way ANOVA or Kruskal–Wallis for normally or nonnormally distributed data or chi-Square for categorical variables. Differences in changes in exhaled NO after the 3-yr follow-up were assessed by general linear model adjusting on confounding factors: gender, age, atopy, physician-diagnosed asthma, and use of asthma medication. We observed a significant difference in changes in exhaled NO; those who remained active significantly increased their levels of eosinophilic airway inflammation independently of their gender, age, atopy, or asthma status (Table 2, Fig. 1). After the 3-yr follow-up, the prevalence of asthma, allergic rhinitis, and use of asthma medication increased significantly in both groups. All subjects increased their overall physical activity levels; however, significant increases in moderate and vigorous physical activity level were only observed in active swimmers. Table 1 Baseline characteristics of participants according with their swimming status at the three-year follow-up Past swimmers (n =39) Active swimmers (n =47) Lost to follow–up (n =19) p Demographics Age in yr, median IQR 14 6.3 13 3.2 15 2.0 0.080* Males 23 (59) 27 (57) 11 (42) 0.990 Swimming h/wk, median (IQR) 12 (9) 12 (7) 14 (4) 0.113* Years of competition, median (IQR) 5 (5) 4 (4) 6 (3) 0.109* Clinical features Atopy 21 (54) 15 (32) 11 (58) 0.056 Allergic rhinitis 13 (33) 11 (23) 3 (16) 0.317 Asthma 6 (15) 6 (13) 0 (0) 0.208 Asthma drugs 5 (13) 9 (19) 0 (0) 0.116 Data presented as n (%) unless otherwise stated. The p value relates to chi-square tests to assess differences between past swimmers, active swimmers, and ‘lost to follow-up’, except otherwise marked. *Kruskal–Wallis test results to assess the differences between past swimmers, active swimmers, and ‘lost to follow-up’. ª2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1 Pediatric Allergy and Immunology Our study has some limitations. First, we had an 18% of losses to follow-up; however, these subjects were similar to others at baseline evaluation. Second, asthma was defined as a positive response to the question, ‘Has a doctor ever said that you have asthma?’ Therefore, misclassification of disease status is possible, although would unlikely differently affect any of the groups and being furthermore independent of airway inflammation assessment. Our study has also important strengths: For the first time, it is reported a prospectively assessment of eosinophilic airway inflammation in non-elite swimmers; it was extensively adjusted for confounders and known risk factors that affect exhaled NO levels; and finally, we used a validated tool to monitor physical activity levels. Only one study prospectively assessed the effect on asthma in those who kept vs. those who retired from swimming (7). In 42 Finnish elite swimmers, after a 5-yr follow-up, airway responsiveness and asthma attenuated or even disappeared in swimmers who stopped high-level training, while mild eosinophilic airway inflammation was aggravated among those who remained active. A detailed knowledge of inflammation remission as assessed by objective testing and natural course of the disease in former elite athletes is also missing and should be investigated (9). Although several studies report an association, the relationship between asthma and swimming remains controversial. Previous data have shown that elite training in chlorinated pools affects airway structure; also, asthma is more commonly found in swimmers than among other high-level athletes (10). Adolescent competitive swimmers with allergic asthma show a mixed type of airway inflammation (11), and the development of airway hyper-responsiveness generally occurs in young adults rather than in adolescent competitive swimmers (3), which supports that it may be the result of the cumulative effects of years of exposure as they develop respiratory disorders during their athletic career. However, a causal relationship between swimming and asthma could not be established as most studies are cross-sectional (3) and the association has never been confirmed among non-competitive swimmers (10). Table 2 Changes in airway inflammation, prevalence of asthma and rhinitis, and physical activity levels in athletes according with their swimming status after the 3-yr follow-up Past swimmers, n =39 Active swimmers, n =47 Past vs. active swimmers†Baseline Follow-up Baseline Follow-up Exhaled NO, p.p.b.‡18 (19.5) 16 (20) 14 (11) 14 (18) 0.008 Asthma, n (%) 6 (15) 9 (23)*6 (13) 9 (19)*0.656 Allergic rhinitis, n (%) 13 (33) 16 (41)*11 (23) 13 (28)*0.305 Asthma drugs, n (%) 5 (13) 12 (31)*9 (19) 12 (26)*0.382 Physical activity‡ Walking 462 (685) 693 (2402) 396 (1188) 739 (1148) 0.016 Moderate 720 (2010) 1320 (1440) 120 (960) 960 (3120) ** 0.065 Vigorous 5760 (1920) 3840 (6120) 5760 (7040) 13440 (11520)*<0.001 Total 7173 (3864) 7242 (5718)*6222 (9159) 17196 (8544)*0.001 Data presented as median (IQR) unless otherwise stated; Physical activity expressed as MET-min/wk; bold figures represent statistically significant differences between comparing groups; Paired sample t-tests or chi-square tests were used as appropriate, and differences in changes between groups after the follow-up were assessed by general linear model adjusting on the following confounders: gender, age, atopy, physician-diagnosed asthma, use of asthma medication for exhaled NO and on gender and age for physical activity levels, with baseline values as covariable. *p<0.001; **p=0.002; p =0.005. †Comparison of exhaled NO variation (Δ) from baseline to 3-yr follow-up among active and past swimmers. ‡Data presented on exhaled NO and physical activity are absolute values, although they were log-transformed for comparison analysis. Past swimmers Active swimmers Exhaled nitric oxide levels (ppb) Baseline 90 80 70 60 50 40 30 20 10 0 Baseline3 years follow-up 3 years follow-up Figure 1 Variation of exhaled nitric oxide at baseline and at 3 yr followup among past and active swimmers. 2ª2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Correspondence In conclusion, our prospective study of competitive swimmers shows that those who remained active at a 3-yr follow-up significantly increased their levels of airway inflammation measured by exhaled NO independently of their gender, age, atopy, or asthma status. Nevertheless, asthma incidence did not increase in active swimmers. In fact, it has been previously shown that intense swimming activity causes a lung growth greater than normal in children and adolescents (12), and that physical training does not increase allergic inflammation in children with asthma (13). Our study supports the recommendation to engage in physical activity to all asthmatics as long as the disease is controlled. Source of funding None. Mariana Couto 1,2 ; Patr ıcia Andrade 1 ; Marta Pereira 1 ;Jo ~ ao Ara ujo 1 ; Pedro Moreira 3 ;Lu  ıs Delgado 1,2 & Andr e Moreira 1,2 1 Faculty of Medicine, University of Porto, Porto, Portugal; 2 Centro Hospitalar Sa ˜o Joa ˜o, Porto, Portugal; 3 Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal E-mail: [email protected] References 1. Bernard A. Chlorination products: emerging links with allergic diseases. Curr Med Chem 2007: 14: 1771–82. 2. Haahtela T, Malmberg P, Moreira A. Mechanisms of asthma in olympic athletes– practical implications. Allergy 2008: 63: 685–94. 3. Pedersen L, Lund TK, Barnes PJ, Kharitonov SA, Backer V. Airway responsiveness and inflammation in adolescent elite swimmers. J Allergy Clin Immunol 2008: 122: 322–7. 4. Bougault V, Turmel J, Boulet LP. Airway hyperresponsiveness in elite swimmers: is it a transient phenomenon? J Allergy Clin Immunol 2011: 127: 892–8. 5. Bougault V, Turmel J, St-Laurent J, Bertrand M, Boulet L. Asthma, airway inflammation and epithelial damage in swimmers and cold-air athletes. Eur Respir J2009: 33: 740–6. 6. Bougault V, Loubaki L, Joubert P, et al. Airway remodeling and inflammation in competitive swimmers training in indoor chlorinated swimming pools. J Allergy Clin Immunol 2012: 129: 351–8, 8 e1. 7. Helenius I, Rytila P, Sarna S, et al. Effect of continuing or finishing high-level sports on airway inflammation, bronchial hyperresponsiveness, and asthma: a 5-year prospective follow-up study of 42 highly trained swimmers. J Allergy Clin Immunol 2002: 109: 962–8. 8. Craig CL, Marshall AL, Sj€ ostr€ om M, et al. International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc 2003: 35: 1381–95. 9. Ali Z, Norsk P, Ulrik CS. Mechanisms and management of exercise-induced asthma in elite athletes. J Asthma 2012: 49: 480–6. 10. Goodman M, Hays S. Asthma and swimming: a meta-analysis. J Asthma 2008: 45: 639–47. 11. Moreira A, Delgado L, Palmares C, et al. Competitive swimmers with allergic asthma show a mixed type of airway inflammation. Eur Respir J 2008: 31: 1139–41. 12. Silvestri M, Crimi E, Oliva S, et al. Pulmonary function and airway responsiveness in young competitive swimmers. Pediatr Pulmonol 2013: 48: 74–80. 13. Moreira A, Delgado L, Haahtela T, et al. Physical training does not increase allergic inflammation in asthmatic children. Eur Respir J 2008: 32: 1570–5. ª2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 3 Correspondence