Effect of Mannitol Dry Powder Challenge on Exhaled Nitric Oxide in Children

Background Fractional exhaled nitric oxide (FENO), a non-invasive marker of eosinophilic airway inflammation, is increasingly used for diagnostic and therapeutic decisions in adult and paediatric asthma. Standardized guidelines for the measurement of FENO recommend performing FENO measurements before rather than after bronchial provocation tests. Objective To investigate whether FENO levels decrease after a Mannitol dry powder (MDP) challenge in a clinical setting, and whether the extent of the decrease is influenced by number of MDP manoeuvres, baseline FENO, atopy and doctor diagnosed asthma. Methods Children aged 6–16 years, referred for possible reactive airway disease to a respiratory outpatient clinic, performed an MDP challenge (Aridol®, Pharmaxis, Australia). FENO was measured in doublets immediately before and after the challenge test using the portable NIOX MINO® device (Aerocrine, Stockholm, Sweden). We analysed the data using Kruskal-Wallis rank tests, Wilcoxon signed rank tests and multivariable linear regressions. Results One hundred and seven children completed both tests (mean±SD age 11.5±2.8 years). Overall, median (interquartile range) FENO decreased slightly by −2.5 ppb (−7.0, −0.5), from 18.5 ppb (10.5, 45.5) before the MDP challenge to 16.5 ppb thereafter (8.5, 40.5; p<0.001). In all participants, the change in FENO was smaller than one standard deviation of the baseline mean. The % fall in FENO was smaller in children with less MDP manoeuvres (e.g. higher bronchial responsiveness; p = 0.08) but was not influenced by levels of baseline FENO (p = 0.68), atopy (p = 0.84) or doctor diagnosed asthma (p = 0.93). Conclusion MDP challenge test influences FENO values but differences are small and clinically barely relevant.


Introduction
Asthma is the most common chronic disease of childhood, and its diagnosis remains clinical [1]. A diagnosis of asthma in children is usually based on history of a characteristic pattern of episodic respiratory symptoms and signs in the absence of an alternative explanation of them. In children, as in adults, tests of airflow obstruction, airway responsiveness and airway inflammation may provide support for a diagnosis of asthma [1]. The frequent finding of exercise-related airway narrowing in many children with asthma has been used as a diagnostic tool for years, especially when the diagnosis of asthma is uncertain [2]. Testing for exercise induced bronchoconstriction is not very sensitive, but highly specific for the diagnosis of pediatric asthma compared to direct bronchial provocation tests using methacholine [3].
Mannitol dry powder (MDP) is a new indirect bronchial provocation test, consisting of a hyperosmolar challenge comparable to hypertonic saline [4,5]. In children, a MDP challenge is easier, quicker to perform and better tolerated than a challenge test with hypertonic saline or methacholine, and it shows comparable accuracy for diagnosing asthma as exercise testing [6][7][8][9]. For these reasons, it is likely to become one of the standard bronchial challenge tests in clinical practice and research [10].
Fractional exhaled nitric oxide (FENO), a non-invasive marker of eosinophilic airway inflammation, is increasingly used for diagnostic and therapeutic decisions in adult and paediatric asthma [11]. Standardized guidelines for the measurement of FENO, published by the European Respiratory Society (ERS) and the American Thoracic Society (ATS) [12], recommend to perform FENO measurements before rather than after exercise testing, because both spirometry and exercise affect FENO levels in children, particularly those with asthma [13,14]. To plan the sequence of different tests for single children, it is important to know whether FENO levels are affected by a MDP challenge, similarly as by exercise tests.
The aim of this study was therefore to determine whether FENO levels decrease after a MDP challenge in a clinical setting. In addition, we assessed whether the extent of this decrease is affected by: baseline FENO, severity of bronchial hyperresponsiveness (BHR, which is inversely proportional to the number of MDP manoeuvres), atopy or a diagnosis of asthma.

Ethics Statement
All parents and children gave informed consent for the study. The local ethics committee and the Swiss agency for the authorisation and surveillance of therapeutic products (Swissmedic) approved the study.

Subjects and Study Design
We studied 107 white children (Caucasian Europeans) aged 6 to 16 years, who had been referred for possible reactive airway disease to respiratory outpatient clinics in two tertiary children's hospitals in Switzerland (St.Gallen, Basel). Details of the study have been published elsewhere [6]. Exclusion criteria were other respiratory diseases (e.g. cystic fibrosis, primary ciliary dyskinesia) and a respiratory tract infection during the preceding four weeks.

Fractional Exhaled NO
We measured FENO in doublets immediately before and after the MDP challenge using the portable multi-gas analyzer (NIOX MINOH, Aerocrine, Sweden), in accordance with published guidelines [12] and previous studies using this device [15,16]. The portable analyzer ensures a constant expiratory flow of 5065 ml/s, has an accuracy of 610% with a minimum of 65 ppb. FENO measurements from this portable analyzer correlate well with those obtained by chemiluminiscence detectors [16].

Mannitol Dry Powder Challenge
The MDP challenge (AridolH, Pharmaxis, New South Wales, Australia) was performed as described previously [5,7]. In brief, subjects were asked to inhale the contents of a MDP capsule through the delivery device (OsmohalerH). Doses were increased step-wise (0, 5, 10, 20, 40, 80, 160, 160, 160 mg) until there was either a $15% fall of forced expiratory volume in 1 second (FEV 1 ) compared to baseline, or a 10% fall in FEV 1 between two doses. After each dose, children were instructed to perform a 5-second breath-hold, and then spirometry was performed in duplicates one minute later, and the higher FEV 1 was recorded. The results of the MDP challenge were reported as a percent decrease of FEV 1 from baseline (FEV 1 value measured after the 0-mg capsule), and the provocative dose inducing a 15% drop of FEV 1 (PD 15 ) was calculated. At the end of the challenge, a short-acting beta 2agonist was given to reverse bronchoconstriction. A negative MDP challenge, defined as no significant fall in FEV 1 after a cumulative dose of 635 mg, included eleven FEV 1 manoeuvres in doublets. BHR severity is defined as mild (155-635 mg, 8-10 manoeuvres), moderate (35-155 mg, 5-7 manoeuvres) and severe (,35 mg, #4 manoeuvres).

Clinical Assessment
The responsible paediatric respiratory physicians made a diagnosis (asthma, or alternative diagnoses) for every child after the first visit, taking into consideration medical history, clinical examination and all conventional test results (including bronchodilator response) before MDP challenge and FENO measurement were performed.

Skin Prick Test
We performed skin prick tests (SPT) using birch, grass, mugwort, alternaria, cat, house dust mites (D. pteronyssinus) and positive and negative controls, considering a wheal of .3 mm as positive. These allergens cover 95% of inhaled allergies in Switzerland [17]. Atopy is defined as mild (1 to 2 positive SPT) and moderate ($3 positive SPT).

Statistics
We analysed the data with STATA, version 11.2 (Stata Corporation, Austin, Texas). Because FENO levels were not normally distributed, we used non-parametric tests: the Kruskal-Wallis rank test to compare FENO levels between groups and the Wilcoxon signed rank test to compare FENO levels before and after MDP challenge. We assessed the association between % difference in FENO and levels of baseline FENO, doctor diagnosed asthma, atopy and BHR severity, using linear regressions.

Discussion
This study showed that FENO levels are indeed slightly reduced after a MDP challenge, compared to baseline. The differences, although statistically significant, were however small and unlikely to affect clinical decisions relating to diagnosis or treatment.
Results were unaffected by baseline FENO levels, the presence of atopy or doctor diagnosed asthma. However, the decrease was larger in children with normal MDP challenge who had performed more MDP manoeuvres (11) compared to those with moderate to severe BHR with less MDP manoeuvres (#7). This suggests that the increased ventilation associated with repeated MDP manoeuvres and associated spirometries might have caused the decrease. The median decrease (22.5 ppb, IQR 27.0, 20.5) was comparable or a bit larger than the decrease reported for spirometry alone [3,4], but smaller than the decrease reported for exercise tests (25 ppb, from 223.5 ppb to 218.5 ppb) [13].
To our knowledge, this is the first study comparing FENO levels before and after MDP challenges. It includes a large number of children (107) and the clinical setting guarantees that results are representative for patients referred to respiratory outpatient clinics for potential reactive airways disease. Because most children were atopic and the majority diagnosed with asthma, results should not be extrapolated to healthy non-atopic children.
In conclusion, this study provides further support for the recommendation that FENO measurements in children, as a rule, should preferably be performed before spirometry and before bronchial provocation testing. This is particularly true for research. However, as the differences are small and clinically barely relevant, FENO measurements can still be interpreted even if performed after a MDP challenge test.