Article Text

Single and multiple breath nitrogen washout compared with the methacholine test in patients with suspected asthma and normal spirometry
  1. Aline Stalder Siebeneichler1,
  2. Desiree M Schumann1,
  3. Meropi Karakioulaki1,
  4. Nora Brachsler1,
  5. Andrei M Darie1,
  6. Leticia Grize1,
  7. Thiago G Heck2,
  8. Michael Tamm1,
  9. Philipp Latzin3 and
  10. Daiana Stolz1,4,5
  1. 1 Clinic of Pneumology, University Hospital Basel, Basel, Switzerland
  2. 2 UNIJUI, Ijui, RS, Brazil
  3. 3 Inselspital, Kinderklinik, University of Bern, Bern, Switzerland
  4. 4 Clinic of Pneumology, University of Freiburg, Freiburg, Germany
  5. 5 Faculty of Medicine, University of Freiburg, Freiburg, Germany
  1. Correspondence to Dr Daiana Stolz; daiana.stolz{at}


Background Methods used to assess ventilation heterogeneity through inert gas washout have been standardised and showed high sensitivity in diagnosing many respiratory diseases. We hypothesised that nitrogen single or multiple breath washout tests, respectively nitrogen single breath washout (N2SBW) and nitrogen multiple breath washout (N2MBW), may be pathological in patients with clinical suspicion of asthma but normal spirometry. Our aim was to assess whether N2SBW and N2MBW are associated with methacholine challenge test (MCT) results in this population. We also postulated that an alteration in SIII at N2SBW could be detected before the 20% fall of forced expiratory volume in the first second (FEV1) in MCT.

Study design and methods This prospective, observational, single-centre study included patients with suspicion of asthma with normal spirometry. Patients completed questionnaires on symptoms and health-related quality-of-life and underwent the following lung function tests: N2SBW (SIII), N2MBW (Lung clearance index (LCI), Scond, Sacin), MCT (FEV1 and sGeff) as well as N2SBW between each methacholine dose.

Results 182 patients were screened and 106 were included in the study, with mean age of 41.8±14 years. The majority were never-smokers (58%) and women (61%). MCT was abnormal in 48% of participants, N2SBW was pathological in 10.6% at baseline and N2MBW abnormality ranged widely (LCI 81%, Scond 18%, Sacin 43%). The dose response rate of the MCT showed weak to moderate correlation with the subsequent N2SBW measurements during the provocation phases (ρ 0.34–0.50) but no correlation with N2MBW.

Conclusions Both MCT and N2 washout tests are frequently pathological in patients with suspicion of asthma with normal spirometry. The weak association and lack of concordance across the tests highlight that they reflect different but not interchangeable pathological pathways of the disease.

  • Asthma
  • Asthma Mechanisms
  • Exhaled Airway Markers
  • Respiratory Measurement

Data availability statement

Data are available upon reasonable request.

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  • The role of the small airway in understanding respiratory diseases, including asthma is been established in the last years. Although technical improvements helped research to flourish in this field, there are still knowledge gaps to be fulfilled before we can implement this method into daily practice


  • To the best of our knowledge, this study is the first to assess nitrogen single breath washout and nitrogen multiple breath washout as well as MCT in patients with clinical suspicion of asthma and normal spirometry. This specific population is a reality in the pneumological practice and one of the most common steps in the investigation of asthma is to proceed to a bronchoprovocation test. There is, however, no gold standard test to diagnose or exclude asthma in this population. This study showed that a substantial part of these patients has a pathological N2washout and that this is not strongly associated to a positive MCT.


  • This study reinforces the importance of investigating the small airway of patients with suspicion of asthma and normal spirometry. Further studies are necessary to understand the impact of this finding in the long term, but given the potential burden associated with untreated asthma N2 washout may become a complementary routine diagnostic tool in the future.


Asthma is a highly prevalent respiratory disease, estimated to affect 262 million people worldwide and leads to 21.6 million disability adjusted life years.1 The definition of the disease is based on a history of respiratory symptoms combined with a variable expiratory airflow limitation.2 There is, however, no single test that is considered as the gold standard to confirm or exclude the diagnosis of asthma.3 4 In clinical practice, the investigation of patients with clinical suspicion of asthma with normal spirometry includes a methacholine challenge test (MCT). MCT is a safe test with a high negative predictive value for the diagnosis of asthma,5 nevertheless, it is time consuming, demands trained personnel and the administration of a medication, resulting in an increased diagnostic cost.3

Small airways play an important role in the pathophysiology of asthma. Histological findings in severe asthmatic patients indicate that small airways present significantly more inflammation than larger airways.6 Therefore, one could postulate that non-invasive methods assessing ventilation heterogeneity of the small airways would be a reasonable diagnostic tool. Several methods analysing different inert gases are available. If nitrogen is the inert gas analysed, there are two different methods available: nitrogen single breath washout (N2SBW) and nitrogen multiple breath washout (N2MBW).7 N2SBW is performed with a forced expiratory manoeuvre, which provides a nitrogen slope of phase III washout (SIII). N2MBW is performed at tidal breathing and provides the lung clearance index (LCI) as well as the evaluation of SnIII slopes from the first breath (Sacin) and from lung turnover 1.5 to 6 (Scond).7 8 Cosio et al, as early as 1978, demonstrated a clear association between histological small airway alterations and the SIII slope (SIII).9 The assessment of small airway involvement by the analysis of inert gas washout in cystic fibrosis, bronchiolitis obliterans in graft versus host disease as well as COPD is gaining momentum,10 11 as these methods become more standardised.7 12

In subjects with diagnosed asthma, Downie et al found a significant association between ventilation heterogeneity in the conducting airways (Scond) and dose response rate (DRR) in the MCT.13 In addition, Kjelberg et al reported a significant association between FEV1, LCI, Sacin and Scond in asthmatic patients.14 SIII was higher in severe when compared with mild/moderate asthmatic patients15 and, more recently, in the ATLANTIS study, Scond showed a positive association with asthma severity.16

Inert gas washout lung function tests are still not incorporated in the daily clinical assessment of the asthmatic patient. It remains unclear whether they are more sensitive and could contribute to an earlier diagnosis of asthma than traditional lung function tests. We hypothesised that N2SBW and/or N2MBW are pathological in patients with suspicion of asthma but normal spirometry. Therefore, our main aim was to assess SIII, LCI, Scond and Sacin in comparison to MCT in this population. In addition, we postulate that an alteration in SIII at N2SBW could be detected before the 20% fall of FEV1 in MCT.

Materials and methods

Study design, setting and participants

This is a single-centre, investigator-initiated, prospective, observational study performed at the University Hospital of Basel.

Patients referred to the Clinic of Respiratory Medicine and Pulmonary Cell Research between April 2019 and January 2020 were included in the study if they fulfilled the following inclusion criteria: aged 18 years or more, clinical suspicion of asthma and with normal spirometry defined as FEV1/forced vital capacity (FVC) ≥70%. The exclusion criteria: exacerbation in the previous 2 weeks, patient unable to perform spirometry, current pregnancy, known aortic aneurysm, heart attack or stroke in the last 3 months, eye surgery in the last month and inability to participate due to language barrier or dementia. Patients included in the study were referred to the respiratory medicine department (in most cases by the general practician (GP)) for further evaluation due to respiratory symptoms. The suspicion of asthma was determined by the GP or by the attending physician at the respiratory department. None of these patients had a previous asthmatic diagnosis. All patients underwent a full lung function testing including spirometry. In case of obstruction, bronchial dilatation with short-acting B2-agonist (Salbutamol 4 puffs 100 μg) was performed and the test repeated after 15 min. Patients presenting FEV1/FVC ratio <0.7 before OR after bronchodilatation were excluded from the study. Patients or the public were not involved in the design, conduct, reporting or dissemination plans of the study.

Study assessments

Patients self-completed the following six questionnaires to ascertain symptoms: Asthma Control Questionnaire (ACQ), Asthma Quality of Life Questionnaire (AQLQ), Work Productivity and Activity Impairment Questionnaire-General Health (WPAI-GH), Reflux Severity Index (RSI), Gastroesophageal Reflux Disease questionnaire (GERDq) and Leicester cough questionnaire. Physical examination assessing vital signs, oedema and lung auscultation was performed and documented by a respiratory physician. A medical history pertaining to family history of asthma, smoking status, comorbidities, concomitant medication, vaccinations and symptoms was also taken. A skin prick test evaluating the response to 16 main regional inhalant allergens was performed (see online supplemental appendix 2).

Supplemental material

Patients under treatment with long-acting beta-agonist (LABA)/LAMA and/or inhaled corticosteroids (ICS) were instructed to discontinue this at least 48 hours prior to the study assessments, antihistamines at least 24 hours before assessments and SABA/SAMA at least 12 hours before lung function testing.

The N2SBW test was performed in all patients followed by an N2MBW test and an MCT. Furthermore, one N2SBW test was performed between each methacholine dose increment. All tests were performed according to the guidelines for inert gas washout measurement of the European Respiratory Society/American Thoracic Society,7 except for the number of repetitions of each test modality, due to a time limitation (see online supplemental appendix 1). In brief, all patients were instructed to sit in an upright position and wear the nose clip. For the N2SBW test, subjects were instructed to place their mouth around the mouthpiece of the device and to make a maximal inhalation to total lung capacity from a source of 100% oxygen concentration and exhaled fully in a constant flow. The ventilation inhomogeneity was assessed by SIII (slope phase of phase III N2 washout calculated between 25%–75% of vital capacity).8 For the N2MBW test, patients were instructed to inhale through the mouth from a source of 100% oxygen from functional residual capacity and perform a series of multiple breaths at tidal volume, until nitrogen (N2) concentration decreased to 1/40th (2.5%) of its initial value. The estimation of the ventilation inhomogeneity was obtained from the LCI (number of lung turnovers needed to achieve the N2 washout) as well as Scond (SnIII between lung turnover 1.5 and 6, due to convection-dependent inhomogeneity (ICD)) and Sacin (SnIII for diffusion-convection-dependent variable from first breath).7 The measurements were performed with the Exhalyzer D (Eco Medics AG, Durenten, Switzerland) using Spiroware V.3.1 software. For analysis, the updated Spiroware V.3.3 was used.17

MCT was performed according to the recommendations of the European Respiratory Society, including the instruction of individuals to discontinue the maintenance inhaler in order to decrease the chance of a false-negative methacholine provocation test.5 18 In brief, a baseline spirometry was performed with the patient instructed to sit in an upright position, with a nose clip closing the nose and the mouth sealed around the mouthpiece. The patient breathed normally, took a fast, full inhalation and exhaled forcefully. The procedure was repeated after inhaling NaCl 0.9%, and then methacholine at a cumulative dose of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL, utilising the Carefusion Spirometry PC Software. A positive methacholine test was reached when FEV1 fell by ≥20%, named here as MCT20. Alternatively, we also looked for a reduction of 40% in specific airway conductance (sGeff), named here as MCT40.5 19 Furthermore, we calculated the DRR for FEV1 fall of 20% predicted.13

Statistical analysis

The sample size was estimated according to the following assumptions: (a) the mean of Sacin in normal population is 0.072±0.025 L−1 1; (b) the mean of Sacin in young asthmatics (mean 33 years old) is 0.080 L−1 20; (c) two-tailed alpha value of 5%; (c) statistical power of 80%. Using these assumptions, it is estimated that 77 cases would be necessary to compare Sacin in the N2 washout test in non-asthmatics to asthmatics. Adjusting for non-compliance and loss to follow-up of 20%, the final sample size required was 97 cases.

The statistical analysis was performed using IBM-SPSS-Statistics V.25 and SAS V.9.4 (SAS Institute, Cary, North Carolina) and the graphics presented in the study were obtained using GraphPad Prism V.9.5.1. A p value <0.05 was considered significant. All tests were two tailed. The χ2 test was used to calculate differences in dichotomous variables and the Mann-Whitney U test to calculate differences in continuous variables. All associations were conducted using Spearman’s correlation test. Furthermore, we performed a McNemar’s test to look for agreement between SIII and MCT during the provocation phases. Results are presented as mean±SD or SEM (respectively, SD and SE of the mean).

An abnormal reading for LCI, Scond, Sacin and SIII was defined as a value >1.96 z-score at baseline. DRR was calculated from the final step in the test as a percentage of decrease in FEV1/methacholine dose (µmol). A constant of 3 was added to allow log transformation of zero and negative values. Higher DRR values indicate more severe airway hyperresponsiveness.13


In total, 182 patients with suspicion of asthma referred to the University Hospital of Basel were screened and 106 were included in the study (figure 1). The average age was 41.8±14 years and there was a majority of women (61%), and never-smokers (57.8%; table 1). Family history of asthma was reported by 41% of patients. The most common reported symptoms were cough (60%) and sputum (19%), physical activity was reported as a trigger for symptoms in 35% of patients, followed by emotional stress in 15%. ICS were already prescribed as treatment in 20% of patients, LABA in 16%, and 81% of the patients were allergic to at least one of the tested inhalant allergens in the skin prick test (table 1).

Figure 1

Study design. MCT, methacholine challenge test; N2SBW, nitrogen single breath washout; N2MBW, nitrogen multiple breath washout; Tiffeneau, forced expiratory volume in the first second(FEV1)/forced vital capacity (FVC) ratio; sGeff, specific airway conductance. *Some curves needed to be excluded after the quality control of washout tests, see online supplemental appendix 1.

Table 1

Study subjects demographics

MCT20 was positive in 48% (51) of the patients with an increase to 50% of subjects with MCT40. N2 washout tests, on the other hand, showed very heterogeneous outcomes: SIII was pathological in only 10.6% of the patients at baseline, Scond in 18%, Sacin in 43% and LCI in 81% of the study population. The distribution of abnormal versus normal N2 washout tests compared with positive versus negative MCT20 is graphically demonstrated in figure 2.

Figure 2

Comparison of N2 washout test results in patients with positive versus negative MCT20. LCI, lung clearence index from N2MBW; MCT20, methacholine challenge test according to≥20% fall of FEV1; N2SBW, nitrogen single breath washout; N2MBW, nitrogen multiple breath washout; SIII, slope III from N2SBW at baseline; Scond, SnIII from lung turnover 1.5–6 in the N2MBW; Sacin, SnIII from first breath in N2MBW. + stands for patients with a positive/pathological test (>1.96 z-score) and – stands for the patients with a negative/normal test.

Patients with positive MCT were more commonly women (74.5% vs 49.1%, p=0.009) with a significantly higher DRR (89.5 vs 5.7% fall FEV1/mmol methacholine+3, p<0.001; table 2). Patients with pathological SIII at baseline presented a lower predicted FEV1 (91% vs 98.5%, p=0.018), and patients with pathological SIII as well as pathological Scond and Sacin were significantly older than their non-pathological counterparts (table 2). Other lung volumes in the spirometry did not differ significantly between the groups. Respiratory symptoms, assessed through ACQ, were mild in all groups21 and the difference in quality of life due to asthma symptoms, chronic cough, reflux symptoms and work impairment (respectively assessed through ACQLQ, Leicester Questionnaire, GERD/RSI and WPAI-GH) between groups was not statistically significant.

Table 2

Population characteristics with normal versus abnormal methacholine challenge tests and normal versus abnormal nitrogen washout tests

Methacholine challenge test (MCT) compared with N2 single breath washout (N2SBW)

SIIII showed a very low sensitivity (12%) and a high specificity (90.7%) for MCT according to 20% decrease in FEV1, without improvement of these when MCT40 was used as reference (table 3). The DRR from MCT depicted a weak correlation with SIII at baseline and a weak to moderate correlation with the subsequent SIII during provocation phases 1 to 6 (table 4). Patients with positive MCT also showed significantly higher SIII at baseline (1.79 /L vs 1.41 /L, p 0.002) (table 2).

Table 3

Sensitivity and specificity of nitrogen washout tests as compared with methacholine challenge tests

Table 4

Correlation between lung function tests

Analysing SIII over the bronchoprovocation phases, there was an increase in the proportion of pathological tests, especially around provocation phases 3–4 with a significant association between the tests in provocation phases 1, 3, 4 and 5 (figure 3). Further analysis for agreement between the tests showed a slight agreement in most provocation phases (1,2 and 6), with phase 3, 4 and 5 reaching a fair to moderate agreement (kappa coefficient 0.238–0.503) (table 5).

Figure 3

Number of patients with MCT20+ and MCT20- in various bronchoprovocation phases. MCT20, methacholine challenge test according to≥20% fall of FEV1; Prov1, bronchoprovocation with methacholine dose 0.1 mg/mL; Prov2, bronchoprovocation with methacholine dose 0.2 mg/mL; Prov3, bronchoprovocation with methacholine dose 0.4 mg/mL; Prov4, bronchoprovocation with methacholine dose 0.8 mg/mL; Prov 5, bronchoprovocation with methacholine dose 1.6 mg/mL; Prov 6, bronchoprovocation with methacholine dose 3.2 mg/mL. + stands for patients with a positive/pathological test (>1.96 z-score) and – stands for the patients with a negative/normal test. *p<0.05.

Table 5

Measuring agreement between MCT20 and SIII z-score (all available pairs for each of the steps)

Methacholine challenge test (MCT) compared with N2 multiple breath washout (N2MBW)

The sensitivity of N2MBW outcomes for a pathologic test ranged from 21.7% with Scond to 80.4% with LCI (table 3). The highest specificity (86.1%) was observed with Scond (table 3). There was no association between DRR and LCI, Scond or Sacin (table 4).

N2 single breath washout (N2SBW) compared with N2 multiple breath washout (N2MBW)

SIII showed a moderate association with LCI at baseline (ρ 0.528) as well as after each methacholine provocation phase (table 4). Sacin showed a moderate correlation with SIII at baseline (ρ 0.548), that persisted except in provocation phase 1, where this was weak (ρ 0.394). Scond showed only a weak correlation to SIII, except at provocation phase 6, where it increased to moderate (table 4).

Patients with pathological SIII at baseline showed a markedly elevated LCI and Sacin than the patients with SIII within normal range, respectively, LCI 10.6 versus 7.5, p<0.001 and Sacin 0.20 versus 0.09/L, p<0.001. Moreover, both pathological Scond and Sacin groups showed significantly higher SIII at baseline (respectively, 2.16 vs 1.46, p=0.012 and 1.59 vs 1.15, p<0.001; table 2).

Nitrogen multiple breath washout test

Within the N2MBW test, Sacin was strongly correlated to LCI (ρ 0.759; table 4). Participants with pathological LCI showed a significantly higher Sacin (0.12 vs 0.05/L, p<0.001; table 2). Scond showed no significant association with LCI, while Sacin and Scond depicted only a weak association (table 4).


To the best of our knowledge, this is the first study that compares MCT, N2MBW and N2SBW (including measurements across methacholine doses) in a large population of patients with suspicion of asthma and with normal spirometry. This study also provided a thorough assessment of symptoms and quality-of-life using various questionnaires (ACQ, AQLQ, WPAI-GH, Leicester cough questionnaire, RSI and GERDq). Our results indicate that both ventilation inhomogeneity, specially LCI and Sacin, assessed by N2 washout, as well as airway hyperresponsiveness, assessed by MCT were present in a significant proportion of the participants. We did not find a significant association of symptoms and spirometric values to pathological nitrogen washout outcomes.

We observed a diverse prevalence of pathological tests across N2SBW and N2MBW, ranging from 10.6% for SIII from N2SBW up to 81% for LCI from N2MBW. There are few data in the literature comparing N2SBW and N2MBW. Our findings reinforce the conclusion from Kjelberg et al 22 suggesting N2MBW to be more sensitive than N2SBW to diagnose small airway disease and, therefore, SIII seems less promising for clinical indications. One could postulate that this difference is due to the fact that CDI and diffusion-convection interaction dependent inhomogeneity, particularly its non-gravitational component, contributes to SIII in a lesser degree then in N2MBW assessments.7 22 We similarly observed a stronger correlation between SIII with Sacin as well as LCI then with Scond.22 What exactly is the contribution of the CDI component in asthma remains a valid but unanswered question. When dividing our study patients in pathological versus non pathological N2 washout groups, patients within pathological LCI and Sacin groups showed increased mean SIII, Sacin and LCI but no increased mean Scond. Zell-Baran et al 23 found that patients with small airway involvement due to different environmental exposures and pulmonary diseases in military deployers also had higher LCI and Sacin but the same was not seen with Scond.

Nevertheless Scond was pathological in 18% of our participants, a similar prevalence then reported in patients with Asthma Global Initiative for Asthma (GINA) class 1 in the ATLANTIS cohort,16 where the authors found that the involvement of small airways, including Scond, increased according to higher GINA stratification groups. Our proportion of pathological Sacin of 43% was, on the other hand, similar to that found in patients with GINA 5 in ATLANTIS (40.9%). We did have a significantly older population in both groups, pathological Sacin and Scond, and this may be a confounding factor, once increased age was previously associated with ventilation inhomogeneity.22 Furthermore, our study included 20.6% of current smokers, while in the ATLANTIS cohort, this proportion was of only 3% and Scond and Sacin are known to be altered in smokers as well, even when spirometry values are normal.24 25

Inert gas washout methods are sensitive tests that do not require the administration of a provocative agent.7 Previous studies have found a correlation between SIII N2SBW and FEV1 in COPD patients,26 thus, they potentially represent an appealing alternative to volume change measurement in MCT. Airway hyperresponsiveness, assessed by MCT, was present in 48% (MCT 20) to 50% of participants (MCT 40). The highest sensitivity for a positive was observed with LCI N2MBW (80.4%) and the highest specificity was reached by SIII N2MBW (90.7%), but looking to the tests correlations, DRR from MCT showed a weak correlation only to SIII from N2SBW and no association with N2MBW outcomes. While repeated SIII N2SBW determinations along the provocation phases depicted a moderate association to DDR of MCT, and the prevalence of pathological SIIIN2SBW increased during the process, the agreement between tests, however, was low in most provocation phases, so it did not add to a simplification or shortening of the MCT test. Methacholine provocation test is a direct method to trigger airway hyper-responsiveness and is considered a characteristic but not a specific feature of asthma, that is less specific than indirect provocation tests, for example.8 Pathophysiological pathways involved in asthma are complex, multifactorial and not yet fully understood.8 12 So far, we know that both ventilation heterogeneity and airway hyperresponsiveness represent important features of the disease, but this study highlights that the patients not necessarily present both of them simultaneously and there might not be used interchangeably in the disease. Further studies focusing in this methods and patient phenotyping could hopefully improve our understanding of the involved mechanisms.

Important limitations of our study include the fact that performance of N2 washout tests could only be compared with methacholine test and not to a definite gold standard to diagnose asthma, for instance, typical remodelling in endobronchial tissue. Therefore, the sensitivity and specificity of these tests are relative. In addition, our study population may be quite heterogeneous as it is expected to include asthmatic patients and healthy subjects. Furthermore, our tests were performed at a single timepoint, and patients with asthma display variable pathology over time. If might be possible that repeated measures of N2washout might add more information than a single test. Nevertheless, one could expect that by causing bronchoconstriction generally one could potentiate pathological Sacin and Scond values during MCT. Finally, patients reaching the threshold for MCT were not further provoked, preventing the additional evaluation of the SIII outcome in N2SBW.

In conclusion, the findings of this study highlight that MCT as well as Sacin and LCI from N2MBW are frequently pathological in patients with suspicion of asthma and a normal spirometry. However, nitrogen washout test cannot yet replace MCT for asthma diagnosis. It is tempting to hypothesise that the weak correlation and lack of concordance between the tests might imply that these tests reflect different but not interchangeable pathological pathways of the disease.

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

The study was approved by the Ethics Committee of Northwest/Central Switzerland (EKNZ 2018-02086). Participants gave informed consent to participate in the study before taking part.


The authors are very thankful to the patients for their willingness to participate in the study. They also appreciate the competence and diligence of the health care professionals of University Hospital of Basel, which made this study possible. ASS is the guarantor of this manuscript’s content.


Supplementary materials

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  • Presented at Prior abstract presentation: 10o Health International Congress Unijuí (April 2023, Ijuí, Brazil), accepted as a poster for the SSC/SSCS–SSP/SSTS Joint Annual Meeting 2023 (June 2023 in Basel, Switzerland) and accepted as a poster for the annual ERS congress (September 2023 in Milan, Italy).

  • Contributors All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. DS is the guarantor who accepts full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish. All authors listed here contributed substantially to the conception and study design, data analysis and interpretation, and the writing of the manuscript.

  • Funding This study was fully supported with the research budget from the Clinic of Respiratory Medicine and Pulmonary Cell Research, University Hospital Basel, Switzerland.

  • Competing interests DS (second author) is currently employed at Tillotts Pharma AG, however, during the period of the study was an employee of the University Hospital of Basel. AMD has received a grant from University Hospital Basel, PL has a grant/contract to Vertex and OM Pharma. DS (last author) reports grants from Astra-Zeneca AG, Curetis AG, BostonScientific, Novartis AG, GSK AG, Roche AG, Zambon, Pfizer, Schwabe Pharma AG, Vifor AG. Other authors have no conflict of interest to declare.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer-reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.