Methacholine challenges: comparison of different tidal breathing challenge methods

Participants

This study recruited male and female asthma patients, with a pre-bronchodilator FEV₁ ≥65% predicted, and a methacholine PD₂₀ value of <1.2 mg. Eligible patients were nonsmokers or ex-smokers with a smoking history of <5 pack-years. All patients were prescribed either inhaled corticosteroid (ICS) and short-acting β₂-agonist (SABA), or SABA only. Patients taking ICS were on a stable dose for ≥4 weeks prior to the study and for the study duration. Patients were excluded using the following criteria: known respiratory disorder other than asthma; current aortic aneurysm; recent myocardial infarction or stroke; hypertension; respiratory infection or use of antibiotics within 4 weeks; or presence of a contraindication to any the procedures, that in the investigator's opinion made it unsafe for the patient to participate. All patients provided written informed consent using a protocol approved by Greater Manchester East Research Ethics Committee (05/Q1402/41).

Study design

The study was conducted in two parts (figure 1). In part 1, participants attended for three visits, where tidal dosimetric challenges were performed. Each patient was randomised to perform a standard dosimetric (SD) challenge, using different methacholine concentrations at each step, at visit 1 followed by an adjusted dosimetric (AD) challenge at visit 2, or vice versa. The AD challenge used fewer methacholine concentrations, but adjusted the nebuliser output (table 1). At visit 3, a repeated AD challenge was performed. In part 2, patients returned to complete two more visits, where a continuous-output breath-actuated tidal breathing challenge was performed at each visit, here referred to as the continuous-output (CO) challenge. All visits were performed ≥3 days apart.

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FIGURE 1

Study design. At visit 1, patients were randomised to perform one of the dosimetric challenges, followed by the alternative at visit 2. SD: standard dosimetric challenge; AD: adjusted dosimetric challenge; CO: continuous-output breath-actuated challenge.

Study procedures

Spirometry was performed on the NDD Easy On-PC system (NDD Medical Technologies, Zurich, Switzerland), according to American Thoracic Society (ATS)/(ERS) standards [10]. During post-challenge time points, FEV₁ only was measured. Predicted values were calculated using the Global Lung Function Initiative 2012 equations [11].

Each patient complied with the ERS pre-test guidelines [2] prior to each challenge. The SD challenge was performed using ProvoX system (Ganshorn, Niederlauer, Germany) with an 8900 nebuliser (Salter Labs, Arvin, CA, USA). This setup provides nebulised methacholine for a defined time during the initial portion of each tidal inhalation. The nebuliser output time was set to 0.4 s per inhalation, with 15 inhalations per dose step, using doubling methacholine concentrations (0.03125–16 mg·mL⁻¹). The AD challenge was performed using the same equipment, with 15 inhalations per dose step, and four dose steps per methacholine concentration (0.25 and 4 mg·mL⁻¹), other than the final concentration (16 mg·mL⁻¹), which was utilised for two dose steps. The nebuliser output time was doubled, as shown in table 1 for each concentration, to achieve a doubling dose challenge that matched that of the SD challenge. Methacholine was diluted to the required concentrations prior to each challenge, using Provocholine (Methapharm, Coral Springs, FL, USA).

In part 2, the CO challenge was performed using the AeroEclipse breath actuated nebuliser II (Trudell Medical International, Ontario, Canada), with a 20-s dose duration, and doubling methacholine concentrations (0.03125–16 mg·mL⁻¹), matching the doses of the part 1 challenges (table 1). This breath-actuated device provides a continuous output of nebulised methacholine during the whole inhalation phase of each tidal breath. Subjects were instructed to perform spontaneous quiet tidal breathing while maintaining a seal on the nebuliser mouthpiece. For all challenges, FEV₁ measurements were performed at 30 and 90 s post-end of administration, with the highest value used to calculate response. Incremental doses were administered at 4-min intervals (between start times of each administration) for all methods.

Statistical methods

The primary end-point was PD₂₀, which was calculated for each challenge by linear interpolation, using cumulative dose, as per standard guidelines [6]. Dose was calculated using nebuliser output, as determined by the manufacturer (ProvoX system) or by testing using methacholine solution (AeroEclipse BAN II), as reported previously [12]. The natural log-transformed PD₂₀ values were calculated and compared between challenge methods using paired t-tests. These values were back-transformed to report the ratio of one challenge method to another. Intraclass correlations (ICC) were performed; these are interpreted as excellent (>0.75), fair to good (0.40–0.75) or poor (<0.40) [13]. Bland–Altman plots were constructed as the difference between the natural log-transformed PD₂₀ values (y-axis) versus the mean untransformed PD₂₀ values (x-axis). Repeatability using doubling dose (DD) difference was calculated as reported previously; [14] briefly, within-subject standard deviation (WSTD) was calculated using the log-transformed mean difference and standard deviation of differences. DD was then calculated using the formula DD=(2(WSTD))/(log10(2)). t-tests were performed using Prism 5 (Graphpad, CA, USA), and ICC analysis was performed using SPSS 25.0 (IBM, Armonk, NY, USA). p<0.05 was considered statistically significant. No formal sample size calculation was performed; however, a sample of 15 was considered suitable given the study design.