Use of a vibrating mesh nebulizer for allergen challenge

Background Allergen inhalation tests are a valuable research tool. The allergen dose producing an early asthmatic response (EAR) can be predicted from methacholine responsiveness and allergen skin test endpoint (STE). The Wright® jet nebulizer, which is both inefficient and increasingly difficult to obtain, has been used historically. We assessed the Solo® vibrating mesh nebulizer as an alternative for allergen and methacholine challenges. Methods Eighteen mild atopic asthmatics completed the study. Doubling concentration allergen prick skin tests were performed to determine the STE in allergen units/mL. The Wright® protocol was used to measure the methacholine provocation dose causing a 20% forced expired volume in one second (FEV1) fall (PD20) (μg) and the allergen PD20 (units). The Solo® protocol (0.5 mL nebulized to completion, tidal breathing inhalation) was used to determine both methacholine PD20 and allergen PD20. The nebulizer order was randomized and separated by ≥ 2 weeks. Results All data were log transformed. The allergen PD20, predicted from the methacholine PD20 and the STE, was within 2 doubling doses of the PD20 measured with the Wright® and 2.64 doubling doses of that measured with Solo®. The Wright® allergen PD20 correlated with the Wright® methacholine PD20 (r = 0.74) and the STE (r = 0.78) and more strongly with the product of the two (Wright® methacholine PD20 × STE, r = 0.91, p < 0.00001). The Solo® allergen PD20 showed similar relationships with the Solo® methacholine PD20 (r = 0.61), the STE (r = 0.75) and the product of the two (Solo® methacholine PD20 × STE, r = 0.83, p < 0.00002). The Wright® and the Solo® methacholine geometric mean PD20s were not significantly different (49.3 and 54.5 μg respectively, p = 0.62). The Wright® allergen PD20 was slightly but significantly lower than the Solo® allergen PD20 (geometric means 6.7 and 10.5 units respectively, p = 0.003). Conclusion The Solo® allergen PD20 showed the same relationship with methacholine responsiveness and STE as did the Wright®. The Solo® allergen PD20 was slightly but significantly higher than the Wright® allergen PD20. The Solo® vibrating mesh nebulizer was well tolerated and is an acceptable alternative for allergen challenge. Trial registration clinicaltrials.gov: NCT03491358


Background
Allergen inhalation challenge is a valuable research tool for the study of asthma pathophysiology and investigational new drug efficacy [1]. The early asthmatic response (EAR) to allergen depends on (non-allergic) airway responsiveness and the level of allergen-specific IgE [2]. It has been previously demonstrated that the concentration/dose of allergen required to produce a threshold EAR of a 20% decline in forced expired volume in one second (FEV 1 ) can be predicted within 2-3 concentrations using the level of airway responsiveness measured by methacholine or histamine provocation and the level of allergen specific IgE assessed by the allergen skin test endpoint titration (STE) [3]. A caveat for this prediction is the requirement for methacholine and allergen to be inhaled in the same fashion using the same type of nebulizer, calibrated to the same weight loss.
Historically, the Wright ® jet nebulizer (Roxon Medi-Tech, St. Leonard, QC) calibrated to run at a weight loss of 0.13 g/min with inhalation performed by two minutes of tidal breathing [4] has been used for both methacholine and allergen inhalation. The Wright ® nebulizer is inefficient (approximately 75% of weight loss is evaporation [5,6]), expensive, non-disposable, and increasingly difficult to acquire. The Aerogen Solo ® vibrating mesh nebulizer, referred to as the Solo ® throughout, (Aerogen Ltd, Galway Ireland) features no evaporation and has been validated for use in methacholine challenge testing [7,8]. The current study was designed to assess the Solo ® vibrating mesh nebulizer for use in the standardized allergen challenge protocol performed in AllerGen National Centres of Excellence (NCE) Clinical Investigator Collaborative (CIC) studies and to compare it to the current Wright ® jet nebulizer protocol.

Participants
Eligible participants had mild atopic asthma requiring only infrequent inhaled β 2 agonist, an FEV 1 > 70% predicted, and a methacholine provocation dose causing a 20% fall in FEV 1 (PD 20 ) ≤ 400 μg. Participants were non-smokers with < 10 pack year cumulative smoking history. Individuals who were pregnant, lactating, who had relevant allergen exposure or respiratory tract infection within the previous 4 weeks or who had significant medical conditions were excluded. Ethics approval was received from each study site and signed informed consent was obtained prior to study entry.

Study design
Participants attended the laboratory on 5 occasions. Visit 1 visit was to assess eligibility, to obtain signed consent, to perform baseline spirometry and screening allergen skin prick tests and from these select the best allergen for inhalation testing. The selected allergen was one which was clinically relevant to the participant and which produced a large (≥ 5 mm) wheal. The STE for the selected allergen was measured at Visit 2 to allow prediction of the starting allergen concentration for inhalation in conjunction with the methacholine response [3]. At Visits 2 and 3 the methacholine PD 20 and the allergen PD 20 were measured respectively both with either the Wright ® or the Solo ® nebulizer. After a minimum 2-week washout, at Visits 4 and 5 these challenges were repeated with the other nebulizer. The order of the nebulizers was randomized.

Skin test endpoint (STE) titration
The STE was determined as previously outlined [9]. Allergens (Omega Laboratories, Montreal QC) were dispensed in protein nitrogen units/mL, biologic allergen units/mL or allergen units/mL. For conformity the allergen dose was expressed in "units". Allergens were diluted two-fold from 1:8 to 1:1024 or beyond if required and the dilutions were labeled as the allergen concentration in units/mL. Duplicate skin prick tests were performed, the mean wheal diameter measured at 10 min, and the STE recorded as the weakest concentration (units/mL) causing a 2 mm mean wheal diameter.

Methacholine inhalation tests
The standard Wright ® nebulizer methacholine test [4,10] was done as follows. The nebulizer was calibrated to a weight loss of 0.13 g/min. Complete spirometry was initially measured in triplicate. Isotonic saline was then inhaled by tidal breathing for 2 min, and the FEV 1 (truncated manoeuvre to avoid fatigue) measured at 30 and 90 s. Doubling concentrations of methacholine were then inhaled in the same manner at 5 min intervals with FEV 1 repeated at 30 and 90 s The available concentrations ranged from 0.031 to 64 mg/mL; the starting concentration for an individual was selected based on previous testing if available or based on validated guidelines [10,11]. Inhalations were stopped when the FEV 1 had fallen ≥ 17% and the provocation concentration causing a 20% FEV 1 fall (PC 20 ) was interpolated from the last 2 data points [10] or extrapolated from the last data point [12]. The PC 20 was converted to a PD 20 (μg) based on several studies documenting that a Wright ® PC 20 of 16 mg/mL equates to a PD 20 of 400 μg [6-8, 13, 14]. The  20 and PD 20 > 16 mg/mL and > 400 μg respectively [15]. The Solo ® methacholine challenge was done as previously described [8]. Doubling doses of methacholine were delivered by nebulizing 0.5 mL of methacholine to completion and inhaling by tidal breathing; this requires 90 to 180 s [8]. A concentration of 2 mg/ mL × 0.5 mL × 0.4 (respiratory duty cycle [16]) exposes the individual to 400 μg. Following saline inhalation appropriate doubling concentrations up to 4 mg/mL (= 800 μg) were used. The remainder of the challenge (timing of FEV 1 measurements, time between doses, calculation of the PD 20 , etc.) was identical to the Wright ® method.

Allergen inhalation tests
Allergen inhalation tests were done as previously described using the Wright ® nebulizer [17]. Spirometry was measured in triplicate. Doubling concentrations of allergen were then inhaled (2 min of tidal breathing, nebulizer calibrated to a weight loss of 0.13 g/min and starting 3 or 4 concentrations below the predicted EAR concentration [3]) at 12 min intervals until the FEV 1 measured at 10 min after inhalation had fallen ≥ 15%. At an FEV 1 fall between 15 and 20% the FEV 1 was repeated 10 min later before giving another concentration if required. The allergen PC 20 (units/mL) was converted to allergen PD 20 (units) assuming a similar relationship as seen with methacholine. After PD 20 measurement, participants received a single inhaled dose of salbutamol 200 μg to reverse bronchoconstriction and a single inhaled dose of fluticasone propionate 500 μg to prevent development of the late asthmatic response [18].
The Solo ® allergen challenge was performed in an analogous manner. Assuming a similar relationship for dose comparison between the Solo ® and the Wright ® nebulizers seen with methacholine, the starting allergen concentration was 3 doubling concentrations below the starting concentration used for the Wright ® (i.e. 6-7 concentrations below the Wright ® prediction). The allergen, 0.5 mL, was nebulized to completion and inhaled by tidal breathing; the remainder of the challenge protocol was identical to the Wright ® protocol; the result was expressed as the allergen PD 20 in units.

Analysis
Statistics were done using a computerized statistics programme (Statistix 9 (Analytical Software, Tallahassee, FL, USA). PD 20

Results
Eighteen participants, all poylsensitized but with no current allergen exposure (except house dust mite), completed the study without adverse events. Three additional enrolled participants did not complete the study; one because the FEV 1 was < 70% at Visit 1, one because the methacholine PD 20 was > 400 μg at Visit 2, and one because of a failure to respond to allergen (1:32 with the Solo ® equating to ~ 1:4 with the Wright ® ) at Visit 3. Anthropometric data, baseline FEV 1 , baseline methacholine PD 20 and allergen used for challenges are shown in Table 1.

Solo ®
The measured Solo ® allergen PD 20 correlated with the predicted allergen PD 20 (r = 0.84, p = 0.000013) and was within 2 doubling does of the predicted allergen PD 20 in 14 of 18 and within 2.64 doubling doses in all 18 (Fig. 3). Similar to the Wright ® , the Solo ® allergen PD 20 correlated with both the Solo ® methacholine PD 20 (r = 0.61) and the STE (r = 0.75) and more strongly with the product of the 2 (r = 0.83, p = 0.00002) (Fig. 4).    Fig. 5. There was no sequence effect (i.e. nebulizer order) nor was there any difference between the three sites.

Discussion
These data indicate that the Solo ® vibrating mesh nebulizer can be successfully used for performance of allergen inhalation tests. Allergen responsiveness showed the same relationship with methacholine responsiveness and level of allergen sensitivity as was seen with the Wright ® nebulizer protocol. The measured Solo ® allergen PD 20 was within 2.64 doubling doses of the prediction. In 1987, an equation was developed to predict the dilution/concentration of allergen that would produce a 20% EAR [3]. This was based on the histamine PC 20 (mg/mL) and the allergen skin test endpoint (dilution) producing a 2 mm wheal. Both allergen and histamine were inhaled with 2 min of tidal breathing from a Wright ® nebulizer calibrated in the same manner. Methacholine was subsequently substituted for histamine in the same concentration since histamine and methacholine PC 20 s are identical in asthmatics [19]. In the original study, the equation successfully estimated the dilution required for an EAR within 2 doubling dilutions in 92% and 3 doubling dilutions in 100% of challenges [3]. The equation and a sample calculation are below: Log predicted allergen dilution = 0.68 log (methacholine PC 20 × STE dilution) For example with a methacholine PC 20 of 2.2 mg/mL and an STE of 1/1024. This prediction is routinely used as a guide for allergen challenge tests. A starting allergen dilution of 3 or occasionally 4 doubling dilutions below the prediction has proved a safe and effective method for allergen challenges performed in AllerGen NCE CIC and other studies. The purpose is to allow some test shortening when compared to methods advocating starting with the weakest allergen dilution causing a 2 mm wheal skin test response [20]. The current study validates this prediction equation when applied to the Wright ® data, since all measured values were within 2 doubling doses of the prediction. Despite the slightly higher measured Solo ® allergen PD 20 vs the Wright ® (i.e. slightly less responsive), 78% of values were within 2 doubling doses and 100% within 2.64 doubling doses. This would suggest that the 1987 prediction equation can be safely and effectively used (with modification for the units and nebulizer differences) until such time as there are enough data to develop a "Solo ® specific" equation.  The major strength of this study is the experienced group of investigators at the three sites. The one weakness is the inability to assess the solute output of the jet (Wright ® ) nebulizer. Based on the known evaporative features [5,6], and both breath simulation testing [6] and clinical challenge testing [7,8,12,13] it is reasonable to equate a methacholine PC 20 of 16 mg/mL to a methacholine PD 20 of 400 μg. The current study validates this, since using this conversion the Wright ® and Solo ® methacholine PD 20 s were essentially identical. However, data are currently lacking for nebulized allergen and it is possible that allergen solutions could be handled differently by the nebulizers.

Conclusion
In summary the Solo ® vibrating mesh nebulizer, proved to be a safe, effective and well tolerated device for administering inhaled allergen. This provides a valuable alternative to the Wright ® jet nebulizer.