ORIGINAL RESEARCH Provocative Dose of Methacholine Causing a 20% Drop in FEV1 Should Be Used to Interpret Methacholine Challenge Tests with Modern Nebulizers Sharon D. Dell1,2,3,4, Sundeep S. Bola1,3, Richard G. Foty1,2, Laura C. Marshall1,2, Kathleen A. Nelligan1,2, and Allan L. Coates1,3 1 Division of Respiratory Medicine, and 2Child Health Evaluative Sciences, The Hospital for Sick Children, Toronto, Ontario, Canada; and 3Department of Pediatrics, and 4Institute of Health, Policy, Management and Evaluation, University of Toronto, Toronto, Ontario, Canada
Abstract Rationale: The American Thoracic Society guidelines (1999) for methacholine challenge tests (MCTs) using the 2-minute tidal breathing protocol were developed for the now-obsolete EnglishWright (EW) nebulizer. In addition, the guideline recommendation to use the provocative concentration of methacholine causing a 20% drop in FEV1 (PC20) rather than the provocative dose of methacholine causing a 20% drop in FEV1 (PD20) for determining the level of bronchial hyperresponsiveness has been challenged. Objectives: To determine if cumulative dose or concentration of methacholine delivered to the airways is the determinant for airway responsiveness and to validate use of the AeroEclipse* II BAN (Aero; Trudell Medical International, London, ON, Canada) nebulizer compared with use of the reference standard EW nebulizer. Methods: Subjects with asthma (10–18 yr old) participated in randomized, controlled cross-over experiments comparing four MCT protocols using standard methacholine concentrations, but varying: (1) methacholine starting concentration (testing for cumulative effect); (2) nebulizer (EW versus Aero); and (3)
inhalation time. PD20 was calculated using nebulizer output rate, inhalation time, and preceding doses delivered. ANOVA analyses were used to compare geometric means of PC20 and PD20 between protocols. Results: A total of 32 subjects (17 male) participated. PC20 differed when starting concentration varied (0.46 vs. 0.80 mg/ml; P , 0.0001), whereas PD20 did not (0.06 vs. 0.08 mg). PC20 differed with the EW versus the Aero nebulzer with 30-second inhalation (1.19 vs. 0.43 mg/ml; P = 0.0006) and the EW versus the Aero nebulizer with 20-second inhalation (1.91 vs. 0.89 mg/ml; P = 0.0027), whereas PD20 did not (0.07 vs. 0.06 mg and 0.11 vs. 0.09 mg, respectively). Conclusions: In MCTs, the cumulative dose (PD 20 ), not the PC 20 , determines bronchial responsiveness. Modern nebulizers may be used for the test if clinical interpretation is based on PD 20 . Clinical trial registered with www.clinicaltrials.gov (NCT01288482). Keywords: asthma; bronchial hyperreactivity; methacholine; diagnosis; nebulizers
(Received in original form September 23, 2014; accepted in final form January 8, 2015 ) Supported by a competitive grant from the government of Ontario Health Technology eXchange program, which required matching funds from industry (Trudell Medical International, London, ON, Canada, which provided the AeroEclipse II nebulizer devices free of charge) and the sponsoring institution (The Hospital for Sick Children, Toronto, ON, Canada); the Hospital for Sick Children donated in-kind funds for the salaries of the investigators and indirect costs related to the conduct of the study. The study protocol was written and conducted, and data analyzed and interpreted, by the study investigators independently of Trudell and the government sponsor. Author Contributions: study concept and design—S.D.D. and A.L.C.; data management—R.G.F. and S.D.D.; acquisition of data—R.G.F., L.C.M., K.A.N., and S.S.B.; analysis and interpretation of data—S.D.D., R.G.F., S.S.B., and A.L.C.; drafting of the manuscript—S.S.B. and S.D.D.; critical revision of the manuscript for important intellectual content—all authors; statistical analysis—R.G.F.; obtained funding—S.D.D. and A.L.C.; administrative, technical, or material support, and study supervision—S.D.D., who had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Correspondence and requests for reprints should be addressed to Sharon D. Dell, M.D., 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: [email protected] Ann Am Thorac Soc Vol 12, No 3, pp 357–363, Mar 2015 Copyright © 2015 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201409-433OC Internet address: www.atsjournals.org
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ORIGINAL RESEARCH The methacholine challenge test (MCT) is the diagnostic test of choice for confirmation of asthma when clinical suspicion is high, but spirometry is ambiguous or normal (1, 2). Although its positive predictive value is less than optimal, a negative test on a symptomatic subject is highly predictive for the absence of asthma. The provocative concentration of methacholine causing a 20% drop in FEV1 from baseline is known as the PC20 (3). Sparse literature discusses the use of a provocative dose of methacholine causing a 20% drop in FEV1 (PD20), instead of PC20, to describe severity of bronchial hyperresponsiveness (4–10). The use of PD20 for histamine and methacholine is supported by evidence suggesting that dose, not concentration, of bronchoprovocation agent is important when assessing airway response (11, 12). The American Thoracic Society (ATS) 1999 guidelines for conducting and interpreting MCTs (3) describe two methods for methacholine delivery:
a 2-minute tidal breathing protocol using an English-Wright (EW) nebulizer (Roxon Medi-Tech, Montreal, PQ, Canada), and a five-breath “dosimeter” method using a Devilbiss 646 nebulizer (DeVilbiss HealthCare, Somerset, PA). The dosimeter method has the problem of underestimating bronchial response, particularly in subjects with mild asthma (13–18). The EW nebulizer has become obsolete, as it is an inefficient nebulizer with many undesirable characteristics. The EW produces aerosol continuously, even with an expiratory filter, so if the patient comes off the nebulizer to cough, as children frequently do, aerosol enters the environment, posing potential safety hazards for technicians (19, 20). The EW nebulizer also poses the theoretical risk of transmission of airborne pathogens if the subject “drools” back into the device. Consequently, many centers have either adopted newer devices not validated for equivalency to conduct a 2-minute tidal breathing protocol, or use the dosimeter AeroEclipse II 30 second (Aero30) protocol and Cumulative effect protocol*
English-Wright Nebulizer (EW) 2 minute protocol 0
Inhalation
1
Inhalation
2 min
Time (min)
2
Spirometry 3
AeroEclipse II 20 second (Aero20) protocol Inhalation
30 s
Spirometry
30s post inhalation
Repeat Spirometry
90 s post inhalation
90 s post inhalation
Next Inhalation . . .
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1.5 min post repeat spirometry
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5 Next Inhalation . . .
20 s
Spirometry
30s post inhalation
Repeat Spirometry
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30 s post inhalation Shake out nebulizer into garbage
Repeat Spirometry
6
method. The practice of using nebulizers not validated for equivalency to the EW could be problematic, as nebulizer output varies from device to device (21, 22). The AeroEclipse* II BAN (Aero; Trudell Medical International, London, ON, Canada) is a newer breath-actuated nebulizer, generating aerosol only during patient inspiration, thus minimizing drug loss to the environment. Using in vitro methods with a breath simulator, it has been estimated that the Aero would deliver the equivalent pulmonary dose of methacholine in 12 seconds compared with the reference standard 120-second EW protocol (23). A shortened MCT inhalation time would provide considerable economic efficiency in the pulmonary function laboratory, although potentially complicate matters if there is a cumulative effect (10, 24, 25), because, if total time of an MCT is reduced, the final inhalation may be increasingly influenced by effects from previous inhalations. In addition, a 12second protocol could be expected to cause
1.5 min post repeat spirometry
Shake out nebulizer into garbage
Next Inhalation . . .
2 min post repeat spirometry
Discard AeroEclipse in garbage at end of procedure
Figure 1. Methacholine challenge protocols: English-Wright (EW) is the current American Thoracic Society/European Respiratory Society reference standard. AeroEclipse* II BAN nebulizer (Aero) 20-second protocol (Aero20) and Aero 30-second protocol (Aero30) use a different nebulizer and inhalation time (20 and 30 s, respectively, vs. 2 min for the standard EW ). *Cumulative effect protocol uses the same procedures as the Aero30 protocol, except that the starting concentration of methacholine in the cumulative effect protocol is the final concentration of methacholine that had been used in the previous Aero30 test for each subject.
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ORIGINAL RESEARCH Table 1. Participant baseline characteristics (n = 32) Variable
Value
Mean age, yr (SD) Mean age of diagnosis, yr (SD) Male sex Median baseline FEV1, L (range)* Median baseline % predicted FEV1 (range)* Allergies i. Environmental ii. Food iii. Eczema iv. Rhinitis Medication use i. ICS (n = 32) a. ICS past 12 mo (n = 21) b. ICS past 2 wk (n = 21) ii. SABA (n = 32) a. SABA in past 12 mo (n = 27) b. SABA in past 2 wk (n = 27) iii. LTRA (n = 32) a. LTRA in past 12 mo (n = 3) b. LTRA in past 2 wk (n = 3) iv. Combination medication (n = 32)† a. Combination medication past 12 mo (n = 10) b. Combination medication past 2 wk (n = 10)
12.56 3.61 17 2.46 94.5%
(1.78) (2.06) (53%) (1.33–4.89) (65–114%)
24 6 15 24
(75%) (18.75%) (46.88%) (75%)
21 13 4 27 21 8 3 1 1 10 9 4
(65.63%) (61.9%) (19.05%) (84.38%) (77.78%) (29.63%) (9.38%) (33.33%) (33.33%) (31.25%) (90.00%) (40.00%)
Definition of abbreviations: ICS = inhaled corticosteroids; ICU = intensive care unit; LTRA = leukotriene receptor antagonist; SABA = short-acting b agonist. *With the reference standard English-Wright nebulizer protocol. † Combination medication was ICS plus long-acting b agonist.
increased variability in MCT results, as the average young adult would only take two to four breaths, resulting in a possible twofold difference of pulmonary deposition, depending on the number of breaths taken. Hence, shortened inhalation times for MCTs need to be carefully tested for equivalency to the reference standard. The primary objective of this study was to determine if cumulative dose or concentration of methacholine delivered to the airways is the determinant for airway responsiveness. The secondary objective was to determine the PC20 and PD20 of the Aero nebulizer with a modified, shortened
inhalation time MCT protocol compared with the reference standard EW protocol for the diagnosis of asthma in children. Some of this work was presented in the form of an abstract at a meeting of the ATS in San Francisco, California, May 18–23, 2012.
Methods Subjects
Subjects with known asthma were recruited from a previous community-based study (26), the ambulatory asthma clinics at The
Hospital for Sick Children (Toronto, ON, Canada), and from members of the general population responding to study advertisements. Inclusion criteria were: age 10–18 years; physician-diagnosed asthma; tidal breathing PC20 of 16 mg/ml or less using the EW reference standard protocol recommended by the ATS; FEV1 greater than 65% predicted; no respiratory tract infection or allergen exposure of 4 weeks or greater before participation; and stable asthma control with no change in inhaled corticosteroid dosing during the study period. Exclusion criteria were: preterm birth (more than 4 wk early of calculated date); chronic health conditions other than asthma; and smokers. Consent was obtained from the subject or a parent with patient assent, as appropriate. The study was approved by the Hospital for Sick Children research ethics board and registered at www.clinicaltrial. gov (NCT01288482). Study Design
A randomized, controlled cross-over design was used to compare PC20 and PD20 outcomes using different MCT protocols in the same subjects. Interventions included four different methacholine challenge protocols (Figure 1): i. EW protocol: standard 2-minute tidal breathing protocol with EW nebulizer, per ATS guidelines, using optional diluent step (3). ii. Aero nebulizer 30-second protocol (Aero30): differed from the EW protocol by using the Aero nebulizer and a 30-second methacholine inhalation time. This would be expected to deliver approximately three times more methacholine than the EW (26). The nebulizer was driven by a 50-psi source (8 L/min) tank.
Table 2. Provocative concentration of methacholine causing a 20% drop in FEV1 and provocative dose of methacholine causing a 20% drop in FEV1 geometric means for four different methacholine challenge test protocols
PC20 mg/ml (range)† PD20 mg (range)
EW (n = 32)
Aero30 (n = 16)
Cumulative Effect (n = 13)
Aero20 (n = 29)
ANOVA P Value*
1.81 (0.14–32.00) 0.10 (0.01–1.52)
0.43 (0.04–3.76) 0.06 (0.005–0.50)
0.80 (0.06–6.06) 0.08 (0.004–0.88)
0.89 (0.08–32.00) 0.09 (0.01–2.72)
0.0028 0.41
Definition of abbreviations: Aero = AeroEclipse II nebulizer; Aero20 = AeroEclipse II BAN nebulizer 20-second protocol; Aero30 = AeroEclipse II BAN nebulizer 30-second protocol; EW = English-Wright nebulizer; PC20 = provocative concentration of methacholine causing a 20% drop in FEV1; PD20 = provocative dose of methacholine causing a 20% drop in FEV1. *P value for overall differences between geometric means of the four protocols. † Negative methacholine challenge tests were set to PC20 = 32 mg/ml.
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ORIGINAL RESEARCH
A
B
p = 0.10 1
1
0.1 PD20 (mg)
PC20 (mg/ml)
p < 0.0001 10
0.1
0.01
0.01
0.001 AeroEclipse 30s
Cumulative Effect
AeroEclipse 30s
Cumulative Effect
Figure 2. Comparison of provocative concentration of methacholine causing a 20% drop in FEV1 (PC20) (A) and provocative dose of methacholine causing a 20% drop in FEV1 (PD20) (B), measured by AeroEclipse II BAN nebulizer 30-second tidal breathing protocol and the cumulative effect protocol in 13 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P , 0.0001), whereas use of PD20 (B) suggests equivalency (P = 0.10).
iii. Cumulative effect protocol: in a subset of patients, the Aero30 protocol was repeated and modified by starting the test in each subject with the final concentration of methacholine that had been used in the previous Aero30 test for that subject. A difference in the PC20 between the Aero30 protocol and the cumulative effect protocol in the same subject suggests that bronchial reactivity is dependent on the accumulating dose during a methacholine challenge rather than the final concentration of methacholine delivered. iv. Aero nebulizer 20-second protocol (Aero20): differed from Aero30 by using a 20-second methacholine inhalation time. The first 16 subjects completed EW and Aero30 protocols at two separate visits. Visits were separated by 24 hours to 2 weeks, at the same time of the day, and during a time of clinical stability. The order of tests was randomized. To determine the presence of a cumulative methacholine dosing effect, the first 13 subjects also completed the cumulative effect protocol at a third visit under the same visit conditions as the first two visits. Early data showed a lower PC20 with the Aero30 protocol versus the EW, so the next 29 subjects (16 new subjects and 12 original) completed the EW and Aero20 MCT protocols with the same visit conditions, again randomizing the order of protocols. Subjects withheld short-acting b agonists for 360
at least 8 hours and long-acting b agonists for at least 36 hours before all study visits. Calculation of PC20 and PD20 for Each MCT PC20 ¼ Antilog logCpre ðlog Cpost log CpreÞ3ð20 %FEV1 fallCpreÞ 1 ð%FEV1 fallCpost %FEV1 fallCpreÞ
where C is concentration of methacholine before and after the 20% fall, and %FEV1 fallCpre and %FEV1 fallCpost are the fall at the dose before and the dose after, respectively (3). The current dose of methacholine delivered at any given concentration was calculated using the known rate of output of each nebulizer (23) and multiplying by the given concentration and inhalation time. For a 16 mg/ml solution, the EW would deliver 0.19 mg/min to the lower airway, and the Aero would deliver 2.05 mg/min (23). The cumulative dose (D) delivered at any given inhalation was calculated by adding all preceding doses to the current dose. The PD20 was then calculated as follows: PD20 ¼ Antilog logDpre ðlog Dpost log DpreÞ3ð20 %FEV1 fallDpreÞ 1 ð%FEV1 fallDpost %FEV1 fallDpreÞ
Statistical Analysis
The PC20 and PD20 obtained from the EW, Aero20, Aero30, and cumulative effect
protocols were log transformed and geometric means were compared using first matched ANOVA (assuming a factorial block design with 32 subjects and four different MCT protocols), and then pairwise comparisons of MCT protocols with paired student t tests (where deemed appropriate based on ANOVA testing) to determine the effects of nebulizer, shortened inhalation time, starting dose, and concentration versus dose. To maximize statistical power in the analysis, negative methacholine tests were conservatively set to have a PC20 of 32 mg/ ml (18). All of the statistical analyses were done using SAS version 9.2 (SAS Institute Inc., Cary, NC).
Results The subjects included in the study were 32 children (17 male) aged 12.6 (61.78 SD) years (Table 1). Matched ANOVA analyses showed overall differences in PC20 between protocols (Table 2); therefore, subsequent analyses focused on pairwise comparisons. Testing for MCT Cumulative Effect: Aero30 versus Cumulative Effect Protocol
PC20 geometric means differed significantly between Aero30 and cumulative effect protocols (0.46 vs. 0.80, respectively; P , 0.0001). Most subjects (8 of 13) differed by one doubling-dose step, two subjects differed by two doubling-dose steps, and three subjects responded with the same PC20 (Figure 2A). In contrast, the calculated PD20, which takes into account accumulation of dose from all inhalations during the test, did not differ between the Aero30 versus cumulative effect protocol (0.06 vs. 0.08, respectively; P = 0.10; Figure 2B). Comparison of EW to Aero Nebulizer Outcomes: PC20 and PD20
PC20 geometric means differed significantly between EW and Aero30 protocols (1.19 vs. 0.43, respectively; P = 0.0006). Most (9 of 16) subjects responded earlier (i.e., had a lower PC20) with the Aero30 protocol by two or more doubling doses, four differed by one doubling dose, and three did not differ by at least one doubling dose. PC20 was lower with the Aero20 protocol in all but one subject (Figure 3A). In contrast, the calculated PD20 did not differ between the
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ORIGINAL RESEARCH
A
B
100
bronchial hyperreactivity (PC20 . 4 mg/ml with EW).
10
p = 0.0006
p = 0.6114
Discussion
1
PD20 (mg)
PC20 (mg/ml)
10
1
0.1
0.1
0.01
0.01
0.001 English-Wright
AeroEclipse 30s
English-Wright
AeroEclipse 30s
Figure 3. Comparison of PC20 concentration (A) and PD20 dose (B), measured by English-Wright 2-minute tidal breathing protocol and the AeroEclipse II BAN nebulizer 30-second protocol in 16 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P = 0.0006), whereas use of PD20 (B) suggests equivalency (P = 0.6114).
EW versus Aero30 protocols (0.07 vs. 0.06, respectively; P = 0.61; Figure 3B). Intraclass correlation coefficient for EW versus Aero30 protocols was 0.49 (95% confidence interval [CI], 0.04–0.78) for PC20 and 0.69 (95% CI, 0.34–0.87) for PD20. Similarly, comparison of PC20 geometric means between EW and Aero20 protocols in 29 subjects differed significantly (1.91 vs. 0.89, respectively; P = 0.0027), whereas PD20 (0.11 vs. 0.09, respectively; P = 0.36) did not. For the
p = 0.0027
B
p = 0.3555
100
10
10
1 PD20 (mg)
PC20 (mg/ml)
A
majority of subjects (21 of 29), PC20 was lower for the Aero20 versus the EW protocol (Figure 4). Intraclass correlation coefficient for EW versus Aero20 protocols was 0.63 (95% CI, 0.36–0.80) for PC20 and 0.73 (95% CI, 0.51–0.86) for PD20. Examination of Bland-Altman plots (Figure 5) confirmed the improved agreement between nebulizer protocols when the PD20 was used as the MCT outcome compared with the PC20, particularly for subjects with milder
1
0.1
We have demonstrated that PD20, but not PC20, has good agreement when MCTs are performed using different nebulizers, inhalation times, and starting concentrations of methacholine. Our results call into question the previous dogma of reporting PC20 for MCTs (3), and suggest that the cumulative PD20 is a superior standard with which to measure bronchial hyperreactivity. Although previous studies have compared the dosimeter method to the EW 2-minute tidal breathing protocol, no previous studies have compared results of the 2-minute tidal breathing protocol with different nebulizers. The novel proof of concept is that, as long as differences in nebulizer output are reconciled by calculating the dose delivered, the EW may be substituted with newer nebulizers. The corollary is that, if pulmonary function laboratories continue to use PC20 as the MCT outcome of choice (instead of PD20) while using newer, more efficient nebulizers, many more patients would be expected to be falsely diagnosed with bronchial hyperreactivity and asthma. Our study has relevant and timely clinical significance, as the EW nebulizer is no longer available for purchase.
0.1
0.01
0.01
0.001 English-Wright
AeroEclipse 20s
English-Wright
AeroEclipse 20s
Figure 4. Comparison of PC20 concentration (A) and PD20 dose (B), measured by English-Wright 2-minute tidal breathing protocol and the AeroEclipse II BAN nebulizer 20-second protocol in 30 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P = 0.0027), whereas use of PD20 (B) suggests equivalency (P = 0.3555).
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A
B Difference in PD20(mg) between EW and Aero20 Protocol
Difference in PC20(mg/ml) between EW and Aero20 Protocol
4 2 0 –2 –4 –6 –8 –10 0.1
1
10
100
Log Mean PC20(mg/ml) of EW and Aero20 Protocol
4 2 0 –2 –4 –6 –8 –10 0.001
0.01
0.1
1
Log Mean PC20(mg) of EW and Aero20 Protocol
Figure 5. Bland-Altman plot: difference between English-Wright (EW) and AeroEclipse 20-second protocol (Aero20) PC20 concentration (A) and PD20 dose (B) plotted against the mean of the two values in 25 of 29 subjects (negative tests excluded).
Our results agree with previous literature that suggests that inhalation time plays an important role during a bronchial provocation test (11, 12). Drotar and colleagues (11) demonstrated that, if you multiplied the inhalation time for methacholine by four, 2 minutes versus 30 seconds, that the 2-minute PC20 multiplied by four would be no different than the 30-second PC20. Cockcroft and Berscheid (12) demonstrated the same to be true for histamine in bronchial provocation. Both of these studies demonstrated that PC20 was a substandard determinant of bronchial response in that, if the inhalation time was changed, the PC20 becomes of limited value. Our results extend the findings of previous studies by providing an alternative and superior measure: the cumulative PD20. It has been previously demonstrated that histamine (8) and methacholine (25) exert a cumulative effect. That is to say that, for any given MCT, the effect seen at the time FEV1 drops by 20% is a function of not only the inhalation causing the drop, but also the previous accumulating inhalations. This finding is explained by previous work that demonstrates that methacholine can have a lingering bronchoconstrictive effect for up to 1–2 hours (27, 28). We have also demonstrated this to be true. When, however, cumulative PD20 was used to report the result of the MCT, we found that there was no statistically significant 362
difference between the cumulative PD20 that is calculated for the two nebulizers (EW and Aero). This would suggest that cumulative PD20 takes the cumulative effect into account, and is thus a more appropriate measure to report an MCT than the PC20. Our findings have the strongest clinical implications for ruling out mild asthma where an MCT with the EW reference standard may be expected to have a PC20 of 4–16 mg/ml (2). If a different nebulizer is substituted with either more or less dose output, one would expect to get false-positive or negative tests, respectively, if the dose difference is not accounted for. This is not an insignificant
problem, because it is precisely the setting for which the MCT is used in clinical practice. To provide meaning in a clinical setting, MCTs using different nebulizers must have defined implications for severity of bronchial hyperreactivity based on the PD20 values generated. For this reason, we have generated values of PD20 that correlate to the PC20 generated by the EW (Table 3). Limitations
We did not study an Aero protocol that was identical to the EW reference standard 120second inhalation protocol. Although it could have added to the strength of our
Table 3. Predicted methacholine dose delivered and provocative concentration of methacholine causing a 20% drop in FEV1 equivalents for standard English-Wright 2-minute tidal breathing methacholine challenge test PC20 (mg/ml) 1 2 4 8 16
Methacholine Dose Delivered* (mg)
PD20 Equivalent† (mg)
0.024 0.048 0.095 0.190 0.380
0.047 0.094 0.189 0.379 0.759
Definition of abbreviations: PC20 = provocative concentration of methacholine causing a 20% drop in FEV1; PD20 = provocative dose of methacholine causing a 20% drop in FEV1. *Methacholine dose delivered is calculated using in vitro performance characteristics of nebulizer output rate (0.012 ml/min 3 PC20 mg/ml) 3 inhalation time (2 min). † PD20 equivalent is calculated by adding up all preceding accumulating methacholine doses assuming the standard English-Wright protocol (starting concentration = 0.03 mg/ml).
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ORIGINAL RESEARCH findings by having an experiment that varied only nebulizer device, we did not want to exhaust our study subjects with an additional experiment, which we felt would be self-evident based on pre-existing literature. The in vitro data that informed our protocol design indicated that the Aero would deliver the same dose in 12 seconds as the EW in 2 minutes. Such a short inhalation time poses the problem that variability in pulmonary deposition of methacholine may occur related to small changes in respiratory rate. For this reason, we used protocol times of 20 and 30 seconds. In addition, the in vitro data from which we calculated our PD20 used a breath
simulator with a tidal volume of 0.75 L, respiratory rate of 15 breaths/min, and inspiratory time–to–total breath time ratio of 0.4, which will underestimate or overestimate dosing, depending on age and size of the subject. Our population was entirely children, who have less comorbidity than adults, and thus may be more homogeneous. The work should be reproduced in adults. Our study results should facilitate a paradigm shift in the MCT from a concentration-based test to a dose-based test. It is possible to continue using the same standard doubling doses of methacholine that are currently recommended and
References 1 National Heart, Lung, and Blood Institute. Expert panel report 3: guidelines for the diagnosis and management of asthma. NIH Publication Number 08-5846. Bethesda, MD: National Heart, Lung, and Blood Institute; 2007. 2 Lougheed MD, Lemiere C, Ducharme FM, Licskai C, Dell SD, Rowe BH, Fitzgerald M, Leigh R, Watson W, Boulet LP; Canadian Thoracic Society Asthma Clinical Assembly. Canadian Thoracic Society 2012 guideline update: diagnosis and management of asthma in preschoolers, children and adults. Can Respir J 2012;19:127–164. 3 Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, MacIntyre NR, McKay RT, Wanger JS, Anderson SD, 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, July 1999. Am J Respir Crit Care Med 2000;161:309–329. 4 Schulze J, Rosewich M, Riemer C, Dressler M, Rose MA, Zielen S. Methacholine challenge—comparison of an ATS protocol to a new rapid single concentration technique. Respir Med 2009;103:1898–1903. 5 Bennett JB, Davies RJ. A comparison of histamine and methacholine bronchial challenges using the DeVilbiss 646 nebulizer and the Rosenthal-French dosimeter. Br J Dis Chest 1987;81:252–259. 6 Siersted HC, Walker CM, O’Shaughnessy AD, Willan AR, Wiecek EM, Sears MR. Comparison of two standardized methods of methacholine inhalation challenge in young adults. Eur Respir J 2000;15:181–184. 7 Hagmolen of ten Have W, van den Berg NJ, van der Palen J, Bindels PJ, van Aalderen WM. Validation of a single concentration methacholine inhalation provocation test (SCIPT) in children. J Asthma 2005;42:419–423. 8 Connolly MJ, Avery AJ, Walters EH, Hendrick DJ. The use of sequential doses of inhaled histamine in the measurement of bronchial responsiveness: cumulative effect and distortion produced by shortening the test protocol. J Allergy Clin Immunol 1988;82:863–868. 9 Connolly MJ, Avery AJ, Walters EH, Hendrick DJ. The relationship between bronchial responsiveness to methacholine and bronchial responsiveness to histamine in asthmatic subjects. Pulm Pharmacol 1988;1:53–58. 10 Mariotta S, Sposato B, Ricci A, De Clementi F, Mannino F. Time intervals (39 or 59) between dose steps can influence methacholine challenge test. Lung 2005;183:1–11. 11 Drotar DE, Davis BE, Cockcroft DW. Dose versus concentration of methacholine. Ann Allergy Asthma Immunol 1999;83:229–230. 12 Cockcroft DW, Berscheid BA. Standardization of inhalation provocation tests: dose vs concentration of histamine. Chest 1982;82:572–575. 13 Prieto L, Lopez V, Llusar R, Rojas R, Marin J. Differences in the response to methacholine between the tidal breathing and dosimeter
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commercially available; however, the output rate of each nebulizer needs to be well characterized to calculate PD20 from PC20. Only then will it be possible to achieve comparable MCT results in pulmonary function laboratories around the world that use different nebulizers and inhalation times. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank Dr. Donald Cockcroft of the University of Saskatchewan for his advice on various aspects of our study design.
methods: influence of the dose of bronchoconstrictor agent delivered to the mouth. Chest 2008;134:699–703. Allen ND, Davis BE, Hurst TS, Cockcroft DW. Difference between dosimeter and tidal breathing methacholine challenge: contributions of dose and deep inspiration bronchoprotection. Chest 2005;128: 4018–4023. Cockcroft DW. Methacholine challenge methods. Chest 2008;134:678–680. Cockcroft DW, Davis BE. Deep inhalation bronchoprotection in asthma: correlation with airway responsiveness. J Allergy Clin Immunol 2006; 117:951–952. Prieto L, Ferrer A, Domenech J, Perez-Franc ´ es ´ C. Effect of challenge method on sensitivity, reactivity, and maximal response to methacholine. Ann Allergy Asthma Immunol 2006;97:175–181. Cockcroft DW, Davis BE, Todd DC, Smycniuk AJ. Methacholine challenge: comparison of two methods. Chest 2005;127:839–844. Shapiro GG, Simon RA; American Academy of Allergy and Immunology. Bronchoprovocation committee report. J Allergy Clin Immunol 1992;89:775–778. Hurst TS, Cockcroft DW, Hannah VD, Davis BE. A comparison of 2 methods of continuous aerosol administration during methacholine challenge testing. Respir Care 2006;51:46–48. Ward RJ, Reid DW, Leonard RF, Johns DP, Walters EH. Nebulizer calibration using lithium chloride: an accurate, reproducible and user-friendly method. Eur Respir J 1998;11:937–941. Massey DG, Miyauchi D, Fournier-Massey G. Nebulizer function. Bull Eur Physiopathol Respir 1982;18:665–671. Coates AL, Leung K, Dell SD. Developing alternative delivery systems for methacholine challenge tests. J Aerosol Med Pulm Drug Deliv 2014;27:66–70. Simard B, Turcotte H, Cockcroft DW, Davis BE, Boulay ME, Boulet LP. Deep inspiration avoidance and methacholine response in normal subjects and patients with asthma. Chest 2005;127:135–142. Juniper EF, Frith PA, Dunnett C, Cockcroft DW, Hargreave FE. Reproducibility and comparison of responses to inhaled histamine and methacholine. Thorax 1978;33:705–710. Dell SD, Foty RG, Gilbert NL, Jerret M, To T, Walter SD, Stieb DM. Asthma and allergic disease prevalence in a diverse sample of Toronto school children: results from the Toronto Child Health Evaluation Questionnaire (T-CHEQ) Study. Can Respir J 2010;17:e1–e6. Cartier A, Malo JL, Begin ´ P, Sestier M, Martin RR. Time course of the bronchoconstriction induced by inhaled histamine and methacholine. J Appl Physiol 1983;54:821–826. Mannino F, Sposato B, Ricci A, Grasso D, De Clementi F, Mariotta S. Induction and recovery phases of methacholine-induced bronchoconstriction using FEV1 according to the degree of bronchial hyperresponsiveness. Lung 2001;179:137–145.
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Abstract Rationale: The American Thoracic Society guidelines (1999) for methacholine challenge tests (MCTs) using the 2-minute tidal breathing protocol were developed for the now-obsolete EnglishWright (EW) nebulizer. In addition, the guideline recommendation to use the provocative concentration of methacholine causing a 20% drop in FEV1 (PC20) rather than the provocative dose of methacholine causing a 20% drop in FEV1 (PD20) for determining the level of bronchial hyperresponsiveness has been challenged. Objectives: To determine if cumulative dose or concentration of methacholine delivered to the airways is the determinant for airway responsiveness and to validate use of the AeroEclipse* II BAN (Aero; Trudell Medical International, London, ON, Canada) nebulizer compared with use of the reference standard EW nebulizer. Methods: Subjects with asthma (10–18 yr old) participated in randomized, controlled cross-over experiments comparing four MCT protocols using standard methacholine concentrations, but varying: (1) methacholine starting concentration (testing for cumulative effect); (2) nebulizer (EW versus Aero); and (3)
inhalation time. PD20 was calculated using nebulizer output rate, inhalation time, and preceding doses delivered. ANOVA analyses were used to compare geometric means of PC20 and PD20 between protocols. Results: A total of 32 subjects (17 male) participated. PC20 differed when starting concentration varied (0.46 vs. 0.80 mg/ml; P , 0.0001), whereas PD20 did not (0.06 vs. 0.08 mg). PC20 differed with the EW versus the Aero nebulzer with 30-second inhalation (1.19 vs. 0.43 mg/ml; P = 0.0006) and the EW versus the Aero nebulizer with 20-second inhalation (1.91 vs. 0.89 mg/ml; P = 0.0027), whereas PD20 did not (0.07 vs. 0.06 mg and 0.11 vs. 0.09 mg, respectively). Conclusions: In MCTs, the cumulative dose (PD 20 ), not the PC 20 , determines bronchial responsiveness. Modern nebulizers may be used for the test if clinical interpretation is based on PD 20 . Clinical trial registered with www.clinicaltrials.gov (NCT01288482). Keywords: asthma; bronchial hyperreactivity; methacholine; diagnosis; nebulizers
(Received in original form September 23, 2014; accepted in final form January 8, 2015 ) Supported by a competitive grant from the government of Ontario Health Technology eXchange program, which required matching funds from industry (Trudell Medical International, London, ON, Canada, which provided the AeroEclipse II nebulizer devices free of charge) and the sponsoring institution (The Hospital for Sick Children, Toronto, ON, Canada); the Hospital for Sick Children donated in-kind funds for the salaries of the investigators and indirect costs related to the conduct of the study. The study protocol was written and conducted, and data analyzed and interpreted, by the study investigators independently of Trudell and the government sponsor. Author Contributions: study concept and design—S.D.D. and A.L.C.; data management—R.G.F. and S.D.D.; acquisition of data—R.G.F., L.C.M., K.A.N., and S.S.B.; analysis and interpretation of data—S.D.D., R.G.F., S.S.B., and A.L.C.; drafting of the manuscript—S.S.B. and S.D.D.; critical revision of the manuscript for important intellectual content—all authors; statistical analysis—R.G.F.; obtained funding—S.D.D. and A.L.C.; administrative, technical, or material support, and study supervision—S.D.D., who had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Correspondence and requests for reprints should be addressed to Sharon D. Dell, M.D., 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: [email protected] Ann Am Thorac Soc Vol 12, No 3, pp 357–363, Mar 2015 Copyright © 2015 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201409-433OC Internet address: www.atsjournals.org
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ORIGINAL RESEARCH The methacholine challenge test (MCT) is the diagnostic test of choice for confirmation of asthma when clinical suspicion is high, but spirometry is ambiguous or normal (1, 2). Although its positive predictive value is less than optimal, a negative test on a symptomatic subject is highly predictive for the absence of asthma. The provocative concentration of methacholine causing a 20% drop in FEV1 from baseline is known as the PC20 (3). Sparse literature discusses the use of a provocative dose of methacholine causing a 20% drop in FEV1 (PD20), instead of PC20, to describe severity of bronchial hyperresponsiveness (4–10). The use of PD20 for histamine and methacholine is supported by evidence suggesting that dose, not concentration, of bronchoprovocation agent is important when assessing airway response (11, 12). The American Thoracic Society (ATS) 1999 guidelines for conducting and interpreting MCTs (3) describe two methods for methacholine delivery:
a 2-minute tidal breathing protocol using an English-Wright (EW) nebulizer (Roxon Medi-Tech, Montreal, PQ, Canada), and a five-breath “dosimeter” method using a Devilbiss 646 nebulizer (DeVilbiss HealthCare, Somerset, PA). The dosimeter method has the problem of underestimating bronchial response, particularly in subjects with mild asthma (13–18). The EW nebulizer has become obsolete, as it is an inefficient nebulizer with many undesirable characteristics. The EW produces aerosol continuously, even with an expiratory filter, so if the patient comes off the nebulizer to cough, as children frequently do, aerosol enters the environment, posing potential safety hazards for technicians (19, 20). The EW nebulizer also poses the theoretical risk of transmission of airborne pathogens if the subject “drools” back into the device. Consequently, many centers have either adopted newer devices not validated for equivalency to conduct a 2-minute tidal breathing protocol, or use the dosimeter AeroEclipse II 30 second (Aero30) protocol and Cumulative effect protocol*
English-Wright Nebulizer (EW) 2 minute protocol 0
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method. The practice of using nebulizers not validated for equivalency to the EW could be problematic, as nebulizer output varies from device to device (21, 22). The AeroEclipse* II BAN (Aero; Trudell Medical International, London, ON, Canada) is a newer breath-actuated nebulizer, generating aerosol only during patient inspiration, thus minimizing drug loss to the environment. Using in vitro methods with a breath simulator, it has been estimated that the Aero would deliver the equivalent pulmonary dose of methacholine in 12 seconds compared with the reference standard 120-second EW protocol (23). A shortened MCT inhalation time would provide considerable economic efficiency in the pulmonary function laboratory, although potentially complicate matters if there is a cumulative effect (10, 24, 25), because, if total time of an MCT is reduced, the final inhalation may be increasingly influenced by effects from previous inhalations. In addition, a 12second protocol could be expected to cause
1.5 min post repeat spirometry
Shake out nebulizer into garbage
Next Inhalation . . .
2 min post repeat spirometry
Discard AeroEclipse in garbage at end of procedure
Figure 1. Methacholine challenge protocols: English-Wright (EW) is the current American Thoracic Society/European Respiratory Society reference standard. AeroEclipse* II BAN nebulizer (Aero) 20-second protocol (Aero20) and Aero 30-second protocol (Aero30) use a different nebulizer and inhalation time (20 and 30 s, respectively, vs. 2 min for the standard EW ). *Cumulative effect protocol uses the same procedures as the Aero30 protocol, except that the starting concentration of methacholine in the cumulative effect protocol is the final concentration of methacholine that had been used in the previous Aero30 test for each subject.
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ORIGINAL RESEARCH Table 1. Participant baseline characteristics (n = 32) Variable
Value
Mean age, yr (SD) Mean age of diagnosis, yr (SD) Male sex Median baseline FEV1, L (range)* Median baseline % predicted FEV1 (range)* Allergies i. Environmental ii. Food iii. Eczema iv. Rhinitis Medication use i. ICS (n = 32) a. ICS past 12 mo (n = 21) b. ICS past 2 wk (n = 21) ii. SABA (n = 32) a. SABA in past 12 mo (n = 27) b. SABA in past 2 wk (n = 27) iii. LTRA (n = 32) a. LTRA in past 12 mo (n = 3) b. LTRA in past 2 wk (n = 3) iv. Combination medication (n = 32)† a. Combination medication past 12 mo (n = 10) b. Combination medication past 2 wk (n = 10)
12.56 3.61 17 2.46 94.5%
(1.78) (2.06) (53%) (1.33–4.89) (65–114%)
24 6 15 24
(75%) (18.75%) (46.88%) (75%)
21 13 4 27 21 8 3 1 1 10 9 4
(65.63%) (61.9%) (19.05%) (84.38%) (77.78%) (29.63%) (9.38%) (33.33%) (33.33%) (31.25%) (90.00%) (40.00%)
Definition of abbreviations: ICS = inhaled corticosteroids; ICU = intensive care unit; LTRA = leukotriene receptor antagonist; SABA = short-acting b agonist. *With the reference standard English-Wright nebulizer protocol. † Combination medication was ICS plus long-acting b agonist.
increased variability in MCT results, as the average young adult would only take two to four breaths, resulting in a possible twofold difference of pulmonary deposition, depending on the number of breaths taken. Hence, shortened inhalation times for MCTs need to be carefully tested for equivalency to the reference standard. The primary objective of this study was to determine if cumulative dose or concentration of methacholine delivered to the airways is the determinant for airway responsiveness. The secondary objective was to determine the PC20 and PD20 of the Aero nebulizer with a modified, shortened
inhalation time MCT protocol compared with the reference standard EW protocol for the diagnosis of asthma in children. Some of this work was presented in the form of an abstract at a meeting of the ATS in San Francisco, California, May 18–23, 2012.
Methods Subjects
Subjects with known asthma were recruited from a previous community-based study (26), the ambulatory asthma clinics at The
Hospital for Sick Children (Toronto, ON, Canada), and from members of the general population responding to study advertisements. Inclusion criteria were: age 10–18 years; physician-diagnosed asthma; tidal breathing PC20 of 16 mg/ml or less using the EW reference standard protocol recommended by the ATS; FEV1 greater than 65% predicted; no respiratory tract infection or allergen exposure of 4 weeks or greater before participation; and stable asthma control with no change in inhaled corticosteroid dosing during the study period. Exclusion criteria were: preterm birth (more than 4 wk early of calculated date); chronic health conditions other than asthma; and smokers. Consent was obtained from the subject or a parent with patient assent, as appropriate. The study was approved by the Hospital for Sick Children research ethics board and registered at www.clinicaltrial. gov (NCT01288482). Study Design
A randomized, controlled cross-over design was used to compare PC20 and PD20 outcomes using different MCT protocols in the same subjects. Interventions included four different methacholine challenge protocols (Figure 1): i. EW protocol: standard 2-minute tidal breathing protocol with EW nebulizer, per ATS guidelines, using optional diluent step (3). ii. Aero nebulizer 30-second protocol (Aero30): differed from the EW protocol by using the Aero nebulizer and a 30-second methacholine inhalation time. This would be expected to deliver approximately three times more methacholine than the EW (26). The nebulizer was driven by a 50-psi source (8 L/min) tank.
Table 2. Provocative concentration of methacholine causing a 20% drop in FEV1 and provocative dose of methacholine causing a 20% drop in FEV1 geometric means for four different methacholine challenge test protocols
PC20 mg/ml (range)† PD20 mg (range)
EW (n = 32)
Aero30 (n = 16)
Cumulative Effect (n = 13)
Aero20 (n = 29)
ANOVA P Value*
1.81 (0.14–32.00) 0.10 (0.01–1.52)
0.43 (0.04–3.76) 0.06 (0.005–0.50)
0.80 (0.06–6.06) 0.08 (0.004–0.88)
0.89 (0.08–32.00) 0.09 (0.01–2.72)
0.0028 0.41
Definition of abbreviations: Aero = AeroEclipse II nebulizer; Aero20 = AeroEclipse II BAN nebulizer 20-second protocol; Aero30 = AeroEclipse II BAN nebulizer 30-second protocol; EW = English-Wright nebulizer; PC20 = provocative concentration of methacholine causing a 20% drop in FEV1; PD20 = provocative dose of methacholine causing a 20% drop in FEV1. *P value for overall differences between geometric means of the four protocols. † Negative methacholine challenge tests were set to PC20 = 32 mg/ml.
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ORIGINAL RESEARCH
A
B
p = 0.10 1
1
0.1 PD20 (mg)
PC20 (mg/ml)
p < 0.0001 10
0.1
0.01
0.01
0.001 AeroEclipse 30s
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AeroEclipse 30s
Cumulative Effect
Figure 2. Comparison of provocative concentration of methacholine causing a 20% drop in FEV1 (PC20) (A) and provocative dose of methacholine causing a 20% drop in FEV1 (PD20) (B), measured by AeroEclipse II BAN nebulizer 30-second tidal breathing protocol and the cumulative effect protocol in 13 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P , 0.0001), whereas use of PD20 (B) suggests equivalency (P = 0.10).
iii. Cumulative effect protocol: in a subset of patients, the Aero30 protocol was repeated and modified by starting the test in each subject with the final concentration of methacholine that had been used in the previous Aero30 test for that subject. A difference in the PC20 between the Aero30 protocol and the cumulative effect protocol in the same subject suggests that bronchial reactivity is dependent on the accumulating dose during a methacholine challenge rather than the final concentration of methacholine delivered. iv. Aero nebulizer 20-second protocol (Aero20): differed from Aero30 by using a 20-second methacholine inhalation time. The first 16 subjects completed EW and Aero30 protocols at two separate visits. Visits were separated by 24 hours to 2 weeks, at the same time of the day, and during a time of clinical stability. The order of tests was randomized. To determine the presence of a cumulative methacholine dosing effect, the first 13 subjects also completed the cumulative effect protocol at a third visit under the same visit conditions as the first two visits. Early data showed a lower PC20 with the Aero30 protocol versus the EW, so the next 29 subjects (16 new subjects and 12 original) completed the EW and Aero20 MCT protocols with the same visit conditions, again randomizing the order of protocols. Subjects withheld short-acting b agonists for 360
at least 8 hours and long-acting b agonists for at least 36 hours before all study visits. Calculation of PC20 and PD20 for Each MCT PC20 ¼ Antilog logCpre ðlog Cpost log CpreÞ3ð20 %FEV1 fallCpreÞ 1 ð%FEV1 fallCpost %FEV1 fallCpreÞ
where C is concentration of methacholine before and after the 20% fall, and %FEV1 fallCpre and %FEV1 fallCpost are the fall at the dose before and the dose after, respectively (3). The current dose of methacholine delivered at any given concentration was calculated using the known rate of output of each nebulizer (23) and multiplying by the given concentration and inhalation time. For a 16 mg/ml solution, the EW would deliver 0.19 mg/min to the lower airway, and the Aero would deliver 2.05 mg/min (23). The cumulative dose (D) delivered at any given inhalation was calculated by adding all preceding doses to the current dose. The PD20 was then calculated as follows: PD20 ¼ Antilog logDpre ðlog Dpost log DpreÞ3ð20 %FEV1 fallDpreÞ 1 ð%FEV1 fallDpost %FEV1 fallDpreÞ
Statistical Analysis
The PC20 and PD20 obtained from the EW, Aero20, Aero30, and cumulative effect
protocols were log transformed and geometric means were compared using first matched ANOVA (assuming a factorial block design with 32 subjects and four different MCT protocols), and then pairwise comparisons of MCT protocols with paired student t tests (where deemed appropriate based on ANOVA testing) to determine the effects of nebulizer, shortened inhalation time, starting dose, and concentration versus dose. To maximize statistical power in the analysis, negative methacholine tests were conservatively set to have a PC20 of 32 mg/ ml (18). All of the statistical analyses were done using SAS version 9.2 (SAS Institute Inc., Cary, NC).
Results The subjects included in the study were 32 children (17 male) aged 12.6 (61.78 SD) years (Table 1). Matched ANOVA analyses showed overall differences in PC20 between protocols (Table 2); therefore, subsequent analyses focused on pairwise comparisons. Testing for MCT Cumulative Effect: Aero30 versus Cumulative Effect Protocol
PC20 geometric means differed significantly between Aero30 and cumulative effect protocols (0.46 vs. 0.80, respectively; P , 0.0001). Most subjects (8 of 13) differed by one doubling-dose step, two subjects differed by two doubling-dose steps, and three subjects responded with the same PC20 (Figure 2A). In contrast, the calculated PD20, which takes into account accumulation of dose from all inhalations during the test, did not differ between the Aero30 versus cumulative effect protocol (0.06 vs. 0.08, respectively; P = 0.10; Figure 2B). Comparison of EW to Aero Nebulizer Outcomes: PC20 and PD20
PC20 geometric means differed significantly between EW and Aero30 protocols (1.19 vs. 0.43, respectively; P = 0.0006). Most (9 of 16) subjects responded earlier (i.e., had a lower PC20) with the Aero30 protocol by two or more doubling doses, four differed by one doubling dose, and three did not differ by at least one doubling dose. PC20 was lower with the Aero20 protocol in all but one subject (Figure 3A). In contrast, the calculated PD20 did not differ between the
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ORIGINAL RESEARCH
A
B
100
bronchial hyperreactivity (PC20 . 4 mg/ml with EW).
10
p = 0.0006
p = 0.6114
Discussion
1
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PC20 (mg/ml)
10
1
0.1
0.1
0.01
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Figure 3. Comparison of PC20 concentration (A) and PD20 dose (B), measured by English-Wright 2-minute tidal breathing protocol and the AeroEclipse II BAN nebulizer 30-second protocol in 16 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P = 0.0006), whereas use of PD20 (B) suggests equivalency (P = 0.6114).
EW versus Aero30 protocols (0.07 vs. 0.06, respectively; P = 0.61; Figure 3B). Intraclass correlation coefficient for EW versus Aero30 protocols was 0.49 (95% confidence interval [CI], 0.04–0.78) for PC20 and 0.69 (95% CI, 0.34–0.87) for PD20. Similarly, comparison of PC20 geometric means between EW and Aero20 protocols in 29 subjects differed significantly (1.91 vs. 0.89, respectively; P = 0.0027), whereas PD20 (0.11 vs. 0.09, respectively; P = 0.36) did not. For the
p = 0.0027
B
p = 0.3555
100
10
10
1 PD20 (mg)
PC20 (mg/ml)
A
majority of subjects (21 of 29), PC20 was lower for the Aero20 versus the EW protocol (Figure 4). Intraclass correlation coefficient for EW versus Aero20 protocols was 0.63 (95% CI, 0.36–0.80) for PC20 and 0.73 (95% CI, 0.51–0.86) for PD20. Examination of Bland-Altman plots (Figure 5) confirmed the improved agreement between nebulizer protocols when the PD20 was used as the MCT outcome compared with the PC20, particularly for subjects with milder
1
0.1
We have demonstrated that PD20, but not PC20, has good agreement when MCTs are performed using different nebulizers, inhalation times, and starting concentrations of methacholine. Our results call into question the previous dogma of reporting PC20 for MCTs (3), and suggest that the cumulative PD20 is a superior standard with which to measure bronchial hyperreactivity. Although previous studies have compared the dosimeter method to the EW 2-minute tidal breathing protocol, no previous studies have compared results of the 2-minute tidal breathing protocol with different nebulizers. The novel proof of concept is that, as long as differences in nebulizer output are reconciled by calculating the dose delivered, the EW may be substituted with newer nebulizers. The corollary is that, if pulmonary function laboratories continue to use PC20 as the MCT outcome of choice (instead of PD20) while using newer, more efficient nebulizers, many more patients would be expected to be falsely diagnosed with bronchial hyperreactivity and asthma. Our study has relevant and timely clinical significance, as the EW nebulizer is no longer available for purchase.
0.1
0.01
0.01
0.001 English-Wright
AeroEclipse 20s
English-Wright
AeroEclipse 20s
Figure 4. Comparison of PC20 concentration (A) and PD20 dose (B), measured by English-Wright 2-minute tidal breathing protocol and the AeroEclipse II BAN nebulizer 20-second protocol in 30 subjects with known asthma. Bold horizontal lines indicate geometric means. When using PC20 to measure bronchial responsiveness (A), the difference in geometric means between protocols is statistically significant (P = 0.0027), whereas use of PD20 (B) suggests equivalency (P = 0.3555).
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A
B Difference in PD20(mg) between EW and Aero20 Protocol
Difference in PC20(mg/ml) between EW and Aero20 Protocol
4 2 0 –2 –4 –6 –8 –10 0.1
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Figure 5. Bland-Altman plot: difference between English-Wright (EW) and AeroEclipse 20-second protocol (Aero20) PC20 concentration (A) and PD20 dose (B) plotted against the mean of the two values in 25 of 29 subjects (negative tests excluded).
Our results agree with previous literature that suggests that inhalation time plays an important role during a bronchial provocation test (11, 12). Drotar and colleagues (11) demonstrated that, if you multiplied the inhalation time for methacholine by four, 2 minutes versus 30 seconds, that the 2-minute PC20 multiplied by four would be no different than the 30-second PC20. Cockcroft and Berscheid (12) demonstrated the same to be true for histamine in bronchial provocation. Both of these studies demonstrated that PC20 was a substandard determinant of bronchial response in that, if the inhalation time was changed, the PC20 becomes of limited value. Our results extend the findings of previous studies by providing an alternative and superior measure: the cumulative PD20. It has been previously demonstrated that histamine (8) and methacholine (25) exert a cumulative effect. That is to say that, for any given MCT, the effect seen at the time FEV1 drops by 20% is a function of not only the inhalation causing the drop, but also the previous accumulating inhalations. This finding is explained by previous work that demonstrates that methacholine can have a lingering bronchoconstrictive effect for up to 1–2 hours (27, 28). We have also demonstrated this to be true. When, however, cumulative PD20 was used to report the result of the MCT, we found that there was no statistically significant 362
difference between the cumulative PD20 that is calculated for the two nebulizers (EW and Aero). This would suggest that cumulative PD20 takes the cumulative effect into account, and is thus a more appropriate measure to report an MCT than the PC20. Our findings have the strongest clinical implications for ruling out mild asthma where an MCT with the EW reference standard may be expected to have a PC20 of 4–16 mg/ml (2). If a different nebulizer is substituted with either more or less dose output, one would expect to get false-positive or negative tests, respectively, if the dose difference is not accounted for. This is not an insignificant
problem, because it is precisely the setting for which the MCT is used in clinical practice. To provide meaning in a clinical setting, MCTs using different nebulizers must have defined implications for severity of bronchial hyperreactivity based on the PD20 values generated. For this reason, we have generated values of PD20 that correlate to the PC20 generated by the EW (Table 3). Limitations
We did not study an Aero protocol that was identical to the EW reference standard 120second inhalation protocol. Although it could have added to the strength of our
Table 3. Predicted methacholine dose delivered and provocative concentration of methacholine causing a 20% drop in FEV1 equivalents for standard English-Wright 2-minute tidal breathing methacholine challenge test PC20 (mg/ml) 1 2 4 8 16
Methacholine Dose Delivered* (mg)
PD20 Equivalent† (mg)
0.024 0.048 0.095 0.190 0.380
0.047 0.094 0.189 0.379 0.759
Definition of abbreviations: PC20 = provocative concentration of methacholine causing a 20% drop in FEV1; PD20 = provocative dose of methacholine causing a 20% drop in FEV1. *Methacholine dose delivered is calculated using in vitro performance characteristics of nebulizer output rate (0.012 ml/min 3 PC20 mg/ml) 3 inhalation time (2 min). † PD20 equivalent is calculated by adding up all preceding accumulating methacholine doses assuming the standard English-Wright protocol (starting concentration = 0.03 mg/ml).
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ORIGINAL RESEARCH findings by having an experiment that varied only nebulizer device, we did not want to exhaust our study subjects with an additional experiment, which we felt would be self-evident based on pre-existing literature. The in vitro data that informed our protocol design indicated that the Aero would deliver the same dose in 12 seconds as the EW in 2 minutes. Such a short inhalation time poses the problem that variability in pulmonary deposition of methacholine may occur related to small changes in respiratory rate. For this reason, we used protocol times of 20 and 30 seconds. In addition, the in vitro data from which we calculated our PD20 used a breath
simulator with a tidal volume of 0.75 L, respiratory rate of 15 breaths/min, and inspiratory time–to–total breath time ratio of 0.4, which will underestimate or overestimate dosing, depending on age and size of the subject. Our population was entirely children, who have less comorbidity than adults, and thus may be more homogeneous. The work should be reproduced in adults. Our study results should facilitate a paradigm shift in the MCT from a concentration-based test to a dose-based test. It is possible to continue using the same standard doubling doses of methacholine that are currently recommended and
References 1 National Heart, Lung, and Blood Institute. Expert panel report 3: guidelines for the diagnosis and management of asthma. NIH Publication Number 08-5846. Bethesda, MD: National Heart, Lung, and Blood Institute; 2007. 2 Lougheed MD, Lemiere C, Ducharme FM, Licskai C, Dell SD, Rowe BH, Fitzgerald M, Leigh R, Watson W, Boulet LP; Canadian Thoracic Society Asthma Clinical Assembly. Canadian Thoracic Society 2012 guideline update: diagnosis and management of asthma in preschoolers, children and adults. Can Respir J 2012;19:127–164. 3 Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, MacIntyre NR, McKay RT, Wanger JS, Anderson SD, 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, July 1999. Am J Respir Crit Care Med 2000;161:309–329. 4 Schulze J, Rosewich M, Riemer C, Dressler M, Rose MA, Zielen S. Methacholine challenge—comparison of an ATS protocol to a new rapid single concentration technique. Respir Med 2009;103:1898–1903. 5 Bennett JB, Davies RJ. A comparison of histamine and methacholine bronchial challenges using the DeVilbiss 646 nebulizer and the Rosenthal-French dosimeter. Br J Dis Chest 1987;81:252–259. 6 Siersted HC, Walker CM, O’Shaughnessy AD, Willan AR, Wiecek EM, Sears MR. Comparison of two standardized methods of methacholine inhalation challenge in young adults. Eur Respir J 2000;15:181–184. 7 Hagmolen of ten Have W, van den Berg NJ, van der Palen J, Bindels PJ, van Aalderen WM. Validation of a single concentration methacholine inhalation provocation test (SCIPT) in children. J Asthma 2005;42:419–423. 8 Connolly MJ, Avery AJ, Walters EH, Hendrick DJ. The use of sequential doses of inhaled histamine in the measurement of bronchial responsiveness: cumulative effect and distortion produced by shortening the test protocol. J Allergy Clin Immunol 1988;82:863–868. 9 Connolly MJ, Avery AJ, Walters EH, Hendrick DJ. The relationship between bronchial responsiveness to methacholine and bronchial responsiveness to histamine in asthmatic subjects. Pulm Pharmacol 1988;1:53–58. 10 Mariotta S, Sposato B, Ricci A, De Clementi F, Mannino F. Time intervals (39 or 59) between dose steps can influence methacholine challenge test. Lung 2005;183:1–11. 11 Drotar DE, Davis BE, Cockcroft DW. Dose versus concentration of methacholine. Ann Allergy Asthma Immunol 1999;83:229–230. 12 Cockcroft DW, Berscheid BA. Standardization of inhalation provocation tests: dose vs concentration of histamine. Chest 1982;82:572–575. 13 Prieto L, Lopez V, Llusar R, Rojas R, Marin J. Differences in the response to methacholine between the tidal breathing and dosimeter
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15 16
17
18 19
20
21
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commercially available; however, the output rate of each nebulizer needs to be well characterized to calculate PD20 from PC20. Only then will it be possible to achieve comparable MCT results in pulmonary function laboratories around the world that use different nebulizers and inhalation times. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank Dr. Donald Cockcroft of the University of Saskatchewan for his advice on various aspects of our study design.
methods: influence of the dose of bronchoconstrictor agent delivered to the mouth. Chest 2008;134:699–703. Allen ND, Davis BE, Hurst TS, Cockcroft DW. Difference between dosimeter and tidal breathing methacholine challenge: contributions of dose and deep inspiration bronchoprotection. Chest 2005;128: 4018–4023. Cockcroft DW. Methacholine challenge methods. Chest 2008;134:678–680. Cockcroft DW, Davis BE. Deep inhalation bronchoprotection in asthma: correlation with airway responsiveness. J Allergy Clin Immunol 2006; 117:951–952. Prieto L, Ferrer A, Domenech J, Perez-Franc ´ es ´ C. Effect of challenge method on sensitivity, reactivity, and maximal response to methacholine. Ann Allergy Asthma Immunol 2006;97:175–181. Cockcroft DW, Davis BE, Todd DC, Smycniuk AJ. Methacholine challenge: comparison of two methods. Chest 2005;127:839–844. Shapiro GG, Simon RA; American Academy of Allergy and Immunology. Bronchoprovocation committee report. J Allergy Clin Immunol 1992;89:775–778. Hurst TS, Cockcroft DW, Hannah VD, Davis BE. A comparison of 2 methods of continuous aerosol administration during methacholine challenge testing. Respir Care 2006;51:46–48. Ward RJ, Reid DW, Leonard RF, Johns DP, Walters EH. Nebulizer calibration using lithium chloride: an accurate, reproducible and user-friendly method. Eur Respir J 1998;11:937–941. Massey DG, Miyauchi D, Fournier-Massey G. Nebulizer function. Bull Eur Physiopathol Respir 1982;18:665–671. Coates AL, Leung K, Dell SD. Developing alternative delivery systems for methacholine challenge tests. J Aerosol Med Pulm Drug Deliv 2014;27:66–70. Simard B, Turcotte H, Cockcroft DW, Davis BE, Boulay ME, Boulet LP. Deep inspiration avoidance and methacholine response in normal subjects and patients with asthma. Chest 2005;127:135–142. Juniper EF, Frith PA, Dunnett C, Cockcroft DW, Hargreave FE. Reproducibility and comparison of responses to inhaled histamine and methacholine. Thorax 1978;33:705–710. Dell SD, Foty RG, Gilbert NL, Jerret M, To T, Walter SD, Stieb DM. Asthma and allergic disease prevalence in a diverse sample of Toronto school children: results from the Toronto Child Health Evaluation Questionnaire (T-CHEQ) Study. Can Respir J 2010;17:e1–e6. Cartier A, Malo JL, Begin ´ P, Sestier M, Martin RR. Time course of the bronchoconstriction induced by inhaled histamine and methacholine. J Appl Physiol 1983;54:821–826. Mannino F, Sposato B, Ricci A, Grasso D, De Clementi F, Mariotta S. Induction and recovery phases of methacholine-induced bronchoconstriction using FEV1 according to the degree of bronchial hyperresponsiveness. Lung 2001;179:137–145.
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