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Comparison of methacholine and mannitol bronchial provocation tests in workers with occupational asthma

Published:October 24, 2011DOI:https://doi.org/10.1016/j.jaci.2011.09.012
      To the Editor:
      Although a specific substance is identified as the cause for the onset of occupational asthma (OA), the removal of this substance does not induce a remission of the disease in the vast majority of subjects. The assessment of impairment/disability of OA after removal from exposure is currently performed in the province of Quebec by performing spirometry and identifying airflow limitation through airway hyperresponsiveness (AHR) to methacholine, and assessing the need for asthma medication 2 years after the initial diagnosis of OA.
      AHR can be assessed by performing bronchial provocation tests (BPTs) with direct (eg, methacholine) or indirect stimuli (ie, exercise, hypertonic saline, AMP, and mannitol). Methacholine acts directly on smooth muscle receptors to cause contraction and airway narrowing. In contrast, mannitol acts indirectly by causing mast cell release of mediators, which are potent bronchoconstrictor agents that act on sensitive airway smooth muscle to cause contraction and airway narrowing. We sought (1) to compare AHR assessed by using methacholine and mannitol in workers with previously diagnosed OA who had been removed from exposure for 2 years or more and (2) to compare the yield and quality of sputum produced after a mannitol BPT and after inhalation of hypertonic saline after a methacholine challenge.
      We performed a crossover study in workers who had been given a previous diagnosis of OA using specific inhalation challenges between 2000 and 2008 in our center and who were removed from exposure after the diagnosis of OA was made. These subjects were contacted to attend 2 separate visits scheduled 2 to 7 days apart in a random order during which a mannitol BPT and a methacholine BPT were performed. The subjects were asked to inhale increasing doses of dry powder mannitol (Aridol; Pharmaxis Ltd, Frenchs Forest, Australia) from 0 mg (empty capsule) to 160 mg (4 × 40-mg capsules). The test was considered positive if the cumulative dose of mannitol inducing a 15% decrease in FEV1 was 635 mg or less.
      • Anderson S.
      • Brannan J.
      • Spring J.
      • Spalding N.
      • Rodwell L.
      • Chan K.
      • et al.
      A new method for bronchial-provocation testing in asthmatic subjects using a dry powder of mannitol.
      Doubling concentrations of methacholine from 0 (diluent) up to 128 mg/mL were administered. The results were expressed as the PC20 value in noncumulative units.
      • Juniper E.
      • Cockcroft D.
      • Hargreave F.
      Histamine and methacholine inhalation tests.
      Two different cutoffs (4 and 16 mg/mL) were studied for defining a positive methacholine BPT result.
      • Crapo R.O.
      • Casaburi R.
      • Coates A.L.
      • Enright P.L.
      • Hankinson J.L.
      • Irvin C.G.
      • 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.
      Asthma control was assessed during each visit by using the validated Asthma Control Questionnaire.
      • Juniper E.F.
      • Bousquet J.
      • Abetz L.
      • Bateman E.D.
      Identifying “well-controlled” and “not well-controlled” asthma using the Asthma Control Questionnaire.
      A sputum sample was obtained after inhalation of increasing concentrations of nebulized hypertonic saline (3%, 4%, and 5%) after the methacholine BPT or during and immediately after the mannitol BPT.
      • Pizzichini E.
      • Pizzichini M.
      • Efthimiadis A.
      • Evans S.
      • Morris M.
      • Squillace D.
      • et al.
      Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements.
      The fraction of exhaled nitric oxide was measured with chemiluminescent analyzers (280i Sievers; GE Analytical Instruments, Boulder, Colo) before performing spirometry.
      ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005.
      The study was approved by the Hôpital du Sacré-Cœur de Montréal research ethics committee. All subjects provided written consent. Health Canada approved the use of dry powder mannitol (Aridol) in Canada for this research project (file no. 145 033).
      We used descriptive statistics to analyze demographic data. A χ2 test was used to compare the number of subjects with AHR based on mannitol or methacholine BPT results.
      The Student t test was used for between-groups comparison, whereas paired analyses were conducted for within-subjects comparison. The mannitol response-dose ratio (RDR)
      • Brannan J.D.
      • Anderson S.D.
      • Perry C.P.
      • Freed-Martens R.
      • Lassig A.R.
      • Charlton B.
      The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline.
      and the methacholine dose-response slope (DRS)
      • Burrows B.
      • Sears M.
      • Flannery E.
      • Herbison G.
      • Holdaway M.
      Relationships of bronchial responsiveness assessed by methacholine to serum IgE, lung function, symptoms, and diagnoses in 11-year-old New Zealand children.
      were calculated as previously described. Intraclass correlation coefficients and κ tests were used for assessing the agreement for sputum cell counts obtained after use of hypertonic saline or mannitol. Significance was accepted at a P value of .05 or less (IBM SPSS Statistical Software, version 19.0.0; IBM Corp, Somers, NY).
      Thirty subjects (mean age, 45.8 ± 11.6 years; 23 male subjects) were enrolled and underwent both mannitol and methacholine BPTs. The subjects had been given a diagnosis of OA 6.1 ± 2.2 years previously and had not been exposed to their offending agent (17 high-molecular-weight agents, 10 low-molecular-weight agents, and 3 unidentified agents) since then. Fifty percent of them were never smokers.
      Nine (30%) subjects had a positive mannitol BPT result, whereas 22 (73.3%) subjects had a PC20 value equal to or lower than 16 mg/mL, and 13 (43.3%) subjects had a PC20 value equal to or lower than 4 mg/mL. No subject with a PC20 value of greater than 16 mg/mL had a positive mannitol BPT result.
      Subjects with a positive mannitol BPT result had lower FEV1 (P = .01), more airflow limitation (P = .01), higher fraction of exhaled nitric oxide levels (P = .03), and higher sputum eosinophil counts (P = .02), as well as a clinically significantly higher Asthma Control Questionnaire score (although not statistically significant at P = .1) compared with values seen in subjects with a negative mannitol BPT result (Table I). There was a positive correlation between mannitol RDR and methacholine DRS (r = 0.6, P = .01), as well as between mannitol RDR and sputum eosinophil counts (rho = 0.4, P = .03), but not between methacholine DRS and sputum eosinophil counts (rho = 0.1, P = .3).
      Table IFunctional and inflammatory characteristics in subjects with positive and negative mannitol and methacholine BPT results
      MethacholineMannitol
      PC20 ≤4PC20 >4PD15 ≤635No PD15
      No.1317921
      Atopy, no. (%)12 (92.1)16 (94.1)8 (88.9)20 (95.2)
      Never smokers. no. (%)8 (53.3)7 (46.7)6 (66.7)9 (42.9)
      FEV1 (% predicted)85.9 ± 13.8101.8 ± 114.4
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      86.6 ± 12.6100.2 ± 12.4
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      FEV1/FVC73.4 ± 14.385.4 ± 12.2
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      70.9 ± 11.484.1 ± 13.0
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      RDR (%/mg)0.06 (0.025-0.12)0.004 (0.007-0.01)
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      0.11 (0.05-0.22)0.005 (0.002-0.01)
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      ACQ score1.4 ± 1.00.9 ± 1.21.6 ± 1.20.9 ± 1.0
      Feno (ppb)19.9 ± 13.716.5 ± 9.826.7 ± 14.116.0 ± 10.6
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      Sp Eos (%)2.5 (4.5)1.2 (2.5)2.0 (3.0)0.7 (2.0)
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      Sp Neu (%)35.0 (40.6)45.7 (83.8)26.7 (44.8)28.5 (35.8)
      Subjects receiving ICSs, no. (%)8 (61.5)5 (29.4)5 (55.5)8 (38.1)
      ACQ, Asthma Control Questionnaire; Feno, fraction of exhaled nitric oxide; FVC, forced vital capacity; ICS, inhaled corticosteroids; PD15, cumulative dose of mannitol inducing a 15% decrease in FEV1; Sp Eos, sputum eosinophil count; Sp Neu, sputum neutrophil count.
      P ≤ .05 between subjects with positive and negative mannitol and methacholine BPT results.
      The success rate for obtaining valid sputum samples was high and similar with hypertonic saline (96.7%) and the mannitol BPT (100%). There was a lower cell viability and greater squamous cell contamination with the mannitol BPT than with hypertonic saline. There were marked differences between the total and differential counts, as well as with the inflammatory phenotypes obtained by using both methods (Table II).
      Table IIResults of sputum cell counts obtained after mannitol and hypertonic saline
      Hypertonic salineMannitolP valueAgreement
      Sputum production, no.2930
       Total cell counts (106/g)2.1 (4.4)1.6 (2.1).020.5 (.02)
      Intraclass correlation coefficient (P value).
       Eosinophils (%)1.5 (3.13)1.5 (2.3).80.4 (.09)
      Intraclass correlation coefficient (P value).
       Neutrophils (%)43.7 (31.7)27.6 (41.9).010.8 (<.01)
      Intraclass correlation coefficient (P value).
       Macrophages (%)42.2 (22.5)54.5 (31.9).20.6 (.04)
      Intraclass correlation coefficient (P value).
       Lymphocytes (%)1.25 (1.75)0.7 (1.3).30.8 (<.01)
      Intraclass correlation coefficient (P value).
       Bronchial cells (%)3.7 (8.1)8.4 (10.2).020.7 (.02)
      Intraclass correlation coefficient (P value).
       Viability (%)80.3 (16.3)70.7 (20.4).0060.8 (<.01)
      Intraclass correlation coefficient (P value).
       Squamous cells (%)3.4 (9.9)15.7 (32.1).0010.4 (.07)
      Intraclass correlation coefficient (P value).
      Inflammatory phenotypes0.2 (.1)
      κ Test (P value).
       Eosinophilic8 (27.6)8 (26.7)0.1 (.9)
      κ Test (P value).
       Neutrophilic6 (20.7)5 (16.7)0.7 (<.001)
      κ Test (P value).
       Paucigranulocytic15 (51.7)18 (60.0)0.1 (.6)
      κ Test (P value).
       Mixed01 (3.3)
      Intraclass correlation coefficient (P value).
      κ Test (P value).
      This study showed that although mannitol BPT results were positive in the minority of subjects (30%), those with a positive mannitol BPT result had more active disease in terms of greater airflow limitation and more eosinophilic inflammation and higher exhaled nitric oxide levels compared with values seen in subjects with a negative mannitol BPT result. Therefore the mannitol BPT might be more relevant than the methacholine BPT for assessing the impairment/disability of subjects with a previous diagnosis of OA. However, the mannitol BPT was not sensitive for identification of AHR to methacholine in this population. The mannitol BPT was able to successfully induce sputum for obtaining valid sputum cell counts. The quality of sputum samples was better with hypertonic saline than with mannitol but still of good quality after using mannitol. There was a significant discrepancy between the cell counts obtained with hypertonic saline and mannitol for sputum induction. Furthermore, there was a substantial difference between the classifications in the different inflammatory phenotypes between the 2 methods, as shown by the low agreement of the eosinophil (κ = 0.1) and paucigranulocytic (κ = 0.1) phenotypes between the methacholine and mannitol BPTs (Table II). These results differ somewhat, with previous results showing similar eosinophil and neutrophil cell counts after hypertonic saline and mannitol.
      • Wood L.G.
      • Powell H.
      • Gibson P.G.
      Mannitol challenge for assessment of airway responsiveness, airway inflammation and inflammatory phenotype in asthma.
      The protocols used to administer hypertonic saline differed significantly between the 2 studies, which might explain these differences.
      In conclusion, the mannitol BPT is a useful test for assessing the impairment/disability and disease activity of workers with a previous diagnosis of OA because this test has the ability to differentiate the subjects according to the severity of their disease. The mannitol BPT allows the assessment of airway responsiveness and collection of sputum to be made at the same time. However, the sputum cell counts obtained with the mannitol BPT or with hypertonic saline substantially differ.

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