Airway hyperresponsiveness to mannitol and methacholine and exhaled nitric oxide: A random-sample population study

Airway hyperresponsiveness to mannitol and methacholine and exhaled nitric oxide: A random-sample population study Asger Sverrild, MD, Celeste Porsbjerg, MD, PhD, Simon Francis Thomsen, MD, PhD, and Vibeke Backer, MD, DMSc Copenhagen, Denmark Background: Studies of selected patient groups have shown that airway hyperresponsiveness (AHR) to mannitol is more specific than methacholine for the diagnosis of asthma, as well as more closely associated with markers of airway inflammation in asthma. Objective: We sought to compare AHR to mannitol and methacholine and exhaled nitric oxide (eno) levels in a nonselected population sample. Methods: In 238 young adults randomly drawn from the nationwide civil registration list in Copenhagen, Denmark, AHR to mannitol and methacholine, as well as levels of eno, were determined, and the association with asthma was analyzed. Results: In diagnosing asthma the specificity of methacholine and mannitol was 80.2% (95% CI, 77.1% to 82.9%) and 98.4% (95% CI, 96.2% to 99.4%), respectively, with a positive predictive value of 48.6% versus 90.4%, whereas the sensitivity was 68.6% (95% CI, 57.1% to 78.4%) and 58.8% (95% CI, 50.7% to 62.6%), respectively. In asthmatic subjects AHR to mannitol was associated with increased eno levels (positive AHR to mannitol: median, 47 ppb [interquartile range, 35-68 ppb]; negative AHR to mannitol: median, 19 ppb [interquartile range, 13-30 ppb]; P 5.001), whereas this was not the case for AHR to methacholine (median of 37 ppb [interquartile range, 26-51 ppb] vs 24 ppb [interquartile range, 15-39 ppb], P 5.13). Conclusion: In this random population sample, AHR to mannitol was less sensitive but more specific than methacholine in the diagnosis of asthma. Furthermore, AHR to mannitol was more closely associated with ongoing airway inflammation in terms of increased eno levels. (J Allergy Clin Immunol 2010;126:952-8.) Key words: Asthma, airway hyperresponsiveness, mannitol, methacholine, diagnosis, exhaled nitric oxide, inflammation, epidemiology, population From the Respiratory Research Unit, Department of Respiratory Medicine, Bispebjerg University Hospital. The Danish Agency for Science, Technology and Innovation, an institution under the Danish Ministry of Science, Technology and Innovation, supported the study with a 1-year scholarship. Pharmaxis provided the research team with an unrestricted grant, with which one of the research assistants was employed. Moreover, mannitol test kits were provided by Pharmaxis Ltd (Frenchs Forest, NSW, Australia). Disclosure of potential conflict of interest: C. Porsbjerg receives honoraria from Pharmaxis and receives research funding for the Danish Agency of Science and Technology. The rest of the authors have declared that they have no conflict of interest. Received for publication December 3, 2009; revised August 18, 2010; accepted for publication August 19, 2010. Available online October 13, 2010. Reprint requests: Celeste Porsbjerg, MD, PhD, Respiratory Research Unit, Department of Respiratory Medicine, Bispebjerg Hospital, Bispebjerg Bakke 23, 2400 Copenhagen NV, Denmark. E-mail: Porsbjerg@dadlnet.dk. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2010.08.028 Abbreviations used AHR: Airway hyperresponsiveness BPT: Bronchial provocation test eno: Exhaled nitric oxide IQR: Interquartile range RDR: Response-dose ratio ROC: Receiver operating characteristic Bronchial provocation tests (BPTs) have a higher sensitivity for the diagnosis of asthma than spirometry or reversibility testing, 1,2 but BPTs have been somewhat limited by being more technically challenging, being more time-consuming, and requiring more equipment. Furthermore, the traditional direct BPTs using histamine or methacholine have a relatively weak relationship with airway inflammation, 3,4 which might account for their relatively low specificity, with false-positive test results in nonasthmatic subjects. Histamine and methacholine act directly on the smooth muscle cells of the airways to cause bronchoconstriction, and airway hyperresponsiveness (AHR) to these agents might be seen in the absence of airway inflammation. 3,4 Direct BPTs are therefore less useful for confirming the presence of asthma with active airway inflammation. 5 In comparison, indirect BPTs, such as use of hypertonic saline and adenosine monophosphate, act through inducing release of bronchoconstricting mediators, such as histamine, prostaglandins, and leukotrienes, from inflammatory cells in the airways, and these tests have been shown to reflect airway inflammation better than direct BPTs. 4,5 However, practical issues, such as the need for specific equipment, have limited the use of indirect tests. An indirect BPT using mannitol powder has been developed that consists of a simple single-use test kit that has the practical advantages of being easy to use, being safe, and requiring less equipment. 6 We have previously shown a closer relationship between AHR to mannitol and markers of airway inflammation (sputum eosinophil percentage and exhaled nitric oxide [eno] level) compared with AHR to methacholine in a selected group of asthmatic subjects who were not receiving anti-inflammatory treatment. 7 This is a random-sample population study of AHR to mannitol and methacholine in teenagers and young adults. Data on the diagnostic properties of mannitol BPTs have recently been published, 8 and the present article compares the diagnostic validity of the mannitol BPT and the methacholine BPT and their relationship with eno level as a marker of airway inflammation in asthmatic and nonasthmatic subjects. METHODS Study design The study is a cross-sectional population study performed at the Respiratory Research Unit, Copenhagen University Hospital Bispebjerg, Denmark. The 952

J ALLERGY CLIN IMMUNOL VOLUME 126, NUMBER 5 SVERRILD ET AL 953 same data were used in the recently published characterization of the diagnostic properties of inhaled mannitol in asthmatic subjects. 8 A sample of 1000 young adults between the ages of 14 and 24 years was randomly drawn from the civil registration list. All subjects received a validated selfadministered asthma and rhinitis screening questionnaire with 20 questions adopted from the American College of Allergy, Asthma, and Immunology screening program extended with questions concerning tobacco consumption. 9 All participants attended 1 study visit, which included (in order of execution) measurement of eno at a rate of 50 ml/min, lung function measurement, a skin prick test, a BPT with inhaled mannitol, a BPT with methacholine, and a reversibility test to an inhaled b 2 -agonist. The results of the methacholine test have been shown not to be influenced by the performance of a previous mannitol test, 10 and this test was performed when the subjects had reached a level of 95% of their baseline FEV 1. In line with international guidelines on bronchial provocation, all participants were told to withhold use of antiasthma drugs before the examination. 11 This included use of inhaled corticosteroids within 12 hours before the visit. All participants completed a semistructured interview at the visit, including questions regarding respiratory symptoms, such as chest tightness, cough, wheezing, exercise-induced dyspnea, nocturnal symptoms, and rhinitis; atopic dermatitis; and familial predisposition to asthma, allergy, or both. A single investigator carried out all tests, measurements, and interviews. A group of nonresponders were contacted by telephone to test differences between responders and nonresponders. Further details on the study population, methods, and guidelines have previously been published. 8 Skin prick tests A skin prick test to 10 aeroallergens (birch [Betula species], grass [Phleum pratense] mugwort, horse, dog, cat [Felis domesticus], house dust mite [Der p 1 and Der f 2], and fungi [Alternaria and Cladosporium species; ALK-Abelló, Hørsholm, Denmark]) was performed according to the European Academy of Allergy and Clinical Immunology s recommendations. 17 Allergic sensitization was defined as a positive skin prick test response to at least 1 of these 10 aeroallergens. Asthma definition A respiratory specialist performed the evaluation to ensure consistent classification of the asthma diagnosis. 8 Interpretation of lung function tests and other paraclinical data followed current international guidelines. 18 A diagnosis of asthma was consistent with symptoms of asthma within the last 12 months in combination with either a eno level of greater than 30 ppb, a history of allergic rhinoconjunctivitis, dermatitis, a positive skin prick test response, a familial predisposition to atopic disease, nonallergic rhinoconjunctivitis, or an FEV 1 /forced vital capacity ratio of less than 75%. The examiner was blinded to the results of the mannitol and methacholine tests but had otherwise free access to the abovementioned clinical information. Subjects with clinical remission of disease for longer than 12 months were classified as having no asthma. The clinical diagnosis of asthma was not objectively confirmed because the results from the bronchial provocation challenges constituted the primary end point of the analysis. Spirometry Spirometry was performed according to European Respiratory Society recommendations. 12 FEV 1 and forced vital capacity were measured with a 7-L dry wedge spirometer (Vitalograph, Buckingham, United Kingdom) calibrated weekly. Mannitol BPT Dry-powder mannitol (Aridol; Pharmaxis LTd, Frenchs Forest, NSW Australia) was administered according to the recommendations of the manufacturer, and FEV 1 was recorded in line with current guidelines. 12 FEV 1 recorded after inhalation of a 0-mg placebo capsule constituted baseline lung function. The challenge was stopped at a decrease in FEV 1 of 15% or greater from baseline values or when the maximum cumulative dose of 635 mg had been administered. A positive challenge response was defined as a decrease in FEV 1 of at least 15% after inhalation of 635 mg of mannitol or less. Methacholine challenge Bronchial provocation with methacholine up to a cumulative dose of 8 mmol was performed with the Spira dosimeter (Spira Respiratory Care Center Ltd, Hämeenlinna, Finland) by using the dosimetric method previously described by Yan et al. 13 A positive challenge response was defined as a decrease in FEV 1 of at least 20% after inhalation of 8 mmolofmethacholineorless. The response-dose ratio (RDR) values for methacholine and mannitol were calculated as the percentage decrease in FEV 1 after the last dose divided by the cumulative dose in micromoles or milligrams. eno eno levels were measured online (rate, 0.05 L/s) with the Nitric Oxide Analyzer (NIOX; Aerocrine AB, Solna, Sweden) and according to American Thoracic Society guidelines. 14 In the overall assessment of whether the participant had asthma, a cutoff of 30 ppb was used (see the section on the diagnosis of asthma for more details). However, for the analysis of the relation between airway inflammation and AHR to mannitol and methacholine, a cutoff of 26 ppb for eno was chosen because this level has been shown to have the highest sensitivity and specificity for predicting a sputum eosinophil percentage of greater than 3%. 15,16 Statistical analysis Data were analyzed with SPSS version 17.0 (SPSS, Inc, Chicago, Ill). Data are reported as means (SDs) for normally distributed variables and as medians (interquartile ranges [IQRs]) for nonnormally distributed variables. For analysis of parametric data, x 2 tests, 2-sample t tests, and ANOVA were used, and the Fisher exact test, the Kruskal-Wallis test, and the Mann- Whitney U test were used for analysis of nonparametric data. A receiver operating characteristic (ROC) curve was constructed, plotting RDRs to mannitol and methacholine against the diagnosis of asthma. The overall accuracy of the test was measured as the area under the ROC curve. RDR values for mannitol and methacholine, as well as eno levels, were nonnormally distributed when assessed with Kolmogorov-Smirnoff analysis and were therefore log transformed. The correlation between the log RDR to mannitol and that to methacholine was analyzed with Pearson correlation analysis. After log transformation, log eno values were still not normally distributed (Kolmogorov-Smirnoff 5 0.04), and the Spearman correlation coefficient was therefore used to assess the correlation between the degree of airway responsiveness to mannitol and methacholine and the level of eno. The proportion of subjects with increased eno levels (>26 ppb) among asthmatic subjects with AHR to mannitol versus AHR to methacholine was compared by calculating the z value as follows: z5ðor mannitol OR methacholine Þ2=ð½SE mannitol Š2 1½SE methacholine Š2Þ 1; with OR defined as the odds ratio. To assess whether the relationship between AHR to mannitol and methacholine and eno level was independent of other factors that might normally influence the level of eno, logistic regression analysis was performed in the 51 asthmatic subjects, including factors that are known to potentially influence eno levels, such as age, height, sex, smoking, use of inhaled steroids, atopy, rhinitis, and AHR to mannitol and methacholine, as independent variables and an eno level of greater than 26 ppb as the dependent variable. Because of the limited number of subjects, the enter method was used, whereby all variables are entered into the model simultaneously. Because there was a significant association between the response to mannitol and methacholine, 2 separate regression analyses were performed, one including AHR to mannitol and one including AHR to methacholine.

954 SVERRILD ET AL J ALLERGY CLIN IMMUNOL NOVEMBER 2010 TABLE I. Baseline characteristics Current asthma (n 5 51) No asthma (n 5 187) P value Age (y), (median [minimum-maximum]) 18 (15-24) 19 (14-24).77 Sex (% female) 61% (31) 60% (113).87 Atopy 77% (39) 32% (60) <.0001 Smoking (current) 29% 24%.32 FEV 1 % predicted, mean (95% CI) 92% (89% to 95%) 94% (92% to 95%).32 FEV 1 /FVC ratio, mean (95% CI) 0.85 (0.82-0.87) 0.88 (0.87-0.89).001 Use of ICS 16% 0% <.0001 FVC, Forced vital capacity; ICS, inhaled corticosteroid. TABLE II. Results of 238 randomly selected adolescents tested with inhaled mannitol and methacholine: 51 asthmatic subjects and 187 nonasthmatic subjects TABLE III. Diagnostic properties of inhaled mannitol and methacholine in 238 randomly selected subjects Sensitivity Specificity PPV NPV Methacholine 69 (57-78) 80 (77-83) 49 (40-56) 90 (87-93) Mannitol 59 (51-63) 98 (96-99) 91 (78-97) 90 (88-91) Numbers are percentages (95% CIs). A positive mannitol test response was defined as a decrease in FEV 1 of 15% or greater at a cumulative dose of 635 mg or less, whereas a positive methacholine test response was defined as a decrease in FEV 1 of 20% or greater at a cumulative dose of 8 mmol or less. The mannitol data have already been published by Sverrild et al. 8 RESULTS Population characteristics Population characteristics are shown in Table I. Most asthmatic subjects had normal or near-normal lung function in terms of FEV 1 and FEV 1 /forced vital capacity ratio, although the percent predicted FEV 1 was slightly lower compared with that seen in nonasthmatic subjects. Most were nonsmokers, and the prevalence of smoking was comparable between asthmatic and nonasthmatic subjects. Atopy was observed in 77% of asthmatic subjects compared with 32% of nonasthmatic subjects. Current asthma was diagnosed in 51 (21%) of the 238 subjects participating in the study, most of whom had normal lung function (FEV 1 ). A further 12 subjects had previously had symptoms of asthma but had not experienced any symptoms within the past 12 months. Inhaled steroids were used by 16% at the time of the study, and an additional 31% (16/51) had previously used inhaled steroids, although only 1 had used them within the last 12 months. Diagnostic properties of mannitol versus methacholine The results of the 2 tests in all 238 subjects are shown in Tables II and III. As recently published, 7 the sensitivity and specificity of mannitol were 59% (95% CI, 51% to 63%) and 98% (95% CI, 96% to 99%), respectively, for a diagnosis of asthma, and the positive predictive value (PPV) and negative predictive value (NPV) were 91% (95% CI, 78% to 97%) and 90% (95% CI, 88% to 91%), respectively. In comparison, methacholine, using 8 mmol as the cutoff, had a sensitivity and specificity of 69% (95% CI, 57% to 78%) and 80% (95% CI, 77% to 83%), respectively, for a diagnosis of asthma, corresponding to a PPV of 49% (95% CI, 40% to 56%) and an NPVof 90% (95% CI, 87% to 93%). The differences in specificity and predictive values reflect that among nonasthmatic subjects, 37 (19.8%) responded to methacholine, whereas only 3 (1.6%) responded to mannitol. In asthmatic subjects the median RDRs for mannitol and methacholine were 0.026 (IQR, 0.014-0.094) and 10.8 (IQR, 1.8-61.7), respectively, whereas in nonasthmatic subjects the median RDRs for mannitol and methacholine were 0.005 (IQR, 0.002-0.009) and 1.0 (IQR, 0.54-2.08), respectively. Fig 1 shows an ROC curve of RDRs to methacholine and mannitol versus the diagnosis of asthma. The area under the curve of methacholine amounts to 84.9% (95% CI, 79.1% to 90.8%), and the area under the curve for mannitol to 89.1% (95% CI, 83.2% to 95.0%). Decreasing the cutoff of a positive methacholine test response to 1 mmol increased the specificity to 97.9% (95% CI, 95.6% to 99.1%) but with a simultaneous decrease in sensitivity to 43.1% (95% CI, 34.8% to 47.8%). Of the 51 subjects with asthma, 37 had experienced symptoms within the last 4 weeks. Using a 4-week cutoff instead of 12 months to define asthma, the sensitivity and specificity of methacholine were 70.3% and 77.1%, respectively, whereas the sensitivity and specificity of mannitol were 56.8% and 94.0%, respectively. Relationship between AHR to mannitol and methacholine Among asthmatic subjects, 26 responded to both mannitol and methacholine, 4 responded only to mannitol, and 9 responded only to methacholine. The Pearson correlation coefficient for log RDR mannitol and log RDR methacholine in asthmatic subjects was 0.73 (P <.001, Fig 2).

J ALLERGY CLIN IMMUNOL VOLUME 126, NUMBER 5 SVERRILD ET AL 955 FIG 1. ROC curve of the degree of AHR to mannitol and methacholine (RDR) for predicting a diagnosis of asthma. FIG 2. Scatter plot of log RDR to mannitol versus log RDR to methacholine in asthmatic subjects. Using inhaled mannitol to predict the outcome of a methacholine challenge, the sensitivity was reduced to 38.9% (95% CI, 32.3% to 42.7%), and the specificity was 97.0% (95% CI, 94.1% to 98.6%). Furthermore, PPV and NPV were 84.8% (95% CI, 70.6% to 93.2%) and 78.5% (95% CI, 76.2% to 79.9%), respectively. AHR and eno levels in subjects with current asthma The association between the degree of hyperresponsiveness and eno levels was similar for mannitol and methacholine, although slightly stronger for mannitol (Spearman rho: log RDR methacholine, 0.43; log RDR mannitol, 0.48; Fig 3). However, a positive response to the mannitol challenge test more clearly distinguished between asthmatic subjects with and without increased eno levels. An eno level of greater than 26 ppb was found in 70% of asthmatic subjects with AHR to mannitol compared with 57% of subjects with AHR to methacholine (P 5.007, Table II). Furthermore, subjects with AHR to mannitol had significantly higher levels of eno than subjects without AHR to mannitol (median eno level, 47 ppb [IQR, 35-62 ppb] vs 19 ppb [IQR, 13-30 ppb]), whereas the level of eno did not differ between subjects with and without AHR to methacholine (median eno level, 37 ppb [IQR, 26-51 ppb] vs 24 ppb [IQR, 15-39 ppb], P 5.13; Table IV). AHR and eno levels in nonasthmatic subjects Only 3 (1.6%) nonasthmatic subjects responded to mannitol; all 3 had an eno level of greater than 26 ppb, and 2 had previously had asthma (Table IV). In the 37 (20%) nonasthmatic subjects with AHR to methacholine, eno levels were slightly higher than in nonasthmatic subjects without AHR to methacholine (median eno level, 17 ppb vs 13 ppb; P 5.03; Table IV). Of these 37 subjects, 6 had previously had asthma, and 3 of them had an eno level of greater than 26 ppb. Another 2 subjects had eno levels greater than 26 ppb but no history of asthma. Regression analysis To control for the effect of other factors potentially influencing the level of eno, a logistic regression analysis, including data on sex, age, height, smoking, allergic sensitization, rhinitis, and steroid use, was performed, which showed that AHR to mannitol predicted an eno level of greater than 26 ppb independently of these factors (Table V). A similar logistic regression analysis including methacholine instead of mannitol showed that this was not the case for methacholine (Table V). DISCUSSION This study compares the diagnostic properties of inhaled mannitol and methacholine in a random population sample and the correlation with the inflammatory marker eno. We found that mannitol was significantly more specific in the diagnosis of asthma than methacholine because 98% of the subjects without asthma had a negative mannitol test response compared with 80% when using methacholine, and the respective PPVs were 91% for mannitol compared with 49% for methacholine. As expected based on previous comparisons of direct and indirect BPTs, methacholine was a more sensitive test because 69% of the asthmatic subjects had a positive test result compared with 59% with mannitol. Importantly, although decreasing the cutoff of methacholine from 8 to 1 mmol did increase the specificity of the methacholine test, it also decreased the sensitivity significantly. We have previously shown that asymptomatic subjects with AHR to methacholine selected from a random population study sample did not respond to mannitol. 19 The present study confirms that mannitol is more specific than methacholine in a random population sample.

956 SVERRILD ET AL J ALLERGY CLIN IMMUNOL NOVEMBER 2010 FIG 3. Scatter plot of log eno versus log RDR to mannitol (A) and log RDR to methacholine (B) in asthmatic subjects. Of the 187 subjects without asthma, 37 (19.8%) had a positive response to methacholine. The reasons other than asthma, which can explain AHR to methacholine, are many and well described (ie, technical, allergy, smoking, and normal variation). Background rates for asymptomatic AHR to methacholine are reported from less than 10% to greater than 40%, depending on selection and protocol. 20 Female subjects are in general more responsive than male subjects (possibly because of differences in airway caliber), and having 60% girls participating in the study, this might be a contributing factor to this outcome. However, it is also a well-described phenomenon that adolescents experiencing clinical remission in their asthma remain hyperresponsive with increased eno levels. 21 Six of the 37 nonasthmatic subjects with AHR to methacholine had a history of asthma, which was also the case for 2 of the 3 nonasthmatic subjects with positive responses to mannitol. The definition of asthma in this study was limited to those who had experienced any symptom of asthma within the past 12 months, and the subjects with previous asthma symptoms and current AHR might have been in remission but with an increased risk of experiencing relapse of symptoms later in life. However, the majority of nonasthmatic subjects with AHR to methacholine denied having any current or previous respiratory symptoms suggestive of asthma. Asthma, especially in young subjects, is often characterized by an intermittent course of disease. Using the epidemiologic definition of asthma (12 months) holds the potential risk of false-negative test results because of AHR returning to normal in periods of clinical remission. However, the diagnostic properties of the 2 tests, when using a 4-week restriction in experienced symptoms to define asthma, showed no appreciable changes compared with a 12-month cutoff. Methacholine has for many years been thought of as a highly sensitive test in the diagnosis of asthma. However, this is not supported by this study in that the sensitivity of methacholine was only slightly higher than that of mannitol. Furthermore, in a recently published phase III study by Anderson et al, 22 the sensitivity of methacholine was similar to that of mannitol. There might be several explanations for this. First, methacholine might in fact not be a highly sensitive test in population samples. This is supported by another study using 8 mmol as the cutoff against a physician s diagnosis of asthma, reporting an average sensitivity of 56% and a specificity of 86%. 23 Second, it has been argued that the dosimeter method for administrating methacholine shows a significantly reduced response in subjects with mild AHR compared with the tidal-breath method. 24 A third possibility is misclassification of disease, which is discussed in further detail below. We found responsiveness to mannitol and methacholine to correlate well, which tells us a high RDR to methacholine in general can be expected in subjects with high RDRs to mannitol. This is also the case in more selected groups of asthmatic subjects. 8 That the 2 tests are correlating is not surprising because both are thought to cover parts of the underlying hyperresponsiveness. However, as suggested by the eno findings in this study, it is unlikely the 2 tests cover the same elements of AHR and the underlying pathobiology. More defined subgrouping of asthmatic subjects and in-depth investigation of underlying mechanisms (ie, inflammatory and genotypic features) are needed in this field. Relationship between eno level as a marker of airway inflammation and AHR There was a similar association between the level of eno and the degree of AHR to mannitol, as well as to methacholine, in asthmatic subjects, but a positive mannitol test response more clearly differentiated between subjects with and without increased eno levels. Furthermore, whereas very few nonasthmatic subjects responded to mannitol, a significant number had AHR to methacholine with or without increased eno levels. The observation that the degree of responsiveness to both mannitol and methacholine were related to eno level might reflect that although the indirect stimuli are dependent on inflammatory cells, such as eosinophils or mast cells, being present and releasing sufficient amounts of bronchoconstricting mediators, there is also an indirect association between the response to methacholine and ongoing airway inflammation in

J ALLERGY CLIN IMMUNOL VOLUME 126, NUMBER 5 SVERRILD ET AL 957 TABLE IV. eno levels in asthmatic and nonasthmatic subjects with and without AHR to mannitol and methacholine Current asthma (n 5 51) No asthma (n 5 187) Mannitol Methacholine Mannitol Methacholine (n 5 21) (n 5 30) (n 5 16) (n 5 35) (n 5 184) (n 5 3) (n 5 150) (n 5 37) eno (ppb), median (IQR) 19 (13-30) 47 (35-62) 24 (15-39) 37 (26-51) 14 (13-15) 46 (10-214) 13 (12-41) 17 (13-21) P value.001.13 <.0001.03 eno >26 ppb, %(n) 29 (6) 70 (21) 44 (7) 57 (20) 11 (21) 100 (3) 11 (16) 22 (8) P value <.0001.07.01.07 terms of increased smooth muscle responsiveness to methacholine, as well as accessibility to the smooth muscle layer through disruption of the basal membrane. 25 Approximately 30% of asthmatic subjects with a positive response to mannitol had a normal eno level and might not have had airway eosinophilia. An explanation for this could be that the response to mannitol is mediated mainly through mast cells that are responsible for the main release of bronchoconstricting mediators, and a response to mannitol might therefore occur in the absence of eosinophils or an increased eno level. 26,27 One third of asthmatic subjects with a negative mannitol test response had an eno level of greater than 26 ppb. However, the median eno level of 19 ppb and upper IQR of 29 ppb in this group indicates that these subjects generally had relatively low eno levels. Comparatively, the group that did respond to the mannitol test had a median eno level of approximately 47 ppb, which is the eno level that has been shown to be optimal for predicting a response to steroid treatment. 28 Clinically, the interpretation of these findings is that in someone with a positive mannitol test response, airway inflammation is likely to be present, whereas the same assumption might not be based on a response to methacholine. Limitations In lack of an operating gold standard of asthma, a clinical diagnosis based on asthma symptoms, paraclinical data, atopic disease, and familial predisposition was used. This strategy holds several strengths, as well as weaknesses. Lack of accuracy is a potential bias in this study design because interobserver variability cannot be assessed. On the other hand, the asthma diagnosis is consistent and can be reproduced. A participation rate of 25% puts the study at risk of selection bias. However, a more detailed analysis of participants and a control group of nonresponders showed no differences in selfreported asthma, respiratory symptoms, and smoking, among others. 8 Altogether, there is no strong evidence that the study subjects were not representative of the population as a whole. The study population was relatively young and in general had a history of low tobacco consumption. This should be kept in mind when extrapolating these results to an older population who might have a longer history of asthma, a larger tobacco consumption, and other potential comorbidities, such as chronic obstructive pulmonary disease, which might all potentially have an effect on the relationship between AHR to direct versus indirect BPTs and eno levels because of airway remodeling, alteration of airway caliber, and elastic recoil, as well as an altered inflammatory response in smokers. 3,6 Regarding the use of eno as a marker of airway inflammation, eno levels have been shown to correlate with the degree of TABLE V. Determinants of eno: Logistic regression analysis, including AHR to mannitol and AHR to methacholine* Mannitol Methacholine OR 95% CI P value OR 95% CI P value Age (y) 0.9 0.7-1.1.19 0.9 0.7-1.1.15 Sex (male 5 1) 2.5 0.3-17.7.37 2.0 0.3-12.2.47 Height (cm) 1.1 0.9-1.2.24 1.1 0.9-1.2.32 Current smoker 0.8 0.2-3.6.75 0.8 0.2-3.4.77 Current ICS use 4.9 0.5-50.0.18 5.9 0.6-57.9.13 Atopy 2.3 0.3-16.7.41 2.6 0.4-15.3.30 Rhinitis 2.1 0.1-33.3.60 2.5 0.2-35.0.51 AHR to mannitol 5.3 1.3-21.0.02 AHR to methacholine 1.8 0.4-7.3.43 ICS, Inhaled corticosteroid; OR, odds ratio. *Because of the association between the response to mannitol and methacholine, 2 separate regression analyses were performed, including RDRs to mannitol and methacholine, respectively. airway eosinophilia in asthmatic subjects, the relationship is not linear, and eosinophilia might be present in spite of a normal eno levels and vice versa. 16 However, any limitations of eno level as a marker of inflammation would be expected to be similar in asthmatic subjects with AHR to mannitol or to methacholine, and we believe it is unlikely that they have any significant influence on the present findings. Other factors might be associated with increased levels of eno, such as allergic sensitization, rhinitis, smoking, age, sex, and height, but after controlling for these factors in a regression analysis, we found that there was still an independent relationship between eno level and AHR to mannitol. Taking these limitations into consideration, the present findings support the concept that, when compared with methacholine, the mannitol BPT is a more specific test for asthma with ongoing airway inflammation. This is also in keeping with previous observations that responsiveness to mannitol decreases after steroid treatment, with an associated improvement in symptom scores, 29,30 and that AHR to mannitol predicts asthma deterioration during steroid dose downtitration. 31 Apracticaladvantageofthe mannitol test is the ease of performance of the test, which, combined with the high positive predictive value for the diagnosis of asthma, might increase the use of BPTs in general. In conclusion, AHR to mannitol is more specific but less sensitive than AHR to methacholine in the diagnosis of asthma in an unselected group of young adults. Furthermore, AHR to mannitol was associated with increased levels of eno, whereas subjects without AHR to mannitol generally had normal eno levels. In comparison, eno levels did not differ significantly between subjects with and without AHR to methacholine.

958 SVERRILD ET AL J ALLERGY CLIN IMMUNOL NOVEMBER 2010 Clinical implications: AHR to mannitol was less sensitive but more specific than methacholine in the diagnosis of asthma and more closely associated with increased eno levels. Responsiveness to mannitol might be more reflective of asthma with ongoing airway inflammation. REFERENCES 1. Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, et al. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med 2000;161:309-29. 2. Backer V, Groth S, Dirksen A, Bach-Mortensen N, Hansen KK, Laursen EM, et al. Sensitivity and specificity of the histamine challenge test for the diagnosis of asthma in an unselected sample of children and adolescents. Eur Respir J 1991;4:1093-100. 3. Henriksen AH, Lingaas-Holmen T, Sue-Chu M, Bjermer L. Combined use of exhaled nitric oxide and airway hyperresponsiveness in characterizing asthma in a large population survey. Eur Respir J 2000;15:849-55. 4. Cockcroft DW, Davis BE. Diagnostic and therapeutic value of airway challenges in asthma. Curr Allergy Asthma Rep 2009;9:247-53. 5. Van Den Berge M, Meijer RJ, Kerstjens HA, de Reus DM, Koeter GH, Kauffman HF, et al. PC(20) adenosine 59-monophosphate is more closely associated with airway inflammation in asthma than PC(20) methacholine. Am J Respir Crit Care Med 2001;163:1546-50. 6. Anderson SD, Brannan J, Spring J, Spalding N, Rodwell LT, Chan K, et al. A new method for bronchial-provocation testing in asthmatic subjects using a dry powder of mannitol. Am J Respir Crit Care Med 1997;156:758-65. 7. Porsbjerg C, Brannan JD, Anderson SD, Backer V. Relationship between airway responsiveness to mannitol and to methacholine and markers of airway inflammation, peak flow variability and quality of life in asthma patients. Clin Exp Allergy 2008;38:43-50. 8. Sverrild A, Porsbjerg C, Thomsen SF, Backer V. Diagnostic properties of inhaled mannitol in the diagnosis of asthma: a population study. J Allergy Clin Immunol 2009;124:928-32, e1. 9. Winder JA, Nash K, Brunn JW. Validation of a life quality (LQ) test for asthma. Ann Allergy Asthma Immunol 2000;85:467-72. 10. Gade EB, Thomsen SF, Porsbjerg C, Backer V. The bronchial response to mannitol is attenuated by a previous methacholine test: but not vice versa. Clin Exp Allergy 2009;39:966-71. 11. Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, 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;161:309-29. 12. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. Eur Respir J 2005;26:319-38. 13. Yan K, Salome C, Woolcock AJ. Rapid method for measurement of bronchial responsiveness. Thorax 1983;38:760-5. 14. ATS workshop proceedings: exhaled nitric oxide and nitric oxide oxidative metabolism in exhaled breath condensate: executive summary. Am J Respir Crit Care Med 2006;173:811-3. 15. Shaw DE, Berry MA, Thomas M, Green RH, Brightling CE, Wardlaw AJ, et al. The use of exhaled nitric oxide to guide asthma management: a randomized controlled trial. Am J Respir Crit Care Med 2007;176:231-7. 16. Berry MA, Shaw DE, Green RH, Brightling CE, Wardlaw AJ, Pavord ID. The use of exhaled nitric oxide concentration to identify eosinophilic airway inflammation: an observational study in adults with asthma. Clin Exp Allergy 2005;35:1175-9. 17. Dreborg S. Skin tests used in type I allergy testing. Allergy 1989;44(suppl 10): 22-30. 18. Pellegrino R, Viegi G, Brusasco V, Crapo RO, Burgos F, Casaburi R, et al. Interpretative strategies for lung function tests. Eur Respir J 2005;26:948-68. 19. Porsbjerg C, Rasmussen L, Thomsen SF, Brannan JD, Anderson SD, Backer V. Response to mannitol in asymptomatic subjects with airway hyperresponsiveness to methacholine. Clin Exp Allergy 2007;37:22-8. 20. Hewitt DJ. Interpretation of the "positive" methacholine challenge. Am J Ind Med 2008;51:769-81. 21. van Den Toorn LM, Prins JB, Overbeek SE, Hoogsteden HC, de Jongste JC. Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyperresponsiveness. Am J Respir Crit Care Med 2000;162:953-7. 22. Anderson SD, Charlton B, Weiler JM, Nichols S, Spector SL, Pearlman DS, et al. Comparison of mannitol and methacholine to predict exercise-induced bronchoconstriction and a clinical diagnosis of asthma. Respir Res 2009;10:4. 23. James A, Ryan G. Testing airway hyperresponsiveness using inhaled methacholine or histamine. Respirology 1997;2:97-105. 24. Cockcroft DW, Davis BE. The bronchoprotective effect of inhaling methacholine by using total lung capacity inspirations has a marked influence on the interpretation of the test results. J Allergy Clin Immunol 2006;17:1244-8. 25. Moller GM, Overbeek SE, van Helden-Meeuwsen CG, Hoogsteden HC, Bogaard JM. Eosinophils in the bronchial mucosa in relation to methacholine dose response curves in atopic asthma. J Appl Physiol 1999;86:1352-6. 26. Porsbjerg C, Lund TK, Pedersen L, Backer V. Inflammatory subtypes in asthma are related to airway hyperresponsiveness to mannitol and exhaled NO. J Asthma 2009;46:606-12. 27. Brannan JD, Gulliksson M, Anderson SD, Chew N, Kumlin M. Evidence of mast cell activation and leukotriene release after mannitol inhalation. Eur Respir J 2003; 22:491-6. 28. Smith AD, Cowan JO, Brassett KP, Filsell S, McLachlan C, Monti-Sheehan G, et al. Exhaled nitric oxide: a predictor of steroid response. Am J Respir Crit Care Med 2005;172:453-9. 29. Brannan JD, Koskela H, Anderson SD, Chan HK. Budesonide reduces sensitivity and reactivity to inhaled mannitol in asthmatic subjects. Respirology 2002;7:37-44. 30. Koskela H, Hyvärinen L, Brannan JD, Chan H-K, Anderson SD. Sensitivity and validity of three bronchial provocation tests to demonstrate the effect of inhaled corticosteroids in asthma. Chest 2003;124:1341-9. 31. Leuppi JD, Salome CM, Jenkins CR, Anderson SD, Xuan W, Marks GB, et al. Predictive markers of asthma exacerbations during stepwise dose-reduction of inhaled corticosteroids. Am J Respir Crit Care Med 2001;163:406-12.