This study included a total of 340 children who visited Severance Children's Hospital between July 2006 and August 2011. The children were divided into 3 groups: those who had atopic asthma and AR (asthma group, n=112), those who had AR without asthma (AR group, n=196) and those who had neither asthma nor AR (control group, n=32). Atopic asthma was defined as a Δ FEV1≥12% in response to a short-acting bronchodilator or BHR in the MCT (PC20≤16 mg/mL) accompanied by any typical asthma symptoms, such as cough or dyspnea.16 Of the 196 children in the AR group, 24 had BHR in the MCT (PC20≤16 mg/mL) without any typical asthma symptoms, such as cough or dyspnea, and the remaining 172 did not have BHR in the MCT (PC20>16 mg/mL). The control group showed normal spirometric results but had neither a history of respiratory disease, parenchymal lung abnormalities, BHR, nor BDR. We excluded non-atopic subjects based on allergy screening tests as well as subjects with asthma and without AR. Subjects had no history of respiratory infection for at least 6 weeks prior to the evaluation. We excluded subjects who had taken inhaled or systemic corticosteroids or leukotriene antagonists within 8 weeks prior to the study. All subjects were instructed to discontinue their β₂-agonist medications for at least 3 days prior to pulmonary function and methacholine challenge tests.

Serum total and specific immunoglobulin E (IgE) levels and peripheral blood eosinophil counts were measured, and skin prick tests were performed at the initiation of the evaluation. IOS and spirometry were performed at the second visit, with IOS being performed prior to spirometry in order to avoid any influence of forced expiratory maneuvers on airway function during breathing at rest. Finally, FeNO was measured before the MCT and the MCT was performed at the third visit because repetitive exhalations of methacholine could decrease the FeNO levels. This study was approved by the Institutional Review Board of Severance Hospital (Seoul, Korea).

Total and specific serum IgE levels were measured using the Pharmacia CAP assay (Uppsala, Sweden). A specific IgE test was performed for 6 allergens commonly encountered in Korea: Dermatophagoides pteronyssinus, Dermatophagoides farina, egg white, cow milk, German cockroach, and Alternaria alternata. Atopy was defined as IgE levels greater than 0.7 kU/L that were specific to more than 1 allergen or total IgE levels greater than 150 IU/mL. Atopy was also defined as a positive skin reaction to more than 1 out of the 12 common aeroallergens, including 2 types of dust mites, cat and dog epithelium, as well as mold and pollen allergens, such as Alternaria, Aspergillus, birch, oak, mugwort, Japanese hop, ragweed, and bermuda grass. A saline solution was used as a negative control and a 0.5% histamine HCL (hydrochloric acid) solution was used as a positive control. Wheal diameters were measured after 15 min; a positive reaction was defined as a wheal diameter greater than 3 mm.17 All subjects had atopy according to these criteria.

A Jaeger MasterScreen PFT (pulmonary function test) system (Jaeger Co, Wurzberg, Germany) was used. Flow-volume curves were obtained before and after bronchodilator (BD) inhalation according to the American Thoracic Society guidelines.18 The MCT was performed according to standardized procedures.16 Each child inhaled increasing concentrations of methacholine (0.075, 0.15, 0.31, 0.62, 1.25, 2.5, 5, 10, 25, and 50 mg/mL) nebulized by a dosimeter (MB3; Mefar; Brescia, Italy) until FEV1 was reduced by 20% from a post-nebulized saline solution value. BHR was expressed as the provocative concentration of methacholine causing FEV1 to decrease by 20% (PC20, mg/mL). This was calculated by linear interpolation of the log dose-response curve.

FeNO was measured using a CLD 88 (Eco Medics, Duernten, Switzerland) at a constant expiratory flow rate of 50 mL/s. The measurements were made according to the ERS/ATS guidelines.19 Since nitrate-rich foods could affect FeNO levels, all children refrained from eating nitrate-rich foods for 2 h before measurement of FeNO. The mean value of the 3 consecutive measurements was calculated and regarded as the actual value.

The Jaeger MasterScreen IOS system (Jaeger Co, Wurzberg, Germany) was used to perform impulse oscillometry. Measurements were made according to the ERS/ATS guidelines and repeated just before spirometry after bronchodilator administration.20 The system was calibrated through a single volume of air (3 L) at different flow rates, with a reference device (0.2 kPa/L/s). The machine was also calibrated to the air temperature and pressure of the saturated gas. The impulse generator produced brief pressure pulses at intervals of 0.2 sec. In this study, mean resistance (R) values were calculated over a measurement period of 60 sec at frequencies of 5 Hz (R5) and 10 Hz (R10). Reactance (X) values were measured at 5 Hz (X5). We also recorded reactance area (AX), an integrated response index for reactance developed by Goldman,20 which reflects the integral of the negative values of reactance from 5 Hz to the resonant frequency. R5 reflects obstruction in the total airways, R20 only reflects the large airways, and the difference between R5 and R20 (R5-R20) is a parameter of the small airway alone. X5 and AX also reflect the degree of obstruction in the peripheral airways.21

During IOS, children sat upright with their heads resting against the back of the chair. They used nose clips and were instructed to breathe quietly through a mouthpiece. To decrease the shunt compliance of the cheeks, an investigator stood behind the patient and supported both the cheeks and the chin with their hands. Observations did not show any artifacts caused by coughing, breath holding, swallowing, or vocalization. Three correct measurements were averaged at each time point. We used acceptable coherence values of ≥0.8 at 10 Hz.21

The values with a parametric distribution in the text and tables are expressed as mean±SD. Values with a non-parametric distribution are expressed as median with interquartile ranges. Patients' characteristics, spirometric results, FeNO levels, and IOS values were evaluated using analysis of variance (ANOVA) for comparison of the 3 groups, using t tests or Fisher's exact tests for comparison of 2 groups if the data were parametric and continuous, and using the Kruskal-Wallis test and the Mann-Whitney test if the data were non-parametric and continuous. If the data were categorical variables, we used chi-square tests. We also compared the AR group with BHR to that without. Correlation coefficients between FeNO and PC20 levels were calculated with Pearson product moment correlation.

A P value of <0.05 was considered statistically significant. The Statistical Package for the Social Sciences (version 18.0, SPSS Inc., Chicago, IL, USA) was used for all analyses.

The clinical characteristics of the patients in this study are shown in Table 1. The 3 groups did not differ in terms of age, sex, and height. Serum total IgE levels and blood eosinophil counts were significantly higher in the AR and asthma groups than in the control group (P<0.05). Skin tests were performed on 224 children. The frequency of positive skin reactions was higher in the AR and asthma groups than in the control group (P<0.05). FEV1, the FEV1/forced expiratory vital capacity (FVC), and forced expiratory flow between 25% and 75% (FEF_25-75) were significantly lower in the asthma group than in the AR and control groups (P<0.0001). There were no significant differences in FEV1, FEV1/FVC, or FEF_25-75 between the AR and control groups.

BHR of the 3 groups as assessed by the MCT and FeNO levels are shown in Table 2. BHR was observed in 96% of the subjects in the asthma group and 12% of those in the AR group; it was significantly higher in the asthma and AR groups than in the control group (P<0.0001 and P=0.031, respectively). FeNO levels was higher in the asthma group than in the AR group (P<0.0001) and in the AR group than in the control group (P=0.005). In the total subjects, FeNO levels significantly correlated with PC20 levels (r=-0.337, P<0.0001) (E-Table 1).

BDR parameters as determined by spirometry and IOS in the asthma, AR, and control groups are shown in Table 3. The Δ FEV1 and % change in FEF_25-75 were higher in the asthma group than in the AR and control groups (P<0.0001). The difference in resistance, as measured in % change, between 5 and 20 Hz (Δ R5-R20) and the differences in R5 (Δ R5), R10 (Δ R10), X5 (Δ X5), and AX (Δ AX) were also higher in the asthma group than in the AR and control groups (the asthma vs. AR groups: Δ R5-R20, P=0.001; Δ R5, P<0.0001; Δ R10, P=0.006; Δ X5, P<0.0001; Δ AX, P<0.0001)(the asthma vs. control groups: Δ R5-R20, P=0.009; Δ R5, P=0.001; Δ R10, P=0.033; Δ X5, P<0.0001; Δ AX, P< 0.0001). The variables Δ X5 and Δ AX were the indices of BDR, which were more elevated in the AR group than in the control group (P=0.012; P=0.022).

Serum total IgE levels and blood eosinophil counts were significantly higher in the AR group with BHR than in the AR group without, as shown in E-Table 2 (P=0.026 and P=0.009, respectively). Skin tests were performed on 135 children of the AR group. There was no significant difference in positive skin reactions between the 2 groups. The AR group with BHR tended to have higher FeNO levels than the AR group without (P=0.08) (E-Table 2, Figure. A). FeNO levels significantly correlated with PC20 levels (r=-0.175, P=0.045) in the AR group (E-Table 1). Since Δ X5 and Δ AX were significantly higher in the AR group than in the control group, comparisons of these values were made between the AR group with BHR and the AR group without; however, no difference was statistically not significant (E-Table 2, Figure. B).